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Annals of Pathology and Laboratory Medicine 2016; Vol. 3, Issue 5 (Supplementary)

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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

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Emergence of Chikungunya infection in North India Krunal D Mehta, Shruti Malik, K P Singh, T N Dhole

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Apoptosis and micronucleus in cervical pap smears: promising assays to increase the diagnostic value of the test. Amoolya Bhat, Vijaya C, Padmasri R

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Utility of modified bleach method technique for the demonstration of acid fast bacilli in the diagnosis of Tuberculous lymphadenopathy in comparison to routine Ziehlneelsen staining Manika Khare, Manju Kaushal

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Contents

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Hepatitis C With or Without Schistosomiasis Afkar Abdel-GhanyBadawy, Olfat Hammam, Mona Moussa, Raafat Atta, Tarek Aboshousha, Noha Said, Nihal El-Assaly, Nawal El-Badrawy

III

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Contents Original Article Phyllodes tumour of Breast: Ki 67 expression in various histological subtypes B R Vani, Deepak Kumar B, Padma Priya K, Sandhyalakshmi B N, Srinivasamurthy .

A362-A367

Prevalence of carbapenemases with detection of NDM-1 gene in nonfermenters isolated from A368-A373 a tertiary care hospital of north India Mohan Lal Gupta, Nidhi Negi, Pradyot Prakash, Malay Ranjan Sen The Histopathological and Epidemiological Study of Pediatric Brain Tumors in a A374-A381 Tertiary Care Hospital, Mumbai Sangita Margam, Nitin Maheshwar Gadgil, Ganesh Ramdas Kshirsagar, Vaishali Prashant Gaikwad, Prashant Vijay Kumavat, Chetan Sudhakar Chaudhari Spectrum of endometrial lesions in patients with abnormal uterine bleeding: A A382-A387 histopathological study Ankita Goel, Vissa Shanthi, Nandam Mohan Rao, Parul Jain, Syam Sundara Byna, Jyothi Conjeevaram Histomorphologic Pattern of Renal Disease in Patients with Acute Nephritic A388-A396 syndrome: A Single Centre South Indian Study Clement Wilfred Devadass, Vijaya Mysorekar V, Gireesh Siddaiah M, Mahesh E, Gurudev Channabasappa K Cytohistological Study of Head and Neck lesions and their Diagnostic Pitfalls Aparna Bhardwaj, Apoorva Pandey, Sanjeev Kishore, Sanjay Kaushik, Tripti Maithani Evaluation of small intestinal biopsies in malabsorption syndromes Ashmeet Kaur, Poojaba Jadeja, Neha Garg, SML Rai, N Mogra

A397-A407 A408-A414

Nosocomial Burn Wound Infections due to Non-Fermenting Gram Negative Bacteria: A415-A420 Our experiences from a tertiary care center in North India Naz Perween, S Krishna Prakash, Prabhav Aggarwal, Lalit Mohan Gupta Clinicopathological evaluation of Prostatic Adenocarcinoma: A unicenter study A421-A426 Clement Wilfred Devadass, Rashmi Krishnappa, Sharath Soman, Vijaya Viswanath Mysorekar, Radhika Kunnavil, Sharon Roshin Reginalt Role of Bone Marrow profile in Cytopenias A427-A432 Neelima Bahal, Brijesh Thakur, Aparna Bhardwaj, Sandip Kudesia, Seema Acharya, Sanjeev Kishore Assessemnt of Biochemical, Serological, Molecular Viral Marker and Histological A433-A440 Parameters in HBeAg Positive Chronic Hepatitis B to Determine Therapy Response Rig Vardhan, Kavita Sahai, Gurvinder Singh Chopra, Mukul Bajpai Etiological Evaluation of Pancytopenia in a Tertiary Care Hospital Anuja Dasgupta, Shetty K Padma, Sajitha K, Jayaprakash Shetty

A451-A450

Histopathological Study of Villous Morphology in Spontaneous First Trimester A451-A457 Abortions Archana Shetty, Aparna Narasimha Prognostic significance of Ki 67 labelling index and P63 immunoreactivity in intra A458-A464 cranial and intra spinal meningiomas Shrinivas Bheemrao Somalwar, Naval Kishore Bajaj, Sujani Krishnaprakash Madbhushi, Ezhil Arasi Nagamuthu Thrombocytopenia in Dengue illness: Destruction, Suppression and Composite A465-A470 platelet index: A Retrospective study Vani Krishnamurthy, Rajalakshmi Rajeshakar, Srinivasa Murthy Doreswamy Blood donor deferral analysis: A tertiary care centre experience. Harjot Kaur, Rahul Mannan, Mridu Manjari, Sonam Sharma, Tania Garg, Tejinder Bhasin

III

A471-A475

Correlation of Her-2/neu Status With Estrogen, Progesterone Receptors and A476-A483 Histologic Features in Breast Carcinoma Anupama dayal, Rupal J Shah, Sadhana Kothari, S M Patel A study of haematological profile in Human Immune Deficiency Virus infection: A484-A489 Correlation with CD4 counts Sheela Devi CS, Suchitha Satish, Manali Gupta

Case Report

Primary Intracerebral Myxopapillary Ependymoma : A Rare Case Report Priyanka Patangia, Naresh N. Rai, Rajeev Saxena, Preetam Singh Mandawat

C236-C240

Co-Occcurence of Two Different Subtypes of Renal Cell Carcinoma in A Unilateral C241-C245 Healthy Kidney Neeraj Dhameja, Hema Goyal, Priyanka Aswal, U S Dwivedi Cytodiagnosis of Schwannoma of parotid gland: a rare entity in a child Nishat Afroz, Shagufta Qadri, Sunanda Chauhan, Syed Abrar Hasan Gastrointestinal Stromal Tumor in a roux en Y limb Naveen Kumar, Saishalini Chinnathambi Narayanan, Prathiba Duvvuru, Babu Elangovan

C246-C249 C250-C253

An unusual case of bladder tumour with diagnostic dilemma; A rare case report C254-C258 Jayaprakash K Shetty, Kishan Prasad, Rajeev T P, Nigi Ross Philip, Anitha Chakravorthy, Prajwal Ravinder An unusual case of follicular variant of Papillary Thyroid Carcinoma with temporal C259-C263 bone metastasis diagnosed by cytology Poonam G Lahane, Prashant Kumavat, Kavita Khedekar, Nitin M Gadgil, Chetan S Chaudhari Small Cell Carcinoma of Gallbladder: A Rare Case Report Nidhi Gupta, Bawana Raina, Arvind Khajuria

C264-C267

Lymphadenopathic form of PAX 3-7/FKHR fusion gene and ALK negative, solid type C268-C272 of alveolar rhabdomyosarcoma in an infant: A rare entity mimicking lymphoma with a review of literature. Chetan Sudhakar Chaudhari, Ganesh Ramdas Kshirsagar, Prashant Vijay Kumavat, Nitin M Gadgil Late relapse of malignant peripheral nerve sheath tumor after 18 years. Case report C273-C278 with differential diagnosis. Csaba Biró, Katarína Macháleková, Štefan Galbavý, Gabriel Bognár, Sarah Catharina Hubinská, Martin Kopáni, Karol Kajo, Dagmar Kalátová

Letter to editor Transitional Cell Carcinoma of Cervix Gazi Naseem Ahmed, M N Baruah

Myelolipoma of spleen: an unusual presentation Malini Goswami

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L20-L221

Original Article Phyllodes Tumour of Breast: Ki 67 Expression in Various Histological Subtypes B.R. Vani1*, Deepak Kumar B1, Padma Priya K2, Sandhyalakshmi B.N.1 and Srinivasamurthy1 Department of Pathology ,ESIC Medical College and PGIMSR, Rajajinagar, Bangalore, India Department of Pathology, Sapthagiri Institute of Medical Sciences and Research Institute, Hesarghatta, Bangalore, India 1

Keywords: Phylloides, Borderline Malignant, Ki 67, Breast

ABSTRACT Background: Phyllodes tumours [PT] are uncommon fibroepithelial tumours with marked stromal proliferation over epithelial component with majority being benign.Along with grade, additional study of proliferative markers such as p 53,Ki 67 are essential to identify those with potential for aggressive behaviour.In this background study was undertaken to asses the histopathological characters and correlate Ki 67 expression in different subtypes of PT. Methods: A total of 21 cases were studied.Clinical details, gross features and primary histologic features were taken into consideration. Immunostaining for Ki 67 were performed. Ki 67 immunoexpression was categorised as 0-10, 11-30, 31 and above depending on the percentage of positive tumor cells in each case. Ki 67 immunoexpression was correlated with histologic grade and clinical features. Result: 14 cases (66.7%) of Benign, 03 cases (14.3 %) of borderline, and 04 cases (19 % ) cases of malignant phylloides tumor were seen. Average Ki 67 expression in BPT and BlPT was 4.5 % (range 1-8%) and 14 % (range 13-15 % ) respectively. MPT exhibited Ki range of 41-80 % with average LI of 56 %. A significant association was seen between expression of Ki 67 in different grades of phyllodes tumour. Conclusion: Inclusion of Ki 67 in routine histopathology reporting of phylloides is mandatory as Ki LI proves to be of paramount importance in sub categorisation of phylloides.

*Corresponding author: Dr B.R. Vani, 169/B, 4th Main, 3rd cross, J P Nagar, # rd phase, Bangalore-78, INDIA. Phone: +91 9945511383 Email: vanibr@yahoo.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

Phyllodes tumours [PT] are uncommon fibroepithelial tumours with marked stromal proliferation over epithelial component.Majority are benign and constitutes 0.3-1.5% of breast neoplasms. [1,2,3,4,5]. Common clinical presentation is of rapidly growing lump in the breast with median age of occurance being 45 years and 10-20 years later than fibroadenoma. [2,6,7,8,9]. PT encompasses benign (BPT), borderline (BlPT) and malignant subtypes (MPT) with varied propensity for recurrence, over all incidence being 8-40 %. [3,5]. Metastasis corresponds with grade of phylloides and is 4%, 22% in BlPT and MPT respectively. [8,9].MPT has poor prognosis with 5 year survival rate of 66% [4]. Histological grade is based on semiquantitative assessment of degree of stromal cellularity, cellular pleomorphism, mitotic activity, and tumour margins. [8] Additional study of proliferative markers such as p53, Ki67 are essential to identify those with potential for aggressive behaviour. [3]. Literature data shows tumour grade corresponds with p53, and Ki 67 expression. [10]. In this background study was undertaken to asses the histopathological characters and correlate Ki 67 expression in different subtypes of PT.

Materials and Methods

The study was undertaken in department of pathology,ESIC MC & PGIMSR,RNR, Bangalore from January 2010-December 2013. Total number of PT cases were 21. Clinical details including age of patient, nature of surgery etc were collected from medical records. Gross features of the specimen and appearance at cut section were noted. During this period cases of PT if recurred were also studied. Primary histologic criteria defining PT considered is stromal overgrowth with absence of epithelial component in atleast one low power field. [1]. Further depending on histological features PT were graded as BPT, BlPT, MPT. [8]. PT with mitotic activity (MA) less than 5/ 10 high power field (hpf) and any one or more of the features as described for BPT were categorised as BPT. Tumours with MA more than 10 / 10 hpf and any one or more of the features describing those of MPT were categorised as MPT. Tumours with MA more than 5/10 hpf but less than 9/10 hpf and intermediate features describing those of BlPT were categorised as BlPT. [8]

background level were scored as positive and labelling index [LI] was determined by counting a minimum number of 1000 cells in the most active areas. Ki 67 immunoexpression was categorised as 0-10, 11-30, 31 and above depending on the percentage of positive tumor cells in each case. Data was analysed using descriptive statistics such as mean and standard deviation.Difference between 2 categories were evaluated by using chi square test of significance for independence. A p value less than 0.05 was considered to be significant.

Result

During the four year study period total phylloides case were 21. Age range was 24-64 years with mean age of 38.2 years. [Table 1]. Majority presented as solitary lump in breast (95.23 %). One patient had synchronous fibroadenoma [FA] in left breast with PT affecting right breast. Lumpectomy was the nature of surgery in 85.7 % cases. Modified radical mastectomy [MRM] was performed in three cases since FNA reported as malignancy. Over all PT size ranged from 2 to 26 cms with mean of 9.7 cms. Average size in BPT is 6.9 cms (2- 14 cms range),BlPT 9.3 cms (7-14 cms range), MPT 19.7 cms (12-26 cms). [Table 2]. Histologically BPT were 14 cases (66.7%), BlPT 03 cases (14.3 %), MPT 04 cases (19 % ). [Table 3]. All BPT exhibited histological features as defined by WHO. In BlPT uniform stromal cellularity was a feature noted in 2 cases. Third case with heterologous stromal expansion ; though had well circumscribed margins, recurred and this recurred tumour had intermediate margins with areas of necrosis and other histological features defining BlPT. IHC for Ki 67 done on this recurred BlPT had same score as primary tumour. All MPT exhibited the classical features defined by WHO with monomorphic or pleomorphic sarcomatous stroma. Axillary lymphnode dissection was accompanied with MRM performed in two cases and all the lymphnodes were free of tumour microscopically.IHC for vimentin was positive, while EMA,SMA,CK was negative . Average Ki 67 expression in BPT, BlPT was 4.5 % (range 1-8%) and 14 % (range 13-15 %) respectively. MPT exhibited Ki range of 41-80 % with average LI of 56 %. [Table 4] [Fig 1,2]

Immunostaining for Ki 67 were performed. Positive and negative controls were run with each batch of IHC for Ki 67. The proportion of nuclear positivity defined by any detectable brown staining of the nucleus above the

Upon statistical analysis, a significant association was seen between tumor size and various grades of phyllodes tumour. Expression of Ki 67 in different grades of phyllodes tumour was also statistically significant.

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Phyllodes Tumour and Ki 67 Expression

Table 1: Age distribution in different grades of phylloides tumor. Age (yr)

Benign PT (N= 14)

Borderline PT (N=3)

Malignant PT (N=4)

< 30

31-40

41-50

51-60

>60

Table 2: Tumor size in different grades of phylloides tumor. Tumor size

Benign PT (N= 14)

Borderline PT (N=3)

Malignant PT (N=4)

< 10 cm

10-20cm

>20cm

P value

0.019

Table 3: Histological features in different grades of phylloides tumor. Histological features

Benign PT (N= 14)

Borderline PT (N=3)

Malignant PT (N=4)

Stromal cellularity

Low (14)

Moderate (3)

High (3)

Cellular pleomorphism

Mild (14)

Moderate (2)

Marked (3)

Mitotic index

<5/10 hpf (14)

5-9/10 hpf

>10/10hpf

Margins

Well circumscribed (14)

Well circumscribed (2) pushing (1)

Infiltrating (3)

Stromal proliferation

Uniform (14)

uniform

Irregular (3)

Table 4: Average[Av] Ki 67 expression in different grades of phylloides tumor. Ki 67 expression

Benign PT cases(Av Ki in % )

Borderline PT cases(Av Ki in % )

Malignant PT cases(Av Ki in % )

< 10

14 (4.5)

11-30

3 (14)

>31

4 (56)

Fig. 1: Benign Phyllodes tumor; (a) Gross ; (b) H&E â&#x20AC;&#x201C; 4x; (c) Ki67 IHC 10x..

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 05, (Suppl) Nov. 2016

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Fig. 2: Borderline Phyllodes tumor; (a) Gross ; (b) H&E – 4x; (c)IHC Ki 67 IHC 40x.

Fig. 3: MalignantPhyllodes tumor; (a) Gross ; (b) H&E – 10x; (c) Ki67 IHC 10x; (d) Ki 67 IHC 40x.

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Phyllodes Tumour and Ki 67 Expression

Discussion

Johnnes Muller in 1838 coined the term cystosarcoma phylloides due to morphologic leafy bulbous protrusion of epithelium covering proliferating stroma and often extending into cystic space. [4,6,7,11] Majority were benign and not all cases exhibited cystic degeneration ,hence WHO renamed the misleading nomenclature as phylloides tumour. [4,7,8,11,12] PT constitutes 0.3 to 1 % of primary breast neoplasm and 2.5 % of fibroepithelial breast lesions. [4,6,12,13]. In our institution breast lesions during the study period constituted 6.7% of total histopathology specimens. Prevalence of PT was 2.3 % of breast lesions and is in concordance with literature available . [6,7]. Median age group of presentation is 40-50 years. [2,6]. Occurance at younger age with average of 25- 30 years reported in Asians. [2,7]. In the present study maximum number of cases were seen in age group of 31-40 years (52.4 %) with median age of presentation of 38.2 yrs ,which is in concordance to thedata available in literature. [6,13]. Phylloides present as painless, palpable mass in upper outer quadrantfrequently affecting right breast[6,13] ; bilaterality reported in 3.4-3.8%.[6]. In our case series all were solitary ,majority occurring in upper outer quadrant with 12 cases affecting right breast and 9 cases involving left breast. One patient presented with synchronous FA of opposite breast .Literature search revealed concomitant FA in 4.2 % to 1/3 rd of women with PT and in proportion of FA somatic mutation result in monoclonal stromal proliferation progressing to PT. [4,6]. In our patient probably an existing FA would have shown monoclonal proliferation and hence patient presented with PT. This under scores the fact that FA and PT represent two ends of a single spectrum with origin being intralobar stroma. In present study tumour size varied from 2-26 cms, with average size of 9.7 cm and is in concordance with average size reported in literature.[6]. Mean tumour size of BPT, BlPT, MPT is 6.9 cm,9.3 cm and 19.7 cm respectively. No single morphologic feature predicted clinical tumour behavior and histologic grade is independent of size, presentation, clinical behaviour.[4,6]. In our study increased size had increased grade and was statistically significant. Tumours were lobulated, fleshy ,grey white to grey tan with cystic change in few.In malignant phylloides, patient presented with sudden growth and tumours were bulky varying in size from 12-26 cm. Areas of haemorrhage, necrosis, microcysts and infiltrating nodules were a feature in MPT.The skin over the tumour exhibited dilated veins; however no ulceration noted in our study. [5,12].

On cytology smears showed large and increased stromal fragments with plump spindle cells in the background, suggesting phylloides, however a differential of fibroadenoma was offered. Smears exhibiting marked nuclear pleomorphism, increased mitotic activity prompted malignancy of breast; epithelial or stromal. Phylloides histology exhibited proliferating fibroblastic stroma lined by ductal epithelial cells projecting into cleft like spaces, appearing morphologically as leaf like pattern . In a typical benign phyllodes, stroma was reminiscent of exaggerated intracanalicular pattern of fibroadenoma.Borderline tumours exhibited heterogeneity with hypercellular and hypocellular areas and mitotic count of < 10/10hpf. The sarcomatous stroma ranged from monomorphic to pleomorphic resembling fibroscaroma, malignant fibrous histiocytoma or liposarcoma. [11] According to various studies malignancy in PT range from 7 to 45 %. [4,6] In our study MPT comprised 1.7 % of all type of primary malignant lesions of breast. Of all the PT ;MPT accounted for 19.04 % and this in concordance with study by Eroglu E et al. [13] A remarkable feature observed in 2 cases [50 %] of MPT was secondary changes comprising of osteoclastic giant cells and areas of necrosis. The stroma in these osteoclast rich lesion showed a high Ki 67 LI of 80 % and 56 % ; the same has been notified as a case report. [15] Metastasis is 25-50 % [6,7] and is via blood often to lungs,pleura,bone,CNS.[6,11] Axillary metastasis is uncommon, while axillary lymphadenitis seen in 10- 20% patients exhibits reactive histology as noted in 2 cases of MPT that we studied. [6,7,11,14]. Chan Y J et alstated that BPT with Ki expression more than 10 % needs to be treated and followed up to avoid recurrence and malignant transformation. [1]. In our study all BPT had Ki less than 8 % with average of 4.5 %. Gatalica et al reported mean Ki of 7.73 and 23.42 in BPT and MPT respectively. [16]. The more obvious increased Ki LI was seen as expected in malignant phylloides category and our study showed a high mean Ki 67 LI of 56 % as compared to Gatalica et al. We conclude that expression of Ki 67 correlated well with morphologic grade and was statistically significant similar to Chan Y J et al.

Conclusion

Inclusion of Ki 67 in routine histopathology reporting of phylloides is mandatory as Ki LI proves tobe of paramount importance in sub categorisation of phylloides.

Acknowledgement Nil

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 05, (Suppl) Nov. 2016

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Funding Competing Interests

8. Tavassoli FA, Devilee P, editors. World Health Organization of Tumors. Pathology and Genetics of Tumors of the Breast and Female Genital Organs. Lyon: IARC Press; 2003.

Reference

9. Rosen PP. Rosen’s Breast Pathology. 3th ed. Philadelphia: Lippincott William and Wilkins; 2009.

None

None Declared

1. Chan YJ, Chen BF, Chang CL, Yang TL, Fan CC. Expression of p53 Protein and ki 67 Antigen in Phylloides Tumor of the Breast. J Chin Med Assoc 2004;67:3-8. 2. Khurshid A, Kayani N, Bhurgri Y. Phylloides Tumors in Adoloscent Girls and Young Women in Pakistan. Asian Pacific J Cancer Prev. 7:563-566. 3. Esposito NN, Mohan D, Bruhky A, Lin Y, Kapali M, Dablo DJ. Phyllodes Tumor. A Clinicopathologic and Immunohistochemical Study of 30 cases. Arch Pathol Lab Med. 2006;130:1516-21. 4. Parker SJ, Harries SA. Phyllodes Tumors. Postgrad Med J 2001;77:428-35. 5. Carter D, Schnitt SJ, Mills RR. The Breast. In: Carter D, Greenson JK, Reuter VE, Stoler MH, Mills SE, Sternberg’s Diagnostic Surgical pathology. 5th ed. Philadelphia: Lippincott Williams and Wilkins:2010, The Breast;285-350. 6. Abdelkrim SB, Trabelsi A, Bouzrara M, Boudagga MZ, Memmi A, Bakir DA, Mokni M. Phyllodes tumors of the Breast: A review of 26 cases. World Journal of Oncology 2010,1(3):129-34. 7. Lester SC. The Breast. In: Kumar V, Abbas AK, Fausto N, Aster JC,editors. Robbins and Cotran Pathologic Basis of Disease. 8th ed. India.Elseivier India Pvt Ltd;2010.

10. Kuruk U, Bayol U, Pala EE, Cumureu S. Importance of P53, Ki-67 expression in the differential diagnosis of benign/malignant phyllodes tumors of the breast. Indian J Pathol and Microbiol 2013; 56(2): 129-34.

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11. Rosai J. Rosai and Ackerman’s Surgical Pathology. 10th ed. New Delhi: Reed Elsevier India Private Limited; 2011. Chapter 20, Breast;1659-1770. 12. Stamatakos M, Tsaknaki S, Kontzoglou K, Gogas J, Kostakis A. Safioleas. Phyllodes tumor of the breast: a rare neoplasm, though not that innocent. Internal Seminars in Surgical Oncology. 2009. 13. Eroglu E. Irkkan C, Bulak H, Kilicoglu B, Oral S. Phyllodes tumor of the breast: case series of 40 patients. Gazi Medical journal 2003;14:29-34. 14. Ahmed S, Rahman MA, Murshed KM, Shirin L. Phyllodes Tumor of Breast: BSMMU Experience. J Bangladesh CollPhySurg 2007;2570-72. 15. Vani BR, Kumar BD, Sandhyalakshmi B N, Geethamala K, Murthy V S, Radha M. Malignant phyllodes tumor with osteoclastic giant cells. Saudi J Health Sci 2014;3:124-6. 16. Gatalica Z, Finkelstein S, Lucio E, Tawfik O, Palazzo J, Hightower B et al.p 53 protein expression and gene mutation in phyllodestumour of the breast.Pathol Res Pract 2001;197:183-7.

Original Article Prevalence of Carbapenemases with Detection of NDM-1 Gene in Nonfermenters Isolated from a Tertiary Care Hospital of North India Mohan Lal Gupta, Nidhi Negi*, Pradyot Prakash and Malay Ranjan Sen Banaras Hindu University, Varanasi, India Keywords: Carbapenemases, Metallo-Î˛-lactamases, Minimal Inhibitory Concentration.

ABSTRACT Background: Increasing multidrug resistance in nonfermenters is of great concern to mankind because the drugs left in pipeline are only a few for the serious infections in hospital ICU settings. The present study was performed to determine the prevalence of carbapenem resistance among nonfermenters and to characterize the most prevalent MBL genes (NDM-1) by polymerase chain reaction, among phenotypically MBL positive isolates in a tertiary care centre of north India. Methods: A total of 383 clinical isolates comprising of 181 A. baumanii and 182 P. aeruginosa were included in this study. The A. baumanii and P. aeruginosa were identified by using standard bacteriological techniques. All these isolates were screened for imipenem and meropenem resistance by Kirby-Bauer disk diffusion method and Minimal Inhibitory Concentration (MIC) methods according to CLSI guidelines. Results: In the present study, the two nonfermenters (A. baumanii and P. aeruginosa) showed highest susceptibility to polymyxin-B (78.2%) followed by meropenem (70.5%), imipenem (66.4%), tigecyclin (57.6%) and combination of piperacillin-tazobactam (51.5%) by using disc diffusion method. Conclusion: Treatment of infections caused by nonfermerters like P. aeruginosa and A. baumanii producing different carbampenemases including NDM-1 are now posing serious challenge as these infections are resistant to almost all available antimicrobial agents.

*Corresponding author: Dr Nidhi Negi, Banaras Hindu University, Varanasi, India Phone: +91 9456569166 Email: nidhinegi28@gmail.com

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Gupta et al.

Introduction

Non Fermentative Gram-Negative Bacilli (NFGNB) are generally saprophytic in nature but can cause a significant number of infections, particularly in the immunocompromised patients. Pseudomonas aeruginosa and Acinetobacter baumanii are the commonest nonfermenters implicated in such infections. Infections caused by other species are relatively infrequent. [1] Resistance to carbapenems is of great concern as these are considered to be the antibiotics of last resort to combat infections by multidrug resistant bacteria in recent days, especially in ICUs and other high risk areas of a hospital. [2] Resistance to carbapenem occurs due to decreased outer membrane permeability, increased efflux system and emergence of carbapenem hydrolyzing enzymes.[3] Out of huge spectrum of such prevalent carbapenemases, metallo beta lactamases (MBL) are one of the most important enzyme. Therefore it seems imperative to see presence of resistance against different antibiotics specially imipenem and meropenem in the two non-fermenters that are notoriously known for very high resistance. Keeping in mind the above factors, this study was planned to see overall prevalence of carbapenem resistance with special reference to MBL including blaNDM in the tertiary level hospital of North India.

Materials And Methods

A total of 383 clinical isolates comprising of 181 A. baumanii and 182 P. aeruginosa were included in this study. These organisms were isolated from specimens like endo-tracheal aspirate, pus, urine, blood, sputum, pleural fluid and ascitic fluid of patients admitted to different wards and OPDs. The A. baumanii and P. aeruginosa were identified by using standard bacteriological techniques. [4] All these isolates were screened for imipenem and meropenem resistance by Kirby-Bauer disk diffusion method and Minimal Inhibitory Concentration (MIC) methods according to CLSI guidelines.[5]

A-369 dispensed to one of the ceftazidime disc. Plates were incubated overnight at 35°C aerobically. The test was considered positive when the diameter of the growthinhibitory zone around ceftazidime disk with boronic acid was 5 mm larger than that around a disk containing the ceftazidime disc alone. [6] Detection of Carbapenemases The detection was done by using Modified Hodge Test (MHT) in which an overnight culture suspension of Escherichia coli ATCC 25922 adjusted to 0.5 McFarland standard was inoculated on MHA agar plate (HI-MEDIA, Mumbai, India) and allowed to dry for 3-5min. After drying 10µg imipenem disc (HI-MEDIA, Mumbai, India) was placed at the centre of the plate and the test strain was streaked from the edge of the disc to the periphery of the plate in a straight line. Plate was incubated overnight at 35°C aerobically for overnight. Presence of “Clover leaf” type indentation at the intersection of the test organism and the E. coli 25922, within the zone of inhibition of the imipenem susceptibility disc was considered positive for carbapenemase production.[7] Detection of class A carbapenemase Phenotypic assays for the identification of KPC and other class A carbapenemases are based on the inhibitory effect of boronic acids, usually (3-aminophenylboronic acid i.e., APB) with either meropenem or imipenem. A cut-off values ≥5mm of zone diameter differences between discs with a carbapenem plus APB and the carbapenem alone is proposed as being indicative of production of KPC or other class A carbapenemase. [8] Detection of metallo β-lactamases (MBL) All the carbapenem resistant isolates were screened for the presence of MBLs by double-disk synergy test (DDST) and combined-disk test (CDT), with Imipenem-EDTA disc.

Detection of AmpC β-lactamase Boronic Acid Disc Tests (Screening Test) Two disc containing 30µg ceftazidime were placed on the plate and 20 µl boronic acid (containing 400µg) was

i) Combined-disk Test (CDT): A suspension of the organism to be tested was prepared in sterile normal saline and its turbidity was matched with 0.5 McFarland standard. A sterile swab dipped into the inoculum was swabbed over MHA plate. Two 10µg imipenem discs were placed on the plate and 5µl EDTA (750µg per disc) stock solution was added to one of the imipenem disc. Plates were incubated overnight at 35°C aerobically. Isolates were identified as metallo β-lactamase positive if the increase in the inhibition zone with the imipenem and EDTA disc was ≥7mm than imipenem disc alone .[9] ii) Double Disk Synergy Test (DDST): MHA plate was inoculated as described in CDT, then an IMP disk(10 μg) was placed near a blank filter paper disk at a centre to centre distance of 10 to 25 mm. 5µl of 0.5 M EDTA was applied to the blank disk (750 μg). After

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Extended Spectrum β Lactamase Detection (ESBL) Combined Disc Diffusion test This test was carried out for all the screened positive isolates against Ceftazidime (30µg) disc with and without Clavulanic acid (10µg). A ≥5mm increase in zone diameter of ceftazidime + clavulanic acid versus ceftazidime alone confirms ESBL production.[5]

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Detection of NDM-1 Gene from a Tertiary Care Hospital

incubation for 16-18 h, the presence of an enlarged zone of inhibition was interpreted as EDTA synergy test positive.[7] Molecular detection of NDM-1 gene PCR was performed to all the isolates which were confirmed phenotypically to be positive for MBL production. For partial gene PCR amplification, primers specific for blaNDM-1 using oligonucleotide sequence blaNDM-1-F 5’- GGGCAGTCGCTTCCAACGGT- 3’ and blaNDM-1-R 5’GTAGTGCTCAGTGTCGGCAT- 3’ was used for reaction with bacterial DNA as template. DNA was extracted by classical method [3]. Reaction condition kept for amplification were same as used by Manoharan et al with the annealing temperature of 55°C. [10] Presence of bands of molecular weight of 475bp suggests the presence of blaNDM-1 gene on gel electrophoresis.

and 37(20.3%) were P. aeruginosa. Similarly, 57(15.7%) showed AmpC β –lactamases production, of which 36(19.9%) were A. baumanii and 21(11.5%) were P. aeruginosa isolates. Carbapenemases Production by The Nonfermenters Isolated: Of the 130 isolates showing imipenem resistance by both Kirby – Bauer method and MIC study, 41 (32%) isolates showed positive for carbapenemase production by modified Hodge test (Table 3). Metallo β-lactamases status among test isolates Of the 130 imipenem resistant isolates tested, 65 (50%) were confirmed to be MBL producers by imipenem- EDTA combined disc method and 56 (43%) were positive for MBL by double disc synergy test (Table 3).

Result

Class A carbapenemases status Imipenem-Boronic acid disc test was used to screen for the presence of KPC and other class A carbapenemases among all 130 imipenem resistant isolates. A total of 43 (33%) isolates were found to produce class A carbapenemases (Table 3).

Mic Study: was performed on all 363 isolates for carbapenems (imipenem and meropenem), Out of which, 130 (36%) isolates showed resistance to imipenem and 121(33%) to meropenem (isolates showing intermediate MIC value were considered resistant) (Table 2).

Co-production of different class of β-lactamases Of the 43 MBL positive isolates of A. baumanii, 12 (28%) showed co-production of MBL+ESBL, 7(16%) MBL+AmpC and 3(7%) MBL+ESBL+AmpC. Similarly of the 22 MBL positive isolates of P. aeruginosa, 9(41%) showed co-production of MBL+ESBL, 4(18%) MBL+AmpC and 4(18%) MBL+ESBL+AmpC.

Antimicrobial Susceptibility Testing: (Kirby Bauer disc diffusion Method) : All the 363 isolates were subjected to commonly used antibiotics and results are shown in the table 1.

ESBL and AmpC β – lactamase status among test isolates Out of 363 isolates of nonfermenters, 55 (15.2%) were ESBL producer, of which 18(9.9%) were A. baumanii

PCR: was performed using primers specific for blaNDM-1 gene among 65 MBL producing isolates, blaNDM-1 could be detected in 13 strains and results are shown in table 4.

Table 1: Antimicrobial susceptibility testing of A. baumanii and P. aeruginosa by disc diffusion method.

Antibiotic (disc conc. in µg)

A. baumanii (n=181, exception in*)

P. aeruginosa (n=182, exception in*)

Total (n= 363)

Number of susceptible isolates

% of susceptible isolates

Number of susceptible isolates

% of susceptible isolates

Number of susceptible isolates

% of susceptible isolates

Carbencillin

10.5

121

66.5

140

36.6

Ceftazidime

9.4

50.0

108

29.8

Gentamicin

18.8

46.2

118

32.5

Amikacin

24.3

48.4

132

36.4

Ciprofloxacin

21*(n=138)

15.2

68*(n=120)

56.7

89*(n=258)

34.5

Levofloxacin

64*(n=138)

46.4

65*(n=120)

54.2

129*(n=258)

50.0

12*(n=43)

27.3

17*(n=62)

27.4

29*(n=105)

27.6

Norfloxcin

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Gupta et al.

Antibiotic (disc conc. in Âľg)

A-371 A. baumanii (n=181, exception in*)

P. aeruginosa (n=182, exception in*)

Total (n= 363)

Number of susceptible isolates

% of susceptible isolates

Number of susceptible isolates

% of susceptible isolates

Number of susceptible isolates

% of susceptible isolates

Cefepime

7.2

14.8

11.0

Imipenem

51.4

148

81.3

241

66.4

Meropenem

136

75.1

120

65.9

256

70.5

Piperacillin/ Tazobactum

48.6

54.4

187

51.5

Polymyxin-B

132

72.9

152

85.5

284

78.2

Tigecycline

135

74.6

40.7

209

57.6

*Norfloxacin was used in isolates from urine specimen while for isolates from other samples ciprofloxacin and levofloxacin were used.

Table 2: Minimum inhibitory concentration (MIC) of clinical isolates A. baumanii and P. aeruginosa against imipenem and meropenem. Sensitive

Organism

Intermediate

Resistant

Imi

Mero

Imi

Mero

Imi

Mero

A. baumanii (n=181)

89 (49.2%)

127 (72.2%)

7 (3.9%)

3 (1.7%)

85 (46.9%)

51 (28.2%)

P. aeruginosa (n=182)

144 (79.1%)

115 (63.2%)

5 (2.7%)

6 (3.3%)

33 (18.1%)

61 (33.5%)

233 (64.2%)

242 (66.7%)

12 (3.3%)

9 (2.5%)

118 (32.5%)

112 (30.8%)

Total

Imi = Imipenem, Mero = Meropenem For both A. baumanii and P. aeruginosa : Imi(R) Vs Mero(R), p <0.001

Table 3: Results of Modified Hodge test, MBL and Class-A carbapenemases positive isolates among imipenem resistant isolates. Carbapenemases detection Organism

Class A carbapenemase producer

MBL producer

MHT

CDT

Percentage No. of positive

DDST

No.

Percentage of positive

CDT

No.

Percentage of positive

No.

Percentage of positive

Acinetobacter baumanii (n=92)

26.1

46.7

40.2

23.9

Pseudomonas aeruginosa (n=38)

44.7

57.9

50.0

55.3

Total= 130

31.5

50.0

43.1

33.1

N= number of positive organisms, %= percentage of positive organisms

Table 4: Detection of NDM-1 gene among in different MBL producing isolates of A. baumanii and P. aeruginosa. NDM-1

Phenotypically MBL positive isolates

Acinetobacter baumanii

16.3

Pseudomonas aeruginosa

27.3

Total

20.0

Organism

N= number of positive organisms, %= percentage of positive organisms

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Detection of NDM-1 Gene from a Tertiary Care Hospital

Discussion

In the present study, the two nonfermenters (A. baumanii and P. aeruginosa) showed highest susceptibility to polymyxin-B (78.2%) followed by meropenem (70.5%), imipenem (66.4%), tigecyclin (57.6%) and combination of piperacillin-tazobactam (51.5%) by using disc diffusion method. Carbapenems which include imipenem, meropenem, ertapenem, dorepenem etc.,are broad spectrum antibiotics having β-lactam nucleus. These molecules are active against gram positive, gram negative and anerobic bacteria. Apart from the decrease membrane permeability and activation of the efflux pump in nonfermenters, production of β-lactamases including carbapenemase enzymes are also known to be responsible for conferring the resistance. Carbapenemases have been classified into class A to D. Amongst them, the class B enzymes are clinically most important. This Metallo beta lactamases (MBL) causing drug resistance has been reported worldwide but commonly in Enterobacteriaceae group of bacteria. [11] However when all the 363 isolates of above two nonfermenters were subjected to determination of MIC value, 35.8% of the isolates was found resistant to the imipenem while 33.3% of isolates were found resistant to meropenem. These rates are almost comparable with that of the disc diffusion method (Imi, 34.1% and Mero, 30.0%) (p value > 0.05). Production of class A carbapenemases could be observed in 11.8% (43/363) of the isolates. Although 17.9% (65/363) of the isolates were found producing Metallo-β-lactamase, only 3.9% (13/363) of the total isolates were found to be producing blaNDM-1 gene. Our study is one of the few reports showing presence MBL including blaNDM in A. baumanii and P. aeruginosa. A. baumanii has been reported producing blaNDM-1 from south India, [12] as well as from other parts of the world like Germany, [13] Egypt, [14] China. [15] However, there is one report from Serbia showing presence of NDM in P. aeruginosa. [16] Interestingly, imipenem was found to be less effective for A. baumanii as compared to meropenem (p value <0.01). Contrary to this for P. aeruginosa imipenem was found to better drug as compared to meropenem (p value <0.01). This difference may be explained on the basis of differential sensitivity to β-lactamases and orp-gene expression for imipenem and meropenem. In case of P. aeruginosa which has stronger efflux pump activity than A. baumanii, imipenem may be better as meropenem has been stated to act as a substrate for Mex AB-oprM efflux pump because of the presence of hydrophobic side chain at position 2 whereas imipenem containing strongly charged

hydrophilic side chains cannot become a substrate for this efflux pump. [17] However, contrary to this, there a report from France showing meropenem as the better drug for P. aeruginosa and imipenem for A. baumanii. [18]

Conclusion

Treatment of infections caused by nonfermerters like P. aeruginosa and A. baumanii producing different carbampenemases including NDM-1 are now posing serious challenge as these infections are resistant to almost all available antimicrobial agents. Now it is the time that uniform antibiotic policy should be implemented through WHO so that the unethical and irresponsible marketing practices by pharmaceutical companies may be stopped. The up to date microbiological services should be available even in developing world. These steps might help us in retaining the relevance of some newer drugs e.g. tigecycline, colistin, polymyxin and aztreonam etc. Moreover, we must explore the alternatives of the antibiotics now.

Acknowledgements None

Funding None

Competing Interests None Declared

Reference

1. Fass RJ, Barnishan J, Solomon MC, Ayers LW. In vitro activities of quinolones, β-lactams, tobramycin and trimethoprim sulfamethoxazol against nonfermentative gram negative bacilli. Antimicrob. Agents. Chemother. 1996; 40: 1412-18. 2. Gladstone P, Rajendra P, Brahmthan KN. Incidence of carbapenem resistant nonfermenting gram negative bacilli from patients with respiratory infections in the intensive care units. Indi J Med Microbiol. 2005; 23: 189-91. 3. Fritsche TR, Sader HS, Toleman MA, Walsh TR, Jones RN. Emerging metallo-beta-lactamase-mediated resistances: a summary report from the worldwide SENTRY antimicrobial surveillance program. Clin Infect Dis. 2005; 41: 276 -278. 4. Collec JG, Miles RS, Wan B. Test for identification of bacteria. In: Collee JG, Marmion BP, Fraser AG, Simmons A, editors. Mackie & McCartney Practical Medical Microbiology. 14th edn. Edinburg: Churchill Livingstone. 1996; 131-150. 5. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility

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testing; Clinical and Laboratory Standards Institute Wayne; (2014); 23rd Informational Supplement M100-S24 Yagi T, Wachino J, Kurokawa H, Suzuki S, Yamara K, Doi Y, Shibata N, Kato H, Shibayama K, and Arakawa Y. Practical methods using boronic acid compounds for identification of class C β-lactamase-producing Klebsiella pneumoniae and Escherichia coli. J Clin Microbiol. 2005; 43: 2551-58. Lee K, Chong Y, Shin HB, Kim YA, Yonng D, Yum JH. Modified Hodge and EDTA-disk synergy tests to screen metallo-beta-lactamase-producing strains of Pseudomonas and Acinetobacter species. Clin Microbiol Infect. 2001; 7: 88–91. Pasteran F, Mendez T, Guerriero L, Rapoport M, Corso A. Sensitive screening tests for suspected class A carbapenemase production in species of Enterobacteriaceae. J Clin Microbiol. 2009; 47: 1631–39. Yong D, Lee K, Yum JH, Shin HB, Rossolini GM, Chong Y. Imipenem–EDTA disk method for differentiation of metallo-beta-lactamase-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol. 2002; 40: 3798–3801. Manoharan A, Chattergee S, Mathai D. Detection of Metallo beta lactamases producing Pseudomonas aeruginosa. Indian J Med Microbio. 2010; 28: 241-44. Nordmann P, Carrer A. Carbapenemases in enterobacteriaceae. Arch Pediatr. 2010; 17(4): 154-62. Kumarasamy KK, Toleman MA, Walsh TR, BagariaJ, Butt F, Balakrishnan R et al. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and

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Original Article The Histopathological and Epidemiological Study of Pediatric Brain Tumors in A Tertiary Care Hospital, Mumbai Sangita Ramulu Margam*, Nitin Maheshwar Gadgil, Ganesh Ramdas Kshirsagar, Vaishali Prashant Gaikwad, Prashant Vijay Kumavat and Chetan Sudhakar Chaudhari Department of Pathology, Lokmanya Tilak Memorial Medical College,Sion, Mumbai, Maharashtra, India Keywords: Astrocytoma, Epidemiology, Medulloblastoma, Pediatric Brain Tumor

ABSTRACT Background: The primary pediatric brain tumors (PBT) are the second most common cause of death due to malignancies in children. This study was done to analyze the histological spectrum of primary brain tumors in children and also to find out the epidemiology of the common pediatric brain tumors. Methods: Data regarding age, gender, topography, and histopathology of 239 pediatric patients (0â&#x20AC;&#x201C;18 years) with brain tumors operated over a period of 15 years (January-2001 to December-2015) was collected and analyzed according to World Health Organization 2007 classification Result: PBTs were more common in males (54.8%) as compared to females (45.2%) with male to female ratio of 1.21:1. Frequency of tumors was higher in childhood age group (>5-<15 years) i.e. (55.6%) when compared to adolescent age group (15.9%). The most common anatomical site was cerebellum (40.5%), followed by hemispheres (21.4%). Supratentorial tumors (51.9%) were predominant than infratentorial tumors (45.2%) and 2.9% at spinal location. Astrocytomas (46.8%) and embryonal tumors (18.4%) were the most common histological types followed by ependymal tumors (12.5%) and craniopharyngiomas (9.2%). Medulloblastoma was the most common histological type with pilocytic astrocytoma being most common astrocytic tumor. In comparison to adults, meningiomas and lymphomas were rare in children. Conclusion: Astrocytomas and medulloblastomas are the most common tumors among children and adolescents in our region. Except for a slightly higher frequency of craniopharyngiomas, the histological profile of pediatric brain tumors in India is similar to that reported in the Western literature.

*Corresponding author: Dr Sangita Margam, A-5, New Hill view Apartment, Opp. Himalaya society, Govind Nagar, Asalpha, Ghatkopar west, Mumbai- 400084, INDIA Phone: +91 7738853976 Email: drsvgoyal@gmail.com

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Margam et al.

Introduction

Tumors of the nervous system are the second most common childhood tumor after leukemia, [1] constituting approximately 25-35% of all childhood malignancies and remain the leading cause of cancer-related deaths in children. [2] Childhood central nervous system (CNS) tumors differ significantly from adult brain tumors in reference to their sites of origin, clinical presentation, tendency to disseminate early, histological features and their biological behaviour [3]. Whereas in adults the predominant CNS tumor types are metastases, glial neoplasms and meningiomas, in children, besides gliomas, other major tumor types including primitive embryonal neoplasms are also common[4].

Materials and Methods

Two hundred and thirty nine paediatric brain tumour (PBT) specimens of patients aged 0 to 18 years were included in this study. The data collected from Department of Pathology of the major tertiary care hospital in Mumbai, India. The case summaries, histopathological records and discharge reports of all the children up to 18 years of age, were reviewed who were operated for brain tumors during the period from January 2001 to December 2015. Only patients with the proven histopathological diagnosis were included in the study. In addition to the location and histological types of the tumor, patient demographics including age and sex were also recorded. Patients with tumor-like cystic lesion (arachnoid cysts, epidermoid cysts and colloid cysts), space occupying lesion of infectious etiology and vascular malformation were excluded from the study. The hemotoxylin and eosin (H and E) stained slides of all 239 cases were studied and grading was done according to the 2007 World Health Organization classification system[5] .

Result

During the 15 year study period, 21% occurred in the pediatric age group [239 pediatric tumors vs. total 1140 brain tumours cases]. Among these 239 cases, 131 were males and 108 were females, with Male: female ratio being 1.21:1. Their ages varied from 5 months to 17 years. Children (5–15 years) accounted for 133 cases, on the other hand, adolescents (15–18 years) accounted for 46 cases. This clears that the frequency of paediatric brain tumours is higher in childhood age group when compared to adolescent age group. In the present study, PBT were commonly located supratentorially (124 cases) as compared to infratentorial one (108 cases) and 7 cases at spinal level. Comparing location under 15 years of age, Infratentorial tumors (100 www.pacificejournals.com/apalm

A-375 cases) were common than supratentorial one (84 cases). The most common anatomical site for PBT was cerebellum (40.5%) followed by cerebral hemispheres (21.4%). The distribution of PBTs according to anatomical location in the present series is shown in figure 1. The most common histological entities encountered were medulloblastomas accounting for 16.3% of all PBTs, and then Grade-I astrocytomas (15.5%), ependymomas (12.5%), Grade-II astrocytomas (10.9%), oligodenrocytoma (10%), & craniopharyngiomas (9.2%). Grade-I astrocytomas included pilocytic astrocytomas (13%) and subependymal giant cell astrocytomas (2.5%). Grade-II astrocytomas included pleomorphic xanthoastrocytomas (1.7%) and diffuse fibrillary astrocytomas (9.2%). Less common entities included mixed gliomas (10 cases), Grade IV astrocytomas- Glioblastoma multiforme, schwanomas & meningiomas (7 cases each), anaplastic astrocytomas (5 cases), whereas, central neurocytomas, hemangioblastomas, germ cell tumor, primitive neuroectodermal tumor and atypical teratoid/rhabdoid tumors (AT/RT) constituted rare histological entities (1.3% each) with choroid plexus tumors (2 cases), and 1 case each of gangliogliomas, pineoblastoma, medulloepithelioma, neuroblastoma. One case each of Wilms’ tumor and Non Hodgkin’s lymphoma had metastasized. Thus, astrocytomas (46.8%) and embryonal tumors (18.4%) were the most common histological types almost contributing more than half of all PBTs as shown in [Figure 2].

Discussion

The management of PBTs is important due to their high incidence, challenging aspects of surgery and high mortality. In developing countries like India, the exact tumor burden of such diseases goes unnoticed and is underestimated [6]. Jain et al (2011)[7] had assessed the hospital-based prevalence of such tumors by collecting data on CNS tumors in the pediatric age group (<18 years of age) from the neuropathology records of seven tertiary hospitals in India, which included GB Pant Hospital, New Delhi; Christian Medical College (CMC), Vellore; Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh; National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore; Tata Memorial Hospital (TMH), Mumbai; Chhatrapati Shahuji Maharaj Medical University (CSMMU), Lucknow, and All India Institute of Medical Sciences (AIIMS), New Delhi. We also compared our data with this study and other studies to know prevalence of PBT. In the present study, we studied the data regarding demographics and histological profile of 239 pediatric patients operated for brain tumors at one of the major eISSN: 2349-6983; pISSN: 2394-6466

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Fig. 1: Distribution of pediatric brain tumors according to anatomical location

Fig. 2: Distribution of different histological types of pediatric brain tumors.

tertiary care hospital, Mumbai, India over a period of 15 years. Pediatric CNS tumors accounted on an average 21% of total intracranial tumors. [Table 1]. Thus the incidence of PBT in our study is comparable with GB Pant, TMH & Harshil Shah Study, while others show relatively lower incidence. We found the slightly higher proportion of brain tumors in males as compared to in females with male to female ratio of 1.21:1 which was consistent with the findings of other studies on PBTs, in which male to female ratio in pediatric patients varied from as low as 1.03 to as high as

2.52[8,9,10,11,12,13,14]. In our study, males and females with PBT were evenly distributed among different age groups and observed differences which was in line with the findings of other studies [8,15,16] . Age-wise and Gender-wise Distribution:The frequencies of PBTs in children (>5-15 yrs.) was 55.6% and adolescents (>15 yrs.) was 19.2%, which were comparable to the frequencies observed in Harshal Shah [8] & other studies, which states higher among childhood age group when compared to the adolescent age group.[16],[17] Pilocytic Astrocytoma and medulloblastomas were most common in posterior fossa. Patients with astrocytomas

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Table 1: Prevalence of paediatric tumours reported in India. Institute /Study

Time Period

PBT %

AIIMS[7]

2002-2007

2003-2007

NIMHANS TMH

[7]

2006-2007

CSMMU[7]

2003-2007

PGIMER

2003-2007

1990-2007

GB Pant

2003-2007

20.1

Shah

2012-2013

10.2

2001-2015

[7]

CMC Vellore[7] [7]

[8]

Present study

presented at a higher age, probably because of greater number of glioblastoma cases. Similar was also reported by Rosenberg [1] who studied epidemiology of 1195 pediatric brain tumors in single institute. Childhood primitive neuroectodermal tumor (PNETs) presented at a mean age of 7.4 years, similar to the observations made by Grotzer et al. and Chan et al. [18,19]. There was equal distribution of boys and girls suffering from brain tumor when all tumors were taken together; however, girls outnumbered boys in case of Pilocytic Astrocytomas, while male predominance was found in medulloblastomas. Similar was observed by Sehgal et al [6]. Location Wise Distribution: A total of 47.7% tumors were located supratentorially, with even distribution among different age groups. In childhood age group (0-15yrs.), infratentorial tumors were bit predominant whereas, in adolescent age group, supratentorial tumors were more common, which was consistent with the findings of other studies [4,16,17]. Topographically, the cerebellum was found to be the most common site for PBTs followed by cerebral hemispheres. Medulloblastomas and most of the pilocytic astrocytomas, which comprise the major histological entities of PBTs, were frequently found involving the cerebellum. These findings were in line with the results of other studies [8,10,16]. Histopathological Distribution: In the present study, medulloblastoma was the single most common histological entity, followed by pilocytic astrocytomas (Grade-I). However, overall, astrocytomas were the most common tumors followed by embryonal tumors including medulloblastomas, supratentorial PNETs, and AT/RT. Kumar et al [20], GB Pant & TMH [7] institute had reported a higher incidence of medulloblastomas amongst Indian children. While other Indian studies like AIIMS, NIMHANS, CSMMU, CMC Vellore, PGIMER, Jain [7], Shah et al [8]and most of Western studies showed predominance of astrocytic tumors [21,22,23,24,25,26][Table 2]. www.pacificejournals.com/apalm

There were one case each of pilocytic astrocytoma with extensive myxoid changes (Figure 3a ) and another with nuclear degeneration and multinucleation. The histology of PXA was confused with high grade gliomas, but CT findings helped for diagnosis (Figure 3b ). Ependymoma formed third most common entity, followed by Oligodendrogliomas and Craniopharyngiomas in our series. Similar was also observed by TMH study. In a large meta-analysis by Rickert and Paulius, [17] it was seen that internationally, ependymomas are the third most common tumors followed by craniopharyngioma occupying the fourth place. This trend can be seen in [Table 4] in the data from Canada, [15] Germany, [10,22] Sweden, [23] and Morocco [9] but contradicted with the multi-institutional study carried out in India and studies from Korea [21] and Brazil [1] which showed craniopharyngiomas as third most common PBT. Data from a single institute in Beijing, China, however, showed craniopharyngiomas to be the second commonest tumor[12,24] [Table 3]. There were one case each of clear cell ependymoma (Figure 3c), with subependymoma and oligodendroglioma like features. In case of Clear cell Ependymoma, the histology was confused with Oligodendroglioma. But CT, MRI findings confirmed the Ependymal origin. While there were oligodendrogliomas with marked microcalcification and prominent microcystic change (Figure 3 d). Four and half year old child presented with vomiting and headache with convulsions. The resected tumor is composed predominantly of elongated cells with eosinophilic cytoplasm, some showing discernible cross striations (indicating myoblastic differentiation) and focally of round cells with scant cytoplasm (indicating neuroectodermal differentiation) (Figure 4). The differential diagnosis thought of were Medullomyoblastoma, Atypical teratoid rhabdoid tumor, and Metastatic rhabdomyosarcoma. Immunohistochemistry staining revealed positive expression of desmin and myogenin eISSN: 2349-6983; pISSN: 2394-6466

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in the elongated eosinophilic cells while the round cells were negative for the same. The round cells were weakly positive for synaptophysin and retained INI-1 protein expression (Figure 5). The diagnosis offered as a rare tumour â&#x20AC;&#x153;medullomyoblastomaâ&#x20AC;? which is more aggressive in nature. Primary tumors of the CNS containing muscle elements are exceptional hence awareness and knowledge of this entity is necessary to make correct diagnosis.

suggesting environmental and/or genetic differences. For germ cell tumour (GCT), the treatment approach depends on the histological subtypes. In general, with the exception of malignant teratoma, GCT is chemo- and RT sensitive. (25) . Thus it is important to recognize it on histomorphology.

In the present study, we had 3 cases of germ cell tumor. Rickert and Paulius found germ cell tumors to be the fifth most common type, [17] as seen from [Table 3]. The frequency of germ cell tumors varies markedly in different countries ranging from just 0.9% in Morocco [9] to 14.3% in Japan [11]. In particular, all the three oriental Asian countries included i.e. Korea, China, & Japan, show higher frequency for germ cell tumors and craniopharyngiomas,

The incidence of various CNS tumors in the current study falls well within the range seen in the international studies for every tumor category. This can have important connotations in the field of pediatric brain tumor research in India, particularly when analyzing differences in their molecular and genetic pathways, which could aid in the development of targeted, individualized therapies and planning treatment protocols and strategies.

We could find few cases (2.9%) of intracranial nerve sheath tumors and meningiomas, which form the predominant entities in adulthood.

Table 2: Percentage breakup of various histological subtypes of pediatric CNS tumors in Indian studies. Tumor Astrocytoma Mb & PNETs Ependymoma Oligodendroglioma Craniopharyngioma Schwanomma Meningioma Neuronal & Mixed neuronal glial Germ cell tumor Chroid plexus tumor Pineal tumors

AIIMS [7]

NIMHANS[7] GB PANT[7] TMH[7] CSMMU[7] CMC[7]

PGIMER[7]

SHAH[8]

PRESENT

33.7 16.8 8.5 0.7 12.7 7 5.6

44.1 19.7 8.5 0.9 7.7 4.3 4.3

22.3 32 12.2 2.9 13.5 1.3 0.3

28.6 29 19.1 1.4 4.5 2.4 3.4

30.6 27.7 9.4 1.5 13.1 2.2 2.2

46.7 10.3 4.8 0 8.5 4.6 3.5

37 21.6 6.3 0 11.5 NA NA

40.8 29.0 6.6 2.6 11.8 2.6 1.3

46.8 18.4 12.5 10 9.2 2.9 2.9

4.1

2.8

5.2

2.1

1.3

1.7

2.2 1.5 0.7

2.2 2.6 1.4

3.3 1.6 1.3

1.7 1.7 1

2.2 1.5 3

NA NA NA

NA 3.5 NA

0 2.6 1.3

1.3 0.8 0.8

0.5

0.3

1.1

0.8

Lymphoma

Table 3: Frequency of various types of pediatric CNS tumors reported in different countries (in percentage). Tumor

Brazil [1]

Korea Germany Canada Bejing Sweden Morocco Japan Hongkong [21] [23] [15] [12] [23] [9] [11] [26]

PRESENT

Astrocytoma

32.5

27.8

41.7

39.4

30.5

37.1

Mb & PNETs

13.9

19.

25.7

15.4

14.6

Ependymoma

7.4

8.1

10.4

5.6

Oligodendroglioma

0.9

2.6

1.1

1.7

6.2

1.7

Craniopharyngioma

9.2

4.4

6.8

18.4

4.6

6.6

10.5

6.0

9.2

Chroid plexus tumor

2.2

2.3

1.8

1.9

0.8

7.6

6.2

3.2

3.1

1.3

1.7

2.6

1.2

3.1

1.6

2.2

1.9

2.9

Schwanomma

0.4

3.1

2.8

1.1

2.9

Germ cell tumor

3.6

8.1

3.1

7.9

1.5

0.9

14.3

2.0

1.3

Pineal tumors

1.3

0.5

0.6

2.7

0.7

0.8

Neuronal & Mixed neuronal glial Meningioma

35.7

57.0

46.8

28.9

23.0

18.4

4.8

8.0

12.5

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Fig. 3: Different Histological variation of pediatric brain tumor viz. (A) Pilocytic astrocytoma with marked myxoid change with cystic degeneration, (B) pleomorphic xanthoastrocytoma showing multinucleated giant cells and mitotic figures, clear cell ependymoma (C) was confused with oligodendroglioma and microcystic pattern of oligodendroglioma (D).

Fig. 4: H & E sections from tumour showing two populations of cells (A) - H & E 100 X, predominantly of spindle shaped elongated strap-like cells with elongated nuclei and eosinophilic cytoplasm showing discernible cross striations(C & D) - H & E 400 X and foci of small round cells with hyperchromatic nuclei with scant cytoplasm.(B) - H & E 400 X Rhabdomyoblastic spindle shaped elongated strap-like cells (E) - H & E 100 X which highlighted on (F) Massonâ&#x20AC;&#x2122;s Trichome stain- 1000 X.

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Fig 5: Immunohistochemistry revealed positive staining for desmin and myogenin in the elongated eosinophilic cells while the round cells were negative for the same ( A & C) 100 X. The round cells were weakly positive for synaptophysin (B) 100 X. The tumor showed retained INI-1 protein expression. (D) 100 X.

Conclusion

From the present series, we conclude that, there is a rising global trend in the incidence of pediatric CNS tumors. The frequencies of major histologic types of PBTs found in the study do not differ substantially from that found in other developed and developing countries. Medulloblastomas and astrocytomas, are the major histologic types in pediatric patients. Epidemiological surveillance of various histological types of PBTs is of great importance which helps in planning the disease management and preventive programs.

Acknowledgements Nil

Funding None

Competing Interests None Declared

Reference

1. Rosemberg S, Fujiwara D. Epidemiology of pediatric tumors of the nervous system according to the WHO 2000 classification: A report of 1,195 cases from a single institution. Childs Nerv Syst 2005;21:940-4. 2. Fleming AJ, Chi SN. Brain tumors in children. Curr Probl Pediatr Adolesc Health Care 2012;42:80-103.

3. Wilne SH, Ferris RC, Nathwani A, Kennedy CR. The presenting features of brain tumours: A review of 200 cases. Arch Dis Child 2006;91:502-6. 4. Nasir S, Jamila B, Khaleeq S. A retrospective study of primary brain tumors in children under 14 years of age at PIMS, Islamabad. Asian Pac J Cancer Prev 2010;11:1225-7. 5. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, editors. World Health Organization Classification of Tumors of the Central Nervous System. Lyon: IARC; 2007. 6. Sengupta S, Chatterjee U, Banerjee U, Ghosh S, Chatterjee S, Ghosh A. A study of histopathological spectrum and expression of Ki-67, TP53 in primary brain tumors of pediatric age group Indian Journal of Medical and Paediatric Oncology 2012;33(1);25-31. 7. Jain A, Sharma MC, Suri V, Kale SS, Mahapatra AK, Tatke M, et al. Spectrum of pediatric brain tumors in India: A multi-institutional study. Neurol India 2011;59:208-11 8. Shah H, Ubhale B, Shah J. Demographic and histopathologic profile of pediatric brain tumors: A hospital-based study. Pediatric Oncology 2015; 4(3):146-148.

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Margam et al. 9. Karkouri M, Zafad S, Khattab M, Benjaafar N, El Kacemi H, Sefiani S, Kettani F, Dey S, Soliman A. Epidemiologic profile of pediatric brain tumors in Morocco. Childs Nerv Syst 2010;26:1021-7. 10. Kaatsch P, Rickert CH, Kühl J, Schüz J, Michaelis J. Population-based epidemiologic data on brain tumors in German children. Cancer 2001;92:3155-64. 11. Makino K, Nakamura H, Yano S, Kuratsu J, Kumamoto Brain Tumor Group. Population-based epidemiological study of primary intracranial tumors in childhood. Childs Nerv Syst 2010;26:1029-34. 12. Zhou D, Zhang Y, Liu H, Luo S, Luo L, Dai K. Epidemiology of nervous system tumors in children: A survey of 1,485 cases in Beijing Tiantan Hospital from 2001 to 2005. Pediatr Neurosurg 2008;44:97-103.

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17. Rickert CH, Paulus W. Epidemiology of central nervous system tumors in childhood and adolescence based on the new WHO classification. Childs Nerv Syst 2001;17:503-11. 18. Grotzer MA, Janss AJ, Fung KM, Biegel JA, Sutton LN, Rorke LB, et al. TrkC expression predicts good clinical outcome in primitive neuroectodermal brain tumors. J Clin Oncol 2000;18:1027-30. 19. Chan GC. Recent advances in childhood brain tumours. HK J Paediatr 2006;11:3-12. 20. Kumar R. Scenario of pediatric CNS tumors in India. JK Sci 2006;8:190-2. 21. Suh YL, Koo H, Kim TS, Chi JG, Park SH, Khang SK, Choe G, Lee MC, Hong EK,Sohn YK, Chae YS, Kim DS, Huh GY, Lee SS. Tumors of the central nervous system in Korea: A multicenter study of 3221 cases. J Neurooncol 2002;56:251-9. 22. Kaatsch P, Rickert CH, Kühl J, Schüz J, Michaelis J. Population-based epidemiologic data on brain tumors in German children. Cancer 2001;92:3155-64. 23. Hjalmars U, Kulldorff M, Wahlqvist Y, Lannering B. Increased incidence rates but no space-time clustering of childhood astrocytoma in Sweden, 1973-1992: A population-based study of pediatric brain tumors. th Cancer 1999;85:2077-90. 24. Makino K, Nakamura H, Yano S, Kuratsu J, Kumamoto Brain Tumor Group. Population-based epidemiological study of primary intracranial tumors in childhood. Childs Nerv Syst 2010;26:1029-34. 25. Chan GCF . Recent Advances in Childhood Brain Tumours. HongKong J Paediatr (New Series) 2006;11:3-12.

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13. Zhang R, Shen WQ, Zhou LF. Primary pediatric central nervous system tumors statistic: Study of 763 cases in a single institution. Zhonghua Yi Xue Za Zhi 2007;87:442-7. 14. Neervoort FW, Van Ouwerkerk WJ, Folkersma H, Kaspers GJ, Vandertop WP. Surgical morbidity and mortality of pediatric brain tumors: A single center audit. Childs Nerv Syst 2010;26:1583-92. 15. Kaderali Z, Lamberti-Pasculli M, Rutka JT. The changing epidemiology of paediatric brain tumours: A review from the Hospital for Sick Children. Childs Nerv Syst 2009;25:787-93.

Original Article Spectrum of Endometrial Lesions in Patients with Abnormal Uterine Bleeding: A Histopathological Study Ankita Goel1*, Vissa Shanthi1, Nandam Mohan Rao1, Parul Jain2, Syam Sundara Byna1 and Jyothi Conjeevaram3 Department Of Pathology, Naryana Medical College, Nellore, Andhra Pradesh, India Department Of Endocrinology, Naryana Medical College, Nellore, Andhra Pradesh, India 3 Department Of Social And Preventive Medicine, Naryana Medical College, Nellore, Andhra Pradesh, India 1

Keywords: Abnormal Uterine Bleeding, Endometrial Curetting, Endometrial Hyperplasia, Endometrial Carcinoma

ABSTRACT Background: Abnormal uterine bleeding (AUB) is the most common menstrual problem in women of developing countries. Endometrial curettage followed by histopathological examination can be used for definitive diagnosis of AUB. This study was conducted to assess endometrial causes of AUB on histopathological examination and its association with different age groups presenting with AUB. Methods: This study was conducted at Department of Pathology, Narayana medical college and hospital, Nellore, India on 264 women presenting with AUB over a period of 1 year from January to December 2015. A statistical analysis between endometrial histopathology and age of presentation was done using chi square test. Result: Most of the patients with AUB presented in 30 -40 years age group (37.5%) with menorrhagia as the commonest bleeding pattern. The cause of AUB was determined 261 out of 264 endometrial samples as 3 specimens were inadequate for evaluation. Out of the remaining 261 cases, 223 (84.47%) cases were due to functional causes while the remaining 38 cases (14.39%) showed definite endometrial pathology (organic AUB). Endometrial causes of AUB and age of presentation showed positive association with p value <0.05. Conclusion: Endometrial curettage is recommended in women of perimenopausal and menopausal age group presenting with AUB to rule out pre-neoplastic conditions and malignancy.

*Corresponding author: Dr. Ankita Goel, Department Of Pathology, Naryana Medical College, Nellore, Andhra Pradesh, India Phone: +91 9988218311 Email: ankig88@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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Introduction

Abnormal uterine bleeding (AUB) is one of the most challenging and common problem for women of all ages consulting a gynecologist. [1] Abnormal uterine bleeding denotes bleeding pattern that does not fall within normal ranges for amount, frequency, duration and cyclicity.[2] It has led to many hysterectomy procedures without definitive diagnosis. Also, early diagnosis and timely treatment of AUB is important to rule out malignancy and to confirm the exact nature of lesion. Various diagnostic techniques like transabdominal/ transvaginal ultrasonography, hysteroscopy & dilatation and curettage are available for evaluation of AUB.[3] Out of these endometrial curettage is considered most cost effective method and also can pick small lesions. With limited resources, in developing countries like India, it is the most commonly used method of assessing AUB .[4] This study was done to evaluate the endometrial causes of AUB and to determine various histopathological patterns associated with AUB in different age groups.

Materials and Methods

This study was conducted by Department of Pathology, Narayana medical college and hospital, Nellore, Andhra Pradesh, India in 264 women presenting with AUB over a period of 1 year from January to December 2015. Inclusion criteria- All endometrial curettage specimens received for AUB in reproductive and postmenopausal women were included in the study. Exclusion criteria- Endometrial curettage done for evaluation of pregnancy related complications (threatened, incomplete or missed abortion, gestational trophoblastic disease, ectopic pregnancy, etc). AUB due to cervical or vaginal pathology. Systemic diseases that may cause abnormal uterine bleeding (hypothyroidism, cirrhosis, and coagulation disorders) were excluded from the study. The endometrial tissue obtained by dilatation and curettage, were preserved in 10% buffered formalin and clinical details of the patient was obtained from the requisition forms and case sheets. Thereafter the specimen was processed by paraffin embedding and hematoxylin and eosin (H&E) staining. Microscopic evaluation and histopathology reporting were done by two pathologists, individually to reduce observer bias.

Result

A total of 264 endometrial curetting from patients with AUB were analyzed for histopathology, age of presentation and cause of bleeding. In our study, age of patients presenting with AUB ranged from 21-62 years. These 264 cases of AUB were categorized into five age groups with most of them present in 30 -40 years (37.5%) and perimenopausal age group (40-50 years, 36%). Most common pattern of bleeding was menorrhagia (62.5%) followed by metorrhagia (14%), postmenopausal bleeding (12%), menometorrhagia (6.1%) and polymenorrhea (5.3%) as depicted in figure 1. The cause of AUB was determined in 261 out of 264 endometrial samples as 3 specimens were inadequate for evaluation. Out of the remaining 261 cases, 223 (84.47%) cases were due to functional causes as no organic pathology was found, while the remaining 38 cases (14.39%) showed definite endometrial pathology (organic AUB). Out of 261 cases of functional AUB (table 1), proliferative pattern (49.62%) was most predominant followed by secretory pattern (23.48%), atrophic pattern (6.81%), irregular ripening (1.13%), irregular shedding (1.13%), luteal phase defect (2.26%). Out of 38 cases of organic AUB (table1), Disordered proliferative endometrium (figure 2) was present in 10 patients. Twenty one cases of hyperplasia were diagnosed with 16 cases without atypia (Figure 3) and five cases showing atypia (Figure 4). Three cases presented with endometritis with one case possessing features of granulomatous endometritis. Two cases were of endometrial polyp with one showing features of hyperplastic polyp and other showing features of functional polyp. Adenocarcinoma endometrium was diagnosed in 2 cases of organic causes of AUB with one case of endometrioid carcinoma and one case of serous carcinoma (Figure 5). Cyclic pattern of endometrium was mostly seen in 3140 years of age i.e. reproductive age group, disordered proliferative pattern, hyperplasia and carcinoma were observed in perimenopausal and menopausal age group. This association is statistically significant with p value - 0.001

Discussion

Data was compiled and analyzed using the statistical package SPSS version 19 for MS- Windows (SPSS Inc., Chicago, IL). Pearson chi square test is used to analyze the data and p-value is calculated wherever required. P-value of 0.05 or less was considered statistically significant.

AUB is the most commonly encountered symptom that confronts the gynecologist, thus posing a considerable health risk. It includes bleeding from structural/ organic causes and dysfunctional uterine bleeding (DUB). Structural causes include ďŹ broids, polyps, hyperplasia, endometritis, endometrial carcinoma, pregnancy complications, etc.

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Table 1: Age wise distribution of cases of AUB with histopathological pattern of endometrium. Age Group (in years)

Histopathology of endometrium

Total

20 -30

31-40

41-50

51-60

61-70

Proliferative

4(3.1%)

56(42.7%)

46(35.1%)

23(17.6%)

2 (1.5%)

131(49.62%)

Secretory

3(4.8%)

27(43.5%)

22(35.5%)

10(16.1%)

62(23.48%)

Atrophic

3(16.7%)

11(61.1%)

4(22.2%)

18(6.81%)

Irregular ripening

1 (33.3%)

2 (66.7%)

3 (1.13%)

Irregular shedding

1 (33.3%)

1(33.3%)

1 (33.3%)

3 (1.13%)

Luteal phase defect

3 (50.0%)

3(50.0%)

6 (2.27%)

1(10%)

2(20%)

7 (70%)

10(3.79%)

Hyperplasia without atypia

7 (43.7%)

4 (25.0%)

4 (25%)

1 (6.2%)

16 (6.06%)

Hyperplasia with atypia

1 (20.0%)

2 (40.0%)

5 (1.89%)

Non specific Endometritis

2 (100%)

2 (0.76%)

Granulomatous endometritis

1 (100%)

1 (0.38%)

Endometrial polyp

1 (50.0%)

2 (0.76%)

Adenocarcinoma

1(50.0%)

1 (50.0%)

2 (0.76%)

Inconclusive

3 (100%)

3 (1.13%)

8 (3.03%)

99 (37.50%)

95 (35.98%)

55 (20.83%)

7 (2.65%)

264

Disordered proliferative

Total p- value

0.001

Fig. 1: Bleeding pattern of abnormal uterine bleeding.

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Fig. 2: Disordered proliferative endometrium, showing focal glandular dilatation of proliferative phase glands. (H&E, x100) .

Fig. 3: Endometrial hyperplasia without atypia showing crowding of endometrial glands with compact stroma (H&E, x400).

Fig. 4: Endometrial hyperplasia with atypia showing closely packed endometrial glands with sparse intervening stroma. Lining epithelial cells show cytological atypia, high nucleo-cytoplasmic ratio and irregular clumping of chromatin. (H&E, x400)

Fig. 5: Serous carcinoma endometrium showing broad papillae lined by atypical cells with irregular nuclei and vesicular nuclei with prominent nucleoli (H&E, x400).

DUB is an abnormal uterine bleeding without any demonstrable organic cause and is diagnosed only after exclusion of structural, iatrogenic and systemic disorders by various diagnostic techniques. DUB, in most occasions, is due to the occurrence of an anovulatory cycle. [6, 7]

was 21 year old female (reproductive age group) and eldest was 67 year old post menopausal women. No case was encountered in adolescent girls. This may be because abnormal bleeding in adolescent girls resolves spontaneously and also invasive procedures are avoided in this age group.

[5]

The etiology of AUB is different for various age groups â&#x20AC;&#x201C; adolescent (12-18 years), reproductive (19-40 years), perimenopausal (41-50 years) and post menopausal age group (>50 years).[8] The youngest patient in this study www.pacificejournals.com/apalm

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Spectrum of Endometrial Lesions in AUB

significantly associated with aggressiveness of the lesion. Cyclic endometrium (90/193; 46.63%) was more common in reproductive age where as disordered proliferative endometrium (7/10; 70%) and hyperplasia (13/21; 61.9%) was commonly encountered in perimenopausal and menopausal women. All cases of endometrial carcinoma were noticed in age group of more than forty years. Similar distribution of cases was observed by Abid et al.[8] Increased prevalence of lesions in higher age group could be due to the fact that endometrium is exposed to estrogen for a longer period of time as compared to patients with younger age group. The most common pattern of bleeding noticed was menorrhagia, followed by metorrhagia. These results are consistent with the studies conducted by Jyotsna et al [9] and Bhosle et al. [10] We evaluated that predominant number of cases showed normal menstrual pattern (73.11%) with proliferative phase in 49.62% patients, secretory phase in 23.48% cases. Similar observations were made by Abdullah [11] et al. Abnormal bleeding in proliferative phase can be due to hormonal imbalance leading to anovulatory cycles and AUB in secretory phase can be due to ovulatory dysfunction like loss of LH surge.[12] Atrophic endometrium was noticed in 6.8% cases. Studies conducted by Ara S [13] et al reported a similar incidence of 7%. The cause of AUB in atrophic phase is not clear. It can be due to diminished number and quality of ovarian follicles, abnormal local hemostatic mechanism or thin walled veins superficial to expanding cystic glands making vessels vulnerable to injury .[12] In 25% patients with AUB, a well-defined structural/ organic abnormality is noticed.[14] While in our study, organic abnormality was found in only 14.39%. Large number of patients with lower age group might be the reason for this lower prevalence. Disordered proliferative pattern of endometrium is seen in 3.79% cases. Similar incidence of 4.8% was seen by Jairajpuri et al [15]. It is thought to be an exaggeration of normal proliferative endometrium without significant increase in gland to stroma ratio. It is due to persistent estrogen stimulation. It differs from proliferative phase of endometrium by presence of non- uniform glands. Pathologically, disordered proliferative pattern simulates a simple hyperplasia but the process is focal in former whereas diffuse in later. [12] In this study disordered proliferative pattern was most commonly seen in perimenopausal women which is similar to the study of Doraiswami et al.[12] Also, in the spectrum of proliferative lesions of

endometrium, it lies at one end with intervening stages of hyperplasia and carcinoma at other end. Hence, early diagnosis of patients at this stage of spectrum will definitely help gynecologists to prevent disease progression.[12] But pathologists must have clear cut rule for interpretation of disordered proliferative pattern and this should emerge as a waste paper basket diagnosis. In our study, endometrial hyperplasia (21/264; 7.95%) was the most frequent structural cause observed for AUB. Diagnosis of endometrial hyperplasia is important as they are precursors of endometrial carcinoma. The calculated risk of progression of hyperplasia to cancer is 5-10%.[18] We classified hyperplasia into hyperplasia without atypia (16/21) and hyperplasia with atypia (5/21) according to the new WHO classification.[16] Incidence is lower as compared to studies by Abdullah et al [11] (9.1%) and Gredmark et al[17] (10%). The possible reason could be that most of the patients in this study belong to younger age group as compared to other studies. Endometritis was diagnosed in 1.14% cases including 0.38% cases of granulomatous endometritis suggestive of tuberculosis. All cases were observed in 40- 50 years age i.e. peri-menopausal age group. The endometritis was diagnosed on the basis of presence of plasma cells and granulomatous endometritis contained epithelioid cells alongwith plasma cells and lymphocytes. Chronic endometritis is often a result of intra uterine contraceptive devices (IUCD), pregnancy and incomplete abortions. [15] This pathology needs to be diagnosed and kept in mind while dealing with a case of AUB because with specific treatment, endometrium can be reverted back to normal state. Endometrial polyps (2/264; 0.76%) were seen in reproductive age group and perimenopausal age group in equal amount. Polyp in reproductive age group was benign functional polyp and in perimenopausal age group was benign hyperplastic polyp. Functional polyp is peculiar to the cyclic nature of endometrium in reproductive age group. There is compelling difference between endometrial polyp and normal endometrium in receptor expression, cell proliferation and apoptosis regulation suggesting that polyp may provide a suitable background for the advancement of malignancy. [12] Other benign lesions noted were irregular shedding (1.14%) and luteal phase defect (2.27%). According to our study, the incidence of carcinoma endometrium is 0.8% including one case of endometrioid carcinoma and one case of serous carcinoma. Lower incidences of endometrial carcinoma 0.4% & 0.47% have

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also been observed by Jairajpuri[15] et al & Khan[19] et al. This lower incidence of endometrial carcinoma in our patients can be due to early child bearing and multiparity as progesterone decreases the proliferative activity of endometrium during pregnancy. The endometrial specimens were inadequate for evaluation in 3/264 (1.14%) cases. The specimen which showed scanty fragmented endometrial glands, stromal tissue and large areas of hemorrhage were labeled as unsatisfactory/ inadequate for reporting.

Conclusion

Histopathological examination of endometrial curetting is an important diagnostic procedure in evaluation of AUB, thus providing specific diagnosis to physician for affluent management of abnormal bleeding. Functional causes of AUB are much more common in reproductive age group whereas in perimenopausal and menopausal age group organic lesion were responsible for AUB. Thus endometrial curettage is recommended in women of perimenopausal and menopausal age group presenting with AUB to rule out pre-neoplastic conditions and malignancy.

Acknowledgements None

Funding None

Competing Interests None Declared

Reference

1. Nicholson WK, Ellison SA, Grason H, Power NR. Patterns of ambulatory care use gynecological conditions: a national study. Am J obstect Gynecol 2001;184: 523-30. 2. Munro MG, Critchley HO, Fraser IS. The FIGO classification of causes of abnormal uterine bleeding in the reproductive years. Fertil Steril 2011;95:2204-8. 3. Kotdawala P, Kotdawala S, Nagar N. Evaluation of endometrium in peri-menopausal abnormal uterine bleeding. Journal of Mid-Life Health. 2013;4(1):16-21. 4. Krampl E, Bourne T, Hurlen-Solbakken H, Istre O. Transvaginal ultrasonography sonohysterography and operative hysteroscopy for the evaluation of abnormal uterine bleeding. Acta Obstet Gynecol Scand 2001;80:616-22.

6. Muzaffar M, Akhtar KA, Yasmin S, Mahmood UR, Iqbal W and Khan MA. Menstrual irregularities with excessive blood loss: a clinico-pathological correlation. JPMA 2005 Nov;55(11):486-9. 7. Ely JW, Kennedy CM, Clark EC, Bowdler NC. Abnormal Uterine Bleeding: A Management Algorithm. J Am Board Fam Med 2006; 19: 590-602 8. Abid M, Hashmi AA, Malik B, Haroon S, Faridi N, Edhi MM et al. clinical pattern and spectrum of endometrial pathologies in patients with abnormal uterine bleeding in Pakistan: need to adopt a more conservative approach to treatment. BMC women’s health 2014, 14:132 9. Jyotsana, Manhas K, Sharma S. Role of hysteroscopy and laparoscopy in evaluation of abnormal uterine bleeding. J K Sci 2004;6:23-7. 10. Bhosle A, Fonseca M. Evaluation and histopathological correlation of abnormal uterine bleeding in perimenopausal women. Bombay Hosp J 2010;52:69-72. 11. Abdullah LS, Bondagji NS. Histopathological pattern of endometrial sampling performed for abnormal uterine bleeding. Bahrain Med Bull 2011; 33: 1-6. 12. Doraiswami S, Johnson T, Rao S, Rajkumar A, Vijayaraghavan J, Panicker VK. Study of endometrial pathology in abnormal uterine bleeding. Journal of obstetrics and gynaecology of India 2011;61(4):426–30. 13. Ara S, Roohi M. Abnormal uterine bleeding: Histopathological diagnosis by conventional dilatation and curettage. Prof Med J 2011;18:587-91. 14. Brenner PF. Differential diagnosis of AUB. Amer J Obstet Gynecol 1996; 175: 766-9. 15. Jairajpuri ZS, Rana S, Jetley S. Atypical uterine bleeding-histopathological audit of endometrium - A study of 638 cases. Al Ameen J Med Sci 2013; 6: 21-8. 16. Emons G, Beckmann MW, Schmidt D, Mallmann P. New WHO Classification of Endometrial Hyperplasias. Geburtshilfe und Frauenheilkunde. 2015;75(2):135-6 17. Gredmark T, Kvint S, Havel G, Mattsson LA. Histopathological ﬁndings in women with post menopausal bleeding. Br J Obstet Gynaecol 1995; 102: 133-6. 18. Baak JP, Mutter GL. EIN and WHO94. J Clin Pathol 2005;58:1-6.

5. Albers JR, Hull SK, Wesley MA. Abnormal uterine bleeding. Amer Fam Phys 2004; 69: 1915-26.

19. Khan S, Hameed S, Umber A. Histopathological pattern of endometrium on diagnostic D&C in patients with abnormal uterine bleeding. Annals 2011; 17: 166-70.

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Original Article Histomorphologic Pattern of Renal Disease in Patients with Acute Nephritic syndrome: A Single Centre South Indian Study Clement Wilfred Devadass1*, VijayaMysorekar V1, GireeshSiddaiah M2, Mahesh E2 and GurudevChannabasappa K2 Department of Pathology, MS Ramaiah Medical College And Hospitals. MSRNagar, Bangalore, India. Department of Nephrology, MS Ramaiah Medical College and Hospitals. MSRNagar, Bangalore. India

1 2

Keywords: Acute Nephritic Syndrome, Glomerulonephritis, Post Infectious Glomerulonephritis, Renal Biopsy.

ABSTRACT Background: The frequency and distribution of primary and secondary glomerular disorders presenting as acute nephritic syndrome (ANS) varies according to the geographic and racial characteristics, renal biopsy practices and patient selection criteria. The present study was carried out to determine the histomorphologic patterns of lesions in renal biopsies from patients presenting with ANS. Methods: The study was conducted on patients presenting with ANS, in a tertiary care hospital in South India, during the period between 2008 and 2015. The renal biopsies performed were studied by light and immunofluorescence microscopy. Results: A total of 112 patients of ANS, with a male: female ratio of 1: 1.5 and mean age of 32.9 Âą16.85 years, were included. Primary glomerular disease (PGD) was present in 77.7% and secondary glomerular disease (SGD) in 22.3% of the cases. The commonest PGD presenting as ANS was postinfectious glomerulonephritis followed by crescentic glomerulonephritis. The commonest SGD causing ANS was lupus nephritis (LN). Conclusion: A range of PGD and SGD can present as ANS. Various studies, done in India and abroad, show variations in the frequency and distribution of PGD presenting as ANS. The distribution pattern of SGD presenting as ANS largely corresponds to the pattern described in other Indian studies with LN being the commonest.

*Corresponding author: Dr Clement Wilfred D, Associate Professor, Department of Pathology, M.S Ramaiah Medical College and Hospitals, MSRIT Post, MSRNagar, Bangalore- 560060, INDIA Phone: +91 9945226314 Email: clement.wilfred@yahoo.com

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

Devadass et al.

Introduction

Acute nephritic syndrome (ANS) is one of the presentations of renal disease that is characterised by hematuria, subnephrotic proteinuria and fall in glomerular filtration rate with azotemia, hypertension, oliguria and edema. [1,2] Varied histopathologic lesions, especially affecting the glomeruli, can cause the syndrome.[1,3] A meticulous histopathologic examination of renal biopsy and clinical correlation is required to distinguish between the various causes of ANS, as they exhibit different clinical behaviour and require different treatment protocols. Early recognition of the underlying cause of ANS and prompt institution of relevant therapy is vital for improvement in or preservation of renal function.[4]Further, the lesions causing ANS vary according to geographic area, socioeconomic conditions, demography and race, and there is paucity of this information in the native south Indian population.[5]

A-389 and serological data [complement levels (C3 and C4), anti-nuclear antibodies, dsDNA and ASLO titers] were retrieved from the patients’ case files. The renal diseases were classified into primary glomerular disease (PGD) and secondary glomerular disease (SGD). The various PGD and SGD, presenting as ANS, were diagnosed on the basis of both clinical and histopathological (LM and IFM) investigations. The following criteria were used to diagnose the various disease entities: Postinfectious glomerulonephritis (PIGN) was diagnosed when acute glomerulonephritis, characterised by diffuse global glomerular endocapillary proliferation with neutrophilic exudation on LM and coarse granular predominantly membranous and a less prominent mesangial deposit of C3 and IgG on IFM, was present related to recent streptococcal / nonstreptococcal infection.[7]

The study was conducted in the department of Pathology in conjunction with the department of Nephrology, M.S Ramaiah Medical College and Hospitals, Bangalore; over a duration of seven years (between 2008 and 2015) and was a cross-sectional, hospital based study. Patients presenting with ANS, who underwent percutaneous renal biopsy were included in the study. ANS was defined as rapid onset of edema, oliguria and hypertension with hematuria (microscopic or macroscopic) and mild to moderate proteinuria (< 3.5 g per day per 1.73m2 surface area).[1,3]Patients with nonglomerular/ urothelial/ urological hematuria caused by interstitial diseases, urolithiasis and trauma, benign or malignant mass lesions and infections of the urinary tract were excluded from the study.[6]The renal biopsy specimens were processed for light microscopy (LM) and immunofluorescence microscopy (IFM) as perstandard protocol. For LM, 3 to 4 μm thick paraffin embedded tissue sections were stained with haematoxylin and eosin (H&E), periodic acid Schiff (PAS), Jones silver methenamine (JMS) and Masson’s trichrome stains. For IFM, cryo-sections were stained with fluorescein isothiocyanate (FITC) conjugated antisera (Bio Genex) specific for immunoglobulin IgG, IgM, IgA and complement component 3 (C3). The staining distribution was described as mesangial or membranous in a granular or linear pattern and the staining intensity was graded semiquantitatively from 0 to +++. The clinical data and relevant investigations, including age, gender, urinalysis, 24 hour proteinuria, serum creatinine, blood urea nitrogen levels

Crescentic glomerulonephritis (CresGN) was diagnosed when 50% or more of the glomeruli showed crescent formation on LM.[8] In addition “anti-glomerular basement CresGN” was diagnosed when linear IgG and C3 staining of glomerular basement membrane was discerned on IFM (usually with associated elevated anti-glomerular basement membrane antibody titer); “immune complex CresGN” was diagnosed when glomerular deposits of immunoglobulins and or complement were clearly present on IFM; “pauci-immune CresGN” was diagnosed when little or no immunoglobulin and complement deposits were discerned on IFM (usually with presence of antineutrophil cytoplasmic antibodies).[8,9] Goodpasture syndrome (GPS) was diagnosed when features of anti-glomerular basement CresGN was present with pulmonary involvement (pulmonary haemorrhage).[8] Membranoproliferative glomerulonephritis (MPGN) was diagnosed when lobular hypercellular glomeruli with thickened capillary walls (with “ tram tracks” on PAS/ JMS stains) and increased mesangial substance was present on LM, and membranous and mesangial deposits of C3 with or without accompanying immunoglobulin deposits was present of IFM with clinical finding of hypocomplementemia.[10] IgA nephropathy (IgAN) was diagnosed when glomerulonephritis with predominantly IgA deposits in the mesangium was present in the absence of systemic disease.[11] Membranous nephropathy (MN) was diagnosed when diffuse global uniform thickening of glomerular capillary wall was present on LM (with subepithelial “spikes” in PAS/ JMS) and diffuse fine granular membranous deposits of IgG, and to a lesser extent C3 deposits were seen on IFM.[12] Focal and segmental glomerulosclerosis (FSGS) was diagnosed when sclerosis with associated synechiae formation and hyalinosis was present, involving the glomeruli in a focal and segmental fashion.[13]

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The aim of the present study was to determine the histomorphologic patterns of lesions in renal biopsies from patients presenting with ANS in a tertiary care hospital in south India.

Material and Methods

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Pattern of Nephritic Syndrome

Lupus nephritis (LN) was diagnosed when glomerular mesangial matrix expansion/ mesangial proliferation/ capillary wall thickening/ endocapillary proliferation/ necrosis/ crescents were present in the appropriate clinical setting (i.e patients fulfilling the American college of Rheumatology revised criteria for classification of Systemic lupus erythematosus), usually with “full house” mesangial or membranous deposits of immunoglobulins and complement.[14] Henoch-Schönlein purpura (HSP) was diagnosed when glomerular IgA immune complex deposits were present with extrarenal manifestations such as purpura.[15] Hemolytic uremic syndrome (HUS) was diagnosed when histological evidence of glomerular/ arteriolar thrombotic microangiopathy (fibrinoid necrosis/ thrombosis) was present in the clinical setting of microangiopathic hemolytic anemia.[16] Malignant nephrosclerosis was diagnosed when histological features of arterio/ arteriolonephrosclerosis was present in the clinical setting of accelerated hypertension, papilledema and retinal hemorrhage.[17]

proportion. The frequency and percentage of each type of renal disease was determined.

Results

A total of 112 cases were included in the study of which 44 were males and 68 were females (male: female ratio = 1: 1.5). The distribution of sex in each decade is shown in Figure 1. The mean age of the patients was 32.9 ±16.85 (range 5-75) years. Majority of the patients were in the second (23.2%, 26/112) and third (21.4%, 24/112) decades. The mean 24 hour proteinuria and serum creatinine were 2.27±0.68 (range: 1.0- 3.3) and 2.91±2.35 (range: 0.713.7) respectively. The frequencies and histologic patterns of renal lesions and some basic data in patients with ANS, are shown in Table 1. PGD was present in 77.7% (87/112) and SGD in 22.3% (25/112) of the cases. The commonest PGD presenting as ANS was postinfectious glomerulonephritis (PIGN) (37.9%, 33/87), followed by crescentic glomerulonephritis (CresGN) (23%, 20/87) (Fig 2). The commonest SGD causing ANS was lupus nephritis (LN) (68%, 17/25) (Fig 3). The distribution of renal disease in each decade is shown in Table 2 and the immunofluoresence findings in the different histomorphological lesions of ANS are depicted in Table 3.

Statistical Analysis: Data was analysed using Statistical Package for Social Sciences version 20.0, (SPSS, IBM, USA). All the continuous parameters were expressed as mean and standard deviation and all qualitative data as

Table 1: Histologic patterns of renal disease and laboratory parameters. Histological diagnosis

Number of cases (%)

Mean age ± SD

Males

Females

Male: Female ratio

Serum 24 hour creatinine proteinuria g/ mg/dl ± SD day ± SD

Primary glomerular disorders PIGN

33 (29.5)

33.7±19.74

1:1.1

2.26±1.08

1.92±0.62

CresGN

20(17.9)

36.2±17.0

1:1.2

5.18±3.61

2.21±0.63

MPGN

17 (15.2)

33.8±13.84

1:1.1

2.16±1.6

2.37±0.74

IgAN

14 (12.5)

31.4±13.41

1.3:1

3.13±2.71

2.67±0.64

2 (1.8)

32.5±3.53

1:1

1.3±0.84

3.1±0.42

FSGS

1 (0.9)

2.2

2.9

2.39±1.56

2.52±0.68

Secondary glomerular disease LN

17 (15.2)

27.1±12.89

MNS

2 (1.8)

27±5.65

1:1

5.1±.14

2.45±0.78

HUS

2 (1.8)

8.5±4.94

1:1

2.75±0.91

1.7±0.28

HSP

2 (1.8)

24.5±19.09

2.05±0.07

2.25±0.35

GPS

1 (0.9)

1.9

1.8

Vasculitis

1(0.9)

1.4

1.9

112

32.9±16.85

1:1.5

Total

CresGN (Crescentic glomerulonephritis), FSGS (Focal and segmental glomerulosclerosis), GPS (Goodpasture syndrome), HSP (Henoch-Schönlein purpura), HUS (Hemolytic uremic syndrome), IgAN (IgA nephropathy), LN (Lupus nephritis), MN (Membranous nephropathy), MNS (Malignant nephrosclerosis), MPGN (Membranoproliferative glomerulonephritis), PIGN (Postinfectious glomerulonephritis).

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Table 2: Distribution of renal disease in each decade. Disease PIGN CresGN MPGN IgAN MN FSGS LN MNS HUS HSP GPS Vasculitis Total

â&#x2030;¤10 4 1 1 6

11-20 10 4 3 2 5 1 1 26

21-30 1 3 5 5 1 8 1 24

31-40 7 3 4 4 1 1 1 1 22

41-50 6 6 2 1 3 18

51-60 1 2 3 2 1 9

61-70 3 1 1 5

71-80 1 1 2

Total 33 20 17 14 2 1 17 2 2 2 1 1 112

Table 3: Immunofluorescence findings in the various histomorphological lesions of ANS. Histological diagnosis PIGN ( N=33) CresGN ( N=20) MPGN( N=17) IgAN( N=14) MN( N=2) FSGS( N=1) LN( N=17) MNS( N=2) HUS( N=2) HSP( N=2) GPS( N=1) Vascuitis( N=1) Total( N=112)

IgG

IgM

IgA

33 12 12 7 2 0 17 0 0 1 1 0 85 (76%)

10 4 8 4 0 1 12 0 0 0 0 1 40 (36%)

5 3 0 14 0 0 10 0 0 2 0 0 34 (30%)

33 8 17 8 1 1 16 0 0 1 0 1 86 (77%)

Fig. 1: Distribution of sex in each decade.

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Pattern of Nephritic Syndrome

Fig 2: (A) Postinfectious glomerulonephritis with global glomerular endocapillary proliferation (H&E, x 200); (B) Postinfectious glomerulonephritis with predominantly membranous deposits of C3 (IFM, x200); (C) Immune complex CresGN with crescent (arrow) formation (H&E, x 200); (D) Immune complex CresGN with membranous deposits of IgG (IFM, x200).

Fig. 3: (A) Lupus nephritis with glomerular endocapillary and mesangial proliferation and wire loop lesions (arrow) (H&E, x 200); (B) Lupus nephritis with glomerular capillary hyaline thrombi (arrow) (H&E, x200); (C) Lupus Nephritis with early crescent (arrow) formation (H&E, x 200); (D) Lupus Nephritis with membranous and mesangial deposits of IgG (IFM, x200).

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Discussion

ANS is a glomerular syndrome that is characterised by inflammatory changes within the glomerulus.[6] The inflammatory changes are associated with complement activation, production of pro-inflammatory cytokines and mediators, infiltration by neutrophils and monocytes, and mesangial and endothelial cell proliferation with swelling which cause i) local hemodynamic alterations and reduced filtration area resulting in decreased GFR with consequent salt and water retention, oliguria, edema and varying degrees of hypertension ii) glomerular hematuria manifested by dysmorphic red blood cells (RBCs) and RBC casts and iii) damage to glomerular filtration barrier with resultant mild to moderate proteinuria.[6,18]A variety of PGD and SGD can present as ANS. The common PGD that present as ANS are PIGN, membranoproliferative glomerulonephritis (MPGN), IgA nephropathy (IgAN) and CresGN (rapidly progressive glomerulonephritis). The SGD that manifest as ANS include LN, Henoch-SchĂśnlein purpura (HSP), hemolytic uremic syndrome (HUS), Goodpasture syndrome (GPS), bacterial endocarditis, shunt nephritis, hypersensitivity vasculitis, Wegener granulomatosis and polyarteritis nodosa.[6,19] Our gender distribution of mild female preponderance was comparable to a study from Bangladesh (M:F= 1: 1.15), however mild male preponderance was found in a west Indian (Mumbai) study (M:F= 1.8:1).[4,20]LN frequently occurs in females and the higher relative frequency of this disease in our series compared to the latter study, may partly explain the female predominance. The age distribution was similar to other Indian studies.[4,20] Even though ANS can occur in any age group, it is most prevalent in children.[4] However in the present study only 18% (20/112) of the cases occurred in children (age â&#x2030;¤15 years). Similarly Rahman et al, in their study, observed that paediatric patients accounted for only 4% of the ANS cases.[4]This discrepancy could due to the fact that paediatric ANS cases generally respond well to therapy and therefore are seldom biopsied.[4]

A-393 constituted the second and third commonest causes of ANS respectively in studies from west India (Mumbai) (25.7%) and Serbia (18.1%).[20,22] However, in a study from Bangladesh PIGN was the least common cause (2.7%).[4] The authors of the latter study attribute the low prevalence of the disease to improvement of personal hygiene and health care. Further, the authors commented that as PIGN cases resolve with treatment they are seldom biopsied. In our institution, we biopsy children with clinically suspected PIGN, only if persistent hypocomplementemia (> 6weeks) or gross hematuria (> 1month) or hypertension (> 2 months) or progressive decline in GFR or occurrence of ANS within 48 hours of pharyngitis is present. However we biopsy adults more liberally as PIGN is not as common in adults as in children and a number of glomerulonephritis may clinically masquerade as PIGN. CresGN constituted the second most common cause of ANS (17.9%) which is similar to the study conducted in Serbia (21.7%).[22] This is in contrast to studies from south India (Hyderabad), west India(Mumbai), Bangladesh and Brazil where the disease was either muchless frequent or was not reported.[4, 5, 20, 21]The reasons for these variations in disease frequency, whether due to differences in patient selection criteria or differing environmental influences, is not clear. Further evaluation of our cases of CresGN, by IFM, revealed 5 cases (20%) of anti-glomerular basement CresGN, 7 cases (35%) of immune complex CresGN and 8 cases (40%) of pauci-immune CresGN. Similarly literature review showed that pauci-immune CresGN is the most frequent category of CresGN (60%) followed by immune complex CresGN(24%), with anti-glomerular basement CresGN (15%) being the least frequent.[8]

The most common cause of ANS in the present study was PIGN (29.5%) which is in synchrony with another south Indian study (Hyderabad) and Brazilian study, where PIGN constituted the commonest cause of ANS, accounting for 56.6% and 17.2 % of the cases respectively.[5,21] PIGN

In the present study MPGN (15.2%) and LN (15.2%) constituted the third most frequent cause of ANS. MPGN (Fig 4) was the commonest cause of ANS in studies conducted in India (Mumbai) and Bangladesh unlike Serbian and Brazilian studies where low frequencies were reported.[4,20,21,22] The latter study attributed the low frequency of MPGN to improved socioeconomic conditions and decline in regional endemic diseases.[21]Similar to other Indian studies and a study from Bangladesh, LN was the single most common secondary cause of ANS. In the present study diffuse LN (class IV) was the most common histologic pattern (53%, 9/17) followed by focal LN (class III) (24%, 4/17), mesangial proliferative LN (class II) (12%, 2/17), membranous LN (class V) (6%, 1/17) and minimal mesangial LN (class I) (6%, 1/17). Similar to our observations, literature review reveals that ANS is a commoner manifestation of proliferative LN (classes III and IV) rather than classes I, II and V.[23]

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The frequency and distribution of the various PGD and SGD presenting as ANS vary according to geographic area, race, socio-economic conditions, prevalence of infectious diseases and policies in renal biopsy practice. Table 4 shows the comparison of our data with a few other national and international published studies.

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Pattern of Nephritic Syndrome

IgAN (Fig 4) was the commonest cause of ANS in a Serbian study (35.7%). It is considered as the most common primary glomerular disease especially in young adult Caucasians and the most common cause of end-stage renal disease.[24]In the current study the frequency of IgAN presenting as ANS was 12.5%, which is slightly higher than the other Indian studies.[4,20] Immunofluorescence studies in the cases presenting with ANS, showed deposits of single or multiple

immunoglobulin and/ or complement component (C3). C3 deposits were the most frequent and were present in 77% of the cases followed by IgG deposits (76%) and IgM deposits (36%). Rahman et al in their study, observed that IgM was the predominant deposit (52%) followed by C3 (48%) and IgG (47%) deposits.[4] The differing immunofluorescence findings could be due to the differing patterns of glomerulonephritis causing ANS and technical factors inherent in the methodology.

Table 4: Comparison of our data with other published studies. Variables Present study Bangladesh4 No of ANS cases 112 73 PIGN 29.5 2.7 CresGN 17.9 0 MPGN 15.2 43.8 IgAN 12.5 5.5 MN 1.8 9.6 FSGS 0.9 0 LN 15.2 11 HUS 1.8 0 HSP 1.8 0

Mumbai20 74 25.6 0 33.8 2.7 9.5 0 0 0 4.1

Hyderabad5* 166 56.6 3.6 1.8 4.8 0 2.4 13.3 0 1.2

Serbia22 83 18.1 21.7 2.4 35.7 0 4.8 -

Brazil21** 789 17.2 3.1 4.7 15.8 3.9 10.2 14.2 -

These figures represent percent of total cases of ANS. * figures have been calculated from Table 2 of the article. ** data obtained from Table 7 of the article. - indicates that data are not available

Fig. 4: (A) Membranoproliferative glomerulonephritis with lobular hypercellular glomeruli exhibiting thickened capillary walls (H&E, x 200); (B) Membranoproliferative glomerulonephritis with membranous and mesangial deposits of C3 (IFM, x200); (C) IgA nephropathy with mesangial hypercellularity (H&E, x 200); (D) IgA nephropathy with mesangial IgA deposis (IFM, x200).

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Devadass et al.

Conclusion

Various studies, done in India and abroad, show variations in the frequency and distribution of PGD presenting as ANS. The reasons for this include varying renal biopsy practices, differing patient selection criteria, differences in the prevalence of infectious diseases and variations in the prevalence of various glomerulonephritis in different regions of the world. The commonest PGD presenting as ANS in the present study, was PIGN followed by CresGN. The distribution pattern of SGD presenting as ANS largely corresponds to the pattern described in other Indian studies with LN being the commonest. The present study represents a contribution to understanding the epidemiology of renal disease manifesting as ANS in South India.

Acknowledgment Nil

Funding Nil

Competing Interests Nil

References

1. Lewis JB, Neilson EG. Glomerular disease. In: Kasper DL, Hauser SL, Jameion LJ, Fauci AS, Longo DL, Loscalzo J, editors. Harrison’s principles of Internal Medicine. 19thed. New York: MC Graw-Hill Education; 2015. p.1831-50. 2. Colvin RB editor. Introduction to Renal Pathology. In: Diagnostic Pathology. Kidney Diseases. 1st ed. Canada: Amirys Publishing Inc; 2011.p.1-2. 3. Iseki K, Miyasato F, Uehara H, Tokuyama K, Toma S, Nishime K, et al. Outcome study of renal biopsy patients in Okinawa, Japan. Kidney International. 2004;66:914-9. 4. Rahaman MA, Kamal M, Rahman MS, Biswas MA, Sarker RA: Histomorphological Patterns of Glomerulonephritis in Patients Presenting with Acute Nephritic Syndrome Dinajpur Med Col J. 2012;5:26-33. 5. Das U, Dakshinamurthy KV, Prayaga A. Pattern of biopsy-proven renal disease in a single center of south India: 19 years experience . Indian J of Nephrol. 2011;21:250-7.

A-395 editors. Brenner and Rector’s The Kidney, 9th ed. Philadelphia: Elsevier; 2012.p.844-67. 7. Satoskar AA, Nadasky T, Silva FG. Acute Postinfectious Glomerulonephritis and Glomerulonephritis caused by persistent Bacterial Infection. Chapter 10. In: Jennette JC, Olson JL, Silva FG, D’Agati VD, editors. Hepinstall’s Pathology of the Kidney, 7th ed.. Lippincott Williams & Wilkins, Philadelphia. 2015;1:367-436. 8. Jennette JC, Nickeleit V. Anti-Glomerular Basement Membrane Glomerulonephritis and Goodpasture Syndrome. Chapter 15. In: Jennette JC, Olson JL, Silva FG, D’Agati VD, editors. Hepinstall’s Pathology of the Kidney, 7th ed. Lippincott Williams & Wilkins, Philadelphia. 2015;1:657-84. 9. Jennette JC, Thomas DB. Pauci-immune and Antineutrophil Cytoplasmic Autoantibody Mediated Crescentic Glomerulonephritis and Vasculitis. Chapter 16. In: Jennette JC, Olson JL, Silva FG, D’Agati VD, editors. Hepinstall’s Pathology of the Kidney, 7th ed. Lippincott Williams & Wilkins, Philadelphia. 2015;1:685-713. 10. Colvin RB. Classification of Membranoproliferative Glomerulonephritis and Complement- related Diseases. In: Colvin RB editor. Diagnostic Pathology. Kidney Diseases. 1st ed. Canada: Amirys Publishing Inc; 2011.p.64-73. 11. Cornell LD. IgA Nephropathy. In: Colvin RB editor. Diagnostic Pathology. Kidney Diseases. 1st ed. Canada: Amirys Publishing Inc; 2011.p.142-155. 12. Chang A. Membranous Glomerulonephritis. In: Colvin RB editor. Diagnostic Pathology. Kidney Diseases. 1st ed. Canada: Amirys Publishing Inc; 2011.p.46-55. 13. Farris AB. Focal Segmental Glomerulosclerosis Classification. In: Colvin RB editor. Diagnostic Pathology. Kidney Diseases. 1st ed. Canada: Amirys Publishing Inc; 2011.p.14-17. 14. Meehan SM. Systemic Lupus Erythematosus. In: Colvin RB editor. Diagnostic Pathology. Kidney Diseases. 1st ed. Canada: Amirys Publishing Inc; 2011.p.170-187. 15. Chang A. Henoch- Schonlein Purpura. In: Colvin RB editor. Diagnostic Pathology. Kidney Diseases. 1st ed. Canada: Amirys Publishing Inc; 2011.p.156-63.

6. Emmett M, Fenves AZ, Schwartz JC. Approach to the patient with Kidney disease. In: Tall MW, Chertow GM, Marsden PA, Skorecki K, Yu ASL, Brenner BM,

16. Kambham N. Haemolytic Uremic Syndrome, Infection related. In: Colvin RB editor. Diagnostic Pathology. Kidney Diseases. 1st ed. Canada: Amirys Publishing Inc; 2011.p.70-73.

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Pattern of Nephritic Syndrome

17. Farris AB. Hypertensive Renovascular Disease. In: Colvin RB editor. Diagnostic Pathology. Kidney Diseases. 1st ed. Canada: Amirys Publishing Inc; 2011.p.106-113. 18. The University of Tennessee Health Science Center Nephrology [Internet]. Tennessee. Renal syndromes leading to abnormal kidney function [updated 2014 March 14] Available from: https://www.uthsc.edu/ nephrology/documents/renal-syndromes-guide.pdf 19. Floege J, Feehally J. Introduction to Glomerular disease: Clinical presentations. In: Johnson RJ, Freehally J, Floege J, editors. Comprehensive Clinical Nephrology. 5th ed. Philadelphia. Elsevier Inc; 2015.p.184-97. 20. Deshpande SA, Kale K, Moulick ND. Clinicohistopathological correlation of Nephritic Syndrome in adults in urban population. International J. of Healthcare and Biomedical Research. 2015;3:34-45.

21. Polito MG, Ribeiro de Moura LA, Kirsztajn GM. An overview on frequency of renal biopsy diagnosis in Brazil: clinical and pathological patterns based on 9617 native kidney biopsies. Nephrol Dial Transplant. 2010;25:490–6 22. Naumovic R, , Pavlovic S, Stojkovic D, BastaJovanovic G, Nesic V. Renal biopsy registry from a single centre in Serbia: 20 years of experience. Nephrol Dial Transplant. 2009;24:877–85. 23. D’Agati VD. Renal Disease in Systemic Lupus Erythematosus, Mixed Connective Tissue Disease, Sjogren’s Syndrome, and Rheumatoid Arthritis, Chapter 12. In: Jennette JC, Olson JL, Schwartz MM, Silva FG, editors. Hepinstall’s Pathology of the Kidney, 6th ed. Lippincott Williams & Wilkins, Philadelphia. 2007;1:518-612. 24. Pesce F, Schena FP. World distribution of glomerular diseases: the role of renal biopsy registries. Nephrol Dial Transplant. 2013;28:334-6.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 05, (Suppl) Nov. 2016

Original Article Cytohistological Study of Head and Neck lesions and their Diagnostic Pitfalls Bhardwaj Aparna1*, Pandey Apoorva2, Kishore Sanjeev1, Kaushik Sanjay1, Maithani Tripti2 Department of Pathology, SGRR Institute of Medical & Health Sciences, Dehradun, India. Department of Otorhinolaryngology, SGRR Institute of Medical & Health Sciences, Dehradun, India. 1

Keywords: Cytology, Lymph Node, Thyroid, Histopathological Examination.

ABSTRACT Background: Head and neck swellings often present with a perplexing diagnostic dilemma and may originate from lymph nodes, thyroid and salivary glands. FNAC is presently employed for both therapeutic and prognostic reasons, thereby helping the surgeon to decide modality of treatment. Thus this study was conducted with an aim to correlate cytological diagnosis of head and neck swellings with histopathology and to compare statistical data employing sensitivity, specificity and diagnostic accuracy. Methods: This was a retrospective study carried out for duration of 3 years and included 1231 cases referred to the Department of Pathology. FNAC was done from palpable head and neck masses and correlated with histopathology findings. Statistical analysis was done to calculate the sensitivity, specificity and diagnostic accuracy of cytological diagnosis. Results: Of 1231 cases for which FNAC was done, 234 cases were available for histopathological correlation. There was an overall male preponderance with male:female ratio being 1.04:1. The age of patients ranged between 16 years to 90 years. The maximum number of aspirates were from lymph nodes (680 cases, 55.23%) followed by thyroid gland (324 cases 26.32%), neck and post-auricular swellings (117, 9.5%) and salivary gland (110, 8.9%). Statistical analysis was done to calculate overall diagnostic accuracy, sensitivity and specificity of FNAC and was found to be 93.4%, 72.8%, 97.3% respectively. Conclusion: FNAC is a fairly accurate, reliable and cost effective method for rapid and reliable diagnosis of palpable lesions in the head and neck region, thus helping the surgeon modify or monitor his surgical approach.

*Corresponding author: Dr. Aparna Bhardwaj, Assosciate Professor, Pathology, House No. 1, Main street, ashiward enclave, Dehradun, Uttarakhand, India- 248001 Phone: +91 9411718270 Email: aparnapande1977@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

The aspirates from head and neck region encompass lesions from salivary glands, thyroid, lymph node, soft tissue, blood vessels and neural tissue and have variable pathology ranging from benign inflammatory lesions to neoplasms. Fine needle aspiration cytology (FNAC) which was originally described by Martin and Ellis is considered an excellent first line method with high specificity for investigating the nature of palpable swellings in the head and neck region, thus obviating the need for surgical intervention. [1,2] It has an important role in diagnosing head and neck tumors that account for almost half of all the tumors at different body sites with high specificity. [3] Further, the lesions can be categorized into inflammatory/ benign and malignant with a high degree of specificity. [4] Thus this study was undertaken to study the spectrum of head and neck lesions and to correlate the cytological results with histopathological examination. Descriptive statistics was done to evaluate our results.

Materials and Methods

This was a retrospective study carried out for duration of 3 years and includes 1231 cases of head and neck swellings referred from department of otorhinolaryngology for pre-operative FNAC and subsequent histopathological evaluation. The study was vetted and approved by Institutional Ethics committee. Prior to FNAC a detailed clinical history and clinicoradiological findings were noted in a preformed proforma and informed consent was taken. Subsequently fine needle aspiration was done using 22-23 G needle attached to a 10 ml plastic disposable syringe. For all the palpable swellings of head and neck region atleast four smears were made and stained by May-Grunwald Giemsa, Hematoxylin & Eosin (H&E), and Papanicolou stains. A few unstained smears were fixed and kept for Ancillary stains and/or immunochemistry as and when required. Out of 1231 cases, surgical biopsies were available for 234 cases for histopathological correlation. The cytology results were classified into the following categories: true negative (absence of malignancy correctly diagnosed); true positive (presence of malignancy correctly diagnosed); false negative (the cytological specimen failed to diagnose as malignancy); and false positive (the cytological specimen was incorrectly considered or suspect of malignancy). Data analysis was based on Galen and Gambino method which calculated sensitivity and specificity of cytology in differentiating benign and malignant lesions.

Results

Total number of cases for which FNAC was done was 1231. Amongst these 630 patients were males and 601 were

females, with an overall male to female ratio of 1.04:1. The youngest patient in this series was 16 years old and the eldest 90 years old with the mean age of 34.24 years. Of all these cases, 234 patients were subjected to surgical intervention and subsequent histopathological examination and were thus available for cytohistological correlation. All 1231 cases were categorized cytologically, on the basis of site of origin into lesions of lymph nodes, thyroid, salivary glands and neck and postauricular swellings. They were broadly classified into inflammatory, benign and malignant lesions (Table 1). Lymph Nodes: Of 680 cases for which FNAC was performed, 72 cases were available for cytohistopathological correlation. The most common group of lymph nodes involved was deep jugular group (63.9%), followed by supraclavicular group (9.3%). Lymph node lesions were categorized into benign and malignant lesions. Of 72 cases the final diagnoses under benign lesions were tuberculosis (18 cases), reactive lymphadenitis (16 cases), sarcoidosis (1 case), inflammatory (1 case) and under malignant conditions were Hodgkins disease (8 cases), Non Hodgkins lymphoma (10 cases), metastatic (16 cases) and angio-immunoblastic (2 cases).There were one false positive and six false negative cases in this group (Table no 2). Thyroid: Total number of patients who presented with thyroid swelling were 324 with commonest clinical complaint of enlargement of thyroid gland associated with voice change and difficulty in swallowing. The female to male ratio for thyroid lesions was 2.4: 1 thus exhibiting a paradox in this group with female preponderance. Of these 324 cases for which FNAC were performed, 69 cases were subjected to surgical intervention hence available for cyto-histopathological correlation. On FNAC, the thyroid lesions were classified as colloid goiter (51 cases), cystic nodule (3 case), lymphocytic thyroiditis (10 cases), granulomatous thyroiditis (1 case), follicular neoplasm (6 cases), medullary carcinoma (2 cases), papillary carcinoma (2 cases), hurthle cell neoplasm (1 case), and Non hodgkins lymphoma (1 case). Considering histopathology as gold standard, 54 cases were benign and 12 cases were malignant. There were 5 false positive in this group (Table no 3). Salivary Glands- Of 110 salivary glands swellings 40 cases were subjected to surgical intervention hence available for cyto-histopathological correlation. Of these 40 cases, 15 cases were female and 25 were males. Parotid was the commonest salivary gland to be involved. Of all the 40 cases, 27 cases were benign and 13 cases were malignant on histopathology. The commonest salivary gland lesion

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was pleomorphic adenoma (18 cases), followed by chronic sialdenitis and Warthin’s tumour (4 cases each). Amongst the malignant salivary gland lesions, Mucoepidermoid carcinoma, Adenoid cystic, and Squamous cell carcinoma accounted for 2 cases each. There were 3 false positive cases in this group (Table no 4). Neck and Post Auricular Swelling- Of 117 cases, which presented with neck and post-auricular soft tissue swellings, 53 cases were sent for histopathology and were available for cyto-histopathological correlation. Of these 53 cases, 37 were males, and 16 were female. Overall 51 cases were diagnosed as benign on histopathology. Castleman’s

disease, a morphologically distinct form of hyperplasia, was cytologically diagnosed as reactive hyperplasia. Two cases of Basal cell carcinoma (BCC) and metastatic deposit of squamous cell carcinoma which were diagnosed as benign cystic lesions on cytology respectively were malignant (Table no 5). There were one false positive and one false negative diagnosis in this group. Statistical analysis was done and sensitivity, specificity and diagnostic accuracy was calculated (Table no 6). Overall diagnostic accuracy was found to be 93.4%, sensitivity 72.8%, specificity 97.3%, positive predictive value 95.5% and negative predictive value 91.9% respectively.

Table 1: Distribution of 1231 cases of head and neck swellings on cytological diagnosis. Site Inflammatory Neoplastic Benign Malignant Lymph node 34 (5%) 531(78.1%) 91 (13.4%) Thyroid 59 (18.2%) 230(70.98%) 20 (6.2%) Salivary glands 42 (38.2%) 47 (42.72%) 16 (14.5%) Neck and postauricular swellings

Inconclusive

Total

24 (3.5%) 15 (4.62%) 6 (5.45%)

680 324 110

16 (13.7%)

79(67.52%)

4 (3.4%)

17 (14.5%)

117

151

887

131

1,231

Total

Table 2: Cyto-Histological Correlation of Lymph Node lesions. Cytological diagnosis Number of cases Histopathological diagnosis Benign (N) Malignant (N) Reactive hyperplasia 21 Reactive 16 Angioimmunoblastic HL NHL TB/granuloma

Inflammatory HL NHL Metastatic

1 5 10 16

Tuberculosis Sarcoidosis Inflammatory

17 1 1

Reactive

2 2 1

Metastatic

Table 3: Cyto- Histological Correlation of Thyroid lesions. Cytological diagnosis Number of cases

Colloid goiter

Cystic nodule

Lymphocytic thyroiditis

Granulomatous thyroiditis

Follicular neoplasm

Medullary carcinoma

Papillary carcinoma

NHL Hurthle cell neoplasm

1 1

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Histopathological diagnosis Benign (N) Malignant (N) Colloid goiter 42 Graves disease 3 Papillary Carcinoma Hashimoto’s thyroiditis 3 Hurthle cell Adenoma 1 Colloid goiter with 2 Papillary Carcinoma cystic degeneration Lymphocytic thyroiditis 7 Colloid goiter 3 Colloid goiter with 1 granulomatous thyroiditis Follicular Adenoma 1 Folicular Carcinoma Adenomatous goiter 3 Medullary carcinoma Papillary Carcinoma Follicular variant of Papillary Carcinoma Follicular Adenoma

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2 1

2 2 3 1

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Table 4: Cyto- Histological Correlation of salivary Gland lesions. Cytological diagnosis Number of cases (n) Histopathological Diagnosis Benign (N) Malignant (N) Lipoma 2 Lipoma 2 Sialadenosis 1 Chronic Sialadenitis 5 Chronic sialadenitis 4 Haemorrhagic cyst 1 Hemangioma 1 Pleomorphic adenoma 18 Mucoepidermoid Basal cell adenoma 1 Carcinoma Pleomorphic Adenoma 21 Adenoid cystic carcinoma Mucoepidermoid Carcinoma

Squamous cell Carcinoma

Oncocytoma Warthinâ&#x20AC;&#x2122;s tumour Oncocytic carcinoma

1 5 1

Warthinâ&#x20AC;&#x2122;s tumour

1 1 1

Mucoepidermoid Carcinoma Squamous cell Carcinoma Adenoid cystic carcinoma Acinic Cell Carcinoma

2 1 1

Salivary duct carcinoma

Table 5: Cyto-Histological Correlation of Neck and Post auricular Swellings. Cytological diagnosis Number of cases (n) Histopathological diagnosis Benign ( N) Malignant (N) Tuberculosis 3 Granulomatous Sarcoidosis 1 5 Inflammation/ TB Castleman disease 1 12

Lipoma

Epidermoid cyst

Epidermoid cyst ProliferatingTrichilemmal Cyst Mature teratoma

19 1 1

Sebaceous cyst Spindle cell lesion Neurofibroma

1 1 1

Sebaceous cyst Neurofibroma Neurofibroma

1 1 1

Basal cell carcinoma

NHL/PNET

Calcifying Epithelioma of Malhurbe (Pilomatricoma)

Benign Vascular lesion

Benign cystic lesion

Hemangioma Thyroglossal cyst Brachial cyst

5 3 1

Lipoma

Basal cell carcinoma

Metastatic deposit of Squamous cell carcinoma

Table 6: Sensitivity, Specificity and Diagnostic Accuracy of FNAC in diagnosis of Head and Neck lesions in this study. Organ of origin

HPE/FNAC

Sensitivity

Specificity

Accuracy

Lymph node

72/680

83.3%

97.2%

90.3%

Thyroid

69/324

58.33%

100%

93.7%

Salivary Glands

40/110

56%

100%

90%

Neck and Post Auricular swelling

53/117

50%

95.23%

95.5%

234/1231

72.8%

97.3%

93.4%

Total

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Fig. 1a: Cytology smears showing only RBC (MGG,100X) suggestive of Heamorrhagic cyst with corresponding tissue section showing dilated cavernous spaces filled with RBC seperated by connective tissue stroma in 1b (H&E,100X) 1c. Cytology smears showing RS cell diagnosed as Hodgkins lymphoma (MGG, 400X) with tissue section exhibiting mononuclear variant of RS cell in 1d (H&E, 400X).

Fig. 2a: Cytology smear from nodule thyroid showing prominent intranuclear inclusion against haemorrhagic background (MGG,100X) with corresponding tissue section showing papillae and cells exhibiting Orphan Annie Eye nuclei in 2b (H&E, 100X). 2c. Cytology smears shwing numerous anucleate squames diagnosed as sebaceous cyst (MGG, 100X) with corresponding tissuesection showing well formed cyst filled with abundant keratin in 2d (H&E, 100X).

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Cyto Histological Correlation of Head and Neck Lesions

Fig. 3a: Cytology smears showing epithelial cells associated with fibrillar myxoid background in Pleomrphic Adenoma (MGG,100X) with corresponding tissue section exhibiting bimodal population of epithelial and myoepithelial cells in 3b (H&E, 100X) 3c Cytology smear showing monolayered sheets of uniform oncocytic cells, lymphocytes against amorphus background in Warthinâ&#x20AC;&#x2122;s tumour (MGG, 100X) with corresponding tissue section showing oncocytic epithelium resting on lymphocytes in 3d (H&E,100X).

Fig. 4a: Smear showing poorly cohesive malignant epithelial cells with hyperchromatic nuclei against necrotic background diagnosed as Squamous Cell Carcinoma (MGG, 400X) with corresponding tissue section in 4b showing Adenoid Cystic Carcinoma (H&E, 400X) 4c. Smear showing thin colloid and follicular epithelial cells diagnosed as colloid goiter (MGG 100X) with corresponding tissue section in 4d showing destruction of thyroid follicles by lymphocytes in Hashimotoâ&#x20AC;&#x2122;s thyroiditis (H&E, 100X)

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Fig. 5a: Smear showing sheets of oxyphill cells diagnosed as Hurthle cell Adenoma (MGG, 400X) with corresponding tissue section in 5b showing well circumscribed Follicular Adenoma(H&E, 100x). 5c Smear showing monotonus population of cells diagnosed as NHL/PNET (MGG,400X) with tissue section corresponding tissue section showing ghost cells diagnosed histologically as Pilomatricoma in 5d (H&E, 100x).

Fig. 6a: Smear showing epithelioid cells cytologically diagnosed as granulomatous lesion (MGG, 400x) with tissue section showing solid granulomas, Arrow showing: giant cell with Asteroid body in Sarcoidosis (H&E, 400X). 6c Smear showing many cyst macrophages against diagnosed as benign cystic lesion (MGG 400X) with Corresponding tissue section in 6d showing Thyroglossal cyst (H&E, 100X).

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Discussion

Head and neck masses are common clinical conditions and FNAC serves an important first line investigation thus providing useful information to surgeon to determine further management. Being minimally invasive it is a preferred investigation particularly in young patients who have low incidence of malignancy and in patients who are in palliative settings. [2] Furthermore, most head and neck masses of distinct cause present in fairly predictable locations within specific age group. This permits a methodical approach to clinch a working diagnosis and a differential diagnosis and appropriate treatment protocol for the patients. Of all the 1231 cases for which FNAC was done, histopathological correlation was available for 234 cases. Evaluation of palpable head and neck swellings demands a systematic and rational approach. Organ wise cytological findings were thus correlated with histopathological diagnosis (Table no 7). Establishing the exact nature of swellings and preliminary identification of lesion as cystic, inflammatory or neoplastic substantially obviates unnecessary surgeries in a significant number of cases. All 1231 cases were divided into inflammatory, benign and malignant lesions. In the present study 62 cases (5.04%) were rendered inconclusive. The inconclusive lesions in head and neck masses ranged between 0-22% and 3-30% which correlates well with our study. [6,7] The inconclusive cases were mainly due to small size of swelling (<1cm), hemorrhagic aspirate and lesions with extensive fibrosis and necrosis. In the present study maximum number of aspirates were from lymph nodes (680 cases, 55.23%) followed by thyroid gland (324 cases 26.32%), neck and post-auricular swellings (117, 9.5%) and salivary gland (110, 8.9%) which Correlates well with other studies. [5,8] Lymph Node: Highest degree of sensitivity (83.3%) was seen in diagnosing lymph node pathologies. Of 72 cases for which histological correlation was available there were one false positive and six false negative cases. Tubercular lymphadenitis was the commonest diagnostic Table 7: Organ wise FNAC diagnosis correlation with HPE. FNAC diagnosis Total Number (n) Lymph Node 72 Thyroid 79 Salivary gland 40 Neck & Post auricular swelling 53

pathology which correlated well with studies by other authors. [9] Tuberculosis is widely prevalent in developing countries with prevalence as high as 1.5% and tubercular cervical lymphadenopathy being the most common form of extrapulmonary tuberculosis. FNAC has been reported to have 90-100 % diagnostic accuracy in detection of tuberculous affliction of nodes. [7,10] The percentage of reactive lymphadenitis (22.2%) was next only to Tubercular lymphadenitis (23.6%) and is comparable to studies by other authors. [11] In our study two cases diagnosed as reactive lymphadenitis were histologically diagnosed as Hodgkinâ&#x20AC;&#x2122;s disease and Non Hodgkin Lymphoma respectively on histopathology. The diagnostic dilemma arising in distinguishing cytologically between reactive lymphadenitis and TB from lymphomas may be due to cluster of epithelioid cells, tingible body macrophages and polymorphous population of lymphocytes found in lymphomas.[13] Thus the presence of these features does not necessarily rule out lymphoma and its classification must be made on adequate tissue biopsy. [9] Likewise one case was diagnosed as NHL on cytology turned out to be reactive lymphadenitis on histopathology. Aspirates from germinal centers from reactive hyperplasia may lead to erroneous results owing to increased number of large cells and increased mitosis and is well known to produce False Positive diagnosis.[13] Two cases of Angioimmunoblastic lymphadenopathy (AILD) were cytologically diagnosed as reactive hyperplasia. AILD is referred by most authors as a form of peripheral T- cell Lymphoma. [14] An exuberant benign reactive host inflammatory reaction is characteristic in producing difficulty in its separation from reactive lymphadenopathy. Initial diagnosis is usually confirmed by tissue biopsy. Ancillary test to confirm diagnosis is by flow cytometry which detects abnormal T- cell antigen expression. [15,16] According to Frable et al cytological diagnosis was used preliminary for the documentation of residual or recurrent lymphomas and to assess the stage of disease. The use of FNAC to render preliminary diagnosis of lymphomas remains controversial. [17] Correlation with Histopatholgy Present 65 74 36 5

FP 1 0 0 1

FP- False Positive FN-False Negative

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FN 6 5 4 1

Aparna et al. The diagnostic accuracy in diagnosing metastatic deposits involving cervical lymph nodes was 100% in our study. However, partial lymph node involvement, micro metastases in sub capsular sinus or scattered single cells are unlikely to be sampled by repeated aspiration hence are the main causes of false negative cytological reports. [13] Thyroid: Out of 324 aspirates of thyroid lesions, 79 surgical biopsies were available for histopathological correlation. Of 79 cases, 67(84.8%) were benign lesions and 12 (15.2%) were neoplastic lesions. Out of these 67 benign lesions 53 cases were diagnosed as colloid goiter (67.1%), which was the commonest pathology observed in this group. Amongst the malignant lesions, Papillary carcinoma thyroid was the commonest neoplasm (7.6%). Two cytologically diagnosed cases of colloid goiter turned out to be papillary carcinoma on histopathology. Such erroneous interpretations have also been reported by other authors. [9] The cause of false negative results may be due to poor cellularity, sampling error in case of small focus of neoplasm, absence of papillary fragments that fail to demonstrate intranuclear inclusions and nuclear grooves.[18] One case diagnosed as thyroid cyst on FNAC on the basis of scant cellularity, foamy macrophages, few follicular cells and colloid was histologically confirmed as Papillary carcinoma. Cystic lesions of thyroid pose diagnostic difficulties which have been well observed by other authors. [19] Recurrent cysts, incompletely decompressed lesions, lesions greater than 3- 4 cm in diameter in which aspiration of several areas does not give evidence of colloid nodule and lesions in young male are indications for surgical excision. [20] Another diagnostic dilemma in FNAC of thyroid lesion is to differentiate follicular adenoma from follicular carcinoma. Similarly, colloid goiter may be difficult to differentiate from adenoma. Similar observations have beencited by other authors who have stressed that the cytological appearance in colloid goiter and follicular adenoma is overlapping and the cytological criteria cannot distinguish between the two. [19] Three cases of Hashimotoâ&#x20AC;&#x2122;s thyroiditis were cytologically diagnosed as nodular colloid goiter. On reviewing slides, they revealed hypocellular smears comprising few follicular cells and scant colloid. Studies by other authors suggest similar diagnostic dilemma. [21] One case in this group demonstrated dual pathologies as colloid goiter with granulomatous thyroiditis on histopathology. Studies by other authors have emphasized the same observation of finding two or more surgical www.pacificejournals.com/apalm

A-405 pathology on histology amongst thyroid lesions. Multiple aspirations in a thyroid swelling in order to obtain representative material from different areas is indicated since the thyroid may be affected by more than one disease process.[22] Salivary Gland: In our study, of all the salivary glands, Parotid were most frequently involved salivary gland and Pleomorphic Adenoma was the commonest lesion reported. There were 4 false negative cases, however no false positive case was seen in this group. A case of Mucoepidermoid carcinoma was diagnosed as pleomorphic adenoma on FNAC. Studies by other authors suggest that low grade mucoepidermoid carcinoma is one of the most difficult entities to diagnose cytologically and is the source of false negative results. One case of Pleomorphic Adenoma was found to be Adenoid Cystic carcinoma on histopathology. On reviewing the smears, cluster of uniform cells in myxoid stromal background were noted with notable absence of hyaline globules. Similar findings were noted by other authors.[23] One case of acinic cell Carcinoma was misdiagnosed as Warthinâ&#x20AC;&#x2122;s tumour on cytology hence reported as false negative. Studies by authors have documented that Acinic Cell carcinoma is most frequently misdiagnosed as Warthins tumour. Such difficulties arise because of close resemblance of acinar cells of acinic carcinoma with sheets of oncocytes. [24] Neck and Post Auricular Swellings: Amongst the miscellaneous lesions from Neck and Posterior auricular swellings 1 case was false positive and 1 false negative. A case of Pilomatricoma was given as NHL on cytology and hence rendered False Positive. On reviewing smears hyperchromatic cells with prominent nucleoli were seen that were misdiagnosed as Lymphoma. Wrong et al in his study of 16 cases on Pilomatricoma observed that a correct diagnosis could be given only in 25% of the cases. The most common diagnostic pitfall was a false positive or suspicious diagnosis of carcinoma. [25] A case of metastatic deposit of well differentiated squamous cell carcinoma was cytologically diagnosed as benign cystic lesion (brachial cyst) since smears revealed only necrotic material due to liquefactive necrosis.[26] Another case of Post auricular swelling cytologically diagnosed as Reactive hyperplasia proved out to be Castlemanâ&#x20AC;&#x2122;s disease on histopathology. On reviewing the smears they showed abundant plasma cells. The plasma cell

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lesion represents an earlier and active stage of disease that may lead to diagnostic errors on cytology. The cytologic characterization of Castleman’s disease may be difficult. [27] The sensitivity, specificity and Accuracy of present study were 72.8%, 97.3%, 93.4% respectively and are comparable with studies by other authors and are fairly accurate.[6,9,28]

Conclusion

FNAC is simple and fairly accurate method of diagnosing palpable head and neck swellings with a high diagnostic accuracy. It can be employed for both the neoplastic and non neoplastic lesions and is a highly effective diagnostic procedure in the diagnosis and management of head and neck masses. In the present study there were only 2 false positive cases and 16 false negative cases.

References:

1. Martin HE, Ellis EB. Aspiration biopsy. Surg Gynecol Obstet 1934;59:578-89.

10. Khan RA, Wahab S, Chana RS, Naseem S, Siddique S. Children with significant cervical lymphadenopathy: clinicopathological analysis and role of fine needle aspiration in Indian setup. J Pediatr (Rio J).2008;84(5):449-454. 11. Javaid M, Niamalulla, Anwar K, Said M. Diagnostic value of fine needle aspiration cytology in cervical lymphadenopathy. JPMI 2006;20:117-120. 12. Wakeley PE. Fine needle aspiration cytopathology in diagnosis and classification of malignant lymphoma: accurate and reliable ? Diagn Cytopathol 2000;22:120-25. 13. Orell SR, Sterrett GF, Walters Max N et al: Lymph nodes. In Manual and Atlas of Fine Needle aspiration of cytology, 4th Edn. New York: Churchill Livingstone 2005:103-114. 14. Rosai J. ed. Rosai and Ackerman’s surgical Pathology 9th ed. Vol. 2, New York Mosby; 2004: 1908- 09.

2. Mitra P, Bharti R, Pandey MK. Role of fine needle aspiration cytology in head and neck lesions of paediatric age group. J Clin Diag Res 2013;7:1055-8.

15. Ng WK, Ip P, Choy C, Collins RJ. Cytologic findings of angioimmunoblastic T-cell lymphoma. Analysis of 16 fine needle aspiration over a 9 year period. Cancer(Cancer cytopathol) 2002;96:166-73.

3. El Hay IA, Chiedozi LC, al Reyees FA, Kollur SM. Fine needle aspiration cytology of head and neck masses. Seven years experience in a secondary care hospital. Acta Cytol 2003;47:387-92.

16. AI Shangeely D, Mourad Wa. Diagnosis of peripheral T-cell lymphoma by fine needle aspiration biopsy: a cytomorphologic and immunophenotypic approach. Diagn Cytopathol 2000;23:375-79.

4. Boccalo P, Altavilla G, Blandamura S. Fine needle aspiration biopsy of salivary gland lesions. A reappraisal of pitfalls and problems. Acta Cytol.1998;42:888-98.

17. Frable WJ, Kardos TF. Fine needle aspiration biopsy. Application in the diagnosis of lymphoproliferative disease. AM J surg Pathol 1988;12:62-72.

5. Singal P, Bal MS, Kharbanda J, Sethi PS. Efficacy of fine needle aspiration cytology in Head and Neck lesions. IJMD 2014; 3(2):131-4. 6. Tandon S, Shahab R, Benton JI, Ghosh SK, Sharad J, Jones TM. Fine needle aspiration cytology in a regional head and neck cancer center: Comparison with a systematic review and meta analysis. Head Neck 2008;30(9):1246-56. 7. Fernandes H, D’souza CR, Thejaswini BN. The role of fine needle aspiration cytology in palpable head and neck masses. J Clin Diagn Res 2009;3:1719-25.

18. Baloch ZW, Livolsi VA. Cytologic and architectural mimics of papillary thyroid carcinoma. Am J Pathol. 2006;125:135-44. 19. Canberk S, Firat p, Schmitt F. Pitfalls in the Cytological Assesment of Thyroid Nodule. Turkish Journal of Pathology 2015;31( Suppl):18-33. 20. Neki NS, Kazal HL. Solitary Thyroid Nodule-An Insight. JIACM 2006;7(4):328-33. 21. Jayram G. Fine needle aspiration cytology study of the solitary thyroid nodule. Profile of 308 cases with histologic correlation. Acta Cytol 1985;29 (6):967-73.

8. Taviad DS, Jadav K, Nikhra P, Panchal A, Patel V. Role of fine needle aspiration cytology in head and neck swelling. Int.J Res Med 2014;3:131-4.

22. Kollur SM, ElL Sayed S, EL Hag IC. Follicular thyroid lesions coexisting with Hashimoto’s thyroiditis: incidence and possible sources of diagnostic errors. Diagnostic Cytopathology 2003; 28(1):35-38.

9. Tilak V,Dhadad Av, Jain R. Fine needle aspiration cytology of head and neck masses. Indian J Pathol Microbiol.2002;4591):23-9.

23. Khafaji BM, Nestok BR, Katz RL. Fine- needle aspiration of 154 parotid masses with histologic correlation. Cancer Cytopathol 1998;84(3):153:59.

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Aparna et al. 24. Pawani AV, Ali SZ. Diagnostic accuracy and pitfalls in fine needle aspiration interpretation of Warthin tumour. Cancer 2003; 99:166-71. 25. Thapliyal N, Joshi U, Vaibhav G, Sayana A, Srivastva AK, Jha RS. Pilomatricoma Mimicking Small Round. Cell Tumor on Fine Needle Aspiration Cytology. A Case Report. Acta Cytologica 2008;52(5):2008.

A-407 Indian J Otolaryngol Head Neck Surg. 2014; 66(2):182-86. 27. Hidvegi DF, Sorensen K, Lawrence JB, Nieman HL, Isoe C. Castlemanâ&#x20AC;&#x2122;s disease. Cytomorphological and cytochemical features of a case . Acta Cytol 1982;26(2):243-46.

26. Poorey VK, Tyagi A. Accuracy of Fine Needle aspiration Cytology in Head and Neck Masses.

28. Balakrishnan K, Castling B, Mc Mahon J, et al. Fine needle aspiration cytology in the management of a parotid mass: a two centre retrospective study. Surgeon 2005;3 (2):67-72.

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Original Article Evaluation of Small Intestinal Biopsies in Malabsorption Syndromes Ashmeet Kaur1*, Poojaba Jadeja2, Neha Garg2, SML Rai2 and N. Mogra2 Department Of Pathology, Santokba Durlabhji Memorial Hospital, India Department of Pathology, Geetanjali Medical College and Hospital, Udaipur, India 1

Keywords: Malabsorption, Celiac Disease, Chronic diarrhea . Non specific duodenitis . Villous atrophy

ABSTRACT Backgound: The gold standard test for evaluation of intestinal malabsorption is biopsy. The varied yet similar, abnormal mucosal architecture patterns in intestinal malabsorption makes it difficult for the pathologist to report its etiological cause Methods: We analyzed the clinical presentation, endoscopic and histological features of 328 consecutive patients with complaints suggestive of malabsorption syndrome. The spectrum of disease in these patients were studied and divided into four groups on the basis of histologic features and correlated with clinical/endoscopic findings Results: Group1 were entities usually associated with a diffuse severe villous abnormality and crypt hyperplasia ,all 57 cases were of celiac disease (17.3%), Group 2 were entities usually associated with a variable villous abnormality and crypt hypoplasia, included 7 cases-malnourished 5(1.5%) and post chemotherapy 2(0.6%), Group 3 were entities usually associated with a nonspecific variable villous abnormality, usually not flat included 252 biopsies- Tropical sprue 16(4.8%), nonspecific duodenitis/jejunitis/ileitis 212 (64.6%), peptic duodenitis 16(4.8%), intestinal tuberculosis 8 (2.43%), and in Group 4 were entities associated with variable villous abnormalities illustrating specific diagnostic changes included 12 cases-giardiasis 2 (0.6%), Crohnâ&#x20AC;&#x2122;s disease 8 (2.4%), intestinal lymphangiectasia 1 (0.3%), lymphoma 1(0.34%).

*Corresponding author: Dr Ashmeet Kaur, Department Of Pathology, Santokba Durlabhji Memorial Hospital, India Phone: +91 9680027604 Email: ashmeetkochar@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

The etiology of malabsorption syndrome varies according to the geographical location, age and vary over time .[1,2,3] While celiac disease, Crohn’s disease, cystic fibrosis, and intestinal lymphangiectasia are the frequent causes of malabsorption syndrome in the West, tropical sprue,parasitic infections, intestinal tuberculosis, and primary immunodeficiency syndromes have been reported to be the commonest causes of malabsorption syndrome in the developing countries.[3] In the past, tropical malabsorption, popularly known as tropical sprue, was a common cause of epidemics in southern Indian villages.[3] Histologically, varied spectrum of morphology is observed. Gastrointestinal tract infections are extremely common in a developing country like India because of poor hygienic conditions but it is also noticed that speciﬁc infections such as giardiasis or tuberculosis are not more frequent. Pathologists often report nonspecific inflammation may be due to chronic gastric mucosal irritation (with drugs),or gastrointestinal tract infections, we tend to give a diagnosis of mild nonspeciﬁc duodenitis if we see even few lymphocytes or plasma cells in the lamina propria . Quite often we miss important diagnosis like celiac disease presuming its low prevalence in adults and in a developing country like India. The aim of this study was to evaluate the histological features of patients with malabsorption at a tertiary care center. We also report the differences in the clinical, endoscopic, and histological features between patients with celiac disease and other causes of malabsorption.

Material and Methods

Overall 328 patients of malabsorption were enrolled in study during Jan 2012-Aug 2015 and were evaluated to ascertain the etiology of disease process and managed as per need. These included all patients consecutively attending Gastroenterology Clinics, referred from medicine & pediatrics-indoors & outdoors after basic workup. The diagnosis of malabsorption was made on the basis of the following features: (1) Diarrhea, vomiting/abdominal pain or clinical features suggestive of nutritional deficiencies (iron, folate, vitamin D), and/or (2) presence of anemia and/or hypoalbuminemia and/or (3) Abnormal endoscopic finding. Patients with lactose intolerance and irritable bowel syndrome and those showing non-compliance to follow up were excluded from this study. Thus, 328 subjects (195 men), in whom all relevant investigations were available, were included in the final analysis.

Hematological and biochemical investigations including hemoglobin, total protein , liver function test and thyroid function test (if required).Upper gastrointestinal endoscopy was done for all the patients, and the status of the duodenal folds was recorded (normal, attenuation, scalloping of mucosal folds, erythema/ulcers and thickened). Biopsies were analyzed for mucosal changes by two pathologists and were divided into four groups on the basis of histologic features. Group 1- diffuse severe villous abnormality and crypt hyperplasia , Group 2variable villous abnormality and crypt hypoplasia Group 3- nonspecific variable villous atrophy , usually not flat and Group 4 -variable villous abnormalities illustrating specific diagnostic changes.4 Data thus collected were entered in excel sheet and were subjected for statistical analysis. Continuous variables were summarized as mean and standard deviation, while categorical and nominal variables were summarized as percentages. Unpaired t test and ANOVA test with Post Hoc Tukey HSD test were used for comparing continuous variable while nominal and categorical variable were compared by chi-square test. Agreement between grades were measured and analysed by using kappa statistics. Diagnostic Criteria for Causes of Malabsorption: The diagnosis of celiac disease was made on the basis of clinical manifestations, biochemical (ttG), endoscopic finding ,specific histologic changes and response to gluten free diet. The diagnosis of celiac disease was graded by Ensari criteria which require presence of duodenal mucosal biopsy changes. Patients with diarrhea, malabsorption of two or more substances, after exclusion of other causes, and a persistent response to antibiotics, were diagnosed to have tropical sprue. Intestinal tuberculosis was diagnosed in presence of acid fast bacilli on mucosal biopsy or presence of caseating granuloma on biopsy specimen. Crohn’s disease was diagnosed using a combination of histology, endoscopic, radiological, histological features and response to treatment. On all the biopsies in this group, stool examination was carried out and parasitosis was detected by microscopic examination of stained stool smears. Specific abnormality like intestinal lymphangiectasia was detected on the basis of clinical history, biochemical parameters and histology. When no other cause was found but lamina showed inflammation, it was labeled as non specific inflammation.

Results

Demographic and clinical features including age, gender, duration of the disease, frequency of stools/day, consistency of stools, abdominal pain, and vomiting were recorded.

Out of the total 328 biopsies, 238 were from duodenum, 74 were from jejunum and 13 were from ileum and 3 resected specimens. The mean duration of diarrhea was 136.48 days (range 10days-720days )

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Etiology of Malabsorption Syndrome: Subjects included in group1 were 57, in group 2 were 7, in group 3 were 252 and in group 4 were 12.The most common cause of malabsorption was nonspecific duodenitis/ jejunitis/ ileitis (n=212,64.6%), followed by celiac disease (n=57,17.3%). Other causes of malabsorption in this study are illustrated in figure 1.

vs 39.6%) (p< 0.001) as in table 3. Total protein although relatively low in celiac disease patients compared to non specific inflammation , statistically insignificant.

From table 2, we concluded that patients with celiac disease were younger than those suffering from any other cause of malabsorption(32.58, p=0.005). Although patients suffering from Tropical sprue, Intestinal tuberculosis and nonspecific inflammation (40.60) presented with almost similar age as those with crohns, lymphoma or lymphangiectasia (38.7), it was not a significant finding.

Duration of diarrhea was observed to be more in patients with celiac disease(219.01 days vs 108.64 days) (p=0.005)

Majority of the subjects presented with diarrhea (52.3% ) and the mean duration of diarrhea was 136.48 days(range=10-720 days).Other minor complaints of presentation were abdominal pain, vomiting, weight loss and dyspepsia. Duration of diarrhea was maximum in subjects of celiac disease, though not significant. Hemoglobin, platelet count and total protein were lowest in malnourished and post therapy patients, although not significant Demographic,Clinical and Laboratory data comparison: Patients with celiac disease were younger than those with nonspecific duodenitis (mean age at presentation 32.58 years vs 40.02 years), though statistically not significant. More number of patients with celiac disease had anemia compared to those with nonspecific duodenitis (42.10%

Associated diseases like hypothyroidism were higher in patients with celiac disease than in those with nonspecific (10.52% vs 7.1%), though not significant.

Endoscopy and Histology: Most patients with nonspecific duodenitis had erythematous duodenal folds with/without ulcers (38.6%) while, the appearance of duodenal folds was scalloped in 54% of patients with celiac disease as seen in table 4 . Scalloping of folds (54.3%) and both scalloping and attenuation (21%) of duodenal folds were more frequently present in patients with celiac disease. On histological examination, while 17 (29.8%)patients with celiac disease in this study had normal/ mild villous atrophy, 55 (25.94%,p) patients with nonspecific duodenitis had normal/mild villous atrophy. 19( 33.34%) and 22 (38.54%) patients with celiac disease had moderate and severe villous atrophy, 29 (13.67%) & 7(3.3%) patients with nonspecific duodenitis had moderate and severe villous atrophy, respectively. Crypt hyperplasia was higher in celiac disease than in non specific duodenitis. (p<0.001). Histologically, IELs were highest in subjects of celiac disease among all groups (32.8,p<0.01)

Table 1: Abnormal mucosal architecture in different diseases causing malabsorption4 I.

Entities usually associated with a diffuse severe villous abnormality and crypt hyperplasia • Celiac sprue (CS) • Other protein allergies • Lymphocytic enterocolitis

II. Entities usually associated with a variable villous abnormality and crypt hypoplasia • Kwashiorkor, malnutrition • Megaloblastic anemia • Radiation and chemotherapeutic effect III. Entities usually associated with a nonspecific variable villous abnormality, usually not flat • Partially treated or clinically latent CS • Infection • Tropical sprue • Peptic duodenitis • Nonspecific duodenitis IV. Entities associated with variable villous abnormalities illustrating specific diagnostic changes • Collagenous sprue • Common variable immunodeficiency • Whipple disease • Intestinal lymphoma • Parasitic infestation • Waldenström macroglobulinemia • Lymphangiectasia • Enteropathy-associated T-cell lymphoma • Abetalipoproteinemia

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Table II: Differentiation between the 4 groups is illustrated in the following table. Category

Mean

Std. Deviation

32.58

17.86

Age

Duration of diarrhoea (days)

Platelets Count

Total Protein

IEL/100

49.42

15.60

III

252

40.38

15.12

38.75

12.8

213.5

151.8

123.75

123.9

III

132

111.69

117.41

97.00

90.53

9.75

3.07

9.32

2.98

III

252

10.71

2.69

10.87

3.09

2.1

0.90

1.68

0.49

III

252

2.24

1.06

2.50

0.716

7.09

0.69

6.26

1.303

III

252

7.20

0.74

6.98

1.13

32.84

8.98

7.57

4.15

III

252

10.59

5.72

16.43

13.59

‘p’ Value*

Significant difference from** II, III I

0.003

I III

0.026

0.716

0.794

0.523

II, III, IV 0.000

I I I

*ANOVA **Tukey HSD

Table III: Comparison between the clinical and lab investigation of celiac disease and non specific inflammation. Parameters

Celiac disease (n=57)

Non specific duodenitis/jejunitis/ileitis (n=212)

Age(yrs)

32.58

40.60

Male:female ratio

1: 1

1.28:1

Duration of diarrhea (days)

219.01

108.64

Clinical features

Abdominal pain Vomiting Weight loss/failure to gain weight Anemia Hypothyroidism

n =9(15.78%) n =4(7.01%) n =14(24.5%) n =24(42.1%) n=6(10.52%)

n =69(32.54%) n =12(5.66%) n=9(4.24%) n=84(39.6%) n =15(7.07%)

Investigation

Hemoglobin (gm/dL) Platelets(lacs) Total protein (g/dL)

9.75 2.11 7.03

12.42 2.29 7.21

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Table I V: Comparison between the endoscopic and histological features celiac disease and non specific inflammation. Parameters

Celiac disease (n=57)

Non-specific duodenitis/jejunitis/ileitis (n=212)

Endoscopic features

Normal folds Attenuated folds Scalloped folds Thickened folds Erythema/ulcers

11 (19.2%) 18(31.5%) 31(54.3%) 2(3.5%) 4(7.01%)

33(15.56%) 9(4.24%) 26(12.26%) 18(8.49%) 82(38.67%)

Histological features IELs/20

<6 >6

0 57 (average=32.84)

55 (25.94%) 157(74.05%)

Villous atrophy

Mild Moderate Severe

17(29.82%) 19(33.33%) 22(38.59%)

55(25.94%) 29(13.67%) 7(3.30%)

Crypt hyperplasia

normal hyperplasia

17(29.82%) 40(70.17%)

203(95.75%) 9(4.24%)

Fig. I: Etiology of Malabsorption.

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Fig. 2: Photomicrographs from a duodenal biopsy with intestinal lymphangiectasia showing distorted ,dilated villi filled with proteinaceous fluid. (Fig.2 H,andE x40).

Fig. 3: Photomicrographs from a duodenal biopsy with intestinal tuberculosis showing granuloma(Fig. 3 H, and E x 40).

Fig. 4: Photomicrographs from a duodenal biopsy with celiac disease show markedly increased IELs, with flattening of villi and crypt hyperplasia(Fig. 4 H, and E x 40).

Fig. 5: Photomicrographs from a duodenal biopsy showing increased intraepithelial neutrophils in peptic duodenitis(Fig. 5 H, and E x 100).

Discussion

In the present study, most common cause of malabsorption was nonspecific inflammation followed by celiac disease. Gastrointestinal tract infections are extremely common in a developing country India because of poor hygienic conditions , may be due to chronic gastric mucosal irritation (with drugs), we tend to give a diagnosis of mild nonspeciďŹ c duodenitis if we see even few lymphocytes or plasma cells in the lamina propria .

Although, our ďŹ ndings are also compatible with the observation of others who argued that celiac disease is common in developing countries where wheat is the major staple diet as our institution caters population of most of Rajasthan, parts of Gujarat and Harayana , where wheat is the staple diet.In most of our patients, the disease was diagnosed in adulthood. As indicated by other investigators ,most adults with celiac disease, may initially have a subclinical form of the disease and not exhibit classical symptoms of celiac

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disease such as diarrhea and weight loss and may only present with subtle manifestations such as an isolated iron deﬁciency anemia, which may delay referral to a gastroenterologist and therefore delay the diagnosis. However, it is surprising that speciﬁc infections such as giardiasis or tuberculosis were not more frequent. In contrast to our study, a study in Mumbai revealed celiac disease (26%) and intestinal tuberculosis (26%) were the two most common causes of Malabsorption syndrome.[5] One possible explanation for the low prevalence of ITB in this study may be due to low prevalence of malabsorption in this disorder and widespread use of anti tubercular antibiotics empirically at primary and secondary health care centres. [6] Similiarly, the low frequency of giardiasis in our series may be due to pretreatment with antibiotics before endoscopy but needs to be further investigated by conducting much larger studies to assess its true incidence and prevalence in our population. The low prevalence of tropical sprue in our study compared to other studies may be presence of endemic and epidemic forms of disease on South India in contrast to only endemic form of tropical sprue.[6 ,7] Crohn’s disease of small intestine can cause malabsorption due to extensive small bowel disease or resection. The emergence of crohn’s disease in india has been postulated to be due to improved sanitation. Crohn’s disease was reported in 9% patients in Lucknow study.[8,9] An interesting observation is the absence of IPSID as a cause of malabsorption in this study.As intestinal microbes have been postulated as trigger factors for IPSID, improved sanitation and nutrition during past decade may be responsible for absence of this disease or it may just be referral bias due to small number of patients.6 In conclusion, Celiac disease has emerged as a common cause of malabsorption in adults in Southern Rajasthan, where wheat is the major staple diet. We need to conduct much larger studies to assess the true incidence and prevalence of Celiac Disease in our population. In a country, where commercial gluten-free products are not easily available, it is a major challenge to maintain patients with Celiac Disease on a permanent gluten-free diet.

Consent

Written informed consent were obtained from all the patients for publication and any accompanying images.

Conflicts of Interest

The authors declare no conflict of interest.

References

1. Makharia G. Where are Indian adult celiacs? Trop Gastroenterol 2006;27:1–3. 2. Ramakrishna BS. Malabsorption syndrome in India. Indian J Gastroenterol 1996;15:135–41. 3. Yadav P, Das P, Mirdha BR, Gupta SD, Bhatnagar S, Pandey RM. Current spectrum of malabsorption syndrome in adults in India. Indian J Gastroenterol 2011; 30(1) : 22-28 4. Petras Robert, Gramlich Terry. Non neoplastic intestinal diseases.In : Stacey E Mills, Darryl Carter , Joel K Greenson, Victor E Reuter , Mark H Stoler et al. Sternberg’s Diagnostic Surgical Pathology. Fifth edition. Philadelphia, US :Lippincott Williams and Wilkin’s; 2009:1313-1368 5. Thakur B, Mishra P, Desai N, Thakur S, Alexander J, Sawant P. Profile of chronic small-bowel diarrhea in adults in Western India: a hospital-based study. Trop Gastroenterol 2006;27(2):84-6 6. Dutta AK, Balekuduru A, Chacko A. Spectrum of Malabsorption in India – Tropical Sprue is Still the Leader. J Assoc Physicians India 2011; 59 : 420-2 7. Ramakrishna BS. Malabsorption syndrome in India. Indian J Gastroenterol 1996 ;15:135-41. 8. Desai HG, Gupte PA. Increasing incidence of Crohn’s disease in India: is it related to improved sanitation? Indian J Gastroenterol 2005;24: 23-4. 9. Ghoshal UC, Mehrotra M, Kumar S. Spectrum of malabsorption syndrome among adults & factors differentiating celiac disease & tropical malabsorption,. Indian J Med Res 2012:136(4):687

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Original Article Nosocomial Burn Wound Infections due to Non-Fermenting Gram Negative Bacteria: Our Experiences From A Tertiary Care Center In North India

Naz Perween1, S. Krishna Prakash2, Prabhav Aggarwal2* and Lalit Mohan Gupta2 Dept. of Microbiology, Superspeciality Pediatric Hospital and Postgraduate Teaching Institute. NOIDA, India 2 Dept. of Microbiology, Maulana Azad Medical College, New Delhi, India

Keywords: Pseudomonas, Acinetobacter, Antimicrobial Resistance, Burns.

ABSTRACT Background: Nosocomial infections contribute to up to 50% mortality in burn patients. Non fermenting Gram negative bacteria, being ubiquitous in nature, can easily colonize the burn site and subsequently cause infections. The objective of the study was to understand the role of two of the most common non-fermenters, Pseudomonas aeruginosa and Acinetobacter baumanii in nosocomial burn wound infections and determine their antimicrobial resistance pattern. Methods: Wound swabs were collected from burn patients and cultured using standard microbiological techniques. Isolates of non-fermenters, Pseudomonas aeruginosa and Acinetobacter baumanii, were identified by conventional biochemical tests. The antibiotic susceptibility testing of these isolates were carried out by disc diffusion method. Result: A total of 248 and 70 clinical strains of Pseudomonas aeruginosa and Acinetobacter baumanii were isolated, respectively. Out of these, 67.62% and 72.05% isolates of Pseudomonas aeruginosa and Acinetobacter baumannii were MDR. Pseudomonas showed highest resistance to gentamicin (83.3%), followed by ceftazidime (80.18%), and Netilmicin (76.89%). Among Acinetobacter baumanii isolates, resistance to cephalexin (98.5%) was highest, followed by gentamicin (94.6%) and cefotaxime (94.12%). Conclusion: The increasing antibiotic resistance shown by these important pathogens leaves us with fewer option to treat severe life-threatening infections, stressing the need of a continuous antibiotic surveillance program and stringent implementation of infection control practices.

*Corresponding author: Dr Prabhav Aggarwal, E-49, Sector-55, NOIDA, Uttar Pradesh-201301, India Phone: +91 9899303626 Email: prabhavaggarwal@yahoo.co.in

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Burn Wound Infections Due to Non-fermenters

Introduction

Infections are an important cause of morbidity and mortality in patients with burns. Wound infections are one of the most common sites of nosocomial infections in burn patients with prevalence of about 60%, followed by blood stream infections (20%), urinary tract infections (20%) and pneumonia (10%). [1] Burn wound infections can lead to scarring, bacteremia, sepsis and multi-organ dysfunction, contributing to 75% mortality in burn patients. [2,3] The occurrence of nosocomial burn infections depends on several factors such as the burn severity, immune status, prolonged stay, invasive procedures and overcrowding leading to cross infections. [4] Disruption of skin and vasculature with resulting compromise in the immune status is the usual pathogenesis. [5,6] Hence the infected burn wound is a frequent source of sepsis. [3] Pseudomonas aeruginosa, Acinetobacter, Staphylococcus aureus, Klebsiella spp. and other Gram negative bacteria are commonly incriminated pathogens in burn patients. [1] Pseudomonas aeruginosa is known to frequently colonize the inanimate surfaces like floors, bed rails, sinks in the hospital and is transmitted by hands of health care workers. [7] Its resistance to commonly used disinfectants and antibiotics are the contributing factors. Hence, Pseudomonas infection is challenging to treat and is associated with a high mortality rate. [8] Acinetobacter baumanii is also rapidly emerging nosocomial pathogen in various countries including India, especially in ICUs, burn units and surgical wards. [4] Like Pseudomonas, Acinetobacter baumanii tends to exist in the hospital environment due to its ability to adhere on inanimate surfaces and is also inherently resistant to several antibiotics. Hence, this species is notorious for causing infections where there is a breach in natural immunity like in the burn wounds. [9] Burn units are often the sites of major and prolonged outbreaks with resistant organisms.10 Both Pseudomonas aeruginosa and Acinetobacter baumanii can be problematic due to their inherent resistance to several drug classes as well as the easy development of acquired resistance. [5] Multidurg resistant Pseudomonas aeruginosa (MDR Pa) in burn units is often associated with significant morbidity and mortality; mostly due to lack of treatment options.[7] Previous studies reveal the fact that the bacterial profile of burn wound infections changes over time in any given burn unit probably due to cross infections, change in antibiotic use, and overcrowding.[11] Hence, it is important to regularly monitor the bacterial species responsible for burn wound infections and their antibiotic susceptibility pattern, to aid

use of appropriate antibiotics both for empirical therapy and specific treatment. Thus, the present study was conducted to determine the role of non-fermenting Gram negative bacteria, namely Pseudomonas aeruginosa and Acinetobacter baumanii in the burn wound infection and assess their prevalent antibiotic susceptibility pattern.

Materials And Methods

The study included 1294 burn wound patients admitted in the burns unit of a 1600-bedded tertiary care hospital in New Delhi, India during the year 2012. Swabs and/or pus discharge were collected from the apparently infected site after decontaminating the wound. The swabs/pus were inoculated onto blood agar and MacConkey’s agar. In addition, pus specimen was inoculated in Brain Heart infusion broth and incubated for 24hrs at 37˚C; subsequent subculture was done on blood agar and MacConkey’s agar if primary plates failed to show bacterial growth. The plates were incubated at 37˚C for 24-48 hours in ambient air. The identification of Pseudomonas aeruginosa and Acinetobacter baumanii was done using the standard biochemical tests. The antibiotic susceptibility testing was carried out by the modified Stokes disc diffusion method against gentamicin, amikacin, ciprofloxacin, piperacillin/tazobactam, impenem, meropenem, netilmicin, polymixin B and colistin. Pseudomonas aeruginosa isolates were also tested against aztreonam, ceftazidime and tobramycin; and Acinetobacter baumanii was also tested against cephalexin, cefotaxime, ceftriaxone and amoxicillin (HiMedia Ltd., India). Pseudomonas aeruginosa isolates were identified as MDR Pa (Multi-drug resistant Pseudomonas aeruginosa), if the isolate was resistant to drug(s) in three or more antimicrobial classes; XDR Pa (Extensively drug resistant Pseudomonas aeruginosa) when resistant to all tested antibiotics except one or two classes; and PDR Pa (Pan drug resistant Pseudomonas aeruginosa) when resistant to all drug classes. [12] MDR Acinetobacter spp. was defined as the isolate resistant to at least three classes of antimicrobial agents: all penicillins and cephalosporins (including inhibitor combinations), fluroquinolones, and aminoglycosides; XDR (Extensively drug resistant) Acinetobacter spp. was defined as isolate that was resistant to the three classes of antimicrobials described above (MDR) and also to carbapenems; and PDR (Pan drug resistant) Acinetobacter spp. was the XDR Acinetobacter spp. that was also resistant to polymyxins and tigecycline.[13]

Result

Wound swabs/ pus specimens were collected from the infected wound sites of 1,294 patients and transported

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to the Microbiology laboratory immediately. The study population included 711 males and 583 females with male: female ratio of 1.22. The median age of these patients was 32 years (range=11â&#x20AC;&#x201C;76 years). On culture, 171 specimens showed no growth; while rest of the wound swabs had bacterial growth, including 160 samples with poly-microbial growth; yielding a total of 883 bacterial isolates. These included 248 (28.08%) isolates of Pseudomonas aeruginosa and 70 (7.92%) of Acinetobacter baumanii. Therefore, the non fermenting Gram negative bacteria constituted 36% (318/883) of the total isolates. Gram positive cocci constituted 15.96% (141/883) of the isolates, while 47.88% (424/883) were members of Family Enterobacteriaceae.

Pseudomonas isolates showed highest resistance towards gentamicin (83.33%) followed by ceftazidime, netilmicin (Table 1). Most of the Acinetobacter isolates were highly resistant to amoxicillin, cephalexin, ceftriaxone and cefotaxime, (100%, 98.51%, 90.91% and 94.12% respectively). Approximately, 50% and 16% of the isolates were resistant to imipenem and Meropenem, respectively (Table 2). We found that 67.62% Pseudomonas and 72.05% Acinetobacter isolates were multidrug resistant (MDR); while approximately 17% strains of both Pseudomonas and Acinetobacter isolates were extensively drug resistant (XDR). None of Pseudomonas or Acinetobacter strains were Pan drug resistant (PDR) (Table 3).

Table 1: Antimicrobial susceptibility pattern of Pseudomonas aeruginosa. Antimicrobial agent

Susceptible (%)

Intermediate susceptible (%)

Resistant (%)

Ceftazidime

16.13

3.69

80.18

Piperacillin + Tazobactam

65.82

18.99

15.19

Aztreonam

30.77

9.62

59.62

Imipenem

85.25

5.74

9.02

Meropenem

70.42

10.83

18.75

Ciprofloxacin

25.16

3.87

70.97

Gentamicin

16.22

0.44

83.33

Amikacin

8.97

66.03

Netilmicin

21.23

1.89

76.89

Tobramycin

17.84

4.87

77.29

Polymyxin B

93.78

1.91

4.31

Colistin

92.13

2.81

5.06

Susceptible (%)

Intermediate susceptible (%)

Resistant (%)

Amoxicillin

100

Cephalexin

1.49

98.51

Ceftriaxone

9.09

90.91

Cefotaxime

6.25

94.12

Table 2: Antibiotic susceptibility pattern of Acinetobacter baumanii. Antimicrobial agent

Piperacillin/tazobactam

37.5

27.5

Imipenem

37.14

14.29

48.57

Meropenem

47.37

36.84

15.79

Ciprofloxacin

17.14

5.71

77.14

Gentamicin

5.41

94.6

Amikacin

6.45

4.84

88.71

Netilmicin

32.35

9.52

61.76

84.85

15.15

Polymixin B Colistin

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Table 3: Patterns of drug resistance in isolates of Pseudomonas and Acinetobacter. Drug resistance

Pseudomonas aeruginosa (n= 244)

Acinetobacter baumanii (n=68)

MDR

165 (67.62)

49 (72.05)

XDR

41 (16.80)

12 (17.64)

PDR

0 (0)

Note: MDR: multidrug resistant; XDR: extensively drug resistant; PDR: Pan drug resistant Figures in parentheses indicate percentages.

Discussion

The prevalence of non-fermenting Gram negative bacteria in our study, namely Pseudomonas aeruginosa and Acinetobacter baumanii was found to be 28.8% and 7.92%, respectively. Few other Indian studies involving burns patients have shown wide isolation rates of Pseudomonas aeruginosa ranging from 18.2 to 59%, as well as that of Acinetobacter baumanii ranging from 7.2-28.6% (Table 4). Our result is comparable to the Tamil Nadu based study done in 2011 where the prevalence of Pseudomonas aeruginosa in burn wound infections was 28%;[16] however, Acinetobacter was not isolated in that study. Results of the previous studies from Varanasi, Mumbai and Delhi corroborate with our findings.[3,17,20] However, global studies report slightly higher isolation rate of Pseudomonas aeruginosa and Acinetobacter baumnaii as compared to those reported by Indian authors, with the

isolation rates ranging from 28-58.3% and 10.4-62.3% respectively (Table 5). In western countries, Pseudomonas aeruginosa is found to be the most prevalent cause of burn wound infection followed by Acinetobacter. In our study, Pseudomonas aeruginosa isolates showed high resistance to resistant to gentamicin (83.33%). Previous studies done in Turkey and Baghdad revealed the lower resistance to gentamicin (40% and 45% respectively).[5,22] However, in Iran 100% sensitivity to gentamicin was seen.[1] Resistance to amikacin in our study was again much higher than reported in other studies done elsewhere in India, where majority of the isolates were sensitive.[3,15] International studies too revealed a much lower resistance rate, except in the Baghdad based study where the amikacin resistance rate was 84.6%.[13] High resistance was also seen towards netilmicin, tobramycin and ciprofloxacin (76.89%, 77.29% and 70.97%, respectively). This is in contrast to the studies

TABLE 4: Indian scenario of the role of Pseudomonas and Acinetobacter in burn wound infection cases. Place of study

Author

Period of study

Pseudomonas spp. (%)

Acinetobacter spp.

Pune14

Bhatt P et al

2013-14

54.9

18.6

Bhat AS et al

2013

18.2

28.6

Karnataka TN

Mumbai3 Chandigarh Varanasi

Valarmathi S et al

2011

Srinivasan S et al

1994-2006

31.84

2002-05

51.5

14.23

Mehta M et al Anupurba S et al

2004-05

Chandigarh18

Agnihotri N et al

1997-2002

7.2

Delhi19

Sharma S et al

1992-1994

53.9

Delhi

Singh NP et al

1997-2002

Revathi G et al

1993-97

1.1

Delhi21

TABLE 5 International scenario of the role of Pseudomonas and Acinetobacter in burn wound infections cases. Place of study

Author

Period of study

Pseudomonas spp. (%)

Acinetobacter spp. (%)

Turkey

YolbaĹ&#x; I et al

2008-09

25.8

62.3

Keen EF et al

2003-08

20.04

22.2

Alkaabi SAG

2011-11

48.14

14.81

Ekrami A

2003-04

37.5

10.4

Oncul O et al

2004-05

Azimi L et al

2010

Hussien IA et al

2010-11

58.3

USA23 Baghdad Iran

Turkey10 Iran

Baghdad

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Perween et al. done in Iran and Baghdad, which showed 0% and 34.3% resistance against tobramycin.[1,5] The resistance rate for ciprofloxacin in our study was much higher as compared to a Karnataka based study where most of the isolates (95.8%) were sensitive.[15] Similarly, in Turkey and Baghdad, 22.829% resistance was seen.[5,22] However, as the resistance pattern differs among different institutions, another study in Baghdad revealed 84.6% of ciprofloxacin resistance.[2] In our study, resistance to ceftazidime was 80.18%, higher as compared to the studies done in abroad, where the resistance of 55-60.25% has been reported.[2,5] Resistance against rest of the antibiotics, imipenem, meropenem, piperacillin + tazobactam , polymyxin B and colistin was lower, though still comparable to most of the international studies.[1,7,10,22] The resistance to gentamicin in Acinetobacter baumanii isolates in our study was 94.6%, as against 55.6% and 96% in studies done abroad.[9,23] An Iranian study revealed 100% sensitivity of Acinetobacter to this drug. [1] Other drugs against which high resistance was shown by Acinetobacter were amikacin (88.71%), ciprofloxacin (77.14%), ceftriaxone (90.91%), cephalexin (98.51%), amoxicillin (100%), and netilmicin (61.76%) while the rest of the antibiotics were relatively more effective. Resistance to amikacin, ciprofloxacin and ceftriaxone was also high in other studies, similar to ours.[2,9,22] Amoxicillin resistance reported from Vietnam and Baghdad was also 100%, similar to our study.[2,9]

A-419 susceptibility pattern at regular intervals, and should also study the trend in the nosocomial spread of the pathogens. This will guide in the administration of empirical antibiotic therapy before the culture results are obtained. There should be a strict infection control policy in place with a stringent antibiotic stewardship program in order to ensure optimum treatment of the patient and to curb the menace of antibiotic resistant organisms. In addition, clear definitions of Multi drug resistance, Extensive drug resistance and Pan drug resistance should be available, to optimize inter-study comparisons.

Acknowledgements None

Funding None

Competing Interests None Declared

References

To conclude, burns units in every hospital should determine the bacterial profile of the burn wounds and the antibiotic

1. Ekrami A, Kalantar E. Bacterial infections in burn patients at a burn hospital in Iran. Indian J Med Res 2007;126:541-4. 2. Alkaabi SAG. Bacterial Isolates and Their Antibiograms of Burn Wound Infections in Burns Specialist Hospital in Baghdad. J Baghdad Sci. 2013;10(2):331-40. 3. Srinivasan S, Vartak AM, Patil A, Saldanha J. Bacteriology of the burn wound at the Bai Jarbai Wadia Hospital for children, Mumbai, India- A 13 year study, Part-I Bacteriological profile. Indian J Plast Surg. 2009:42(2):213-218. 4. Mehta M, Dutta P, Gupta V. Bacterial isolates form burn wound infections and their antibiograms: A eight year study. Indian J Plast Surg. 2007;40(1):25-8. 5. Hussien IA, Habib KA, Jassim KA. Bacterial Colonization of Burn Wounds. J Baghdad Sci. 2012:9(4);623-31. 6. Church D, Elsayed S, Reid O, Winston B, Lindsay R. Burn Wound Infections. Clin Microbiol Rev. 2006;19(2):403-34. 7. Japoni A, Anvarinejad M, Farshad S, Giammanco GM, Rafaatpour N, Alipour E. Antibiotic Susceptibility Patterns and Molecular Epidemiology of Metallo-βLactamase Producing Pseudomonas Aeruginosa Strains Isolated from Burn Patients. Iran Red Crescent Med J. 2014;16(5):1-6. 8. Branski LK, Al-Mousawi A, Rivero H, Jeschke MG, Sanford AP, Herndon DN. Emerging Infections in Burns. Surg Infect. 2009;10(5):389-97.

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Although there is no consensus regarding the definition of the terms ‘multidrug resistant’, ‘extensively drug resistant’ and ‘pan drug resistant’ used for these bacteria, we used the previously described and commonly used definitions to report the results of our study.[12.13] Approximately twothirds of the Pseudomonas strains and three-fourths of the Acinetobacter baumanii strains were resistant to three or more anti-microbial classes defined for each bacteria, making them MDR. Seventeen percent strains of both showed extensive drug resistance. Pan drug resistance was not seen in our study. A USA based study had reported MDR Pa as 15% and MDR Acinetobacter as high as 53%; while there was only 1 isolate each of Pan DR Pa and Pan DR Acinetobacter.[23] A Turkish study has previously shown 7.7% and 13.2% pan drug resistance in these isolates from burn wound infections.[10] These are much low as compared to our study; however, comparison with these studies may not be accurate due to lack of standard definitions for these terminologies; thus emphasizing the urgent need for clearcut definitions.

Conclusion

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Burn Wound Infections Due to Non-fermenters

9. Van TD, Dinh Q, Vu PD, Nguyen TV, Pham CV, Dao TT et al. Antibiotic susceptibility and molecular epidemiology of Acinetobacter calcoaceticusbaumannii complex strains isolated form a referral hospital in northern Vietnam. J Glob Antimicrob Resist. 2014;2(4):318-21. 10. Oncul O, Ulkur E, Acar A, Turhan V, Yeinz E, Karacaer Z, Yildiz F. Prospective analysis of nosocomial infections in a Burn Care Unit, Turkey. Indian J Med Res. 2009;130:758-64. 11. Liwimbi, OM, Komolafe IOO. Epidemiology and bacterial colonization of burn injuries in Blantyre. Malawi Med J. 2007;19(1):25-7. 12. Falagas ME, Karageorgopoulos DE. Pandrug Resistance (PDR), Extensive Drug Resistance (XDR), and Multidrug Resistance (MDR) among GramNegative Bacilli: Need for International Harmonization in Terminology. Clin Infect Dis. 2008;46(7):1121-2. 13. Manchanda V, Sanchaita S, Singh NP. Multidrug resistant Acinetobacter. J Global Infect Dis. 2010;2:291-304. 14. Bhatt P, Rathi KR, Hazra S, Sharma A, Shete V. Prevalence of multidrug resistant Pseudomonas aeruginosa infection in burn patients at a tertiary care centre. Indian J Burns 2015;23:56-9. 15. Bhat AS, Vinodkumar CS. Prevalence of burn wound infections with aerobic and anaerobic bacteria and their antimicrobial susceptibility pattern at tertiary care hospital. Int J Sci Res. 2013;2(11):410-3.

16. Valarmathi S, Pandian MR, Senthilkumar B. Incidence and screening of wound infection causing microorganisms. J Acad Indus Res. 2013;1(8):508-10. 17. Anupurba S, Bhattacharjee, A, Garg A, Sen MR. Antimicrobial susceptibility of Pseudomonas aeruginosa isolated from wound infections. Indian J Dermatol. 2006;51(4):286-8. 18. Agnihotri N, Gupta V, Joshi RM. Aerobic Bacterial Isolates from burn wound infections and their antibiograms- a five year study. Burns. 2004;30(3):241-3. 19. Sharma S, Hans C. Bacterial Infections in Burn patients: A three years study at RML Hospital, Delhi. J Commun Dis. 1996;28(2):101-6. 20. Singh NP, Goyal R, Manchanda V, Das S, Kaur I, Talwar V. Changing Trends in bacteriology of burns in burns unit, Delhi, India. Burns. 2003;29(2):129-32. 21. Revathi G, Puri J, Jain BK. Bacteriology of Burns. Burns. 1998;24(4):347-9. 22. Yolbaล I, Tekin R, Kelekรงi S, Selรงuk CT, Okur MH, Tan I, et al. Common pathogens isolated from burn wounds and their antibiotic resistance patterns. Dicle Med J. 2013;40(3):364-8. 23. Keen EF, Robinson BJ, Hospenthal DR, Aldous WK, Wolf SE, Chung KK, et al. Incidence and bacteriology of burn infections at a military burn center. Burns. 2010;36(6):461-8. 24. Azimi L, Motevallian A, Ebrahimzadeh Namvar A, Asghari B, Lari AR. Nosocomial Infections in Burned Patients in Motahari Hospital, Tehran, Iran. Dermatol. Res. Pract. 2011; doi: 10.1155/2011/436952.

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Original Article Clinicopathological Evaluation of Prostatic Adenocarcinoma: A Unicenter Study Clement Wilfred D1*, Rashmi Krishnappa1, Sharath Soman1, Vijaya Viswanath Mysorekar1, Radhika Kunnavil2 and Sharon Roshin Reginalt1 Department of Pathology, M. S. Ramaiah Medical College, Bangalore, India Department of Community medicine, M.S. Ramaiah Medical College, Bangalore, India 1

Keywords: Gleason Score; Prostatic Adenocarcinoma; Prostate Specific Antigen; Transurethral Resection of Prostate.

ABSTRACT Background: Prostate cancer is a significant cause of morbidity and mortality. It ranks fifth in cancer incidence and fourth in cancer mortality in India. As the literature on the issue in India is limited, we undertook the study with the objective of evaluating the histopathological features of prostatic adenocarcinoma (PCa) and correlating these with certain clinicopathological variables. Methods: All the cases of PCa diagnosed on transurethral resection (TURP) specimens and core needle biopsies, over a period of three years (between January 2013 and January 2016), were evaluated. The clinicopathological data obtained was subjected to statistical analysis to discern correlations. Results: The study included 55 cases of PCa comprised of 29.1% of moderately differentiated, 18.2% of moderate to poorly differentiated and 52.7% of poorly differentiated cases. The mean patient age was 69 years with mean preoperative serum PSA level of 162.9 ng/ml. The three commonest clinical presenting symptoms were increased frequency of micturation (45.5%), incomplete voiding (40%) and dysuria (38.2%). Gleason score 8 was the most frequent [15(27.3%)] followed by Gleason score 9 [13(23.6%)]. The average tumour volume in TURP specimens and needle biopsies was 52.5 % and 58.1% respectively. Conclusions: A positive correlation was found between high Gleason score and increased PSA levels and tumour volume. Majority of our patients had poorly differentiated PCa with high PSA levels suggesting that the disease is advanced at the time of diagnosis.

*Corresponding author: Dr Clement Wilfred D, Associate professor, Department of Pathology, M.S. Ramaiah Medical College, MSR Nagar, MSRIT Post, Mathikere-560054. Phone: +91 9945226314 Email: clement.wilfred@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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Prostatic Adenocarcinoma

Introduction

Prostate cancer is a common malignancy and has become a major health problem in industrialized countries.[1] Globally it is the second most frequent cancer in men and fifth most common cancer overall.[2] An increasing trend in the incidence of prostate cancer has been revealed by several Indian registries.[2] It ranks fifth in cancer incidence and fourth in cancer mortality in India.[3] As the literature on malignant prostatic lesions in India is limited, and as the magnitude of the problem is significant, we conducted the present study with the objective of evaluating the various histopathological features of prostatic adenocarcinoma. Further we correlated the histopathological findings with significant clinicopathologic variables like age, serum PSA (Prostate specific antigen) levels, Gleason score and volume of tumour in the specimen.

Material and Methods

This was a single centre prospective study of all the cases of prostatic adenocarcinoma (PCa) diagnosed on transurethral resection (TURP) specimens and core needle biopsies, conducted in the department of Pathology, M.S Ramaiah Medical College and Hospitals, Bangalore over a period of three years (between January 2013 and January 2016). The formalin fixed prostate specimens were processed as per standard protocol and 4- 5 µm paraffin sections were obtained that were stained with Haematoxylin and Eosin in the standard manner. The sections were viewed in detail and presence of perineural invasion, tumour volume [proportion (%) of prostatic tissue involved by tumour] and Gleason score were determined for each case. Gleason score was determined using the modified Gleason system. [4,5,6] Primary grade ( most common) and secondary grade (second most common) patterns were assigned to each case and Gleason score was obtained by summing up the grades. In needle biopsies, when different cores exhibited different Gleason grades, the overall worst Gleason score was given. [5,6] Posthormonal therapy and postradiotherapy cases were excluded. Clinical details and relevant investigations including age, pre-operative serum PSA levels were obtained from the records. Statistical Analysis: Continuous data was summarised using descriptive statistics. Qualitative variables were summarized using frequency and percentage. One way ANOVA and Kruskal-Wallis followed by post hoc test was employed to test the differences in the mean/median values of variables like PSA, tumour volume and age. Fisher’s exact test was used to compare the tumour differentiation across different age groups. Spearman’s correlation was used to find the correlation between Gleason score and other parameters. A level of p < 0.05 was considered as statistically significant.

Results

A total of 55 cases of prostatic acinar adenocarcinomas (PCa) from 55 previously untreated patients were diagnosed on prostate specimens comprising of 33 TURP specimens and 22 needle biopsies over a duration of 3 years. Prostatectomy specimens were not received during this period. The mean patient age was 69± 8.9 years (age range: 52 to 90 years) and the median age was 68 years, with most of the cases occurring in the 6th decade (43.6%) followed by 7th decade (32.7%) [Table 1]. The indications for prostatic tissue sampling were elevated PSA (53%, 29/55), abnormal digital rectal examination (DRE) (20%, 11/55) and abnormal DRE + elevated PSA (27%, 15/55). The clinical presenting symptoms were increased frequency of micturation (45.5%, 25/55), incomplete voiding (40%, 22/55), dysuria (38.2%, 21/55), nocturia (12.7%, 7/55), hematuria (9.1%, 5/55) and acute urine retention (9.1%, 5/55). Of the 55 cases of PCa, 16 (29.1%) were moderately differentiated (Gleason score 5- 6) (Fig 1), 10 (18.2%) were moderate to poorly differentiated (Gleason score 7) (Fig 2) and 29 (52.7%) were poorly differentiated (Gleason score 8- 10) (Fig 3). There was no statistically significant correlation between the degree of tumour differentiation and age (p= 0.128). The commonest Gleason score was score 8 (27.3%) followed by score 9 (23.6%) [Table 2]. Gleason score 7 was present in 18.2% (10/55) of the cases of which 70% (7/10) had Gleason grade 4+3 and 30% (3/10) had Gleason grade 3+ 4. Gleason pattern 4 was the most frequent primary pattern, occurring in 54.5% (30/55) of the cases. The most common secondary pattern was also Gleason pattern 4 (43.6%; 24/55) [Table 3]. Tertiary pattern was not identified. A higher Gleason score was associated with increased PSA levels (p= 0.000) and tumour volume (p= 0.003). No correlation was found between age and Gleason score (p= 0.181) [Table 4]. 37.5% (6/16) of moderately differentiated PCa, 40% (4/10) of moderately to poorly differentiated PCa and 51.7% (15/29) of poorly differentiated PCa exhibited perineural invasion. Overall, 45.5 % (25/55) of the cases showed perineural invasion. High grade PIN was present in 7.3% (4/55) of the cases. The average volume of TURP specimen was 13.2 ± 9.4 (range: 2 to 40 ml). The average tumour volume in TURP specimen was 52.5± 26.4 % (range: 10 to 95%). The average number of cores in needle biopsies was 7.0 ± 3.2 (range: 2 to 12 cores) and the average tumour volume was 58.1 ± 19.7 with the average number of cores involved being 4.6 ± 2.77. The overall average tumour volume (TURP + needle biopsies) was 54.7± 23.9 %.

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Wilfred D et al.

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Table 1: Age incidence and distribution of prostatic adenocarcinoma. Moderately Age group No. of cases (%) differentiated tumour 50- 59 6 (10.9%) 5 60-69 24 (43.6%) 5 70-79 18(32.7%) 4 80- 89 6(10.9%) 2 90- 99 1(1.8%) 55 16 (29.1%)

Moderate to poorly differentiated 7 3 10 (18.2%)

Poorly differentiated tumour 1 12 11 4 1 29 (52.7%)

Table 2: Distribution of Gleason score, age, tumour differentiation, tumour volume and PSA levels. Tumour Average tumour Gleason score No. of cases (%) Age (mean ± SD) differentiation volume (%) ± SD 5 7(12.7%) 67.4 ± 10.2 Moderate 21.4± 10.3 6 9(16.4%) 66.9 ± 10.4 Moderate 55.6± 15.3 7 10(18.2%) 67.5 ± 5 Moderate to Poor 60.2± 21,4 8 15(27.3%) 74 ± 8.7 Poor 62.7± 24.9 9 13(23.6%) 69.2 ± 8.5 Poor 63.6± 21.8 10 1(1.8%) 80.0 Poor 60

Mean PSA (ng/ml) ± SD 41.3± 9.9 65.2± 23.9 77.4± 33.6 178.5± 165.5 329.3± 242.8 310

PSA, Prostate specific antigen.

Table 3: Gleason Pattern. Gleason pattern 1 2 3 4 5

Primary pattern (N= 55) No. of cases (%) 0 7 (12.7) 12(21.8) 30(54.5) 6(10.9)

Secondary pattern (N= 55) No. of cases (%) 0 0 22 (40) 24(43.6) 9(16.3)

Table 4: Comparison of Gleason score with age, PSA levels and tumour volume. Gleason score vs. PSA Gleason score vs. Age level Spearman’s correlation 0.183 0.703 coefficient p value 0.181 (not significant) 0.000 (significant)

Gleason score vs. Tumour volume 0.392 0.003 (significant)

PSA, Prostate specific antigen

Fig. 1: Prostatic adenocarcinoma, moderately differentiated Gleason score 3+3= 6/10. (a) Pattern 3- single separate very small, small and medium sized glands of variable shape and size with elongated, angular and twisted forms. (b) Pattern 3 with ill-defined infiltrating edges. (H&E, 100X).

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Prostatic Adenocarcinoma

Fig. 2: Prostatic adenocarcinoma, moderate to poorly differentiated Gleason score 3+4= 7/10. (a) Primary pattern 3- single separate small to medium sized glands of variable shape and size. (b) Secondary pattern 4- fused microacinar formations, cords and diffusely permeative tumour cells, many with cleared cytoplasm (hypernephroid pattern). (H&E , 200X).

Fig. 3: Prostatic adenocarcinoma, poorly differentiated Gleason score 4+5= 9/10. (a) Primary pattern 4- cords and fused Illdefined glands with poorly formed glandular lumina . (b) Secondary pattern 5- Expansile solid masses of tumour cells with central necrosis (arrow) (comedocarcinoma) (H&E, 100X).

The mean preoperative serum PSA was 162.9 ng/ml (range: 22 to 800) and median PSA was 85 ng/ml. PSA levels increased with the average tumour volume (p< 0.0001).

Discussion

Prostatic adenocarcinoma occurs predominantly in older men. In the present study the mean age was 69 years which is similar to that reported by Jackson et al (68.5 years) and Shirish et al (66.07 years). [7, 8] Majority of our cases (43.6%) occurred in the 6th decade which is similar to that reported by Shirish et al (37%).[7] In studies conducted by

Jackson et al and Anushree et al peak incidence of PCa was seen in 7th decade.[3, 7] A south Indian study conducted by Anushree et al revealed that increased frequency of micturation (38.5%), dysuria (38.5%) and incomplete voiding (38.5%) were the three most common clinical presenting symptom of PCa.[3] Similarly, in another south Indian study by Atchyuta et al, increased frequency (26.7%), dysuria (26.7%) and incomplete voiding (26.7%) were the commonest present symptoms.[8] Our findings are in synchrony with the above studies.

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Wilfred D et al. Poorly differentiated PCa comprised the largest group (52.7%) in the present series followed by moderately differentiated PCa (29.1%) and moderate to poorly differentiated PCa (18.2%). A west Jamaican retrospective study on 191 PCa cases found that moderately differentiated PCa was the largest group (35.29%) followed by moderate to poorly differentiated PCa (34.39%).[7] A south Indian study on 17 PCa cases revealed that moderate to poorly differentiated PCa (52.94%) was most frequent followed by poorly differentiated PCa (29.4%).[9] Study conducted in Pakistan on 190 PCa revealed that poorly differentiated PCa (Gleason score 8- 10) (52.7%) was the commonest followed by moderate to poorly differentiated PCa ( Gleason score 7) ( 33.1%) and moderately differentiated PCa ( Gleason score 5- 6) (17%).[10] The higher frequency of poorly differentiated PCa in our study is most likely a reflection of the patient’s late presentation at the time of diagnosis. Differences in the geographic area, demography and race may possibly have a role in such differences between our study and other studies. Further for needle biopsies, there are several sources of grading discrepancies, including observer variability, pathologist expertise, tissue distortion and sampling error.[11] Similar to our study none of the other studies quoted above encountered well differentiated PCa.[7,9,10] The possible explanation is that Gleason patterns 1 and 2 are almost never diagnosed on needle biopsy specimens as the calibre of needle cores does not enable the edges of nodules to be seen.[4,5,6,9] These tumours are small, tend to be located anteriorly in the prostate and are usually clinically asymptomatic and thus may not be sampled.[3,9] Well differentiated PCa are rare, exhibit poor diagnostic reproducibility among experts and are often an incidental finding in prostatectomies and TURP specimens performed due to a clinical diagnosis of benign prostatic hyperplasia. [9,11] In a clinicopathological study conducted in Saudi Arabia, which included prostatectomies, well differentiated PCa was found in only 3.8% of the 94 PCa cases and in another study conducted in Sultanate of Oman only 0.08% of the PCa cases were well differentiated with Gleason score between 2 and 4.[12,13] Gleason grading of PCa is the single most important predictor of biological behaviour and one of the most significant factors determining the therapy of PCa.[14]. Cases with Gleason score 6 or lower are candidates of active surveillance (“watchful waiting therapy”), cases with Gleason score 8- 10 are candidates for radiation therapy or adjuvant therapy and cases with Gleason score 7 usually require some form of definitive therapy.[6] Since its inception by Donald Gleason in 1966, it has remained the cornerstone in the diagnosis and management of PCa and has been www.pacificejournals.com/apalm

A-425 endorsed by the World Health Organisation.[6] It is solely based on architectural pattern with all tumours falling into a 5 grade system representing a continuum of progressively complex morphologies.[6,13] Studies have consistently shown that there is a positive correlation of Gleason score to tumour volume, more extensive tumours, positive surgical margins and advanced pathologic stage.[7,10,14] Our experience also is similar in that a higher Gleason score was associated with increased tumour volume (p= 0.003). PSA is a 33-kd single chain glycoprotein that is a highly sensitive serum biomarker of PCa.[15] Despite debate on the specificity, positive predictive value and utility of PSA in population screening, it still continues to play an indispensible role in the diagnosis and management of PCa.[16] Availability of PSA levels and prostate biopsy has markedly increased the diagnosis of PCa.[7] Further it has a role in determining the long term risk of a particular PCa and monitoring of patients following hormonal/definitive therapy.[16] A study on 67 Nigerian African men with PCa revealed a positive correlation between serum PSA and Gleason grade and score (Spearman’s correlation coefficient= 0.40, p= 0.001).[17] Jackson et al, in their study, found that mean PSA of PCa cases with Gleason score of 6 was 50.11ng/ml compared with 70.8 ng/ml, 136.5 ng/ml and 140.5 respectively in Gleason scores 7, 8 and 9.[7] They concluded that PSA levels increases with Gleason score. Another study conducted on 200 PCa patients, in Brazil, revealed positive correlation of high Gleason scores to higher preoperative PSA .[14] These studies are in synchrony with our study, where higher Gleason score was associated with higher PSA levels ( Spearman’s correlation coefficient= 0.703, p= 0.000). Poorly differentiated PCa tend to be larger and of more advanced stage; thus even though they produce less PSA per cell as compared to well differentiated PCa, they are associated with increased PSA levels.[7,14] Similar to our study (p< 0.0001) other studies have shown that serum PSA levels correlate with tumour volume.[7,14] Various studies have quoted different incidence of perineural invasion, ranging from 7% to 47%.[7, 8, 9] In the present study the incidence of perineural invasion was 45.6%. In 7.3% of the our PCa cases, the adjacent prostatic tissue showed high grade PIN which is slightly higher than a west Jamaican study(Jackson et al) (4.7%) and significantly lower than another south Indian study (Anushree et al) (50%). [3, 7]

Conclusion

In the present study we found positive correlation between PSA levels, tumour volume and Gleason score. Majority of eISSN: 2349-6983; pISSN: 2394-6466

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Prostatic Adenocarcinoma

our patients had poorly differentiated PCa with high PSA levels suggesting that the disease is advanced at the time of diagnosis. Serum PSA screening in middle and old age group of men, with skilful clinical examination and prompt prostate biopsy is required to diagnose PCa early enough for a favourable prognosis.

Acknowledgement Nil

Funding Nil

Competing Interests

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Nil

References

1. Paker SK, Kilicarslan B, Cyftcyoglu AM, et al. Relationship Between Apoptosis Regulator Proteins ( bcl-2 and p53) and Gleason Score in Prostate Cancer. Pathology Oncology Research 2001;7:209-212. 2. Lalitha K, Suman G, Pruthvish S, Mathew A, Murthy NS. Estimation of Time Trends of Incidence of Prostate Cancer – an Indian Scenario. Asian Pacific J Cancer Prev 2012;13: 6245-6250. 3. Anushree C.N, Venkatesh K. Morphological Spectrum of Prostatic Lesions- A Clinicopathological Study. Medica innovatica.2012;1:49-54. 4. Epstein JI, Allsbrook WC Jr, Amin MB, Egevad LL; ISUP Grading Committee. The 2005 International Society of Urological Pathology (ISUP) Consensus Conference on Gleason Grading of Prostatic Carcinoma. Am J Surg Pathol. 2005;29:1228-42. 5. Epstein JI. An Update of the Gleason Grading System. J Urol. 2010;183:433-40. 6. Shah RB. Current Perspectives on the Gleason grading of prostate Cancer. Arch Pathol Lab Med.2009;133:1810-16. 7. Jackson LA, Mc Growder DA, Lindo RA. Prostate Specific Antigen and Gleason Score in men with Prostate Cancer at a private Diagnostic Radiology

11. 12.

13. 14.

15. 16. 17.

Centre in Western Jamaica. Asian Pacific J cancer Prev. 2012;13:1453-56. Atchyuta. M, Krishna R, Latha PP, Renuka IV, Tejaswini V, Vahini G. Histological Spectrum of Prostatic Adenocarcinomas in Correlation with PSA Values. Indian Journal of Pathology and Oncology. 2016;3:1-6. Shirish C, Jadhav PS, Anwekar SC, Kumar H, Buch AC, Chaudhari US. Clinico-pathological study of benign and malignant lesions of prostate. Int J Pharm Bio Sci.2013;3:162-78. Arshad H, Ahmad Z. Overview of Benign and Malignant Prostatic Disease in Pakistani Patients : A Clinical and Histopathological Perspective. Asian Pacific J Cancer Prev.2013;14:3005-10. Humphrey PA. Gleason grading and prognostic factors in carcinoma of the prostate. Modern Pathology.2004;17:292-306. Mosli HA, Abdel-Meguid TA, Al-Maghrabi JA, Kamal WK, Saadah HA, Farsi HM. The clinicopathologic patterns of prostatic diseases and prostate cancer in Saudi patients. Saudi Med J.2009;3:1049-53. George E, Thomas S. A Histopathologic Survey of Prostate Disease in the Sultanate of Oman. The Internet Journal of Pathology; 2009;3. Guimaraes MS, Quintal MM, Meirelles LR, Magna LA, Ferreira U, Billis A. Gleason Score as Predictor of Clinicopathologic Findings and Biochemical (PAS) Progression following Radical Prostatectomy. Int braz J Urol.2008;34:23-9. Altuwaijri S. Role of Prostate Specific Antigen (PSA) in Pathogenesis of Prostate Cancer. Journal of Cancer Therapy.2012;3:331-36. Huang JG, Campbell N, Goldenberg SL. PSA and beyond: Biomarkers in prostate cancer. BC Medical Journal.2014;56:334- 41. Okolo CA, Akinosun OM, Shittu OB, et al. Correlation of Serum PSA and Gleason Score in Nigerian Men with Prostate Cancer. African Journal of Urology.2008;14:15-22.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 05, (Suppl) Nov. 2016

Original Article Role of Bone Marrow Profile in Cytopenias Neelima Bahal, Brijesh Thakur, Aparna Bhardwaj*, Sandip Kudesia, Seema Acharya and Sanjeev Kishore Department of Pathology, SGRR Institute of Medical & Health Sciences, Patel Nagar, Dehradun, India Keywords: BMA, BMB, Pancytopenia, Bicytopenia

ABSTRACT Background: Cytopenia occurs usually due to some underlying haematological medical disorder. There is a long list of causes for pancytopenia and bicytopenia. Bone marrow examination is an effective way of evaluating various causes of cytopenia. Method: Relevant clinical history and examination findings along with routine hematological, serological and biochemical investigations were recorded in all included 129 cases. Bone marrow aspiration smears and biopsy slides were studied. Special stains like Reticulin, PAS, Perlâ&#x20AC;&#x2122;s stain were used when required. Results: Megaloblastic anemia was the commonest cause of pancytopenia as well as bicytopenia. Other significant causes were leukemia, lymphoproliferative disorders, hypoplastic marrow and metastatic lesions. Conclusion: Both bone marrow aspiration and biopsy are complementary to each other to give the correct diagnosis.

*Corresponding author: Dr. Aparna Bhardwaj, Professor, Department of Pathology, SGRR Institute of Medical & Health Sciences, Patel Nagar, Dehradun, India- 248001. Phone: +91 9411718270, 11 352760439 Email: aparnapande1977@gmail.com

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Introduction

Cytopenia occurs due to failure of normal hematopoiesis. It is a presentation of some underlying medical disorder most of which are primarily haematological.[1] Pancytopenia is defined as the decrease in the number of all the three formed elements of the blood below their normal reference values while bicytopenia is a disorder in which any two cell lineages are suppressed.[2] There is a considerable overlap between the causes of pancytopenia and bicytopenia in India as well as other countries, which may be attributable to geographical factors, socioeconomic status, diagnostic criteria, genetic causes and various cultural taboos. Bone marrow examination involving the study of bone marrow aspirates, imprint smears and trephine biopsy is an effective way of diagnosing and evaluating hematologic and metastatic neoplasm as well as non hematological disorders responsible for cytopenia. These three procedures are complementary to each other and superiority of one method over the other depends on the specific disease process.[3] The current study is intended to evaluate the various causes of bicytopenia and pancytopenia in adult and pediatric patients as well as their clinico-hematological correlations in a tertiary care centre of Uttarakhand.

Materials and Methods

This study was carried out in haematology section of the department of Pathology and included all cases diagnosed as bicytopenia or pancytopenia by routine hematological investigations. The inclusion criteria (all three or any two) used for selection of cases were1. Hb < 10gm/dl 2. TLC < 4000 cells/µl 3. Platelet count < 1.0 lac/µl Relevant clinical history and examination findings along with routine hematological, serological and biochemical investigations were recorded. Bone marrow aspiration was done in all included 129 cases using Salah needle from posterior superior iliac spine. All the aspirate smears were stained with May-Grunwald-Giemsa stain (MGG) and special stains like Myeloperoxidase (MPO), Periodicacid Schiff (PAS), Perl’s Prussian blue stain were done as required. Bone marrow trephine biopsy was done in 90 cases from posterior superior iliac spine by using Jamshidi needle. Core biopsy was then used to make imprint smears, before placing it in Bouin’s fluid for fixation. Routine histopathological processing was done after decalcification in 5% nitric acid for 24 hours. Trephine biopsy sections were stained with haematoxylin and eosin (H&E) and

special stains like Reticulin, PAS, Perl’s stain were used when required.

Results

A total of 129 cases of cytopenia (bicytopenia and pancytopenia) were included in the present study. The age of the patients ranged from 7 months to 93 years with maximum number of cases observed in the 2nd and 3rd decade. There were just 13 cases (10.07%) presenting at the age of more than 60 years. Males accounted for 73 (56.59%) cases, females for 56 (43.41%) cases and the male to female ratio was 1.3:1. (Table 1) Most of the cases presented with a combination of two to three or more clinical features. As per the frequency, pallor was seen in 82.0% cases followed by fever in 50.38% cases, splenomegaly in 24.8% cases, weakness in 23.25% cases and hepatomegaly in 16.6% cases (fig. 1). Hemoglobin values in the present study varied from 2.2 gm/dl to 12.7 gm/dl. Majority of cases (87 cases, 67.43%) had hemoglobin values less than 8 gm/dl with 33.33% (43 cases) having hemoglobin less than 6 gm/dl. The lowest hemoglobin was 2.2 gm/dl seen in a case of acute leukemia. Total leucocyte count ranged from 400 to 95000 cells/ µl and 60.46% (78 cases) had leucocyte count between 1000 and 4000/µl. Lowest count of 400/µl and highest count of 95000/µl were noted in case of acute leukemia. Platelet count ranged from 5000 to 2.2 lac/µl. The lowest platelet count of 5000/µl was seen in three cases of hypoplastic marrow. Anemia was seen 96.12% (124) cases followed by thrombocytopenia in 83.73% (108) cases and leucopenia in 69.76% (90) cases. Pancytopenia was noted in 60 (46.51%) cases and bicytopenia in 69 (53.49%) cases of cytopenia. Thrombocytopenia and anemia were the commonest form of bicytopenia seen in 40 cases (57.97%) followed by anemia and leucopenia in 25 cases (36.26%) and thrombocytopenia and leucopenia only in 4 cases (05.79%). Megaloblastic anemia was the commonest cause of pancytopenia seen in 46.66% (28) cases followed by leukemia in 20.00% (12) cases and hypoplastic marrow in 18.33% (11) cases in the present study. Megaloblastic anemia and leukemia were also the most common causes of bicytopenia seen in 28.98% (20) and 23.18% (16) cases respectively. Myelofibrosis, multiple myeloma, lymphoproliferative disorders, infections and metastasis were among the occasional and isolated causes of

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pancytopenia and bicytopenia. Among the cases of megaloblastic anemia, pancytopenia was seen in 58.33% cases and bicytopenia in 41.67 % cases and among the cases of leukemia, pancytopenia was seen in 42.85% cases and bicytopenia in 57.15% cases. (Table 2)

Discussion

This incidence of cytopenia was 55.6% (129/232) during the study peroid. This correlated with the studies done by Naseem et al. and Bushra et al. from Pakistan, where the incidence of cytopenia was observed to be 53.3% and

Table I: Distribution of the cases of cytopenia according to age and sex (n=129).

Pancytopenia

Age

Bicytopenia

Total (129)

Male

Female

Male

Female

0 - 10

11 - 20

21 - 30

31 - 40

41 - 50

51 - 60

61 - 70

71 - 80

81 - 90

91 - 100

Table II: Distribution of cases of cytopenia according to the causes (n = 129) diagnosed on bone marrow aspiration and/or biopsy.

Pancytopenia

Causes

Bicytopenia

BMA

BMB

BMA

BMB

Megaloblastic anemia

Leukemia

Normoblastic

Hypoplastic marrow

Multiple myeloma

Lymphoproliferative disorders

Megakaryocytic thrombocytopenia with nutritional anemia

Myelofibrosis

Metastasis

Others (Leishmaniasis & Haemophagocytosis)

No opinion possible

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Role of Bone Marrow Profile...

Fig. 1: Bar diagram showing distribution of cases of cytopenias according to clinical features.

Fig. 2 a: Bone marrow aspiration smears showing megaloblasts with sieve like chromatin. (MGG 1000x) b)Bone marrow biopsy showing megaloblastic erythroid hyperplasia. (H&E 400x) c) Bone marrow aspiration smears comprising large no. of blasts with inset showing myeloperoxidase positivity in blast. (MGG 1000x) d) Bone marrow biopsy showing blasts. (H&E 1000x)

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Fig. 3 a: Bone marrow aspiration smears showing sheets of tumor cells exhibiting an ill defined glandular pattern. (MGG 400x) b) Bone marrow biopsy showing infiltration of marrow by the tumor cells forming glands & tubules. (H&E 400x) c) Bone marrow aspiration smears showing Leishman Donovan bodies within macrophage. (MGG 1000x) d) Bone marrow aspiration smears showing numerous plasma cells. (MGG 400x) e) Bone marrow biopsy showing a cluster of myeloma cells with eccentric nucleus & prominent nucleoli. (H&E 1000x).

57.7% respectively.[1,4] However, studies conducted in India show a lower incidence of cytopenia varying from 20.0% to 37.6%. [3,5,6,7] The higher incidence of cytopenia in the study can be explained by the fact that the cases included were mainly referred cases from remote hilly areas, the study center being a tertiary care hospital. Moreover, the cases reached the centre after a considerable time gap, possibly due to lack of awareness about the urgency of the disease entities. Similar to our study, clinical features have been reported by other authors also, although the frequency varies. In a recent study by Javalgi et al. generalized weakness was the most common symptom seen in 92% cases followed by pallor in 83% cases, splenomegaly in 17% cases and lymphadenopathy in 6% cases.[8] In another report by Hasan et al. from Pakistan, pallor was seen in 82% cases, hepatomegaly in 44.8%, splenomegaly in 37.2% and lymphadenopathy in 22.2% cases.[9] A study by Bhatnagar et al. from India reported incidence of pancytopenia seen in 54.5% cases and bicytopenia seen in 45.5% cases which is almost similar to our study.[10] However, the observations of Naseem et al. from Pakistan in their study were very different from the results of present study with pancytopenia seen in 40% cases and bicytopenia www.pacificejournals.com/apalm

seen in 17% cases.[1] This difference in the incidence of cytopenia can be attributed to difference in methodology, selection of diagnostic criteria, socio-economic status and nutritional status of cases included in the study. There are very few studies in the literature which have considered pancytopenia and bicytopenia as different entities. Most of the studies have been done for pancytopenia. In this study pancytopenia and bicytopenia were separately evaluated. The commonest cause of pancytopenia reported in various studies throughout the world is aplastic anemia.[11,12,13,14] However, we observed megaloblastic anemia as the commonest cause of pancytopenia followed by leukemia and hypoplastic marrow in the study. Some studies conducted in India have also reported megaloblastic anemia to be the major cause of pancytopenia.[15] The incidence of megaloblastic anemia as a cause of pancytopenia has been reported to be 44% and 40.3% in reports by Khodke et al. and Verma et al. which is correlating with the findings of this study.[4,6] In the present study, megaloblastic anemia was the commonest cause of bicytopenia followed by leukemia. Only 8.69% cases of hypoplastic marrow were seen to present as bicytopenia. Neoplastic conditions, as the most common cause of pancytopenia, are seen more commonly in western eISSN: 2349-6983; pISSN: 2394-6466

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countries. Higher incidence of megaloblastic anemia in Indian subcontinent can be attributed to low socioeconomic status, poor hygiene, inadequate nutrition and some cultural taboos. A high frequency of aplastic anemia can be related to environmental factors such as exposure to toxic chemicals rather than genetic factors. Farming being a major occupation in Indian subcontinent leading to increased exposure to pesticides also contributes to aplastic anemia. Easy availaibility of over the counter medicines can also lead to increased risk of aplastic anemia. All these risk factors are not seen in the western countries making neoplastic conditions as a common cause of pancytopenia rather than megaloblastic anemia or aplastic anemia. The higher incidence of leukemia and other marrow infiltrative disorders in this study can be attributed to the inclusion of referred and high risk cases in the study as the study center is a tertiary care hospital. The interpretation of this study is that megaloblastic anemia, leukemia and hypoplastic marrow can present either as pancytopenia or bicytopenia. However, it was observed that megaloblastic anemia and hypoplastic marrow present more commonly as pancytopenia while leukemia usually presents as bicytopenia. More frequent association of leukemia with bicytopenia was also observed by Nassem et al. in their study from Pakistan where bicytopenia was associated with malignant conditions in 69.5% cases as compared to 26.6% cases of pancytopenia, which is relatively higher as compared to findings in our study.[1] This signifies that although pancytopenia is taken with greater clinical concern than bicytopenia, timely evaluation of bicytopenia is also very important. Another Indian study by Bhatnagar et al. observed similar results regarding the frequent association of leukemia with bicytopenia.[10] Gupta et al. in their study have reported that bone marrow aspiration and trephine biopsy are complementary to each other to arrive at a definitive diagnosis.[2] This was observed in our study also and was evident in the following cases: A case of Plasmodium falciparum infection presenting as pancytopenia was diagnosed by peripheral blood film examination. It was not evident on the bone marrow examination, signifying the complementary role of peripheral blood film examination to arrive at a diagnosis. Similarly a case diagnosed as metastasis on trephine biopsy was later found to be case of leukemia on immunohistochemistry, which correlated with the aspiration findings. Thus both bone marrow aspiration and biopsy are complementary to each other to give the correct diagnosis.

References

1. Naseem S, Varma N, Das R, Ahluwalia J, Sachdev Marwah R. Pediatric patients with bicytopenia/ pancytopenia: Review of etiologies and clinico-

10.

11.

12.

13.

14. 15.

16.

haematological profile at a tertiary centre. Indian J Pathol and Microbiol. 2011; 54: 75-80. Gupta N, Kumar R, Khajuria A. Diagnostic assessment of bone marrow aspiration smears, touch imprints and trephine biopsy in hematological disorders. Jkscience. 2010; 12(3): 130-33. Tilak V, Jain R.Pancytopenia: A clinicohematological analysis of 77 cases. Indian J Pathol Microbiol 1999;42:399-404. Bhushra A, Khalid H, Naghmi A. Diagnostic Utility of bone marrow examination in chronic liver disease patients referred for evaluation of haematological derangements. Int J Pathol. 2012; 10(1): 13-6. Khodke R, Marwah S, Buxi G, Yadav RB, Chaturvedi NK. Bone marrow examination in cases of pancytopenia. J Indian Acad Clin Med. 2001; 2: 55-9. Wintrobe M. Haematology, the Blossoming of a Science: A Story of Inspiration and Effort. Philadelphia: Lea and Febiger; 1985. Verma N, Malik H, Sharma V, Agarwal A. Etiology of pancytopenia in and around Meerut. Journal of advance research in biological sciences. 2012; 4(2): 145-51. Tilak V, Jain R. Pancytopenia: A clinicohematological analysis of 77 cases. Indian J Pathol Microbiol. 1999; 42: 399-404. Javalgi AP, Dombale VD. Clinico-hematological analysis of pancytopenia: A bone marrow study. Natl J Lab Med. 2013; 2(4): 12-7. Khan FS, Hasan RF. Bone marrow examination of pancytopenic children. J Pak Med Assoc. 2012; 62(7): 660-63. Bhatnagar SK, Chandra K, Narayan S, Sharma S, Singh V, Dutta AK. Pancytopenia in children: Etiological Profile. J Trop Pediatr. 2005; 51(4): 236-39. Khan TA, Khan IA, Mahmood K. Clinicohematological spectrum of pancytopenia in a tertiary care hospital. JPMI. 2013; 27(2): 143-47. Jalbani A, Ansari IA, Chutto M, Gurbakhshani KM, Shah AH. Proportion of megaloblastic anemia in 40 cases with pancytopenia at CMC hospital Larkana. Medical channel. 2009; 15: 34-7. Tariq M, Khan NU, Basri R, Said A. Aetiology of pancytopenia. Prof Med J. 2010; 17: 252-56. Hossain MA, Akond AK, Chowdhary MK. Pancytopenia â&#x20AC;&#x201C; a study of 50 cases. Bangladesh J Pathol. 1992; 1: 9-12. Gayathri BN, Rao KS. Pancytopenia: A clinicohematological study. J Lab Physicians. 2011, 3(1): 15-20.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 05, (Suppl) Nov. 2016

Original Article Assessemnt of Biochemical, Serological, Molecular Viral Marker and Histological Parameters in HbeAg Positive Chronic Hepatitis B to Determine Therapy Response Rigvardhan1*, Kavita Sahai2, Gurvinder Singh Chopra3 and Mukul Bajpai4 Dept of Pathology, Command Hospital (Central Command), Lucknow, India Department of Pathology, Army Hospital (Research & Referral), Delhi Cantt, India 3 Department of Microbiology and Molecular Biology, Christian Medical College and Hospital, Ludhiana, India 4 Department of Molecular Biology, Command Hospital (Central Command), Lucknow, India 1

Keywords: HBeAg, Chronic Hepatitis B, Therapy, Serological and Viral Markers, Histology

ABSTRACT Introduction: About 300 million people worldwide have chronic hepatitis B virus (HBV) infection with varying degree of liver damage. The presence of continuing viral replication correlates with continuing disease activity and is associated with Hepatitis B e antigen (HBeAg) and hepatitis B virus DNA (HBV-DNA) in serum. Subsequently, the patient may undergo a spontaneous or therapy induced remission, which is accompanied by loss of HBV-DNA and HBeAg. This prospective study was undertaken to correlate all the above parameters so as to have an insight to monitoring of therapy in HBeAg positive chronic hepatitis B (CHB). Aims and objectives: 1. To determine changes in biochemical, serological and virological profile in HBeAg positive CHB with therapy. 2. Determination of histology in liver biopsies in all cases and correlation with immunohistochemical detection of HBsAg and HBcAg with above parameters. Methods: 42 patients of HBeAg positive CHB were enrolled and were followed up for 24 months. Blood samples were collected for alanine aminotransferase (ALT), hepatitis B surface antigen (HBsAg), Anti-hepatitis B core antigen (HBcAg), HBeAg and HBV-DNA. Liver biopsies were done in all individuals. Immunohistochemical staining for HBsAg and HBcAg were done where indicated. The statistical analysis was done using SPSS (Statistical Package for Social Sciences) Version 15.0 statistical Analysis Software. Results: There was a statistically significant improvement in the biochemical, serological and virological profile of the patients after therapy. However, the necroinflammatory activity showed improvement but was not statistically significant. Immunohistochemistry showed good correlation with viral load.

*Corresponding author: Dr. Rigvardhan, Asso. Professor, Dept of Pathology, Command Hospital (Central Command), Lucknow â&#x20AC;&#x201C; 226002; India Phone: +91 9455125354 Email: vardhanrig@yahoo.com

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Markers in HBeAg Positive Chronic Hepatitis B

Introduction

Hepatitis B Virus (HBV), a DNA virus accounts for more than 300 million cases of chronic infection and about 600,000 deaths each year worldwide and is a major health problem in Asia. [1,2] Progression to long term HBV infection occurs in approximately 15-40% of infected patients resulting in chronic hepatitis B (CHB) and depends on age, sex, immune status of the individual, viral load, replication of HBV and other factors. [3,4,5] The natural course of CHB starts as Hepatitis B e Antigen (HBeAg) positive, immune tolerant phase which progresses to a HBeAg-positive immune-reactive phase, HBeAgnegative, inactive HBV carrier state, HBeAg-negative CHB phase and HBsAg negative phase (occult infection). [6,7] Histopathological changes include necroinflammatory activity and fibrosis, which are correlated with HBeAg, anti-HBe, alanine aminotransferase (ALT), and HBV DNA levels. [8] In CHB, HBeAg is an important marker of viral replication, infectivity and ongoing liver injury. Loss of HBeAg and acquisition of anti-HBe tends to be associated with biochemical and histological improvement. [9] During acute phase of infection, anti- hepatitis B core (HBc) of IgM class predominates. As the infection evolves, antiHBc IgM levels gradually decline and often become undetectable within six months. [3, 9] Antiviral therapy against HBV plays a pivotal role in determining the outcome of CHB as it can achieve control of viral replication, ALT normalization, HBeAg loss and seroconversion, and a small number of patients may achieve HBsAg seroconversion.[10,11] Numerous definitions have been used to assess response to antiviral therapy such as biochemical response (normalization of ALT); virological response (decrease in serum HBV DNA or loss of HBeAg with or without the development of antiHBe); histological response (improvement in the histology activity index by at least 2 points without worsening of fibrosis score as compared to pretreatment biopsy); and complete response (biochemical and virological response with loss of HBsAg). [12,13] Thus the aims and objectives of this study were to determine changes in ALT levels, serological and molecular viral marker profile along with histological parameters in HBeAg positive CHB to assess therapy response.

Material and Methods

In this prospective study, 42 cases of HBeAg positive CHB were followed for 24 months. Age and sex was no bar. History of any concomitant illness was taken into consideration but was not an exclusion criterion. Informed consent was taken and Institutional ethical clearance was

obtained. They were treated with lamivudine with or without peginterferon. They were evaluated for: 1. Biochemical Parameters: ALT were measured using ERBA kits in opERA system (BAYER) in accordance with principle based on International federation of clinical chemistry. method, kinetic. Quality control measures were strictly ensured. 2. Serological Parameters: HBsAg, HBeAg and IgManti HBc were performed by enzyme immunoassay (Milano, Italy). Positive and negative controls were run simultaneously to check the validity of test. 3. Molecular Viral Marker: Extraction of DNA was done on unhemolysed serum samples using AccuPrep Genomic DNA Extraction Kit by BIONEER which is a column-based assay. Quantitative PCR assays were carried out using HB V RG Real-ArtTM reagents in cycling A.FAM of the Rotor-Gene 2000 instrument. The detection occurs via the fluorescence labeling of oligonucleotide probes that bind specifically to the PCR amplicate and fluorescence intensity during the course of Real time PCR enables verification as well as quantification of the accumulating product. Samples with more than 105 copies/ml were considered positive. 4. Histological Evaluation: Specimens were fixed in 10% buffered formalin, processed by routine methods, embedded in paraffin and sections cut to 3-4 um in thickness. Sections were subjected to hematoxylin and eosin stain and reticulin stain to study architecture; interface hepatitis; portal inflammation and lobular inflammation and fibrosis. Scores were accorded as per modified Knodell-Ishaak scoring system. [14] Immunohistochemical (IHC) staining was done in selected cases. IHC for HBsAg and HBcAg was done with monoclonal antibodies and ready to use kit manufactured by SEROTEC (USA). Interpretation of IHC staining: (a) HBsAg: strong brown staining of cytoplasm or membranous or both pattern of staining. (b) HBcAg: strong brown staining of nucleus, cytoplasm or mixed pattern. Statistical Analysis The statistical analysis was done using SPSS (Statistical Package for Social Sciences) Version 15.0 statistical Analysis Software. The values were represented in Number (%) and Mean±SD (Standard deviation). Wilcoxan assigned rank test was used to test the significance of two means. The level of significance “p” value was considered statistically significant if <0.05.

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Results

All the 42 cases in this study were males with mean age of 34.5 (± 6.45 SD) years. Clinical symptoms of the patients were anorexia (73%) and nausea (70%), discomfort in hypochondrium and epigastrium (53%), weight loss (35%) and yellowish urine (27%). 1. Biochemical Profile: The mean value of ALT before treatment was 112.8 ±139.6 SD IU/L (Range= 26 to 696 IU/L) which reduced to 63.11 ± 62.4 SD IU/L (Range= 23-428 IU/L). Pre-treatment, 15 (35.7%) cases had normal ALT levels, 12 (28.6%) cases in subgroup 41-80, 08 (19%) in the subgroup 81-160 and 07 (16.7%) in the subgroup >160 IU/L. Post therapy, 23 (54.8%) cases had normal ALT levels, 08 (19%) were in the subgroup 41-80, 06 (14.3%) in the subgroup 81-160 and 05 (11.9%) in the subgroup of more than 160 IU/L. The decrease in ALT level was statistically significant (p value = 0.002).

Summary of Pre- and Post-treatment ALT profile of HBeAg positive cases is shown in table no.1. Amongst these 16 cases (38.09%) had normalized, 05 (11.9%) remained static, 10 (23.8%) cases worsened whereas 11 (26.19%) cases improved but did not normalize.

2. Serological Profile: Before treatment, 39 (92.8%) were HBV-DNA positive and 03 (7.2%) cases were HBV-DNA negative. IgM anti-HBc was carried out in 32 cases, of which 12 (37.5%) cases were positive and 20 (62.5%) cases were negative. Out of 39 HBV-DNA positive cases, 23 (59%) became negative whereas 16 (41%) continued to remain positive. Of the 03 HBVDNA negative cases, 01 case became positive whereas 02 cases continued to remain negative. Quantitative determination of HBV-DNA was done who were HBV-DNA positive, the mean viral load was 711,403, 098.35 -copies/ ml which reduced to the mean HBV-DNA level of 267,064,433.7-copies/ ml with treatment. Out of 12 IgM anti-HBc positive cases, 07 (58.3%) became negative whereas 05 (41.7%) cases remained positive. Of the 20 IgM anti-HBc negative cases, 02 (10%) became positive whereas 18 (90%) cases remained negative. HBsAg seroconversion was seen in 08 (19%) cases. All cases were HBeAg positive before therapy, post-treatment 35 (83.3%) became negative whereas 07 (16.7%) continued to remain HBeAg positive. Summary of Pre- and Posttreatment serological profile of HBeAg positive cases is as per table 2.

Statistical correlation by Wilcoxan assigned ranks test shows statistically significant HBsAg seroconversion (Z value of – 4.38 and p value of 0.002), HBeAg www.pacificejournals.com/apalm

seroconversion (Z value of – 5.96 and p value of 0.000), IgM anti-HBc seroconversion (Z value of – 4.45 and p value of 0.002) and HBV-DNA seroconversion (Z value of – 5.86 and p value of 0.000). 3. Histological Profile: Before therapy, 24 (57.1%) cases had KI score of less than 4, 10 (23.9%) were in the subgroup of KI score 5-8 and 08 (19%) in the subgroup of KI score more than 8. Post therapy liver biopsy was done in 15 cases of which 09 (60%) were in the subgroup less than 4, 04 (26.6%) in the subgroup 5-8, whereas 02 (13.4%) were in the subgroup more than 8. Pre-and post-treatment histological profile of HBeAg positive cases is shown in table 3.

Four patients deteriorated histologically (i.e. > 2 points increase in activity). Of these, two patients were found to be was HIV positive. One patient did not show significant improvement or deterioration. Ten patients showed significant improvement (i.e. > 2 points improvement in histological activity). Statistical correlation by Wilcoxan assigned ranks test shows a Z value of – 1.605 and p value of 0.12.

Immunohistochemistry 1. HBsAg: Two patterns of staining were noted Cytoplasmic and cytoplasmic + Membranous. Cytoplasmic positivity was seen in 20 (47.6%) of cases. The mean KI score in these cases was 3.8/22. Cytoplasmic and Membranous positivity was seen 16 (38.09%) cases with mean KI score of 6.2/ 22. This pattern was associated high titres of HBV-DNA (mean-633,807,112.75 copies/ml). Negative staining for HBsAg was seen in 6 (14.3%) cases. 2. HBcAg: Three patterns of staining are usually seen nuclear, nuclear + cytoplasmic and only cytoplasmic. In this study following was observed: (a) Nuclear staining: This pattern of HBcAg staining was seen in 21 (50%) cases. (b) Nuclear and cytoplasmic pattern: This pattern of immunohistochemistry staining was observed in 18 (42.8%) cases. This pattern of staining was seen in cases with very high levels of HBV-DNA (mean=513,654,388.5 copies/ml). These cases had a mean KI score of 8/22.

Only cytoplasmic pattern of staining was not seen.

Discussion

The presence of HBeAg in serum correlates with the presence of viral replication in the liver. It is recommended that detectable HBeAg should be taken as a surrogate marker for HBV DNA in hepatitis B virus carriers with raised serum ALT in case HBV PCR testing is not available. [8, 15] eISSN: 2349-6983; pISSN: 2394-6466

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Markers in HBeAg Positive Chronic Hepatitis B

Table 1: ALT Profile. Pre-treatment ALT Profile (IU/L) n=42

Post-treatment ALT Profile (IU/L) n=42

0-40

41-80

81-160

>160

0-40

41-80

81-160

>160

Table 2: Serological Profile. HBeAg Positive cases (n=42) HBV-DNA (n=42) IgM anti-HBc (n=32)

Post-Treatment Profile

Pre-Treatment Profile

Remained Positive

Became Negative

Positive

Negative

Positive

Negative

HBsAg (n=42)

All cases positive

HBeAg (n=42)

All cases positive

Table 3: Histological Profile. Pre-treatment Profile (n=42)

Post-treatment Profile (n=15)

0-4

5-8

0-4

5-8

A. Biochemical Profile: ALT is not specific to hepatocytes and may be increased with injury to other organs; however, the most common cause of elevated ALT is liver disease. [16] In this study, 16 (38.1%) cases normalized and HBeAg seroconversion was seen in 35 (83.3%) cases. There was a significant decrease in mean post-treatment ALT levels (112.8 ±139.6 SD IU/L which reduced to 63.11 ± 62.4 SD IU/L). However using serum ALT alone as marker for therapy response has limited value as patients have shown no improvement in necroinflammatory score in spite of achieving biochemical response.[17] In a recent study large scale study of CHB patients, significant number of patients who had persistently normal ALT levels (<40 IU/L) showed significant inflammation or fibrosis on biopsy.[18] The reason of this decreasing response rates with time can occur due to accumulation of YMDD mutants (Substitution of Isoleucine for Methionine at position 552) a virological breakthrough, which are always persistent.[19] B. Serological Response: HBsAg seroconversion was seen in 08 (19%) cases. Serum HBsAg levels are known to reflect the presence of covalently closed circular DNA (cccDNA) in the hepatocytes and its clearance is thought to be the limiting factor for the elimination of infection.[20] Although, HBsAg seroconversion is most durable treatment endpoint it correlates poorly with therapy.[11] It occurs in 3-8 % of patients receiving interferon or peg-interferon and less than 2% of patients taking nucleoside analogues.[12]

HBeAg seroconversion was seen in 35 (83.3%) out of 42 cases. HBeAg is used as an indicator of active underlying liver disease and high degree of infectivity. In contrast, the clearance of HBeAg from sera is associated with reduction in viral replication and normalization of transaminases.[21] Long term lamivudine therapy even after HBeAg seroconversion has shown additional benefit where relapse rate after stoppage of therapy was 13% at one year and 16% at two years, suggesting that long-term therapy might increase the durability of response.[22] IgM anti-HBc is an indirect marker for acute phase of hepatitis and is the only marker for HBV detection in the “window period”.[1,23] Out of 12 IgM anti-HBc positive cases, 07 (58.3%) became negative whereas 05 (41.7%) cases remained positive. Of the 20 IgM anti-HBc negative cases, 02 (10%) became positive whereas 18 (90%) cases remained negative. Thus, total 07 cases were positive after therapy. Out of these, liver biopsy was done in 02 cases, all of whom showed worsening of KI score compared to pre-treatment KI score. They were also found to be HIV positive. Semiquantitative measurement of IgM anti-HBc has shown that antibody titer below 0.2 has 75% predictive of a mild necroinflammatory activity and rules out severe activity (29% sensitivity and 91.6% specificity) whereas antibody titer between 0.2 to 0.5 and more than 0.5 was associated with moderate and severe necroinflammatory activity, respectively. Although necroinflammatory activity correlates with IgM anti-

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 05, (Suppl) Nov. 2016

Rigvardhan et al. HBc levels, fibrosis was unrelated to IgM anti-HBc antibodies.[24] Quantitative IgM-anti HBc can be a novel biomarker for predicting treatment response in HBeAg-positive patients receiving therapy. [23] C. Molecular Viral Marker Profile: HBV-DNA is the hallmark of active viral replication as it has been found in the liver biopsies of cases, which were HBsAg and HBeAg negative on serological examination. Molecular hybridization techniques have demonstrated HBV-DNA in liver biopsies in cases, which were antiHBe positive and HBsAg negative. [25]

In this study, 23 (59%) cases became HBV-DNA negative whereas 16 (41%) cases remained HBVDNA positive. Serial HBV DNA level profile can alter the course of therapy as studies suggest that initial viral kinetics during therapy can predict the sustained virological response in CHB. [11,15,26] Regarding cases, which became HBeAg negative but remained HBVDNA positive are those which harbor mutations in the precore promoter i.e. A to G substitution at position 1896 in HBV genome.[27] Emergence of drug resistance conferred by mutations in the YMDD motif of HBV-DNA reverse transcriptase is a major problem in therapy. The prevalence of YMDD mutations increases with duration of antiviral therapy and has been detected in 20% of immunocompetent patients per year of treatment.[20,27] In our study, HBVDNA positivity post-therapy is also probably due to emergence of mutant strains.

Histological Response We used modified Knodell-Ishaak scoring system which includes interface hepatitis and bridging necrosis, lobular inflammation, portal inflammation and fibrosis. [14] In HBeAg positive and HBV DNA positive cases, 01 case remained static, 04 cases worsened while 10 cases improved. The improvement was seen in interface hepatitis, lobular inflammation, as well as in portal inflammation. Of the cases that improved, extent of fibrosis also improved in 07 (43.7%) cases (fig 1 A,B). Amongst the cases, which worsened histologically, 03 cases continued to remain HBV-DNA positive after therapy whilst HBeAg seroconversion was seen in all cases. 02 of the cases, which worsened after treatment, were found to be HIV positive. Other case which worsened histologically may be case of some other chronic infection or reaction to drugs. The case, which became HBV-DNA negative with therapy but worsened histologically, might be harboring HBV-DNA mutants, which could not be detected during routine screening using conventional primers. www.pacificejournals.com/apalm

A-437 Immunohistochemistry Profile We found that Cytoplasmic positivity for HBsAg (fig 2A) was present in 20 (47.6%) cases. Mean KI score in these cases was 3.8/22. Cytoplasmic and membranous pattern for HBsAg (fig 2B) was seen in 16 (38%) cases. Mean KI score in these cases was 6.2/22. These cases also had high viral load with mean HBV-DNA levels of 633,807,112.75 copies/ml. Negative staining for HBsAg was seen in 6 (14.3%) cases. Thus, overall 85.69 % cases were positive for HBsAg. HBcAg profile was nuclear (fig 2C) in 21 out of 42 HBeAg positive cases. These cases had a mean KI score of 3.6/22. Nuclear and cytoplasmic pattern (fig 2D) was seen in 18 out of 26 HBeAg positive cases. These cases had a mean HBV-DNA level of 513,654,388.5 copies/ml and a mean KI score of 8/22. 03 HBeAg positive cases did not show positive staining for HBcAg. The reason for negative can be explained on the basis of sequencing analysis of integrated viral DNA which suggested that the HBsAg gene remains intact whereas the HBcAg gene gets either deleted or rearranged resulting in impaired synthesis of HBcAg in the liver with integrated HBV-DNA. [28] Significant correlation has been found between intrahepatic HBcAg expression with HBeAg and HBV-DNA (p<0.001) with highest levels of HBV-DNA found in the cases with nuclear and cytoplasmic pattern of staining (mean= 106 viral genomes/ml). Significant link between HBVDNA and membranous pattern of HBsAg staining has also been found (p=0.001).[29] According to pathogenetic theory the immune response to HBeAg (membrane bound nucleocapsid antigen) is responsible for liver damage, while the immune response to free HBcAg has no apparent antiviral effect since the nucleocapsid is always masked within the HBsAg envelope of the virion.[30] Furthermore, the data suggests that HBcAg expression is not associated with integrated form of HBV-DNA and HBsAg staining can be seen in integrated as well as episomal forms of HBV-DNA. [28]

Conclusion

Ours was a prospective study, which evaluated 42 cases of HBeAg positive CHB cases. After treatment, we found statistically significant decrease in mean ALT levels and statistically significant HBsAg, HBeAg, IgM antiHBc and HBV-DNA seroconversion. Improvement was observed in histological profile but was not statistically significant. Based on these findings we conclude that single most reliable marker for assessing therapy-induced response is HBV-DNA. Persistence of IgM anti-HBc post eISSN: 2349-6983; pISSN: 2394-6466

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Fig. 1: Improvement in fibrosis (A-pre-treatment; B-post treatment) with therapy (Reticulin stain, 20x).

Fig. 2: Cytoplasmic (A) and cytoplasmic + membranous (B) pattern of HBsAg immunohistochemistry staining. Nuclear + cytoplasmic (C) and nuclear (D) pattern of HBcAg immunohistochemistry staining.(Immunohistochemistry stain, 20x).

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therapy denotes continuing necroinflammatory activity. Histological profile did show improvement but was not statistically significant in our study. In IHC, nuclear and cytoplasmic pattern of HBcAg staining is associated with high levels of viremia and marked necroinflammatory activity.

11. Andersson KL, Chung RT. Monitoring During and After Antiviral Therapy for Hepatitis B. Hepatology. 2009 ; 49: 166–173.

Acknowledgement

13. Lok AS, Heathcote EJ, Hoofnagle JH. Management of hepatitis B: 2000--summary of a workshop. Gastroenterology. 2001; 120:1828–1853.

None

Source of Funding Nil

Conflict of Interests

12. Hoofnagle JH, Doo E, Liang TJ, Fleischer R, Lok AS. Management of hepatitis B: summary of a clinical research workshop. Hepatology. 2007; 45:1056–1075.

14. Ishak K, Baptista A, Bianchi L, Callea F, De Groote J, Gudat F et al. Histological grading and staging of chronic hepatitis. J Hepatol. 1995 Jun;22(6):696-699.

References

15. Hussain AB, Karamat KA, Anwar M, Kazmi SY, Tariq WU. Correlation of HBV DNA PCR and HBeAg in hepatitis carriers. J Coll Physicians Surg Pak. 2004; 14: 18-20.

2. World Health Organization: WHO: Hepatitis B. Fact Sheet No.204. (http://www.who.int/mediacentre/ factsheets/fs204/en/).

16. Pincus MR, Tierno PM, Fenelus M, Bowne WB, Bluth MH. Evaluation of liver function and injury. In Henry JB: Clinical diagnosis and management by laboratory methods, El siever. 22th edition; Chapter 21: 296-311.

All authors have none to declare 1. Lee WM. Hepatitis B virus infection. New Eng J Med 1997;24:1733-1745.

3. Lok AS: Chronic hepatitis B. N Engl J Med. 2002;346(22):1682-3. 4. Koyuncuer A. Associations between HBeAg Status, HBV DNA, ALT Level and Liver Histopathology in Patients with Chronic Hepatitis B. Science Journal of Clinical Medicine. 2014;3:117-123. 5. Sharma SK, Saini N, Chawla Y: Hepatitis B virus: inactive carriers. Virol J. 2005; 2:82. 6. Squadrito G, Spinella R, Raimondo G: The clinical significance of occult HBV infection. Ann Gastroenterol. 2014;27(1):15-19. 7. Yim HJ, Lok AS. Natural history of chronic hepatitis B virus infection: what we knew in 1981 and what we know in 2005. Hepatology. 2006; 43(2 Suppl 1):S173-81. 8. Geller SA, Petrovic LM. Chronic Hepatitis (Chronic Necroinflammatory Disease of the Liver)-Grading and Staging. Biopsy Interpretation of the Liver. 2nd edition. Philadelphia: Lippincott Williams & Wilkins; 2009;97-120. 9. Badur S, Akgun A. Diagnosis of hepatitis B infections and monitoring of treatment. J Clin Virol. 2001; 21: 229-37. 10. Dienstag JL: Benefits and risks of nucleoside analog therapy for hepatitis B. Hepatology 2009, 49:S112–S121. www.pacificejournals.com/apalm

17. Lai CL, Chien RN, Leung NW, Chang TT, Guan R, Tai DI, Ng KY, et al. A one-year trial of lamivudine for chronic hepatitis B. Asia Hepatitis Lamivudine Study Group. N Engl J Med. 1998; 339:61–68. 18. Kumar M, Sarin SK, Hissar S, Pande C, Sakhuja P, Sharma BC, Chauhan R, et al. Virologic and histologic features of chronic hepatitis B virus-infected asymptomatic patients with persistently normal ALT. Gastroenterology. 2008; 134:1376–1384. 19. Zeng Y, Yang B, Wu Y, Chen J, Shang H, Chen X et al. Clinical significance of periodic detection of hepatitis B virus YMDD mutation by ultrasensitive real-time amplification refractory mutation system quantitative PCR during lamivudine treatment in patients with chronic hepatitis B. Journal of Medical Microbiology (2015), 64, 237–242. 20. Feld JJ, Wong DK, Heathcote EJ: Endpoints of therapy in chronic hepatitis B. Hepatology 2009, 49:S96–S102. 21. Yang J, Chen J, Ye P, Jin L, Wu W, Sheng G, Li L. HBsAg as an important predictor of HBeAg seroconversion following antiviral treatment for HBeAg-positive chronic hepatitis B patients. Journal of Translational Medicine 2014, 12:183-191. 22. Ryu SH, Chung YH, Choi MH, Kim JA, Shin JW, Jang MK, Park NH, et al. Long-term additional lamivudine eISSN: 2349-6983; pISSN: 2394-6466

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therapy enhances durability of lamivudine-induced HBeAg loss: a prospective study. J Hepatol. 2003; 39:614–619. 23. Hou FQ, Song LW, YuanQ, Fang LL, Ge SX, Zhang J et al. Quantitative Hepatitis B Core Antibody Level Is a New Predictor for Treatment Response In HBeAgpositive Chronic Hepatitis B Patients Receiving Peginterferon. Theranostics 2015;5:218-226. 24. Colloredo G, Bellati G, Sonzogni A, Zavaglia C, Fracassetti O, Leandro G et al. Semiquantitative assessment of IgM antibody to hepatitis B core antigen and prediction of severity of chronic hepatitis B. J viral Hepatitis 1999;6:429-434. 25. Harrison TJ, Anderson MG, Murray-Lyon IM, Zuckerman AJ. Hepatitis B virus in hepatocytes: a series of 160 biopsies. J Hepatol 1986;2:1-10. 26. Lai CL, Gane E, Liaw YF, Hsu CW, Thongsawat S, Wang Y, Chen Y, et al. Telbivudine versus lamivudine

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in patients with chronic hepatitis B. N Engl J Med. 2007; 357:2576–2588. Yuan HJ, Yuen MF, Ka-Ho Wong D, Sum SM, Sablon E, Oi-Lin Ng I, Lai CL. Impact of precore and core promoter mutations on hepatic histology in patients with chronic hepatitis B. Aliment Pharmacol Ther. 2005;22:301-307. Omata M, Yokosuka O, Imazeki F, Ito Y, Mori J, Uchiumi K et al. Correlation of hepatitis B virus DNA and antigens in the liver. Gastroenetrology 1987;92:192-196. Ramalho F, Brunetto MR, Rocca G, Piccari GG, Batista A, Chiaberge E et al. Serum markers of hepatitis B virus replication, liver histology and intrahepatic expression of hepatitis B core antigen. J Hepatol 1988;7:14-20. Lindh M, Savage K, Rees J, Garwood L, Horal P, Norkrans G et al. HBeAg immunostaining of liver tissue in various stages of chronic hepatitis B. Liver 1999;19:294-298.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 05, (Suppl) Nov. 2016

Original Article Etiological Evaluation of Pancytopenia in A Tertiary Care Hospital Anuja Dasgupta*, Shetty K Padma, Sajitha K and Jayaprakash Shetty Dept. of Pathology, K.S Hegde Medical Academy, NITTE University, Deralakatte, Mangalore, India Keywords: Pancytopenia, Hypersplenism, Malaria, Alcohol Liver Disease

ABSTRACT Background: Pancytopenia is not a disease by itself, however it is a common hematological problem characterized by simultaneous presence of anemia, leucopenia and thrombocytopenia. The disease pattern associated with pancytopenia varies with geographic location, age group, nutritional status, drug intake and prevalence of infective disorder. This prospective study was to investigate and identify different causes of pancytopenia with frequency, to ascertain percentage of occurrence of pancytopenia, to determine its incidence in relation to sex and age, and to compare findings with those of other similar studies. Methods: 80 patients diagnosed with pancytopenia were clinically evaluated, with complete blood count, peripheral smears, and bone marrow aspiration-biopsy whenever possible in Justice K. S. Hegde Hospital attached to Nitte University, Deralakatte, Mangalore, from June 2012 to June 2014. Results: Among the 80 cases analyzed, most of the cases were seen in the age group of 41-50 years, with male predominance. Hypersplenism (28.75%), malaria (16.25%) and megaloblastic anemia (13.75%) were the three commonest causes in our hospital. In 13 cases of malaria, Plasmodium vivax (8 cases) was most commonly noted. Conclusion: The present study concludes that varied causes of pancytopenia can be attributed to the geographic area, nutritional and drug intakes, personal habits, infective causes, stringency of diagnostic criteria, and differences in methodology used. Hence, a detailed clinical history and meticulous examination along with hematological investigations provide invaluable information in the complete workup of patients with pancytopenia for understanding the disease processes, planning further investigations and management, and ascertain the cause.

*Corresponding author: Dr. Anuja Dasgupta, Dept. Pathology, K.S Hegde Medical Academy, NITTE University, Deralakatte, Mangalore -575018, India Phone: +91 9902336642 Email: dasgupta.anuja@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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Evaluation of Pancytopenia

Introduction

Pancytopenia is defined as reduction of the cellular elements of blood with hemoglobin <10gm/dl, white blood cell count <4000/mm3 and platelet count <100,000/mm3. Hence, is not a disease entity but rather a triad of findings that may result from a number of disease processes.[1,2] The etiology of pancytopenia varies in different populations depending on the age, nutritional status, geographic location and the prevalence of infections. Some causes of pancytopenia with prompt diagnosis are curable. However, in certain circumstances where complete cure is not possible, early diagnosis and supportive treatment can improve the quality of life by decreasing morbidity and mortality.[3,4] The present study was carried out in patients diagnosed with pancytopenia attending to our hospital in order to find the incidence of various etiological factors with clinical details, hematological findings and bone marrow (BM) aspiration-biopsy whenever possible.

Materials and Methods

This prospective study was carried out over a period of two years, June 2012 to June 2014 in the Clinical Diagnostic Laboratory, Justice K. S. Hegde Charitable Hospital, NITTE University, Deralakatte, Mangalore. Approval from institutional ethical committee of K. S. Hegde Medical Academy (KSHMA) was obtained to conduct this study. The institutional ethical committee felt an informed consent was not required to conduct this study and thus deemed it to be not applicable. Criteria for inclusion: i. All age groups and both sexes ii. Hemoglobin: <10g/dL iii. Total leukocyte count: <4000/ÂľL, and iv. Platelet Count: <100,000/ÂľL. Relevant history was taken in every patient including their habits and intake of drugs, along with meticulous clinical examination. In all patients, complete blood count and peripheral smear study, and BM study wherever possible. Other investigations which whenever indicated included: chest and bone radiographs, ultrasound / C.T. Scan, urine and stool examination, liver function test, urinary Bence Jones proteins and serum electrophoresis, serological investigations and ELISA tests, and tests for serum ferritin, cobalamin, folic acid, lactate dehydrogenase and antinuclear antibody. A clinico-pathological correlation was done in all cases before reaching a definitive diagnosis and the findings of this study was compared with similar other studies.

Result

A total of 80 patients were studied with a male to female ratio of 1.2:1. The age ranged from 2-90 years, with the maximum number of cases observed in the 41-50 years age group followed by 31-40 years (Fig 1). The causes of pancytopenia with age distribution of the patients are shown in Table 1. Hypersplenism was observed in 23 (28.75%) cases constituting 14 males and 9 females. Hypersplenism was due to alcohol liver disease (ALD) (12 of 23 cases), and chronic liver disease (CLD) of idiopathic type (8 cases) and other causes (3 cases). ALD was most encountered in males between 31-70 years although one case was noted in a 42 year old woman. CLD was mostly noted in women of age group 41-50 years. The other three cases included: portal vein thrombosis with cavernous transformation, compensated portal hypertension caused by multiple liver metastasis from carcinoma rectum and Hodgkins lymphoma. Of the thirteen (16.25%) cases of malarial infestation with an age range of 5-50 years, Plasmodium vivax was the predominant parasite seen in 8 cases, mixed malarial infestation (Plasmodium vivax and Plasmodium falciparum) in 4 cases and 1 case of Plasmodium falciparum infestation (Fig 2). Megaloblastic anemia (MA) was seen in 11 (13.75%) cases with the commonest age groups being between 31-50 years (54.55%) and 61-80 years (45.45%). Females were affected more than males. BM examination was performed in 6 cases and showed hypercellular marrow with megaloblastic features and giant metamyelocytes (Fig 3). There were 2 cases with history of alcohol consumption. We encountered seven (8.75%) cases of acute leukemia (AL) which included 4 children and 3 adults. Among the children, 3 developed Acute Lymphoblastic Leukemia (ALL) with monotonous population of lymphoblasts and 1 had Acute Myeloid Leukemia (AML) (M2). In the adults, all were diagnosed with AML (M2, M3, M4). The case with AML (M4) had increased number of monoblasts (Fig 4). In our study we observed six (7.5%) cases of pancytopenia following intake of drugs over a long duration of time or as a transient presentation and if when discontinued the blood counts would either come to normal range or remained in low counts. There were two cases of anti-platelet drug intake, clopidogrel which was administered to patients with Ischemic Heart Disease and Non ST elevation Myocardial Infarction. Other cases constituted two cases of chemotherapy-induced, one case of anti-rheumatic drug leflunamide which lead to myelofibrosis grade 2 and a case of Break Bone Fever by Chickungunya virus with four years of self- administered analgesic Indomethicin.

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We noted two (2.5%) alcoholic male patients of ages 48 years and 70 years. Both were on heavy alcohol consumption for over 20 years. Ultrasound of both showed features of liver cirrhosis without splenomegaly.

The age of the patients ranged 2 - 90 years with the commonest age group being 41 - 50 years (Table 2). Majority of the cases presented in 4th and 5th decade (Fig 1). Few other studies too have reported 4th and 5th decade as the commonest age group for presentation of pancytopenia.[5,6,7,8] A male predominance was noted in our study which was similar to other studies. With social taboos in our society, this could make health care facilities more readily available to males in comparison to females therefore showing an increased in male presentation at hospitals.

The other causes of pancytopenia included in our study were three (3.75%) cases each of multiple myeloma, aplasitc anemia (AA) and myelodysplastic syndrome (MDS) of Refractory Anemia with Excessive Blasts (RAEB) type; two (2.5%) cases of dengue fever and one (1.25%) case each of myelofibrosis, iron deficiency anemia (IDA), lymphoma (lymphoplasmacytic), HIV infection, systemic lupus erythematous (SLE), multi-focal bone metastasis from adenocarcinoma of prostate, and secondary to autoimmune cause.

The commonest cause of pancytopenia, reported in various studies throughout mostly in the subcontinent area had been MA.[9] This is in sharp contrast with the results of our study, where the commonest cause of pancytopenia was found to be hypersplenism (n = 23, 28.75%). In other similar studies the incidence varies from 3 to 68%.[5] This contrast could suggest that there is an increasing trend of alcoholism in todayâ&#x20AC;&#x2122;s society; hence most patients attending to our hospital present with ALD, and hypersplenism being one of the consequences.

Discussion

The present study with a total of 80 patients of pancytopenia was conducted to analyze the incidence of various causes of pancytopenia, age pattern, gender-wise along with their clinical findings, hematological and BM spectrum. The statistical data hence obtained were compared with previous published literature.

Table :. Age distribution of the patients and causes of pancytopenia.

Age group (in years)

Causes

Total

2-10

11-20

21-30

31-40

41-50

51-60

61-70

71-80

81-90

Hypersplenism

Malaria

Megaloblastic anemia

Acute leukemia

Drug induced

Multiple myeloma

Aplastic anemia

MDS

Dengue

ALD without splenomegaly

Myelofibrosis

IDA

Lymphoma

HIV

SLE

Bone mets*

Autoimmune cause

MDA: myelodysplastic syndrome; IDA: iron deficiency anemia; ALD: alcohol liver disease; HIV: human immunodeficiency virus; SLE: systemic lupus erythematous; Bone mets*: Bone involvement by metastatic adenocarcinoma prostate

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Evaluation of Pancytopenia

Table 2: Comparison of number of cases, age and sex distribution and three most different studies conducted in different countries. Study No. of Age group Country (Year) M : F ratio Commonest cause cases (years) Tilak V et al[6] India (1999) 77 5 to 70 1.1 : 1 MA (68%) Savage DG et al[20] Zimbabwe (1999) 134 All MA (35.8%)

common causes of pancytopenia in

Khodke K et al[27]

India (2001)

3 – 69

1.3 : 1

MA (44%)

Khunger et al [7] Niazi M et al[2] Hamid GA et al[10] Devi PM et al[21] Jalbani A et al[4] Santra G et al[14] Ashraf S et al[11] Aziz T et al[32] Gayathri BN et al[9] Tariq M et al[40] Weinzierl EP et al[22] Tareen SM et al[16]

India, 2002 Pakistan (2004) Yemen (2008) India (2008) Pakistan (2010) India (2010) Pakistan (2010) Pakistan (2010) India (2011) Pakistan (2012) California (2012) Pakistan (2012)

200 89 75 50 40 111 150 88 104 50 250 180

2 - 70 1 to 75 3 – 85 3 – 80 12 – 70 13 – 65 15 – 60 15 – 60 2 – 80 15 – 70 All All

1.2 : 1 1.7 : 1 1.03 : 1 1.5 : 1 2.6 : 1 1.5 : 1 1.1 : 1 2.6 : 1 1.2 : 1 1.7 : 1 1.8 : 1

MA ( 72%) BM Aplasia (38.3%) HS (45.3%) HA (22%) AA (32.5%) AA (22.72%) HS (68%) MA (40.9%) MA (74.04%) AA (36%) AL (34.4%) Malaria (29.44)

2nd common cause AA (7.7%) AA (26.1%) AA (14%) NEH (14%) KA (14%) AA ( 28%) MA (24.7%) MA (14.7%) MA (18%) HS (22.5%) HS (15%) MA (25.4%) AA (31.9%) AA (18.3%) MA (16%) MDS (22.4%) Leukemia (17.78)

Kumar DB et al[27]

India (2012)

10 – 70

1.0 : 1.8

HM (33.33%)

NEH (27.08%)

Metikurke SH et al[1]

India (2013)

All

3:2

Jain A et al[5]

India (2013)

250

All

2.6 : 1

HS (29.2%)

Present study

India

2 – 90

1.2 : 1

HS (28.75%)

MA (50%)

3rd common cause Other causes (24.3%) AIDS (17.2%) Multiple myeloma (04%) SL (5%) HS (18.4%) AA (13.3%) MDS (18%) MA (15%) DI (13%) HM (6.6%) HS and CM (11.4%) SL (3.8%) MDS (14%) AA (7.6%) Tuberculosis (16.11) Megaloblastic marrow (18.75%)

Nutritional anemia AA (13.04%) (23.9%) Myelosuppressants Infections (25.6%) (16.8%) Malaria (16.25%) MA (13.75%)

MA: Megaloblastic anemia; AA: Aplastic anemia; AIDS: Acquired immune deficiency syndrome; NEH: Normoblastic erythroid hyperplasia; KA: Kala azar; BM: Bone marrow; HS: Hypersplenism; HA: Hypoplastic anemia; MDS: Myelodysplastic syndrome; DI: Drug induced; HM: Hypoplastic marrow; CM: Chronic malaria; SL: Subleukemic leukemia; AL: Acute leukemia

Fig. 1: Age and gender distribution of patients with pancytopenia.

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Fig. 2: Peripheral smear of malarial infestation showing Plasmodium falciparum cresentric gametocyte with centrally placed chromatin (Leishman stain 1000X).

Fig. 3: BM aspiration of MA showing giant metamyelocytes displaying nuclear-cytoplasmic asynchrony with bud-like protrusion and sieve-like chromatin of the nuclei (Leishman stain 1000X).

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Evaluation of Pancytopenia

Fig. 4: Buffy coat of AML (M4; Acute Myelomonocytic Leukaemia) shows monoblasts with bluish-gray cytoplasm, pseudopods and cytoplasmic vacuoles. (Leishman stain 1000X).

Fig. 5: BM biopsy of myelofibrosis (grade 2) showing increase in reticulin with intersections (Reticulin stain 400X).

In hypersplenism there is peripheral pooling or trapping and destruction of cells in an enlarged spleen which results in cytopenias. Increasing in severity of the condition leads to pancytopenia, commonly seen in patients with liver disease.[5]

Pakistan[16] found malaria (29.44%) to be the commonest cause which probably attributed to the poor sanitation among the low income group. Thakkar BB et al in India[3] reported 19% malaria cases of 100 patients and was the second common cause of pancytopenia.

Hypersplenism was most encountered among males between 31-70 years with the most affected age group being 31-40 years and a case noted in a 42 year old woman. We believed that this could be reflected by the changes in lifestyle as well as the increased consumption of alcohol from a younger age. Hypersplnism was also the commonest cause of pancytopenia in studies conducted by Hamid GA et al[10] with 45.3%, Ashraf S. et al[11] 68% and Jain A et al[5] 29.2%.

In our study we noted a higher incidence of Plasmodium vivax malaria parasite causing pancytopenia. However, studies done by Tareen SM et al, Arya TV et al and Hemmer R et al reported that malaria due to Plasmodium falciparum has been implicated as a cause of pancytopenia. [16,17,18] This could be explained by the difference in the geographic distribution of malaria parasites.[19] No deaths have been reported among these patients and have recovered completely.

Malaria related cytopenia was noted in literatures done by Cannard and Aouba et al.[12,13] Malaria may cause pancytopenia by direct bone marrow invasion of the parasite, immune hemolysis, disseminated intravascular coagulation, hypersplenism, bone marrow necrosis or hemophagocytosis.[14] Malaria (n = 13, 16.25%) causing pancytopenia was the second commonest cause in our study, which was in contrast to previous literature. The data observed in our study may be explained by the geographic location since Malaria is endemic in Mangalore, Karnataka. [15] Therefore when malaria is diagnosed the clinicians treat the acute illness without advising BM examination. The incidence was similar to the study done by Hamid GA et al in Yemen[10] (14.7%) wherein Malaria was the second common cause of pancytopenia. However Tareen SM et al,

In our study MA (n=11, 13.75%) stood next to Hypersplenism and Malaria as a cause for pancytopenia which was similar to the study done by Hamid GA et al[10] with 14.7%. However, the data obtained here was in sharp contrast to other previous studies done in India wherein, MA was the most commonest cause of was due to nutritional deficiency.[6,7,9,20,21] Of the seven (8.75%) cases in leukemia, 4 cases were in the pediatric age group and 3 cases in the adult group. This was comparatively similar to Savage DG et al[20] with eleven cases (8.2%) of AL from the 134 patients with pancytopenia. Jain A et al[5] received 7 leukemia cases out of 250 pancytopenic cases (2.8%) and Metikurke SH et al[1] with five cases (8.8%) of 57 pancytopenic patients. In contrast Weinzierl EP et al[22] from California obtained

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a total of 86 cases of AL out of 250 cases of pancytopenia (34.4%). ALL in childhood is the most common association, however pancytopenia preceding AML has been described in the literature and adults are occasionally known to be affected.[5]

myeloma cells in S phase may suggest that the bone marrow cytokine milieu, permissive for myeloma cell proliferation, is not conducive to efficient erythropoiesis. In patients with more advanced disease, there may be thrombocytopenia and neutropenia along with anemia.[28,29]

We found six cases (7.5%) of drug induced pancytopenia in our study. Two cases of anti-platelet drug Clopidogrel induced, 2 cases of Chemotherapy induced, and one case each of antirheumatic drug Leflunamide and analgesic Indomethacin. Weinzierl EP et al[22] obtained 8 cases of drug induced pancytopenia including chemotherapeutic regimens out of 250 cases of pancytopenia. Santra G et al[14] found 15 cases of pancytopenia secondary to drug induced including chemotherapy cases out of total 111 cases of pancytopenia.

We had 3 cases (3.75%) of AA. Internationally, AA occurs more commonly in the East than in the West with a higher prevalence in the Indian subcontinent which may be attributed to environmental factors such as exposure to toxic chemicals, viral infections and drugs rather than genetic factors.[22] The incidence of AA usually varies from 7.7% to 43% among pancytopenic patients.[8] However, the incidence in our study was lower to those studies reported by Khunger JM et al[7] (14%), Gayathri BN et al[9] (19%) and Hamid GA et al[10] (13.3%). Fortunately, this was similar to an incidence of 4.8% reported by Jain A et al.[5]

Clopidogrel, an anti-platelet drug, has major adverse effects causing marrow suppression.[23] Previous reports have suggested clopidogrel may be a potential cause of pancytopenia irrespective of the duration of treatment, though rarely seen. Hence, careful clinical and hematological monitoring should be carried out in the course of treatment with clopidogrel.[24] In the present study the two cases were of transient bone marrow suppression due to intake of Clopidogrel. Once the treatment was stopped the blood counts were normalized. Leflunomide, a disease-modifying antirheumatic drug, inhibits pyrimidine synthesis in lymphocytes (T-cells) and other rapidly dividing cells. It may rarely be associated with life-threatening pancytopenia. Time of onset pancytopenia after the drug intake is variable.[25] On discontinuation of the treatment the patient in our study had improved with time. Pancytopenia following intake of analgesic drugs have a delay of 2-3 months between BM injury and the onset of pancytopenia.[26] In the present study a case of self-administered non-steroid anti-inflammatory drug (NSAID) Indomethacin for about four years following the diagnosis of Break Bone Fever caused by Chickungunya virus. Unfortunately after withdrawal of the drug there was no improvement in the hematological parameters. Santra G et al[14] found 2 patients out of 111 cases (1.8%) developing pancytopenia following intake of NSAIDs (ibuprofen and diclofenac). We received three (3.75%) multiple myeloma presenting with pancytopenia, which was in comparison to the incidences found in Tilak V et al[6] (1.3%), Gayathri BN et al[9] (0.96%) and Khodke K et al[27] (4%). Hemoglobin concentration correlated directly with the percentage of www.pacificejournals.com/apalm

MDS is characterized by ineffective hematopoiesis by its increased apoptosis in early and matured forms of hematopoietic cells. MDS may thus be suspected in cases of pancytopenia.[7] Three cases of pancytopenia with MDS of RAEB (3.75%) were noted in the present study. The incidence of MDS as reported in other similar studies varies from 0 to 18%.[5] Khunger et al[7] reported 2% of MDS causing pancytopenia and Santra G et al[14] with 2.7% cases in 111 adult pancytopenia patients. Jain A et al[5] reported 0.4% case of pancytopenia due to MDS of RAEB type. MDS are most common in the elderly and should be included in the differential diagnosis of elderly patients with pancytopenia, even if mild. Pancytopenic presentation is more common with MDS-RAEB type.[5] Dengue was found in 2 cases (2.5%) in our study. Santra G et al[14] had a single case of dengue (0.09% of 111 cases) with hypoplasia of BM due to hemophagocytosis. One patient in our study expired due to Dengue Hemorrhagic Fever with shock. We noted 2 cases (2.5%) of ALD without splenomegaly. Alcohol is known to cause suppression of hematopoeisis especially seen in severe alcoholism, and also may lead to nutritional deficiencies of folic acid and other vitamins that play a role in the hematopoietic cell development.[30] A study done by CF Weston et al[31] from UK observed 3 patients of alcohol liver disease without splenomegaly who presented with pancytopenia. We found a single case of myelofibrosis (1.25%) in our study. Literature also reveals pancytopenia in myelofibrosis in studies done by Tilak V et al[6] (1 of 77 cases, 1.29%) and Khunger JM et al[7] (2 of 200 cases, 1%). eISSN: 2349-6983; pISSN: 2394-6466

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IDA is usually associated with thrombocytosis due to platelet production by erythropoietin.[32] However, some patients with severe or long standing IDA may develop mild thrombocytopenia possibly due to complicating factors such as folate deficiency or splenic sequestration and the role of iron being required in the late stage of thrombopoiesis.[21] Some studies believed that the increasing severity of iron deficiency leads to normalization and occasionally even decreases the platelet count. The exact mechanism of this is unclear but could be related to the alteration in the activity of iron dependent enzymes in thrombopoiesis and leucopoiesis.[33] IDA was seen in a single case (1.25%) of our cases of pancytopenia. An incidence of 1.3% by Hamid GA et al[10] and 8% by Devi PM et al[21] were reported. Non-Hodgkin lymphomas can lead to pancytopenia by BM replacement, autoimmune cytopenias / splenomegaly. [31] The incidence of NHL in other similar studies varies from 0.9 to 10%.[5] We observed a case of Non-Hodgkin lymphoma- Lymphoplasmacytic Lymphoma (1.25%). Weinzierl EP et al[22] observed 5 cases of large B-cell lymphoma out of 125 cases of adults with pancytopenia. Khunger JM et al[7] found 2 cases of Non-Hodgkins Lymphoma from 200 cases of pancytopenia. One case of HIV infection (1.25%) was noted in the present study. Pancytopenia occurs late during the course of HIV infection.[27] Previous literature reveals an incidence of 1.8% (2 of 111 cases) in Santra G et al[14], 6% (3 of 50 cases) in Devi PM et al[21] and 2% (1 of 50 cases) in Khodke K et al[27]. SLE presenting with pancytopenia was seen in one case (1.25%) in our study. This was similar to the study by Devi PM et al[21] with one case (2%) of SLE with pancytopenia. Santra G et al[14] diagnosed 8 cases of SLE with pancytopenia from 111 total cases. Previous literature has suggested that SLE can present with pancytopenia and bone marrow fibrosis. The non-characterized autoimmune diseases with positive autoimmune serology can also lead to modest reticulin fibrosis.[34] We received a case (1.25%) of pancytopenia associated with metastatic adenocarcinoma of prostate without prior history of chemotherapy. Weinzierl EP et al[22] observed 2 cases (1.6%) of neoplastic diagnoses among 125 adults with pancytopenia were attributed to metastatic carcinoma involving bone marrow. Devi PM et al[21] also reported a case of pancytopenia following squamous cell carcinoma of nasal septum. There was one case (1.25%) of pancytopenia secondary to autoimmune cause. Studies done by Bailey FA et al, Roffe

C et al and Sumimoto S et al have found the presence of an antibody or suppressor cells directed against hematopoietic stem cells.[35,36,37] Abrams EM et al[38] have suggested the possibility of an underlying relationship of cytotoxic T lymphocytes in the pathogenesis of autoimmune diseases and aplastic anemia. A study by Kritharis A et al[39] had observed a case of idiopathic autoimmune pancytopenia and was screened for rheumatologic, infectious and autoimmune disorders but had a negative work-up, although BM biopsy was consistent with autoimmune destruction of peripheral blood cells with panhyperplasia and mild perivascular plasmacytosis. It was suggested pancytopenia due to autoimmune dysregulation could be secondary to lymphoid hyperactivity or deficiency in lymphocyte apoptotic pathway eg Fas, but the exact cause was unknown.[35]

Conclusion

Pancytopenia is a laboratory diagnosis and is not an uncommon hematological problem. A large proportion of causes for pancytopenia are treatable and reversible, therefore accurate diagnoses and timely intervention maybe lifesaving and will have impact on the morbidity and mortality. Physical findings and peripheral blood picture provide valuable information in the work up of pancytopenic patients and further helps in planning the investigations as well as management of the patients. In the present study BM examination was deferred in many of the cases especially diagnosed with hyperrsplenism and positive for malarial parasites. Hypersplenism, especially due to ALD, was the commonest cause of pancytopenia in this study, indicating a change in the trend of lifestyle in the patients who are attending our hospital. The next common cause was malaria. Plasmodium vivax species was a more common parasite, which indicates that pancytopenia depends on geographic location. Present study concludes that stringent diagnostic criterion and a general conceptual framework for ascertaining the cause of pancytopenia is very valuable and a demand of time.

Acknowledgements Nil

Funding None

Competing Interests None

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 05, (Suppl) Nov. 2016

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Reference

1. Metikurke SH, Bhavika R. Correlation of Bone Marrow Aspirate, Biopsies and Touch Imprint Findings in Pancytopenia. Journal of Hematology. 2013:13;2:8-13. 2. Niazi M, Raziq F: The incidence of underlying pathology in pancytopenia – an experience of 89 cases. J Postgrad Med Inst. 2004; 18:76–79.

A-449 15. Malaria in India – Malaria site. Alvailable at: http:// www.malariasite.com/malaria-india/ 16. Tareen SM, Tariq MM, Bajwa MA, Awan MA, Ahmad Z, Javed Y. Study of pancytopenia in Balochistan, Pakistan. Gomal Journal of Medical Sciences 2012;10(2). 17. Arya TV, Prasad RN. Fatal pancytopenia in falciparum malaria. J Assoc Physicians India. 1989;37:469-70.

3. Thakkar BB, Bhavsar UN, Trivedi NJ, Agnihotri AS. A study of pancytopenia in adult patients more than 12 years of age in North West region of saurashtra. Nat J Med Res. 2013;3:48-52.

18. Hemmer R. Airport malaria in Luxembourg. Euro Surveillance Weekly. 1999;3:790-819.

4. Jalbani A, Ansari IA, Shah AH, Gurbakhshani KM, Chutto M, Solangi GA. Pancytopenia; Study of 40 patients at CMC Hospital Larkana. Professional Med J. 2010;17:105-10.

20. Savage DG, Allen RH, Gangaidzo IT, Levy LM, Gwanzura C, Moyo A et al. Pancytopenia in Zimbabwe. Am J Med Sci. 1999;317:22-32.

5. Jain A, Naniwadekar M. An etiological reappraisal of pancytopenia-largest series reported to date from a single tertiary care teaching hospital. BMC Hematology. 2013;13:1-9. 6. Tilak V, Jain R. Pancytopenia- A clinic-hematologic analysis of 77 cases. Indian J Pathol Microbiol. 1999;42:399-404. 7. Khunger JM, Arulselvi S, Sharma U, Ranga S, Talib VH. Pancytopenia- A clinicohaematological study of 200 cases. Indian J Pathol Microbiol. 2002;45:375-9. 8. Kumar DB, Raghupathi AR. Clinicohematologic analysis of pancytopenia: Study in a tertiary care centre. Basic and Applied Pathology. 2012;5:19-21. 9. Gayathri BN, Rao KS. Pancytopenia: a clinico hematological study. J Lab Physicians. 2011;3:15-20. 10. Hamid GA, Shukry SA. Patterns of pancytopenia in Yemen. Turk J Hematol. 2008;25:71-4. 11. Asharaf S, Naeem S: Frequency of hypersplenism in chronic liver disease patients with pancytopenia. Annals of King Edward Medical University, North America, Special Edition Annals 2010, 16(1):108–110. 12. Latger Cannard V, Bibes B, Dao A, et al. Malaria related cytopenia. Ann Biol Clin. 2002;60:213-216.

19. Malaria in Mangalore – Malaria site. Available at: http://www.malariasite.com/category/epidemiology/

21. Devi PM, L Rajesh Singh, Sharma PS, Singh AM, Singh MK, Singh YM. Clinico-hematological Profile of Pancytopenia in Manipur, India. Kuwait Med J 2008;40:221-4. 22. Weinzierl EP, Arber DA. Bone marrow evaluation in new-onset pancytopenia. Human pathology. 2013;44:1154-64. 23. M Montalto, I Porto, A Gallo, C Camaioni, RD Bona, A Grieco et al. Clopidogrel-Induced Neutropenia after Coronary Stenting: Is Cilostazol a Good Alternative? International Journal of Vascular Medicine. 2011;2011. 24. Uz O, Kardesoglu E, Aparci M, Yiginer O, Isilak Z, Ozmen N. Nonfatal aplastic anemia associated with clopidogrel/Klopidogrel ile iliskili olumcul olmayan aplastik anemi. The Anatolian Journal of Cardiology (Anadolu Kardiyoloji Dergisi). 2010;10:291-2. 25. Chan J, Sanders DC, Du L, Pillans PI. Leflunomideassociated pancytopenia with or without methotrexate. Ann Pharmacother 2004;38:1206-11. 26. EC Gordon Smith. Aplastic anemia and pure red cell aplasia. In: Anna Porwit, Jeffrey McCullough, Wendy N. Erber, editors. Text Book of Blood and Bone Marrow Pathology, 2nd ed. Britain: Churchill Livingstone Elsevier; 2011. 213-24.

14. Santra G, Das BK. A cross-sectional study of the clinical profile and aetiological spectrum of pancytopenia in a tertiary care centre. Singapore Med J 2010;51:806-12.

27. Khodke K, Marwah S, Buxi G, Yadav RB, Chaturvedi NK. Bone marrow examination in cases of pancytopenia. J Indian Acad Clin Med 2001;2:55-9. 28. Angela Dispenzieri, Martha Q. Lacy, Philip R. Greipp. Multiple Myeloma. . In: Jonathan W. Pine, editor. Text Book of Wintrobe’s Clinical Hematology, 12th ed. Philadelphia: Lippincott Williams & Wilkins; 2009. 2372-438.

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13. Aouba A, Noguera ME, Clauvel JP, Quint L. Haemophagocytic syndrome associated with plasmodium vivax infection. Br J Haematol. 2000; 108: 832-833.

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29. Plasma cells neoplasms. In: Barbara J Bain, David M Clark, Bridget S Wilkins, editors. Text Book of Bone Marrow Pathology, 4th ed. UK: Wiley-Blackwell; 2010. 421-60.

35. Bailey FA, Lilly M, Bertoli LF, Ball GV. An antibody that inhibits in vitro bone marrow proliferation in a patient with systemic lupus erythematosus and aplastic anemia. Arthritis Rheum 1989;32:901-5.

30. Ballard HS. The hematological complications of alcoholism. Alcohol health and research world 1997;21:42-52.

36. Roffe C, Cahill MR, Samanta A, et al: Aplastic anaemia in systemic lupus erythematosus: A cellular immune mechanism? Br J Rheumatol 1991;30:301.

31. CF Weston and MJ Hall. Pancytopenia and folate deficiency in alcoholics. Postgrad Med J 1987;63:117â&#x20AC;&#x201C;120.

37. Sumimoto S, Kawai M, Kasajima Y, Hamamoto T. Aplastic anemia associated with systemic lupus erythematosus. Am J Hematol 199;38:329-31.

32. Aziz T, Ali L, Ansari T, Liaquat HB, Shah S, Ara J. Pancytopenia: Megaloblastic anemia is still the commonest cause. Pak J Med Sci 2010;26:132-6.

38. Abrams EM, Gibson IW, Blydt-Hansen TD: The concurrent presentation of minimal change nephrotic syndrome and aplastic anemia. Pediatr Nephrol 2009;24:407.

33. Ganti AK, Shonka NA, Haire WD. Pancytopenia due to iron deficiency worsened by iron infusion: a case report. J Med Case Reports. 2007;1:1.

39. Kritharis A, Bastola S, Zhang X, Scott Z. Idiopathic Autoimmune Pancytopenia Treated With Rituximab. Available at: http://www.northshorelij.com/ ccurl/341/39/Kritharis.pdf

34. Weinzierl EP, Arber DA. The Differential Diagnosis and Bone Marrow Evaluation of New-Onset Pancytopenia. Am J Clin Pathol 2013;139:9-29.

40. Tariq M, Khan NU, Basri R, Said A. Aetiology of pancytopenia. Professional Med J 2012;17:252-6.

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Original Article Histopathological Study of Villous Morphology in Spontaneous First Trimester Abortions Archana Shetty and Aparna Narasimha* Department of Pathology, Sapthagiri Institute of Medical Sciences and Research Center, Bangalore, India Keywords: Chorionic Villi, Fibrosis, Vascularity, Stromal Fibrosis

ABSTRACT Background: First trimester abortions are seen in 10 – 20 % of pregnancies. Sending the tissue evacuated after miscarriage for histopathological examination is a topic of debate till date, as some professionals feel it is a waste of time and expensive, while on the other hand a school of thought still persists on doing the same. Methods: We studied the various histopathological changes seen in the abortus tissue in first trimester spontaneous abortions over a period of over a period of one year. A total of hundred slides and requests for histopathological examination of first trimester abortions were retrieved and studied in detail by two pathologists for the following featuresVillous size, contour, vasculature, trophoblastic proliferation, perivillous and intervillous hemorrhage, perivillous fibrin deposition, stromal fibrosis, inflammation and decidual change. The observed changes were also categorized according to the weeks of abortion. Appropriate statistical tests were employed. Results: Our study showed many dysmorphic features in the villi like reduced vessels per villous (72%), fibrosis (21.3%), hydropic change (32%) and abnormal trophoblastic proliferation (49%). Other features noted were villous hydrops, inter and perivillous fibrin deposition, inflammation, decidual change and Arias Stella reaction. Reduced vessels per villi and hydropic change were significantly associated with abortions happening at 8 - 10 weeks, while reduced patency of vessels, abnormal villous contour, ghost villi and trophoblastic proliferation were more associated with earlier dated abortions. Conclusion: Cases with dysmorphic features as seen in the present study are known to be associated with clinically significant conditions like diabetes, eclampsia and with certain chromosomal abnormalities. Such cases can not only be filtered for cytogenetic work up, but documentation of these features can also aid in counselling and planning of future pregnancies. Thus histopathological examination of abortus material is highly recommended.

*Corresponding author: Dr. Aparna Narasimha, No. 22, “Moyenvilla”, Moyenville Road, Langford Town, Bangalore – 25, INDIA Phone: +91 9632140850 Email: sonrichie14@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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Villous Morphology in 1st Trimester Abortions

Introduction

The commonest complication of pregnancy is abortion, its incidence being around 15%.[1] Though genetic aberrations are associated with higher frequency of spontaneous abortions the exact etiology is still unknown.[2] The development of fetus and placenta occurs simultaneously and hence fetal developmental abnormalities will be reflected with the changes observed in the foetal part of the placenta.[3] This aberrant growth of the placenta is recognized by the uterus as abnormal and thus it expels such products giving rise to spontaneous abortions. Thus the study of histological aspects of chorionic villi in spontaneous abortions may be imperative in understanding the etiology and pathogenesis of abortions.[4] Our study aims to explore the various histological changes occurring in the chorionic villi in first trimester abortions.

Materials and Methods

Around 100 cases of curetted material of first trimester abortions sent for histopathological examination were studied from January 2015 to December 2015. Relevant clinical history was obtained from the requisition forms sent along with the curetted material. The specimens underwent routine histopathological processing and were stained with Haematoxylin and Eosin stains. Special stains were employed wherever necessary. The slides were examined by two pathologists independently. The following histological parameters suggestive of villous maldevelopment or degeneration along with a few other features were studied and categorized. [5, 6] 1. Villous size- small, intermediate, large 2. Villous contour – round, irregular, scalloping 3. Villous vasculature Grade I: normal (8- 10vessels per villous). Vessels with nucleated blood cells are present in almost every (at least nine of 10) villus, have a very clear appearance and are located centrally as well as peripherally (in contact with the trophoblastic layer). In some villi, the number of vessels are even numerous (>5).

Grade IIA: mild hypoplasia (5 – 8 vessels per villous). Vessels with nucleated blood cells are not present in all villi, less numerous and predominantly located centrally.

Grade IIB: severe hypoplasia ( 3 – 4 vessels per villous). Villi are predominantly avascular, however, in a single villous, a vessel is present with one or more nucleated blood cells.

Grade III: avascular < 3 vessels per villous. All villi are avascular, although sporadically a very small

vessel, with or without a nucleated blood cell may be present.[4] 4. Trophoblastic proliferation 5. Perivillous and intervillous haemorrhage 6. Perivillous fibrin deposition – none , few , intermediate, abundant, 7. Ghost villi 8. Hydrops : marked villous edema > 25% of villi 9. Stromal fibrosis: grade 1 < 6 % grade II > 6% 10. Hoffbauer macrophages 11. Arias Stella Reaction 12. Decidualised tissue 13. Inflammation Exclusion Criteria: Curetted samples of spontaneous first trimester abortions, having risk factors and /or associated or with proven morbid conditions like diabetes, hypertension, infections etc were excluded from the present study. Statistical Analysis: Descriptive and inferential statistical analysis was carried out in the present study. Results on continuous measurements were presented as Mean ± SD (Min-Max) and results on categorical measurements were presented in Number (%). Significance was assessed at 5% level of significance. P value < 0.5 was considered significant. The following assumptions on data were made. Assumptions 1. Dependent variables are normally distributed. 2. Samples drawn from the population are random. Chisquare/Fisher Exact test has been used to find the significance of study parameters on categorical scale between two or more groups. Statistical Software: The Statistical software namely SAS 9.2, SPSS 15.0, Stata 10.1, MedCalc9.0.1, Systat 12.0 and R environment ver.2.11.1 were used for the analysis of the data and Microsoft word and Excel were used to generate graphs, tables etc.

Results

A total of 100 cases were retrieved, of which 25 were excluded. The cases excluded were those of voluntary medical termination of pregnancy (MTP), cases which on microscopy showed no villi, and which had areas of extensive hemorrhage and necrosis to the extent of obscuring the morphology. The age group of the females ranged from 18 to 37 years, with 77.3% falling in the 20 – 30 age group. The types of abortions were as categorized as follows [Table 1].

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The patients were categorized on the basis of weeks of abortions as follows.

features were compared with the weeks of abortion [Table 3 and Table 4 ].

Histopathology: Sizes of majority of the villi were intermediate (47%) with large villi seen in 17.3% of the cases and the rest being small sized villi.[Figures 1A, 1B and 1C].

Surprisingly, 72 % of the villi had less than 3 vessels per villi. Intervillous haemorrhage was seen in 44 % of the villi. A total of 21.3% of the villi showed abundant fibrin deposition. [Fig 2A and 2B]. The fibrin deposition was further highlighted using Masson’s Trichome stain.

Regular villous contour was seen in 54.7% of the villi. Ghost villi were seen in 50.7% with hydropic change in 32% of the cases. Increased trophoblastic proliferation was seen in 49% of cases. Decidual change was seen in 53.4% of cases. Significant stromal fibrosis was seen in 23.5% of the cases.

When categorized the changes according to the weeks of abortion it was seen that reduced vessels per villi and hydropic change [Fig 8A] were predominantly features of abortions seen during 8 – 10 weeks, while reduced patency of vessels, abnormal villous contour, ghost villi and trophoblastic proliferation were predominantly seen in earlier dated abortions. However, there was no statistically significant co- relation between the weeks and variables. Other features observed were increased Hoffbaeur cells [Fig 8B] in about 34.6% of cases, Arias -Stella reaction, decidualised tissue and endometrial glands.

Severe inflammatory reaction comprising of a mixture of neutrophils along with lymphocytes and plasma cells were seen in 5.9% of the cases. Ghost villi or hyalinized villi were seen in 50.7% of the cases. The various morphological Table 1: Types of abortion. Abortion type Incomplete Abortion Missed abortion Inevitable abortion Total

% 88 9.3 2.7 100

TABLE 2:Categorization of the abortion according to weeks: Weeks of abortion <6 6-8 8-10 10-12 Total

% 22.7 36 28 13.3 100

Table 3: Correlation of weeks of abortions and study morphological variables. Variables

Weeks of Abortion

No. of patients (n=75)

<6.0 (n=17)

6.0-8.0 (n=27)

8.0-10.0 (n=21)

10.0-12.0 (n=10)

P value

Size (mm) •

Intermediate

47(62.7%)

9(52.9%)

14(51.9%)

17(81%)

7(70%)

•

Large

13(17.3%)

2(11.8%)

6(22.2%)

3(14.3%)

2(20%)

•

Small

15(20%)

6(35.3%)

7(25.9%)

1(4.8%)

1(10%)

2(2.7%)

2(9.5%)

0.21

Vessels per Villi •

8-10

•

5-8

•

3-4

19(25.3%)

5(29.4%)

9(33.3%)

4(19%)

1(10%)

•

54(72%)

12(70.6%)

18(66.7%)

15(71.4%)

9(90%)

0.05

Contour •

Nil

•

Regular

•

Scalloped

1(1.3%)

1(5.9%)

41(54.7%)

8(47.1%)

14(51.9%)

13(61.9%)

6(60%)

33(44%)

8(47.1%)

13(48.1%)

8(38.1%)

4(40%)

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Variables

Villous Morphology in 1st Trimester Abortions Weeks of Abortion

No. of patients (n=75)

<6.0 (n=17)

6.0-8.0 (n=27)

8.0-10.0 (n=21)

10.0-12.0 (n=10)

P value

Haemorhage •

Perivillous

17(22.7%)

5(29.4%)

6(22.2%)

4(19%)

2(20%)

•

Intervillous

33(44%)

8(47.1%)

11(40.7%)

10(47.6%)

4(40%)

0.6

Fibrin Deposits •

Nil

2(2.7%)

1(3.7%)

1(10%)

•

Fewer

21(28%)

6(35.3%)

7(25.9%)

5(23.8%)

3(30%)

•

Intermediate

24(32%)

3(17.6%)

10(37%)

9(42.9%)

2(20%)

•

Abundant

16(21.3%)

3(17.6%)

5(18.5%)

6(28.6%)

2(20%)

0.4

Table 4: Correlation of weeks of abortions and other findings. Other findings

Weeks of Abortion

No. of patients (n=75)

<6.0 (n=17)

6.0-8.0 (n=27)

8.0-10.0 (n=21)

10.0-12.0 (n=10)

P value

Ghost Villi •

Nil

37(49.3%)

10(58.8%)

11(40.7%)

11(52.4%)

5(50%)

•

38(50.7%)

7(41.2%)

16(59.3%)

10(47.6%)

5(50%)

•

0.7

Hydropic change •

Nil

51(68%)

11(64.7%)

20(74.1%)

14(66.7%)

6(60%)

•

24(32%)

6(35.3%)

7(25.9%)

7(33.3%)

4(40%)

•

0.6

Stromal Fibrosis •

Nil

30(40%)

13(76.5%)

10(37%)

4(19%)

3(30%)

•

<6%

21(28%)

3(17.6%)

13(48.1%)

2(9.5%)

3(30%)

•

>6%

24(32%)

1(5.9%)

4(14.8%)

15(71.4%)

4(40%)

<0.001

Decidual change •

Nil

35(46.7%)

5(29.4%)

11(40.7%)

11(52.4%)

8(80%)

•

38(50.7%)

12(70.6%)

15(55.6%)

10(47.6%)

1(10%)

•

2(2.7%)

1(3.7%)

1(10%)

Fig. 1: Microphotography showing varying sized villi a) small b) medium and c) large (H&E, X100).

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Fig. 2: Microphotograph showing a) intervillous hemorrhage b) perivillous fibrin deposits (H&E,X100).

Fig. 3: Microphotograph showing fibrosis- arrows a) (H&E, X100) b) Massonâ&#x20AC;&#x2122;s trichome stain (MTS, X100).

Fig. 4: Microphotograph showing a )Hofbauer macrophages b) large hydropic villi (H&E,X100).

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Villous Morphology in 1st Trimester Abortions

Discussion

An early pregnancy loss or first trimester miscarriage is the most common complication of human reproduction, with an incidence ranging between10 - 20 % of all conceptions. [5, 6] In most centers it is a routine practice to send the tissue obtained from uterine evacuation after miscarriages for histopathological examination, not only for confirmation of pregnancy but also for medico legal purposes and for ruling out ectopic and molar pregnancies which necessitate special follow up.[5] However till date both school of thoughts pertain, stating the necessity and no necessity of histopathological examination following abortions. The main morphological villous criteria used to investigate early pregnancy loss are regularity of the villous contour, stromal oedema or fibrosis, foetal vasculature and intervillous fibrin deposition.[6] Irregular villous contour with small villous size are often related to trisomies. In the present study also small villi were seen in 20% of the cases.[7] Large bulbous villi are characteristic of first trimester abortions. Hydropic villi, poorly vascularised villi, villous haemorrhages and increased syncitial knotting points to abnormal villous shapes, usually found under hypoxic conditions.[8] Although perivillous fibrin is common, intravillous fibrin is abnormal, seen in diabetic pregnancies. [8] Fibrosis is a final common pathway for nearly all forms of diseases that progress towards end organ failure. The resident cells can often stimulate factors producing basement membrane material and cellular matrix molecules like collagen type I, II and 1V fibronection and proteoglycans. [9] Fibrosis is a feature associated with pre- eclampsic and diabetic pregnancies. Edematous villi can be seen in infections like syphilis, CMV and a variety of cases of hydrops. Extensive stromal fibrosis can be seen in cases of intra uterine growth retardation. [8] Cross section of the vessels in first trimester villi on an average showed 8 â&#x20AC;&#x201C; 10 vessels per villous. The cases in our study showed significantly reduced number of vessels per villous. The reduced villi may be either due to their de novo poor formation or secondary to the fibrosis as mentioned above. Also vascular compromise is a key factor in contributing to miscarriage. Studies have documented the differences in vascularity between villi of spontaneous versus induced abortions. [9] The poor villous vasculature can also explain the stromal oedema, which results from accumulation of water normally drained by the trophoblast before it becomes non â&#x20AC;&#x201C; functional. [6] Vascular compromise was a key feature in our study. Vascular changes in first trimester chorionic villi are due to defective vasculogenesis than due to post-mortem changes as proven by studies done by Hakvoort et al.[10] Reduced vessels per villi was the most consistently seen feature in our study also. A detailed study of the role of

Hoeffbaeur cells in abortions have documented their increase in number in cases of missed abortions.[11] The limitation of our study to count the exact number of macrophages was that we have not employed the immunohistochemical marker CD68. Their identification was only based on the routine Hand E stain Cytogenetics and Villous Morphology

Chromosomal abnormalities are known to be associated with abortions. Trisomies are related to smaller villous size, reduced capillaries per villi and increased perivillous fibrin deposition Villous hydrops is also associated with trisomy. [7] But cytogenetic evaluation of miscarriage specimens is expensive and not always successful. [10] A study on karyotyping of abortus villi by Genest et al [7] has proved that although spontaneous first trimester abortions do have abnormal histomorphological features, not many were diagnostic of a particular abnormal karyotype. [12] In our study we have observed that some morphological features were seen consistently in all abortions like reduced villous size, compromised vasculature and stromal fibrosis. Studies have proven that dysmorphic villous features although not specific, can still be indicators of chromosomal abnormalities. Although histopathological examination may not be mandatory, documentation of these abnormal features may filter cases for cytogenetic evaluation and other special work up. Specimens of recurrent abortions have features of chronic inflammation. These many have associated genetic and also non genetic abnormalities like abnormalities in tryptophan metabolism. Factor V mutation and Antiphospholipid antibody syndromes.[13] Chronic intervillositis is associated with recurrent foetal demise.[14] Histopathological examination of abortions achieves following goals--confirmation of an intrauterine pregnancy, exclusion of hydatidiform mole, recognition of phenotypic clues that warrant a complete cytogenetic study.[7] Review of 670 cases at Charing cross hospital of London , Pathology department showed 120 (18%) of patients with unsuspected molar pregnancy were diagnosed as moles on account of their abundant trophoblast in early pregnancy or by presence of hydrops, thereby favoring histopathological examination of the abortus material.[15] However till date here is no general agreement about the value of submitting tissue for histopathological examination [HPE]. One explanation of poor predictive value of histology is because the features assessed are variable and there is poor inter and intra observer reproducibility. Histopathological features of partial hydatidiform mole overlap with spontaneous abortions harbouring trisomies. [12] Another postulated reason for poor predictive value of histology would be the retention time of products in the

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Shetty et al. uterine cavity for about 3 -8 weeks after foetal demise, leading to post mortem artifacts. [11,12]

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1. Crum, Christopher P. “The Female Genital Tract” In Ramzi S. Cotran, Vinay Kumar, and Tucker Collins: Robbins Pathologic Basis of Disease, 8th ed. Philadelphia: W.B. Saunders, 2007:1079- 80. 2. Kalousek DK, Law AE. Pathology Of spontaneous abortion In Dimmick JE, Kalousek DK: Developmental Pathology of the embryo and Fetus: Phiiladelphia: JB Lippincot, 1992: 62-68. 3. Regan L, Clifford K. Sporadic and recurrent misccairage In Geofferry Chamberlain, Philip J Steer. Turnbull’s Obstetrics. 3rd edition Philadelphia: Churchill Livingstone 2001: 17 – 25.

4. Novak RF. A brief review of anatomy, Histology and ultrastructure of the full term placenta. Archives of Pathology and Laboratory Medicine 1991;115:654- 9. 5. Alsibiani SA. Value of Histopathologic Examination of Uterine Products after First-Trimester Miscarriage. BioMed Research International, 2014;1 :1- 5 6. Jauniaux E, Burton GJ. Pathophysiology of histological changes in early pregnancy loss. Placenta .2005;26:114-23. 7. Genest DR, Roberts D, Boyd T, Bieber FR. Fetoplacental histology as a predictor of karyotype: a controlled study of spontaneous first trimester abortions. Hum Pathol. 1995; 26: 201-9. 8. Ventura F, Rutigliani M, Bellini C, Bonsignore A, Fulcheri E. Clinical difficulties and forensic diagnosis: histopathological pitfalls of villous mesenchymal dysplasia in the third trimester causing foetal death. Forensic Sci Int. 2013;10: 229(1-3): e35–e41 9. Haque AU, Siddique S, Jafari MM, Hussain I, Siddiqui S. Pathology of Chorionic Villi in Spontaneous Abortions .International. Journal of pathology 2004;2: 5–9. 10. Hakvoort RA, Lisman BA, Boer K, Bleker OP, van Groningen K, van Wely M, Exalto N. Histological classification of chorionic villous vascularization in early pregnancy. Hum Reprod. 2006;1 :1291-4. 11. Karakaya YA, Ozer E. The role of Hofbauer cells on the pathogenesis of early pregnancy loss. Placenta. 2013;34: 1211-5. 12. Norris-Kirby A, Hagenkord JM, Kshirsagar MP, Ronnett BM, Murphy KM. Abnormal villous morphology associated with triple trisomy of paternal origin. J Mol Diagn 2010;12: 525-9. 13. Redline RW, Zaragoza M, Hassold T Prevalence of developmental and inflammatory lesions in non molar first-trimester spontaneous abortions. Hum Pathol. 1999; 30 :93-100. 14. Weber MA, Nikkels PG, Hamoen K, Duvekot JJ, de Krijger RR. Co-occurrence of massive perivillous fibrin deposition and chronic intervillositis: case report. Pediatr Dev Pathol. 2006; 9:234-8. 15. Fram KM. Histological analysis of the products of conception following first trimester abortion at Jordan University Hospital. Eur J Obstet Gynecol Reprod Biol. 2012;105:147-9. 16. Heath V, Chadwick V, Cooke I, Manek S, MacKenzie I Z. “Should tissue from pregnancy termination and uterine evacuation routinely be examined histologically?” British Journal of Obstetrics and Gynaecology 2000; 107:727–30.

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Study by Heath et al recommended HPE only when pre -operative diagnosis was uncertain or trophoblastic tissue was seen during evacuation. [16] In Asia the incidence of Hydatidiform mole is as high as 1 in 80 pregnacies, unlike the western countries, highlighting the importance of a routine HPE of the abortus tissue.

Recommendations

The cytogenetic analysis of spontaneous first trimester abortions, showing the above mentioned dysmorphic features on histopathological examination will be taken up as the next part of the study after obtaining the patients consent.

Conclusion

Our study has analyzed many dysmorphic villous features which may be the effect or the cause of the first trimester abortions. Histopathological examination of abortus material not only confirms the documentation of pregnancy, but can also pick up unsuspected molar pregnancies, and gives clue to associated with clinical conditions like diabetes and eclampsia. Various dysmorphic features like small villous size and reduced vessels per villi, helping to filter the cases for cytogenetic evaluation, which on the other hand is expensive and not easily affordable by patients in a developing country like ours. More standardization is required on the aspect of submitting the aborted material for histopathological examination.

Acknowledgements

We would like to thank the staff of the Department of Obstretics and Gynaecology for regularly sending us the required material for our study.

Funding None

Competing Interests None Declared

References

Original Article Prognostic Significance of Ki 67 Labelling Index and P63 Immunoreactivity in Intra Cranial and Intra Spinal Meningiomas Shrinivas. B Somalwar*, Naval Kishore Bajaj, Sujani Madabhushi and Ezhil Arasi Dept. of Pathology, Osmania Medical College and Hospital, Hyderabad, India Keywords: Meningeal tumors, Meningiomas, Cytology, Smear, Tumor grade, Ki67, P 63

ABSTRACT Background: Meningiomas are indolent tumors and their grading is based on histopathological parameters which have inherent limitations. Adjunct immunohistochemical markers are often used to overcome these limitations. Objective: To investigate the role of Ki67 and P63 immunoexpression in various grades of meningiomas. Methods: A retrospective analysis of 144 biopsies of intracranial and spinal meningothelial and nonmeningothelial tumors operated from January 2011 to May 2016 was carried out. Intraoperative squash smears were stained by 1% aqueous toluidine blue and rapid H&E method. Ki67 and p63 immunohistochemical staining was performed on paraffin sections of 125 histologically proven meningiomas cases. Grading was done according to the modified 2016 World Health Organization classification of CNS neoplasms .The results were analysed using statistical methods. Results: The total number of biopsy samples received was from144 cases. There was a female preponderance accounting for 68%of total cases. Positive cytohistological correlation was seen in 86.14% (125/144 cases). 112 out of 125 cases were found to be WHO grade I tumors out of which 20 showed a high Ki-67 LI and p63expression. 6 out of 7 cases of WHO Grade II meningiomas showed strong ki 67 and p63 nuclear expression whereas 3 anaplastic meningioma cases out of 6 WHO grade III meningiomas showed strong ki 67 and p63 nuclear positivity . Conclusion: The expression of P63 is variable in different grades of meningiomas. There was statistically significant increase of P63 protein expression and Ki 67 LI between the grades I to II and grades II to grade III. In the present study it was observed that 20/112 grade I meningiomas expressed high p 63 immunoreactivity contrary to other studies. High grade meningiomas showed increased Ki67 labelling index. P63 immunoexpression was high with predictability of recurrence across all the grades.

*Corresponding author: Dr Shrinivas B Somalwar, 3-4-756/1, Flat no 303, Sai Raghavendra Apartments, Barkatpura, Hyderabad. 500027, INDIA Email: somalwar73@gmail.com

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

Somalwar et al.

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Introduction

and subsequently resected tumor tissue and stained using routine H & E method. A total of 144 cases received over a five year period from Jan 2011 to May 2016 were analyzed A total of 144 cases were reported as meningiomas on squash cytosmears. 125/144 cases had cyto-histological correlation remaining 19 cases had varied diagnosis. A total of 130 cases were reported as meningiomas Available clinical data, including patient age, sex, clinical and radiological findings were retrieved from the records. Ki67 and P63 IHC was carried out on 125 cases of histologically proven meningiomas. Five cases were omitted from the present study for various technical reasons

Meningiomas constitute 24 to 30% of all the Central Nervous System tumors.[1]They are known to have a female preponderance and are more commonly seen in patients who are in the fourth decade of their life.[2] The current World Health Organization (WHO) 2016 classification has identified 15 histopathological subtypes of meningiomas ,which have been graded on the basis of their recurrence potential and net growth rate.[3] Morphological features such as increased mitoses, geographic necrosis, and invasion into the brain parenchyma are not always reliable, in representing the overall biologic nature of lesion.Inspite of total resection 7-20% of benign (Grade I), 29- 40% of atypical (Grade II), 50-78% of anaplastic (Grade III) meningiomas are known to recur.[4] All radiologically and clinically suspected meningeal tumors may at times histopathologically prove to be rare non meningothelial tumors of meninges. Their histological recognition is a major concern as it has an effect on the clinical outcome and further treatment modality. Several immunohistochemical (IHC) biomarkers such as Ki-67[1,7,8] P63,[9],Claudin[6] , and the absence of progesterone receptors have been proposed to assist conventional methods of tumour grading.[5] Use of one single IHC marker has often been found to be inadequate in definitively predicting the grades of these meningiomas. P63 is a structural homologue of the p53 tumor suppressor gene has been suggested to have a role in oncogenesis[7] . P63 is not only involved in maintaining epithelial stem-cell populations but also known to play an important role in cellular differentiation and neoplasia. So far p63 has been frequently used in the diagnosis of prostate and breast carcinoma.[8,9] Additionally the p63 protein has been found to have a strong association with squamous-cell carcinoma.[9] In the available literature only few studies [10] have used p63 immunostaining along with Ki-67 LI expression in various grades of meningiomas. We thus report this study which was carried out to assess the correlation between p63 and Ki 67 immunoexpression across all grades of meningiomas.

Material and Methods

In this retrospective study squash cytosmears were prepared from the fresh biopsy samples of intracranial and intraspinal meningothelial and non meningothelial lesions sent to the Department of Neuropathology, Osmania Medical College for intraoperative pathological consultation. The squash smears prepared were stained using 1% aqueous toluidine blue and rapid H&E method. Paraffin block sections were prepared from the residual www.pacificejournals.com/apalm

Ki-67 and P63 Immunohistochemistry Technique: Sections of 4-micron thickness using Leica 2255 microtome collected on poly-L-lysine coated slides were subjected to IHC by routine indirect immunoperoxidase technique using primary antibody (Ki67, monoclonal antibody, DakoCytomation, Glostrup Denmark, dilution 1;50) and p63 (clone; Dak-p63 DakoCytomation, Glostrup Denmark dilution 1:50), followed by incubation in secondary antibody for 30 min (Polymer link detection kit, DakoCytomation, Glostrup, Denmark).At least 1000 tumor nuclei were counted at high magnification (40Ă&#x2014; objective) without recounting same areas and the average was expressed as percentage by two trained pathologist. Foci of hemorrhage and necrosis were excluded. Appropriate positive and negative controls were used for both the antibodies. Myoepithelial cells of the breast and basal cells of prostate were taken as positive controls. The primary antibody was omitted in negative controls. Immunoreactivity of p63 was scored semi quantitatively based on nuclear positivity within neoplastic cells as, Grade 0 : no staining, Grade 1+: <10% cells, Grade 2+ : 10 -50 % cells, Grade 3+: >50% cells. Immunoreactivity for Ki-67 was assessed by counting at least 1000 tumor nuclei at high magnification (40x) without recounting the same area and the average was expressed as percentage. Foci of necrosis were excluded Statistical Analysis: The relationship between P63 expression and the Histological grades were analyzed by using ANOVA, independent t-test and by Chi-square test using SPSS 14.1 version. The results were considered statistically significant if the P value was <0.05.

Results

This study encountered 19 discordant cases on squash cytology out of a total 144 CNS samples. They had varied histopathological diagnosis. 4 tumors were diagnosed as hemangiopericytoma, 3 as schwannoma, 2 as hemangioblastomas,1 case each of hemangioma, PNET, eISSN: 2349-6983; pISSN: 2394-6466

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Ki 67 Labelling Index and P63 Immunoreactivity in Meningiomas

mesenchymal chondrosarcoma and neuricysticercosis.5 cases of non meningeal tumors were histologically confirmed as meningiomas. In 125 histologically proven meningiomas the demographic distribution was found to be statistically non significant with a female preponderance 85 (68.1%) vs 40 ( 31.9%) males. There were 108 (86.4%) intracranial meningiomas as compared to 17(13.6%) intraspinal meningiomas. The age of the patients ranged from 4-75 years including 7 children below the age of 18years. The maximum number of cases were seen in the age group of 40-49 years (35.4%) followed by 30-39 years (18%).The least number of cases were seen in patients of age group 0-9 years ( 1%).

rhabdoid features was 22.33 and a lone case of anaplastic meningioma expressed mean of 97.16 being the highest in our series. Thus meningothelial meningiomas in WHO grade I category and tumors in WHO grade II and grade III categories showed higher P63 expression.

Geographically 62% meningiomas were located on brain convexities, 15% at skull base, 5%at Cerebello-pontine angle, 5%of cases at various locations and the remaining 13%cases at spinal location.

Meningiomas are common in middle aged woman with only few cases being reported in males and pediatric age group. Age and Gender of the patient has no influence on the proliferative activity and recurrence potential of meningiomas. Our series noted no statistical significance of Ki67 and P63 expression with respect to age and gender. Recurrence was noted in only four cases, three of which were males and all four recurred meningioma cases were located in the skull base. Tumors occurring in other locations showed no statistical significance across various grades with respect to Ki67 LI and P63 immunoexpression. Other studies compared to in literature either used P63 or Ki67immunoexpression as an adjunct to conventional histopathological grading techniques. This study has used a combined grading system of P63 and Ki67 immunoexpression along with conventional grading method as per classification of CNS WHO tumor guidelines in all the 125 cases.

Histological grading of meningiomas was done according to WHO CNS tumor classification.112 out of 125 tumors were classified as WHO grade I of which meningotheliomatous subtype being the commonest accounting for 27%,transitional 26% ,fibroblastic 25%,psammomatous 10%,angiomatous 4%,microcystic 0.8%,secretory and metaplastic 1.6% each. 7(5.6%) tumors were classified as WHO grade II. Six cases in this grade were diagnosed as atypical meningiomas which infiltrated the brain parenchyma as depicted in figure G and figure H.A single case of clear cell variant of meningioma was also reported. Grade III tumors accounted for 6 (4.8%) cases contributed by papillary, rhabdoid and anaplastic meningiomas characterized by high degree of anaplasia, foci of necrosis and its infiltrative nature. It was observed that mean P63 expression in the 6th decade was highest at 12.5 with total mean in all age groups at 8.13 similarly mean Ki67 LI was at 1.725 being highest in the 6th decade .The mean P63 expression in males was 6.82 and 8.74 in females .The mean Ki67 LI in males was 1.495 and 1.55 in females. Age and sex related correlation for test of significance could not be established. Meningothelial meningiomas were the most commonly occurring grade I tumors with a mean Ki 67 LI of 3.212 whereas secretory, microcystic subtype showed 0.608 mean Ki67 LI. The mean P63 positivity index in the meningothelial meningiomas was highest at 23.06 amongst Grade I category and lowest at 0.234 in secretory micro cystic type respectively. The mean P 63 expression in WHO grade II subtypes of atypical and clear cell meningioma was observed as 92.8. The mean P63 expression in papillary meningioma with

Discussion

A total number of 144 CNS tumor cases undergoing neurosurgical intervention were assessed using routine intraoperative cyto-pathological methods.19 cytohistological discordant tumors were analyzed. Discordance resulted due to varied cytomorphology with increased cellularity, vascularity and technical limitations.

In the present study84% meningiomas in WHO grade I category did not show P63 immunoexpression, tumors in meningotheliomatous meningiomas subtype expressed more than 10-50% of nuclear positivity in the same group. All the meningiomas in WHO grade II and WHO grade III category showed strong P63 and Ki 67 nuclear expression. Value of significance between by using Independent t-test between grades I and II, grades II and III for P63 and Ki67 immunoexpression are 0.0001 and 0.001 respectively. Whereas comparison between grades I and III was 0.001 and 0.243 respectively, thus the p value is significant when meningiomas in WHO grade I category were compared with tumors in WHO grade II & WHO grade III categories. In this present study four meningioma cases out of a total of one twenty five meningioma cases recurred clinically. A total of twenty meningothelial meningioma cases out of 112 WHO grade I meningiomas showed a high Ki-67 LI and P63immunoexpression which was also suggested by Guarnaschelli et al [14].Three cases were histologically

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categorized to WHO grade I with minimal atypia and no evidence of brain invasion; however these meningioma cases showed focal hypercellularity. The remaining single case was histologically categorized into WHO grade II.

The mean Ki-67 LI and P63immunoexpression scoring of recurrent meningiomas was 8.6% and 62.5% respectively. Period of recurrence ranged from 2.1 to 9.5 years with a mean of 6 years. Among the recurrent tumors the p value for

Table1: Correlation of P63 and Ki67 with respect to Age, Sex, Histological grade and morphological subtypes: Mean SD Standard Error 95% CI (Lower Statistical P value bound) test Age P63 Ki67 P63 Ki67 P63 Ki67 P63 Ki67 P63 Ki67 1-10yrs

0.0

0.6

0.0

11-20yrs

10.72

1.6

21.36

1.43

8.721

-11.70

21-30yrs

04.76

1.37

11.79

1.42

3.151

-2.04

31-40yrs

7.94

1.43

17.77

1.85

3.094

1.64

41-50yrs

7.66

1.59

16.99

1.8

2.505

51-60yrs

12.5

1.725

25.53

2.11

6.383

-1.10

61-70yrs

8.88

1.65

16.53

1.64

5.848

-4.95

71-80yrs

0.0

1.4

0.0

17.85

1.597

4.97

Total 08.13 Histological subtypes

2.61

independent t-test NA

0.998

Grade 1 Meningothelial

23.06

3.212

25.07 2.398 4.364

0.417 14.17

0.417

Fibroblastic

0.36

0.569

0.903 0.447 0.177

0.036

7.08

1.421

16.23

1.072 2.825

0.093 -0.01 0.204 1.33

0.54

0.510

1.168

0.510 0.268

0.0

-0.03

0.575

0.32

0.63

0.921

0.436 0.212

0.234

0.608

0.212 -0.021 0.139 1.253

Transitional Psammomatous Angiomatous Secretory and Microcystic Grade 2 Atypical and Clear

Anaplastic Total (125 cases) Sex

1.0

4.62

92.8

Grade 3 Papillary with Rhabdoid

0.483 0.197

1.414

1.0

Metaplastic

0.781

97.16 8.13

0.301

11.23

17.86

4.219

190.3

0.673

-11.71

21.36

13.79

22.33

1.005

1.597

4.97

Male (40 cases)

6.82

1.495

17.06 1.837 2.698E0

0.290

Female (85 cases)

8.74

1.55

18.28 1.720 1.983E0

0.188

independent 0.56 0.85 t-test

Table 2: Comparison of present study with other studies. Rushing et al[11] (n=37)

Sharifi et al[12] (n=54)

Jain et al[13] (n=85)

Present study (n=125)

GRADE I

51%

35%

16%

GRADE II

92%

71%

62%

100%

GRADE III

75%

100%

64%

100%

Grades

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Ki 67 Labelling Index and P63 Immunoreactivity in Meningiomas

Fig. 1: Variants of Meningioma, (H & E Stain). A:Meningothelial,B: Fibroblastic, C:Transitional, D:Angiomatous, Psammomatous, F: Metaplastic, G&H :Atypical with brain parenchymal invasion, I:Papillary.

Figure cont...

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cont... Figure

Fig. 2:A Grade I Meningothelial Meningioma low Ki67 LI(3%),P63<10%,B: Grade I High Ki67 8% LI and P63(3+) Positivity,C: Grade 2 Atypical Meningioma,Raised Ki67LI(8%), P63(3+),D:Grade 2 Atypical Meningioma Low Ki 67 LI (1%) with high P63(3+),E: Papillary Meningioma,High Ki67 LI(9%) and P63(3+).

Fig. 3: Radiologic images depicting various sites of Meningiomas. A: T2W MRI Saggital section showing fronto-temporal lesion with dural enhancement. B:T1W MRI Coronal section showing parieto-temporal lesion with dural enhancement, C: CT image showing basally located dural enhancement. D:T1W MRI Sagittal section showing parieto â&#x20AC;&#x201C;occipital dural lesion. E:T1W MRI Coronal section showing tentorial enhancement. F: T1W MRI Coronal section showing falx cerebri dural enhancement.

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Ki-67 and p63 were highly significant as 0.0043 and 0.0009 respectively. Remaining 16 meningioma cases of WHO grade I category with high Ki-67 LI and correspondingly high P63 immunoexpression are under follow-up for the past two years and have showed no recurrence till the time of this report.

Conclusion

The present study has made an effort to grade meningiomas by using conventional as well as adjunct immunohistochemical markers P63 and Ki67.The expression of P63 is variable in different grades of meningiomas.p63 alone cannot make a distinction between low and higher grades of meningiomas. High grade meningiomas showed increased Ki67 labeling index and high P63 immunoexpression is a predictor of recurrence.There was statistically significant increase in P63 protein immunoexpression and Ki67LI between the grades hence further molecular characterization and gene profiling studies may define the biological behaviour of meningiomas.

Acknowledgements None

Funding None

Competing Interests

10.

None declared

References

1. Ostrom QT, Gittleman H, Farah P, Ondracek A, Chen Y, Wolinsky Y, et al. CBTRUS Statistical Report: Primary Brain and Central Nervous System Tumors Diagnosed in the United States 2006-2010. NeuroOncol. 2013; 15(2):ii1-ii56. 2. Kasuya H, Kubo O, Tanaka M, Amano K, Kato K, Hori T. Clinical and radiological features related to the growth potential of meningioma. Neurosurg Rev 2006; 29:293-7. 3. Lloyd RV, Kovacs K, Young Jr WF, Farrel WE, Asa SL, Trouillas J, Kontogeorgos G, Sano T, Scheithauer B, Horvath E. Tumours of the pituitary gland. In: DeLellis RA, Lloyd RV, Heitz PU, Eng C, editors.

11.

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14.

World Hearth Organization Classification of Tumours. Pathology and genetics of tumours of endocrine organs. Lyon: IARC Press; 2004. 9-47. Karamitopoulou E, Perentes E, Melachrinou M, et al Proliferating cell nuclear antigen immunoreactivity in human central nervous system neoplasms. Acta Neuropathol 85:316-322, 1993 Nakasu S, Nakasu Y, Nakajima M, Matsuda M, Handa J. Preoperative identification of meningiomas that are highly likely to recur. J Neurosurg 1999; 90:455-62 Hahn HP, Bundock EA, Hornick JL Immunohistochemical staining for claudin-1 can help distinguish meningiomas from histologic mimics. Am J Clin Pathol. 2006 Feb; 125(2):203-8 Hopf NJ, Bremm J, Bohl J, Perneczky A. Image analysis of proliferating cells in tumors of the human nervous system: an immunohistological study with monoclonal Antibody Ki67. Neurosurg 1994; 35:917-23 LoMuzio L, Campisi G, Farina A. Effect of p63 expression on survival in oral squamous cell carcinoma. Cancer Invest. 2007; 25:466-469. Sharifi N, Katebi M. An immunohistochemical study of p63 protein expression in meningioma. Iranian J Pathol 2008; 3: 146-150. Devaprasath A, Chack G. Diagnostic validity of the Ki-67 labeling index using the MIB-1 monoclonal antibody in the grading of meningiomasNeurology India2003;51:336-340 Rushing EJ. Correlation of P63 immunoreactivity with tumor grade in meningiomas.Inter J Surg Pathol 2008; 16(1):38-42 Sharifi N, Katebi M. An immunohistochemical study of p63 protein expression in meningioma. Iranian J Pathol 2008; 3:146-150. Jain D. Correlation of P63 protein expression with histological grade of meningiomas: An immunohistochemical study. Inter J Surg Pathol 2012; 20(4):349-354. Guarnaschelli JJ, Stawicki SP. Brief communication: Recurrent brain meningiomas. OPUS 12 Scientist 2008; 2:32-4.

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Original Article Thrombocytopenia in Dengue Illness: Destruction, Suppression and Composite Platelet Index: A Retrospective Study Vani Krishnamurthy*, Rajalakshmi Rajeshakar and Srinivasa Murthy Doreswamy Dept. of Pathology, JSS Medical College, JSS University, Mysuru, Karnataka, India Keywords: Composite Platelet Index, Dengue Fever, Mean Platelet Volume, Platelet Distribution Width, Platelet Indices, Platelet Large Cell Ratio

ABSTRACT Background: Dengue is a common viral illness which can be fatal. Thrombocytopenia is the common hematological abnormality which can result in catastrophic bleeding. The present study attempts to look at the magnitude of different mechanisms causing thrombocytopenia with the help of platelet indices. Methods: This was a retrospective study undertaken in a tertiary care center in south India. Of all the adult patients admitted for the treatment of Dengue illness between June 2013 to May 2014, Platelet indices such as Mean platelet volume (MPV) and Platelet large cell ratio (P â&#x20AC;&#x201C; LCR) of 98 patients were collected and analyzed. Proportion of patients with increased MPV (> 11.5) and P â&#x20AC;&#x201C; LCR (>37) was then computed. Result: Among the dengue patients, 56.1% had raised MPV (>11.5) and 59.2% had raised Platelet large cell ratio (>37). This suggested that more than half of the patients had platelet destruction and the rest had bone marrow suppression. There was significant increase in Mean Platelet Volume (MPV), Platelet Distribution Width (PDW) and platelet Large Cell Ratio (LCR) in Dengue patients compared to the controls. We devised a composite platelet index (CPLI) by multiplying platelet count with MPV which we believe could be a better indicator of bleeding than either alone. In our study we found the median composite platelet index in dengue patients was 5.9 compared to 27. 2 in the controls. Conclusion: Peripheral destruction and bone marrow suppression is seen in equal proportion of patients with dengue illness.

*Corresponding author: Dr Vani Krishnamurthy, 70, Prakruthi, BEML 2nd stage, Rajarajeshwarinagara, Mysuru. INDIA Phone: +91 9980334076 Email: vanidrsri@gmail.com

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Introduction

Dengue is a common epidemic illness seen in both pediatric and adult population. The severity of the disease can vary from mild fever to death. The most common hematological abnormalities observed are hemoconcentration and thrombocytopenia. Thrombocytopenia in dengue is due to multiple mechanisms such as peripheral destruction and bone marrow suppression to a variable degree. Gupta et.al have extensively reviewed dengue infection in Indian scenario [1] Platelet counts below normal value define thrombocytopenia in dengue patients but do not indicate the underlying pathomechanism. Automated analyzers measure various platelet indices such as Mean platelet volume (MPV), Platelet distribution width (PDW) and Platelet large cell ratio (P-LCR), and their clinical usefulness has recently been recognized. [2,3,4] With these indices, it is possible to assess the functional state of the bone marrow. Increased MPV and platelet large cell ratio indicate that the bone marrow is active and releasing the immature larger platelets at a higher rate than usual. Conversely, in thrombocytopenia due to hypo functional bone marrow, these indices remain unchanged. [5] With the help of these indices, we can infer the predominant mechanism of thrombocytopenia operating in dengue patients without bone marrow examination. So far studies have suggested different mechanisms of thrombocytopenia in dengue illness but we do not have the information about the proportion of patients developing thrombocytopenia due to various mechanisms. This study was undertaken to estimate the magnitude of different mechanisms of thrombocytopenia based on the different platelet parameters. Primary objective: To estimate the magnitude of peripheral platelet destruction and bone marrow suppression suggested by depression of two or more cell lines in peripheral blood smear, causing thrombocytopenia in dengue patients. Secondary objectives: 1. To compare platelet indices between dengue patients and controls 2. To compare the gender differences in platelet indices in dengue patients 3. To assess the correlation between MPV and platelet count 4. To derive a composite platelet index for prediction of bleeding in dengue patients

Materials and Methods

Study design: This is a retrospective analytical study undertaken in a tertiary care referral hospital in south India.

Inclusion criteria: Patients who were detected positive for dengue NS1 antigen, IgM, or both IgG and IgM antibodies by Immunochromatography (Standard Diagnostic inc. Bioline, Dengue duo, Gyeibggu-do, Republic of Korea) and who were evaluated for platelet indices were included in the study. Subjects: We screened 368 patients of seropositive dengue cases over a period of one year from June 2013 to May 2014. Out of these, 98 patients satisfying the inclusion criteria were selected. One hundred and sixteen age matched controls were selected from normal healthy persons enrolled in master health checkup. Sample Processing: The blood sample was collected in EDTA vacutainer and transported to the laboratory via chute system. As soon as the sample was received in the laboratory, initial identification formalities were completed and subjected to analysis immediately. The average time taken from collection of sample to start processing is 40 mins in our hospital. The platelet count, PDW, MPV and P-LCR were analyzed by automated analyzer Sysmex XN1000 (Sysmex America, Inc. in Lincolnshire, Illinois) and the results were compared. Data Collection: All the results after approval is stored in hospital database. This was accessed using the inpatient number obtained from medical record section. Composite Platelet index (CPLI). This is a conceptual index which we derived by multiplying platelet count (as decimal) with the Mean platelet volume (e.g. Platelet count of 1.5 X 105 mm3and MPV of 10 fl, CPLI = 1.5 X 10 = 15). The rationale behind this index is, fresh and large platelets would be more functional and hence counter the disadvantage of lower platelet count to certain extent. This means for a given platelet count, higher CPLI indicates higher MPV and hence better function and protection from bleeding. Sample Size: In one of the study, the prevalence of severe neutropenia is reported to be 11.8% among dengue patients. [6] Assuming this to be due to suppression of bone marrow, confidence level of 90%, margin of error of 5%, we needed 113 patients. We had a total of 116 patients who fulfilled the inclusion criteria. However, 18 of them were below 12 years of age and constituted pediatric population. Since we did not have any control in that age group, we excluded them from the study. Thus 98 cases remained in the study. One hundred and sixteen patients from master health checkup who were healthy without any medical or surgical illness and not on any medication that could affect platelet counts. We had a total of 214 subjects.

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Statistical Analysis: Continuous variables were analyzed with Student t test for comparison of means. In case of skewed distributions, the data was summarized as Median and interquartile range. Mann Whitney U test was used for comparison. Categorical variables were reported as proportions. Comparison of categorical variables was done using either Chi square test or Fischer exact test as appropriate. Analysis was done using Microsoft Excel 2013 and SPSS 20. A p value of less than 0.05 was considered statistically significant. Simple linear correlation between platelet count and mean platelet volume was calculated as Pearson product moment correlation.

Our control population had a mean MPV of 9.9 fl, with a standard deviation of 0.81. Mean plus 2 standard deviation yielded a value of 11.5 fl which was considered as upper limit in our study. Similarly, the value for platelet large cell ratio was obtained, which was 37. There were 53.4% of Patients with MPV more than 11.5fl and 56% of patients with P – LCR greater than 37 (Table 3). This was significantly higher than in control population (P = 0.0001). The proportion of patients with thrombocytopenia due to bone marrow suppression was just less than half. The new Composite platelet index (CPLI) was computed by multiplying the platelet count and MPV. The median CPLI in dengue patients was 5.9 and in controls was 27.2 (Fig 1). Comparison of the medians using Mann Whitney U test yielded a Z value of -13.1, U of 0 and P of 0.0001 suggesting statistically significant difference.

Ethics: This study was approved by institutional ethical committee.

Result

Median age of cases was 27 years and that of controls was 31 years. Males were higher in number among cases with 63.8%( Male: Female = 1: 0.57)) compared to control group which was 57.8% (Male: Female = 1: 0.73). Table 1 depicts the baseline characteristics of the study population. Mean platelet count in Dengue patients was 0.53 X105 mm3 which was significantly lower compared to control group (mean 2.84 X 105mm3). Other platelet indices such as platelet distribution width, mean platelet volume and platelet large cell ratio (P – LCR) were significantly lower in the control group. Table 2 depicts the comparison of platelet indices between cases and controls.

Our study did not find any significant difference in platelet indices between males and females. The mean difference in platelet count between the genders was 0.04 X 105, being low in males with a 95% Confidence interval of -0.14 to 0.06. Similarly, the mean difference in platelet distribution width was -0.9 fl (95% CI -0.97 to 0.17), mean platelet volume was zero (95% CI -0.35 to +0.35) and platelet large cell ratio was -0.9 (95% CI -3.62 to 1.82). We did not find any significant linear correlation between platelet count and mean platelet volume in both the cases and the control groups. The correlation ‘r’ was 0.12 in cases and -0.1 in control group.

Table 1: Baseline characteristics. Age Median(IQR) Males, n (%) Females, n (%)

Cases (N = 98) 29.5 (21 – 40.8) 64 (65.3) 34 (34.7)

Control (N = 116) 31 (26 - 38) 67 (57.8) 49 (42.2)

P value 0.234 0.258

Table 2: Comparison of Platelet indices between Dengue cases and controls. Platelet indices Mean (SD)

Cases

Control

Mean diff

95% CI

P value *

Platelet count ( X 105mm3)

0.53 (0.28)

2.84 (0.75)

-2.31

- 2.45 to -2.16

0.0001

Platelet distribution width

15.1 (2.9)

10.8 (1.6)

4.3

3.7 to 4.9

0.0001

Mean platelet volume (Femtolitre)

11.7 (0.97)

9.9 (0.81)

1.8

1.57 to 2.02

0.0001

Platelet large cell ratio

38.3 (7.5)

23.7 (6.3)

14.6

12.8 to 16.3

0.0001

* Student ‘t’ Test. SD - Standard deviation, CI – Confidence interval Table 3: Comparison of proportion of patients with high MPV and P – LCR. Cases (%)

Control (%)

P Value *

MPV > 11.5

54 (55)

4 (3.4)

0.0001

MPV < 11.5

44 (45)

112 (96.6)

P - LCR > 37

56(57)

4 (3.4)

P - LCR < 37

42 (43)

112 (96.6)

0.0001

* Fischer exact test, MPV – Mean platelet volume, P –LCR – Platelet large cell ratio

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Fig. 1: Comparison of Composite Platelet Index in Dengue patients and controls.

Discussion

Dengue fever is commonly associated with thrombocytopenia. [7, 8] Our study also reaffirmed such a finding. Out of the 350 patients screened, 98 (28%) had thrombocytopenia with platelet count of less than 1X105 hence necessitating request for platelet indices. Anti-platelet antibodies are shown to increase during the acute phase of dengue infection resulting in thrombocytopenia. [9] A study by Bashir AB et al on platelet indices in dengue patients had low platelet count, high PDW and high P-LCR. [10] Our study showed similar results. It is well known that the platelets are destroyed stimulating the bone marrow activity. This results in varied sized platelets and hence higher PDW. Hitherto, many authors have suggested multiple mechanisms of thrombocytopenia in Dengue patients. [11, 12] However, there is no sufficient information on the magnitude of different mechanisms of thrombocytopenia in these patients. In our study we have addressed this issue. We found that both peripheral platelet destruction and bone marrow suppression to be equally predominant mechanisms in dengue patients. This could help clinicians in adopting strategies to counter immune mechanism with drugs such as Steroids or using blood products as temporary measure. [13, 14] Several authors have studied the mechanism of thrombocytopenia in experimental and in vitro studies. [15,16,17,18] Viral infection of the non-erythrocytic cells and

release of cytokines which suppress the bone marrow is documented in these studies. In our study we studied this phenomenon with the help of platelet indices. The composite platelet index proposed by us is based on very simple rationale that active bone marrow as noticed by increased MPV could be protective against bleeding in dengue patients. However, this has to be corrected to the platelet count for standardization. In this study we have not correlated this index with bleeding. In future, studies have to be designed to look into this potential predictive indicator, which will throw more light on the utility of this index in regular clinical scenarios. MPV raises in thrombocytopenia due to peripheral platelet destruction. Hence we expected a negative linear correlation between platelet count and MPV. However, in our study we did not find any significant linear correlation between platelet count and MPV. This could be explained by the fact that there could be variable amount of bone marrow suppression even in patients with peripheral platelet destruction. It is interesting to note that corticosteroid use in dengue infection has produced inconsistent results in clinical studies. Extensive review by Rajapakse and team have detailed 12 studies with five of them favoring the use of steroids and rest seven not showing any benefit. [19] This raises the possibility of steroids being effective in a subset of patients who possible have immune mediated thrombocytopenia. Our new index CPLI could be of use to

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categorize these patients with thrombocytopenia as due to peripheral destruction or otherwise. This could set a stage for further evaluation of benefits of steroids in dengue patients. Recently it has been shown that Dengue virus bind to platelet and become more infectious. [20] The role of such a mechanism on platelet indices, and therapeutic value of immunosuppressant is yet to be studied.

3. Elsewefy DA, Farweez BA, Ibrahim RR. Platelet indices: consideration in thrombocytopenia. Egyptian Journal of Haematol. 2014;39:134-8.

In a large retrospective study in New Delhi, it has been shown that there is poor correlation between thrombocytopenia alone and clinical bleeding. [21] Other authors have shown that it is possible to predict bleeding with the help of platelet indices instead of platelet count alone, which are now easily available from most of the analyzer. [4] Our effort is to further fine tune these indices to identify the subset of patients with dengue illness and thrombocytopenia who could develop clinical bleeding.

5. Khaleed JK, Abeer Anwer AA, Maysem Alwash AA. Platelet indices and their relations to platelet count in hypoproductive and hyper-destructive Thrombocytopenia. Karbala J. Med. 2014;7:1952-8.

Limitations: Sample did not include the patients where platelet indices were not requested and hence not available.

Conclusion

Platelet indices suggest that both peripheral destruction of platelets and possible bone marrow suppression play role in causing thrombocytopenia in Dengue patients. In nearly half of the Dengue Patients Mean platelet volume is raised suggesting platelet destruction. In rest of the patients, hematological parameters suggest decreased production which could account for thrombocytopenia. Dengue patients have decreased platelet count, increased MPV, PDW and P-LCR compared to controls. No statistical significance was found between the platelet indices in men and women with dengue infection No linear correlation was found between MPV and platelet count in dengue patients, which suggests variable suppression of the marrow.

Acknowledgements

We thank Dr Manjunath GV, HOD department of Pathology for his encouragement and guidance.

Infection in Red Sea State, Sudan. International Journal of Science and Research. 2015;4:1573-6.

4. Jayashree K, Manasa GC, Pallavi P, Manjunath GV. Evaluation of Platelets as Predictive Parameters in Dengue Fever. Indian J Hematol Blood Transfus. 2011;27:127-30.

6. Thein T-L, Lye DC, Leo Y-S, Wong JGX, Hao Y, Wilder-Smith A. Severe Neutropenia in Dengue Patients: Prevalence and Significance. Am J Trop Med Hyg. 2014;90:984-7 7. Narayanan M, Aravind MA, Ambikapathy P, Prema, Jeyapaul MP. Dengue Fever â&#x20AC;&#x201C; Clinical and Laboratory Parameters Associated with Complications. Dengue Bulletin. 2003;27:108-15. 8. Santosh ST, Chincholkar VV, Kulkarni DM, Nilekar SL , Ovhal RS, Halgarkar CS. A study of NS1 antigen and platelet count for early diagnosis of dengue infection. Int.J. Curr. Microbiol. App.Sci. 2013;2:40-4. 9. Saito M, Oishi K, Inoue S, Dimaano EM, Alera MTP, Robles AMP, et al. Association of increased plateletassociated immunoglobulins with thrombocytopenia and the severity of disease in secondary dengue virus infections. Clin Exp Immunol. 2004;138:299-303. 10. Bashir AB, Mohammed BA, Saeed OK, Ageep AK. Thrombocytopenia and bleeding manifestations among patients with dengue virus infection in Port Sudan,Red Sea State of Sudan. J infect Dis.Immun. 2013;7:7-13.

Funding

11. Murgue B, Cassar O, Guigon M, Chungue E. Dengue virus inhibits human hematopoietic progenitor growth in vitro. J Infect Dis. 1997;175:1497-501.

Competing Interests

12. Mitrakul C. Bleeding problem in dengue hemorrhagic fever: platelets and coagulation changes. Southeast Asian J Trop Med Public health. 1987;18:407-12.

None

None Declared

Reference:

1. Gupta N, Srivastava S, Jain A, Chaturvedi UC. Dengue in India. The Indian J Med Res. 2012;136:373-90. 2. Bashir AB, Saeed OK, Mohammed BA, Ageep AK. Role of Platelet Indices in Patients with Dengue www.pacificejournals.com/apalm

13. Dinesh N, Patil VD. Persistent Thrombocytopenia after Dengue Hemorrhagic Fever Indian Pediatr. 2006;43:1010-11. 14. Sellahewa KH, Samaraweera N, Thusita KP, Fernando JL. Is fresh frozen plasma effective for eISSN: 2349-6983; pISSN: 2394-6466

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Platelet Indices & CPLI in Dengue Illness

thrombocytopenia in adults with dengue fever? A prospective randomised double blind controlled study. Ceylon Med J. 2008;53:36-40. 15. Murgue B, Cassar O, Guigon M, Chungue EJ. Dengue virus inhibits human hematopoietic progenitor growth in vitro. Infect Dis. 1997;175:1497-501. 16. Clark KB, Noisakran S, Onlamoon N, Hsiao HM, Roback J, Villinger, F. et al. Multiploid CD61+ Cells Are the Pre-Dominant Cell Lineage Infected during Acute Dengue Virus Infection in Bone Marrow. PLoS ONE. 2012;7:e52902. 17. Noisakran S, Onlamoon N, Pattanapanyasat K, et al. Role of CD61+ cells in thrombocytopenia of dengue patients. Int J Hematol. 2012;96:600-10.

18. La Russa VF1, Innis BL. Mechanisms of dengue virus-induced bone marrow suppression. Baillieres Clin Haematol. 1995;8:249-70. 19. Rajapakse S, Rodrigo C, Maduranga S, Rajapakse AC. Corticosteroids in the treatment of dengue shock syndrome. Infect and Drug Resist. 2014;7:137-43. 20. Simon AY, Sutherland MR, Pryzdial ELG. Dengue virus binding and replication by platelets. Blood. 2015;126:378-85. 21. Sharma S, Sharma SK, Mohan A, Wadhwa J, Dar L, Thulkar S, Pande JN. Clinical profile of dengue hemorrhagic fever in adults during 1996 -outbreak in Delhi, India. Dengue Bulletin. 1998; 22:20-7.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 05, (Suppl) Nov. 2016

Original Article Blood Donor Deferral Analysis: A Tertiary Care Centre Experience

Harjot Kaur1, Rahul Mannan1, Mridu Manjari1, Sonam Sharma2*, Tania Garg1 and Tejinder Bhasin1 Department of Pathology, Sri Guru Ram Das Institute of Medical Sciences and Research, Amritsar , Punjab, India 2 Department of Pathology, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India

Keywords: Blood Bank, Deferral, Donors, Punjab, Repeat , First Time

ABSTRACT Background: Blood donor selection is important to ensure the safety of both donors and recipients. There is a paucity of data on reasons for blood donor deferral in Punjab. The aim of this study was to identify the reasons for predonation deferral at a blood collection site at a blood bank of a tertiary care teaching hospital. Method: The investigators conducted retrospectively of data pertaining to donor deferral for blood donors from January 1, 2014 to December 31, 2014. Results: Among 8523 donors reporting to our blood bank during the one year period, 91 % were males. Of these, 7927 (93%) attempted donation, while 596 (6.9%) were deferred. Among the deferred, 342 (57.4%) were deferred for temporary reasons and 254 (42.6%) for permanent or semi-permanent reasons. Main reasons for temporary deferral were low hemoglobin (17.25%), alcohol abuse (13.15% ) and tattooing (12.18%). A positive history of cardiac abnormalities with high blood pressure ( 37.4%) followed by age disparity ( age , 18 years or > 65 years)- 25.98 % and practicing unsafe sex or drug abuse (13.77%) were the main reasons for permanent/semi-permanent deferrals. Statistically it was noted that the overall proportion of deferral was higher in males (5.3% vs 1.1%, p < 0.0001), and first-time donors (4.97% vs 2.01%, p < 0.0001) Conclusion: First time donors are more frequently deferred than repeat donors, especially because of interview decisions and presence of high risk behaviour. The study suggests the importance of recruiting repeated blood donors for better hemovigilance and blood safety practices.

*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

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

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Blood Donor Deferral Analysis

Introduction

whole blood once more than five years previously), repeat donor (donors who donated whole blood at least twice in the previous 12 months), and sporadic donors (donors who donated whole blood at least twice in an interval greater than 12 months and less than five years). Some of the things, which complete the donor selection, are enquiry against number of whole blood donations, past medical history, history of high-risk occupations and behavior. Additional laboratory test hemoglobin with cyanmethemoglobin method was utilized wherever the pallor was noted clinically or in case of doubt.

Appropriate donor selection is an important step in ensuring safe supply of blood and blood products. Availability of safe blood is the main goal of blood transfusion center. The aim of Blood Transfusion Services (BTS) should be to provide effective blood and blood products, which are as safe as possible and adequate to meet patients need.[1] In this context the donor screening process is very critical to ensure the safety of the blood supply.[2,3] Screening, usually of two types: temporary or permanent deferral, is a double edged sword because on one hand where it is necessary for safe blood while on the other hand it can itself lead to relatively lower donation rates and in Indian scenario can add onto already existing blood shortage.

Data of all blood donors and pre donation deferrals over the stated period were obtained using a pre tested structure questionnaire designed for the study. The data was presented as simple descriptive studies by means of tables. Donor deferral rates were compared using chi-square test with p < 0.5 being considered as significant.

Another problem associated with a rigorous screening/ deferral program is change in donor behavior as it has been noted that there was less chance of first time donors who were once temporarily deferred to return for subsequent donation than those who donated successfully.[4] This deferral can also have a bearing on an overall cost benefit ratio of a blood bank.[5]

Results

Among 8523 donors reporting to our blood bank during one year period, 91 % were males. Of these, 7927 (93%) attempted donation, while 596 (6.9%) were deferred (TABLE 1). The overall average age was 35 years, ranging from 18 to 55 years. Most of the prospective donors were replacement (69.3%) and were first time donors (24%).

Thus knowledge of the rate and reasons for donor deferral is quite essential as this can guide future donor recruitment strategy. The objectives of the present study were to determine the proportion and reasons of donor deferral in a blood bank of a tertiary care teaching hospital. This in turn helps ensure a more informed process of donor recruitment and selection and forestall unnecessary donor deferrals.

Among the deferred, 342 (57.4%) were deferred for temporary reasons and 254 (42.6%) for permanent or semipermanent reasons. Main reasons for temporary deferral were low hemoglobin (17.25%), alcohol abuse (13.15% ) and tattooing (12.18%) (TABLE 2).

Materials and Methods

A positive history of cardiac abnormalities with high blood pressure ( 37.4%) followed by age disparity ( age , 18 years or > 65 years)- 25.98 % and practicing unsafe sex or drug abuse (13.77%) were the main reasons for permanent/ semi-permanent deferrals (TABLE 3).

The retrospective data was retrieved from the records of Sri Guru Ram Das Institute of medical Sciences and Research, Amritsar (Punjab) during the period from 1st January – 31st December 2014. The donor selection criteria enumerated by WHO are based on age, body weight, blood pressure, and hemoglobin level according to sex. Other factors include status of the donor i.e., first-time donor (never donated or donated

Statistically it was noted that the overall proportion of deferral was higher in males (5.3% vs 1.1%, p < 0.0001), and first-time donors (4.97% vs 2.01%, p < 0.0001) (TABLE 4).

Table 1: Demographic characteristics of donors- successful donation and deferral. Total

Donated

Deferred

Sex Male

8125

7625

500

Female

398

302

Donor Status Repeat

6052

5628

172

First Timer

2471

2299

424

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Table 2: Causes of Temporary Deferral with their relative proportions. Cause Low hemoglobin Alcohol Tattoo & needle pricking Tuberculosis Dog bite Fever Jaundice Low weight Medication Malaria Previous Surgery Previous blood donation Typhoid Miscellaneous

Numbers 59 45 42 25 26 25 21 20 18 15 12 12 08 15

Percentage of temporary deferral 17.25% 13.15% 12.28% 7.30% 7.60% 7.30% 6.14% 5.84% 4.38% 4.38% 3.50% 3.50% 2.33% 4.38%

Table 3: Causes of Permanent Deferral with their relative proportions. Causes

Number

Percentage of deferrals

High Blood pressure and other cardiac problems

19.6%

AGE >65 yr < 18 yrs

25.97%

High risk behavior

13.77%

Thyroid dysfunction

12.99%

Epilepsy

7.87%

Table 4: Comparison of deferral rates and most common reasons for deferral in our study with other studies. Study

Deferral rate

Present study

Shiraz Blood Transfusion Center, Iran 2009[18]

Nigeria 2014 [19]

6.99%

30.90%

16%

Most common reason for deferral Age<65 and <18 yrs, High blood pressure Low Hb Having risk factors that may be related to HIV or hepatitis infections (43.60%) Underlying diseases (31.90%) Non-eligible general conditions (13.50%) Low Hb 39% Hypertension 30% Weight < 45 2.4%

India 2014 [20]

11.6%

Low Hb 33.5% Hypertension 11% Alcohol intake 10.8%

Turkey 2007 [11]

14.6%

Low hematocrit (20.07%) Common cold (17.70%) High-risk sexual activity (16.70%)

Singapore 1993 [12]

14.4%

Drug consumption Influenza Low hemoglobin

United States 2004 [9]

13.6%

Low hemoglobin (60%) Emigration from an area with malaria (59%) Tattoo or needle exposure (29%)

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Blood Donor Deferral Analysis

Discussion

Donor selection is the most important step in improving the safety of blood and blood products in donor selection. Insight into the reasons of deferral is very important to avoid any permanent loss of the donor as donor donation program. In view of blood unit scarcity owing due to shortage of safe blood donors in our country, which mirrors the situation prevalent in many of the resource-challenged countries of Asia and Africa, it is important to understand the reasons for deferral. Identification of reasons for deferral can lead to changes in blood safety programs and can lead to informed and well-directed intervention. There have been few publications describing the reasons for deferral in northern part of India, specifically for state of Punjab. Our study evaluated reasons for deferral in a population of voluntary and non-remunerated donors reporting to a blood bank of a tertiary care teaching hospital. In this study, the deferral rate was 6.99%. The deferral rate in other Indian and international studies have a reportable range of 4 % to 16.4%.[6,7,8] These have been compared in Table 4. The deferral rate in our center was much lower than other studies conducted elsewhere such as in United states, Europe, Turkey, Singapore where these have been reported as 12.8%, 10.8%, 14.6% & 14.4% respectively.[9,10,11,12] Temporary deferrals accounted for 57.28% of all deferrals in our study. This is in accordance with the studies done by Custer et al [4] and Shaz et al [13] who have a reported range of short-term deferral similar to us as 68.5% and 68%. However, in a study by Lawson Ayayi et al in France the temporary deferral rate was unusually very high as 91.3 % of all deferrals.[10] The current study had a higher percentage of male donors compared with some of the other Asian studies.[14] In the study conducted the rate of temporary deferrals varied according to the sex of the donor and type of donor (p < 0.05). Deferral rates-stratified by donor sex-differed for both temporary and permanent deferral. The literature published shows studies differ according to a particular geographical area, with Custer et al [4] having concluded that the rates of deferral do vary by sex and LawsonAyayi [10] having reported that permanent deferral was not sex related. The study also concluded that deferral rates in first time donors was higher as compared to the repeat donors and this relationship was statistically significant. The finding of more chances of deferral in first time donors in comparison to repeat donors is explained by the fact that the repeat donors have greater awareness about blood donation

criteria.[5] Hence, it is logical for any successful blood donation program that regular/ repeat donors encouraged or felicitated by some means and increased community based health education and information programs be initiated to encourage and educate the general population regarding the eligibility criteria for blood donation.[15] The most common cause of temporary deferral overall in our study was low hemoglobin (16.08%), alcohol abuse(13.15%) and needle exposure (acupuncture) / tattooing (12.25%). These findings are often different in different centers due to cultural and socioeconomic indicators. This is illustrated by the fact that while the most common reasons of temporary deferral were low hemoglobin (46%), common cold (19%) elevated temperature (10%) in work done by Halperin D et al [16] ; yet in another study by Lim et al [12], the most frequent reasons for deferral were drug consumption followed by influenza, low hemoglobin, hypertension and recent high risk sexual activity. The observations such as these are illustrated by the fact that while in most of the studies low hemoglobin (low Hct) was the most common cause of temporary deferral but the next common cause varies according to the various geographical areas ; for example study by Custer et al recorded emigration from an area with malaria & tattoo / needle exposure as next common reason of deferral [4] and another study on Turkish population shows the main reasons for short term deferral were common cold in men and low hematocrit in women , while low hematocrit was the most common reason overall (20.07) followed by common cold (17.7%) , high risk sexual activity (16.7%) , hypertension (5.6%) and polycythemia (2.8%).[11] In the present study 42.6 % donors were deferred for permanent reasons and most common reason for permanent deferral was cardiac causes most notably hypertension followed by age related deferrals ( >65 years and < 18 years). Arsalan et al [11] reported permanent deferral rate of around 10.0 % long term and 1.5 % permanent deferrals and Custer et al [4] reported a rate of 10.5% in their study. In accordance with the study conducted even Di Lorenzo Oliveria et al also found hypertension as the most common cause of permanent donor deferral. [17] Donor deferral is a noteworthy problem in the setting of blood banks as it may discourage potential/ first time donors as it has been noted that the temporary deferral due to any reason genuine or trivial often do not return to blood bank in future for further donations. Many researchers especially, Zou et al [9] and Halparin et al have documented a negative impact of temporary deferral on the donor return rates.[16] All the potential donors deferred due to temporary reasons should be informed at the time of deferral. These donors

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should be appropriately counseled and managed to improve the efficiency of the donor program. Health authorities should also implement policies for the preventive measures to decrease the incidences of common deferral causes as this reflects the health status of the society.

Conclusion

First-time donors are more frequently deferred than repeat donors, especially because of interview decisions and presence of high risk behavior ( recreational drug abuse/ multiple partners/ unprotected sexual intercourse). In this study (in similarity with many of the national and global blood bank based deferral studies), the most common reason for deferral among first-time donors was the high risk behavior that might be related to HIV or hepatitis infection. This often was not the case with repeat donors. Thus the study emphatically emphasizes the importance of recruiting repeated blood donors for better hemovigilance and blood safety practices. Apart from not only the shrinkage of the donor pool the deferrals also inflicts an expenditure so called cost implication for blood collection organizations.

References

1. World Health Organization. Towards 100% voluntary blood donation. Available online: www.who.int/entity/ bloodsafety/publications/9789241599696_eng.pdf 2. Eder A: Evidence-based selection criteria to protect blood donors. J Clin Apher 2010;25:331-337. 3. Goncalez TT, Sabino EC, Schlumpf KS, Wright DJ, Mendrone A, Lopes M, et al . NHBLI Retrovirus Epidemiology Donor Study –II (REDS-II), International Component. Analysis of Donor Deferral at three blood centres in Brazil. Transfusion 2013; 53:531-8. 4. Custer B, Johnson ES, Sullivan SD, et al. Quantifying losses to the donated blood supply due to donor deferral and miscollection. Transfusion 2004; 44(10):1417-26. 5. Smith GA, Fischer SA, Doree C, Roberts DJ. A systematic review of factors associated with the deferral of donors failing to meet low hemoglobin thresholds. Transfusion Med 2013;23(5): 309-20. 6. Agnihotri N. Whole blood donor deferral analysis at a center in Western India. Asian J Transfus Sci 2010;4(2):116-22. 7. Chaudhary RK, Gupta D, Gupta RK. Analysis of donor-deferral pattern in a voluntary blood donor population. Transfus Med 1995;5(3):209-12. 8. Bahadur S, Jain S, Goel RK, Pahuja S, Jain M. Analysis of blood donor deferral characteristics in www.pacificejournals.com/apalm

Delhi, India. Southeast Asian J Trop Med Public Health. 2009;40(5):1087-91. 9. Zou S, Musavi F, Notari EP, et al. Donor deferral and resulting donor loss at the American Red Cross Blood Services, 2001 through 2006.Transfusion 2008;48(12):2531-9. 10. Lawson-Ayayi S, Salmi LR. Epidemiology of blood collection in France. Eur J Epidemiol 1999;15(3):285-92. 11. Arslan O. Whole blood donor deferral rate and characteristics of the Turkish population. Transfus Med 2007;17(5):379-83. 12. 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;22(3):326-31. 13. Shaz BH, James AB, Hillyer KL, Schreiber GB, Hillyer CD. Demographic variations in blood donor deferrals in a major metropolitan area. Transfusion. 2010;50(4):881-7. 14. Noma AM, Goto A, Nollet KE, Sawamura Y, Ohto H, Yasumura S. Blood Donor Deferral among Students in Northern Japan: Challenges Ahead. Transfus Med Hemother 2014; 41(4): 251-6. 15. 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;4(2):112-5. 16. Halperin D, Baetens J, Newman B. The effect of shortterm, temporary deferral on future blood donation. Transfusion 1998;38(2):181-3. 17. Di Lorenzo Oliveira C, Loureiro F, de Bastos MR, Proietta FA, Cameiro-Proietti AB. Blood donor deferral in Minas Gerais State, Brazil: blood centers as sentinels of urban population health. Transfusion 2009;49(5):851–7. 18. Kasraian L, Negarestani N. Rates and reasons for blood donor deferral, Shiraz, Iran. A retrospective study. Sao Paulo Med J 2015; 133(1):36-42. 19. Ekwere TA, Ino-Ekanem M, Motilewa OO, Iquo Augustine Ibanga. Pattern of blood donor deferral in a tertiary hospital, South-south, Nigeria: A three-year study review. Int J Blood Transfus Immunohematol 2014;4:7–13. 20. Gajjar H, Shah FR, Shah NR. Whole blood donor deferral analysis at General Hospital Blood BankA Retrospective study . NHL Journal of Medical Sciences 2014; 3(2):71-6. eISSN: 2349-6983; pISSN: 2394-6466

Original Article Correlation of Her-2/neu Status With Estrogen, Progesterone Receptors and Histologic Features in Breast Carcinoma Anupama Dayal, Rupal J Shah*, Sadhana Kothari and S. M. Patel Pathology Department, GCS Medical College Hospital and Research Centre. Ahmedabad. India Keywords: Breast carcinoma, Her-2/neu, ER/PR

ABSTRACT Background: Breast cancer is the commonest cancer in urban Indian females, and the second commonest cancer in the rural Indian women. One of the hallmark of the disease is expression of Estrogen Receptor (ER), Progesterone Receptor (PR) and Her-2/neu that ultimately drives prognosis & treatment modalities. Aims: This study is to evaluate the expression of ER and PR and Her-2/neu in Breast Carcinoma & to compare them with other prognostic parameters: histological type and grade, tumor size, patientsâ&#x20AC;&#x2122; age and lymph node metastases. Method: 80 cases of Invasive Breast Carcinoma received as MRM (Modified Radical Mastectomy) over a period of 6-8 months were included. Routine H&E staining and IHC analysis for ER, PR and Her-2/neu was carried out in all cases. Result: Out of 80 cases, 69 (86%) cases were of Infiltrating Ductal Carcinoma with mean age of 53 years. ER, PR & Her-2/neu expression were seen in 56.9%, 35.5% & 21.3% respectively. ER, PR expressing tumours tend to be of lower grade I(p<0.05). 94.1% Her-2/neu expressing tumours presented with higher tumour grade (II, III) but statistically not significant. Lymph node metastases was found to be significantly associated with PR and Her-2/neu positive status (p=0.03, p=0.01 respectively). Her-2/neu expression was inversely related to ER and PR expression (p<0.05). Conclusion: Higher number of grade 1 tumors showed ER, PR positivity as compared to grade 3 tumors. Inverse relationship was observed between Her-2/neu expression and ER, PR receptor status. Her-2/neu expression was associated with higher rate of axillary metastasis but did not show any significant correlation with age, tumour size and grade.

*Corresponding author: Dr. Rupal. J. Shah. 401, Ananya Residency, Near Jay Ambe Gruhudyog. Paliyad Nagar, Naranpura. Ahmedabad. Gujarat. India. 380013. Phone: +91 9825586560 Email: rupu_desai@yahoo.co.in

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

Dayal et al.

Introduction

With rising incidence and awareness, breast cancer is the commonest cancer in urban Indian females, and the second commonest cancer in the rural Indian women.[1] In India, Breast Cancer is second to cancer of the cervix among women, but is considered the leading cancer in certain metros such as Mumbai and Bangalore. It is estimated that approximately 80,000 cases occur annually; the age adjusted incidence rates varying between 16 and 25/100,000 population. [2] Breast cancer survival is linked to early detection, timely appropriate treatment and genetic predisposition. Prognosis is related to a variety of clinical, pathologic and molecular features which include classical prognostic factors viz. histologic type, grade, tumor size and lymph node metastases. Estrogen and progesterone receptors (ER, PR) and more recently, Her-2/neu have with increasing importance influenced the management of the malignancy. [3] Her-2/neu, also known as c-erbB-2, a protooncogene located on chromosome 17, is amplified and/or the protein (Her-2) overexpressed in 15% to 25% of invasive breast carcinomas and is associated with a worse clinical outcome.[4,5] In contrast, ER is expressed in 70% to 95% of invasive lobular carcinomas and in 70% to 80% of invasive ductal carcinomas, and PR is expressed in 60% to 70% of invasive breast carcinomas.[6,7] It is known that ER and PR expression are the only predictive factors with proven usefulness in selecting patients who are likely to respond to adjuvant endocrine therapy.[8,9] Patients lacking these receptors tend to have shorter disease free survival and earlier recurrences than those expressing these receptors.[9] The interrelationship of ER, PR, and Her-2/neu has come to have an important role in the management of breast cancer. It has been shown that patients with breast carcinoma overexpressing Her-2/neu do not respond to Tamoxifen therapy. [4]The prognostic effects of Her-2/neu expression appear to be stronger in node positive carcinomas than in node negative carcinomas.[10] Her-2/neu has gained an even greater deal of attention lately after the introduction of a humanized monoclonal antibody known as trastuzumab that can be effective in the treatment of cases in which this oncogene product is overexpressed.[11,12]

Materials and Methods

A-477 specimens was carried out for tumor size, location and nodal metastases. All tissues were fixed in 10% buffered formalin for 12 hours. Representative tumor tissue was submitted for processing, 4 μm thick, paraffin embedded sections were cut and stained by Hematoxylin & Eosin (H&E) for routine histological examination & diagnosis. Sections from the cases were reported by two pathologists independently with consensus and invasive tumors were divided into two major categories- ductal type and lobular type- acknowledging the existence of mixed and intermediate forms.[13] Other morphological types like Metaplastic carcinoma, Medullary carcinoma , Papillary carcinoma, Tubulo-lobular carcinoma were also included. For invasive carcinomas, Nottingham’s Modified Bloom Richardson (B-R) combined histologic system was used for grading.[13,14] (Table 1) Final combined Bloom-Richardson (B-R) grade: Add score for tubule formation, mitoses, and nuclear pleomorphism. If B-R score is 3, 4 or 5, then B-R grade is low (I). If B-R score is 6 or 7, then B-R grade is intermediate (II). If B-R score is 8 or 9, then B-R grade is high (III). Representative sections of tumor and normal breast tissue (as internal control) were processed for ER, PR and Her-2/ neu immuno-histochemical staining. The positive control slides were prepared from breast carcinoma known to be positive for the antigen under study. For ER, PR and Her-2/ neu staining, sections were taken on poly L lysine coated slides. Antigen retrieval was done by citrate buffer and the slides stained with monoclonal antibodies against estrogen and progesterone receptors by LSAB (labeled streptavidin biotin) system (BioGenex). The ER and PR results were screened and interpreted according to the published guidelines by The American Society Of Clinical Oncology & The College Of American Pathologist (ASCO/CAP).[13] This considers both the proportion and intensity of stained cells. Tumours having 1% or higher invasive cancer cells are considered positive and based on above findings final interpretation was given whether the sample is positive or negative.(Fig.1) For interpretation of Her-2/neu staining, following scoring system was used. [13] Score 0: No staining is observed or membrane staining is observed in <10% of the tumor cells.

The present study was carried out in the Department of Pathology, tertiary care centre of Ahmedabad, Gujarat, India, over a period of 6-8 months. 80 cases of Invasive Breast Carcinoma received as MRM (Modified Radical Mastectomy) were included. This study has been done as per standard ethics & by maintaining confidentiality of reports. Detailed gross examination of all received MRM

Score 1+: Faint/barely perceptible membrane staining is detected in >10% of tumor cells. The cells exhibit incomplete membrane staining.

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Score 2+: A weak to moderate complete membrane staining is observed in >10% of tumor cells. It is considered as weak positive.

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IHC and Histopathological Prognostic Markers in Breast Carcinoma

Score 3+: Strongly Positive. A strong complete membrane staining is observed in >30% of tumor cells. Score 3+ was considered as strong positive immunostaining for Her-2/ neu. (Fig. 2). The equivocal cases (score 2+) were not subjected to FISH due to funding problem. Results were analyzed statistically and p value calculated using chi-square test.

Result

Total 80 cases of Breast Carcinoma were studied. The age of patients ranged from 26 to 85 yrs with mean age of 53.0 yrs. Most of the patients (85%) were of more than 40 yrs and majority (65%) belonged to 41- 60 yrs age group. (Table 2) The most common histopathological pattern was Invasive Ductal Cell Carcinoma (IDC)(86.3%). Majority of tumours were of grade II (64.4%) and measured 2-5 cm in size (77.5%). 90% of tumours showed necrosis, fibrosis and calcification while 61.3% were having axillary lymph node involvement (≥1). IHC profile showed 56.9% positivity for ER, 35.5% positivity for PR and 21.3% positivity for Her2/neu. (Table 3) Table 4 shows correlation of age and tumour characteristics with ER, PR and Her-2/neu. Mean age of patients with strong (+3) Her-2/neu overexpression was 51 yrs which is comparatively younger (54 yrs) than those lacking Her-2/ neu expression. Similarly 25% of patients ≤ 40 yrs of age were Her-2/neu positive as opposed to 20.5% of patients > 40 yrs of age. Patients with strong (+3) Her-2/neu overexpression presented with smaller tumour size (mean size 3.5cm) compared to Her-2/neu negative tumours (mean size 3.8cm). 24.2% of T2 tumours (2-5cm) were Her-2/neu positive as compared to 8.4% of T3 tumours ( >5cm). Patients with strong Her-2/neu expression (94.1%) presented with higher tumour grade (II, III) compared to Her-2/neu negative patients (78.1%). In grade II and III tumours, 26.5% were strong positive for Her-2/neu

as compared to 8.4% of grade I tumours. In lymph node status, 82% of Her-2/neu positive patients had lymph node metastasis (≥1) whereas only 55%of Her-2/neu negative patients had same. Mean age for ER positive and ER negative patients were 55.5 and 49.6 yrs respectively. Among ER positive patients, 6.7% were ≤40 yrs of age as compared to 93.3% of patients >40yrs (p = 0.03). Mean age for PR positive and PR negative patients were 53.9 and 52.5 yrs respectively. 7.1% of PR positive patients were ≤40 yrs of age as opposed to 93% of patients >40yrs.These findings were similar to ER status. Mean tumour size in ER positive patients was 3.4cm versus 4.2cm in ER negative patients. In T2 tumours (2-5cm), 58% were ER positive whereas 42% of T3 tumours (>5cm) showed ER positivity. In PR positive patients, mean tumour size was 3.6cm versus 3.8cm in PR negative patients. 37% of T2tumours showed PR positivity compared to 33% of T3tumours. Amongst grade II and III tumours, 50.8%were ER positive whereas 91.6% of grade I tumours were positive for ER (p = 0.005) i.e. lower grade tumours show more ER expression. 29.5% of grade II and III tumours were PR positive compared to 66.7% of grade I tumours (p = 0.02). Patients with PR negative status had more incidence of lymph node metastasis (66.6%) than those with PR positivity (50%)(p = 0.03). There was no statistical significant difference in lymph node metastasis detected in relation to ER status (Table 4). Table 5 shows inverse relation between Her-2/neu expression and ER, PR. Of 17 Her-2/neu positive cases, 64.7% were ER negative and 88.2% were PR negative (p<0.05). 32.3% of ER negative and 29.4% of PR negative tumours were Her-2/neu positive. A positive correlation between ER and PR was also detected. Out of 45 ER positive cases, 28 were also PR positive whereas all 34 ER negative cases were also PR negative.

Table 1: Microscopic Grading of Breast Carcinoma: Nottingham modification of the Bloom – Richardson System.

Tubule formation

Nuclear pleomorphism

> 75% of cells arranged in tubules

score 1

> 10 to 75% of cells arranged in tubules

score 2

< 10%

score 3

cell nuclei are uniform in size and shape, relatively small, have dispersed chromatin patterns, and are without nucleoli

score 1

cell nuclei are somewhat pleomorphic, have nucleoli, and are of intermediate size

score 2

cell nuclei are relatively large, have prominent nucleoli or multiple nucleoli, coarse chromatin patterns, and vary in size and shape

score 3

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Mitotic count Via low power scanning (10x), locate the most mitotically active area of tumor and proceed to high power (40x)

< 10 mitoses per 10 high power fields(hpf)

score 1

≥ 10, and < 20 mitoses per 10 hpf

score 2

≥ 20 mitoses per 10 hpf

score 3

Table 2: Distribution of patients according to age group Age Group(yrs)

Cases(%)

21 - 30

1(1.3)

31 - 40

11(13.7)

41- 50

25(31.3)

51- 60

27(33.7)

61 - 70

10(12.5)

> 70

6(7.5)

Total

80(100)

Table 3: Clinicopathological features and receptor status. Histopathological Features

69(86.3)

ILC

5(6.3)

Medullary

4(5)

Papillary

1(1.2)

Metaplastic

1(1.2)

Positive

45(56.9)

Negative

34(43.1)

Positive

28(35.5)

Negative

51(64.5)

Positive

17(21.3)

Negative

63(78.7)

12(16.4)

47(64.4)

14(19.2)

<2cm

6(7.5)

2-5cm

62(77.5)

>5cm

12(15)

Necrosis

69(92)

Fibrosis

67(89.3)

Calcification

71(94.7)

Lymphocytic Infiltrate

43(57.3)

Positive

49(61.3)

Negative

31(38.7)

≤40

12(15)

> 40

68(85)

Subtype ER PR Her 2/Neu Tumor Grade Tumor Size Tumor Characteristics

No. (%) IDC

Lymphnode Status Age

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IHC and Histopathological Prognostic Markers in Breast Carcinoma

Table 4: Her-2/neu Status And Estrogen And Progesterone Receptor Expression In Invasive Breast Carcinoma Patients.

Her 2 strong positive(+3)

Her 2 weak positive(+2)

Her 2 negative

ER positive

ER negative

PR positive

PR negative

Mean Age (yrs)

50.5

55.5

49.6

53.9

52.5

No Of Patients ≤ 40 yrs

No Of Patients > 40 yrs

Mean Size(cm)

3.5

3.8

3.4

4.2

3.6

3.8

No of T1 Tumors(<2cm)

No of T2 Tumors(2-5cm)

No of T3 Tumors(>5cm)

No Lymphnodal Metastases

1-3 LN Metastases

>3 LN Metastases

Tumor Grade

Table 5: Relation of Her-2/neu expression with hormone receptor status. Hormone Receptor

Her-2/neu Negative Patients No.(%)

Her-2/neu Positive Patients No.(%)

Chi-Square (p-value)

Estrogen Receptor Status Positive

Negative

0.0784

Progesterone Receptor Status Positive

Negative

Fig. 1: ER positivity (+++) in tumour cells of Invasive Ductal Carcinoma, Breast (10x).

0.0436

Fig. 2: Her-2/neu positivity (+++) in tumour cells of Invasive Ductal Carcinoma, Breast (40x).

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Discussion

Breast carcinoma is a disease with a tremendous heterogeneity in its clinical behavior. Clinical and pathological variables such as tumor size, histologic grade, histologic type, lymph node metastases, vascular space invasion, tumor cell proliferation, tumor necrosis, extent of ductal carcinoma in situ, age, and pregnancy may help in predicting prognosis and the need for adjuvant therapy. [15] The presence of hormone receptors (ER and PR) in the tumor tissue correlates well with the response to hormone therapy and chemotherapy. [16] Tumors that are better differentiated are more likely to be ER and PR positive and have a relatively better prognosis.[17] The overall survival was significantly better for patients without Her2/neu receptor markers compared to patients with Her-2/ neu overexpression .[18] Present study comprised of 80 cases of primary breast carcinoma. Infiltrating ductal carcinoma(86%) was the most common histological type which is comparable to Azizun N et al (85.3%)[19] and Nidal MA et al (84%).[20] The mean age at diagnosis in present study was 53.0 yrs comparable to studies done by Azizun N et al,[19] Nidal MA et al [20] and Pathak TB et al.[21] The left breast was more commonly involved (55%).[19] T2 tumours comprised the majority of cases in our study as seen in other studies with frequency ranging from50% to 65%.[19,20] Tumor grade is one of the prognostic factors in the breast cancer, tumors expressing higher grade tend to have poor prognosis. In this study, grade II tumors constitute the highest number of cases 64.4% followed by grade III and grade I. This is similar to studies done by Azizun N et al [19] and Pathak TB et al.[21] Our study showed 56.9% of cases with positive ER status while 35.5% PR positive status. The study by Nidal MA et al [20] showed 53.0% and Fatima S et al [22] showed 55% positivity for ER, Bhagat VM et al [23] reported 37.9% positivity for PR. In this study, we found that 21.3% cases were Her-2/neu positive. Although there is a wide variation in Her-2/neu overexpression and amplification, our figure appears to be within the commonly accepted range of 20% to 30% reported in literature.[19,20,23,24] In our study significant association was found between age and ER status (p=0.03). The mean age of ER positive patients was 6 years more than ER negative patients. These findings are in agreement with other reports in the literature, which show an association between ER expression in breast carcinoma patients and older age at the www.pacificejournals.com/apalm

A-481 time of diagnosis.[20,25] In literature, Her-2/neu expression tend to decline with age as opposed to ER expression which tends to increase with age.[26] In present study also Her-2/neu positive patients were younger than those lacking expression; mean age being 3 yrs less than Her-2/ neu negative patients. In present study, significant association was found between tumour grade and ER, PR expression (p=0.005, p=0.01 respectively). This is in accordance with studies done by Azizun N et al [19] and Fatima S et al[22] which also reported decreased expression of ER and PR in the higher grade III tumours. However no significant correlation could be established between Her-2/neu and the grade. There was no significant association found between tumour size and Her-2/neu, ER and PR expression. Axillary lymph node metastases (â&#x2030;Ľ1) was found in 82% of Her-2/neu overexpressing tumours compared to 55% of Her-2/neu negative tumours(p=0.01). A direct relationship between lymph node metastases and Her-2/neu expression was also reported by Hoff ER et al.[27] A significant inverse relation between Her-2/neu overexpression and ER, PR expression was found in present study (p=0.04 and p= 0.02 respectively). Similar results were found in studies done by Azizun N et al,[19] Bhagat VM et al,[23] Arafah M,[28] MS Al-Ahwal.[29] A strong correlation between ER and PR expression was also noted in our study.

Conclusion

Breast cancers usually presented in 4th to 6th decade of life with mean age being 53 years. IDC was the most common histopathological type. No significant association of Her-2/ neu. PR expression was found with age and tumour size. Better differentiated grade I tumours showed higher ER, PR positivity compared to grade III tumours in contrast, Her-2/neu expression increases with increasing grade of tumour. Her-2/neu expressing tumours were significantly associated with axillary lymph node involvement. Inverse relationship was observed between Her-2/neu expression and ER, PR status. As ER, PR and Her-2/neu are important prognostically as well as for the selection of best therapy for better survival of breast cancer patients, their assessment is strongly advocated in each case.

Funding None

Competing Interests None Declared

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IHC and Histopathological Prognostic Markers in Breast Carcinoma

Reference

1. National Cancer Registry Program: Ten year consolidated report of the Hospital Based Cancer Registries, 1984–1993, an assessment of the burden and care of cancer patients. Indian Council of Medical Research, New Delhi, 2001. 2. Harrison AP, Srinivasan K, Binu VS, Vidyasagar MS, Nair S. Risk factors for breast cancer among women attending a tertiary care hospital in southern India. Int J of Collaborative Research on Internal Med & Pub Health. 2010; 2(4):109-116. 3. Rampaul RS, Pinder SE, Elaston CW, Ellis IO. Prognostic and predictive factors in primary breast cancer and their role in patient management; the Nottingham breast team. Eur J Surg Oncol. 2001; 27: 229-238. 4. Kaptain S, Tan LK, Chen B. Her-2/neu and breast cancer. Diagn Mol Pathol. 2001;10:139-152. 5. Bundred NJ. Prognostic and predictive factors in breast cancer. Cancer Treat Rev. 2001;27:137-142 6. Sastre-Garau X, Jouve M, Asselain B, et al. Infiltrating lobular carcinoma of the breast; clinicopathologic analysis of 975 cases with reference to data on conservative therapy and metastatic patterns. Cancer. 1996;77:113-120. 7. Zafrani B, Aubriot MH, Mouret E, et al. High sensitivity and specificity of immunohistochemistry for the detection of hormone receptors in breast carcinoma: comparison with biochemical determination in a prospective study of 793 cases. Histopathology. 2000;37:536-545. 8. Allred DC, Harvey JM, Berardo M, Clark GM. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol. 1998; 11:155-168. 9. Early Breast Cancer Trialists’ Collaborative Group. Tamoxifen for early breast cancer: an overview of the randomised trials. Lancet. 1998; 351:1451-1467. 10. Hanna W, Kahn HJ, Trudeau M. Evaluation of Her-2/ neu (erbB- 2) status in breast cancer: from bench to bedside. Mod Pathol.1999; 12:827-834. 11. Hortobagyi GN. Overview of treatment results with trastuzumab (Herceptin) in metastatic breast cancer. Semin Oncol. 2001; 28:43-47. 12. McKeage K, Perry CM. Trastuzumab: a review of its use in the treatment of metastatic breast cancer overexpressing HER2. Drugs. 2002; 62:209-243.

13. Rosai J. Breast. In: Houston M, Scott J, editors. Rosai and Ackerman’s Surgical Pathology. 10th ed (2). Missouri: Elsevier; 2012:1696-1720. 14. Elston CW. Grading of invasive carcinoma of the breast. In: Page DL, Anderson TJ, editors. Diagnostic histopathology of the breast. Churchill Livingston;1987: 300-311. 15. Tavassoli FA, Devilee P. World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of the Breast and Female Genital Organs Lyon. IARC, in press; 2004. 16. Barnes DM, Hanby AM. Oestrogen and progesterone receptors in breast cancer: past, present and future. Histopathology. 2001;38: 271-274. 17. Hilf R, Feldstein ML, Savlov ED, Gibson SL, Seneca B. The lack of relationship between estrogen receptor status and response to chemotherapy. Cancer.1980; 46: 2797-2800. 18. Jakesz R, Hausmaninger H, Kubista E, Gnant M, Menzel C, Bauernhofer T, et al. Randomized adjuvant trial of tamoxifen and goserlin versus cyclophosphamide, methotrexate and fluorouracil: evidence for the superiority of treatment with endocrine blockade in premenopausal patients with hormone responsive breast cancer - Austrian Breast and Colorectal Cancer Study Group. J Clin Oncol. 2002;20:4621-4627. 19. Azizun N, Yasmin B, Farrukh R, Naila K. Comparison of ER, PR & Her-2/neu (C-erb B) Reactivity Pattern with Histologic Grade, Tumor Size and Lymph Node status in Breast Cancer. Asian Pacific Journal of Cancer Prevention. 2008; 9:553-556. 20. Nidal MA, Mohammad AH. Immunohistochemical evaluation of human epidermal growth factor receptor 2 and estrogen and progesterone receptors in breast carcinoma in Jordan. Breast Cancer Research. 2005; 7 (5): 598-604. 21. Pathak TB, Bashyal R, Pun CB, Shrestha S, Bastola S, Neupane S, Poudel BR, Lee MC. Estrogen and progesterone receptor expression in breast carcinoma. Journal of Pathology of Nepal. 2011; 1: 100-103. 22. Fatima S, Faridi N, Gill S. Breast cancer. Steroid receptors and other prognostic indicators. J Coll Physicians Surg.2005; 15: 230-233. 23. Bhagat VM, Jha BM, Patel PR. Correlation of hormonal receptor and Her-2/neu expression in breast cancer: a study at tertiary care hospital in south Gujarat. National Journal Of Medical Research.2012; 2( 3):295-298.

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24. Lal P, Lee K et al. Correlation of HER-2 Status With Estrogen and Progesterone Receptors and Histologic Features in 3,655 Invasive Breast Carcinomas. Am J Clin Pathol. 2005; 123:541- 546. 25. Ashba J, Traish AM. Estrogen and progesterone receptor concentrations and prevalence of tumor hormonal phenotypes in older breast cancer patients. Cancer Detect Prev. 1999; 23:238-244. 26. Eppenberger-Castori S, Moore DH, Thor AD, Edgerton SM, Kueng W, Eppenberger U, et al. Ageassociated biomarker profiles of human breast cancer. Int J Biochem Cell Biol. 2002; 34: 1318-1330.

27. Hoff ER, Tubbs RR, Myles JL, Procop GW. HER2/neu amplification in breast cancer: stratification by tumor type and grade. Am J Clin Pathol. 2002; 117:916-921. 28. Arafah M. Correlation of Hormone Receptors with Her 2/neu Protein Expression and the Histological Grade in Invasive Breast Cancers in a Cohort of Saudi Arabia. Turkish Journal of Pathology. 2010; 26(3):209-215. 29. Mahmoud S Al-Ahwal. HER-2 Positivity and Correlations with other Histopathologic Features in Breast Cancer Patients - Hospital Based Study. J Pak Med Assoc. 2006; 56(2):65-68.

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Original Article A study of Haematological Profile in Human Immune Deficiency Virus Infection: Correlation with CD4 Counts Sheela Devi CS1*, Suchitha Satish1, Manali Gupta2 Department of Pathology, JSS Medical college, JSS university, Mysore, India 2 Consultant Pathologist, Chennai, India

Keywords: HIV, CD4 Counts, Haematological Parameters, Anemia, Thrombocytopenia

ABSTRACT Introduction: Hematologic defects are a common complication of human immunodeficiency virus (HIV) infection and result from several influences on the hematopoietic tissue. These abnormalities reflect the underlying immune status and may be prevented or corrected by the use of highly active antiretroviral therapy (HAART). Anemia commonly occurs during HIV infection and has been associated with increased progression to acquired immune deficiency syndrome (AIDS) and reduced survival. Aims: The aim of this study was to study the hematologicalprofile in HIV patients, to evaluate the various hematological parameters and their association with CD4 counts. Methods: One hundred HIV-positive patients, symptomatic as well as asymptomatic, diagnosed by enzyme-linked immunosorbent assay (ELISA) method according to the National AIDS Control Organization (NACO) guidelines were included. Complete hematologic profile was recorded. Relationship between CD4 counts and various hematologic parameters was analyzed. Descriptive statistics were applied. Association between two attributes was calculated by chi-square test and P value less than 0.05 was considered statistically significant. Results: The most common hematologic abnormality was anemia, seen in 85% (n=85) of the patients. Normocytic normochromic anemia was seen in 57 patients,which was the most common type. A strong association between anemia and CD4 counts was observed. The hemoglobin, red blood cell count and hematocrit also showed corresponding low values with decreasing CD4 counts. Thrombocytopenia was the second common abnormality and had no significant relationship with CD4 counts. Conclusion: HIV affects virtually all organ systems, with well-known abnormalities related to hematopoietic system. Anemia being the most common abnormality has strong correlation with CD4 counts and therefore could be used to predict the development of a more advanced disease.

*Corresponding author: Dr. Sheela devi CS, #362, Adarsha, TK layout, Mysore-570009, Karnataka, India Phone: +91 9845274440, 8212547340 Email: devi.sheela1@gmail.com

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

Devi CS et al.

Introduction

Acquired immunodeficiency syndrome (AIDS) is a caused by the HIV virus and characterized by severe immunosuppressionthat leads to opportunistic infections, neoplasms and neurological manifestations. [1,2] It is a major health challenge in the modern world causing damage in the resource poor south-east Asian countries. It involves almost all the systems in human body. [3,4]More people than ever are living with HIV, largely due to greater access to treatment. It is fast cbecoming a major health hazard in the Indian subcontinent, with an estimated 3.7 million persons being infected with HIV. [5] Hematologic abnormalities are among the most common complications of HIV. These involve all the lineages of the blood cells. Anemia is the most common hematologic abnormality affecting 60% to 80% of patients in the late stage disease with high viral load. [1,6]Neutropenia is caused by inadequate production and thrombocytopenia by immune-mediated destruction of platelets in addition to inadequate production. [1,7]As the infection progresses, there is development of pancytopenia. [3] HIV associated hematologic expressions seem to be dependent on the level of viral replication, as these abnormalities are severe in AIDS patients with high viraemia and decreased CD4 counts. [1]There are a few studies on haematological changes in HIV and a very few have correlated results with CD4 count. [8]In the present study, the haematologic changes have been studied and correlated with CD4 cell counts to highlight these manifestations in disease progression.

Materials and Methods

One hundred patients, seropositive for HIV by ELISAwere included in the study. Ethical clearance from the institutional ethical committee was obtained.Demographic and clinical informationwere recorded. Complete hemogramusing automated cell counter –Sysmex XN-1000 was done and various parameters including hemoglobin, total leucocyte count (TLC), Absolute neutrophil count (ANC) and absolute lymphocyte count (ALC) differential leucocyte count, platelet count, red blood cell indices, red cell distribution width (RDW), hematocrit (HCT) and reticulocyte count were noted.Prothrombin Time (PT) and activated partial thromboplastin time (aPTT) were done using semi automaticcoagulometeranalyser CA 50 SYSMEX.CD4 count was available in seventy patients. CD4 lymphocyte count was done in BD FACS Caliburflowcytometer. Anemia was defined as hemoglobin <13 g/dl (Men) and <12 g/dl (women, non-pregnant). Leucopenia was defined as TLC of less than 4000 cells/mm3. Neutropenia was defined www.pacificejournals.com/apalm

A-485 as ANC of <1000 cells/mm3. Lymphopenia was considered when ALC was <800 cells/mm3 Thrombocytopenia was defined as total platelet count < 150 × 103/mm3 [1]. Statistical Analysis Descriptive statistics were expressed as Mean ± Standard Deviation and results on categorical measurements as numbers (%). Comparison between data was done by chi-square test. A P-value <0.05 was taken as statistically significant. Statistical software namely SPSS 15.1, Stata 8.0, MedCalc 9.0.1 and Systat 11.0 was used for analysis of data.Microsoft word and Excel were used to generate graphs and tables.

Results

Demographic Data: The youngest patient was seven years old and the oldest was 80 years old and the mean age was 39.8 ±10.7 years. Most patients (45%) were in the age group of 31-40 years. Sixty five patients were males and thirty five were females with a male to female ratio of 1.9:1. Clinical Presentation and CD4 Counts: Seventy eight patients were symptomatic with fever (55%), generalized weakness (35%), diarrhea (20%), loss of weight /appetite (23%), and productive cough (26%). Patients were divided into three categories depending on CD4 counts; category 1 –less than 200 cells /µL,category 2 - 200 to 499cells/µL and category 3 –more than 500cells/µL [8].Most patients belonged to category 1 (51.4%).Twenty one patients were on antiretroviral therapy. However, majority of them were taking irregular treatment. Anemia: Most of the patients presented with anemia (85%) and was more common in symptomatic patients, accounting for 83% (n=71). Anemia was graded based on hemoglobin level into severe anemia with hemoglobin <8 gm/dl and mild to moderate anemia with hemoglobin >8gm/dl. Severe anemia was seen in 20% (n=17)) and mild to moderate anemia in 80% (n=68) of the cases. In relation with CD4 counts, 53% (n=32) of category-1 patients had anemia while among category-2 and category-3 patients, anemia was seen in 33% (n=24) and 14 % (n=10) patients respectively, which was statistically significant (P<0.005). The mean hemoglobin, HCT and RBC countsalso showed statistically significant Pvalue when compared with CD4 counts (Table1). The commonest type of anemia was normocytic normochromic anemia (NNA), which accounted to 67.1% (n= 57). Microcytic hypochromic anemia (MHA) was seen in 23.5% patients (n =20) and macrocytic anemia (MA) in six patients. Leukopenia: Leukopenia was seen in 20% (n=20) of the patients. Neutropenia was seen in three patients. Twenty eISSN: 2349-6983; pISSN: 2394-6466

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three patients had lymphocytopenia. TLC, ANC and ALC also showed corresponding low values with a decrease in CD4 count. However the P value of these parameters was not statistically significant (Table 1). Thrombocytopenia: Thirty two patients had thrombocytopenia. There was a reduction in the platelet count with a decrease in CD4 count. However, the P value was not statistically significant (Table/figure1). Eight patients had pancytopenia. Coagulation profile did not show significant changes.

Discussion

The most common source of HIV disease throughout the world is HIV-1 which was first identified in 1981 in West Africa. The first AIDS case in India was detected in Chennai and since then HIV infection has been reported in all the states and union territories. [3,9]The spread of HIV in India has been irregular with more severe epidemics being reported in the southern India and the north-east. 3 Worldwide, the phenomenon of HIV/AIDS is best viewed as a pandemic affecting nearly all the countries of the world. [10] Hematological abnormalities are among the most common complications of HIV. These involve all the lineages of blood cells. [11] HIV associated haematological abnormalities appear to be dependent on the level of virus replication, as these abnormalities are severe in late-stage AIDS patients with high viremia. The mechanism underlying these abnormalities is still equivocal. A specific diagnosis of the cause and mechanism must be required because specific treatment may be needed for its correction.[11] Anemia is the most common cytopenia in HIV-infected individuals, occurring in 10-20 % of patients at initial presentation and diagnosed in approximately 70 -80 % of patients over the course of disease. The incidence is strongly related with the progression of the disease and is common in the symptomatic group. [1,12,13,14,15,16,17] In the present study, severe anemia was observed in 20% (n=17) as compared to 7%, 18.5% and 33% in various studies. [1,6,12]Thirty six (53%) patients were anemic with a CD4 count of <200 cells/µL. [1Attili et al[7] found a strong correlation between CD4 count and severity of anemia in their hospital- based cohort study. The cumulative incidence and severity of anemia was highest among patients who had CD4 count of < 200 cells/ µL and was lowest with CD4 count of >500 cells/ µL. Sullivan et al, in their study reported that the incidence of anemia was strongly and dependably associated with the progression of HIV disease as measured by diagnosis of AIDS defining opportunistic illness and measurement of CD4 count < 200 cells/μl. [19]

In the present study, the presence of anemia correlated with disease progression as most of the patients with anemia belonged to category 1 (CD4 counts of <200cell/ mm3). This association is most likely explained by the increasing viral burden as HIV disease progresses, which leads to cytokine-mediated myelosuppression and anemia. [19] Inflammatory cytokines released by lymphocytes such as tumour necrosis factor (TNF), interleukin-1 (IL-1) and interferon gamma play an important role in the pathogenesis of anaemia. These cytokines inhibit erythropoiesis in vitro. [20] TNF levels were found to be consistently elevated in HIV infection and this condition is correlated with viral load.21Studies also clearly indicate that anemia does affect the survival of HIV infected patients. [1,3,7,18,22] The HGB, RBC and HCT levels also showed correspondingly low values with decrease in CD4 counts. The P value was statistically significant. The results of a study by Mocroftet al[23]showed that hemoglobin levels provided prognostic information independent of that provided by CD4 count. The overall frequency of anemia in this study group was comparable with other studies (Table2). [24,25,26,27,8,28] Risk factors currently associated withanemia in HIV infection include clinical AIDS, CD4 cell count of <200 cells/µL, women, plasma viral load, black race, zidovudine use, lower body mass index, increasing age, bacterial pneumonia, oral candidiasis and fever. [29] The increased frequency of normocytic normochromic anemia (67.1%) in the present study was in accordance with some studies and discordant with others(Table 3). [1,6,25,30,8,28] In the present study, we did not find any patient with hemolytic anemia. Macrocytosis has been well described in patients undergoing antiretroviral therapy with zidovudine. [12,18,20] In the present study group, the six patients with macrocytic anemia were on irregular ART, which could explain anemia in this group. Leukopenia typically involves granulocytes and lymphocytes, although monocytopenia has also been reported in patients with the diagnosis of AIDS. Neutropenia of less than 1000 cells/μL is reported in approximately 10% of patients with early, asymptomatic HIV infection and in more than 50% of individuals with advanced HIV related immunodeficiency.[3,13,20,29]Neutropenia is often caused or exacerbated by concomitant myelosuppressive drugs. Adverse drug reactions and their complications can also cause neutropenia in patients with HIV/AIDS. [1] Various studies have reported neutropenia in 13% to 44% of cases with progression of disease from HIV to AIDS. [7] In the present study, only three patients were detected to have neutropenia. Dikshith et al [1] did not identify any case

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Table 1: Correlation of haematological parameters with CD4 counts. CD 4 count ( cells/µL)

<200

200-499

500+

P value

HGB (mean±SD) g/dl

9.6±2.2

9.5±2.2

12.07±1.5

0.007**

RBC count (Mean±SD) millions/µL

3.3±0.7

3.5±0.8

4.3±0.6

0.003**

HCT (Mean ± SD) %

30.63±6.04

31.74±6.6

36.65±4.8

0.02*

TLC (cells/µL)

6358±3774

6237±3028

8870±4419

0.126

ANC(cells/µL)

4897±3505

4737±3107

6184±4040

0.51

ALC(cells/µL)

1383±694

1554±1059

1953±725

0.16

1.83±1.17

1.89±0.98

2.17±1.41

0.7

Platelets L/ mm (Mean±SD) 3

(* significant P value) Table 2: Comparison of number of anaemia cases in the present study with other studies. Authors

No of anaemia cases

Total cases

Percentage

Karcher et al

175

197

89 %

Tripathi et al

82.4 %

Sitalakshmi et al

64.2 %

Kaloutsi et al

85 %

Parinitha et al

210

250

84 %

Rahman et al

103

204

50.5%

Present study

100

85%

Table3: Comparison of morphological patterns of anemia in the present study with other studies. Patterns of blood picture

Tripathi et al

Khandekar et al

Parinitha et al

Rahman et al

Present study

Number

Normocytic normochromic anaemia

72.9

48.57

101

40.4

13.7

67.1

Microcytic hypochromic anaemia

5.4

10.71

7.2

4.9

23.5

Macrocytic anemia

4.1

22.86

7.1

of neutropenia which differed from a study by Attili et al in which 22.7% of patients had neutropenia. [7] The correlation between the level of ANC and the need for hospitalization for bacterial infections has also been proven in some trials. [31]Thus, it could be important to recognize patients with neutropenia, who are at increased risk of developing these infections. HIV infection can directly result in lymphopenia as the infection evolves, leading to a decrease in CD4+ lymphocytes and is one of the most important prognostic indicators for the risk of developing opportunistic infections. [32]

the present study, lymphopenia was defined as when the patients had an absolute lymphocyte count of <800cells/ mm3. However, the cut-offs in the other studies was in the range of <1000-1500 cells/mm3. This could probably explain this variability. The TLC, ANC and ALC did not show a statistically significant association with CD4 count, although the counts were low with decreasing CD4 counts. The sample size and the cut-off of lymphopenia might elucidate this.

In the present study, lymphopenia was seen in 23/100 (23%) of patients. There has been a variation in the frequency of lymphopenia reported in different studies which includes 28.9% (59/204), 65.2%(163/250), 70% (14/20) and in 25.6% (19/74) of the cases. [28,8,33,25] A significant association of absolute lymphocyte count with CD4 cell counts was observed in some studies. [8,28]In

Thrombocytopeniais reasonably common during the course of HIV infection, occurring in approximately 40% of patients. It serves as the first symptom or sign of infection in approximately 10% of individuals. [34]Presence of thrombocytopenia is independent of the disease progression. The mechanism of thrombocytopenia in HIV infection is mainly due to ineffective platelet production and at the same time increased platelet destruction. [35]

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Prevalence of thrombocytopenia is reported to be higher among persons with AIDS, homosexuals, older persons and injecting drug users.[36] In the present study,thrombocytopenia was seen in thirty two patients.Various studies have reported thrombocytopenia in 18% (45/250), 13% (65/500), 13% (121/925) and 3.4% (7/204) of the cases.[8,37,38,28] However, Karcher et al [24] reported thrombocytopenia in 88/196 (45%) cases, which was in accordance with the present study. It was the most frequent cytopenia observed by Kasturi et al and a feature seen in advanced disease. [12]However, in the present study, no significant relationship with CD4 counts was observed,endorsing the data of the previous studies. [1,7] Our study reported pancytopenia in eight patients.In the present study, coagulation abnormalities were not seen. Though the increased risk of venous thrombosis is known in HIV infection, coagulation abnormalities are not well established. In a CDC study, it was found that overall incidence of thrombotic episode was 2.6 per 1000 HIVinfected persons. [7]

Conclusion

Hematologic abnormalities are the common manifestations in patients with HIV/AIDS. All the cell lines are affected by HIV, resulting in anemia, thrombocytopenia and leukopenia. The recent surge of HIV infection has led to an increase in the incidence of hematologic abnormalities. Anemia is the most common hematologic abnormality which is more prevalent among patients with declining CD4 count and is strongly associated with progression of the disease. Hence, it is prudent to investigate and find the cause of anemia for instituting specific treatment. The severity of other peripheral cytopenias is related to the disease burden and has got significant impact on clinical outcome. Early detection, exact cause and appropriate treatment of these abnormalities will reduce morbidity and mortality in HIV/AIDS patients. The spectrum of these problems has been recognized in certain parts of the world. The study of nature of these hematological abnormalities is therefore relevant in a tertiary care centre in a south Indian population.

Acknowledgement: Dr Manjunath GV References

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A-489 Infected Persons with Correlation to CD4 Cell Count. J Bangladesh Coll Phys Surg2014;32:130-6. 29. Lichtman MA, Beutler E, SeugsohnU, KaushanskyK, Kipps TO. Hematologic Aspects of Human Immunodeficiency Syndrome: Overview. Williams Hematology. 7th ed. United States of America: The McGraw-Hill Companies; 2007:1109-34. 30. Khandekar MM, Deshmukh SD, Holla VV, Rane SR, Kakrani AL, Sangale SA, et al. Profile of bone marrow examination in HIV/AIDS patients to detect opportunistic infections, especially tuberculosis. Indian J PatholMicrobiol 2005;48:7-12. 31. Jacobson MA, Liu RC, Davies D, Cohen PT. Human immunodeficiency virus disease-related neutropenia and the risk of hospitalization for bacterial infection. Arch Intern Med 1997;157:1825-31. 32. Cingolani A, Gastaldi R, Fassone L, Pierconti F, Giancola ML, Martini M, et al. Epstein-Barr virus infection is predictive of CNS involvement in systemic AIDS-related non-Hodgkin’s lymphomas. J ClinOncol 2000;18:3325-30. 33. Treacy M, Lai L, Costello C, Clark A. Peripheral blood and bone marrow abnormalities in patients with HIV related disease. Br J Haematol 1987;65:289-94. 34. Ballem PJ, Belzberg A, Devine DV, Lyster D, Spruston B, Chambers H,et al. Kinetic studies of the mechanism of thrombocytopenia in patients with human immunodeficiency virus infection. N Engl J Med 1992;327:1779-84. 35. Kuter DJ, Phil D, Gernsheimer TB. Thrombopoietin and Platelet Production inChronic Immune Thrombocytopenia.HematolOncolClin North Am 2009;23:1193–1211. 36. Kouri YH, Borkowsky W, Nardi M, Karpatkin S, Basch RS. Human megakaryocytes have a CD4 molecule capable of binding human immunodeficiency virus-1. Blood1993;81:2664-70. 37. Patwardhan MS, Gowlikar AS, Abhyankar JR, Atre MC. Hematologic profile of HIV positive patients. Ind J PatholMicrobiol 2002;45:147-50. 38. Costello C. Haematological abnormalities in human immunodeficiency virus (HIV) disease. J ClinPathol 1988;41:711-15.

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Case Report Primary Intracerebral Myxopapillary Ependymoma: A Rare Case Report Priyanka Patangia*, Naresh N. Rai, Rajeev Saxena and Preetam Singh Mandawat Department of Pathology, Government Medical College, Kota, Rajasthan, India. Keywords: Myxopapillary Ependymoma; Cerebral Hemisphere; Frontal Lobe

ABSTRACT Myxopapillary ependymoma (MPE) is a variant of ependymoma occurring almost exclusively in the conus medullaris or filum terminale. Myxopapillary ependymoma primarily in the brain is extremely rare. Here we report the case of a 21 year female with a right frontal myxopapillary ependymoma which did not demonstrate any connection to the lateral ventricles. Patient is presented with complaint of headache and vomiting with no history of convulsion, loss of consciousness. On radiological examination, MRI of brain revealed an ill-defined, cystic mass lesion in frontal lobe. On histopathological examination it was reported as myxopapillary ependymoma. This is the sixteenth reported case of histologically proven primary myxopapillary intracranial ependymoma and fourth cases of supratentorial intaparenchymal lesion.

*Corresponding author: Dr Priyanka Patangia, Department of Pathology, Government Medical College, Kota, Rajasthan, India. 324005 Phone: +91 9461656729 E-mail: drpriyanka1406@gmail.com

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

Patangia et al.

Introduction

Myxopapillary ependymoma was first described by Kernohan in 1932 [1], as a variant of ependymoma occurring in the conus medullaris or ďŹ lum terminale exclusively [2]. Although it can affect patients of all ages, they are most frequent in young adults. Bailey and Cushing recognised ependymomas as an independent entity in their first brain tumour classification (1926). Myxopapillary ependymoma occurring primarily in the brain parenchyma is extremely rare and only Fifteen cases have been reported in the literature till date.[3-16] The characteristic histological feature of myxopapillary ependymomas are the juxtaposition of pseudo-papillary structures consisting of a centrally located vessel surrounded by a ring of mucin lined by cuboidal ependymal cells. The most characteristic histological feature is the abundance of intercellular and perivascular mucin and the arborizing vasculature, which tends to form papillae. Myxopapillary ependymoma should be a possible differential diagnosis whenever an intracranial cystic tumor is found.

Case History

A 21 year female patient presented with complaint of headache and vomiting with no history of convulsion, loss of consciousness. On radiological examination, MRI of brain revealed an ill-defined, cystic mass lesion measuring 6.2 x 5.9 x 4.8cm involving cortical and sub cortical region of right frontal lobe; these areas appeared hypointense to gray matter on T1, hyperintense on T2W and FLAIR images; on post contrast images cystic lesion show rim enhancement with central non-enhancing areas. Small mural nodule also seen. Faint calcification seen within the lesion. There is mild perilesional white matter edema not

C-237 showing enhancement thus favouring vasogenic edema. Ventricles were normal but there was a mid-line shift to left side. (Figure 1 & 2) Based on MRI findings differential diagnosis given were primary brain tumor, ganglioglioma/ cystic oligodendroglioma/ low grade astrocytoma. Patients complete blood counts and serum biochemistry were within normal limits. Patient was subjected to excision of mass with duroplasty. On gross examination multiple soft tissue pieces were received of which largest tissue measured 4 x 3 x 2.5 cm with presence of normal brain tissue. On microscopic examination sections revealed characteristic finding of myxopapillary ependymoma (Figure 3 & 4) i.e. presence of papillary architecture with abundant perivascular mucin and areas of calcifications (psammoma bodies) (Figure 5 & 6). Alcian blue staining demonstrated perivascular mucin. X-Ray spine excluded a primary tumor in filum terminale region which is the most common site thus ruling out the possibility of intracerebral metastasis. Intervention: The lesion was totally resected. After surgery, the patient was neurologically intact and had an uneventful recovery. On post-operative follow up CT scan of brain revealed acute extradural hemorrhage in right fronto-parietal region causing mass effect over adjacent cerebral parenchyma. Mild shift to left is present. Other regions of cerebral parenchyma, cerebellum and brainstem appears normal.

Discussion

Ependymomas are tumors having origin from the ependymal cells lining cerebral ventricles, spinal central canal, and cortical rests.[17,18] The most common location for ependymomas is the 4th ventricle, followed by the spinal

Fig. 1: MRI scan showing cystic lesion in right frontal lobe which is well separated from lateral ventricle.

Fig. 2: MRI Scan reveals a cystic lesion with sparing of ventricles and midline shift.

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Fig 3. H&E section (10x) showing pseudopapillary structures consisting of a centrally located vessel surrounded by a ring of mucin lined by cuboidal ependymal cells.

Fig. 4: H&E section (20x) showing pseudopapillary structures consisting of a centrally located vessel surrounded by a ring of mucin lined by cuboidal ependymal cells.

Fig. 5: Alcian blue (20x)staining showing perivascular mucin.

Fig 6. Alcian blue staining (10x) showing abundant mucin and psammoma bodies are also present.

cord, ďŹ lum terminale/cauda equina, and supratentorium of the brain [19]. Occasionally it may be seen in the subcutaneous tissue overlying the sacrum. [20]

myxopapillary ependymoma before making a diagnosis of primary intracranial myxopapillary ependymoma.

Myxopapillary ependymoma is a variant of ependymoma, characterized microscopically by a papillary arrangement of neoplastic cells surrounding a fibrovascular core containing both hyalinized blood vessels and myxoid degeneration. Histologically, it is a benign tumor [12] (WHO grade 1) with a peak incidence in the 4th decade of life which is generally conďŹ ned to the lumbosacral region of the spinal cord. It constitutes 27% of ependymomas occurring in the spinal cord. [1,2] It may rarely disseminate via the CSF pathways into the brain and be mistaken as a primary intracranial myxopapillary ependymoma. [22-24] It is therefore important to rule out the possibility of metastasis from a spinal

Including the presented case, 16 primary intracranial myxopapillary ependymomas have been reported [3-16]. Present case is the fourth reported case of intracranial intraparenchymal myxopapillary ependymomas.[4,7,9] Nine tumors were located supratentorially while seven were infratentorial. Of the supratentorial group, four were intraventricular( 3 from lateral ventricle and 1 from third ventricle), four were intraparenchymal and one was transependymal. Among infratentorial group 3 were in fourth ventricle, 2 in cerebellopontine angle, one in each medulla and cerebellum. (Table 1) All of them were treated by total surgical resection. Radiological features of these tumors were all well-

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Table 1: Reported cases of Intracranial Myxopapillary Ependymoma. Author (Year)

Age

Sex

Site

Location

Right Lateral Ventricle

Right occipital lobe

Sato et al (1983)3 Maruyama R et al (1992) Warnick RE et al (1993)

Lateral Ventricle

Matyja E et al (2003)6

Right Lateral Ventricle

Ralte et al (2004)

Left temporal lobe

Tseng Y-C et al (2004)8

7 20

F F

Fourth ventricle Bilateral cerebral falx

IT ST

Tzerakis et al (2004)9

Left frontal lobe

Lim SC et al (2006)10

Fourth ventricle

Left CPA

DiLuna ML et al (2010)12

Medulla

Shapey J et al (2011)

Right CPA

Sparaco M et al (2009)

Margetis K et al (2011)

–

Cerebellar

Chakraborti S et al (2012)15

Fourth ventricle

Wang M et al (2013)

Third ventricle

Right Frontal lobe

Present study

CPA- cerebellopontine angle; ST-supratentorial; IT- infratentorial

demarcated primary cystic masses with strong enhancement at their solid part and/or cystic walls. Intratumoral calciﬁcation or hemorrhage, commonly seen in ependymomas were also seen. However, these imaging features are still not speciﬁc enough to differentiate from other types of ependymomas or other primary cystic intracranial tumors such as astrocytoma, primitive neuroectodermal tumor, and choroid plexus papilloma. In conclusion, myxopapillary ependymoma can occur as a primary in the brain parenchyma or even extra-axial space.

Conclusion

This is the sixteenth reported case of histologically proven primary myxopapillary intracranial ependymoma and fourth case of intaparenchymal MPE. In conclusion, myxopapillary ependymoma can occur in the brain parenchyma or even extra-axial space so whenever a primary cystic intracranial mass is suspected, myxopapillary ependymoma should be always be a differential diagnosis.

Acknowledgement

Dr. S.N.Gautam, Assisstant Professor, Department of Neurosurgery, Government Medical College, Kota. Dr. Sangeeta Saxena, Professor, Department Radiodiagnosis, Government Medical College, Kota.

Funding None

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Competing Interests None Declared

Refrences

1. Kernohan JW. Primary tumors of the spinal cord and intradural ﬁlum terminale.In:PenﬁeldW,editor. Cytology and cellular pathology of the nervous system, vol.3. New York: Paul B. Hoeber; 1932. p.993–1025. 2. Sonneland PR, Scheithauer BW, Onofrio BM. Myxopapillary ependymoma. A clinicopathologic and immunocytochemical study of 77 cases. Cancer 1985; 56:883–93. 3. Sato H, Ohmura K, Mizushima M, et al. Myxopapillary ependymoma of the lateral ventricle. A study on the mechanism of its stromal myxoid change. Acta Pathol Jpn 1983;33:1017–25. 4. Maruyama R, Koga K, Nakahara T, et al. Cerebral myxopapillary ependymoma. Hum Pathol 1992;23: 960–2. 5. Warnick RE, Raisanen J, Adornato BT, et al. Intracranial myxopapillary ependymoma: case report. J Neurooncol 1993;15:251–6. 6. Matyja E, Naganska E, Zabek M, et al. Myxopapillary ependymoma of the lateral ventricle with local recurrences: histopathological and ultrastructural analysis of a case. Folia Neuropathol 2003;41:51–7. eISSN: 2349-6983; pISSN: 2394-6466

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7. Ralte AM, Rao S, Sharma MC, Suri A, Gaikwad S, Sarkar C. Myxopapillary ependymoma of the temporal lobe--report of a rare case of temporal lobe epilepsy. Clin Neuropathol. 2004 Mar-Apr;23(2):53-8. 8. Tseng YC, Hsu H-L, Jung SM, Chen CJ. Primary intracranial myxopapillary ependymomas: report of two cases and review of the literature. Acta Radiol 2004;45: 344–347. 9. Tzerakis N, Georgakoulias N, Kontogeorgos G, Mitsos A, Jenkins A, Orphanidis G. Intraparenchymal myxopapillary ependymoma: case report. Neurosurgery. 2004 Oct;55(4):981. 10. Lim SC, Jang SJ. Myxopapillary ependymoma of the fourth ventricle. Clin Neurol Neurosurg. 2006 Feb;108(2):211-4. 11. Sparaco M, Morelli L, Piscioli I, et al: Primary myxopapillary ependymoma of the cerebellopontine angle: Report of a case. Neurosurg Rev 32:241-244; discussion 244, 2009 12. DiLuna ML, Levy GH, Sood S, Duncan CC. Primary myxopapillary ependymoma of the medulla: case report. Neurosurgery 2010;66(6):E1208–E1209 13. Shapey J, Barazi S, Bodi I, Thomas N. Myxopapillary ependymoma of the cerebellopontine angle: Retrograde metastasis or primary tumour? Br J Neurosurg 2011;25:122-3. 14. Margetis K, Koutsarnakis C, Stranjalis G: Primary cerebellar myxopapillary ependymoma. J Neurooncol 104:839-840, 2011 15. Chakraborti S, Govindan A, Alapatt JP, Radhakrishnan M, Santosh V. Primary myxopapillary ependymoma

of the fourth ventricle with cartilaginous metaplasia: a case report and review of the literature. Brain Tumor Pathol. 2012 Jan;29(1):25-30. 16. Wang M, Wang H, Zhou Y, Zhan R, Wan S. Myxopapillary ependymoma in the third ventricle area and sacral canal: dropped or retrograde metastasis? Neurol Med Chir (Tokyo). 2013;53(4):237-41. 17. Chamberlain MC. Ependymomas. Curr Neurol Neurosci Rep 2003;3:193–9. 18. Mork SJ, Loken AC. Ependymoma: a follow-up study of 101 cases. Cancer 1977;40:907–15. 19. Helwig EB, Stern JB. Subcutaneous sacrococcygeal myxopapillary ependymoma. A clinicopathologic study of cases 30 cases. Am J Clin Patholsemi 1984;81:156–61. 20. Hanchey RE, Stears JC, Lehman RA, et al. Interhemispheric ependymoma mimicking falx meningioma. Case report. J Neurosurg 1976;45:108–12. 21. Marks JE, Adler SJ. A comparative study of ependymomas by site of origin. Int J Radiat Oncol Biol Phys 1982;8:37–43. 22. Moutaery K, Aabed MY, Ojeda VJ. Cerebral and spinal cord myxopapillary ependymomas: a case report. Pathology 1996;28:373–6. 23. Kittel K, Gjuric M, Niedobitek G. Metastasis of a spinal myxopapillary ependymoma to the inner auditory canal. HNO 2001;49:298–302. 24. Liborio R, Pais RF, Soares GB, et al. Medullary and intracranial metastases of myxopapillary ependymoma. Acta Med Port 2001;14:133–8.

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Case Report Co-occcurence of Two Different Subtypes of Renal Cell Carcinoma in A Unilateral Healthy Kidney Neeraj Dhameja1, Hema Goyal1*, Priyanka Aswal1 and U. S. Dwivedi2 Department of Pathology, Institute of Medical sciences, Banaras Hindu University, UP, India 2 Department of Surgery, Institute of Medical sciences, Banaras Hindu University, UP, India

Keywords: Renal Cell Carcinoma(RCC), Clear Cell Carcinoma, Papillary RCC(PRCC), Translocation Associated RCC

ABSTRACT Renal cell carcinoma (RCC) is a malignant neoplasm of renal tubular origin of adult age group which presents as a mass in a kidney with abdominal pain and hematuria. There are various subtypes of RCC, each having distinct morphological features, prognosis and response to targeted therapy. Development of two RCC of different subtypes in the same kidney is rare because each RCC arises from a particular genetic abnormality. here we discuss two different RCC subtypes in a healthy kidney, one showing features of Papillary RCC and other showing mixed features with papillae formation and clear cells.

*Corresponding author: Dr. Hema Goyal, Room no.44, Lady doctor hostel, IMS, BHU, Varanasi, UP, INDIA Phone: +91 7379140789 E-mail: ankush.hema@yahoo.in

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Introduction

Renal cell carcinoma is the commonest malignant epithelial tumor of the kidney. It is a tumor of adult population with peak in the sixth and seventh decades though some recently described entities like translocation associated RCC are common in children. Male predominance is seen. There are different histopathological types of RCC having different morphological, immunohistochemical and cytogenetic features and variable prognosis. RCC presents as a single mass in the kidney, though some RCC like papillary RCC may be multifocal [1]. Simultaneous occurrence of two different types of RCC in the kidney is rare and there are only few case reports and that too either in diseased kidney or with genetic predisposition. Here we describe two different subtypes of RCC in a single healthy kidney.

Case Report

A 50 year male patient presented to our hospital with mass in the abdomen. CT scan showed a mass in the left kidney measuring 13x10.5x9 cm. Radical nephrectomy was done and sent to the pathology department of IMS, BHU. Gross Findings: The radical nephrectomy specimen was measured 13x10.5x9 cm. Part of the ureter was identified on the medial aspect of specimen and measured 3cm in length. The capsule of the specimen was intact. On sectioning, a tumor was found in the upper pole of the kidney measuring 12x8x8 cm. On cut, the tumor was variegated in appearance with yellow, hemorrhagic and

Fig 1 : Gross specimen: Radical nephrectomy specimen shows two distinct tumors - black shaded arrow shows larger dominant tumor at the upper pole & Open arrow shows smaller tumor at the lower pole of kidney.

grayish white areas. No cystic areas were seen. The part of ureter, perinephric fat, gerota fascia and renal sinus were found free of tumor grossly. A separate tumor was seen in the lower pole separate from the main tumor and measured 2.2x2cm. Cut surface was solid grayish white. No hemorrhagic and necrotic areas were identified in the lower pole tumor. (Figure 1). Part of normal parenchyma was also identified in between the two tumors. Microscopic Examination: Histopathological examination of the main (larger) tumor showed predominantly clear cell in acinar pattern and focal areas of papillary pattern. The acinar pattern showed nests of tumor cells separated by thin fibrovascular septae (Figure 2). The papillary pattern showed papillae lined by cells with abundant clear cytoplasm along with thin fibrovascular core. (Figure 3). Nuclei were basally placed and were of high nuclear grade. Few psammoma bodies and hyalinized areas were also seen. No subnuclear vacuolation, stromal metaplasia, foamy macrophages or hemosiderin in the epithelial cells were seen. IHC showed few CK positive cells (Figure 4). The second tumor at the lower pole of kidney showed a papillary tumor with many foamy macrophages within the fibro vascular cores (Figure 5,6). The papillae were lined by single layer of cells with scant basophilic cytoplasm. Based on these findings it was reported as papillary RCC type I. CK7 was strongly positive (Fig7). On follow up at 6 months, patient was symptom free.

Fig. 2: Dominant tumor shows acinar pattern â&#x20AC;&#x201C; nests of tumor cells(clear cells) separated by fibrovascular septa. ( H&E , 10x10).

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Fig. 3: Dominant tumor shows papillary pattern â&#x20AC;&#x201C; long papillae lined by polygonal clear cells with a thin fibrovascular core.( H&E, 10x10).

Fig. 4: Immunohistochemistry (IHC), Cytokeratin : Papillary areas of dominant tumor shows membranous cytokeratin positivity (IHC CK, 10x10).

Fig 5 : Smaller tumor : Sections from smaller tumor shows features of Papillary RCC ( H&E ,10x10) .

Fig. 6: Smaller tumor : High power view shows papillae with prominent fibrovascular core containing foamy macrophages (arrow) (H&E, 20X).

Fig. 7: IHC (CK7) : Smaller tumor shows strong diffuse membranous positivity for CK 7 (IHC,CK7, 10x10).

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Discussion

is a member of microphthalmia associated transcription (MITF) family[6,7]. This carcinoma is the commonest RCC subtype in children and associated with prior chemotherapy. It can also develop in adult population and one study found an incidence of 4.2% in adult population[8]. Morphologically it shows papillae and acinar pattern, clear cells, psammoma bodies and hyalinized nodules. Strong nuclear positivity for TFE3 is seen on IHC. In the present case, Xp11 translocation may be one possibility, however, without specific IHC and molecular studies, it cannot be confirmed.

Clear cell RCC is the commonest RCC subtype characterized by acinar or nesting pattern with thin fibrovascular septae and abundant clear cytoplasm. Papillae formation is rare but pseudopapillae may be seen in high grade tumors. Immunohistochemistry shows diffuse membranous positivity for CAIX and CD 10. 3p deletion is detected on cytogenetic evaluation[4].

Few reports of concurrent RCCs of different histological types have been described[9,10]. In these reports, the patients had some medical or genetic disease and PRCC was the dominant tumor, whereas, in the present case, the patient was healthy and PRCC was the smaller tumor. Since these cases are rare, the prognosis is uncertain. In the present case, the patient underwent radical nephrectomy and is currently symptom free after a follow up of six months. Synchronous 2 different types of RCC in same kidney should not change the management approach.

RCC includes different types of tumors arising from the tubular epithelium from the kidney. Usually a single type of RCC develops in a kidney. Simultaneous development of two different RCCs in the same kidney is rare, though association of RCC with benign tumors like oncocytoma and angiomyolipoma has been described[2]. WHO classifies RCC into different types based on characteristic morphological features [3]. Each RCC subtype has specific immunohistochemical and cytogenetic features.

Papillary RCC is the next common RCC subtype comprising around 10% of RCC and characterized by papillae formation with foamy macrophages within the fibrovascular cores. Cytologically, PRCC is divided into type I PRCC characterized by cells with single layer of nuclei and scant amphophilic cytoplasm and type II PRCC by cells showing nuclear stratification with abundant eosinophilic cytoplasm. Clear cells are rare. IHC shows strong diffuse membranous positivity for CK7. Cytogenetics shows trisomy of chromosomes 7 and 17 and loss of chromosome Y[5]. In the present case, the smaller tumor showed features of PRCC and the dominant tumor showed acinar and papillary pattern with abundant clear cytoplasm, psammoma bodies and high nuclear grade. WHO classifies these tumors under category, Renal cell carcinoma, unclassified. H Ross et al., described four subtypes for tumors showing both clear cell and papillary pattern as Clear cell RCC, Papillary RCC, Clear cell papillary RCC and Xp11 translocation carcinoma[6]. Clear cell RCC and Papillary RCC may show focal papillae formation and clear cells respectively, but the nuclear grade is low in case of clear cell RCC and foamy macrophages are seen in papillary RCC. Moreover, the characteristic IHC and cytogenetics distinguish these tumors. Clear cell papillary RCC is a recently described entity showing papillae lined by clear cells of low nuclear grade, subnuclear vacuolation and smooth muscle stromal metaplasia[6]. Xp11 translocation carcinoma is characterized by chromosomal translocation involving TFE3 gene on Xp11.2 locus with resultant fusion of TFE3 with multiple partner genes like ASPL, PRCC, NonO, PSF. This results in overexpression of TFE3 which

Conclusion

In conclusion, we here describe a rare finding of simultaneous occurrence of two different RCCs subtypes in a unilateral healthy kidney. There are very few case reports describing the same. To the best of our knowledge, this is the 13th case of the literature that had these two different subtypes of RCC in the same kidney [11]

Acknowledgements

I want to acknowledge my whole department of pathology for the great support.

Funding None

Competing Interests None Declared

Reference

1. Reuter VE, Tickoo S. Sternbergâ&#x20AC;&#x2122;s Diagnostic Surgical Pathology (5th edition). Philadelphia: Lippincott Williams & Wilkins; 2010.p. 1757-98. 2. Petrolla AA, MacLennan GT. Renal Cell Carcinoma and Other Concurrent Renal Neoplasms. The Journal of Urology. 2007;178:2163. 3. Eble JN, Togashi K, Pisani P: Renal cell carcinoma In: World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of the Urinary System and Male Genital Organs (JN Eble, G Sauter, JI Epstein, IA Sesterhenn Eds.) IARC Press, Lyon, 2004, pp. 12-14.

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Dhameja et al. 4. Licht M.R., Novick A.C., Tubbs R.R., Klein E.A., Levin H.S., Streem S.B. Renal oncocytoma: clinical and biological correlates. J Urol. 1993;150:1380–1383. 5. Jimenez RE, Eble JN, Reuter VE, Epstein JI, Folpe AL, de Peralta-Venturina M, Tamboli P, Ansell ID, Grignon DJ, Young RH, Amin MB: Concurrent angiomyolipoma and renal cell neoplasia: a study of 36 cases. Mod Pathol. 2001, 14: 157-163. 10.1038/ modpathol.3880275. 6. Ross H, Martignoni G, Argani P. Renal cell carcinoma with clear cell and papillary features. Arch Pathol Lab Med. 2012;136:391–399. 7. Armah HB, Parwani A. Xp11.2 Translocation Renal Cell Carcinoma. Arch Pathol Lab Med 2010;134:124-29. 8. Zhong M, Osborne L, Merino MJ, Hameed M, Aisner S. The study of Xp11 translocation renal cell

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C-245 carcinoma (RCC) in adults by TMA, IHC and FISH [abstract]. Mod Pathol. 2010;23(suppl 1):232A. 9. Simhan J, Canter DJ, Sterious SN, Smaldone MC, Tsai KJ, Li T, et al. Pathological concordance and surgical outcomes of sporadic synchronous unilateral multifocal renal masses treated with partial nephrectomy. J Urol 2013;189:43-47. 10. E. Capaccio, V. Varca, A. Simonato, C. Toncini, G. Carmignani, and L. E. Derchi, “Synchronous parenchymal renal tumors of different histology in the same kidney,” Acta Radiologica, 2009, vol. 50 (10),1187–1192. 11. Ustuner M, Yaprak B, Teke K, Ciftci S, Kart M, Yildiz K, et al. Coexisting papillary and clear renal cell carcinoma in the same kidney. Case Rep Urol 2014;2014:575181.

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Case Report Cytodiagnosis of Schwannoma of Parotid Gland: A Rare Entity in a Child Nishat Afroz1, Shagufta Qadri*1, Sunanda Chauhan1 and S Abrar Hasan2 Department of Pathology, Jawaharlal Nehru medical College, Aligarh Muslim University, Aligarh, U.P., India Department of Otorhinolaryngology, Jawaharlal Nehru medical College, Aligarh Muslim University, Aligarh, U.P., India 1

Keywords: Intra-parotid, Schwannoma, FNAC, Cytological Findings

ABSTRACT Schwannoma of parotid gland in a child is rare. We report a rare case of schwannoma in parotid gland in a 10-year-old child who presented with a gradually enlarging swelling in parotid region for the last I year. Color doppler study showed increased vascularity in the lesion with venous return pattern, suggestive of a hemangioma. However, fine needle aspiration cytology showed features consistent with schwannoma, comprising of sheets of uniform spindled cells with nuclei having tapering ends and exhibiting subtle nuclear palisading within a hypocellular fibrillary stromal background. The parotid mass was then excised. Histopathological and immunohistochemical examinations further yielded the diagnosis of schwannoma, confirming the cytological diagnosis. This case report emphasizes that the cytodiagnosis of schwannoma is difficult due to many pitfalls encountered in fine needle aspirate. Therefore, schwannoma should always be considered in differential diagnosis of spindle-cell lesion in salivary glands even in children for early diagnosis and appropriate treatment. Unless schwannoma is included in the cytologic differential diagnosis, the surgeon may fail to recognize it at operation and may inadvertently transect the facial nerve.

*Corresponding author: Dr Shagufta Qadri, Assistant Professor , Department of Pathology, Jawaharlal Nehru medical College (JNMC), Aligarh Muslim University (AMU), Aligarh, U.P., India PIN: 202002 Phone: +91 9897532879 E-mail: qadridrshagufta@gmail.com

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Afroz et al.

Introduction

Schwannomas (neurilemmomas) are common benign peripheral nerve sheath tumors composed of a relatively uniform spindle cells showing schwannian differentiation. Occurrence of schwannoma in parotid gland is extremely rare, accounting for only 0.2â&#x20AC;&#x201C;1.5 % of all parotid tumors.[1] The cytological diagnosis of schwannoma in parotid gland is rarely reported in literature and is difficult usually due to low yield of cells or paucicellularity in fine needle aspirates, combined with its varied cytomorphology, contributing to its wide differential diagnosis, mimicking a reactive or inflammatory process with spindle cell morphology or even a malignant spindle cell neoplasm.[2,3,4] This case is being reported for its rare occurrence at unusual site in a child and its distinctive cytological appearance.

Case report

A 10-year old boy presented with a slow growing painless swelling in the left parotid region from the last 1 year. There was no significant family history or hereditary predisposition. No history suggestive of systemic infection, any autoimmune disease or trauma at the site of swelling was present. Clinical examination revealed an ill-defined, firm, non-tender, mobile mass, measuring 3 Ă&#x2014; 3 cm in size and extending from the zygomatic arch to short of angle of mandible and from pre-auricular region to anterior border of masseter in vertical and horizontal planes, respectively (Figure 1a). Facial nerve functions were found to be normal. Ultrasound imaging ofthe mass showed a welldefined heterogenous lesion in the left parotid gland. Color Doppler study revealed increased vascularity with multiple vascular channels showing venous flow pattern (Figure 1b), suggestive of a hemangioma. Fine

C-247 needle aspiration (FNA) smears were obtained with a 22 gauge needle and stained with Hematoxylin and Eosin (H&E) and Papanicolaou (Pap) stains. The FNA cytology revealed high cellularity smears, comprising of sheets of uniform spindle cells with nuclei having tapering ends and exhibiting subtle nuclear palisading within a hypocellular fibrillary stromal background (Figure 2a and 2b). There was no significant nuclear atypia, mitoses or any evidence of necrosis. The diagnosis of schwannoma was made on cytomorphological features. The mass was then excised and submitted for histopathological examination, which showed features consistent with schwannoma, comprising of well-circumscribed, encapsulated tumor with cells having morphology varying from round, oval-to-spindle shape with slender wavy nuclei, and exhibiting prominent nuclear palisading (Verocay bodies) focally (Figure 2c), without necrosis and infiltrative growth pattern. The tumor sections showed strong immuno-histochemical positivity to S-100 (Figure 2d), and non-reactivity to cytokeratin, smooth muscle actin (SMA), calponin, P-63, desmin, neurofilament and CD-34, further affirming the diagnosis of schwannoma. The post-operative recovery of patient was uneventful. No recurrence of the tumor was seen during the follow-up period of past 8 months.

Discussion

Schwannomas are slow growing solitary, firm, wellcircumscribed, encapsulated round or ovoid tumors, located in the head, neck, and flexor surfaces of the extremities and usually seen in adults with age ranging from 2nd to 5th decade.[5] Occurrenceof schwannoma in intraparotid location in a child is rare and reported to arise from the intraparotid branches of the facial nerve.[6 ]

Fig. 1: 1a) showing mass (3 x 3 cm) in left parotid region in a child, 1b) color doppler image of the mass showing increased vascularity with vascular channels showing venous flow pattern.

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Cytodiagnosis of Parotid Schwannoma

Fig. 2:2a) and 2b) FNAC of parotid mass showing high cellularity smears comprising of uniform spindled cells with tapered nuclei, exhibiting subtle nuclear palisading within a fibrillary stromal background (a x H&E x 50, b x H&E x 125), 2c) histological section showing cellular areas comprising of palisaded spindled cells with tapered nuclear end (H&E x 125), 2d) showing strong S-100 positivity of tumor cells ( x 50).

Several pitfalls may be encountered in FNA smears for the diagnosis of neural lesions due to varied cytomorphologicalappearance of different variants, associated with usually a low yield of cells or paucicellularity.[3] The differential diagnosis in the present case included other spindle-cell lesions of parotid gland including myoepithelial tumors like myoepithelioma, cellular pleomorphic adenoma, benign lesions like nodular fasciitis, fibromatosis, solitary fibrous tumor, neurofibroma, leiomyoma andhemangioma. The most common diagnostic pitfalls in the evaluation of spindle cell aspirates of the salivary gland are myoepithelial tumors. Myoepitheliomas are also encapsulated tumors and have been occasionally reported in children.[7] Theyshowvaried cell population like plasmacytoid, epithelioid, stellate or clear cells, but it is the spindled-cell form of myoepithelioma that pose a diagnostic challenge. However, the myoepithelial cells in the spindle cell type myoepithelioma can be distinguished morphologically as they are elongated with uniform ovoid to fusiform nuclei with rounded ends in contrast to schwannoma which have wavy nuclei with pointed ends. The fibrillary stroma and subtle nuclear palisading, (Verocay body) can be characteristically identified in FNA smears of schwannoma and is scant or absent in myoepitheliomas. Moreover, cells of myoepithelioma show cytokeratin, SMA and calponin immuno-expression in contrast to schwannoma.[8] Cellular

or myoepithelial predominant pleomorphic adenoma may resemble a schwannoma clinically as it also presents as gradually enlarging painless mass. However, cytologically, the cells show similar features as that of myoepithelioma i.e. have spindle nuclei with rounded ends.While cellular pleomorphic adenomas can very rarely exhibit nuclear palisading causing confusion, but the presence of focal nuclear palisading associated with fibrillar areas, favors a diagnosis of schwannoma. Neurofibromamay closely resemble schwannoma on FNA. It is unencapsulated and can be distinguished cytologically as it shows cells with heterogenous pattern of oval and wavy nuclei, lacking palisading. Immunohistochemically, it shows the presence of scattered neurofilament positive axons. Nodular fasciitis is another pitfall in the diagnosis of spindle-cell lesions of salivary gland.[9] It is a reactive myofibroblastic proliferative lesion, characterized by loosely cohesive groups of spindle-shaped and stellate myofibroblasts admixed with collagen. Cells have plump oval to elongate nuclei and wispy, tapering bipolar cytoplasmic processes in mucoid background. Positive SMA immuno-expression and non-reactivity to S-100, distinguishes nodular fasciitis from schwannoma.[10] Fibromatosis can occur in head and neck region in young individuals. FNA shows uniform polygonal to elongate

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Afroz et al. plump fibroblasts admixed within myxoid or collagenous tissue.[11] Immunohistochemically, the fibroblasts and myofibroblasts comprising the lesion are positive for SMA and sometimes desmin. Solitary fibrous tumor is another rare neoplasm that occurs in the head and neck, and occasionally involving the parotid gland. Aspirates from SFT show hypercellular cohesive groups of haphazardly arranged monotonous tapered spindle cells. Immunohistochemically, it is distinguishable from schwannoma by its non-reactivity to S-100 and reactivity to CD-34 immuno-marker.[12] Similarly aspirates from a leiomyoma show loose fascicular groups of spindle cells with moderate amounts of eosinophilic cytoplasm, and elongated, blunt-ended “cigar-shaped” nuclei that lack atypia and mitotic activity. Immunohistochemically, leiomyoma show reactivity to SMA and desmin.[13] Another pitfall in the diagnosis of schwannoma is granulomatous inflammation. Epithelioid histiocytes may sometimes resemble cells of spindle-cell tumor. However, the background shows suppuration or necrosis in infective conditions. Also, rarely schwannomas may show increased vascularity on ultrasonography, as was seen in the present case and hence can mimic a vascular neoplasm. However, distinct cytomorphological features distinguish schwannoma from hemangioma or other vascular neoplasms. The malignant tumors like myoepithelial carcinoma, spindle cell-carcinoma and spindled malignant melanoma demonstrates substantial nuclear atypia, prominent nucleoli in cells, significant mitotic figures, and tumor necrosis, and thus can be excluded with ease on cytologic grounds.

Conclusion

Intraparotid schwannoma in a child is a rare entity. The cytological diagnosis of schwannoma pose diagnostic challenge as several pitfalls may be encountered in FNA smears due to its varied cytomorphological appearance simulating other spindle-cell lesions of salivary gland. However, meticulous close examination of FNA smears can exclude other closely resembling spindle-cell lesions of salivary gland, leading to early diagnosis and appropriate treatment.

Conflicts of interest

Nil - No conflict of interest with any.

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References

1. Oncel S, Onal K, Ermete M, Uluc E. Schwannoma (neurilemmoma) of the facial nerve presenting as a parotid mass. J LaryngolOtol 2002;116:642–3. 2. Jain R, Gupta S, Borkataky S, Agarwal R, Singh S, Gupta K et al. Rare Diagnosis on aspiration cytology of parotid gland schwannoma. ActaCytol 2010;54:112-4. 3. Henke CA, Salomao DR, Hughes JH. Cellular schwannoma mimics a sarcoma: an example of a potential pitfall in aspiration cytodiagnosis. DiagnCytopathol 1999;20:312-6. 4. Chhieng DC, Cohen JM, Cangiarella JF. Fine-needle aspiration of spindle cell and mesenchymal lesions of the salivary glands. DiagnCytopathol 2000;23:253–9. 5. Hui-Chi KU, Chi–Wei Yeh. Cervical Schwannoma. A case report and eight years review. Journal of Laryngology and Otology 2000;114(6):414–7. 6. Falcioni M, Russo A, Taibah A, Sanna M. Facial nerve tumors. OtolNeurotol 2003;24:942-7. 7. Yaman H, Gerek M, Tosun F, Deveci S, Kiliç E, Arslan HH. Myoepithelioma of the parotid gland in a child: a case report. J PediatrSurg2010;45:E5-7. 8. Politi M, Toro C, Zerman N, Mariuzzi L, Robiony M. Myoepithelioma of the parotid gland: Case report and review of literature. Oral Oncology Extra 2005;41:104-8. 9. Saad RS, Takei H, Lipscomb J, Ruiz B. Nodular fasciitis of parotid region: a pitfall in the diagnosis of pleomorphic adenomas on fine-needle aspiration cytology. DiagnCytopathol2005;33:191-4. 10. Peng WX, Kudo M, Yamamoto T, Inai S, Fujii T, Teduka K et al. Nodular fasciitis in the parotid gland: a case report and review of the literature. DiagnCytopathol2013;41:829–33. 11. Iyer VK. Cytology of soft tissue tumors: Benign soft tissue tumors including reactive, nonneoplastic lesions. J Cytol 2008;25:81-6 12. Bauer JL, Miklos AZ, Thompson LD. Parotid gland solitary fibrous tumor: a case report and clinicopathologic review of 22 cases from the literature. Head Neck Pathol2012;6:21-31 13. Tao LC, Davidson DD. Aspiration biopsy cytology of smooth muscle tumors. A cytologic approach to the differentiation between leiomyosarcoma and leiomyoma. ActaCytol 1993;37:300-8.

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Case Report Gastrointestinal Stromal Tumor in a Roux en Y limb Naveen Kumar1, Saishalini Chinnathambi Narayanan1*, Prathiba Duvvuru1 and Babu Elangovan2 Department of Pathology, Sri Ramachandra Medical college and research institute. Chennai, Tamil Nadu, India 2 Department of Surgical Gastroenterology, Sri Ramachandra Medical college and research institute. Chennai, Tamil Nadu, India

Keywords: CD 117, DOG1, Gastrointestinal Stromal Tumor, Roux-en-Y Anastomosis

ABSTRACT Our patient, a 54 year old male came with abdominal distension and pain for 5 months with on and off constipation. Per abdomen examination revealed a midline scar with an ill-defined mass in the epigastrium and right hypochondrium. Endoscopy revealed a bulge in the posterior wall of the stomach. Contrast enhanced CT scan suggested a lesion arising from the posterior wall of stomach. But, laparotomy showed the mass to be arising from the blind end of the jejunum, post roux-en-Y anastomosis. We received a globular mass measuring 14x8x7.5 cm attached to one end of the intestine. External surface was grey tan to grey brown with multiple nodules of varying sizes. Cut surface was variegated with solid and cystic areas filled with blood. Our initial differential diagnoses were leiomyosarcoma, angiosarcoma, inflammatory fibroid polyp and GIST.Microscopy revealed spindle cells showing mild to moderate pleomorphism admixed with lymphocytes and plasma cells with mitotic count of four per 50 high power fields. By immunohistochemistry the spindle cells were CD117 and DOG1 positive which prompted a diagnosis of gastrointestinal stromal tumor. We present this case for the uncommon location of GIST in a roux en Y limb.

*Corresponding author: Dr. C.N. Saishalini, Assistant professor, Department of pathology, Sri Ramachandra Medical College and Research Instittue, Porur, Chennai- 600116, Tamilnadu, India Phone: +91 9940217743 E-mail: saishalini_cn14@yahoo.com

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Kumar et al.

Introduction

Gastrointestinal stromal tumor (GIST) arises from the interstitial cell of Cajal. They are the commonest mesenchymal tumor of the gut. The median age of presentation is 60 years with no specific predilection. [1]GIST can arise anywhere from the wall of GIT most commonly from stomach, small intestine, colon and esophagus. [2]Omentum, mesentery, retroperitoneum and pelvis may also give rise to GIST. CD 117 and PDGFRA are the most common genes mutated in GIST. [3]Difficulty in their interpretation arises due to variation in their differentiation. Morphological variations include tumors with smooth muscle differentiation, with neural differentiation (GANT), both neural and smooth muscle like and those lacking differentiation. Other variants are those with prominent myxoid matrix, signet ring features, granular cell change, oncocytic, rhabdoid, crystalloid formation, heavy inflammatory infiltrate and tumor giant cells. [4]

C-251 leiomyosarcoma, angiosarcoma, inflammatory fibroid polyp and GIST. Microscopy revealed spindle cells with mild to moderate pleomorphism admixed with plasma cells and lymphocytes with mitotic count of four per 50 high power fields (Figs 2, 3). By immunohistochemistry, the spindle cells were diffusely positive for CD117 (Fig 4), vimentin, and DOG 1 (Fig 5). In addition they were focally positive for SMA and hence a diagnosis of unifocal low grade gastrointestinal stromal tumor with focal smooth muscle differentiation was given.

Case Report

54 year old male presented with complaints of abdominal distension and pain for 5 months with on and off constipation. On inspection his vitals were stable. Per abdomen revealed a midline scar of length ten centimeters. The patient gave a history of surgery, the details of which were not available with him. An ill-defined mass was palpated in the epigastrium and hypochondrium. Endoscopy and CT scan suggested a tumor arising from posterior wall of stomach. But laparotomy revealed a globular mass arising from the jejunal limb of a previous Roux en Y anastomosis. The tumor was resected with the jejunal limb and a jejuno-jejunal anastomosis was done. We received irregular globular mass measuring 14x8x7.5 cm attached to one end of the intestine (Fig 1). External surface was grey-tan to grey-brown with multiple nodules of varying sizes. Cut surface was variegated with solid and cystic areas filled with blood. Our initial differential diagnoses were

Fig. 1: Gross- variegated lesion attached to one end of jejunum.

Fig. 2: Microscopy-100x- H&E- Spindle cells admixed with dilated spaces some of them filled with red blood cells.

Fig. 3: H&E- 400x-spindle cells admixed with lymphocytes, plasma cells and red blood cells.

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Roux en Y GIST

Fig. 4: Immunohistochemistry-400x-CD 117.

Fig. 5: Immunohistochemistry- 400x- DOG1.

Discussion

A meta-analysis done by Xiaofei Zhi et al from 12 studies concluded that, microscopic margin positive resections with or without rupture proved to be an adverse prognostic factor which improved with imatinib treatment.[5]GIST commonly metastasizes to liver and peritoneum followed by lymph nodes, bone, lung and soft tissue. Metastasis to heart has also been reported. [13]

Gastrointestinal stromal tumors (GIST) are found to be the commonest mesenchymal neoplasm of gut. They have the ability to behave benign or malignant. Recent advances in the molecular characterization of GIST have helped revolutionizing targeted treatment. [5]The frequent clinical symptoms include anemia, gastrointestinal bleeding, abdominal distension and early satiety.[6]They may also be asymptomatic due to noninvasive behavior and sub mucosal location.[7]These tumors are most frequently seen in stomach followed by small intestine and colon. Extra gastrointestinal GISTs have also been reported.[8,9] CD 117 and DOG1 positivity as in our case are useful in identification of GIST in 95% of adults. In addition PDGFRA, BRAF, NF1, HRAS, NRAS and succinate dehydrogenase complex mutations were also found in GIST. Though c-KIT inhibitors like imatinib perform well as an adjuvant therapy, resistance is emerging to be a problem in the management. Wild type GISTs that are negative for KIT and PDGFRA mutations are also imatinib resistant. Among the KIT mutations exon 11 is most frequent then follows mutations in exons 9, 13 and 17. Mutations in exons 11 and 13 are naive to imatinib while exon 9 mutations are associated with resistance. Autosomal dominant inheritance is well established in GIST occurring in young individuals. Modified NIH risk stratification which includes size, location, mitosis and rupture is currently being followed for treatment.[10]Recent studies propose molecular studies to be included for risk stratification scoring, as certain mutations like exon 11 if present do not require additional treatment with KIT inhibitors while others like CDKN2A/B/C alteration leads to recurrence and treatment resistance.[11,12]

In our case the patient presented with abdomen distension and pain with a previous history of surgery in the abdomen, the details of which were not known. Laparotomy revealed a globular mass arising from jejunal limb of a previous roux en Y anastomosis. Grossly the tumor was a 14 cm globular mass with variegated appearance on cut surface. Based on this, we had differential diagnosis of leiomyosarcoma, angiosarcoma and GIST. Microscopy showed predominantly spindle cells which were mildly pleomorphic, admixed with blood filled spaces, lymphocytes and plasma cells, so inflammatory fibroid polyp was added to our differentials. The mitotic count was low with no evidence of necrosis. Immunohistochemistry for CD 117, DOG1 and CD 34 was performed. Strong positivity for CD 117 and DOG1 favored a diagnosis of GIST. Leiomyosarcoma was ruled out in our case as the tumor had a low mitotic count and was positive for CD 117, DOG 1 and only focally for SMA which can happen if GIST has smooth muscle differentiation.[14]CD 34 negativity in the spindle cells ruled out the possibility of angiosarcoma and inflammatory fibroid polyp even though microscopy showed lymphocytes and plasma cells within the tumor.[15] Epithelial malignancies have also been reported following post gastric bypass surgeries. A case of small bowel adenocarcinoma has been reported by Abraham Yacoub

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Kumar et al. in 2015 in a patient who underwent roux en y procedure for weight loss.[16]Around the same time Deepa Magge and Matthew P. Holtzman reported 2 cases of gastric adenocarcinoma in those who underwent gastric bypass surgery.[17]Adenocarcinoma was not considered as a differential diagnosis in our case because of predominant spindle cell morphology in microscopy. Reports have shown that the gastric GISTs have the lowest rates of recurrence while rectal and duodenal GISTs have the highest rates. So far recurrence in gastro-jejunal anastomosis site has been reported in one case of a low grade GIST in 71 year old male in 2010.[1] As the details of previous surgery were not available, we hypothesize that the initial surgery could have been for a gastrointestinal stromal tumor which had recurred.

Conclusion

It is important to keep GIST as a differential diagnosis in recurrent tumors arising from post gastric bypass surgery. GIST can present as a solid and cystic mass with hemorrhage. Careful follow up is essential to monitor recurrence even in low grade GIST. Treatment with c-Kit inhibitors should be initiated after mutational studies to rule out the risk of drug resistance.

Acknowledgements

Dr.J.Thanka, Head of Department of pathology, Sri Ramachandra Medical College and Research institute

Funding

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6. 7.

9. 10.

11.

12.

None

Competing Interests None Declared

Reference

1. Papalambros A, Petrou A, Brennan N, Bramis K, Felekouras E, Papalambros E. GIST suture-line recurrence at a gastrojejunal anastomosis 8 years after gastrectomy: can GIST ever be described as truly benign? A case report. World J Surg Oncol. 2010 Oct 14;8(1):1. 2. Liegl-Atzwanger B, Fletcher JA, Fletcher CDM. Gastrointestinal stromal tumors. Virchows Arch. 2010;456(2):111–27. 3. Jaison J, Joshi SR, Pathak S, Tekwani D, Nagare M. Gastrointestinal Stromal Tumour at An Unusual Site Jejunum: A Case Report. Int J Sci Stud 2014; 2(4):80-83. 4. Rosai Juan, Lauren V. Ackerman. Gastrointestinal Tract. Rosai and Ackerman’s Surgical Pathology. Edinburgh: Mosby, 2011.p. 638-644. 5. Zhi X, Jiang B, Yu J, Røe OD, Qin J, Ni Q, et al. Prognostic role of microscopically positive margins for www.pacificejournals.com/apalm

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15. 16.

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primary gastrointestinal stromal tumors: a systematic review and meta-analysis. Sci Rep [Internet]. 2016;6:21541. Joensuu H, Hohenberger P, Corless CL. Gastrointestinal stromal tumour. Lancet (London, England) [Internet]. 2013 Sep 14 [cited 2016 Jun 2];382(9896):973–83. Nishida T, Blay J-Y, Hirota S, Kitagawa Y, Kang Y-K. The standard diagnosis, treatment, and follow-up of gastrointestinal stromal tumors based on guidelines. Gastric Cancer [Internet]. 2016 Jan [cited 2016 May 16];19(1):3–14. Fagkrezos D, Touloumis Z, Giannila M, Penlidis C, Papaparaskeva K, Triantopoulou C. Extragastrointestinal stromal tumor of the omentum: a rare case report and review of the literature. Rare Tumors [Internet]. 2012 Jun 26 [cited 2016 Jun 1];4(3):e44. Behammane H, Aggouri Y, Oussaid M, et al. Stromal tumor of the lesser omentum : a case report. Pan Afr Med J [Internet]. 2014 Jan [cited 2016 Jun 1];17:236. Bhatt NR, Collins D, Crotty P, Ridgway PF. Prognosis and management of adult Wild Type GastroIntestinal Stromal Tumours (GISTs): A pooled analysis and review of literature. Surg Oncol [Internet]. 2016 May [cited 2016 May 16]; Joensuu H, Rutkowski P, Nishida T, et al. KIT and PDGFRA mutations and the risk of GI stromal tumor recurrence. J Clin Oncol [Internet]. 2015 Feb 20 [cited 2016 May 18];33(6):634–42. Chojecki A, Moss RA, Zhong H, et al. Abstract 08: Role of upfront genomic analysis of gastrointestinal stromal tumors to identify imatinib-insensitive or resistant cases. Clin Cancer Res [Internet]. 2016;22(1_ Supplement):08 Cauchi C, Trent JC, Edwards K, et al. An unusual site of metastasis from gastrointestinal stromal tumor. Rare Tumors [Internet]. 2010 Jan;2(4):e58. Hilal L, Barada K, Mukherji D, Temraz S, Shamseddine A. Gastrointestinal (GI) leiomyosarcoma (LMS) case series and review on diagnosis, management, and prognosis. Med Oncol [Internet]. 2016;33(2):20. De Petris G, Leung ST. Pseudoneoplasms of the gastrointestinal tract. Arch Pathol Lab Med [Internet]. 2010;134(3):378–92. Yacoub A, Bank L, Sidhu JS. First Case of Small Bowel Cancer in a Post Roux-en-Y Gastric Bypass WeightLoss Surgery: A Case Report [Internet]. Journal of Medical Cases. 2015 [cited 2016 May 16]. p. 137–9. Magge D, Holtzman MP. Gastric Adenocarcinoma in patients with Roux-en-Y Gastric bypass: A case series. Surg Obes Relat Dis; 2015;11(5):e35–8. eISSN: 2349-6983; pISSN: 2394-6466

Case Report An Unusual Case of Bladder Tumour with Diagnostic Dilemma: A Rare Case Report Jayaprakash Shetty K1*, Kishan Prasad HL1, Rajeev TP2, Nigi Ross Philip1, Anitha Chakravarthy1 and Prajwal Ravinder2, Department of Pathology, K S Hegde Medical Academy of Nitte University, Mangalore, Karnataka, India. 2 Department of Urology. K S Hegde Medical Academy of Nitte University, Mangalore, Karnataka, India.

Keywords: Bladder, Leiomyosarcoma, Nonurothelial Neoplasm, ALK 1

ABSTRACT Urinary bladder tumours rank ninth in the worldwide cancer incidence. Urothelial carcinomas are the fourth most common malignancy in men. It is very rare in younger age group, but it can occur at any age group. We report a case of a 28 yr old male who presented with dysuria and suprapubic pain with Contrast enhanced computerised tomography revealed polypoidal lesion arising from the dome of the urinary bladder. In light microscopy showed, pleomophic spindle tumours cells arranged in fascicles with increased atypical mitosis along with scattered chronic inflammatory cells. The possibility of leiomyosarcoma or inflammatory myofibroblastictumor was considered in differential diagnosis. The immunohistochemistry was positive for Vimentin, Desmin, Smooth muscle actin, Ki 67 and myo D1 and negative for Cytokeratin, ALK 1, H-Caldesmon, S 100 and CD 34. Hence a diagnosis of leiomyosarcoma was made. This case highlights the unusual tumour with its diagnostic dilemma of the case.

*Corresponding author: Dr. Jayaprakash Shetty K, Professor and Head, Department of Pathology, K S Hegde Medical Academy of Nitte University, Deralakatte, Mangalore, Karnataka, India. Phone: +91 9845085182 E-mail: drjpshetty9@yahoo.com

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Shetty K et al.

Introduction

Nonurothelial neoplasms of the bladder account for <5% of all bladder tumors with leiomyosarcoma comprising 0.1% of all bladder cancers.[1,2] Due to its low incidence rate very little is known about the natural history and prognosis of the tumour and there is a lack of the consensus of the standard treatment. While on the other hand Inflammatory myofibroblastictumor (IMFT) of the urinary bladder is also a very uncommon spindle cell tumor that is said to have undetermined malignant potential. Differentiating between these two lesions is important as the treatment modality varies. We encountered a similar case with histology and immunohistochemistry closely mimicking causing diagnostic dilemma.

C-255 myofibroblastic tumour was made. For further confirmation of the diagnosis, immunohistochemistry was suggested. The immunohistochemistry was positive for vimentin[Fig 3A], desmin[FIG 3B], smooth muscle actin[Fig 3C], Ki 67 and myo D1[Fig 3D] while it was not negative for Cytokeratin, ALK 1[Fig 3E], H-Caldeson, S 100 and CD 34 confirming the above diagnosis. Patient was closely observed and on regular follow up since 6months without any recurrence or metastasis.

Discussion

A 28 year old male, presented with dysuria and suprapubic pain since 3 months. Past, family and personal history was unremarkable. The per rectal examination revealed Grade 1 prostatomegaly. The ultrasound abdomen has showed a hypoechoic polypoidal lesion measuring 6.7x6.9x7.2cm in the urinary bladder. A Contrast enhanced computerised tomography [CECT] was also done which showed a polypoidal mass arising from the dome of the bladder (Fig1A&B). Cystoscopy revealed a large pedunculated mass arising from the left superolateral wall of the urinary bladder. Biopsy of the same showed inflammatory lesion favouring chronic cystitis. This was followed by a partial cystectomy that included the excision of the tumour along with the pedicle and attached bladder wall. On Gross examination, the mass was measuring 7x5cm and the outer surface was smooth with whorled appearance on cut surface as shown in the Fig 1C&D. On microscopy, in H&E stain, the bladder growth with the pedicle and bladder wall showed infiltrative tumour composed of spindly cells in fascicles[Fig 2A] and sheets having mild to moderate eosinophilic cytoplasm with increased N:C and pleomorphic hyper chromatic nucleus.[Fig 2B] Many binucleated and multinucleated tumour giant cells were seen. Atypical mitosis-1-2/hpf. Also noted admixed chronic inflammatory cells. The tumour cells were invading the bladder wall and at places, reached the perivesicle fat. Tumour cell near the bladder wall has epitheloid cell morphology having polygonal appearance with hyperchromatic nucleus.[Fig 2C, D] Focal tumour necrosis was also seen. The perivesicular tissue shows thick walled blood vessels with pleomorphic tumour cells surrounding these. Masson Trichrome stain shows tumour cells with muscle differentiation. Right and lateral pelvic wall were free tumor. A diagnosis of low grade pleomorphic sarcoma probably leiomyosarcoma versus inflammatory

Urinary bladder leiomyosarcoma is relatively rare, with few large series reported in the literature. According to Rosseret al., the most common clinical presentation is gross hematuria(81%) followed by frequent micturition at day time referred to as pollakiuria (28%) and dysuria(19%). [2-5]Our case had symptoms of dysuria and suprabpubic pain. The Inflammatory myofibroblastictumor (IMFT) is a rare spindle cell neoplasm of the urinary bladder which is characterized by atypical spindle cell proliferation accompanied by inflammatory cell infiltrate comprised primarily of lymphocytes and plasma cells. The first case was reported by Roth in 1980.[4-6] It is also known as pseudo sarcoma, atypical fibromyxoidtumor, atypical myofibroblastic and plasma cell granuloma.[5] The morphologic and immunophenotypic features of IMFT overlaps with that of malignant spindle cell tumors of the urinary bladder and hence a diagnostic distinction from these tumors can create a dilemma The other differentials include psuedosarcomatous appearing transitional cell carcinoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma and osteosarcomas.[12] Immunohistochemical staining useful in such cases are for Anaplastic lymphoma kinase (ALK), vimentin, cytokeratin. Leiomyosarcomas show a fascicular growth pattern and myofibrillar elements. Pseudosarcomatous transitional cell carcinoma is a neoplasm in which an in situ or superficially invasive transitional cell carcinoma is associated with a bizarre sarcoma-like, reactive stromal response which can be easily misdiagnosed as carcinosarcoma. Although necrosis is described in 30% or more of IMFTs, the presence of necrosis at the tumorâ&#x20AC;&#x201C;detrusor muscle interface in muscleinvasive cases was one criterion present in sarcoma that distinguished it from inflammatory pseudotumor. [6] Some leiomyosarcomas of the bladder display myxoid zones and can also express cytokeratin. The histopathological features like lack of a delicate vascular network, interspersed inďŹ&#x201A;ammatory cells, and red blood cells are usually observed in IMT while the marked cytologic atypia, nuclear hyperchromasia, and atypical mitoses are present in leiomyosarcomas that help in the dierential

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Case Report

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An Unusual Case of Bladder Tumour

Fig. 1: A&B] CECT showing polypoidal mass lesion arising from the dome of bladder[Coronal and sagittal view] 1C&D- Gross examination of the bladder mass showing grey white tumour with smooth outer surface and the cut surface with whorled appearance.

Fig. 2A: Histopathology of the Infiltrative tumour showing spindly cells in fascicles[H&E, X100]; 2B- Histopathology showing tumour cells with mild to moderate eosinophilic cytoplasm and pleomorphic hyper chromatic nucleus. [H&E, X100]; 2C&DHistopathology showing epitheloid cell morphology having polygonal appearance, hyperchromatic nucleus.[ H&E, X400].

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Fig. 3: Immunohistochemistry showing reactive for vimentin[3A], Smooth muscle actin[3B], Desmin[3C], Myo-D 1[3D] and negative for ALK -1[3E].

diagnosis. Leiomyosarcomas and leiomyomas lack ALK protein. Myogenin or MyoD1is a potent marker that will help in exclusion of rhabdomyosarcoma.[7] The ALK-1 positivity confirms the chances of local recurrence and muscle invasion.[6-8] Originally it was identified as a protein overexpressed in anaplastic largecell lymphoma, ALK-1 has subsequently been shown to be overexpressed in a substantial proportion of IMFTs of various anatomic locations, including the urinary bladder. [8-9] In IMFT of the urinary bladder, positivity for ALK1 by immunohistochemistry ranges from 33% to 89%, whereas ALK-1 protein expression in leiomyosarcoma and sarcomatoid urothelial carcinoma has not been reported, suggesting that ALK-1 immunohistochemical studies may be useful in the differentiation of IMFT from other spindle cell lesions in the urinary bladder. In our case, ALK 1 was negative and other immunomarkers positivity ruling out IMFT and favoured the diagnosis of leiomyosarcoma.

surgical extirpation, and radical cystectomy with wide margins should be performed whenever possible. [9-11] In most reported cases of IMFTs of the urinary bladder, surgical resections, including transurethral resection and partial/radical cystectomy were done. However in our case, in view of aggressive histological nature, patient was regularly monitored and is on close follow up without any recurrence and metastasis.

Conclusion

In conclusion, we agree with previous literaturethat due to limited experience with this rare tumour, there are insufficient data to suggest an optimum management strategy and prognosis, and as we are in an exciting period of discovery about tumours further options might be available in future.[11]

References

Urinary bladder leiomyosarcomas have always been considered highly aggressive tumors that require aggressive

1. Yamada T, Nagai S, Kanimoto Y. Rapid progression of a urinary bladder leiomyosarcoma: Report of a case. Case Reports in Urology 2011;532081: 3.

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2. Bjergaard JP, Jonsson V, Pedersen M, Jensen KH. â&#x20AC;&#x153;Leiomyosarcoma of the urinary bladder after cyclophosphamideâ&#x20AC;?. J Clin Oncol 1995;13(2):532-3. 3. Rosser CJ, Slaton JW, Izawa JI, Levy LB, Dinney CPN. Clinical presentation and outcome of high-grade urinary bladder leiomyosarcoma in adults. Urology 2003;61(6):1151-5. 4. Roth JA. Reactive pseudosarcomatous response in urinary bladder. Urology 1980;16:635-7. 5. Zones EC, Clement PB, Young RH. Inflammatory pseudotumour of the urinary bladders. A clinicopathologic, immunohistochemical, ultrstructural and flowcytometric study of 13 cases. Am J SurgPathol 1993;17:264-74. 6. Coffin CM, Fletcher JA. Inflammatory myofibroblastic tumour. In: Fletcher CD, Unni KK, Mertens F, editors. World Health Organization Classification of Tumours: Tumours of Soft Tissue and Bone. Lyons, France: IARC Press; 2002. p. 91-3. 7. Montgomery EA, Shuster DD, Burkart AL, Esteban JM, Sgrignoli A, Elwood L, et al. Inflammatory myofibroblastictumors of the urinary tract: A clinicopathologic study of 46 cases, including a

malignant example of inflammatory fibrosarcoma and a subset associated with high-grade urothelial carcinoma. Am J Surg Pathol 2006;30:1502-12. 8. Cook JR, Dehner LP, Collins MH, Ma Z, Morris SW, Coffin CM, et al. Anaplastic lymphoma kinase (ALK) expression in the inflammatory myofibroblastictumor: A comparative immunohistochemical study. Am J Surg Pathol 2001;25:1364-71. 9. Spiess PE, Kassouf W, Steinberg JR et al., Review of the M.D. Anderson experience in the treatment of bladder sarcoma.Urologic Oncol 2007:25(1); 38-45. 10. Ricciardi E, Maniglio P, Schimberni M, Moscarini M. A case of high-grade leiomyosarcoma of the bladder with delayed onset and very poor prognosis. World J Surgical Oncol 2010;8. 11. Hamadalla NY, Rifat UN, Safi KC, Mohammed M, Farsakh HA. Leiomyosarcoma of the urinary bladder: A review and a report of two further cases. Arab J Urol 2013;11:159-64. 12. Yagnik V, Chadha A, Chaudhary S, Patel K. Inflammatory myofibroblastic tumour of the urinary bladder. Uro Ann 2010;2(2):78-9.

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Case Report An Unusual Case of Follicular Variant of Papillary Thyroid Carcinoma with Temporal Bone Metastasis Diagnosed by Cytology Poonam G Lahane*, Prashant Kumavat, Kavita Khedekar, Nitin M Gadgil and Chetan S Chaudhari Dept. of Pathology, LTMMC & LTMGH, Sion, Mumbai. India Keywords: FNAC, Follicular, Metastases, Papillary, Thyroid, Skull

ABSTRACT Skull metastasis of thyroid cancer is rare and majorities are of the follicular subtype, rather than papillary thyroid carcinoma. Here, we present a case of follicular variant of papillary thyroid carcinoma (FVPTC) with temporal metastasis. A 45yrs female presented with thyroid swelling since 8 months and scalp swelling since few days. Fine needle aspiration of thyroid and scalp swelling as well showed features of FVPTC. On CT examination, there was focal osteolytic erosion suggestive of lytic bone metastasis. Histopathological examination after total thyroidectomy confirmed a diagnosis of papillary thyroid carcinoma follicular variant involving regional lymph nodes. Skull metastasis should be considered at the outset of the clinical course of thyroid carcinoma and all patients should be meticulously investigated with a multidisciplinary approach as if detected early it improves the prognosis. Fine needle aspiration cytology (FNAC) and histopathology plays key role in diagnosis along with radiological and clinical examination correlation.

*Corresponding author: Dr. Poonam G. Lahane, F2/5 D.A.D Residential Complex, Kanjurmarg West, Mumbai, Maharashtra, INDIA. Pincode:400078 Phone: +91 9920825352 E-mail: poonamlhn2@gmail.com

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Follicular Variant of PTC with Temporal Bone Metastasis

Introduction

Thyroid carcinomas are relatively less common comprising approximately 1.1% of all carcinomas, and 0.2% of all carcinoma deaths.[1] Among these, differentiated thyroid carcinomas (DTC) comprising papillary and follicular thyroid carcinoma subtypes, represent more than 90% of all thyroid carcinomas. Papillary thyroid carcinoma (PTC) is the most common type of DTC and FVPTC is one of the variants of PTC. PTC is indolent in nature, as it is typically low-grade and slowly progressive. Hence, the prognosis is usually favorable with high survival rate. FVPTC shows similar prognosis as that of PTC but with greater risk of vascular invasion and distant metastasis. The most frequently occurring metastasis affects the cervical and mediastinal nodes while distant metastasis goes to lung and bone. Bone metastases are most likely to occur in the scapula, sternum and ilium and rarely in the skull.[1] Differential diagnosis of osteolytic skull metastases are often from primaries of lung, breast and prostate malignancies rather than thyroid carcinoma. Around 2.5% to 5% of cases of thyroid cancers may present with skull metastasis.[2] Here we report an unusual case of FVPTC with temporal bone metastasis at initial presentation.

Case Report

45year female patient presented to us with anterior neck swelling since 6 years (Fig.1) which was progressively increased in size since last 8 months with scalp swelling over left temporal region since few days. Scalp swelling was soft 2cm in diameter hemispheric tumor. Patient had no history suggestive of hypothyroidism, hyperthyroidism, or blood pressure symptoms. Neck swelling was more

prominent on left side with firm to hard on palpation and moving with deglutition. Thyroid function test was normal. Ultrasound of neck showed enlarged left lobe with a heterogeneous hypoechoic nodule (size of 3.1 cm) and two such nodules in right lobe of thyroid (largest of size 1.4 cm) reported as colloid nodules, and left cervical lymph node mass likely to be metastases. Subsequently patient underwent fine needle aspiration cytology (FNAC) of hypoechoic thyroid nodule which showed thyroid epithelial cells arranged in papillae and in microfollicles at places with nuclear overlapping, granular chromatin, nuclear grooves and nuclear clearing, diagnosis offered as FVPTC Bethesda VI. (Fig.2). To map the disease burden, contrast enhanced computerized tomography(CT) of skull to pelvis was done which showed solitary lytic bone metastasis in squamous part of temporal bone with left cervical level V lymph node metastases (lung was normal). FNAC of cervical lymph nodes and temporal bone lesion (scalp swelling) shown the similar picture as that of thyroid FNAC, suggesting the diagnosis of FVPTC metastases (Fig.3). Total thyroidectomy with left sided modified neck dissection was done. Histopathology report of thyroid specimen given was differentiated papillary carcinoma thyroid follicular variant involving right and left lobe with regional lymph node metastases (Fig.4). For the management of metastatic lesion patient received radioactive iodine therapy (RAI). After receiving RAI therapy size of temporal bone lesion was reduced significantly.

Fig. 1: Neck swelling more on left side.

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Fig. 2: Thyroid FNAC-(Papanicolaou stain) Thyroid follicular epithelial cells arranged in papillae and follicles with nuclear enlargement, fine powdery chromatin, nuclear groves and intranuclear clearing.

Fig. 3: Scalp swelling FNAC â&#x20AC;&#x201C;(Papanicolaou stain) Thyroid follicular epithelial cells arranged in papillae and microfollicles shows nuclear overlapping, powdery fine chromatin, nuclear groves, and intranuclear inclusions.

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Fig. 4: H&E stained section from thyroid nodules shows tumour composed of follicles with nuclear features of PTC i.e nuclear clearing, powdery chromatin, nuclear groves, and intranuclear inclusions. Occasional psammoma bodies seen.

Discussion

Thyroid tumours are more prevalent in females with a female to male ratio of 2.6:1.[2] Papillary carcinoma of thyroid is the most common type of thyroid cancer, accounting for 70%-90% of well differentiated thyroid malignancies. While incidence of FVPTC is difficult to determine as it was misdiagnosed in past as follicular adenoma or follicular carcinoma in some cases. Usually, thyroid carcinoma presents with a neck mass which can be solitary or multinodular. The incidence of metastases to the bone from thyroid carcinoma has been reported from 1% to more than 40%, forming the second most common site following lung involvement. The initial presentation of distant metastases (3-4%) in patients with DTC is a rare event. However, as reflected in this case report, thyroid carcinoma may present with asymptomatic bone metastases and should be considered amongst the potential differential diagnoses.[3] Skull metastases from thyroid cancers are usually soft, hemispheric tumours resting on the skull as seen in our case. These tumours are usually highly vascular, with evident osteolytic changes in the skull.[2] In literature, follicular carcinomas have been reported to show a greater prevalence to distant metastases than other subtypes of DTC. This may be attributed to the generic use of the term follicular carcinoma prior to the recognition of specific sub-types, including FVPTC. Although, FVPTC is thought to behave in a similar clinical manner to true papillary thyroid carcinoma, in some cases it may present with the pathologic features and clinical behavior similar to that of follicular carcinoma.[3]

However, the relatively recent studies by Wood et al,[4] Ruegemer et al,[5]and Mizukami et al[6] report a much higher relative incidence of papillary carcinoma among their cases with bone metastases (41%– 77%), as seen in our case. This relatively higher incidence may be due to overwhelming majority of PTCs amongst thyroid cancers. [7]

10-year survival rate of patients with DTC is 80–95% while it is reduced to 40% in presence of distant metastases.[8] Early detection of bone metastasis improves the prognosis. [3] This means presence of bone metastasis worsens the prognosis which can be prevented by early detection. The mean period from the initial diagnosis of thyroid papillary carcinoma until the detection of skull metastasis has been reported as 23.3 years.[9] in contrast to our case where we diagnosed both the conditions simultaneously. Skull is a rare site for metastases, which when they occur, are most commonly located in the occipital region presenting as a soft, painless lump [9] while in present case temporal bone involvement by metastasis indicates the rarity of presentation. Diagnosis of a skull metastasis from thyroid carcinoma is usually based on clinical judgment, in conjunction with results of radiological investigations and biopsy findings. Current guidelines advise bone metastases should be treated with a combination of surgery, external beam radiotherapy, and 131Iodine therapy.[10]

Conclusion

In summary, although this is a rare case, papillary thyroid malignancy follicular variant should be considered as a

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Lahane et al. potential primary malignancy in patients who present with suspicious skull metastases on initial presentation. Prompt diagnosis and appropriate treatment are essential keys to successful management. FNAC and Histopathology plays important role in the diagnosis of primary as well as metastatic skull bone tumors in case of thyroid cancer.

Abbreviations and Symbols: 

      

FVPTC- Follicular variant of papillary thyroid carcinoma CT- computerized tomography FNAC- Fine needle aspiration cytology DTC- Differentiated thyroid carcinoma PTC- Papillary thyroid carcinoma Fig.- Figure RAI- Radioactive iodine H & E – Haematoxylin and eosin

Acknowledgements

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None

Funding

None

Competing Interests None Declared

Reference

1. Li X1, Zhao G, Zhang Y, et al. Skull metastasis revealing a papillary thyroid carcinoma. Chin J Cancer Res. 2013 Oct;25(5):603-7. 2. Manju P. Antony, Meer M. Chisthi, Tessy P. Joseph, et al. Follicular carcinoma thyroid presenting as skull

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10.

metastasis: a rare case report. Int J Otorhinolaryngol Head Neck Surg. 2015 Jul;1(1):40-44. Siddiq S, Ahmad I, Colloby P, et al. Papillary thyroid carcinoma presenting as an asymptomatic pelvic bone metastases. JSCR.2010: 3:2. Wood W,. Singletary SE, Hickey RC, et al. Current results of treatment for distant metastatic welldifferentiated thyroid carcinoma. Arch Surg 1989;124:1374–1377 Ruegemer, JJ, Hay ID, Bergstralh EJ, et al. Distant metastases in differentiated thyroid carcinoma: a multivariate analysis of prognostic variables. J ClinEndocrinol Metab 1988; 67:501–508. Mizukami Y T. Mishigishi, Nonomura A. et al. Distant metastases in differentiated thyroid carcinomas: a clinical and pathologic study. Hum Pathol 1990;21:283–290. Tickoo S, Pittas A, Adler M, et al. Bone metastases from thyroid carcinoma: a histopathologic study with clinical correlates. Arch Pathol Lab Med 2000;124:1440-1447. Muresan M, Olivier P, Leclere J, et al. Bone metastases from differentiated thyroid carcinoma. EndocrRelat Cancer 2008;15:37-49. Nigam A, Singh AK, Singh SK, et al. Skull metastasis in papillary carcinoma of thyroid: A case report. World J Radiol2012 June 28; 4(6): 286-290 British Thyroid Association, Royal College of Physicians. Guidelines for the management of thyroid cancer. 2nd edition. Perros P,(ed). Report of the Thyroid Cancer Guidelines Update Group. London: Royal College of Physicians, 2007.

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Case Report Small Cell Carcinoma of Gallbladder: A Rare Case Report Nidhi Gupta*, Bawana Raina and Arvind Khajuria Department of Pathology, Acharya Shri Chander College of Medical Sciences and Hospital, Jammu. India Keywords: Gallbladder, Small Cell Carcinoma, Neuroendocrine Carcinoma

ABSTRACT A small cell carcinoma of the gallbladder is being reported from a young lady of 31 years of age, for the first time from Jammu and Kashmir state. The SCC, also called Neuroendocrine carcinoma of gallbladder are comparatively very rare and are reported usually from elderly ladies but in the present case the lady was very young. The patient presented with clear cut symptoms of cholecystitis and was thus diagnosed at early stage of life, which however is not true with most of the cases, which depict symptoms very late at older age leading to their low survival rate after diagnosis. The present report is a case of pure SCC without any association with any other disease. The patient had multiple gallstones, a mass each in fundus and neck of gallbladder. The left hepatic duct was partially blocked and dilated. Microscopy of stained sections of gallbladder showed tumour cells, with hyperchromatic nuclei and high N:C ratio, arranged in nests, cords and trabeculae extending up to serosa (or features suggestive of SCC). The diagnosis of SCC was also confirmed immunohistochemically. Cholecystectomy along with removal of portahepatic lymphnodes was done. The patient is undergoing chemotherapy and is showing signs of improvement.

*Corresponding author: Dr. Nidhi Gupta, B 43, Vasant Vihar, Talab Tillo, Jammu, Jammu & Kashmir, India. Pin â&#x20AC;&#x201C; 180002. Phone: +91 9858225553 E-mail: guptanidhi769@gmail.com

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Gupta et al.

Introduction

In this paper, small cell carcinoma (SCC), of the gallbladder, is being reported from a 31 year old female. On perusal of the available literature, this seems to be the first report of SCC from Jammu region of Jammu and Kashmir State. This is a neoplasm, referred to as, carcinoid tumor or endocrine cell carcinoma but the more preferred term for this is small cell carcinoma (SCC). This tumor was first reported and described by Albores Saavedra et al. in 1981 [1]. The most important characteristics of SCC include, its clinicopathological distinctness from other tumours, its rarity, highly aggressive nature, that tends to metastasize early, its poor prognosis and being more prevalent in elderly ladies. [2] However, a few cases have been reported in elderly males also. [3] Sometimes, it also shows association with endocrine manifestations and chemosensitivity. [2] All these clinicopathological features have been recognized to make it a distinct and separate neoplasm from other ordinary gall bladder carcinomas. [4] This carcinoma is reported to be having high malignant potential leading to its low survival rate. The other reason being very late presentation of symptoms. [5]

C-265 -5x3.5 cm and 3x2cm was seen.(Fig -2) Microscopy revealed intact mucosa, with tumor cells arranged in nests , cords and trabeculae extending up to serosa.(Fig- 3,5) .The tumour cells were round to oval,with hyperchromatic nuclei, high N:C ratio, indistinct nucleoli, scanty cytoplasm and nuclear moulding, in desmoplastic stroma . (Fig- 4 ) There were foci of necrosis, however, peritoneal surface was free of tumour and there was no invasion to liver and no vascular invasion . There was metastasis to lymph node with total replacement of lymph node tissue by tumour but there was no pericapsular invasion. Hence diagonosis of Small cell Carcinoma was made which was later

The present case of neuroendocrine carcinoma of gallbladder is peculiar in that it was diagnosed and resected in a comparatively a very young lady with cholelithiasis.

Case Report

A 31 year old female presented to the OPD, in the Department of Surgery with chief complaints of loss of appetite, abdominal fullness and right upper quadrant abdominal pain for 3 months. On per abdominal examination, a palpable mass was felt over the lower border of liver along the mid-clavicular line.

Fig. 1: CT guided FNAC showing cells with round and dense nuclei ,40X Giemsa.

Serum total bilirubin was 4.1mg/dl, direct 3.8mg/dl, SGOT 211 U/L, SGPT 225 U/L and Alkaline phosphatase 659 U/L. An ultrasound revealed multiple stones (Average, 8-10mm) in the lumen of gallbladder, a mass measuring 42 x 33 mm at the gallbladder fundus and, another mass measuring 33 x 25 mm at the neck of gallbladder, partially blocking the left hepatic duct, which was dilated (8mm). There were multiple enlarged lymph nodes at porta. A CT guided FNAC was done which revealed few clusters of cells with round and dense nuclei. (Fig- 1) Open cholecystectomy was carried out. The gallbladder along with stones, and three porta hepatic lymphnodes were resected out. Liver biopsy was also done. All the three specimens were thoroughly examined in the Department of Pathology. On gross examination , gallbladder measuring -13x4x3cm with growth at fundus and neck measuring www.pacificejournals.com/apalm

Fig. 2: Gross images.

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Small Cell Carcinoma of Gallbladder

confirmed by Immunohistochemically, the tumour cells were positive for NSE (neuron specific enolase). (Fig- 6) The diagnosis was revised to be small cell neuroendocrine carcinoma of gallbladder. Currently, the patient is receiving chemotherapy and is doing well.

has advanced and affected the adjacent organ systems including the obstruction of biliary system leading to poor prognosis. Secondly, the survival rates of this carcinoma are very low than other gallbladder malignancies, causing death before they are properly diagnosed.

Discussion

Although gallbladder carcinomas are fifth most common gastrointestinal malignancies, with seven thousand new cases being reported every year in the United States of America, [6] yet the small cell carcinomas or neuroendocrine carcinomas of gallbladder are exceedingly rare, comprising 0.2% of all the gastrointestinal carcinomas. [7] But according to Henson, 1992, [8] it was 0.5% of all the gallbladder carcinomas. According to Peter Nau et al, 2010 [9] and Amit Mahipal, 2011, [10] 40 and 73 cases only were respectively, reported in English medical journals. However, Moskal et al, 1999, [11] have reported a higher incidence of these carcinomas being of 3.5%. The reasons for its rarity can be two-fold. Firstly, that this tumour is discovered very late when the disease process

As per the available literature, small cell carcinoma of gallbladder affects elderly ladies from whom it has been mostly reported, [11] but exceptions are there, where it has been reported from elderly males also [3, 10]. According to Amit Mahipal et al, (2011), the age of these ladies ranges from 25-86 years with median age of 67years. The present case is peculiar in that the patient belonged to a very younger age group, who was 31 yrs of age. The simple reason is that she was, exceptionally, presenting well defined symptoms and was thus diagnosed at an early stage, that is why she is surviving and doing well after cholecystectomy and is presently undergoing chemotherapy. One important feature of small cell carcinoma is that it is usually associated with cholelithiasis, reported to be 68% [10] which has been confirmed in the present case also.

Fig. 3: Cells showing positivity for Neuron Specific enolase, 40X

Fig. 4: Small cell carcinoma, low power view, 10X, H& E

Fig. 5: small, Hyperchromatic cells with molding, 40 X , H&E

Fig. 6: Tumor cells infiltrating upto serosa, 40X, H&ENSE,40X.

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Gupta et al. In some cases, Small cell carcinoma has been reported in combination with adenocarcinoma of gallbladder with transitions between the two types of carcinomas, [10] however, in present case such combination and transitions were not observed. 28% cases are reported as combined small cell carcinoma and adenocarcinoma or squamous cell carcinoma, whereas, pure Small cell carcinoma is reported in 72%.[10] The present case was pure Small cell carcinoma without any association with other carcinomas. In some cases, Small cell carcinoma has been found associated with AIDS [10] but in present case, the patient did not suffer from any other ailments except Small cell carcinoma. Such combined cases are thought to be incidental findings and has no clinically significant association.[10]

C-267 Clinicians and especially pathologists must be aware of this entity to avoid misdiagnosis and erroneous treatment.

Acknowledgements None

Funding None

Competing Interests None Declared

References

Gallbladder neuroendocrine carcinomas remain an exceptionally rare malignancy with a strikingly poor prognosis. Early diagnosis with prompt surgical intervention provides the patient with the best long term outcome.

1. Albores-Saavedra J, Molberg K, Henson DE. Unusual malignant epithelial tumours of the gallbladder. Semin Diagno Pathol. 13:326-38,1996. 2. Ron IG, Wigler N, Ilie B, et al. Small cell carcinoma of the gallbladder: clinical course and response to chemotherapy. Tumori 78:207-10, 1992. 3. Chaung SS, Lin CN, Chu CH, Chen FF. Small cell carcinoma of the gallbladder : Report of two cases. Pathol Oncol. 5:235-8,1999. 4. Nishihara K, Nagai E, Tsuneyoshi M, et al. Small cell carcinoma combined with adenocarcinoma of the gallbladder. Arch Pathol Lab Med. 118:177-81,1994. 5. Nishime C, Ohnishi Y, Suemizu et al. In vivo chemotherapeutic profile of human gallbladder small cell carcinoma. Biomed Researc. 29:251-6,2008. 6. Lai CHE, Lau WY. Gallbladder cancer â&#x20AC;&#x201C; a comprehensive review. Surgeon 6:101-110,2008. 7. Jun SR, Lee JM, Han JK, Choi BI. High grade neuroendocrine carcinomas of the gallbladder and bile duct report of four cases with pathological correlation. Journal of Computer Assisted Tomography. 30:604-9,2006. 8. Henson DE, Albores-Saavedra J, Corle D. Carcinoma of the gallbladder : histologic types, stage of disease, grade and survival rates. Cancer 70:1493-7,1992. 9. Nau P, Liu J, Dilhoff M, Forster M, Hazey J, Melvin S. Two cases of small cell carcinoma of the gallbladder. Hindawi Publication Corporation, 2010. 10. Mahipal A, Gupta S. Small cellcarcinoma of the gallbladder : Report of a case and literature review. Gastrointestinal Cancer Research. 4:135-6,2011. 11. Moskal TL, Zhang PJ, Nava HR. Small cell carcinoma of the gallbladder. J Surg Oncol 70:54-9, 1999. 12. Chuang SS, Lin CN, Chu CH, Chen FF, Small cell carcinoma of the gallbladder: report of two cases. Pathol and Oncol Research. 5:235-8,1999. 13. Matsuo S, Shinozaki T, Yamaguchi S et al. Small cell carcinoma of the gallbladder: report of a case. Surgery Today. 30:89-93,2000.

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66% cases have been diagnosed at stage IV and the remaining at stage I â&#x20AC;&#x201C;stage III. [10] The present case was at stage II B according to TNM. In the present study, the metastasis had taken place only to porta hepatis lymph nodes where as, metastasis has been reported maximum (70%) in lymph node, liver (64%), lungs (10%) by Amit Mahipal et al, 2010. [10] . The median survival has been given as nine months, ranging from 1-18 months. [10] However, the patient in present study is surviving since last six months, and is on regular observation. Regarding the origin of SCC the accepted view is that it arises from metaplastic epithelium of gallbladder wall. [12] This concept is based on the fact that no neuroectodermal cells are found in the gallbladder mucosa. [13 ]This has been confirmed by work of some investigators who have reported metaplasia in the gallbladder with chronic cholecystitis. However,in the present study although chronic cholecystitis with cholelithiasis was confirmed but no metaplasia or dysplasia was observed in the gallbladder mucosa.This confirms the views of Shih Sung [3] who support a common endodermal, multipotent stem cell origin of Small cell carcinoma ,which was also advocated by Nash. [3, 10] The diagnosis of SCC is difficult and cannot be relied upon a single criterion like radiological findings. Immunochemistry along with histological studies are necessary to confirm the diagnosis.

Conclusion

Case Report Lymphadenopathic form of PAX 3-7/FKHR Fusion Gene and ALK Negative, Solid Type of Alveolar Rhabdomyosarcoma in an Infant: A Rare Entity Mimicking Lymphoma with a Review of Literature Chetan Sudhakar Chaudhari*, Ganesh Ramdas Kshirsagar, Prashant Vijay Kumavat and Nitin M.Gadgil Dept of Pathology, Lokmanya Tilak Municipal Medical College & General Hospital, Sion, Mumbai, India Keywords: Lymphadenopathic form, Solid Alveolar Rhabdomyosarcoma, ALK-Fusion Gene Negative

ABSTRACT Alveolar rhabdomyosarcoma is a high-grade neoplasm, which forms about 30% of rhabdomyosarcomas. A rare solid variant has been described. Cervical and axillary lymph node enlargement due to metastatic rhabdomyosarcoma, without an obvious primary tumor, is a rare finding. We report a case of multiple cervical and axillary lymphadenopathies in a 6 month old infant, clinically suspected as lymphoma. On histopathology examination diagnosed to be a case of solid variant of alveolar rhabdomyosarcoma, which was confirmed by immunohistochemical and genetic studies.Tumour was negative for anaplastic lymphoma kinase (ALK) gene aberrations and for PAX 3-7/FKHR fusion gene studies on RT-PCR, a rare subset of alveolar rhabdomyosarcoma. The pediatric and adolescent cases of this rare tumor reported in English language literature are reviewed.

*Corresponding author: Dr. Chetan Sudhakar Chaudhari, 11, Jagannath darshan, M.D. Keni Road, Near RBI quarters, Bhandup village(East),Mumbai, India Phone: +91 9819133606 E-mail: drchetanchaudhari26@yahoo.co.in

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Chaudhari et al.

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Introduction

nuclear-cytoplasmic ratio, hperchromatic irregular nuclei and deeply eosinophilic cytoplasm (Fig 2). Morphologic evidence of rhabdomyoblastic differentiation including strap cells or cells with cross-striations was seen focally (Fig 3). A differential diagnosis of RMS metastatic to cervical lymph nodes was considered along with nonHodgkin lymphoma(ALCL), extra renal rhabdoid tumor, Ewing’s sarcoma (ES)/primitive neuroectodermal tumor (PNET),alveolar soft part sarcoma. Non-Hodgkin lymphoma(ALCL) may often come in the differential diagnosis of Round cell sarcomas such as RMSs. It shows broad spectrum of morphology and small cell type may mimic RMS. The presence of strap cells may help in such cases for establishing the diagnosis of RMS.Extra renal rhabdoid tumour shows sheets of poorly cohesive large polygonal cells with large eccentric vesicular nuclei, abundant eosinophilic cytoplasm with keratin positivity and strict negativity for muscle markers on IHC. Ewing’s sarcoma(PNET) is a small cell neoplasm with a spectrum of appearance from undifferentiated to forming rosettes and alveolar pattern.No reosttes were noted in this case.Alveolar soft part sarcoma usually shows distinct regular pseudo alveolar pattern and specific intracytoplasmic crystals,which were not seen in this case. Immunohistochemical staining was performed for leukocyte common antigen (LCA), cytokeratin, desmin and myogenin. LCA and cytokeratin were negative, whereas there was strong nuclear positivity for desmin, myogenin and Myo D1 (Fig 4). Thus, a diagnosis of alveolar RMS, solid variant was made. Based on clinical presentation and histopathology features, supported by Immunohistochemistry, diagnosis of solid variant of alveolar RMS was made. No obvious primary was found at time of diagnosis. Tumor was negative for anaplastic lymphoma kinase gene aberrations (ALK) and for PAX 3-7/FKHR fusion gene studies on RT-PCR, which is a rare subset of alveolar rhabdomyosarcoma and has relatively good prognostic values.

Rhabdomyosarcoma (RMS) is one of the most common pediatric soft tissue sarcoma, likely results from dysregulation of the precursor cells during skeletal myogenesis. RMS can be classified into three subtypes histologically: the most common embryonal rhabdomyosarcoma (ERMS), the less common but more aggressive alveolar rhabdomyosarcoma (ARMS), and the rare adult variant pleomorphic rhabdomyosarcoma (PRMS). [1] Although these tumors can arise almost anywhere, the most common locations for these tumors to develop are in the structures of the head and neck (nearly 40% of all cases), the male or female genitourinary tract (about 25% of all cases), and the extremities (about 20% of all cases). Alveolar RMS not otherwise specified (NOS) has a poor prognosis with a 5 years survival rate of about 53%. It occurs frequently in extremities, perirectal and perineal regions mainly in adults. These tumors are composed of sheets of uniform, dyscohesive malignant small rounded cells with a tendency to attach to thin fibrous septae. Multinucleated tumor giant cells with eosinophilic cytoplasm may be seen in some cases. Tumor cells may show a thin rim of eosinophilic cytoplasm. Morphologic evidence of rhabdomyoblastic differentiation including strap cells or cells with cross-striations and multinucleate myoblasts may be seen in 30% of cases. In a rare solid type, predominantely sheets of tumors cells divided by thin fibrovascular septae but without an obvious alveolar pattern seen. [2] Presentation with multiple peripheral lymph node enlargements without an obvious clinical primary lesion is a rare presentation of alveolar RMS, with only a few cases has been reported in a literature.

Case Report

A 6 month old infant presented with multiple axillary and cervical lymph node enlargement since 1 month. Lymph nodes were measuring from 3x2 cm to 1x1 cm, firm, nontender and mobile. There was no other significant history or a clinical finding at time of initial presentation. There was no positive family history of similar lesions in past. Hematological and biochemical investigations were within normal limits. Radiological examination on CT scan showed non necrotic bilateral axillary, left supraclavicular & infraclavicular lymphadenopathy which on PET scan showed hyper metabolic nature. Clinically provisional diagnosis of lymphoma was made and lymph node excision biopsy was received for histopathological examination.

Discussion

On microscopy examination, it revealed tumor was composed of sheets of uniform; dyscohesive malignant small round to oval cells with a tendency to attach to thin fibrous septae at places (Fig 1). Tumor cells showed high

Alveolar rhabdomyosarcoma (RMS) is usually found in adolescents with a mean age of 15–20 years, and typically arises in the deep musculature of the extremities. Within the head and neck region, embryonal RMS is the most common RMS, whereas alveolar and pleomorphic tumors are more common in the extremities. It is estimated that RMS accounts for approximately 8% of cancers in children and 2–5% of all adult sarcomas. [1] Pediatric RMS has been classified by The International Pediatric Sarcoma Working Classification into prognostically useful histological categories, including embryonal botryoid, embryonal spindle, embryonal NOS, alveolar NOS or solid variant and

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Fig. 1: Tumor was composed of sheets of uniform, dyscohesive malignant small round to oval cells with a tendency to attach to thin fibrous septae at places (H & E 100x).

Fig. 2: Tumor cells showed high nuclear-cytoplasmic ratio, hperchromatic irregular nuclei and deeply eosinophilic cytoplasm (H & E 400x).

Fig. 3: Morphologic evidence of rhabdomyoblastic differentiation including strap cells or cells with cross-striations was seen focally ( Massonâ&#x20AC;&#x2122;s trichrome 400 x).

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Fig. 4:Immunostaining: strong nuclear positivity for desmin,myogenin and Myo D1.

undifferentiated sarcoma. [1,2] Certain genetic conditions such as Li-Fraumeni syndrome,Neurofibromatosis type 1 (NF1),Beckwith-Wiedemann syndrome,Costello syndrome,.Noonan syndrome increase the risk of childhood rhabdomyosarcoma. In most cases, the cause of rhabdomyosarcoma is not known. Approximately two-thirds of children with RMS have the more common embryonal type (or the spindle-cell or botryoid variants). These tumors are more common in younger children, particularly those with tumors arising in the head and neck sites (including parameningeal sites) and the genitourinary system (including the bladder and prostate). [3] About 2025% of children with RMS have the less common alveolar type (or solid alveolar variant). These tumors are much more common in teenagers, and most commonly arise in the extremities. The tumor cells tend to be smaller and rounder, often with a denser cellularity, and are so named because of their resemblance to the appearance of the “alveoli”. Alveolar tumors are often considered more “aggressive”, or “higher risk”, than embryonal tumors Immunohistochemical staining for desmin, MyoD1, and myogenin (myogenic markers) assist in the diagnosis. The alveolar variant exhibits the greatest propensity for lymph node metastasis; 33% are associated with positive regional nodes on initial examination and 75–85% develops either regional or distant nodal deposits. Recently, there have been case reports of alveolar rhabdomyosarcoma masquerading as hematologic malignancies, and there have been several cases of alveolar rhabdomyosarcoma with lymph node involvement. [4, 5] Lymphadenopathic form of alveolar RMS shows lymph node involvement as first clinical manifestation in absence of recognizable primary tumor. There are a few studies reporting this form of alveolar RMS. Solid variant of alveolar RMS is a rare variant in which the alveolar pattern is not seen. Although, this can have a strong resemblance with lymphoma, presence of strap cells and multinucleated giant cells with deeply eosinophilic cytoplasm is a characteristic finding www.pacificejournals.com/apalm

in alveolar RMS. Solid variant of alveolar RMS with unknown primary has been reported in few studies. [6, 7, 8] In approximately 90% of cases of alveolar RMS, a portion of one of the PAX genes (most commonly the PAX 3 gene located on chromosome 2, less commonly the PAX 7 gene located on chromosome 1) fuses with a portion of the FKHR gene (located on chromosome 13) to create a new “hybrid” gene (PAX-FKHR) that turns on growthstimulatory genes that would otherwise be “inactive” and turns off growth-inhibitory genes that are normally active. Since this abnormal “hybrid” gene is found only in cases of alveolar RMS, it can be used for diagnostic purposes and, potentially in the future, as a target for immunemediated cancer therapies. This abnormality is usually tested for using a technique known as RT-PCR (reverse transcriptase polymerase chain reaction). Alveolar RMS have chromosomal translocations (2; 13) (q35; q14) or (1; 13) (p36; q14). Approximately 20% of Alveolar RMS are translocation negative, and have allelic imbalance and Loss of heterozygocity patterns that are indistinguishable from conventional Embryonal RMS cases. [9] The clinical behavior and molecular characteristics of Alveolar RMS without a fusion gene are indistinguishable from Embryonal RMS cases and significantly different from fusion-positive Alveolar RMS cases. Thus, fusion gene status may play a role as a factor in risk stratification in RMS, irrespective of histology. The proper treatment and the exact nature of PAX fusion-negative alveolar RMS are currently debated, so that histological diagnosis remains the primary determinant for therapeutic protocol assignment. [10]

Conclusion

Our case attempts to highlight the rare occurrence of Lymphadenopathic form of solid alveolar RMS. A possibility of metastatic alveolar RMS must be kept in mind in cases presenting with isolated lymphadenopathy. A rare possibility of solid variant cannot be overemphasized to allow timely diagnosis and management. eISSN: 2349-6983; pISSN: 2394-6466

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Acknowledgements Nil

Funding None

Competing Interests None Declared

Reference:

1. Weiss SW, Goldblum JR. Enzinger and Weiss’s soft tissue tumors. 5th edn. Mosby Press, 2008:595–9. 2. Rosenblum MK, Bibao JM, Ang LC. Neuromuscular system: Central nervous system. In: Rosai J, editor. Rosai and Ackerman’s Surgical Pathology. 9 th ed., Vol. 2. India: Thomson Press, Mosby, Elsevier 2004: 2301-6. 3. Parham DM. Pathologic classification of rhabdomyosarcomas and correlations with molecular studies. Mod Pathol 2001; 14:506-14. 4. Reinecke P, Gerharz CD, Thiele KP, et al: Alveolar rhabdomyosarcoma presenting as acute leukemia. Verh Dtsch Ges Pathol 1998;82:336-339. 5. Duran Padilla MA, Rodriguez Martinez HA, Chavez Mercado L, et al: Atypical biological behavior of

10.

alveolar rhabdomyosarcoma in five patients. Rev Invest Clin1999;51:17-22. Mekni A, Bouraoui S, Boussen H, et al: Lymphadenopathic form of alveolar rhabdomyosarcoma: A case report. Tunis Med 2004;82:241-244. Chen L, Shah HO, Lin JH: Alveolar rhabdomyosarcoma with concurrent metastases to bone marrow and lymph nodes simulating acute hematologic malignancy. J Pediatr Hematol Oncol 2004;26:696-697. Ganesan P, Thulkar S, Rajan A, Bakhshi S. Solid variant of alveolar rhabdomyosarcoma mimicking non-Hodgkin lymphoma: Case report and review of literature. J Pediatr Hematol Oncol 2008;30:772-4. Parham DM, Qualman SJ, Teot L, Barr FG, Morotti R, Sorensen PH, Triche TJ; Soft Tissue Sarcoma Committee of the Children’s Oncology Group. Correlation between histology and PAX/FKHR fusion status in alveolar rhabdomyosarcoma: a report from the Children’s Oncology Group. Am J Surg Pathol. 2007 Jun;31(6):895-901. Egas-Bejar D, Huh WW. Rhabdomyosarcoma in adolescent and young adult patients: current perspectives. Adolesc Health Med Ther. 2014; 5: 115–125.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 05, (Suppl) Nov. 2016

Case Report Late Relapse of Malignant Peripheral Nerve Sheath Tumor After 18 Years. Case Report with Differential Diagnosis. Csaba Biró1*, Katarína Macháleková1, Štefan Galbavý2, Gabriel Bognár1, Sarah Catharina Hubinská3, Martin Kopáni4, Karol Kajo1 and Dagmar Kalátová5 Department of Pathology Institute of Oncology St. Elizabeth, Bratislava, Slovakia 2 Institute of Forensic Medicine of Comenius University, Bratislava, Slovakia 3 European Parliament Brussels, Belgium 4 Institute of Medical Physics, Biophysics, Informatics and Telemedicine Medical Faculty of Comenius University, Bratislava, Slovakia 5 St. Elizabeth University of Health and Social Work, Department of St. Jan Nepomuk Neumann, Příbram, Czech Republic 1

Keywords: MPNST, Immunohistochemistry, Differential Diagnostics

ABSTRACT Malignant mesenchymal tumors represent a heterogeneous group of neoplasms. Diagnostics is based on a comprehensive clinical and pathological assessment that takes into account the results of imaging methods and molecular genetics. The exact diagnosis of these neoplasms is important for their subsequent therapeutic management. Authors describe a case of a 47-year-old female patient with poorly differentiated sarcoma in the left neck region which they, based on histomorphological features, a wide immunohistochemistry panel, and detailed history, diagnosed as a late recurrence of MPNST 18 years after the primary diagnosis. Tumors of the Ewing sarcoma/PNET, synovial sarcoma, which have similar histomorphological features, were excluded in the process of differential diagnostics. MPNST is characterized as a malignant tumor with unpredictable biological behavior, and as the case described above indicates, it may even return many years after the primary diagnosis.

*Corresponding author: MUDr. Csaba Biró, PhD., Department of Pathology Institute of Oncology St. Elizabeth , Heydukova 10, 812 50 Bratislava, Slovakia, Phone: +421 904948473 E-mail: csaba.biro@ousa.sk

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Introduction

The diagnosis of soft-tissue sarcomas, although much rarer than carcinomas, is much more difficult as it requires a careful and comprehensive evaluation of clinical, microscopic, histomorphological, and immunohistochemistry features supplemented by an analysis of molecular genetic alterations. Moreover, many cell and growth characteristics overlap between individual subjects, which makes their exact diagnosis more difficult. Advancements in cancer treatment result in the relative healing of some malignancies, which often manifest as a late recurrence many years later. The authors describe an interesting case of a late recurrence of a malignant peripheral nerve sheath tumor (MPNST), whose histomorphological and immunohistochemistry features were the same as in Ewing sarcoma/primitive neuroectodermal tumor (PNET), synovial sarcoma (SS), in differential diagnostics.

Case Report(S)

We have examined a tumor in the left supraclavicular region of the neck in a 47-year-old female patient. As far as the medical history is concerned, in 1997 the patient underwent surgery of the left axilla due to a plexus brachialis tumor with a diagnosis of “neurofibrosarcoma” (malignant peripheral nerve sheath tumor, MPNST). Following surgery, the patient had 6 chemotherapy cycles and was given local radiotherapy. The patient was observed until 2004, when she stopped coming for follow-up evaluations.

She returned in 2015 with severe lymphadenopathy and infiltration in the left supraclavicular region. Clinically speaking, there was a tumor resistance of 30x25x30 cm in size, from which several small fragments were taken for biopsy. In terms of histomorphology, it was a heterologous, poorly differentiated or even anaplastic tumor proliferation mainly of a histoid type and to a lesser degree, organoid in origin. The growth of cell population was solid and focally storiform, with the presence of cells of a round, oval, and sporadically even fusiform shape, while cells in other regions were literally small and round and densely pressed together. Focally, they were epithelioid elements with eosinophilic cytoplasm, as well as cells with lighter cytoplasm or even intracytoplasmic vacuoles that resembled lipoblasts. The tumor cell nuclei prevailed over the cytoplasm with visible nucleoli, irregular fragmented chromatin masses, numerous mitoses and abnormal mitoses (up to 40 mf/10 HPF), anisokaryosis, and with the presence of anaplastic cells. In the interstitial space, there were obvious plexiform vascular formations. In terms of immunohistochemistry, the cells were diffuse positive for vimentin, proto-oncogene B cell lymphoma 2 (Bcl-2).[4] The expression of acidic protein (S100 protein), cell to cell adhesion glycoprotein (CD56), synaptophysin, and epithelial membrane antigen (EMA) was a bit weaker and focal. The receptor for kit protein (CD117) was focally positive. The expression of cell proliferation marker Ki67 was 35%.

Table 1: Antibody information Antibody

Source

Pretreatment

Dilution

Incubation (min.)

CK5/6 CK7 CK20 EMA p63 Vimentin α-Aktin (SMA) Desmin CD117 S100 protein LCA (CD45) CD34 Class II CD56 Chromogranin Synaptophysin CD99 Bcl-2 Ki67

Dako Novocastra Novocastra Dako Dako Dako Dako Novocastra Dako Dako Dako Dako Dako Dako Novocastra Dako Dako Dako

Tris-EDTA,pH9 Tris-EDTA,pH9 Tris-EDTA,pH9 Tris-EDTA,pH9 Tris-EDTA,pH9 Citrat, pH6 Tris-EDTA,pH9 Tris-EDTA,pH9 Tris-EDTA,pH9 Citrat, pH6 Tris-EDTA,pH9 Tris-EDTA,pH9 Tris-EDTA,pH9 Tris-EDTA,pH9 Tris-EDTA,pH9 Tris-EDTA,pH9 Tris-EDTA,pH9 Citrat, pH6

1:70 1:100 1:50 1:75 1:70 1:150 1:420 1:80 1:370 1:420 1:80 1:100 RTU 1:170 1:140 RTU 1:100 1:120

30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30

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Table 2: The results and differential diagnosis within immunohistochemistry. MPNST ES/PNET CK5/6 CK7 CK20 EMA +/+/p63 Vimentin + + Aktin Desmin CD117 +/S100 focal + + LCA CD34 CD56 +/Chromogranin Synaptophyzin +/+/CD99 + cytopl. + membr. BCL-2 + + Ki 67 35%

SS + + +

+ + + cytopl. and membr. +

MPNST: Malignant peripheral nerve sheath tumor, ES/PNET: Ewing sarcoma/Primitive neuroectodermal tumor, SS - Synovial sarcoma, + positivity, negativity, +/- focal positive to negative, + cytopl.: cytoplasmatic positivity, + membr.: membranous positivity;

Fig. 1: The proliferation of poorly differentiated round, oval, spindle-shaped , epithelioid cells, H&E, 10x.

Fig. 2: The area of the proliferated round, oval cells with mitosis, H&E, 20x.

Fig. 3: The area of the proliferated cells resembling lipoblasts, H&E, 20x.

Fig. 4: Proliferated cells - storiform architecture development, H&E, 10x.

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Fig. 5: EMA, focal positivity, 20x.

Fig. 6: Vimentin, diffuse positivity, 10x.

Fig. 7: CD117, focal positivity, 20x.

Fig. 8: S100 protein, weak focal positivity, 20x.

Fig. 9: CD34, negative reaction in the cells of the tumor, positive reaction in the blood vessels , 10x.

Fig. 10: CD56, positive reaction, 10x.

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Fig. 11. Synaptophysin, positive reaction, 10x.

Fig. 12: Bcl-2, diffuse positivity, 10x.

Tumour samples were fixed in 10 % buffered formalin and embedded in paraffin. Histological sections were studied after staining with hematoxylin and eosin (H&E) and 18 antibodies by methods of standard immunohistochemical techniques. Information about antibody with pre-treatment and incubation are shown in Table 1, were used for visualization systems DAKO EnVision : K5007 and Bond Polymer Refine DS9800 Leica Biosystems.

destructive, with a 5-year survival rate of 70% in patients with metastases and relapses; in treatment-resistant forms, the survival rate drops to 20-30%.

Discussion

Malignant peripheral nerve sheath tumors (MPNST, neurofibrosarcoma) most frequently occur between 35 and 45 years of age and between 60 and 80 years of age. MPNST affects women more than men and is most common in the proximal parts of the extremities, in the paraspinal region of the trunk and the neck.[3] The fiveyear survival rate ranges from 15 to 40%; patients with neurofibromatosis type 1 (NF1) have a worse prognosis. Local relapses are frequent, occurring in more than half of cases. MPNST metastasizes to the lungs, bones, pleura, liver, and brain meninges. MPNST is difficult to diagnose; its various histomorphological forms may mimic synovial sarcoma (SS) or Ewing sarcoma (ES/PNET) and in 15% of cases may contain epithelioid or other heterologous components such as a rabdomyoblast, smooth muscle, cartilage, bone, glandular structures, liposarcoma, and neuroendocrine components.[1]

Synovial sarcoma (SS) affects mostly men around the age of 30 and is relatively frequent in children as well, but rarely occurs under 5 years of age and at an older age. It grows in deep structures, e.g., in intramuscular areas, most often in the proximal parts of extremities, in the groin area, abdominal wall, neck region, hypopharynx, in the temporal, orofacial region of the head, in the retroperitoneum, mediastinum, femoral veins, heart, oesophagus, duodenum, and in the kidneys. If not completely removed surgically, it recurs with typical distant metastases, most frequently to the lungs, liver, and brain. Metastases to the lymph nodes are rare. The five-year survival rate of patients with welldifferentiated SS is 60-70%; five-year survival for patients with poorly differentiated SS is 20-30%.

Tumors of the Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET) group occur most frequently in children and adolescents (so called pediatric tumors).[2] They develop mostly in bone diaphysis and are most frequently located in the paravertebral area, in the thoracic basket. In 15% of cases, they develop extra-ostially (e.g., in the retroperitoneal region) and epidurally, where they can mimic MPNST, since MPNST may also contain PNET-like foci. Bone lesions are

The best marker in the immunohistochemistry diagnosis of MPNST is the S100 protein, although it is known that it is positive in only 50% of cases.[4] The S100 protein is also positive in 40% of SS and ES/PNET cases. Another usable marker is the marker of hematopoietic progenitor cells and endothelial cells (CD34), which is only focally positive in MPNST, and anti-Nestin (NES)[5], with a sensitivity of 78% and specificity of 96%. In the diagnosis of MPNST, it is best to combine NES and S100 protein, since this combination is specific and nearly 100% positive and can safely differentiate MPNST from SS and ES/PNET. It has been established that there are various rare and poorly differentiated histomorphological forms in the MPNST group. These forms are associated with neurofibromatosis and are S100 protein and NES negative, but focally positive for CD117.[6] The primary marker for the diagnosis of ES/ PNET is the E2 surface glycoprotein of T cells (CD99),

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although nowadays we know that it can be positive in other tumors as well, such as in synovial sarcoma. ES/PNET is characterized by significant membrane positivity for CD99. Today, the Friend leukemia integration-1 protein (Fli-1), with significant strong nuclear positivity and 70% sensitivity, is successfully used. The combination of CD99/Fli-1 is recommended for the diagnosis of ES/ PNET versus MPNST and SS. Important markers in the diagnosis of synovial sarcoma (SS) include EMA, keratin cocktail (AE1/AE3), type II keratin of simple nonkeratinizing epithelia (CK7), Bcl-2, CD56, and CD99. In terms of specificity and sensitivity, the combination of CK7 and EMA with a sensitivity of 50% and an almost 100% specificity is most suitable. In the synovial sarcoma, Bcl-2 is 85% positive, CD56 70% positive, and CD99 up to 70% positive, with 30% of that strongly in the membranes. This may be useful in differential diagnostics with regard to MPNST, in which this form of CD99 positivity is not demonstrated.[5] In biphasic SS, there were cases with a strongly positive reaction to EMA and AE1/AE3 and also cases of monophasic SS with EMA positivity, AE1/ AE3 negativity, and positivity for S100 protein and CD99, thus mimicking MPNST and ES/PNET. SS, which is negative for AE1/AE3, is significantly positive for CD56 (in 30%). The summary of the results of all MPNST immunohistochemistry exams with differential diagnostics for ES/PNET and SS is provided in Table 2.

Conclusion

During the assessment of clinical results, medical history, and morphological and immunohistochemistry results, the tumor was diagnosed as a poorly differentiated malignant peripheral nerve sheath tumor with a focal epithelioid and

neuroendocrine component, recurring after 18 years from the time when it was primarily diagnosed.

Funding None

Competing Interests

The authors declare that they have no conflict of interests.

Reference

1. Guo A, Liu A, Wei L, Xin Song. Malignant peripheral nerve sheath tumors: Differentiation patterns and immunohistochemical features – a mini review and our new findings. J Cancer. 2012 :3, 303-9. 2. Anderson JL, Denny CT, Tap WD, Federman N. Pediatric sarcomas: translating molecular pathogenesis to disease to novel therapeutic possibilities. Pediatr Res 2012;72: 112-21 3. Miettinen M. Modern soft tissue pathology : tumors and non-neoplastic conditions, Cambridge University Press, 2010, 703-716, 4. Stephen HO, Thomas DG, Lucas DR. Cluster analysis of immunohistochemical profiles in synovial sarcoma, malignant peripheral nerve sheath tumor, and Ewing sarcoma. Modern Pathology 2006:19; 659 5. Shimada STT, Nagasaka T, Kuroda M, et al. Nestin expression in malignant peripheral nerve sheath tumor (MPNST) and other soft tissue tumors. Mod Pathol 2005;1:22A. 6. Sandberg AA, Stone JF. The Genetics and Molecular Biology of Neural Tumors. Springer Science & Business Media, 2008.

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Letter to Editor Transitional Cell Carcinoma of Cervix Gazi Naseem Ahmed* and M.N. Baruah North East Cancer Hospital and Research Institute, Amerigog, Jorabat, Guwahati, Assam, India Keywords: Transitional cell carcinoma, Cervix, Cytokeratin 7, Cytokeratin 20.

Dear Sir,

Transitional carcinoma of the cervix is a rare neoplasm of recent description that probably represents a subgroup of squamous cell carcinoma of cervix.[1,2] This tumor has a propensity for late metastatsis and local recurrence, inspite of the fact that histologically it could be misinterpreted as CIN grade III with a papillary configuration or as a squamous cell papilloma.[3,4] A 53 years old lady presented in the OPD with a history of post menopausal bleeding and abdominal pain for the last 5 months. There was no significant past history. Ultrasonography of the abdomen showed a heterogeneous lesion of the cervix. Urinary bladder, ureters and renal pelvis appeared normal. On speculum examination an ulcerated lesion was noted in the anterior lip of cervix from which a punch biopsy was taken using a biopsy forceps. Sections showed papillary growth invading into the underlying stroma and are lined by eight or more layers of oval to spindle, darkly stained cells arranged vertically similar to urothelial transitional cell carcinoma. Moderate pleomorphism and loss of polarity noted in these cells. Atypical mitotic figures are seen (Fig 1). Cytokeratin profile was CK7 positive and CK20 negative (Fig 2). A diagnosis of transitional cell carcinoma of cervix was made. Transitional cell carcinoma of cervix is a rare neoplasm that probably represents a subgroup of squamous cell carcinoma of cervix.[2,3] The cytologic characteristics of the tumor have not been published to date. Some authors think that transitional carcinoma of cervix is a metaplastic variant of squamous cell carcinoma of cervix. Support for this hypothesis is the presence of squamous and transitional areas in the same neoplasm, epidemiologic and risk factors of transitional carcinoma of cervix similar to those of

squamous cell carcinoma of cervix and causal relationship with the HPV 16 genome, found in 67% of cases of transitional carcinoma of cervix.[2] This tumor occurs mainly in post menopausal females. However, the affected age group ranges from 34 to 81 years.[3] In our case, age at presentation was 53 years. It often resembles transitional cell carcinoma of the urinary tract.[3,4] It remains unclear whether papillary carcinomas of the cervix represents two clinicopathologically distinct groups of tumors (squamous and transitional) or if they reflect morphologic continuum within a single clinically homogenous entity. The Armed Forces Institute of Pathology fascicle uses the term “squamous” and “transitional” interchangeably to refer to these papillary carcinomas of cervix.[4] In our case there was no squamous components. Our case showed papillary architecture with fibrovascular core and showed an “inverted” endophytic pattern, as reported by other authors as well.[2,3] The cells formed cohesive groups in a multilayered fashion and had an oval or spindle shape with tapered ends. The nuclei were hyperchromatic, with coarse and medium sized granules that frequently displayed a wrinkled membrane, nuclear grooves which are similar to the features observed by Ortega – Gonzalez P et al.[2] Microscopic features were similar to those originating in urinary bladder or ovary as noted by Albores – Saavedra J et al.[5] Cytokeratin profile is similar to that of squamous cell carcinoma of cervix; positive for CK7, negative for CK 20.[1,3] Similar findings were seen in our case. This profile differs from metastatic urothelial transitional cell carcinoma where both CK20 and CK 7 is positive.[6] To conclude, Transitional cell carcinoma is a rare neoplasm that probably represents a subgroup of squamous cell carcinoma of cervix and carries a similar prognostic outcome as that of squamous cell carcinoma of cervix. It is

*Corresponding author: Dr Gazi Naseem Ahmed, North East Cancer Hospital and Research Institute, 11th Mile, Amerigog, Jorabat, Guwahati – 781023 Assam, India Phone: +91 9854884861 E-mail: drgnahmed@gmail.com

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Fig. 1: Low and high powered views showing darkly stained oval to spindle malignant transitional cells arranged vertically with a fibrovascular core. Low powered views A,B â&#x20AC;&#x201C; (H&E 100X), High powered views C,D â&#x20AC;&#x201C; (H&E 400X).

Fig. 2: Immunohistochemistry shows cytokeratin 7 positive and cytokeratin 20 negative transitional cells (400X).

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Ahmed et al. important to recognize its pathologic features to establish prognostic differences from those of other types of non squamous cell malignancies of the cervix.

Acknowledgements None

Funding None

Competing Interests None

Reference

1. Prasad KK, Devgan R, Kaur S, Bhatia AK. Transitional cell carcinoma of uterine cervix complicated by pyometra. Indian J Pathol MIcrobiol. 2004 Jan; 47(1): 71 – 3. 2. Ortega – Gonzalez P, Chanona – Vilchis J, Dominguez – Malagon H. Transitional cell carcinoma of the uterine cervix. A report of six cases with clinical, histologic and cytologic findings. Acta Cytol. 2002; 46(3): 585 – 90.

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L-19 3. Kaur A, Singh J, Bansal R, Kaur R et al. Transitional cell carcinoma: A case report with clinical, histological and cytological findings. J Clin Diagn Res. 2013; 7(12): 2954 – 2955. 4. Anand M, Deshmukh SD, Gulati HK. Papillary squamotransitional cell carcinoma of the uterine cervix: A histomorphological and immunohistochemical study of nine cases. Indian J Med Paediatr Oncol. 2013; 34: 66-71. 5. Albores – Saavedra J, Young RH. Transitional cell neoplasms (carcinomas and inverted papillomas) of the uterine cervix. A report of five cases. Am J Surg Pathol. 1995 Oct; 19(10): 1138-45. 6. Jiang J, Ulbright TM, Younger C, Sanchez K, et al. Cytokeratin 7 and Cytokeratin 20 in Primary Urinary Bladder Carcinoma and Matched Lymph Node Metastasis. Arch Pathol Lab Med. 2001;125:921–923.

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Letter to Editor Myelolipoma of Spleen: An Unusual Presentation Malini Goswami Department of pathology, Rajiv Gandhi Cancer Institute And Research Centre, New Delhi, India Keywords: Myelolipoma, Splenic

Dear Sir,

A 56 year old male presented with pallor, multiple episodes of vomiting and abdominal swelling. He had a past history of jaundice and also received 2 units of blood earlier. His blood parameters at presentation were: haemoglobin -8.5g/ dl, MCV - 76.4 fl, MCH- 24pg, MCHC-31.5% and RDWCV-23.6%, TLC-8200/cumm. CT scan revealed a well-defined retroperitoneal mass with areas of internal fat density-likely neoplastic in the left paraaortic region extending from just below the left renal vessels till L4 vertebra. The mass was abutting and displacing the left kidney, aorta, mesenteric vessels, small bowel loops with no sign of obstruction. Excision of the mass was performed and grossly it was a large well circumscribed mass measuring 15 x 10 x 8 cm ,cut section of which exhibited grey red solid appearance and firm consistency. Histopathological examination (figure 1) revealed an encapsulated mass, the parenchyma of which seemed to be partitioned by fibrous trabeculae. Focal lymphoid follicles with central hyalinised blood vessels were seen around the trabeculae. The entire morphology was reminiscent of spleen, however almost the entire splenic parenchyma was overrun and replaced by trilineal haematopoiesis with normal morphology and mature adipocytes. A diagnosis of myelolipoma of spleen was rendered. The patient was followed up later and is doing well. Myelolipoma (myelo- marrow; lipo-meaning of, or pertaining to, fat; -oma, meaning tumor or mass) is a rare benign tumor composed of mature lipomatous and hematopoietic tissue which was first described in the adrenal gland by Gierke in 1905.[1] They are mostly encountered in persons older than 40 years.[2] Small tumors tend to be asymptomatic and often are detected

incidentally, but rarely these tumors can grow to huge sizes causing pressure effects, retroperitoneal haemorrhage and abdominal swelling.[3] They occur most commonly in the adrenal gland with rare incidences in extra adrenal locations including lung, liver, retroperitoneum, mediastinum and testes.[4] Splenic myelolipomas are more commonly seen in other species, but rarely in humans with only 7 cases reported so far.[4] They must be differentiated from extramedullary hematopoiesis, myeloid sarcoma, lipoma and well differentiated liposarcoma. While EMH is a more diffuse process with less chance of mass formation, myelolipoma may also sometimes involve the spleen diffusely and completely overrun the parenchyma.[5] However presence of entrapped mature adipose tissue is the defining feature favouring myelolipoma over EMH or myeloid sarcoma. Since adipose tissue is a defining component of myelolipomas, well differentiated liposarcomas and lipomas are included in the differential diagnosis however absenceof a haemic component helps in eliminating them. [5] Surgery is curative and prognosis is good.

Abbreviations and Symbols:

MCV: mean corpuscular volume MCH: mean corpuscular haemoglobin MCHC: mean corpuscular haemoglobin concentration RDW-CV: red cell distribution width-coefficient of variation TLC: total leucocyte count CT: computed tomography EMH: extramedullaryhematopoiesis L4:lumbar 4 Pg: picogram Fl: femtolitre Cumm: cubic milimetre

*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

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

Goswami et al.

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Fig. 1: A-Residual lymphoid follicle of white pulp of spleen (arrow)with hyalinised central vessel,H&E,100X. B-Thick capsule(double arrows)of the spleen and underlying tumor, H&E,100X. C- The tumor with its trilineage haematopoietic elements along with intervening adipose tissue(arrow), H&E,200X. D-Higher magnification of the tumor with trilineage haematopoietic elements (megakaryocyte marked by a circle) along with intervening adipose tissue,H&E,400X.

Acknowledgements

2. Weiss & Goldblum. Benign lipomatous tumors In: Schmitt W, Black S, editors. Enzinger and Weiss’s Soft Tissue Tumors.5th edition. China: Elsevier Inc; 2008.

Funding

3. Meyer A, Behrend M. Presentation and therapy of myelolipoma. Int J Urol 2005;12:239

Competing Interests

4. Aguilera NS, Auerbach A. Extra-adrenal myelolipoma presenting in the spleen: A report of two cases.Human Pathology: Case Reports 2016;6:8-12.

I would like to thank Dr.Anurag Mehta,who is the head of department,pathology for his encouragement and support. None

None Declared

References

1. Wood WG, Restivo TE, Axelsson KL, SvahnJD. Myelolipoma in the spleen: a rare discovery of extraadrenal hematopoietic tissue, JSurg Case Rep 2013;3

5. Cina SJ, Gordon BM, Curry NS. Ectopic adrenal myelolipomapresenting as a splenic mass. Arch Pathol Lab Med 1995;119:561-3.

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