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Annals of Pathology and Laboratory Medicine December. 2016; Vol. 3, Issue 6 (Suppl)

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DOI : 10.21276/apalm

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Co-Editor-in-Chief Dr Prashant Goyal Dr Shelly Sehgal

Annals of Pathology and Laboratory Medicine Co-Editor in Chief

Dr Rajan Chopra King Fahad Hospital, Hufof, Saudi Arabia Dr Niti Singhal Abu Dhabi, United Arab Emirates Dr (Prof) Severino Rey Quiron Hospitals and Pontifical Catholic University, Ecuador Dr Rajeshwar Reddy Prof. & Head, Dept. of Microbiology, Gandaki Medical College, Pokhara, Nepal Dr Nasser Said-Al-Naief ODRP/ Anatomic Pathology, Loma Linda Medical Center, Loma Linda, CA, United States Dr Hoda A Hagrass Clinical Pathology dept, Faculty od Medicine Zagazig University, Sharkyia, Egypt Dr Kemal Turker UlutaĹ&#x; Kadirli State Hospital, Central Laboratory, Osmaniye, Turkey Dr Dennis P Oâ&#x20AC;&#x2122;Malley Pathologist, Clarient Pathology Services, Columbia, Aliso Viejo, CA, United States Dr Parthasarathi Pramanik Consultant Forensic Pathologist, Forensic Science Laboratory, Kingston, Jamaica Dr Arvind Rishi Asst. Prof., Dept of Pathology, Hofstra North Shore-LIJ School of Medicine, New York, United States Dr Ahmad Mohammad Ragab - Senior Consultant Pathologist, Kameda Hospital & Oncology Center - JAPAN - National Medical Institute, Egypt Dr Amado Ona Tandoc III Research Institute for Tropical Medicine, Muntinlupa City, Philippines Dr Shamim Sheikh Dept. of Pathology, M.P. Shah Medical College, Jamnagar, Gujarat, India Dr Viral M Bhanvadia Asst. Prof. Dept. of Pathology, Shri M.P. Shah Medical College, Jamnagar, Gujarat, India Dr Navin K Sinha Director-Lab, Artemis Health Institute, Gurgaon, India Dr Soumyesh Ghosh Dept. of Pathology, SDN Hospital, Delhi, India Dr Deepti Mittal Pathologist, Haryana, India Dr Amit Agravat Asso. Prof. Dept. of Pathology, PDU Medical College, Rajkot, Gujarat, India

Dr Prashant Goyal Director-Laboratory, Accuprobe Healthcare and Diagnostics, Delhi, India Dr Shelly Sehgal Specialist Pathologist, Department of Pathology, SDN Hospital, Delhi, India

Associate Editor

Dr Asitava Mondal Clinical Cytologist and Oncopathologist, Kolkata, West Bengal, India Dr Roque G. Wiseman Pinto Professor and Head of Dept. of Pathology, Goa Medical College, Bambolim, Goa, India Dr Sompal Singh Specialist Pathologist, Dept. of Pathology, N D M C Medical College & Hindu Rao Hospital, Delhi, India Dr Ruchika Gupta Pathologist (Scientist-C), Institute of Cytology & Preventive Oncology (ICPO), Delhi, India Prof. Vatsala Mishra HOD, Dept. of Pathology Moti Lal Nehru Medical College, Allahabad, India Dr Anil Parwani Vice Chair, Anatomical Pathology; Director of Pathology Informatics and Digital Pathology The Ohio State University Wexner Medical Center, Columbus, Ohio, United States Dr. Mohammad Zillur Rahman HOD & Associate Professor, Department of Pathology, Chittagong Medical College, Chittagong, Bangladesh Dr Manu Noatay Head Operations, Niche Theranostics, New Delhi, India Dr Manjusha Biswas Consultant Histopathologist & Oncopathologist, Bangalore, India, India Dr Mudit Agarwal Director Lab Services, Nishtha Pathology Lab, New Delhi, India Dr A S Ramaswamy Specialist Pathologist, Lifeline Hospital, Salalah, Sultanate of Oman Dr Harsh Vardhan Singh Senior Biochemist, N D M C Medical College & Hindu Rao Hospital, Delhi, India

Editorial Board Members

Dr Sarah Iqbal Ch Faculty of Pathology King Edward Medical University, Lahore, Pakistan Dr Jerad M Gardner Asst Prof, Pathology and Dermatology, University of Arkansas for Medical Sciences, Little Rock, AR, United States Dr Naila Atif Associate Prof., Histopathology, Central Park Medical College, Lahore, Pakistan

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Contents

Original Article Immunohistochemical characteristics of Breast Cancer patients with the comparative A490-A494 study of BRCA1, ER, PR, BCL2, P53 and Ki-67 immunohistochemical markers: A population based study. Manisha Sharma, Menka Khanna, Mridu Manjari, Manas Madan, Taranveer Singh, Tania Garg A study of histopathological changes seen in chronic plaque psoriasis, before and after treatment with narrow band Ultraviolet B Amoolya Bhat, Subramanya H, Murthy PS, Santosh KV Utility of Image Guided Fine needle aspiration in the Diagnosis Of Ovarian Masses: A Cytohistopathological Correlation Menka Khanna, Manisha Sharma, Mridu Manjari, Manas Madan, Kiranjot Kaur, Shruti Gupta Ki-67 and p53 immunohistochemical expression in prostate carcinoma: An experience from a tertiary care centre of North India. Harjot Kaur, Mohit Paul, Mridu Manjari, Sonam Sharma, Tejinder Singh Bhasin, Rahul Mannan p53 as a prognostic marker in carcinoma breast in correlation with conventional estrogen and progesterone hormone receptors Sanjay Piplani, Manas Madaan, Mridu Manjari, Rahul Manan Correlation of Fine needle aspiration cytology and Core Needle Biopsies in Breast carcinoma: Our experience Parul Garg, Harjot Kaur, Navtej Singh, Arun Puri, Ishwer Tayal Study of Glycated hemoglobin level in Non-Diabetic Iron deficiency anemia Bhavesh Ramji Faldu, Chetankumar Maganlal Dharaiya, Harshid L Patel Correlation Of Ki67 with ER, PR, Her2neu and other Prognostic Factors In Breast Carcinoma. Menka Khanna, Manisha Sharma, Mridu Manjari, Kiranjot Kaur, Saumil Garg, Saloni Goyal Expression of Hypoxia Inducible Factor-1Îą in Esophageal Squamous Cell Carcinoma and its correlation with clinicopathological parameters Manisha Sharma, Menka Khanna, Manas Madan, Mridu Manjari, Taranveer Singh, Komalpreet Kaur Evaluation of various discrimination indices in differentiating Iron deficiency anemia and Beta Thalassemia trait: A practical low cost solution. Sanjay Piplani, Manas Madaan, Rahul Mannan, Mridu Manjari, Taranveer Singh, Monika Lalit Role of autologous platelet rich plasma in treating symptomatic patients with osteoarthritis via double spinning technique Harjot Kaur Arora, Rohit Sharma, Mridu Manjari, Parampreet Singh Mean Platelet Volume In Diabetes Mellitus Type II Anupama Dayal, Sadhana Kothari, Rupal J Shah, S M Patel Evaluation of Fine needle aspiration cytology in the diagnosis of suspicious/gray zone lesions in breast lesions and its histopathological correlation Manas Madan, Manisha Sharma, Sanjay Piplani, Mridu Manjari, Neha Sharma, Saloni Goyal Comparative Study of Fine Needle Non-Aspiration and Fine Needle Aspiration in Cytological Diagnosis of Thyroid Lesions Manisha Sharma, Mridu Manjari, Manas Madan, Taranveer Singh, Akanksha Singh, Tarunpreet Saini Cytological-Radiological Correlation of Image Guided FNAC Of Hepatic Mass Lesions: Our Experience In Tertiary Care Centre Menka Khanna, Kiranjot Kaur, Manisha Sharma, Ashish Khanna, Mridu Manjari, Saumil Garg, Amritpal Kaur Distribution of genital HPV infection in women of reproductive age group in a north Indian city screened for cervical cancer by Pap smears Manas Madan, Sanjay Piplani, Manisha Sharma, Mridu Manjari, Akanksha Singh Cytohistologic correlation study of Thyroid lesion: insight into the causes of discordance Archana S Bhat, Hilda Fernandes, Leena Priya

III

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Case Report

Letter to editor

Importance of bone marrow examination in cases of pancytopenia: A morphological study. Rumpa Das, Gorakh Nath Evaluation of blood donor deferral causes: a retrospective study from South India Mourouguessine Vimal, Srinivasan Sowmya, Anandabaskar Nishanthi, Gandhi Ramya Role of Fine Needle Aspiration Cytology in Diagnosis of Epidermal Inclusion Cysts of Breast: A Clinical & Radiological Dilemma Richa Bhartiya, Sujata Mallick, Mahasweta Mallik, Nawanita Kumari, Ran Vijoy Narayan Singh Evaluation of platelet indices in Acute Coronary Syndromes and Diabetes Mellitus. Mathu Mithra, Roopa Urs, Shreya ., Bhavani S, Dhananjay Kotasthane Papillary Renal Cell Carcinoma Type-II: a distinct clinicopathological subtype of renal epithelial neoplasm Sunil Jagtap, Atul Beniwal, Swati Jagtap, Anil Huddedar A very rare occurrence of synchronous tumors of variable histology in bilateral ovaries Gokul Kripesh, Sandhya Sundaram, Palaniappan N, Naveen Kumar Case of Peutz-Jeghers Syndrome with Multiple Intussusceptions Prathipaa Rajavel, J Thanka, M Susruthan, Lawrence D Cruze A rare case of primary leiomyosarcoma of thyroid in a young adult with literature review Rushabh Jitendra Shah, Rachana Amit Chaturvedi, Leena Pravin Naik Signet Ring Cell Carcinoma Colon in A Twelve Year Old Child: A Case Report with Review of Literature Amita Jain Gupta, Jenna Blah Bhattacharya, Vijaya Vaishnav, Richa Gupta, Vivek Manchanda Primary fallopian tube carcinoma presenting as a pelvic mass: A rare case report. Gauri A Chavan, Abhijit G Bhanji, Chandralekha S Tampi Combined stromal smooth muscle tumour of endometrium: report of two cases Sakshi Sirswal, Charanjeet Ahluwalia, Sufian Zaheer, Amit Kumar Yadav, Ashish Kumar Mandal Pseudochylous Pleural Effusion in a case of Rheumatoid Arthiritis Harsh Malik, Priyageet Kaur Kalsi, Puneet Kaur Osteosarcoma of the talus: A rare location of the tumour with literature Thin Thin Win, NoorulBalqisChe Ibrahim, Wan FaishamNuâ&#x20AC;&#x2122;man Wan Ismail An unusual case of Nerve sheath myxoma with schwannomatous and perineural differentiation Smitha Surendra Masamatti, Aparna Narasimha, Jayalakshmi Valligari Janardhan Primary vocal cord histoplasmosis: a rare presentation impersonating laryngeal malignancy Malini Goswami Pilomatrixoma with extensive ossification: a rare case report Chidambharam Choccalingam, Vijayshree Raghavan

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Original Article Immunohistochemical Characteristics of Breast Cancer Patients with the Comparative Study of BRCA1, ER, PR, BCL2, P53 and Ki-67 Immunohistochemical Markers: A Population Based Study Manisha Sharma*, Menka Khanna, Mridu Manjari, Manas Madan, Taranveer Singh and Tania Garg Dept of Pathology, SGRDIMSR Amritsar, India Keywords: Breast Carcinoma, Immunohistochemistry, BRCA1, ER, PR, Ki-67

ABSTRACT Background: To correlate clinicopathological and immunohistochemical profile of BRCA1 positive and non BRCA1 breast cancer patients with ER, PR, BCL2, P53 and Ki-67 to gain more insight into biological characteristics of breast cancer to emphasise the need of genetic testing for BRCA1 in blood relatives of patients with BRCA1 mutation. Methods: The study was conducted in 70 randomly selected cases of breast carcinomas received in the Department of Pathology, SGRDIMSR, Amritsar. Clinical History was taken as per proforma and formalin fixed paraffin embedded tissue was studied for histopathological typing and grading after staining with haematoxylin-eosin. All cases were subjected to immunohistochemistry for BRCA1, ER, PR, P53, BCL2 and Ki-67 expression. Results: Grade II Tumors constituted the maximum (67%). The most common age group was 41-60 years (62%). BRCA1 positivity was seen in 27/70 cases (38%). BRCA1 positive cases tend to present at higher stage than BRCA1 negative cases showing significantly greater tumor size (p< 0.001) and lymph node involvement (p =0.001). Similarly BRCA1 positivity was associated with poor prognostic factors significantly as with high grade of tumor (p=0.015), hormonal receptors negativity (81.5% vs 18.5%, p <0.001) and high proliferative index (71% vs 29%, p<0.007), BRCA1 related cases had significantly high P53 positivity (67% vs 33%, p<0.008) and lower BCL2 expression (78% vs 2.2%, p <0.005) Conclusion: Our study proves that BRCA1 positive tumors have a higher grade and are associated with poor clinicopathological and immunohistochemical prognostic markers. Further studies are needed to justify more aggressive treatment in BRCA1 positive cases.

*Corresponding author: Dr Manisha Sharma, Associate Professor, Dept of Pathology, SGRDIMSR Amritsar, Punjab India Phone: +91 9876842942 Email: manisha_salwan@yahoo.com

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Sharma et al.

Introduction

Breast Cancer is the leading cause of death among women suffering from cancer. In the year 2000, there were about 796,000 new breast cancers diagnosed and about 314,000 deaths due to breast cancer around the world.[1,2] Earlier BRCA1 and BRCA2 were estimated to be responsible for 75% of familial breast cancers. However, recent data shows this percentage to be much less and depends upon the population studied.[2] It has been analysed that the cancer arising in carriers of mutation in BRCA1 differs from non BRCA1 mutation carriers. BRCA1 mutation positive carriers have poorly differentiated morphology with higher mitotic count and pleomorphism.[3] Immunophenotypically BRCA1 tumours are more frequently ER, PR negative, BCL2 negative, P53 positive and have high proliferation index (Ki-67 positivity).[4] Most familial breast cancers are not associated with BRCA1 mutation, so in an attempt to better define the clinical features and outcome of such patients it is important to define the immunohistochemical features and their relation to BRCA1 positivity to carry out genetic testing more effectively and to know the biological character of such tumours better. This study in 70 breast cancer patients has been done to attempt to gain insight better relationships of BRCA1 with other immunohistochemical markers like ER, PR, BCL2, P53 and Ki-67.

Material and Methods

Haematoxylin–Eosin (H&E) stained sections from randomly selected 70 formalin fixed breast cancer specimens received as mastectomy and lumpectomy specimens were taken and studied among all the breast cancer cases received over the period of one year (Nov 2013 –Oct 2014) in the Department of Pathology, Sri Guru Ram Das Institute of Medical Sciences and Research, Amritsar. The tumours were graded from grade I to grade III according to Nottingham Modification of BloomRichardson method taking into account the parameters – tubule formation, nuclear grading and number of mitosis/ HPF. The tumours were evaluated for the histological types, lymphocytic stromal response, nuclear chromatin pattern and nucleoli. Lymph nodes recovered were evaluated for the presence of metastatic deposits.

A-491 the wash buffer and Diaminobenzene tetrahydrochloride (DAB) was used as the chromogen. The endogenous activity was blocked by using hydrogen peroxide. After protein blocking, the slides were incubated overnight with the available ER, PR, BRCA1, BCL2, P53 and Ki-67 primary antibodies and were conjugated with streptavidin Horse Radish Peroxidase (HRP). The slides were counterstained with hematoxylin and were examined by light microscopy. ≥10% nuclei stained brown were taken positive for ER and even 1% stained were taken positive for PR as taken by other researchers in their studies.[5,6] For BRCA1 this value for positive stained nuclei was ≥ 30% as per other studies. [7,8] For BCL2 cytoplasmic staining in >1% cells was taken as positive. P53 immunostaining was characterized by the percentage of immunostained nuclei. >5% of nuclei stained were taken positive. Ki-67 was evaluated by the percentage of immunostained nuclei, <10% staining was scored low, 10-20% intermediate and >20% as high as done in the study by Jaramillo et al.[9]

Results

The patients age varied from 28-70 years with the maximum number of cases belonging to group 41-60 years (62% of patients). The size varied from 1-5 cms with maximum cases with the size >2cms (68%). All the tumours diagnosed were infiltrating ductal carcinoma NOS (not otherwise specified). Grade II cases were maximum consisting of 67% of cases followed by Grade III (28.5%) and then Grade I (4.5%). Lymph node involvement was seen in 39 cases out of 60 cases in which lymph nodes were recovered. BRCA1 positivity was significantly higher in grade II and grade III tumour (p=0.013). Similarly BRCA1 positive cases showed significant lymph node involvement as compared to BRCA1 negative cases (p=0.001). BRCA1 positivity was higher in cases where tumour size was >2 cms (p<0.001) (Table 1). 22 cases were ER+ and PR+ and 3 cases were ER+ and PR-. They were taken together as positive for ER PR (25 cases-36%). BRCA1 positivity was seen in 27/70 cases (38%). Out of 27 BRCA1 positive only 5 cases showed positivity for ER PR. Rest 20 positive ER PR cases were BRCA1 negative. Correlating ER PR expression with BRCA1 it was concluded that BRCA1 expression is significantly correlated with lower ER PR expression (p<0.001).

IHC was performed by using antibodies against the estrogen receptors (ER), the progesterone receptors (PR) (Diagnostic Biosystem) and BRCA1 (Biocare Medical), P53, BCL2, Ki-67 (Diagnostic Biosystem). The antigen retrieval was done by using pressure cooker method with 10mmol citrate buffer at pH 6. Tris buffer was used as

BCL2 positivity was observed in 37% (26/70) of the cases with percentage of cell positivity varying from 2671%. While correlating BCL2 expression with BRCA1 expression it was found that out of 27 BRCA1 positive only 6 cases were immunopositive for BCL2, therefore BRCA1 expression is associated with low BCL2 expression significantly (p<0.005).

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P53 positivity was observed in 40% cases (28/70). P53 expression was found to be directly correlated to BRCA1 expression. Out of 27 positive BRCA1 cases 18 were also positive for P53. BRCA1 expression was associated with increased P53 expression significantly (p<0.008). Ki-67 positivity was seen in 28 cases (40%). Out of 28 positive cases 19 cases of high and intermediate

proliferation rate were BRCA1 positive. So BRCA1 positivity was associated with increased proliferation index significantly. (p<0.007) (Table 2)

Discussion

Breast cancer is becoming number one cancer in Indian population like western world thus making cervical cancer as second common.[10] Breast cancer diagnosis,

Table 1: Pathological characteristics of the breast cancer patients tested for BRCA1 mutation analysis. Parameter

No mutation

Mutation

Histological Grade

Grade I Grade II Grade III

03 36 04

00 11 16

Lymph Node Status

N0 N1(1-3) N2(4-9) N3(>10)

20 03 07 03

01 07 14 05

Size

<2cm >2cm

17 26

05 22

Table 2: Immunohistochemical characteristics of the breast cancer patients tested for BRCA1 mutation analysis. Parameter

No mutation

Mutation

p-value

Estrogen And Progesterone Receptor Status

Positive Negative

20(46.5%) 23(53.5%)

05(18.5%) 22(81.5%)

<0.001

BCL2 receptor status

Positive Negative

20(46.5%) 23(53.5%)

06(22%) 21(78%)

<0.005

P53 receptor status

Positive Negative

10(23%) 33(77%)

18(67%) 9(33%)

<0.008

Ki-67 receptor status

Intermediate/ High 9(21%) Low proliferation/Negative 34(79%)

19(71%) 08(29%)

<0.007

Fig. 1: Immunohistochemistry showing IHC (400X) (A) ER positivity (Nuclear) (B) : P53 positivity (Nuclear) (C): BRCA1 positivity (Nuclear), (D): BCL2 positivity (Cytoplasmic).

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 06, (Suppl) December, 2016

Sharma et al. treatment and prognosis have been found to be associated with various morphological immunohistochemical and genetic variables. The morphological findings in BRCA1 positive cases were correlated to various studies conducted in past. 16/20 (80%) of grade III cases were BRCA1 positive as compared to 4/20 (20%) of grade III cases which showed BRCA1 negativity. This result is similar to previous studies which have shown that BRCA1 related cancers were of higher histological grade. [3,11] In a study conducted in Jewish women, 76.5% of BRCA1 related tumours had a higher nuclear grade as compared to only 23.5% of BRCA1 negative tumours.[11] The individuals with BRCA1 mutation were significantly less likely to present with stage1 disease. BRCA1 positive cases had higher number of lymph nodes involvement (p= 0.001) and tumour size >2 cms at the time of presentation (p<0.001). These results are in concordance with the results reported previously in the literature.[11,12] As in this study, morphological differences were significant among BRCA1 positive and BRCA1 negative cases, this study was an appropriate sample to compare various immunohistochemical variables as well. In our study BRCA1 positive cancers were less frequently ER PR positive as (22/27-81.5%) cases were ER PR negative. Thus BRCA1 expression was associated with lower ER PR expression significantly (p<0.001) correlating with various previous studies conducted where 60-85% of BRCA1 mutations were diagnosed ER PR negative as compared to 20-40% of BRCA1 non mutations.[12,13,14] BRCA1 positivity was associated with lower BCL2 expression as out of 27 BRCA1 positive cases only 6 had BCL2 expression as well (p<0.005). This finding is in accordance with the results of other studies conducted where BRCA1 was found to be associated with decreased expression of BCL2.[11,15,16] Intermediate and higher proliferation rate (Ki-67 score) was observed in 19/27 (71%) cases of BRCA1 related tumours (p<0.007). Similar results were observed by others researchers in their study where this percentage varied from 60-78%.[16,17] Our study revealed a higher positivity for P53 immunostaining in BRCA1 positive cases. Out of 27 positive cases 18 cases had positivity for P53, so associated with P53 expression significantly (p<0.008). Most previous studies have demonstrated a higher positivity of P53 in tumours in BRCA1 mutation carriers with significant correlation between two varying from 0.005 to 0.001.[11,15,16,17] www.pacificejournals.com/apalm

A-493 It was proposed that there is some correlation between BRCA1 and other markers at molecular level. BRCA1 mutation is followed by P53 dysfunction and cancer cells to be ER PR negative, therefore favoring some mechanism of interaction among BRCA1 and other molecular markers. [17,1819,20]

Several studies have proved the BRCA1 positivity relation with poor prognostic markers but have not demonstrated worse clinical outcome in the terms of five year relapse free survival, five year event free survival and five year overall survival. But the association of BRCA1 mutation with younger age of presentation and increased chances of development of contralateral breast cancer and ovarian cancer was proved.[21] In the above study, BRCA1 positivity showed a statistically significant association with poor prognostic and clinical variables such as high histological grade, higher stage at presentation, P53 positivity, Ki-67 proliferation index and ER PR and BCL2 negativity. Thus, proving that although BRCA1 positive tumours are heterogeneous from a genetic point of view, but they share common characteristics. Blood relatives of these patients should be screened for BRCA1 gene mutation as they may show 50% increased chances of its expression and 87% of life time risk for developing breast cancer.[22]

Conclusion

Our study proves that BRCA1 positive tumors have a higher grade and are associated with poor clinicopathological and immunohistochemical prognostic markers showing ER, PR and BCL2 negativity with high proliferation index (Ki-67 expression) and increased P53 expression. Whether the selection and delineation based upon morphological, immunohistochemical and molecular features not only justifies the aggressive treatment approach but also selection of candidate patient and blood relatives for BRCA gene studies is still a matter of debate. Further studies should address the treatment outcome difference in familial breast cancer cases (BRCA1 carriers than non BRCA1 carriers).

Abbreviations ER

- Estrogen Receptor

HPF - High Power Field HRP - Horse Radish Peroxidase PR

- Progesterone Receptor

Funding None

Competing Interests None Declared

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11. Palacios J, Honrado E, Osorio A, et al. Immunohistochemical Characteristics Defined by Tissue Microarray of Hereditary Breast Cancer Not Attributable to BRCA1 or BRCA2 Mutations: Differences from Breast Carcinomas Arising in BRCA1 and BRCA2 Mutation Carriers. Clin Cancer Res. 2003;9:606–14.

22. Ford D, Easton DF, Stratton M. Genetic heterogeneity and penetrance analysis of the BRCA1 BRCA2 genes in breast cancer families: The Breast Cancer Linkage Consortium. Am J Hum Genet. 1998;62:676-89.

21. Robson M, Gilewski T, Haas B, Levin D, Borgen P, Rajan P. BRCA-associated breast cancer in young women. J Clin Oncol. 1998;16:1642-9.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 06, (Suppl) December, 2016

Original Article A Study of Histopathological Changes Seen in Chronic Plaque Psoriasis, Before and After Treatment with Narrow Band Ultraviolet B Amoolya Bhat1*, Subramanya H2, PS Murthy3 and Santosh KV4 Department of Pathology, Sapthagiri Institute of Medical Sciences and Research Centre. India 2 Department of Pathology, Vydehi Institute of Medical Sciences and Research Centre. India 3 Department of Dermatology Bengaluru, Karnataka, India 4 Department of Pathology, Apollo Hospital, Bengaluru, Karnataka, India

Keywords: Histopathology, Narrow Band Ultraviolet B, PASI Score, Psoriasis

ABSTRACT Background: Psoriasis is a multifactorial chronic inflammatory skin disease characterized by infiltration of T cells in dermis and hyper proliferation of keratinocytes in epidermis. Narrow band ultraviolet B (NB UV-B) therapy utilizes the narrow band of UV light frequencies peaking around 311 – 313 nm. Very few histopathological studies have been done, especially on Asian skins which are predominantly that of Fitzpatrick type IV to V to assess the of effectiveness of narrow band ultraviolet B (NBUVB) for psoriasis. Methods: Severity of psoriasis was graded clinically using Psoriasis area severity index (PASI) score and the Success Rate and Effective Rate of UVB therapy were calculated. Two 4 mm punch biopsies were taken before and after therapy from the affected area of the skin of each patient. Each patient was given a histopathological score based on certain histological parameters such as parakeratosis, thickness of granular layer and mitotic figures. The difference between pre and post treatment histopathological score was calculated. The changes in histopathological parameters among the responders were compared with that of non responders. Result: Success rate of NBUVB therapy in our study was 91.52%. The mean (±SD) PASI scores before and after treatment were 12.44±2.23 and 2.83±4.49 respectively. The mean (±SD) histopathological score before and after treatment were 9.25± 0.44 and 1.81±1.93 respectively. These differences in the scores were found to be statistically significant (p<0.001). Conclusions: A significant difference was observed between the responders and non responders in the mean change in PASI score and histopathological scores. Percentage of parakeratosis, thickness of granular layer and mitotic figures per mm of the surface can be used as markers of progression to remission following therapy.

*Corresponding author: Dr. Amoolya Bhat, Assistant Professor, Department of Pathology, Sapthagiri Institute of Medical Sciences and Research Centre. Phone: +91 9480315066 Email: simpathologist@gmail.com

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

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Introduction

Psoriasis is a multifactorial skin disease with hereditary component that affects 1%-3% of world population. It is characterized by infiltration of T cells and hyperproliferation of keratinocytes in focal skin areas. Narrow Band Ultra Violet B (NB UV-B) is a modality of phototherapy that utilizes the Narrow Band of UV light frequencies peaking around 311 – 313 nm. It has immunosuppressive properties and also inhibits cutaneous delayed type of hypersensitivity responses to haptens. Very few authors have evaluated the histopatholgical changes after the NBUVB therapy for chronic plaque psoriasis in patients with Fitzpatrick skin type IV-V [1-4]. This prospective study aims to document the histopathological changes in lesions of chronic plaque psoriasis in the tertiary care centre before and after treatment with narrow band ultraviolet B (UVB) with 30 exposures spread over the period of three months.

Materials And Methods

Source of data: Consenting patients, clinically diagnosed to have chronic plaque psoriasis and willing to undergo NBUVB therapy at the Department of Dermatology of a teaching hospital in Bangalore, India, during the period April 2009 to March 2010, were the subjects for this study. Method of collection of data: This was a single centre, prospective, time bound study. The duration of study was one year from April 2009 to March 2010. Ethical clearance was obtained. The four clinical criteria used in confirming the presence of psoriasis were cutaneous plaques clinically suggesting psoriasis, bilateral symmetry of lesions, involvement of characteristic skin areas, presence of nail changes [5,6] Patients’ personal particulars such as age, sex, religion and history of presenting illness, past medical/ surgical history, treatment history were recorded. The inclusion and exclusion criteria were as follows: Inclusion criteria OO Consenting patients having chronic plaque psoriasis aged between 18 to 60 years with no co morbid illness or any medications. Exclusion criteria OO The patients with history of photodermatitis, e.g.: Geno photodermatoses, vitiligo, history of lupus erythematosus, dermatomyositis, melanoma and non-melanoma skin cancer, diabetes mellitus ,concomitant photo sensitising drugs and topical or systemic immunosuppressive agents, currently or in the recent past.

Pregnant women and patients who have been on any modality of therapy for psoriasis for preceding three months. Patients were assessed clinically thrice a week. After informed written consent, Psoriasis Area Severity Index (PASI) scoring was done as shown below [7,8]. a. Divide the body into four areas (A): Head (H), Upper extremities (U), Trunk to groin (T), and Legs to top of buttocks (L). b. Generate an average score (Scale 0-4) for the Erythema (E), Induration (I), and Scale (S) for each of the 4 areas. (0 = none, 1 = slight, 2 = mild, 3 = moderate, 4 = severe) c. Sum scores of Erythema (E), Induration (I), and Scale (S) for each area. i.e., Head (H) = (EH+IH+SH) Trunk to groin (T) = (ET+IT+ST) Upper extremities (U) = (EU+IU+SU) Legs to top of buttocks (L) = (EL+IL+SL) d. Generate a percentage for skin covered with psoriasis for each Area (A) and convert that to a 0–6 scale. (0 = 0%; 1 = <10%; 2 = 10–<30%; 3 = 30 – <50%; 4 = 50–<70%; 5 = 70–<90%; 6 = 90–100%). e. Multiply score of item (c) with item (d) above for each area and multiply that by 0.1, 0.2, 0.3, and 0.4 for head, arms, trunk, and legs, respectively. f. Add these scores to get the PASI score. i.e., PASI= 0.1(EH+IH+SH) AH + 0.3(ET+IT+ST) AT + 0.2(EU+IU+SU) A U + 0.4(EL+IL+SL) AL

PASI score from 0-72 was then calculated. A 4mm punch biopsy was taken from the active psoriatic lesions before treatment. Patients were treated with NBUVB as per guidelines recommended for their type of skin by the British Association of Dermatology. They were reviewed thrice a week before and after each phototherapy session and the dosage increased as per the guidelines till 30 sessions were completed. A repeat punch biopsy was taken close to the site of previous biopsy. Based on the clinical response to NBUVB, Patients were classified as “Good”, “Moderate”, “Mild” and “No Improvement” categories when they showed ≥90%,60% to 89%,25% to 59%,≤ 25% reduction in PASI scores respectively [Figure 1a&b]. “Effective rate” [9] = [(cases with very good improvement + cases with moderate improvement)/ total number of

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cases] × 100% and “Success rate” was calculated with the formula: Success rate = [Number of improved cases / total number of cases] X 100% The following histopathological findings were specially looked for and given score of (’’0 ‘‘) when absent or (”1”) when present. They include: Parakeratosis, Munro micro abscesses in keratin layers, spongiform pustules of Kogoj in epidermis, reduction or absence of granular layer, acanthosis, regular elongation of rete ridges and clubbing of their lower ends, thinning of suprapapillary epidermis, edema and distension of papillae, dilated tortuous capillaries, perivascular lymphocytes in dermis[Figure 1c] [10,11]. Ocular micrometer was used to measure the thickness of stratum malpighii, thickness of stratum granulosum, and average height of dermal papilla[12,13]. Thickness of stratum malpighii was measured starting from under surface of stratum corneum to stratum basalis at ten different high power fields in two separate sections lying at least 6 sections apart and an average was taken[12]. The height of the dermal papilla was measured starting from lowest point of rete pegs to the highest point on dermal papilla in

ten different high power fields in two histological sections lying at least six sections apart and an average was taken [12] . Estimation of mitosis: The mitotic figures were counted in all the layers of epidermis, in the histopathology sections which represented 3 mm of epidermal surface. Two sections lying at least six sections apart were utilized in determination of mitotic index. Then the average number of mitotic figures per millimeter of epidermal surface was calculated [Figure 1d][14]. Treatment Protocol: Patients were given 30 exposures of NBUVB over the period of three months, on thrice a week basis. The phototherapy unit was a whole body irradiation unit fitted with 18 Philips TL01/100W fluorescence lamps. The minimal erythema dose (MED) is first determined before treatment. All the patients belong to the Fitzpatrick skin type IV and V. The preferred MED test site was the upper back. The template was made of a thin sheet of leather with 8 windows of 2 x 2 cm with Velcro tapes stitched to the margin for manual closure of windows after each dose. Patients’ upper backs were irradiated after fixing this template using a test dose ladder of 250-1500 mJ/cm 2 (250, 500, 750, 1000, 1100, 1200, 1300 and 1500). The rest

Fig. 1: showing a) Pretreatment case of chronic plaque psoriasis, b) post treatment case of chronic plaque psoriasis,c) photomicrographs showing acanthosis, papillomatosis, edema of dermal papilla, Munroe Microabscess (arrow)(H&E;x100), d)increased suprabasal mitosis (H&E;x400).

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of the body was adequately protected from irradiation by using a protective gown and goggles.

Side Effects Observed After NBUVB Therapy: Itching was observed following NBUVB therapy in 2(3.38%) patients. None of the patients had a burn. The rest 57(96.61%) did not have any side effects.

MED were read at 24 hours after NBUVB exposure. A test was considered to have 1+ erythema if faint uniform erythema was present, but margination was not required. MED for NB-UVB varied from 500 to 1100 mJ/cm2 (average 893 mJ/cm2 ), and the median MED was 1000 mJ/cm2. According to the skin type, the MEDs were 7501100 mJ/cm2 in type IV skin (Median , 1000 mJ/cm2 ) and 500-1100 mJ/cm2 in type V skin (Median, 750 mJ/cm2 ) and the starting irradiation dose is set at 70% of MED[15]. The subsequent dosages were adjusted based on the level of erythema after previous treatment. Twenty percent dose increments were given with each subsequent exposure, till 30 exposures are completed over the period of 3 months. This was followed by re- biopsy at the same lesion. The histopathological changes were recorded as above and compared with pre treatment histological findings. Post treatment PASI score was calculated and compared with pre treatment PASI score. PASI change value was calculated by the formula [15] , PASI change value = [(pretreatment total score of PASI – post-treatment total score of PASI)/ pretreatment total score of PASI] ×100%. Statistical Analysis: The data collected was entered in the Microsoft excel sheet. All the statistical analyses was performed using SPSSV version 18(Statistical Package for Social Science) after importing data to SPSS. An (Alfa) error of 5% was considered to test the significance or p < 0.05 was considered as statistically significant. Comparison of study variables before and after treatment was done using Wilcoxson signed rank test (non-parametric test). Comparison of change produced after treatment in various study variables was done using an independent ‘t’ test.

Result

Histological comparison of pre & post treatment biopsy specimen and PASI score: In this study a statistically significant(p<0.001) improvement was noted following narrow band ultraviolet B therapy in PASI score, histopathological score, percentage of parakeratosis, number of mitotic figures per millimeter of surface, height of dermal papilla, thickness of granular layer and stratum malphigii[Table 1]. Patient categorization based on reduction in PASI score is shown in Table 2. Success Rate of UVB = [Total number responders/ total number of non responders] X 100 = { [54 / 59] X 100}= 91.52%. Effective Rate of UVB = [(number of patients with good improvement+ number of patients with moderate improvement) / total number of patients] X 100 = { [(29+23)/ 59] }X 100 = 88.1%.

Comparison of Responders and Non Responders: The patients were divided into two groups based on PASI change value as responders (those with more than 25% improvement in PASI score.) and non responders (those with less than or equal to 25% improvement in PASI score). There were 54 responders (29 cases with good improvement, 23 cases with moderate improvement, and 2 cases with mild improvement) and 5 non responders. Of the 54 improved cases 32 (59.3) were males and 22 (40.7) were females. Most of them were in 31-40 year age group. Only 5 cases were in 51-60 year age group. Most of these patients 28 (51.9%) had the disease for a duration of less than 10 years. Among the 5 non responders, there were 4 females and 1 male patient. Two patients were in the age group of 31-40 and one case each was there in remaining age groups. Two of the patients had the disease for more than 20 years, and 2 had it for less than 10 years. A significant difference was observed between the responders and non responders in the mean change value of following variables: PASI score, histopathological score, percentage parakeratosis, thickness of granular layer, thickness of the stratum malpighii , height of dermal papilla , mitotic figures per mm of the surface[Table 4]. The percentage change observed in the PASI score, histopathological score, percentage parakeratosis, thickness of granular layer, thickness of stratum malpighii, height of the dermal papilla have shown considerably good improvement in responders as compared to non responders and therefore can be taken as reliable markers predictive of good response to therapy. Percentage reduction in mitotic figures per mm of surface is unsuitable to be used as marker to predict progression towards resolution of psoriasis following NBUVB.

Discussion

This was a prospective study conducted to evaluate the histopathology in cases of chronic plaque psoriasis and to document the changes after therapy with narrow band UVB. Basic data such as age, sex, past history, treatment history, and family history were noted down. Clinical features such as pruritis, redness, exacerbating factors were noted. Baseline PASI score was calculated followed by a skin punch biopsy which was analyzed and compared with the post treatment biopsy. All the patients were treated with 30 exposures of narrow band ultraviolet B. Success Rate: Success rate of ultraviolet B therapy in the present study was 91.52%. The success rates were 90% and

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Table 1: Clinical and histological comparison of the pre & post treatment cases. Pre treatment biopsy Post treatment biopsy Improvement

Parameter

Mean

PASI score

12.44

2.23

2.83

4.49

9.61

2.94

Histopathological score

9.25

0.44

1.81

1.93

7.44

Percentage parakeratosis

68.39

19.37

5.95

17.85

Mitotic figures per mm

2.96

1.33

0.81

0.64

Thickness of stratum malpighii (µm)

448.27

123.77

140.80

Average height of dermal papilla (µm)

425.24

127.11

Height of granular layer in micrometer (µm)

5.05

9.16

Percentage change

p- value

Mean

<0.001

80.71

24.79

1.63

<0.001

81.04

18.90

62.44

20.72

<0.001

93.37

19.56

2.15

0.87

<0.001

73.04

11.85

77.93

307.47 117.29 <0.001

68.07

12.49

113.14

84.59

312.10 121.64 <0.001

73.32

13.86

37.66

16.06

32.61

86.03

24.21

16.10

<0.001

Table 2: Distribution of patients based on reduction in PASI score. Group Responders Non responders

Category

Number of cases

Percentage

Good improvement

49.15

Moderate improvement

38.98

Mild improvement

03.38

No improvement

08.49

100

Total Table 3: Side effects observed after NBUVB therapy. Side effects

Number of cases

Percentage

3.38

Itching Erythema

No side effects

96.61

Total

100

Table 4: Comparison of the responders and non responders based on improvement in clinical and histological parameters:. Mean change observed Sl.No: Parameter

Responders (n= 54)

Non responders (n=5)

Percentage change observed Responders (n=54)

Non responders p – value (n=5)

Mean

PASI score

10.34

1.76

0.62

87.23

12.64

10.24

3.78

<0.001

Histopathological score

7.89

0.69

2.60

0.55

86.13

8.74

26.00

5.48

<0.001

Percentage of Parakeratosis

65.63

18.50

28.00

8.37

99.16

2.98

30.87

9.62

<0.001

Mitotic figures per mm of surface

1.98

0.70

3.92

0.38

74.29

11.57

59.53

3.39

0.001

Thickness of stratum malpighii(µm)

314.80 119.54 228.40

39.89

70.88

8.55

37.80

6.26

< 0.001

Height of dermal papilla(µm)

320.85 123.11 217.60

39.13

76.67

8.48

37.08

6.01

<0.001

Thickness of granular layer(µm)

35.59

0.89

87.31

22.47

3.33

7.45

<0.001

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13.29

0.40

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86% respectively in the studies conducted by Green C and Dogra S [2] et al.

[16]

Reduction Observed in PASI Score: It has been observed in this study that the mean (±SD) PASI score before treatment was 12.44±2.23 and after treatment was 2.83±4.49 with a percentage reduction of 80.71 ±24.79. This difference in the PASI score before and after treatment was found to be statistically significant (p<0.001). Hiong and coworkers (2002) [1] treated 26 patients diagnosed with chronic plaque psoriasis with narrow band UVB therapy. The median pretreatment PASI score was 14.7 (range 4.8-30). At the end of therapy 68% patients achieved greater than 70% reduction in PASI scores. The median improvement after the completion of 20 exposures was 75% (means 71%; range 8-98%). Tanew A and colleagues (1999) [17] showed that the median pretreatment PASI score of 16 (range 6.2-23.4) was reduced by 84% to 2.5 (range 0-12.6) by narrow band UVB treatment and by 89% to 1.8 range (0-8.2) by PUVA treatment. Coven TR and coworkers [18] showed that clinical resolution of psoriasis was achieved in 86% cases treated with NBUVB. On an average disease severity scores following NBUVB were reduced by 38% after 1week, 66% after 2 weeks, 80% after 3 weeks, and 92% after 4 weeks. Picot E and colleagues (1992) [19] showed that the percentage reduction of psoriatic lesions was 78.5% with NBUVB.Thus the results of the current study are in concurrence with the results of these studies.

Highest thickness of stratum malpighii observed before the therapy was 708 micrometers and lowest value was 254 micrometers. The maximum height of dermal papilla before therapy was 687 micrometers. The highest of mitotic figures prior to therapy was 7.2 per mm of epithelial surface. The maximum percentage of parakeratosis before the treatment was 95%. The highest pretreatment thickness of granular layer was 38 micrometer and lowest value was 0 (Absent granular layer). Gordon M et al [20], studied histopathological sections from 100 untreated psoriasis cases and 3 normal patients which served as controls. On the sections from psoriatic skin, the thickness of stratum malpighii varied from 242 micrometers to 448 micrometers and average was 303 micrometers. The height of the dermal papillae varied from 151 to 408 micrometers. Average was 250 micrometers. On the sections from normal skin, the distance between under surface of stratum corneum and bottom of rete ridges varied from 73-105 micrometers. The height of the dermal papillae varied from 36 to 55 micrometers. Thus the results of this study are comparable to that of above mentioned studies.

Grades of Improvement: In the present study 29 (49.15%) cases showed good improvement, 23 (38.98%) cases showed moderate improvement, 2 (3.38%) cases showed mild improvement, and in 5 (8.47%) there was no improvement. The effective rate was 88.13%. Yuehua Y and colleagues (2010) [9] treated 73 patients with chronic plaque psoriasis with narrow band ultra violet B and found out the change in PASI score. In their study 11 (25.6%) cases showed good improvement, 25 (58.1%) cases showed moderate improvement, 5 (11.6%) cases showed mild improvement and 2 (4.7%) cases showed no improvement. In their study the effective rate was (83.7%). Thus the results of the present study are in concurrence with the above mentioned study.

The mean (±SD) thickness of stratum malpighii before treatment was 448.27 ± 123.77 micrometers and after treatment was 140.80 ± 77.93 with a percentage reduction of 68.07±12.4. The maximal thickness of stratum malpighii in the study conducted by Soltani K [21] et al was 600 micrometers, and at that level the height of the dermal papilla was 250 micrometers, thickness of granular layer was 10 micrometers, and mitotic count was 9.0. Walters I.B and coworkers (1999) [22] treated eleven patients with narrow band UVB for 6 weeks on a three times a week basis. Mean thickness of stratum malpighii before treatment was 242.53 micrometers and following narrow band therapy was 149.66 micrometers, percentage reduction was 36.83%. UV B induced lymphocyte reduction in dermis was 45.63%. Coven TR and colleagues(1997) [18]compared the therapeutic effectiveness of daily exposure to narrow band UVB and broad band UVB employing 22 patients with chronic plaque psoriasis. Mean thickness of stratum malpighii measured 265 +/- 13.4 micrometer before and 145 +/- 10.9 micrometer after NB UVB treatment. Thus the findings of this study are comparable to other studies mentioned above.

Histopathological Improvement: In the present study that the mean (±SD) histopathological score before treatment was 9.25 ± 0.44 and after treatment was 1.81 ± 1.93 with a percentage reduction of 81.04 ± 18.90. This difference in the score before and after treatment was found to be statistically significant (p<0.001).

In the present study that the mean (±SD) number of mitotic figures per millimeter of surface before treatment was 2.96 ± 1.33 and was 0.81 ± 0.64 after treatment with a percentage reduction of 73.04 ± 11.85. The mean (±SD) percentage of parakeratosis before treatment was 68.39 ± 19.37and after treatment was 5.95 ± 17.85 with a percentage reduction of

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93.37 ± 19.56. Cox A.J and Watson W (1972) [23] studied histological sections from 107 cases of psoriasis. Among the 10 patients who had 0-20% parakeratosis, average mitotic figures per millimeter of the surface was 1.7 and among the 35 patients with 91-100% parakeratosis average mitotic figures per millimeter of the surface was 8.7.

Reference

In this study the mean (±SD) height of dermal papilla was 425.24 ± 127.11 micrometers and after treatment was 113.14±84.59 with a percentage reduction of 73.32 ±13.86. The mean (±SD) thickness of granular layer was 5.05 ± 9.16 micrometers and after treatment was 37.664 ± 16.06 with a percentage reduction of 86.03 ± 24.21.

2. Dogra S, Kanwar AJ. Narrow band UVB phototherapy in dermatology Indian Journal of Dermatology, Venereology, Leprology, 2004;70:205-209.

Comparison of Responders and Non Responders: The clinical and histological parameters that can be used as reliable markers to identify the responders were: Percentage reduction in PASI score, percentage reduction in histopathological score, percentage reduction in percentage of parakeratosis, percentage reduction thickness of granular layer, percentage reduction in thickness of stratum malpighii, percentage reduction in height of dermal papilla. In all these parameters the improvement observed in responders was significantly more than that seen in non responders. Percentage reduction in mitotic figures per mm of surface showed considerable improvement even in non responders; therefore cannot be used as marker to predict resolution following NBUVB therapy.

Conclusion

Majority of the patients with chronic plaque psoriasis showed “Good Improvement” following the NB UVB therapy with a very high Success Rate of 91.52% and Effective Rate of 88.1%. A significant difference was observed between the responders and non responders in the mean change of following variables: PASI score, histopathological score, percentage of parakeratosis, thickness of granular layer and mitotic figures per mm of the surface. Therefore these can be used as markers of progression to remission following therapy.

Acknowledgements

We thank Dr. A K Jaiswal Professor, Department of Dermatology, VIMS & RC for the help and support provided throughout the study.

Funding None

Competing Interests None Declared

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1. Hiong HLT, Shih-Wee LK. Narrow band ultra violet B for psoriasis: A preliminary report of a prospective study performed at national skin centre. Bulletin for medical practitioners. 2002; 13(2). National Skin Centre.htm (5.1.2002)

3. Bedi TR. Clinical profile of psoriasis in North India. Indian J Dermatol Venereol Leprol 1995;61:202-5 4. Kaur I, Handa S, Kumar B. Natural history of psoriasis: a study from the Indian subcontinent. J Dermatol. 1997; 24:230-4. 5. Bedi TR. Clinical profile of psoriasis in North India. Indian J Dermatol Venereol Leprol 1995;61:202-5. 6. Farber EM., Bright RD, Nall ML. Psoriasis: A Questionnaire Survey of 2,144 Patients.1968; 98:248 -259. 7. Domyati M E, Barakat M, Abdel-Razek R, Expression of Apoptosis Regulating Proteins, P53 and Bcl-2 in Psoriasis. J Egypt wom Dermatol Soc, 2006; 3:46-50. 8. Bos JD. The pathomechanisms of psoriasis; the skin immune system and cyclosporine. British journal of dermatology, 1988; 18: 141-155. 9. Yuehua Y, Khalaf A T, Xiaoxiang Z, Xinggang W. Narrow-band ultraviolet B and Conventional UVB phototherapy in Psoriasis: A Randomised Controlled Trial. American Journal of Applied Sciences 2008; 5 (8): 905-908. 10. Velazquez EF, Murphy GF. Histology of the Skin. In:Elder D E, Murphy G. F, Elenitsas R, Johnson B.L, Xiaowei Xu editors. Lever’s Histopathology of the Skin: Tenth edition, Philadelphia: Lippincott Williams & Wilkins, 2008; pp: 8- 69. 11. Mckee P.H, Colonje E, Granter S, R. Pathology of the skin with clinical correlations. China: Elsevier Mosby, An imprint of Elsevier Limited, 3rd edition. Volume 1, 2005; pp:195-205. 12. Trozak D.J. Histologic grading system for psoriasis vulgaris. International Journal of Dermatology. 1994; 33:380-381. 13. Fry L, McMinn M.H. N. Observations on mitosis in psoriatic epidermis. British Journal of Dermatology. 1970; 82:19-22. eISSN: 2349-6983; pISSN: 2394-6466

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superior clinical and histopathological resolution of moderate to severe psoriasis in patients compared with the broad band UV-B. Archives of Dermatology 1997; 133 (12): 1514 – 22.

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19. Picot E, Meuinier L, Picot MC – Debeze, Peyron JL, Meynadier. Treatment of psoriasis with a 311 –nm UVB lamp. British journal dermatology1992; 127:509-512.

16. Green C, Lakshmipathy T, Johnson B, E, Ferguson J. A comparison of the efficacy and relapse rates of narrow band UVB (TL01) monotherapy vs. etretinate (re- TL-01) vs. etretinate –PUVA (re-PUVA) in the treatment of psoriasis. 1992; 127:5-9.

20. Gordon M, Johnson WC. Histopathology and histochemistry of psoriasis. Archives of dermatology 1967; 95:402-407. 21. Soltani K, Van Scott E.J. Patterns and sequences of tissue changes in incipient and evolving lesions of psoriasis. Arch Dermatol.1972; 106: 484 – 490.

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22. Walters IB, Ozawa M, Cardinale I, Trepicchio, Kruger JG. Narrowband UVB (312-nm) suppresses Interferon Gamma and Interleukin 12 and increases Interleukin 4 transcripts. Archives of dermatology.2003; 139:155-161.

18. Coven T R. Burack LH, Gilleaudeau R, Keogh M, Ozawa M, Krueger JG. Narrow band UV-B produces

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Annals of Pathology and Laboratory Medicine, Vol. 03, No. 06, (Suppl) December, 2016

Original Article Utility of Image Guided Fine Needle Aspiration in the Diagnosis of Ovarian Masses: A Cytohistopathological Correlation Menka Khanna*, Manisha Sharma, Mridu Manjari, Manas Madan, Kiranjot Kaur and Shruti Gupta Department of Pathology, SGRDIMSAR, Amritsar, India Keywords: Image Guided FNAC, Ovary, Neoplasms, Histopathological Correlation

ABSTRACT Background: The incidence of ovarian masses has increased over the past two decades and is one of the leading causes of cancer-related deaths globally. Although histopathology remains the gold standard, in recent times, imageguided aspiration is being increasingly used as a rapid, inexpensive and efficient method for the pre-operative diagnosis of ovarian masses. The present study was performed to evaluate the efficacy of image guided FNAC in diagnosis of ovarian masses in comparison with histopathology and to assess the limitations of cytological interpretation. Methods: The study was conducted on 92 cases of ovarian masses which were evaluated by image guided FNAC. Sensitivity, specificity and diagnostic accuracy were calculated using histopathology as gold standard. Results: Cytological diagnosis was obtained in all the 92 patients with ovarian masses followed by histopathological examination. The cytological diagnosis was grouped into 2 categories- Benign/Possibly Benign (60 cases) and Malignant/Suspicious of malignancy (32 cases). On cytohistological correlation it was found that a correct diagnosis was made in 86 cases, resulting in a diagnostic accuracy of 93.4%, sensitivity of 84.3 % and specificity of 98.3%. Conclusion: Image guided FNAC seems to be a relatively safe, simple, fast and cost-effective procedure where most ovarian malignancies can be correctly diagnosed with a high accuracy.

*Corresponding author: Dr. Menka Khanna, Department of Pathology, SGRDIMSAR, Amritsar, India Phone: +91 9464986355 Email: mona74_khanna@yahoo.co.in

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

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Introduction

Ovarian neoplasms are a heterogenous group comprising of benign and malignant tumors of epithelial, stromal and germ cell origin. [1] The incidence of malignant ovarian masses has increased over the past two decades and is one of the leading causes of cancer-related deaths globally. The majority of ovarian masses are benign but almost two-thirds of malignant ovarian tumours present at an advanced stage. [2] Fine-needle aspiration cytology (FNAC) of these ovarian masses is offer the potential to decrease the need for surgical procedures in these women. The use of radiological techniques can ensure greater accuracy and reduction in false negativity in the assessment of tumors by increasing the yield of cytological specimens. Differing from blind procedure, ultrasound or CT guided FNAC can localize the most suspicious areas in tumor mass and therefore help in taking the sample from the most representative site. [3,4,5] Geier and StreckerÂ have suggested that FNAC should be used for (1) recurrent and metastatic tumors, (2) suspected benign ovarian cysts and (3) when the patientâ&#x20AC;şs condition is unsuitable for laprotomy. [6] Â However, till today, gynaecologists all over the world are hesitant to accept the role of FNAC on pelvic masses because of the controversial opinion about the potential risk of intraperitoneal tumor implantation, particularly of ovarian tumors, although the risk of carcinoma cell seeding within the abdominal cavity due to contamination by needles is overestimated and has not been documented. [7] Although histopathology remains the gold standard, in recent times, image-guided aspiration is being increasingly used as a rapid, inexpensive and efficient method for the pre-operative diagnosis of ovarian masses.[8 ]The present study was performed to evaluate the role of image guided FNAC in pre-operative cytological diagnosis of ovarian masses in comparison with histopathology and to assess the discrepancies and limitations of cytological diagnoses.

Materials and Methods

The study was conducted on 92 patients who presented with the ovarian masses diagnosed clinically or radiologically. Following the clinical examination, informed consent was taken from the patients. USG/CT guided FNAC was done using 22-23 gauge needle attached to a 20 ml syringe. Smears were prepared from the aspirate, fixed in 95% alcohol and stained with May Grumwald Giemsa and Hematoxylin and eosin stains. In the cases where cyst fluid was aspirated, it was subjected to cytocentrifugation and the sediment was stained by the similar methods. No major complications were observed in any of the patients. The cytological diagnosis was compared with histological diagnosis. Taking histological diagnosis as the gold standard, various statistical calculations were done.

Result

A total of 92 cases of ovarian mass lesions were evaluated on cytological smears. The most common presenting feature was lower abdominal pain (80.5%), followed by abdominal distension (50%), menstrual irregularity (27.7%) and weight loss (8.3%). The age of the patients range from 18 to 65 years, with maximum patients in 4th decade of life. USG / CT helped in the assessment of type of mass lesion (cystic/solid) along with its size, location and extent and thus augmented the cytological diagnosis. The cytological diagnoses were made as benign/possibly benign and malignant/suspicious of malignancy. Considering the histopathological diagnosis as gold standard, the cyto-histopathological correlation was done in these cases. On histopathology 60 cases were diagnosed as non malignant, out of which 8 cases were of non-neoplastic cysts and 52 cases were of benign ovarian tumors. Serous cystadenomas were the most common (27) benign ovarian tumor followed by mucinous cystadenoma (14) , mature teratoma (8) and fibroma (2).There was one case each of thecoma and sclerosing stromal tumor.( Table 1). A histopathological diagnosis of malignancy was given in 32 cases. Serous cystadenocarcinoma was the most common (16) histopathology diagnosis among malignant lesions followed by mucinous cystadenocarcinomas (5) and serous borderline tumor (3). There were 2 cases each of mucinous borderline tumor, dysgerminoma and granulosa cell tomor and one case each of immature teratoma and mixed germ cell tumor. (Table 2) Mature teratomas on histopathology show cyst lined by keratinising stratified squamous epithelium along with differentiated glandular epithelium,neuroglia and other elements.(Figure 1) However in immature teratoma islands of neuroepithelium were clearly identified. The sections in cystadenomas showed cysts lined by single layer of columnar epithelium which was ciliated in serous tumors, where as in borderline cases complex papillary structures lined by epithelium with stratification and nuclear atypia were seen; clear cut stromal invasion was identified in cystadenocarcinomas. (Figure 2) Sclerosing stromal tumor showed lobular arrangement with alternate hyper and hypocellular areas comprised of oval to spindle shaped cells with many dilated blood vessels in hypocellular areas. The sections in granulosa cell tumor showed sheets of oval to spindle cells with nuclear grooves and forming Call Exner bodies. (Figure3) FNAC from serous cystadenoma mostly yielded straw coloured fluid and the smears prepared

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from the centrifuged deposits showed papillary fragments with bland nuclei and cyst macrophages. Aspirates from benign cystic teratoma showed mature squamous cells in a dirty background (Figure1 ). Mucinous cystadenomas showed isolated clusters of columnar epithelial cells with basally placed nuclei against mucinous background. Serous cystadenocarcinoma showed papillary fragments comprising of hyperchromatic pleomorphic cells having high N/C ratio (Figure2). The mucin producing cells with malignant nuclear features against mucinous background were diagnosed as mucinous cystadenocarcinoma. Cytology of granulosa cell tumor comprised of uniform round nuclei with microfillicle formation and nuclear grooving (Figure3 ).

including 3 cases of borderline tumors, one case each of mucinous cystadenocarcinoma and immature teratoma) were reported to be falsely negative for malignancy. In borderline tumors the smears showed few papillae lined by cells lacking much of atypia, conspicuous nucleoli or mitotic figures.(Figure.4) One case of mucinous cystadenocarcinoma on cytology showed mainly mucin and scattered epithelial cells and the solid area of the tumor was not sampled adequately. The cytological smears in immature teratoma just showed few squamous and round to oval cells along with eosinophilic agranular material. The smears in case of sclerosing stromal tumor showed atypical oval to spindle cells along Call-Exner bodies like structures.

Of the 60 cases which were histopathologically diagnosed as non-neoplastic cysts (8) or benign neoplasms (52), 59 cases were diagnosed as benign or possibly benign on cytology and one case of sclerosing stromal tumor was falsely reported to be suspicious of malignancy. Of the 32 cases diagnosed as malignant tumors a cytological diagnosis of malignancy was made in 27 cases and 5 cases(

For all the 92 cases, the sensitivity and specificity of cytological diagnosis considering the histopathological diagnosis as gold standard were 84.3 % and 98.3% respectively with overall diagnostic accuracy of 93.4 %. The positive predictive value and negative predictive value were 96.4% and 92.1% respectively.

Table 1: Comparative analysis of confirmed benign ovarian lesions (on histopathology) with their corresponding cytological diagnosis. Cytological Diagnosis Benign/Possibly benign False positive for malignancy

Histopathological Diagnosis

Non Neoplastic Cysts

Benign Neoplasms

Follicular cyst ( n=4)

Endometriotic cyst (n=2)

Corpus luteal cyst (n=2)

Fibroma (n=2)

Thecoma (n=1)

Sclerosing stromal tumor (n=1)

Serous cystadenoma (n=27)

Mucinous cystadenoma (n=14)

Benign cystic teratoma (n=8)

Table 2: Comparative analysis of confirmed Malignant ovarian lesions (on histopathology) and their corresponding cytological diagnosis. Histopathological diagnosis

Cytological diagnosis Malignant / suspicious of malignancy

False negative for malignancy

Serous cystadenocarcinoma (n=16)

Mucinous cystadenocarcinoma (n=5)

Serous borderline tumors (n=3)

Mucinous borderline tumors (n=2)

Dysgerminoma (n=2)

Mixed germ cell tumor (n=1)

Immature teratoma (n=1)

Granulosa cell tumor (n=2)

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Table 3: Correlation of cytological diagnosis with histopathological diagnosis in ovarian lesions Histopathology

FNAC Malignant

Benign

Total

Benign

Total

Malignant

Fig. 1: Benign cystic teratoma; benign squamous cells against a dirty background of keratinous debris (MGG100X) with inset showing cyst lined by keratinizing suamous epithelial cells ( H and E-100X).

Fig. 2: Papillary fragments lined by malignant glandular cells.(MGG 400X)Inset shows histopathology withcomplex papillary structures lined by anaplastic cells along with stromal invasion.(H and E 100X).

Fig. 3: Granulosa cell tumor;sheetsof monomorphic cells with increased nucleocytoplasmic ratio and nuclei with finely granular chromatin forming microfollicular pattern.(MGG400X) Inset shows histopathology sections monomorphic cells forming Call-Exner bodies (H and E -100X).

Fig. 4: Scanty cellularity in case of borderline serous tumor showing scattered groups of small epithelial cells with mild nuclear atypia. (MGG-100X) Inset showing histopathology of borderline serous tumor with hierarichal pattern of papillae lined by atypical cells. (H and E -400X).

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Discussion

detect with needle aspiration. Tumors of low malignant potential or borderline tumors were difficult to diagnose with accuracy on cytological examination and often could not be clearly distinguished from well-differentiated cystadenocarcinomaÂ or even cystadenomas, as was seen in 3 of our cases. [12,13,15,16] This category of ovarian neoplasms constitutes a grey zone and is subject to inter-observer variations. Histopathology is a pre-requisite for assessing the presence or absence of stromal invasion and for the sub-typing of a tumor as borderline. [1]

Aspiration cytology has been widely used for both primary diagnosis of ovarian lesions and the follow up of recurrent malignancies. Developing radiologic guidance techniques have also contributed to the higher accuracy of FNAC in recent years. [8] Due to complexity of lesions and wide spectrum of diagnoses, the cytological diagnosis of ovarian lesions is a challenging process. However, by careful evaluation of the cytoarchitecture and background features, the differentiation into benign and malignant lesions is still possible. [2] In many cases, the pinpoint diagnosis can also be made. The critical issue of this procedure is that FNAC can lead to rupture and spillage of tumor cells into peritoneal cavity and can potentially cause upstaging of malignant tumor. [7] The magnitude of rise of tumor spillage is unknown and not substantiated by convincing evidence as most of the malignant ovarian tumors present in an advanced stage, the threat of tumor dissemination seems less important when weighed against the effective and economical diagnostic tool available in the form of FNAC. [4] The sensitivity and specificity of cytology in the diagnosis of ovarian masses in our study was 84.3% and 98.3% with overall diagnostic accuracy of 93.4%. Our observation corroborate closely with other investigations, indicating that FNAC can have appreciable sensitivity, specificity and accuracy in the diagnosis of ovarian masses. Gupta and Rajwanshi found a sensitivity of 85.7% and a specificity of 98.0%. [9] Cole and co workers found FNAC to be highly specific (100%) but conversely with a very low sensitivity of 50%. [10] Ganjei et al and Roy et al in their study found sensitivity and specificity of cytology in diagnosis of ovarian lesions as 94.2%, 91.4%, and 75% , 100% respectively. [11,12]Nazoora et al reported sensitivity of 79.25%, specificity of 90.6% and overall diagnostic accuracy of 89.9%. [13] The overall diagnostic accuracy in this study was 92.8% compared to overall diagnostic accuracy of 96% described by Moran et al. [14] Wojcik & Selvaggi also reported that the majority of cystic ovarian lesions can be diagnosed accurately; however, they did not correlate FNAC with histology in 53% of their cases. [5] Several factors may explain the erroneous diagnosis seen in 5 of our cases. Many tumors ovarian cyst fluid may have an inadequate number of cells to accurately assess. Second, malignant cells in an ovary may not be uniformly distributed in the ovary. The number of malignant cells within ovarian cysts may be insufficient or absent to www.pacificejournals.com/apalm

Only one false positive result was reported in our study and thus have a high positive predictive value. However, among benign tumours only, one case of Sclerosing tumour Stromal tumour was falsely reported to be suspicious of malignancy on cytology as it showed atypical oval to spindle cells along with formation of Call Exner bodies like structures. Similar findings were also reported by other authors. [17] Moreover, radiological findings in this tumour were suggestive of malignancy because of both cystic and solid areas. Other researchers have also reported incapability of USG and CT in identifying this particular tumour as benign .[18] Our study and the view of other experts suggest that FNAC is more specific than sensitive in detecting ovarian malignancy and therefore, its use, as a reliable initial diagnostic test, cannot be overemphasized. The cytopathologists should be aware of the potential diagnostic pitfalls and the interpretational errors that can be reduced further, if the aspirates are obtained from different portions of the mass with the use of proper radiological guidance,with expert cytopathologists to perform and interpret the aspirates, and with the use of immunohistochemical and molecular markers.

Conclusion

To conclude the image guided FNAC despite the potential disadvantages, when combined with the available, clinical, radiological and laboratory findings, is a quick, easy, fairly sensitive, specific and cost effective modality for the preoperative diagnosis of malignant as well as benign ovarian masses with minimal morbidity. Accurately identifying borderline tumours and false negative cytological analysis due to low cellularity or secondary degenerative changes may be its limitations but it has high positive predictive value.

Abbreviations CT

- Computed Tomography

FNAC - Fine Needle Aspiration Cytology USG - Ultrasound eISSN: 2349-6983; pISSN: 2394-6466

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Acknowledgements

ovarian tumors: An assessment of diagnostic efficacy. J Cytol 2010;27(3):91-5.

None

Funding None

Competing Interests None Declared

Reference

1. Goel S, Agarwal D, Goel N, Naim M, Khan T, Ekrammulah. Ultrasound Guided Fine Needle Aspiration Cytology In Ovarian Neoplasms: An Assessment Of Diagnostic Accuracy And Efficacy And Role In Clinical Management. Int J Pathol 2010;11(2):1-6. 2. Uguz A, Ersoz C, Bolat F, Gokdemir A, Vardar MA. Fine needle aspiration cytology of ovarian lesions. Acta Cytol 2005;49:144–8. 3. Hajdu S, Melamed MR. Limitations of aspiration cytology in the diagnosis of primary neoplasms. Acta cytol 1984;28:337-45. 4. Ray S, Gangopadhyay M, Bandyopadhyay A, Majumdar K, Chaudhury N. USG guided FNAC of ovarian mass lesions: A cyto-histopathological correlation, with emphasis on its role in pre-operative management guidelines. J Turk Ger Gynecol Assoc 2014; 15(1):6–12. 5. Wojcik EM, Selvaggi SM. Fine needle aspiration cytology of cystic ovarian lesions. Diagn Cytopathol1994;11:9-14. 6. Geier GR, Strecker JR. Aspiration Cytology and E2 content in ovarian tumors. Acta Cytol 1981;25:400-6. 7. Kreuzer GF, Paradowski T, Wurche KD, Flenker H. Neoplastic or Nonneoplastic ovarian cyst? The role of cytology. Acta Cytol 1995;39:882–6. 8. Mehdi G, Maheshwari V, Afzal S, Ansari HA, Ansari M. Image guided fine needle aspiration cytology of

9. Gupta N, Rajwanshi A, Dhaliwal LK, Khandelwal N, Dey P, Srinivasan R, Nijhawan R. Fine needle aspiration cytology in ovarian lesions: an institutional experience of 584 cases. Cytopathology 2012;23:300–7. 10. Cole L, Mount S, Nuzzo E, Wong C. Aspiration cytology of ovarian cystic masses: histologic correlation and review of the literature. Acta Cytol 2011;55:19–25. 11. Ganjei P, Dickinson B, Harrison TA, Nassiri M, Lu Y. Aspiration cytology of neoplastic and non-neoplasic ovarian cysts: Is it accurate? Int J Pathol1996;15:94–101. 12. Roy M, Bhattacharya A, Roy A, et al. Fine needle aspiration cytology of ovarian neoplasms. J Cytol 2003;20:31–5. 13. Khan N, Afroz N, Aqil B, Khan T, Ahmad I. Neoplastic and nonneoplastic ovarian masses: Diagnosis on cytology J Cytol 2009;26(4):129–33. 14. Moran O, Menczer J, Gijlad BB, Lipitz S, Goor E. Cytologic examination of ovarian cyst fluid for the distinction between benign and malignant tumors. Obstet Gynecol 1993;82:444–6. 15. Nadji M, Greening SE, Sevin BU. Fine needle aspiration cytology in gynaecologic oncology II, morphologic aspects. Acta Cytol 1979;23:380-8. 16. Kjellgren O, Angstrom T, Bergman F. Fine needle aspiration biopsy in diagnosis and classification of ovarian carcinoma. Cancer1971;28:967-76. 17. Banik T, Gupta N, Rajwanshi A, Dhaliwal LK. Fine Needle Aspiration Cytology in Sclerosing Stromal Tumour of Ovary. A series of 3 cases. Diagnostic Cytopathology 2012:40(4); 342-5. 18. Joja I, Okuno K, Tsunode M et al. Sclerosing Stromal Tumour of Ovary. US, MR and Dynamic MR Findings. J Comput Assist Tomo 2001: 25(2);201-6.

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Original Article Ki-67 and p53 Immunohistochemical Expression in Prostate Carcinoma: An Experience from a Tertiary Care Centre of North India

Harjot Kaur1, Mohit Paul2, Mridu Manjari1, Sonam Sharma3*, Tejinder Bhasin1 and Rahul Mannan1 Department of Pathology, Sri Guru Ram Das Institute of Medical Sciences and Research, Amritsar, Punjab, India 2 Civil Hospital, Garhshankar (Hoshiarpur), Punjab, India. 3 Department of Pathology, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India

Keywords: Prostate Carcinoma, p53, Ki-67, Punjab, Significance

ABSTRACT Background: Carcinoma of the prostate poses a considerable medical and public health challenge in many parts of the world. Despite advances in screening and multimodal management of this disease, overall survival remains poor. The need to identify tumor markers as prognostic indicators and as targets for new therapeutic strategies, still remains a major challenge in prostate carcinoma research. Material: The study was conducted on 50 histopathologically proven cases of prostate carcinoma received in the Department of Pathology, Sri Guru Ram Das Institute of Medical Sciences and Research, Amritsar, Punjab, India. The aim was to evaluate the immunohistochemical expression of Ki-67 antigen and p53 protein in prostate carcinoma and to find their correlation with other clinicopathological parameters. The results obtained were tabulated and statistically analyzed. A p value less than 0.05 was considered statistically significant. Results: Maximum cases (74%) were observed in the age group of 61 â&#x20AC;&#x201C; 80 years. The most common pattern of Gleason grade seen was ( 5+4) constituting 32% of the total cases. The most common Gleason score seen was 7 - 8 constituting 48% of the total cases. The Ki-67 positivity was observed in 80% of cases with percentage positive cells varying from 3-84% with moderate and strong staining intensity. It was observed that with increase in the grade and score the number of cases showing positivity also increased but no statistical significance was seen with the same. The p53 positivity was observed in 58% of cases with percentage positive cells varying from 5-80% with mild, moderate and strong staining intensity. It was observed that with increase in the grade and score the number of cases showing positivity also increased but no statistical significance was seen with the same. 24 cases (48.0%) were positive for both Ki-67 and p53. Of the 24 cases of intermediate grade tumors positivity for both Ki-67and p53 was noted in 11 cases (45.8%) but no statistical significance was observed with increase in grade and score. Conclusion: The present study highlights that the immunohistochemical expression of Ki-67 and p53 should be assessed in all the cases of prostate carcinoma as these markers allow identification of tumors with a higher rate of cell growth. They also permit development of prognostic factors as their expression increases with increase in the grade and these patients can be benefited with the appropriate targeted treatment leading to increase in the survival time.

*Corresponding author: Dr. Sonam Sharma, B-5, Varun CGHS Ltd., Plot No. GH-03, Sector - 52, Gurgaon - 122003, Haryana, India. Phone: +91 9999841393 Email: drsonamsharma@gmail.com

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Ki-67 and p53 Expression in Prostate Carcinoma

Introduction

Prostate carcinoma is a global health problem and the second leading cause of cancer related deaths in the western world with the consensus reaching to nearly the same level in the Indian subcontinent. Its incidence is continuously rising with over 200000 new cancer cases and 35000-40000 deaths per year.[1] Despite advances in screening and multimodal management of this disease, overall survival remains poor. Hence, there is a need to identify various prognostic markers for developing new therapeutic strategies for better management of prostate carcinoma patients. There are various benign mimickers of prostate carcinoma such as benign hyperplasia, prostatitis, atrophy, adenosis, atypical adenomatous hyperplasia (AAH) and nephrogenic adenoma, which makes the diagnosis of prostate carcinoma challenging.[2]Therefore, many pathological parameters are required for its proper assessment. Among them, Gleason grading of the cancer is the most widely used, and accepted histopathological method for providing information about the prognosis of prostate carcinoma. This grading system is based entirely on the histological pattern of differentiation and arrangement of carcinoma cells and cell groups in Hematoxylin and Eosin (H&E) stained sections. [3] Other being , Prostate-specific antigen (PSA),which is the most useful tumor marker in the diagnosis of prostate carcinoma[4] but suffers from many fallacies such as borderline levels and also supuriously high levels in many reactive/ inflammatory conditions. Therefore, IHC plays a major role besides PSA and the important tumor markers are p53, Ki-67 and Bcl-2 to predict the prognosis and to decide the treatment modalities. p53 is a tumor suppressor gene, mutations of which can result in uninhibited cellular growth and have been implicated in numerous malignancies.[5] In most human cancers, its increased immunohistochemical expression is associated with point mutations in one allele of p53 gene and loss in the other. Thomas et al and Shurbaji et al evaluated the immunohistochemical detection of p53 protein in prostate cancer and its utility as a prognostic indicator. They concluded that mutations of p53 gene, which have long half-life, are involved in carcinogenesis of prostate cancer, and that p53 reactivity marks an aggressive subset of prostate cancer.[6,7] Ki-67 is one of several cell-cycle-regulating proteins, which can be demonstrated by IHC.[8,9] It is a DNA-binding protein, which is expressed in all phases of cell cycle but undetectable in resting cells.[10,11] McLoughlin et al studied that Ki-67 index (fraction of Ki-67 positive nuclei in IHC) was higher for carcinomas than for hyperplastic glands

while within the group of carcinomas, Ki-67 indices in patients with metastatic disease were significantly higher than in those without metastasis and that high Ki-67 index could define a group of patients with poor prognosis.[10] Borce et al showed that the accumulation of p53 had a special correlation with patientâ&#x20AC;&#x2122;s survival in their studied population. The presence and activity of p53 was greatly associated with the cell proliferation marker Ki-67 and the level of p53 activity was an important independent prognostic factor that was inversely associated with patient survival.[12] The present study was conducted at a tertiary care teaching hospital at Punjab (North India) to assess the expression of Ki-67 and p53 in histologically proven cases of carcinoma prostate and to evaluate any correlation between the two. An attempt was also made to correlate the expression of both the markers with other histopathological parameters such as grading and scoring.

Material and Methods

50 cases of carcinoma prostate diagnosed in needle biopsies and prostatic chips at the Department of Pathology, Sri Guru Ram Das Institute Of Medical Sciences And Research, Amritsar, Punjab, India were included in the present study. Detailed clinical data of the patient was recorded. Routine histopathological processing was done, followed by staining with H & E. The slides were observed under the light microscope, and Gleason scoring was done using the 2005 ISUP (International Society of Urological Pathology) Modified Gleason Scoring System. IHC was done on formalin fixed and paraffin embedded 4 micron meter sections of representative blocks of each tumor and were mounted on poly-lysine coated slides. Antigen retrieval was done in a pressure cooker using sodium citrate buffer solution at pH 6.0. Peroxidase inhibition was then done, followed by washing in tris buffer saline, and protein block. To evaluate for p53 expression, the primary antibody used was CME298BK and for Ki-67 the primary antibody used was CRM325B; both procured from Biocare Medical, India. Positive and negative controls were run with every batch. For both p53 and Ki-67: Brown nuclei were taken as positive [Figure 1and 2]. Following scoring pattern was utilized. SCORING of p53 Percentage positivity <5 % 5â&#x20AC;&#x201C;25 % 25â&#x20AC;&#x201C;50 % >50 %

Score 0 1 2 3

Staining intensity Nil Mild Moderate Severe/Strong

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Fig. 1: a) Closely packed and irregularly separated glands (Pattern 3a) [H&E;200X]; b) Raggedly outlined fused glands (Pattern 4a)[H&E;100X] , Inset : Hypernephroid pattern with large pale cells ( Pattern 4b)[H&E;400X]; c) Comedocarcinoma (Pattern 5a) [H&E;100X]; d) Tumor cells showing strong intensity (3+) p-53 expression [IHC; 400X].

Fig. 2 : a) Diffuse growth pattern [H&E;400X]; b) Tumor cells showing moderate intensity Ki-67 expression [IHC; 200X]; c) Mild intensity nuclear expression of Ki-67 by the tumor cells [IHC; 200X]; d) Strong intensity Ki-67 immunoexpression [IHC; 200X].

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Index for Ki-67 +1=Index less than equal to 25% +2=Index between 26-50% +3=Index between 51-75% +4=Index between 76-100% Statistical Analysis: The relationship between IHC expression and clinicopathological parameters was compared using Chi Square test/ Fisher’s exact test wherever applicable. A value of p <0.05 was considered statistically significant.

Results

The majority of the patients were in the age group of 61-80 years constituting 74% of the total. The youngest patient was 55 years old and the eldest was 88 years. All the cases were adenocarcinoma with Gleason score varying between 6 and 10.The majority of patients had Gleason score of 7-8 constituting 48% of the total while the least common score was 6 constituting 10% of the total. Ki-67: 40 cases were positive out of the fifty cases and 10 cases were negative for Ki-67 constituting 80% and 20% respectively. The percentage of positive cells varied between 3% to 84%. The staining intensity varied from moderate to strong with 26 cases showing strong staining intensity.

score the number of cases showing positivity also increased but it was not statistically significant (p value=0.989) (TABLE 2). p53: Out of the total 50 cases p53 was positive in 29 cases and negative in 21 constituting 58% and 42% respectively. The percentage of cells positive varied between 5% and 80%.The staining intensity varied from mild to strong with 18 cases showing moderate intensity. Correlation of Gleason’s grade with p53 expression: Of the 16 cases of most common pattern (5+4), 10 were positive for p53 and 6 were negative for p53. It was observed as the grade increased p53 positivity also increased but it was not statistically significant (TABLE 3). Correlation of Gleason’s score with p53 expression: Majority of patients of Gleason’s score 7-8 showed positivity for p53 (62.5%). It was observed that with increase in the score the number of cases showing positivity also increased but it was not statistically significant (p value=0.647) (TABLE 4). Combined Ki-67 and p53 expression: Out of the 50 cases, the majority of patients showed combined positivity for both Ki-67 and p53 constituting 48% of the total.

Correlation of Gleason’s grade with Ki-67 expression: Of the 16 cases of most common pattern (5+4), 13 were positive for Ki-67 and 3 were negative for Ki-67.Thus showing an increase in positivity with increase in grade but it was not statistically significant (TABLE 1).

Correlation of Gleason’s grade and score with combined Ki-67 and p53 expression: Out of the 16 cases of grade (5+4), combined Ki-67 and p53 positivity was seen in 12 cases (75%) (TABLE 5).Of the all cases of Gleason score 7-8, 11 cases showed both Ki-67 and p53 positivity but no statistically significant correlation was seen with increase in grade (p value = 0.648) (TABLE 6).

Correlation of Gleason’s score with Ki-67 expression: Majority of patients of Gleason score 7-8 showed positivity for Ki-67(79.2%). It was observed that with increase in the

Thus, it was observed that Ki-67 and p53 have got a direct correlation with the Gleason grade and Gleason scoring but in the present study it was not statistically significant.

Table 1: Correlation of Ki-67 expression with Gleason grade. Gleason Grade Ki-67 –VE CASES(%) 4+4 1(9) 3+3 1(20) 3+4 3(33.3) 5+4 3(18) 5+5 1(20) 4+3 1(25) TOTAL 10(20) Table 2: Correlation of Gleason score with Ki-67 expression. GRADING (According to score) Ki- 67 –VE cases (%) Gleason score 2-6 (Low Grade) 1 (20) Gleason score 7-8 (Intermediate Grade) 5(20.8) Gleason score 9-10 (High Grade) 4(19.0) TOTAL 10(20)

Ki-67+VE CASES(%) 10(91) 4(80) 6(66.7) 13(82) 4(80) 3(75) 40(80) Ki-67 +VE cases (%) 4 (80) 19(79.2) 17(81.0) 40(80)

TOTAL (%) 11(100) 5(100) 9(100) 16(100) 5(100) 4(100) 50(100) TOTAL (%) 5(100) 24(100) 21(100) 50(100)

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Table 3: Correlation of p53 expression with Gleason grade. GLEASON GRADE

p53 –VE CASES(%)

p53+VE CASES(%)

TOTAL (%)

4+4

5(45.4)

6(54.6)

11(100)

3+3

3(60)

2(40)

5(100)

3+4

3(33.3)

6(66.7)

9(100)

5+4

6(37.5)

10(62.5)

16(100)

5+5

3(60)

2(40)

5(100)

4+3

1(25)

3(75)

4(100)

TOTAL

21(42)

29(58)

50(100)

Table 4: Correlation of Gleason score with p53 expression. GRADING (According to score)

p53 –VE CASES (%)

Gleason score 2-6 (Low Grade)

p53+VE CASES (%)

TOTAL (%)

3( 60)

2 (40)

5 (100)

Gleason score 7-8 (Intermediate Grade)

9 (37.5)

15 (62.5)

24 (100)

Gleason score 9-10 (High Grade)

9 (42.9)

12 (57.1)

21 (100)

TOTAL

21 (42)

29 (58)

50 (100)

Table 5: Gleason grade correlation with both p53 and Ki-67 expression. GLEASON GRADE

BOTH p53 & Ki-67 -VE(%)

p53-VE AND Ki-67 +VE(%)

p53+VE AND Ki-67-VE(%)

BOTH p53 & Ki-67 +VE(%)

TOTAL(%)

4+4

0(0)

4(36.5)

2(18.1)

5(45.4)

11(100)

3+3

1(20)

2(40)

1(20)

5(100)

3+4

2(22.2)

3(33.4)

9(100)

5+4

2(12.5)

0(0)

12(75)

16(100)

5+5

0(0)

3(60)

0(0)

2(40)

5(100)

4+3

0(0)

3(75)

0(0)

1(25)

4(100)

TOTAL

5(10)

16(32)

5(10)

24(48)

50(100)

Table 6: Gleason score correlation with both p53 and Ki-67 expression. GRADING (According to score)

p53-VE & Ki- 67 –VE cases (%)

p53 –VE & Ki-67 +VE cases (%)

p53 +VE & Ki-67 –VE cases (%)

p53 +VE AND Ki67 +VE cases (%)

TOTAL

Gleason score 2-6 (Low Grade)

1 (20)

2 (40)

0(0)

2(40)

5(100)

Gleason score 7-8 (Intermediate Grade)

1(4.2)

8(33.3)

4(16.7)

11(45.8)

24(100)

Gleason score 9-10 (High Grade)

3(14.3)

6(28.6)

1(4.8)

11(52.4)

21(100)

5(10)

16(32)

5(10)

24(48)

50(100)

TOTAL

Discussion

Prostate carcinoma is the second most common cause of cancer and the sixth leading cause of cancer death among men worldwide. Now-a-days more patients are diagnosed at earlier stages. Increased early detection of the disease is due to increased availability of PSA measurement and also because of using other diagnostic methods such as cystoscopy, transurethral ultrasonography, biopsy and tumor markers. Various markers such as p53 and www.pacificejournals.com/apalm

Ki-67 are expressed immunohistochemically in prostate cancer. Mutations of p53 tumor suppressor gene can result in uninhibited cellular growth while Ki-67 proliferation marker provides a reliable index of cellular expression and expression of both reflect poor prognosis in carcinoma patients.[13,10] The present study showed that prostate carcinoma usually affects the elderly age group. Similar results have been seen in studies conducted by Cartar et al and Malti et al eISSN: 2349-6983; pISSN: 2394-6466

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who demonstrated that prostate cancer is more common in men > 65 years.[14, 15] Gleason scoring was done and the most common score was 9, which was followed by 7 occurring in 32% and 26% of cases respectively. Similar results have been demonstrated by Petrescu et al and Madani et al who in their respective studies showed Gleason score of 8-10 in 43% and 51% of cases respectively.[16, 17] p53 positivity was found in 29 cases (58%) while p53 negativity was found in 21 cases (42%). Percentage positive cells varied from 5-80% and exhibited mild, moderate and strong staining intensity. Moul et al and Sasor et al have found a higher p53 expression of 69.1% and 62.2 % respectively whereas Madani et al demonstrated a positive p53 expression in the range of 48%- 68% expression in high grade prostate cancer.[17, 18, 19] Although expression and intensity of p53 increased with increase in grade; no statistically significant correlation was found between the two. This was also reflected by Sasor et al and Yaman et al who have statistically proven that there is no significant difference between the presence of p53 in low and high grade tumor.[19, 20] On the other hand, Kallakury BV et al and Petrescu et al have demonstrated a positive correlation between p53 immunopositivity and higher Gleason grades with expression of 21% and 39% respectively.[21, 16] Ki-67 positive cells varied from 3-84% and moderate and strong staining intensity was found in 80% of cases in the present study. Similar higher positivity and strong staining intensity was also recorded by Madani et al, Thompson et al and Zhong et al in 71.4%,76.0% and 67.76% of cases respectively whereas a very high Ki-67 expression of 93% was observed by Makarewicz et al.[17, 22, 23, 24] Out of the 32 cases of Gleasonâ&#x20AC;&#x2122;s score of 8 and above, Ki67 expression was noted in 81% cases while 78% cases of Gleasonâ&#x20AC;&#x2122;s score 6 and 7 showed Ki-67 expression. Thus, showing increase in Ki-67 positivity with increase in grading but it was not statistically significant. Madani et al also observed that Ki-67 expression increased from 62.2% to 88% as the grade increased.[17] Marian Sulik et al and Bettencourt et al have also stated significant correlation between immunopositivity of Ki-67 and higher Gleason grade.[25,18]Although Munroz et al have not found a statistically significant difference between Ki-67 expression and higher Gleason grade.[26] The present study revealed 48% cases (24/50) exhibiting positivity for both of these markers. In a study done by Madani et al high Ki-67 expression was observed in 71 %

cases of prostate cancer while p53 expression was noted in 42.9 % cases.[17] Moul et al showed Ki-67 expression in 38.3% of cases and p53 expression in 69% of cases.[18] Out of the 32 cases of Gleason score 8 and above, combined Ki-67 and p53 expression was noted in 46% of cases while 50 % cases of Gleason score 6 and 7 showed combined p53 and Ki-67 expression. A study by Rashed et al demonstrated a positive relationship between the expressions of Ki-67 and p53 in patients with low grade prostate cancer.[29] Borce et al have also showed that accumulation of p53 had a special correlation with patients survival. The presence and activity of p53 was greatly associated with the cell proliferation marker Ki-67 and the level of p53 activity was an important independent prognostic factor that was inversely associated with patient survival.[19] Moul et al have also recommended the clinical use of Ki-67 and p53 immunohistochemical protein expression in the primary tumor as combined predictors of disease progression.[18] In the present study antibodies were not employed to find an association between benign proliferative or insitu lesions. Various studies Indian and abroad have documented that immunoexpression of p53 and ki-67 is up regulated in cancerous tissue as compared to benign prostatic ones such as nodular hyperplasia , adenosis and stromal hyperplasia and in situ lesions such as prostatic intraepithelial lesion (PIN). While many researchers have documented the rate of immunoexpresssion of p53 in benign tissue to be low as 2 % to 10 %[27,28]; there are many (notably amongst them Petrescue A et al) who have reported a lack of immunoreactivity in all such cases. [16] The same work by Petrescue A also documented that the immunoexpression in PIN ( in-situ lesion) especially the high grade (HPIN), as the insitu lesion moves from low to high; the expression increases. Similar findings have been recorded by researchers worldwide while studying Ki-67 expression, which is significantly low in benign prostatic conditions and PIN as compared with the cancerous tissue.[29] This leads to the hypothesis whether the occurrence of p53 mutations and Ki-67 expression in prostatic carcinoma is an early event.[16, 27]

Acknowledgements None

Funding None

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Competing Interests None Declared

Conclusion

Ki-67 and p53 were performed on a small cohort of Punjabi population (North India) which showed an increase in the expression of these markers with increasing grade and score. Hence, immunostaining with both these markers should be done in all cases of prostate carcinoma as these markers allow identification of tumors with a higher rate of cell growth, allowing the development of prognostic factors and new targeted therapeutic strategies for increased survival in these patients.

11.

12.

13.

References

1. Zhong WD, Li JL, He HC. Study on the expression of BPH and prostate cancer related genes using real-time quantitative RT-PCR. Chin J Urol 2005;26:615-61. 2. Srigley JR. Benign mimickers of prostatic adenocarcinoma. Mod Pathol 2004;17:328-48. 3. Humphrey PA. Gleason grading and prognostic factors in carcinoma of the prostate. Mod Pathol 2004;17:292-306.

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4. Radiae S. Prostate cancer molecular staging. State of –the – Art in prostate and breast cancer treatment.Euro Sch Oncol 2002;50:101-4.

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5. Stricker HJ, Jay JK, Linden MD, Tamboli P, Amin MB. Determining prognosis of clinically localized prostate cancer by immunohistochemical detection of mutant p53.Urology1996;47:366-9.

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6. Thomas DJ, Robinson M, King P, Hasan T, Charlton R, Martin J. p53 expression and clinical outcome in prostate cancer.Br J Urol 1993;72:778-81.

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7. Shurbaji MS, Kalbfleisch JH, Thurmond TS. Immunohistochemical detection of p53 protein as a prognostic indicator in prostate cancer. HumPathol1995;26:106-9. 8. Guillaud P, duManoir S, Seigneurin D. Quantification and topographical description of KI-67 antibody labelling during the cell cycle of normal fibroblastic (MRC-5) and mammary tumor celllines(MCF-7). Anal Cell Pathol 1989;12:568-72. 9. Gerdes J, Li L, Schlueter C, Duchrow M, Wohlenberg C, Gerlach C. Immunological and molecular biologic characterisation of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67.Am J Pathol 1991;138:867-73. 10. Rajeswari K, Meenakshisundaram K, Anushuya G, Rajalaxmi J. Ki 67 as a prognostic marker in www.pacificejournals.com/apalm

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comparison with Gleason’s grading system in prostatic carcinoma. Ind J Pathol Oncol 2016; 3: 92-5. Bettencourt MC, Bauer JJ, Sesterhenn IA, Mostofi FK, McLeod DG, Moul JW.Ki-67 expression is a prognostic marker of prostate cancer recurrence after radical prostatectomy. J Urol1996;156:1064-8. Borce M, Stausbat-Gron B, Overgaard J. P53 accumulation associated with bcl-2, the proliferation marker MIB-1 and survival in patients with prostate cancer subjected to watchful waiting. J Urol 2000;164:716-21. Lumachi F, Marino E, Lacobone M. Immunohistochemical markers PCNA-LI and Ki67, and DNA index in patients with Parathyroid carcinoma. Oral Presentations 2006;26:316-8. Carter HB, Epstein JI, Partin AW. Influence of age and prostate-specific antigen on the chance of curable prostate cancer among men with nonpalpable disease. Urology 1999 ;53(1):126-30. Malati T, Kumari GR, Reddy PVLN, Prakash BS. Prostate specific antigen in patients of benign prostate hyperplasia and carcinoma prostate. Indian J Clin Biochem 2006;21:34-40. Petrescu A, Marzan L, Codreanu O, Niculescu L. Immunohistochemical detection of p53 protein as a prognostic indicator in prostate carcinoma. Rom J Morphol Embryol 2006;47:143-6. Madani SH, Ameli S, Khazaei S, Kanani M, Izadi B. Frequency of Ki-67(MIB-1) and P53 expressions among patients with prostate cancer. Indian J Pathol Microbiol 2011;54:688-91. Moul JW, Bettencourt MC, Sesterhenn IA, Mostofi FK, McLeod DG, Srivastava S, et al. Protein expression of p53, bcl-2, and KI-67 (MIB-1) as prognostic biomarkers in patients with surgically treated, clinically localized prostate cancer. Surgery 1996;120:159-66. Sasor A, Wagrowska-Danilewicz M, Danilewicz M. Ki 67 antigen and p53 protein expression in benign and malignant prostatic lesions. Immunohistochemical quantitative study. Pol J Pathol 2000;51:31-6. Yaman O, Ozdiler E, Orhan D, Sak SD, Baltaci S, Tulunay O, et al. Immunohistochemical determination of p53 protein in prostatic cancer and prostatic intraepithelial neoplasms. Urol Int 1997;58:199-202. Kallakury BV, Figge J, Ross JS, Fisher HA, Figge HL, Jennings TA. Association of p53 immunoreactivity with high gleason tumor grade in prostatic adenocarcinoma. Hum Pathol 1994;25:92-7. eISSN: 2349-6983; pISSN: 2394-6466

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22. Thompson SJ, Mellon K, Charlton RG, Marsh C. p-53 and Ki-67 immunoreactivity in human prostate cancer and benign hyperplasia. Br J Urol 1992;69:609-13. 23. Zhong W, Peng J, He H, Wu D, Han Z, Bi X, et al. Ki-67 and PCNA expression in prostate cancer and benign prostatic hyperplasia. Clin Invest Med2008;31:E8-E15. 24. Makarewicz R, Zyromska A, Andrusewicz H. Comparative analysis of biological profiles of benign prostate hyperplasia and prostate cancer as potential diagnostic, prognostic and predictive indicators. Folia Histochemica Et Cytobiologica 2011;49:452-7. 25. Sulik M, Maruszak K, Puchalska J, Misiukiewicz M. Expression of KI-67 as a proliferation marker in prostate cancer.Pol Ann Med 2011;18:12-9.

26. Munoz E, Gomez F, Paz JI, Casado I, Silva JM, Corcuera MT, et al. Ki-67 immunolabeling in pre-malignant lesions and carcinoma of the prostate. Histological correlation and prognostic evaluation. Eur J Histochem 2003;47:123-8. 27. Verma R, Gupta V, Singh J, et al. Significance of P53 and ki-67 expression in prostate cancer. Urol Ann 2015; 7(4): 488-93. 28. Wang Y, Zhang YX, Kong CZ, Zhang Z, Zhu YU. Loss of P53 facilitates invasion and metastasis of prostate cancer cells. Molecular and Cellular Biochemistry 2013; 384: 121-7. 29. Rashed HE, Kateb MI, Ragab AA, Shaker SS. Evaluation of minimal prostate cancer in needle biopsy specimens using AMACR (p504s), p63 and Ki-67. Life Sci 2012;9:12–21.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 06, (Suppl) December, 2016

Original Article p53 as a Prognostic Marker in Carcinoma Breast in Correlation with Conventional Estrogen and Progesterone Hormone Receptors Sanjay Piplani*, Manas Madaan, Mridu Manjari and Rahul Manan Department of Pathology, Sri Guru Ram Das Institute of Medical Sciences and Research, Amritsar, India Keywords: Estrogen Receptor, Progesterone Receptor, p53, Tumor Grading, Lymph Node

ABSTRACT Background: Carcinoma breast is the most common cancer in women worldwide. The present study was done with the objective to find expression and correlation of Estrogen Receptors(ER), Progesterone Receptors(PR) and p53 in breast carcinoma and to correlate expression of these tumor markers with histological type, grade and other parameters. Methods: The study was conducted on 65cases of breast cancer in the department of Pathology of a tertiary health care center. Histopathological and IHC studies were done for ER, PR and p53 expression, brown nuclei were taken as positive results and IHC Scoring was done. Results: All the cases were of Infiltrating Ductal Carcinomas(NOS) between 30-70years of age with varying tumor size1-6cm. Maximum cases were of grade III followed by grade II. Metastatic carcinomatous deposits in Lymph nodes were seen in 42cases. On the basis of ER PR positivity, tumors were separated into 4 categories with category IV having maximum number of cases. ER/PR expression in grade III cases was significantly low. With increasing grade, the expression of p53 increased which was statistically significant p=0.007. Lymph nodes showing metastatic deposits were highest in ER PR negative cases which was statistically significant(p=0.028). p53 expression was higher in category IV cases(32.3%) as compared to category I(15.4%). Conclusion: In the present study it was observed that as the grade increases, ER PR decreases and p53 positivity increases. Thus ER PR status is inversely proportional to p53 expression and emphasizes the need to find out the prognosis, survival and line of treatment.

*Corresponding author: Dr. Sanjay Piplani, 24, Lane-5, Gopal Nagar, Majitha Road, Amritsar, Punjab 143001, India Phone: +91 9914785777 Email: sanjaymikki@gmail.com

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

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Introduction

Breast cancer, second only to carcinoma lung, is the most common cancer in women worldwide. The incidence of breast cancer which was about 12% in 2012 is expected to increase by 26% by 2020 and most of these will be seen in developing countries.[1] One in every eight women stands the chance of being diagnosed with breast cancer in their lifetime.[2] Incidence of breast cancer is strongly related to age, with the highest incidence rates being in older women, supporting a link with hormonal status.[3] In Asia however, breast cancer incidence peaks among women in their forties whereas in the United States and Europe, it peaks among women in their sixties.[4] In India premenopausal women constitutes about 50% of all breast cancer patients. Breast cancer risk in India revealed that lifetime duration of breast feeding was inversely associated with breast cancer risk among premenopausal women. [5,6] Higher education level and income are also shown to be significant reasons for an increased risk of breast cancer.[7,8] This is because economic independence may encourage women to remain single or marry late thereby increasing their risk of getting the disease.[1] Histopathology, either excision or tru-cut biopsy, is the gold standard to confirm the breast malignancies and also to type and grade it. In recent years, for therapeutic and prognostic purposes breast cancer once diagnosed, is then subjected to immune-histochemical studies (IHC) which commonly include estrogen receptor (ER) and progesterone receptor (PR). With advent of molecular classification of breast carcinoma along with these, other ancillary cytokeratin prognostic markers (BRCA 1, BRCA 2, p53, Bcl 2 and Ki 67) have markedly revolutionized the research for breast cancer.[9] Estrogen receptors (ER) are specific proteins located mainly in the cytoplasm of cells of target tissue for estrogen action.[10] Progesterone receptor is an intracellular steroid receptor that specifically binds progesterone expressed by a single gene.[11] Recent studies also suggest that assessment of PR are equally or more valuable than those of ER in predicting the diseasefree interval in patients with breast cancer. The patients expressing ER and PR positivity respond well to the hormonal treatment and have a better prognosis as well as better survival rate.[12] p53 is the main regulator of genomic stability through regulation of the cell cycle. Over expression of p53, which is caused by TP 53 mutation, is the most frequent genetic alteration in not only breast cancers but also in various malignancies such as ovarian, esophageal and GIT.[13] Breast tumors expressing a high amount of p53 are more frequently ER-negative and PR-negative and are also

associated with a high proliferation rate, high histological and nuclear grades, aneuploidy and poorer survival.[14,15] The present study was done with the objective to find expression and correlation of ER, PR and p53 in breast carcinoma as well as to correlate expression of these tumor markers with histological type, grade and other parameters in a small cohort of north Indian female population suffering from breast cancer.

Materials and Methods

The study was conducted on 65 cases of breast cancer received as mastectomy specimens in the department of Pathology of a tertiary health care center. The patients were divided into four groups according to age. These were labeled as Group I, II, III & IV which included patients between 31-40 years of age, 41-50 Years, 51-60 Years and 61-70 Years respectively. Depending upon the tumor size, they were also classified as those having size less than 2 cm, size between 2 – 5 cm and size more than 5 cm. (Table 1) Exclusion criteria is only cases of infiltrating ductal carcinoma NOS were included in the study. Rest of the histo-pathological variants such as medullary, tubular, etc. were not included in the study. The tissue was formalin fixed and paraffin embedded with sections obtained and stained for Haematoxylin and Eosin for histopathological confirmation, typing and grading. In all the cases IHC studies were done for ER, PR, and p53 expression. The sections were taken on Poly-L-Lysin coated slides and antigen retrieval was done by conventional heat extraction system on pressure cooker and protein block was obtained. Protein Block was incubated for 30 min. and 2 washes in tris buffer were given for 5 minutes each. Primary antibody for ER(Monoclonal rabbit antibody RMA B001from diagnostic biosystem India), PR(Monoclonal rabbit antibody RMA B002from diagnostic biosystem India) and p53 (CME298BK from biocare medical) were employed and sections were kept for 1 hour in the moist chamber followed by 2 washes in tris buffer for 5 minutes each. The post primary block was then applied for 30 minutes, 2 buffer washings given for 5 minutes each, incubation done with Polymer for 30 minutes. Again after 2 washings with tris buffer slides were covered with DAB for 2-3 min and haematoxyllin Counterstaining was done. For ER, PR and p53, brown nuclei were taken as positive results. In all the cases positive and negative controls were run for ER, PR and p53. Scoring of Estrogen Receptor: For number of positively staining cells less than 10%, 10-50%, 50-75% and more than 75% a score of 1, 2, 3and 4 was given respectively. Similarly a score of 1, 2and 3 was given for cells staining with low, medium and strong intensity respectively.(Fig.1)

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Scoring of Progesterone Receptor: Zero score was given for no nucleus staining brown. For nuclei ranging between 1-25%, 26-50%, 51-75% and 76-100%, a percentage score (PS) of 1, 2, 3 and 4 was given. Similarly an intensity score of 0,1, 2 and 3 was given for cells showing No staining, weak, medium and high intensity staining respectively. Total score was calculated according to the formula (TS=PS+IS) Range 0-7. Low score was graded as

quick score 2-3, Medium as quick score 4-5 and High as quick score 6-7. (Fig.2) Scoring of p53: It was also done on a similar pattern with less than 5% cells getting a score of zero, 5-25% score of 1, 25-50% 2 and more than 50% being awarded a score of 3. Similarly a score of 1, 2 and 3 was awarded for mild moderate and strong staining intensity. (Fig.3)

Table 1: Showing Correlation of ER, PR & P53 with Age of Patients and Tumor Size. Groups According to Age

Groups According to Tumor Size

Group I 31-40 Yrs.

Group II 41-50 Yrs

Group III 51-60 Yrs.

Group IV 61-70 Yrs.

Group I <2cm

Group II 2-5cm

Group III >5cm.

No. of Cases

ER+

ER -

PR+

PR-

p53+

p53-

Fig. 1: ER positivity (Nuclear) – IHC.

Fig. 2: PR positivity (Nuclear) – IHC.

Fig. 3: p53 positivity Grade III – IHC.

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Results

All the patients included in the study were females betweenÂ 30-70 years of age with most of the cases in the fifth and sixth decade of life (67%). Right breasts were more commonly involved (63%) with tumor more commonly seen in upper and outer quadrant (84%). Tumor size varied from 1-6 cm with maximum number of cases ranging between 2-5 cm (44/65; 67.7%) i.e. in the T2 stage. As per exclusion criteria of the study all the 65 cases were infiltrating ductal carcinomas (NOS). Grading: Nottingham modification of RBB scoring when employed showed that maximum cases were of grade III (42 cases; 64.6%) followed by grade II (17 cases; 26.2%) and then grade I (6 cases; 9.2%). Lymph Node Status: Lymph nodes were recovered in all 65 cases with metastatic carcinomatous deposits Â noted in 42 cases(64.6%) which included 14 cases of N1 stage (1-3 nodes), 18 cases of N2 stage (4-9 nodes) and 10 cases of N3 stage ( >10 nodes). All the nodes recovered in 6 grade I tumors were reactive. Out of 17 cases of grade II carcinoma metastatic deposits were seen in 10 cases (59%) with 7 cases (41%) showing reactive hyperplasia. 32/42 (76.2%) grade III tumor cases showed metastatic carcinomatous deposits. Immuno-Histochemical Expression: Estrogen receptor (ER) positivity was seen in 27/65 cases (41.6%) while Progesterone Receptor (PR) positivity was seen in 26/65 cases (40%). p53 expression was noted in 38/65 cases (58.5%). On the basis of ER/PR positivity, tumors were separated into 4 categories :- Category I as ER+ve PR+ve, Category II as ER+ve, PR-ve, Category III as ER-ve, PR+ve, and Category IV as ER-ve, PR-ve. It was noted that maximum number of cases were of category IV accounting for 50.7% of total cases (Table 2). Correlation of ER, PR with Age of the patients: From the Table 1 it can be seen that maximum number of cases were in group II (25/65) followed by group III (19/65). Out of the 25 cases in group II only 4 (16%) were ER positive whereas 21 (84%) cases were ER negative. In group III, out of 19 cases 11 (57.9%) showed ER Positivity while 8 cases (42.1%) were negative for ER expression. (p=0.003; Significant). The ER positivity in group I and group IV patients was 63.7% and 50 % respectively. When we evaluated the PR expression, the group II patients showed a PR positivity of 28% as compared to group III patients where it was 26.3%. (p=0.901; Not Significant). PR positivity in group I patients was 91% and in group IV patients was 40%. Correlation of p53 with Age of the patients: The group II cases showed a high p53 expression of 76% as compared to group III patients where the p53 expression was 52.6%.

The p53 expression in group I and group IV was 36.4% and 50 % respectively. Correlation of ER, PR with Size of the Tumor: ER positivity was 52.6% (10/19) cases with tumor size less than 2 cm while it was 36.4% (16/44) patients having a tumor size between 2-5 cm. The two cases having tumor size more than 5 cm showed an ER positivity of 50%. PR positivity for the three groups was 57.9%, 31.8% and 50% respectively. Correlation of p53 With Size of the Tumor: Out of 38 cases showing a positive p53 expression 10 cases (26.3%) had tumor size less than 2 cm, 26 cases (68.4%) had tumor size ranging between 2-5cm and 2 cases (5.3%) had tumor size more than 5cm. Correlation of ER, PR with Grade of Tumor: All the 6 grade I tumor cases were of category I. The 17 grade II cases expressed an ER positivity of 53% (9/17) and PR positivity of 58.8% (10/17). ER and PR expression in grade III cases was significantly low with ER positivity of 28.6 % and PR positivity of 23.8% only. (Table 3) Correlation of p53 with Grade of Tumor: p53 immunoexpression was 38/65 (58.5%). None of the grade I tumors was positive for p53 expression. In the study conducted, with increasing grade of the tumor, the expression of p53 also increased (58.8% in grade II & 67.4% in grade III). This was statistically significant p=0.007. (Table 4) Correlation of ER and PR with Lymph Nodes Status: In the present study, out of 19 ER, PR positive cases, 8 cases (42.1%) showed lymph nodes having metastatic carcinomatous deposits while in 11 cases (57.9%) nodes showed reactive hyperplasia. When the number of cases showing lymph nodes with metastatic deposits was calculated in ER PR negative cases, it rose to 24/33 accounting for 72.8% involvement. Only 9/33 (27.3%) cases showed reactive lymph nodes. This correlation was found to be statistically significant (p=0.028). (Table 5) Correlation of p53 With Lymph Nodes Status: In the present study out of 38/65 p53 positive cases, 26 (68.42%) had metastatic carcinomatous deposits while 12 cases (31.6%) showed reactive lymph nodes. The percentage of cases showing metastatic deposits in 27 p53 negative cases was 59.3% whereas 40.7% of p53 negative cases did not show any metastatic deposits. The results were however were not found to be statistically significant. (Table 5) Correlation of ER, PR and p53: p53 expression was found to be higher in category IV cases which were both ER & PR-ve (Hormonal -ve) 32.3% as compared to patients who were hormonally positive i.e. both ER & PR +ve category I (15.4%) but the correlation was not significant. (Table 6)

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Table 2: Showing Combination of ER and PR Cases. Combination of ER and PR

Number of cases (n=65)

Percentage

ER+PR+ (Category I)

29.3%

ER+PR- (Category II)

10.7%

ER-PR+ (Category III)

9.3%

ER-PR- (Category IV)

50.7%

Table 3: Showing Correlation of ER & PR with Grade of Tumor. Grade of Tumour

ER+

PR+

ER+ PR+

ER- PR+

ER+ PR-

ER- PR-

III

Table 4: Showing Correlation of P53 with Grade of Tumor. Grade Of Tumor

P53 Positive Score 0

P53 Positive Score 1

P53 Positive Score 2

P53 Positive Score 3

Total P53 No. Of Positive Negative Cases

III

Total No. Of Cases

% Of Positivity

58.8%

67.4%

Table 5: Showing Correlation of ER PR and P53 with Lymph Node Status. ER+ PR+

ER+PR-

ER- PR+

ER- PR-

P53+

P53-

Metastatic Lymph Nodes

Reactive Lymph Nodes

No. of Cases

Table 6: Showing Correlation of ER, PR and p53. ER PR status

p53 Positive

p53 Negative

Total

Category I (ER + PR+)

Category II (ER + PR-)

Category III (ER- PR+)

Category IV (ER- PR-)

Discussion

Carcinoma breast is the most frequent cancer in females throughout the world with 1.6 million cases diagnosed and 4,25,000 deaths reported in 2010.[16] In India though ranked second after carcinoma cervix at the moment, it is all set to overtake cervical cancer and become the most common cancer in Indian women by 2020. An ICMR release in 2011 showed an increase in incidence from 10 per 100,000 to 23 per 100,000 in just 10 years.[17] Breast cancer incidence and death rates generally increase with age. In the present study, the maximum numbers of the patients were in the age group of 41-60 years comprising 67% of the total cases. This is in accordance with work done by Kaur et alÂ who noted a peak age of 45 - 55 years in a study done on 177 cases.[18]

In the present study, right side was more commonly involved (60% of cases)Â than the left side (40% of cases) which is in accordance with work done by Saleh & Abdeen on 166 patients of breast carcinoma.[19] In the present study, there was definite preponderance for the upper outer quadrant(84%). Possible explanation is that the upper outer quadrant has a relatively larger volume of breast tissue. [20,21,22] One of the most important and well established prognostic factors in carcinoma breast is tumor size.[23] In the present study, the size of the tumor varied from 1 cm to 6 cm and in 67.7% cases tumor size was between 2-5 cm constituting the largest group. This is in concurrence with results obtained by Kaur et al and Saleh & Abdeen [18,19] Where as in the western countries, Taucher et al reported that the tumors were predominantly less than 2 cm in size

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which could be due to early detection programs prevalent in the western countries.[24] Tumor grade is another well established prognostic marker in case of breast carcinoma.[23] Most of the cases in present study were of grade III (64.6%) which is in contrast to findings noted by Muhammad Hanif, who reported maximum number of tumors (59%) to be of grade II.[25] Maximum number of grade II cases were also noted in studies done in Singapore, Malaysia, Pakistan and India. [26-30] This difference in the tumor grades may be due to lack of routine mammographic screening in our population coupled with the lack of awareness. Correlating tumor grade with lymph node involvement in the present study, it was observed that grade III tumors demonstrated the highest frequency (64.8%) of lymph node involvement. The findings are in concurrence with the work done by Shokouh et al (Grade 3 tumors 73.5%).[31] Kaur and others noted that in grade III tumors lymph node metastasis was present in 57.6% cases.[18] When correlation of tumor size with lymph node involvement was attempted, it was seen that in the present study, 6 cases (28.57%) out of a total 21 with size <2 cm showed metastatic carcinomatous lymph node deposits and this percentage increased to 70.5% (31/44 cases) when the tumor size was between 2-5 cm. This shows a direct relationship between increased tumor size and lymph node involvement. This was in concordance with the studies done by Shokouh et al and Bojić et al who showed a strong correlation between the two.[31,32] Indian literature reports estrogen receptor positivity varying between 30-50%.[33] It was also reflected in the present study with 27 cases (41.5%) showing ER positivity. Out of 38 ER negative cases in the present study, 28 cases were below the age of 55 years and 10 above the age of 55 years. Manjunath et al demonstrated that ER negative disease occurred at a younger age, at a mean of 50.2 years (SD 10.28), whereas the mean age of ER positive disease was 55.7years.[34] Progesterone receptor expression also showed a similar trend with 26 cases comprising 40% of the total cases showing PR positivity. Desai et al. from India have documented the prevalence of 46.1% for PR-positive breast cancers.[35] Ambroise et al. in their study from South India have showed 51% PR positivity.[30] Similarly, Mudduwa, in a study from Srilanka documented a prevalence of 48.3% PR-positive tumors.[36] Maximum number of cases in the present study were in category IV (49.2%) followed by category I (30.7%) as shown in Table 2. The prevalence of hormones receptor positive breast cancer in Asian countries has been found to be lower than those in the western world [37] because of lesser use of OCP’s and increased hormonal replacement

therapy after menopause. The percentage of tumors expressing PR but negative for ER was 9.23% in the present study. The results are in concurrence with the study done by Patnayak et al. [38] In the present study, when we compared the ER expression according to age in two groups with highest number of cases (Group II & III), it was seen that group II patients did not express ER in 84% of cases as compared to group III patients where this negative expression was seen in 42.1% of the cases. This correlation was found to be significant (p=0.003; Significant). Furthermore when p53 expression was evaluated in these 29 ER negative cases, it was seen that out of 21 ER negative cases in group II, 18 cases showed a positive p53 expression. On the other hand only 3 out of 8 ER negative cases in group III exhibited a p53 positivity (37.5%). This correlation was again found to be significant (p=0.009; Significant). Similar results were echoed by Mohammed et al who reported that patients younger than 50 years of age with Grade II and III infiltrating ductal carcinoma had significantly lower levels of estrogen receptors than patients older than 50 years of age (P <0.001).[39] Breast tumors arising in older patients have slower growth rates, are more likely to be ER-positive, and are less likely to be p53-positive. In the present study, the ER and PR expression in patients with tumor size less than 2 cm was higher than that seen in patients with tumor size between 2-5cm (52.6% & 57.9% vs 37% & 32.6% respectively). P53 expression however demonstrated a reverse relation with patients having tumor size varying between 2-5cm showing a higher positivity (73.3%). The results however were not statistically significant. Similar results were obtained by Ahmed et al in their study done on 157 Yemeni women.[15] In the present study ER positivity decreased from 100% to 53% to 28.6% as the grade increased from first to third, though the results were not statistically significant. Manjunath et al, Jovicić-Milentijević et al and Barnes et al also showed the same results, as the grade of the tumor increases ER positivity decreases.[34,40,41]Progesterone receptor expression also showed a similar trend that as the grade increased PR positivity decreased although the results were not statistically significant. Similar results were noted in various other studies.[37,41,42] None of grade 1 tumors in the present study showed p53 positivity. Out of 17 cases of grade II, 10 cases (58.2%) showed p53 positivity and out of 43 cases of grade III, 29 cases (67.4%) showed p53 positivity which was found to be

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Piplani et al. statistically significant (p=0.007). This shows as the grade increases p53 positivity increases. A retrospective study done by Shokouh et al from 2008 to 2014 on 566 patients of breast carcinoma also showed that higher grades had greater p53 positivity.[31] In the present study the percentage of cases showing metastatic carcinomatous deposits was highest in ER PR negative group (category IV; 72.8%) as compared to the ER PR positive category I (42.1%) (p=0.028; Significant). However no significant correlation was noted for p53 expression where metastatic lymph node involvement was seen in 68.4% p53 positive cases and 59.3% p53 negative cases. The findings of the present study are in concurrence with the work done by Ali et al who reported that the ER PR negative cases have 2.8 times increased risk of metastasis in comparison to ER PR positive breast cancer cases.[43] Dunnwald et al in their study demonstrated the association between ER/PR status and breast cancer specific mortality with in subgroups of women defined by tumor characteristics like axillary lymph node metastasis. They observed a higher risk of lymph node involvement in ER PR negative tumors relative to ER PR positive tumors. [44] When we attempted a correlation between ER, PR and p53 in the present study, it was seen that 21/33 (63.6%) of ER, PR negative cases showed p53 positivity. This constituted the largest group of p53 positive cases (21cases) and showed that ER PR and p53 are inversely related. An inverse association between hormones receptors and p53 has been demonstrated by Ahmed et al on Yemini women with breast cancer.[15] This was further confirmed by Sirvent et al.[45] In the present study, it was observed that out of 42 cases of grade III tumors, 29 cases showed p53 positivity (66.7%) and 28 cases showed ER PR negativity (66.6%). In grade II tumors, out of 17 cases, 10 showed p53 positivity (58.8%) with 5 ER PR negative cases (29.4%). This shows that as the grade of the tumor increases p53 positivity increases and ER PR positivity decreases suggesting further that ER PR are inversely related to P53 status. However this difference was not statistically significant. Marc Lacroix et al showed that breast tumors expressing a high amount of p53 (as measured by IHC) are more frequently ERnegative and PR-negative. They are also associated with a high proliferation rate, high histological and nuclear grades, aneuploidy and poorer survival.[14]

Conclusion

A-523 of lymph nodes showing metastatic deposits was more in ER PR negative cases as compared to patients showing a positive ER PR expression. It was also concluded that as the grade of the tumor increased, p53 positivity increased and ER, PR expression decreased suggesting an inverse relationship between the two. It was further observed that ER, PR positivity is high in low grade tumor and p53 positivity is associated with high grade tumors. Thus it shows that ER and PR status are inversely proportional to p53 expression and emphasizes the need to find out the prognosis, survival and line of treatment.

Acknowledgements

I acknowledge the efforts of Dr. Monika Lalit in providing the technical help, statistical help and writing assistance. i state that there was no conflict of interests.

Funding None

Competing Interests None Declared

References

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It was concluded in the present study that ER expression was significantly low in patients younger than 50 years of age as compared to those older than 50 years. The number

8. Tavani A, Gallus S, La-Vecchia C, et al. Risk factors for breast cancer in women under 40 years. Eur J Cancer. 1999;35(9):1361-7.

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9. Jeffery SR. Predictive and prognostic molecular markers in breast cancer. In: Lowe DG, Underwood JCE, editors. Recent Advances in Histopathology. London: Royal Society of Medicine Press Ltd; 2005. p. 31-50. 10. Rosai J. The Breast. In: Rosai and Ackerman’s Surgical Pathology. 10th Edition (Vol.2). New York: Mosby (Elsevier); 2012. p.1719-20. 11. Gadkar-Sable S, Shah C, Rosario G, Sachdeva G, Puri C. Progesterone Receptors, various forms and functions in reproductive tissues. Frontier Biosciences. 2005;10:2118-30. 12. Onitilo AA, Engel JM, Greenlee R T, Mukesh BN. Breast Cancer Subtypes Based on ER/PR and Her2 Expression: Comparison of Clinicopathologic Features and Survival. Clin Med Res. 2009;7(1-2):4–13. 13. Lee SK, Bae SY, Lee JH, et al. Distinguishing LowRisk Luminal A Breast Cancer Subtypes with Ki-67 and p53 Is More Predictive of Long-Term Survival. PLoS One. 2015;10(8):e0124658. 14. Lacroix M, Toillon RA, Leclercq G. p53 and breast cancer, an update. Endocr Relat Cancer. 2006;13(2):293-325. 15. Ahmed HG, Al-Adhraei MA, Al-Thobhani AK. Correlations of Hormone Receptors (ER and PR), Her2/neu and p53 Expression in Breast Ductal Carcinoma Among Yemeni Women.The Open Cancer Immunology Journal. 2011;4:1-9. 16. Komen SG. Breast Cancer Facts [Internet]. Dallas: Susan G. Komen; [cited 2013 Nov 14]. Available from: http://ww5.komen.org/uploaded Files/Content_ Binaries/806-316a.pdf. 17. Shetty P. India faces growing breast cancer epidemic. The Lancet. 2012;379(9820):992-3. 18. Kaur G, Ismail R, Kam LS, Sabaratnam S, Ahmad N. Assessment of correlation between Clinicopathological Features and Lymph Node Metastasis in Breast Cancer. The Internet J Pathol. 2007;5:2. 19. Saleh F, Abdeen S. Pathobiological features of breast tumours in the State of Kuwait: a comprehensive analysis. J Carcinog. 2007;6:12. 20. Hussain MA, Ali S, Tyagi SP, Reza H. Incidence of cancer breast at Aligarh. J Indian Med Assoc. 1994;92(9):296-7. 21. Lee AH. Why is carcinoma of the breast more frequent in the upper outer quadrant? A case series based on needle core biopsy diagnoses. Breast. 2005;14(2):151-2.

22. Shaheen G, Arshad M, Shamim T, et al. Prevalence Of Breast Cancer In Punjab. The Internet Journal of Public Health. 2011;1(1):88-93. 23. Lester SC. The Breast. In: Kumar V, Abbas AK, Fausto N, Aster JC, editors.Robbins and Cotran Pathologic Basis of Disease. 8th Ed. Philadelphia.Elsevier; 2010. p. 1066-90. 24. Taucher S, Rudas M, Mader RM, Gnant M, Dubsky P, Bachleitner T. Do we need Her-2/ neu testing for all patients with primary breast carcinoma? Cancer. 2003;98(12):547-53. 25. Hanif M, Sabeen B, Maqbool A, Ahmed A, Nadeem F, Habib S. Breast Cancer: Incidence (Thirteen Year Data Analysis) and (OneYear Clinicopathological Data) of Patients In a Tertiary Care Cancer Hospital. 2015;12(3):373-79.23. 26. Siddiqui MS, Kayani N, Sulaiman S, Hussainy AS, Shah SH, Muzaffar S. Breast carcinoma in Pakistani females: a morphological study of 572 breast specimens. J Pak Med Assoc. 2000;50(6):174-7. 27. Tan EY, Wong HB, Ang BK, Chan MYP. Locally advanced and metastatic breast cancer in a tertiary hospital. Ann Acad Med Singapore 2005;34(10):595-601. 28. Nisa A, Bhurgri Y, Raza F, Kayani N. Comparison of ER, PR and HER-2/ neu (C- erb B2) reactivity pattern with histologic grade, tumor size and lymph node status in breast cancer. Asian Pac J Cancer Prev. 2008;9(4):553-6. 29. Kamil M, Khalid I, Hashim H, Biswas M, Kaur G, Islam R. Association of carcinoma breast: grade and estrogen progesterone receptor expression. J Coll Physicians Surg Pak. 2010;20(4):250-2. 30. Ambroise M, Ghosh M, Mallikarjuna VS, Kurian A. Immunohistochemical profile of breast cancer patients at a tertiary care hospital in South India. Asian Pac J Cancer Prev. 2011;12(3):625-9. 31. Shokouh TZ, Ezatollah A, Barand P. Interrelationships Between Ki67, HER2/neu, p53, ER, and PR Status and Their Associations With Tumor Grade and Lymph Node Involvement in Breast Carcinoma Subtypes. Medicine (Baltimore). 2015;94(32):e1359. 32. Bojić T, Djordjević N, Karanikolić A, Filipović S, Granić M, Poultsidi AA. Assessment of axillary lymph nodes involvement in patients with breast cancer depending on the tumor size and its histological and nuclear grades. Vojnosanit Pregl. 2012;69(5):414-9. 33. Fisher ER, Redmond CK, Liu H, Rockette H, Fisher B. Correlation of Estrogen Receptor and pathologic

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Original Article Correlation of Fine Needle Aspiration Cytology and Core Needle Biopsies in Breast carcinoma: Our experience Parul Garg1*, Harjot Kaur2, Navtej Singh1, Arun Puri1 and Ishwer Tayal3 Department of Pathology, Guru Gobind Singh Medical College, Faridkot (Punjab), India Department of Pathology, Sri Guru Ram Das Institiutue of Medical Sciences and Research, (Punjab), India 3 Department of Forensic Medicine, Guru Gobind Singh Medical College, Faridkot (Punjab), India 1

Keywords: Breast, Cytology, Core needle Biopsy, Grading, Histopathology

ABSTRACT Background: The management of breast carcinoma relies heavily on the diagnostic parameters. Over the years there has been a steady decline in the usage of fine needle aspiration cytology (FNAC) over more popular core needle biopsy (CNB). The present study was conducted in a tertiary care teaching hospital to evaluate the role of FNAC and CNB in diagnosis of suspicious breast cancer by keeping mastectomies- surgical excision (SE) as gold standard. Methods: A total of 60 patients were subjected to simultaneous on site FNAC and CNB. Histopathological correlation (SE) was available in half of the cases only (30 cases). While the Cytological grading was done according to Robinson grading system; the histopathological grading was done by Nottingham Modification of Scarff-Bloom Richardson method. All the findings were compared and statistical analysis was done. Result: In contrast to FNAC where 29 cases were diagnosed as malignant only 26 cases were diagnosed as malignant on CNB with 3 cases signed out as non diagnostic for malignancy owing to non-sampling from the representative areas. Cytological grading assessment was higher with most of the cases kept in grade 3 (46.6 %) as compared to lower grading ( grade 2) on CNB- 62.07% cases. On comparing, FNAC and CNB; 22 showed agreement but this agreement was not statistically significant (p=0.14) Comparison of CNB and SE; in 17 cases agreement were noted for both CNB and SE and statistically the values were highly significant (p=0.0001). On comparison of FNAC and SE in 13 cases agreement was noted for both FNAC and SE and the data was statistically significant (p=0.035). Conclusion: Thus it was concluded in the present study that FNAC and CNB are comparable and also showed high sensitivity and pick up rate of FNAC as compared to CNB.CNB is however; more accurate for eventual grading and has an advantage that immunohistochemical studies can be applied on them.

*Corresponding author: Dr Parul Garg, Assistant Professor, Department of Pathology, Guru Gobind Singh Medical College, Faridkot (Punjab) India Email: parultayal45@gmail.com

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

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Introduction

Women worldwide seek medical opinion in setting of breast related problems such as breast masses, nipple discharge, breast pain and abnormal mammograms. Discrete palpable masses are the second most common breast symptom, following mastalgias, which need to be comprehensively distinguished from neoplastic transformation vis-àvis the normal nodularity of breast. [1] The likelihood of palpable mass being malignant increases with age of the patient. About 80% of palpable breast lumps are benign but malignancy can develop in benign breast lesions like fibrocystic diseases. The most common palpable lesions are cysts, fibroadenomas, and invasive carcinomas. Only 10% of breast masses in women younger than age 40 are malignant as compared with 60% of masses in women older than age 50.[2] So every woman presenting with a breast mass should be evaluated to exclude/establish a diagnosis of cancer.[3] Breast cancer is the commonest female malignancy worldwide.[4] In India, twelve population based cancer registries have shown it to be the most common followed by cervical cancer (ICMR-2004).[5] Increased reporting of breast cancer has led to the changes in modern management for the sole purpose of rapid preoperative diagnosis to allow the planning of a comprehensive therapeutic plan for the patient [6] FNAC and radiological imaging-mammography and ultrasonography as conjunction to the clinical examination (triple test) have become the standard approach to the investigation of palpable breast lumps and this has to some extent overcome the limitations of each individual method. [7] Minimally invasive biopsy refers to percutaneous techniques of sampling or removing suspicious areas of the breast without open surgery and excisional biopsy. These techniques include fine needle aspiration cytology and core needle biopsy. They are used for preoperative diagnosis of breast mass and has led to reduction of radical mastectomies in a large cohort of patients.[8] Extensive literature search fails to define the superiority of one method over other has not been established and may depend on the nature of the lesion, skill of the individual obtaining the sample and the skill of the pathologist interpreting the specimens. [9]It is computed by the studies done elsewhere that FNA biopsy of the breast is a diagnostically accurate procedure, having an average sensitivity of 87% (range of 72–99%), specificity of 98– 100%, negative predictive value of 87–99%, and efficiency of 89–99%. Technical difficulties resulting in less than optimal or unsatisfactory FNA specimens include the size of the mass, since both small and large lesions have been www.pacificejournals.com/apalm

associated with an increased false-negative rate. Breast masses smaller than 1 cm have a false-negative rate with a range of 6–24%. [10, 11] Though core biopsy has some limitations, it has gained popularity over last years, replacing FNAC as preferred modality prior to excision. Some authors have advocated the complementary role of synchronous FNAC and CNB in the management of palpable breast masses.[10] The present study was conducted in a tertiary care teaching hospital to evaluate the role of FNAC and CNB in diagnosis of suspicious breast cancer by determining the accuracy and compare the results of fine needle aspiration cytology (FNAC) and CNB in the diagnosis of the same.

Materials and Methods

This was a prospective study done in the department of Pathology of a tertiary care teaching hospital catering to the urban as well as rural population in northern part of India. A total of 60 patients with clinically suspicious lumps in the breast were included in this study. These patients were subjected to simultaneous on site Fine needle aspiration cytology and CNB. Histopathological correlation in the form of follow up excision biopsies (lumpectomies) and mastectomies- surgical excision (SE) were available in half of the cases only (30 cases). The cytological findings were compared with histopathological findings of both CNB and SE. The cytology slides were stained with May- Grunwald - Giemsa and Pap stains while the biopsy slides were stained with Hematoxylin and Eosin stain. The sensitivity, specificity positive and negative predictive value of FNAC was calculated and compared with that of CNB. Overall efficacy of both procedures was calculated statistically keeping SE as the gold standard. While the Cytological grading was done according to Robinson grading system [24] with score 6-11 kept as Grade I; while the scores 12-14 and 15-18 were kept as Grade II and III; the histological grading of H&E stained tissue sections was done by Nottingham Modification of Scarff -Bloom Richardson method[12] with score 3-5 kept as Grade I and scores 6-7 and 8-9 kept as Grade II and III respectively. Statistical Analysis: All the findings were compared and statistical analysis was done for sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy. The findings were deemed significant in case p value was < 0.05 by employing chi- square test.

Result

After noting the brief history and clinical findings in all patients, FNAC and trucut biopsy was performed under aseptic conditions. Out of 60 cases histopathological eISSN: 2349-6983; pISSN: 2394-6466

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specimens were available in only 30 cases as lumpectomy and mastectomy specimens for comparison. Overall material was satisfactory for evaluation in all the cases of FNAC while it was not satisfactory/ inconclusive in 3 cases of CNB. Apart from procedural pain and post procedural swelling ; no major complication was recorded in the present study. The age range in the present study was 21-80 years with all females and the mean lump size > 2 cm ( 3.8 cm). On FNAC, 29 cases were diagnosed as malignant breast lesion. One case was diagnosed as fibroadenoma on FNAC despite clinically suspicious presentation of the lump (lump in a 50 year old patient; not freely mobile); this was also noted on CNB. In contrast to FNAC, 26 cases were diagnosed as malignant on CNB with 3 cases signed out as non diagnostic for malignancy owing to non-sampling from the representative areas. On SE specimens 29 cases reported as malignant on FNAC, there was 100 % concurrence with the finding of fibroadenoma confirmed. (Table 1) . FNAC vs. SE: Comparing FNAC with SE (keeping it as gold standard diagnostic procedure), FNAC showed 100% agreement with accuracy with 100% sensitivity and 100% specificity. CNB vs. SE: Comparing CNB with SE (keeping it as gold standard diagnostic procedure) , although specificity was 100 % but sensitivity of CNB procedure was 89.67 %. This implies that although PPV was 100 % in CNB procedure but NPV was only 25 %. Grading on FNAC: Out of 58 cases, maximum cases were graded as grade 3 (46.55 %) followed grade 2 (36.21 %) and grade 1 (17.24 %). [Figures 1 A, 1B, 2 A, 3 A and 3B] Grading on CNB: Maximum number of cases i.e.36 (62.07%) was seen in grade 2 followed by 9 cases (15.52%) in grade 3 and 7 cases (12.07%) in grade 1. The grading was not done in 6 cases ( 3 cases of non –diagnostic material and 3 cases of pauci-cellular material where grading was

not feasible to account for the third parameter of mitotic count. [Figures 1 C, 2B, 3 C]. Grading on SE: When grading was done on lumpectomy or mastectomy specimens, maximum cases were in grade2 ie14 cases (48.27%), followed by 9 cases (31.03%) in grade1 and 5 cases (17.24%) cases in grade 3.[Figures 1 D and 3 D]. In one case grading was not possible as there were only few tumor cells because the patient underwent chemotherapy for de-bulking after FNAC and CNB diagnosis. Comparison of FNAC and CNB: Out of 58 cases, 10 cases were diagnosed as grade 1 on FNAC but the number was reduced to 7 cases on CNB whereas the grade 2 cases increased markedly from 21 cases on FNAC to 36 cases on CNB. Grade 3 was given in 27 cases on FNAC, which got reduced to 9 cases on CNB. Out of 58 cases in diagnostic yield was adequate in both the procedures, 22 showed agreement but this agreement was not statistically significant (p=0.14) (Table 2). Comparison of CNB and SE: Out of 29 cases, grade 1 was given in 6 cases on CNB but it increased to 9 cases on SE. Thirteen cases were given grade 2 on CNB but it was 14 cases on SE. Grade 3 was given in 6 and 5 cases on CNB and SE respectively. Despite material being non diagnostic in one case on CNB, in 17 cases agreement were noted for both CNB and SE and statistically the values were highly significant (p=0.0001). (Table-3). Comparison of FNAC and SE: Out of 29 cases, cytological grade 1 was seen in 7 cases whereas on SE, it increased to 9. Seven cases were given grade 2 on FNAC but number was increased to 14 on SE whereas grade 3 was given in 15 cases on FNAC and it was reduced to 5 cases on SE. Despite owing to pauci -cellular nature of one of the biopsies on CNB, in 13 cases agreement was noted for both FNAC and SE and the data was statistically significant (p=0.035). (Table 4).

Table 1: Showing comparison of FNAC, CNB and histopathology (SE). FNAC 29 1 0 30

Malignant Fibroadenoma Material non diagnostic Total

CNB 26 1 3 30

SE 29 1 0 30

Table 2: Showing comparison of FNAC and CNB grading. FNAC Grade 1 Grade2 Grade 3 Total

Grade 1 3 2 2 7

Grade 2 5 14 17 36

Grade 3 2 2 5 9

CNB Non Diagnostic 0 2 1 3

Grading Not Possible 0 1 2 3

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Total 10 21 27 58

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Table 3: Showing comparison of CNB and SE grading. CNB Grade1 Grade 2 Grade 3 Grading not possible Material non diagnostic Total

Grade 1 5 2 1 1 0 9

Grade 2 1 9 2 1 1 14

Grade3 0 1 3 1 0 5

Grading not possible 0 1 0 0 0 1

Total 6 13 6 3 1 29

Table 4: Showing comparison of FNAC and SE grading. FNAC Grade 1 Grade2 Grade3

Grade 1 4 2 3 9

Grade2 2 5 7 14

Grade3 1 0 4 5

Grading not possible 0 0 1 1

Total 7 7 15 29

Fig. 1: A and 1 B Carcinoma Breast –FNAC Grade 1 (MGG 100X)Vs CNB Grade 1 (H&E 40 X).

Fig. 1: C and 1 D Carcinoma Breast –CNB Grade 1 (H&E 400X) and SE (mastectomy) Grade 1 ( H& E 100 X).

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Fig. 2: A Carcinoma Breast –FNAC Grade 2 (MGG 100X).

Fig. 2: B Carcinoma Breast –CNB Grade 2 (MGG 200X).

Fig. 3: A and 3 B Carcinoma Breast –FNAC Grade 3 (MGG 100X) Vs CNB Grade 1 (H&E 40 X).

Fig. 3: C and 3 D Carcinoma Breast –CNB Grade 3 (H&E 400X) and SE (mastectomy) Grade 1 ( H& E 400 X).

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Discussion

In the present study on morphological typing, 28 cases were typed as ductal carcinoma-not otherwise specified (IDC-NOS) and 1 was typed as mucinous carcinoma. All of these cases were confirmed on CNB and SE except for mucinous carcinoma, which was not diagnosed correctly on CNB as no mucin could be demonstrated on CNB but at the same time it was diagnosed correctly on FNAC. Several studies have compared the utility of both FNA and CNB in differentiating benign from malignant mucinous breast lesions such as Lam et al who have shown FNAC as 56% sensitive and CNB as 100% accurate in diagnosis of mucinous carcinoma but this was not in our study.[13] The fact that there was only one case of mucinous carcinoma in the spectrum of 60 breast malignancies is also to be kept in mind and probably no conclusion can be drawn regarding accuracy of CNB vis a vis mucinous carcinoma in our study. Although in the present study conducted of consecutive 60 cases of clinically suspicious breast carcinoma the diagnostic yield was 100 % but in large case series review for clinical audit/ quality assurance program especially the one undertaken by the College of American Pathologists (CAP) which had included a retrospective assessment of more than 13,000 FNA biopsy specimens of the breast from 294 institutions, some 82% of the cases were satisfactory for evaluation, with one-third having histologic correlation that served as the basis for determining diagnostic accuracy. The cases for diagnostic accuracy in our study were a little higher accounting for half of the cases.[11] Statistical parameters of FNAC Various studies have been done to determine the efficacy and usefulness of both FNAC and CNB; and the results vary. When SE was taken as diagnostic procedure, sensitivity of FNAC was calculated as 100% which was similar to those seen by Ballo and Sneige et al[10] and others[14, 15] but was much higher in comparison than those reported by others[4, 16, 17] [Table-5]. Using similar statistical parameters the specificity calculated in the present study of FNAC was 100% which is in concordance with the work done by Khanna et al [15] but higher when compared with other researchers. Positive predictive value (PPV) of 100% also was similar to that reported by Homesh et al[4] and Scopa et al.[18]Negative predictive value (NPV) of 100% which is much higher when compared with other studies. [4, 18] [Table no 5] False positivity on FNAC was seen in 0% cases, which were same to that seen by Westenend et al 17Berner et al showed false positivity of 1.7%, which is much higher than present study. False negativity was reported as 0%, www.pacificejournals.com/apalm

which was much lower when compared with others.[16, 19, 20] [Table-5] Other researchers such as Ducatman et al who have extensively reviewed the various studies conducted world wide state that false-positive results in FNAC of breast occur in 0 to 2% of cases while False-suspicious result rates are higher, ranging from 1% to 13%.[21] Statistical parameters of CNB: Taking SE as diagnostic procedure, sensitivity of CNB was calculated as 89.67% which was similar to that reported by many researchers [Table 6] but was lower when compared with the study of Mohammed et al[22] who reported sensitivity of 97%respectively. While specificity and PPV of CNB calculated in present study was 100% which is in concordance with the work conducted by many researchers.[10, 15, 17, 18, 22, 23, 24, 25, 26]but calculated NPV value of 25 % in comparison was much lower than studies done elsewhere. [Table-6]. No false positive case was reported on CNB which was comparable with study done by Cusick et al[23] and Berner et al.[20] Gukas et al[27] however has reported false positive cases to be higher (4.8%). 10% cases were reported as false negative which was similar to study done by other author [19] but was much higher than reported by Berner et al.[20] Statistical Comparison FNAC and CNB: In the present study, it was found that FNAC and CNB do equally well for specificity (100% in both) and PPV (100% for both). But statistical difference was found in sensitivity (100% for FNAC vs 89.67% for CNB), NPV (100% for FNAC and 25% for CNB) and diagnostic accuracy (100 % for FNAC and 90 % for CNB).These were comparable with studies done by Ballo and Sneige et al[10]and Scopa et al.[18] [Table 7]. As noted above in the present study no major life threatening complication was encountered apart from pain and swelling, mild bleeding and bruising associated with the procedure. However, a major life threatening complication attributable to the breast FNAC is pneumothorax owing to its location is described in literature, which was first described by Orr and Margarey in 1978. [28] The complication rate of pneumothorax has been reported as 1 in 10,000 cases (0.01%) in a large Italian study of 2,00,000 FNAC procedures to 1 in 1000 by Gateley to as high as 1 in 417 by Kaufman. [29] However; it is also thought that these figures could be just a tip on the proverbial iceberg due to many unrecognized and asymptomatic cases of pneumothoraces occurring post FNAC or CNB procedures.

Conclusion

Thus it was concluded in the present study that FNAC and CNB are comparable and as it was evident that none of the eISSN: 2349-6983; pISSN: 2394-6466

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Correlation of FNAC Vs CNB in Ca Breast

Table 5: Comparison of various parameters of FNAC with other studies. Reference

Sensitivity (%)

Specificity (%)

Accuracy

PPV

NPV

Karin et al6

96.8

100

Khanna et al

Ballo and Sneige et al

97.5

100

Scopa et al16

100

Aristo et al12

94.6

99.9

98.8

Kim et al14

64.5

71.9

98.4

66.66

81.8

75.7

100

56.1

80.9

63.8

100

Homesh et al Randa et al

Present study

Table 6: Comparison of various parameters of CNB with other studies. Reference Cusick et al

Year of publication

Sensitivity

Specificity

Accuracy

PPV

NPV

1990

100

Khanna et al

1991

100

Scopa et al16

1996

100

Caruso et al23

1998

100

Gukas et al

2000

88.9

96.8

93.5

95.2

Homesh et al3

2005

92.3

94.8

93.4

100

Mohammed et al19

2008

100

89.67

100

Present study

case was negative on FNAC but three cases had a false negative report on CNB, which shows high sensitivity and pick up rate of FNAC as compared to CNB. Hence in the todayâ&#x20AC;&#x2122;s diagnostic world where CNB is replacing FNAC, we still recommend that FNAC can be an important quick, cost effective and relatively painless procedure in comparison to CNB. In FNAC one can perform the procedure from various directions even in smaller lumps whereas CNB in very small lump is not feasible, local anesthesia is required, the needle may not hit the representative area especially if it is small, freely mobile and patient is non cooperative. Thus it was concluded in the present study that as FNAC gives 100% results, it is quite reliable investigation and CNB should be done as an adjunct if needed especially for grading of the tumor and for immunohistochemistry.

Acknowledgements

No Acknowledgment required.

Funding None

Competing Interests None Declared

Reference

1. Lester SC. The breast. In: Kumar V, Abbas AK, Fausto N. Robbins and Contran Pathologic Basis of disease. 9th ed. New Delhi: Reed Elsevier India Private Limited; 2014. p.1143-73. 2. Anders CK, Johnson R, Litton J, Phillips M, Bleyer A. Breast carcinoma before age 40 years. SeminOncol. 2009; 36:237-9. 3. Parker SH, Percutaneous large core breast biopsy. Cancer 1994; 74(1):256-62. 4. Homesh NA, Issa MA, El-Sofiani HA. The diagnostic accuracy of fine needle aspiration cytology versus core needle biopsy for palpable breast lump(s). Saudi Med J 2005; 26 (1):42-6. 5. Dutta DC. Genital malignancies. In: Dutta DC. Textbook of Gynaecology. 5thed. New Delhi: New central book agency (P) Ltd; 2009 .p. 321-72 6. Clarke D, Sudhakaran N, Gateley CA. Replace fine needle aspiration cytology with automated core biopsy in the triple assessment of breast cancer. Ann R CollSurgEngl 2001; 83(2):110-2. 7. Karin L. Breast In: Orell SR, Sterrett GF, Whitaker D. Fine needle aspiration cytology, 4th ed. Churchill Livingstone Co 2005; 165-9.

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8. Hernan IV, Shahla M. Implementation of a minimally invasive breast biopsy program in countries with limited resources. The Breast 2003;9(2):81-5. 9. Shannon J, Douglas Jones AG, Dallimore NS. Conversion to core biopsy in preoperative diagnosis of breast lesions: is it justified by results? Clinical Pathology 2001; 54: 762-5. 10. Ballo MS, Sneige N. Can core needle biopsy replace fine-needle aspiration cytology in the diagnosis of palpable breast carcinoma? A comparative study of 124 women. Cancer 1996 15; 78 (4):773-7. 11. Comprehensive Cytopathology. Breast. In: Bibbo M, Wilbur DC. 4thed. New Delhi: Reed Elsevier India Private Limited; 2015. Pp 583. 12. Rosai J. Breast. In: Rosai and Ackerman’s Surgical Pathology. 9th ed. Noida: Reed Elsevier India Private limited; 2009. p. 1787-1827. 13. Lam WW, Chua WC, Tseb GM, Mac TK, Tango AP. Role of fine needle aspiration and trucut biopsy in diagnosis of mucinous carcinoma of breast-from a radiologist’s perspective. Clinical Imaging 2006; 30 (1): 6-10. 14. Aristo R, Cuccorese C, Accinelli G, Mano MP, Bordon R, Fessia L. Role of fine-needle aspiration biopsy in breast lesions: Analysis of a series of 4,110 cases. 1998; 18(6):462–7. 15. Khanna AK, Singh MR, Khanna S, Singh AK.Fine needle aspiration cytology, imprint cytology and trucut needle biopsy in breast lumps: a comparative evaluation. J Indian Med Assoc1991; 89(7):192-5. 16. Kim A, Lee J, Choi JS, Won NH and Koo BH. Fine Needle Aspiration Cytology of Breast. Experience at an outpatient breast clinic. ActaCytologica 2000; 44:361-7 17. Khair RZA, Musa M T, Al-Arabi YA. Evaluation of cytopathology in diagnosing breast cancer in Khartoum. Khartoum Medical Journal 2009; 02(1):169 – 71. 18. Scopa CD, Koukouras D, Spiliotis J, Harkoftakis J, Koureleas S, Kyriakoupoulou D, Tzoracoleftherakis E. Comparison of fine needle aspiration and Tru-Cut biopsy of palpable mammary lesions.Cancer Detect prev1996; 20(6):620-4.

19. Westenend PJ, Sever AR, Beekman-De Volder HJ, Liem SJ. Comparison of aspiration cytology and core needle biopsy in the evaluation of breast lesions. Cancer 2001; 93(2):146-50.

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20. Berner A, Sigstad E, Reed W, Risberg B. Fine-needle aspiration cytology or core biopsy when diagnosing tumours of the breast. TidsskrNorLaegeforen 2003; 123(12):1677-9. 21. Wang HH. Cytology. Diagnostic Principles and Clinical Correlates. In: Cibas ES, Ducatman BS. 4thed. New Delhi: Reed Elsevier India Private Limited; 2014. Pp 233. 22. Bdour M, Hourani S, Mefleh W, Shabatat A, Karadheh S, Nawaiseh O et al. Comparison between fine needle aspiration cytology and trucut biopsy in the diagnosis of breast cancer. Journal of Surgery Pakistan (international) 2008; 13 (1): 19-21. 23. Cusick JD, Dotan J, Jaecks RD. The role of Tru-Cut needle biopsy in the diagnosis of carcinoma of the breast. SurgGynecolObstet1990; 170(5):407-10. 24. Minkowitz S, Moskowitz R, Khafif RA, Trucut needle biopsy of the breast, an analysis of its specificity and sensitivity. Cancer. 1986; 57(2): 320-3. 25. Vega A, Garijo F, Ortega E. Core needle aspiration biopsy of palpable breast masses 1995; 34(1): 31-4. 26. Caruso ML, Gabrieli G, Marzullo G, Pirrelli M, Rizzi E, Sorino F. Core biopsy as alternative to fine needle aspiration biopsy in diagnosis of breast tumors. The Oncologist 1998; 3: 45-9. 27. Gukas ID, Nwana EJ, Ihezue CH, Momoh JT, Obekpa PO. Tru-cut biopsy of palpable breast lesions: a practical option for pre-operative diagnosis in developing countries. The Central African Journal of Medicine 2000; 46 (5): 127-30. 28. Orr KB, Margarey CJ. Pneumothorax after aspiration of breast cysts. Med J Aust. 1978;1:101. 29. Salhab M, Sarakbi WA, Perry N, Mokbal K. Pneumothorax after a clinical breast fine-needle aspiration of a lump in a patient with Poland’s syndrome. IntSeminSurgOncol. 2005; 2:14-7.

Original Article Study of Glycated hemoglobin level in Non-Diabetic Iron Deficiency Anemia Bhavesh Ramji Faldu1, Chetankumar Maganlal Dharaiya2 and Harshid L Patel3* Department of Pathology, B.J.Medical College, Ahmedabad, Gujarat, India Department of Pathology, GMERS Medical College, Sola, Ahmedabad, India 3 Department of Pathology, GMERS Medical College, Gandhinagar, Gujarat. India 1

Keywords: Anemia, Glycated hemoglobin, Non-diabetic, HbA1c

ABSTRACT Background: The traditional role of HbA1c analysis has been for assessing glycaemic control in patients with diabetes. The results of seminal studies demonstrated that early, intensive glycaemic control could significantly reduce the risk of a range of diabetes-related complications, and permitted the establishment of precise HbA1c target values for treatment goals. Methods: Study involves total Total 140 partcipants and among them 70 are non-diabetic, anaemic patients and 70 agematched healthy subjects. Haematologic investigations were done and the fasting and postprandial glucose and HbA1c levels were measured in all the subjects. Result: The mean HbA1c (9.1 Âą 1.7%) level in the patients with IDA was higher than that in the control group (5.5% Âą 0.8) (p < 0.05). There were no differences in the levels of fasting and postprandial glucose between the IDA and the control groups (p > 0.05). Conclusion: HbA1c is not affected by the blood sugar levels alone, and there are various confounding factors when HbA1c is measured, especially that of iron deficiency, which is the commonest of the deficiency diseases worldwide. It is hence important to rule out IDA before making a therapeutic decision, based on the HbA1c levels.

*Corresponding author: Dr Harshid L Patel, 9, Aditi Tenament, B/H Deswali society, K.K Nagar Road, Umiya Hall,Ghatlodiya, Ahmedabad, Gujarat, Pin-382481 INDIA Email: harshidpatel11111@gmail.com

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Introduction

Diabetes mellitus is a metabolic disease which is caused by absolute or relative insulin deficiency. About 10% of the Indian population suffers from this disease. Various factors play a role in the aetiopathogenesis and in the glycaemic control among the type 2 diabetic patients.[1] The term HBA1C refers to glycated haemoglobin. It develops when haemoglobin, a protein within red blood cells that carries oxygen throughout your body, joins with glucose in the blood, becoming ‘glycated’. By measuring glycated haemoglobin (HbA1c), clinicians are able to get an overall picture of what our average blood sugar levels have been over a period of weeks/months. HbA1c is likely to be affected by iron deficiency and iron deficiency anemia with a spurious increase in HbA1c values, the researchers wrote. This may lead to confusion when diagnosing diabetes using HbA1c. This review clearly identifies the need for more evidence, especially in identifying the types and degrees of anemia likely to have significant impact on the reliability of HbA1c. [2] According to the American Diabetes Association (ADA) guidelines, the value of HbA1c should be kept below 7% in all the diabetics .[3] The values which are greater than 7% indicate an increased chance of progression to the dia-betic complications, especially the microvascular ones. When plasma glucose is consistently elevated, the nonenzymatic glycation of haemoglobin increases; this altera-tion reflects the glycaemic history over the previous 2–3 months, since erythrocytes have an average lifespan of 120 days.[4] Evidence has accumulated, which supports the hypothesis that the glycation reaction, apart from the traditional chronic hyperglycaemia, can be modulated by the iron status of the patient. If the degree of glycation of other proteins in anaemic patients was similar to that of the glycated haemoglobin, it would have important clinical implications. Thus, the main aim of the present study was to determine whether the HbA1c levels were increased among the anaemic patients without diabetes. If so, the iron deficiency has to be corrected before any diagnostic or therapeutic decision was made based on the HbA1c level. [5]

Materials and Methods

This study was conducted at B.J. Medical College, Ahmedabad, Gujarat. from 2012-2013 in department of pathology. Blood samples (3ml) were obtained from 70 anaemic patients of the mean age, 45±5 years, among which 35 were males and 35 were females and 70 age-matched healthy subjects. The anaemic patients were recruited from the Medicine Outpatients Department of our institute. www.pacificejournals.com/apalm

All collected sample were analysed for complet blood count (CBC), Diabetic profile (FBS, PP2BS) and HbA1c. CBC was done in 3 part Nihon kohden Celtac Alpha hematology analyser. The anaemic patients were selected, based on their haemoglobin levels (Hb < 11 g/dl), and on their peripheral blood smears (mostly microcytic hypochromic), which suggested iron defiiciency anaemia and on their haematologic investigations(Like MCV,MCH,MCHC) and serum fasting and postprandial glucose levels. The HbA1c levels were determined by turbidimetric immunoinhibition.The peripheral blood smears were examined in all the patients.Estimation of blood sugar was done by GOD-POD method in semi automated biochemiatry analyser alon with Biorad Quality control sera. Exclusion Criteria: Patients having Diabetes mellitus and any type of hemoglobinopathies were excluded from our study.

Result

All the parameters which were tested in both the groups have been reported in [Table 1]. The fasting and the postprandial blood glucose levels confirmed the non-diabetic status. The peripheral blood smears showed a hypochromic microcytic picture. The HbA1c levels were signifiicantly increased among the IDA patients as compared to those in the controls.[Table 2] The mean HbA1c (9.1 ± 1.7%) level in the patients with IDA was higher than that in the control group (5.5 ± 0.8%) (p < 0.05) [Table 3]. There were no differences in the levels of fasting and postprandial glucose between the IDA and the control groups (p > 0.05). Table 1:Age wise distribution of participant. Group Test (IDA) Control

Number(n) 70 70

Age(year) Mean ± SD 45±5 43±4

Table 2: Laboratory data of study group. Test Group (IDA) (N=70)

Control Group (N=70)

Hemoglobin gm/dl

9.9±1.5

12.1±1.3

PCV %

30.1±4.3

41.4±2.7

MCV fl

79.1±4.9

83.2±4.6

MCH pg

24.5±2.2

32.9±1.7

Fasting blood Glucose mg/dl

96.5±9.4

93.5±9.8

Postprandial blood sugar Mg/dl

114.8±6.1

116.5±5.8

9.1±1.7

5.5±0.8

Parameter

HbA1C %

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HbA1c Level in Iron Deficiency Anemia

Table 3: Comparisons of various parameter between Test(n=70) and control(n=70) Group. Parameter Hb (gm/dl) Fasting blood sugar (mg/dl) Postprandial blood sugar (mg/dl) HbA1C %

Group

Number(n)

Result(Mean)

Test

9.9

Control

12.1

Test

96.5

Control

93.5

Test

114.8

Control

116.5

Test

9.1

Control

5.5

P-value <0.05 >0.05 >0.05 <0.05

Graph 1: Showing comparison of HbA1c (%) concentration between test and control group.

Discussion

The results of the present study show that Hb concentrations are positively correlated with HbA1c concentrations, that HbA1c concentrations tended to be higher in the presence of iron deficiency, and that participants with IDA had similar HbA1c concentrations as participants with normal concentrations of Hb and a normal iron status. The positive association between HbA1c concentrations and Hb may have offset the small inverse association between HbA1c concentrations and iron status. We observed erythrocyte size and Hb to have a negative correlation with HbA1c, compatible with the hypothesis that iron deficiency increases Hb glycation, perhaps related to an association between insulin resistance and higher glucose levels. Our results shows that higher concentration of HbA1c is found in iron deficiency patients as compared to control group. Similarly, Brooks et al., showed higher HbA1c concentrations in iron-deficient nondiabetic adults, which decreased to normal after iron replacement. [6] Hansen et al.showed normal HbA1c concentrations in

iron deficiency, which dropped to subnormal levels after iron supplementation. [7] In contrast with several previous studies, our results suggest that IDA has little population effect on concentrations of HbA1c or on diabetes prevalence. In an early study from the US, the mean HbA1c concentration for four patients with IDA was 4.9% compared with a mean HbA1c concentration of 5.3% among 14 healthy adults. [8] In a subsequent study from the UK, the mean total HbA1 concentration was 9.9% among 35 non-diabetic men and women with IDA, levels higher than the normal mean of 7.9%.[9] A case report from Australia noted that IDA was associated with a rise in the concentration of HbA1c. [10] A subsequent report of 14 non-diabetic patients with IDA from the UK noted a mean concentration of HbA1c of 6.9% compared with a normal mean of 7.0%.[11] The authors attributed the differences in findings with previous studies to differences in methodology for measuring HbA1c. Another study from India that included 15 nondiabetic patients with IDA and 12 controls also failed to find a difference in mean concentrations of HbA1c. [12]

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The exact reason for that is not found till date but it was suggested that in iron deficiency, the quaternary structure of the hemoglobin molecule was altered, and that glycation of the globin chain occurred more readily in the relative absence of iron . Sluiter et al. tried to provide an explanation for the above findings. They proposed that the formation of glycated hemoglobin is an irreversible process and hence, the concentration of HbA1 in 1 erythrocyte will increase linearly with the cell’s age. [13] Several limitations should be noted. First, the results from the present study may not be fully generalizable to individuals with severe degrees of IDA or non-IDA. Second, although the sample size in the present study was large, the number of participants with IDA was nevertheless limited. Thus, additional studies with larger numbers of participants with IDA would be helpful in examining the impact of IDA on measurements of HbA1c. Saudek et al considered measurements of HbA1c to be invalid in the presence of anemia. [14] Although these warnings are sound, our analyses suggest that anemia is unlikely to be a major concern in diagnosing diabetes using concentrations of HbA1c in the US. The difference in concentrations of HbA1c between extremes of concentrations of Hb is in the order of an absolute 0.2%. This would suggest that primarily people with anemia who are close to the diagnostic threshold may require retesting or the use of another diagnostic method. Therefore, consideration should be given to performing glucose testing in patients who have low Hb concentrations and an HbA1c concentration just below the diagnostic threshold for diabetes and prediabetes or who have high Hb concentrations and an HbA1c concentration just above the diagnostic threshold for diabetes and prediabetes. [14] According to some investigators, the increase in the glycated haemoglobin levels in non-diabetic anaemic patients has been mainly attributed to the decrease in the haemoglobin levels in these patients But studies which have investigated the glycation levels of other proteins have not been carried out. This study has got a significant relevance because iron deficiency anemia is very highly prevalent in a tropical country like India. IDA, being a common variable, influences the HbA1c levels when they are estimated by the most commonly employed methods like immunoturbidometry and so, the IDA must be corrected before making any diagnostic or therapeutic decision based on the HbA1c levels. HbA1c is commonly used to www.pacificejournals.com/apalm

assess the long-term blood glucose control in the patients with diabetes mellitus, because the HbA1c value has been shown to predict the risk for the development of many of the chronic complications in diabetes.

Conclusion

From our study we would like to conclude that iron deficiency was associated with higher proportions of HbA1c, which could cause problems in the diagnosis of uncontrolled diabetes mellitus in iron-deficient patients.

Acknowledgements

We Acknoweldge whole Department of pathology and medicine of our Institute.

Funding None

Competing Interests None Declared

Reference

1. Mangukiya K, Sharma N. Clinical correlation between diabetes mellitus type 2 and Vitamin D3 (25 OHCholecalciferol) Level, I.J.S.N., 2014;5 (3) 4402. Telen MJ, Kaufman RE. The mature erythrocyte. In: Greer JP, Forester J, et al., editors. Wintrobe’s clinical hematology. 11th ed. Lippincot: Williams and Wilkins; 2004. pp. 230. 3. American Diabetes Association. Position statement: Standards of medical care in diabetes-2007. Diabetes Care 2007;30 S1:S9 4. Shekhar H, Mangukiya K, Kaur A, Jadeja P. Effect of iron deficiency anemia on glycation of haemoglobin in nondiabetics, I.J.S.N., 2014; 5(3) 477-79 5. El-Agouza I, Abu Shola A, Sirdah M. The effect of iron defiiciency anaemia on the levels of the haemoglobin subtypes: the possible consequences in a clinical diagnosis. Clin Lab Haematol.; 2002;24:285e9 6. Brooks AP, Metcalfe J, Day JL, Edwards MS. Iron defiiciency and glycosylated haemoglobin A1. Lancet. 1980;19(ii):141 7. Hansen PG, Eriksen J, Andersen TM, Olesen L. Glycosylated hae-moglobin (HbA1c) in iron and vitamin B12 deficiency. J Intern Med. 1990; 227:133–36 8. Horton BF, Huisman TH. Studies on the heterogeneity of haemoglobin. VII. Minor eISSN: 2349-6983; pISSN: 2394-6466

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HbA1c Level in Iron Deficiency Anemia

haemoglobin components in haematological diseases. Br J Haematol. 1965; 11: 296–304. 9. Brooks AP, Metcalfe J, Day JL, Edwards MS. Iron deficiency and glycosylated haemoglobin A. Lancet.1980;2:141 10. Davis RE, McCann VJ, Nicol DJ. Influence of irondeficiency anaemia on the glycosylated haemoglobin level in a patient with diabetes mellitus. Med J Aust. 1983; 1: 40–1. 11. Van Heyningen C, Dalton RG. Glycosylated haemoglobin in iron-deficiency anaemia. Lancet. 1985; 1:874.

12. Rai KB, Pattabiraman TN. Glycosylated haemoglobin levels in iron deficiency anaemia. Indian J MedRes.1986;83:234–6. 13. Sluiter WJ, van Essen LH, Reitsma WD, Doorenbos H. Glycosylated haemoglobin and iron deficiency. Lancet 1980;2:531–532. 14. Saudek CD, Herman WH, Sacks DB, Bergenstal RM, Edelman D, Davidson MB. A new look at screening and diagnosing diabetes mellitus. J Clin Endocrinol Metab. 2008; 93: 2447–53.

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Original Article Correlation of Ki67 with ER, PR, Her2neu and other Prognostic Factors In Breast Carcinoma Menka Khanna*, Manisha Sharma, Mridu Manjari, Kiranjot Kaur, Saumil Garg and Saloni Goyal Department of Pathology, SGRDIMSAR, Amritsar, India Keywords: Breast Cancer, Immunohistochemistry, Ki67, ER, PR, Her2neu

ABSTRACT Background: Breast cancer is the most common malignancy occurring in females, accounting for 23% of all malignant tumors.1 Various predictive and prognostic factors affect tumor progression. In addition to estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (Her2neu) overexpression, recently Ki67, a proliferative marker has been recognised as an important predictive and prognostic marker in many studies. The aim of the present study is to find the correlation of Ki67 proliferation index with ER PR and Her2neu status and with other clinicopathological parameters. Methods: The study was conducted on 70 cases of breast cancer received as mastectomy or lumpectomy specimens. The histopathological grading of the breast carcinoma was done according to the Nottingham modification of the Bloom Richardson grading system. IHC was performed by using antibodies against the estrogen receptors (ER), the progesterone receptors (PR), Her2neu and Ki67. An attempt was made to find a correlation between ER, PR, Her2neu and Ki67 expression with the various clinicopathological parameters. Result: ER PR expression was seen to be decreased with increase in Ki67 positivity. Ki67 proliferation index when correlated with the various clinicopathological parameters (grade, lymph node metastasis and size of tumor) it was concluded that as the Ki67 positivity increased the prognosis became poorer. Conclusion: It was concluded that Ki67 can be considered as a new prognostic marker in addition to ER PR and Her2neu and is associated with a poor prognosis.

*Corresponding author: Dr. Menka Khanna, Department of Pathology, SGRDIMSAR, Amritsar, India Phone: +91 9464986355 Email: mona74_khanna@yahoo.co.in

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Ki67, ER, PR, Her2neu Expression in Breast Carcinoma

Introduction

Breast cancer is the most common malignancy occurring in females, accounting for 23% of all malignant tumors. [1] Over one lakh new breast cancer patients are estimated to be diagnosed every year in India. It is expected to increase by 26% by 2020 in developing countries. It is the second most common carcinoma in rural areas. However, in urban India it is the commonest cancer among women, where it constitutes >30% of all cancers in females.[2,3] Various predictive and prognostic factors affect tumor progression.[4,5] Predictive factors determine the response to treatments and prognostic factors are measurable and are associated with the nature of the disease.[4] Some factors are both prognostic and predictive, including estrogen receptor (ER) and progesterone receptor (PR) status and human epidermal growth factor receptor (Her2neu) overexpression. Prognostic factors include the type of tumor, number of involved lymph nodes at the time of tumor diagnosis, size of the tumor, tumor grade and the patient’s age.[6,7] Classification of breast carcinoma according to immunohistochemistry (IHC) are luminal A (ER +ve and PR +ve, Her2neu –ve ), luminal B (ER+ve, and/or PR +ve, Her2neu +ve or-ve ), triple negative or basal like (ER-ve, PR-ve and Her2neu-ve) and Her 2 type (ER-, PR-, Her2neu+).[8] Estrogen receptor (ER) is the most important prognostic and predictive marker for breast cancer.[9 ]Presence of both ER and PR is related to better prognosis and responsiveness to hormonal therapy.[10] Typically Her2 amplified tumors are associated with high grade and often extensive ductal carcinoma but respond well to specific chemotherapy. The expression of the nuclear proliferating antigen, Ki67 has been observed to reflect the proliferation rate of malignant tumors. It is associated with the development and metastasis of a variety of malignant tumours, as well as with the prognosis of patients.[11] Many studies found Ki67 to be a predictive and prognostic marker for either clinical and/or pathological response. This will be particularly helpful in cases which are triple negative.[12] The aim of the present study is to find the correlation of ER, PR, Her2neu and Ki67 expression with the various clinicopathological parameters and also determine correlation of Ki67 proliferation index with ER PR and Her2neu status.

Materials and Methods

The study was conducted on 70 cases of breast cancer received as mastectomy or lumpectomy specimens in the Department of Pathology, Sri Guru Ram Das institute of medical sciences, Sri Amritsar. The clinical history of the patients was taken. The tissue was formalin fixed and

paraffin embedded and was then stained for Haematoxylin and Eosin for histopathological typing and grading. The histopathological grading of the breast carcinoma was done according to the Nottingham modification of the Bloom Richardson grading system. IHC was performed by using antibodies against the estrogen receptors (ER), the progesterone receptors (PR) (Diagnostic Biosystem) and Her2neu, Ki67 (Biocare Medical). The antigen retrieval was done by using pressure cooker method with 10mmol citrate buffer at pH 6. Tris buffer was used as the wash buffer and diaminobenzene tetrahydrochloride (DAB) was used as the chromogen. The endogenous activity was blocked by using hydrogen peroxide. After protein blocking, the slides were incubated overnight with the available ER, PR, Her2neu and Ki67 primary antibodies and were conjugated with streptavidin Horse Radish Peroxidase (HRP). The slides were counterstained with hematoxylin and were examined by light microscopy. For ER ≥10% nuclei stained brown were taken positive and 10% stained were taken positive for PR.[13] For Her2neu, membranous staining in >25% cells was taken as positive. Ki-67 immunoreactivity was based on the proportion of positive tumor cells (0-100%) regardless of staining intensity. levels of Ki-67 are quantified as high (immunostaining ≥30%), low (immunostaining <15%) and intermediate (between 16 to 30%) approach adopted by St Gallen International Expert Consensus.[ 14]In our study for statytical analysis Ki67 was evaluated by the percentage of immunostained nuclei with 1-14% staining scored as low and >14% as high.[15]

Result

The age of the patients varied from 29-68 years with the maximum number of cases belonging to 41-60 (65% of the patients). The tumor size varied from 1.5 to 4.5 cm in size with 54.2% cases with size more than 2cm. All the cases were of infiltrating ductal carcinoma NOS (not otherwise specified). Grade III tumors were maximum consisting of 65.7% (46/70) followed by Grade II tumors which were 32.8% (23/70) and Grade I tumors consisted of only one case. Lymph nodes were recovered in 63 cases out of which metastasis were seen in 40 cases. ER and PR positivity was observed in 31.5% cases (22/70). All of these cases were Her2neu negative. Amongst the 48 ER PR negative cases, 20 cases came out to be positive for Her2neu and 28 cases were triple negative. On correlating the Ki67 proliferation index with the various clinicopathologial parameters it was found that Ki67 proliferation index significantly correlated with higher grade of the tumor. 90% of the cases with high proliferation index were of Grade III as compared to only 3 cases (10%)

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being of Grade II (p= 0.001). Ki67 proliferation index was higher in the cases with size >2cm (24/30= 80% cases) with the rest of the cases being less than 2cm in size (p= 0.004). 22 out of 40 cases with lymph node metastasis showed high Ki67 proliferation index. Thus, Ki67 proliferation index being significantly correlated with lymph node metastasis (p=0.008). (Table 1) Similarly, ER PR and Her 2neu status were correlated with grade of tumor and it was concluded that as the grade increased ER PR expression decreased. 25 out of 28 triple negative cases were of Grade III. 13 out of 20 Her2neu positive cases were of Grade III as compared to 7/20 cases which were of Grade II. (Table 2) While correlating the Ki67 expression with ER PR Her2neu expression, it was observed that 23 out of 28 (82%) triple negative cases showed high proliferation index as compared to 2/24 ER PR positive cases giving a significant correlation between Ki67 positivity and ER PR negativity (p=0.002).

Discussion

Breast cancer is one of the most common malignancies among women. Despite the advanced technology in

early diagnosis and treatment methods, it is among the leading cause of death from cancer in women worldwide. Different predictive and prognostic markers are involved in tumorigenesis and its progression. The outcome and response of tumor to the treatment varies from patient to patient depending upon the interplay of such various factors involved. The present study was conducted to have better insight to elucidate the correlation between these factors. ER, PR, Her2neu and Ki67 expression were correlated with various clinicopathologial parameters like grade, size of tumor and lymph node metastasis. We also determined the correlation of Ki67proliferation index with ER, PR and Her2neu status. In our study maximum number of cases were from 45-60 years which corroborates the findings in studies conducted in Indian subcontinent which is a decade earlier than results observed in European countries.[15] All the cases were of infiltrating ductal carcinoma breast – NOS with grade III cases (65.7%) outnumbering the grade II cases (32.8%). ER PR positivity was observed in 31.5% of the cases which is a bit lower than the studies conducted in this part of the continent where this percentage varied from 3847%.[5,8] Researchers in the western world had found this

Table1: Correlation of Ki67 with other clinicopathological prognostic factors. PARAMETERS

Ki67 –ve

Ki67 (1-14%)

Ki67 > (14%)

Histological grade

Grade I Grade II Grade III

01(5%) 12(60%) 07(35%)

0 8(40%) 12(60%)

0 03(10%) 27(90%)

Lymph node status

NR N0 Nx (metastatic)

2(10%) 10(50%) 8(40%)

2(10%) 8(40%) 10(50%)

3(10%) 5(16.7%) 22(73.3%)

=< 2 cm >2 cm

16(80%) 4(20%)

10(50%) 10(50%)

6(20%) 24(80%)

Size

Table 2: Correlation of ER ,PR and Her2neu status with the grade of the tumor Grade 1

Grade 2

Grade 3

TOTAL

Triple negative

ER PR +ve Her2neu -ve

ER PR –ve Her2neu +ve

Table 3: Correlation of ER, PR and Her2neu status with Ki67 status. Ki67 –ve

Ki67 (<14%)

Ki67(>14%)

TOTAL

Triple negative

4(20%)

1(5%)

23(76.67%)

28(40%)

ER PR +ve Her2neu –ve

12(60%)

8(40%)

2(6.67%)

22(31.43%)

ER PR –ve Her2neu +ve

4(20%)

5(16.67%)

11(55%)

20(28.57%)

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Ki67, ER, PR, Her2neu Expression in Breast Carcinoma

Fig 1 (A): ER positivity (Nuclear) – IHC (400X) (B): PR positivity (Nuclear) – IHC (400X) (C): Ki67 positivity (Nuclear) – IHC (400X) (D): Her2neu positivity (Cytoplasmic) – IHC (400X).

percentage to be even higher varying from 40-55%.[1,4] All ER PR positive cases showed Her2neu negativity. Among 48 ER PR negative cases, 20 cases had Her2neu positivity and 28 cases (40%) being triple negative. Similar findings had been recorded in literature with this percentage varying from 30-40% depending upon the population studied.[1,4,16] Ki67 positivity was noted in 50 cases with 30 cases showing high proliferation index (>14%). Ki67 is a non histone nuclear protein closely linked to the proliferation of the cells. In the present study high Ki67 proliferation index was significantly correlated with higher grade of the tumor (p=0.001), increasd size (p=0.004) and lymph node metastasis (p=0.008). Previous studies had reported similar results where high Ki67 expression was associated with high histological grade, large tumor size and the presence of axillary lymph node metastasis.[17,18,19] Among the Her2neu positive cases, 80% cases showed Ki67 positivity. In contrast to Her2neu negative cases which showed Ki67 positivity in only 68% cases. So there was direct

correlation between Her2nu positivity and Ki67 positivity. Previous studies found similar correlation between Her2neu and Ki67.[19] In triple negative cases, high Ki67 proliferation index (>14%) was present in 76% patients. Many researchers also reported similar results.[21,22,23] Ki67 has repeatedly been confirmed as an independent predictive and prognostic factor in breast cancer.[24] Breast cancer with high Ki67 expression responds better to chemotherapy but is associated with poor prognosis.[25,26] Triple negative cases also have poor survival despite a higher response to neoadjuvant chemotherapy.[22,27] Keam et al further reported that triple negative breast cancer patients with high Ki67 expression had poor survival than triple negative breast cancer patients with low Ki67 expression.[28]

Conclusion

It was concluded that Ki67 can be considered as a new prognostic marker in addition to ER PR and Her2neu. The

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breast cancers with a high Ki67 index are associated with poor prognosis and it may be helpful in triple negative cases for deciding a better treatment protocol.

Acknowledgements None

12.

Funding None

Competing Interests

13.

None

Reference

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1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer staristics. CA Cancer J Clin 2011;61:69–90. 2. Khokhar K. Breast Cancer in India: Where do we stand and where do we go. Asian Pacific J Cancer Prev 2012;13(10):4861-6.

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3. Shetty P. India faces growing breast cancer epidemic. The Lancet 2012;379(9820):992-3. 4. Moutafoff C, Coutant C, Bezu C. Prognostic and predictive factors in multifocal breast carcinoma. Gynecol Obstet Fertil 2011;39:425–32. 5. Mahmood H, Faheem M, Mahmood S. Impact of age, tumor size, lymph node metastasis, stage, receptor status and menopausal status on overall survival of breast cancer patients in Pakistan. Asian Pac J Cancer Prev 2015;16:1019–24.

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6. Ariga R, Zarif A, Korasick J. Correlation of her-2/ neu gene amplification with other prognostic and predictive factors in female breast carcinoma. Breast J 2005;11:278–80. 7. Baulies S, Cusido M, Gonzalez-Cao M. Hormone receptor and HER2 status: the only predictive factors of response to neoadjuvant chemotherapy in breast cancer. J Obstet Gynaecol 2015:35(5):485-9. 8. Barnard ME, Boeke CE, Tamimi RM. Established breast cancer risk factors and risk of intrinsic tumor subtypes. Biochim Biophys Acta 2015;1856(1):73-85. 9. Osborne CK. Steroid hormone receptors in breast cancer management. Breast Cancer Res Treat 1998;51:227–38.

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10. Wong SY, Kernohan NM, Walker F. Breast cancers with extremely high estrogen receptor protein status. Histopathology 1990;16:125–32.

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11. Liu M, Lawson G, Delos M. Predictive value of the fraction of cancer cells immunolabeled for

22.

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proliferating cell nuclear antigen or Ki67 in biopsies of head and neck carcinomas to identify lymph node metastasis: comparison with clinical and radiologic examinations. Head Neck 2003;25:280–8. Fasching PA, Heusinger K, Haeberle L. Ki67, chemotherapy response, and prognosis in breast cancer patients receiving neoadjuvant treatment. BMC Cancer 2011;11(486):6-8. Gobbin H, Rocha RM, Nunes CB. Predictive factors of breast cancer evaluated by immunohistochemistry. J Bras Pathol Med Lab 2008;44(22):131-40. Goldhirsch A, Wood WC, Coates AS, et al (2011). Strategies for subtypes-dealing with the diversity of breast cancer: highlights of the St Gallen International Expert Consensus on the primary therapy of early breast cancer. Ann Oncol 2011;22:1736-47. Shokouh TZ, Ezatollah A, Barand P. Interrelationships Between Ki67, HER2/neu, p53, ER, and PR Status and Their Associations With Tumor Gradeand Lymph Node Involvement in Breast Carcinoma Subtypes: Retrospective-Observational Analytical Study. Medicine 2015;94(32):1359-65. Khokhar A. Breast cancer in India: where do we stand and where do we go? Asian Pac J Cancer Prev 2012;13:4861–6. Peng Y. Potential prognostic tumor biomarkers in triple-negative breast carcinoma. Beijing da Xue Xue Bao 2012;44:666-72. Han JS, Cao D, Molberg KH, Sarode VR, Rao R. Hormone receptor status rather than HER2 status is significantly associated with increased Ki-67 and p53 expression in triple-negative breast carcinomas, and high expression of Ki-67 but not p53 is significantly associated with axillary nodal metastasis in triplenegative and high-grade non-triple-negative breast carcinomas. Am J Clin Pathol 2011;135:2307-12. Rhee J, Han SW, Oh DY, Kim JH, Im SA. The clinicopathologic characteristics and prognostic significance of triple-negativity in node-negative breast cancer. BMC Cancer 2008.8:307-9. Lukashina MI, Glukhova EI, Zhukova LG, Ermilova VD, Bogatyrev VN, Baryshikov A. Her-2/Nu, Ki67 expression and ploidy in breast carcinoma. Arkh Pathol 2003;65(5):25-9. Li H, Han X, Liu Y, Liu G, Dong G. Ki67 as a predictor of poor prognosis in patients with triple-negative breast cancer. Oncol Lett 2015;9(1):149-52. Keam B, Im SA, Kim HJ, Oh DY, Kim JH, Lee SH, Chie EK, Han W, Kim DW, Moon WK. Prognostic eISSN: 2349-6983; pISSN: 2394-6466

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impact of clinicopathologic parameters in stage II/ III breast cancer treated with neoadjuvant docetaxel and doxorubicin chemotherapy: paradoxical features of the triple negative breast cancer. BMC Cancer 2007;7:203-6. 23. Rhee J, Han SW, Oh DY, Kim JH, Im SA, Han W, Park IA, Noh DY, Bang YJ, Kim TY. The clinicopathologic characteristics and prognostic significance of triplenegativity in node-negative breast cancer. BMC Cancer 2008;8:307-10. 24. Urruticoechea A, Smith IE, Dowsett M. Proliferation marker Ki-67 in early breast cancer. J Clin Oncol 2005;23:7212–20. 25. Jones RL, Salter J, Hern RA, Nerurkar A, Parton M, Reis-Filho JS, Smith IE, Dowsett M. Relationship between oestrogen receptor status and proliferation in predicting response and long-term outcome to

neoadjuvant chemotherapy for breast cancer. Breast Cancer Res Treat 2010;119:315–23. 26. Brown RW, Allred CD, Clark GM, Osborne CK, Hilsenbeck SG. Prognostic value of Ki-67 compared to S-phase fraction in axillary node-negative breast cancer. Clin Cancer Res 1996;2:585–92. 27. Liedtke C, Mazouni C, Hess KR, Andre F, Tordai A, Mejia JA, Symmans WF, Gonzalez-Angulo AM, Hennessy B, Green M. Response to neoadjuvant therapy and long-term survival in patients with triplenegative breast cancer. J Clin Oncol 2008;26:1275–81. 28. Keam B, Im SA, Lee KH, Han SW, Oh DY, Kim JH, Lee SH, Han W, Kim DW, Kim TY, Park IA, Noh DY, Heo DS, Bang YJ. Ki-67 can be used for further classification of triple negative breast cancer into two subtypes with different response and prognosis. Breast Cancer Res 2011 Mar 2;13(2):R22.

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Original Article Expression of Hypoxia Inducible Factor-1α in Esophageal Squamous Cell Carcinoma and its correlation with clinicopathological parameters Manisha Sharma*, Menka Khanna, Manas Madan, Mridu Manjari, Taranveer Singh and Komalpreet Kaur Dept of Pathology, Sri Guru Ramdas Institute of Medical Sciences And Research, Amritsar, Punjab, India Keywords: Esophageal Carcinoma, Immunohistochemistry, HIF-1α, Chemoradiotherapy

ABSTRACT Background: Esophageal cancer is an aggressive tumour carrying a very poor prognosis. HIF-1α (Hypoxia induced factor) is an oxygen sensitive transcription factor which activates transcription of proangiogenic cytokines like VEGF (Vascular Endothelial Growth Factor) responsible for angiogenesis. In the present study we examined the HIF-1α expression in esophageal carcinoma cases and its correlation with various clinicopathological parameters. We also determined the clinical outcomes in such patients with chemoradiotherapy. Methods: The study was conducted on 50 cases of Esophageal Squamous Cell Carcinomas diagnosed in SGRDIMSAR, Amritsar. Detailed clinical data of the patient was recorded as per proforma attached. Tissue were formalin fixed, paraffin embedded and were studied for histopathological grading after staining with haematoxylin-eosin. All cases were subjected to immunohistochemistry for HIF-1α expression. Results: The maximum incidence was seen in age group of 41-60 years. Most of the cases were moderately differentiated 34 cases (68%) followed by poorly differentiated and well differentiated consisting of 20% and 12% respectively. The HIF-1α positivity was observed in 34 cases. 16/34 cases were classified as high score group. High HIF-1α expression significantly correlated with poor degree of differentiation (P=0.005), with the lymph node metastasis(P=0.015), lymhpatics invasion(P=0.018) and depth of invasion. Out of 20 patients resistant to therapy, 12 showed high HIF-1α score. Conclusion: The present study concluded that expression of HIF-1α was significantly correlated with poorer clinicopathological variables including higher grade, lymph node metastasis, lymphatics invasion and increased depth of tumour invasion. It was also determined that HIF-1α expression significantly correlates with an unfavourable prognosis in such patients treated with adjuvant chemoradiotherapy.

*Corresponding author: Dr Manisha Sharma, B-241, Ranjit Avenue, Amritsar, Punjab, India Phone: +91 9876842942 Email: manisha_salwan@yahoo.com

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

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Expression of HIF-1α in Esophageal SSC

Introduction

Esophageal cancer is an aggressive tumour carrying a very poor prognosis. Esophageal cancer is the eighth most common cancer around the world. In 2008, an estimated 4,82,000 new esophageal cancer cases were diagnosed and 4,07,000 related deaths occurred globally.[1] Esophageal cancers have many associations and risk factors. In Indian studies the role of nitrosamines and nitroso compounds, tobacco, alcohol and dietary/nutritional factors in the causation of esophageal cancer have been evaluated by various researchers.[2,3] Hypoxia in tumour tissues induces serial changes that promote tumor growth, invasion and metastasis among which the over-expression of HIF-1α (Hypoxia induced factor) is the most predominant regulator of oxygen homeostasis. HIF-1α is an oxygen sensitive transcription factor which activates transcription of various proangiogenic cytokines like VEGF (Vascular Endothelial Growth Factor) responsible for angiogenesis. Evidence has been provided that over-expression of HIF-1α is associated with a malignant phenotype. It has been shown that HIF-1α is a key player in the cancer cells response to low-oxygen tension in a variety of physiologic processes including angiogenesis, tumorigenesis and metastasis. Moreover, hypoxic regions have been shown to be both chemo- and radiation resistant.[4,5,6] HIF-1α expression has been found to be associated with poor clinicopathological factors in various malignancies. HIF-1α positivity can predict lymph node metastasis thus can be useful in the choice of therapeutic strategy in esophageal cancers as lymph node involvement is the most important determinant in such cases.[7] It is seen that the pretreatment evaluation of HIF-1α is a useful and sensitive indicator of response to radiation and chemotherapy in esophageal cancer thus targeting HIF-1α could represent a novel approach to cancer therapy.[7,8] In the present study we examined the HIF-1α expression in esophageal carcinoma cases and its correlation with various clinicopathological parameters. We also determined the clinical outcomes in such patients with chemoradiotherapy.

Material and Methods

The study was conducted on 50 cases of Esophageal Squamous Cell Carcinomas diagnosed in Sri Guru Ramdas Institute of Medical Sciences And Research (SGRDIMSAR), Amritsar. Detailed clinical data of the patient was recorded as per proforma attached. Histopathological examination of the tissues obtained was done after processing them to prepare paraffin blocks. Blocks were cut and stained with Haematoxylin

and Eosin stain and studied under light microscope for histopathological grading. All the cases were subjected to immunohistochemistry for HIF-1α expression. Immunohistochemistry was performed by using antibodies against HIF -1α ( Diagnostic Biosystem ). The sections were taken on Poly – L – lysine hydrobromide (0.1%) pre coated slides. The antigen retrieval was done by using pressure cooker method with 10 mmol citrate buffer at ph 6.0 . Tris buffer was used as wash buffer . Endogenous activity was blocked by using hydrogen peroxide. After Protien blocking slides were incubated overnight with anti HIF-1α antibodies and were conjugated with Streptavidin Horse Radish Peroxidase (HRP). DAB ( Diaminobenxene tetra hydrochloride ) was used as the chromogen. The slides were counterstained with hematoxylin and were examined by light microscopy. For HIF-1α brown nuclei and cytoplasmic staining were taken positive. 1 = Nuclear staining < 1% of cells 2 = Nuclear staining 1-10% of cells and /or with weak cytoplasmic staining 3 = Nuclear staining 10-50% of cells and/or with distinct cytolasmic staining 4 = Nuclear staining >50 % of cells and /or with distinct cytoplasmic staining HIF-1α 3+ and 4+ were considered high expression and while the others were taken as HIF-1α low expression.

Results

The maximum incidence was seen in age group of 4160 years. The youngest patient was 28 years old and the oldest was 89 years old. The incidence of esophageal cancer in females was found to be slightly higher than the males. Most of the patients presented with complaints of 3-6 months duration. The lower segment of esophagus was involved in 56% of cases followed by middle segment with upper segment showing least involvement. The most commonly observed growth was of polypoidal type which was seen in 28 cases (56%) with proliferative and ulcerative showing equal frequency. Most of the cases were found to show moderate differentiation 34 cases (68%) (Fig A) followed by poorly differentiated (Fig B) and well differentiated consisting of 20% and 12% respectively. Out of 50 cases 33 were esophagectomy specimens while remaining 17 were endoscopic biopsies. Among the 33 cases in which lymph nodes were recovered the metastasis was seen only in 21 cases (63.7%) while 12 cases (36.3%) showed reactive pathology. All the tumours from these 33 cases of esophagectomy specimen were divided into four stages depending upon the depth

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of invasion. There were 15 cases (45.5%) of T4 (tumour invading adjacent structures), 8 cases (24.5%) of T3 (tumour invading the adventitia), 7 cases (21%) of T2 ( tumour invading the muscularis propria), 3 cases (9%) of T1 (tumour invading the lamina propria or submucosa). Mean follow up of the patients were 18 months ( median 12 months; range 1-36 months).

On comparing the response of the patients to chemoradiotherapy it was observed that out of 20 patients who were resistant to therapy and had reccurence within six months after therapy 12 showed high HIF-1α as compared to eight patients of low HIF-1α score and negative cases.

Discussion

Nationally, the incidence of esophageal cancer in western, southern and northern India is 4.48, 3.50 and 2.36 per 1,00,000 respectively with south and western India contributing 55% of all the cases.[2] Despite advances in screening and multimodal management of this disease, overall survival for esophageal cancer remains poor with median survival of being less than one year after diagnosis. [9] Various factors thought to influence survival are stage of the tumour, lymph node metastasis, tumour length, grade of the tumour, lymphatic invasion, tumour necrosis, involvement of the surgical margins, response to therapy and various molecular markers like Ki-67 proliferation index, p53, growth factors and their receptors, cell cycle regulator, heat shock proteins and HIF-1α expression.[10.11]

The HIF-1α positivity was observed in 34 cases comprising 68% of the total cases. 16 out of these 34 cases were classified as high score group- HIF-1α 3+ and 4+ (Fig C). Rest of 18 cases were of low score group for HIF-1α expression- HIF-1α 1+ and 2+ (Fig D). Correlating the HIF-1α expression with the degree of differentiation it was observed that 8/10 (80%) cases of poorly differentiated squamous cell carcinoma showed high HIF-1α expression as compared to 8/34 (23.5%) cases of moderate differentiation showing high HIF-1α positivity. It was concluded that high HIF-1α expression was significantly correlated with the higher grade (poor differentiation) with P=0.005 (Table 1). High HIF-1α expression correlated with the lymph node metastasis as compared to cases showing no metastasis ((12/21- 57.2% vs 02/12-16.5% respectively) with significant P=0.015 (Table 1). Similarly high HIF-1α expression correlated with lymphatic invasion with 12/2352.2% cases positive for lymphatic invasion showed high HIF-1α expression as compared to 2/10-20% cases without lymphatic invasion (P=0.018) (Table1). The frequency of high HIF-1α expression increased with increased depth of invasion. No case of T1, 14.2% of T2 ( one of seven cases), 50% of T3 ( 4 of 8 cases) and 60% of T4 ( 9 of 15 cases) had high HIF-1α.

In the current study most common age group was 41-60 years with incidence being slightly higher in females. The findings concur with other studies where most of carcinoma esophagus patients were in their 50s with mean age of esophageal resection at an age of 64 years (range, 42–79 years).[12] Indian data reveals a low sex ratio as compared to worldwide ratio, all major registries with a national average of 1.2:1. The possible explanation for this could be a high prevalence of betel quid chewing habit amongst Indian women.[13,14] The lower segment of esophagus was found to be affected in majority of the cases (constituting about 56 % of the total cases) followed by middle and

Tasble 1:Correlation of HIF 1- α expression with various clinicopathological features of Esophageal carcinoma. Parameter

HIF 1- α High score

HIF 1- α low score

HIF 1- α negative

Differentiation a)Well differentiated b)Moderately differentiated c)Poorly differentiated

00 08 08

01 16 01

05 10 01

2. Lymph node involvement a)Positive b)Negative c) Not recovered

12 02 02

06 04 08

03 06 07

3. Lymphatics/ blood vessel involvement a)Positive b)Negative c)Not identified in biopsy

12 02 02

08 04 06

03 04 09

Invasion depth T1 T2 T3 T4

00 01 04 09

01 01 03 05

02 05 01 01

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Fig. 1 (A): Squamous cell carcinoma-Moderately differentiated ( H&E 100X); (B) : Squamous cell carcinoma-Poorly differentiated ( H&E 100X); (C): Immunohistochemistry showing HIF -1α Positivity (Nuclear Strong Intensity) (IHC, 400X); (D): Immunohistochemistry showing HIF -1α Positivity (Nuclear Moderate Intensity) (IHC, 400X).

upper segments. Similar observation was made by other researchers who have reported lower esophageal cancers out numbering the mid esophageal cancers.[15,16] Most of the cases were found to show moderate differentiation 34 cases(68%) corroborating the findings of other studies where this percentage varied from 55-70%.16 The lymph nodes were recovered from esophagectomy specimens, 21 (63.7%) showed metastasis and remaining 12 cases (36.3%) showed reactive pathology. Other studies had calculated metastatic lymph node percentage varying from 60-96.7%.[8,17] The HIF-1α positivity was observed in 34 cases comprising 68% of the total cases. Other workers have reported a variable HIF-1α positive expression ranging from 39 to 95% cases.[7,8,18,19] 16 cases (32% ) showed high HIF1α expression which is comparable to 30.8% observed by Kurokawa et al in their study.[8] Correlating the HIF1α expression with the degree of differentiation it was observed that 8/10 (80%) cases of poorly differentiated

squamous cell carcinoma showed high HIF-1α expression as compared to 8/34 (23.5%) cases of moderate differentiation showing high HIF-1α positivity with significant P=0.005. Similar results have been elicited by workers elsewhere who have reported an expression of 53.9% in poorly differentiated carcinomas with a lower incidence of HIF-1α immunoexpression in moderately differentiated tumors (12%).[7] High HIF-1α expression correlated with the lymph node metastasis as compared to cases showing no metastasis ((12/21- 57.2% vs 02/1216.5% respectively) with significant P=0.015. Same was reflected in the results deduced by other researchers where lymph node metastasis showed significant correlation with high HIF-1α expression.[7,8] HIF-1α expression also correlated with lymphatic invasion with 12/23-52.2% cases positive for lymphatic invasion showed high HIF1α expression as compared to 2/10-20% cases without lymphatic invasion (P=0.018). Other studies also observed such significant correlation between two with P value ranging from 0.005-0.02.[7,8] The frequency of high HIF-

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1α expression increased with increased depth of invasion. No case of T1, 14.2% of T2 ( one of seven cases), 50% of T3 ( 4 of 8 cases) and 60% of T4 ( 9 of 15 cases) had high HIF-1α. Similar findings were observed by Matsuyama et al in their study where high HIF-1α immunopositivity was seen in T4 and T3 cases as compared to T2 and T1.[7] It was observed that out of 20 patients who were resistant to chemoradiotherapy and had reccurence within six months after therapy 12(60%) showed high HIF-1α as compared to eight patients(40%) of low HIF-1α score and negative cases. Similar results were seen in other studies where patients with HIF-1α expression had significantly poorer local control than those with low expression P=0.0322.[20]

References

A high expression of HIF-1α has been found associated tumour aggressiveness and unfavourable prognosis in variety of tumours including carcinomas of breast, cervix, colon, lung, ovary and esophagus. Furthermore its expression is associated with poorer response to adjuvant therapy and poorer overall and disease free survival as well.[7,8,20] Hypoxia has been proved to compromise the beneficial effects of radiotherapy and interfere with the response of tumour to chemotherapy.[8]

5. Benizri E, Ginouves A, Berra E. The magic of the hypoxia-signaling cascade. Cell Mol Life Sci. 2008;65:1133-49.

Conclusion

The present study concluded that expression of HIF-1α was significantly correlated with poorer clinicopathological variables including higher grade, lymph node metastasis, lymphatics invasion and increased depth of tumour invasion. It was also determined that HIF-1α in esophageal carcinoma significantly correlates with an unfavourable prognosis in such patients treated with adjuvant chemoradiotherapy. Preoperative studies on endoscopic biopsies might guide clinicians to design better therapeutic decision in patients with HIF-1α expression. Further studies are required to address the role of introduction of more intensive therapy in such patients with high HIF-1α expression who are poor responders to conventional therapy.

Abbreviations

HIF-1α - Hypoxia induced factor VEGF - Vascular Endothelial Growth Factor HRP

- Horse Radish Peroxidase

DAB - Diaminobenxene tetra hydrochloride

Funding None

Competing Interests None Declared

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1. Jemal A, Bray F, Center MM. Global cancer statistics. Cancer J Clin. 2011;61:69-90. 2. Rao DN, Sanghvi LD, Desai PB. Epidemiology of esophageal cancer. Semin Surg Oncol. 1989;5:351-4. 3. Ammigan, Nair UN, Lalitha VS, Bhinde SV. Carcinogenicity studies of masheri: pyrolysed tobacco product in vitamin A deficient Sprague-Dawley rats. J Cancer Res Clin Oncol. 1991;117:5-9. 4. Semenza GL, Roth PH, Fang HM, Wang GL. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J Biol Chem. 1994;269(38):23757–63.

6. Gordan JD, Simon MC. Hypoxia-inducible factors: central regulators of the tumor phenotype. Curr Opin Genet Dev. 2007;17:71-7. 7. Matsuyama T, Nakanishi K, Hayashi T, Yoshizumi Y, Aiko S, Sugiura Y, Tanimoto T, Uenoyama M, Ozeki Y, Maehara T. Expression of hypoxia-inducible factor1α in esophageal squamous cell carcinoma. Cancer Sci. 2005;96(3):176-82. 8. Kurokawa T, Miyamoto M, Kato K, Cho Y, Kawarada Y, Hida Y, Shinohara T, Itoh T, Okushiba T, Kondo S, Katoh H. Overexpression of hypoxia-inducible-factor 1a (HIF-1a) in oesophageal squamous cell carcinoma correlates with lymph node metastasis and pathologic stage. Br J Cancer. 2003;89:1042–7. 9. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer. 2010;127(12):2893-917. 10. Rohatagi P, Swisher SG, Cornea AM, Wu TT, Liao Z, Komaki R, Walsh GL, Vaporciyan AA, Rice DC, Roth JA, Ajani JA. Characterization of pathologic complete response after preoperative chemotherapy in carcinoma of the esophagus and outcome after pathologic complete response. Cancer. 2005;104:2365-72. 11. Eloubeidi MA, Desmond R, Arguedas MR, Reed CE, Wilcox CM. Prognostic factors for the survival of patients with oesophageal carcinoma in the U.S.: the importance of tumor length and lymph node status. Cancer. 2002;95:1434-43. 12. Youssef EM, Matsuda T, Takada N, Osugi H, Higashino M, Kinoshita H, Watanabe T, Katsura Y, Wanibuchi eISSN: 2349-6983; pISSN: 2394-6466

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13.

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Expression of HIF-1α in Esophageal SSC

H, Fukushima S. Prognostic significance of the MIB1 proliferation index for patients with squamous cell carcinoma of the esophagus. Cancer. 1995;76:358-66. Parkin DM, Muir CS, Whelan SL, Gao YT, Ferlay J, Powell J. Cancer incidence in five continents. Lyon: International Agency for Research on Cancer; 1992. Department of Health. National Cancer Registry Programme Annual report. New Delhi: Indian Council of Medical Research; 1990. Cherian JV, Sivaraman R, Muthusamy AK, Jayanthi V. Carcinoma of the Esophagus in Tamil Nadu (South India): 16-year Trends from a Tertiary Center. J Gastrointestin Liver Di. 2007 Sep;16(3):245-9 Delima SL, McBride RK, Preshaw PM, Heasman PA, Kumar PS. Response of subgingival bacteria to smoking cessation. J Clin Microbiol. 2010;48(7):2344–9. Wang DY, Xiang YY, Tanaka M, Li XR, Li JL, Shen Q, Sugimura H, Kino I. High prevalence of p53 protein

overexpression in patients with esophageal cancer in Linxian, China and its relationship to progression and prognosis. Cancer. 1994;74:3089–96. 18. Takala H, Saarnio J, Wiik H, Ohtonen P, Soini Y. HIF-1α and VEGF are associated with disease progression in esophageal carcinoma. J Surg Res. 2011;167(1):41-8. 19. Koukourakis MI, Giatromanolaki A, Skarlatos J, Corti L, Blandamura S, Piazza M, Gatter KC, Harris AL. Hypoxia inducible factor (HIF-1α and HIF-2α) expression in early esophageal cancer and response to photodynamic therapy and radiotherapy. Cancer Res. 2001;61:1830–2. 20. Ogawa K, Chiba I, Morioka T, Shimoji H, Tamaki W, Takamatsu R, Nishimaki T, Yoshmini N, Murayama S. Clinical Significance of HIF-1α Expression in Patients with Esophageal Cancer Treated with Concurrent Chemoradiotherapy. Anticancer Res. 2011;31:2351-60.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 06, (Suppl) December, 2016

Original Article Evaluation of Various Discrimination Indices in Differentiating Iron Deficiency Anemia and Beta Thalassemia Trait: A Practical Low Cost Solution Sanjay Piplani1*, Manas Madan1, Rahul Mannan1, Mridu Manjari1, Taranveer Singh1 and Monika Lalit2 Department of Pathology, Sri Guru Ram Das Institute of Medical Sciences and Research, Amritsar, India Department of Anatomy, Sri Guru Ram Das Institute of Medical Sciences and Research, Amritsar, India

Keywords: Iron Deficiency Anemia, Beta Thalassemia Trait, Microcytosis, Discrimination Indices

ABSTRACT Background: Mild microcytic hypochromic anemias due to Iron deficiency (IDA) and beta thalassemia trait(β-TT) continue to be a cause of significant burden to the society, particularly in the poorer developing countries. The objective of the present study was to evaluate the validity of 12 different discrimination indices to distinguish β-TT from IDA. Methods: A total of 225 patients diagnosed with mild microcytic hypochromic anemia on complete blood count and peripheral blood film were included in the study. HB, RBC count, MCV, MCH and RDW obtained from the electronic cell counter were used to calculate 12 discrimination indices by various mathematical formulae. Sensitivity, specificity, positive predictive value(PPV), negative predictive value(NPV) and Youden’s index(YI) were calculated. Result: The Shine & Lal and Ricerca et al indices exhibited the highest sensitivity of 100% each while the England & Fraser index demonstrated the lowest sensitivity of 61.1%. The England & Fraser and Mentzer indices demonstrated the highest specificities of 90.2% and 86.9% respectively. The highest and the lowest PPV were found for Mentzer index (77.3%) and MCHD (32.71%) respectively. The Ricerca et al. and Shine & Lal indices demonstrated the highest NPV of 100% each and the lowest NPV was exhibited by MCHD (69.8%). The highest and the lowest values for Youden’s index were shown by Mentzer index (81.3%) and MCHD (2.36%) respectively. Conclusion: Though HBA2 estimation is the gold standard for diagnosing β-TT, in developing countries, Mentzer index followed by Ehsani et al. index can be used to screen mild microcytic hypochromic anemia cases to eliminate as many false positive cases as possible to reduce the financial cost.

*Corresponding author: Dr. Sanjay Piplani, 24, Lane-5, Gopal Nagar, Majitha Road, Amritsar, Punjab 143001, India Phone: +91 9914785777 Email: sanjaymikki@gmail.com

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

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Discrimination Indices in Differentiating IDA & β-TT

Introduction

Iron deficiency anemia (IDA) and beta thalassemia trait (β-TT) are the two most frequent disorders presenting clinically with mild microcytic hypochromic anemia.[1] Lack of sufficient dietary iron resulting in IDA is the most common hematological disorder. It has been estimated that 30% of the world population suffers from IDA with majority of the affected people living in developing countries. In beta thalassemia trait there is impaired globin chain synthesis resulting in decreased hemoglobin leading to microcytic hypochromic anemia. 1.5% of world population carries genes for beta thalassemia.[2] Thalassemia traditionally has a high prevalence in some parts of the world (Mediterranean regions, up to 8%; countries of the Middle East, up to 10%;India, 3%-15%; Southeast Asia, up to 9%), where it represents a major public health problem. Non endemic countries such as in Northern Europe and North America are also involved in thalassemia related problems as a result of demographic changes caused by migration and intermarriages of different ethnic populations.[3,4] Nowadays population migration has spread thalassemia genes over nearly the entire globe.[5] In India, certain communities such as Sindhis, Kutchis, Lohanas, Bhanusalis, Punjabis, Mahars, Agris, Gauds, Saraswats, Gowdas etc. have a higher frequency.[6] It is important to differentiate between thalassemic and nonthalassemic microcytosis as both conditions share many characteristics and have important clinical implications.[7] Thus a correct diagnosis in patients with microcytic anemia can provide an indication for supplementing iron to IDA patients, for avoiding unnecessary iron therapy in thalassemia carriers and of course also for preventing severe and lethal forms of thalassemia syndromes in the framework of premarital counseling in high-prevalence areas.[8] A definitive differential diagnosis between β-TT and IDA is based on the result of HBA2 electrophoresis, serum iron levels, and ferritin calculations.[9] However, these investigations are money and time consuming and moreover areas where thalassemia is endemic often have low health care resources and these assays may not be generally available.[8] Thus, various discrimination indices have been proposed to distinguish between β-TT and IDA. These indices are derived from several simple red blood cell (RBC) indices, like RBC count, mean cell volume (MCV), mean corpuscular hemoglobin (MCH), RBC distribution width (RDW) and hemoglobin (HB) as these are provided by electronic cell counters.[10] The purpose of using indices

to discriminate anemia is to detect subjects who have a high probability of requiring appropriate follow-up and to reduce unnecessary investigative costs. An ideal discrimination index has high sensitivity and specificity; that is, it can detect the maximum number of patients with β-TT (high sensitivity) while eliminating patients with IDA (high specificity). Not much work is available from Northern India where the incidence of β-TT is high (3-15%). [3,4] The present study was undertaken, to evaluate the ability of 12 different discrimination indices to distinguish β-TT from IDA by calculating their sensitivity, specificity and Youden’s index values in a population group of North India and to compare its findings with studies done in other parts of the world.

Materials and Methods

The present study was conducted in the department of Hematology in a tertiary health care center in North Indian state of Punjab. A total of 225 patients (167 children and 58 adults) attending the outpatient departments of various specialties and diagnosed with mild microcytic hypochromic anemia on complete blood count and peripheral blood film were included in the study. Family members (Parents and siblings) of known cases of beta thalassemia major coming for blood transfusion of the affected child were also included in the study group. They were selected because parents of a beta thalassemia major patient carry beta thalassemia trait gene (100%) and the chances of carrying this gene in the siblings is also 50%. Venous blood was drawn with the usual precautions after the patient had been lying quietly for at least 20 minutes. The hematological analyses were carried out with a Sysmex XS-800i particle counter. HB concentration, RBC count, MCV, MCH and RDW were recorded. High performance liquid chromatography technique was used for determination of HbA2 [11] Serum iron (SI), serum iron binding capacity (SIBC), transferrin saturation (TS), serum ferritin, and HbA2 values were determined in all. The mean values and standard deviation of various hematological and biochemical parameters are shown (Table 1). Patients with hemoglobin concentration between 8.611.5g% and MCV less than 80fl were included in the study. Those having HB less than 8.6g% were excluded. The cut off value for HBA2 was kept at 3.5%. Patients with HBA2 levels more than 3.5% were labelled as β-TT group and those with value less than 3.5% were labelled as IDA group. Values of HB, RBCcount, MCV, MCH and RDW obtained from the electronic cell counter were used to calculate 12 discrimination indices by various mathematical formulae

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as given in (Table2). Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and Youden’s index (YI) were calculated for all the 12 discriminants as follows: Sensitivity = True Positive / True Positive+False Negative × 100. Specificity=True Negative/TrueNegative+FalsePositive × 100. PPV = Truepositive / True Positive+FalsePositive × 100. NPV = True Negative/ True Negative+ False Negative × 100. Youden’s index = (sensitivity+specificity) – 100. P values less than 0.05 were considered significant. The differential values for each discrimination index were applied as defined in the original published reports. [12-20] The number and proportions of correctly identified patients (True positive) were calculated (Table 3). Sensitivity, specificity, PPV, NPV and Youden’s index for each discrimination index were calculated (Table 4).

Result

In the present study, the maximum number of patients belonged to the 5-15 years age group (134, 59.5%) with range being 1.5- 39 years. Majority of the patients in the present study had no obvious complaints related to anemia (57%) but weakness (38%) followed by fatigue (5%) were the next common complaints received. On the basis of HBA2 levels, 72 patients having HBA2 more than 3.5% were grouped into β-TT group. Rest 153 patients having HBA2 less than 3.5% were included in the IDA group. The mean values for HB in β-TT group were 10.7+ 0.8 and those in the IDA group were 9.9+ 1.43 (p>0.05; not significant).

In β-TT group, the mean MCV and MCH were 63.6+5.49 and 18.4+2.15 respectively which were lower than those found in the IDA group (69.4+5.16 and 20.67+1.34 respectively). (p<0.05; significant). The mean RBC count in β-TT group was definitely higher 5.79+0.76 as compared to the IDA group 4.77+0.53. (p<0.05; significant). The mean RDW values were increased in both the groups, but the IDA group showed the values to be higher (17.2+2.96) as compared to the β-TT group (15.9+1.62). (p>0.05; not significant) The mean values for serum iron, transferrin saturation and serum ferritin were much lower in IDA group as compared to those in the β-TT group but TIBC showed an inverse trend. [Table 1]. As is evident from the results shown in Table 4; none of the indices showed 100% efficiency in recognizing beta thalassemia trait. The Shine and Lal and Ricerca et al indices exhibited the highest sensitivity of 100% each but failed miserably when it came to specificity which was very low to the tune of 6.25% and 16.9% respectively. The England and Fraser index demonstrated the lowest sensitivity of 61.1% which means it missed about 39% of β-TT cases. The England and Fraser and Mentzer indices demonstrated the highest specificities of 90.2% and 86.9% respectively. The highest and the lowest PPV were found for Mentzer index (77.3%) and MCHD (32.71%) respectively. The Ricerca et al. and Shine and Lal indices demonstrated the highest NPV of 100% each and the lowest NPV was exhibited by MCHD (69.8%). The highest and the lowest values for Youden’s index were shown by Mentzer index (81.3%) and MCHD (2.36%) respectively. None of the indices was completely sensitive and specific in differentiating between BTT and IDA.

Table I: Showing the Mean Values and Standard Deviation of Various Hematological and Biochemical Parameters. β-TT

IDA

S. No.

Parameter

9-11.5

10.7+0.8

8.6-11.3

9.9+1.43

RBC

4.6-6.4

5.79+0.76

3.7-5.3

4.77+0.53

MCV

56.4-71.1

63.6+5.49

54.2-78.4

69.4+5.16

MCH

16.1-22.4

18.4+2.15

14.2-26.7

20.67+1.34

RDW

14-22.9

15.9+1.62

12.7-27.1

17.2+2.96

26.1-178

78.2+18.3

5.3-27.4

21.1+9.4

SIBC

261-398

312+26.2

279-473

386+38.1

Range

Mean+ SD

Range

Mean+ SD

7.1-62

23.7+7.1

1.1-7.9

6.4+2.1

S Ferritin

14.7-87

36.1+18.4

2.3-12.8

6.92+2.9

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Discrimination Indices in Differentiating IDA & β-TT

Table 2: Showing Different RBC Indices and Mathematical Formulae Used to Differentiate Between Β-Tt and Ida. Hematological index

Year

Formula

Mentzer index

1973

MCV/RBC

England and Fraser Index

1973

MCV − (5 × Hb) − RBC − 3.4

Srivastava Index

1973

MCH/RBC

Shine and Lal Index

1977

MCV × MCV × MCH/100

RDW Index

1987

MCV × RDW/RBC

Ricerca et al Index

1987

RDW/RBC

Green and King Index

1989

MCV × MCV × RDW/Hb × 100

MDHL Index (Mean Density of Hb/liter of Blood)

1999

(MCH/MCV) × RBC

MCHD Index (Mean Cell Hb Density)

1999

MCH/MCV

Sirdah Index

2007

MCV − (5 × Hb) − RBC − 3.4

Ehsani et al Index

2009

MCV − (10 × RBC)

Table 3: Showing Number of Correctly Identified Patients By Each Discrimination Index. S.No.

INDICES

β-TT

IDA

TOTAL No. of Correctly Diagnosed Cases

PERCENTAGE

Mentzer β-TT<13 IDA>13

68 04

20 133

68+133=201

89.3

RBC β-TT>5 IDA<5

67 05

55 98

67+98=165

73.3

England & Frazer β-TT<0 IDA>0

44 28

15 138

44+138=182

80.9

Srivastva β-TT<3.8 IDA>3.8

63 09

55 38

63+38=101

44.9

Shine & Lal β-TT<1530 IDA>1530

72 00

144 09

72+9=81

36.0

RDWI β-TT<220 IDA>220

58 14

24 119

58+119=177

78.6

Ricerca et al β-TT<4.4 IDA>4.4

72 00

127 26

72+26=98

43.6

Green & King β-TT<65 IDA>65

57 15

46 107

57+107=164

72.9

MDHI β-TT>1.63 IDA<1.63

54 18

70 83

54+83=137

60.9

10.

MCHD β-TT>0.3045 IDA<0.3045

53 19

109 44

53+44=97

43.1

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S.No.

INDICES

β-TT

11.

Sirdah β-TT<27 IDA>27

54 18

12.

Ehsani et al β-TT<15 IDA>15

67 05

IDA

TOTAL No. of Correctly Diagnosed Cases

PERCENTAGE

25 128

54+128=182

80.9

26 127

67+127=194

86.2

Table 4: Showing Sensitivity, Specificity, Positive Predictive Value (Ppv), Negative Predictive Value (Npv), and Youden’s Index for all Twelve Discrimination Indices Between Β-Tt and Ida. S.No.

INDICES

SENSTIVITY

SPECIFICITY

PPV

NPV

Youden’s Index

Mentzer β-TT IDA

94.4% 86.9%

86.9% 94.4%

73.3% 97.1%

97.1% 77.3%

81.3%

RBC β-TT IDA

93.05% 64.05%

64.05% 93.05%

54.9% 95.1%

95.1% 54.9%

57.1%

England & Frazer β-TT IDA

61.1% 90.0%

90.2% 61.1%

76.6% 83.1%

83.1% 76.6%

51.3%

Srivastva β-TT IDA

87.5% 64.05%

64.05% 87.5%

53.4% 90.7%

90.7% 53.4%

51.55%

Shine & Lal β-TT IDA

100% 6.25%

6.25% 100%

33.3% 100%

100% 33.3%

6.25%

RDWI β-TT IDA

80.5% 77.7%

77.7% 80.5%

70.7% 89.47%

89.47% 70.7%

58.2%

Ricerca et al β-TT IDA

100% 16.9%

16.9% 100%

39.2% 100%

100% 39.2%

16.9%

Green & King β-TT IDA

79.2% 69.9%

69.9% 79.2%

55.3% 87.7%

87.7% 55.3%

49.1%

MDHI β-TT IDA

75.0% 54.24%

54.24% 75.0%

43.54% 82.17%

82.17% 43.54%

29.4%

10.

MCHD β-TT IDA

73.61% 28.75%

28.75% 73.61%

32.71% 69.84%

69.84% 32.71%

2.36%

11.

Sirdah β-TT IDA

75.0% 85.65%

83.65% 75.0%

68.4% 87.6%

87.6% 68.4%

58.65%

12.

Ehsani et al β-TT IDA

93.05% 83.0%

83.0% 93.05%

72.04% 96.2%

96.2% 72.04%

76.05%

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Discussion

Correct identification of β-TT is especially important, as the management of a patient with beta thalassemia major is not only expensive (strains on the limited resources of developing countries),[21] but also causes extreme misery to the patient and the family due to compromised quality of life. Also as the red cell morphology in β-TT is microcytic hypochromic; these patients are often misdiagnosed as those suffering from IDA and given unnecessary iron medication. In this setting a reliable discrimination index becomes a need of the hour. The best index for beta thalassemia trait should have a very high sensitivity as well as a reasonably high specificity so that the number of false positive cases can be reduced to minimum. Since 1973, several indices have been introduced in an attempt to distinguish these two conditions in a cheaper and easier way. According to the original published papers by the authors of these indices, their sensitivity in the detection of β-TT from IDA is approximately 100%. [12,13,17,22-26] However later studies failed to confirm these results and estimated these indices’ sensitivity to be between 61-91%.[27-31] These indices incorporate MCV, MCH, RBC Count, RDW and HB in various combination. Table I shows that there were significant differences between the haematological and biochemical parameters of β-TT and IDA but these did not reflect in the indices’ reliability in differential diagnosis of β-TT and IDA. RBC count has been considered as a valuable index.[32] Majority of the β-TT patients in the present study had a high RBC count of more than 5 million. However a significant number of IDA patients (55/153) also had an RBC count of >5 million. So though the sensitivity of RBC count for beta thalassemia trait was 93.05%, it showed a specificity and Youden’s index of only 65.05% and 57.1% respectively. So according to our results, RBC count alone cannot be taken as a reliable index to distinguish between of beta thalassemia trait and IDA. RDW, a measure of the degree of variation in red cell size, has been reported to be a good discrimination index to differentiate between β-TT and IDA[33-36]. But in our study the values were elevated in both the groups, though they were slightly higher in the IDA group. In the present study, RDW index showed the sensitivity of 80.5%, specificity of 77.7 % and youden’s index of 58.2%. Our results concur well with results of[37-39] who found that RDW alone is reasonable but not sufficiently specific or sensitive enough to differentiate between β-TT and IDA. In the present study, the Mentzer index showed a reasonably high sensitivity, specificity and good Youden’s

index of 94.4%, 86.9% and 81.3% respectively. This was followed by Ehsani et al index which showed sensitivity, specificity and Youden’s index of 93.05%, 83.0% and 76.05% respectively. Ehsani et al. in 2009 showed that the best discrimination index according to Youden’s criteria was the Mentzer index (90.1%), followed by the Ehsani et al.index(85.5%). In their study, the Mentzer and Ehsani et al. indices were able to correctly diagnose 94.7% and 92.9% of cases, respectively.[20] Rahim and Keikhaei in 2009 examined the diagnostic accuracy of 10 indices in 153 patients with β-TT and 170 patients with IDA. They found that the Mentzer index had 85% sensitivity, 93% specificity, and 79% Youden’s index.[40] Similar results (Mentzer index: sensitivity, 90.9%;specificity,80.3%)were found by Ghafouri et al. [41] In 2007, AlFadhli et al.[42] in their research work applied and compared nine well-documented discriminant functions in 153 confirmed cases of microcytic anemia and measured validity using Youden’s index. They showed that the England & Frazer index had the highest Youden’s index value (98.2%) in correctly differentiating between IDA and α - and ß-thalassemia minor, while the Shine & Lal index was found ineffective in differentiating between microcytic anemias in their study. Ferrara et al. in 2010 demonstrated that the England and Fraser index had the highest specificity of 99.1 % and a Youden’s index of 64.2%.[43] In the present study, the England and Frazer index had the highest specificity of 90.2 %, but the Youden’s index was 51.3%. Shine and Lal index showed a Youden’s index of only 6.25% and proved to be ineffective in differentiating between β-TT and IDA. In the present study, the Sirdah index demonstrated a sensitivity, specificity and youden’s index of 75.0%, 83.6% and 58.6% respectively. The findings are comparable with the results obtained by Vehapoglu et al 2014[44] who found these to be 85.7%, 79.4% and 65.0% respectively. In the present study, the Srivastva index showed a sensitivity, specificity and Youden’s index of 87.5%, 64.05% and 51.5% respectively. Alfadhli et al compared nine discriminant functions in patients with microcytic anemia and found a Youden’s index of 54.9% for Srivastva index.[42] In 2007, Ntaios et al. reported that the Green and King index was the most reliable index, as it had the highest sensitivity (75.06%), efficiency (80.12%), and Youden’s index (70.86%) for detecting β-TT.[10] A similar result for the Green and King index (Youden’s index, 80.9%) was found by Urrechaga et al.[45] However in the present study, though the sensitivity was 79.2%; it showed a low specificity and Youden’s index of 69.9% and 49.1% respectively. The

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 06, (Suppl) December, 2016

Piplani et al. Ricerca index also exhibited a sensitivity of 100% but the specificity was very low (16.9%) and a Youden’s index of 16.9% ruling it out as a reliable indicator. Similar results were obtained by Vehapoglu et al 2014[44] who found a youden’s index of 14.7%. In the present study, the MDHL index showed a sensitivity of 75.0% and a youden’s index of 29.4%. The MCHD index exhibited a very low Youden’s index of 2.36% only. These findings are in coherence with the works done by Vehapoglu et al 2014.[44] So in the present study, according to the Youden’s index which is a measure of high performance of the discrimination index, following rankings of various discrimination indices were obtained. Mentzer> Ehsani> Sirdah> RDWI> RBC> Srivastva> England & Fraser> Green & King> MDHL> Ricerca> Shine & Lal> MCHD. However one important point needs a special mention in β-TT. Literature documents more than 200 mutations noted in β-TT so far, and also according to the extent of the reduction of beta chain output, these mutations have been divided in to severe, mild and silent.[46] It is these silent mutations (measurable only by special genetic studies) which are characterized by near normal hematological indices and borderline HBA2 levels leading to mislabeling of these individuals as phylogenetically normal which may result in genotypic abnormalities in the next generation leading to much avoidable morbidities. But these high end and expensive genetic studies are not widely available in resource challenged countries where applications of these hematological indices are practically implementable.

Conclusion

From the above data, it is clear that Mentzer index followed by Ehsani et al. index are highly sensitive and reasonably specific in differentiating β-TT from IDA and none of the indices is 100% sensitive and specific. Though HBA2 estimation continues to be the gold standard for diagnosing a case of β-TT, a practical approach in developing countries with limited health care resources would be to screen mild microcytic hypochromic cases by these 2 indices and try to eliminate as many false positive cases as possible to reduce the financial cost.

Acknowledgements

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Reference

1. Brittenham GM. Disorders of iron metabolism: iron deficiency and overload. In: Hoffman R, Benz EJ Jr, Shattil SJ et al (eds) Hematology basic principles and practice, 2nd edn. 1995. Churchill Livingstone, New York. 2. RathodD A, Kaur A, Patel V. Patel K, Kabrawala R, Patel V, Patel M and Shah P. Usefulness of cell counterbased parameters and formulas in detection of β-thalassemia trait in areas of high prevalence. American Journal of Clinical Pathology. 2007;128(4):585–589. 3. Angastiniotis M, Modell B. Global epidemiology of hemoglobin disorders. Ann N Y Acad Sci. 1998;850:251-269. 4. Weatherall DJ, Clegg JB. Inherited haemoglobin disorders: An increasing global health problem. Bull World Health Organ. 2001;79:704-712. 5. Milman N. Anemia–still a major health problem in many parts of the world! Ann Hematol 2011;90:369 – 77. 6. Lokeshwar MR, Shah N, Kanakhiya S, Manglani M. Thalassemia. In: IAP Textbook of Paediatrics. 4th edition . Parthasarthy A, Menon PSN, Agarwal Rk, et al (eds): Jay Pee Brothers Medical Publishers Pvt Limited, New Delhi; 2009; 794-815. 7. Hallberg L. Iron requirements: comments on methods and some crucial concepts in iron nutrition. Biol Trace Elem Res. 1992;35:25-45. 8. Hoffmann J JML, Eloísa Urrechaga E, Aguirre U. Discriminant indices for distinguishing thalassemia and iron deficiency in patients with microcytic anemia: A meta-analysis. Clin Chem Lab Med. 2015; 53(12): 1883–1894. 9. Thomas C, Thomas L. Biochemical markers and hematologic indices in the diagnosis of functional iron deficiency. Clinical Chemistry. 2002;48(7):1066–1076. 10. Ntaios G, Chatzinikolaou A, Saouli Z, et al. Discrimination indices as screening tests for β-thalassemic trait. Ann Hematol. 2007 86:487–491. 11. Turpelnen U. Liquid-chromatographic determination of hemoglobin A2. Clin Chem 1986;3216:999-1002.

I acknowledge the contribution of paramedical staff and all the patients.

12. Mentzer JWC. Differentiation of iron deficiency from thalassaemia trait. The Lancet 1973;1(7808):882.

Funding

13. England JM, Fraser P M. Differentiation of iron deficiency from thalassaemia trait by routine bloodcount. The Lancet. 1973;1(7801):449–452.

Competing Interests

14. Srivastava PC. Differentiation of thalassemia minor fromiron deficiency. The Lancet. 1973;2:154–155.

None

None Declared

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15. Shine I, Lal S. A strategy to detect β thalassaemia minor. The Lancet. 1977;1(8013):692–694. 16. Ricerca BM, Storti S, d’Onofrio G, et al. Differentiation of iron deficiency from thalassaemia trait: A new approach.,” Haematologica. 1987;72(5):409–413. 17. Green R, King R. A new red cell discriminant incorporating volume dispersion for differentiating iron deficiency anemia from thalassemia minor. Blood Cells. 1989;15(3):481–495. 18. Telmissani OA, Khalil S, George T R. Mean density of hemoglobin per liter of blood: a new hematologic parameter with an inherent discriminant function. Laboratory Haematology. 1999;5:149–152. 19. Sirdah M, Tarazi I, Najjar EA, Haddad RA. Evaluation of the diagnostic reliability of different RBC indices and formulas in the differentiation of the β-thalassaemia minor from iron deficiency in Palestinian population. International Journal of Laboratory Hematology. 2008;30(4):324–330. 20. Ehsani M A, Shahgholi E, Rahiminejad M S, Seighali F, and Rashidi A. A new index for discrimination between iron deficiency anemia and beta-thalassemia minor: Results in 284 patients. Pakistan Journal of Biological Sciences. 2009;12(5):473–475. 21. Piplani1 S, Manan R, Lalit M, Manjari M, Bhasin T, Bawa J. NESTROFT - A Valuable, Cost Effective Screening Test for Beta Thalassemia Trait in North Indian Punjabi Population. JCDR. 20137(12):2784-2787. 22. Klee GG, Fairbanks VF, Pierre RV, Virgh D, O’Sullivan MB. Routine erythrocyte measurements in diagnosis of irondeficiency anemia and thalassemia minor. Am J Clin Pathol. 1976; 66:870–8777. 23. Bessman JD, Feinstein DI. Quantitative anisocytosis as a discriminant between iron deficiency and thalassemia minor. Blood. 1979;53:288–293. 24. England JM, Fraser P. Discrimination between iron deficiency and heterozygous-thalassaemia syndromes in differential diagnosis of microcytosis. Lancet. 1979;1:145–148. 25. Marti HR, Fischer S, Killer D, Bürgi W. Can automated haematology analysers discriminate thalassaemia from iron deficiency? Acta Haematol.1987;78:180–183. 26. Jayabose S, Giavanelli J, Levendoglu-Tugal O, Sandoval C, Özkaynak F, Visintainer P. Differentiating iron deficiency anemia from Thalassemia minor by using an RDW-based index. J Pediatr Hematol. 1999;21:314.

27. Flynn MM, Reppun TS, Bhagavan NV. Limitations of red blood cell distribution width (RDW) in evaluation of microcytosis. Am J Clin Pathol. 1986;85:445–449. 28. Miguel A, Linares M, Miguel A, Miguel-Borja JM. Red blood cell distribution width analysis in differentiation between iron deficiency and thalassemia minor. Acta Haematol. 1988;80:59. 29. Bentley SA, Ayscue LH, Watson JM, Ross DW. The clinical utility of discriminant functions for the differential diagnosis of microcytic anemias. Blood Cells. 1989;15:575–584. 30. Perutelli P. Red blood cell distribution width in microcytosis. Haematologica. 1989;74:221–222. 31. Van Zeben D, Bieger R, Van Wermeskerken RK, Castel A, Hermans J. Evaluation of microcytosis using serum ferritin and red blood cell distribution width. Eur J Haematol. 1990;44:106–109. 32. Demir, Yaralı N, Fısgın T, Duru F and Kara A. Most reliable indices in differentiation between thalassemia trait and iron deficiency anemia. Pediatrics International. 2002; 44(6):612–616.. 33. Novak RW. Red blood cell distribution width in pediatric microcytic anemias. Pediatrics. 1987;80:251–4. 34. Junca J, Flores A, Roy C, Alberti R and Milla F. Red cell distribution width, free erythrocyte protoporphyrin, and England–Fraser index in the differential diagnosis of microcytosis due to iron deficiency or betathalassemia trait: A study of 200 cases of microcytic anemia. Hematol Pathol. 1991;5:33–6. 35. Yermiahu T, Ben-Shalom M, Porath A. Quantitative determinations of microcytic–hypochromic red blood cell population and glycerol permeability in irondeficiency anemia and b-thalassemia minor. Ann Hematol 1999;78:468–71. 36. Clarke G, Higgins T. Laboratory investigation of hemoglobinopathies and thalassemia: review and update. Clin Chem. 2000;46:1284–90. 37. Roberts GT, El-Badawi SB. Red cell distribution widths index in some hematologic diseases. Am J Clin Pathol. 1987;83:222–6. 38. Marsh WL, Jr, Bishop JW, Darcy TP. Evaluation of red cell volume distribution width (RDW). Hematol Pathol.1987;1:117–23. 39. Burk M, Arenz J, Giagounidis AA, et al. Erythrocyte indices as screening tests for the differentiation of microcytic anemias. Eur J Med Res. 1995;1:33–7. 40. Rahim F, Keikhaei B. Better differential diagnosis of iron deficiency anemia from beta-thalassemia trait. Turkish Journal of Hematology. 2009;26(3):138–145.

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41. Ghafouri M, Sefat LM, Sharifi L. Comparison of cell counter indices in differention of beta thalassemia trait and iron deficiency anemia. The Scientific Journal of Iranian Blood Transfusion Organization. 2006;2(7):385–389. 42. AlFadhli S M, Al-Awadhi A M and AlKhaldi D. Validity assessment of nine discriminant functions used for the differentiation between Iron deficiency anemia and thalassemia minor. Journal of Tropical Pediatrics. 2007;53(2):93–97. 43. Ferrara M, Capozzi L, Russo R, Bertocco F and Ferrara D. Reliability of red blood cell indices and formulas to discriminate between β thalassemia trait and iron deficiency in children. Hematology. 2010;15(2):112–115.

44. Vehapoglu A, Ozgurhan G, Demir A D, Uzuner S, Nursoy M A, Turkmen S and Kacan A. Hematological Indices for Differential Diagnosis of Beta Thalassemia Trait and Iron Deficiency Anemia. Hindawi Publishing Corporation Anemia. 2014, Article ID 576738, 7 pages http://dx.doi.org/10.1155/2014/576738

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Original Article Role of Autologous Platelet Rich Plasma in Treating Symptomatic Patients with Osteoarthritis Via Double Spinning Technique Harjot Kaur Arora1*, Rohit Sharma2, Mridu Manjari1 and Parampreet Singh2 Dept of Pathology, Sri guru Ram Das Institute of Medical Sciences And Research, Amritsar Punjab, India Dept of Orthopedics, Sri guru Ram Das Institute of Medical Sciences And Research, Amritsar Punjab, India 1

Keywords: Blood Bank, Intra-articular, Osteoarthritis, Platelet Rich Plasma

ABSTRACT Background: Platelet rich plasma (PRP) is a hemoderivative containing a platelet concentration well above the normal baseline rich in platelet degranulation products such as cytokines/vasoactive amines and growth factors, which make it a good therapeutic tool for repair and healing in many speciality fields of orthopaedics, dentistry, plastic surgery (including cosmetic and burn therapy) and dermatology. The present study was conducted with aim of seeing the response of PRP on symptomatic alleviation and functional restoration in patients of osteoarthritis. Methods: The present study was undertaken in the department of transfusion medicine in conjunction with department of orthopedics at a tertiary care teaching hospital on 50 cases of OA graded according to Kellgren-Lawrence grading system. In all the cases single intra-articular injection of PRP made by double spin technology was injected and response of the patient followed up at 3, 6 and 12 months with visual analogue score (VAS) and Western Ontario & Mc Master Universities Arthritis (WOMAC) index. Apart from simple statistics of mean and averages; the means of paired samples were compared for the VAS with Studentâ&#x20AC;&#x2122;s t parametric test. The WOMAC index was tested by the non-parametric Wilcoxonâ&#x20AC;&#x2122;s rank sum test. In both the settings a value < 0.05 was deemed significant. Result: Statistically significant differences (p<0.001) between pre-treatment and follow up values were found for pain, stiffness and functional restoration of the joint as measured by WOMAC index and VAS pain scores. There were no adverse events related to PRP infiltration. Conclusion: The study hence concludes that intra-articular infiltration of autologous PRP prepared by double spinning approach in OA of the knee according to the established protocol is a safe, tolerable and effective tool which results in reduction of pain and is also associated with functional improvement of the joint as well.

*Corresponding author: Dr Harjot Kaur, Dept of Transfusion Medicine , Sri Guru Ram Das Institute of Medical Sciences And Research, Vallah Amritsar Punjab 143501, INDIA Phone: +91 9781516166 Email: harjotbhandari@gmail.com

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Arora et al.

Introduction

Platelet rich plasma (PRP) is a hemoderivative containing a platelet concentration well above the normal baseline. [1,2] According to application of the technique utilized it is classified conventionally as pure platelet rich plasma (P-PRP), leucocyte and platelet rich plasma (L-PRP), pure platelet rich fibrin (P-PRF), leucocyte and platelet rich fibrin (L-PRF) based upon the technique used to prepare and the final content of the product.3 Different derivatives have different physiological responses for example PRF is reportedly has a better therapeutic response in comparison to PRP owing to better platelet degranulation [3,4] and leucocyte poor PRP are better in treatment response owing to pauci-immune response in such types of PRP. [5] Since late 90’s its usage is increasing and getting more and more popular in field of orthopedics, dentistry, ophthalomology, wound healing, plastic surgery including burn management and maxillofacial surgeries which require active tissue regeneration and repair .[6] The therapeutic effect of PRP is attributed to the rich plethora of cytokines/vasoactive amines and growth factors which are released into it via the alpha granules of the platelets, namely; platelet-derived growth factor (PDGF), transforming and fibroblastic growth factors-β, insulin-like growth factor-1/2 (IGF-1/2), vascular endothelial growth factor, epidermal growth factor amongst others with these growth factors acting as inexpensive, efficient, biologically safe (autologous hemoderivative) adjunct therapy to maintain physiological hemostasis ( in case of orthopedic sciences as an aid to repair and remodel the skeletal and soft tissue compartment). Thus PRP itself is coming up in various fields as an alternative to the conventional pharmacological and instrument based therapies which have proven associated side effects or morbidity. [7-12] The rationale behind the injection of autologous blood preparations lies in the exploitation of body’s own natural response to injury and to release growth factors [13,14] or the production of anti-inflammatory cytokines by blood components as explained above [11,15] Blood is an important and unique source of cellular and protein products that has been explored more intensively over the last three decades for the production of biomaterials for clinical use .[10]

A-561 In field of orthopaedics, Osteoarthritis (OA) is a chronic degenerative condition of hyaline cartilage accounting for profound morbidity, pain and health care expenses. The consequences to the individual and to the population as a whole are very significant, particularly with our aging population. There are few validated interventions that improve the clinical condition of a patient once the degenerative process becomes symptomatic. Given the lack of response of the body’s healing mechanisms to degenerative conditions generally, injection of growth factors and cytokines is sensible. Lab and animal models exist for using PRP in OA with generally favorable results.[15] A recent article by Kon et al indicates improved functional outcomes. [16] It is still unknown whether PRP acts by local paracrine factors to alter pain, by new hyaline or fibrocartilage formation or a combination of both or neither.[17]

Materials and Methods

The study was conducted in Department of Transfusion Medicine in conjunction with department of orthopedics between November 2013- December 2014 at a tertiary care teaching hospital. The hospital ethical committee approved the study. Patients included in the study were between the age group 39-60 years who attended the orthopedics outpatient department with complains associated with joint pain and ultimately diagnosed as suffering from osteoarthritis (OA) according to the criteria’s laid down by American College of Rheumatology (ACR). [18] The aim of the present study was to judge the response of PRP injected intra-articularly in knee joint by double spin technique on pain relief and functional restoration of the joint by measureable clinical indicators. Study: Patients included in the study were those having a body mass index (BMI) < 30, non responsive to antiinflammatory drugs > 3 months without any previous therapeutic invasive procedure or history of fracture. A pre-study questionnaire designed at the start of the study to record patient details such as age, BMI, current work status (active/passive/retired), dominant extremity and the two tests VAS (Visual analogue score) and WOMAC (Western Ontario and Mc Master Universities Arthritis) index were duly filled up.

In the field of transfusion medicine apart from supply of the “safe blood and its components” and apheresis an added responsibility of provision of PRP has been incorporated. As a recent addition the safety and standaridization protocols are still being evolved targeting the specific fields of medicine and surgery where they are being utilized.

Exclusion Criteria: Patients with thrombocytopenia (count < 1,00,000), anemia (< 10 gm/dl hemoglobin), immunodeficiency states/ immunosuppressive therapy, positive for transfusion transmitted infections (TTI’s), advanced tri-compartmental OA, possible infective arthritis of knee joint and patients with skeletal knee deformities as a squeal of advanced OA were excluded.

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Autologous Platelet Rich Plasma in Osteoarthritis

Also excluded were patients using consistent NSAID’s or use of local corticosteroid injection and systemic usage of corticosteroids within 2 weeks. Informed consent of all the patients was obtained. In total 50 patients were finally included in the study of OA knee having grade 1, grade 2 and grade 3 criteria of KellgrenLawrence grading system [19] done on anterio-posterior / lateral knee and skyline/ standing 45 degree flexion knee radiograms. [Table-1] All the 50 patients were treated with one intra articular injection of autologous PRP produced by double spinning approach with pre-procedural complete blood count (CBC), coagulation profile and tests for transfusion transmissible infections (TTI’s). Technique for Preparation of PRP: To prepare 20 ml of PRP with platelet concentration of 4-6 times the average normal value; 100 ml of venous blood from antecubital vein under aseptic precaution was collected in an adult double bag (Polymed/J.Mitra) with 14ml of citrate phosphate dextrose and adenine as an anticoagulant. Blood with CPDA -1 was centrifuged twice- first at 1800 rpm for 15 min to separate RBC and a second at 3500 rpm for 10 min to concentrate platelets. The final PRP was extracted for intra-articular injection in two 10 ml syringes (approximately 8 ml per knee) and 0.5 ml PRP was sent to laboratory for platelet counting and validation of the procedure. Procedure: Under aseptic conditions, PRP thus obtained were injected through a classic supra lateral approach using a 22 G needle in a supine position with knee in full extension. Immediately post procedure, the patients were told to actively flex and extend their knees a few times to allow the PRP to spread throughout the joint before it coagulates spontaneously (gelling). Patients were sent home after 15-20 min of rest with instruction to have rest, to limit weight bearing and to use cold packs 3-4 times a day for 10 min for the next 48 hours. Patients were prohibited from using NSAIDS, steroids or any medications, which might influence platelet count or function. Exercise or physical treatment were also restricted during the study period to eliminate any synergistic effects. Post-procedural Follow Up: All the patients were followed up for a minimum period of one year. Follow up was done on a regular interval (3 months, 6 months and 12 months) with both clinical and functional evaluation for pain relief as per VAS (Visual analogue score) and WOMAC (Western Ontario and Mc Master Universities Arthritis) index. VAS assessment was done with numbers from “0” to“10”, equidistantly marked on a 10 cm line. The patients were

explained that “0” meant they were experiencing no pain, “5” moderate pain and “10” unbearable pain, and they were asked to mark the appropriate score on the line describing their own pain during rest and physical activity. [20] The WOMAC osteoarthritis index is a disease-specific questionnaire for the disease, which assesses pain, stiffness and physical functions of OA patients. It consists of 24 questions in total: 5 on pain, 2 on stiffness and 17 on physical functions. Individual subgroups score or the total score can be calculated. A Likert scale (1: none, 2: low, 3: medium, 4: high, 5: very high) is used to assess all parameters on the WOMAC OA index. High WOMAC scores are indicative of intense pain and stiffness and impairment of the physical function. In our study, the patients’ WOMAC sub-scores (pain, stiffness, function) and total WOMAC score were calculated.[21] Both the scores were done pre-procedure and after procedure on follow up visits and backed up with the standard blood investigation such as repeat CBC, ESR and CRP levels. Statistical Analysis: Apart from simple statistics of mean and averages; the means of paired samples were compared for the VAS with Student’s t parametric test. The WOMAC index was tested by the non-parametric Wilcoxon’s rank sum test. In both the settings a value < 0.05 was deemed significant.

Result

In the study conducted on 50 patients there was a male preponderance (29 cases) with ages ranging from 39 years to 60 years with mean age of 49.8 years.PRP was injected mostly in right knees (32 cases; 62%). The chondropathic grade distribution in the study according to the Kellgren-Lawrence grading system was mostly skewed towards grade 3 ( 40 % ) cases followed by grade 2 (36 % cases) and grade 1 ( 24 % cases) respectively. No grade 4 lesions were included in the study as per predetermined exclusion criteria. [Table -2] It was noted in the study that the VAS score for pain which was 9.21 at pretreatment declined subsequently to 3.32 at 3 months and again showed a small fluctuation of 3.88 at 6 months and slight increase of 5.51 at 12 month follow-up, with statistically significant differences at each time point (P<0.0001). An improvement was documented in large group of cohorts (87.8% of patients). [Table-3] The scores for WOMAC Index in the present study for pain, stiffness and functional capacity, respectively, were18.4, 7.44 and 54.4 at pretreatment, 3.21, 2.13 and 15.31 at

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3-month, 4.55, 2.84, 18.5 at 6- month, and 8.81, 4.11, 28.8 at 12-month follow up with significant differences for all three periods (P<0.0001). The patients followup improvement was 84.4%, 67.7% and 81.1% for pain, stiffness and functional capacity respectively thus showing affable patient satisfaction overall. [Table-3]

In the study conducted no major signs or symptoms (adverse reactions) were recorded related to the PRP injections (both intra or post procedure); although in some cases slight pain was present during first 2 or 3 days but it regressed spontaneously without any administration of analgesics.

Table 1: Kellgren-Lawrence Classification.

Description

Grade Grade 0

Normal

Grade 1

Doubtful narrowing of joint space and possible osteophytic lipping

Grade 2

Definite osteophytes and possible narrowing of joint space

Grade 3

Moderate multiple osteophytes, definite narrowing of joint space, some sclerosis, and possible deformity of bone ends

Grade 4

Large osteophytes, marked narrowing of joint space, severe sclerosis, and definite deformity of bone ends

Table 2: Patient demographic data and Kellgren- Lawrence grades. No. of Patients

Male

Female

Right

Left

Grade 1

Grade 2

Grade 3

Table 3: Global results: pre/post treatment average values, percentage of improvement, statistical significance rates. VAS

WOMAC Pain

WOMAC Stiffness

WOMAC Functional capacity

Pretreatment

9.21

18.4

7.44

54.4

3 month

3.32

3.21

2.13

15.31

6month

3.88

4.55

2.84

18.52

12 month

5.51

8.81

4.11

28.80

87.8%

84.4%

67.7%

81.1%

<0.0001

%age of improvement P value

Discussion

The present study was done to determine the effect of intra-articular injection of platelet rich Plasma (PRP) in symptomatic patients with knee osteoarthritis and to educate patients regarding importance of early intervention. Platelet-rich plasma (PRP) is a simple, efficient, and minimally invasive method of obtaining a natural concentration of autologous growth factors. Generation of PRP involves centrifugation of autologous blood to separate and extract the plasma and buffy coat portion of the blood, which contain high concentrations of platelets. The application of PRP has been documented in many fields. First promoted by Ferrari in 1987 as an autologous transfusion component after an open-heart operation to avoid homologous blood product transfusion, now its application is in many fields.[22]

Many factors are contributive to the outcome in the patient, which are documented in literature such as increased concentration of platelet population from a baseline (lower type: 2.5-3.0 times or higher: 5.0-9.0 times), proportion of leucocytes within PRP (leukocyte rich or poor) and type of anti-coagulant.[23] It would seem intuitive that a higher platelet count would yield more growth factors and better clinical results, however, this has not yet been determined. Graziani et al suggested that the optimal concentration of PRP was 2.5x baseline and above this there may be an inhibitory effect. [23] OA is the leading cause of disability in the world and more than 10 % of the elderly population having symptomatic disease.[24] By mediating chondroprotective action PRP therapies can delay joint deterioration by interfering with the early catabolic and

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Autologous Platelet Rich Plasma in Osteoarthritis

inflammatory events and by promoting anabolic responses subsequently reducing OA pain. The central role of metalloproteinases is now well documented as the source of chondro-degeneration which is additionally aggravated by other physical and intrinsic elements notably amongst them an increase in BMI > 25.[25] In the present study maximum patients were of grade 3 (40 %) with very few patients of grade 1 is explained by the fact that in grade 1 the symptoms are very much mild and patient hence alleviates his/her pain with over the counter medications and doesn’t present to the medical specialist. PRP Technique and its correlation with Pain Improvement: Although there are 3 protocols for PRP production: selective blood filtration, single-spinning and double spinning procedures.[26] The latter two are associated with better results and patients compliance and is also cost effective. Double spinning is thought to increase the baseline platelet levels by 4- to 8- fold in comparison to 1 to 3 times in single spin technique. The method used in the present study was double spinning and there was significant improvement in all 50 patients at 3 and 6 months follow up (p <0.0001), although from 6 months to 12 months patients with grade 2 and 3 started experiencing slight pain and stiffness but still the results were statistically significant (p<0.001) from baseline evaluation. This is in concordance with work done by researchers notably amongst them Kon et al who have employed intra-articular injections of PRP obtained after double spin technique in a cohort of 100 patients of OA with a two year evaluation period [16,27] .In a clinical trial conducted it was noted that technique of intra-articular autologous double-centrifuged PRP had better therapeutic efficacy than the hyaluronic acid (HA) injections in alleviating patient symptoms and also resulted in recovery of joint movements even in those patients afflicted by severe chondropathies. [28] VAS score: In our study baseline VAS score of patient was taken at 100 at the time of presentation to outpatient department. The reduction of mean VAS score at 3 month, 6 month and 12 month with increased proportion of percentage of improvement was found to be statistically significant with p value <0.0001. The percentage improvement of 87.8 % in our study was better than those seen in Kon et al , Wang et al and Sanchez et al who have reported them to be 80 % , 73.4 % and 33.3 % respectively.[16,29,30] WOMAC score: The scores for WOMAC Index in the present study for pain, stiffness and functional capacity, respectively, were lesser in the 3 month follow up period than at the 6 and 12 month follow up period but still

were lower than the pre-treatment levels. The finding was statistically significant in all three follow up periods (p<0.001). The patient’s follow-up improvement was higher than the work conducted by Spakova et al, Patel et al and Creza et al.[31,32,33]

Conclusion

The study hence concludes that intra-articular infiltration of autologous PRP prepared by double spinning approach in OA of the knee according to the established protocol is a safe, tolerable and effective tool which results in reduction of pain in the long run. The study also proves emphatically that reduction of pain is also associated with functional improvement of the joint as well (measured in the study by WOMAC score). Apart from the improved clinical findings an additional benefit of PRP is absence of systemic toxic / adverse drug reactions/ idiosyncratic reactions associated with the conventional pharmaco-therapeutic agents. Although blood banks in past apart from providing transfusion services have been entrusted additional services of therapeutic procedures such as aphaeresis with PRP being the new entrant. In the coming years blood banks are going to be an intrinsic and integral part of therapeutic interventions in many specialties. Hence work has to be done in framing of standardization for making PRP as a therapeutic tool in various specialties.

Acknowledgements NA

Funding None

Competing Interests None Declared

Reference

1. Russell RP, Apostolakos J, Hirose T, Cote MP, Mazzocca AD. Variability of platelet-rich plasma preparations. Sports Med Arthrosc. 2013;21:186–90. 2. Marques LF, Stessuk T, Camargo IC, Sabeh Junior N, dos Santos L, Ribeiro-Paes JT. Platelet-rich plasma (PRP): Methodological aspects and clinical applications. Platelets. 2015;26:101–13. 3. Dohan Ehrenfest DM, Rasmusson L, Albrektsson T. Classification of platelet concentrates: From pure platelet-rich plasma (P-PRP) to leucocyteand platelet-rich fibrin (L-PRF) Trends Biotechnol. 2009;27:158–67. 4. Passaretti F, Tia M, D’Esposito V, et al . Growth promoting action and growth factor release by

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Arora et al. different platelet derivatives . Platelets 2014 ;25:252-6 5. Dhillon MS, Behera P , Patel S, Shetty V. Orthobioloics and platelet rich plasma . Indian J of Orthop. 2014 ;48:1-9 6. Sampson S, Gerhardt M, Mandelbaum B. Platelet rich plasma injection grafts for musculoskeletal injuries: A review. Curr Rev Musculoskelet Med. 2008;1:165–74 7. Ziltener JL, Allet L, Sclison P, Grosclaude M. How effective are injections of platelet-rich plasma (PRP) for the treatment of sports injuries: a critical review of the literature. J Sports Med Doping Studie 2012; doi:10.4172/2161-0673.S2-003. 8. Brossi PM, Baccarin RY, Massoco CO. Do blood components affect the production of reactive oxygen species (ROS) by equine synovial fluid cells? Pesq Vet Bras. 2012;32:1355–60. 9. Moreira JJ, Moraes APL, Brossi PM, Machado TSL, Michelacci YM, Massoco CO, et al. Autologous processed plasma: cytokine profile and effects upon injection in healthy equine joints. J Vet Sci. 2015;16:47–55. 10. Burnouf T, Goubran HA, Chen TM, Ou KL, El E, et al. Blood-derived biomaterials and platelet growth factors in regenerative medicine. Blood Rev. 2013;27:77–89. 11. Textor J. Autologous biologic treatment for equine musculoskeletal injuries: platelet-rich plasma and IL-1 receptor antagonist protein. Vet Clin North Am Equine Pract. 2011;27:275–98. 12. Chevalier X. Intraarticular treatments for osteoarthritis: new perspectives. Curr Drug Targets. 2010;11:546– 60. 7. Alsousou J, Thompson M, Hulley P, Noble A, Willett K. The biology of platelet-rich plasma and its application in trauma and orthopaedic surgery: a review of the literature. J Bone Joint Surg (Br). 2009;91:987–96. 13. Fortier LA, Barker JU, Strauss EJ, McCarrel TM, Cole BJ. The role of growth factors in cartilage repair. Clin Orthop. 2011;469:2706–15. 14. Gobbi G, Vitale M. Platelet-rich plasma preparations for biological therapy applications and limits. Oper Tech Orthop. 2012;22: 10–5.

A-565 produced favorable results on degenerative cartilage lesions. Knee Surg Sports Traumatol Arthrosc. 2010;18:472-79 17. Mulvaney, S. Treatment of peripheral nerve entrapments with real time ultrasound guided percutaneous hydro-neurolysis. 2010. Presented at AMSSM annual meeting. 18. Misso ML, Pitt VJ, Jones KM, et al . Quality and consistency of clinical practice guidelines for diagnosis and management of osteoarthritis of knee and hip :a descriptive overview of published guidelines . Med J Aust, 2008, 189:394-399. 19. Kijowski R, Blankenbaker D, Stanton P, et al:Arthroscopic validation of radiographic grading scales of osteoarthritis of the tibiofemoral joint.AJR Am J Roentgenol, 2006, 187:794-799. 20. Dixon JS, Bird HA, :Reproducibility along a 10 cm vertical visual analogue scale. Ann Rheum Dis, 1981 , 40 :87-89. 21. Yim Chiplis PK , Talbot LA. Defining and measuring balance in adults. Res Nurs , 2000 , 1 :321-31 . 22. Ferrari Met al. A new technique for hemodilution, preparation of autologous platelet-rich plasma and intraoperative blood salvage in cardiac surgery. Int J Artif Organs. 1987 Jan;10(1):47-50. 23. Graziani et al. The in vitro effect of different PRP concentrations on osteoblasts and fibroblasts. Clin Oral Implants Res. 2006 Apr;17(2):212-9. 24. Loeser RF . Age related changes in the musculoskeletal system and the development of osteoarthritis. Clin Geriatr Med . 2010 Aug ;26 (3):371-86. 25. Dawson J, Jaszczak E, Thorogood M, Marks SA, Dodd C, Fitzpatrick R. An investigation of risk factors for symptomatic osteoarthritis of the knee in women using a life course approach . J Epidemiol Community Health . 2003 Oct ;57(10):823-30 26. Mei –Dan O, Lippi G, Sanchez M, Andia T, Maffulli N. Autologous platelet rich plasma: a revolution in soft tissue sports injury management ? Phys Sportsmed. 2010;38:127-135

15. Akeda K et al. Platelet rich plasma stimulates porcine articular chondrocyte proliferation and matrix biosynthesis. Osteoarthritis Cartilage. 2006;14(12):1272-1280.

27. Filardo G, Kon E, Buda R, et al. Platelet-rich plasma intrarticular knee injections for the treatment of degenerative cartilage lesions and osteoarthritis. Knee Surg Sports Traumatol Arthrosc. 2011Apr; 19(4):528-535.

16. Kon E, Buda R, Filardo G, Di Martino A, Timoncini A. Platelet-rich plasma: intra-articular knee injections

28. Kon E, Buda R, Filardo G, Timocini A, Marcacci M, Giannini S. The treatment of severe chondropathies

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of the knee: platelet rich plasma vs hyaluronic acid. Presented at 8th world congress of the international cartilage repair society (ICRS), Miami, USA, 23â&#x20AC;&#x201C;26 May 200. 29. Wang-Saegusa A, Cugat R, Ares O, Seijas R, Cusco X, Wenu Y. Infiltration of plasma rich in growth factors for osteoarthritis of the knee short term effects on function and quality of life. Arch Orthop Trauma Surg. 2011;131:311-317. 30. Sanchez M, Anitua E, Cugat R, et al. Nonunions treated with autologous preparation rich in growth factors. J Orthop Trauma. 2009;23:52-59.

31. Sapoka T, Rosocha J, Lacko M, Harvanova D, Gharaibek A. Treatment of knee joint osteoarthritis with autologous Platelet rich plasma in comparison with hyaluronic acid. Am J Phys Med Rehabil. 2012;91(5):411-7 32. Patel S, Dhillon MS, Aggarwal S, Marwaha N, Jain A. Treatment with platelet rich plasma is more effective than placebo for knee osteoarthritis. a prospective double blind randomised trial . Am J Sports Med. 2013:41 (2); 356-64 33. Cerza F, Carni S, Carcangui A et al. Comparison between hyaluronic acid and platelet rich plasma, intra articular infilteration in the treatment of gonoarthrosis. Am J Sports Med, 2012, 40:2822-27.

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Original Article Mean Platelet Volume In Diabetes Mellitus Type II Anupama Dayal*, Sadhana Kothari, Rupal J Shah and S.M.Patel Pathology Department, GCS Medical College Hospital and Research Centre, Ahmedabad. Gujarat. India Keywords: Mean Platelet Volume, Diabetes Mellitus, HbA1c.

ABSTRACT Background: Diabetes Mellitus (DM) is a metabolic disorder and a major health problem because of its high prevalence and morbidity. Platelets have a key role in development of its thrombogenic complications. Platelet size is considered as a marker and determinant of platelet function with younger, larger platelets exhibiting more reactivity. This study was carried out to compare Mean Platelet Volume (MPV) in subjects with diabetes and non diabetic controls. Method: Total 211 subjects, 105 diabetics and 106 nondiabetic controls were included. MPV, platelet count, blood glucose levels, lipid profile and HbA1c levels were measured and data analyzed by independent “t” test. Result: Mean MPV was significantly higher in the diabetic group compared to the controls (9.94 ± 1.07 fl versus 9.36 ± 0.96 fl; p=.00003). Mean BMI, cholesterol and triglycerides were also significantly higher in the diabetic group. Within the diabetic group, mean MPV, cholesterol and triglycerides were significantly higher in subjects with HbA1c ≥ 6.5% (10.30±0.95fl vs 9.82±1.08fl, p=.03; 206.28±37.17 mg/dl vs 185.07±41.91 mg/dl, p=.026; 176.96 ± 88.54 mg/dl vs 133.19 ± 46.30 mg/dl, p=.002 respectively). Conclusion: High MPV in diabetics may indicate platelet hyperactivity, which may contribute to the vascular complications of type II DM. Thus MPV can be used as a simple, costeffective parameter to assess the probability of developing vascular complications in diabetes.

*Corresponding author: Dr. Anupama Dayal , B-62, Shakuntal Bungalows, Nr. Rajhans Cinemas, Nikol-Naroda road.Nikol. Ahmedabad. 382350.Gujarat.India Phone: +91 9898264571 Email: dayal1.anupama.ad@gmail.com

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

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Introduction

Diabetes mellitus (DM) is the most common endocrine disease characterized by metabolic abnormalities, hyperglycemia, and by long term complications. There are two major subgroups of DM, type I insulin dependent (IDDM) and type II (NIDDM). [1] Thrombosis, atherosclerosis and other vascular diseases are common complications of diabetes mellitus. [2] Platelets play an important role in the normal hemostasis; the Mean Platelet Volume (MPV), an accurate measure of the platelet size is considered a marker and determinant of platelet function.[3] Larger platelets with higher MPV are hemostatically more reactive and produce higher amounts of the prothrombotic factor Thromboxane A2, increasing a propensity to thrombosis.[3] Platelets from subjects with DM, particularly from those with type 2 diabetes, exhibit increased reactivity. Factors that may contribute to this greater platelet reactivity are not completely elucidated and include metabolic abnormalities such as hyperglycemia, hyperlipidemia, insulin resistance, and conditions as oxidative stress, inflammation, and endothelial dysfunction. [4] MPV, a determinant of platelet function, is a newly emerging risk factor for atherothrombosis. A large proportion of persons with type 2 DM suffer from preventable macrovascular complications. There is a need to develop risk factor modification interventions to reduce the impact of longterm complications.[5] MPV can be easily measured during routine haematological analysis by automated hematology analyzers. Thus, MPV can emerge as an important, simple, effortless, and cost-effective tool for monitoring and for early recognition of patients that could possibly benefit from preventive treatment.

Materials and Methods

This study was done in a tertiary care hospital of Ahmedabad, Gujarat, India over a period of 6 – 8 months after approval of the IEC. A total of 211 subjects were included in this study, 105 were type II diabetic patients, 106 were healthy individuals matched for age and gender. Height and weight of all subjects was recorded. Patients with anemia, hypertension, hypo-hyperthyroidism, congestive heart failure, leukocytosis, thrombocytopenia or history of stroke, antiplatelet medication were excluded as they may affect platelet size. Venous blood samples were collected in EDTA & Plain vacuities. Blood samples were taken after an 8–12 hour period of fasting for estimation of glucose and lipid profile. Haemoglobin (Hb), MPV, platelet count was done by

automated hematology analyzer(KX- 21) & serum was analyzed for fasting (FBS) and post parandial blood sugar (PPBS), glycosylated haemoglobin (HbA1c) and lipid profile. Glucose level was measured by the oxidase method, HbA1c level was analyzed by immunoturbidimetric method, total cholesterol, triglyceride, low density lipoprotein (LDL) cholesterol and high density lipoprotein (HDL) cholesterol were measured by commercial enzymatic methods by fully automated biochemistry analyzer (XL- 640). All the investigations were carried out within one hour of collection of samples to avoid error due to sample ageing. The statistical analysis of the results was performed by using the Statistical Package for Social Sciences (SPSS) for IBM version 20.0. Independent “t”-test was used for testing difference significance, P value < 0.05 considered statistically significant.

Result

A total of 211subjects (105 patients with type 2 diabetes and 106 healthy controls) were included in the study. There were 55 males and 50 females in the diabetic group and 49 males and 47 females in the nondiabetic group. Mean age of diabetic patients was 53.5 ± 9.2 yrs whereas that of nondiabetic population was 47.9 ± 11.7 yrs. Table 1 shows the comparison of laboratory data between the study and control groups. The mean FBS level in the diabetic group was 152.13 ± 57.7 mg/dl as compared to 95.34 ± 8.3 mg/dl in the non-diabetic group (p= <.00001). In the diabetic group the mean PPBS was 229.1 ± 90 mg/dl while it was 122.2 ± 15.5 mg/dl in the non-diabetic group (p < .0001). The mean HbA1c in the diabetic group was 8.61 ± 2.43% as compared to 5.87 ± .39% in the non-diabetic group (p < .00001). The mean MPV was significantly higher,9.94 ± 1.07 fl, in the diabetic group as compared to 9.36 ± 0.96 fl in the non-diabetic group (p=.00003). Mean BMI, Cholesterol and triglycerides (TG) were also significantly higher in the diabetic group. Platelet count was lower in the diabetic group though not statistically significant. There was no significant difference in the value of HDL between the two groups. Diabetic patients were further grouped according to the HbA1c level into two groups: HbA1c < 6.5% (n=76) and HbA1c ≥ 6.5% (n=29) (Table 2). Individual variables were analyzed by independent student’s “t” test. Mean MPV (10.29±0.95fl) in patients with HbA1c ≥ 6.5% was significantly higher than that of HbA1c < 6.5% (9.82±1.08fl)(p=.03). Similarly the mean FBS and PPBS were also significantly higher in patients with HbA1c ≥ 6.5% (p< .0001). There was no significant difference in the platelet count between the two groups.

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Dayal et al.

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Data was available in 100 patients for lipid profile. The mean cholesterol in patients with HbA1c ≥ 6.5% (n=71) was 206.2±37.17 mg/dl was significantly higher than that of patients with HbA1c < 6.5% (n=29) (185.07±41.91 mg/dl) (p=.026).Similarly the mean triglyceride level was also significantly high in the patients with HbA1c

≥ 6.5% (176.96 ± 88.54 mg/dl vs 133.19 ± 46.30 mg/dl) (p=.002). However there was no significant difference in the BMI and HDL between the two groups. Within the diabetic group no significant pearsons correlation could be found between MPV and FBS, PPBS, HbA1c or BMI.

Table 1: Comparison of general characteristics and laboratory parameters between study group(Diabetic) and controls. Diabetic group

Parameters

Controls

p-value

mean

Std. Deviation

mean

Std. Deviation

Age (years)

105

53.50

9.250

106

47.97

11.71

Males

Females

Height (cm.)

105

159.90

9.27

156.91

8.94

Weight (kg)

105

71.74

13.02

64.16

14.39

Body Mass Index (Kg/m²)

105

28.22

4.82

25.22

4.81

0.0025

MeanPlateletVolume (fl)

105

9.94

1.07

106

9.36

0.96

0.00003

Platelet Count (x 10 /L)

105

265.44

74.48

106

283.84

80.82

0.087

FastingBloodSugar (mg/dl)

105

152.13

57.74

106

95.34

8.29

<0.00001

PostParandialBloodSugar (mg/dl)

105

229.11

90.05

105

122.25

15.51

<0.0001

HbA1c (%)

105

8.61

2.43

102

5.87

0.39

<0.00001

Cholesterol (mg/dl)

100

199.39

39.34

105

186.90

35.68

0.018

Triglyceride (mg/dl)

100

164.29

80.33

105

128.06

85.45

0.004

High Density Lipoprotein (mg/dl)

100

48.47

13.60

105

49.20

8.57

0.65

Table 2: Comparison of individual variables in diabetic population according to HbA1c levels. Parameters Body Mass Index(Kg/m²) Platelet Count (x109/L)

HbA1c (%) ≥ 6.5 < 6.5 ≥ 6.5 < 6.5 ≥ 6.5

MPV(fl)

< 6.5

FBS(mg/dl) PPBS(mg/dl) Cholesterol(mg/dl) Triglycerides(mg/dl) HDL(mg/dl)

≥ 6.5 < 6.5 ≥ 6.5 < 6.5 ≥ 6.5 < 6.5 ≥ 6.5 < 6.5 ≥ 6.5 < 6.5

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Total 105 105 105 105 105 100 100 100

Mean

Std. Deviation

p - value

27.72

4.18

0.503

28.38

4.21

265.23

71.21

0.938

263.78

86.59

9.82

1.08

0.038

10.30

0.95

169.37

60.12

<0.0001

107.00

10.98

262.16

86.35

<0.0001

144.48

15.59

206.28

37.17

0.026

185.07

41.91

176.96

88.54

0.002

133.19

46.30

49.03

15.22

0.683

47.96

9.57

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Discussion

Diabetes mellitus (DM) is associated with macrovascular and microvascular complications (coronary artery disease, ischemic stroke, peripheral arterial disease, nephropathy, and retinopathy). Platelets have a “key role” in atherogenesis and its thrombotic complications in subjects with DM.[6] Countries with the highest absolute number of diabetics are in India(19 million), China(16 million), and the United States(14 million).[7] MPV was found to be significantly higher in the diabetic group as compared to the non-diabetic group in the present study. This is in agreement with the studies done by Hekimsoy et al,[8] Zuberi et al,[9] Kodiatte et al,[10] Ulutas et al.[11] Larger platelets are more active hemostatically and enzymatically, and they contain more prothrombotic molecules, such as platelet factor 4, serotonin, and plateletderived growth factor, and possess greater aggregability in response to ADP.[12] Mean platelet volume (MPV), which is used to measure platelet size, can reflect platelet activity.[13] Increased MPV may lead to a prothrombotic condition with increased thromboxane A2 (TXA2) and B2 and adhesion molecule expression, such as P-selectin and glycoprotein IIb/IIIa, and β-thromboglobulin release.[12] Platelets of diabetic patients are characterized by dysregulation of several signaling pathways and have been suggested to be hyperreactive, showing increased adhesion, activation, and aggregation.[14] Several mechanisms may account for the increased platelet activity in diabetes. The glycation of platelet surface proteins reduces membrane fluidity and increases platelet adhesion, causing incorporation of glycated proteins into the thrombi. An increase in calcium mobilization from intracellular storage pools, resulting in increased intracellular calcium levels, has been correlated with reduction in membrane fluidity.[15]

HbA1c similar to studies done by Sharpe et al,[19] Lutfullah Cakir et al,[20] Unubol M et al,[21] E.C.Yenigun et al.[22] Kim JH found that MPV was only positively correlated with FPG in newly diagnosed diabetic women.[23] This could explain why no significant correlation was found in this study as the patients selected were having diabetes for a longer duration and were also taking treatment for the same. If vascular damage was only due to increased number of large and reactive platelets, then the rate of damage would have been constant for the duration of disease independent of glycemic control. This clearly shows that platelet reactivity alone cannot explain the progression of vascular complications in DM since there are other vascular risk factors that may be influenced by the degree of control of diabetes. [10] This is supported by the nonsignificant correlation between MPV and HbA1c. Platelet count was lower in the diabetic patients similar to results of E.C.Yenigun et al[22] and Tschöpe et al,[24] this difference was not statistically meaningful. In the diabetic group a negative linear relationship between MPV and the number of platelets was seen (p=0.018). Decreased count could be the result of small platelets being consumed in order to maintain a constant platelet functional mass. [25] Association of increased platelet volume and reduced platelet survival in diabetic patients has been reported by Jones et al.[26]

MPV was found to be significantly increased in patients with HbA1c ≥ 6.5% as in other studies. [10, 16] Hyperglycemia can increase platelet reactivity by inducing nonenzymatic glycation of proteins on the surface of the platelet, by the osmotic effect of glucose and activation of protein kinase C.[10] Mean platelet volume was significantly decreased at the 3-month follow-up period, compared to baseline MPV, in diabetic patients who achieved improved glycemic control with intensive diet and pharmacotherapy.[17] Significant positive correlation between the reduction in thrombus formation and the reduction in HbA1c has also been repor ted.[18] These findings suggest that glycemic control may be helpful in decreasing the platelet reactivity and thus may prevent or delay possible vascular complications.

Lipid abnormalities significantly contribute to the increased risk of cardiovascular disease and other morbidity in diabetics. In this study the level of total cholesterol, TG, were increased in diabetics as compared to controls and also significantly higher in diabetics with HbA1c ≥ 6.5%. Increased total cholesterol, TG and LDL in diabetics was reported by Farah Jabeen et al[27] and Vijaya C et al.[28] Kamilla R Alhadas[29] found higher levels of Triglycerides only in diabetics as compared to controls. Within the diabetic group higher cholesterol was reported by Dindar et al[16] while Kadic et al[30] found significantly higher TG in diabetics with poor glycemic control(HbA1c > 7%). These findings are in accordance with the present study. Diabetes mellitus is a complex disease where the carbohydrate, protein and fat metabolism are impaired. [31] Insulin stimulates synthesis of fatty acid in liver, adipose tissue and in the intestine and synthesis of cholesterol.[32] In diabetes mellitus abnormal increased levels of lipid, lipoprotein and lipid peroxides in plasma may be due to the abnormal lipid metabolism.[33] Poor glycemic control associated with abnormal lipid profile can hasten the development of vascular complications.

No positive pearsons correlation could be established between MPV and glycemic indices especially FBG and

Micro and macrovascular complications are often seen in DM and represent the main causes of morbidity and

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 06, (Suppl) December, 2016

Dayal et al. mortality, which are closely associated with glycaemic control.[34] Mean platelet volume is usually established to associate with macrovascular complications such as myocardial infarction and restenosis after percutaneous coronary intervention.[25] Many studies[16, 22] have shown association between higher MPV and vascular complications. This underlines the fact that MPV can be used as a parameter to assess platelet function and activity and can provide an indication to the development of complications. Considering that odds of inadequate glycemic control are 2 times increased per femtoliter greater MPV, its use, especially in the primary health care centers, could improve the screening of high-risk individuals for vascular complications. Early diagnosis and appropriate treatment could thereby delay their onset or progression.[30] Several measurements of platelet activity have emerged as potential contributors to atherothrombosis. Many of the parameters are timeconsuming, expensive, use a high sample volume, or require specialty training.[35,36] MPV is an easily available, economical, clinical marker of platelet reactivity done by routine automated hemograms. A small sample size and subjects were known diabetics on treatment may be the two limitations of this study. This study could not establish a causal relationship between MPV and vascular complications in diabetes but it supports a link between platelet activity, glucometabolic state and glycemic control.

A-571 edition. Lippincott Williams and Wiklins: Philadelphia, London, New York, Tokyo, Hong Kong, 2000; 220. 2. UK Prospective Diabetes Study Group (UKPDS): Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). Lancet. 1998; 352:837 –853. 3. Vizioli L, Muscari S, Muscari A: The relationship of mean platelet volume with the risk and prognosis of cardiovascular diseases. Int J Clin Pract. 2009; 63:1509–1515. 4. Tschoepe D, Roesen P, Schwippert B, Gries FA. “Platelets in diabetes: the role in the hemostatic regulation in atherosclerosis,” Seminars in Thrombosis and Hemostasis.1993; 19: 122–128. 5. Khuwaja AK, Rafique G, White F, Azam SI. Macrovascular complications and their associated factors among persons with type 2 diabetes in Karachi, Pakistan--a multi-center study. J Pak Med Assoc. 2004; 54:60-6. 6. Tufano A, Cimino E, Di Minno M.N.D, et al. “Diabetes Mellitus and Cardiovascular Prevention: The Role and the Limitations of Currently Available Antiplatelet Drugs.” International Journal of Vascular Medicine. vol. 2011; Article ID 250518: 1-5.

Conclusion

7. King H, Aubert RE, Herma WH. Global burden of diabetes,1995-2025: prevalence, numerical estimates, and projections. Diabetes Care 1998; 21:1414-31.

Acknowledgement

9. Zuberi BF, Akhtar N, Afsar S. Comparison of mean platelet volume in patients with diabetes mellitus, impaired fasting glucose and non-diabetic subjects. Singapore Med J 2008; 49: 114-116

This study shows significantly high MPV in diabetics as compared to controls thus establishing that MPV is strongly and independently associated with diabetes. Glycemic control plays a role in the reactivity of platelets and thus MPV can be used as a simple cost effective tool for monitoring diabetic patients.

8. Hekimsoy Z, Payzin B, Ornek T, Kandoğan G. Mean platelet volume in Type 2 diabetic patients. J Diabetes Complications 2004; 18:173-6.

We would like to express our thanks for the valuable help and support provided by the department of community medicine in particular Ms Jayshree Tolani for the statistical evaluation.

10. Kodiatte TA, Manikyam UK, Rao SB, Jagadish TM, Reddy M, Lingaiah HK and Lakshmaiah V. Mean platelet volume in Type 2 diabetes mellitus. J Lab Physicians 2012; 4: 5-9.

Funding

11. Ulutas KT, Dokuyucu R, Sefil F, Yengil E et al. Evaluation of mean platelet volume in patients with type 2 diabetes mellitus and blood glucose regulation: a marker for atherosclerosis?. Int J Clin Exp Med 2014;7(4):955-961

None

Competing Interests None Declared

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systematic review and meta-analysis. J Thromb Haemost 2010; 8: 148-156. 26. Jones R L, Paradise C, Peterson C M. Platelet survival in patients with diabetes mellitus. Diabetes 1981;30:486-489. 27. Jabeen F, Rizvi HA, Aziz F, Akhtar Y. Effect of Glycemic Control on Lipid Profile, Platelet Indices and Antioxidant Enzymes (Catalase and Superoxide dismutase) Activities in Type 2 Diabetics. International Journal of Advanced Research 2013; 1: 207-215. 28. Vijaya C, Shetty A, Parikshith. Comparative Study of Significance of Lipid Profile, Platelet Count and MPVDiabetics and Non-Diabetics. Sch. J. App. Med. Sci. 2014; 2(5B):1584-1588 29. Alhadas KR, Santos SN, S. Freitas MM, Viana SMAA, Ribeiro LC, Mônica B. Costa MB. Are platelet indices useful in the evaluation of type 2 diabetic patients? Bras Patol Med Lab, April 2016 ;52(2): 96-102. 30. Kadić D, Hasić S, Spahić E. Mean platelet volume predicts the glycemic control deterioration in diabetes mellitus type 2 patients. Medicinski Glasnik 2016; 13(1): 1-7. 31. Altamer E, Vendemisle G, Chicco D; Increased lipid peroxidation in Type-II poorly control Diabetic patients. Diabetes Etab., 1991; 18(4):264-671. 32. Jain AP, Gupta DP; Study of blood lipids in Diabetics without any manifest vascular complications. J Dia Asso Ind., 1980; 20: 29-34. 33. Suckling KE., Brian J; Animal Models of Human lipid Metabolism. Prog Lipid Res.1993; 32(1): 124–124. 34. Stratton IM, Adler AI, Neil HA, Matthews DR, Manley SE, Cull CA et al. Association of glycaemia with macrovascular and microvascular complications of type 2 diabetes (UKPDS 35): prospective observational study. BMJ 2000; 321: 405−12. 35. Michelson AD; Methods for the measurement of platelet function. Am J Cardiol., 2009;103(3Suppl.): 20A–26A. 36. Nicholson NS, Panzer-Knodle SG, Haas NF, Taite BB, Szalony JA, Page JD et al.; Assessment of platelet function assays. Am Heart J., 1998;135(5 Pt 2 Su):S170–S178.

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Original Article Evaluation of Fine Needle Aspiration Cytology in the Diagnosis of Suspicious/gray Zone Lesions in Breast Lesions and its Histopathological Correlation Manas Madan*, Manisha Sharma, Sanjay Piplani, Mridu Manjari, Neha Sharma and Saloni Goyal Dept. of Pathology, SGRDIMSR, Amritsar, Punjab, India Keywords: FNAC, Malignant, Breast, Histopathology

ABSTRACT Introduction: Fine needle aspiration cytology (FNAC) is an established tool in the diagnosis of palpable lesions. FNAC is a sensitive and rapid method in differentiating benign breast lesions from malignant ones. But there exist some gray areas in which this differentiation becomes difficult. Problem arises in categories C3 and C4 in which there exists significant interobserver variation in the diagnosis. The study was done to evaluate the usefulness of FNAC in diagnosis of C3 and C4 categories and correlate it with histopathological diagnosis. Methods: This Study was conducted on 512 cases of breast FNAC in from January 2014 to December 2014. FNAC diagnosis of C3 and C4 categories were selected. The cytological diagnosis was compared with histopathological diagnosis in the cases where biopsy was done subsequent to FNAC diagnosis. Results: C3 and C4 categories constituted 28 (5.46%) and 48 (9.3%) cases respectively. Histopathology was available in 13 cases of C3 (46.4%) and 30 cases of C4 (62.5%). Among C3 category, 10/13 cases showed benign lesions (77%) and 3/13 cases were malignant (23%). Among C4 category, 4/30 cases showed benign lesion (13.3%) and 26/30 cases were malignant (86.7%). There was a significant statistical difference between benign and malignant diagnosis of C3 & C4 categories (p< 0.001). Conclusion: Clinicians and pathologists should understand the limitations of FNAC. C3 and C4 categories should still be continued with, as there was a statistically significant difference in benign & malignant diagnosis for these categories in our study.

*Corresponding author: Dr Manas Madan, 21 A, Sandhya Enclave Majitha Road Amritsar (143001), Punjab, India Phone: +91 9888015365 Email: manasmadaan@gmail.com

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-574

FNAC in Suspicious Breast Lesions

Introduction

Fine needle aspiration cytology (FNAC) is an established tool in the diagnosis of various palpable lesions and it correlates well with histopathological diagnosis in most of the cases. The major utility of FNAC is in differentiating benign and malignant lesions of various tissues. Breast is one of the organs, which is routinely subjected to FNAC to diagnose malignant lesions. Breast carcinoma is the second commonest cancer among Indian females after carcinoma cervix and FNAC is a very cost-effective, sensitive and rapid diagnostic method in differentiating benign breast lesions from malignant ones. [1,2] But there exist some gray areas in breast lesions in which this differentiation becomes difficult. Although needle core biopsy (CNB) is now being preferred over FNAC, FNAC still has a lot to offer as a first line diagnostic procedure, particularly in developing countries with economic restrictions. Moreover FNAC remains almost as accurate as CNB in determination of malignancies. [1,2,3] Breast FNAC diagnosis are characterized in to inadequate (C1), benign (C2), atypical, probably benign (C3), suspicious, favor malignancy (C4) and malignant (C5). These were recommended by the National Cancer Institute (NCI) to bring about uniformity in diagnosis of breast FNAC in a way similar to the Bethesda classification for reporting of thyroid cytology. Among these categories, C1, C2 and C5 do not pose much of a difficulty to the pathologists and leads to a good interobserver comparability. The shortcomings in FNAC are to differentiate invasive from in situ cancers and the presence of gray zone categories where definite diagnosis of benign or malignant is difficult. This problem arises in categories C3 and C4 in which there exists significant interobserver variation in the diagnosis, as no strict criteria are present for the diagnosis of these categories. [1,2,3 4] Some authors have suggested the use of term â&#x20AC;&#x153;equivocalâ&#x20AC;? for such inconclusive diagnosis (C3 & C4) on FNAC. [5] The present study was done in order to evaluate the usefulness of FNAC in diagnosis of C3 and C4 categories and to correlate it with histopathological diagnosis.

Materials and Methods:

The study was a retrospective one conducted in the department of pathology SGRDIMSR, Amritsar from January 2014 to December 2014 after taking the necessary approval from the institution. Slides of the entire breast FNAC conducted during the above period were surveyed and those fitting in to C3 and C4 categories were selected for study. The cytological diagnosis was compared with histopathological diagnosis in the cases where biopsy was done subsequent to FNAC diagnosis. The statistical

significance of benign and malignant lesions in both these categories was calculated.

Results

A total of 512 breast FNAC were done in the abovementioned period. The age of the patients ranged from 16 to 87 years. The commonest age group was 21-30 years. Out of these, C3 and C4 categories constituted 28 (5.46%) and 48 (9.3%) cases respectively. All patients belonging to these two categories were females. Among these categories, the age ranged from 29-61 years. Histopathological diagnosis was available in 13 cases of C3 (46.4%) and 30 cases of C4 (62.5%). All the malignant cases in both these categories were infiltrating ductal carcinomas (29 cases). Benign histological diagnosis between both categories included fibroadenoma (07 cases), fibrocystic disease (03 cases) and proliferative breast disease (04 cases). Among C3 category, 10/13 cases in which histopathological examination was available showed benign lesions (77%) and 3/13 cases were malignant (23%). Among C4 category, 4/30 cases available for histopathological examination showed benign lesion (13.3%) and 26/30 cases turned out to be malignant (86.7%) (TABLE1). There was a s4gnificant statistical difference between benign and malignant diagnosis of C3 & C4 categories (p< 0.001). TABLE 1: CYTO-Histological Correlation. HISTOPATHOLOGICAL DIAGNOSIS

FNAC C3

FNAC C4

TOTAL

BENIGN

MALIGNANT

TOTAL

Discussion Breast FNAC is a widely accepted safe, rapid and an effective diagnostic modality for diagnosis o breast lesions especially malignancy. Out of the various diagnostic categories, C3 and C4 pose challenges to the pathologists and are also known as gray zone/indeterminate where a definite diagnosis of benign or malignant is difficult. [1,2,3,4,5] An interpretation of C3 is given when the aspirates show benign characteristics but have some features not present usually in benign aspirates. These include any or a combination of nuclear pleomorphism, loss of cell cohesion, nucleocytoplasmic changes resulting from treatment/hormonal influences and increased cellularity. C4 category diagnosis is given when the aspirates have cells with features of malignancy however the material is not very cellular to be diagnostic, poorly preserved or spread. These also include samples showing malignant

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 06, (Suppl) December, 2016

Madan et al. features of a greater degree than seen in C3 without the presence of overtly malignant cells. [2,4,5,6] In this study, the age ranged from 29-61 years for both the above-mentioned categories and is comparable to many other studies conducted. [1,2] C3 and C4 categories combined constituted 76/512 (14.8%) cases subjected to breast FNAC during the study period. This percentage of C3 and C4 categories correlated well with various other studies, which give a range of 4-17.7% for both, these categories combined. [1,2,4,5,7,8] This is essential because there should not be erroneous overuse of these categories in reporting of breast FNAC. In C3 category, where 13/28 cases were available for histopathological examination, 3 cases turned out to be (IDC) infiltrating duct carcinomas (3/13= 23%) and thus were considered false negative (FN). [Fig 1] This result also corroborated well with the range established by other studies (8.6-52%). [1,2,5,7,9] The reasons for these false negative cases can be sampling error, small tumor size. Low-grade tumor, less cellularity or low grade well differentiated carcinomas arising in cystic lesions. These 3 FN cases were again reviewed after histopathological diagnosis. All of these cases showed mainly cohesive sheets of ductal epithelial cells, bare nuclei with few clusters showing cellular crowding and lack of cohesiveness. Thus patients with C3 diagnosis need not undergo a surgical procedure if the proper clinical and mammographic correlation is done and they too suggest a benign lesion. In C4 category, 26/30 cases (86.7%) available for histopathological examination showed malignant

A-575 pathology (IDC). 4/30 cases (13.3%) showed benign pathology on histopathological examination and were considered false positive (FP). [Fig 2a,2b] Of these 2 were highly cellular fibroadenomas and 2 were proliferative breast disease. These cases showed dyscohesive clusters, cellular overlapping and moderate cellular and nuclear pleomorphism. These results also correlate with the other studies, which show a range of 81-97% for malignancies in this category. [1,7,9,10,11,12,13] Some degree of atypia, dyscohesion and nuclear pleomorphism can be seen in fibroadenomas and along with increased cellularity can cause diagnostic difficulty. Most of these cases are conventional fibroadenoma although a few may be associated with proliferative lesions especially when atypical changes are present. Proliferative breast lesion is another gray zone lesion and can be called as the nightmare of the pathologistsâ&#x20AC;&#x2122; especially radial scar and complex sclerosing lesions. These lesions can be hypercellular with dyscohesive cell clusters, atypia and absence of myoepithelial cells in few clusters. Thus all the patients with cytological diagnosis of C4 lesion on breast FNAC should undergo surgery, as the percentage of malignancies in these lesions is very high. Thus to conclude, FNAC of breast is a simple, safe, rapid and inexpensive diagnostic modality and plays a very important role in diagnosis of breast lesions especially malignancies. However it is important that clinicians understand the limitations of FNAC. C3 and C4 categories should still be continued with, as there was a statistically significant difference in benign & malignant diagnosis for these categories in our study.

Fig. 1: FNAC diagnosis C3 showing clusters of ductal epithelial cells showing mild overlapping and pleomorphism along with few bare nuclei in the background (MGG 100X). Inset showing histopathology of the same case with features of infiltrating ductal carcinoma (H&E 100X).

Fig. 2 (a): FNAC diagnosis C4 showing ductal epithelial cells arranged in groups (MGG 100X), inset showing overlapping cells exhibiting pleomorphism (MGG 400X). Note the lack of bare nuclei in background.

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FNAC in Suspicious Breast Lesions 4. Mitra S, Dey P. Grey zone lesions of breast: potential areas of error in cytology. J Cytol. 2015;32:145-52. 5. Goyal P, Sehgal S, Ghosh S, Aggarwal D, Shukla P, Kumar A et al. Histopathological correlation of Atypica; (C3) and Suspicious (C4) categories in Fine Needle Aspiration Cytology of the breast. Int J Breast Cancer. 2013;2013: 965498. 6. Yu YH, Wei W, Liu JL. Diagnostic value of fine-needle aspiration biopsy for breast mass: a systematic review and meta-analysis. BMC Cancer. 2012;25:12-41.

Fig 2. (b): Histopathology of same case exhibiting fibrocystic disease with epithelial hyperplasia (H&E 100X), inset showing high power of hyperplastic epithelium (H&E 400X).

Funding None

Competing Interests None Declared

References

1. Arul P, Masilamani S, Akshatha C. Fine needle aspiration cytology of atypical (C3) and suspicious (C4) categories in the breast and its histopathologic correlation. J Cytol. 2016;33:76-9. 2. Shabb NS, Boulos FI, Abdul-Karim FW. Indeterminate and erroneous fine-needle aspirates of breast with focus on the ‘true gray zone’: a review. Acta Cytol. 2013;57(4):316-31. 3. Chaiwun B, Sukhamwang N, Lekawanvijit S, Sukapan K, Rangdaeng S, Muttarak M et al. Atypical and suspicious categories in fine needle aspiration cytology of the breast: histological and mammographical correlation and clinical significance. Singapore Med J. 2005;46(12):706-9.

7. Kanhough R, Jorda M, Gomez-Fernandez C, Wang H, Mirzabeigi M, Ghorab Z, et al. Atypical and suspicious diagnoses in breast aspiration cytology-is there a need for two categories? Cancer. 2004;102:164-7. 8. Howell LP. Equivocal diagnoses in breast aspiration biopsy cytology: Sources of uncertainty and the role of “atypical/indeterminate”  terminology. Diagn Cytopathol. 1999;21:217-22. 9. Deb RA, Matthews P, Elston CW, Ellis IO, Pinder SE. An audit of “equivocal” (C3) and “suspicious” (C4) categories in fine needle aspiration cytology of the breast. Cytopathology. 2001;12:219-26. 10. Lim JC, Al-Masri H, Salhadar A, Xie HB, Gabram S, Wojcik EM. The significance of the diagnosis of atypia in breast fine-needle aspiration. Diagn Cytopathol. 2004;31:285-8. 11. Yusuf I, Atanda AT, Imam MI. Cyto-morphologic correlationof equivocal C3 and C4 breast lesions. Arch Int Surg. 2014;4:131-5. 12. Tikku G, UMAP P. Comparative Study of Core Needle Biopsy and Fine Needle Aspiration Cytology in Palpable Breast Lumps: Scenario in Developing Nations. Turk Patoloji Derg. 2016;32:1-7. 13. Omi Y, Yamamoyo T, Okamoto T, Nishikawa T, Shibata N. Fine needle aspiration versus core needle biopsy in the diagnosis of the intraductal breast papillary lesions. World Journal of Pathology. 2013;2:11.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 06, (Suppl) December, 2016

Original Article Comparative Study of Fine Needle Non-Aspiration and Fine Needle Aspiration in Cytological Diagnosis of Thyroid Lesions Manisha Sharma*, Mridu Manjari, Manas Madan, Taranveer Singh, Akanksha Singh and Tarunpreet Saini Dept of Pathology, SGRDIMSAR Amritsar, Punjab, India Keywords: Thyroid, FNAC, FNNAC, Cytology

ABSTRACT Background: Fine needle aspiration (FNA) has been proven to be an important, simple, safe and first line investigation in evaluation of various thyroid lesions. But its efficacy is limited by presence of significant amount of blood in smears diluting the cellular yield. An alternate method fine needle non aspiration cytology (FNNAC) was developed which is less painful, easy to perform and shows better patient compliance. The present study was undertaken to compare the efficacy and quality of FNNAC with FNAC to determine whether it is a superior and the procedure of choice in vascular organs like thyroid. Methods: The study was done on 120 patients attending the cytology section in department of Pathology SGRDIMSAR, Amritsar. All of these patients had undergone FNAC and FNNAC simultaneously by single operator. The smears were scored using five parameters- background blood or clot, amount of cellular material, degree of cellular degeneration, degree of cellular trauma and retention of appropriate architecture by the pathologist without the prior knowledge of technique used. All the results were tabulated and were statistically interpreted using student t test. Results: Significant higher score favoured FNNAC for background blood (P<0.001), for cellular material and retention of architecture (P<0.012) over FNAC. For degree of cellular trauma and degeneration of material insignificant higher scores were observed. Total and average score favoured FNNAC over FNAC (P<0.001). Though FNAC yielded more diagnostically adequate samples but diagnostically superior case were higher in FNNAC smears. Conclusion: It was concluded that FNNAC offers better quality smears. FNAC yielded more adequate samples. Both the techniques are complimentary to each other and diagnostic accuracy can be enhanced by combining both the techniques.

*Corresponding author: Dr Manisha Sharma, B-241, Ranjit Avenue, Amritsar, Punjab, India Phone: +91 9876842942 Email: manisha_salwan@yahoo.com

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

A-578

FNNAC vs FNAC in Thyroid Lesions

Introduction

Fine needle aspiration (FNA) has been proven to be an important, simple, safe and first line investigation in evaluation of various thyroid lesions. FNAC along with clinical evaluation and imaging by ultrasound is the mainstay in diagnosis of thyroid lesions. But its efficacy is limited by presence of significant amount of blood in smears in aspiration of vascular organs like thyroid therefore decreasing the cellular yield and limiting the diagnostic efficacy of the technique. An alternate method known by many names like non-aspiration cytology, fine needle capillary sampling (FNCS), cytopuncture and fine needle non-aspiration cytology (FNNAC) was developed in France in 1982.[1] and was applied in thyroid lesions in 1988.[2] This technique is less painful, easy to perform and shows better patient compliance. But its usefulness and efficacy as compared to FNAC needs to be validated. The present study was undertaken to compare the efficacy and quality of FNNAC with FNAC to determine whether it is a superior and the procedure of choice in vascular organs like thyroid.

Materials and Method

The study population consisted of 120 patients attending the cytology section in department of Pathology, Sri Guru Ram Das Institute of Medical Sciences and Research (SGRDIMSAR), Amritsar. All of these patients had undergone FNAC and FNNAC simultaneously by single operator. Each slide was evaluated without the prior knowledge of technique used. Thus making the study single blinded to prevent the observer bias. FNAC was done with 22 gauge needle and 10 cc of plastic syringe. FNNAC was done with 22 gauge needle only by holding it in finger tips and moving it back and forth in various directions at different depths. The material was expelled on the slide with the help of air filled syringe. Smears were air dried for May Grunwald Giemsa stain and fixed in 95% ethyl alcohol for Papanicolaou staining.

The smears were analysed using a scoring system developed by Mair et al.[3] Five parameters used for scoring were background blood or clot, amount of cellular material, degree of cellular degeneration, degree of cellular trauma and retention of appropriate architecture ( Table 1). A cumulative score was obtained and were categorized into 3 categories 1. Unsuitable for cytodiagnosis ( Score 0-2 ) 2. Adequate for cytological diagnosis ( Score 3-6 ) 3. Diagnostically superior (Score 7-10) All the results were tabulated and were statistically interpreted using student t test .

Results

Five parameters studied objectively in all 120 cases. It was noted that background blood was more in aspiration smears than non aspiration smears with statistical significant difference (p <0.001). Similarly amount of cellular material and retention of architecture favoured FNNAC by a significant difference of P<0.012. On comparison for the degree of cellular degeneration and degree of cellular trauma it was observed that scores were higher in non aspiration smears than aspiration ones but this difference was not statistically significant. On analysing the total and average score for each technique it was observed that non - aspiration was significantly superior than aspiration (P< 0.001) ( Table 2). On comparison for the performance of both the techniques, it was seen that diagnostically superior cases were more in FNNAC- 65 cases (54.2%) vs. 40 cases (33.3%) of FNAC. But FNAC yielded more diagnostically adequate cases- 60 cases (50%) as compared to 48 cases (40%) of non- aspiration. It was observed that 20 cases (16.7 %) were diagnostically inadequate by FNAC as compared to 7 cases (5.8%) of FNNAC (Table 3). Frequency of various thyroid lesion was as per given in (Table 4).

Table 1: Methodology of a Point Allocation. S.No Criterion Quantitative Description Large amount; great compromise in diagnosis. Background blood or 1 Moderate amount; diagnosis possible clot Minimal; diagnosis easy, specimen of textbook quality Minimal to absent; diagnosis not possible Amount of cellular 2 Moderate; Sufficient for cytodiagnosis material Abundant; diagnosis easy Marked; diagnosis impossible Degree of cellular 3 Moderate; diagnosis possible degeneration Minimal;good preservation; diagnosis simple Marked; diagnosis impossible Degree of cellular 4 Moderate; diagnosis possible trauma Minimal; diagnosis easy

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 06, (Suppl) December, 2016

Point score 0 1 2 0 1 2 0 1 2 0 1 2

Sharma et al. S.No

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Criterion Retention of appropriate architecture

Quantitative Description Point score Minimal to absent; diagnosis not possible 0 Moderate; some preservation of e.g Follicle, papillae, acini, flat sheets, 1 syncytia or single cell patterns. Excellent architecture display closely reflecting histology; diagnosis obvious 2

Table 2: Comparison of FNAC and FNNAC for Various Parameters. S.No Criterion Mean FNAC + SD 1. Background Blood/clot 0.62 + 0.490

Total 72

Mean FNNAC + SD 1.69 + 0.482

Total 192

P value <0.001

2. 3.

Amount of cellular material Degree of cellular degeneration

1.15+ 0.389 0.92 + 0.462

132 98

1.74 + 0.632 1.08+0.478

179 120

<0.012 <0.08

4. 5.

Degree of cellular trauma Retention of appropriate architecture

0.95 + 0.412 1.07 + 0.6480

125 110

1.13+ 0.518 1.65 + 0.5431

142 162

<0.08 <0.012

4.71+ 2.401

537

7.29+ 2.653

795

<0.001

Total Score

Table 3: The Performance of FNAC and FNNAC Technique in Thyroid Lesions. S. No 1 2 3

Technique

Performance Diagnostically inadequate Diagnostically adequate Diagnostically superior

Table 4: Frequency of Various Thyroid Lesions. S No Type of lesion 1 Colloid goitre 2 Adenomatous goitre 3 Thyroiditis 4 Follicular neoplasm 5 Papillary carcinoma 6 Medullary carcinoma Total

FNAC FNNAC 20(16.7%) 60(50%) 40(33.3%)

7(5.8%) 48(40%) 65(54.2%)

No of cases 30 35 24 22 8 1 120

Percentage 25% 29.2% 20% 18.4% 6.6% 0.8% 100

Fig. 1 (A): FNNAC smear of Adenomatous goite showing high cellularity and minimal blood; (B) : FNAC smear of Adenomatous goite showing less cellularity and blood contamination; (C): FNNAC smear of Throiditis showing high cellularity and minimal blood; (D): FNAC smear of Thyroiditis showing less cellularity and blood contamination (MGG,100X).

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FNNAC vs FNAC in Thyroid Lesions

Fig:. 2(A): FNNAC smear of Follicular Neoplasm showing high cellularity and minimal blood; (B) : FNAC smear of Follicular Neoplasm showing less cellularity and blood contamination; (C): FNNAC smear of Papillary Carcinoma Thyroid showing high cellularity and minimal blood; (D): FNAC smear of Papillary Carcinoma Thyroid showing less cellularity and blood contamination (MGG,400X).

Discussion

Fine needle aspiration of thyroid has been proved to be widely accepted, accurate, simple, safe and cost effective technique in evaluation of thyroid lesions.[4] But the common challenge remains the haemorrhage material in the smears diluting the cellularity specially in a vascular organ like thyroid.[5] To overcome this, modified technique FNNAC came into the picture obviating the need of suction as the material yield depends upon the capillary action in narrow channel. The fluid and semifluids ascends in inverse proportion of the diameter of capillary.[2] FNNAC has its advantage of being easily operable with better control over the procedure due to good perception of the lesion. Patients tolerate the procedure as it is less traumatic and less painful.[6] On comparison for blood contamination between two techniques, it significantly favoured non aspiration (P<0.001). This was in accordance with the results of various previous studies conducted.[7,8] However some of the researchers did not notice significant difference between the two techniques for background blood and clot.[9] Similarly cellularity and retention of architecture in the smears of FNNAC was significantly better than FNAC (P<0.012) corroborating the findings of the other studies. [7,8] Akhtar et al and other researchers observed the high cellular yield and retention of architecture in non aspiration samples though the difference was not statistically significant.[9,10] Some other studies revealed higher yield in FNAC smears than non aspiration.[11]

Lesser degree of cellular trauma and lesser degree of cellular degeneration was seen by non aspiration technique; however the difference was statically insignificant. Similar results had been observed by the workers in previous studies. [12,13] Total and average score was significantly higher for non aspiration as compared to FNAC (<0.001) which was in agreement with the findings of previous studies.[14,15] On comparing the performance of both the techniques, it was observed that FNNAC yielded more diagnostic superior cases than FNAC (65 cases vs 40 cases) and diagnostically adequate cases were more frequent in FNAC as compared to FNNAC (60 cases vs 48 cases). Similar findings had been observed in other studies.[16,17] Diagnostically inadequate cases were 16.7% (20 cases) in FNAC while this percentage was only 5.8% (7 cases) in FNNAC agreeing the results obtained by Rizvi et al and Ciatto el al.[16,18] Dilution of the cellularity with the blood is the cause of higher inadequacy rate in FNAC as it involves high suction pressure filling the needle with the blood leading to early withdrawal of the needle. So, less of time is available to manipulate the needle leading to less cellular yield. On the contrary in FNNAC no suction is applied and spontaneous ascent of the material with capillary action is allowed thus yielding high cellularity with less blood contamination. Specially in thyroid malignancies dilution of the cellularity poses more of the difficulty as these tumours are highly vascular with increased fragility of the malignant cells. FNNAC offers less contamination

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 06, (Suppl) December, 2016

Sharma et al. and better preservation of cellularity in such cases. An adequate material in the smears is the mainstay of accurate diagnosis and subsequent treatment of the patient.

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1. Zajdela A, Zillhardt T, Voillemet N. Cytological diagnosis by fine needle sampling without aspiration. Cancer. 1987;59(6):1201-5. 2. Santos JE, Leiman G. Non aspiration fine needle cytology: Application of a new technique to nodular thyroid diseases. Acta Cytol. 1988;32(3):353-6. 3. Mair S, Dunbar F, Becker PJ, Du Plessis W. Fine needle cytology - is aspiration suction necessary? A study of 100 masses in various sites. Acta cytol. 1989;33(6):809-13. 4. Hamberger B, Gharib H, Melton Lj. Fine needle aspiration of thyroid nodules : impact on thyroid practice and cost of care. Am j Med.1982;73:381-4. 5. Santos JEC, Leiman G. Non aspiration fine needle cytology : Application of a new technique to nodular thyroid diseases. Acta Cytol. 1988;32:353-6.

6. Meherbano MK, Arjune DG, Kulkarni HR. Comparative study of fine needle aspiration and fine needle capillary sampling of thyroid lesions. Acta cytol. 2000;46:30-4. 7. Pinki P, Alok D, Ranjan A, Chand MN. Fine needle aspiration cytology versus fine needle capillary sampling in cytological diagnosis of thyroid lesions. Iranian J Pathol. 2015;10(1):47-53. 8. Ghosh A, Misra RK, Sharma SP, Singh HN, Chaturvedi AK. Aspiration vs nonaspiration technique of cytodiagnosis- A critical evaluation in 160 cases. Indian J Pathol Microbiol. 2000;43(2):107-12. 9. Akhtar SS, Huq IU, Faiz-U-Din M, Reyes LM. Efficacy of fine needle capillary biopsy in the assessment of patients with superficial lymphadenopathy. Cancer cytopathol. 1997;81(5):277-80. 10. Maurya AK, Mehta A, Mani NS, Nijhawan VS, Batra R. Comparison of aspiration vs non aspiration techniques in fine needle cytology of thyroid lesions. J cytol. 2010;27(2):51-4 11. Kamal MM, Arjune DG, Kulkarni HR. Comparative study of fine needle aspiration and fine needle capillary sampling of thyroid lesions. Acta Cytol. 2002;46(1):30-4. 12. Raghuveer CV, Leekha I, Pai MR, Adhikari P. Fine needle aspiration cytology versus fine needle sampling without aspiration. A prospective study of 200 cases. Indian J Med Sci. 2002;56(9):431-9. 13. Pontheir DD, Narula AA. Should we apply suction during needle cytology of thyroid lesions? A systematic review and meta-analysis. Ann R Coll Surg Engl. 2006;88(7):643-5. 14. Dey P, Shashirekha, Ray R. Fine needle sampling without suction in intra-abdominal lesion. Acta Cytol. 1994;38:495-7. 15. Romitelli F, Di Stasio E, Santoro C, Iozzino M, Orsini A, Cesareo R. A comparative study of fine needle aspiration and fine needle non aspiration biopsy on suspected thyroid nodules. Endocr Pathol. 2009;20:108-13. 16. Rizvi SA, Husain M, Khan S, Mohsin M. A comparative study of fine needle aspiration cytology versus non aspiration technique in thyroid lesions. Surgeon. 2005;3(4):273-6. 17. Mahajan P, Sharma PR. Fine needle aspiration versus non- aspiration technique in cytodiagnosis in thyroid lesions. JK science. 2010;12(3):120-2. 18. Ciatto S, Lossa A, Cicchi P. Non aspiration fine needle cytology of thyroid tumors. Acta Cytol. 1983;33(5):939.

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Though on ultimate analysis for total and average score for each criteria for both techniques FNNAC scored significantly better than FNAC (P<0.001), diagnostically adequate cases were more frequent in FNAC as compared to FNNAC.

Conclusion

It was concluded that FNNAC offers better quality smears with less of contamination with blood, high cellular yield and better preservation of the architecture and FNAC yields more of diagnostically adequate smears. FNNAC has extra advantage of easy operability, better control over the procedure with more perception of the swelling. It also offers better patient compliance of being less traumatic and less intimidating by innocuous needle, syringe and handle. So both the techniques have their own advantages and disadvantages. Both the techniques are supplementary to each other and can be substitutive in some cases. Though this study proves more efficacy and quality superiority for FNNAC over FNAC, further studies are required with interobserver comparison using two evaluators for the study to have the better insight of role and efficacy of both the techniques. We recommend that both the techniques are complimentary to each other and diagnostic accuracy can be enhanced by combining both the techniques.

Funding None

Competing Interests None Declared

References

Original Article Cytological-Radiological Correlation of Image Guided FNAC of Hepatic Mass Lesions: Our Experience In Tertiary Care Centre Menka Khanna1*, Kiranjot Kaur1, Manisha Sharma1, Ashish Khanna2, Mridu Manjari1, Saumil Garg1 and Amritpal kaur1 Department of Pathology, SGRDIMSAR, Amritsar, Punjab, India Department of Microbiology, SGRDIMSAR, Amritsar, Punjab India 1

Keywords: Hepatocellular Carcinoma, FNAC, Image Guided, Metastasis

ABSTRACT Background: Fine needle aspiration cytology is of latest and extensively utilized in the diagnosis of lesions aided by image guidance, it is fast becoming an initial investigation of choice for various hepatic lesions. It is safe, less traumatic, quick and cost effective tool. The present study was undertaken to know the pathological spectrum of hepatic mass lesions and to correlate the cytoradiological findings. Methods: The present study was conducted in a tertiary care centre of North India on 169 patients with hepatic lesions over a period of two years. Image guided FNAC was carried out and aspirates were examined, and compared with radiological diagnoses. Results: Metastatic malignancies were more frequent (58%) than hepatocellular carcinoma (HCC). By cytology, the most common metastases was of adenocarcinoma (49.1%) followed by squamous cell carcinoma, small cell carcinoma and poorly differentiated carcinoma, malignant melanoma and lymphoma. HCV was seen to be associated with hepatocellular carcinoma in 31.3% cases. Multiple SOLs was more common finding in radiology for HCC than single SOL. Cytoradiological correlation was found to be 92% in the present study. Conclusion: Present study thus concludes that image guided FNAC of hepatic lesions is a simple, safe, economically prudent technique associated with low morbidity and leading to quick and early diagnosis.

*Corresponding author: Dr. Menka Khanna, 538, Basant Avenue, Amritsar-143001, Punjab, India Phone: +91 9464986355 Email: mona74_khanna@yahoo.co.in

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Khanna et al.

Introduction

Liver is involved in many non-neoplastic and neoplastic diseases. The diagnosis of various hepatic mass lesions is a common clinical problem and their appropriate management depends on accurate diagnosis. [1]Despite recent improvement, radiological imaging does not always allow exact characterization of the lesions. Serological markers like alpha fetoprotein can be useful in narrowing the differential diagnosis when their levels are markedly elevated but a substantial number of patients unfortunately do not have high levels of these markers at the time of presentation. Therefore, a cytological diagnosis is often required to guide subsequent management. Fine needle aspiration cytology under image guidance has gained increasing acceptance as the diagnostic procedure of choice for patients with focal hepatic lesions. [2]

A-583 looking hepatocytes along with blood and few inflammatory cells were seen. Majority of the patients were in the age group between 4th to 7th decade (83.7%) with the age ranging from 22 to 87 years. No significant male or female preponderance was noted in this study as the M: F ratio of 1 : 1.13. (Table 1) Non neoplastic lesions comprised minority of cases (9.6%) and included pyogenic abscess, fatty change , cirrhotic nodule and granulomatous hepatitis. The smears from pyogenic liver abscess predominantly showed neutrophils, necrotic cells and debris. The smears from the granulomatous hepatitis showed epitheloid cell collection with Langhanâ&#x20AC;&#x2122;s type multinucleated giant cell. (Table 2)

The diagnostic yield of image guided FNAC liver in our study was 96.4% , as the material was inadequate for interpretation in six cases (3.6%), where only benign

Malignancy was the most common cytological diagnosis (88.7%). Metastatic tumors were the most common (58%) among the malignant liver lesions. Among the metastatic lesions, the most common was adenocarcinoma (49.1%), followed by squamous cell carcinoma (1.2%) small cell carcinoma (0.6%) one case each of malignant melanoma and lymphoma. The smears from the adenocarcinoma revealed hypercellularity with columnar to cuboidal cells arranged in monolayered sheets, palisade forms, acinar pattern and in singles having vacuolated or granular and eosinophilic cytoplasm.(fig.1) The cells showed altered N:C ratio, anisonucleosis with central or eccentrically placed nucleus and fine-coarse dispersed chromatin. Many showed benign hepatocytes in the background. Most of the metastatic adenocarcinoma were from gall bladder, followed by colon, ovary and breast. In metastatic squamous cell carcinoma, smears showed squamoid, tadpole-like and spindle-shaped cells with well-defined abundant keratinized cytoplasm and pleomorphic and hyperchromatic nuclei.(fig.2) Smears from small cell metastatic deposits showed mainly small monomorphic cells with finely granular nuclear chromatin, inconspicuous or absent nucleoli and scanty cytoplasm. Nuclear moulding and streaking were also noted. (fig 2) Metastatic melanoma showed large tumor cells with abundant cytoplasm, pleomorphic nuclei and prominent nucleoli, intranuclear cytoplasmic inclusions and melanin granules in the cytoplasm. Smears from metastatic non Hodgkin lymphoma showed dispersed monotonous cells with granular nuclear chromatin and scanty cytoplasm with presence of lymphoglandular bodies in the background. These diagnoses were made in correlation with clinical, radiological evidences of primary lesion. (Table 2).The commonest primary hepatic lesion was hepatocellular carcinoma (HCC) 51 (30.2%). Hepatocellular carcinoma was further differentiated into three grades, grade I (well differentiated) were 25% cases grade II (moderately differentiated) were 56.3% and grade III (poorly

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Material and Methods

The aim of present study is to know the pathological spectrum of hepatic mass lesions and to correlate the radiological findings and cytological findings obtained in image guided FNAC. One hundred sixty nine patients with clinical, biochemical and radiological evidence of liver diseases and/or space occupying lesion (SOL) with normal prothrombin time were subjected to image guided FNAC over a period two years from January 2014 to December 2015. Ultrasound guidance was usually preferred for its simplicity, real-time monitoring and flexible needle placement. CT guidance was reserved for lesions that were not demonstrated by ultrasound. The patients with hemangioma and hydatid disease of liver diagnosed radiologically were excluded to prevent undue complications. Proper aseptic care was taken by cleaning the skin surface with povidone iodine before every FNAC. Aspiration was done using 21G, 88mmlong spinal needle attached to a 10mL disposable which was introduced into the lesion under radiological guidance. The cytological material obtained were smeared on clean glass slides, wet-fixed or air-dried and stained by Papanicolaou (Pap), May-Grunwald Giemsa (MGG), and haematoxylin and eosin stains. The slides were screened for adequacy of the material. The diagnoses rendered by full consensus by three experienced cytopathologists were taken as final. The radiological opinion of each individual lesion was also recorded. Both cytological and radiological opinions were tabulated and compared statistically.

Results

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Cytoradiological Correlation of Hepatic Lesions

differentiated) were 18.7% cases. The main cytological features in grade I- HCC were hypercellularity with broad trabeculae, endothelial rimming/transgression of vessels in the cell clusters, bare atypical nuclei, large polygonal cells with abundant eosinophilic granular cytoplasm, intracytoplasmic bile, increased nucleus to cytoplasm (N:C) ratio, central round nucleus and intranuclear inclusions. (fig 4) Grade II HCC had many features of Grade I HCC. Endothelial rimming or transgressing of cell clusters, eccentric nuclei, multinucleation, multiple nucleoli and macronucleoli were more associated with Grade II HCC. Grade III HCC showed cells in sheets, small groups and

singles. Anisocytosis, anisonucleosis, irregular nuclear chromatin, hyperchromasia, multiple nuclei, macronucleoli and bare atypical nuclei were seen in all the patients. HCC showed multifocal space occupying lesions (SOL) in 56.6% and single SOL was seen in 44.4 % cases where as among multiple SOLs, metastasis was the commonest (83.6%) cytological diagnosis. Hepatitis C positivity was associated with HCC in 31.3% cases. No hepatitis B case was seen during this study. On correlating cytological diagnosis with radiological findings the cytoradiological correlation was seen in 92 % cases. (Table 3).

Table 1: Demographic and Cyto-radiological details. Subject

Subheading

Total Number

Age

<40 40-70 >70

15 128 26

Sex

Male Female

79 80

Radiological Diagnosis

Benign/Inflammatory HCC Metastasis

16 47 100

Cytological Diagnosis

Benign/Inflammatory HCC Metastasis

19 52 92

Table 2: Spectrum of lesions in the present study. Cytological diagnosis Non neoplastic

Number

Percentage

Pyogenic abscess

3.6%

Granulomatous hepatitis

1.8%

Fatty change

2.4%

Cirrhotic nodules

1.8%

Hepatocellular Carcinoma

30.2%

Cholangiocarcinoma

0.6%

Adenocarcinoma

49.1%

Small cell carcinoma

0.6%

Squamous cell carcinoma

1.2%

Malignant melanoma

0.6%

Lymphoma

0.6%

Poorly differentiated carcinoma

2.4%

Benign Hepatic adenoma Malignant primary

Neoplastic

Metastatic

Inadequate Total

3.6%

169

100%

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Table 3. Radiological findings in cytologically diagnosed malignant liver masses. Radiology

Primary

Metastases

Single SOL

Multiple

Total

Fig. 1: Metastatic deposits of adenocarcinoma showing hyperchromatic pleomorphic cells forming acinar pattern.( MGG 400X).

Fig. 2: Metastatic deposits of squamous cell carcinoma showing tadpole cells and neoplastic squamous cells with hyperchromatic enlarged nuclei and abundant keratinised cytoplasm.

Fig. 3: Metastatic small cell carcinoma with sheets of small round cells showing nuclear moulding and nuclear streaking.(MGG 400X).

Fig. 4: Hepatocellular carcinoma showing neoplastic cells with nuclear psuedoinclusions along with endothelial rimming around the small group of cells.

Discussion

by Nasit et al reported remarkable difference between sensitivity of diagnosis of malignant (87.3%) and benign (40%) lesions. [6]

FNAC is a sufficiently accurate, simple, rapid, safe and relatively painless and cost-effective technique, rendering it an attractive alternative to percutaneous biopsy. [3] Other points in favour of FNAC is that FNAC has high degree of sensitivity in cases of malignancy which is reported to be as high as 99.5% and 95.3% by various researchers. [4,5] Diagnosis is easier on FNAC in malignant hepatic lesions as compared to benign lesions as recent work done www.pacificejournals.com/apalm

The diagnostic yield in our study was 98.4% which is high as compared to other researchers, as the study employed the technique of immediate cytological assessment by means of rapid staining procedures and joint presence of both radiologists and pathologists which is recommended eISSN: 2349-6983; pISSN: 2394-6466

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Cytoradiological Correlation of Hepatic Lesions

by various researchers. The reported diagnostic yields in other studies range from 83.4% -90.4%. [1,7] Malignancy was the most common cytological diagnosis seen in 88.7% of cases. This corroborates with the work done by other researchers. [5,7] Metastatic deposits were seen more frequently (58%) as compared to HCC (30.2%) which was in concordance with the work done by other researchers. [7-10] In contrast to this, a study carried out in Pakistan reported HCC as the most common malignant lesion in liver (60.49%) because of high prevalence of Hepatitis B and C in the country. [5] Among the HCC cases on cytology, grade II was the commonest (56.3%). Other researchers also reported similar findings.[7,11] However, Tariq et al reported well differentiated HCC as the most common grade (44.9%). [5] Although the most common presentation of HCC in imaging studies in most studies is a single SOL, the tumor may also form multiple smaller nodules or a dominant mass with satellite nodules, thus mimicking the more common metastatic cancer. In our study, 56% of the HCC cases presented as multiple lesions on radiology. Hepatitis C positivity was seen in 31.3% cases of the HCC cases. No hepatitis B case was seen during this study. In contrast, many other studies reported 15.4% to 66.7% of Hepatitis B positivity in HCC cases.[11,12] Kaur et al reported a higher seropositivity of HCV in the blood donors of Amritsar as compared to that found in the studies from other parts of India. [13] Although imaging techniques have helped greatly with the early and accurate diagnosis, the appearances are often non-specific. There is some overlap between the radiologic features of liver abscesses, HCC and metastases.[11] Cytoradiological correlation in our study was established in 92% cases. Discordance was noted in eight (4.7%) cases of HCC were diagnosed as metastatic deposits on radiology as these were multicentric HCC. The same was reported by other researchers where secondaries liver reported by radiology turned out to be HCC on FNAC.[9] Thus, the exclusion of metastases in these cases prevented further search for a primary, saving the patient from needless investigations. Two cases of hepatic adenoma and one case of pyogenic abscess were misdiagnosed as HCC on radiology. HCC can be small and focal, solitary and large, multifocal or diffuse, and infiltrating, thereby, mimicking benign lesions on one hand and metastases on the other, especially in imaging studies. [14] Tumors, primary or secondary, may undergo extensive necrosis, with the resultant radiologic image of the cavitary neoplasms mimicking abscesses; abscesses are accompanied by proliferative reactive changes, making

radiologic differentiation from a neoplastic process almost impossible. In these situations FNAC plays an essential complementary role.[11] Complications of hepatic FNAC are rare with about 0.5% minor complications, 0.05% major complications requiring surgery and less than 0.01% mortality.[15] In our study, complications were limited to hemorrhage in two cases .The present study thus concludes that Image-guided FNA in hepatic lesions is a simple, safe, and economically prudent technique associated with low morbidity, leading to quick diagnosis. Present study also concludes that it leads to fairly accurate diagnosis in conjunction with radiological guidance enabling direct visualization leading to greater degree of predicting true positive malignant cases. It should be used as a first line of investigation. Hence it helps in early diagnosis and has increased chance of effective intervention and formulating immediate effective management of hepatic lesions.

Funding None

Competing Interests None Declared

Refrences

1. Mane A, Kanetkar SR, Saini S, Saini N. Role of image guided fine needle aspiration cytology in cases of hepatic mass lesions. Int J H Biomed Res 2015;3:149-55. 2. Chhieng DC. Fine needle aspiration biopsy of liver an update. World J Surg Oncol 2004;2:5. 3. Kramer H, Koeter GH, Sleijfer DT, van Putten JW, Groen HJ. Endoscopic ultrasound-guided fine-needle aspiration in patients with mediastinal abnormalities and previous extrathoracic malignancy. Eur J Cancer 2004;40:559-62. 4. Soyuer I, Ekinci C, Kaya M, Genc Y, Bahar K. Diagnosis of hepatocellular carcinoma by fine needle aspiration cytology. Cellular features. Acta Cytol 2003;47:581-9. 5. Nazir RT, Sharif MA, Iqbal M, Amin MS. Diagnostic accuracy of fine needle aspiration cytology in hepatic tumours. J Coll Physicians Surg Pak 2010;20:373-6. 6. Nasit JS, Patel V, Parikh B, Shah M, Davara K. Fine-needle aspiration cytology and biopsy in hepatic masses: A minimally invasive diagnostic approach. Clin Cancer Investig J 2013;2:132-42. 7. Rasania A, Pandey CL, Joshi N. Evaluation of FNAC in diagnosis of hepatic lesion. J Cytol 2007;24:51-4.

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8. Singh S, Sen R, Kumar S, Gupta S, Chhabra S. Profile of liver fine needle aspiration in tertiary care hospital. Euroasian J Hepatogastroenterol 2013;3:31-5. 9. Ahmad SS, Akhtar K, Akhtar SS,Abari A, Nasir A, Khalid M. Ultrsound guided fine needle aspiration biopsy of abdominal masses. JK Science 2006;8:200-4. 10. Adhikari RC, Tuladhar A, Shrestha S, Sharma SK. Deep seated thoracic and abdominal lesions : usefulness of ultrasound guided fine needle aspiration cytology, a 3 year experience. Nepal Med Coll J 2010;12:20-5. 11. Swamy MCM, Arathi C, Kodandaswamy C. Value of ultrasonographyguided fine needle aspiration cytology in the investigative sequence of hepatic lesions with

an emphasis on hepatocellular carcinoma. J Cytol 2011;28:178-84. 12. Sumana BS, Muniyappa B. Ultrasonography Guided Fine Needle Aspiration Cytology with Preparation of Cell Blocks in the Diagnosis of IntraAbdominal Masses. J Clin Diag Res. 2015;9:08-12. 13. Kaur H, Manjari M, Thaman RG, Singh G. Prevalence of markers of Hepatitis C virus among the blood donors. J Clin Diagn Res 2012;6:959-62. 14. Wee A. Fine needle aspiration biopsy of the liver: Algorithmic approach and current issues in the diagnosis of hepatocellular carcinoma. Cytojournal 2005;2:7. 15. Ji XL. Fine-needle aspiration cytology of liver diseases. World J Gastroenterol 1999;5:95-7.

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Original Article Distribution of Genital HPV Infection in Women of Reproductive Age Group in a North Indian City Screened for Cervical Cancer by Pap Smears Manas Madan*, Sanjay Piplani, Manisha Sharma, Mridu Manjari, Akanksha Singh Dept of Pathology, SGRDIMSR, Amritsar, Punjab. India Keywords: Cervical Carcinoma, HPV, Pap Smears, PCR

ABSTRACT Introduction: Carcinoma cervix is one of the leading causes of morbidity and mortality worldwide but has a benefit of being identified at a precancerous stage before the invasion starts and hence is amenable to early detection and treatment. HPV infection as the etiological agent of carcinoma cervix and its precursor lesions is well established. High risk HPV (16,18) is most commonly associated with invasive cervical carcinomas worldwide. The aim of this study is to find the prevalence of genital HPV infection in women of reproductive age group in a north Indian city that were screened for cervical cancer by Pap smears. Methods: The was a prospective study conducted from Jan 2014 to June 2016 on women in reproductive age group (< 49 years) who were screened for cervical cancer by Pap smears and HPV testing was done by PCR (sample was sent to higher center for testing). HPV was classified into high risk HPV DNA and low risk HPV DNA. Proper informed consent was taking from the participating women. Results: A total of 1549 tests were received during the above period.1068 (69%) were CPS and 481 (31%) were LBC. HPV testing was carried out in 169 cases of which 20 cases were found to be positive for HPV DNA (11.8%). High risk DNA was found in 17 cases (85%) and low risk DNA in 03 cases (15%). The annual rate of HPV detection showed a gradual slight increase from 10.3% in 2014 to 12.8% in 2016. Abnormal cytological findings were detected only in 5/20 cases (25%), which were subjected to HPV DNA testing pressing home the advantage of HPV testing in the reproductive age group. Conclusion: The study still establishes the important role of HPV as an etiological agent in carcinoma cervix and the need to get HPV testing included in the cervical cancer-screening program so as to diagnose the precursor lesions at an early stage leading to early treatment and reduction in morbidity and mortality from a world wide prevalent and preventable cancer.

*Corresponding author: Dr Manas Madan, 21 A, Sandhya Enclave Majitha Road, Amritsar (143001), Punjab, India Phone: +91 9888015365 Email: manasmadaan@gmail.com

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Madan et al.

A-589

Introduction

Carcinoma cervix is one of the leading causes of morbidity and mortality worldwide, more so in the developing countries. However one major advantage for clinicians is that this is one cancer that can be identified at a precancerous stage before the invasion starts and hence is amenable to early detection and treatment. Papanicolaou (Pap) smear was introduced way back in 1940 and has successfully led to a reduction in cervical carcinoma load.[1,2] Initially introduced as Conventional pap smear (CPS), it is gradually being replaced by Liquid based cytology (LBC) with the latter offering substantial advantages over CPS most important one being the ability to use the same sample for Human Papilloma Virus (HPV) DNA testing. This negates the need for reexamination and resampling for HPV testing in case it needs to be done.[1,3] HPV infection as the etiological agent of carcinoma cervix and its precursor lesions is well established and is one of the commonest sexually transmitted infections. Although there are huge variations in the genotypes of HPV, high risk HPV (16,18) are most commonly associated with invasive cervical carcinomas worldwide. Other factors i.e. use of oral contraceptives, high parity, smoking also play their role. HPV infection occurs in most of the sexually active people at some stage of life but usually this is a transient infection and subsides gradually. However in a few women, this persists possibly due to impaired immunity, which slowly but surely affects the cervical epithelium and can lead to cervical intraepithelial lesions and in later stages invasive cervical carcinoma in case it is not detected and treated at an early stage.[1,2,3] According to updated guidelines, women aged 21-29 years should be screened with a Pap test every 3 years. Women between 30-65 years can then be screened every 5 years with Pap and HPV co-testing or every 3 years with Pap test alone. For women younger than 30 years, HPV screening does not seem to confer any additional benefit. There also seems to be no benefit in reducing cancer incidence and mortality by screening women < 21 years of age. Same applies to women > 65 years or those who underwent hysterectomy provided they were not diagnosed with a high-grade precancerous lesion. Thus, no screening needs to be done in such women. [4]

The aim of this study is to find the prevalence of genital HPV infection in women that were screened for cervical cancer by Pap smears.

Materials and Methods

The was a prospective study conducted from Jan 2014 to June 2016 done in a private medical laboratory on women in reproductive age group (<49 years) who were screened for cervical cancer by Pap smears and HPV testing was done by Polymerase chain reaction (PCR) (sample was sent to higher center for testing). The pap smears were collected either as CPS or by LBC. HPV DNA co-testing was done as and when asked by the clinician and if the patient agreed to be a part of the study. Informed consent was taken from the patients. The detection rates of HPV by PCR were expressed as percentages. HPV was classified into high risk HPV DNA and low risk HPV DNA. Proper informed consent was taking from the participating women. Pregnant women, women that had undergone hysterectomy and those >49 years were excluded from the study.

Results

A total of 1549 tests were received during the above period.1068 (69%) were CPS and 481 (31%) were LBC. The age of the patients ranged from 23 to 49 years. HPV testing was carried out in 169 cases of which 20 cases were found to be positive for HPV DNA (11.8%).High risk DNA was found in 17 cases (85%) and low risk DNA in 03 cases (15%) (Table 1). High risk DNA were type 16 (11 cases), type 18 (3 cases), type 35 (1 case), type 45 (1 case) and type 39 (1 case) (Table 2). Low risk DNA were type 6 (2 cases), and type 11 (1 case) (Table 3). Age wise distribution of genital HPV detection was 1 case (<25 years) (5%), 12 cases (25-34 years) (60%) and 7 cases (35-49 years) (35%) (Table 4). The annual rate of HPV detection showed a gradual slight increase from 10.3% in 2014 to 12.8% in 2016 (Table 1). Abnormal cytological findings were detected only in 5/20 cases (25%) (Table 1), which were subjected to HPV DNA testing pressing home the advantage of HPV testing in the reproductive age group. Out of these, 4 were detected on LBC and only 1 on CPS. This further proves the superiority of LBC over CPS in diagnosing intraepithelial lesions of cervix.

Table 1: Year wise rates of HPV detection. Period Jan 2014-Dec 2014 (1 year) Jan 2015-Dec 2015 (1 year) Jan 2016-June 2016 (6 months) Total

Cases for HPV screening 58 72 39 169

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Abnormal findings in Cytology 1 (CPS) 2 (LBC) 2 (LBC) 5

HPV detected in 6 (10.3%) 9 (12.5%) 5 (12.8%) 20 (11.8%)

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HPV Infection in Reproductive Age Group Females

Table 2: High risk HPV types. TYPES

CASES DETECTED

Total

Table 3: Low risk HPV types. TYPES

CASES DETECTED

Total

Table 4: Age distribution of 20 HPV positive cases. Age

CASES DETECTED

<25 years

1 (5%)

25-34 years

12 (60%)

34-49 years

7 (35%)

Discussion

Cervical carcinoma is distributed through out the world and is one of the leading causes of death in females. Pap smear was developed in order to identify the women at risk while still in the precancerous stage and thus reduces the cancer mortality. HPV is a proven etiological agent for cervical carcinoma and hence its detection at an early stage of infection can be beneficial for the patient due to the early treatment given to her.[1,2,4] In this study, HPV testing was carried out by PCR in 169 cases of which 20 cases were found to be positive for HPV DNA (prevalence rate of 11.8%). This finding correlates well with various other studies done. [1,2,3]High risk DNA detected were type 16 (11 cases), type 18 (3 cases), type 35 (1 case), type 45 (1 case) and type 39 (1 case). Low risk DNA were type 6 (2 cases), and type 11 (1 case). Most of HPV positive cases were in the age group of 2534 years comprising 60% of the cases. This was slightly different from other studies that established the peak age of HPV infection in the younger age group.[1,2,3,5,6] This could be probably due to small sample size in our study and hence the findings need to be corroborated with study on a larger population. The annual rate of HPV detection showed a gradual slight increase from 10.3% in 2014 to 12.8% in 2016 in this study. This could be due to more awareness among general public and increase initiative on the part of the clinicians to convince the women to undergo HPV testing.

HPV can be divided into high risk and low risk types. Major high-risk HPV types include type 16, 18, 31, 33, 35, 39, 45, 51, 52, 56,58, 68 and 69. Of these types 16 and 18 account for a majority of the infections. Similar results were obtained in our study in which both of these accounted for 14/17 (82.3%) high risk HPV detected. Low risk HPV types include type 6, 11, 40, 42, 43, 44, 53, 54, 61, 72, 73 and 81. Among these, types 6 & 11 are the commonest to cause infection, which was also seen in our study where all the low risk HPV DNA detected were either type 6 or 11.[1,2,8,9,10,11,12] On comparing HPV results with cytology, it was found that abnormal cytological results were obtained in only 5/169 cases (2.95%) that underwent HPV detection. Of these, 1 was diagnosed on CPS and 4 on LBC. Other studies done showed comparable results to our study [2,3,11,12]This further underlines the importance of HPV testing to be incorporated in to the cervical cancer screening program and also highlights the superiority of LBC over CPS in diagnosing pre invasive cervical lesions. There are numerous known strains of HPV and the association of high risk HPV 16,18 with invasive cervical carcinoma is well known. Most of the HPV types lead to rather innocuous lesions on the skin commonly known as warts. The high-risk strains also require other environmental, genetic and other molecular factors to aid in the transformation of normal cervical epithelium to dysplastic and ultimately invasive carcinoma. The appropriate diagnostic method can arrest the transformation at a precursor level and prevent the occurrence of fullblown invasive carcinoma cervix in susceptible women. [13] These lesions can be recognized at an early stage by various methods that include CPS, LBC and MPV testing. Among these, HPV testing is the most sensitive method as it can detect HPV strains even when recognizable morphological changes have not yet been produced to be recognized by CPS or LBC. In addition, if the technique is faulty and the squamocolumnar junction has not been adequately sampled, the precursor lesions are very likely to be missed even if they are present.[12,13] Same findings were noted in our study in which the sensitivity of HPV detection by PCR was much more as compared to LBC and CPS. This gains more importance, as now the vaccination against HPV is available which is a major advancement in the prevention against cervical carcinoma. Thus the screening methods can provide us with more conclusive data on the effectiveness of HPV vaccination for prevention against cervical carcinoma And HPV detection should be a part of it due to its effectiveness in detection of low risk and

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high risk HPV strains as against CPS and LBC.[13,14,15] The higher cost of HPV detection however can be a deterrent in this especially in cost sensitive developing countries like India.

6. Marks MA, Gupta S, Liaw KL, Tadasse A, Kim E, Phongnarisorn C. Prevalence and correlates of HPV among women attending family-planning clinics in Thailand. BMC Infect Dis 2015;15:159-68.

Conclusion

7. Thomas JO, Herrero R, Omigbodun AA, et al. Prevalence of papillomavirus infection in women in Ibadan, Nigeria: a population-based study. Br J Cancer. 2004;90(3):638-45.

Although our study is limited by a small sample size and limited age group included in the inclusion criteria, the study still establishes the important role of HPV as an etiological agent in carcinoma cervix and the need to get HPV testing included in the cervical cancer screening program so as to diagnose the precursor lesions at an early stage leading to early treatment and reduction in morbidity and mortality from a world wide prevalent and preventable cancer.

Funding None

Competing Interests None Declared

References

8. Saslow D, Solomon D, LawsonHW, et al. American Cancer Society, American Society for Colposcopy and Cervical Pathology, and American Society for Clinical Pathology Screening Guidelines for the Prevention and Early Detection of Cervical Cancer. AJCP. 2012;137:516-542.PMID: 22431528. 9. Stoler MH. HPV for cervical cancer screening: is the era of the molecular pap smear upon us. J Histochem Cytochem. 2001; 49:1197-1198. PMID: 11511693. 10. Denny L. Cervical cancer: prevention and treatment. Discov Med 2012;14(75):125-31.

1. Bell KL, Luciani S, Unger ER, Hariri S, McFarlane S, Steinau M et al.Genital human papillomaviruses among women of reproductive age in Jamaica.Rev Panam Salud Publica 2013;33 (3):159-65. 2. Anthony SNA, Famooto AO, Dareng EO, et al. Age-specific prevalence of human papilloma virus infection among Nigerian women. BMC Public Health 2014;14:656. 3. Rama CH, Martins CMR, Derchain SFM, Filho AL, Gontijo RC, Sarian LOZ. Prevalence of genital HPV infection among women screened for cervical cancer. Rev Saude Publica 2008; 42(1):1-7. 4. Moyer VA. Screening for Cervical Cancer: U.S. Preventive Services Task Force Recommendation Statement. Ann Intern Med2012;156:880-91. 5. Sreedevi A, Javed R, Dinesh A. Epidemiology of cervical cancer with special focus on India. Int J Womens Health 2015;7:405-14.

11. Walboomers JM, Jacobs MV, Manos MM, FX Bosch, Kummer JA, Shah KV. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999;189(1):12-9.

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12. Clifford GM, Smith JS, Plummer M, Munoz N, Franceschi S. Human papillomavirus types in invasive cervical cancer worldwide: a meta-analysis. Br j Cancer 2003;88(1):63-73. 13. Burd EM. Human Papillomavirus and cervical Cancer. Clin Microbiol Rev 2003;16(1):1-17. 14. Kaarthigeyan K. Cervical cancer in India and HPV vaccination. Indian J Med Paediatr Oncol 2012;33(1):7-12. 15. Brotherton JM, Fridman M, May CL. Early effect of the HPV vaccination programme on cervical abnormalities in Victoria, Australia: an ecological study. Lancet 2011;377:2085-92.

Original Article Cytohistologic Correlation Study of Thyroid Lesion: Insight into the Causes of Discordance Archana S Bhat1*, Hilda Fernandes1 and Leena Priya2 1

Department Of Pathology, Father Muller Medical College, Mangalore, India 2 Consultant Pathologist, Private Lab, Abu Dhabi Keywords: FNAC, Thyroid, Cytohistology Correlation

ABSTRACT Background: Fine needle aspiration cytology (FNAC) is a popular diagnostic as well as therapeutic test in thyroid pathology. Like any other diagnostic modality, FNAC also has its limitations and pitfalls. This study was aimed at finding the causes for cytohistologic discordance, thereby minimizing the false positive and negatives. Methods: 1091 patients underwent thyroid FNA in a period of 2 years in our institute. Histopathological diagnosis was available in 313 cases. Data of these cases was collected and statistically analysed. Cases showing cytohistologic disparity were reevaluated to unmask the causes of discordance. Result: Of 313 cases with histopathological diagnosis, 19.48%(n=61) were neoplastic out of which 73.77%(n=45) were malignant. Mean age of study population was 42.48 years. 86.58% (n=271) were females. We found that sensitivity and specificity of FNA was 54.83% and 98.40% respectively. Positive predictive value was 89.47% and negative predictive value was 89.81%. Cytohistologic concordance was achieved in 89.78% of the cases. Of the discordant cases, false positive accounted for 1.2%(n=4) and false negative for 8.94%(n=28).Too much emphasis on cellularity and amount of colloid, failure to appreciate subtle nuclear features and sampling error were the most common causes for failure. Conclusion: Proper representative sampling, if needed guided by imaging modalities and meticulous examination of all the smears are the key points in reducing the number of discrepant cases.

*Corresponding author: Dr Archana S Bhat, Assistant Professor, Department Of Pathology, Father Muller Medical College, Mangalore. India Phone: +91 9035572134 Email: archibhat3@gmail.com

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

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Introduction

Fine needle aspiration cytology (FNAC) is a popular diagnostic as well as therapeutic test in thyroid pathology. Given the ease of technique and relatively minimal invasive nature of the procedure, it has become a popular diagnostic modality. It is being widely used to differentiate benign from malignant lesions. But there is some “gray zone” of thyroid FNAC where the diagnostic efficacy becomes low, making it less reliable in categorizing the nature of the lesion leading to discrepant cases.[1] We conducted this study to determine the diagnostic efficacy of thyroid FNAC by comparing it with histopathology which is considered gold standard. We also reviewed the discordant cases and looked at causes for cytohistologic discordance. This may help in minimizing the false positive and false negative cases.

Materials and Methods

This retrospective study was conducted in the Department of Pathology at Father Muller Medical College, Mangalore. A total of 1091 patients with thyroid swelling were aspirated during a period of two years from 2011 to 2013 using a 23-gauge needle and a 5-ml syringe. The slides were both air-dried and wet-fixed for May-Grünwald Giemsa and Papanicolaou stains, respectively. A concise clinical history, examination and details of relevant investigations were also obtained. Surgical follow-up was available for 313 aspirations. The concordance between cytological and histological diagnoses was determined. Cases with cytohistological discrepancy were selected and cytological smears of these cases were reevaluated for the detection of possible causes of failure. The aims of the study were: 1) To find the sensitivity, specificity, positive and negative predictive values of thyroid FNAC. & 2) To critically evaluate the discordant cases and find out causes of discordance.

Result

Among the 1091 were divided into 1) and neoplastic included nodular

patients, the cytological diagnoses 2 categories: non neoplastic (Table (Table 2). Non neoplastic category goitre, adenomatous goiter and

different cases of thyroiditis like hashimoto’s thyroiditis, lymphocytic thyroiditis and de quervain’s thyroiditis. Neoplastic category included indeterminate cases, papillary carcinoma, medullary carcinoma and anaplastic carcinoma. The lesions suspicious of malignancy, follicular and hurthle cell neoplasms were also included in the indeterminate category. In 313 cases surgical follow up with histopathological diagnosis was available. Mean age of these cases was 42.48 years. 86.58% (n=271) were females. 19.48% (n=61) of the total cases were neoplastic out of which 73.77%(n=45) were malignant. Cytological diagnoses were then compared with histopathological diagnoses. Among the non neoplastic lesions, histopathology was available in 252 cases. Cyto-histo concordance was obtained in 244 cases, whereas remaining 4 showed malignant histology. Conversely, among the neoplastic category, histopathology was available for 61 cases(Table 3). Papillary carcinoma was the most common neoplasm(n=25). Complete cytohisto concordance was obtained in 33 cases. Of the 28 discordant cases, 22 were diagnosed as goiter, 2 as hashimoto’s thyroiditis, 2 as cystic colloid nodules and 2 as follicular neoplasms. Evaluation of 32 discordant cases identified, 4 false positive and 28 false negative cases, which were missed on cytological examination. A false negative diagnosis of goitre was made in 22 cases of which 8 turned out to be follicular adenoma, 7 were follicular variant of papillary carcinoma, 4 cases were papillary carcinoma and 3 turned out to be papillary micro carcinoma. The possible causes for misdiagnosis of these cases which were found on reviewing the cytology smears and histopathology sections are tabulated below (Table 4). Overall, we found that cytohistologic concordance was achieved in 89.78% of the cases. Positive predictive value was 89.47% and negative predictive value was 89.81%. Of the discordant cases, false positive accounted for 1.28%(n=4) and false negative for 8.94%(n=28).

Discussion

FNAC has become a primary diagnostic tool for evaluating thyroid nodules. A correct cytologic diagnosis obviates

Table 1 Non neoplastic lesions (Cytology)

Number of cases

Goitre

739

Thyroiditis

290

Total

1029

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Table 2 Neoplastic lesions (Cytology) Papillary carcinoma Anaplastic carcinoma Medullary carcinoma Indeterminate total

Number of cases 29 6 3 24 62

Type of neoplasm (Histopathology)

Number of cases

Follicular adenoma

Hurthle cell adenoma

Follicular carcinoma

Papillary micro carcinoma

Table 3

Papillary carcinoma

Follicular variant of papillary carcinoma

Total

Table 4: False negative diagnosis of goiter and possible reasons. Cytology diagnosis

Goitre (n=22)

Histopathology diagnosis

Possible reasons for discordance

Colloid rich follicular adenoma (n=4)

Abundance of colloid in the background

Follicular adenoma (n=4)

Small lesion in a thyroid with multiple nodules leading to sampling error, misinterpretation as hyperplastic goiter.

Follicular variant of papillary carcinoma (n=7)

Subtle nuclear features, background hemorrhage, focal nuclear features

Papillary carcinoma (n=4)

Focal and subtle nuclear features, grooves thought to be a part of adenomatous hyperplasia

Papillary microcarcinoma (n=3)

Sampling error

Fig 1: Focal nuclear features of papillary carcinoma can be easily missed. Note the elongated, grooved nuclei with pale chromatin. (PAP 400X).

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unnecessary thyroid surgeries. The percentage of patients undergoing thyroidectomy has decreased by 25%, and the yield of carcinoma in patients who undergo surgery has increased from 15% to at least 30%. Fine-needle aspiration has decreased the cost of care by 25%.[2] However, FNA has few inherent limitations which are related to inadequate sampling and its inability to distinguish between benign and malignant follicular lesions in the absence of nuclear features of papillary carcinoma.[3] Another limitation of FNAC is its inefficiency in diagnosing group of lesions which have overlapping benign and malignant features. For instance, the distinction between a cellular colloid goiter and a follicular neoplasm may be impossible.[4] The important steps in FNAC thyroid are careful sample procurement, appropriate sample preparation and accurate interpretation. Meticulous and carefully scrutinization for various cytologic features may reduce the number of discrepant cases. In the present study, we have studied the efficacy and diagnostic ability of FNAC and made an attempt to ascertain the possible reasons for discrepancies and ways to minimize them. The value of a diagnostic test lies in its ability to detect the presence of disease when it is present (sensitivity) and reliably verify the absence of disease when it is not present (specificity). The reported sensitivity and specificity of the thyroid FNAC ranges from 43 to 99% and 72 to 100% respectively. Our findings are similar to those reported in other series.[5,6] False negative rates in our study are in accordance with other studies which report a range from 1 to 11%.[2,7] This high rate of failure to diagnose neoplasm could be attributed to the failure of aspiration from the representative site. This is of special concern since false negative diagnosis results in a delay in treatment and thereby adversely affecting the outcome in patients with thyroid cancer.[8] Most of the discordant cases in our study were those of follicular lesions of thyroid namely follicular adenoma and follicular variant of papillary carcinoma. These cases belong to the gray area of cytology due to the overlapping features. Greaves, et al studied 92 cases of follicular lesions of which in 63 cases there were no distinguishing features predictive of the histologic outcome.[9] We reviewed these cases to find out the potential clues that can help us in differentiating benign from malignant lesions and also at the same time we attempted to delineate the features which can be potentially misleading. Syncytial pattern, papillary pattern and microfollicular pattern are more common in neoplasms and thyroiditis. The predominant cell pattern gives us a clue as to what we are dealing with and aids in diagnosis in combination with cell morphology and background details in the cytological www.pacificejournals.com/apalm

diagnosis of thyroid lesions.[10] In a study conducted by Pandey, et al on 112 cases, they got 6 false negative cases which were reported as colloid goiter 4 of which turned out to be follicular adenoma and 2 were cystic papillary carcinoma. The cytological smears of all the 4 cases showed small groups of thyroid follicular cells and moderate amount of colloid which was misleading towards the diagnosis of nodular colloid goiter.[11] In our study too, we misdiagnosed few cases due to overemphasis on the amount of colloid. Also cytologically it is difficult to distinguish nodular goiter and colloid rich follicular adenoma.[12] Occurrence of cystic change is another known pitfall in thyroid cytology. In our study, 2 cases of papillary carcinoma were misdiagnosed as cystic colloid nodule. This type of misinterpretation can be minimized by selective sampling from solid areas under the guidance of imaging modalities and also by strict adherence to adequacy criteria. We missed 7 cases of follicular variant of papillary carcinoma (FVPTC). This is also a known pitfall due to failure to recognize the nuclear features of FVPCT (Figure 1). Cytological diagnosis is difficult again due to overlapping features with follicular lesions and masked or absent nuclear features in the sampled cells. A finding of 2 different population of follicular cells or cells with 2 different types of nuclear chromatin should raise a strong suspicion of neoplastic lesion. Also another pointer towards neoplastic lesion is the different looking or altered nature of the colloid. False positive cases constituted 1.28% of the total cases in our study. 3 of the false positive cases reported as follicular neoplasms were goiter with adenomatous hyperplasia which is a known pitfall as described earlier. The feature of hypercellularity outshadowed the uniform cytomorphology of the follicular cells and paved the way for misdiagnosis of follicular neoplasm. Presence of few number of bare nuclei is another indicator towards neoplastic lesion. Too much emphasis on cellularity and amount of colloid, less emphasis on subtle nuclear features, nature of colloid and bare nuclei together with sampling error were the most common causes for failure.

Conclusion

FNAC is a very important diagnostic method in thyroid pathology and helps in picking up neoplastic lesions and avoiding unnecessary thyroidectomies. The sensitivity and specificity of this method can be increased by strict adherence to adequacy criteria and relying on combination of various cytological features rather than emphasizing on only one feature. Proper representative sampling, if needed eISSN: 2349-6983; pISSN: 2394-6466

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guided by imaging modalities and meticulous examination of all the smears are the key points in reducing the number of discrepant cases.

6. Ergete W, Abebe D. Discordance rate between thyroid fine needle aspiration cytology and histopathologic diagnosis. Ethiop J Health Dev 2002;16:227-31.

Acknowledgements

7. Bakhos R, Selvaggi SM, DeJong S, Gordon DL, Pitale SU, Herrmann M, et al. Fine-needle aspiration of the thyroid: Rate and causes of cytohistopathologic discordance. Diagn Cytopathol 2000;23:233-7.

None

Funding None

Competing Interests None Declared

Reference

1. Somma J, Schlecht NF, Fink D, Khader SN, Smith RV, Cajigas A. Thyroid fine needle aspiration cytology: follicular lesions and the gray zone. Acta Cytol 2010;54:123-31. 2. Gharib H, Goellner J R. Fine-needle aspiration biopsy of the thyroid: An appraisal. Ann Intern Med 1993;118:282-9. 3. Sinna EA, Ezzat N. Diagnostic accuracy of fine needle aspiration cytology in thyroid lesions. Journal of the Egyptian National Cancer Institute 2012;24: 63–70 4. Hall TL, Layfield LJ, Philippe A, Rosenthal DL. Source of diagnostic error in the fine needle aspiration of the thyroid. Cancer 1989;63:718–25. 5. Caraway NP, Sneige N, Samaan NA. Diagnostic pitfalls in thyroid fine-needle aspiration: A review of 394 cases. Diagn Cytopathol 1993;9:345-5

8. Yeh MW, Demircan O, Ituarte P, Clark OH. False negative fine-needle aspiration cytology results delay treatment and adversely affect outcome in patients with thyroid carcinoma. Thyroid 2004;14:207-15. 9. Greaves TS, Olvera M, Florentine BD, Raza AS, Cobb CJ, Tsao-Wei DD, et al. Follicular lesions of thyroid: A 5 year fine-needle aspiration experience. Cancer 2000;90:335-41. 10. Bommanahalli BP, Bhat RV, Rupanarayan R. A cell pattern approach to interpretation of fine needle aspiration cytology of thyroid lesions: A cytohistomorphological study. J Cytol. 2010; 27(4): 127–132. 11. Pandey P, Dixit A, Mahajan NC. Fine-needle aspiration of the thyroid: A cytohistologic correlation with critical evaluation of discordant cases. Thyroid Res Pract 2012;9:32-9 12. Suen KC. How does one separate cellular follicular lesions of the thyroid by fine needle- aspiration biopsy? Diagn Cytopathol 1988;4:78-81.

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Original Article Importance of Bone Marrow Examination in Cases of Pancytopenia: A Morphological Study Rumpa Das1* and Gorakh Nath2 1

Department of Pathology. Hind Institute of Medical Sciences. Barabanki, India 2 Department of Otorhinolaryngology, Nath ENT Centre. Faizabad, India

Keywords: Pancytopenia, Bone Marrow Examination, Megaloblastic Anemia, Aplastic Anemia, Acute Leukemia

ABSTRACT Background: Pancytopenia is a pathological manifestation resulting from various disease processes affecting the bone marrow. Therefore, bone marrow examination is an important diagnostic tool to evaluate the underlying causes of pancytopenia. Methods: Present study was done in 64 cases fulfilling the criteria of pancytopenia over a period of two years. Bone marrow examination including bone marrow aspiration and bone marrow biopsy (only in cases of dry tap on aspiration) was performed in all the cases and morphological study along with cytochemical staining was done. The findings were correlated with peripheral blood examination. Diagnostic value of bone marrow examination in cases of pancytopenia was established. Result: 61 out of 64 cases were successfully diagnosed with the morphological examination of bone marrow. The most common cause of pancytopenia in the present study was megaloblastic anemia (57.81%) followed by aplastic anemia (18.75%) and acute leukemia (9.37%). Other causes of pancytopenia were dimorphic anemia due to mixed deficiency, lymphoma, multiple myeloma, infectious disease and storage disease. The diagnostic value of bone marrow examination in cases of pancytopenia in the present study was 95.31%. Conclusion: Morphological examination of bone marrow is an extremely helpful and specific diagnostic tool in cases of pancytopenia. Bone marrow aspiration and biopsy complement each other but diagnosis can solely be made in most of the cases by examining only the bone marrow aspiration smears.

*Corresponding author: Dr Rumpa Das, Department Of Pathology. Hind Institute of Medical Sciences. Barabanki- 225003, India Phone: +91 918795132000, 8756860132 Email: dr.rumpadas@gmail.com

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

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Introduction

Pancytopenia is described as a simultaneous process of anemia, leucopenia and thrombocytopenia. It exists in adults when the hemoglobin level is less than 13.5 gm/dl in males or 11.5 gm/dl in females, leukocyte count is less than 4 x 109 /L and platelet count is less than 100 x 109 /L. Pancytopenia is not a disease entity but a pathological manifestation resulting from a variety of disease process affecting the bone marrow either primarily or secondarily. It usually results due to infections, toxins, malignant cell infiltration, chemotherapy and radiotherapy. However, pancytopenia can be due to peripheral destruction of blood cells as seen in cases of hypersplenism. Patients of pancytopenia can present with a variety of clinical symptoms resulting from anemia, leucopenia and thrombocytopenia. Peripheral blood smears of patients with pancytopenia usually show a normochromic normocytic or macrocytic red blood cells (RBCs). Bone marrow cellularity and composition in cases of pancytopenia usually differ in relation to underlying etiological condition. Bone marrow cellularity is reduced in cases of pancytopenia caused by primary production defect. Bone marrow is normocellular or hypercellular in cases of pancytopenia resulting from ineffective hematopoiesis, increased peripheral destruction and bone marrow invasion. Therefore, bone marrow examination is extremely helpful to identify the cause of pancytopenia. The most common cause of pancytopenia in worldwide is aplastic anemia [1] but the most common cause of pancytopenia in India is megaloblastic anemia.[2] Although it is a common problem, but the prevalence of the causes of pancytopenia is reported in very limited number of studies. Present study will highlight upon the prevalence, hematological parameters and utility of bone marrow examination in different causes of pancytopenia.

Materials and Methods

The study was conducted in the department of Pathology, G.S.V.M Medical College, Kanpur, Uttar Pradesh, India from December 2008 to September 2010. A total number of 64 patients with a diagnosis of pancytopenia referred from department of medicine, pediatrics and otorhinolaryngology (patients having epistaxix with pancytopenia) were included in the study while patients having malignancy with pancytopenia developed after radiotherapy and chemotherapy were not included in the study. Informed consent was obtained from each patient prior to their enrolment. Relevant history and clinical findings of the patients were noted and all patients were subjected to complete blood count (CBC), reticulocyte count, peripheral blood smear examination including general blood picture

(GBP) and bone marrow aspiration and bone marrow biopsy when there is a dry tap on aspiration. CBC was performed by ‘Sysmex XP100’. Peripheral blood smears were stained with Leishman’s stain. Bone marrow aspiration was done from anterior superior iliac spine or posterior iliac spine and bone marrow biopsy was done from posterior superior iliac spine under proper aseptic measure and local anesthesia with Sala’s bone marrow aspiration needle. Approximately 0.3 ml of marrow fluid was aspirated and smears were prepared on a glass slide. Bone marrow aspiration smears were stained with Leishman’s stain, Myeloperoxidase (MPO), Prussian blue and Periodic Acid-Schiff (PAS). Smears were then examined under scanner and low power to assess the cellularity, megakaryocytes and metastatic carcinoma cells. The area where the cells were well spread out was selected and under oil immersion at least 500 marrow cells were differentially counted. Bone marrow biopsy was done from posterior superior iliac spine whenever there is a dry tap on aspiration. Jamshidi needle was used for bone marrow biopsy. 1.5 to 2.0 cm long bone marrow biopsy specimen was obtained and fixed in bouin’s fixative. Biopsy specimen was decalcified processed and sections were stained with Hematoxylene and Eosin (H&E) stain.

Result

Among the 64 cases of pancytopenia, the most common cause was megaloblastic anemia (57.81%) followed by aplastic anemia (18.75%), Acute leukemias (9.37%), dimorphic anemia due to mixed deficiency (06.25%), pancytopenia due to bone marrow involvement by primary lymphoma (3.12%), multiple myeloma (1.56%), pancytopenia due to infectious disease (1.56%) and pancytopenia due to storage disease (1.56%). (Table : 1) Total 06 cases of acute leukemias were diagnosed on bone marrow examination, out of which four cases were further typed as acute lymphoblastic leukemia (ALL) and two cases as acute myeloid leukemia (AML). Males were more commonly affected than females (ratio 1.2:1) and most common affected age group was 1630 years, followed by 00-15 years, 31-45 years and >60 years (Chart 1). Females of age group 16-45 years were commonly found to have pancytopenia due to megaloblastic anemia and dimorphic mixed nutritional deficiency. The most common clinical presentations were pallor and weakness; whereas other clinical features were dyspnea, bleeding tendency, fever, splenomegaly, hepatomegaly and lymphadenopathy. (Table: 2) Nine out of 12 patients of aplastic anemias were transfusion dependent.

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Details of peripheral blood findings are given in table no 3 (Table 3). Peripheral blood smears of patients with megaloblastic anemia showed macro-ovalocytic red cells with presence of occasional basophilic stippling, cabot ring and Howell-Jolly bodies. Hypersegmented neutrophils were noted in 83.78% (31/37) cases. Peripheral smears of 10 out of 12 cases of aplastic anemia showed normocytic normochromic red cells; however the rest showed macrocytosis. Cases of dimorphic anemia due to mixed deficiency showed severe anisopoikilocytosis with presence of microcytic hypochromic red cells, hypochromic macrocytes and few pencil cells. Cases of ALL showed pancytopenia with presence of few immature lymphoid cells. Presence of occasional plasma cells was seen in the case of multiple myeloma. Bone marrow cellularity was increased in all the cases of pancytopenia caused by megaloblastic anemia, dimorphic anemia and leukemias; however bone marrow cellularity was decreased in cases pancytopenia caused by aplastic anemia, bone marrow involvement in lymphomas, multiple myeloma and storage disease. Myeloid / Erythroid ratio (M/E ratio) was reversed in all cases of megaloblastic anemia and dimorphic anemia; but M/E ratio was increased in cases of AML; however it was normal in cases of aplastic anemia. M/E ratio could not be determined in cases of ALL and lymphoma. Lymphoid / Erythroid ratio was taken in these cases and it was found to be increased. Megaloblastic reaction with presence of megaloblasts having sieve like nuclear chromatin was noted in all cases of megaloblastic anemia. (Fig 1a) Features of dyserythropoiesis in the form of nuclear budding, nuclear fragmentation and irregular nuclei were also noted. (Fig 1b) Morphologic aberration in myeloid series was also noted in the form of giant myelocytes. (Fig 1c) Prussian blue stain showed increased iron content in 30% cases (Fig 1d) and normal iron content in 70% cases of megaloblastic anemia. Bone marrow aspiration (BMA) smears of aplastic anemia were diluted with peripheral blood and showed severe Table 1: Different causes of pancytopenia. Diagnosis Megaloblastic anemia Aplastic anemia Acute leukemia Dimorphic anemia due to mixed deficiency Bone marrow infiltration by lymphomas Multiple myeloma Infectious disease Storage disease Total

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reduction of hematopoietic precursors with increased marrow fat. (Fig 2a)There were focal cellular areas with presence of lymphocytes, plasma cells and occasional mast cells. (Fig 2b) Gelatinous transformation of bone marrow was noted. (Fig 2c) Bone marrow iron content was increased in all of the cases. (Fig 2d) BMA smears of ALL were hypercellular with severe reduction of erythroid, myeloid and megakaryocytic series with presence of >20% lymphoblasts (Fig 3a), showing PAS positivity. The cases of AML showed severe reduction of erythroid and megakaryocytic series with presence of >20% myeloblasts (Fig 3b), showing positivity for MPO. Cases of dimorphic anemia due to mixed deficiency showed erythroid hyperplasia with presence of micronormoblasts and megaloblsts. Features of dyserythropoiesis were also observed. Prussian blue stain showed decreased iron content in all the cases of dimorphic anemia. The case of multiple myeloma showed increased number of lymphoid cells and presence of atypical plasma cells with severely hypocellular marrow. Patients with bone marrow involvement by lymphoma showed severe suppression of all three lineages with presence of medium sized lymphoid cells.(Fig 3cd) A possibility of bone marrow involvement by lymphoproliferative disorder was suggested in those cases. One case of tuberculosis with bone marrow involvement showed presence of granulomas in the bone marrow aspiration smears. Bone marrow aspiration revealed dry tap in the case of storage disease which was diagnosed on bone marrow biopsy. Bone marrow biopsy of the case showed large foamy Nieman Pick cells. Among the 64 cases of pancytopenia; 61 cases was diagnosed on the basis of bone marrow examination; whereas three cases including two cases of lymphomas and a case tuberculosis needed further diagnostic workup. So, the diagnostic value of morphological examination of bone marrow in cases of pancytopenia in the study was 95.31%. Number of cases 37 (57.81%) 12 (18.75%) 06 (9.37%) 04 (6.25%) 02 (3.12%) 01 (1.56%) 01 (1.56%) 01 (1.56%) 64

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Table 2: Details of clinical manifestation: Diagnosis (total number) Pallor Weakness Megaloblastic anemia (37) 37 37 Aplastic anemia (12) 12 12 Acute leukemia (06) 06 06 Dimorphic anemia (04) 04 04 Lymphoma (02) 02 02 Multiple myeloma (01) 01 01 Infectious disease (01) 01 01 Storage disease (01) 01 01 Total = 64 64 64

Bleeding 11 10 04 01 01 27

Fever Splenomegaly Hepatomegaly lymphadenopathy 07 08 02 04 01 03 04 03 04 01 01 01 01 01 01 01 01 01 18 15 06 06

Table 3: Details of peripheral blood findings. Diagnosis (total number)

Megaloblastic anemia (37) Aplastic anemia (12) Acute leukemia (06) Dimorphic anemia (04) Lymphoma (02) Multiple myeloma (01) Infectious disease (01) Storage disease (01) Total = 64

Anisocytosis 35 02 04 01 01 01 01 45

Dimorphic Hypersegmented Immature Erythroblasts Lymphocytosis smear neutrophils cells 18 07 31 04 01 04 06 04 02 01 01 01 01 01 22 12 10 31 06

Fig. 1: Megaloblastic anemia showing (a) Megaloblasts having sieve like nuclear chromatin (Leishman, X1000), (b) Features of dyserythropoiesis in the form of nuclear fragmentation and irregular nuclei (arrow) (Leishman, X1000), (c) Giant myelocyte (arrow) ) (Leishman, X1000), (d) Increased bone marrow iron (Prussian blue X400).

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Fig. 2: Aplastic anemia showing (a) severe reduction of hematopoietic precursors with increased marrow fat(Leishman, X40), (b) Focal cellular area with presence of lymphocytes, plasma cells and mast cell (arrow) (Leishman, X1000),, (c) Gelatinous marrow transformation(Leishman, X1000), , (d) Increased bone marrow iron (Prussian blue X100).

Fig. 3: (a) ALL with presence of lymphoblasts (arrow) (Leishman, X1000), (b) AML with presence of myeloblasts (arrow) (Leishman, X1000), (c) Marrow involvement with lymphomas showing severe suppression of all three lineages with presence of medium sized lymphoid cells and a megakaryocyte (arrow) (Leishman, X400), (d) lymphomas with presence of medium sized lymphoid cells and a megakaryocyte (arrow) (Leishman, X1000).

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Chart 1: Age and sex distribution.

Discussion

The most common cause of pancytopenia in the present study was megaloblastic anemia (57.81%). Different studies conducted in India also showed megaloblastic anemia as the most common cause of pancytopenia.[2,3,4] Megaloblastic anemia was also the most common cause of pancytopenia in other study conducted by Devid et al in 1999 in Zimbabwe.[5] It was the second most common cause of pancytopenia in other studies conducted by Jha et al and Tariq et al in Nepal and Pakistan respectively.[6,1] Etiological factors of megaloblastic anemia in a developing country like India could be a diet poor in cobalamine and folate, increased requiremint during growth period and pregnancy and also the use of acid supperessing medication.[7] Other contributing factors for development of megaloblastic anemia could be geographical factor, socio-economic factor, food habit and religious factor. Findings of CBC like MCV, MCH and MCHC were not well correlated with the bone marrow morphology in the cases of megaloblastic anemia. This finding was similar to the findings of Bhatiya et al where they conducted a study in 117 patients of vit- B12 deficiency to see the usefulness of MCV in cases of vit-B12 deficiency and concluded that the MCV as a screening parameter in cases of macrocytic anemia may be misleading.[8] Increased marrow cellularity with decreased M/E ratio, presence of megaloblasts, features of dyserythropoiesis and giant myeloid precursor were the diagnostic features in cases of megaloblastic anemia in the present study and were also mentioned as diagnostic features of megaloblastic anemia in other studies.[7] Aplastic anemia was the second most common cause of pancytopenia in the present study. Similarly, it was the second most common cause of pancytopenia in different studies conducted by Devid et al in Zimbabwe[5] and Khunger et al in India[3]. However; it was the most common

cause of pancytopenia in studies conducted by Gupta et al[9] and Tariq et al[1]. No definite etiological factor was found in the cases of aplastic anemia. Increased incidence of aplastic anemia may be due to environmental factors like increased exposure to toxic chemicals like pesticides, hydrocarbons from motor vehicles, viral infection, radiation exposure, use of toxic ban-drugs by quacks or some genetic factors.[10] Cases of aplastic anemia were diagnosed solely upon the findings of bone marrow examination; however clinical features helped us to reach our diagnosis. Diluted smears with increased marrow fat along with severe reduction in all the lineages and gelatinous marrow transformation were the features favoring aplastic anemias; which were also mentioned in other studies similar to our study.[11] Increased marrow iron in cases of aplastic anemia was due to repeated blood transfusion and previously received iron therapy for anemia. Acute leukemia was the third most common cause of pancytopenia in the present study. The cases of ALL and AML presented with pencytopenia were diagnosed in bone marrow smears with the presence of >20 lymphoblasts and myeloblasts respectively. Peripheral blood smears of these cases showed pancytopenia with presence of occasional blasts. The findings were similar to the findings described by Shon et al for ALL [12] and Marcovic et al for AML [13] . Pancytopenia in cases of AML could be due to bone marrow necrosis due to immune T cell response in cases of subclinical AML.[13] Leucocyte cytochemistry was helpful in these cases. Acute leukemias including ALL and AML were third most common cause of pancytopenia in the present study which were also an important cause of pancytopenia in other studies.[1,2,14] Dimorphic anemia due to mixed deficiency was the next common cause of pancytopenia in our study. It is

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predominantly seen among the women of child bearing age suggesting the nutritional deficiency of the women of that age group. Menon et al and Chen et al also mentioned about the coexistence of megaloblastic and iron deficiency anemias in their studies.[11, 15] Cases of dimorphic anemias showed presence of microcytic hypochromic red cells and hypochromic macrocytes in the peripheral blood smears with normal to decreased reticulocyte count. Bain BJ conducted a study on hypochromic macrocytes and mentioned that an increased hypochromic macrocytosis could be seen in cases having strong drive to erythropoiesis leading to iron deficient erythropoiesis; indicating the possibility of reticulocytosis or dyserythropoiesis present in cases of megaloblastic anemia or myelodysplastic syndrome.[16] All the cases of dimorphic anemia in the present study showed the presence of hypochromic macrocytes which could be due to increased erythropoietic drive in a iron deficient state. Erythroid hyperplasia along with presence of micronormoblasts and megaloblasts were noted in all of the cases. We did not include these cases into the category of megaloblastic anemia because all of these cases had low bone marrow iron content. Other studies also mentioned the similar bone marrow findings in cases of dimorphic anemia.[11,15] Other causes of pancytopenia were lymphoma, multiple myeloma, infectious disease and storage disease which were mentioned as the cause of pancytopenia in different other studies.[1,2, 4-6,9] Severely hypocellular marrow was noted in the case of multiple myeloma with increased lymphoid cells and atypical plasma cells. The finding was similar to the findings described by Medhi et al in 2008.[17] The most common affected age group was between 16-30 years and overall males were more commonly affected than females. However; females were more commonly affected between the ages of 16- 45 years. Megaloblastic anemia and dimorphic anemia due to mixed deficiency was the most common cause of pancytopenia among the females of 16-45 years. The most common presenting features were pallor and weakness followed by dyspnea in cases of megaloblastic anemia and bleeding in cases of aplastic anemia. Other studies also mentioned the most common clinical features to be pallor, weakness and dyspnea in cases of megaloblastic anemia.[2,11,18] Bleeding, fever and dyspnea were the other associated clinical presentation mentioned in other studies in cases of aplastic anemia.[4,18] History of transfusion dependency was also present in 09 out of 12 cases of aplastic anemia. Overall; the bone marrow examination in cases of pancytopenia was diagnostic in 95.31% cases. Diagnostic www.pacificejournals.com/apalm

value of morphological examination of bone marrow in cases of pancytopenia was 55%, 84.26% and 76.5% in the studies done by Imbert et al,[19] Prajuli et al,[20] and Jha et al[21] respectively. All the cases of megaloblastic anemia, aplastic anemia, dimorphic anemia due to mixed deficiency, ALL, AML, multiple myeloma and storage disease were diagnosed by bone marrow examination. Findings of bone marrow examination were not conclusive in two cases of lymphomas and one case of tuberculosis. Bone marrow smears of the cases of lymphomas suggested a possibility of bone marrow involvement by lymphoproliferative disorder and the case of tuberculosis suggested a possibility of granulomatous lesion and only these three cases needed further diagnostic workup.

Conclusion

The present was designed to analyze the underlying pathology, different clinic-hematological features and importance of bone marrow examination in cases of pancytopenia. High percentage of bone marrow examination for pancytopenia in our study may be due to unavailability of specified sophisticated investigations. As a consequence we had to rely on bone marrow examination and morphological features. The most common cause of pancytopenia in our study was megaloblastic anemia and there was no case of myelodysplastic syndrome present in our study; therefore most of the cases were diagnosed only on the bone marrow aspiration smears without the need of topographical examination of hematopoietic cells in bone marrow biopsy, as morphological details are best studied in bone marrow aspiration smears. Our observation supports the morphological examination of bone marrow as an extremely helpful and specific diagnostic tool in the diagnosis of pancytopenia.

Acknowledgements

I want to express my deep sense of gratitude towards my teachers Dr. Mahendra Singh, Dr. P.K Singh, Dr. Asha Agrawal and my friend Dr. Usha Dubey who helped and wished for the successful completion of this work. I am equally thankful to the technical staff Mr. Balram Singh, Mr. B.K Dubey, Mr. G.N Bajpai and Mr. N.P Shukla for their valuable advice and worthy guidance.

Funding None.

Competing Interests None

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Reference

Importance of BM Examination in Pancytopenia

1. Tariq M, Khan N, Basri R, Amin S. Aetiology of pancytopenia. Professional Med J 2010;17(2):252-256 2. Khodke K, Marwah S, Buxi G, Yadav RB, Chaturvedi NK. Bone Marrow Examination in Cases of Pancytopenia. Indian Academy of Clinical Medicine 2001;2(1-2): 55-59 3. Khunger JM, Arulselvi S, Sharma U, Ranga S, Talib VH. Pancytopenia. Indian J Pathol Microbiol 2002;45(3):375-9 4. Bhatnagar SK, Chandra J, Narayan S, Sharma S, Dutta AK. Pancytopenia in children: etiological profile. J Trop Pediatr 2005; 51(4):236-9 5. David G, Allen, Robert H, Ganaidzo, Innocent T, Levy, Lorraine M, Gwanzura, Christine F, Moyo, Alpha F, Mudenge, Boniface F, Kiire, Clement, Mukiibi, Joshua, Stabbler, Sally P, Lindenbaum, John. Pancytopenia in Zimbabwe. American Journal of the Medical Sciences 1999;317(1):22-32 6. Jha A, Sayami G, Adhikari RC, Panta AD, Jha R. Bone marrow examination in cases of pancytopenia. J Nepal Med Assoc 2008;47(169):12-7 7. Khanduri U, Sharma A. Causative and prevelance of megaloblastic anemia. Natl Med J India 2007;20(4):172-5 8. Bhatia P, Kulkarni JD, Pai SA. Vitamin B12 deficiency in India: Mean corpuscular volume is an unreliable screening parameter. The national medical journal of india 2012; 25(6): 336-38 9. Gupta V, Tripathi S, Singh TB, Tilak V, Bhatia BD. A study of bone marrow failure syndrome in children. Indian Journal of Medical Sciences 2008;62(1):13-18 10. Hayat AS, Khan AH, Baloch GH, Shaikh N. Pancytopenia; study for clinical features and etiological pattern of at tertiary care settings in Abbottabad. Professional Med J 2014;21(1): 060-065. 11. Memon S, Shaikh S, Nizamani MA. Etiological spectrum of pancytopenia based on bone marrow

examination in children. J Coll Physicians Surg Pak 2008;18(3):163-7 12. Sohn SK, Suh JS, Lee J, Lee KB. Pancytopenic Prodrome(pre-ALL) of Acute Lymphoblastic Leukemia in Adults : Possible Pathogenesis. The Korean Journal of Internal Medicine 1998;13(1):64-67 13. Markovic SN, Phyliky RL, Li CY. Pancytopenia Due to Bone Marrow Necrosis in Acute Myelogenous Leukemia: Role of Reactive CD8 Cells. American Journal of Hematology 1998;59:74-78 14. Alkhouri N, Ericson SG. Aplastic Anemia: Review of etiology and treatment. In Practice Stragies 1999;46:52 15. Chen SH, Hung CS, Yang CP, Lo FS, Hsu HH. Coexistence of megaloblastic anemia and iron deficiency anemia in a young woman with chronic lymphocytic thyroiditis. Int J Hematol 2006;84(3):238-41 16. Bain BJ, Cavil IAJ. Hypochromic macrocytes: are they reticulocytes? J Clin Pathol 1993;46:963-64 17. Medhi K, Kalita D, Chopra A, Anand M, Raina V, Kumar R. Multiple myeloma presenting with coexisting severe marrow hypoplasia. Indian Journal of Pathology and Microbiology 2008;51(4):543-545 18. Osama I, Haider ZB, Anwer F, Nisar H. Pattern of pancytopenia patients in a gerneral medical ward and a proposed diagnostic approach. J Ayub Med Coll Abbottabsd 2004;16:8-13 19. Imbert M, Scoazec JY, Mary JY, Jouzult H, Rochant H, Sultanc. Adult patients presenting with Pancytopenia. Hematol Pathol 1989;3(4):159-67 20. Parajuli S, Tuladhar A. Correlation of bone marrow aspiration and biopsy findings in diagnosing hematological disorders â&#x20AC;&#x201C; a study of 89 cases. Journal of Pathology of Nepal (2014) Vol. 4, 534 â&#x20AC;&#x201C; 538 21. Pathak R, Jha A, Sayami G. Evaluation of bone marrow in patients with pancytopenia. Journal of Pathology of Nepal (2012) Vol. 2, 265-271

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 06, (Suppl) December, 2016

Original Article Evaluation of Blood Donor Deferral Causes: A Retrospective Study From South India

Mourouguessine Vimal1*, Srinivasan Sowmya1, Anandabaskar Nishanthi2 and Gandhi Ramya1 Department of Pathology, Sri Manakula Vinayagar Medical College and Hospital, Puducherry, India Department of Pharmacology, Jawarhalal Institute of Postgraduate Education and Research, Puducherry, India 1

Keywords: Blood Donation, Donor Deferral, Selection Criteria, Prospective Donors

ABSTRACT Background: Detailed analysis of various causes for deferral of blood donors may help medical personnel to curb the barriers that impede the blood donation. This study aims to evaluate the deferral reasons of a blood donor in our centre and to uncover the hindering factors behind the target of achieving 100% acceptance of blood donors Methods: This is a retrospective record based study over a time period of 4 years from January 2012 to December 2015.Data were collected from the Donor deferral registry with respect to age and Gender. The causes of deferral were categorized as temporary and permanent. Result: A total of 9557 registered donors were screened and 1421 (14.87%) were deferred. Overall, men (1258; 88.5%) were deferred more than the women (163; 11.5%). But women had higher deferral rate (163/236; 69.1%) compared to Men (1258/9321; 13.5%). Temporary deferral (78.7%) was common than the permanent deferral (21.3%). Anemia with low hemoglobin was the commonest cause of temporary deferral (31.5%) in both males and females. Hypertension was the commonest cause of permanent deferral (75.8%) constituting 16.2% of the total deferrals. Conclusion: Ensuring the confidentiality of history taking and examination of donors will help them to answer the questions honestly and to curb the barriers that impede the donation. Defining and educating the donor selection criteria to the community by the medical professionals and awareness and self motivation from the donors are the key factors behind achieving cent percent acceptance of blood donors.

*Corresponding author: Dr Mourouguessine Vimal, No.21, Narmatha street, Vasanth nagar, Muthialpet, Puducherry â&#x20AC;&#x201C; 605003, India Phone: +91 9994083575 Email: drvimalm@gmail.com

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

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Introduction

Blood Transfusion Services (BTS) is the vital part of modern health care system without which efficient medical care is impossible. The reports of National AIDS Control Organisation state that in India the annual rate of blood donation is only 7.4 million against the annual requirement of 10 million units. [1] The statistics from World Health Organisation show that annually there are over 81 million units of blood are collected, but the developing nations contributes to only 39% of this magnitude where around 82% of worldâ&#x20AC;&#x2122;s population is living. [2] The success of screening for blood donor lies in the fact that the balance should be maintained of the fact that neither the donor should pose risk by the donation nor the recipient should be exposed to the risk by receiving it. [3] Further ensuring the safety of the donors by medical examination, their confidence and trust can be won and this increase their future willingness for donation. [4] Deferral of a donor results in loss of atleast one precious unit from a truly willing person. Further the deferral of a first time donor may have a significant impact on his attempt for subsequent donations. Hence detailed analysis of various causes for deferral of blood donors may help medical personnel and doctors to curb the barriers that impede the blood donation. As there is a changing trend in the deferral causes as well as deferral rate in different parts of the globe, this study aims to evaluate the deferral reasons in our center and compare these reasons and rates among different parts of the nation and the world, and tries to uncover the hindering factors behind the target of achieving 100% acceptance of blood donors.

Materials and Methods

This is a retrospective record based study carried out on donors who presented to Blood bank in a Tertiary care

center in southern part of India over a time period of 4 years from January 2012 to December 2015. Donors were selected in our Blood bank based on Drugs and Cosmetic Act 1940 which is supplemented by Guidelines of Directorate General of Health Services guidelines, Ministry of Health & Family Welfare (2003) and National AIDS Control Organisation (NACO). Predonation screening of the donors were done using Donor questionnaire followed by physical examination, Haemoglobin estimation and Blood grouping. Data were collected from the Donor deferral registry with respect to age and Gender. The causes of deferral were categorized as temporary and permanent. All repeat donations were considered as independent attempts and for simplicity sake, they were not segregated from first time donations.

Results

A total of 9557 registered donors were screened during the study period. Out of them, 8136 (85.13%) were selected for blood donation and 1421 (14.87%) were deferred. Regarding the Gender distribution among the donors donated blood, 8063 (99.1%) were males and 73 (0.9%) were females. Overall, Men (1258; 88.5%) were deferred more than the women (163; 11.5%). But Women had higher deferral rate (163/236; 69.1%) compared to Men (1258/9321; 13.5%). Most of the deferred donors were in the age group of 18-30 in both males (63.75%) and females (56.44%). Overall among the deferral, Temporary deferral (78.7%) was common than the Permanent deferral (21.3%). The causes of Temporary and Permanent deferrals and their relative proportions were shown in Tables 3 and 4. Anemia with low hemoglobin was the commonest cause of temporary deferral (31.5%) in both males and females constituting 24.8% of the total deferrals. Hypertension was the commonest cause of permanent deferral (75.8%) constituting 16.2% of the total deferrals.

Table 1:Gender distribution of Registered, Selected and Deferred donors. Donors Males Females Registered Selected Deferred

9321 8063 1258

Table 2: Age group of Deferred Donors and their percentage. MALES Age in years Number of donors Percentage of deferrals <18 18 1.43 18-30 802 63.75 31-40 288 22.89 41-50 125 9.95 51-60 25 1.98 Total 1258 100

236 73 163

Total 9557 (100%) 8136 (85.13%) 1421 (14.87%)

FEMALES Number of donors Percentage of deferrals 0 0 92 56.44 55 33.74 15 9.2 1 0.61 163 100

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Table 3: Causes of Temporary Deferrals and their relative proportions. Cause Low Hemoglobin Alcohol intake Hypotension Drug intake Low Weight Typhoid Tuberculosis Other Infections Jaundice Recent donation Tatooing Vaccination Surgery Menstruation Fever Low age Allergy Snake bite Dog bite Miscellaneous Total

Males 226 226 130 99 42 39 6 24 11 26 12 41 8 0 20 18 14 3 3 13 961

Females 127 0 8 4 4 0 0 2 1 0 0 1 2 7 1 0 0 0 0 6 163

Total Number 353 226 138 103 46 39 6 26 12 26 12 42 10 7 21 18 14 3 3 9 1123

% of Temporary deferrals 31.5 20.2 12.3 9.2 4.1 3.5 0.5 2.3 1.1 2.3 1.1 3.8 0.9 0.6 1.9 1.6 1.3 0.3 0.3 1.2 100

% of total deferrals 24.8 15.9 9.7 7.3 3.4 2.8 0.4 1.7 0.8 1.8 0.8 3 0.7 0.5 1.5 1.3 1 0.2 0.2 0.9 78.7

Table 4: Causes of Permanent Deferrals and their relative proportions. Cause Hypertension Asthma Seizures High risk behaviour Diabetes Heart disease Thyroid disease Total

Males 226 20 10 13 10 5 7 291

Females 3 1 2 0 0 4 1 11

Total Numbers % of Permanent deferrals 229 75.8 21 7 12 4 13 4.3 10 3.3 9 3 8 2.6 302 100

% of Total deferrals 16.1 1.6 0.8 0.9 0.7 0.6 0.6 21.3

Table 5: Comparison of Total Donor deferral rate of similar studies from various countries. Study Bahadur et al[33] Gajjar et al[13] Taneja et al[14] Sareen et al[12] Rehman et al [34] Chauhan et al[2] Rabeya et al[15] Kasraian et al[8] Custor et al[5] Lawson et al[7] Arslan et al[10] Lim et al[29] Present study

Study period 2 years 2.6 years 1 year 3 months 5 years 1.7 years 1 year 1 year 1 year 4 months 5 years 4 years 4 years

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Total donors screened 34373 24062 8700 53950 14347 4138 141820 116,165 57,003 95,317 278401 9557

Total Deferral rate 9 11.16 17.1 22.36 12.4 4.6 5.6 30.9 13.6 10.8 14.6 14.4 14.87

Country India India India India India India Malaysia Iran USA France Turkey Singpore India

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Discussion

Selection of a correct donor is the key for the success of safe Transfusion practice. Effective recruiting guidelines for blood donor can avoid unnecessary wastage of precious blood products time and human efforts. The overall deferral rate (14.8%) of donor in this study is similar to that of similar studies from India and other countries. The increased deferral rate in our studies is because of the stringent pre donor selection criteria in our blood bank and this is reflected in our very negligible seroprevalence rate of transfusion transmissible infections (2.9%) among our blood donors compared to similar other studies. In our study, temporary deferrals (78.7%) were common than the permanent deferral (21.3%). This finding is similar to that of other studies Custer et al[5] (68.5%), Shaz et al[6] (65%) Lawson et al[7] (91.3%) and Kasraian et al[8] (95.5%). In our study, most of deferred donors (56.4%) were of the age group 18-30. Many similar studies, like Lawson et al[7] reported 50%, Radhiga et al [9] reported 59%. But Arslan et al[10] reported more deferral rate in 50 -60 age group. In our study the deferral rate in 50-60 age groups is 2.59% and this is because most of our donors were of younger age group. In this study, females had a higher deferral rate (69.1%) compared to men (13.5%). Similar deferral figures were reported by Kasraian et al,[8] Newman et al, [11] Arslan et al, [10] Shaz et al.[6] But Lawson et al[7] showed though temporary deferral rate is high among females because of increased prevalence of anemia in them, actually there is no gender difference in the permanently deferred donors. Anemia with low hemoglobin was the commonest cause of temporary deferral in our study in both males and females and this was the commonest cause of temporary deferral in most similar studies from India. [1,2,12-13] Most studies [5,14-17] report low hemoglobin as the commonest cause for deferral. This finding supports the fact that in developing countries like India, the prevalence of clinical and subclinical anemia is very high. Anemia is common in females of reproductive age group because of menstrual disorders and pregnancy related iron loss. But anemia in males and post-menopausal females signifies underlying medical illness and warrants proper evaluation and treatment.[18] Implementation of screening programmes and treatment for anemia at community blood centers can reduce the burden of low hemoglobin deferrals and can produce more healthy donors. [19-21] We had set a cut of value of 12.5% for hemoglobin as per NACO guidelines and this can be the reason for deferring many donors whose hemoglobin was between 12 to 12.4 gm%. Thus minimal reduction in the hemoglobin level of 12gm% for accepting atleast female donors can help blood banks to accept many donors who are at this range and they can fulfill the ever rising demand of blood products to some extent. [22]

Frequent donors are at risk of depleted iron stores [23] and donation of one unit of blood results in loss of 236mg of Iron. [24] Hence implementing an iron conservation programme for the blood donors may prevent the depletion of iron stores in frequent donors. [25-27] While anemia remains the most common cause of donor deferral in most donor deferral studies, the cause was different in Lim et al [28] - recent ingestion of medication, Kasraian et al[8] and Maghsoudlou et al[29] – High risk behaviour, Arslan et al[10] – Sore throat/Common cold, Cheraghali et al[30] – Hypotension, Reikvam et al[25] - Intercurrent illness. The second most common cause remained diverse among different studies depending on the community based demographical factors. In our study uncontrolled Hypertension (16.11%) remained the second most common cause of deferral and most common cause of permanent deferral and it constituted to 75.8% of the total permanent deferrals. This is because most of our donors are from the rural community and they lack awareness about signs and symptoms of hypertension and seldom goes for annual checkup. Girish et al[31] reported highest hypertensive donor deferrals (39.95%) and Gajjar et al, [12] Taneja et al, [13] Bahadur et al, [32] Sundar et al[16] also came with similar findings. This should be taken as alerting signal to initiate the need for starting a screening programme at a community level to detect this underdiagnosed epidemic which is often called as a “Silent Killer”. Rehman et al[33] reported Malaria and Arslan et al[10] reported Common cold to be the second most common cause of deferral and Bobati et al, [34] Bahadur et al, [32] Shah et al[35] reported alcohol consumption within 72 hours was the second leading cause of deferral. However in our study this was the third most common cause contributing to 15.9%. The incidence of alcohol consumption has risen in the past and almost 11% of the total Indian population is indulged in binge drinking. [34] This has to be viewed seriously and steps has to be taken at the root level inculcating moral values to the children and educating the adult population about the ill effects of alcohol. 138 (9.7%) of our donors were deferred because of hypotension. Hypotension and bradycardia can occur as a complication of blood donation as a result of vasovagal reaction. [36] Though there is no current evidence that hypotensive donors have higher risk of adverse events, [37] because of higher incidence of syncope attacks following blood donation may have negative impact on a donor and the relatives about blood donation. Intake of medications either for an acute or chronic illness constituted 7.3% of our deferred donors and nearly half of them were taking native medications for chronic diseases.

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Vimal et al. Unnikrishnan et al[38] reported a highest percentage of medication related deferrals (15.15%). We deferred 3.4% of our donors because of low weight and this denotes the poor nutrition and health status of the donors from low socio economic status. Among the total number of deferred donors, 1.3 % were because of low age and they were encouraged by us to come for blood donation after attaining 18 years of age. This indicates that there is enthusiasm among the younger population about blood donation, but the awareness about the criteria for blood donation is lacking in them. Hence steps must be taken to bridge the gap between enthusiasm and knowledge. We had deferred 1% of our donors with recent history of tattooing. Tatooing has been associated with higher magnitude of transfusion transmissible association because of its association with drug addiction and high risk behaviour. [34] In India, the deferral period for Tatooing is 1 year. But the American Red cross criteria accepts tattooed donor if sterile needle and ink was applied during the procedure. [8] But in developing countries like India, this may not be applicable because the medical personnel cannot judge the sterility of the needle used and often have difficulty in judging the time duration of the tattooing based on the donor’s statement. Other causes for temporary deferral were Infections including tuberculosis, typhoid, recent donation within 3 months, jaundice, Vaccination, recent major surgery, menstruation, allergy, snake and dog bites. Miscellaneous causes which constituted minor percentage were recent smoking, old age and unwillingness for donation. We reported an overall permanent deferral rate of 21.3%. The permanent deferral rate in various other studies were - Rehman et al[33] – 36.3%, Custer et al [5] – 10.6%, Arslan et al[10] – 10%. Seizures, chronic heart disease, thyroid diseases and asthma contributed to the other major causes of permanent deferrals in our study which was comparable with these studies.

A-609 Though the donor deferral rate of this study is similar to that of other studies, the individual causes and their proportions are different and this is because of variation in the demographic profile of the donors and varying donor selection criteria among different centres. There should be a regular follow up and efficient management of temporary donors and they can be compensated for the increasing demand of the blood donors.

Conclusion

To meet the ever rising demand for blood products, blood banks should effectively recruit new donors and maintain the existing donors. Though donor education alone cannot produce better donor educating the community about the donor selection criteria and its significance and curbing the myths and social stigma about blood donation may help to reduce the deferral rate to a great extent. Also ensuring the confidentiality of history taking and examination of donors will help them to answer the questions honestly and to curb the barriers that impede the donation. Similar studies at different parts of the nation with a large sample size will help to identify the deferral rate and its root cause at a national level. Defining and educating the donor selection criteria to the community by the medical professionals and awareness and self-motivation from the donors are the key factors behind achieving cent percent acceptance of blood donors.

Funding None

Competing Interests None Declared

References

Kasraian et al[8] found a higher deferral rate among first time donors (48.1%) when compared to repeated donors(13.1%). This may be because repeat donors will have more awareness about blood donation screening criteria and take more precautionary steps before coming to donate blood. On the contrary there is a hidden danger of hiding the true history to avoid being rejected.

1. Sareen R, Gupta GN, Dutt A, Sareen R, Gupta GN, Dutt A. Donor awareness: key to successful voluntary blood donation. F1000Research 2012. 1;29 2. Chauhan D, Desai K, Trivedi H, Agnihotri A. Evaluation of blood donor deferral causes: a tertiarycare center-based study. Int J Med Sci Public Health. 2015;1. 3. Van der Bij AK, Coutinho RA, Van der Poel CL. Surveillance of risk profiles among new and repeat blood donors with transfusion-transmissible infections from 1995 through 2003 in the Netherlands. Transfusion (Paris). 2006;46(10):1729–36. 4. Nguyen DD, Devita DA, Hirschler NV, Murphy EL. Blood donor satisfaction and intention of future donation. Transfusion (Paris). 2008 Apr;48(4):742–8. 5. Custer B, Johnson ES, Sullivan SD, Hazlet TK, Ramsey SD, Hirschler NV, et al. Quantifying losses

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As most of the deferrals in our study is because of temporarily modifiable factors, the deferred donors were counselled regarding the cause for deferral and they were encouraged to return back for blood donation after correcting the deferred cause.

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Cent Percent Acceptance of Blood Donors: Mission or Mirage ?

to the donated blood supply due to donor deferral and miscollection. Transfusion (Paris). 2004 Oct;44(10):1417–26. 6. Shaz BH, James AB, Hillyer KL, Schreiber GB, Hillyer CD. Demographic variations in blood donor deferrals in a major metropolitan area. Transfusion (Paris). 2010 Apr;50(4):881–7. 7. Lawson-Ayayi S, Salmi LR. Epidemiology of blood collection in France. Eur J Epidemiol. 1999 Mar;15(3):285–92. 8. Kasraian L, Negarestani N, Kasraian L, Negarestani N. Rates and reasons for blood donor deferral, Shiraz, Iran. A retrospective study. Sao Paulo Med J. 2015 Feb;133(1):36–42. 9. Radhiga ST, Kalpana S, Selvakumar, Natarajan MV. Evaluation of Deferral Causes Among Voluntary Blood Donors in Chennai –A Retrospective Study. Int J Med Health Sci. 1357925221;2(1):42–7. 10. Arslan O. Whole blood donor deferral rate and characteristics of the Turkish population. Transfus Med Oxf Engl. 2007 Oct;17(5):379–83. 11. Newman B. Blood donor suitability and allogeneic whole blood donation. Transfus Med Rev. 2001 Jul;15(3):234–44. 12. Gajjar H, Shah FR, Shah NR, Shah CK. Whole blood donor deferral analysis at General hospital blood bank–A retrospective study. Medicine (Baltimore). 2014;235:6–13. 13. Taneja K, Bhardwaj K, Arora S, Agarwal A. Analysis of the reasons for deferral of prospective blood donors in a Tertiary Care Hospital in North India. J Appl Hematol. 2015;6(4):154. 14. Rabeya Y, Rapiaah M, Rosline H, Ahmed SA, Zaidah WA, Roshan TM. Blood pre-donation deferrals--a teaching hospital experience. Southeast Asian J Trop Med Public Health. 2008 May;39(3):571–4. 15. Charles KS, Hughes P, Gadd R, Bodkyn CJ, Rodriguez M. Evaluation of blood donor deferral causes in the Trinidad and Tobago National Blood Transfusion Service. Transfus Med. 2010 Feb 1;20(1):11–4. 16. Sundar P, Sangeetha SK, Seema DM, Marimuthu P, Shivanna N. Pre-donation deferral of blood donors in South Indian set-up: An analysis. Asian J Transfus Sci. 2010 Jul;4(2):112–5. 17. Agnihotri N. Whole blood donor deferral analysis at a center in Western India. Asian J Transfus Sci. 2010;4(2):116.

18. Mast AE. Low hemoglobin deferral in blood donors. Transfus Med Rev. 2014;28(1):18–22. 19. Radtke H, Tegtmeier J, Röcker L, Salama A, Kiesewetter H. Daily doses of 20 mg of elemental iron compensate for iron loss in regular blood donors: a randomized, double-blind, placebo-controlled study. Transfusion (Paris). 2004 Oct;44(10):1427–32. 20. Gordeuk VR, Brittenham GM, Bravo J, Hughes MA, Keating LJ. Prevention of iron deficiency with carbonyl iron in female blood donors. Transfusion (Paris). 1990 Mar 4;30(3):239–45. 21. Magnussen K, Bork N, Asmussen L. The effect of a standardized protocol for iron supplementation to blood donors low in hemoglobin concentration. Transfusion (Paris). 2008 Apr 1;48(4):749–54. 22. Gandhi MJ, Duffy K, Benike M, Jenkins S, Stubbs JR. Effect of increasing hemoglobin cutoff in male donors and increasing interdonation interval in whole blood donors at a hospital-based blood donor center. Transfusion (Paris). 2012 Sep;52(9):1880–8. 23. Mittal R, Marwaha N, Basu S, Mohan H, Ravi Kumar A. Evaluation of iron stores in blood donors by serum ferritin. Indian J Med Res. 2006 Dec;124(6):641–6. 24. Cançado RD, Chiattone CS, Alonso FF, Langhi Júnior DM, Alves R de C. Iron deficiency in blood donors. São Paulo Med J Rev Paul Med. 2001 Jul 5;119(4):132–134; discussion 131. 25. Reikvam H, Svendheim K, Røsvik AS, Hervig T, Reikvam H, Svendheim K, et al. QuestionnaireRelated Deferrals in Regular Blood Donors in Norway, Questionnaire-Related Deferrals in Regular Blood Donors in Norway. J Blood Transfus J Blood Transfus. 2012 Jan 17;2012, 2012:e813231. 26. Røsvik AS, Hervig T, Wentzel-Larsen T, Ulvik RJ. Iron status in Norwegian blood donors: comparison of iron status in new blood donors registered in 1993-1997 and in 2005-2006. Vox Sang. 2009 Jan;96(1):49–55. 27. Røsvik AS, Ulvik RJ, Wentzel-Larsen T, Hervig T. The effect of blood donation frequency on iron status. Transfus Apher Sci Off J World Apher Assoc Off J Eur Soc Haemapheresis. 2009 Dec;41(3):165–9. 28. Lim JC, Tien SL, Ong YW. Main causes of predonation deferral of prospective blood donors in the Singapore Blood Transfusion Service. Ann Acad Med Singapore. 1993 May;22(3):326–31. 29. Maghsudlu M, Makipour M, Nasizadeh S. Evaluation of deferral causes of blood donors and relevant factors. The Scientific Journal of Iranian Blood Transfusion Organization. 2006;3(1):9-16.

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30. Cheraghali A. Overview of blood transfusion system of iran: 2002-2011. Iran J Public Health. 2012;41(8):89–93. 31. Girish CJ, Chandrashekhar TN, Ramesh BK, Kantikar SM. Pre-donation deferral of whole blood donors in district transfusion centre. J Clin Diagn Res. 2012;6(1):47–50. 32. Bahadur S, Jain S, Goel RK, Pahuja S, Jain M. Analysis of blood donor deferral characteristics in Delhi, India. Southeast Asian J Trop Med Public Health. 2009 Sep;40(5):1087–91. 33. Rehman S, Arif SH, Mehdi G, Mirza S, Saeed N, et al. The Evaluation of Blood Donor Deferral Causes: A Tertiary Care Centre-based Study. J Blood Disorders Transf. 2012;3:131. 34. Bobati SS, Basavraj V, Prakash P, others. Analysis of predonation loss of blood donors due to deferrals-in a tertiary care hospital set up. Int J Health Allied Sci. 2016;5(1):15.

35. Shah SD, Shah MC, Bhatnagar NM, Gajjar MD, Soni SA, Patel TR. Analysis of blood donor deferral characteristics in a tertiary care hospital in a Blood Bank – A review. Southeast Asian J Case Rep Rev. 2013;2(5):389–95.

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36. Crocco A, D’Elia D. Adverse reactions during voluntary donation of blood and/or blood components. A statistical-epidemiological study. Blood Transfus. 2007 Jul;5(3):143–52. 37. Pauwels NS, Cusack L, De Buck E, Compernolle V, Vandekerckhove P. The effect of pre-donation hypotension on whole blood donor adverse reactions: a systematic review. J Am Soc Hypertens. 2014 Jun;8(6):429–36. 38. Unnikrishnan B, Rao P, Kumar N, Ganti S, Prasad R, Amarnath A, et al. Profile of blood donors and reasons for deferral in coastal South India. Australas Med J. 2011 Jul 31;4(7):379–85.

Original Article Role of Fine Needle Aspiration Cytology in Diagnosis of Epidermal Inclusion Cysts of Breast: A Clinical and Radiological Dilemma

Richa Bhartiya1*, Sujata Mallick2, Mahasweta Mallik1, Nawanita Kumari1 and Ran Vijoy Narayan Singh3 Dept. of Pathology, Patna Medical College & Hospital (PMCH), Patna, Bihar, India 2 Dept. of Pathology, KPC Medical College, Kolkata, West Bengal, India 3 Dept. of Pathology, Vardhman Institute of Medical Sciences (VIMS), Pawapuri, Nalanda, Bihar, India 1

Keywords: Benign breast lesion, Fine Needle Aspiration Cytology, Epidermal Inclusion Cyst.

ABSTRACT Background: An epidermal inclusion cyst is a common benign lesion which can occur anywhere in the body. However, EIC occurring in the breast (EICB) is very rare.This study is aimed to analyze the incidence, clinical factors, cytomorphology with the differential diagnosis and cytohistopathological correlation with diagnostic & treatment options of this lesion & to evaluate the role of Fine Needle Aspiration in its diagnosis. Methods: This study was done for a span of 6 years in Department of Pathology at Tertiary Teaching Hospital. In this study, patients with firm breast lump who had undergone FNAC in this duration were analyzed and the cases with one of the diagnosis of EICB were included. Smears from these cases were re-evaluated by two cytopathologists and clinico-radiological and cyto-histopathological correlation was done. Result: This study included 8 female and 1 male patients with age ranging from 26-60 years. Patients presented with firm breast lump measuring from 0.5 to 2.0 cm in periaerolar region. Radiological findings were suggestive of benign breast lesion in six cases and two infective lesion in female. FNAC was performed &cytodiagnosis of EICB was made. Cytohistopathological correlation was available in eight cases. Conclusion: EIC of breast is a rare entity & these lesions are often mistaken as benign or malignant tumours both clinically &radiologically. However, FNAC plays a pivotal role in providing accurate pre-operative diagnosis.

*Corresponding author: Dr. (Mrs.) Richa Bhartiya, C/o Shri Vinay Kumar Shrivastava, Bungalow No. 882, Railway Officersâ&#x20AC;&#x2122; Colony, Danapur (KHAGAUL) Patna, Bihar (INDIA) 801105 Phone: +91 97714-50000 Email: richabhartiya1972@gmail.com

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Introduction

Epidermoid Cyst of a breast (EICB) is a rare condition which develops due to proliferation and implantation of epidermal elements within a circumscribed space in the dermis.[1]An epidermal inclusion cyst is a benign cyst of Pilosebaceous origin. It is also known as follicular infundibular cysts, epidermal cysts and epidermoid cysts. Epidermal Inclusion Cyst (EIC) may occur anywhere in the body, although they are most prevalent on the face, trunk, neck, extremities and scalp.[1]EICB manifests as palpable lump that is primarily localized in periaerolar region.[2] Published cytological literature on EIC breast is scanty & there are only eleven published reports in the cytological literature.[1, 2, 3, 4, 5, 6, 7] The aim of this study is to analyze the incidence, clinical features, cytodiagnosis with differential diagnosis, cytohistopathological correlation with diagnostic treatment options of this lesion.

Materials and Methods

This is a study over a period of 6 years from Feb 2010 to Jan 2016 which included patients with breast lump, who had undergone FNAC.A total no. of 4260 breast lumps were analyzed. Out of which, 1670 were non-neoplastic in nature on cytological examination.

Among the Non-neoplastic group, benign cystic lesions were 680. Whereas, 14 cases had one of the cytological diagnosis of EICB. However, among this, only 9 lesions were deep-seated, located inside the breast parenchyma. Rest five cases present on the breast skin were excluded from the study. These nine were reevaluated by two Cytopathologists and both clinic-radiological and cyto-histological findings were reviewed. Result There were 8 female and one male (F:M8:1) with age ranging from 26 to 60 years. Patient presented

with breast lump in right side in 6 cases and 3 in left side, size measuring from 0.5 cm to 2 cm firm, mobile and non-tender [7] with tender [2] and sinus discharging pus [2]. There was no change in overlying skin, retraction of nipple or nipple discharge. Contra-lateral breast and bilateral axilla were normal in all patients and there was no history of trauma, previous surgery, breast infection, hormone intake or a family-history of breast disease. No patient had associated EIC at other body-locations. No information regarding weight, Body Mass Index (BMI) and biochemical tests were available. Radiological diagnosis was suggestive of benign breast disease in females(Fig 1). Patients were subjected to FNAC using 22-gauge disposable needle under aseptic condition. Aspirates were pultaceous in all except in two cases where it was purulent. Smears were airdried and stained with Giemsa stain. Cytosmears revealed many anucleatesquames(AS),nucleated benign squamous epithelial cells (NBSC) along with few adipocytes, benign ductal epithelial cells in the clear background in all cases except in case Nos. 3 & 7 (Fig 2). In case Nos. 3& 7, cytosmears showed few anucleatesquames in acute inflammatory background (Fig 3). Cytological diagnosis of EIC was made under light microscopy in all cases except in case Nos. 3& 7 in which it was infected EICB, as shown in Table-1 below. Histopathological correlation was made in eight cases except in one male case (case No. 5) where the patient refused to undergo surgery. Surgical excision of the mass was performed in eight patients. Specimens were submitted for histopathological examination and case Nos. 1, 2, 4, 6, 8 & 9 were diagnosed as EICB (Fig 4) whereas lumpectomy specimen from Case Nos. 3& 7 were diagnosed as infected EICB. Postoperative course was uneventful & patients remained alive without signs & symptoms of recurrence at a follow-up of 24 months whereas in last two cases, it was only 18 & 11 months respectively of follow-up.

Table 1: Detailed Clinical findings with Clinic-radiological &cyto-histological correlations. Case Age/Sex No. 1.

Clinical findings

Cytological findings & diagnosis

Radiological diagnosis

Histological diagnosis

37/F

1.0X1.0 cm firm, mobile & non-tender lump in periareolar area of left breast from 4 years

Thick pultaceous aspirate many anucleated squames and nucleated benign squamous cells against clean background. Cytological diagnosis: EIC

Well-defined, hyperdense mass. Diagnosis: Benign lesion

EICB

28/F

1.5X1.0 cm firm, mobile & non-tender lump in periareolar area of right breast from 2 years

Thick pultaceous aspirate. Many anucleated squames and few clusters of benign squamous cells, few adipocytes with clean background. Cytological diagnosis: EIC

Well-defined, hyperdense mass. Diagnosis: Benign lesion

EICB

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Case Age/Sex No.

34/F

58/F

26/M

60/F

31/F

40/F

29/F

Clinical findings

Cytological findings & diagnosis

1.5X1.0cm firm, mobile & tender pus-discharging sinus in periareolar area of left breast from 2 months 1.0 X 1.0 cm firm, mobile & non-tender lump in periareolar area of right breast from 1 month 1.0 X 0.5 cm firm, mobile & non-tender lump in periareolar area of right breast from one month

Purulent aspirate many polymorphs few histiocytes and many anucleatedsquames. Cytological Diagnosis: Abscess? TB, infected EIC Thick pultaceous aspirate many anucleated squames and nucleated benign squamous cells against clean background. Cytological diagnosis: EIC Thick pultaceous aspirate many anucleatedsquames and nucleated benign squamous cells against clean background. Cytological diagnosis: EIC Thick pultaceous aspirate many anucleated squames and nucleated benign squamous cells against clean background. Cytological diagnosis: EICD/D: FA/FCD with SM, MCa Purulent aspirate many polymorphs few histiocytes & many anucleated squames. Cytological Diagnosis: Abscess? TB, infected EIC Thick pultaceous aspirate many anucleatedsquames and nucleated benign squamous cells against clean background. Cytological diagnosis: EIC Thick pultaceous aspirate. Many anucleatedsquames and few clusters of benign squamous cells, few adipocytes with clean background. Cytological diagnosis: EIC

1.5 X 1.0 cm firm mobile and non-tender lump inperiareolar area of right breast from 1 month 1.0 X 1.0 cm firm mobile and tender pus discharge sinus in right breast from 15 days with past history of ATT. 1.0 X 1.0 cm firm, mobile & non-tender lump in periareolar area of left breast from 4 years 1.5 X 1.0 cm firm, mobile & non-tender lump in periareolar area of right breast from 2 years

Radiological diagnosis

Histological diagnosis

Well-defined mass with mixed density. Diagnosis: ?Benign Lesion

Infected EICB

Well-defined, hyperdense mass. Diagnosis: Benign lesion

EICB

Well-defined hyper-dense mass Diagnosis Benign lesion

EICB

Well-defined mass with mixed density. Diagnosis: ? Benign Lesion Well-defined, hyperdense mass. Diagnosis: Benign lesion Well-defined, hyperdense mass. Diagnosis: Benign lesion

Infected EICB

EICB

Fig. 1: Mammogram (Case 1). Left cranio-caudal (left) and medio-lateral oblique (left) shows solid cystic soft tissue density mass lesion in periareolar region.

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Discussion

EIC of breast is an uncommon lesion. Only a few casereports have been described in the literature.[1] To date, the international literature has reported 90 cases of patients who have been affected by EIC of the breast.[8]To the best of our knowledge, the first histologically definedcase of EIC of the breast was reported in December 1990 at John Hopkins Hospital, Baltimore, MD, USA.[9]

Fig. 2: Cyto-smear shows many anucleate squames and nucleated benign squamous cells (Giemsa x100).

Fig. 3: Cyto-smear shows many anucleatesquames and a fewnucleated benign squamous cells in a background containing neutrophils and fewbenign ductal epithelial cells (Giemsa x400).

Fig 4Section showing a cyst cavity lined by keratinzed squamous epithelium and contents of cyst comprising of lamellated keratin (H&E x100).

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Several hypothesis have been postulated regarding their etiology within the breast.[4] They can develop due to variety of mechanisms that may result in damage to epidermis which gets implanted within the breast tissue and it can occur after trauma, surgical changes, like core needle biopsy or reduction mammoplasty or developed following squamous metaplasia(SM)of normal columnar cells within an ecctatic ducts in fibroadenoma(FA), fibrocystic disease(FCD)or phyllodestumour(PT).[4, 10, 11] In our study, the possible etiology appears to be obstruction of hair follicle as there was no history of previous trauma, surgery or lump in breast.EIC of breast may be congenital, arising from the cell nests that remain from specific cells including the embryonic mammary ridge. EIC of breast may also develop from obstructed hair follicles. FNAC aspirate of EIC yield pultaceous material &cytosmears reveal numerous anucleate squamous as well as nucleated benign squamous cells in case of superadded infection, aspirate become usually turbid and shows inflammatory cells such as neutrophils and histiocytes. Radiologically on mammography well circumscribed non-calcified mass with homogeneous increased density was noted(Fig 1). Ultrasonography shows solid well circumscribed and complex mass.[4] An â&#x20AC;&#x153;onion ringâ&#x20AC;? appearance, with alternating concentric hyperechoic and hypoechoic rings corresponding to the multiple layers of laminated Keratin in EICB.[12] They are often confused clinically and radiologically with any of the benign or malignant lesions of the breast and accurate preoperative diagnosis may be difficult as breast consists of flexible fat and mammary gland tissue under its skin.[12] EIC in Breast grows deep inside the subcutaneous tissue making difficult to differentiate with breast tumours. Kapila et al in their study found five cases of EIC, in 424 benign breast aspirates from 651 males over a period of 22 years. Cyto-histological correlation was not available in any of the cases.[6] Lilleng et al found only one case of EIC in 779 histologically documented benign diagnoses from surgical biopsies of male breast masses over a 10 year period.[7] Das et al reported three cases of EIC/ pilar cysts in fine needle aspirates from 188 males with eISSN: 2349-6983; pISSN: 2394-6466

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breast lesions, only one of the three had histopathology available; it indicated a pilomatrioxma.[5] Singh et al in their study reported six cases of EICB in 2 years duration with histological correlation in five cases.[2] In present study, we reported nine cases of EICB in 6 years duration with histological correlation in eight cases. EIC yields a dirty whitish aspirate, which on smears shows numerous AS or NBSC better seen on Giemsa stain. Due to the intraparenchymal nature of the lesion, sometimes adjacent normal breast epithelial cells and adipocytes can also be included. Since EICB is rare, diagnosis of squamous metaplasia is Fibroadenoma, Fibrocystic disease, phyllodes tumour and rarely metaplastic carcinoma (MCa), depending upon the age of the patient, is also kept as differential diagnosis(D/D) in such lesions.[2] In present study, differential diagnosis included Fibrocystic disease (Case 1, 2, 8 & 9) due to fluid aspirate and presence of a few benign epithelial cells. Breast abscess (Case 3) due to purulent aspirate and tuberculous breast abscess (Case 7) due to purulent aspirate and past history of ATT. Carcinoma Case (4, 6) since the patient was old and gynaecomastia (Case 5). EICB can cause severe complications as spontaneous rupture leading to release of non-absorbable Keratin, which acts as an irritant and subsequently leads to secondary foreign body reactions, granulomatous reactions or abscess formation. Another rare but important complication is malignant transformation into squamous cell carcinoma. Malignant change occurs more in EICB, as compared to EIC at other sites and it may be due to squamous metaplasia of mammary duct epithelium.[2] The reported malignant transformation of cyst wall varies from 0.045% to 19%.[9, 13] Overall, the incidence of EICB is less, reports are variable on its malignant transformation and thus actual percentage is uncertain. Pagets disease can rarely occur in EIC arising from nipple &perinipple epidermis. [14] According to Paliotta et al in their extensive review of literature noted a significant correlation between tumour size and malignant transformation.[8]Small sized (<2 cm diameter), asymptomatic lesions can be followed by imaging and clinically whereas symptomatic ones require surgical excision through an elliptical incision. The removal of entire cyst wall is recommended for histopathological examination to prevent any complication like recurrence or malignant transformation.[15]

Conclusion

EIC of the breast is a rare entity and FNAC plays an important role in providing a quick & definitive preoperative diagnosis. This is being reported to create awareness among clinicians & Pathologists about such

unusual occurrence of a common benign lesion that may potentially be malignant and to reemphasize the role of FNAC in the diagnosis of a breast lesion. However, studies with focus on identification of predisposing factors, which may influence the development of this disease, is advocated.

Acknowledgements NA

Funding None

Competing Interests None Declared

Reference

1. Lam SY, Kasthoori JJ, Mun KS, Rahmat K. Epidermal inclusion cyst of the breast: A rare benign entity. Singapore Med J. 2010;51:e191–4. 2. Singh M, Maheshwari B, Khurana N, Jain S. Epidermal inclusion cyst in breast: Is it so rare? J Cytol. 2012;29:169–72. 3. Sharma S, Pujani M. Epidermoid cyst of breast: A clinical and radiological dilemma resolved by FNAC. J Cytol 2012;29:155-6. 4. Chantra PK, Tang JT, Stanley TM, Bassett LW. Circumscribed fibrocystic mastopathy with formation of an epidermal cyst. AJR Am J Roentgenol. 1994;163:831–2. 5. Das DK, Junaid TA, Mathews SB, Ajrawi TG, Ahmed MS, Madda JP, et al. Fine needle aspiration cytology diagnosis of male breast lesions. A study of 185 cases. ActaCytol. 1995;39:870–6. 6. Kapila K, Verma K. Fine needle aspiration cytology of epidermal inclusion cysts in the male breast. ActaCytol. 2003;47:315–7. 7. Lilleng R, Paksoy N, Vural G, Langmark F, Hagmar B. Assessment of fine needle aspiration cytology and histopathology for diagnosing male breast masses. ActaCytol. 1995;39:877–81. 8. Paliotta A, Sapienza P, D’ermo G, Cerone G, Pedulla G, Crocetti D, DeGori A, DeToma G. Epidermal inclusion cyst of the breast: A literature review. Oncology Letters 2016;11:657-660. 9. Menville JG. Simple dermoid cysts of the breast. Ann Surg. 1936;103:49–56. 10. Morris PC, Cawson JN, Balasubramaniam GS. Epidermal cyst of the breast: detection in a screening programme. AustralasRadiol 1999;43:12-5.

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Bhartiya et al. 11. Davies JD, Nonni A, D’Costa HF. Mammary epidermoid inclusion cysts after wide-core needle biopsies. Histopathology. 1997;31:549–51.

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12. Crystal P and Shaco-Levy R: Concentric rings within a breast mass on sonography: Lamellated keratin in an epidermal inclusion cyst. AJR Am J Roentgenol 184 (Suppl): S47S48, 2005.

13. Cameron DS, Hilsinger RL., Jr Squamous cell carcinoma in an epidermal inclusion cyst: Case report. Otolaryngol Head Neck Surg. 2003;129:141–3. 14. Stephenson TJ, Cotton DW. Paget’s disease in an epidermal cyst. Dermatologica 1987;174:186-90. 15. Phukan JP, Sinha A, Pal S and Sinha R: Cytological diagnosis of epidermal inclusion cyst of breast: A rare benign lesion. J Nat SciBiol Med 2014;5: 460-462.

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Original Article Evaluation of Platelet Indices in Acute Coronary Syndromes and Diabetes Mellitus Mathumithra. T1*, Roopa Urs1, Shreya2, Bhavani S1 and Dhananjay Kotasthane1 1

Department of pathology, Mahatma Gandhi Medical College and Research Institute, Puducherry, INDIA. 2 Mahatma Gandhi Medical College and Research Institute, Puducherry, INDIA. Keywords: Acute Coronary Syndromes, Mean Platelet Volume.

ABSTRACT Background: Acute Coronary Syndrome (ACS) is the most common cause of adult mortality worldwide. Atheromatous plaque with superimposed thrombus is the commonest underlying cause. Platelets play a pivotal role initiating a thrombus, larger platelets being metabolically more active than the smaller ones. Hence the aim is to study the changes in platelet volume indices and platelet count in acute coronary syndromes and diabetes mellitus. Methods: This was a comparative study of 120 people (60 controls, 60 with ACS). ACS patients were subdivided as those with and without diabetes. Blood was collected in EDTA anticoagulated tubes. The platelet indices and platelet count were assayed using 8 part hematology analyzer (HORIBA) and compared among the groups. Result: Mean platelet volume (MPV) and platelet distribution width (PDW) were significantly raised in ACS patients, being higher in ST elevation Myocardial infarction(STEMI) followed by Non-ST elevation Myocardial infarction ( NSTEMI), unstable angina(UA) and controls. The mean values of MPV and PDW were 8.86fl and 15.24% in STEMI, 8.76fl and 14.62% in NSTEMI, 8.17fl and 13.54% in UA, 8.04fL and 12.74% in controls respectively. Plateletcrit and platelet count did not show significant variation among the groups. Both MPV and PDW were higher in diabetic ACS patients (8.66 fl and 15.71% ) than the non-diabetics(8.17 fl and 14.2% ). Conclusion: Patients with acute coronary syndromes and diabetes had higher MPV and PDW compared to the controls. Measurements of platelet volume indices may be of some benefit in detecting those patients at higher risk for acute coronary events.

*Corresponding author: Dr. Mathumithra.T, No.89, High school road, Kirumampakkam, Puducherry, 607402 India. Phone: +91 8870181486, 0413-2611123 Email: madhumithra59@yahoo.com

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Introduction

Thrombosis and its complications like embolism are a global pandemic contributing significantly to adult mortality worldwide.[1] In the new era of non-communicable diseases, the problem of coronary artery disease(CAD) and its related emergency like acute coronary syndromes(ACS) balloons out huge afflicting significantly a large proportion of Indians.[2] The spectrum of ACS includes unstable angina (UA), Non-ST Elevation Myocardial infarction (NSTEMI), ST Elevation myocardial infarction (STEMI). The most common cause underlying ACS is a ruptured or complicated atherosclerotic plaque with a superimposed thrombus.[3,4] It is well known that platelets are activated along with the coagulation cascade at the onset of thrombosis. During activation, the platelets change their morphology, develop pseudopodia and release a number of substances enhancing the formation of thrombus. Larger platelets are haemostatically more active and are a risk factor for developing coronary thrombosis, leading to myocardial infarction. Platelet indices also vary in diseases like diabetes, hypertension, obesity, hyperlipidemia etc. [5] Hence it is wise to study the distribution of platelet indices in patients with established clot activation and analyze whether they change significantly in comparison to healthy adults. Hence our aims were to study and compare the distribution of platelet count and platelet indices OO

In patients with acute coronary syndromes, unstable angina (UA), Non-ST Elevation Myocardial infarction (NSTEMI), ST Elevation myocardial infarction(STEMI)) in comparison to the age and sex matched controls. In diabetic ACS patients Vs non-diabetic ACS patients

Materials and Methods

2. Patients more than 18 years of age Exclusion Criteria: Patients with bleeding diathesis, major operations or trauma (in past two weeks), malignant hypertension (>180/110mm of Hg). Patients presenting with Acute Coronary Syndromes within 24 hours were inclued. All subjects (both cases and controls) were inter-viewed as per the pre-prepared proforma. The blood samples of the patients on admission (before treatment) were collected as a part of emergency protocol and sent for complete blood count(CBC) and cardiac marker analysis. Samples for CBC were collected in EDTA coated vaccutainers. The sample was run within two hours of venepuncture using the 8 part differential automated Hematology analyzer (HORIBA) and complete blood count analysis of the sample was made including the platelet indices (MPV, PDW, Plateletcrit). Relevant investigations like electrocardiogram and cardiac enzymes (Creatine Kinase-MB and Troponin T) were analysed for confirmation of the diagnosis. Trop T Sensitive kit was used as an aid in the diagnosis of myocardial injury. Detection of rise or fall in cardiac biomarker Troponin T with at least one value above 99th percentile of upper limit was considered diagnostic. All parameters were compared between the two groups of cases and controls. Statistical Analysis: The categorical variables were expressed as frequencies or percentages. Quantitative variables were expressed as mean Âą standard deviation. The means of continuous variables were compared using independent samples t-test (between two groups ACS and controls, diabetics and non-diabetics). One way ANOVA was used for comparing the continuous variables among the various subgroups of ACS. All the tests were twotailed; p<0.05 was considered statistically significant.

Result

A prospective comparative hospital-based study was carried out on 60 ACS patients and 60 matched controls for a period of three months (from July 2016 to September 2016). All patients diagnosed with ACS were included in the study and compared with the controls. Sex and age matched non-diabetic patients admitted in the hospital for other non-thrombotic diseases with no cardiac symptoms, no history of ischemic heart disease and normal electrocardiogram were included as controls. The study was approved by the Institute Ethics Committee.

Of the 60 cases, 46 were males and 14 females. The mean age of patients was 57Âą11.6years. Among the ACS patients, 13(21.6%) had unstable angina, 20(33.3%) had NSTEMI and 27(45%) had STEMI. 38/60(63.3%) patients had diabetes mellitus. The cardiac markers, Troponin T was increased in 26 (43.3%) patients and CKMB in 31(51.6%) patients. The most common risk factor for ACS syndromes, next to diabetes was hypertension followed by smoking. 34(56.6%) patients were hypertensives and 23(38.3%) were smokers.

Inclusion criteria 1. Patients diagnosed with unstable angina (UA), ST segment elevation myocardial infarction (STEMI), non-ST segment elevation myocardial infarction (NSTEMI) irrespective of the diabetic status.

The platelet count, Mean platelet volume (MPV), Platelet distribution width (PDW) and Platelet large cell ratio (P-LCR) were studied among patients with ACS and compared with age and sex matched controls. The mean of MPV and PDW for the control group, for unstable angina,

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for STEMI & NSTEMI is shown (Table 1). The MPV(in fl) was highest in ST-Elevation Myocardial Infarction group (8.86±0.96)followed by Non-ST-Elevation Myocardial Infarction(8.76±1.04) and unstable angina (8.17±0.77) when compared to the control group(8.04±.59) and this was statistically significant (p-0.000).Similarly, the PDW (in %) was highest in ST-Elevation Myocardial Infarction group (15.24±2.295)followed by Non-ST-Elevation Myocardial Infarction(14.62±2.14) and unstable angina (13.74±.89) when compared to the control group(12.54±0.81). The p value was 0.000.

Similarly platelet count and plateletcrit was compared among the groups .The platelet count was lowest in STEMI compared to the NSTEMI, unstable angina and controls but was not statistically significant (Table 1). In contrast, Plateletcrit was lower among the controls than the cases. The ACS patients were subcategorized as those with and without diabetics and their platelet indices were compared and also with the controls. Among the indices, MPV and PDW was higher in the diabetics compared to the non-diabetics and controls (p value-0.000) whereas the platelet count and plateletcrit failed to prove such significance (Table 2).

Table 1: Distribution of platelet indices. Parameters

Controls (n=60)

Unstable angina (n=13)

NSTEMI* (n=20)

STEMI** (n=27)

p value (among Cases 4 groups) (n=60)

Platelet count(lakhs/cmm)

2.8±1.06

2.9±0.5

2.87±0.91

2.6±0.71

0.791

2.78±0.75

MPV(fl)

8.04±0.59

8.17±0.77

8.76±1.04

8.86±0.96

0.000

8.68±0.97

PDW(%)

12.54±0.8

13.74±.897

14.62±2.14

15.24±2.295 0.000

14.7±2.07

PLCT(%)

0.22±0.07

0.24±0.07

0.26±0.08

0.25±0.06

0.25±0.076

0.2

*- Non-ST segment elevation Myocardial Infarction.; **- ST segment elevation Myocardial Infarction.

Table 2: Distribution of platelet indices. Parameters

Controls (n=60)

Diabetic ACS* (n=38)

p value Non-diabetic ACS* p value (among 2 groups) (n=22) (among 3 groups)

Platelet count (lakhs/cmm)

2.84±1.06

2.67±0.56

0.31

2.98±0.99

0.43

MPV(fl)

8.04±0.59

8.66±0.94

0.000

8.17±1.04

0.000

PDW(%)

12.54±0.81

15.71±1.9

0.000

14.2±2.3

0.000

Plateletcrit(%)

0.22±0.07

0.24±0.06

0.17

0.27±0.09

0.06

*- acute coronary syndromes.

Discussion

The burden of cardiovascular diseases is increasing globally, accounting for approximately 12 million deaths annually.[6] Though multifactorial in etiology, many risk factors are strongly associated with ischemic heart disease (IHD) and henceforth atherosclerotic plaque formation, during which platelets play a vital role. Studies prove larger platelets have more prothrombotic activity, because of more dense granules.[7] In such a scenario (Acute coronary syndromes in our study) it is logical to reason out the increase in MPV and PDW. Inspite of the small sample size of our study, our results corroborate findings in previous studies emphasizing MPV and PDW as hematolological markers to assess thrombotic risk. In our study, MPV and PDW was highest among the MI patients compared to those of unstable angina and age and sex matched controls. Studies in literature support the same. In a study by Manchanda et al, [6] MPV and PDW was highest in NSTEMI, followed by STEMI and unstable angina. In our study no significant reduction in

plateletcrit was obtained. In a study by Costa et al,[8] similar findings were obtained whereas a study by Pipliwal et al[9 ] showed significant lowering of plateletcrit in ACS patients compared to the controls. Platelet large cell ratio is yet another controversial platelet index measured only in some hematology analyzers. Discordant results were obtained in literature search, with few studies demonstrating increase in P-LCR in ACS patients with reference to the controls and others showing nil significance.[6,9,10] Though controversial, platelet count was evaluated along with the platelet indices in most such studies. Our study showed no significant difference in platelet count between the controls and ACS patients and among the sub-categories of ACS. A study by Assiri et al[11] shows similar results whereas many studies also show significant lowering of platelet count.[8,9, 12] Most studies show MPV as a reliable marker of predicting cardiovascular risk and also the treatment outcomes. [5,13,14] Chu et al. present a systematic review and a metaanalysis emphasising the value of MPV as a predictor of

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cardiovascular risk.[15] In a study by Slavka et al, MPV is shown to predict the vascular mortality following ischemic heart disease.[16] Another study showed MPV as a predictor of acute stent thrombosis in ACS patients.[14] In our study, follow up of the patients was not done. Inspite of so many studies demonstrating the utility of MPV as a predictive marker, in reality, it is a parameter which is subjected to biological and technical variations.[5]The platelet parameters derived by the automated cell counter are highly specific to the individual technologies developed for each type of analyzer.[5] With impedance counting, the MPV increases over time as platelets swell in EDTA, with increases of 7.9% within 30 min having been reported and an overall increase of 13.4% over 24 hours, although the majority of this increase occurs within the first 6 hours.[17] Conversely when MPV is measured by an optical light scatter system derived from the modal platelet size, the MPV decreases over time, possibly as a result of the dilution of cytoplasmic contents leading to a decrease in light scatter. [5] Second, MPV values can be influenced by type of anticoagulant used and the delay in time from sampling to analysis. In a study by Vagdatli et al,[18] where MPV was recorded hourly for four hours, there was a steady increase in MPV in EDTA anticoagulated blood. The reason is that platelets swell with time in EDTA blood. Hence, by standardizing the time delay between sampling and analysis and by using a alternative anticoagulant (like citrate), MPV can be reliably measured. [5]Over the past 20 years, platelet analyzers have been developed. Additional analyzer derived platelet parameters have been developed, such as the measurement of an immature platelet fraction and platelet large cell ratio, may provide information comparable to the MPV.[19]

indices on the treatment outcome and overall morbidity and mortality was not assessed. Platelet function tests was not done to support the prothrombotic nature of the disease.

Conclusion

We conclude saying MPV and PDW is increased in ACS patients compared to the controls, values being higher in MI group than those with unstable angina. Both the parameters were also higher in the diabetic patients than the non-diabetics. Hence, the results of the present study appear to substantiate that increased MPV can serve as a predictive marker of prothrombotic state in ACS patients.

Acknowledgements

Dr. Dhananjay Kotasthane, Professor and head, Department of Pathology, Mahatma Gandhi Medical College and Research Institute, Puducherry-607 402, India

Funding None

Competing Interests None Declared

References

Our study has some limitations. Owing to the short duration of study period, sample size was small. Follow up of the patients was not done hence implication of the platelet

1. Lippi G, Filippozzi L, Salvagno GL, Montagnana M, Francwhini M et al. Increased Mean Platelet Volume with Acute Coronary Syndromes. Arch Pathol Lab Med 2009;133:1441-3. 2. Gupta R. Burden of coronary heart disease in India. Indian Heart J 2005;57:632-8 3. Obeidi SR, Ahmedm SH, Obeid FA. Evaluation of Platelet Indices in Patients with Acute Coronary Syndrome. Mustansiriya Medical Journal 2013; 12 :58 -64. 4. Kumar V, Abbas AK, Fauselto N, Aster JC. Robbins and Cotran Pathologic Basis of Disease. Eighth edition. New Delhi.2010:547-558. 5. Machin SJ, Briggs C. Mean platelet volume: a quick, easy determinant of thrombotic risk? J Thromb Haemost 2010;8:146â&#x20AC;&#x201C;7. 6. Manchanda J, Potekar RM, Badiger S, Tiwari A.The study of platelet indices in acute coronary syndromes. Annals of pathology and laboratory medicine 2015;2:30-5. 7. Bray PF .Platelet Hyperreactivity: Predictive and intrinsic properties. Hematol Oncol Clin North Am. 2007;21:633â&#x20AC;&#x201C;6. 8. Costa SC, de Matos Vinagre CG, Chacra APM , de Azevedo MRA. Platelet Indices in Patients with Acute Coronary Syndrome. Journal of Biosciences and Medicines 2015;3:71-6.

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In addition to the technical variation, MPV is influenced by a variety of hematological (eg-platelet function disorders, ITP etc) and non-hematological disorders like diabetes, hypertension, COPD, drug intake etc.[5,20,21] Studies prove increased MPV values in patients with diabetes mellitus compared to the controls. Diabetics with complications had a higher MPV compared to those without complications.[22]Our study also proves the same. When these variations are given a thought, appropriate measures taken and then analyzed in special settings like acute coronary syndromes, it will definitely prove a specific and reliable assessement marker.

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9. Pipliwal PS, Singh G, Ishran R, Bansal S. Mean platelet volume and other platelet volume indices in patients with acute myocardial infarction: A case control study. Journal of Dental and Medical Sciences Volume 2015;14: 35-8. 10. Khandekar MM, Khurana AS, Deshmukh SD, Kakrani A L, Katdare A D, Inamdar A K Platelet volume indices in patients with coronary artery disease and acute myocardial infarction: an Indian scenario. J Clin Pathol 2006;59:146–9. 11. Assiri AS, Jamil AM, Mahfouz AA, Mahmoud ZS, Ghallab M. Diagnostic importance of platelet parameters in patients with acute coronary syndrome admitted to a tertiary care hospital in southwest region, Saudi Arabia.J Saudi Heart Assoc 2012;24:17–21. 12. Yaghoubi A, Golmohamadi Z, Alizadehasl A, Azarfarin R. Role of platelet parameters and haematological indices in myocardial infarction and unstable angina. J Pak Med Assoc 2013;63:1133-7. 13. Balli M, Taşolar H, Çetin M, Cagliyan CE, Gözükara MY, Yilmaz M et al. Relationship of platelet indices with acute stent thrombosis in patients with acute coronary syndrome. Postep Kardiol Inter 2015;11:224–9. 14. Ranjith MP, DivyaRaj R, Mathew D et al. Mean platelet volume and cardiovascular outcomes in acute myocardial infarction. Heart Asia 2016;8:16–20. 15. Chu SG, Becker MD, Berger PB, Bhatt DL, Eikelboom JW, Konkle B et al. Mean platelet volume as a predictor of cardiovascular risk: a systematic review and metaanalysis. J. Thromb Haemost 2009;8:148–56.

16. Slavka G, Perkmann T, Haslacher H, Greisenegger S, Marsik C,Wagner OF .Mean Platelet Volume May Represent a Predictive Parameter for Overall Vascular Mortality and Ischemic Heart Disease. Arterioscler Thromb Vasc Biol. 2011;31:1215-8. 17. Bowles KM, Cooke LJ, Richards EM, Baglin T. Platelet size has diagnostic predictive value in patients with thrombocytopenia. Clin Lab Haematol 2005;27:370-3. 18. Vagdatli E, Gounari E, Lazaridou E, Katsibourlia E, Tsikopoulou F, Labrianou I. Platelet distribution width: a simple, practical and specific marker of activation of coagulation. Hippokratia 2010;14:28-32. 19. Briggs C, Kunka S, Hart D, Oguni S, Machin SJ. Assessment of an immature platelet fraction (IPF) in peripheral thrombocytopenia. Br J Haematol 2004;126:96–9. 20. Papanas N, Symeonidis G, Maltezos E, Mavridis G, Karavageli E, Vosnakidis T. Mean platelet volume in patients with type 2 diabetes mellitus. Platelets 2004;15:475-8. 21. Hekimsoy Z, Payzinb B, Ornek T, Kandogan G. Mean platelet volume in type 2 diabetic patients. J Diabetes Complications. 2004;18:173–6. 22. Badiger R, Hosalli A, Ashwin S, Dinesha SV, Abhishek, Vijayalakshmi. Platelet indices in patients of acute coronary syndrome patients in tertiary care hospital. International Journal of Applied Research 2015;1:731-4.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 06, (Suppl) December, 2016

Case Report Papillary Renal Cell Carcinoma Type-II: A Distinct

clinicopathological Subtype of Renal Epithelial Neoplasm Sunil Vitthalrao Jagtap1*, Atul Beniwal1, Swati Sunil Jagtap1 and Anil Huddedar2 Department of Pathology, Krishna Institute of Medical Sciences University, Karad, India Department of Urosurgery, Krishna Institute of Medical Sciences University, Karad, India

1 2

Keywords: Malignant Renal Tumors, Renal Cell Carcinoma, Papillary Tumors.

ABSTRACT Papillary Renal Cell Carcinoma (PRCC) has been recognized as a distinct clinicopathologic subtype of Renal Cell Carcinoma (RCC). Herewith we present a case of 45 year old male patient having complaints of left lower flank pain and hematuria of 1 month duration. Ultrasonography showed large homogenous mass measuring 9.1x9x6.1cm at upper pole of left kidney. Doppler showed mild to moderate vascularity with low resistance to flow. Small cystic areas with calcifications were noted. On radiological finding, diagnosis suggestive of renal cell carcinoma was given. Left radical nephrectomy was done. On histopathology evaluation; diagnosed as PRCC Type-II. As the prognosis of PRCC Type-II is poorer than Type-I, these tumors should be properly evaluated.

*Corresponding author: Dr Sunil Vitthalrao Jagtap. Professor of Pathology, Krishna Institute of Medical Sciences University, Karad-415110, India. Phone: +91 9960628672 E-mail: drsvjagtap@gmail.com

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Introduction

PRCC comprises about 10-20% of RCC and it is distinct morphological entity. [1,2] The term chromophil RCC has been used to describe these tumour. [ 3] PRCC having distinct histologic criteria for its diagnosis and grading which is helpful for clinical outcome of these malignancies when treating the patient.

Case Report

A 45 year old male patient came with history of pain in abdomen and left abdominal palpable mass since 1 month which gradually increased in size. He is tobacco chewer since 15 years. On routine hematological examination Hb-10.5gm%, polymorphs-78%, lymphocytes 20%, eosinophils 2%, platelets-3lacs/cu3. Routine urine examination showed mild hematuria and pyuria. Renal function tests were normal. X-ray chest showed no evidence of metastases. Ultrasonography pelvi-abdomen showed large homogenous mass measuring 9.1x9x6.1cm at upper pole of left kidney. Doppler showed mild to moderate vascularity with low resistance to flow. Small cystic areas with calcifications noted. Radiological diagnosis -suggestive of renal cell carcinoma was given. Other kidney was normal and rest of pelvi-abdominal organs showed no significant abnormality. No any other significant contributory personal, past or family history was there. Left radical nephrectomy was done and specimen was sent for histopathological examination. The postoperative period was uneventful.

700g. Renal surface was irregular, thickened and showed two nodules. Also perinephric fatty tissue at hilum showed three nodular structures measuring approximately 1cm in diameter. Cut section of kidney showed large, circumscribed grey brown to dark tan colored mass with variegated appearance measuring 9x6x5 cm. Areas of hemorrhage, necrosis were prominent ( Figure-1). Tumor was replacing almost all renal tissue and extending over renal capsule, pelvis and perinephric fat. Renal vessels were not involved. Microscopy: Multiple sections showed renal tissue with a tumor composed of predominantly papillary pattern (90%) with solid areas (Figure-2). The papillae were large, discrete forming fronds and lined by one-two layered large round to elongated cells having moderately pleomorphic nuclei and 1-2 prominent nucleoliand abundant eosinophilic cytoplasm (Figure-3). In areas pseudostratification was noted. Papillary core was infiltrated by inflammatory cells. Large areas of hemorrhage, necrosis was noted. Few psammoma bodies were seen. Tumor was infiltrating renal capsule, pelvi-ureter and nodules in the perinephric fat. Lymph nodes were negative for tumor infiltration. The histopathological diagnosis was given as papillary renal cell carcinoma type-II grade-III of left renal mass.

Discussion

Gross: We received left nephrectomy specimen with perinephric fat, totally measuring 15x11x10 cm and weighing

The incidence of RCC is rising worldwide. RCCs on histopathology has been classified into approximately clear cell (70%), papillary (15%), granular cell (8%), chromophobe (5%), sarcomatoid (1-2%) and collecting duct type (0.5%). [3]

Fig. 1: Left nephrectomy specimen, cut section showing large yellow to brown tumor mass with areas of hemorrhage and necrosis.

Fig. 2: photomicrograph showing renal tissue with a tumor composed of predominantly papillary pattern. (H&E stain, x100).

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C-281 Type-II with nuclear grade III according to Fuhrman’s grading. [8] PRCC predominantly shows papillary pattern on histomorphology, however areas tubular, solid and spindle cell (sarcomatoid) component were also reported. Tumors with cytoplasmic features of eosinophilia (42%), basophilia (34%) and mixed (24%) were distingusied . [5] Eosinophilic tumors were predominantly of high grade. As necrosis and phagocytic activity within a tumor become quiet extensive, cytoplasmic clearing in some tumors may cause morphologic confusion with clear cell RCC.

Fig. 3: photomicrograph showing large papillae forming fronds and lined by one-two layered large round to elongated cells having moderately pleomorphic nuclei and 1-2 prominent nucleoli and abundant eosinophilic cytoplasm (H&E stain, x400)

PRCC represent about 10-20% of all RCCs, with male predominance (male: female ratio 2 to 5:1). [4] The mean age of patient presentation is 61.8 years (range 22-83). [5] Various etiological factors includes hereditary, autosomal dominant transmission, heavy metal exposure, drugs, tobacco etc. have been described. Development of multiple tumors and bilateral renal masses are noted in PRCC. [6] In our case the other kidney was normal. Clinically mostly they present with abdominal mass, pain and hematuria. On gross examination PRCC are usually well circumscribed and may show a fibrous pseudocapsule. On cut section tumor appears yellow to brown with large areas of hemorrhage and necrosis, occasional cases shows cystic change. PRCC has been recognized as a distinct clinic-pathologic subtype of RCC. Diagnostic histological pattern given for PRCC is carcinoma of kidney with a predominant papillary pattern. The two types have been identified on the types of cells lining papillae.[7 ] In type-I papillae are lined by small cells with clear to basophilic cytoplasm. The other associate frequent findings are foamy macrophages, edema in papillary cores, few psammoma bodies and glomeruloid papillae and these are usually called low grade PRCC. While in type-II papillae are lined by large cells with abundant eosinophilic cytoplasm. The nuclei show frequently pseudo stratification and prominent nucleoli. These are considered high grade PRCC. Our case was PRCC www.pacificejournals.com/apalm

The differential diagnosis for PRCC is renal adenoma, clear cell RCC with foci of papillary areas, clear cell tubopapillay carcinoma, papillary oncocytoma etc. In papillary adenoma it shows similar morphology and tumor with or without fibrous capsule but size of tumor is 5mm or less by WHO criteria. In case of Clear cell carcinoma with focal papillary architecture the neoplasm have dyshesive tumor areas may have pseudopapillary appearance. Papillary renal cell carcinoma with clear cells is a novel entity with a unique clinical and histopathological features.. The presence of clear cells is associated with aggressive pathological characteristics and poorer prognosis.[9] In case papillary Oncocytoma on microscopy no classic areas of chromophobe carcinoma, no prominent cell membranes, no crinkle or raisinoid nuclei, no binucleation , no abundant microvesicles. These lesions should be kept in mind and carefully evaluated on routine H&E histopathology for proper diagnosis and typing of the tumor. In most of these cases histopathological features are sufficient to make correct diagnosis. The cases of PRCC usually present at an early stage and has better 5 year survival rates (82-90%) than does RCC of the same stage (65-70%).[5] The comparison of the type 1 PRCCs with type 2 PRCCs revealed that type 2 tumors were associated with a greater stage and grade and microvascular invasion significantly. [10] The overall and disease-free survival rate was 89% and 92% in type 1 tumors and 55% and 44% in type 2 tumors, respectively. [10] Our patient received surgical resection and in on regular follow-up and doing well.

Conclusion

The diagnosis of RCC with both papillary architecture and cell with clear cytoplasm can be challenging for pathologist. Histologic subtype of renal carcinoma as PRCC Type-II appears to have poor prognostic implications and therefore it is important to diagnose and grade this tumor for better management of the patients.

Acknowledgements None

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FUNDING

evaluation of conventional pathologic prognostic parameters in 62 cases. Am J Surg Pathol.1997, 21 (6): 621-35.

None

Competing Interests None Declared

Reference

1. Weiss LM, Gelb AB, Medeiros LJ. Adult renal epithelial neoplasm. Am J ClinPathol 1995; 103: 624-63. 2. Amin MB, Tamboli P, Javidan J, et al. Prognostic impact of histopathologic subtyping of adult renal epithelial neoplasm: an experience of 405 cases. Am J SurgPathol 2002; 26: 281-291. 3. Eble JN, Sauter G, Epstein JI, et al. eds. Tumors of the kidneys. Lyon France: International Agency for Research on cancer Press, 2004. 4. Kovas G. Papillary renal cell carcinoma. A morphologic and cytogenetic study of 11 cases. Am J Pathol 1989; 134: 27-34. 5. Amin MB, Corless CL, Renshaw AA, Tickoo SK, Kubus J, Schultz DS.Papillary (chromophil) renal cell carcinoma: histomorphologic characterstics and

6. Henn W, Zwergel T, Wullich B, et al. Bilateral multicentric papillary renal tumors with heteroclonal origin based on tissue specific karyotype instability. Cancer 1993; 72: 1315-1318. 7. Delahunt B, Eble JN. Papillary renal cell carcinoma: a clinicopathologic and immunohistochemical study of 105 tumors. Mod Pathol 1997; 10: 537-544. 8. Fuhrman SA, Lasky LC, Limas C. Prognostic significance of morphologic parameters in renal cell carcinoma. Am J SurgPathol. 1982; 6(7): 655-63. 9. Klatte T, Said JW, Seligson DB, Rao PN, et al. Pathological, immunohistochemical and cytogenetic features of papillary renal cell carcinoma with clear cell features. J Urol. 2011; 185(1):30-5. 10.

Pignot G, Elie C, Conquy S, et al. Survival Analysis of 130 Patients with Papillary Renal Cell Carcinoma: Prognostic Utility of Type 1 and Type 2 Subclassification. Urology.2007;69(2):230-35.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 06, (Suppl) December, 2016

Case Report A Very Rare Occurrence of Synchronous Tumors of Variable Histology in Bilateral Ovaries Gokul Kripesh1*, Sandhya Sundaram1, Palaniappan N2 and Naveen Kumar1 1

Department of Pathology, Sri Ramachandra Medical College, Porur, Chennai, India 2 Dept of OB & G, Sri Ramachandra Medical College, Porur, Chennai, India Keywords: Synchronous, Ovarian Tumours, Immunohistochemistry.

ABSTRACT Introduction: Ovarian tumors make up 30% of all cancers of the female genital system. Surface epithelial tumors of the ovary are by far the commonest variety (90%) followed by germ cell tumors (30%) and sex cord stromal tumors (8%).We present a case of a 46 year old female with bilateral ovarian mass showing two distinct histopathological types of ovariantumor. Case Report: A 46 year old female came with complaints of irregular menstrual cycles, abdominal pain and distension since 8 months. Serum CA125 level was elevated. Ultrasonography revealed a complex cyst arising from the left adnexa. Patient underwent staging laparotomy. Left ovary sent for frozen section which was reported as malignant was a solid and cystic mass measuring 14x12x6 cm with the inner surface showing a solid area measuring 10x10x5 cm. Right ovary measured 5x4.4x4 cm with a 4x4x4cm grey white solid area seen on the cut surface. Microscopy revealed a surprising histopathological picture of a clear cell carcinoma of the left ovary and a granulosa cell tumor of the right ovary which was confirmed by immunohistochemistry. Both the tumors were confined to the ovaries without lymph node involvement. Conclusion: Morphologically different tumors arising from both ovaries is a rare occurrence with there being no case report of a synchronous clear cell carcinoma and granulosa cell tumordocumented in literature.

*Corresponding author: Dr Gokul Kripesh, Room No 27, A block, Gents hostel, Sri Ramachandra medical college, Porur, Chennai- 600116. INDIA Phone: +91 9884167858 E-mail: gokul.kripesh@hotmail.com

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Synchronous Ovarian Tumors

Introduction

Ovarian tumours make up 30% of all cancers of the female genital system. Surface epithelial tumours of the ovary are by far the commonest variety followed by germ cell tumours and sex cord stromal tumours. [1,2] Mixedtumours of ovaries are well documented, however the occurrence of two distinct types of ovariantumours in each of the ovaries is very rare and only few cases have been documented. We present a case of a 46-year-old female with bilateral ovarian mass showing two distinct histopathological types of ovarian tumor

Case Report

A 46 year old female came with complaints of irregular menstrual cycles, abdominal pain and distention since 8 months. Serum CA125 level was elevated. Ultrasonography revealed a complex cyst arising from the left adnexa.Patient underwent staging laparotomy. Left ovary sent for frozen section had a solid and cystic mass measuring 14x12x6 cm with the inner surface showing a solid area measuring 10x10x5 cm.[Fig 1] Microscopy revealed a surprising histopathological picture of a cellular infiltrating lesion in the left ovary with solid, glandular and microcystic spaces lined by atypical tumour cells with clear cytoplasm and features of hobnailing corresponding to the histopathological picture of a clear cell adenocarcinoma. [Fig 3]Following this she underwent a trans abdominal hysterectomy with bilateral salpingo-oophrectomy and pelvic node dissection. Right ovary measured 5x4.4x4 cm with a 4x4x4cm grey white solid area seen on the cut surface.[Fig 2] Histologically it showed a well-defined

Fig. 1: Cut surface of left ovarian mass.

lesion consisting of monotonous population of tumour cells arranged in a trabecular pattern with scant eosinophilic cytoplasm and hyperchromatic nuclei with many cells showing nuclear grooving indicating a granulosa cell tumour.[Figs 6]Immunohistochemistry was done to confirm the diagnosis and to exclude the differential diagnosis of a mucinous adenocarcinoma. The clear cell adeno carcinoma was positive for cytokeratin 7[Fig 4], epithelial membrane antigenand negative for cytokeratin 20 with the background normal ovarian stroma showing positivity for WT-1.[Fig 5] Inhibin immune marker was done to confirm the diagnosis of a granulosa cell tumour which was diffusely and strongly positive. [Fig 7] Both the tumours were confined to the ovaries without lymph node involvement.Patient had no further management and was discharged. The patient is on regular follow up without any complications till date.

Discussion

The incidence of ovarian cancer is second only to uterine and cervical malignancies based on demographical locations.[1,2]Factors responsible for the development of ovarian cancers include excessive estrogen, dietary habits and obesity.[3,4] The tumor promoting role of estrogen in the development of ovarian cancers is of importance which acts through gene transcription causing cell proliferation and differentiation through the estrogen receptors and indirect free radical injury and mutagen production form estrogen metabolism.[5]The role of mutations such as KRAS, BRAF in type-1 and p53 in type-2 ovarian cancers is of prognostic importance.[5] Synchronousprimary tumors if present in

Fig. 2: Cut surface of right ovarian mass.

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Fig. 3: left ovary (H&E): Clear cells with nuclear atypia and hobnailing.

Fig. 4: left ovary: Tumor cells positive for cytokeratin 7.

Fig. 5: left ovary: Tumor cells negative for WT-1 with the background stroma showing nuclear positivity.

Fig. 6: right ovary (H&E): Tumor cells with features of nuclear grooving.

Fig. 7: right ovary: Tumor cells positive for inhibin.

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the female genital tract is usuallyseen in the age group of 25-45 years with the most common being an endometrial adenocarcinoma and a surface epithelial ovarian carcinoma. [6,7] Primary synchronous ovarian tumors of variable histology is a rare presentation with occasional case reports of a serous carcinoma and clear cell adenocarcinoma and a mature teratoma with a mucinous cystadenoma .[8,9] Our case presented with a clear cell carcinoma of the left ovary and a surprising incidental finding of a granulosa cell tumor in the right ovary. Clear cell adenocarcinoma is a malignant tumor which is classified under the surface epithelial tumors of the ovary. Macroscopically the lesion has a mean size of 15cm and may have a unilocular or multiloculated cyst with solid and fleshy nodules. These tumors arise from an endometriotic cyst and may contain chocolate brown fluid. Microscopically the tumor cells are polygonal with abundant clear cytoplasm arranged in sheets or forms the lining of tubulocystic or papillary structures showing features of hobnailing separated by a fibrovascular or hyalinised stroma.Clear cell adenocarcinomas are high grade tumors with poor prognosis. [10]Granulosa cell tumor which is classified under the sex cord-stromal tumors which are composed of granulosa cells in a fibrothecomatous background. They are further subtyped into adult and juvenile granulosa cell tumors based on the age of presentation and cytological features. [11] The histological picture is that of tumor cells arranged in variety of patterns with scant cytoplasm, round to oval nucleus with a nuclear groove along with the typical CallExner bodies. The tumors are hormonally active and can also have atypical clinical presentations and morphology.[12] Granulosa cell tumors have potential for recurrence and the most important prognostic factor is the stage of the tumor. [13] In our case the grading and staging of both the ovarian tumors was done according to the AJCC/UICC TNM, 7th edition, and FIGO 2014.[14]Immunohistochemistry for clear cell carcinoma of the ovary shows positivity for keratins, CA125 and hormone receptors while granulosa cell is positive for inhibin and calretinin.[15]

Conclusion

We document the rare synchronous occurrences of clear cell carcinoma in the left ovary and incidentally detected granulosa cell tumour of the right ovary. This case is documented to highlight the fact that histologically diverse tumours can occur in the ovaries and immunohistochemistry is proven useful in distinguishing them.

Acknowledgements

Dr. J Thanka, Head of the department of pathology, Sri Ramachandra medical college.

Funding None

Competing Interests None Declared

Reference

1. Greenlee RT, Hill-Harmon MB, Murray T, Thun M. Cancer statistics, 2001. CA Cancer J Clin. 2001;51(1):15–36. 2. Levi F, Lucchini F, Negri E, Franceschi S. Cervical cancer mortality in young women in Europe: patterns and trends. Eur J [Internet]. 2000 [cited 2016 Aug 24]; Available from: http://www.sciencedirect.com/ science/article/pii/S0959804900003464 3. Garg PP, Kerlikowske K, Subak L, Grady D. Hormone replacement therapy and the risk of epithelial ovarian carcinoma: a meta-analysis. ObstetGynecol. 1998;92(98):472–9. 4. Calle EE, Rodriguez C, Walker-Thurmond K, Thun MJ. Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults. N Engl J Med. 2003;348(17):1625–38. 5. Mungenast F, Thalhammer T. Estrogen biosynthesis and action in ovarian cancer. Front Endocrinol (Lausanne) [Internet]. 2014 [cited 2016 Aug 24];5(NOV). Available from: http://journal.frontiersin. org/article/10.3389/fendo.2014.00192/full 6. Walsh C, Holschneider C, Hoang Y, Tieu K. Coexisting ovarian malignancy in young women with endometrial cancer. Obstet. 2005;106:693-9 7. Saatli B, Yildirim N, Ozay AC, Koyuncuoglu M, Demirkan B, Saygili U. Synchronous tumors of the female genital tract: a 20-year experience in a single center. Ginekol Pol. 2014;85:441-5. 8. Preeti A, Arunachalam KA, Pradeep Y, Mati GM. Bilateral synchronous high-grade serous carcinoma and clear cell carcinoma in right and left ovaries with immunohistochemical confirmation: an exceptional finding. Indian J PatholMicrobiol. 2014;57:623-5 9. Sule A, Ochicha O, Yusuf I. Bilateral synchronous mature ovarian teratoma and mucinous cystadenoma. Arch IntSurg. 2015;5:174-6 10. Del Carmen MG, Birrer M, Schorge JO. Clear cell carcinoma of the ovary: A review of the literature. Gynecologic Oncology. 2012;126: 481–90.

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11. Koukourakis G V, Kouloulias VE, Koukourakis MJ, Zacharias GA, Papadimitriou C, Mystakidou K, et al. Granulosa Cell Tumor of the Ovary: Tumor Review. Integr Cancer Ther . 2008;7:204–15. 12. Kanthan R, Senger J. The multifaceted granulosa cell tumours—myths and realities: a review. ISRN Obstet. 2012: 878635 13. Ayhan A, Salman MC, Velipasaoglu M, Sakinci M, Yuce K. Prognostic factors in adult granulosa cell

tumors of the ovary : a retrospective analysis of 80 cases. J. 2009;20(3):158–63. 14. Otis CN. Protocol for the Examination of Specimens From Patients With Primary Tumors of the Ovary or Fallopian Tube Protocol applies to all primary borderline and malignant epithelial tumors. 2016 15. Mittal K, Soslow R. Application of immunohistochemistry to gynecologic pathology. Arch Pathol. 2008;132(3):402-23

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Case Report A Case of Peutz-Jeghers Syndrome with Multiple Intussusceptions R. Prathipaa*, J. Thanka, M. Susruthan and Lawrence D Cruze Department of Pathology, Sri Ramachandra Medical College and Research Institute, Sri Ramachandra University, Porur, Chennai, India. Keywords: Peutz-Jeghers Syndrome, Polyp, Intussusception, Jejunum.

ABSTRACT Peutz-jeghers syndrome (PJS) is an autosomal dominant cancer syndrome with variable penetrance and characterised by hamartomatous polyp in gastrointestinal tract with mucocutaneous pigmentation. Approximately one third of PJS patients present in the first decade of life, the rest of them present by second or third decade with equal predilection for both males and females.The most common site of Peutz-jeghers polyp is jejunum. Most of the cases are associated with germline mutations in LKB1/STK11 located on chromosome 19p13.3.The most frequent presentation is intermittent abdominal pain due to intestinal obstruction or intussuception. However intusussception in adults are rare compared to children. Here we present a case of 22 years female who presented with Peutz-jeghers syndrome with multiple intussusceptions.

*Corresponding author: Dr R. Prathipaa, Department of Pathology, Sri Ramachandra Medical College and Research Institute, Sri Ramachandra University, Porur, Chennai, India. Phone: +91 9789819775 E-mail: prathipaa.priya@gmail.com

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Prathipaa et al.

Introduction

In 1921, Johannes Peutz, a dutch physician noted a relationship between intestinal polyps and mucucutaneous pigmented macules. Harold Jeghers , an American physician was the first person to publish the description of the term “Peutz-jeghers syndrome” (PJS). PJS is a rare inherited autosomal dominant cancer syndrome with variable penetrance. Approximately one third of PJS patients present in the first decade of life, the rest of them present by second or third decade with equal predilection for both males and females. It is characterised by hamartomatous polyps throughout the gastrointestinal tract and mucocutaneous pigmentation. Less than 5% of cases with PJS lack abnormal mucocutaneous pigmentation and less than 5% with pigmentation lack polyps. Its incidence is 1 in 300,000 births[1].

C-289 or arborescent- like appearance at low power (fig: 2). The smooth muscle fibres of muscularis mucosa are thick and broad centrally and thin peripherally.The smooth muscle fibres separating rhe glands gives a lobulated appearance in some foci (fig: 3). In high power, epithelium lining the glands are benign looking without any atypia and the diagnosis of Peutz-jeghers polyp was made (fig: 4).

Case Report

22years female presented with complaints of intermittent abdominal pain and abdominal distension for 1week and two to three episodes of vomiting with past history of surgery for ileocolic intussusceptions 10 years back. On examination, pigmentation over mucutaneous junction of lips ,fingers and toes were noted. The patient had severe anemia with haemoglobin of 5.4 gm%. CECT –abdomen showed intussusceptions at various levels involving ileoileal and jejunojejunal segments with partial, subacute intestinal obstruction and few calcified mesenteric lymph nodes. UGI scopy showed multiple gastric and duodenal sessile polyps and biopsy taken from these showed H.Pylori associated chronic gastritis and hyperplastic polyp. Colonoscopy showed pedunculated polyps in rectum, sigmoid colon, descending colon and mucosal thickening noted in caecum. Biopsies were taken and it showed tubular adenoma in rectum and caecum, hyperplastic polyps in descending and sigmoid colon. Patient was taken up for diagnostic laparoscopy which showed multiple intussusceptions at ileoileal, 30 cm from duodenojejunal flexure, jejunojejunal levels. Also multiple adhesions from omentum to small bowel noted. Laparotomy was done. After removing the adhesions, intusussception was reduced followed by small bowel resection and anastomosis. Resected segment sent for histopathological examination. Gross: The resected small bowel segments were sent as two separate specimens. One segment measuring 13.5cm and the other measuring 11.5 cm in length. Thickness of the bowel wall is around 0.5 to 0.8 cm. Both segments showed sessile polyp on mucosal surface measuring 4×3×3 cm and 3×3×2.5 cm respectively (fig: 1) .

Fig. 1: Gross: Segments of small intestine showing polyp.

Microscopy of polyps showed glandular epithelium resting on a tree -like branching smooth muscle framework that arises from muscular mucosa which gives ‘Christmas tree’

Fig. 2: (a,b): 40x H&E- “Christmas tree appearance”Glands resting on branching tree like smooth muscle fibres of muscularis mucosa. The smooth muscle is broad and thick centrally and thin peripherally.

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P-J Syndrome with Multiple Intussusceptions

Fig. 3: 40x H&E-Smooth muscle fibres separating group of glands giving a lobular apperance.

Fig. 4: 100x H&E- Glands shows no atypia.

Discussion

with symptoms of obstruction and abdominal pain whereas those with large intestinal polyps present with bloody stools. However the most frequent presentation is intermittent abdominal pain due to intussusception. Intussusception is rare in adults compared to children. Also the site of intussusception vary in both. In chidren, it involves colon whereas small intestine is the common site in adults. Other rare presenting features include anemia, hematochezia, hematemesis, prolapse of polyp[4]. In a series of 222 patients with Peutz-Jeghers syndrome (PJS), Utsunomiya et al noted the following distribution of presenting gastrointestinal symptoms: Obstruction – 42.8% of patients ,Abdominal pain caused by infarction – 23% of patients , Rectal bleeding caused by ulceration – 13.5% of patients , Extrusion of polyp – 7% of patients.

Peutz-jeghers syndrome, an autosomal dominant syndrome is characterised by hamartomatous polyp involving the GIT and mucocutaneous pigmentation. In GIT, most common site of hamartomatous polyp is small intestine followed by colon and stomach. In small intestine, polyps involve jejunum followed by ileum and duodenum[2]. Apart from GIT, hamartomatous polyps can also occur at extraintestinal sites such as nose, bronchi, gallbladder, bile duct, urinary bladder, ureter. The polyps can have adenomatous component in 16 % of cases. Our patient also had adenomatous component in rectum and caecum. Mucocutaneous pigmentation is characterised by flat , pigmented freckle -like lesions most commonly seen around vermilion border of lips in 95% of cases followed by buccal mucosa in 80% of cases. Other sites include hands, feet, genitalia, around nose and eyes. These lesions are benign and have no malignant potential. The genetic basis for PJS is germline mutations in LKB1/ STK11 located on chromosome 19p13.3. However, up to 25% of PJS have no family history but represent spontaneous mutation. LKB1/STK11 encodes serinethreonine kinase that phosphorylates and activates the members of the AMPK(AMP-activated protein kinase)related subfamily of protein kinases. LKB1/STK11 is a tumor suppressor gene, which plays important role in G1 cell cycle arrest, cell polarity, p53-dependent apoptosis, and cellular energy levels. 80% of patients with PJS had mutations involving this gene. Common mutation are frameshift and nonsense mutations in exons 1–6[3].

At the time of presentation , 3 to 6% of patients with PJS are found to have neoplastic change. They have a 15-fold increased risk of developing intestinal cancer compared with that of the general population[5]. The hamartomatous polyps in PJS can have adenomatous foci that can evolve in to cancer as the age progresses. .The evolution of hamartoma-adenoma-carcinoma has been demonstrated for stomach, small bowel, and colorectal polyps in PJS. The other associated malignancies which occur outside the gastrointestinal tract include lung, breast, uterus, gonads, prostate, thyroid, multiple myeloma, skin, and pancreas. Sex cord tumors with annular tubules and adenoma malignum of uterine cervix in women and testicular sertoli cell tumors in male are other uncommon tumors seen with this syndrome[6].

The clinical presentation depends upon the site of location of polyps. Patients with small intestinal polyp present

The clinical diagnosis of PJS is made if any one of the following criteria is present:

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1. Two or more histologicaly confirmed Peutz -jeghers polyp 2. Any number of Peutz-jeghers polyp with a family history 3. A family history of PJS mucocutaneous pigmentation.

with

characteristic

Acknowledgements

I acknowledge Dr. J. Thanka, Professor and Head, Department of Pathology, Sri Ramachandra Medical College and Research Institute, Sri Ramachandra University, Porur, Chennai, for guiding me in publication of this article.

4. Characteristic mucocutaneous pigmentation with any number of Peutz-jeghers polyp[7].

Funding

The polyps may lack characteristic diagnostic features when ischemic necrosis occurs secondary to intussusceptions. Also in the colon and rectum, prolapse may cause histopathological changes that can mimic peutz-jeghers polyp which can be a diagnostic problem in case of solitary polyps.Epithelial structures in smooth muscle fibres can misled to diagnosis of invasion in cancer.Occasionally, there is herniation of cysticaly dilated epithelial structures in to the bowel wall extending up to serosa and forms tumor masses analogous to colitis cystica and jejunitis cystica profunda. This is known as pseudoinvasion which is reported in 10 % of PJS polyps. Epithelial misplacement can be differentiated from neoplasia under microscopy by the presence of benign looking glands, presence of lamina propria, absence of desmoplastic response around the glands.

Competing Interests

None

None Declared

Reference

We are presenting this case of Peutz-Jeghers Syndrome for its rarity and to highlight the diagnostic challenges in histopathology. The appropriate diagnosis of Peutz-Jeghers polyp will help in proper screening of intestinal and extraintestinal malignancies associated with it, facilitating the early diagnosis and further management.

1. Choudhury T, Enam S, Begum F, Taufiq M, Kamal M. Peutz-Jeghers polyp: a retrospective study on twelve cases received at the department of Pathology, Bangabandhu Sheikh Mujib Medical University. Bangabandhu Sheikh Mujib Medical University Journal. 2012;5(1):12-7. 2. Sreeramulu PN, Agrawal VP. A rare case of multiple intussusceptions in Peutz-Jeghers syndrome. Journal of Medical & Allied Sciences. 2013 Feb 28;3(1):26. 3. Volikos E, Robinson J, AittomĂ¤ki K, Mecklin JP, JĂ¤rvinen H, Westerman AM, de Rooji FW, Vogel T, Moeslein G, Launonen V, Tomlinson IP. LKB1 exonic and whole gene deletions are a common cause of Peutz-Jeghers syndrome. Journal of medical genetics. 2006 May 1;43(5):e18-. 4. McGarrity TA, Amos C. Peutz-Jeghers syndrome: clinicopathology and molecular alterations. Cellular and Molecular Life Sciences CMLS. 2006 Sep 1;63(18):2135-44. 5. Weng MT, Ni YH, Su YN, Wong JM, Wei SC. Clinical and genetic analysis of Peutz-Jeghers syndrome patients in Taiwan. Journal of the Formosan Medical Association. 2010 May 31;109(5):354-61. 6. Hearle N, Schumacher V, Menko FH, Olschwang S, Boardman LA, Gille JJ, Keller JJ, Westerman AM, Scott RJ, Lim W, Trimbath JD. Frequency and spectrum of cancers in the Peutz-Jeghers syndrome. Clinical Cancer Research. 2006 May 15;12(10):3209-15. 7. Bhattacharya S, Mahapatra SR, Nangalia R, Palit A, Morrissey JR, Ruban E, Jadhav V, Mathew G. Melaena with Peutz-Jeghers syndrome: a case report. Journal of medical case reports. 2010 Feb 8;4(1):1. 8. Kopacova M, Tacheci I, Rejchrt S, Bures J. PeutzJeghers syndrome: diagnostic and therapeutic approach. World J Gastroenterol. 2009 Nov 21;15(43):5397-408.

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Surveillance in patients with PJS is done to detect early cancers and to prevent morbidity due to its complications. Hence, periodic surveillance for gastric and small-bowel polyposis should begin at the age of 8-10 years and continue for every 2-year intervals. Screening should include all possible investigations to detect both intestinal and extraintestinal malignancies in all the patients with PJS such as colonoscopy, upper endoscopy, if possible wireless capsule endoscopy, CT, MRI or ultrasound of the pancreas, chest X-ray, mammography and pelvic examination with ultrasound in women, testicular examination in men, carbohydrate antigen 19-9 (CA-19-9), and cancer antigen (CA 125)[8]. Surgical excision of lesions include the following : Endoscopic polypectomy for diagnosis and control of symptoms. Laparotomy and resection are reserved for repeated intussusception or persistent intestinal bleeding.

Conclusion

Case Report A Rare Case of Primary Leiomyosarcoma of Thyroid in a Young Adult with Literature Review Rushabh Jitendra Shah, Rachana Amit Chaturvedi* and Leena Pravin Naik Department of Pathology, Seth GS Medical College & KEM Hospital, Parel, Mumbai, India Keywords: Thyroid Gland, Thyroid Neoplasms, Primary Leiomyosarcoma Of Thyroid, Young Male

ABSTRACT Primary leiomyosarcoma of the thyroid gland (PLT) is an extremely rare neoplasm and still rarer is its occurrence in younger age group. It is one of the most aggressive malignancies with high metastasizing potential. They can be difficult to diagnose on histopathology and can mimic anaplastic or medullary thyroid carcinoma. Only 22 cases of PLT have been reported in the literature and the youngest patient was a 39 years old male. However we report a case of PLT in a 23 years male, being the youngest patient reported till date (23rd in series). He presented with progressively increasing anterior neck swelling since two months and underwent total thyroidectomy. Histopathology and immunohistochemistry showed features suggestive of leiomyosarcoma of the thyroid gland. Two months later, the tumour recurred at subglottis for which he underwent total laryngo-pharyngectomy followed by radiotherapy. Patient was on regular follow up for one and half year with no recurrence and lost to follow up later.

*Corresponding author: Dr Rachana Amit Chaturvedi, 1203 Erica, Dosti Acres, Wadala-East, Mumbai-37, INDIA Phone: +91 9967017267 E-mail: rachanachaturvedi@yahoo.co.in

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Introduction

Primary leiomyosarcoma of thyroid gland (PLT) is an extremely rare neoplasm and it represents 0.014% of primary thyroid cancers. It usually occurs in older individuals with a slight female preponderance and its exact etiology is not known.[1, 2] Two paediatric cases of thyroid leiomyosarcoma have been reported, none of them were primary and one was associated with Epstein Barr Virus. PLT is a malignant mesenchymal tumour arising from smooth muscles of the intraglandular blood vessels and is composed of spindle shaped cells which are positive for smooth muscle markers on immunohistochemistry (IHC). It has aggressive nature with low survival rates and needs to be differentiated from medullary and anaplastic thyroid carcinomas, solitary fibrous tumours (SFT) and other thyroid tumours including metastatic leiomyosarcoma (LMS).[2]Surgery is the main stay of treatment; however prognosis remains poor despite therapy. [1, 2]To the best of our knowledge only 22 cases of PLT are reported till date, and none from India. We hereby report a unique case of PLT in a young male with subsequent recurrence at subglottis.

and TTF-1. Right lobe and isthmus were unremarkable. Thus the diagnosis of PLT was made. Patient was stable in the immediate post-operative period and his routine investigations were within normal limits. He later developed mild vocal cord paresis for which speech therapy was advised. Patient was discharged on fifth postoperative day without any fresh complaints. Subsequently he was referred to a cancer hospital and after an asymptomatic period of two months; follow up CT scans showed a mass in paratracheal and retrotracheal region (figure 2) with no evidence of cervical lymphadenopathy or lung metastases. Subsequently a total laryngo-pharyngectomy was performed and a diagnosis of high grade leiomyosarcoma was made on histopathology for which patient underwent radiotherapy. He did not show any recurrence till 18 months but was lost to follow up later on.

Case Report

A 23 years male presented with progressively increasing neck swelling since two months which recently became painful and was associated with dysphagia, voice hoarseness and weight loss. There was no past history of radiation exposure, surgery or mass elsewhere in the body. Patient did not have signs and symptoms of hypothyroidism or hyperthyroidism. General and systemic examinations were unremarkable. Local examination revealed a hard anterior neck swelling, moving with deglutition with mobile vocal cords on indirect laryngoscopy. Haematological and biochemical investigations, T3, T4, TSH, thyroid antibody, serum calcium and parathyroid hormone, were within normal limits. USG showed a hypoechoic mass in left thyroid lobe. Fine needle aspiration cytology (FNAC) requested was inconclusive due to scanty material showing very occasional lymphocytes without any thyroid follicular cells. Patient underwent total thyroidectomy. Gross examination showed an enlarged left lobe with a firm white mass (figure 1a) which on microscopy showed pleomorphic spindle cells (figure 1b) showing hyperchromatic nuclei, brisk mitosis (5/high power field) with atypical mitotic figures without any necrosis. Focal infiltration of thyroid parenchyma (figure 1c) and skeletal muscle at the periphery was also seen (figure 1d). Multiple sections studied did not show any epithelial differentiation or vascular invasion. On IHC, tumour cells were positive for vimentin, desmin and smooth muscle actin (figure 1e) and negative for pankeratin, chromogranin, Bcl-2, CD 117

Fig. 1: (a)Bosselated left lobe without any capsular breech or soft tissue attachment on external aspect and a well circumscribed, grey white fleshy mass with areas of whorling on cut surface, (b) Tumour cells with eosinophilic cytoplasm, cigar shaped, blunt-ended hyperchromatic nuclei, high nuclear:cytoplasmic ratio and brisk mitosis (inset) (HE x 400); (c) Interlacing fascicles of spindle shaped cells invading the thyroid parenchyma (HE x 100); (d) Invasion of adjacent skeletal muscle fibers (HE x 100); (e) Tumour cells showing strong positivity for desmin and smooth muscle actin (SMA) (x100)

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Primary Leiomyosarcoma of Thyroid except a focus of skeletal muscle invasion seen only on microscopy. Moreover, metastases are usually multiple in numbers while our case showed a solitary lesion confined to the thyroid.

Fig. 2: Elongated mass in retro tracheal region, indenting the posterior tracheal wall and oesophagus (arrow).

Discussion

Thyroid tumours represent most common malignancies of the endocrine system.[1] Thyroid carcinomas are more common while stromal tumours are relatively rare which include smooth muscle tumour, angiosarcoma, peripheral nerve sheath tumour, paraganglioma, SFT, follicular dendritic cell tumour, Langerhans cell histiocytosis and spindle cell tumour with thymus like differentiation. PLT is rare and its differential diagnoses includes other primary tumors like medullary and anaplastic carcinoma, SFT, spindle cell tumor with thymus like differentiation and other primary as well as metastatic sarcomas (specially LMS) from elsewhere in the body. Diagnosis of thyroid sarcoma should only be made when there is a complete lack of all epithelial differentiation with definite evidence of specific sarcomatous differentiation. [3] Our case did not show any epithelial component in spite of extensive sampling and was negative for pankeratin on IHC with strong positivity for smooth muscle actin (SMA), vimentin and desmin, thus favoring the diagnosis of LMS. Nevertheless further IHC done was negative for TTF-1, chromogranin and Bcl-2, thus ruling out the possibility of other stromal tumours. Thyroid gland can be secondarily involved by locoregional spread of LMS arising from head and neck soft tissue or by distant metastasis. Surgical explorations and pathological examinations are indispensable for correct identification of primary site of tumour. It is believed that approximately 1% of thyroid cancers are metastatic and in autopsy series, thyroid metastases are seen in up to 24% of patients of cancer in general.[4] In our case points favoring primary thyroid tumour over metastasis included absence of any significant previous history or mass elsewhere in the body, along with clean resection of the specimen without any adhesions and no capsular breach on gross examination

First case of PLT was described in 1969 by Adachi et al which showed metastasis to the heart and brain.[2]Only 22 cases have been reported in the literature till date, which are summarized in Table 1 along with our case. [1,2,4-9] On analyzing, we can see that more than 50% (n = 12) of patients are females (male: female ratio 3:4). Their ages range from 23 to 90 years, with the mean age of 60.1 years in males and 68.7 years in females. Majority of the patients are in the seventh decade (n=7), only four patients are younger than 50 years of age, our patient being the youngest. Duration of symptoms available ranges from few days to seven months and many have history of rapidly increasing mass (n=10). Primary complaint of neck mass was present in all the patients along with various other symptoms, commonest being hoarseness of voice, dysphagia and dyspnea. Thyroid function tests available in 12 cases[1-2, 4-6]were within normal range as in our case. FNAC details were available in five cases, of which one was suspicious for malignancy[2], two reported as spindle cell/mesenchymal malignancy[1, 6]while two other were inconclusive[5]similar to our case. Both gross as well as microscopic details were available in 12 cases. Grossly tumour ranged in size from 1.9 to 16 cm, more commonly involving the left lobe with either an irregular/well demarcated intra-thyroidal nodule or completely replacing the entire lobe as in our case. Rest of the gross features along with microscopy were also similar except the vascular invasion, which was seen in 5 cases but was not observed in our patient. IHC available in 18 cases, including our case, were positive for vimentin and SMA, with or without desmin positivity (focal or diffuse) and few also showing positivity for caldesmon (not performed in our case) and epithelial markers were negative in all cases. Data regarding metastasis and/or local recurrence was available in 16 cases (Table 1) of which 13 (seven males, five females, gender not known in one) had metastasis, commonest being in lung followed by liver and bone. The age of these patients ranged from 43 to 83 years with variable duration (few weeks to 47 months) between first presentation and occurrence of metastasis. Thus a wide inter-patient variability regarding clinical presentation was noted with no definite age or gender predilection. Also no definite correlation between the tumour size and metastasis was observed as both large (case 19) as well as smaller (case 21) tumours showed metastasis within few

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weeks irrespective of their sizes. Local recurrence was observed in three including our case which showed spread to subglottic region. Follow up details regarding survival and deaths were available in 19 cases (Table 2); age and sex details were not available in one. Thirteen patients succumbed of which 10 died within six months, suggesting a very poor survival with a fatal outcome. Most (9/10) of them were elderly (seventh decade or above) and showed no definite gender predilection (six females, four males) for aggressive behavior. Longer survival (more than one year) was noted in seven, of which three patients survived for four to five

years (case no 6, 13 and 18). One of them was a 72 years female, which had metastasis at 47 months, succumbed to death 51 months post surgery. The other patient was a 65 years male with no recurrence and metastasis at 60 months post surgery and third patient was relatively young male (39 years), who survived at 48 months without any recurrence and metastasis. In addition, two other young patients (case no 19 and 23), who developed metastasis within few weeks, also survived for about one year. Thus it may be possible that men affected at a relatively younger age can have better survival and prognosis. This needs to be investigated in future by studying much larger series.

Table 1: Review of literature. Case No

Size

Lobe

Metastasis / local recurrence

RIM, dysphagia, dyspnoea

6.5cm

Rt.

Lung mets-bilateral

RIM, dyspnoea, TO

RIM

Lt.

2 mth

RIM

5[2]

Lung mets, local recurrence

6[2]

4 mth

NM, hoarseness

3.5cm

Rt.

At 48 mth-no mets

7[4]

8[4]

NM, Lt. arm pain

Lt.

Adjacent vertebrae mets

9[4]

2mth

RIM (painful), skin fistula

8.5cm

Lt.

10[5]

3mth

RIM, dysphagia, TO, weight loss, odynophagia

7cm

Lt.

At 2 mth-lung, 5 mth- liver, bone, peritoneal mets

11[5]

4mth

RIM, hoarseness, dysphagia

3cm

Lt.

At 8 mth-lung mets

12[7]

Lung, liver mets

13[8]

5mth

NM, Lt.arm pain

16cm

Lt.

At 60 mth-no mets

14[9]

RIM

12cm

15[9]

1 mth

RIM, hoarseness, tracheal deviation

5.5cm

Rt.

Recurrence at submandibular region

16[9]

Disseminated mets

[9]

3.5cm

At 15 mth-no mets

18[9]

7 mth

3cm

Rt.

At 47 mth-bone mets

19[9]

3mth

RIM, TO, weight loss

9cm

Lt.

At few weeks-lung mets

20[9]

RIM, dysphagia

5.5cm

At few weeks-lung mets

21[9]

few days

NM, hoarseness

1.9cm

Lt.

At few weeks-lung mets

22[9]

7.5cm

Rt.

At 2 mth-lung, at 5mth-liver, peritoneal mets

23(our case)

2mth

NM, dysphagia, hoarseness

5.5cm

Lt.

At 3.4 mth- mets to subglottis region

Age- years Sex Dura-tion Presenting complaints

[1]

2[2]

3[2]

4[2]

Abbr. â&#x20AC;&#x201C;F-female, Lt-left, M-male, mets-metastasis, mth-months, NA-not available, NM-neck mass, RIM-rapidly increasing mass, Rt.-right, TO-tracheal obstruction

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Table 2: Survival and Death (n=19). Case No

Age- years

Sex

Follow up

14[9]

Death within 1mth of presenting

1[1]

Death within 1.3mth post surgery

[4]

Death within 2mth post surgery

8[4]

Death within 2mth post surgery

[7]

Death within 3mth post surgery

20[9]

Death within 3mth of presenting

15[9]

Death within 4mth of presenting

[9]

Death within 5mth post surgery

10[5]

Death within 5mth post surgery

Death within 6mth post surgery

[6]

Death within 8mth post surgery

21[9]

Death within 18mth post surgery

18[9]

Death within 51mth post surgery

[9]

Alive at 11mth post surgery

17[9]

Alive at 15mth post surgery

6[2]

Alive at 48mth post surgery

13[8]

Alive at 60mth post surgery

Lost to follow up -after 12m (Alive at 12 mth)

Lost to follow up-after 18m (Alive at 18 mth)

[2]

[9]

23 (our case)

Acknowledgements Nil

Funding None

Competing Interests None Declared

Reference

1. Conzo G, Candela G, Tartaglia E, Gambardella C, Mauriello C, Pettinato G, et al. Leiomyosarcoma of the thyroid gland: A case report and literature review. OncolLett 2014; 7(4):1011-4. 2. Bertelli AA, Massarollo LC, Volpi EM, Ueda RY, Barreto E. Thyroid gland primary leiomyosarcoma. Arq Bras Endocrinol Metabol 2010; 54(3):326-30. 3. Hedinger C, Williams ED, Sobin LH. Histologic typing of thyroid tumors. In: World Health Organization International histologic classiﬁcation of tumors. 2nd ed. Berlin: Springer; 1988. pp. 13–5.

4. Amal B, El Fatemi H, Souaf I, Moumna K, Affaf A. A rare primary tumor of the thyroid gland: report a new case of leiomyosarcoma and literature review. DiagnPathol 2013; 27;8:36. 5. Mansouri H, Gaye M, Errihani H, Kettani F, Gueddari BE. Leiomyosarcoma of the thyroid gland. ActaOtolaryngol 2008; 128(3):335-6. 6. Ege B, Leventoğlu S. Primary leiomyosarcoma of the thyroid. J Korean Surg Soc 2013; 85(1):43-6. 7. Tanboon J, Keskool P. Leiomyosarcoma: a rare tumor of the thyroid. EndocrPathol 2013; 24(3):136-43. 8. Mouaqit O, Belkacem Z, Ifrine L, Mohsine R, Belkouchi A. A rare tumor of the thyroid gland: report on one case of leiomyosarcoma and review of literature. Updates Surg 2014; 66(2):165-7. 9. Thompson LD, Wenig BM, Adair CF, Shmookler BM, Heffess CS: Primary smooth muscle tumors of the thyroid gland. Cancer 1997; 79: 57987.

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Case Report Signet Ring Cell Carcinoma Colon in A Twelve Year Old Child: A Case Report with Review of Literature

Amita Jain Gupta1, Jenna B Bhattacharya1*, S Vijaya1, Richa Gupta1, Vivek Manchanda2 Department of Pathology, Maulana Azad Medical College, New Delhi, India Department of Paediatrics, Maulana Azad Medical College, New Delhi, India

1 2

Keywords: Signet Ring Cell Carcinoma, Colon, Paediatric Colon Carcinoma

ABSTRACT A twelve year old male patient presented with pain abdomen and vomiting on and off since 4 months. Patient had abdominal distension and a lump was palpable in abdomen on examination. The gross examination of the resected sigmoid colon revealed a stricture which on microscopy revealed the features of signet ring carcinoma colon. This case is being presented here due to rarity of this type of carcinoma in children.

*Corresponding author: Dr Jenna. B. Bhattacharya, C-2102, Amrapali Eden Park , Appts, Block-F, Sector-50, Noida. U.P. 201301 India Phone: +91 9899840023 E-mail: jennamamc@gmail.com

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Introduction

Signet ring cell carcinoma colon is aggressive tumour [1] with an overall prognosis worse than conventional adenocarcinoma due to high rate of metastases and local spread. This tumour constitutes 0.01 to 0.9% of patients of colon and rectal carcinoma. [1] The occurrence of these tumours is even rare with very few cases reported till date. We present a case of signet ring cell carcinoma in a twelve year old child.

Case Report

A 12 year old male patient presented with pain abdomen and vomiting on and off since four months. Patient had abdominal distension and constipation since three days. There was history of weight loss and weakness. Patient had pallor and was cachexic. An abdominal mass was palpable on examination. There was no palpable lymphadenopathy. Clinically tuberculosis was suggested. X ray abdomen showed dilated bowel loops. The patient had to be operated in emergency as he had sudden severe pain abdomen along with obstipation and features of acute obstruction. Operative findings revealed sigmoid stricture with peritoneal white deposit in pelvis and extensive dilated loops of colon. A provisional diagnosis of tubercular stricture with caseous material and tubercles in peritoneum was thought of in view of the age and presentation of the patient.

open a stricture was indentified 4 cm from the smaller resected end comprising of sigmoid colon with dilation of the proximal colon. The mucosa over the stricture was completely denuded and ulcerated and the bowel wall was thickened over the entire length of the stricture to 0.8 cm. The stricture measured 6 cm in length (Fig.1A) and both the resected ends were uninvolved. The colon proximal to the stricture was completely dilated and the bowel wall had become thin however no area of perforation was seen. The mucosa over the dilated segment was maintained. Externally intestine was congested. Mucosa was predominantly flattened out and partially ulcerated. Lymph nodes were isolated in mesocolon were firm and homogenous. Microscopic Examination: Sections from the stricture revealed complete mucosal ulceration and presence of sheets of atypical cells spread transmurally in a dyscohesive pattern. The cells were signet ring type with peripherally placed hyperchromatic nucleus. The presence of intracellular mucin was confirmed by mucicarmine stain. Also present were focal areas (comprising approximately 10%) with extracellular mucin deposits and malignant glands.

Gross Examination: Colonic segment measured 40 cm in length comprising of sigmoid colon and proximal large intestine. No growth was seen grossly. On cutting

The tumor cells reached the serosa. A diagnosis of signet ring cell carcinoma was given. Fourteen lymph nodes were isolated out of which one showed tumor deposit. Resected ends and intervening mucosa were uninvolved. Extensive sampling of the peritoneum revealed a peritoneal deposit. The colon cancer was classified as Duke stage C2 in this patient. There was no lymphovascular invasion.

Fig. 1A: Gross appearance of intestine showing presence of stricture.

Fig. 1B: H&E section showing microscopic appearance at low power view (10x) of signet cells in the submucosa.

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Fig. 2A: H&E section showing presence of signet cells at high power view (40x).

Fig. 2B: Mucicarmine stain showing positivity in both the signet ring cells and extracellular mucin.

Discussion

components. Signet ring cell carcinoma recurs more frequently compared to mucinous adenocarcinomas colon. [7]

Signet ring cell carcinoma can be defined by the presence of signet cells comprising more than 50 percent of total tumour cells with prominent intracytoplasmic mucin. This tumour occurs across all age groups suffering from colon and rectal carcinoma.[1] The median age of presentation is 57.1 years.[1] This tumour is rarely reported in children and adolescent age group.[2,3,4,5] Right hemicolon is more frequently involved followed by left colon and rectum.[1] The presenting features of tumor are rapid history of weight loss, rectal bleeding, abdominal pain, abdominal mass, vomiting, constipation and abdominal fullness. [5] The obstructive symptoms are more common due to stricture formation similar to clinical presentation of our case. In young age group, this can be often confused with tuberculosis due to obstructive signs and symptoms and can lead to a delay in diagnosis. Colorectal signet cell carcinoma has a poor prognosis compared to conventional adenocarcinoma.[1,3] This is usually detected at advanced stages as the symptoms manifest in a very late stage with few cases detected at early stage.[1] Also there are higher chances of metastasis due to late manifestation, diagnosis and surgery. [6] The five year survival rate on review of various studies is 0% (median 15 months). [7] Thus the differential diagnosis of colorectal carcinoma especially signet ring cell carcinoma must be considered even in children and adolescent age group as delayed diagnosis will result in poorer survival of this disease which already has such a grave prognosis. In addition, in small biopsies, the diagnosis may be missed because the signet ring cancer cells may be misinterpreted as foamy macrophages. An important differential diagnosis is mucinous adenocarcinoma with 50% or more mucinous www.pacificejournals.com/apalm

It has been postulated in few articles that Signet ring carcinoma may also have precursor lesions [pre existing adenomatous polyp / denovo cancer] and may have multiple synchronous association.[8] Our patient did not have any family history of adenocarcinoma. On review of various studies in the literature in signet ring carcinoma K-ras mutation occurs at a lower frequency and B-RAF mutation has higher frequency compared to conventional adenocarcinomas. [9,10] In conclusion in this case report, rare occurrence of primary signet ring cell carcinoma of the colon occurring in twelve year old child has been described as this is associated with poor survival and must be kept as differential diagnosis in children and adolescent age group. Earlier diagnosis with earlier surgery is necessary as the local and distant spread of this tumor is very rapid and responsible for the dismal prognosis.

Abbreviations

USG- ultrasonography

Acknowledgement None

Funding None

Competing interests None Declared

References

1. Fu KI, Sano Y, Kato S, Saito H, Ochiai A, Fujimori T, Saito Y, Matsuda T,Fujii T, Yoshida S. Primary eISSN: 2349-6983; pISSN: 2394-6466

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signet-ring cell carcinoma of the colon at early stage: a case report and a review of the literature. World J Gastroenterol. 2006 Jun 7;12(21):3446-9. Hamazaki M, Kono S, Mimaya J, Ishihara A. Signet ring cell carcinoma in a polyp of the colon. A case report of a six-year-old boy. Acta Pathol Jpn. 1987 Oct; 37(10):1679-84. Thota R, Fang X, Subbiah S. Clinicopathological features and survival outcomes of primary signet ring cell and mucinous adenocarcinoma of colon: retrospective analysis of VACCR database. J Gastrointest Oncol. 2014 Feb;5(1):18-24. Ko YS, Lin LH, Chen DF. Carcinoma of the colon in a child. Zhonghua Min Guo Xiao Er Ke Yi Xue Hui Za Zhi. 1995 May-Jun;36(3):227-30. Marone J, Patel S, Page M, Cheriyath P. Signet ring cell carcinoma of the colon in a 17year old child. J Surg Case Rep. 2012 Sep; 2012(9): 3. Tung SY, Wu CS, Chen PC. Primary signet ring cell carcinoma of colorectum: an age- and sex-

matched controlled study. Am J Gastroenterol.1996 Oct;91(10):2195-9. 7. Secco GB, Fardelli R, Campora E, Lapertosa G, Gentile R, Zoli S, Prior C.Primary mucinous adenocarcinomas and signet-ring cell carcinomas of colon and rectum. Oncology. 1994 Jan-Feb;51(1):30-4. 8. Nakamura T, Nakano G, Sakamoto K. Adenoma of the rectum with multiple foci of signet-ring cell carcinoma. Report of a case. Dis Colon Rectum. 1983 Aug;26(8):529-32. 9. Sim HL, Tan KY, Poon PL, Cheng A. Primary rectal signet ring cell carcinoma with peritoneal dissemination and gastric secondaries. World J Gastroenterol. 2008 Apr 7;14(13):2118-20. 10. Ogino S, Brahmandam M, Cantor M, Namgyal C, Kawasaki T, Kirkner G, Meyerhardt JA, Loda M, Fuchs CS. Distinct molecular features of colorectal carcinoma with signet ring cell component and colorectal carcinoma with mucinous component. Mod Pathol. 2006 Jan; 19(1):59-68.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 06, (Suppl) December, 2016

Case Report Primary Fallopian Tube Carcinoma Presenting as a Pelvic Mass: A Rare Case Report Gauri A. Chavan*, Abhijit G. Bhanji and Chandralekha S. Tampi Department of Histopathology, Lilavati Hospital and Research Centre, Mumbai, India Keywords: Fallopian Tube Carcinoma, Pelvic Drop Metastasis, Transcoelomic Migration

ABSTRACT Fallopian tumours are rare gynaecologic malignancies and are often clubbed together with ovarian cancers due to their frequent ovarian involvement at presentation. Most are serous in nature. Modes of spread consists of direct extension, lymphatics, hematogenous and also transcoelomic . We hereby present a rare case of primary fallopian tube carcinoma presenting as cystic pelvic mass in the pouch of Douglas representing a transcoelomic drop metastasis.

*Corresponding author: Dr Chavan Gauri A., Sai darshan, 3rd floor, Paranjape Scheme B, Road no. 3, Vile parle east, Mumbai, Maharashtra-400057, India. E-mail: gaurisawant18@gmail.com

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Introduction

Bonafide fallopian tube tumours are rare gynaecologic malignancies accounting for about 0.14-1.8% of all female reproductive system cancers. It was first reported by Renaud in 1847 and thereafter only around 2000 cases have been reported till date, most being subsumed within the Tubo-ovarian mass category. Tumours limited to the fallopian tube are rarely diagnosed pre-operatively, the rate of diagnosed being 3-4%.[1] They are also missed intraoperatively in about 50% of cases. These tumours are predominantly serous tumours. They secondarily spread onto the ovary and peritoneal surfaces. Apart from direct extension, lymphatic or hematogenous modes of spread are known. Transcoelomic migration is also known. We hereby present a case of a large cystic pelvic mass which had its origin in a small intraluminal fallopian tube carcinoma (FTC) & represents a transcoelomic drop metastasis.Â

Case Report

A 77 yrs old married women was incidentally detected to have a unilocular cystic mass, posterior to the uterus measuring 8.9x8.6x8.5 cms with multiple enhancing solid mural nodules favouring a left ovarian carcinoma. Investigations revealed CA-125=11.41 U/mL (Normal values: 0 to 35 (U/mL), CEA=3.71 ng/mL (Normal values: less than or equal to 3 ng/mL), bHCG=2.56 IU/L (Normal values in postmenopausal females: <9.5 IU/L), AFP=1.56 ng/mL (Normal values: 0-40 ng/mL). However, intra-operatively both ovaries were far and free. The mass was in the pouch of Douglas and adherent to anterior wall of the rectum.Â The cyst was received for frozen section analysis in two flat pieces measuring 7.5 x 7.5 cms and the wall thickness was 2-6 mms. The outer surface showed fatty tags while inner showed multiple small soft papillaroid excrescences measuring 3-4 mms in thickness. These excrescences represented areas of proliferating high grade malignant epithelium arranged as sheets, clusters and fused papillae. No invasion into the fibrous wall was seen. With no intraoperative detected primary in the uterus and adnexae, the frozen section was reported as a mullerian cyst with in situ carcinoma and unlikely to be from rectum. Total abdominal hysterectomy with bilateral salpingo-oophorectomy and omentectomy was carried out. Grossly, uterus and cervix appeared unremarkable. On opening the specimen, two small grey white bits measuring 2-3 mm were seen lying loose in the endometrial cavity. The right fallopian tube

measured 1cm in diameter throughout its length and felt boggy in its midportion. Its cut section showed, lumen occupied by whitish friable tumour. The ostium was open and unremarkable. The left fallopian tube and both the ovaries were grossly unremarkable. Both the fallopian tubes and ovaries were serially sectioned and submitted entirely. The microscopic examination of the right fallopian tube revealed a small primary intraluminal serous papillary carcinoma formed by complex and fused papillae lined by stratified cubo-columnar cells exhibiting moderately pleomorphic hyperchromatic nuclei and was similar in morphology to the cystic pelvic lesion. Intraluminal detached tumour buds were seen. There was no invasion of the muscular wall and the serosa was unremarkable. The bits lying loose in the endometrial cavity also showed similar histomorphology of the tumour as described above and thus, could represent dissociated tumour bits due to retrograde propulsion into the uterine cavity from the fallopian tube, either due to intraoperative handling of specimen or retrograde spread of tumour which in some cases leads to positive Pap smear for malignant cells. The endometrium, cervix, both ovaries and left fallopian tube did not show tumour, hyperplasia, atypia or dysplasia. The omentum was also free of tumour. Immunohistochemistry(IHC) studies performed on fallopian tube tumour and cystic pelvic mass revealed identical staining patterns with strong positivity for CA125, WT-1& ER, weak positivity for CK-7 and negativity for CK-20 & CEA. Thus, a diagnosis of primary serous papillary carcinoma of fallopian tube with pelvic drop metastasis was made. She was pathologically staged as pT2b as per TNM stage and II B according to FIGO stage, and was offered chemotherapy.

Discussion

FTCs per se are rare gynaecologic malignancies of elderly women with peak incidence in early sixties. Our patient was in her late seventies. Their true incidence may be underestimated as they are often diagnosed as ovarian carcinoma when the ovaries are extensively involved. They are similar in histomorphology and IHC profile to the epithelial ovarian cancers and hence the line of management of epithelial cancers of both these organs is essentially same. Serous carcinomas are the most common occurring in >50% cases followed by endometrioid in one fourth, transitional cell carcinomas in one eighth (0.20.5%) and undifferentiated in a remaining few.[2,3] The most common presenting symptom is post menopausal bleeding,[2] & Pap smears positivity is seen in 10-36% cases.[4] Abdominal distension, colicky abdominal pain

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Fig. 1: (A) Pelvic mass showing papillaroid excrescences (H and E, x100) and (B) Fallopian tube with small intraluminal serous papillary carcinoma (H and E, x100), (C & D)Both lesions are formed of complex and fused papillae lined by proliferating high grade malignant epithelium (H and E, x400). (E) Intraluminal dissociated tumour buds (H and E, x400).

Fig. 2: Both pelvic mass and fallopian tube tumour showing identical IHC: strong WT-1 positivity (A & B) and weak CK-7 positivity (C & D).

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followed by watery serosanguinous discharge called the hydrops tubae profluens is seen in <10% cases.[2] Our case showed a rare presentation of a solitary cystic pelvic mass between rectum and vagina. A thorough research of literature revealed no report of any such case of noninvasive FTC presenting with solitary encysted pelvic drop metastasis. Although one case of a solitary metastasis in a Spigelian hernia was reported in an elderly lady, it was due to spread from ruptured tube.[5] The FTC spreads by direct invasion of surrounding structures via ruptured tubal wall and by lymphatic and hematogenous routes. FTCs are also known to spread via transtubal migration of neoplastic cells through the tubal ostia into the peritoneal cavity. Here, they follow natural flow of the peritoneal fluid.[1,6] Even endometrial carcinoma is known to have transtubal spread to peritoneal cavity.[7] FTC with extensive upper abdominal metastasis was reported by Carolina et al.[1] Thus even a small noninvasive FTC bears a metastatic potential due to exfoliation and trascoelomic spread of malignant cells.

Conclusion

The cells of coelomic epithelium are known to undergo metaplastic change into tubal like lining causing a condition known as endosalpingiosis.[8] The pelvic peritoneum is known to undergo neoplastic transformation ranging from low grade to high grade serous carcinomas.[9] These tumours have identical IHC profiles to the tubal or ovarian epithelial serous carcinoma. Hence, simultaneous carcinomatous transformation in a mullerian rest in the pelvis was our another differential diagnosis. She had no evidence of endometriosis in her pelvic cavity or ovaries. However, presence of prominent intraluminal budding in the fallopian tube tumour favoured a diagnosis of FTC with a pelvic drop metastasis. We refuted the differential diagnosis of two synchronous primary tumours as these phenomenon described above are extremely rare as compared to the presence of FTC with drop metastasis.

Competing Interests

A second remote possibility of a primary peritoneal serous carcinoma (PPSC) with a synchronous primary tubal carcinoma was considered. However, PPSC rarely present as solitary lesions and we came across only one case of solitary PPSC mimicking as liver tumour.[10] Also, PPSC usually show peritoneal dissemination and ascites which was absent in our case. Most fallopian tube carcinomas drop cells into the pouch of Douglas, thus a PFTC with drop metastasis into the retrovaginal peritoneum (POD) seems more favourable diagnosis as compared to PPSC with origin in FT.

Fallopian tube carcinomas per se are rare malignancies & their true incidence may be underestimated as they are often diagnosed as ovarian carcinoma. They are similar in histomorphology and IHC profile to the epithelial ovarian cancers and hence the line of management is essentially same. Apart from the other modes of spread, the fallopian tube carcinomas are also known to spread through transtubal migration to implant into the pelvic cavity and this can occur even when the fallopian tube carcinoma is intraepithelial in nature. We present a case of a large cystic pelvic mass which had its origin in a small intraluminal fallopian tube carcinoma (FTC) & represents a transcoelomic drop metastasis.

Acknowledgements Nil

Funding Nil

None

Reference

1. Oliveira C, Duarte H, Bartosch C, Fernandes D.. Small fallopian tube carcinoma with extensive upper abdominal dissemination: a case report. Journal of Medical Case Reports. 2013; 7:252. 2. Robert H. Young, Philip B. Clement. The fallopian tube and broad ligament. In:Stacey E. Mills, editor. Sternberg’s Diagnostic Surgical Pathology. 5th ed. UK: Lippincott Williams & Wilkins; 2010. 2373-2391. 3. Ghosh S, Goyal P, Sehgal S, Shukla P, Kumar A, Singh S. Primary Fallopian-Tube Carcinoma. Journal of Gynecologic Surgery. April 2014, 30(2): 111-113. doi:10.1089/gyn.2013.0075. 4. Kos Z, Broaddus RR, Djordjevic B. Fallopian tube high-grade serous carcinoma with intramucosal spread and presenting as a malignancy on pap smear. Int J Gynecol Pathol. Jul 2014; 33:443-8. 5. Sultana A, Schembri M, Scerri AP, Wismayer DS. Primary fallopian tube carcinoma presenting with a solitary metastasis in a Spigelian hernia. Malta Medical Journal. 2008;20:39-42. 6. Markman M, Zaino RJ, Fleming PA, Gemignani ML. Carcinoma of the fallopian tube. In: Hoskins WJ, editor. Principles and practice of Gynecologic oncology, Fourth edition. Philadelphia; Lippincott Williams & Wilkins; 2005. 957-1054.

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7. Stewart CJ, Doherty DA, Havlat M, Koay MH, Leung YC, Naran A et al. Transtubal spread of endometrial carcinoma: correlation of intra-luminal tumour cells with tumour grade, peritoneal fluid cytology, and extra-uterine metastasis. Pathology. 2013;45,:382-7. 8. Bermejo R, Gómez A, Galiana N, et al., “Peritoneal Mullerian Tumor-Like (EndosalpingiosisLeiomyomatosis Peritoneal): A Hardly Known Entity,” Case Reports in Obstetrics and Gynecology, 2012, Article ID 329416, doi:10.1155/2012/329416

9. Carrick KS, Milvenan JS, Albores-Saavedra J. Serous tumor of low malignant potential arising in inguinal endosalpingiosis: report of a case. Int J Gynecol Pathol. 2003; 22,:412-5. 10. Iimuro Y, Ohashi K, Suzumura K, Hai S, Tanaka S, Nakamura I et al. Rare form of extraovarian primary peritoneal papillary serous carcinoma with solitary cystic lesion mimicking a liver tumor; report of a case. Clinical Journal of Gastroenterology. 2013;6:145-149.

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Case Report Combined Stromal Smooth Muscle Tumour of Endometrium: Report of Two Cases Sakshi Sirswal*, Charanjeet Ahluwalia, Sufian Zaheer, Amit Kumar Yadav and Ashish Kumar Mandal Department of Pathology, Vardhman Mahavir Medical College & Safdarjung hospital, New Delhi, India Keywords: Cellular Leiomyoma, Endometrial Stromal Nodule, Endometrial Stromal Sarcoma, Immunohistochemistry, Myometrium

ABSTRACT Endometrial stromal tumours account for < 10% of all mesenchymal uterine tumours. If smooth muscle differentiation comprises â&#x2030;Ľ30% of the tumour mass, the neoplasm is referred to as combined stromal smooth muscle tumour. We report here two cases of benign combined stromal smooth muscle tumour of endometrium, each discovered as an incidental finding in a hysterectomy specimen with a clinical diagnosis of fibroid uterus, where each of the two componentsstromal and smooth muscle comprised more than 30% of the tumour. Although none of them showed features of malignancy. This report of two cases of benign combined stromal smooth muscle tumours of endometrium adds to the morphological spectrum of the endometrial mesenchymal tumours.

*Corresponding author: Dr Sakshi Sirswal, room number 409, fourth floor college building, Vardhman Mahavir Medical College & Safdarjung hospital, New Delhi-110029, INDIA Phone: +91 9968529822 E-mail: sakshisirswal@gmail.com

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Sirswal et al.

Introduction

Endometrial stromal tumours (ESTs) are the second most common mesenchymal tumours of the uterus, even though they account for < 10% of all such tumours. [1] Uterine tumours exhibiting both endometrial stromal and smooth muscle differentiation are relatively rare. [2-8]Â Small areas of smooth muscle differentiation are commonly seen in otherwise typical endometrial stromal neoplasms, combined smooth muscleâ&#x20AC;&#x201C;stromal tumors were arbitrarily deďŹ ned as neoplasms having at least 30% of each component. [2, 9, 10] We are reporting here the morphological features of two cases of combined endometrial stromal- smooth muscle tumour.

Case Report(S)

Case 1: A 43-year-old lady, P2L2 presented with menorrhagia of 6 months duration following which she underwent total abdominal hysterectomy and right sided salphingo-oophorectomy with a clinical diagnosis of dysfunctional uterine bleeding. During surgery, a diagnosis of intramural fibroid was made. Grossly, the uterus contained a well-circumscribed intramural tumour measuring 2.5 cm in diameter which on cut section showed homogenous grey white soft to firm areas. For light microscopy, conventional haematoxylin and eosin (H&E) stained slides were examined. Sections revealed a well delineated tumour in the myometrium that contained areas of both endometrial stromal and smooth muscle differentiation. The endometrial stromal component, which made up approximately 30% of the tumour, was composed of uniform small ovoid to spindle shaped cells with scanty cytoplasm and round nuclei (fig1). Small thin-walled blood vessels were scattered uniformly throughout the stromal component. The remainder of the neoplasm was comprised of smooth muscle component with the smooth muscle cells arranged in interlacing fascicles, individual cells being spindle shaped with abundant eosinophilic cytoplasm and a spindle shaped nucleus with blunt ends (fig2). Large, thickwalled blood vessels featured the smooth muscle areas. The two components were well demarcated from each other. The stromal component showed absence of nuclear pleomorphism and mitotic activity. On immunohistochemistry, the stromal component showed positivity for CD-10 and the smooth muscle component showed positivity for SMA and the final diagnosis of benign combined stromal smooth muscle tumour was made.

C-307 of 9 months. On clinical examination, the uterus was found to be bulky and firm. Ultrasound revealed multiple leiomyomas (ranging from 7x7cm to 4.5cm in diameter). Based on these features, a clinical diagnosis of fibroid uterus was made. Total abdominal hysterectomy with left sided salpingo-oopherectomy was performed. The postoperative course was uneventful. we received one container containing an already cut open uterus with cervix with two intramural tumours measuring 1.5x1x0.6cm and 1x1x0.5cm. Each of these tumours were well circumscribed which on cut showed grey white homogenous surface with whirling pattern. Second container had a globular soft tissue piece measuring 5x4x3cm, the cut surface showed grey white homogenous tumour with whirling pattern and few focal cystic areas. The left ovary and fallopian tube were received in the third container. On light microscopic examination, the two intramural tumours were identified as leiomoyomas. H&E sections from the globular soft tissue received in the second container revealed a well delineated tumour composed of stromal and smooth muscle components, each comprising more than 30% of the tumour (fig3). There was cystic change in the stromal component with a cyst lined by flattened epithelium and surrounded by stromal cells. The tumour was not invading the adjacent myometrium and the part of cervix attached to it, though the two components merged with each other. No atypia, necrosis or abnormal mitosis was seen. On immunohistochemistry, the stromal cells were positive for CD-10 (fig4) and the smooth muscle cells were positive for SMA and desmin (fig5). The final diagnosis of benign combined stromal smooth muscle tumour was made.

Case 2: A 49 year old lady P2L2, presented with history of heaviness in lower abdomen and menorrhagia for duration

Fig. 1: (H&E, 40X) The tumour showing stromal component comprised of uniform small ovoid cells with scant amount of cytoplasm and a large round nucleus.

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Fig. 2: (H&E, 40X) The tumour showing smooth muscle component with spindle cells arranged in interlacing fascicles.

Fig. 3: (H&E, 40X) The tumour, showing the two componentsstromal in the top left corner and smooth muscle in the bottom right corner which are merging with each other.

Fig. 4: (CD10, 40X) The tumour cells showed immunopositivity for CD-10 (stromal component).

Fig. 5: (SMA, 40X) The tumour cells showed immunopositivity for smooth muscle actin (smooth muscle component).

Discussion

according to the type of margin as benign (endometrial stromal nodule), having pushing margins and malignant (endometrial stromal sarcoma), having infiltrating margins. [4] Endometrial stromal nodules (ESNs) appear grossly as solitary, sharply circumscribed masses of soft consistency, with the characteristic yellow to orange colour and tend to bulge above the surrounding myometrium. They do not show lymphovascular invasion or invasion into myometrium. Microscopically, these tumours are composed of cells that closely resemble normal proliferative phase endometrial stromal cells, with uniform, small darkly staining round or oval nuclei, finely granular chromatin, occasional mitosis and scant cytoplasm. The stromal nodules are highly vascular, with small arterioles distributed throughout them. Extensive hyalinization is a

The term “stromomyoma” was proposed by Tang etal. [4] to designate a peculiar uterine tumor with ultrastructural characteristics of both endometrial stromal and smooth muscle cells. They opined that these tumours developed due to differentiation of multipotential mesenchymal cells toward myoblasts and stromal cells. They further put forth an alternative explanation that both stromal cells and smooth muscle cells respond to the same stimuli resulting in simultaneous neoplastic proliferation. However, at present, the diagnosis of a combined tumour is based solely on the presence of signiﬁcant amounts of both elements as recognized by routine light microscopy. Endometrial stromal tumours (ESTs) have been classified

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Sirswal et al. common feature in most of these tumours. The prognosis is excellent, with no recurrences. [10] Focal areas of smooth muscle differentiation are commonly observed in ESTs, but tumours with extensive areas of both smooth muscle and endometrial stromal differentiation are rare. A certain number of mesenchymal uterine tumours show features of both endometrial stromal and smooth muscle differentiation. To establish the diagnosis of mixed endometrial stromal tumour with smooth muscle differentiation, the smooth muscle component should occupy at least 30% of the neoplasm, as seen by haematoxylin and eosin staining. [9] The smooth muscle component characteristically shows nodules with central hyalinization (starburst pattern) as seen in our cases, which merge with disorganized short fascicles or long mature fascicles of the smooth muscle, a feature which is almost never encountered in conventional smooth muscle tumours. [2] Behaviourally, these tumours seem to be closer to endometrial stromal than smooth muscle tumour and on the whole, very indolent. Hysterectomy is thus the appropriate therapy and the periphery of the tumour must be thoroughly evaluated to be certain that it is completely circumscribed and noninvasive. The differential diagnosis of this benign neoplasm includes low-grade endometrial stromal sarcoma (ESS) and highly cellular leiomyoma. Microscopically, the most important single criterion for the diagnosis of ESN is a non infiltrative border of the tumour. Focal irregularities in the form of lobulated or finger like projections into the adjacent myometrium that are not â&#x2030;Ľ3mm. and are not >3 in number may be seen; which contrasts with the permeative invasion of the myometrium as well as the myometrial vessels seen in low-grade ESS. [11] From the prognostic point of view, it is extremely important to distinguish between these two tumours, as ESNs do not relapse and low-grade ESSs have a low malignant potential characterized by late recurrences. Hence, extensive sampling of the tumour myometrial interface should be done. This is to evaluate the degree of infiltration of the tumour into the myometrium and to detect vascular invasion, which is extremely important to distinguish the two. Highly cellular leiomyomas are composed of cells with spindle shaped nuclei with a fascicular growth pattern, thick muscular-walled blood vessels, cleft like spaces and show focal merging with the adjacent myometrium. [9, 12] In our cases, the tumour did not exhibit any of the above mentioned features. Differentiating highly cellular leiomyoma from ESN is important in a curettage or myomectomy specimen, if a spindle cell cannot be classified into that of smooth muscle or stromal cell origin. In cases where the diagnosis is difficult to be established by light microscopy, immunohistochemical analysis may be helpful in arriving at the correct diagnosis, www.pacificejournals.com/apalm

C-309 which is crucial, owing to the differences in treatment and prognosis. [12] A panel of antibodies including desmin, h-caldesmon, CD10 and inhibin may be very useful in such a scenario, since cellular leiomyomas express h-caledsmon in addition to desmin, while CD10 and inhibin expression is a feature of stromal cells. [13] Finally, oxytocin receptor, a neurohypophysial peptide which is associated with muscle contraction during labour, stains all conventional leiomyomas and highly cellular leiomyomas as well as leiomyosarcomas, but is not expressed in ESTs. [14] However, this antibody is not used in daily practice at present. In the above mentioned cases, the characteristic morphological features of the stromal cells and the smooth muscle components were identified by light microscopy. The presence of both the components was confirmed by IHC using CD-10 and SMA for the stromal and smooth muscle components respectively. Further, the gross appearance and well defined non infiltrative microscopic tumour margins emphasized the benign nature of these lesions.

Conclusion

In summary, we have described an uncommon, benign mesenchymal uterine tumour, i.e. ESN with smooth muscle differentiation, which needs to be distinguished from tumours with similar morphological features i.e. lowgrade ESS and highly cellular leiomyomas. These cases are being reported because of their rare occurrence and to create awareness amongst pathologists about these lesions regarding their differential diagnosis.

Abreviations

EST: Endometrial stromal tumour ESN: Endometrial stromal nodule ESS: Endometrial stromal sarcoma H&E: Haematoxylin and eosin SMA: Smooth muscle actin

Acknowledgements

We would like to thank all the technical staff of pathology department for their co-operation and help.

Funding None

Competing Interests None Declared

Reference

1. Moinfar F, Kremser M L, Man Y G, et al. Allelic imbalances in endometrial stromal neoplasms: frequent genetic alterations in the nontumorous eISSN: 2349-6983; pISSN: 2394-6466

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normal- appearing endometrial and myometrial tissues. Gynecol Oncol 2004; 95:662-71. 2. Oliva E, Clement PB, Young RH, et al. Mixed endometrial stromal and smooth muscle tumours of the uterus: a clinicopathologic study of 15 cases. Am J Surg Pathol1998:22:997–1005. 3. Schammel DP, Silver SA, Tavassoli FA. Combined endometrial stromal/smooth neoplasms of the uterus: a clinicopathological study of 38 cases [abstract]. Mod Pathol 1999:12:124A. 4. Tang C, Toker C, Ances IG. Stromomyoma of the uterus. Cancer 1979:43:308–16. 5. Chang KL, Crabtree GS, Lim-Tan SK, et al. Primary uterine endometrial stromal neoplasms. A clinicopathologic study of 117 cases. Am J Surg Pathol 1990:14:415–38. 6. Evans HL. Endometrial stromal sarcoma and poorly differentiated endometrial sarcoma. Cancer 1982:50:2170–82. 7. Fekete PS, Vellios F. The clinical and histologic spectrum of endometrial stromal neoplasms: a report of 41 cases. Int J Gynecol Pathol 1984:3:198–212. 8. Khalifa MA, Hansen CH, Moore JL, Jr, et al. Endometrial stromal sarcoma with smooth muscle differentiation: recurrence after 17 years: a followup report with discussion of the nomenclature. Int J Gynecol Pathol 1996:15:171–6.

9. Clement PB. The pathology of uterine smooth muscle tumours and mixed endometrial stromal-smooth muscle tumours: a selective review with emphasis on recent advances. Int J Gynecol Pathol 2000:19(1):39–55. 10. Tavassoli FA, Norris HJ (1981) Mesenchymal tumours of the uterus. VII. A clinicopathological study of 60 endometrial stromal nodules. Histopathology 5:1–10 11. Baker P, Oliva E. Endometrial stromal tumours of the uterus; a practical approach using conventional morphology and ancillary techniques. J Clin Pathol. 2007; 60:235-243. 12. Oliva E, Young R H, Clement P B, et al. Cellular benign mesenchymal tumours of the uterus. A comparative morphologic and immunohistochemical analysis of 33 highly cellular leiomyomas and six endometrial stromal nodules, two frequently confused tumours. Am J Surg Pathol 1995; 19:757-68. 13. Oliva E, Young RH, Amin MB, et al. An immunohistochemical analysis of endometrial stromal and smooth muscle tumours of the uterus: a study of 54 cases emphasizing the crucial importance of using a panel because of overlap in immunoreactivity for individual antibodies. Am J Surg Pathol 2002; 26:403-12. 14. Loddenkemper C, Mechsner S, Foss H D, et al. Use of oxytocin receptor expression in distinguishing between uterine smooth muscle tumours and endometrial stromal sarcoma. Am J Surg Pathol 2003; 27:1458-62.

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Case Report Pseudochylous Pleural Effusion in a Case of Rheumatoid Arthiritis Harsh Malik1*, Priyageet Kaur Kalsi2 and Puneet Kaur1 Pathology Department, Gian Sagar Medical College, Banur, Patiala, India Pathology Department, Government Medical College, Chandigarh, India

Keywords: Pseudochylous, Rheumatoid Arthritis, Cholesterol, Pleural Effusions

ABSTRACT Chyliform (pseudochylous) pleural effusions accumulate gradually due to the breakdown of cellular lipids in long standing pleural effusions, such as Rheumatoid Arthritis (RA) and Tuberculosis. These are also known as cholesterol effusions due to high cholesterol levels in pleural fluid and presence of cholesterol crystals on wet mount. We present the case report of a 60 year old lady, who came to the medicine out patient department with breathlessness and generalized body ache. Radiological investigation revealed a left sided pleural effusion with atelectasis. Based on the characteristic biochemical and cytological findings in the pleural fluid, a diagnosis of pseudochylous effusion was made. Test for rheumatoid factor was done subsequently due to the presence of joint pains, and it was positive.

*Corresponding author: Dr Harsh Malik, Room no 27, New Block 2, Married Doctors Hostel, PGIMER, Chandigarh, India Phone: +91 8872010326 E-mail: drharshmalik30@gmail.com

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

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Pseudochylous Pleural Effusion

Introduction

A chyliform (pseudochylous) pleural effusion is an uncommon disease condition developing as a sequela of a long standing exudative effusion.[1] The most common cause of this pleural reaction is Tuberculosis.[2] The other reported underlying diseases include rheumatoid lung disease, syphilis, diabetes, alcoholism, Meig’s syndrome, paragonimiasis, malignancy, and trauma or hemothorax.[3] This entity was so named because unlike chylous effusion, it is not due to disruption of the thoracic duct, but due to a high lipid content, which causes it to be turbid.[2] The exact pathogenesis of chyliform pleural effusions is unknown.

Case Report

A 60 year old lady presented with breathlessness, generalized body ache, joint pains and loss of appetite since two months. She was a known case of bronchial asthma. There was no history of diabetes, hypertension, tuberculosis or any drug allergy. On complete blood count, the patient had hemoglobin of 12.9 gm/dl and a high white blood cell (WBC) count of 19400/cumm, with neutrophilic predominance (76%). The platelet count was 3.7 lakh/cumm. ESR was 56 mm in the first hour. CRP (C Reactive protein) showed an elevated value of 40.2 mg/L. The RA factor was 35 IU/ml (normal value less than 30 IU/ml). On radiological evaluation, x ray showed left sided pleural effusion and a homogeneous opacity in the left lower zone,

Fig. 1: Chest XRay Postero-Anterior(PA) view showing left sided pleural effusion.

with rising level and blunting of left CP angle (Figure 1). Ultrasound chest showed a moderate pleural effusion on left side. HRCT chest was also done, which confirmed the above findings and showed basal atelectasis on left side along with few fibrotic lesions in the right upper lobe. Thereafter, thoracocentesis was done and 30 ml of pleural fluid was sent for cytological evaluation. The fluid was turbid and slightly hemorrhagic. Pleural fluid WBC count done by Modified Neubauer’s chamber was 50 cells/µl. Manual differential count revealed lymphocytic predominance. Smears showed numerous macrophages, some of which were epithelioid (Figure 2), along with few multinucleated giant cells and mesothelial cells (Figure 3). The background showed amorphous extracellular granular debris. On wet mount, cholesterol crystals with characteristic “shattered glass appearance” were also noted and confirmed with the help of a polarizer (Figure 4). Pleural fluid protein was raised at 9.7 gm/dl; while glucose levels were reduced at 16 mg/dl (normal reference value for pleural fluid protein is less than 3 gm/dl, while that for glucose is 70-210 mg/dl). Adenosine Deaminase (ADA) levels were done to rule out tuberculosis; the value was normal (25 U/L). Pleural fluid cholesterol levels were raised (230 mg/dl), while triglyceride levels were normal (70 mg/dl). Pleural fluid culture was negative. The patient was managed conservatively with thoracocentesis and anti-rheumatoid drugs. Subsequently, the patient was discharged in a satisfactory condition.

Fig. 2: High power view of wet mount of pleural fluid showing amorphous debris in the background, against which are present cholesterol crystals having a characteristic shattered glass appearance. (X400).

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Fig. 3: High power view of cytological smear of pleural fluid showing an epithelioid multinucleated giant cell and few histiocytes. (LeishmannX400).

Fig. 4: High power view of cytological smear of pleural fluid showing many foamy histiocytes and a multinucleated giant cell. (LeishmannX400).

Discussion

to their minimal pleural thickening. The median duration of symptoms (or arthritis, in the case of asymptomatic effusions) in their study was 15 months. The absence of pleural thickening in their patients suggested that cholesterol accumulates due to an acute or subacute process, rather than cellular breakdown and subsequent release of cholesterol within the pleural space. These unusual findings were a deviation from the pseudochylothorax seen in RA. Hence, it was suggested that pseudochylothorax should be considered clinically even in short duration nonfibrotic pleural effusions .[4]

Pseudochylothorax (cholesterol pleurisy or chyliform effusion) is a cholesterol-rich pleural effusion that is commonly associated with chronic inflammatory disorders like Tuberculosis and Rheumatoid Arthritis.[4] It is usually seen in long standing exudative pleural effusions, in which the effusion is encapsulated in a fibrotic part of the thickened pleura. The precise pathogenesis of chyliform pleural effusions is unknown. But various hypotheses have been postulated. In one of these, the lipid in chyliform effusions is thought to arise from breakdown of blood cells.[2] The fibrotic scar tissue, which walls off the effusion, is poorly vascularized. [5] This is thought to result in an abnormally slow transfer of cholesterol and other lipids out of the pleural space and lead to the accumulation of cholesterol in the pleural fluid. [2] In contrast to the cholesterol in acute exudates, which is mostly bound to Low Density Lipoproteins (LDL), cholesterol in Pseudochylous effusions is bound to High Density Lipoproteins (HDL). This would imply that it is derived from serum lipoproteins rather than cellular debris. To explain this anomaly, it has been hypothesized that cholesterol which enters the pleural space with acute inflammation, becomes trapped there and undergoes change in lipoprotein binding characteristics .[6]

Pseudochylous effusions need to be differentiated from the true chylous effusions and empyema. All three entities have in common, a thick milky white and opalescent fluid. Empyema can be differentiated from the other two entities by performing centrifugation. In lipid effusions, the fluid remains uniform unlike the clear supernatant that develops in empyema. Pseudochylothorax can be differentiated from chylothorax by adding 1â&#x20AC;&#x201C;2ml of ethyl ether. The milkiness disappears in the former . [7] Differentiating features of various types of turbid pleural effusions are summarized in Table 1.

Wrightson et al. presented six well-characterized cases of arthritis-associated pseudochylothorax, each notable due

In our case, the diagnosis of rheumatoid pleurisy was confirmed on the basis of increased protein, decreased glucose, raised cholesterol, decreased triglyceride level, characteristic cytology and presence of cholesterol crystals

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Table 1. Differentiating features of turbid pleural effusions Type of effusion Gross appearance Appearance after centrifugation

Chief biochemical feature of effusion fluid

Empyema

Turbid

Clear supernatant

Glucose<60mg/dl

Chylous

Turbid

Fluid remains uniformly milky white

Triglycerides >110mg/dl

Pseudochylous

Turbid

Fluid remains uniformly milky white

High content of cholesterol>200mg/dl with no triglycerides

on wet mount preparation.[9,10] Tuberculosis was ruled out on the basis of negative ADA levels and culture. Management of the underlying disease process is the mainstay of treatment. Although at times, the effusions may not accumulate after initial thoracocentesis, the majority require repeated drainage (12). Decortication should be considered in symptomatic patients with restrictive lung function .[11]

Conclusion

A pseudochylous pleural effusion is an uncommon disease condition developing as a sequela of a long standing exudative effusion, commonly seen in Tuberculosis and Rheumatoid Arthritis. The conventional pathogenesis is cholesterol accumulation as a result of cellular breakdown, leading to elevated cholesterol levels in the pleural fluid. The diagnosis is confirmed by demonstration of raised cholesterol and reduced triglyceride levels in the fluid, along with visualization of characteristic cholesterol crystals on wet mount. Radiologically, in most cases, including ours, the pleura are markedly thickened. It is important to differentiate cholesterol effusion from chylothorax and empyema because each of these entities are managed differently. In pseudochylous effusions, the mainstay of treatment is management of the underlying cause. Repeated thoracocentesis and decortications may be required in selected cases.

Acknowledgements Nil

Funding Nil

Competing Interests None

Reference

1. González R, Ramírez-Rivera J. Chyliform (pseudochylous) pleural effusion. Bol Asoc Med P R 1994; 86:50-2. 2. Hillerdal G. Chyliform pleural effusion. Chest 1985; 88:426-8. 3. Hamm H, Pfalzer B, Fabel H. Lipoprotein analysis in a chyliform pleural effusion: implications for pathogenesis and diagnosis. Respiration 1991; 58:294-300. 4. Wrightson JM, Stanton AE, Maskell NA, Davies RJ, Lee YC. Pseudochylothorax without pleural thickening: time to reconsider pathogenesis? Chest 2009;136:1144-7. 5. Hillerdal G. Chylothorax and pseudochylothorax. Eur Respir J 1997; 10:1157-62. 6. Hamn H, Pfalzer B, Fabel H. Lipoprotein analysis in a chyle form pleural effusion: implications for pathogenesis and diagnosis. Respiration 1991;58:294-300. 7. De Beer HG, Mol MJ, Janssen JP. Chylothorax. Neth J Med 2000; 56:25–31. 8. Staats BA, et al. The lipoprotein profile of chylous and nonchylous pleural effusions. Mayo Clin Proc 1980; 55:700–4. 9. Highland KB, Heffner JE. Pleural effusion in interstitial lung disease. Curr Opin Pulm Med 2004;10. 10. Genzen JR, Motin A. Cholesterol crystals in pleural fluid. American Journal of Respiratory and Critical care medicine 2012; 185:586. 11. Huggins JT. Chylothorax and cholesterol pleural effusion. Semin Respir Crit Care Med 2010; 31:743-50.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 06, (Suppl) December, 2016

Case Report Osteosarcoma of The Talus: A Rare Location of the Tumour with Literature Review

Thin Thin Win1*, Noorul Balqis Che Ibrahim1 and Wan FaishamNuâ&#x20AC;&#x2122;man Wan Ismail2 Department of Pathology, School of Medical Sciences, University Sains Malaysia, 16150 KubangKerian, Kelantan, Malaysia 2 Department of Orthopaedics, School of Medical Sciences, University Sains Malaysia, 16150 KubangKerian, Kelantan, Malaysia 1

Keywords: Osteosarcoma, Primary Malignant Bone Tumor, Talus, Giant Cell Tumour

ABSTRACT Osteosarcoma is the commonest primary malignant bone tumor. Most of them arises de novo in the metaphyseal area of the long bones. Osteosarcoma arising distal to the wrist or ankle is extremely rare and most often secondary to a premalignant process such as Paget disease. We reported arare case of conventional osteosarcoma of the talus. A 40-year-old lady presented with painless left foot swelling for the past two years with gradually increasing in size within 6 months. Tru-cut tissue biopsy was reported as giant cell tumour. However histopathology of resected talus bone revealed that tumour was conventional osteosarcoma with focal features of giant cell rich osteosarcoma.

*Corresponding author: Dr. Thin Thin Win, Department of Pathology, School of Medical Sciences University Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia. Phone: +6 09 767 6445 E-mail: safiya@usm.my

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

C-316

Osteosarcoma of The Talus

Introduction

Osteosarcoma (OS) is the commonest primary malignant bone tumor. Most of them arises de novo in the metaphyseal area of the long bones, particularly the lower end of the femur, the upper end of the tibia, and the upper end of the humerus[1].OS arising distal to the wrist or ankle is extremely rare and most often secondary to a premalignant process such as Paget disease, radiation exposure and preexisting benign bone lesions [2].It usually occurs in patients between 10 and 25 years of age and is exceptionally rare in preschool children. Another peak age incidence occurs after 40, in association with other disorders. There is a slight male predominance.The large majority of OS arise within the medullary cavity, from which they extend into the cortex [1] .Although there are some variants of OS, conventional type is the commonest. When OS occurs in the foot bones, tarsal bones are commonest and calcaneus is the most involving [3].We reported arare case of conventional OS in the talus with no history of previous preexisting lesion. A 40-year-old lady presented with painless left foot swelling for the past two years which gradually increasing in size within 6 months. Magnetic resonance imaging (MRI) report was suggestive of giant cell tumour (GCT) and Tru-cut tissue biopsy was also reported as GCT. However histopathology of resected talus bone revealed that tumour was conventional OS with foci of giant cell-rich OS area.

Case report

40-year-old, Malay lady presented to orthopaedic clinic for painless left foot swelling for the past two years. It was gradually increasing in size within 6 months. She had no history of trauma and any problem at left foot before the appearance of the swelling. On examination, there was a swelling over the medial and lateral aspect of left ankle, irregular in shape, firm to hard in consistency, slightly tender on palpation.

Plain radiograph of ankle (Figure: 1a) showed osteoblastic lesion occupying the talus. MRI (Figure: 1b) and tru-cut tissue biopsy werereported as suggestive of GCT; as the lesion was mainly composed of osteoclastlike multinucleated giant cells. Talus bone was excised together with tumour, navicular bone, lower end of tibia, upper part of calcaneus, surrounding soft tissue and overlying skin medially. Grossly, specimen consisted of talus with tumor, navicular bone, lower end of tibia, upper part of calcaneus, surrounding soft tissue and overlying skin medially. On cut section (Figure 2), there was an infiltrating tumor occupying most of the talus and navicular bone invading anteriorly and posteriorly into soft tissue. Both superior and inferior surgical cut margin were involved by the tumor. Calcaneus bone and its articular cartilage were invaded by the tumor. Tibia and its articular cartilage were not invaded grossly. Lateral surgical margin was also involved by tumor grossly; however skin medially was not involved. Cut surface of the tumor was grayish white and firm in consistency. Small areas of hemorrhage were seen. Histopathologically, there was an infiltrating bone forming tumor originating in the talus. Tumor was mainly composed of spindle to epithelioid looking pleomorphic cells. Mitoses were frequent varying 5-20/10HPF depending on cellularity. Most of the area in between the tumor cells showed pink, amorphous lace like pattern of osteoid tissue. (Figure: 3a) Osteoclast like multinucleated giant cells were seen admixed with tumor cells. (Figure: 3b) Areas of dense collagenization were also seen in the stroma. Small cystic-like spaces with haemorrhages were also seen. With those histopathological findings, we concluded that it was conventional OS, originated in the talus bone. After excision of the tumour, CT (computerizes tomography) thorax showed three lung nodules suggestive of metastasis. Patient was on chemotherapy and radiotherapy; responding well with rehabilitation.

Fig. 1: (1a. Plain radiograph) & (1b. MRI) showed heterogeneous lesion (asterisk) involving the whole talus with surrounding soft tissue mass.

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Fig. 2: Tumour arising in talus (T) invading navicular bone (N) and calcaneus (C). Tibia (Tb) and its articular cartilage are not invaded. Cut surface of the tumour is solid, grayish white and firm to hard in consistency.

Fig. 3: (a). Giant cell rich area of the tumour which mimic GCT. (H&E 200X) (b). Pleomorphic tumour cells and pink amorphous tumour osteoid forming lace like pattern. (H&E 400X).

Discussion

OS arising distal to the wrist or ankle is extremely rare and most often secondary to a pre-malignant process such as Paget disease [2].In a study of 52 cases of osteosarcoma of the foot, tarsal bones are commonly involved. Among them, calcaneus is the commonest followed by the talus and navicular bone [3]. In a study of OS of hands and feet, 62 % of the OS presented in the metacarpals and 23 % in the phalanges, and only two cases occurred in the carpal bones. Distribution in the foot was 56 % in tarsal bones, 33 % in metatarsal bones and 11 % in phalanges [4]. In a study of 1929 cases of OS, only 12 cases were located in the foot with the commonest in the calcaneus followed by the talus [5]. Among the tumours of the foot and ankle, GCT is the commonest [6]. www.pacificejournals.com/apalm

Most of OS affects children and adolescents. However, some of the case studies on OS affecting the foot showed that most of the patients were older than 30 years [5,6]. There were some reported cases of talar OS in elder age without any underlying pathology [7]. In addition, patients with OS of the foot commonly have a long delay from the time of onset of symptoms to diagnosis, with the mean interval greater than 2 years [5]. Patient in this case was middle age woman and onset was slow without any underlying pathology. In a study of OS of hands and feet, 3 cases were seen in the talus out of 27 cases of osteosarcoma feet. That study also found out that patients with OS of the distal extremities are older, have a different gender distribution, and differ eISSN: 2349-6983; pISSN: 2394-6466

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in symptoms, history and grade of malignancy [4]. Most of the OS of hands and feet present with pain with or without swelling and the average duration of symptoms were 13 months [4]. Some of the cases of OS foot may present with painful swelling which lead to misdiagnosis of inflammatory condition [8]. Clinical findings of most of OS of the talus are atypical and it leads to misdiagnosis and delayed correct diagnosis [5]. This case presented with painless swelling for 2 years duration. In this case, Tru- cut biopsy before excision of the tumour was suggestive of GCT as it was mainly composed of multinucleated giant cells with few mononuclear cells. The biopsy was seemed to be taken from the site of giant cell-rich area of the tumour. When we correlated with plain radiograph images, it was osteoblastic lesion rather than osteolytic lesion. The case was not associated with any premalignant bone lesion or prior irradiation. Generally, histopathological diagnosis of OS is mainly based on identification of osteoid and/or bone (calcified osteoid) produced directly by the tumor cells without interposition of cartilage [1]. Immunohistochemical expression of specific proteins produced by tumour cells of OS is supportive in the diagnosis. Extracellular matrix metalloproteinase inducer (CD147) is expressed in human osteosarcoma tumor tissues [9]. Proteins specifically associated with bone metabolism such as osteonectin, osteocalcin, osteopontinand bone morphogenetic protein have been identified immunohistochemically in the cells of osteosarcoma and may be useful in the differential diagnosis of OS [1]. For differentiation between OS and GCT, specific proteins of GCT are useful. A study on GCT showed that strong immunohistochemical expressions of osteprotegerin and osteoprotegerin ligand are seen in the giant cells and background stromal cells [10]. However, these special immunohistochemical stains are not routinely used and not performed in this case.GCT is distinguishable from giant cell-rich OS in haematoxylin and eosin stained sections. GCT does not produce typical lacy tumour osteoid and their stromal cells do not exhibit pleomorphism. In conclusion, OS of the talus bone is rare. Accurate diagnosis can be complicated by subtle clinical features. Thorough radiological correlation in histopathological diagnosis is very important. Representative tissue biopsy is essential to provide thorough histopathological examination to reach correct diagnosis.

Funding None

Competing Interest None

References

1. Rosai J. Bone and Joint. In: Rosai and Ackermanâ&#x20AC;&#x2122;s Surgical Pathology, 10th Edition, Mosby, Edinburgh, 2011, pp. 2137-2235. 2. Fechner R.E; Mills S.E. In: Osseous Lesions. Atlas of Tumor Pathology: Tumors of the Bones and Joints. Washington D.C: Arm Forces Institute of Pathology, 1993. pp 25-77. 3. Choong PFM, Qureshi AA, Sim FH, Unni K.K. Osteosarcoma of the foot: A review of 52 patients at the Mayo Clinic. ActaOrthopScand 1999; 70:361-4. 4. Anninga JK, Picci P, Fiocco M, Kroon HM, Vanel D, Alberghini M, et al. Osteosarcoma of the hands and feet: a distinct clinico-pathological subgroup. Virchows Arch 2013; 462:109-20. 5. Biscaglia R, Gasbarrini A, BĂśhling T et al. Osteosarcoma of the bones of the foot. An easily misdiagnosed malignant tumor. Mayo Clin Proc1998; 73:842-7. 6. Chou LB, Ho YY, Malawer MM. Tumors of the foot and ankle: experience with 153 cases. Foot Ankle Int 2009; 30:836-41. 7. Ellison BS, Potter M, Morrison C, Mayerson J. Talar osteosarcoma in an elderly woman. Am J Orthop (Belle Mead NJ) 2008; 37:198-203. 8. Chaudhary S, Singh D, Sen R, Deviraju C, Bhagwat K. Osteosarcoma of calcaneum, A case series of rare tumour presenting a diagnostic dilemma. WebmedCentral ORTHOPAEDICS 2010; 1(12):WMC001273. 9. Lu Q, Lv G, Kim A, Ha J-M, Kim S. Expression and clinical significance of extracellular matrix metalloproteinase inducer, EMMPRIN/CD147, in human osteosarcoma. Oncology Letters. 2013;5(1):201-207. 10. Yu X, Kong W, Zheng K. Expression of osteoprotegerin and osteoprotegerin ligand in giant cell tumor of bone and its clinical significance. Oncology Letters. 2013; 5(4):1133-1139.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 06, (Suppl) December, 2016

Case Report An Unusual Case of Nerve Sheath Myxoma with Schwannomatous and Perineural Differentiation Smita Surendra Masamatti, Aparna Narasimha* and Jayalakshmi Valligari Janardhan Department of Pathology, Sapthagiri Institute of Medical Sciences and Research Centre, Bangalore. India Keywords: Benign Tumour, Nerve Sheath Myxoma, S100 protein, EMA

ABSTRACT Nerve sheath myxoma (NSM) is a rare benign tumour of peripheral nerves. It is seen in middle aged adults with slight female predilection and presents as a slow growing painless mass. The commonest sites are extremities, scalp, back and neck. Microscopically it has a typical morphological appearance. It is multilobulated and is composed of spindle and stellate shaped cells set in an abundant myxoid stroma. The cells show strong positivity for S-100 protein and are EMA negative or it stains only few perineural cells, indicating its close relationship with schwannoma or neurofibroma. But we report a rare case of nerve sheath myxoma in a 40 year old woman which showed typical microscopic features of NSM. However it showed an unusual co-expression of S100 and EMA indicating a bidirectional schwannomatousperineural differentiation. The clinicopathological features, various differential diagnosis, its histogenesis and brief review of literature are discussed below.

*Corresponding author: Dr. Aparna Narasimha, No.22, â&#x20AC;&#x153;Moyenvillaâ&#x20AC;?, Moyenville Road, Langford Town, Bangalore-560025. INDIA Phone: +91 9632140850 E-mail: sonrichie14@gmail.com

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Nerve Sheath Myxoma with Bidirectional Differentiation

Introduction

Nerve sheath myxoma is a rare benign tumour which arises from the peripheral nerves. Harkin and Reed were the first to define the nerve sheath myxoma in the year 1969. Subsequently Gallager and Helwig named this lesion as Neurothekeoma in 1980. [1] Other synonyms for this tumour are cutaneous lobular neuromyxoma, myxomatous perineuroma, bizarre cutaneous neurofibroma, myxoma of nerve sheath and dermal nerve sheath myxoma.[2] Here we present an unusual case of nerve sheath myxoma in a 40 year old female which showed bidirectional schwannomatous and perineural differentiation.

Case Report

pseudocapsule, located in the dermis (Figure 2a). It was seen reaching upto the subcutaneous tissue. The lobules were of varying sizes, separated by thin fibrous septae and are composed of many scattered spindle shaped cells and stellate cells embedded in abundant myxoid matrix (Figure 2b). Mild atypia was noted (Figure 2c). Many multinucleated giant cells were noted, few showed presence of intranuclear inclusions (Figure 2d). Immunohistochemical study (IHC) showed that the spindle cells and stellate cells were diffusely positive for S100 (Figure 3). Epithelial membrane antigen (EMA) staining showed diffuse positivity for the perineural cells scattered at the periphery of the tumour nodules (Figure 4).

A 40 year old female presented with a slow growing mass over the right lower leg since 1 year. It was gradually increasing in size, not associated with pain, soft to firm in consistency. There was no history of antecedent trauma. Patients past medical history was unremarkable and all clinical investigations were within normal limits. No history of similar swelling elsewhere in the body. Systemic examination was within normal limits. A clinical diagnosis of lipoma was given. It was completely excised and sent for histopathological examination. Gross examination: Received skin covered mass which measured 3.5x2x1.5cms. The cut surface showed multiple small well demarcated, translucent, glistening, mucoid nodules of varying sizes. (Figure 1). Microscopic examination: Sections studied showed a skin covered multilobulated mass having a fibrous

Fig. 1: Gross photography showing cut surface of the mass with multiple small well demarkated, translucent, mucoid nodules of varying sizes.

Fig. 2: Microphotography showing a) skin covered multilobulated mass with apseudocapsule. (H&E,X40) b) lobules composed of spindle and stellate shaped cells lying within the mucinous stroma. (H&E,X100) c) Cells with mild nuclear atypia. (H&E, X1000) (Red arrows) d) multinucleated giant cells with intranuclear inclusions. (H&E, X1000) (Red arrows).

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 06, (Suppl) December, 2016

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Fig. 3: Microphotography showing S100 positive spindle and stellate cells. (S100,X100).

Fig. 4: Microphotography showing Epithelial membrane antigen (EMA) positivity in the perineural cells around the tumour micronodules. (EMA,X1000).

Discussion

will be absent or positive in small number of perineural cells.[7] In our case, S100 was diffusely positive in spindle cells as well as EMA showed diffuse positivity among the perineural cells, which were present more in number at the periphery of the lesion, indicating bidirectional schwannomatous differentiation. Similar case with bidirectional differentiation was reported previously by Zamecniket al in his case report.[2] Electron microscopic findings include single or duplicated external lamina investing the cells, desmosome like junctions, cytoplasmic microfilaments, myelin figures and interdigitating cytoplasmic processes.[8] Recent study was done on gene expression profiles of NSM and neurothekeoma. The neurothekeoma expressed gene that primarily encoded glycoprotein and metalloproteinase from macrophages and fibroblasts. The S100 B gene was differentially expressed gene between NSM and NTK. Hence this gene expression study strongly supports that NSM are of peripheral nerve sheath origin and are distinct neoplasm from NTK. [9]

Nerve sheath myxoma was originally described by Harkin and Reed in 1969. Later on in 1980 Gallager and Helwig described a series of tumour under the name â&#x20AC;&#x153;Neurothekeomaâ&#x20AC;?. Many authors suggested neurothekeoma as a variant of nerve sheath myxoma.[3] The clinical and histological pattern of neurothekeoma and nerve sheath myxoma have many overlapping features, which have been discussed in the differential diagnosis and these lesions should be differentiated from each other as they are entirely different entities. NSM is commonly seen in middle aged adults with a male to female ratio are approximately 1:2. It commonly arises from fingers, knee, pretibial region, hips, thigh, lower leg, ankle, foot, trunk, head and neck region.[4] Other rare sites are oral mucosa, gingival, hallux and CNS.[5,6] In our case, a 40 year old female patient presented with a mass the over anterior aspect of right lower leg. It typically presents as a firm, slow growing, painless nodule or mass with size ranging from 0.4 to 4.5 cms. On cut section, the lesion shows multiple tiny well demarcated, translucent whitish mucoid nodules. Microscopically the lesion is located in the dermis or sometimes in dermis and subcutaneous tissue. It is characterised by multilobular structure and each lobule is composed of spindle and stellate shaped cells arranged in swirling, lamellar and concentric patterns, embedded in abundant myxoid stroma. Mild nuclear pleomorphism and rare mitosis may be seen. [4] In our case all these typical features were seen, however mitosis was not seen. Immunohistochemically, the tumour cells are strongly positive for S100 protein, GFAP and Vimentin. EMA

NSM can be confused with many myxoid soft tissue tumours and spindle cell tumours. The myxomatous nature of NSM can be confused with other myxoid tumours such as myxoid neurothekeoma, superficial angiomyxoma, myxoid MFH, myxoid liposarcoma, myxoid neurofibroma, focal mucinosis, soft part chondromas, metastatic mucinous carcinoma etc. Other differential diagnosis for spindle cell lesion includes schwannoma, perineuroma, desmoplastic melanoma, cellular and mixed neurothekeoma. The clinical appearance, gross and microscopic picture of NSM is indistinguishable from that of myxoid variant of NTK. However NSM are S100, GFAP, and Vimentin positive while EMA is negative of focally positive. NTK are

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Nerve Sheath Myxoma with Bidirectional Differentiation

S100 negative and EMA positive. Recent ultrastructural studies have differentiated these two entities which are already discussed. All malignant myxoid tumours are characterised by increased cellularity, vascular pattern and specific cellular elements such as lipoblast etc. [10] Superficial angiomyxoma consists of spindle and stellate shaped fibroblasts admixed with thin walled capillaries. The tumour cells are vimentin positive. Schwannoma shows presence of verocay bodies. Myxoid neurofibroma lacks the lobular pattern, is unencapsulated and may have fibrillary background. Tumour cells may be admixed with collagen bundles. Cellular and mixed neurothekeoma have less myxoid stroma, more spindly plumper epithelioid cells and greater mitotic activity. They are S100 and GFAP negative. Focal mucinosis have no sharp circumscription, paucity of cells and complete absence of vascular structures. Perineuroma has distinct corded basket weave like whorled pattern. Cells are EMA positive, S100 negative. Soft part chondromas have areas of typical hyaline cartilage/ calcification. They are S100 positive but lack GFAP. Superficial acral fibromyxoma is composed of spindle or stellate cells with a storiform & fascicular pattern embedded in a collagenous or fibromyxoid stroma. It is CD 34, CD 99, EMA positive, but S100 negative. [2, 5] Treatment: Complete surgical excision with margins is the mainstay of treatment as NSM has got high chances of recurrence.

Conclusion

To conclude, we report a very rare case of nerve sheath myxoma exhibiting bidirectional differentiation. Pathologist should be aware of the possible EMA expression in NSM. Since NSM may show focal moderate nuclear atypia and mitosis, it should be differentiated from benign and malignant myxoid neoplasms. The use of terms nerve sheath myxoma and neurothekeoma as synonyms or as variants of the same tumour should be avoided because they represent two distinct lesions.

Acknowledgements

Dr.Vijaya, HOD,Department of Pathology

Funding None

Competing Interests None Declared

Reference

1. Spadari F, Guzzi G, Bombeccari GP, Mariani U, Gianatti A, Ruffoni D et al. Nerve sheath myxoma of the tongue. Acta Dermato venerol Croat. 2014; 22(1):52-6. 2. Zamecnik M, Sedlacek T. Nerve sheath myxoma with bidirectional schwannomatous and perineural differentiation. Cesko-slovenskapatologie. 2010;46(3):73-6. 3. Reed RJ, Paulitzer DR. Tumors of neural tissue. In: Elder DE, Editor. Leverâ&#x20AC;&#x2122;s Histopathology of the skin. 10th ed. Philadelphia: Lippincott Williams & Williams; 2009, p 1126-27. 4. Fathaddin A, Fatani R. Palmar nerve sheath myxoma: A case report. Oman Med Jr. 2012;27(3):212-6. 5. Gehrke JC, Hamson KR, Havey AD. Dermal nerve sheath myxoma of the hallux: a case report. Foot Ankle Int. 1994; 15(12):666-8. 6. Malkoc M, Ormeci T, Keskinbora M, Yilmaz A, Korkmaz O, Tanik CB. Nerve sheath myxoma of the dorsal paravertebral space. Int J Surg Case Rep. 2014;5(11):858-60. 7. Bhat A, Narasimha A, CV, VK S. Nerve sheath myxoma: report of a rare case. Journal of clinical and diagnostic research. 2015;9(4):7-9. 8. Hamodat M. Nerve sheath myxoma presenting as finger nodule in 39 year old female. The internet journal of pathology. 2009:10(1). 9. Sheth S, Li X, Binder S, Dry SM. Differential gene expression profiles of neurothekeomas and nerve sheath myxomas by microarray analysis. Mod Pathol. 2011;24(3):343-54. 10. Suh YL, Song KY, Kim JM. Nerve sheath myxoma (Neurothekeoma)- a case report. J Korean Med Sci.1992;7(1):85-9.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 06, (Suppl) December, 2016

Letter to Editor Primary Vocal Cord Histoplasmosis: A Rare Presentation Impersonating Laryngeal Malignancy Malini Goswami Department of pathology, Rajiv Gandhi Cancer Institute and Research Centre, New Delhi, India

Dear Sir,

A 53 year old male presented with gradual onset of hoarseness of voice. However there was no history of dyspnoea, dysphagia, haemoptysis, weight loss or fever. He was a diabetic and a chronic smoker since 20-25 years with no past history of tuberculosis. His general condition was good and local examination of oral cavity, oropharynx and neck was within normal limits. Direct laryngoscopy was done and showed a growth on the free edge of right true vocal cord with retained mobility of the cord. The false vocal cords showed mild edema bilaterally, however the rest of larynx was within normal limits. MRI showed an altered signal intensity soft tissue thickening involving the entire length of right true vocal cord. No other lesion in any other organ was detected. An incisional biopsy of the vocal cord lesion was taken and subjected to histopathological examination. Microscopic examination revealed ulcerated mucosa with dense myofibroblastic proliferation along with interspersed yeast forms resembling histoplasma capsulatum lying predominantly intracellularly. The basophilic cytoplasm of the fungal cells showed retracted appearance. Special stains with Gomoriâ&#x20AC;&#x2122;smethanamine silver highlighted the fungal yeast forms (Figure 1-A). The unusual finding was absence of granulomatous response in an immunocompetent host. A diagnosis of vocal cord histoplasmosis was rendered and the patient was subsequently treated with amphotericin and recovered in 6 weeks Histoplasmosis, a disease caused by the dimorphic fungus Histoplasma capsulatumis endemic in certain parts of the world including Asia.[1]It usually infects the lung however a disseminated disease may also occur with involvement of bone marrow, lymph nodes, adrenal glands

Fig. 1: Histoplasmacapsulatum (marked (Gomoriâ&#x20AC;&#x2122;smethanamine silver, 1000X).

arrows

,gastrointestinal tract, tongue and oral mucosa.[2]The primary histoplasmosis of larynx with no lung involvement is extremely rare with no evidence of dissemination even a rarer phenomenon. Laryngeal involvement occurs in the mucocutaneous form of the chronic disease.[3]As per data histoplasmosis of head and neck is more common in adults and exclusively in males.[3] Laryngeal histoplasmosis is a benign and easily curable disease. However due to the unusual location and deceptive clinical presentation, it may pose a few diagnostic challenges. It may clinically mimic a malignancy and microscopically resemble blastomycosis (due to similar morphology),tuberculosis(due to presence of necrosis and granulomas) and squamous cell carcinoma because of atypical epithelial response (pseudo-epitheliomatous

*Corresponding author: Dr Malini Goswami, c/o Pranab Goswami , House number 11, Sundarpur, Bye lane 1(left), RG Barua road, Guwahati-781005, Assam, India Phone: +91 9654488079, 9599875132 E-mail: malinig87@gmail.com

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Goswami et al. hyperplasia).[4] Therefore an adequate history coupled with a proper histopathological as well as microbiological examination is a necessity. Moreover all biopsy negative suspicious lesions of the upper airways should be always dealt with carefully keeping in mind a differential diagnosis of histoplasmosis.

Abbreviations and Symbols MRI: magnetic resonance imaging

Acknowledgements

I would like to thank Dr. Anurag Mehta, who is the Head of Department, Pathology, for his encouragement and support.

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Competing Interests None Declared

References

1. Benevides CF, Duraes RO, Aquino B,et al. Bilateral adrenal histoplasmosis in an immunocompetentman. RevSocBrasil Med Trop 2007;40:230-233. 2. Habib SK, Patwary SA, Khan MAI,etal. PrimaryHistoplasmosis of Vocal Cord in an 3. immunocompetent elderly Man- A case Report with Literature Review. J Medicine 2012;13:77-81.

Conflict of Interest

4. Sonkhya N, Mehta R, Sonkhya D, et al. Primary Histoplasmosis of larynx: A Case Series and Review of Literature. International Journal of Otolaryngology and Head & Neck Surgery 2013;2:47-51.

FUNDING

5. Katoch P, Bhardwaj S. Primary Laryngeal Histoplasmosis. JK Science 2009;11:89-90.

None None

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Letter to Editor Pilomatrixoma with Extensive Ossification: A Rare Case Report Chidambharam Choccalingam* and Vijayshree Raghavan Chettinad Medical College, Kelambakkam, India

Dear Sir,

Pilomatroxoma is a benign skin adnexal tumour with differentiation towards hair matrix. It is usually a deep seated firm nodule, found mostly in the head and neck region and upper extremities.[1, 2] Histopathological examination of an established pilomatrixoma shows masses of epithelial cells with intervening connective tissue stroma. The epithelial cells are of two types namely basophilic and eosinophilic cells. The basophilic cells are seen in the periphery and are characterized by indistinct cell borders, minimal cytoplasm, hyperchromatic nucleus and increased mitosis. The eosinophilic cells are labelled as eosinophilic shadow cells and are seen at the centre with distinct cell borders, abundant eosinophilic cytoplasm and no nuclear staining. The eosinophilic shadow cells arise from the basophilic cells and the transition may be smooth, characterized by transitional eosinophilic cells with eosinophilic cells and pyknotic nucleus or rarely abrupt. Calcification is commonly seen either in the eosinophilic shadow cells or stroma, which partly imparts to its other name calcifying epithelioma of Malherbe. The stroma may also show hemosiderin deposition, melanin deposition and ossification of stroma in decreasing order of frequency.[1-3] We present a 32 year old male, with pilomatrixoma having extensive osseous differentiation of stroma. A 32 year old south Indian male presented with painless swelling in the left cervical region for a period of 9 years. The swelling measured 3 x 2 cms and was firm to hard in consistency. The swelling was mobile and overlying skin was normal. A clinical diagnosis of calcified cyst/ calcified lymph node was made. On fine needle aspiration of the swelling attempted twice, no cellular aspirate was obtained. Subsequently, the swelling was excised and sent for histopathological examination. On gross

examination, a nodular mass measured 2 x 2 cms with smooth outer surface. On cut section, grey white hard to gritty areas were noted. On microscopic examination, well circumscribed lesions with islands of eosinophilic ghost cells and interspersed transitional epithelial cells were seen. The intervening stroma showed fibrofatty tissue with areas of calcification and areas of extensive ossification characterized by bony trabaculae with osteocytes and rimmed by few giant cells. No haematopoietic cells were seen within the trabaculae. Based on the histologial findings, a diagnosis of pilomatrixoma with extensive ossification was made (Figure 1). Pilomatrixoma with osseous metaplasia is a rare occurrence. Occasionaly extra-medullary haematopeoisis can occur, which was not noticed in our case.[4, 5] It has been shown that bone morphogenetic protein (BMP-2) localized in epithelial cells plays a role in osseous differentiation.[1] The osseous differentiation was highlighted by birefringence under polarized light microscopy.[6] The morphology of the lesion varies with the age of the lesion. Early lesions have predominantly basaloid cells with brisk mitotic activity, whereas the established old lesions have islands of both basaloid and eosinophilic shadow epithelial cells with intervening stroma containing blood vessels, inflammatory infiltrate, giant cells and occasionally hemosiderin, melanin deposition or bony metaplasia.[2] Typical pilomatrixoma cytology contains clusters and scattered basophilic small cells, anucleate squames and nucleated squamous cells. [7] The extensive ossification in our case would be the reason for the dry tap during fine needle aspiration. Although pilomatrixoma and the various stromal changes documented are benign skin adnexal tumours, occasionally atypical forms with unknown malignant potential are seen. The atypical forms are characterized by loss of polarity,

*Corresponding author: Dr. Chidambharam Choccalingam, Chettinad Medical College, Kelambakkam, India E-mail: Chidambharam@gmail.com

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Choccalingam et al.

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Fig. 1: Microscopic examination showing (A) islands of epithelial cells with adjacent bony trabaculae with osteocytes (H&E, 10x); (B) Transitional epithelial cells and adjacent ossified stroma (H&E, 40x).

Fig. 2: Ossified stroma showing birefringence under polarizing light microscope (H&E, 20x).

increased mitosis, and basaloid cell pleomorphism.[2] Surgical resection is curative and our patient with an established old lesion had no recurrence in follow up of 6 months.[1] In conclusion, pilomatrixoma with extensive ossification is rare. Awareness about the nature of entity as benign, knowledge in technical difficulties while performing fine needle aspiration is essential to reduce anxiety in patients with pilomatrixoma with osseous metaplasia and in subjects with dry tap aspirates.

1. Weedon, D., Weedon’s Skin Pathology. 3 ed.; Churchill Livingstone Elsevier: 2010.

2. Calonje E, Brenn T, Lazar A, Mckee PH., McKee’s Pathology of the Skin. 4 ed.; Elsevier Saunders: 2012; Vol. 2. 3. Hombal P., An unusual presentation of pilomatrixoma. Journal of the Scientific Socirty 2013, 40 (1), 37-38. 4. Khuraijam B, Sobti P, Shangpliang D, Khurana N. Extramedullary Haematopoiesis in a Case of Pilomatricoma. Journal of Clinical and Diagnostic Research 2016, 10 (6), ED17-ED18. 5. Kambale, T.; Iqbal, B.; Jain, A.; Sawaimul, K., Pilomaricoma with florid osseous metaplasia: a rare case report. Mnoufia Medical Journal 2015, 28, 780-782. 6. Suvarna, K. S.; Layton, C.; Bancroft, J. D., Bancroft’s THEORY and PRACTICE of HISTOLOGICAL TECHNIQUES. Churchill Livingstone: 2013. 7. Koss LG, Melamed MR. Koss Diagnostic Cytology and Its Histopathologic Bases. 5 ed.; Lippincott Williams and Wilkins: 2006.

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