APALM 3.2 (2016) by PaGe

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Annals of Pathology and Laboratory Medicine April-June 2016; Vol. 3, Issue 2

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

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Annals of Pathology and Laboratory Medicine Co-Editor in Chief

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

Associate Editor

Dr Asitava Mondal Clinical Cytologist and Oncopathologist, Kolkata, West Bengal, India Dr Sompal Singh Specialist Pathologist, Dept. of Pathology, N D M C Medical College & Hindu Rao Hospital, Delhi, India Dr Ruchika Gupta Pathologist (Scientist-C), Institute of Cytology & Preventive Oncology (ICPO), Delhi, India Prof. Vatsala Mishra HOD, Dept. of Pathology Moti Lal Nehru Medical College, Allahabad Dr Manjusha Biswas Consultant Histopathologist & Oncopathologist, Kolkata, India Dr Mudit Agarwal Director Lab Services, Nishtha Pathology Lab, New Delhi, India Prof. A S Ramaswamy Prof. & HOD, Pathology, P E S Institute of Medical Sciences and Research, Kuppam, India Dr Harsh Vardhan Singh Senior Biochemist, N D M C Medical College & Hindu Rao Hospital, Delhi, India Dr Manu Noatay Head Operations, Niche Theranostics, New Delhi, India Dr Anil Parwani Vice Chair, Anatomical Pathology; Director of Pathology Informatics and Digital Pathology The Ohio State University Wexner Medical Center, Columbus, Ohio, USA

Editorial Board Members

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Contents Original Article

New Diagnostic Strategy for Atypical Pituitary Adenomas: Clinical and Histopathological Score

Francisco Tortosa, Susan M Webb Assessment of Cell Proliferation in Helicobacter Pylori Associated Gastric Epithelial Diseases

Neeraj Dhameja, Ritambra Nada, Deepak Kumar Bhasin, Kusum Joshi Intraoperative Squash Cytology of Cns and Spinal Cord Lesions with Histologic Correlation

Sanjeev Kishore, Aparna Bhardwaj, Anuradha Kusum, Brijesh Thakur, Sanjay Kaushik, Neetika Sharma Placental Pathology in Spontaneous Abortions: A Study with Review of Literature.

Kriti Chauhan, Manish Chaudhary, Monika Garg, Ridhima Auplish, Karuna Sangwan, Nanak Mahajan CD4 Cell Count Analysis Among HIV Patients At A Tertiary Hospital in South India

Vanisri HR, Vadiraja N Correlation of Bone Marrow Aspiration, Touch Imprint Findings and Bone Marrow Biopsy Findings in Pancytopenia.

R Sindhu, Pramita Sahu, Debi Prasad Mishra, Samir Kumar Behera Study of Fine Needle Aspiration Cytology of Breast Tuberculosis

Pragnesh J Patel Histological Classification of Atherosclerosis and Correlation with Ischemic Heart Disease: A Autopsy Based Study

Vandana Porwal, Shweta Khandelwal, Deepali Jain, Seema Gupta Analysis of Whippleâ&#x20AC;&#x2122;s Resection Specimens: A Histopathological Perspective

Shifa Ibrahim, Meena Kumari G

Case Report

Angiomyomatous Hamartoma: A True Lesion or A Vascular Compensatory Hyperplasia with Muscularization? : An Interesting Case Report

Rakesh K Gupta, Kavita Gaur, R K Jindal, Ravindra Kumar Saran, Sachin Agrawal Adenoid Cystic Carcinoma of Floor of The Mouth with Periumblical Cutaneous Metastasis

Reena Sinha, Pallavi Agrawal, Akash Kumar Singh Non-Hodgkin Lymphoma of Cervix: An Unusual Presentation

Mriganka Mouli Saha, Chhanda Das, Gourisankar Kamilya, Madhumita Mukhopadhyay Ovotesticular Disorder of Sexual Development with Rare Karyotype

Ritika Singh, Charanjeet Ahluwalia, A K Mandal Orbital Lymphoid Lesions: Short Series with Cytohistological Correlation

Neelam Sood, Priyanka Bhatia Soni A Rare Case of Juvenile Fibromatosis Infiltrating Neck Subcutis in A 3-Year Old Girl

Amit Kumar, Rakesh Mehra, Tanushree Narain, Neha Garg, Pallavi Agrawal Carcinomatosis Peritonei of Prostatic Adenocarcinoma: Incidental Finding in A Case of Obstructed Umbilical Hernia

Rupesh Prakashrao Gundawar, Umesh Sidheshwar Kanade, Shubhada Yadavrao Bansodde, Rahul Suryakantrao Abhange, Shivaji Dadarao Birare Role of Cytology and Radiology in Diagnosis of Lacrimal Gland Pleomorphic Adenoma

Kamal Kant Gupta, Swayam Prava Pradhan, Ashok Kumar Dash, Debi Prasad Mishra

A45-52 A53-60 A61-72 A73-80 A81-86 A87-93 A94-98

A99-104 A105-113 C56-59 C60-63 C64-67 C68-71 C72-77 C78-81 C82-85 C86-89

Congenital Hepatic Fibrosis : Report on Two Cases and Its Clinicopathological Correlation

Neeraj Dhameja, Varnika Rai, Rajeev Singh, Vineeta Gupta, O P Mishra Squamous Cell Carcinoma of Gall Bladder: A Rare Presentation

Chhanda Das, Koushik Sarkar, Madhumita Mukhopadhyay, Bedabrata Mukhopadhyay Interdigitating Dendritic Cell Sarcoma Arising in The Cervical Node

Shifa Ibrahim, Sharmila Thilagavathy, Meena Kumari Fine Needle Aspiration as A Diagnostic Tool for Metastasis to Thyroid: A Dilemma

Prashant Vijay Kumavat, Anitha Padmanabhan, Manisha Khare, Chetan Sudhakar Chaudhari, Nitin Gadgil Reactive Lymphocytosis: A Diagnostic Dilemma in Pleural Effusion

ma Kumar, Shelly Sehgal

Letter to editor

Burkholderia Cepacia: An Unusual Cause of Post Surgical Endopthalmitis

Vibha Bhargava, Menka Kapil, Rohit Jain, G N Gupta Intraparotid Schwannoma on Cytology

Lakshmi Agarwal, Deepti Sukheeja, Vinny Gupta, Swati Namdev, Naresh N Rai

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C90-93 C94-97

C98-102

C103-106 C107-111 L7-8

L9-10

Original Article New Diagnostic Strategy for Atypical Pituitary Adenomas: Clinical and Histopathological Score Francisco Tortosa1,2*, Susan M Webb2 Department of Pathology, Centro Hospitalar Lisboa Norte, EPE - Hospital de Santa Maria. Lisbon (Portugal) 2 Department of Medicine / Endocrinology, Hospital Sant Pau, IIB-Sant Pau, Universitat AutĂ˛noma de Barcelona (UAB), Barcelona (Spain)

Keywords: Diagnosis, Pituitary Neoplasm, Prognosis

ABSTRACT Background: Currently, prognosis remains the major challenge of the adenomatous pituitary pathology. According to the World Health Organization (WHO), pituitary tumours are classified into typical adenoma, atypical adenoma and carcinoma. Given that the prediction of the behaviour of these tumours remains a major clinical and anatomopathological challenge, we propose a new diagnostic strategy to orient prognosis and therapy of these tumours, based on a multiparameter system, as well as a simple clinico-laboratory and radio-histopathologic diagnostic algorithm. Methods: To validate the method, we have applied it retrospectively to a series of 243 pituitary adenomas (diagnosed according to the 2004 WHO classification on tumours of endocrine organs), operated by transsphenoidal via between 2004 and 2014, at Centro Hospitalar Lisboa Norte, the largest reference centre in Portugal. Result: A hundred twentynine had a follow-up of at least 5 years in order to evaluate recurrences. While 6.2% of typical adenomas recurred, among the atypical the recurrence rate was 68.8%. Conclusion: With this work we intend to provide a more specific differentiating system of possible malignancy, to early identify probable cases of poor evolution, which could be very useful in clinical practice.

*Corresponding author: Francisco Tortosa, Department of Pathology, CHLN, EPE - Hospital de Santa Maria, Av. Prof. Egas Moniz, 1649-035 Lisbon (Portugal). Telephone: +351 968383939 / Fax: +351 217805602 Email: franciscotortosa.pathology@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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New Diagnostic for Pituitary Adenomas

Introduction

(Ki67) lower than 3%. The mechanism of PA progression to more aggressive and invasive tumours is not fully elucidated; in fact a continuum from “typical” to “atypical” adenoma and carcinoma has not been demonstrated, as is well stablished for other types of epithelial tumours, like the adenoma-carcinoma intestinal sequence. Atypical PA exhibit a borderline or uncertain behaviour, with atypical morphological characteristics suggestive of aggressive behaviour (such as locally invasive growth), a high mitotic index, a cell proliferation index (Ki67) above 3% and extensive immunostaining for p53 protein.[11] They are not as uncommon as previously thought.[13,14]

Although considered as benign, some PA are locally invasive and cause significant morbidity and mortality.[7,8] Other epithelial tumours classified as malignant neoplasm, for instance, skin basal cell carcinoma, although widely invasive rarely metastasize. In contrast, some aggressive pituitary tumours cause significant morbidity related to hormonal hypo or hypersecretion, may invade brain structures, cause blindness and cranial nerve paralysis; some may require radiation therapy and, ultimately, may be lethal, despite being considered histologically benign. [9] More than a decade after the last classification of the World Health Organization (WHO), a reassessment of the definition, classification and malignancy criteria of pituitary neoplasms seems appropriated, specifically for PA considered “atypical”.

Pituitary carcinomas are rare, representing 0.2% of pituitary tumours, in part this is due to a highly restrictive definition of the WHO,[11] or previous classifications,[15] since the sine qua non condition is the demonstration of cerebrospinal and/or systemic metastases, once there are no morphological criteria of malignancy. The time period between the initial diagnosis of adenoma to carcinoma is approximately 7 years, and the average survival, after confirmation of malignancy, is reported to be approximately 1.9 years,[16] or 1 year in two-thirds of the patients.[17] Since the suspicion of pituitary carcinoma is only confirmed by the existence of metastasis, this delays a more aggressive therapeutic approach, reducing its potential effectiveness. Due to the latency between initial diagnosis and appearance of metastases, it is often too late to treat the patient when spread appears. Earlier diagnosis and referral to specialized reference centres are fundamental to optimize short and long-term outcomes and prognosis in these patients.[6]

Tumours of the pituitary gland and sellar region represent approximately 10 to 15% of all brain tumours.[1] In fact, pituitary adenomas (PA) represent the third most common primary intracranial tumour in neurosurgery, outnumbered by gliomas and meningiomas.[1] As a result of the extensive use of neuroimaging studies, asymptomatic and incidental PA (“incidentalomas”) are increasingly common.[2,3] In a recent review of autopsy and magnetic resonance imaging (MRI) studies, the estimated overall prevalence of PA was 16.7%.[4] Recent studies show an increase in the PA prevalence up to four times above that previously thought.[5,6]

Since the first morphological classification proposed by Cushing in 1912, many attempts to histologically classify PA have been made. Initial classifications were based on the cellular tinctorial properties distinguishing acidophilic, basophilic and chromophobic adenomas; however, this staining classification does not correlate clinically with the functional characteristics of these tumours. Currently, classification of PA is based on histological criteria, mainly immunohistochemical (the gold standard of diagnosis) and ultrastructural, also taking into account clinical presentation, biochemical information, imaging techniques and surgical findings. Electron microscopy, an expensive and time-consuming technique, is rarely performed today.[10] The current WHO classification of endocrine tumours of the pituitary gland, classifies them as typical adenoma (ICD 8272/0), atypical adenoma (ICD 8272/1) and pituitary carcinoma (ICD 8272/3).[11] However, differences between “typical” and “atypical” adenoma are not clearly established, and there are no morphological criteria to distinguish locally aggressive atypical adenomas from carcinomas, when the tumour is limited to the sella turcica. [12] Most of PA are typical, with “bland” histological features, rare mitotic figures and a proliferative index

Differential diagnosis between an aggressive benign tumour and a malignant tumour in initial stage can be very difficult. The prediction of this type of tumours behaviour remains a challenge for both clinicians and pathologists; it seems necessary an early diagnosis, to allow an aggressive treatment of thosetumours, that do not reveal cytomorphologic features of malignancy ab initio and have worse prognosis. The aim of this study is to propose a new diagnostic strategy to orient prognosis and therapy of these tumours, based on a clinico-laboratorial and radio-histopathologic multiparameter system, as well as a simple diagnostic algorithm. This strategy derives from the retrospective analysis of the PA casuistic operated in the last 11 years at the largest hospital centre in Portugal.

Material and Methods

To validate the method, we applied this new clinicopathological classification retrospectively to patients diagnosed and operated by endonasal transsphenoidal via, with histological confirmation of PA, between

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01/01/2004 and 31/12/2014, at Centro Hospitalar Lisboa Norte, consisting of Hospital Universitario de Santa Maria and Hospital Pulido Valente. The procedures followed were in accordance with the ethical standards of the responsible institutional committee and with the Helsinki Declaration of 1975, as revised in 2000. PA were classified according to the 2004 version of the WHO on tumours of endocrine organs.[11] The rate of recurrence in those patients followed up for at least 5 years has been evaluated. We have designed a simplified, practical and easy to apply diagnostic algorithm for the distinction between “typical” adenoma (which we propose naming endocrine pituitary tumour -PET- of biological behaviour most likely benign) vs “atypical” adenoma (which we propose naming, based on their aggressiveness, PET of uncertain malignant potential or PET of biological behaviour most likely malignant). This algorithm is based on a multiparameter system, none of which is an absolute criterion of malignancy if used alone, and uses a numeric score based on the association of a specified threshold for each parameter of malignancy. It includes criteria related to the cytological appearance, cellular proliferation index, expression of a tumour suppressor gene, invasion and tumour recurrence (Table 1). For each tumour, the points for each parameter must be added to reach the total of score (minimum score: 0; maximum score: 10). Therefore: 0 to 3 points is consistent with: “typical” PA (according to WHO, 2004). We propose to call it: PET grade 1 (lowgrade malignancy) / PET of biological behaviour most likely benign. 4 to 7 points is consistent with: “atypical” PA (according to WHO, 2004). We propose to call it: PET grade 2 (intermediate grade of malignancy) / PET borderline / PET of uncertain malignant potential.

8 to 10 points is consistent with: “atypical” PA (according to WHO, 2004). We propose to call it: PET grade 3 (highgrade malignancy) / PET of biological behaviour most likely malignant (carcinoma in situ or pre-metastatic). In the presence of cerebrospinal and/or systemic metastases, the two ranking systems (WHO, 2004 and our proposal) call these tumours pituitary carcinoma. For this, we define a few parameters, some of which are already used by the WHO in its classification for this type of tumours however without cut-off point referred. We calculated the number of mitoses in representative high-power fields (HPF), according to the average per 10 HPF (HPF of 0.30 mm2, x400 magnification). The cell proliferation index (Ki67) was calculated as the percentage of positive nuclei within a minimum of 500 tumour cells in the areas of strongest immunostaining, analysed in optical microscope with x400 magnification. In equivocal cases, it was estimated with the help of an image processor software for immunohistochemical analysis, a method that compared with the performance of an experienced pathologist is matching 89.7% of cases.[18] As for p53, it is important that the dial intensity is moderate/intense, excluding the nuclei with weak dial (here the contribution of the software can be very valuable, by enabling to create a threshold of intensity). Due to the occasional misdetection of p53 and the absence of avalidated prognostic cut-off value by WHO, this was calculated as for Ki67, considering as a positive a value ≥2, according to the proposal made by the German working group members on pituitary tumours.[19] The tumour size and the extent of invasion are determined by MRI before surgery.[20] Tumours are classified as microadenomas (≤1 cm), macroadenomas (>1 and ≤4 cm) or giant adenomas (>4 cm). Following the WHO criteria,

Table 1: Proposed guide to assess malignant potential of PA (minimum score: 0; maximum score: 10). Parameters:

Score 0

Absent or rare (<2 / 10 HPF)

Present but uncommon (2-5 / 10 HPF)

Present (and/or with atypical mitotic figures) (>5 / 10 HPF)

Ki67 (%)

≤3

>3 and ≤20

>20

p53 (%)

Negative

≥2

Radiological classification

Grade 0-1

Grade 2-3

Grade 4

Yes

Yes (2 or more)

Number of mitoses

Tumour recurrence

HPF = High-power field (x400). 0-3 points: PET grade 1 (low-grade malignancy) / PET of biological behaviour most likely benign. 4-7 points: PET grade 2 (intermediate grade malignancy) / PET borderline / PET of uncertain malignant potential. 8-10 points: PET grade 3 (high-grade malignancy) / PET of biological behaviour most likely malignant (carcinoma in situ or pre-metastatic).

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New Diagnostic for Pituitary Adenomas

microadenomas are radiologically classified as grade 0 (intrasellar adenomas with normal appearance of the sella turcica) or grade 1 (intrasellar adenomas with enlargement of the sella turcica); macroadenomas are graded as grade 2 (tumours with diffuse sellar enlargement without bone erosion), grade 3 (tumours with focal bone erosion) and grade 4 (tumours with extensive bone erosion including the base of the skull and extrasellar structures).[11] We define a postoperative recurrence during followup, as tumour recurrence with imaging studies for non-functioning as functioning adenomas, as well as clinical evidence of postsurgical disease by hormonal hypersecretion for functioning tumours. In addition to the 5 parameters mentioned (mitotic index, Ki67 proliferative index, p53 immunostaining, tumour invasion, and recurrence), other criteria must be considered relevant, including cytomorphologic features, hormonal immunohistochemical subtypes, functionality of these tumours (clinical presentation), rapid progression of neurological signs or intra-operative observed invasion. Cytological atypia must be graded with the x100 objective, according to the following degrees: Without atypia/minimal atypia: round-to-ovoid uniform nuclei, with fine chromatin, inconspicuous nucleoli and a moderate quantity of cytoplasm. Moderate atypia: large nuclei, with some pleomorphism, and open chromatin; recognizable nucleoli. Marked atypia: pleomorphic nuclei, with rude chromatin, and large nucleoli.

Results

Of 243 operated patients, in 214 of them (88.1%) the tumour showed characteristics of “typical” adenoma and in 29 (11.9%) the characteristics of the tumour were of “atypical” adenoma. Then we apply our diagnostic algorithm to these tumours (Fig. 1). Two hundred and sixteen cases (88.9%) were diagnosed as PET of biological behaviour most likely benign (2 of the tumours, that had been diagnosed as atypical with the WHO classification, both clinically “silent” ACTH-producing macroadenomas, presented with score 3 according to our classification system, having shown no recurrence of disease after 9 and 10 years of follow-up) (Fig. 2); 27 cases (10.7%) were diagnosed as PET of uncertain malignant potential (Fig. 3) and 1 case (0.4%) was diagnosed of PET of biological behaviour most likely malignant (Figs. 4 and 5) (Table 2 ).

In 129 of the 243 PA, the follow-up lasted more than 5 years; 113 of these 129 adenomas (87.6%) were diagnosed as PET of biological behaviour most likely benign and the remainder (16; 12.4%) as PET of uncertain malignant potential. Seven of the PET of biological behaviour most likely benign (7/113, 6.2%) had recurrence; of these, 5 were clinically non-secreting macroadenomas (71.4%), with positive immunostaining for prolactin in one case, gonadotrophin in 3 and TSH in the remaining; one case (microadenoma) presented clinically with Cushing’s disease positive to ACTH, and there was a GH-secreting macroadenoma with acromegaly. Eleven of the PET of uncertain malignant potential (11/16, 68.8%) had recurrence; of these, 9 were clinically non-secreting macroadenomas (81.8%), with positive immunohistochemistry for prolactin in 2, ACTH in 2 (“silent”), gonadotrophin in 3 and TSH in 2; 2 cases (a microadenoma and a macroadenoma with pituitary apoplexy) presented clinically as Cushing’s disease, positive for ACTH.

Discussion

Although there are verified differences between adenomas and carcinomas, the usual parameters cannot distinguish conclusively between benign and malignant pituitary neoplasms. With this work we intend to provide a more specific malignancy differentiating system, with a capacity to early identify cases of possible poor evolution, something that could be of great clinical utility. We believe that the proposed strategy for the diagnosis of PA, new and easy to use, can help firstly pathologists in the diagnostic decision, and secondly, clinicians choosing the best post-operative therapy, since that “uncertain” malignant potential tumours would require periodic monitoring, whereas those considered potentially “malignant”, would require a more aggressive treatment. In any case, the multidisciplinary

H&E = Hematoxylin-Eosin.

Fig. 1: Simple algorithm for the primary proliferation of adenopituitary cells.

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Fig. 2: Photomicrographs of a PET of biological behaviour most likely benign positive for GH. A) There is a proliferation of monomorphic cells with round-to-ovoid nuclei and moderate amounts of eosinophilic cytoplasm (H&E x200). B) A reticulin stain demonstrates effacement of the usual adenohypophysis acinar architecture (Gomori Reticulinx 200). C) Cell proliferation index is low (<1%, Ki67 x200). D) Tumour shows cytoplasmic immunoreactivity for GH (GH x200).

Fig. 3: Photomicrographs of a PET of uncertain malignant potential positive for GH. A) This is a moderate to densely cellular tumour, composed of large and occasionally pleomorphic cells, prominent nucleoli and a moderate quantity of pale eosinophilic cytoplasm (H&E x200). B) Scattered mitotic figures are seen (arrow) (H&E x400). C) The tumour shows high proliferative index (5%, Ki67 x200) and diffuse cytoplasmic immunoreactivity for GH (D - GH x200).

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Fig. 4: Preoperative post contrast coronal T1 MRI, obtained in a patient with an â&#x20AC;&#x153;atypical macroadenomaâ&#x20AC;? classified as PET of biological behaviour most likely malignant. Note the high propensity for bilateral invasion to the cavernous sinus, with compression of the aqueduct and incipient hydrocephalus.

Fig. 5: Photomicrographs of a PET of biological behaviour most likely malignant that showed no immunoreactivity for any hormone. A) It is a densely cellular tumour composed of large and pleomorphic cells, prominent nucleoli and moderate amounts of eosinophilic cytoplasm (H&E x200). B) Abundant and sometimes atypical mitotic figures can be observed (arrow) (H&E x400). C) The tumour shows high proliferative index (39%, Ki67 x200) and extensive nuclear immunoreactivity for p53 (D - p53 x200).

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New Diagnostic for Pituitary Adenomas

Table 2: Comparative diagnostic study. No. of patients (%) Patients n = 243 (100 %)

According to the WHO (2004)

Typical adenoma 214 (88.1)

According to the new classification proposal

PET “benign” (grade 1) 216 (88.9)

Atypical adenoma 29 (11.9) PET of “uncertain” malignant potential (grade 2) 26 (10.7)

PET “malignant” (grade 3) 1 (0.4)

PET = Pituitary Endocrine Tumour.

consensus on the best therapeutic decision, also requires a personalized medicine for each patient. Mitoses are rare in adenomas and particularly in microadenomas, where they were found in only 3.9% of invasive adenomas in one of the largest studies to date. [21] Mitosis can be seen in 21.4% of invasive adenomas and 66.7% of carcinomas.[12] It is not established in the WHO classification the number of mitoses that favours the diagnosis of atypical adenoma, being subjectively referred “(…) an elevated mitotic index (…)”. A recent study conducted in Germany suggests a higher number than 2 per 10 HPF to consider invasive a PA, with a sensitivity of 0.90 and a specificity of 0.74, being one of the data that will require future consensus.[19] The use of immunohistochemical studies with Ki67 and p53 for PA has been controversial. Ki67 is a commonly examined antigen in PA, as it can contribute to define a group of adenomas with locally more aggressive behaviour. Increased levels of this antigen are correlated with growth speed, invasion and tumour recurrence.[22] In 1996, the study of Thapar et al. showed that the increase of Ki67 above of 3% is significant to differentiate invasive from non-invasive PA, and this threshold was accepted by the WHO. Their studies reported a Ki67 proliferative index of 1.4%, 4.7% and 11.9% in the non-invasive adenomas, invasive adenomas and carcinomas, respectively. The 3% threshold was used to distinguish non-invasive adenomas of invasive adenomas with 97% specificity and 73% sensitivity.[23] However, studies of cell proliferation with Ki67, unfortunately did not show a consistent correlation with invasiveness or tumour recurrence,[24,25] although three recent publications[26-28] support the concept that only a Ki67 proliferative index higher than 20-30%, suggests the presence of an in situ pituitary carcinoma,[29] or a premetastatic pituitary carcinoma in “sellar phase”;[30] this would be independent of the tumour size and the presence or absence of local invasion. P53 immunoreactivity has been found in all pituitary carcinomas.[10] It is not established in the WHO classification the percentage of positive nuclei and intensity of immunohistochemical staining for tumour suppressor

gene p53, also being subjectively indicated “(…) as well as extensive nuclear staining for p53 immunoreactivity”. A recent study recommends a cut-off value in the definition of this type of tumours in upcoming editions, suggesting a ≥2% cut-off .[19] In spite of this, routine use of Ki67 and p53 immunohistochemistry is not a common practice in many experienced laboratories, because it is not clear for the clinical team that treats the patient, how to evaluate the information that an adenoma is histologically “atypical”. In addition, factors such as size and tumour extension at the time of surgery may seem more relevant than the cellular proliferation. Therefore, the clinical usefulness of this category to identify eventually metastatic tumours is yet to be establish. Invasiveness is defined as the extension to the bone of the sellar floor, cavernous sinus and/or sellar diaphragm,[21] according to the assessment in preoperative neuroimaging studies. Although some studies have shown that invasion itself does not correlate with recurrence or with a worse prognosis, the majority of patients who die because of tumours of the pituitary gland have invasive adenomas. [31] Some experts have pointed out that the WHO classification of 2004 did not take into account the state of the invasive tumour.[22] To date, there are hardly any studies that indicate that “typical” PA has lower rates of surgical remission, or that the PA called “atypical” shows higher rates of recurrence. [22] In our study, while 6.2% of PET of biological behaviour most likely benign presented recurrence, 68.8% of those which we classify as being of uncertain malignant potential did (the probability of postsurgical tumour recurrence in a follow-up longer than 5 years is eleven times higher; p<0.0001). In the recurrent tumours, we also observed an increase in the cell proliferation index (Ki67), 2.73% for PET of uncertain malignant potential compared to 0.29% for PET of biological behaviour most likely benign. The standard morphological characteristics associated with malignancy, including hypercellularity, nuclear and cellular pleomorphism, increased mitotic activity, necrosis and dural/bone invasion, are commonly present in

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carcinoma, although, as in other endocrine organs, they are not necessarily diagnostic.

Funding

Some PA are “intrinsically” aggressive (such as prolactinomas in postmenopausal women and those that occur in young men, sparsely granulated GH-producing adenomas or “silent” ACTH adenomas).The majority of pituitary carcinomas are hormonally active, representing prolactinomas and ACTH-secreting tumours two thirds of the same,[17] although any histologic type and secretory pattern it has been described. Recent studies reveal that 91% of prolactinomas are invasive and 55% show a Ki67>3%. Other proliferative adenomas are gonadotroph/ null and corticotroph.[25,32] Pituitary tumours in patients with multiple endocrine neoplasia syndrome type 1 (MEN1) tend to be larger, invasive and symptomatic, although differences between these tumours and the rest of PA has not been demonstrated.

Competing interests

Conclusion

Early identification of aggressive endocrine tumours would allow the implementation of an intensive treatment that could prevent the recurrence or metastasis. Similarly to other endocrine tumours with problems in defining the histological criteria of malignancy, we present here our proposal for clinicopathological classification, based on a multiparameter grading system, which may incorporate additional clinical and pathological factors. This clinicopathological classification, that evaluates and categorizes the endocrine pituitary tumours in degrees or potential for malignancy, presents advantages such as: 1) assign a prognostic value in predicting a postoperative evolution free of disease or recurrence for each type of tumour; it is more precise than the current system of the WHO and has been shown to have relationship with the biological behaviour of the tumour; 2) is an objective, practical, easy to use and reproducible classification system, with potential to decrease the interobserver variability and, 3) identify the tumours that require a more aggressive treatment, as well as those indolent that might be more consensual. The importance of early identify potential immunohistochemical and molecular markers of invasion and malignancy, enable us to develop therapeutic aimed at improving the prognosis of affected patients. Finally, it would be desirable to reassess the definition, classification and criteria of malignancy that should be applied to pituitary neoplasms, specifically to the PA called atypical.

None.

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. Sanno N, Oyama K, Tahara S, Teramoto A, Kato Y. A survey of pituitary incidentaloma in Japan. Eur. J. Endocrinol. 2003;149:123-127. 3. Vernooij MW, Ikram MA, Tanghe HL, Vincent AJ, Hofman A, Krestin GP, et al. Incidental findings on brain MRI in the general population. N. Engl. J. Med. 2007;357:1821-1828. 4. Ezzat S, Asa SL, Couldwell WT, Barr CE, Dodge WE, Vance ML, et al. The prevalence of pituitary adenomas: a systematic review. Cancer. 2004;101(3):613-619. 5. Daly AF, Rixhon M, Adam C, Dempegioti A, Tichomirowa MA, Beckers A. High prevalence of pituitary adenomas: a cross-sectional study in the province of Liege, Belgium. J. Clin. Endocrinol. Metab. 2006;91:4769-4775. 6. Fernandez A, Karavitaki N, Wass J. Prevalence of pituitary adenomas: a community-based, crosssectional study in Banbury (Oxfordshire, UK). Clin. Endocrinol. 2010;72:377-382. 7. Al-Brahim NY, Asa SL. My approach to pathology of the pituitary gland. J. Clin. Pathol. 2006;59:1245-1253. 8. Asa SL. Practical pituitary pathology: what does the pathologist need to know?. Arch. Pathol. Lab. Med. 2008;132:1231-1240. 9. Al-Shraim M, Asa SL. The 2004 World Health Organization classification of pituitary tumors: What is new? ActaNeuropathol. 2006;111(1):1-7. 10. Trouillas J, Roy P, Sturm N, Dantony Rudelli C, Viennet G, et al. A new clinicopathological classification of adenomas: a multicentric case-control 410 patients with 8 years post-operative ActaNeuropathol. 2013;126:123-135.

E, Cortetprognostic pituitary study of follow-up.

We thank Professor Dra. Yasmin Fernandes for the courtesy of providing the magnetic resonance imaging.

11. 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.

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Acknowledgments

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World Hearth Organization Classification of Tumours. Pathology and genetics of tumours of endocrine organs. Lyon: IARC Press; 2004. 9-47. 12. Pernicone PJ, Scheithauer BW. Invasive pituitary adenoma and pituitary carcinoma. In: Thapar K, Kovacs K, Scheithauer BW, Lloyd RV, editors. Diagnosis and management of pituitary tumors. Totowa NJ: Humana Press; 2001. 369-386. 13. Yildirim AE, Divanlioglu D, Nacar OA, Dursun E, Sahinoglu M, Unal T, et al. Incidence, Hormonal Distribution and Postoperative Follow Up of Atypical Pituitary Adenomas. Turk. Neurosurg. 2013;23(2):226-231. 14. Tortosa F, Webb SM. Atypical pituitary adenomas: 10 years experience in a reference centre of Portugal. Neurología. 2015; doi.org/10.1016/j.nrl.2015.06.010.

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15. Kovacs K, Horvath E, Vidal S. Classification of pituitary adenomas. J. Neurooncol. 2001;54:121-127. 16. Pernicone PJ, Scheithauer BW, Sebo TJ, Kovacs KT, Horvath E, Young WF Jr, et al. Pituitary carcinoma: A clinicopathological study of 15 cases. Cancer. 1997;79:804-812. 17. Ragel BT, Couldwell WT. Pituitary Carcinoma: A Review of the Literature. Neurosurg. Focus. 2004;16(4):E7. 18. Borrecho G, Ortiz S, Tortosa F. Estudo da actividade proliferativa com Ki67 em adenomas hipofisários: O homem e a máquina. XIII Congresso Técnico de Anatomia Patológica; 2012 May 25-27; Figueira da Foz, Portugal: Associação Portuguesa de Técnicos de Anatomia Patológica. 19. Miermeister CP, Petersenn S, Buchfelder M, Fahlbusch R, Lüdecke DK, Hölsken A, et al. Histological criteria for atypical pituitary adenomas - data from the German pituitary adenoma registry suggests modifications. ActaNeuropatholCommun. 2015; doi: 10.1186/ s40478-015-0229-8. 20. Nosé V, Ezzat S, Horvath E, Kovacs K, Laws ER, Lloyd R, et al. Protocol for the examination of specimens from patients with primary pituitary tumors. Arch. Pathol. Lab. Med. 2011;135:640-646. 21. Saeger W, Ludecke DK, Buchfelder M, Fahlbusch R, Quabbe HJ, Petersenn S. Pathohistological classification of pituitary tumors: 10 years of

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experience with the German Pituitary Tumor Registry. Eur. J. Endocrinol. 2007;156:203-216. Zada G, Woodmansee WW, Ramkissoon S, AmadioJ, Nose V, Laws RE. Atypical pituitary adenomas: Incidence, clinical characteristics, and implications. J. Neurosurg. 2011;114:336-344. Thapar K, Kovacs K, Scheithauer BW, Stefaneanu L, Horvath E, Pernicone PJ. Proliferative activity and invasiveness among pituitary adenomas and carcinomas: An analysis using the MIB-1 antibody. Neurosurgery. 1996;38:99-107. Amar AP, Hinton DR, Krieger MD, Weiss MH. Invasive pituitary adenomas: significance of proliferation parameters. Pituitary. 1999;2:117-212. Aranda FI, Niveiro de Jaime M, Peiró G, Alenda C, Picó A. Adenoma hipofisario: estudio de la actividad proliferativa con Ki-67. Rev. Esp. Patol. 2007;40(4):225-231. Dudziak K, Honegger J, Bornemann A, Horger M, Mussig K. Pituitary carcinoma with malignant growth from first presentation and fulminant clinical course - case report and review of the literature. J. Clin. Endocrinol. Metab. 2011;96:2665-2669. Mamelak AN, Carmichael JD, Park P, Bannykh S, Fan X, Bonert HV. Atypical pituitary adenoma with malignant features. Pituitary. 2011;14:92-97. Pasquel FJ, Vincentelli C, Brat DJ, Oyesiku NM, Ioaquimescu AG. Pituitary carcinoma in situ. Endocr. Pract. 2012;19(3):69-73. Heaney AP. Clinical review: Pituitary carcinoma: difficult diagnosis and treatment. J. Clin. Endocrinol. Metab. 2011;96:3649-3660. Scheithauer BW, Gaffey TA, Lloyd RV, Sebo TJ, Kovacs KT, Horvath E, et al. Pathobiology of pituitary adenomas and carcinomas. Neurosurgery. 2006;59:341-353. Lopes MBS. Diagnostic controversies in pituitary tumor pathology. ANNP Companion Meeting. USCAP; 2013 March 2-8; Baltimore, USA: United States & Canadian Academy of Pathology. Pizarro CB, Oliveira MC, Coutinho LB, Ferreira NP. Measurement of Ki-67 antigen in 159 pituitary adenomas using the MIB-1 monoclonal antibody. Braz. J. Med. Biol. Res. 2004;37:235-243.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 02, April - June 2016

Original Article Assessment of Cell Proliferation in Helicobacter Pylori Associated Gastric Epithelial Diseases Neeraj Dhameja1*, Ritambra Nada2, D.K.Bhasin3, Kusum Joshi2 Dept. of Pathology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India Dept of Histopathology, Post Graduate Institute of Medical Education and Research, Chandigarh, India 3 Dept of Gastroenterology, Post-Graduate Institute of Medical Education and Research, Chandigarh, India 1

Keywords: Atrophy , Carcinoma, Chronic Active Gastritis, Dysplasia, Intestinal Metaplasia, Helicobacter Pylori, Proliferative Index

ABSTRACT Background: Alterations in cell proliferation and apoptosis as a result of Helicobacter Pylori infection can contribute to carcinogenesis. This study was planned to assess cellular proliferation during chronic active gastritis with or without H. Pylori, atrophy, intestinal metaplasia, dysplasia and carcinoma. Effect of H. Pylori eradication on cell proliferation was also studied. Methods: Ki67 immunostaining was done to calculate proliferative index (PI) in H. pylori associated gastric diseases. Gastric biopsies of 160 patients with dyspepsia were selected comprising of 20 cases each of following groups 1) Normal control 2) H .pylori positive chronic active gastritis before and after treatment 3)H. pylori negative chronic active gastritis 4) Atrophy 5) Intestinal metaplasia 6) Dysplasia 7) Gastric adenocarcinoma Result: There was increased proliferative index (PI) in H. pylori positive and negative chronic active gastritis as compared to normal controls. However, the PI in H. pylori positive chronic active gastritis was significantly higher than that of H. pylori negative chronic active gastritis. Increased proliferation was persistent in atrophy, intestinal metaplasia, dysplasia and carcinoma. There was significant increase in PI in foveolar regions in chronic active gastritis, intestinal metaplasia and dysplasia . After H. pylori eradication, there was marked reduction in PI. Conclusion: H. pylori infection results in increased gastric epithelial cell proliferation which persists in the premalignant stages of atrophy, intestinal metaplasia, dysplasia and carcinoma. Presence of H. pylori augments proliferation resulting from inflammation and eradication of H. pylori reduces proliferation.. This increased proliferation may be one of the mechanism of H. pylori associated carcinogenesis.

*Corresponding author: Dr. Neeraj Dhameja, Assistant Professor, Dept of Pathology, Institute of Medical Sciences, Banaras Hindu University, Varanasi- 221005, India Telephone: +91 - 7379645390 Email: doctorneeraj146@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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Cell Proliferation in H Pylori Associated Gastric Diseases

Introduction

against a background of atrophic gastritis and intestinal metaplasia. Increasing severity of dysplasia is associated with an expanding proilferating zone. Similarily Fan et al[15] , Tseng et al[16], Ren et al[17] and Peek RM et al[18] found increased gastric epithelial cell proliferation in H.pylori infected gastritis. This increased cell proliferation is also present in the premalignant conditions like atrophy and IM. In the gastritis stage, the proliferation is reversible by eradication of H.pylori[19], however, in the stages of atrophy and intestinal metaplasia, it cannot be reverted back to normal levels.

Long term H.pylori infection leads to multifocal atrophic gastritis characterized by loss of glandular tissue. Atrophy of the antral mucosa leads to the development of intestinal metaplasia (IM) which is linked to increased risk of gastric cancer. [3-7] Hence, atrophy and IM are considered as premalignant lesions.[8] Gastric carcinomas are classified as intestinal and diffuse types and H.pylori is related to both. Various bacterial and host factors have been described in the pathogenesis of H.pylori associated gastric epithelial diseases. The bacterial factors include tissue injury inducers like Cag A and Vac A (vacuolating cytotoxin). These factors act as proinflammatory substances and cause more intense gastric inflammation and greater cytokine production. Exact pathogenesis of gastric carcinoma is not known, however, it has been reported that H.pylori effects the cell kinetics with increased cell proliferation which may lead to development of carcinoma.[9]

Since H.pylori has high prevalence in India, in this study, we evaluated cell proliferation in different gastric epithelial diseases associated with H.pylori by immunostaining with ki67.

Helicobacter pylori is a spiral shaped gram negative bacillus first described by Warren and Marshal in 1983 in gastric biopsies of patients with peptic ulcer and chronic active gastritis.[1] It resides in and beneath the surface mucous layer of the gastric epithelium and secretes urease which protects it against acid by catalyzing urea hydrolysis to produce buffering ammonia. It is present worldwide but more frequent in developing countries and is associated with gastritis, peptic ulcer disease, gastric adenocarcinoma and lymphoid neoplasms like MALTOMA.[2]

In the normal stomach, the proliferative compartment is located in the neck region of glands in the body, fundus and antrum. From this zone, the cells move towards the surface and deeper mucosal glands. Various studies have shown increased proliferation in H.pylori positive biopsies. Brenes et al [10] analyzed H.pylori positive gastric biopsies and demonstrated that gastric mucosa infected with H.pylori is in a state of hyperproliferation which decreased after eradication of H.pylori. Bechi et al [11] analyzed gastric corpus biopsies with H.pylori but no inflammation using tritiated thymidine and showed excessive replication suggesting direct effect of H.pylori on cell proliferation. Peek et al[12] showed increased epithelial cell proliferation in H.pylori infection by immunohistochemical analysis of the proliferation associated antigen ki67. Cahill RJ et al[13] identified an increased gastric epithelial cell proliferation associated with H.pylori infection which is reversed when the organism is eradicated. Murakami et al[14] showed that gastric epithelial cell proliferation in the antrum and corpus is increased in H.pylori associated gastritis and eradication of the bacteria leads to the reversal of cell proliferation to normal. Cell proliferation is increased when atrophy is present and gastric dysplasia develops

Materials and Methods

All adult patients presenting with dyspeptic symptoms were enrolled for this study . After complete clinical evaluation , upper GI Endoscopy (Olympus GEX) was performed after explaining the procedure to the patient and obtaining a written consent . Detailed medical history was taken and patients on NSAIDS , steroids were excluded. The patients who were diagnosed to have chronic active gastritis due to H. pylori were treated using triple drug regimen and a repeat biopsy was taken after six weeks of therapy. Endoscopic antral biopsies were oriented on pieces of filter paper , fixed in 10% buffered formalin and sent to the Histopathology department. The biopsies were processed using standard processing protocol and multiple serials were stained with Haematoxlin & eosin. Biopsies were evaluated by a group of three pathologists independently. Each biopsy was assessed for activity, chronic inflammation , atrophy , intestinal metaplasia, dysplasia and H. pylori according to the updated Sydney system.[20] Biopsies with proper orientation were selected for further evaluation . The biopsies were grouped into eight groups of twenty each as follows 1) Normal control (NC, as per updated Sydney system) 2) Chronic active gastritis (CAG) without H. pylori 3) Chronic active gastritis with H.pylori (Pretreatment biopsies) 4) Chronic active gastritis with H. pylori (Post treatment biopsies) 5) H.pylori gastritis with atrophy 6) H.pylori gastritis with intestinal metaplasia 7) H. pylori gastritis with dysplasia 8) Gastric adenocarcinoma of the intestinal type Proliferation was assessed by immunoperoxidase staining for ki67, a proliferation associated antigen using the MIB-

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Dhameja et al. 1 monoclonal antibody (Novacastra, dilution1:100). At least 1000 cells in the three consecutive gastric foveolar epithelia, neck and glandular regions were counted in each case for assessing topographical distribution of proliferative cells. Cells showing distinctive brown staining of the nuclei were counted as positive. The mean number of positive cells per total of 1000 epithelial cells for each region and combination of these regions (foveolae F, neck N and glands G) was expressed as the Proliferative Index (PI). Proliferative Index (PI) = ki67 positive cells x100/Total cells (1000) Statistical Analysis: Students unpaired t test was used for comparison of normal control and other groups (CAG with H. pylori , CAG without H.pylori , atrophy , intestinal metaplasia , dysplasia and carcinoma). Students paired t test was used for comparison of chronic active gastritis with H. pylori (pretreatment biopsies) and antral biopsies after H. pylori eradication therapy. P value less than 0.05 was considered significant.

Results

There were 120 males and 40 females in the age range of 20 to 75years (mean 45.65yrs). Endoscopically , duodenal ulcers were present in 83 , gastric ulcers in 16 and 133 patients had antral gastritis . Biopsy Rapid urease test of patients with H. pylori was positive The proliferative indices (PI) of different groups were calculated topographically for foveolar , neck and gland regions and combination of these regions. The cumulative PI of control group was 0.6% to 7.5% (mean 3.9%) ( Fig. 1). The cumulative PI of chronic active gastritis without H. pylori was 1.03% to 22.3% (mean 7.57%) (Fig.2) and with H. pylori was 9.06% to 29.54% (mean 16.68%) (Fig.3) which was significantly higher (p0.02 ) and (p0.00000) respectively than the cumulative PI of control group. However , the cumulative PI of chronic active gastritis with H. pylori was significantly more than the cumulative PI of chronic active gastritis without H. pylori (p0.00002). The cumulative PI of chronic active gastritis with H. pylori reduced significantly (1.04% to 17% , mean 7.10% , p0.00000) (Fig.4) after eradication therapy when compared with pretreatment PI. However, post treatment PI was still higher than that of normal control.

A-55 The PI was also calculated topographically for foveolae, neck and gland region . It was found that in H. pylori positive CAG, the PI in the foveolar region (1% to 6.6%, mean 3.29% ) was significantly higher than that of normal control (0.1% to 3.7%, mean 0.92%, p0.00000) and H. pylori negative chronic active gastritis (0.4% to 4%, mean 1.62%, p0.001) This increased PI in the foveolar region was persistent and further increased in the stages of intestinal metaplasia (0.2% to 22.2% , mean 4.7%) and dysplasia (0.66% to 56.6% , mean 22.16% ). (Table.1, Fig.9).

Discussion

Gastric carcinoma has been associated with H.pylori based on epidemiological and clinical investigation. Correa[21] has suggested that H.pylori associated gastric carcinoma develops through a multistep process from chronic active gastritis (CAG) to atrophy, intestinal metaplasia, dysplasia and carcinoma. H.pylori infected gastric mucosa of patients with CAG is characterized by the infiltration of neutrophils and lymphocytes and is associated with varying degrees of atrophy and intestinal metaplasia in long standing infection which are premalignant lesions. Exact pathogenesis of H.pylori associated gastric carcinoma is not known, however, it has been hypothesized that alteration of cellular turnover may be one of the mechanism of H.pylori associated carcinogenesis. The present study was therefore designed to quantitate the cellular proliferation of gastric epithelial cells in various topographic regions of gastric mucosa through the spectrum of gastric epithelial lesions associated with H.pylori infection.

The cumulative PI of premalignant lesions i.e. atrophy (2.56% to 17.56%, mean 8.08%) (Fig.5), intestinal metaplasia (7.1% to 34.23%, mean 15.7%) (Fig.6), dysplasia (9.53% to 45.56%, mean 25.2%) (Fig.7) and adenocarcinoma (33.18% to 71%, mean 38.60%) (Fig.8) was significantly higher than that of normal controls and H pylori negative CAG.

Cellular turnover in a living body consists of a balance between apoptosis and cell proliferation. Imbalance with increased proliferation and decreased apoptosis disrupts homeostasis and may be an early event in malignant transformation. Various studies have shown that H.pylori is associated with increased cell proliferation which reduces to normal after eradication of H.pylori.[10,13,22,23] The increased proliferation is itself not sufficient for carcinoma, but the proliferating cells are susceptible to mutations which may lead to carcinoma. In this study, we used ki67 immunostaining to identify cells in the proliferative phase of the cell cycle. The monoclonal antibody ki67 reacts with human nuclear antigen (expressed only in cycling cells, not in quiescent cells) and the ki67 labelling index corresponds to the growth fraction of a cell population. Our data shows that there is a significant increase in proliferative index (PI) in biopsies with H.pylori positive CAG as compared to normal biopsies. On topographical analysis, it was observed that there was expansion of proliferative zone with increased PI in the foveolar and neck regions. Similar results were shown by Lynch et al[22] and Panella et al[24].

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Cell Proliferation in H Pylori Associated Gastric Diseases

Fig. 1: Ki67 immunostaining in normal gastric antral biopsies

Fig.4. Ki67 immunostaining in posttreatment biopsies

Fig.2: Ki67 immunostaining in H pylori negative CAG

Fig.3: Ki67 immunostaining in H pylori positive CAG

Fig.5: Ki67 immunostaining in antral biopsy with atrophy

Fig.6: Ki67 immunostaining in antral biopsy with intestinal metaplasia

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Dhameja et al.

Fig.7: Ki67 immunostaining in antral biopsy with dysplasia

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Fig.8: Ki67 immunostaining differentitiated adenocarcinoma

moderately

Fig. 9: Comparison of normal and all other groups

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Cell Proliferation in H Pylori Associated Gastric Diseases

Table 1: Proliferative Indices (mean, topographical and cumulative) of Ki67 immunostained antral biopsies of all the groups. S.No

Groups

Foveolae

Neck

Gland

Cumulative

Normal control

0.92 ± 0.9

9.97 ± 5.35

0.83 ± 1.10

3.90 ± 2.20

CAG without H pylori

1.62 ± 1.19

21.29 ± 15.50

1.04 ± 0.78

7.57 ± 6.06

CAG with H pylori (Pre Treatment)

3.29 ± 1.87

45.4 ± 14.62

1.82 ± 1.10

16.68 ± 5.98

Post Treatment

0.77 ± 1.26

19.44 ± 10.81

0.9 ± 1.73

7.10 ± 4.09

H pylori Gastritis with atrophy

1.50 ± 2.09

24.2 ± 11.75

1.39 ± 1.68

8.08 ± 4.9

H pylori Gastritis with IM

4.7 ± 4.99

40.06 ± 17.91

1.77 ± 3.16

15.70 ± 7.54

H pylori Gastritis with Dysplasia

22.16 ± 16.45

48.65 ± 16.63

4.98 ± 9.62

25.24 ± 10.47

Gastric Adenocarcinoma

Our data shows significant increase in PI in CAG without H.pylori with a similar expansion of proliferation compartment as compared to normal controls. However, there was significant difference in PI between H.pylori positive CAG and H.pylori negative CAG. These observations suggest that inflammation increases proliferation both in H.pylori positive and H.pylori negative CAG as compared to normal control, but H.pylori infection has a role in further augmentation of the proliferative response. Bechi et al[11] using ³H tritiated thymidine labelling found higher labelling index in H.pylori positive CAG biopsies compared to H.pylori negative CAG biopsies, however, the difference was not statistically significant. The result of the present study as well as the study by Lynch et al[22] are contrary to the above. This difference may be due to the different methods used to evaluate cell proliferation. ³H thymidine labelling quantitates only the S phase fraction whereas ki67 quantitates growth fraction (all cycling cells in a population). It is apparent that increased cell turnover with a proportionate shortening of all phases of the cell cycle will not be reflected by a higher S phase fraction. Hence ki67 labelling may be considered a better proliferation marker than ³H thymidine labelling. Therefore, our findings along with those of Lynch et al[22] support an association of H.pylori infection with both increased epithelial cell proliferative activity and expansion of the mucosal proliferative compartment. In the present study, PI in antral biopsies showing atrophy was also quantitated. PI (combined and neck) was higher in areas of atrophy as compared to normal biopsies. The PI of foveolae and gland region was also higher in atrophic areas when compared to the normal but the difference was not statistically significant. However, the PI of atrophic region was statistically less than that of H.pylori

38.60 ± 16.65

positive CAG. These observations suggest that chronic inflammation in atrophic areas is responsible for persistent increased proliferation. In the present study, PI in areas of IM was found to be higher in all topographical areas (foveolae, neck and gland) of mucosa as compared to normal controls and was comparable to that of H.pylori positive CAG. Similar results were shown by Tseng et al[16] . Ierardi et al[25] demonstrated that hyperproliferation associated with IM is not reversed by H.pylori eradication, suggesting that proliferation of the metaplastic cells is no longer dependent on bacterial factors. We also studied antral biopsies showing dysplasia and the PI (combined and topographical) was significantly higher in these dysplastic regions when compared with normal control and H.pylori positive CAG. However, PI was statistically increased in the foveolar region only when compared with H.pylori positive CAG and areas of IM respectivley. This suggests that as there is progression from IM to dysplasia, the proliferative compartment shifts to the foveolar region (superficialisation of the proliferative compartment). We also studied PI in gastric adenocarcinomas of intestinal type and found higher PI in the carcinomatous areas as compared to NC and other groups. The combined PI in carcinoma was significantly higher than H.pylori positive CAG, atrophy, IM and dysplasia. Our data to assess the response of proliferation to H.pylori eradication therapy performed on post treatment biopsies showed significant decrease in PI as compared to pretreatment biopsies. However, proliferation in post treatment biopsies was as high as H.pylori negative CAG, thereby suggesting a lowering of proliferation after eradication of H.pylori. The continuing chronic

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 02, April - June 2016

Dhameja et al. inflammation that persists in post treatment biopsies could be responsible for the PI higher than normal controls. Lynch et al[22] and Brenes et al[10] have shown similar results.

Conclusion

In summary, H.pylori associated gastric carcinoma is a multistep process and evolves through the stages of chronic active gastritis to atrophy, intestinal metaplasia and dysplasia. The present study has shown that H.pylori infection is associated with increased proliferation in all regions of gastric mucosa as compared to normal controls. As there is progression from CAG to IM, dysplasia and carcinoma, cell proliferation increases progressively. Eradication of H.pylori markedly reduces cell proliferation but does not bring it down to normal levels, suggesting also a role of inflammation in proliferation.

Funding None

Competing Interests None

References

1. Warren JR, Marshall B. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. The Lancet 1983;321:1273-75. 2. Eurogast study group. An international association between Helicobacter pylori and gastric cancer. The Lancet 1993;341:1359-63. 3. Sipponen P. Gastric cancer- A Long Term Consequece of Helicobacter pylori infection? Scand J Gastroenterol Suppl. 1994;201:24-7. 4. Sipponen P, Hyvärinen H. Role of Helicobacter pylori in the Pathogenesis of Gastritis, Peptic ulcer and Gastric cancer. Scand J Gastroenterol Suppl. 1993;196:3-6. 5. Sipponen P, Kosunen TU, Valle J, Riihelä M, Seppälä K. Helicobacter pylori infection and chronic gastritis in gastric cancer. J Clin Pathol 1992;45:319-23. 6. Rokkas T, Filipe MI, Sladen GE. Detection of an increased incidence of early gastric cancer in patients with intestinal metaplasia type III who are closely followed up. Gut 1991;32:1110-13. 7. Jass JR. Role of intestinal metaplasia in the histogenesis of gastric carcinoma. J Clin Pathol 1980;33:801-10. 8. Correa P. Is gastric carcinoma an infectious disease? N Engl J Med 1991;325(16):1170-71.

A-59 of Cancer Prevention in Human Subjects. Cancer Res 1988;48:235-45. 10. Brenes F, Ruiz B, Correa P, Hunter F, Rhamakrishnan T, Fontham E,et al. Helicobacter pylori causes hyperproliferation of the gastric epithelium: pre and post eradication indices of proliferating cell nuclear antigen. Am J Gastroenterol 1993;88(11):1870-5. 11. Bechi P, Balzi M, Becciolini A, Maugeri A, Raggi CC, Amorosi A,et al. Helicobacter pylori and cell proliferation of the gastric mucosa: possible implication for gastric carcinogenesis. Am J Gastroenterol 1996;91:271-6. 12. Peek RM, Moss SF, Tham KT, Perez-Perez GI, Wang S, Miller GG, et al. Helicobacter pylori Cag A+ strains and dissociations of gastric epithelial cell proliferation from apoptosis. J Natl. Cancer Inst. 1997;89(12):8638. 13. Cahill RJ, Xia H, Kilgallen C, Beattie S, Hamilton H, Colm O’ Morain. Effect of Eradication of Helicobacter pylori Infection on Gastric Epithelial Cell Proliferation. Dig Dis Sci 1995;40:1627-31. 14. Murakami K, Fujioka T, Kodama R, Kubota M, Tokieda M, Nasu M. Helicobacter pylori infection accelerates human gastric mucosal cell proliferation. J Gastroenterol 1997;32:184-8. 15. Fan XG, Kelleher D, Fan XJ, Xia HX, Keeling PW. Helicobacter pylori increases proliferation of gastric epithelial cells. Gut 1996;38:19-22. 16. Tseng HH, Hsu PI, Chen HC, Lai KH, Lo GH, et al. Compartment theory in Helicobacter pylori associated gastric carcinogenesis. Anticancer Res. 2003;23:3223-9. 17. Ren H, Yi C. Role of Helicobacter pylori infection in pathogenesis of gastric adenocarcinoma. J Tongji Med Uni. 1999;19:127-30. 18. Peek RM, Wirth HP, Moss SF, Yang M, Abdalla AM, Tham KT, et al. Helicobacter pylori alters gastric epithelial cell cycle events and gastrin secretion in Mongolian gerbils. Gastroenterology 2000;118:48-59. 19. Hirasawa R, Tatsuta M, Iishi H, Yano H, Baba M, Uedo N, et al. Increase in apoptosis and decrease in ornithine decarboxylase activity of the gastric mucosa in patients with atrophic gastritis and gastric ulcer after successful eradication of Helicobacter pylori. Am J Gastroenterol 1999;94:2398-02.

9. Lipkin M. Biomarkers of Increased Susceptibility to Gastrointestinal Cancer . New Application to Studies

20. Dixon MF, Genta RM, Yardley JH, Correa P. Classification and grading of gastritis. The Updated

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Cell Proliferation in H Pylori Associated Gastric Diseases

Sydney system. International Workshop on the Histopathology of Gastritis. Am J Surg Pathol 1996;20:1161-81. 21. Correa P. A human model of gastric carcinogenesis. Cancer Res 1988;48:3554-64. 22. Lynch DA, Mapstone NP, Clarke AMT, Cobala GM, Jackson P, Morrison L, et al. Cell proliferation in Helicobacter pylori associated gastritis and the effect of eradication therapy. Gut 1995;36:346-50. 23. Fraser AG, Sim R, Sankey EA, Dhillon AP, Pounder RE. Effect of eradication of Helicobacter pylori on

gastric epithelial cell proliferation. Aliment Pharmacol Ther 1994;8:167-73. 24. Panella C, Ierardi E, Polimeno L, Balzano T, Ingrosso M, Amoruso A, Â¨et al. Proliferative Activity of Gastric Epithelium in Progressive Stages of Helicobacter pylori Infection. Dig Dis Sci 1996;41:1132-38. 25. Ierardi E, Francavilla R, Panella C. Effect of Helicobacter pylori eradication on intestianl metaplasia and gastric epithelium proliferation. Ital J Gastroenterol Hepatol 1997;29:470-5.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 02, April - June 2016

Original Article Intraoperative Squash Cytology of Central Nervous System and Spinal Cord Lesions with Histological Correlation Kishore Sanjeev1, Bhardwaj Aparna1*, Kusum Anuradha2, Thakur Brijesh1, Kaushik Sanjay1, Sharma Neetika1 Department of Pathology, SGRR Institute of Medical & Health Sciences, Dehradun, India. Department of Pathology, Himalyan Institute of Medical &Health Sciences, Dehradun, India. 1

Keywords: Central Nervous System, Histopathology, Squash Cytology.

ABSTRACT Aims: Squash Cytology is now a well established and universally accepted technique in diagnosing a wide range of Central Nervous System (CNS) lesions and is presently being employed for both therapeutic and prognostic reasons. This study was conducted with an aim to correlate squash smears with histopathology and to compare statistical data employing sensitivity, specificity and diagnostic accuracy of squash cytology. Methods: The present study was a retrospective study comprising 369 lesions of central nervous system and spinal cord that were retrieved from archives. All the cases for which Intraoperative squash cytology and subsequent histopathology was available were included in the study. Cytology smears were stained with May- Grunwald- Geimsa (MGG), Hematoxylin & Eosin (H&E) and Pap stain. Histopathology smears were made from formalin fixed tissue sent separately and stained with H&E. Results: Of 369 cases, 86.4% were neoplastic and 13.6% nonneoplastic on histopathology. Amongst neoplasms, Astrocytic tumors constituted 24.7% of cases followed by Meningiomas comprising 17.8%. Amongst the benign lesions Tuberculoma was seen most frequently (3.25%). Overall diagnosticAccuracy of squash was 95.25%. On statistical analysis Sensitivity, Specificity, Positive Predictive value (PPV) and Negative Predictive Value (NPV) of squash cytology were 94.3%, 95.6%, 95.3% and 95.1% respectively. On applying student T test, for statistical correlation between squash cytology and histopathology p value was 0.363347 (p>0.05) hence errors in diagnosis by squash were insignificant. Conclusion: Intraoperative squash cytology is fairly accurate, reliable and cost effective method for rapid diagnosis of CNS lesions. Acknowledgement: We would like to acknowledge Dr. Pankaj Arora, Consultant Neurosurgery,SGRRIM&HS for his kind cooperation.

*Corresponding author: Dr. Aparna Bhardwaj, Assosciate Professor, Department of Pathology, SGRR Institute of Medical & Health Sciences, Patel Nagar, Dehradun, India- 248001. Phone: +91 - 911352760439, 09411718270 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

Lesion within the central nervous system are diverse entities ranging from inflammatory conditions to neoplasms and remain one of the most challenging domains of neuropathologists. The accurate assessment of the diseased tissue is fundamental to the diagnosis and management of central nervous system lesion. [1] With the advent of neuroimaging techniques like computed tomography (CT) and magnetic resonance imaging (MRI), the diagnosis of very small, slow growing and minimally symptomatic lesion can be easily discovered. Diagnosis of these lesions based only on clinical and radiological findings is difficult and many a time incomplete with regards to infiltration. Accurate diagnosis of CNS lesions is further required for choosing between surgery and alternative therapeutic strategies. [2] Overall accuracy of preoperative radiological diagnosis ranges between 10%30% as per literature. [3] In the neurosurgical practice, although histopathology is the gold standard, intraoperative cytological diagnosis is now well established and continues to gain momentum. The biggest advantage of cytological diagnosis is rapid intraoperative diagnosis which further helps the surgeon to plan the extent of surgery and modify it accordingly. [4] The squash smear technique has gained importance because of its technical simplicity and its ability to display abnormal cellularity, nuclear and cytoplasmic details and even tissue architecture. Furthermore, squash smear technique can be applied not only to neoplastic lesions but also to non neoplastic lesions. [5] Thus, this study was undertaken to compare the efficacy of intraoperative squash smear cytology with histopathological diagnosis.

Materials and Methods

The present study was a retrospective study conducted in the Department of Pathology for a period of 3 years. As such, the Institutional policy does not envisage an Institutional Ethical clearance for such studies as no Ethical issues were involved. Three hundred and sixty nine cases of CNS neoplasm or lesions sent for intraoperative smear cytology (squash preparation) diagnosis for which subsequent histopathological examination was done were retrieved from the archives. Also relevant clinical and radiological data were retrieved and noted. The biopsy samples obtained at the time of surgery were transported immediately to the neuropathology laboratory in isotonic saline for processing. Smears were prepared by placing 1-2 mm of biopsy material at one edge of clean, dry and labeled slide

and crushing with another slide with just enough pressure to spread the tissue into thin film. Gross and naked eye examination was done to evaluate nature of spread. In most cases 6-8 cytological smears were availaible. Out of which half were immediately fixed in method for staining with Hematoxylin and Eosin (H and E). Remaining smears were air dried and stained with May-Grunewald-Giemsa stain (MGG). Smears were mounted with DPX. Paraffin H&E stained sections were prepared from the residual tissue as well as additional tissue sent for histopathology. Smear cytology diagnoses were correlated with the histopathological findings. Squash 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). The tumors were classified according to the World Health Organization classification of CNS neoplasm 2007.[7] Study design included a comparison between results of squash cytology with final histopathological diagnosis. Data analysis was based on Galen and Gambino method which calculated sensitivity and specificity of cytology in differentiating benign and malignant lesions (Table 1). Table 1: Galan and Gambino method Sensitivity

a/a+c X100

Specificity

d/b+d x100

Positive predictive value

a/a+b x100

Negative predictive value

d/c+d x100

Accuracy

a+d/a+b+c+d x100

a = True Positive b= False Positive c= False Negative d= True Negative Statistical Analysis was done and student T- Test was applied to calculate p value to evaluate the errors in diagnosis by squash cytology technique

Results

A total of 369 cases referred from Neurosurgery department for squash cytology and subsequent histopathology were included in the study. Demographic Details: The age of the patients varied from 4 to 80 years. Maximum number of cases (22.8%) were seen in the age group of 41-50 years comprising 22.8%.

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Sanjeev et al. Only one case was seen after the age of 70 years. Male to female ratio of all patients irrespective of age group was 1.8:1. Clinical Characteristics: Clinically, the patients had variable presentations. Most of the patients presented with seizures, headache, nausea, vomiting, vertigo, tinnitus and difficulty in hearing. A few patients also presented with weakness on one side of the body, ataxic gait and drowsiness. Clinico-Radiological Diagnosis: Out of 369 cases, a definitive diagnosis could not be offered radiologically for 25 cases(6.7%). A provisional clinicoradiological diagnosis could be given in remaining 344 cases. Of these, 303 cases (82.11%) were neoplastic and 41 cases (11.1%) were non neoplastic or inflammatory. Of 303 neoplasms, 151 cases (40.9%) were malignant and 152 cases (14.1%) were benign. Radio imaging techniques showed maximum number of lesions in the cerebral hemisphere (41.5%). Amongst the lesions with more precise location the frontal lobe had the largest number of cases (15.3%) followed by supratentorial location (14.2%).

SQUASH CYTOLOGY

The cytological features of 369 lesions sent for squash cytology were studied (Table 2). On squash cytology 312 cases (84.55%) were designated as neoplastic and 46 cases (12.4%) as non neoplastic. Eleven cases were inconclusive on squash cytology. Of all the 312 neoplastic lesions, 178 cases (48.2%) were malignant and 134 cases (36.3%) were benign. Overall, gliomas was the most frequent cytological diagnosis (97 cases, 26.28%), followed by meningiomas (62 cases, 16.82%). Amongst the non neoplastic benign lesions Epidermoid cyst and Tuberculoma were the most frequent lesions constituting 12 cases each (3.25%).

HISTOPATHOLOGY:

Out of 369 cases histopathological diagnosis was rendered in 368 cases. Only one case could not be diagnosed histologically due to inadequate material. Of all the 368 cases, 318 cases (86.4%) were neoplastic and 50 cases (13.6%) were non neoplastic. All the CNS neoplasms were classified according to WHO classification of CNS tumors (Table 3).

A-63 second largest group of neoplasms in our study. Besides, Schwannomas (32 cases, 8.7%), pituitary Adenoma (27 case, 7.3 %), Glioblastoma Multiforme (23 cases, 6.23%) and metastatic lesion (23 cases, 6.23 %) were the other most frequently reported lesions in our study. Amongst the various non neoplastic lesions, Tuberculomas were seen most frequently (12 cases, 3.25%) followed by Epidermoid cysts (11 cases, 2.9 %). Cytological diagnosis was correlated with histopathological diagnosis (Table 4). Overall diagnostic accuracy of squash cytology was 95.25% excluding 11 inconclusive cases. Diagnostic accuracy for neoplastic lesion was higher (87.82%) in contrast to benign non neoplastic lesion (82.6%). Total discrepancy between cytological and histological diagnosis was seen in thirty three cases (Table 5). Complete correlation was considered when the intraoperative squash cytology diagnosis was same as the histological diagnosis without grading deviation. The results were statistically analyzed. Sensitivity, specificity, positive predictive value and negative predictive value of squash smear cytology were 94.3%, 95.6%, 95.3%, 95.1% respectively (Table 6). Further false negative rate and false positive rate of squash cytology when calculated were 5.2% and 4.32%. Student T-test for correlating cytology and histological findings was applied and T value (0.35113) and P value (0.363347) were calculated. Thus, a significant correlation was found between intraoperative squash cytology diagnosis and histopathology (P > 0.05) and errors in diagnosis by squash cytology technique were found to be statistically insignificant on applying student T-test.

Discussion

Presently, the stereotactic biopsies are employed as a primary diagnostic tool in the evaluation of both neoplastic and more recently non neoplastic intracranial lesions and to differentiate metastatic from primary lesions. [8,9] The present study included a total of 369 CNS lesions comprising both neoplastic and non neoplastic lesions. All the CNS lesions were subjected to detailed squash cytology and the final results were then compared with histopathological diagnosis as gold standard.

The most frequently identified lesions on histopathology were Astrocytic tumors (91 cases, 24.7%). Amongst all the Astrocytic tumors maximum number of cases of Astrocytoma (64 cases, 17.34%) were reported. Meningiomas constituted (66 cases, 17.8%) and were the

The submitted small biopsies for squash cytology were analyzed employing H&E, PAP and May Grunewald Giemsa. The best cytological details were seen with MGG and the best nuclear details including nuclear membrane outline and chromatin pattern were seen with PAP stain. Mouriquand et al, Kontozogtu et al and Fri Piaton in their studies have found a combination of MGG and PAP stain

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to reveal complementary information and better images as compared to H&E stain. [10,11,12]. Besides, the use of H&E and Giemsa stains in contrast to the supravital stains have facilitated a permanent preparation and these smears in concert with paraffin sections have been employed for accurate interpretation. Of 369 lesions, subjected to squash cytology a cytological diagnosis could not be rendered in 11 cases. Common causes for no opinion on cytology were fibrosis, inflammation, calcification, necrosis and lack of definite cytological details. Also the lesions with increased fibrous component, tissue from cyst wall and tissue containing calcified material were difficult to smear, hence rendered inconclusive. Other authors have attributed similar causes for misinterpretation or no opinion on cytological evaluation. [13] In our study, the median age of presentation of patients was 45 years and M: F ratio was 1.8:1 which corresponds to studies by other authors such as Cappabianca et al and Nguyen et al who have reported the median age of about 40-45years. [3, 14] A higher incidence of lesions in males has been reported cross sectionally in all the age groups. [10, 11] Of 358 cases, for which a final cytological diagnosis could be offered 51 cases were non neoplastic and 307 cases neoplastic. Of 307 neoplastic lesions 164 cases (53.4%) were malignant and 177 cases (57.6%) cases were benign. Eight false positive cases and 9 false negative cases on squash cytology in malignant cases were seen in our study which corresponds to studies by other authors. [15] Diagnostic accuracy achieved in present study was 95.25% and was comparable with series of other studies varying from 85.4% to 97%. [16, 17, 18] Also the sensitivity and specificity of squash cytology in our study 94.79% and 95.67% respectively are consistent with studies by other authors. [15]

Such grading deviations have been reported by Marshall et al. [20] Two cases of reactive gliosis were rendered false positive on cytology as low grade astrocytoma in our study. Similar misinterpretation of diagnosing reactive gliosis as low grade astrocytoma has been reported by others authors due to similar cytological features. [21] On review of the smears, reactive astrocytes had abundant cytoplasm and more prominent numerous, long and symmetrical processes. As compared to neoplastic astrocytes these lack hyperchromatic and lobulated nuclei, progressive atypia and mitosis. Smears of low grade Astrocytoma had minimal anisocytosis with finely to coarsely granular chromatin and inconspicuous nucleoli; the cytoplasm was scanty and showed variable processes with fibrillary background. Two cases of Ependymomas were misdiagnosed as high grade Astrocytoma due to absence of true rosettes on squash. Similar opinion has Table 2: Intraoperative Squash cytology Diagnosis

Broad Squash Benign cytology n=134 categorization

Neoplastic n=312

The cyto-histological concordance rates were 87.82% for neoplastic lesions and 82.6% for non neoplastic lesion in our study. In a retrospective study conducted by Jaiswal et al in 2010, that included 326 cases of CNS lesions, concordance of 83.7% was achieved between the intraoperative diagnosis and final diagnosis. [19] The largest number of cases on cytology was of glial tumors (97 cases, 27.1 %) which could be easily identified in the smears due to fibrillary background (Fig.1-a,b). However, a complete correlation could not be offered due to sampling error as a high grade tumor may be undergraded because of avoidance of necrotic tissue deliberately, while preparing a crush smear which is the key diagnostic features on histology.

Non neoplastic n =46

Malignant n=178

Cytological typing (N) Pituatary adenoma 26 Craniopharyngioma 05 Meningioma 62 Schwannoma 23 Neurofibroma 03 Spindle cell lesion 12 Choroid plexus Papilloma 03 Gliomas 97 GBM 07 Oligodendroglioma 08 Ependymoma 11 DCG 01 Central Neurocytoma 01 Pineoblastoma 01 Neuroblastoma 01 Metastatic 22 Vascular lesion 10 Chondroma 02 Round cells 11 Melanoma 01 Medulloblastoma 02 Hemangioblastoma 03 Epidermoid cyst 11 Dermoid cyst 01 Arachnoid cyst 02 Tuberculoma 12 Aspergillosis 03 Abscess/Reactive gliosis 02 Inflammatory 07 Hydatid cyst 01 Neurocysticercosis (NCC) Fibrous

Inconclusive 11

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Table 3: Distribution of CNS neoplasms among various WHO categories ofCNS tumours on Histopathology: Diffuse Astrocytoma

Anaplastic Astrocytoma

Glioblastoma Multiforme (GBM)

Gliosarcoma

Sub Ependymal Giant cell Astrocytoma (SEGA)

Ependymoma

Myxopapillary Ependymoma

Oligodendroglial tumor

Oligodendroglioma

Choroid plexus tumors

Choroid plexus papilloma

Choroid plexus carcinoma

Gangliocytoma

Dysplastic Cerebellar Gangliocytoma (DCG)

Central Neurocytoma

Tumors of pineal region

Pineoblastoma

Embryonal tumor

Medulloblastoma

CNS neuroblastoma

Ewings/PNET

Schwannoma

Neurofibroma

Tumors of meningothelial cells Meningioma

Astrocytic tumors

Ependymal tumors

Neuronal tumor

Neuroepithelial

Tumors of cranial and paraspinal nerves

Tumors of the Meninges

Mesenchymal tumors Chondroma

Chondrocarcoma

Primary Melanocytic lesions Malignant melanoma

Mesenchymal tumor Hemangioma

Lymphomas & Hematopoietic Neoplasm

Tumors of sellar region

Metastatic tumours& local extension of regional tumor

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Other neoplasm related to meninges

Hemangioblastoma

Hemangiopericytoma

Malignant lymphoma

Plasmacytoma

Plasma cell granuloma

Craniopharyngioma

Pituatry adenoma

Metastasis

Squamous Cell Carcinoma (SCC)

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Correlating Squash Cytology with Histopathology

Table 4: Correlation of Histopathological Versus cytological Diagnosis Histopathological Diagnosis Total number(Histopath) Cytological Diagnosis(N) Neoplastic Astrocytoma (Grade I,II,III) GBM Oligodendroglioma Ependymoma Choroid plexus papilloma DCG Central Neurocytoma Pineoblastoma Neuroblastoma Medulloblastoma Round cell tumor Schwannoma Neurofibroma Meningioma Chondroma Primary CNS Melanoma Hemangioma Hemangioblastoma Craniopharyngioma Pitutary Adenoma Metastatic BENIGN LESION Epidermoid cyst Dermoid cyst Arachnoid cyst Hydatid cyst Tuberculoma Aspergillosis NCC Abscess Inflammatory Fibrous Inconclusive Table 5-Discordant cases on cytology: Squash cytology Diagnosis Gliomas/Astrocytoma

Accuracy (%)

85.56%

06 06 06 02 01 01 01 01 02 09 31 04 58 01 01 08 03 05 26 21

07 08 11 03 01 01 01 01 02 11 33 05 62 02 01 10 03 05 26 22

85.7% 75 54.54 66.66 100 100 100 100 100 81.8 93.93 80 93.54 50 100 80 100 100 100 95.95

11 01 02 01 12 03 03 01 04 00 01

11 01 02 01 12 03 03 02 07 04 11

100 100 100 100 100 100 100 50 57 -

Number (N) 5

Histopathology Diagnosis Reactive gliosis GBM Ependymoma

Number (N) 2 2 1

GBM Oligodendroglioma

1 2

Ependymoma

Meningioma

Gliosarcoma Astrocytoma Pitutary Adenoma Astrocytoma PilocyticAstrocytoma Ganglioma Ependymoma Gliosarcoma Hemangiopericytoma Squamous Cell Carcinoma

1 1 1 3 1 1 1 1 1 1

Neurofibroma Choroid Plexus Papilloma Metastatic deposit

1 1 1

Fibrous Meningioma Choroid plexus carcinoma Meningioma

1 1 1

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Squash cytology Diagnosis Inflammatory

Number (N) 3

Reactive gliosis

Round cell malignancy Hemangioma Fibrous

2 2 4

Chondroma Total

1 33

Histopathology Diagnosis Metastatic deposit Adenocarcinoma Astrocytoma Oligodendroglioma Meningioma Medulloblastoma Hemangioblastoma Choroid plexus papilloma Meningioma Tuberculoma Chondrosarcoma

Number (N) 1 1 1 1 2 2 1 2 1 1 33

Table 6: Sensitivity, specificity, Positive predictive value and Negative predictive value of squash smear Squash

Cytological Diagnosis

Final Diagnosis Benign Malignant

Sensitivity

Specificity

Malignant

172

164

94.79%

95.67%

Benign

186

177

Accuracy

PPV NPV

95.25%

95.34%

95.16%

been expressed by Rossler et al who reported the absence of rosettes or rosettoid appearance on cytology leading to erroneous diagnosis on cytology. [4] A diagnostic accuracy of 85.7% was achieved for Glioblastoma Multiforme (GBM) (Fig.1-c,d). A case of gliosarcoma was misdiagnosed as GBM, as the sarcomatous element was missed on cytology. Similar errors have been encountered by Mouriquad et al in their study. [10] Diagnostic accuracy for Oligodendroglioma was 75% in our study (Fig.1e,f). One case of Astrocytoma and another of pituitary adenoma were erroneously diagnosed as oligodendroglioma on squash cytology. The erroneous misdiagnosisdiagnosis on cytology may be due to interpretation of edema as perinuclear halo and neovascularisation as chicken wire pattern that are pathognomic of Oligodendroglioma. Misdiagnosing pituitary adenoma as Oligodendroglioma leading to diagnostic dilemma has been pointed out by Nguyen et al and Bonner.[14,21] The problem arises due to overlapping cytological features, absence of clear halo around nuclei in oligodendroglial cells and pituitary tumor extending beyond the confines of sella turcica. For Ependymomas, the diagnostic accuracy was 54.54%. Smears showing close mixture of cuboidal epithelial cells merging with spindle fibrillated cells clinch the diagnosis in favour of Ependymona. Two cases of astrocytoma were erroneously diagnosed as Ependymomas on squash cytology. Similar opinion was given by Ho-keung Ng who

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said that low grade Astrocytomas and mixed gliomas are important differential diagnosis in case of Ependymomas. [22] One case of Pilocytic Astrocytoma was misdiagnosed as Ependymoma on Squash cytology and stays one of the major differential diagnoses. The principal challenge is to distinguish between these tumors since they belong to different grades. On retrospective analysis of crush smear it was found that diffuse fibrillary Astrocytomas were more cellular with a fibrillary back ground as compared to Pilocytic Astrocytoma which showed fine fibrillarity and absence of well defined bipolar cells (Fig.1 g,h). Teo et al have also described similar findings in their study.[23] One case of Ganglioma was misdiagnosed as Ependymoma due to decreased number of Ganglion cells with poorly preserved outline. The cytohistological correlation for Meningiomas was 93.54% (Fig.-2a,b). In most cases meningothelial cells were easily identifiable as oval to round cells containing vesicular nuclei and conspicuous nucleoli. We misdiagnosed a case of ependymoma as meningioma due to absence of perivascular Rosette. Another case of gliosarcoma was also diagnosed as meningioma due to absence of necrosis and sarcomatous mesenchymal elements in the cytological smears simulating spindle cells leading to diagnostic error. Furthermore, Hemangiopericytoma was misdiagnosed as Meningioma due to presence of spindle cells. A case of moderately differentiated Squamous Cell carcinoma was also misdiagnosed as Meningioma due to smearing difficulty and presence of scattered spindle cells only on crush smear. Two cases of Meningioma were diagnosed as fibrous lesions eISSN: 2349-6983; pISSN: 2394-6466

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on crush cytology due to the presence of elongated spindle cells and absence of cellular whorls in the smear.

elaborated the close resemblance of Medulloblastoma with Round cell malignancy. [25]

The cytohistological correlation for Schwannoma was 93.93% (Fig.-2c,d). One case of fibrous meningioma was diagnosed as Schwannoma. Kobayashi also described similar misinterpretation in their result. [24]

For metastatic lesions, the cytohistological correlation was 95.45% in our study (Fig-3a,b). One case of Meningioma was misinterpretated as metastatic deposit where the atypia was over diagnosed cytologically. In literature misdiagnosis has been reported in cases of metastatic carcinoma, undifferentiated malignant tumor and rare types of histology such as gliomatous and xanthomatous lesions that may be responsible for misdiagnosis. [26, 27]

A 100% correlation was seen in the diagnosis of pituitary adenoma(Fig.-2e,f). The cells appeared uniform with well defined cell border and round to oval nuclei. Pseudorosette like arrangement and vascular proliferation was seen occasionally. The cytohistological correlation for Neurofibroma was 80%. One case of fibrous meningioma was misdiagnosed as Neurofibroma since the tumor cells showed elongated nuclei, long cytoplasmic processes and absence of whorled pattern. Similar opinion has been expressed by Kobayashi. [24] Cytologically diagnosed two cases of Round cell malignancy were histopathologically diagnosed as Medulloblastoma thus accounting a diagnostic accuracy of 81.8%. On reviewing cytology, the smears were highly cellular with small round dyscohesive cells, high N/C ratio, nuclear hyperchromasia and structures appearing as lympho glandular bodies. Folkerth et al in their study have

Of two cytologically proven cases of Chondroma, one of the case was histologically diagnosed as Chondrosarcoma and the discordance was due to sampling error. One case of choroid plexus carcinoma was misinterpretedas choroid plexus papilloma on crush cytology thus giving a cytohistopathological correlation of 66.66%. The reason for erroneous diagnosis may be attributed to inadequate sample. Likewise, a diagnosis of fibrous lesion was given cytologically which on histopathology was diagnosed as Choroid plexus papilloma and may be attributed to improper smear preperation on review.

Fig. 1a: Squash cytology of high grade Astrocytoma showing vascular proliferation and astrocytes with nuclear atypia in a fibrillary background (H&E, 400X) with corresponding tissue section in showing nuclear atypia and pleomorphism in 1b(H&E, 400X) . Fig. 1c: Squash cytology of GBM showing glomeruloid appearance of blood vessels (MGG, 100X) with corresponding tissue section showing palisading of neoplastic astrocytes around necrosis in 1d (H&E ,100X). Fig. 1e: Squash smears showing sheets of neoplastic oligodendrocytes (MGG,100X) with corresponding tissue section showing sheets of uniform round cells with perinuclear halo exhibiting (Inset: calcification) in 1f (H&E, 100X). Fig. 1f: Squash preparation showing bipolar hair like processes of astrocytes in a fibrillary background (MGG,400X) with corresponding tissue section showing pilocytic cells,Rosenthal fibres and eosinophillic granular bodies in 1g (H&E,400X)

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Fig. 2a: Squash cytology of Meningioma showing psammoma bodies (H&E, 100X) with corresponding histological section showing psammoma bodies in 2b (H&E, 100X). Fig. 2c: Squash showing thick fragment of spindle cells (MGG 100X) with tissue section exhibiting Verocay bodies 2d (H&E, 400X) Fig. 2e: Squash cytology of Pitutary Adenoma showing monomorphic appearance of cells (H&E, 400X) with corresponding histological section showing uniform arranged in acini and sheets in 2f (H&E,100X)

A complete 100% cytohistological correlation was seen in cases of Dysplastic cerebellar gangliocytoma, Pineoblastoma, Neuroblastoma, Medulloblastoma (Fig.-3c,d), Primary CNS Melanoma (Fig-3e,f) and Hemangioblastoma (Fig.-3g,h).

Benign Lesion

In our study, 12 case of tuberculoma were reported on cytology which showed a 100% correlation on histology. The smears showed mainly caseous necrotic background, fibrous tissue, few clusters of epitheloid cells, Langhanâ&#x20AC;&#x2122;s multinucleate giant cell and amorphous calcified debris. ZN stain should be mandatory to demonstrate acid fast bacilli (AFB), however in absence of AFB the diagnosis of consistent with tuberculoma can be given. These findings are consistent with Burger et al.[28] Two cases diagnosed cytologically as Fibrous lesion were histologically diagnosed as Choroid Plexus papilloma and Tuberculoma respectively. The discordance was due to improper smear preparation revealing only spindle cells on crush cytology. www.pacificejournals.com/apalm

Eleven cases of epidermoid cyst and one case of dermoid cyst diagnosed cytologically showed a 100% correlation with histology. Epidermoid cyst showed a large number of keratinized mature squamous cells with keratin having lamellated appearance. The Dermoid cysts showed mature squamous epithelium with fibrous tissue and fragments of adipose tissue against a background of amorphous debris, macrophages and having similar morphological findings have been described by Smith et al who further opined histopathology to be done to differentiate between Epidermoid cyst and cystic teratomas. [29] Three cases diagnosed as inflammatory lesions on squash cytology were histologically proved as metastatic deposit of Adenocarcinoma, Astrocytoma and Oligodendroglioma respectively.These false negative diagnosis on squash cytology may be attributed to improper sampling. There were 11 cases (2.98 %) in our study for which a cytological definite opinion could not be given. These eISSN: 2349-6983; pISSN: 2394-6466

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Correlating Squash Cytology with Histopathology

Fig. 3a: Squash cytology of metastatic tumour showing pleomorphic cells in clusters (MGG,100X) with corresponding tissue section showing tumour cells infiltrating into brain tissue in 3b (H&E,400X) Fig. 3c: Smear showing uniform round cell in sheets and clusters (MGG, 400X) with corresponding tissue section exhibiting rosettes in Medulloblastoma in 3d (H&E, 400X) Fig. 3e: Squash smear of Malignant Melanoma showing elongated cells in clusters along with pigment (H&E, 400X) with coressponding histological sections showing tumour cells ( Inset: tumour cells and pigment) in 3f (H&E, 100X) Fig. 3g: Smear showing cellular Hemangioblastoma with small capillaries (MGG 100X) with corresponding tissue section showing many capillaries and cellular stroma in 3h (H&E, 100X)

include 1 case each of pituitary adenoma, dermoid cyst, bronchogenic cyst, vascular lesion, inflammatory lesion and plasma cell granuloma. In these cases the opinion was inconclusive due to low cellularity, crush artifacts, increased fibrous component from the wall, predominantly fluid aspirate with low cellularity and morphology obscuring inflammation.

be offered on cytology due to lack of clear cut cytological criteria in the available literature.

There were 4 cases of seizure related lesions for which surgery was done. These include 2 cases (0.54%) of microdysgenesis and 2 cases (0.54%) of medial sclerosis.

Conclusion

The smears of microdysgenesis showed brain tissue with fibrillary background along with mature ganglion cells and few oligodendroglial like cells that were confirmed on histology and correlated with clinical findings. Similarly, there were 2 cases of medial sclerosis in our study which were characterized by large neuronal cells and astrocytic cells along with intermediate cells with ballooned glassy cytoplasm. Both the cases were subsequently confirmed by histopathology. In all the 4 cases, definite opinion could not

The correlate percentage of cytohistological correlation reported by other authors are, 92.2% by Tores et al, 87.5% by Mouriquand et al, 88% by Willems et al and are comparable with our cytohisto correlation.[5,10,30] To conclude, this study shows a very high degree of cytohistological correlation. With better and precise radioimaging and stereotactic biopsies, the percentage of cytohistological correlation can improve and increase further. Some cases will always require histopathological study and/ or immunohistochemical markers for definitive diagnosis but for most of the lesions, cytology can render a definite opinion.

Acknowledgement:

We would like to acknowledge Dr. Pankaj Arora, Consultant Neurosurgery,SGRRIM&HS for his kind cooperation.

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Sanjeev et al.

Funding None

Competing Interests None declared

References

1. Silverberg SG, De Lellis RA, Frable WJ, Li Volsi VA, Wick MR. Silverberg’s principles and practice of surgical pathology and cytopathology. 4th ed.Philadelphia: Churchill Livingstone; 2006. 2. Kondziolka D, Lunsford LD, Martinez AJ. Unreliability of contemporary neurodiagnostic imaging in evaluating suspected adult supratentorial (low grade) astrocytoma. JNS 1993;79(4):533-36. 3. Cappabinaca P, Spaziante R, Caputi F et al. Accuracy of the analysis of multiple small fragments of glial tumours obtained by stereotactic biopsy. Acta Cytol 1991,35:505-11. 4. Roessler K, Dietrich W, Kitz K. High Diagnostic Accuracy of Cytologic smears of Central Nervous Tumors. A 15- year experience based on 4172 patients. Acta Cytol 2002; 46:664-7. 5. Torres LF, Noronha LD, Gugelmin ES et al. Accuracy of smear technique in the cytological diagnosis of 650 lesions of the central nervous system. Diagn Cytopathology 2001;24:293-95. 6. Eisenhardt L, Cushing H. Diagnosis of intracranial tumor by supravital technique. Am J Pathol 1930;6(5):641-52. 7. WHO classification of tumours of CNS edited by David N Louis et al, 4th edition 2007. 8. Klienhues P, Vol B, Anagnostopoulos J, Kiessnlg M. Morphological evaluation of Stereotactic brain tumour biopsies. Neuro chirurgica Suppl. 1984;33:171-81. 9. Feiden W, Bise K, Steude U, Pfister HN, Molter AA. The sterotactic brain biopsy of focal intracerebral lesion in AIDS patient . Acta Neurol Scand 1993;87:228-33. 10. Mouriquand C, Benabid AL, Breyton M. Stereotactic cytology of brain tumours: review of an eight year experience. Acta Cytol 1991,31:756-64. 11. Kontozoglu TE, Cramer HM. The advantage of intraoperative cytology: Analysis of 215 smears and review of the literature. Acta Cytol 1991;35:154-64. 12. Eric Piaton. Cytology of tumour of Central Nervous System: letters to the editors. Acta Cytol 1996;40:846-48.

A-71 Intraoperative Squash Smear Technique in the Rapid diagnosis of CNS lesions. Bombay Hospital Journal 2010;52(2):153-60. 14. Nguyen Gk, Johnson ES, Mielke BW. Cytology of Neuroectodermal tumours of the brain in crush preparation: A review of 56 cases of deep seated tumours sampled by CT-guided sterotactic needle biopsy. Acta Cytol 1989;33:67-75. 15. Sharma N, Misra V, Singh PA, Gupta SK, Debnath S, Nautiyal A. Comparative Efficacy of Imprint and squash Cytology in Diagnosing Lesions of the Central Nervous System. Asian Pacific J Cancer Prev 2011;12:1693-96. 16. Pawar N, Deshpande K, Sarase S, D’ costa G, Balgi S, Goel A. Evaluation of the squash smear Technique in the Rapid Diagnosis of Central Nervous Tumours: A cytomorphological Study. The Internet Journal of Pathology 2009;11:1. 17. Sidway MK, Jannilla FS. Intraoperative cytological diagnosis of lesions of central nervous system. AJCP 1997;108:56-66. 18. Iqbal M, Shah A, Wani MA, Kirmani A, Ramzan A. Utility of Crush Smear cytology in Intraoperative Diagnosis of Central Nervous System Lesions. Acta Cytol 2006;57:608-16. 19. Jaiswal S, Jaiswal AK, Behari S et al. Intraoperative squash cytology of central nervous system lesions: A single center study of 326 cases. Diag Cytopathol 2010;40(2):104-12. 20. Marshall LF, Adams H, Doyle D, Graham DI. The histological accuracy of the smear technique for neurosurgical biopsies. J Neurosurg 1973;39:82-8. 21. Bonner JM. Central Nervous System. In: intraoperative cytology. An adjunct to frozen section. Edited by JA Welkerson, JM Bonner, New York; IgakiShoin;1989:p.119 22. Ho-Keing Ng. Cytological feature of Ependymomas in smear preperations. Acta Cytol 1994;38:331-34. 23. Teo J, Ho- Keung Ng. Cytodiagnosis of pilocytic astrocytoma in smear preperations. Acta Cytol 1998;42:673-78. 24. Kobayashi S. Meningiomas, neurilemmoma and astrocytoma specimens obtained with the squash method for cytodiagnosis. A cytological and immunohistochemical study. Acta Cytol 1993;37:913-22.

13. Deshpande K, Sarase S, Shedge R, D’ costa G, Bharambe B. Accuracy and Diagnostic Yield of

25. Folkerth RD. Smears and Frozen section in intraoperative diagnosis of CNS lesions. Neuro Surg Clin N Am 1994;5(1):1-18.

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26. Goeld D, Sundaram C, Paul TR et al. Intraoperative cytology (squash smear) in neurosurgical practicepitfalls in diagnosis experience based on 3057 samples from single institution. Cytopathology Official Journal of the British Society for Clinical Cytology.2007;18(5):300-8. 27. Ud Din N, Memon A, Idress R, Ahmad Z, Hasan S. Central nervous system lesions: correlation of intraoperative and final diagnosis, six year experience at a referral centre in developing country,Pakistan.

Asian Pacific Journal of Cancer Preservation: APJCP 2011;12(6):1435-37. 28. Burger PC, Scheithaein BW, Vogel FS. Ed. Surgical Pathology of the Nervous System and its coverings. 4th ed, London:Churchill Livingstone;2002:p.122-26. 29. Smith AR, Eisheik TM, Silverman JF. Intraoperative cytologic diagnosis of supra sellar and sellar cystic lesion. Diagn Cytopathol 1999;20:137-47. 30. Willems JG, Alva-Willems JM. Accuracy of cytologic diagnosis of central nervous system neoplasms in stereotactic biopsies. Acta Cytol 1984;28:243-9.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 02, April - June 2016

Original Article Placental Pathology in Spontaneous Abortions: A Study with Review of Literature Kriti Chauhan, Manish Chaudhry, Monika Garg*, Ridhima Auplish, Karuna Sangwan, Nanak Mahajan Department of Pathology, MM Institute of Medical Sciences & Research, MM University Mullana, Haryana, India

Keywords: Abortion, Retained, Perfusion Defects, Pregnancy, Trophoblasts, Infertility

ABSTRACT Introduction: â&#x20AC;&#x2DC;Retained products of conceptionâ&#x20AC;&#x2122; are quite a frequent specimen received for histopathological examination. Apart from confirmation of pregnancy, a careful examination can provide some additional information about the cause or the conditions associated with abortion. Methods: A total of 53 cases were studied over a period of 1 year (from June 2014 to May 2014). The specimens were received in formalin, kept for fixation and stained with Haematoxylin and Eosin. Results: Thirty out of fifty-three cases showed significant positive findings. Maternal perfusion defects were the most common (80%); of which, ischemia related defects were the most frequent ones (87.5%). The other findings included maternal floor infarction, chronic histiocyticintervillositis and persistent muscularization of basal plate arteries. Fetal perfusion defects constituted of three cases (10%) in which avascular villi were seen in two cases and chorangiosis was seen in one case. One case each of partial mole, complete mole and gestational trophoblastic neoplasm was also found in the study. Conclusion: The present study highlights few of the histopathological findings observed in such specimens, which might help in predicting future pregnancy outcome and in planning the family accordingly.

*Corresponding author: Dr Monika Garg, Professor, Department of Pathology, MM Institute of Medical Sciences & Research, MM University Mullana, Haryana, India 133203 Phone: +91 - 9417378514 Email: monikakash7@yahoo.co.in

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

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Placental Pathology in Spontaneous Abortions

Introduction

Spontaneous abortions are quite prevalent. The initial pathologic assessment is used to confirm the presence or absence of pregnancy tissue and identification of a molar pregnancy. However the pathologists have strived to add more than a diagnosis of products of conception. A careful histopathological examination offers an understanding of aetiology and pathogenesis of abortions, thereby helping in risk assessment for the neonate and in reproductive planning in the family.[1] In this study we focus on determining the various abnormalities of maternal perfusion, fetal perfusion and chorionic villi in routine samples of spontaneous abortions which might help in determining the aetiology of abortion and in planning further pregnancy.

Materials and Methods

The study was carried out on all the specimens received as â&#x20AC;&#x2DC;Retained products of conceptionâ&#x20AC;&#x2122; in our department over a period of one year (June 2014 to May 2015). A total of 53 cases were received, out of which 30 cases showed significant pathological findings. The pathological findings were divided as maternal perfusion defects (MPDs), foetal perfusion defects (FPDs) and others. The remaining 23 cases were either unsatisfactory or showed no specific finding and were excluded from the study. All specimens were fixed in formalin and stained with haematoxylin and eosin stain. The slides were then examined under the microscope.

Results

Out of the 30 cases studied, the majority were first trimester abortions (19). 8 cases were second trimester abortions and only 3 cases were third trimester abortions. 24 cases showed MPDs. Only one case showed FPD and gestational trophoblastic tumor each. Out of the MPDs, ischemia related changes were found to be the most frequent ones (21 cases) of which acute ischemia comprised of 13 and chronic ischemia comprised of 9 cases. One case showed both acute and chronic ischemic changes. The remaining 3 cases were of maternal floor infarction (MFI), chronic histiocyticintervillositis (MCI) and gestational trophoblastic tumor each. In the category of FTDs, two cases were encountered which showed avascular villi and one case showed chorangiosis. One case each of partial and complete mole was also noted.

Discussion

Placenta is an organ with circulation from the mother and foetus. Hence a histopathological examination must be carried out for aetiologies from both.[2] The maternal side disorders that affect the conceptus are numerous and have important foetal sequelae or recurrence risk. Few of

them are infarcts, ischemia, chorangiosis, maternal floor infarction (MFI) and massive chorionic intervillositis. Infarcts: They occur due to ischemic damage to the placenta seen histologically as collapsed villi with empty intervillous space and undergoing necrosis. In addition to infarcts which are really absent maternal perfusion, there are findings which correspond to chronic ischemic damage to placenta. These include small villi, large syncytial knots and absence of midsized villi.[3]Acute infarcts show villous crowding, increased perivillous fibrin deposition and ghost villi. They are found much more commonly in patients with hypertension or preeclampsia.[4]In our study acute infarcts were seen in 13 cases (50%) and chronic ischemia was seen in 7 cases (26.9%). One case (3.84%) showed both acute and chronic ischemic changes. Figure 1a and 1b show changes in chronic ischemia and acute ischemia (infarct) respectively.

Fig. 1a: Low power view showing hypermature small sized villi with increased syncytial knots. (H&E 100X). Figure 1b: Low power view showing infarct necrosis with ghost villi and increased perivillous fibrin. (H &E 100X).

Maternal Floor Infarction (MFI): It is characterised by markedly increased perivillous fibrin and extracellular matrix fibrinoid surrounding distal villi with large number of associated intermediate trophoblast cells. The entrapped villi may become infarcted due to diminished perfusion. [4] .Fibrinoid material is derived from haemorrhages. The haemorrhage seeps through adjacent decidua and forms a membrane like sheet around chorionic villus. In some cases it may be due to maternal thrombophilia leading to increased fibrin deposition at the villous maternalfetalinterface. In addition, compromised villous trophoblast integrity exposes tissue factor and activates the clotting cascade locally on the villous surface. Grossly, the placenta is firm and marbled.It has been found to be associated with autoimmune diseases, preeclampsia, thrombophilias, increased risk of renal tubular dysgenesis,[5] cystic renal dysplasia[6] and anti fetal rejection.[7]It is also associated with IUGR, prematurity, diabetes, Rh-incompatibility recurrent abortion, preterm delivery, fetal death, adverse

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Fig. 2: Low power view showing normal villi (right side) and increased perivillous fibrin deposition around distal villi with trophoblasts (left side). (H&E 100X).

neurodevelopmental outcome, CNS injury and elevated levels of maternal serum alpha fetoprotein.[8-10] We found one case of MFI histologically showing increased perivillous fibrin around distal villi with areas of infarct.

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Fig. 3a: Low power view showing a focal collection of lymphocytes and histiocytes in the intervillous area. (H&E 100X). Figure 3b: High power view showing histiocytes, lymphocytes with admixed trophoblastic cells. (H&E 400X).

Marchaudon et al[13] observed a very substantial increase in alkaline phosphatase levels in MCI associated with an immune phenomenon. Mekinian et al have suggested an autoimmune association such as antiphospholipid syndrome.[14]

Another feature of maternal vascular perfusion defect is persistent â&#x20AC;&#x2DC;Muscularization of Basal Plate Arteriesâ&#x20AC;&#x2122; also called as failure of physiological transformation which means failure of extra-villous trophoblast invasion of the maternal spiral arterioles. This results in persistent vaso-reactivity to circulating vasoactive mediators decreasing the blood flow into intervillous bed. The end result is chronic ischemia and villous infarcts.[2] The term physiological transformation is used to describe the disappearance of normal muscular and elastic structures of arteries and their replacement by fibrinoid material in which trophoblasts are embedded. Preeclampsia and fetal growth retardation have been found to be strongly associated with failure of spiral artery remodelling[15] because it results in the failure of development of a low resistance arteriolar system which dramatically decreases the blood supply to the growing fetus. In preeclampsia, invasion of the uterine spiral arteries is limited to proximal decidua with 30% to 50% of the spiral arteries escaping endovascular trophoblast remodelling.[16] Maternal and fetal complications associated with preeclampsia include abruption, renal failure, IUGR, preterm delivery and death. Kam et al[17] have stressed upon the role of trophoblast in the physiological change in decidual spiral arteries. According to them, the likely sequence of events leading to remodelling is that first the interstitial trophoblasts home to the spiral arteries and destroy the vessel media as a priming process which is subsequently followed by migration of endovascular trophoblasts down the arterial lumen causing destruction of endothelial cells accompanied by deposition of fibrinoidnaterial. Figure 4a shows physiologically transformed decidual arteries showing endovascular and interstitial trophoblastic infiltration in vessel wall. Figure 4b shows thickened untransformed spiral arterioles.

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Chronic (Histiocytic) Intervillositis also called as Massive Chronic Intervillositis (MCI): is a striking finding in which the maternal space is filled by CD68 positive histiocytes with admixed or nodular increase in fibrin.[2]The absence of villous inflammation and large number of histiocytes in chronic intervillositis distinguishes this lesion from chronic lymphohistiocyticvillitis with perivillous extension and fibrin deposition. The intensity of intervilloushistiocytic burden increases with gestational age. It is known to be caused by maternal T lymphocytes, predominantly CD-8 positive, that inappropriately gain access to the villous stroma. Fetal antigen presenting cells (Hofbaeur cells) expand and are induced to express class II major histocompatibility complex molecules. Maternal monocyte â&#x20AC;&#x201C;macrophages in the perivillous space likely amplify the immune response .This lesion is more commonly seen in spontaneous abortions especially in first trimester and is rare in later pregnancy. It has reportedly the highest recurrence risk and causes fetal death.[11, 12]It is a strong risk factor for neonatal encephalopathy and cerebral palsy. MCI can also be observed in malaria infection. However, the histiocytes contain material pigment and show no villous damage. [11]Parant et al[12] has classified MCI into two histological levels of severity, viz moderate and severe. We report one case of MCI which falls in moderate category showing focal intervillouslymphohistiocytic inflammatory infiltrate with mild fibrinoid deposition (Figure 3a and 3b).

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Placental Pathology in Spontaneous Abortions or is a result of impairment of placental circulation due to different causes. Ceratinforms of fibrosis may develop via stromal edema (e.g. diabetes mellitus, blood group incompatibility, immunological disorders).

Fig. 4a: Low power view showing endovascular trophoblastic remodeling in decidual arterioles. (H&E 100X). Figure 4b: Low power view showing persistent muscularization of decidual arterioles. (H&E 100X).

In our study, we found one such case showing lack of physiological transformation. The fetal perfusion defects: affecting the conceptus are 1) Fetal thrombotic vasculopathy, 2) Hemorrhagicendovasculitis, 3) Recanalization of stem vessels, 4) Chorangiosis. The end result of long standing fetal vascular occlusion is avascular villi. (Figure 5)

Hemorrhagicendovasculitis is a distinctive histopathological finding of damage to endothelium of villous vessels resulting in extravasation of fetal red blood cells and recanalized stem vessel lumens. Obstruction to fetal vascular blood flow results in IUGR, neurologic injury, cardiac failure, oligohydramnios and fetal death. [19, 20] We found two cases of intrauterine death showing avascular villi consistent with fetal transfusion defects. Chorangiosis: Chorionic villi rarely contain more than five capillaries in a term placenta. Villous hypervascularity in which individual terminal villi contain an excessive number of vessels has been called as chorangiosis by Altshuler,[21] who has defined it as the presence of more than 10 capillaries per villous in 10 medium power fields in at least 3 non infarcted areas of placenta. Chorangiosis has to be differentiated from chorangioma, chorangiomatosis, placental congestion and ischemia. Chorangioma and chorangiomatosis are commonly seen before 32 weeks of gestation and involves proximal elements of villous structures whereas chorangiosis is a diffuse process commonly seen after 37 weeks of gestation involving the terminal villi and having numerous capillaries with intact basement membrane. [22] Placental congestion shows a numerically normal vasculature and ischemic placenta shows shrinkage of villi with hyper-maturation. The pathogenesis of chorangiosis is thought to be hypoxic stimulus which causes excessive villous capillary and connective tissue proliferation.[21] In our study, we found one case of chorangiosis. (Figure6). We also found one case each of partial mole, complete hydatiform mole and gestational trophoblastic neoplasm

Fig. 5: Low power view showing avascular term villi with hyalinised stroma. (H&E 100X)

They appear as collagenized pink villi without intravillous vessels.Initially the collagen is soft which later gets condensed and hyalinised. The term is reserved for the group with 15 or more affected terminal villi per section. The finding is well described in preeclampsia and diabetic pregnancies Scattered foci of avascular villi (and/or villous stromal vascular karyorrhexis) could be used to describe less severe cases.[18]Stromal fibrosis of terminal villi is a consequence of regression after intrauterine fetal death

Fig. 6: Low power view showing hypervascular term villi. (H&E 100X)

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Table 1.The findings in all the 30 cases. No. Age (yrs)

Obstetric History

POG of present Time of previous pregnancy abortion (if any)

Histological findings

Diagnosis

G4P1A3

2 months

2 month

Infarct necrosis with ghost villi

Findings are consistent with acute ischemic damage to placenta

G3P2A1

11 weeks

Hypermature villi with increased syncytial knots and empty intervillous space

Chronic ischemic damage to placenta

G2P0A2

7 weeks

2 months

Villous crowding with increased perivillous fibrin deposits

Acute ischemic damage

G3P2A1

8 weeks

Villous crowding with increased perivillous fibrin deposits

Acute ischemic damage

G1P0A1

11 weeks

Infarcted ghost villi with increased Acute ischemic damage fibrin

G5P4A1

4 months

Infarcted ghost villi with increased Acute ischemic damage fibrin

G1P0A1

11 weeks

Hypermature villi with increased syncytial knots and empty intervillous space

Chronic ischemic damage

G6P2A4

3 weeks

2 months

Infarct with ghost villi and increased fibrin deposits

Acute ischemic damage to placenta

G1P0A1

16 weeks

Proliferation of trophoblasts with atypical features in a background of necrosis, haemorrhage, infiltrating muscle bundles. No villi seen.

Gestational trophoblastic tumor (?choriocarcinoma / PSTT)

10.

G2P1A1

3 months

Hypermature villi with sclerosis along with intervening infarcted areas

Both acute and chronic ischemic damage

11.

G2P1A1

10 weeks

Areas of increased perivillous fibrin, trophoblastic cells and infarct

Maternal floor infarction

12.

G9P7A2

15 weeks

2 months

Hypermature villi with increased syncytial knots and sclerotic villi

Chronic ischemic damage

13.

G1P0A1

24 weeks

Infarcted villi only. No viable area

Acute ischemic damage

14.

G1P0A1

8 weeks

Hypermature villi with increased syncytial knots

Chronic ischemic damage

15.

G1P0A1

10 weeks

Persistent muscular layer around spiral arterioles in decidua

Lack of physiological transformation of decidual arteries

16.

G5P4A1

4 month

Ghost villi with increased fibrin

Acute ischemic damage

17.

G3P2A1

8 weeks

Infarct with ghost villi and increased fibrin

Acute ischemic damage

18.

G5P3A2

7 weeks

10 weeks

Ghost villi with increased fibrin

Acute ischemic damage

19.

G2P1A1

11 weeks

Increased syncytial knots and sclerotic hypermature villi

Chronic ischemic damage

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Placental Pathology in Spontaneous Abortions

No. Age (yrs)

Obstetric History

POG of present Time of previous pregnancy abortion (if any)

Histological findings

Diagnosis

20.

G3P1A2

10 weeks

8 weeks

Sclerotic hypermature villi and empty intervillous spaces

Chronic ischemic damage

21.

G2P1A1

18 weeks

Sclerotic hypermature villi and empty intervillous spaces

Chronic ischemic damage

22.

G2P1A1

7 weeks

Collection of histiocytes and lymphocytes in the intervillous space

Chronic (histiocytic) intervillositis

23.

G1P0A1

9 weeks

Large areas of infarct with ghost villi

Acute ischemic damage

24.

G2P1A1

39 weeks (IUD)

Occluded vessels in stem villi with avascular villi

Hemorrhagicendovasculitis

25.

G1P0A1

6 weeks

Areas of infarct with ghost villi

Acute ischemic damage

26.

G1P0A1

20 weeks

Areas of infarct with ghost villi

Acute ischemic damage

27.

G3P1A2

11 weeks

12 weeks

Both normal and hydropic villi with trophoblastic proliferation

Partial mole

28.

G4P3A1

13 weeks

Large hydropic villi with cistern formation and circumferential trophoblastic proliferation

Complete hydatiform mole.

29.

G4P2A2

27 weeks

24weeks

Avascular villi

Consistent with fetal transfusion defects

30.

G1P0A1

34 weeks

Villi with increased number of capillaries filled with blood

Chorangiosis

(GTN) (?choriocarcinoma / ?PSTT) in our study. Microscopically, partial mole showed both normal and hydropic villi (Figure 7) whereas complete mole showed only hydropic villi with cistern formation and circumferential trophoblastic proliferation. The two established risk factors associated with it are extremes of maternal age and prior molar pregnancy. Compared to women aged 21-35 years, the risk of complete mole is 1.9 times higher for women both >35 years and <21 years as well as 7.5 times higher for women >40 years. Prior hydatidiform mole predisposes to another molar pregnancy. The risk of repeat molar pregnancy after 1 mole is about 1%, or about 10-20 times the risk for the general population. Complete hydatidiform moles undergo early and uniform hydatid enlargement of villi in the absence of an ascertainable fetus or embryo, the trophoblast is consistently hyperplastic with varying degrees of atypia, and villous capillaries are absent. Approximately 90% of complete moles are 46, XX, originating from duplication of the chromosomes of a haploid sperm after fertilization of an egg in which the maternal chromosomes are inactive. The other 10% of complete moles are 46, XY, or 46, XX, as a result of fertilization of an empty ovum by 2 sperm (dispermy). Partial hydatidiform moles show fetal tissue,

chorionic villi with focal edema, scalloping and prominent stromal trophoblastic inclusions, a functioning villous circulation, as well as focal trophoblastic hyperplasia with mild atypia only. Most partial moles have a triploid karyotype (usually 69, XXY), resulting from the fertilization of an apparently normal ovum by 2 sperm.[23]

Fig. 7: Scanner view showing both normal and hydropic villi. (Inset showing circumferential trophoblastic proliferation in a hydropic villus). (H&E 40X)

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Chauhan et al. GTN showed complete absence of chorionic and presence of atypical cytotrophoblasts syncytiotrophoblasts in a background of necrosis haemorrhage. An occasional mitotic figure was identified.

A-79 villi and and also

Conclusion

Ischemia in maternal transfusion defects has been found to be the most common disorder affecting placenta in spontaneous abortions. We stress upon careful sampling and inspection of all products of conception in cases of spontaneous abortion and report any significant pathological finding which predicts the nature of future pregnancies and fetal outcome so that a required intervention, planning, counselling may be done at the right time.

References

1. Lathi RB, Gray Hazard FK, Heerema-McKenney A, Taylor J, Chueh JT. First trimester miscarriage evaluation. SeminReprod Med. 2011;29(6):463–9. 2. Roberts DJ. Placental pathology, a survival guide. Arch Pathol Lab Med. 2008;132(4):641–51. 3. Redline RW, Boyd T, Campbell V, Hyde S, Kaplan C, Khong TY, et al., Society for Pediatric Pathology, Perinatal Section, Maternal Vascular Perfusion Nosology Committee. Maternal vascular underperfusion: nosology and reproducibility of placental reaction patterns. PediatrDevPathol. 2004 Jun;7(3):237–49. 4. Baergen RN. Manual of pathology of the human placenta. 2nd Ed. New York: Springer; 2011. 544 p. 5. Linn RL, Kiley J, Minturn L, Fritsch MK, Dejulio T, Rostlund E, et al. Recurrent massive perivillous fibrin deposition in the placenta associated with fetal renal tubular dysgenesis: case report and literature review. PediatrDevPathol. 2013 Oct;16(5):378–86. 6. Taweevisit M, Thorner PS. Maternal floor infarction associated with oligohydramnios and cystic renal dysplasia: report of 2 cases. PediatrDevPathol. 2010 Apr;13(2):116–20. 7. Romero R, Whitten A, Korzeniewski SJ, Than NG, Chaemsaithong P, Miranda J, et al. Maternal floor infarction/massive perivillous fibrin deposition: a manifestation of maternal antifetal rejection? Am J ReprodImmunol. 2013 Oct;70(4):285–98.

definitions, association with intrauterine fetal growth restriction, and risk of recurrence. PediatrDevPathol. 2002 Apr;5(2):159–64. 9. Andres RL, Kuyper W, Resnik R, Piacquadio KM, Benirschke K. The association of maternal floor infarction of the placenta with adverse perinatal outcome. Am J Obstet Gynecol. 1990 Sep;163(3):935–8. 10. Adams-Chapman I, Vaucher YE, Bejar RF, Benirschke K, Baergen RN, Moore TR. Maternal floor infarction of the placenta: association with central nervous system injury and adverse neurodevelopmental outcome. J Perinatol. 2002 May; 22(3):236–41. 11. Contro E, deSouza R, Bhide A. Chronic intervillositis of the placenta: a systematic review. Placenta. 2010 Dec;31(12):1106–10. 12. Parant O, Capdet J, Kessler S, Aziza J, Berrebi A. Chronic intervillositis of unknown etiology (CIUE): relation between placental lesions and perinatal outcome. Eur J ObstetGynecolReprod Biol. 2009 Mar;143(1):9–13. 13. Marchaudon V, Devisme L, Petit S, Ansart-Franquet H, Vaast P, Subtil D. Chronic histiocyticintervillositis of unknown etiology: clinical features in a consecutive series of 69 cases. Placenta. 2011 Feb;32(2):140–5. 14. Mekinian A, Revaux A, Bucourt M, Cornelis F, Carbillon L, Fain O. Fetal death in primary SS associated with chronic intervillositis. Rheumatology (Oxford). 2012 Jun;51(6):1136–7. 15. Lyall F, Robson SC, Bulmer JN. Spiral artery remodeling and trophoblast invasion in preeclampsia and fetal growth restriction: relationship to clinical outcome. Hypertension. 2013 Dec;62(6):1046–54. 16. Granger JP, Alexander BT, Llinas MT, Bennett WA, Khalil RA. Pathophysiology of hypertension during preeclampsia linking placental ischemia with endothelial dysfunction. Hypertension. 2001 Sep; 38:718–22. 17. Kam EP, Gardner L, Loke YW, King A. The role of trophoblast in the physiological change in decidual spiral arteries. Hum Reprod. 1999 Aug;14(8):2131–8.

8. Katzman PJ, Genest DR. Maternal floor infarction and massive perivillous fibrin deposition: histological

18. Redline RW, Ariel I, Baergen RN, Desa DJ, Kraus FT, Roberts DJ, et al. Fetal vascular obstructive lesions: nosology and reproducibility of placental reaction patterns. PediatrDevPathol. 2004 Oct;7(5):443–52.

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19. Redline RW, Pappin A. Fetal thrombotic vasculopathy: the clinical significance of extensive avascular villi. Hum Pathol. 1995 Jan;26(1):80–5. 20. Saleemuddin A, Tantbirojn P, Sirois K, Crum CP, Boyd TK, Tworoger S, et al. Obstetric and perinatal complications in placentas with fetal thrombotic vasculopathy. PediatrDevPathol. 2010 Dec;13(6):459–64. 21. Altshuler G. Chorangiosis. An important placental sign of neonatal morbidity and mortality. Arch Pathol Lab Med. 1984 Jan;108(1):71–4.

22. Manjarkhede A, Joshi A, Gowardhan V. Chorangiosis of placenta: Report of two cases. Panacea J Med Sci. 2014;4(2):50–1. 23. Lurain JR. Gestational trophoblastic disease I: epidemiology, pathology, clinical presentation and diagnosis of gestational trophoblastic disease, and management of hydatidiform mole. AJOG. December 2010; 203(6):531–539.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 02, April - June 2016

Original Article CD4 Cell Count Analysis Among HIV Patients at a Tertiary Hospital in South India Vanisri H R1*, Vadiraja N2 Dept of Pathology, Chamarajanagar Institute of Medical Sciences, Chamarajanagar, India Dept of Community Medicine, Mysore Medical College & Research Institute, Mysore, India 1

Keywords: CD4 Count, HIV, ART

ABSTRACT Background: HIV (Human Immunodeficiency Virus) infection is characterized by a gradual deterioration of immune function. CD4 cell counts generally decrease as HIV progresses in untreated HIV-infected subjects .The present study was undertaken to know the effect of ART (Anti retroviral therapy) and the status of CD4 counts in study patients and among the genders in HIV patients. Methods: This was a cross sectional study on secondary data maintained in the ART centre for the period of 6 months from March 2015 to October 2015. The patient reports (150 cases) in the ART centre during the period of the study (6 months) were selected. CD4 cell count was done using Flow Cytometry Absolute Cell Count System. Result: Out of 150 patients 82 were male and 68 were female patients. The mean value for base line CD4 count for general group lies between those of female and male groups, with mean female (126.88cells/cmm) was lesser than that of male group(i.e. 141cells/cmm) indicating females were at higher risk of being Immunocompromised compare to males. Also that the standard deviation of female (5.4) was lesser to male (48.43) supporting the above statement. After 6 months of antiretroviral therapy, the mean CD4 counts was in the relation 352.74 cells/cmm(Male) < 378.82 cells/cmm(general) < 410.26cells/cmm (Female) with the standard deviation relation 162.68 (male) < 179.49(general) < 194.46 (Female). Conclusion: The results of this study indicated that antiretroviral therapy plays an important role in maintaining the CD4cell counts and CD4 counts in female patients are much lower compared to the males and they respond well to therapy compared to the male population.

*Corresponding author: Dr Vanisri H R, (Asso. Prof. Pathology) 59/D5 2nd main , 2nd cross, Yadavagiri Mysore 20, India Phone: +91 - 821 2514284 Email: drvanisri16@gmail.com

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CD4 Cell Count Analysis

Introduction

HIV (Human Immunodeficiency Virus) infection is characterized by a gradual deterioration of immune function. Most notably, crucial immune cells called CD4 cells are disabled and killed during the typical course of infection. [1] HIV is persistent and ultimately progressive in the vast majority of untreated hosts, there is increasing evidence that HIV have specific cellular immune responses playing a major role in determining the tempo of viral replication and thus the clinical outcome of infection. [2] At the end of 2010, an estimated 34 million people [31.6 millionâ&#x20AC;&#x201C;35.2 million] were living with HIV worldwide, up 17% from 2001. This reflects the continued large number of new HIV infections and a significant expansion of access to antiretroviral therapy (ART), which has helped reduce Acquired Immunodeficiency Syndrome (AIDS) - related deaths, especially in more recent years. [3] In humans, evidence supporting a protective effect of cellular immune responses in HIV infection is less direct. Long-term non progressive infection has been associated with both strong virus-specific CTL and with robust gag p24-specific CD4 cell proliferative responses. [4]Some studies have reported a direct inverse correlation between viral load and HIV-specific T-cell responses in untreated HIV-infected subjects. CD4 cell count is used to determine how well the immune system is working in people who have been diagnosed with HIV. The pattern of CD4 cell counts over time is more important than any single CD4 cell value because the values can change from day to day. The CD4 cell pattern over time shows the effect of the virus on the immune system. CD4 cell counts generally decrease as HIV progresses in untreated HIV-infected subjects. [5] Chamarajanagar is located in southernmost part of Karnataka. The population of Chamarajanagar is 10.20 Lakhs with a sex ratio of 989 females for every 1,000 males, and a female literacy rate of 54.32% with an overall literacy rate of 61.12% (Census 2011). According to 2012 PPTCT data, the level of HIV positivity was low (0.19%) among the PPTCT attendees, with a declining trend. [6] We are on the verge of a significant breakthrough in the AIDS response living with and affected by HIV. The vision of a world with zero new HIV infections, zero discrimination, and zero AIDS-related deaths has captured the imagination of diverse partners, stake holders and people living with and affected by HIV. New HIV infections continue to fall and more people than ever are starting treatment. With research giving us solid evidence that antiretroviral therapy can prevent new HIV infections, it is encouraging that 6.6 million people are now receiving treatment in low- and middle-income countries: nearly half those eligible. [3]

Hence, the present study was undertaken to clearly understand about the effect of ART and the status of CD4 counts in study patients and among the genders. This gives the insight into the need and urgency of antiretroviral therapy to HIV/AIDS infected people.

Materials and Methods

This was a cross sectional study on secondary data maintained in the ART centre for the period of 6 months from March 2015 to October 2015. Permission was obtained from the district surgeon, district hospital Chamarajanagar, to collect the secondary data therefore there were no ethical issues. Subjects for the study were the reports of HIV patients registered in the centre during March 2015 to October 2015. Methodology: Sample size: According to 2012 ICTC (Integrated Counseling and Testing Centre)data for Chamarajanagar district, the HIV prevalence was low among male (2.13%) and female (1.70%) attendees. With level of significance 5% and absolute allowable error 5%using â&#x20AC;&#x153;estimation set up techniqueâ&#x20AC;? the inflated sample size for male and female were 34 and 27 respectively and the total sample size was 61. However all the available patient reports (i.e.150) in the ART centre during the period of the study (6 months) were selected. Regimen of ART used was Zidovudine Lamivudine and nevirapine and was the same to all subjects in this study. CD4 cell count was done using Flow Cytometry Absolute Cell Count System in the hospital ART centre laboratory and values were maintained in the hospital records. The results of the test were recorded in excel-sheet and analyzed using standard statistical tool called R software.

Result

CD4 count base line value and after 6 months on ART value follow normality assumption. (Figure1&2) The base line CD4 values for the general population group were minimum 11cells/cmm, maximum 297cells/cmm and the mean of 135cells/cmm. The minimum value corresponds to the female and maximum value corresponds to the male. The minimum and maximum values for female were lesser than those of male group. The mean value for base line CD4 count for general group lies between those of female and male groups, with mean female (126.88cells/cmm) was lesser than that of male group(i.e. 141 cells/cmm) indicating females were at higher risk of being Immunocompromised compare to males. Also that the standard deviation of female (5.4) was lesser to male (48.43) supporting the above statement. The standard deviation of female group indicates that the entire group behaves in the similar manner indicating ART was crucial for the group, in other

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wards the female group should be prioritized to male in giving therapy. The standard deviation of female indicates that the sharper 95% confidence interval to female than that of male (Table 1&2)

group. On the whole, all the groups after ART had shown better CD4 counts. (Table 3) Thus there was a significant improvement in the CD4 counts in the patients after ART was initiated. (Figure 4& 5)

The mean CD4 counts was in the relation 352.74cells/ cmm (Male) < 378.82cells/cmm (general) < 410.26cells/ cmm (Female) with the standard deviation relation 162.68 (male) < 179.49(general) < 194.46 (Female). The 6 months post ART therapy indicates that the risky female group was behaving well with wider variability compared to the male

However when we compare the SD for base line and after 6 months groups , it was more in the after 6 months group than baseline group indicating the wide variation among the 150 patients and was supported by coefficient of variation also.

Table 1: Descriptive values for CD4 counts CD4 cell count

Baseline Male

Baseline Female

Baseline Combined

After 6 months Male

After 6 months Female

After 6 months Combined

150

Minimum

17.00

11.00

11.000

116.00

99.00

99.000

Maximum

297.00

200.00

297.000

876.00

1029.00

1029.000

No of cases

Median

136.500

340.000

Mean

141.80

126.88

135.040

352.74

410.26

378.820

95% CI Upper

152.45

137.66

142.646

388.49

457.33

407.779

95% CI Lower

131.16

116.10

127.434

317.00

363.19

349.861

Std. Error

5.35

5.40

3.849

17.96

23.58

14.655

Standard Dev

48.43

44.53

47.140

162.68

194.46

179.489

C.V.

0.349

0.474

SW Statistic

0.97

0.971

0.94

0.93

0.937

SW P-Value

0.03

0.07

0.003

0.00

0.000

Table 2:Paired t test table for equality of CD4 counts Mean difference 95.00% CI

Male

Female

Combined

-210.94

-283.38

-243.780

-244.30 to -177.57

-328.83 to -237.93

-271.583 to -215.977

SD of difference

151.85

187.76

172.327

-12.58

-12.45

-17.326

149

p-value

0.00

0.000 (indicating the significance)

Bonferroni adj p-value

0.00

0.000

Table 3: Two-sample t-test on CD4 after 6 Months grouped by SEX against Alternative = Female â&#x20AC;&#x2DC;greater than â&#x20AC;&#x2DC;Male Pooled variance: Difference in means

57.5208

95.00% confidence bound

9.2591

1.9728

148

p-value

0.0252

Bonferroni adj p-value

0.0252

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The absolute value of mean difference CD4 counts satisfying the relation 210.94cells/cmm (male) < 243.78 cells /cmm < 283.38 cells/cmm (Female) indicating better increase after treatment.(Table 2) The p value of paired t test and Bonferron adjusted p value show the significance mean difference for all individual groups. (Table 2& Figure3) Most of the patient values for CD4 counts are <200 cells/cmm after treatment ,it shows that more number of percentage of patients have better CD4 counts with minimum variability indicating patients are responding in a better way to treatment. (Figure 4 and 5)

The Equality of Variance test was passed. The t test for independent sample means was adopted with the alternative â&#x20AC;&#x153;Female is greater than the male CD4 countsâ&#x20AC;? to know whether females responding well to the treatment. (F=1.43, DF=67, 81, p-value =0.12) The p-value indicates that the data do not provide sufficient information to reject the insignificance difference and hence the alternative hypothesis was accepted. (Table 3) That is Females were responding well to ART than males. (Figure 6)

Discussion

As per NACO guidelines, currently in India, absolute CD4 cell count is being used as the basis for initiation of ART.

Fig. 1 : Probability plot showing normality for CD4 before treatment

Fig. 3: indicates that there is an increase in the CD4 count after ART supporting the p-value

Fig. 2: Probability plot showing normality for CD4 after treatment

Fig. 4: Indicates that there is an increase in the CD4 count after ART supporting the p-value in Males

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Fig. 5: Indicates that there is an increase in the CD4 count after ART supporting the p-value in Females

Fig. 6: Graph comparing Female and Male groups after ART

In the present study, baseline mean CD4 cell count was 135 cells/cmm, with mean of 126.88cells/cmm in females and 141 cells/cmm in males, indicating females were at higher risk of being Immunocompromised compare to males. After 6 months of ART , the mean CD4 cell count increased to 378.82 cells /cmm, with 352.74cells/cmm in Males and 410.26 cells/cmm in Females. After 6 months of ART therapy the female patients showed an increase in the CD4 counts compared to the male patients with wider variability. These findings were similar to the findings in the previous studies with base line mean CD4 count being 155cells/cmm and154.2+/-47.3 cells/cmm respectively and the 6 months follow up counts of 297cells/cmm and 325.4 +/- 96.1 cells/cmm. [7&8] These findings suggest that the ART therapy was effective. Earlier studies reported that the gradual CD4 cell count rise are likely to reflect the generation of new cells by peripheral expansion of pre existing T-cell clones or generation of thymically derived naĂŻve cells among ART patients . [9&10] There is a continuous increase of CD4 cell count among the HIV/ AIDS subjects who receive highly active antiretroviral therapy. [11] According to a previous study, lower percentages of CD4 T-lymphocytes are associated with adverse clinical outcomes among children and adolescents infected with human immunodeficiency virus (HIV). CD4 lymphocytes percentage generally increases with receipt of highly active antiretroviral therapy, but long term follow-up is required to assess whether these increases in CD4 cell percentages are maintained and whether they lead to normal CD4 cell percentages in children and adolescents with severe immunosuppression.[12] This finding indicated that the treatment was effective. It was reported that, the initial

increases in CD4 cell percentage observed in the first year after ART initiation are sustained for at least 5 years after ART initiation among children and adolescents infected with HIV and that greater increases occur among those with the greatest degree of immunosuppression. [12]

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[7]

The CD4 cell count decreased due to the disruption of the cell membrane as HIV buds from the surface or the intracellular accumulation of heterodisperse RNAs and unintegrated DNA.[ 7] Intracellular complexing of CD4 cell and viral envelope products can result in cell killing. [8] Similarly, other investigators proposed that the untimely induction of a programmed cell death as an additional mechanism for CD4 cell loss in HIV infection. [13]

Conclusion

HIV patients should be educated about the importance of continous intake of antiretroviral therapy which will prolong their survival and decrease the viral load and transmission of the disease hence, it can be concluded that ART is effective enough in slowing the progression of HIV infection to AIDS and increasing the survival rate of patients with good performance. Special reference is to be given to female patients since their CD4 counts are much lower compared to the males and they respond well compared to the male population

Acknowledgement

We acknowledge the guidance of Dr Chandrashekher T N, Dean and Director of Chamarajanagar Institute of Medical Sciences, Chamarajanagar.

Funding None

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Competing Interests None declared

Reference

1. Allen TM, O’Connor DH, Jing P, Dzuris JL, Mothé BR, Vogel TU et al . Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia. Nature 2000; 407: 386-90. 2. Metzner KJ , Jin X, Lee FV, Gettie A, Bauer DE, Di Mascio M et al . Effects of in vivo CD8 T cell depletion on virus replication in rhesus macaques immunized with a live, attenuated simian immunodeficiency virus vaccine. J Exp Med 2000; 191: 1921-31. 3. UN AIDS, World AIDS Day Report, 2011. 4. Pitcher CJ, Quittner C, Peterson DM, Connors M, Koup RA, Maino VC et al. HIV-1-specific CD4 T cells are detectable in most individuals with active HIV-1 infection, but decline with prolonged viral suppression. Natural Med 1999; 5: 518-25. 5. Ogg GS, Jin X, Bonhoeffer S, Dunbar PR, Nowak MA, Monard S, et al. Quantitation of HIV-1- specific cytotoxic T lymphocytes and plasma load of viral RNA. Science 1998; 279: 2103-6. 6. NACO District HIV/AIDS epidemiological profiles developed through data triangulation, National AIDS Control Organization, Ministry of Health & Family Welfare, Government of India, 2014, Chapter 2, p 24. 7. K. Suresh Babu, Ch. Ram Babu, N. Baratha Jyothi K. Sunita. A retrospective study on status of CD4 counts and effect of ART in patients attending VCTC of MGM Hospital, Warangal, Andhra Pradesh, India

10.

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IOSR Journal of Nursing and Health Science (IOSRJNHS) 2014;3: 25-35. Tiwari BR, Ghimire P, Malla S. Study on CD4 cell responses in HIV infected subjects in Nepal. Nepal Med Coll J 2008; 10: 45-47. Gulick RM, Mellors JW, Havlir D, Eron JJ, Gonzalez C, McMahon D, et al . Treatment with indinavir, zidovudine and lamivudine in adults with human immunodeficiency virus infection and prior antiretroviral therapy. New England Journal of Medicine, 1997;337: 734-739. Pakker NG, Notermans DW, De Boer RJ, Roos MTL, De Wolf F, Hill A, et al. Biphasic kinetics of peripheral blood T cells after triple combination therapy in HIV-I infection; A composite of redistribution and proliferation. Nature Medicine, 1998:4; 208-214. Hunt PW , Deeks SG, Rodriguez B, Valdez H, Shade SB, Abrams DI, et al. Continued CD4 cell count increases in HIV-infected adults experiencing 4 years of viral suppression on antiretroviral therapy. AIDS 2003; 17: 1907-15. Patel K, Hernán MA, Williams PL, Seeger JD, McIntosh K, Van Dyke RB et al. Pediatric AIDS Clinical Trials Group 219/219C Study Team, Long-term effects of Highly Active Antiretroviral Therapy on CD4+ cell evolution among children and adolescents infected with HIV: 5 Years and Counting. Clinical Infectious Diseases, 2008;46:1751-1760. Terai C, Wasson DB, Carrera CJ, Carson DA. Dependence of cell survival on DNA repair in human mononuclear phagocytes. J Immunol 1991; 147: 4302-6.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 02, April - June 2016

Original Article Correlation of Bone Marrow Aspiration, Touch Imprint Findings and Bone Marrow Biopsy Findings in Pancytopenia. R. Sindhu*, Pramita Sahu, Debi Prasad Mishra, Samir Kumar Behera Dept. of Pathology, MKCG Medical College, Berhampur, Odisha, India

Keywords: Pancytopenia, BMA, BMI and BMB.

ABSTRACT Background: Pancytopenia is an important hematological entity encountered in our day to day clinical practice.Bone marrow examination is a useful and cost effective diagnostic approach in evaluating these disorders. Although bone marrow aspiration alone is sufficient in diagnosing most of these disorders, the comparative evaluation of bone marrow aspiration (BMA), imprint (BMI) and biopsy (BMB) is needed for more rapid and efficient diagnosis. Many studies have been done in evaluating the importance of each of these procedures in various hematological disorders, with very few studies evaluating the importance of all three procedures in pancytopenia specifically.So, the objective of our study was to compare and correlate all three procedures in cases of pancytopenia. Methods: All patients who were found to be pancytopenic were evaluated and bone marrow aspiration, imprint and biopsy were done in all the cases simultaneously at the same site using two needle technique. Result: A total of 148 cases of pancytopenia were studied and most common cause of pancytopenia was hypoplastic/ aplastic anemia (33.8%), followed by nutritional anemia (30.4%). We found a positive correlation of 72.04% in BMA with BMB and 91.20% in BMI with BMB. Conclusion: Trephine biopsy has greater value in providing information about the architecture, pattern of cellularity, and presence of infiltrates and granulomas. At the same time morphological features of individual cells may be identified by aspiration and by imprint in cases of dry tap. Hence, both BMA and BMB along with imprint cytology are complementary to each other.

*Corresponding author: Dr. R. Sindhu, Department of Pathology, M.K.C.G Medical College, Berhampur, Odisha 760004 Phone: +91 - 7854831852 Email: Sindhushankar51@gmail.com

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Bone Marrow Aspiration, Imprint and Biopsy in Pancytopenia

Introduction

Pancytopenia is an important hematological entity encountered in our day to day clinical practice. A spectrum of disorders, primarily or secondarily affecting the bone marrow may manifest with pancytopenia.[1] Evaluation of the bone marrow is an indispensable adjunct to the study of hematopoietic disorders. Marrow can be examined by aspiration, or trephine biopsy and preparing a clot section from aspiration material orpreparing imprint smears from biopsy. Each procedure has its own advantages and disadvantatges. The present study was therefore conducted to compare and correlate the role of bone marrow aspirate cytology, touch imprint cytology and trephine biopsy sections to formulate an effective and rapid method for diagnosing the hematological and non hematological causes of pancytopenia.

Material and Methods

A prospective study was done in our department. All patients who were found to have Hb <10 gm/dl, TLC < 4.00x103 Cells/µl and platelet count <100x103 cells/ µl [2] (done through automated five part cell analyzer Sysmex xt-2000i) were considered to be pancytopenic and included in our study. Diagnosed patients who are already under chemotherapy were excluded from the study. After getting a written consent from the patient both bone marrow aspiration and biopsy were done simultaneously (biopsy being done first) using 2 needle technique at the same skin incision site. A trephine length of 1.5 – 2cm was considered to be adequate. At least 4 good smears were prepared from the aspirate material. Imprint smears were also prepared by touch and roll technique in all cases before fixation of biopsy core. Special stains like Perl’s

Fig. 1: Microphotograph of BMB in metastatic squamous cell carcinoma from carcinoma cervix.H&E (X400x).

stain for iron, reticulin stain, PAS, MPO, and CAE were done wherever required. The bone marrow aspiration and biopsy were reported according to the ISCH Protocol.[3] Both touch imprint and aspiration were correlated with trephine biopsy. The findings were tabulated in MS Excel 2010 and analysed using the statistical software InStat Graph Pad (Trial Version). Some of the distinct cases with special stains are shown in the figure ( figure 1 to 6).

Result

A total of 148 cases of pancytopenia of varying ages were included in the study. Most of the cases affected were in the age group of 11-20 years (22.29%). The most common presenting clinical feature was pallor (98%), followed by easy fatiguability, fever, hepatomegaly, splenomegaly, lymphadenopathy, bleeding tendency, petechiae, sternal tenderness.Most of the cases were nonneoplastic (74.3%), neoplastic ones constituted 25.6% of total cases. Most common cause of pancytopenia in our study was hypoplastic/aplastic anemia (33.8%), followed by nutritional anemia (30.4%), acute leukemia (11.4%), MDS (6.08%), metastatic carcinoma (2.7%), MPN (2.7%), malaria (2.7%), multiple myeloma (2.02%). Other rare causes include primary lymphoma of bone marrow, hemophagocytic lymphohistiocytosis, storage disorders, granulomas. In contrast, most common cause of pancytopenia in children <10 years was found to be ALL. We also found 10 cases of dry tap in BMA where BMB and imprint helped in diagnosis. Imprint was inadequate/not suitable in 4 cases and biopsy was inadequate in 10 cases. The various cause of pancytopenia diagnosed through each procedure is tabulated in detail (TABLE 1). We also

Fig. 2: Microphotograph of BMA in Gaucher’s disease. Leishman Stain (X1000x).

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Fig. 3: Microphotograph of BMB and in myelofibrosis. H&E(X400x).

Fig. 4: Microphotograph of BMA in malaria with hemophagocytosis. Leishman Stain (X100x).

Fig. 5: Microphotograph of BMA showing Grade 4 iron stores in megaloblastic anemia.Perlâ&#x20AC;&#x2122;s stain (X400x).

Fig. 6: Microphotograph of BMI in MDS Chloracetate Esterase Stain.(X100x).

correlated both aspiration and imprint with trephine biopsy (TABLE 2). We found a positive correlation of 72.04% in BMA with BMB and 91.20% in BMI with BMB. We were unable to correlate aspiration and imprint with biopsy in certain cases like nutritional anemia, malaria and some miscellaneous cases because biopsy material was

inadequate and aspiration and imprint was more helpful in diagnosing those cases.

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Discussion

To report a reliable diagnosis, an adequate biopsy material is the most important one.It depends upon various factors

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Table 1: CASES DIAGNOSED ON BMA, BMI, BMB S. NO CAUSES TOTAL CASES 1 Nutritional anemia 45 (30.4%) 2 Hypoplastic/aplastic anemia 49 (33.1%) 3 Acute leukemia 17 (11.4%) 4 Myelodysplastic syndrome 09 (6.08%) 5 Multiple myeloma 03 (2.02%) 6 Metastatic carcinoma 04 (2.70%) 7 Myeloproliferative neoplasms 04 (2.70%) 8 Lymphoma 01 (0.6%) 9 Hemophagocytic disorders 02 (1.35%) 10 Storage disorders 01 (0.6%) 11 Granulomatous lesion 01 (0.6%) 12 Malaria 04 (2.70%) 13 Normal bone marrow 02 (1.35%) 14 Dry tap/ scanty aspirate --15 Inadequate biopsy/imprint --16 Miscellaneous causes 06 (4.05%) TOTAL CASES 148 (100%)

BMA DIAGNOSIS

BMI DIAGNOSIS

BMB DIAGNOSIS

45 39 13 05 2 02 1 00 02 01 00 04 12 10 --06 148

45 49 17 05 03 03 01 00 02 01 00 04 02 --10 06 148

43 49 17 09 03 03 04 01 02 01 01 03 02 ---04 05 148

Table 2: CASES SHOWING POSITIVE CORELATION OF BMA AND BMI WITH BMB S. NO CAUSES TOTAL Positive correlation of CASES BMA cytology and BMB (% of cases) 1 Nutritional anemia 45 ---2 Hypoplastic/ aplastic anemia 49 39 (79.5%) 3 Acute leukemia 17 13 (76.4%) 4 Myelodysplastic syndrome 09 5 (56.5%) 5 Miscellaneous causes 06 ---6 Multiple myeloma 03 2 (66.66%) 7 Metastatic carcinoma 04 2 (50%) 8 Myeloproliferative neoplasms 04 1 (25%) 9 Lymphoma 01 0 10 Hemophagocytic disorders 02 2(100%) 11 Storage disorders 01 1(100%) 12 Granulomatous lesion 01 0 13 Malaria 04 ---14 Normal bone marrow 02 2(100%) TOTAL % OF POSITIVE 148 67 (72.04%) CORRELATION

like the person who does the procedure, the technique used, the site of biopsy etc. We used Islamâ&#x20AC;&#x2122;s two needle technique[4] for all our procedures. Both BMA and BMB were done at the same skin incision site with change of the position of the needle after one procedure to get the maximum material as also done by Pampa ch Toi et al.[5] Around 97.2% of our biopsy samples were found to be adequate with the criteria for adequacy considered as average length of biopsy core to be 1.5 cm or specimen which shows at least 10 partially preserved intertrabecular areas, likewise P. T. Dambhareaet al.[6]who found 96. 55% adequacy.

Positive correlation of BMI cytology and BMB (% of cases) ---49 (100%) 17 (100%) 5 (56.5%) ---3 (100%) 3 (75%) 1 (25%) 0 2 (100%) 1 (100%) 0 ---2 (100%) 83 (91.20%)

In the present study, we had 10 cases of (6.75%) dry tap out of which 2 were metastatic carcinomas to bone marrow (1 from cervix, 1 from unknown primary), 1 came out to be primary non Hodgkin lymphoma of BM, 4 cases of acute leukemia presenting as dry tap because of packed marrow and 3 cases of myelofibrosis in late stage (with reticulin stain showing GRADE 3 fibrosis in all the cases). All these cases were diagnosed by BMB. Thus, all the 10 dry tap cases in aspiration were found to be abnormal in biopsy. This was in accordance with the study done by Navone R et al.[7] andAmrishPandya et al.[8] who also found 100%

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Sindhu et al. abnormal biopsy in dry tap cases. But study done by Engest A et al.[9]andHumphries JEet al.[10] found only 77%, 93% abnormalities in their studies respectively. In the present study 10 cases of hypoplastic anemia were diagnosed by biopsy which were misdiagnosed as normocellular marrow in BMA. This was in accordance with other studies done by P. Ch Toi et al.,[5]Vidisha et al.,[11] Patel Set al.,[12 ]Niranjan et al.[13] who observed that this discordance is because of dry tap in aspiration, or dilution with peripheral blood or aspiration of the hematopoietic foci yielding a normocellular marrow which may be the cause in our study. Cetto et al[14] observed that most of the hypercellular marrows are better identified in BMA as hypercellular but discordance occurs in interpretation of normocellularity and hypocellularity. Coming to myelodysplastic syndromes, in the present study 4 out of 9 cases were diagnosed by biopsy which was misdiagnosed as megaloblastic anemia in BMA. It is a well-known fact that topography is assessed by biopsy and ALIP which is specific for MDS is assessed only through biopsy which was also observed by Gupta N et al.[15] Out of 17 cases of acute leukemia 4 cases were diagnosed by BMB. The reason for this may be due to extreme cellularity and compactness of marrow as observed by Niranjan et.al.[13] According to Bird AR et al.[16]marked increase in reticulin also leads to dry tap in acute leukemia. Out of 4 cases of MPN, 3 cases were myelofibrosis which were diagnosed by biopsy and 1 case was mastocytosis diagnosed in BMA and confirmed by BMB. The diagnosis of myelofibrosis needs BMB with reticulin grading in BMB as BMA is dry tap in almost all cases. A single case of multiple myeloma was diagnosed in BMB which was misdiagnosed as simple reactive plasmacytosis in BMA. The discordance is because identification of sheets of mature and immature plasma cells is more in favor of multiple myeloma than a reactive condition which was observed by Sabharwal BD et al.[17] A single case of primary Non-Hodgkin lymphoma of bone presenting as pancytopenia was diagnosed through BMB. A study done by Gupta N et al.,[15] Menon NC et al.,[18] observed that bilateral biopsy is required for diagnosis of NHL since unilateral biopsies can miss the diagnosis. But in our study we found only a single case of Non Hodgkin lymphoma which was diagnosed with unilateral biopsy. According to Ellman L.[19] the reason for dry tap in cases of lymphoma are, that nodules and cluster of lymphoma cells can be quite dense and adherent and may be difficult to aspirate. A study done bySurbhiGhoyalet al.[20] found that lymphoma diagnosis sensitivity increases with trephine length. www.pacificejournals.com/apalm

A-91 2 out of 4 cases of metastatic carcinoma wasdiagnosed through BMB where BMA was dry tap. This again reiterates the importance of biopsy in cases of dry tap as also observed by other studies.Whereas a single case of metastatic neuroblastoma in the present study was missed because of marrow necrosis, but aspiration showed small round tumor cells, rosettes and neuropil material with suppression of hematopoiesis. This was also observed by Pampa ch Toi et al.[5]who encountered a single case of metastatic carcinoma in BMA where BMB shows only infarction. This indicates the importance of both the procedures in metastatic carcinomas and shows they are complementary to each other. We also encountered a single case of tuberculous granuloma which was later confirmed by ZiehlNeelsen staining for AFB in BMB specimen. P. Ch. Toi et al.[5]observed that BMB along with good imprint alone is sufficient in diagnosis of granulomas. The present study did not show a positive correlation between BMA and BMB in diagnosis of nutritional anemia because aspirate is more useful in diagnosis of macro/ micronormoblasts. A study done by P. ch Toi et al.[5] shows that megaloblasts look like leukemic blast in BMB. Also grading of iron stores are better appreciated and interpreted in BMA than biopsy as decalcification leeches out the iron. Also in cases of reactive marrow, infections like malaria we found that BMA is more helpful than biopsy. A study done by Sabharwal et al.[17] shows that plastic embedding and semi thin sections of undecalcified bone marrow, can be expected to improve the cytological details of tissue obtained by biopsy. While there are many studies showing the importance of BMB, very few studies in the literature compared the efficacyand importance of BMI in diagnosis of hematological disorders. In the present study we found a 91.20% positive correlation between BMI and BMB which was quite higher when compared with other studies. (TABLE 3)The percent of positive correlation in case of imprint is also higher when compared with BMA, thus indicating that a well smeared and well stained imprint smears increase the sensitivity in diagnosis of hematological disorders

Conclusion

To, conclude, BMA and BMB along with imprint cytology are complementary to each other. Better results are obtained when both the procedures are performed simultaneously as BMA gives better morphology of the cells and BMB gives a good picture regarding the pattern of distribution of cells, and cellularity of marrow. BMB remains the gold eISSN: 2349-6983; pISSN: 2394-6466

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Bone Marrow Aspiration, Imprint and Biopsy in Pancytopenia

TABLE 3: POSITIVE CORRELATION OF BMA AND BMI WITH BMB IN VARIOUS STUDIES POSITIVE CORRELATION WITH BMB OTHER STUDIES NO. OF CASES BMA (%) BMI (%) P. Ch Toi et al (2010) 160 61.25% ---S Chandra (2011) 565 78% 84.3% Vidisha et al (2013) 460 94% ---V. Tilak et al (2013) 182 ---87.3% Niranjan et al (2015) 140 75.8% --Present study (2013-15) 148 72.04% 91.20%

standard for diagnosing cases when BMA is dry tap, and in cases like hypoplastic/ aplastic anemia and granulomatous inflammation. But, assessment of iron status by Perl’s stain is better in BMA than biopsy. A meticulously prepared BMI provides a better information regarding cellularity of marrow than BMA. Also metastatic tumors can be quickly diagnosed in a well prepared imprint smears and unnecessary delay caused by decalcification and processing of BMB specimens in histopathological laboratories can be avoided. Both BMA material and biopsy material can be used for higher studies according to the specific disease, if needed.

Acknowledgement NIL

Competing Interest NIL

Funding

7. Navone R, Colombano MT. Histopathological trephine biopsy findings in cases of ‘dry tap’ bone marrow aspirations. ApplPathol 1984; 2(5): 264-71. 8. Pandya A, Patel T, Shah N. Comparative Utility Of Bone Marrow Aspiration And Bone Marrow Biopsy. Journal of Evolution of Medical and Dental Sciences.2012;1(6): 987-93. 9. Engeset A, Nesheim A, Sokolowski J. Incidence of ‘dry tap’ on bone marrow aspirations in lymphomas and carcinomas. Diagnostic value of the small material in the needle. Scand J Haematol 1979; 22(5): 417-22. 10. Humphries JE. Dry tap bone marrow aspiration: clinical significance. Am J Hematol. 1990; 35(4):247-50.

NIL

References

6. Dambharea PT, Toteb VD, Wasnikc PN, Kumbhalkard DT. Role of Bone Marrow Trephine Biopsy in Diagnosing Hematological Disorders Which Shows Bone Marrow Aspiration Failure-Two Year Observational Study of 58 Cases. Online International Interdisciplinary Research Journal.2015;5(2):66-75.

1. Khodke K, Marwah S, Buxi S, Yadav RB, Chaturvedi NK. Bone Marrow Examination in Cases of Pancytopenia. Journal of Indian Academy of Clinical Medicine 2001;2:16-19. 2. Singh T. Bone Marrow Failure Syndromes and Iron Overload Disorders. In:Atlasand Text book of Haematology. 2nd ed. New Delhi, Avichal Publishers: 2011.p.106. 3. Lee SH, Erber WN, Porwit A, Tomonaga M, Peterson LC. ICSH guidelines for the standardization of bone marrow specimens and reports. Int. Jnl. Lab. Hem. 2008;30:349–364. 4. Islam A. Bone marrow aspiration prior to bone marrow core biopsy using the same bone marrow biopsy needle. A good or bad practice. J Clin Pathol. 2007; 60(2):212–215. 5. Toi PC, Varghese RG, Rai R. Comparative Evaluation of Simultaneous Bone Marrow Aspiration and Bone Marrow Biopsy: An Institutional Experience. Indian J Hematol Blood Transfus.2010; 26(2):41–4.

11. Mahajan V, Kaushal V, Thakur S, Kaushik R. A comparative study of bone marrow aspiration and bone marrow biopsy in haematological and non haematological disorders – An institutional experience JIACM 2013; 14(2): 133-5. 12. Patel S, Nathani P, Shah N, Shah CK. Diagnostic Role of Bone Marrow Aspiration and Trephine Biopsy in Haematological Practice. Gujarat Medical Journal. 2015;70(2):37-41. 13. Mainali N, Homagai N, Tiwari PS, Giri A. A Comparative study of bone marrow aspiration and bone marrow biopsy in hematological diseases. Journal of Nobel Medical College.2015;4(7):12-14. 14. Cetto GL., Iannucci A., Perini A. Bone marrow evaluation : the relative merits of particle sections and smear preparations. Appl Pathol.1983;1:181-193. 15. Gupta N, Kumar R, Khajuria A. Diagnostic Assessment of Bone Marrow Aspiration Smears, Touch Imprints and Trephine Biopsy in Haematological Disorders. JK Science. 2010;12(3):130-13.

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16. Bird AR, Jacobs P. Trephine biopsy of bone marrow. S Afr Med J.1983; 64: 271-276. 17. Sabharwal BD, Malhotra V, Aruna S, Grewal R. Comparative evaluation of bone marrow aspirate particle smears, imprints and biopsy sections. J Postgrad Med 1990; 36(4):194-8. 18. Menon NC, Buchanan JG. Bilateral trephine bone marrow biopsies in Hodgkinâ&#x20AC;&#x2122;s and non-Hodgkinâ&#x20AC;&#x2122;s lymphoma. Pathology 1979;11(1):53-57.

19. Ellman L. Bone marrow biopsy in the evaluation of lymphoma, carcinoma and Granulomatous disorders. Am J Med.1976;60:1- 6.

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20. Goyal S, Singh UR, Rusia U. Comparative Evaluation of Bone Marrow Aspirate with Trephine Biopsy in Hematological Disorders and Determination of Optimum Trephine Length in Lymphoma Infiltration. Mediterr J Hematol Infect Dis 2014; 6(1).

Original Article Study of Fine Needle Aspiration Cytology of Breast Tuberculosis Pragnesh J. Patel* Department of Pathology, GMERS Medical College, Gandhinagar, Gujarat, India

Keywords: Breast, Cytology, Granuloma, Mastitis, Necrosis, Tuberculosis

ABSTRACT Background: Characteristic feature of tuberculous mastitis like pus discharging sinus appear late in the disease. It commonly misinterpreted for malignancy and pyogenic abscess. Fine needle aspiration cytology (FNAC) is rapid and less invasive tool to diagnose tuberculous mastitis in initial phase of disease. Methods: Tuberculous mastitis reported on FNAC of breast during the study period of two years were analyzed. Data were collected for physical examination and microscopic presence of granuloma, necrosis, inflammatory cells and giant cells. Result: During the study period of two years, seven cases were reported as tuberculous mastitis. No case of axillary lymph node or opposite breast involvement detected. Pus discharging sinus was present in one case. On cytology, presence of granuloma, caseous type of necrosis and Langhans giant cells were reported in 100%, 71% and 43% respectively. Conclusion: FNAC of breast is a useful tool to diagnose tuberculous mastitis and differentiate same from malignancy and pyogenic abscess. Cytological diagnosis help to identify tuberculous mastitis early in the course of disease and start of anti tuberculosis therapy eliminate need for radical surgical intervention.

*Corresponding author: Dr. Pragnesh J. Patel; 548 / A / 1, Sector:8-B, Gandhinagar, Gujarat, India â&#x20AC;&#x201C; 382008 Phone: +91 - 98254 32281 Email: drpragnesh_patel@yahoo.com

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Patel et al.

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Introduction

India is a endemic zone for the tuberculosis with prevalence of 211 cases per 1,00,000 population.[1] Fine needle aspiration cytology (FNAC) which is important tool in diagnosis of breast lump, also helpful to diagnose tuberculosis by demonstrating granuloma with or without presence of necrosis.[2] Tuberculous mastitis often misdiagnose as malignancy or pyogenic abscess.[3] In such conditions patient often underwent numerous investigation before definitive diagnosis is made. FNAC in such circumstances help to make diagnosis in early course of disease and institution of specific therapy.Â Clinically early diagnosis is challenging as characteristic sinus formation occur late in the disease. Mantoux test comes positive frequently in endemic areas as India and does not provide much help. Radiological investigations help only to determine extent of disease rather than in diagnosis. Modern radiological methods like mammography, computed tomography, magnetic resonance imaging of the breast have been extensively studied for diagnosis of breast tuberculosis but of no reward.[3] FNAC is the main diagnostic tool to investigate tuberculous mastitis with 73% cases diagnosed by it.[4] Even though high prevalence of tuberculosis, extra pulmonary tuberculosis occurring in the breast is rare.[4] Main objective of this study was to examine cytomorphology of tuberculous mastitis.

Materials and Methods

All the patients came for FNAC of the breast during last 2 years from July 2013 to June 2015 in our cytology department were included in this study. Before aspiration, consent for FNAC was taken and data were collected regarding age, gender, patientâ&#x20AC;&#x2122;s complain, site and size of lump, any lump in contralateral breast and both axilla. Universal safety precautions were taken during procedure. Aspirations were done with 22 or 24 Gauge needle and 10 ml syringe with or without applying negative pressure. Sample material put on glass slide. Second slide was taken and put on first slide to pull in opposite direction in horizontal position. Slides were immediately put in to alcohol for wet fixation and stained with Hematoxylin and Eosin. Cover slip mounted with DPX (Di-n-butyl phthalate in xylene) and reported. During microscopic examination presence of granuloma, necrosis, inflammatory and ductal cells as well as giant cells were noted.

years with mean of 33 years of age. Left side of breast more commonly involved than right. Symptoms of acute inflammation like pain and fever were present in 29% cases. Axillary lymphnode and opposite breast involvement not found in any patient however same were not documented in 3 patients. Pus was aspirated in one case, whitish material in two cases and remaining were blood mixed. None of the aspirate had cheesy material. All the cases on cytology showed granuloma (Fig.1,2). Cellularity on aspiration was adequate for reporting in all cases. Caseous type of necrosis which is characteristic of tuberculosis was present in 71% cases while remaining cases had nonspecific necrosis (Fig.3). 43% cases had Langhans type giant cells (Fig.4), 14% had foreign type giant cell and 43% had no giant cells. No case had nipple discharge. Patient C was on anti tuberculosis therapy during procedure. Patient D and F were diagnosed clinically as carcinoma. Patient E and F were having breast feeding at time of FNA.

Discussion

Considering minimal invasion, cost effectiveness and rapid interpretation, FNAC is the important tool to diagnose breast lump. [5] It remain important modality for diagnosis of tuberculosis of breast.[2] Our study reported tuberculous mastitis in 7 cases out of 218 FNAC of breast lump. In literature reported incident vary from 0.64% to 3.59%.[6] Incident rate in our study was 3.2%, which is consistent with reported in other studies. Tuberculosis of breast in western countries are rare, being less than 0.1% of breast lesions examined histologically.[7,8] In present study duration of symptoms were from 1 week to 1.5 years. Dubey et al and Shukla et al noted history of symptoms varies from few weeks to several years.[9,10] Bilateral breast involvement is uncommon(3%).[11] In present study none of patient had bilateral involvement. Considering objective of present study, which was to study cytomorphology of breast tuberculosis, follow up of the patients after making diagnosis of tuberculous mastitis was omitted as patients were put under consultation of district tuberculosis center for further treatment.

Total of 218 FNAs of the breast were done during the study period. Out of which seven were reported as tuberculous mastitis as shown in table-1. Age ranges from 16 to 53

Major characteristic of diagnosis of tuberculosis on FNAC are presence of granuloma and caseous type of necrosis. All patients under study had granuloma formation detected by FNAC study. 6 out of 7 patientsâ&#x20AC;&#x2122; cytology study indicate presence of caseous type of necrosis and/or Langhans type giant cells considered as feature of tuberculosis. One patient had granuloma without caseous type of necrosis and Langhans type giant cells, but presence of predominant lymphocytes in smear rule out acute suppuration. As only small quantity of tissue is available for examination during FNAC, absence of necrosis on cytology of breast does

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Result

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Cytomorphological Study of Breast Tuberculosis

Table 1: Findings of patients reported as tuberculous mastitis. Case

Patient A

Patient B

Patient C

Patient D

Patient E

Patient F

Patient G

Age in years

Complain duration 7 days

1 week

10 days

2.5 months

25 days

1 month

1.5 years

C/o Pus discharge Yes

C/o Fever

Yes

C/o Pain

Yes

USG/ Mammography

Well define lesion

Abscess

Complex mass

Well define lesion

Not done

Well define lesion

On examination Area of involvement

Right breast; Periareolar

Right breast; Left breast; Lateral half UI quadrant

Right breast; Lower half

Left breast; LO Left breast; Left breast; quadrant UO quadrant Upper half

Axillary LN involvement

Not documented

Opposite breast involvement

Not documented

Lump

Soft

Firm

Ill define thickening

Hard

Firm

Size in cm

2*1

2*2

4*2

4*4

0.5*0.5

5*5

4*2

FNA aspiration

Pus

Blood mixed Yellow whitish

Blood mixed

Whitish

Blood mixed

Microscopy Inflammatory cells Neutrophils

Lymphocytes Neutrophils

Neutrophils

Lymphocytes

Neutrophils

Lymphocytes

Granuloma

Present

Necrosis

Caseous

Present

Caseous

Present

Caseous

Giant cells

Langhans

Present

Ductal cells

Benign

(C/o: complain of; USG: ultrasonography; UI: upper inner; LO: lower outer; UO: upper outer; LN: lymph node)

Fig. 1: Microphotograph of granuloma (H&E, X400)

Fig.2: Microphotograph of granuloma (H&E, X100)

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Fig. 3: Microphotograph of necrosis with neutrophilic inflammatory cells (H&E, X100)

Fig. 4: Microphotograph of Langhans type of giant cell (H&E, X100)

not rule out diagnosis of tuberculosis.[2,3,4,12] Kakkar et al demonstrated 74% cases of cytology had granulomas along with caseous type of necrosis.[4] In present study 71% of cases had similar findings on FNAC. Granulomatous mastitis occurs in diverse pathology including infections like Mycobacterium tuberculosis, Blastomycosis, Corynebacterium; autoimmune process like Wegener granulomatosis and foreign body reaction; diabetes mellitus and sarcoidosis. In India as the prevalence of tuberculosis is high, granulomatous mastitis even in absence of acid fast bacilli(AFB) on Ziehl-Neelsen (ZN) stain, first choice is antituberculosis therapy, and in case no response to it, then consider alternate diagnosis.[13] Detection of AFB on ZN stain not mandatory as extrapulmonary tuberculosis likely to be paucibacillary and to able to detect bacilli on microscopy, number of bacilli must be more than 10,000/ ml of material.[14] Polymerase chain reaction (PCR) is not mandatory for diagnosis in AFB negative patients and false negative reports are a possibility. Considering paucibacillary nature of condition, microscopy, culture and PCR do not have the same diagnostic utility as in pulmonary tuberculosis.[15]

FNA play important role when provisional clinical diagnosis of breast lump is malignancy. Kakkar et al describe a case where pre operative FNA examination not carried out and had been operated for malignancy and on histology turn out to be tuberculosis.[4] In present study there had been two cases, which were clinically diagnosed as malignancy but turn out tuberculous mastitis on cytology examination. Out of these two patients, one was reported having complex mass on ultrasound. Lump in both cases were large ( 4 and 5 cm diameter on physical examination). Pre operative FNA examination helps to avoid unnecessary surgical interventions.

Tewari et al demonstrate 22/30(73%) lump, 11/30(37%) ulcer, 4/30(13%) sinus, while present study had 7/7(100%) lump, 0/7(0%) ulcer and 1/7(14%) sinus during presentation of patients.[16] Multiple lumps are less frequent, comparable to present study having no such presentation.[17] It is unlikely for breast tuberculosis patients to have pulmonary or systemic symptoms.[16,18] Mammography and other radiology techniques play limited role as findings are often indistinguishable from carcinoma of breast.[19,20] In present study complex mass, abscess and well defined mass reported by radiologist in 1,2 and 3 cases respectively. www.pacificejournals.com/apalm

Mckeown and Wilkinson classified tuberculous mastitis as primary and secondary.[21] Vassilakos stated primary condition is quite rare and was diagnose because the clinician was unable to detect true nidus of the disease. [22] However for primary condition, likely possibility is ascending infection from dilated lactiferous duct in lactating women. Two cases were lactating at the time of diagnosis in present study. Tuberculosis of breast is a disease of young female.[12] In this study also mean age was 33 years. In older patients tuberculosis may mimic carcinoma while in younger patients it resemble pyogenic abscess.[23] While treatment for tuberculous mastitis significantly differ from carcinoma and pyogenic abscess, it is important to diagnose condition appropriately and in timely manner. Combination of drug therapy and limited excision of diseased breast is a treatment of choice.[24,25]

Conclusion

Effectiveness of FNAC in diagnosis of breast lump is well established. Same usefulness of FNAC also applied for eISSN: 2349-6983; pISSN: 2394-6466

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Cytomorphological Study of Breast Tuberculosis

diagnosis of tuberculous mastitis and to differentiate from malignancy and pyogenic abscess. Characteristic feature of tuberculosis like sinus formation appear late in the disease. Emphasis should be made for early diagnosis by cytology to initiate antituberculosis therapy and eliminate need for more radical surgical interventions.

Acknowledgements Nil

Funding None

Competing Interests None declared

Reference

1. World Health Organization. (2015, April 15). India: Tuberculosis profile - 2013. Retrieved from http:// www.who.int/tb/country/data/profiles/en/ 2. Kishore B, Khare P, Gupta RJ, Bisht SP. Fine needle aspiration cytology in the diagnosis of inflammatory lesions of the breast with emphasis on tuberculous mastitis. J Cytol 2007;24:155–6. 3. Kant S, Dua R, Goel MM. Bilateral tubercular mastitis. Lung India. 2007;24:90–3. 4. Kakkar S, Kapila K, Singh MK, Verma K. Tuberculosis of the breast. A cytomorphologic study. Acta Cytol 2000;44:292-6. 5. Patel PJ, Chauhan S. Sensitivity, specificity and accuracy of fine needle aspiration cytology of breast lump. Int J Int Med Res. 2015;2(1):32-5 6. Gupta R, Gupta AS, Duggal N. Tubercular mastitis. Int Surg 1982;67:422-4. 7. O’Reilly M, Patel KR, Cummins R. Tuberculosis of the breast presenting as carcinoma. Mil Med 2000;165:800-2. 8. Al-Marri MR, Almosleh A, Almoslmani Y. Primary tuberculosis of the breast in Qatarr ten year experience and review of the literature. Eur J Surg 2000;166:687-90. 9. Dubey MM, Agrawal S. Tuberculosis of the breast. J Indian Med Assoc 1968;51:358-9. 10. Shukla HS, Kumar S. Benign breast disorders in nonwestern populations: Part II - Benign breast disorders in India. World J Surg 1989;13:746-9.

11. Banerjee SN, Ananthakrishnan N, Mehta RB, Prakash S. Tuberculous mastitis: a continuing problem. World J Surg 1987;11:105-9. 12. Sharma PK, Babel AL, Yadav SS. Tuberculosis of breast (study of 7 cases) J Postgrad Med. 1991;37:24–6. 13. Gupta D, Rajwanshi A, Gupta SK, et al. Fine needle aspiration cytology in the diagnosis of tubercular mastitis. Acta cytol 1999;43(2):191-4. 14. Pagel W, Simmonds F, Macdonald J, Nassan E. Pulmonary tuberculosis. 4th ed. London: Oxford University Press; 1964. 15. Pai M, Riley LW, Colford JM Jr. Interferon-gamma assays in the immunodiagnosis of tuberculosis: a systematic review. Lancet Infect Dis 2004;4:761. 16. Tewari M, Shukla HS. Breast tuberculosis: diagnosis, clinical features and management. Indian J Med Res 2005;122:103-10. 17. Dharkar RS, Kanhere MH, Vaishya ND, Baisarya AK. Tuberculosis of the breast. J Indian Med Assoc 1968;50:207-9. 18. Akcakaya A, Eryilmaz R, Sahin M, Ozkan O: Tuberculosis of the Breast. The Breast Journal 2005;11:85-6. 19. Shinde SR, Chandawarkar RY, Deshmukh SP. Tuberculosis of the breast masquerading as carcinoma: a study of 100 patients. World J Surg 1995;19:379-81. 20. Jaideep C, Kumar M, Khanna AK. Male breast tuberculosis. Postgrad Med J 1997;73:428-9. 21. Mckeown KC, Wilkinson KW. Tuberculous diseases of the breast. Br J Surg 1952;39:420. 22. Vassilakos P. Tuberculosis of the breast: cytological findings with fine-needle aspiration. A case clinically and radiologically minicking carcinoma. Acta Cytol 1973;17:160-5. 23. Schwartz GF. Benign neoplasms and ‘inflammations’ of the breast. Clin Obstet Gynecol 1982;25:373-85. 24. Alagaratnam TT, Ong GB. Tuberculosis of the breast. Brit J Surg 1980;67:125-6. 25. Hale JA, Peters GN, Cheek JH. Tuberculosis of the Breast: rare but still extant. Review of the literature and report of an additional case. Amer J Surg 1985;150:620-4.

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Original Article Histological Classification of Atherosclerosis and Correlation with Ischemic Heart Disease: A Autopsy Based Study Vandana Porwal, Shweta Khandelwal*, Deepali Jain, Seema Gupta Dept. of Pathology, JLN Medical College, Ajmer, Rajasthan, India

Keywords: Coronary Artery Disease, Acute Myocardial Infarction, Myocardial Fibrosis, Coronary Artery

ABSTRACT Background: There is an ever increasing trend of rise of coronary artery disease (CAD) in India and globally. The grading of coronary atherosclerotic lesions is important as it correlates with significant cardiac lesions like acute myocardial infarction (MI), myocardial fibrosis and left ventricular hypertrophy (LVH) etc. To study the same in living subjects is difficult, hence autopsy studies are done. Our 1 year retrospective study aims at grading the coronary atherosclerotic lesions and correlating the results with simulating studies in India and abroad. Method: The present study was conducted in the Department of Pathology, JLN Medical College, Ajmer (Rajasthan). Duration of study was from January 2011 to December 2011. Total 103 post-mortem heart specimens, irrespective of cause of death were examined grossly and microscopically for extent of coronary artherosclerosis and associated cardiac lesions. Results and Conclusion: Of the 103, males affected was 74.75% and females was 25.24%. Commonest type of atherosclerosis was type III (preatheroma) 40.7%. 55.33% showed significant coronary artery disease (type IV -VIII) with atheroma (type IV) as the commonest lesion (23.30%). Left anterior descending artery (LADA) was most frequently involved vessels (46.6%) followed by right coronary artery (RCA) involvement (41.71%). Least frequently involved vessel was left circumflex artery (LCX) (38.83%). 15% had single vessel involvement whereas 37% & 40% cases had two vessels and three vessels involvement respectively. Maximum cases of significant cardiac lesions were associated with advanced atherosclerotic lesions ie. type VI & type VII lesions. Eccentric lesions were more common than concentric lesions.

*Corresponding author: Dr.Shweta Khandelwal., Raja Cycle Colony, Street No. 2, Srinagar Road, Ajmer (Raj.) India Phone: +91 - 9950099334 Email: drshwetakhandelwal0103@gmail.com

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Atherosclerosis and Ischemic Heart Disease

Introduction

Ischemic heart disease (IHD) is defined as acute or chronic form of cardiac disability due to imbalance between supply and demand of oxygenated blood. In more than 90% cases the cause of myocardial ischemia is reduced blood flow due to coronary atherosclerosis. Thus IHD is often termed coronary artery disease (CAD) or coronary heart disease (CHD). [1] CAD is the leading cause of global deaths with about 80% of burden occurring in developing countries [2,3]. In India, CAD has emerged as the single largest disease accounting for nearly one third of all deaths. The incidence of coronary artery disease has doubled during past three to four decades. A total of nearly 6.4 crore cases of coronary artery disease are likely in the year 2015, nearly 96% would be coronary heart disease cases. An estimated 1.3 million Indian died from this in 2000 [4]. The projected deaths from coronary artery disease by 2015 is 2.95 million, of which 14% will be under 30 years and 31% will be under 40 years[5]. The exact global incidence of atherosclerosis is not possible to calculate because it can exist without producing any signs & symptoms. An autopsy study gives a good measure of the prevalence, grading and distribution pattern of atherosclerotic lesions. In order to assess the magnitude of coronary atherosclerosis, a retrospective study of autopsy cases for the presence of atherosclerotic lesions of coronary arteries and associated ischemic cardiac lesions like acute myocardial infarction (MI) / myocardial fibrosis MF) / left ventricular hypertrophy (LVH) was under taken for a period of 1 year from January 2011 to December 2011.

Materials and Methods

The present study was conducted in the Department of Pathology, JLN Medical College, Ajmer (Rajasthan) for a period of 1 year from January 2011 to December 2011. Total 103 postmortem heart specimens, irrespective of cause of death (natural, unnatural or sudden death) were examined grossly and microscopically for presence & extent of coronary atherosclerosis and evidence of acute myocardial infarction / myocardial fibrosis / left ventricular hypertrophy. Autolytic specimens were excluded from the study. The medical history and clinical diagnosis before death in view of the cases were unavailable. The heart was fixed in 10% formalin, weighed and biopsied within 4-24 hours of fixation using standard autopsy protocol of heart grossing. Thickness of right & left ventricular wall was measured. The three main coronary arteries were dissected out, exposed transversely and longitudinally and examined for thickening (concentric/ eccentric), yellow streaks calcification, rupture, etc.

Ventricles were sectioned transversally at 10 mm intervals starting from apex. Gray-white areas suspicious of infarction / fibrosis were identified and taken. After routine processing & paraffin embedding 4Âľ sections were taken and stained with H&E. All the sections were examined microscopically for the presence of atheroma and myocardial infarction / fibrosis. According to American Heart Association, Typing of atherosclerotic plaques was done [6,7]. Type I: Isolated intimal foamy cell (minimal change) Type II: Numerous intimal foamy cells often in layers (fatty streaks) Type III: Pools of extracellular lipids without a well defined core (intermediate lesion or preatheroma) Type IV: Well defined lipid core with luminal surface covered by normal intima (atheroma or fibroplaque ) Type V: Lipid core with a fibrous cap with or without calcification (fibroatheroma) Type VI: Fibroatheroma with cap defect such as haemorrhage or thrombosis Type VII: Calcification prominent Type VIII: Fibrous tissue change prominent The stenosis of coronaries is graded based on the luminal narrowing of the coronaries and is graded from grade 0 (normal) to grade IV (complete obstruction). Grade 0

Normal

Grade I

1-25% stenosis

Grade II

26-50% stenosis

Grade III

51-75% stenosis

Grade IV

76-100% stenosis

Results

Total heart specimens received in the Department of Pathology were 131. Out of these, 26 specimens were autolysed. All the subjects were grouped into specific age groups based on the age at the time of death. The different age groups and sexes were correlated with the degree of atherosclerosis (Table-1) The youngest case was 15 years female and the oldest was 80 years male forming a age range of 15-80 years. Mean age was 39.38 years. Mean age for males was 40.92 years and for females was 35.59 years. (Table-1). Of the 103 heart studied, 74.75% were males quite higher than females (25.24%) forming a ratio of 3:1. (Fig.1).

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Commonest prevalent atherosclerotic lesion was type III (preatheroma) 40.7%. Significant coronary artery disease (type IV -VIII) was seen in 55.53% cases (Fig. 2, 3) with atheroma (type IV) as a common lesion 23.30%. (Table-1) In cases with significant coronary artery disease LADA (left anterior descending artey) was most frequently involved vessels in 46.6% cases followed by RCA (right coronary artery) in 41.71% cases. Least frequently involved vessel was LCX (left circumflex artery) in 38.83%. (Table-2) Among the 103 cases 15% had single vessel involvement whereas two vessels and three vessels were involved in 37% & 40% cases respectively. The effect of myocardial ischemia due to coronary atherosclerosis in terms of acute myocardial infarction was 19 cases (18.44%) (Fig. 4), myocardial fibrosis 15 cases (14.56%) (Fig. 5) & left ventricular hypertrophy 24 cases (23.30%). (Table-3) Maximum cases were seen between 41-60 years of age with a mean age of 49.79 years. Male to female ratio was 3.75:1. Theses cardiac lesions were associated with advanced atherosclerotic lesions ie. grade

VI & grade VII (Table 4). Eccentric lesions were more common than concentric lesion with a ratio of 1:2.16. The mean weight of cardiac heart was 303.88 gms, slightly more in males 305.04 gms than females 291.29 gms.

DISCUSSION

There is an alarming increase in the morbidity and mortality due to coronary atherosclerosis in India. There is no valid method for sampling of living population. The autopsy study provides a means of understanding the basic process which sets a stage for clinically significant atherosclerotic cardiovascular disease. Though our study involved only a small number of cases, most of our observation correlated with many similar studies. In the present study, the overall incidence of significant atherosclerosis was found to be 55.33%, comparable to Shirani et.al [8] (65%) and McGill et al[9] (58%) study. Atherosclerosis begins in childhood & progress to form the lesions that causes coronary heart disease. Males have a relatively preponderance of coronary artery disease than females until the menopause. Oestrogen in females

TABLE 1: Correlation of typing of coronary atherosclerosis with age Age Group (Years)

Type II n

Type III n

Type IV n

Type V n

Type VI n

Type VII n

Type VIII n

11-20

21-30

31-40

41-50

51-60

61-70

71-80

Total

3.8

40.7

23.3

13.5

10.6

6.7

0.97

Table 2: Frequency distribution of atherosclerosis in three major coronary vessels TYPE

LADA

RCA

LCX

7.76%

10.67%

14.56%

III

45.63%

47.57%

46.60%

22.33%

21.35%

19.41%

9.70%

12.62%

10.67%

2.91%

6.79%

VII

2.91%

4.85%

1.94%

VIII

0.97%

IV to VIII Grade

46.60%

41.71%

38.83%

TOTAL

103

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Fig. 1. Age and sex distribution of 103 autopsied cases

Fig. 3 : Fibroatheromatous plaque showing organized thrombus in the lumen (Type VI,Grade IV) H&E, 100X

Fig. 2: Concentric atheromatous plaque showing well defined lipid core (Type IV, Grade II) H&E, 40X

Fig. 4 : Myocardial infarction showing mycytonecrosis with abundant neutrophils H&E, 400X

Fig. 5 : Myocardial fibrosis showing areas of fibrosis in myocardium H&E 400X

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Table 3 : Correlation of significant cardiac lesions with age Age Group (Yrs)

Myocardial Infarction

Myocardial fibrosis

Left ventricular hypertrophy

21-30 31-40 41-50 51-60 61-70 Total

1 2 7 6 3 19 (18.44%)

1 1 5 5 3 15 (14.56%)

3 4 8 5 4 24 (23.30%)

TABLE 4 : Correlation of significant cardiac lesions with degree of atherosclerosis Atherosclerosis typing

Myocardial Infarction

Myocardial Fibrosis

Left ventricular hypertrophy

Total

Percentage

Type II

1.72%

Type III

3.44%

Type IV

20.68%

Type V

13.79%

Type VI

36.20%

Type VII

20.68%

Type VIII

3.44%

have protective effect against atherosclerosis. Moreover, males are more indulged in alcoholism and drinking explaining male preponderance towards development of atherosclerosis. In our study males were affected more (74.75%) than females (25.24%) comparable to other studies done. Garg M et al[4] found coronary atherosclerosis lesions in 80.9% males (93 cases) as compared to 19.1% females (22 cases). Bhargava & Bhargava [10] also reported more prevalence in male 74.8% than females 24.2%. Age group in different studies varied from neonates to 90 year. In our study it was 15 year to 80 year. No case was found in the first decade. The 3rd decade of life appeared to be a watershed line in the pathogenesis of coronary vascular atherosclerosis. Dhruv et al.[11], Garg M et al [4] also reported increased frequency of atherosclerosis from 3rd decade onwards. Stressful, sedentary lifestyle, lack of exercise and poor dietary habits are the important factors for early initiation and development of atherosclerosis in young generation.

But significant coronary artery disease showed atheroma as the commonest lesion (23.30%). These findings correlate to those reported by Virmani et al (37.5%) [12] and Farb et al. (33%) [13] Evidence of coronary involvement in LADA, RCA & LCA was 46.60%, 41.71% and 38.83% respectively which was in concordance with the data given by Sudha et al.[14] who showed LADA as the commonest site for plaque formation (47%), followed by RCA (40%) and LCA (38.1%). Garg M et al [4] also showed the most common involvement of LADA (38.1%) followed by RCA (35.1%) and LCA (34%). In our study triple vessel involvement was the commonest (40%) followed by double and single vessel involvement 37% &15% respectively. It was correlated with the study given by Garg M et al. [4] who also reported triple vessels was the most commonly involved vessels (44.4%). However, Virmani et al. [12] showed single vessel involvement was greater than others (44%).

American Heart Association classified atherosclerotic lesion from type I to type VIII. It was proposed that these lesions progressed from one type to the next. In our study commonest prevalent lesion was preatheroma (40.7%) similar to study of Garg M et al who also showed preatheroma as a commonest lesion (30.9%) [4]. However Dhruv et al [11] showed more cases of atheroma (27.4%) followed by preatheroma (23.8%).).

Ischemic heart disease due to CAD is mainly caused by atherosclerosis . In general slowly developing coronary atherosclerosis of high grade (fibroatheroma) may not cause acute lesions but complications in atherosclerotic plaque in the form of superimposed coronary thrombosis due to plaque rupture or thrombosis may lead to acute attack. Those patients who survived attack showed changes of chronic myocardial ischemia in the form of

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focal or diffuse myocardial fibrosis, so small areas of fibrous scarring are found in elderly patients who have history of attacks of MI years back. Hearts from patients with chronic IHD are usually enlarged and heavy, due to left ventricular hypertrophy and dilation. Myocardial hypertrophy represents compensatory mechanism to meet the cardiac demand as hypoxia is produced by significant coronary atherosclerosis. In our study maximum cases of ischemic heart diseases were seen between 4th-5th decade of life with a mean age of 49.79 years. These lesions were associated with advanced atherosclerotic lesions ie. grade VI & grade VII. Acute MI was seen in 18.44% cases comparable with the observation reported by Bharti Jha et al. (12.3%) [15] and Shiladaria P et al (22.5%)[16]. The average weight of the heart was greater in atherosclerotic heart (303.88 gms) comparable to 327 g by Bertomeu et al. [6] . This is due to increased cardiac work and compensatory myocardial hypertrophy. As atherosclerosis was found to be more in males, the average weight of the male hearts was more than those of female hearts. In our study it was found to be 305.04 gms for males and 291.29 gms for females. Our finding are comparable to the similar finding of Dhruva et al. [11] and Garg M et al.[4] who too found that the average heart weight in males was higher as compared to females.

Conclusion

This study showed unexpectedly high prevalence of atherosclerosis in Ajmer, Rajasthan especially in the relatively young population. Though the incidence of atherosclerois is more common in males as compared to females but in both sexes it is alarming. The study of human atherosclerotic lesion is an extremely difficult task in a living subject and an autopsy study is the best possible way to work on it. Our study aids valuable data to the literature regarding the morphology of atherosclerotic lesion and its relation to the significant cardiac lesion. This study highlights a need of life style change in general public and also calls for screening programmes and prevention and control measures against atherosclerosis from early age.

References

1. Kumar V, Abbas A, Aster J. The Heart. Robbins And Cortans Pathological Basis Of Disease 2015; 9th Ed:523-578. 2. Gaziano TA. Cardiovascular disease in the developing world and its cost effective management. Circulation 2005;112:3547-3553.

3. Beaglehole R, Reddy S, Leeder S. Poverty and human development. The global implications of cardiovascular disease. Circulation 2007;116:1871-1873. 4. Garg M, Aggarwal A, Kataria S. Coronary Atherosclerosis and Myocardial Infarction. An autopsy study. J Indian Acad Forensic Med.2011;33:971-973 5. Indrayan A. Forecasting vascular disease cases and associated mortality in India. NCMH Background Papers : Burden of Disease in India. National commission on macroeconomics and Health, Government of India; 2005.p.197-215. 6. Bertomeu A, García-Vidal O, Farré X, Galobart A. Preclinical coronary atherosclerosis in a population with low incidence of myocardial infarction: cross sectional autopsy study. BMJ 2003;327:591- 2. 7. Stary HC. Natural History and Histological Classification of Atherosclerotic Lesions: An Update. Arterioscler Thromb Vasc Biol 2000;20;1177-1178. 8. Shirani J, Youseti, Roberts WC. Major cardiac findings at necropsy in 366 American octogenarians. Am J Cardiol, 1995:75,151-156. 9. Mcgill HC, Brown BW. Gorel. et al. Grading stenosis in the right coronary artery. Circulation, 1968;37:460-468. 10. Bhargava MK, Bhargava SK. Coronary atherosclerosis in North Karnataka. Indian J Pathol Microbiol. 1975; 18:65-79. 11. Dhruva GA, Agravat AH, Sanghvi HK. Atherosclerosis of coronary arteries as predisposing factor in myocardial infarction: An autopsy study. Online J Health Allied Scs. 2012 11:1 12. Virmani R, Kolodgie FD, Burke AP, Farb A, Schwartz SM. Lessons from sudden coronary death– A comprehensive morphological classification scheme for atherosclerotic lesions. Arterioscler Thromb Vasc Biol 2000; 20: 1262-75 13. Farb A, Tang AL, Burke AP, Sessums L, Liang Y, Virmani R. Sudden coronary death – Frequency of active coronary lesions, inactive coronary lesions and myocardial infarction. Circulation 1995; 92:1701-9 14. Sudha ML, Sundaram S, Purushothaman KR, Kumar PS, Prathiba D. Coronary atherosclerosis in sudden cardiac death: An autopsy study. Indian J Pathol Microbiol 2009; 52: 486-9. 15. Bharti Jha et al. Incidence of coronary atherosclerosis in different coronary arteries and its relation with myocardial infarction: a randomized study in 300 autopsy heart in tertiary care hospital .International journal of Medical Science and Public Health. 2013;2: 836-839. 16. Shiladeria P et al .Coronary atherosclerosis and myocardial infarction, an autopsy study. NJIRM. 2013;4: 106-108.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 02, April - June 2016

Original Article Analysis of Whipple’s Resection Specimens: A Histopathological Perspective Shifa Seyed Ibrahim*, Meena Kumari. G. Department of Pathology, Madurai Medical College, Madurai, India

Keywords: Periampullary, Pancreas, Whipple’s Resection

ABSTRACT Background: Pancreaticoduodenectomy or Whipples’s procedure is done for pancreatic carcinoma, bile duct carcinoma, duodenal carcinoma and periampullary carcinoma. About 5% of the gastrointestinal malignancy is constituted by the ampullary and periampullary carcinoma. Histopathological studies related to the diagnosis, grade, stage, nodal status, marginal status, prognosis and incidence of these tumors are analyzed from the received Whipple’s specimen in our study. Aim of this study is to analyze the incidence of various tumors we encounter in the Whipple’s specimen, to calculate the sex ratio, to grade and to stage the tumors based on the WHO grading system. And to compare the incidence with other studies. Methods: Histopathology records of all the patients who had Whipple’s resection during September 2013 - September 2015 were analyzed. The slides were reviewed and the parameters were calculated. Results: Out of thirty cases, on which Whipple’s resection was done, twenty one had ampullary and periampullary carcinoma, The mean age incidence of ampullary carcinoma calculated was 44 years. The sex ratio of ampullary carcinoma was 1:1. Three had pancreatic tumors and six had chronic pancreatitis. Out of the three cases with pancreatic tumor, two had pancreatic endocrine tumors. They both were female. One had a Solid pseudopapillary pancreatic tumor. Literatures were reviewed and the predominance of ampullary carcinoma was noted in our study in contrast to other studies. Conclusion: In the analysis of the Whipple’s specimen we found out that ampullary adenocarcinoma predominates and there were an equal sex incidence. This is in contrast to other published literatures. This variable needs further evaluation.

*Corresponding author: Dr.Shifa.S. 82,J.N.Nagar, Old Natham Road, Madurai- 625017 TamilNadu, India. Phone: +91 - 9486669274 Email: shifafrin@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

Pancreatoduodenectomy otherwise called Whipple’s surgery was first demonstrated by Allen. O Whipple in 1935.[1] This procedure is done for periampullary carcinoma, ampullary carcinoma, pancreatic tumors, tumors of the pancreatic duct, tumors of the common bile duct, duodenal carcinoma and sometimes for nonmalignant conditions. [2] 80% of the tumors in this region are adenocarcinoma and other malignancies form the rest. Ampullary carcinoma has the histological features of duodenal mucosa and the ducts. Tumors in this region are mostly seen among elderly age group around seventh decade and surgery is the only means of curing them. Because of the intimate location of many structures in this area even a benign lesion can cause obstructive symptoms. [2] Whipple’s surgery had been done on those benign conditions as they mimick malignancy. Histopathology is the gold standard when such situation arises. This study was done to analyze these Whipple’s specimens histopathologically by retrieving the old records and slides and critically analyze and sort them according to the site, size, type, and grade, nodal and marginal status. Along with that, the age and sex incidence was analyzed. The prognostic significance of all these characters was analyzed to get the overall picture of these cases in our hospital setup. Comparison of the incidence of our hospital with other literatures was also attempted.

Materials and Methods

This is a retrospective study of the cases done during September 2013- September 2014. All the cases on which Whipple’s surgery was done for both the malignant and nonmalignant reasons were retrieved from the old records. The details about the gross examination of the specimen were taken from the records. Protocols used in the gross examination: ● When most part of the tumor is located in the ampullary region and bulges into the duodenal mucosa stretching it, it is taken as ampullary carcinoma. Adsay V et al in their study had mentioned that they designate ampullary carcinoma if more than 75% of the tumor was seen in the ampullary region.[3] ● A tumor that involved the circumference of the ampulla was taken as periampullary carcinoma. ● A tumor that involved the circumference of the common bile duct [CBD] was taken as common bile tumors. Longitudinal thickening of the bile duct and granular mucosal surface were taken as clues. Gonzalez RS et al in their study had mentioned that common bile duct tumor constitutes 5% among the

●

● ● ●

tumors of pancreatoduodenal origin. [4] The incidence of CBD tumor is higher among Asians.[3] A tumor with the base or the epicenter in the duodenum and not involving the ampulla was taken as duodenal carcinoma. Non ampullary duodenal carcinoma is different from its duodenal counterpart and the plaque like growth of the non ampullary carcinoma is associated with microsatellite instability. [5] Other gross features like cystic neoplasms of the duct, spongy areas in serous cystadenoma of the pancrea were noted. Tumor size, color, consistency, gross invasion and measurements were noted. Nodes- Number and size were noted. Homogenous white gross appearance was taken as a clue for pseudo tumors. Most of the benign lesion occurs around the pancreatic head and the periampullary region. They cause obstructive symptoms mimicking carcinoma leading on to Whipple’s surgery.[2]

The slides were reassessed. Histopathological categorization, grading, tumor budding, staging, nodal status, perineural invasion, angioinvasion and marginal status were assessed. The grading of adenocarcinoma was done based the percentage of glands seen in the tumor tissue. If there were >95% glands it was taken as well differentiated, 5095% glands as moderately differentiated grade, 5-49% as poorly differentiated grade and ,5% as undifferentiated adenocarcinoma. The staging of the Ampullary carcinoma was based on AJCC TNM classification.T1 – If the tumor is limited to the ampulla or sphincter of Oddi. T2- If the tumor invades the duodenal wall.T3- If the tumor invades the pancreas and T4- If the tumor invades the peripancreatic soft tissue or adjacent structures.N1- If there is regional nodal metastasis. In the case of Endocrine neoplasm, the following staging was followed. T1- If the tumor is limited to the pancreas and it is less than 2cm in diameter. T2 -If the tumor is restricted to the pancreas and size is between 2-4 cm. T3 - If the tumor is more than 4 cm diameter if it is limited to the pancreas or if the tumor invades the duodenum or the bile duct. T4- If the tumor invades the adjacent organs. N1- If the regional nodes are involved by the tumor. In case of the solid pseudo papillary tumor T1- When the tumor is limited to the pancreas and was less than 2cm in diameter.T2- When the tumor is limited to the pancreas and more than 2cm in diameter. T3- When the tumor invades duodenal, peripancreatic tissue and the bile duct. T4- When the tumor invades the other structures.

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N1a- When a single node is involved. N1b- Multiple regional nodes were involved.

38% of the cases were well differentiated grade and 62% were moderately differentiated grade. Poorly differentiated grade was not observed in our study [Table 3].

Result

In our study, 68% were in stage two [Table4]. Only 10% of the cases showed metastatic deposits in the nodes and 10% of the ampullary carcinoma showed angioinvasion [Fig 3] [Table 5]. Margins were free of tumor invasion in all the cases.

Thirty Whipple’s specimen was received during our study period. Out of that, twenty one had ampullary carcinoma and periampullary carcinoma. It constitutes around 70% of the tumors in our study. When the age incidence of ampullary carcinoma was calculated the mean age of occurrence in our study was 44 years [Table 1]. The youngest case in our study was a 35 year old female. Neither familial clustering nor familial syndromes were seen in our study. When the sex ratio was analyzed among the patients with ampullary carcinoma, the male to female ratio in our study group was almost 1:1 [Table 1].

Pancreatic endocrine tumor was the second commonest tumor we encountered while analyzing the Whipple’s specimen. In our study, both the cases with the pancreatic endocrine tumor were females [Fig 4]. They were 40 and 42 years old with the mean age of 41 years. Both the tumor was more than 2cm and they were in stage T2 [Table 2]. Both were nonfunctional and showed angioinvasion and neural invasion [Fig 5]

The mean size of the ampullary tumor in our study was 2.4cm [Table 2]. In 89.5% of the cases of the ampullary carcinoma was of intestinal type [Fig1] and 10.5% of the cases were of pancreatobilliary type [Fig 2]. Among them, Table 1: Age and Sex distribution Lesions Ampullary Carcinoma Periampullary Carcinoma Pancreatic Endocrine Tumor Solid Pseuopapillary Tumor Chronic Pancreatitis

35-40 Years 3 1 1 -

41-50 Years 8 1 -

Whipple’s surgery done in six cases presumed of malignancy was diagnosed as chronic pancreatitis in our study. 51-60 Years 3 1 2

Table 2: Tumor size distribution in the Whipple’s specimen Tumors 1-2cm Ampullary carcinoma 9 Periampullary carcinoma 1 Pancreatic Endocrine Tumor Solid Pseudo Papillary tumor -

61-70 Years 2 1 2

>70 Years 3 -

2.1-3cm 7 1 -

3.1-4cm 2 1 1 1

Male

Female

9 2

10 2 2 1 4

>4.1cm 1 -

Table 3: The distribution of type and different grades among the ampullary carcinoma Type Intestinal Pancreatobilliary

Well Differentiated grade 8 -

Moderately differentiated grade 9 2

Poorly differentiated grade -

Table 4: Distribution of stages in Ampullary carcinoma [No. Of Cases] T1

Table 5: Angioinvasion, Neural invasion and Nodal status Tumors

Angio Invasion

Neural Invasion

Ampullary

Peri Ampullary

Endocrine Solid Pseudo papillary tumor

1 1

1 -

2 1

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Fig. 1: Shows tumor cells arranged in villoglandular pattern – Ampullary carcinoma – Intestinal type [10x H&E].

Fig. 3: Shows angioinvasion in ampullary carcinoma [4x H&E] Inset shows a closer view [40x H&E].

Fig. 2: Shows Pancreatobilliary type of ampullary carcinoma in which tumor cells are arranged in a glandular pattern in a desmoplastic stroma infiltrating the pancreas [4x H&E]

Fig. 4: Shows endocrine tumor of the Pancreas in which the tumor cells are arranged in cords, nests and trabeculae [4x, H&E].

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Fig. 5: Shows neural invasion [10x H&E].

Fig. 6&7: Shows tumor cells arranged around the blood vessels and in some areas they show pseudopapillary arrangementSolid Pseudopapillary tumor [10x and 40x respectively H&E].

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Discussion

Whipple’s surgery is done for the tumors involving the ampullary region, periampullary region, common bile duct, duodenum and the pancreas and its ducts. Ampullary region as such is unique as it contains both duodenal and the ductal epithelium. The normal ampullary mucosa is identified as paler columnar cells admixed with many goblet cells that indent the duodenal mucosa. It is composed of more complex and branched submucosal glands. Distinguishing the intramucosal or insitu involvement from the invasive ampullary carcinoma is difficult for the young pathologists. Lobular glandular architecture with lamina propria surrounding the glands, the rounded glands and absent stromal response are the clues to rule out the invasive component when such dilemmas arise. In our study ampullary carcinoma was more prevalent among the Whipple’s specimen [70%]. According to Saraee et al and Landis S H et al, pancreatic adenocarcinoma was most common among the Whipple’s specimen in the western population.[1, 6] Ampullary carcinoma constitutes 16-20% of all the carcinomas of the periampullary region, according to Duffy et al, Talamini et al and Howe et al which is contrast to our study. [7-9] Ampullary adenocarcinoma constitutes about 0.2% of all the gastrointestinal tumors. [10] It constitutes 6% among the tumor of the periampullary region. [9] It is associated with Familial Polyposis syndrome and neurofibromatosis and K ras mutation.[11-14] Klimstra et al had proposed the diagnostic criteria for ampullary carcinoma.[15] According to them, the tumor should be called as ampullary when the epicenter is in the ampulla and there should be a pre invasive lesion in the ampulla. The tumor that grows circumferentially around the ampulla is called periampullary carcinoma. Ampullary tumors are predominantly seen in males in their seventh decade. Henson et al in their study had mentioned that the age incidence of ampullary carcinoma was 69.7 years. [16] Howe et al in their study had mentioned that mean age incidence in their study was 65.6 years. [9] The youngest case in Howe et al’s study was 28.3 years old with a history of familial adenomatosis syndrome.[9] In Yeo JC et al’s study, the mean age of occurrence was 64 years with a male predominance.[17] In contrast, the mean age incidence in our study was 44 years and there was equal sex incidence. Yeo JC et al in their study had mentioned that tumor diameter was smaller for ampullary carcinoma [17] In Howe et al’s study, mean size of the ampullary tumor was 2.7cm which correlated with our study.[9] Histopathologically, the ampullary carcinoma can be of intestinal type, pancreatobilliary type, mixed type and undifferentiated type. Categorization is important

because the prognosis of the intestinal type is better than pancreatobilliary type. [18] 66% of ampullary carcinoma seen in Howe et al’s study was of intestinal type and 27% was of pancreatobilliary type which is in correlation with our study.[9] In their study well and moderately differentiated graded tumors predominated as seen in our study.[9] According to Yeo JC et al’s study, well differentiated tumors were uncommon.[17] The nodal involvement was 10% in our study. In contrast, the nodal metastasis ranged from 29% to 52% in Warren KW et al’s study and 40% in Allema JH et al’s study. [19,20] Carcinoma mimics include adenomyoma of the ampulla, papillary hyperplasia, sclerosing papillitis and anatomic pancreatitis. The adenomyoma or adenomyomatous hyperplasia of the ampullary is larger than 0.5cm and contains complex glands, arranged in a lobular architecture surrounded by the lamina propria and muscle bundle. Its presentation in the elderly individual and its obstructive symptoms may point towards malignancy, but it is distinguished by its architecture and lack of dysplasia and mitosis. [21,22] Other differential includes papillary hyperplasia. Again, this one also lacks atypia and it is an incidental finding and does not produce any symptoms. But when papillary hyperplasia is secondary to cholelithiasis or ampullary inflammation, then reactive atypia may be seen mimicking carcinoma. Sclerosing papillitis is associated with IgG4 group mediated autoimmune disorder. It causes swelling in the ampullary region. [23] Marked atypia seen in the duodenal mucosa surrounding this lesion can mimic carcinoma. Paraduodenal pancreatitis otherwise called anatomical pancreatitis is another symptomatological mimicker for ampullary carcinoma. It causes obstructive symptoms and it is common in the middle aged alcoholic. [24] Treatment for insitu carcinoma and micro invasive carcinoma of the ampullary region is transduodenal ampullectomy and Whipple’s surgery is for the invasive tumors. [25] The prognosis of the ampullary carcinoma is better when compared with ductal carcinoma or pancreatic carcinoma. [26,27] Other prognostic indicators are the stage of the disease, tumor budding, margin free status, MIB index, invasion and DNA ploidy. [28-30] In Howe et al’s study, size more than 2cm, pancreatobilliary type, perineural invasion, angioinvasion, positive margins and positive nodal involvement were associated with decreased in the median survival rate. [9] The incidence of pancreatic endocrine tumor is 5.25/100 000/year. [20] The incidence of pancreatic endocrine neoplasm is less than 3% of all the pancreatic neoplasms according to Henson et al. [16] They have also

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mentioned that ductal carcinoma is more common among the pancreatic neoplasm. But in our study, we did not come across a single pancreatic ductal carcinoma. Mean age of pancreatic endocrine tumors was 55 – 60 years and younger age incidence are associated with MEN syndrome and VHL disease according to Oberg et al. [31] Male predominance was seen according to Oberg et al. [31] It may be a functional or nonfunctional tumor and the majority is nonfunctional. The prognosis of the nonfunctional tumor was inferior according to Halfdanarson et al. [32] Histologically, it is composed of uniform round cells with dispersed chromatin arranged in nests, trabeculae and in festoons. Psammoma bodies and amyloid like material are seen. Mitosis are less than 10/HPF and there is no necrosis in well differentiated tumors. Worst prognostic factors include poor differentiation, extremes of age, functionality, increased size, metastasis, necrosis, increases mitosis, vascular and neural invasion, CD10 and CD19 expression. Resection is the treatment of choice. It is an indolent tumor with malignant potential. Compared with the adenocarcinoma of the pancreas, endocrine tumors have better prognosis. [32]

Six cases were diagnosed as chronic pancreatitis on the Whipple’s specimen suspected as periampullary carcinoma. De la Fuente SG in their study had mentioned that in the Whipple’s specimen they have received they have encountered 7% benign cases.[36] Endoscopic biopsies have limited diagnostic accuracy in case of ampullary carcinoma. [37] FNAC and other investigative modalities have limited application in accurately diagnosing the tumors in this area because of the complex and intimate anatomy of the ampullary area. [2]

Solid Pseuopapillary tumor is a tumor of the young female and middle aged women. Patel et al in their study had mentioned that this tumor is prevalent in young female with the median age of 20 years. [33] Martin RC et al in their study had mentioned the medial age as 39 years, which correlated with our study. [34] It is a cystic and solid neoplasm involving the head and tail of the pancreas. Histopathologically, this tumor has an appearance of the endocrine neoplasm composed of small round cell crowding around the blood vessels. The extensive necrosis of the cells that are away from the blood vessels gives it a pseudo papillary appearance. Individual cells are smaller with oval and folded nucleus. Mitosis is few in number. This tumor expresses beta catenin, Vimentin, CD10 and CD56. [35] This is a tumor of intermediate malignant potential with frequent metastasis to the liver. [33] For localized tumor surgery is the treatment and for metastatic tumors, aggressive management is required.

Conclusion

When the incidence of various carcinomas diagnosed on the Whipple’s specimen by us was compared to Yeo et al, Chan C et al and Michelassi et al’s study pancreatic ductal carcinoma were more prevalent in their study groups in contrast to our study. [17, 38, and 39] [Table 6]. The age incidence of their study groups was around 65 years. But in our study it was 44 years. Equal sex incidence was noticed in our study in contrast to the male predominance in their study. This may be due to the difference in the genetic makeup or other etiological factors which needed further studies. As we analysed the Whipple’s specimens, many questions were raised. All the reviewed literatures had mentioned that pancreatic ductal carcinoma as the commonest tumor in the periampullary area. But ampullary carcinoma was predominant in our study. Ampullary carcinoma was seen in a relatively younger age group than the global age incidence and there was an equal sex incidence in contrast to the literature reports of male predominance. Surprisingly, pancreatic ductal carcinoma was not seen in our study. More studies are needed to analyze the genetic makeup, dietary or environmental factors among our people that are responsible for this contrast.

Acknowledgements Funding None

Table 6: A Comparative analysis of our study with other’s studies Studies

Our study Howe et al9 Yeo et al17 Chan C et al38 Michelassi F et al39

Ampullary carcinomaIntestinal type 63% 76%

Ampullary carcinomaPancreatobilliary type 6.7% 27%

Duodenal Bile duct Pancreatic carcinoma carcinoma ductal carcinoma -

19% 76%

7% 3% 2.5%

4.3%

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Pancreatic Benign Median Endocrine lesions age tumor

Sex predominance

Solid Pseudo Papillary tumor 3.3% -

6.7% -

20% -

Equal sex ratio Male

12% 5%

62% 15%

44 years 65.6 years 64 Years 65 years

6.2%

85%

60.5 years

Male

Male Male

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Whipple’s Resection Specimens

Competing interests None

Reference

1. Saraee A, Vahedian-Ardakani J, Saraee E, Pakzad R and Wadji M B. Whipple procedure: a review of a 7-year clinical experience in a referral center for hepatobiliary and pancreas diseases. Saraee et al. World Journal of Surgical Oncology 2015; 13:98 2. Crothers JW, Zhao L, Wilcox R .Benign is a Relative Term: the Whipple Resection in Non-Oncologic Cases. Ann Clin Pathol 2014:2:1019. 3. Adsay NV, Basturk O, Saka B, Bagci P, Ozdemir D, Balci S, et al. Whipple Made Simple For Surgical Pathologists. Am J Surg Pathol. 2014; 38(4): 480–493. 4. Gonzalez RS, Bagci P, Kong KT, et al. Distal common bile duct adenocarcinoma: analysis of 47 cases and comparison with pancreatic and ampullary ductal carcinomas Mod Pathol. 2012; 25:109. 5. Saka B, Bagci P, Krasinskas A, et al. Duodenal carcinomas of non-ampullary origin are significantly more aggressive than ampullary carcinomas. Mod Pathol. 2013; 26(2S):176. 6. Landis SH, Murray T, Bolden S, et al. Cancer statistics. CA Cancer J. Clin.1999; 49:8. 7. Duffy JP, Hines OJ, Liu JH, Ko CY, Cortina G, Isacoff WH, et al. Improved survival for adenocarcinoma of the ampulla of vater: fifty-five consecutive resections. Arch Surg. 2003; 138:941-950. 8. Talamini MA, Moesinger RC, Pitt HA, Sohn TA, Hruban RH,Lillemoe KD, et al. Adenocarcinoma of the ampulla of vater. A 28-year experience. Ann Surg. 1997; 225: 590-600. 9. Howe JR, Klimstra DS, Moccia RD, Conlon KC, Brennan MF. Factors predictive of survival in ampullary carcinoma. Ann Surg. 1998; 228(1): 87-94. 10. Roder JD, Schneider PM, Stein HJ, Siewert JR. Number of lymph node metastases is significantly correlated with survival in patients with radically resected carcinoma of the ampulla of Vater. Br J Surg 1995; 82: 1693-1696. 11. Costi R, Caruana P, Sarli L, Violi V, Roncoroni L, Bordi C. Ampullary Adenocarcinoma in Neurofibromatosis Type1. Case Report and Literature Review. Mod Pathol 2001; 14(11):1169–1174. 12. Pauli RM, Pauli ME, Hall JG. Gardner syndrome and periampullary malignancy. Am J Med Genet 1980; 6:205-219.

13. Colarian J, Pietruk T, LaFave L, et al. Adenocarcinoma of the ampulla of Vater associated with neurofibromatosis. J Clin Gastroenterol 1990;12:118-119. 14. Howe JR, Klimstra DK, Cordon-Cardo C, et al. K-ras mutations in adenomas and carcinomas of the ampulla of Vater. Clinical Cancer Research 1997; 3: 129-134. 15. Albores-Saavedra J, Henson DE, Klimstra DS. Tumors of the Gallbladder, Extrahepatic Bile Ducts, and Ampulla of Vater. Washington, DC: Armed Forces Institute of Pathology.Atlas of Tumor Pathology2000; 3rd series, fascicle 27. 16. Henson E D, Schwartz M A, Nsouli H, AlboresSaavedra J, Carcinomas of the Pancreas, Gallbladder,Extrahepatic Bile Ducts, and Ampulla of Vater Share a Field for Carcinogenesis. Arch Pathol Lab Med. 2009; 133:67–71. 17. Yeo J C, Sohn A T, Cameron L J, Hruban H R, Uiemoe DK, Pitt A H, Periampullary Adenocarcinoma analysis of 5-Year Survivors. Ann. Surg. 1998; 227(60): 821-831. 18. Westgaard A, Tafjord S, Farstad N I, Cvancarova M, Eide J T, Mathisen O et al. Pancreatobiliary versus intestinal histologic type of differentiation is an independent prognostic factor in resected periampullary adenocarcinoma. BMC Cancer 2008, 8:170 19. Warren KW, Choe DS, Plaza J, Relihan M. Results of radical resection for periampullary cancer. Ann Surg 1975; 181: 534-540. 20. Allema JH, Reinders ME, van Gulik TM, et al. Results of Pancreaticoduodenectomy for ampullary carcinoma and analysis of prognostic factors for survival. Surgery 1995; 117: 247-253. 21. Al Jitawi SA, Hiarat AM, Al-Majali SH. Diffuse myoepithelial hamartoma of the duodenum associated with adenocarcinoma. Clin Oncol 1984; 10: 289-93. 22. Bergdahl L, Andersson A. Benign tumors of the papilla of Vater. Am Surg 1980; 46: 563-6. 23. Sahin P, Pozsar J, Simon K, et al. Autoimmune pancreatitis associated with immune-mediated inflammation of the papilla of Vater: report on two cases. Pancreas 2004; 29: 162-166. 24. Adsay NV, Basturk O, Klimstra DS, et al. Pancreatic pseudotumors: non-neoplastic solid lesions of the pancreas that clinically mimic pancreas cancer. Semin Diagn Pathol 2005; 21: 260-267. 25. Shutze WP, Sack J, Aldrete JS: Long-term followup of 24 patients undergoing radical resection for ampullary carcinoma, 1953 to 1988. Cancer 1990; 66:1717-1720.

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32. Halfdanarson TR, Rabe, K, Rubin J, Petersen GM. Pancreatic endocrine tumors (PETs): Incidence and recent trend toward improved survival. Presented at the 2007 Gastrointestinal Cancers Symposium; Orlando, FL. 2007

33. Patil T B, Shrikhande S V, Kanhere H A, Saoji R R, Ramadwar M R, Shukla PJ, Solid pseudopapillary neoplasm of the pancreas: a single institution experience of 14 cases HPB, 2006; 8: 148-150. 34. Martin RC, Klimstra DS, Brennan MF, Conlon KC. Solid-pseudopapillary tumor of the pancreas: A surgical enigma? Ann Surg Oncol. 2002; 9:35-40. 35. Abraham SC, Klimstra DS, Wilentz RE, Yeo CJ, Conlon K, Brennan M, Cameron JL, Wu T-T, Hruban RH: Solid–pseudopapillary tumors of the pancreas are genetically distinct from pancreatic ductal adenocarcinomas and almost always harbor betacatenin mutations. Am J Pathol 2002; 160:1361-1369. 36. De la Fuente SG, Ceppa EP, Reddy SK, Clary BM, Tyler DS, Pappas TN. Incidence of benign disease in patients that underwent resection for presumed pancreatic cancer diagnosed by endoscopic ultrasonography (EUS) and fine-needle aspiration (FNA). J Gastrointest Surg. 2010; 14: 1139-1142. 37. Asbun HJ, Rossi RL, Munson JL. Local resection for ampullary tumors. Is there a place for it? Arch Surg 1993; 128: 515-20. 38. Chan C, Herrera F M, de la G, Quintanilla-Martinez L, Vargas-Vorackova F, Richaud-Patin Y. Clinical Behavior and Prognostic Factors of Periampullary Adenocarcinoma. Annals of surgery 1995; 222(5): 632-637. 39. Michelassi F, Erroi F, Dawson J P, Pietrabissa A, Noda S, Handcock M et al. Experience with 647 Consecutive Tumors of the Duodenum, Ampulla, Head of the Pancreas, and Distal Common Bile Duct. Ann. Surg. 1989; 210(4): 544-554.

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27. Ohike N, Coban I, Kim GE, Basturk O, Tajiri T, Krasinskas A et al: Tumor budding as a strong prognostic indicator in invasive ampullary adenocarcinomas. Am J Surg Pathol 2010; 34:1417-1424. 28. Sessa F, Furlan D, Zampatti C, Carnevali I, Franzi F, and Capella C: Prognostic factors for ampullary adenocarcinomas: tumor stage, tumor histology, tumor location, immunohistochemistry and microsatellite instability. Virchows Arch 2007; 451:649-657. 29. Shyr Y, Su C, Wu L, Fen-Yau Li A, Chiu J, Wu C et al. Prognostic Value of MIB-1 Index and DNA Ploidy in Resectable Ampulla of Vater Carcinoma. Annals of surgery 1998; 229, (40): 523-527. 30. Carriaga MT, Henson DE. Liver, gallbladder, extrahepatic bile ducts, and pancreas. Cancer 1995; 75:171–190. 31. Öberg K, Knigge U, Kwekkeboom D, Perren A on behalf of the ESMO Guidelines Working Group. Neuroendocrine gastro-entero-pancreatic tumors: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up Ann Oncol 2012;23 (7): 124-130.

Case Report Angiomyomatous Hamartoma; A True Lesion or a Vascular Compensatory Hyperplasia with Muscularization? : An Interesting Case Report Rakesh Kumar Gupta1, Kavita Gaur1, R K Jindal3, Ravindra Kumar Saran1*, Sachin Agrawal2 Department of Pathology, G B Pant Institute of Postgraduate Medical Education and Research, Delhi, India Department of Radiology, G B Pant Institute of Postgraduate Medical Education and Research, Delhi, India 3 Department of Surgery, Maulana Azad Medical College, New Delhi, India

Keywords: Angiomyomatous, Hamartoma, Lymph Node, Lymphedema.

ABSTRACT Angiomyomatous hamartoma is a rare entity of unknown etiology preferentially involving lymph nodes of the inguinal region. We report a case of 20-year-old young male presenting with unilateral lower limb lymphedema. Doppler study of the limb revealed venous flow in the node. Histopathology of the lymph node showed a peculiar replacement of normal hilar anatomy by a haphazard conglomerate of thick walled vessels containing red blood cells and lymphocytes. The present report explores the pathogenesis of angiomyomatous hamartoma with review of literature.

*Corresponding author: Dr. Ravindra Kumar Saran, MD, DNB (Pathology) Professor, Dept of Pathology, Academic Block, G B Pant Institute of Post Graduate Medical Education and Research, Jawaharlal Nehru Marg, New Delhi -110002. INDIA. Phone: +91 97185 99074 E-mail: ravindraksaran@hotmail.com

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Gupta et al.

Introduction

Angiomyomatous hamartoma (AMH) was first described by Chan et al in 1992.[1] It is a rare entity primarily involving inguinal lymph nodes and characterized by replacement of lymphoid follicles by haphazardly arranged combination of smooth muscle bundles, fibrous tissue and thick walled blood vessels. Other less commonly involved lymph nodes are popliteal, cervical, and sub-mandibular. [2-4] Allen et al [5] first described the presence of an adipose tissue component in AMH, while Magro et al [6] termed such variant as an angiomyolipomatous hamartoma (AMLH).

Case report

A 20-year-old young male presented to our hospital with the chief complaints of recurrent subcutaneous swelling of the left lower limb and mild pain in both the limbs since six years. The frequency of these episodes gradually increased over time. No history of injury or filariasis was documented. The patient is a professional tailor and uses his right leg vigorously to propel the footpad of his sewing machine. On examination, bilateral superficial inguinal lymph nodes were palpable, non-tender, and firm in consistency. Right sided lymph node was solitary and measured 4x3 cm; while left sided node measured 1.5x1 cm.Varicose veins were absent. A fine needle aspiration was attempted which yielded inadequate material for any conclusive remark. The colour doppler revealed enlarged lymph nodes with thickened capsule displaying venous flow within the nodes (Fig 1). Bilateral femoral vessels were patent and showed normal flow. As the right inguinal nodes were much larger in comparison to the left, excision was planned on right side. Pathological findings: A lymph nodal specimen measuring 3x1.5x0.4 cm was received. Externally the capsule was intact and thickened. The cut-section was firm and fibrosed. Microscopy showed thickening of lymph node capsule and replacement of the hilum by a disorganized mass comprising of vascular channels with thick smooth muscle bundles traversing haphazardly in fibro-adipose tissue. Both RBCs and lymphocytes were noted in the lumina of vessels. These vessels were interpreted as abnormal thick walled lymphatic afferent channels. Normal calibre veins and thick walled veins were also noted along with many capillaries. Fibrosis was extending into the peripheral cortex with few remnant follicles in the sub-capsular region (Fig 2). No necrosis, granulomas or parasite were found. CD34 and HMB-45 immunohistochemistry for assessing number of pericytes and to rule out angiomyolipoma respectively were non-contributory. CD31 and smooth muscle actin (SMA) were positive in endothelial cells, and smooth muscle cells of the abnormal vessels respectively (Fig 3). www.pacificejournals.com/apalm

A-57 The case was reported as hyperplastic muscularization of large afferent lymphatics of inguinal node secondary to recurrent lower limb edema with ultrasound doppler evidence of veno-lymphatic anastamosis.

Discussion

AMH is a rare benign entity of unknown aetiology. Till date about 35 cases have been reported in medical literature. Earlier cases of inguinal node AMH with concomitant lymphedema of ipsilateral limb have been reported. [7, 8] Bourgeois et al [7] suggested that the destruction of nodal sinuses by vascular and stromal alterations may represent impairment of the lymphatic flow and contribute to the development of lymphedema. However, Sakurai et al [9] hypothesized that impaired lymphatic flow is not just a consequence of hamartoma but may actually play a role in its pathogenesis. We also support this hypothesis as in our case due to higher levels of physical activity in the right limb for occupational reasons this may have led to an increased overall venous and lymphatic drainage. Consequently, right side inguinal lymph nodes were markedly enlarged to compensate the flow, resulting in less edema of the right leg. Channer et al postulated that a haphazard smooth muscle component within the hilum reÂsults from the reparative reaction to inflammation which was later supported by Chan et al. [1, 10] Kim et al [11] in their case reported a popliteal nodal AMH in association with pigmented villonodular synovitis (PVNS) and concluded that macrophages, cytokines and growth factors released from the PVNS leÂ sion reached the adjacent popliteal lymph nodes through the drainÂing lymphatics leading to the development of AMH. However, none of the aforementioned case reports described any evidence of inflammatory reaction within the lymph nodes. We too have not found inflammatory reaction within the lesion. In this case the colour doppler showed prominent dilated

Fig. 1: Colour doppler image showing a large lymph node measuring 3x1.5 cm with echogenic centre and cluster of color signals in the hilum of the node.

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Angiomyomatous Hamartoma; is it a True Entity?

Fig. 2: Photomicrographs of the lymph node biopsy showing compressed peripheral remnant lymphoid follicles and expanded hilum with numerous haphazardly arranged dilated and thickened lympho-vascular channels surrounded by smooth muscle bundles, fibrosis and adipose tissue (a HEx40), high power image of the hilar area showing dilated tortuous vessels filled with both RBCs and lymphocytes (b HEx100).

Fig 3: Immunohistochemistry images showing. a: CD31 positivity in the endothelial cells of the capillaries and small vascular channels, b: Desmin stain highlighting traversing thick smooth muscle bundles, c: SMA stain showing positivity in both smooth muscle and endothelial cells, d: HMB-45 stain negativity in all the components.

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Gupta et al. vasculature with increased flow within the node which on histopathology revealed dilated haphazard muscularized afferent lymphatics, likely causing lymphedema.

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1. Chan JK, Frizzera G, Fletcher CD and Rosai J. Primary vascular tumors of lymph nodes other than Kaposi’s sarcoma. Analysis of 39 cases and delineation of two new entities. Am J Surg Pathol. 1992;16:335-350.

2. Ghosh P, Saha K, Ghosh AK. Vascular transformation of bilateral cervical lymph node sinuses: a rare entity masquerading as tumor recurrence. J Maxillofac Oral Surg. 2015;14:397-400. 3. Prusac IK, Juric I, Lamovec J, Culic V. Angiomyomatous hamartoma of the popliteal lymph nodes in a patient with Klippel-Trenaunay syndrome: case report. Fetal Pediatr Pathol. 2011;30:320-4. 4. Barzilai G, Schindler Y, Cohen-Kerem R. Angiomyomatous hamartoma in a submandibular lymph node: a case report. Ear Nose Throat J. 2009;88:831-2. 5. Allen PW, Hoffman GJ. Fat in angiomyomatous hamartoma of lymph node. Am J Surg Pathol. 1993;17:748-749. 6. Magro G, Grasso S. Angiomyomatous hamartoma of the lymph node: case report with adipose tissue component. Gen Diagn Pathol. 1997;143:247-249. 7. Bourgeois P, Dargent JL, Larsimont D, et al. Lymphoscintigraphy in angiomyomatous hamartomas and primary lower limb lymphedema. Clin Nucl Med. 2009;34:405-9. 8. Piedimonte A, De Nictolis M, Lorenzini P, Sperti V, Bertani A. Angiomyomatous hamartoma of inguinal lymph nodes. Plast Reconstr Surg. 2006;117:714-6. 9. Sakurai Y, Shoji M, Matsubara T, et al. Angiomyomatous hamartoma and associated stromal lesions in the right inguinal lymph node: a case report. Pathol Int. 2000;50: 655-659. 10. Channer JL, Davies JD. Smooth muscle proliferation in the hilum of superficial lymph nodes. Virchows Arch A Pathol Anat Histopathol. 1985;406:261-70. 11. Hyun-Soo Kim, Ki Yong Na Jae-Hoon Lee, Nam Su Cho, Gou Young Kim, Sung-Jig Lim. Angiomyomatous hamartoma of popliteal lymph nodes occurring in association with diffuse pigmented villonodular synovitis of knee. The Korean Journal of Pathology. 2011;45:S58-61. 12. Dzombeta T, Francina M, Matković K, et al. Angiomyolipomatous hamartoma of the inguinal lymph node--report of two cases and literature review. In Vivo. 2012;26:459-62.

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Adipose tissue within AMH may represent an associated additional hamartomatous component or result from metaplastic change. Lymphangiomyomatosis usually involve lungs but may also rarely involve lymph nodes. Angiomyolipoma (AML) usually involves retroperitoneal lymph nodes, which should be differentiated from AMH. [12] In comparison to AMH, the smooth muscle cells show positive immunostaining for HMB-45 in lymphangiomyomatosis and AML. Surgical resection is the best treatment and till date no recurrence has been reported. Though earlier reports have not highlighted the exact clinical significance, AMH should always be considered as a remote possibility causing inguinal swelling with or without lymphedema. To conclude, it is difficult to put this case in a watertight category of AMH. This lesion might result from physiological compensatory changes in lymphatic flow. The resultant pathology is same and accompanied with alteration in lympho-vascular flow, compensatory hyperplasia and muscularization of hilar lymphatic vessels. Present knowledge and literature review did not support clearly one or the other theory. The pathway of draining channels and changes thereafter acknowledged both the view points and hence it should not be put in one category.

Acknowledgement

We would like to acknowledge Miss Dolly for technical help in immunohistochemistry.

Funding None

Competing Interests Not declared

References

Case Report Adenoid Cystic Carcinoma of Floor of The Mouth with Periumbilical Cutaneous Metastasis Reena Sinha, Pallavi Agrawal*, Akash Kumar Singh Department of Pathology, Mahavir Cancer Institute & Research Centre Patna (Bihar), India

Keywords: Adenoid Cystic Carcinoma, Cutaneous Metastasis, Differential Diagnosis, Immunehistochemistry

ABSTRACT Cutaneous metastasis from salivary gland adenoid cystic carcinoma (ACC) is extremely rare. We report a rare case of cutaneous metastasis from ACC of floor of the mouth. The presentation of cutaneous metastasis is nonspecific. Fine needle aspiration cytology becomes a quick and effective tool for the diagnosis in such cases. The clinical, histopathological differential diagnosis and immune-histochemical features are described and discussed. This brief report highlights the importance of clinical awareness of cutaneous metastasis after a long time lag which may mimic adnexal lesions.

*Corresponding author: Dr. Pallavi Agrawal, Department of Pathology, Mahavir Cancer Sansthan, Phulwarisharif, Patna â&#x20AC;&#x201C; 801505 (Bihar), India Phone: +91 7739165410 E-mail: dr.pallavimamc@gmail.com

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Sinha et al.

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Introduction

Adenoid cystic carcinoma (ACC) is a salivary gland tumor characterized by slow and infiltrative growthwith a relatively favorable survival rate of 5 years, but poor long term prognosis due to frequent late, local and distant recurrences[1]. Distant metastasis involves mainly the lungs followed by bone, liver and brain[2]. Cutaneous metastasis is very rare. This case highlights the importance of detailed clinical and dermatological examination in patients treated for salivary gland adenoid cystic carcinoma.

Case Report

A 45-year old female presented with a painless swelling in the floor of the mouth for past one year. It was gradually progressive. There was no cervical lymphadenopathy or any other complaints. Fine needle aspiration cytology (FNAC) was performed which revealed moderately cellular smears with groups of monomorphic cells associated with round to oval globules of matrix material (Fig 1). The background showed scattered single cells. Based on this morphology a diagnosis of adenoid cystic carcinoma was considered. The patient underwent segmental mandibulectomy with extended supra-omohyoid dissection (SOHD) with composite resection of tumor. Gross examination revealed a poorly circumscribed and non-capsulated solid mass measuring 4Ă&#x2014;4Ă&#x2014;3cm. Cut section was solid and grey white in color. On microscopic examination the tumor cells showed cribriform pattern with islands of basaloid cells surrounding variably sized cyst like spaces (Fig 2). Individual tumor cells were angulated with hyperchromatic nucleus and scant amount of eosinophilic cytoplasm. Perineural invasion was not observed.

Discussion

ACC is one of the commonest malignant salivary gland tumors representing approximately 7.5% of the salivary gland malignancies[3]. It is characterized by an indolent clinical course and poor long term prognosis. The incidence of distant metastasis varies between 24% and 52%[4]. The most common sites for distant metastasis are lungs, liver and brain however subcutaneous metastasis is extremely rare. The incidence of subcutaneous metastasis is 2% in men and 1% in women [5]. Sometimes subcutaneous tissue may be involved due to direct extension of the tumor.The exact mechanism of cutaneous metastasis in ACC of salivary gland is incompletely understood. Several hypotheses have been postulated. The cutaneous

Fig. 1: Cytomorphological photograph of the cutaneous nodule showing round to oval globules of matrix material; (MGG, 40X).

Immuno-histochemical analysis revealed diffuse immunoreactivity for cytokeratin 7 and c-kit (CD117) in the epithelial cells and p63,S-100 andcalponin in the myoepithelial cells. Based on these findings a final diagnosis of adenoid cystic carcinoma was attained. She was given external beam radiation therapy and was on regular follow up for two years. The disease was locally controlled except for few side effects of radiation therapy like mucosal dryness of mouth. The patient assumed to be cured was lost to follow up.She presented 3 years later with a periumblicalnodule (Fig 3). FNAC from the nodule revealed the features of adenoid cystic carcinoma. In view of cutaneous metastasis further work up was done. Computerized tomography thorax and abdomen revealed multiple nodules in lungs and liver.The patient was given palliative chemotherapy. She is alive at present without any clinical and radiological evidences of tumor recurrence.

Fig. 2: Histopathological section showing basaloid cells in cribriform pattern, surrounding gland like spaces; (H&E, 40X).

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Adenoid Cystic Carcinoma

Fig. 3: Clinical picture of the metastatic peri-umbilical nodule.

metastasis may evolve through three possible mechanisms including direct spread,local spread and distant spread [6]. Direct extension is due to contiguous spread via tissue planes. Local spread can be ascribed to spread through dermal lymphatics with resultant implantation in the skin. Distant metastasis is the result of hematogenous spread. This route of hematogenous spread could be either through pulmonary circulation or bypassing pulmonary circulation via azygous and vertebral venous plexus [7]. The common differential diagnoses of ACC includes tumors that exhibit tubular and cribrform pattern such as polymorphous low grade adenocarcinoma (PLGA), tumors with basaloid cellular morphology such as basal cell adenoma, basal cell adenocarcinoma, basaloid squamous cell carcinoma with adenoid cystic pattern and tumors with dual population of ductal and myoepithelial cells such as pleomorphic adenoma.Pleomorphic adenoma can be identified by cells with plasmacytoid appearance and abundant cytoplasm. In contrast most ACC show little cytoplasm. Hyaline spherical globules are seen in both the neoplasms. Basal cell adenoma can be identified by the presence of a capsule and lack of stromal and perineural invasion. Adenoid basal cell carcinoma should be differentiated by the presence of peripheral nuclear palisading, stromal retraction clefts, epidermal connection and absence of perineural spread. They are usually CEA and S-100 negative. PLGA occurs almost exclusively in the minor salivary gland and has overlapping histopathologic features with ACC having ductal,tubular or cribriform pattern. Perineural invasion is also common. However,

the presence of cuboidal or columnar cells with pale and ovoid nuclei and eosinophilic cytoplasm is in contrast with the hyperchromatic and angulated cells of ACC. In addition PLGA lacks a dual population of ductal and myoepithelial cells and typically has negative or low (less than 50% of cells) expression of c-KIT compared with the high c-KIT expression in ACC. Basaloid squamous cell carcinoma with adenoid cystic pattern shows well demarcated nests of basaloid tumor cells invested by hyaline basement membrane material often creating cystlike spaces between the cells, to simulate a cribriform pattern. On immunohistochemistry these are positive for high molecular weight cytokeratins while ACC is positive for cytokeratin7 and myoepithelial markers like actin, p63, calponin and S-100. This immunoprofile was seen in the present case as well [4,8]. Another close mimicker of cutaneous ACC is mucinouscarcinoma of the skin which typically shows islandsof basaloid eccrine cells embedded in lakes or pools ofmucin separated by fibrous septa. The mucin ishyaluronidase resistant and sialidase labile, indicating that itis a sialomucin, as apposed to the hyaluronic acid seen incutaneous ACC. Primary cutaneous cribriform apocrine carcinoma should also be included in the differential diagnosis as it is a non-encapsulated dermal tumor with an extensivecribriform pattern formed by multiple interconnectedbasophilic epithelial cells that are arranged in solid nests ortubular structures, and many small round spaces in between. As opposed to cutaneous ACC, basophilic aggregationsas well as spaces within are oftenmore varied in size andshape, cells are more interconnected, true elongated tubules,but no deposition of basement membrane material, areobserved, and neoplastic cells contain pleomorphic ratherthan monomorphous nuclei. No perineural or intravascularinvasion is seen. When ACC arises directly in the skin it is considered as primary cutaneous adenoid cysticcarcinoma(PCACC) which has to be differentiated from a metastasis or direct extension of a salivary gland ACC to the skin. Cutaneous ACC is a rare tumor with histological features closely resembling ACC of the salivary glands. Therefore, cutaneous and metastatic ACC can only be distinguished based on clinical grounds and the diagnosis of cutaneous ACC can only be established by a lack of any history or current evidence of ACC from an extracutaneous source. It is important to differentiate between primary and metastatic ACC as the ACC of salivary glands is an aggressive tumor in which local recurrence and widespread metastases result in death in the majority of patient; whereas cutaneous ACC tends to run an indolent course despite a high tendency for local recurrence. PCACC is characterized by an indolent

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butprogressive course, frequent involvement of the scalp, perineural infiltration, a tendency torecur locally, and rare nodal or metastatic spread. Cutaneous ACC often presents as a firm, slow-growing, ill-definednodule or tumor that may be asymptomatic, or with symptoms including tenderness, pruritus, and secondaryalopecia [9]. Treatment of patients with distant metastasis remains unresolved. ACC is unresponsive to anti-neoplastic drugs generally used for chemotherapy. A transmembrane receptor tyrosine kinase has been found in ACC which is the product of the proto-oncogene c-kit. It has been hypothesized that a high percentage of c-KIT positive ACC suggest that this proto-oncogene could be associated with the development of the tumor[10].Recently, a specific inhibitor of the bcrablautophosphorylation, platelet-derived growth factor receptor (PDGFR), and c-KIT tyrosine kinases, named imatinibmesylate has been proved to show good results in cases of unresectableor metastatic ACC that overexpresses CD117. If there is isolated metastasis surgical resection is considered generally [11].

Conclusion

Both primary and metastatic cutaneous ACC are very rare. The clinical presentation of cutaneous metastasis is nonspecific and may mimic benign lesions. Subcutaneous nodules that are rapidly progressive, eruptive, firm, painful and have non-healing ulceration should remind the clinicians the possibility of cutaneous metastasis of ACC. Since there is no histological difference between primary and cutaneous ACC metastasis so a complete clinical history is very important in such cases. To avoid delay in the treatment of such cases FNAC becomes a quick and effective tool for the early diagnosis and treatment.

Conflict of interest

The authors declare that they have no conflict of interest related to the publication of this manuscript.

FUNDING NIL

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References

1. Khan AJ, DiGiovanna MP, Ross DA, Sasaki CT, Carter D, Son YH, Haffty BG: Adenoid cystic carcinoma: a retrospective clinicalreview. Int J Cancer 2001;96:149-158 2. Spiro RH: Distant metastasis in adenoid cystic carcinoma of salivary origin. Am J Surg 1997;174:495-498 3. Ellis GL, Auclair P: Tumors of the salivary glands: Atlas of Tumor Pathology. Washington, DC: Armed Forces Institute of Pathology, 3rd Series, Fascicle 17, 1996 4. Perez DE, Magrin J, de Almeida OP, Kowalski LP. Multiplecutaneousmetastases from a parotidadenoid cystic carcinoma.PatholOncol Res. 2007;13(2):167-9. Epub 2007 Jul 3. 5. Schwartz RA: Cutaneous metastatic disease. J Am AcadDermatol 1995;33:161-182 6. Kmucha ST, Troxel JM. Dermal metastasis in epidermoid carcinoma of the head and neck. Arch Otolaryngol Head Neck surg. 1993;119:326-30 7. Batson OV. The role of the vertebral veins in metastatic processes. Ann Intern Med. 1942;16:38-45 8. Jaso J, Malhotra R. Adenoid cystic carcinoma.Arch Pathol Lab Med. 2011;135:511-5. doi: 10.1043/20090527-RS.1. 9. Dores GM, Huycke MM, Devesa SS, Garcia. CA. Primary cutaneous adenoid cystic carcinoma in the United States: incidence, survival, and associated cancers, 1976-2005. J Am AcadDermatol. 2010; 63(1): 71â&#x20AC;&#x201C;78. 10. Holst VA, Marshall CE, Moskaluk CA and Frierson HF Jr: KIT protein expression and analysis of c-kit gene mutation in adenoidcystic carcinoma. Mod Pathol 1999;12:956-960 11. Hotte SJ, Winquist EW, Lamont E, MacKenzie M, Vokes E, ChenEX, Brown S, Pond GR, Murgo A, Siu LL: Imatinibmesylate inpatients with adenoid cystic cancers of the salivary glandsexpressing c-kit: A Princess Margaret Hospital phase II consortiumstudy. J ClinOncol 2005; 23:585-90

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Case Report Non-Hodgkin Lymphoma of Cervix: An Unusual Presentation Mriganka Mouli Saha1, Chhanda Das2*, Gourisankar Kamilya1, Madhumita Mukhopadhyay2 Dept of Obst and Gynae, Institute of Post Graduate Medical Education & Research, Kolkata, India 2 Dept of Pathology, Institute of Post Graduate Medical Education & Research, Kolkata, India

Keywords: Non-Hodgkin Lymphoma, CD 20, Cervix, Diffiuse large B cell.

ABSTRACT Non-Hodgkin Lymphoma is diverse and often subdivided into diffuse large B-cell lymphoma, peripheral T-cell lymphoma, Burkittâ&#x20AC;&#x2122;s lymphoma, mantle cell lymphoma and AIDS-related lymphoma. Primary lymphoma involving the uterine cervix is very rarely encountered. We are presenting here a case with post menopausal bleeding with provisional diagnosis of poorly differentiated cervical carcinoma. Radical hysterectomy (type II) with bilateral pelvic lymph node dissection was done . Specimen is sent for histopathological examination and it was reported as diffuse round cells proliferating in an apparently unorganized fashion, with hyperchromatic nuclei and scanty cytoplasm and prominent nucleoli with possibility of poorly differentiated carcinoma or Non-Hodgkin lymphoma involving cervix. Immunohistochemistry study showed expression of CD 20 and negative expression of CD3 there by confirmed the diagnosis of diffuse large B cell type (DLBCL) Non-Hodgkin lymphoma of uterine cervix. The incidence of extranodal Non-Hodgkin Lymphoma is increasing. So, Clinicians and pathologists should be aware of this diagnosis in patients presenting with abnormal vaginal bleeding, and negative papanicolaou test.

*Corresponding author: Dr Chhanda Das, 31 Eastern park ,First Road ,Santoshpur .Kol 75 , India Phone: +91 9433116223 E-mail: chhhdas@gmail.com

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Introduction

Non-Hodgkin Lymphoma (NHL) is diverse and often subdivided into diffuse large B-cell lymphoma (DLBCL), peripheral T-cell lymphoma (PTCL), Burkitt’s lymphoma, mantle cell lymphoma (MCL) and AIDS-related lymphoma. Primary lymphoma involving the uterine cervix is very rarely encountered. It is varying in presentation due to associated cervicitis for inflammatory reaction and nonspecific symptoms like foul smelling discharge or vaginal bleeding and may mimic like cervical carcinoma.[1] Isolated genital tract extranodal disease accounts for less than 1% of NHL[2]. We are describing here a case presented with postmenopausal bleeding with provisional diagnosis of cervical carcinoma but ultimately proved to be a case of primary involvement of cervix of non-Hodgkin Lymphoma.

Case Report

A 66 years old multiparous woman presented with post menopausal bleeding for 2 months. There was no history of fever, night sweats, or weight loss. She was hypertensive on medication. She attained menopause 17 years back and had no episode of bleeding per vagina before this event. On general physical examination, there was mild pallor. There was no lymphadenopathy or hepatosplenomegaly. Other systemic examination was normal. Per speculum examination extremely soft and hypertrophied cervix which bleeds on contact. Internal bimanual examination revealed bulky uterus but no other obvious pelvic abnormality. Ultrasonography showed bulky uterus measuring 10.15cmX5.89cm X4.72cm with large collection in endometrial cavity. Her routine biochemical investigations (including HIV serology) and chest X ray were normal. She underwent examination under anesthesia and proceeded for cervical and endometrial biopsy.. Histopathological examination of biopsy specimen revealed poorly differentiated carcinoma or non-Hodgkin’s lymphoma of cervix. Laparotomy was performed under general anesthesia. After opening the abdomen uterus was diffusely enlarged, soft in consistency with multiple enlarged parametrial, internal iliac, external iliac and obturator lymph nodes. There was a large, soft, vascular mass in the cervical region. (fig 1) Radical hysterectomy (type II) with bilateral pelvic lymph node dissection was done. There was no palpable para-aortic lymph node. Other intra abdominal structures and peritoneum were healthy. Specimen was sent for histopathology. It reported as diffuse round cells proliferating in an apparently unorganized fashion, with hyperchromatic nuclei and scanty cytoplasm and prominent nucleoli, possibility of poorly differentiated carcinoma or Non Hodgkin’s lymphoma involving cervix. (fig2&3) Immunohistochemistry study showed expression of CD 20 and negative expression of CD 3(fig4&5) there www.pacificejournals.com/apalm

by confirmed the diagnosis of diffuse large B cell type nonHodgkin’s lymphoma of uterine cervix.

Discussion

Primary malignant lymphoma of the female genital tract, a rare form of extranodal non-Hodgkin’s lymphoma (NHL), can occur in the ovary, uterine corpus, cervix,vagina, and vulva. The ovary is the most frequent site for NHL involvement of the gynecological tract[3]. Primary malignant lymphoma of the cervix is a rare disease. It is accepted that the diagnosis of primary lymphoma of the cervix must fulfill the criteria proposed by Fox and More (1965). The case at hand fulfilled these criteria, which included a lesion confined to the cervix on diagnosis, and no evidence of lymphoma in other organs for at least several months during follow up [4] Cervical lymphoma presents either as primary or systemic involvement. In our study only cervix was involved. The diagnosis is difficult to make clinically. NHL arising from the cervix is mostly high grade diffuse large B cell type, but other types are also encountered such as follicular lymphoma. Abnormal vaginal bleeding is a common presenting symptom.[1] Other presenting complains include vaginal

Fig. 1: Gross picture showing mass in cervix

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NHL of Cervix

Fig. 2: Picture showing diffuse large B cell lymphoma involving cervix (x100 H&E)

Fig. 3: Diffuse sheets of tumor cells with round nuclei, scanty cytoplasm and prominent nucleoli (H&E, 400x)

Fig. 4: Immunohistochemical stain showing diffuse positivity of tumor cell for CD20 (x100).

Fig. 5 :Showing negative expression of CD3 (100X)

discharge, dyspareunia or pelvic pain. Systemic symptoms like fever, weight loss and peripheral lymphadenopathy was not a feature in our case. The etiology of NHL has been hypothesized to include infectious agents such as the human immunodeficiency virus (HIV), immunosuppressive therapies, environmental exposures to pesticides and pollutants, and improved diagnostic technique .[5]. Our case was not associated with HIV. An important differential diagnosis is with lymphoma like lesions resulting from focally florid lymphoid proliferations associated with chronic cervicitis or as an expression of infectious mononucleosis.[6]These are identified by polymorphic nature of infiltrate including mature plasma cell, small lymphocytes and neutrophils, surface ulceration. Other frequent misdiagnosis in cervical lymphomas includes sarcoma, poorly differentiated carcinoma, neuroendocrine tumors and chronic inflammation.[7] The cervical cytology in these patients with lymphoma is negative,which is

probably due to the fact that most lymphomas in this location are subepithelial unless there is ulceration [8] Immunohistochemistry is of paramount importance to confirm the diagnosis. Though any single marker is not specific but a panel of markers which includes leukocyte common antigen (LCA), B-cell markers (CD20 and CD79a), T-cell markers (CD3 and CD5) and other markers like CD23, bcl-2, bcl-6, CD10, cyclinD1, CD15, CD30, ALK-1, CD138 (based on cyto-architectural pattern) may be expressed by tumor cells .[9] Our case showed positive expression of CD 20 ,and negative expression of CD3 . So it is a NHL of B-cell type. Cervical lymphoma generally has a good prognosis as compared to nodal lymphomas, with an overall median survival of four years. Extent of disease, size of primary tumor and the type of lymphoma are significant prognostic features. However, Stroh et al. developed an the International Index score consisting of age of the patient,

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Ann Arbor stage, number of extranodal sites, performance status, and serum lactic dehydrogenase values, which could be used to predict the outcome for uterine and cervical lymphomas .[10] Patients with advanced-stage diffuse large B-cell lymphoma are primarily treated with chemotherapy. Radiotherapy is considered, if there is bulky disease during presentation. [10]

1. Vasudev DS, Kaler AK. Non-Hodgkin’s Lymphoma of the Uterine Cervix. Online J Health Allied Scs. 2012; 11(1):13.

2. Komaki R, Cox JD, Hansen RM, Gunn W, Greenberg M. Malignant lymphoma of the uterus and cervix. Cancer 1984; 54: 1699-1704. 3. Vang R, Medeiros LJ, Fuller GN, Sarris AH, Deavers M. Non-Hodgkin’s lymphoma involving the gynecologic tract: a review of 88 cases. Adv Anat Pathol,2001:8, 200-17 4. Fox H, More JRS. Primary malignant lymphoma of the uterus. J Clin Pathol, 1965:18, 723-728. 5. Trenhaile TR, Killackey MA. Primary pelvic Non-Hodgkin’s Lymphoma. Obstetrics and Gynecology.2001 ;97:717-720 6. Young RH, Harris NL, Scully RE. Lymphoma like lesions of the lower female genital tract: a report of 16 cases. Int J Gynecol Pathol 1985, 4: 289-99. 7. Charlton I, Norris J, King FM. Malignant reticuloendothelial disease involving the ovary as a primary manfestation. Cancer. 1974; 34:397-407. 8. King J, Elkhalifa M, Michael C. Malignant lymphoma identified on cervical cytologic smear with Immunophenotypic analysis. Acta Cytologica 1996;40:1228 9. Rao IS. Role of immunohistochemistry in lymphoma. Indian Journal of Medical and Paediatric Oncology : Official Journal of Indian Society of Medical & Paediatric Oncology. 2010; 31(4):145-147. 10. Stroh EL, Besa P, Cox D et al. Treatment of patients with lymphomas of the uterus or cervix with combination chemotherapy and radiation therapy. Cancer.1995; 75:2392-2399.

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Conclusion

Although primary non Hodgkin’s lymphoma is rare, the disease may be encountered more frequently as the incidence of extranodal non-Hodgkin’s Lymphoma is increasing. Cervical cytology may be non-diagnostic in such cases. Therefore, Clinicians and pathologists should be aware of this diagnosis in patients presenting with abnormal vaginal bleeding and negative papanicolaou test and to include cervical lymphomas in the differential diagnosis of gynaecological cancers.

Acknowledgements Funding None

Competing Interests None

Reference:

Case Report Ovotesticular Disorder of Sexual Development with Rare Karyotype Ritika Singh*, Charanjeet Ahluwalia, A K Mandal Department of Pathology Vardhaman Mahavir Medical College and Safdarjung Hospital, New Delhi, India Keywords: Ovotestis , Primodial Follicles, Mosaic Karyotype, Mixed Gonadal Dysgenesis, Germ Cell Tumour.

ABSTRACT Ovotesticular disorder of sexual development (OT-DSD) is a rare disorder of sexual differentiation. It is associated with variable genotype of which the most common karyotype is 46,XX. A 2 year-old boy presented with severe penoscrotal hypospadias and unilateral right side cryptorchidism. The right gonad was atrophic , present in the right inguinal region and showed presence of ovarian tissue with mature ovarian follicles and testicular tissue with distinct seminiferous tubules in the same gonad (ovotestis) on histopathology and a 45,XO/46,XY karyotype.

*Corresponding author: Dr Ritika Singh. F-6 Police Station Chanakya Puri New Delhi110021. India Phone: +91 9958492397 E-mail: ritika84singh@gmail.com

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

Singh et al.

Introduction

Ovotesticular disorder of sexual development (OT-DSD) is a rare disorder of sexual differentiation . It is characterized by the presence of ovarian tissue with mature ovarian follicles and testicular tissue with distinct seminiferous tubules in the same gonad (ovotestis).[1] It constitutes 3% â&#x20AC;&#x201C; 10% of all sexual disorders. Both Wolffian and Mullerian duct derivatives are seen, and most affected individuals commonly present with ambiguous external genitalia as neonates or infants.[2] In individuals with OTDSD, the ovotestis is the most common histological gonad type. There is an increased risk of developing germ cell tumor is the gonads of such individuals.[3,4,5] OT-DSD is also associated with variable genotype of which the most common karyotype is 46,XX. [1]

Case Report

A 2 year-old boy presented with severe penoscrotal hypospadias .Physical examination revealed unilateral right side cryptorchidism with left gonad palpable in left hemi scrotum and normal in size. No other physical abnormality was noted. Ultrasound examination showed a heterogenous ovoid structure present in the right inguinal region. Left gonad had homogenous echotexture .Pelvic ultrasound examination showed no evidence of a uterus or ovaries. The testosterone levels were measured both were in the normal range (3-10ng/ml). Cytogenetic analysis performed on peripheral blood lymphocytes revealed a 45,XO/46,XY karyotype. Right sided gonadectomy and hypospadias repair was performed. The right gonad was submitted along for histopathological evaluation. Histopathological findings: Gross examination revealed a tubular grey white structure measuring 3.5x1x0.5 cms. Histopathologic examination of the right gonad showed the ovarian and testicular tissue. Ovarian tissue consisted of ovarian stroma with primordial follicles along with ipsilateral fallopian tubal lumen and endometrial stromal tissue. The testicular tissue consisted of numerous solid seminiferous tubules filled with immature sertoli cells and a few primitive germ cells. The immature sertoli cells had a regular, round to ovoid nucleus with inconspicuous nucleoli. The germ cells were found adjacent to the basement membrane and those were distinguishable from the immature sertoli cells because of their larger nuclei and abundant cytoplasm. Also identified was vas deferens. A histopathological diagnosis of ovotestis was given.

A-69 (TH) is the rarest form of intersexuality and the term is applied to an individual who has both well-developed ovarian and testicular tissues[6] .It accounts for less than ten percent of intersex patients.[7]. Krob et al examined the histopathological structures of the gonads in 283ovotesticular DSD cases and found that the most common gonad type was ovotestis (44.4%), followed by ovary (21%) and testis (12.5%).[5] Ovotestes are usually compartmentalized, with connective tissue separating the ovarian components from the testicular components. However, on rare occasions, an intermixture of these elements may occur.Testicular tissue in OT-DSD is defined by the presence of immature seminiferous tubules lined by immature sertoli cells and primitive germ cells, and ovarian tissue is defined by the presence of numerous primordial and/or maturing follicles within the ovarian stroma.[6] Various types of chromosomal abnormalities have been described in OT-DSD with ovotestis such as 46, XX; 46, XY; 46, XX/46, XY, 45, X/46, XY.[5] This diagnostic nomenclature is applied regardless of the peripheral karyotype. 45XO/XY is a very rare genotype as reported in the present case to be associated with ovotestis .The clinical phenotype associated with 45, X/46,XY mosaicism is broad, ranging from women, with or without Turner syndrome stigmata, to apparently normal males, with intervening variable ambiguous phenotypes[8]. Gonad histology associated with 45, X/46,XY mosaicism is also variable with partial, complete, mixed, or asymmetric gonadal dysgenesis showing streak gonads.[9] In patients with ovotesticular DSD,the rate of occurrence of neoplasia is estimated at 2.6% [4] However some studies estimate that the risk of germ cell tumor development in OT-DSD ranges from 4% among those with the 46,XX karyotype to up to 10% in those with 46,XY and 46,XX/ XY chimerism .[10] The removal of the opposite gonad from the assigned gender and a biopsy of remaining gonadal tissue for histological evaluation may be appropriate.[6]

Ovotesticular disorder of sexual differentiation formerly known as true hermaphroditism

Ovotesticular disorder also needs to be differentiated from mixed gonadal dysgenesis (MGD) with which it may show histological and genotypic overlapping.MGD has varying degree of histological presentations such as streak testis,streak ovaries but unlike OT-DSD maturing primordial follicles are not identified in gonads of MGD [6]. Also various structural and systemic anomalies which need early medical attention are seen in patients of MGD unlike OT-DSD. MGD carries a high risk of tumor development at 12% and possibly at more than 30% if gonadectomy had not been performed. In patients with mosaic karyotype

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Discussion

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Fig. 1. (a). Ovarian tissue identified in the gonad revealed characteristic ovarian stroma and the presence of scattered primordial follicles (H&E ×40). (b). Numerous solid seminiferous tubules filled with immature sertoli cells and a few primitive germ cells along with rudimentary epididymis (H&E ×20). (c). Endometrium and myometrium from the uterine part of gonad (H&E ×20). (d). Fallopian tube (H&E ×40).

the prevalence ranges between 15 and 40%. [4] Bilateral gonadectomy is recommended in all individuals with MGD containing Y-chromosome material.[11] In the present case no such abnormalities were present supporting the histological diagnosis OT-DSD.

Conclusion

The most common genotype associated with ovotesticular DSD is 46,XX . 45,XO/46XY mosaic karyotype is rarely seen with it. This case highlights the importance of histological finding in ovotesticular disorder of sexual development as clinical features, cytogenetic results, hormonal profiles do not appear to be useful in a differentiating it from mixed gonadal dysgenesis.

Acknowledgements Nil

Funding None

Competing Interests None

Refrences

1. Sperling MA, editor. Pediatric endocrinology, 3rd ed.Philadelphia: WB Saunders; 2008:138. 2. Hughes IA, Houk C, Ahmed SF, Lee PA; Lawson Wilkins Pediatric Endocrine Society/European Society for Paediatric Endocrinology Consensus Group. Consensus statement on management of intersex disorders. J Pediatr Urol .2006;2:148 – 62. 3. Bhansali A, Mahadevan S, Singh R, Rao KL et al. True hermaphroditism: clinical profile and management of six patients from North India. J Obstet Gynaecol . 2006;26:348–50. 4.

Pleskacova J, Hersmus R, Oosterhuis JW, Setyawati BA,et al. Tumor risk in disorders of sex development. Sex Dev. 2010;4:259–69.

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Singh et al. 5. Krob G, Braun A, Kuhnle U. True hermaphroditism: geographical distribution, clinical findings, chromosomes and gonadal histology. Eur J Pediatr .1994;153:2– 10. 6. K.-R. Kim et al.Hermaphroditism and Gonadal Dysgenesis.Mod Pathol. 2002;15:1013-9. 7. Damiani D, Fellous M, McElreavey K, Barbaux S et al. True hermaphroditism: clinical aspects and molecular studies in 16 cases. Eur J Endocrinol. 1997;136(2):201-4. 8. Moussaif NE, Haddad NE, Iraqi N, Gaouzi A. 45,X/46,XY mosaicisme: report of five cases and clinical review. Annales d’Endocrinologie. 2011 ;72: 239–243.

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Ocal M. Berberoglu Z. Siklar et al. The clinical and genetic heterogeneity of mixed gonadal dysgenesis: does “disorders of sexual development (DSD)” classification based on new Chicago consensus cover all sex chromosome DSD? Eur J Pediatric .2012;171:1497–1502.

10. Wettasinghe K, Sirisena N, Andraweera P, Jayasekara R et al. A Case Series of Five Sri Lankan Patients with Ovotesticular Disorder of Sex Development. Clin Pediatr Endocrinol. 2012 ; 21(4): 69–73. 11. Hughes IA, Houk C, Ahmed SF, Lee PA. Consensus statement on management of intersex disorders. Arch Dis Child .2006;91: 554–63.

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Case Report Orbital Lymphoid Lesions: Short Series with Cytohistological Correlation Neelam Sood, Priyanka Bhatia Soni* Department, Pathology, Deen Dayal Upadhyay Hospital, New Delhi, India. Keywords: Orbital lymphoid hyperplasia, Pseudolymphoma, lymphoma.

ABSTRACT Orbit and ocular adnexae are common sites (10-15%) for developing lymphoid lesions but have diagnostic and staging difficulties similar to other extranodal sites. Out of 15 fine needle aspirates from orbital lesions received in last 5 years three female patients of age 56, 46 and 72 years presented to FNAC clinic with upper eyelid swellings of 2 years, 3 years and 3 months duration. Systemic examination was unremarkable. Hematological and Serum protein electrophoresis was normal. On MRI case 1 was suggestive of infective/neoplastic lesion. On CECT case 2 was suggestive of pseudotumor/ lymphoma while case 3 was suggestive of pseudotumor. F.N.A.C of case 1 showed polymorphous population of lymphoid cells, without any significant mitosis. FNAC of case 2 showed a monotonous population of lymphoid cells and Case 3 showed a relatively monotonous population in a background of lymphoglandular bodies. Case 1: Responded to steroids. Case 2: was advised a biopsy for confirmation, Case 3: Excision done in view of the recurrent nature with no response to steroids. Histopathology in Case 2 showed a non encapsulated mass with proliferation of lymphoid cells forming nodules, IHC showed a monoclonal pattern while Case 3 showed a nonencapsulated mass with nodular proliferation of lymphoid cells involving the surrounding adipose tissue with formation of primary and secondary follicles and Immunohistochemistry (IHC) showed a polyclonal population. Three cases are being discussed to highlight the diagnostic dilemmas in orbital lymphoid lesions and relevance of IHC in such cases.

*Corresponding author: Dr. Priyanka Bhatia Soni, Department, Pathology, Deen Dayal Upadhyay Hospital, Hari Nagar, New Delhi, India.

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Introduction

A total of 15 Fine needle aspiration cytology (FNAC) lesions of orbit having eyelid swellings were reviewed over a period of five years of which three patients diagnosed as having lymphoid lesions have been taken for the present study. Lymphoid proliferations comprise 1015% of all space occupying lesions of the orbit and pose a diagnostic challenge for clinicians and diagnosticians alike.[1] Dileneation between benign and malignant orbital lymphoid lesions is difficult, in absence of clearcut architectural differentiating criteria as defined in a lymph node. This is due to presence of only few scattered lymphoid cells in the orbit.[2,3] There have been attempts to categorize orbital lymphoid lesions into different categories dividing them into Lymphoma , indeterminate lesions and reactive lymphoid hyperplasia.[2,4] Few authors divided them as Lymphoma, Benign pseudolymphoma, Orbital pseudotumor, Orbital lymphoid hyperplasia and Chronic inflammatory lesions. [1,5] Some authors divided them immunologically into monoclonal and polyclonal .[4,5] Further attempts have been made to extend the utility of Fine Needle Aspiration Cytology (FNAC) in orbital lesions, [6,7] wherein overlaps between pseudolymphoma and lymphoma have been reported invariably, although 100% concordance has also rarely been reported.[8] The cases where atypical features are present, one has to rely on Immunohistochemistry (IHC) or Polymerase chain reaction for further dileneation .[2,4,6,7,9] and through a short series of three cases we aim to highlight these diagnostic dilemmas.

Case Reports

Case 1: 58 year old female presented with a slowly progressive painless right upper eyelid mass of 2 years duration. Systemic and hematological examination revealed no abnormality. Serum electrophoresis showed mild increase in gamma globulins. B-scan orbit was normal. MRI showed a well defined soft tissue signal intensity lesion measuring 21 x 17 x 19 mm in the right upper quadrant of orbit inseperable from the right lacrimal gland and closely abutting the right lateral rectus muscle giving a probable diagnosis of ? neoplastic ?? infective lesion. F.N.A.C done showed polymorphous population comprising of mature and transformed lymphocytes along with occasional plasma cells. Mitosis was absent. A diagnosis of Orbital Lymphoid Hyperplasia was given and a biopsy was advised, however the patient responded to steroids.

Systemic, Hematological and Serum electrophoresis revealed no abnormality. B-scan and visual acuity was normal. The patient did not have any constitutional or other significant symptoms. However experienced slight eye pain and heaviness of head. CECT orbit showed a 27 x 21 x 20 mm ill defined homogenous intraconal mass in superior compartment of left orbit encasing the optic nerve with patchy enhancement, however sparing the surrounding muscles, fat and bones. F.N.A.C done showed highly cellular smears comprising of singly lying pleomorphic cells larger than the mature lymphocytes having round to oval vesicular nucleus with indistinct nucleoli and stripped off cytoplasm in most of the cells , however wherever preserved was scant and pale blue. Few cells showed irregular nucleus with lobulation and clefting along with binucleation. Infrequent mitosis and lymphoglandular bodies were noted in the hemorrhagic background. A diagnosis of high grade lymphoma was given and a biopsy advised. Histopathology showed a nonencapsulated mass with proliferation of lymphoid cells forming nodules at places. The lymphoid cells were larger than the mature lymphocytes with irregular nuclear contours with scant to moderate cytoplasm and vesicular nuclear chromatin. Fine fibrosis with focal areas of necrosis was noted. Mitosis was infrequent. Amongst IHC markers CD 20 positivity was seen mainly in the larger cells with no T cell positivity and thereby confirming the diagnosis. Case 3: 46 year old female a native of Arunachal Pradesh presented with a progressive, painless and recurrent left upper eyelid swelling since 3 years. Systemic, hematological examination and serum electrophoresis revealed no abnormality. B-scan and visual acuity was normal. CECT showed 27 x 14 x 12 mm extraconal soft tissue density in upper outer aspect of left orbit, in periorbital location in continuity with lacrimal gland with minimal surrounding infiltrates sparing extraocular muscles, fat, optic nerves and bony margins with a likely diagnosis of pseudotumor. F.N.A.C showed a relatively monotonous population in a background of lymphoglandular bodies and very very occasional plasma cells. A diagnosis of Orbital lymphoid hyperplasia was given. However biopsy was advised to rule out lymphoma.

Case 2: A 72 year old female presented with a gradually progressive protrusion of the left eyeball since three months.

On Histopathology variable sized nodules separated by fibrous bands showing polymorphous lymphoid proliferation along with formation of germinal centres in many of the nodules was seen. Peripheral areas showed a diffuse pattern with entrapped adipose tissue. The lymphoid cells were larger than mature lymphocytes showing angulated nuclear borders and scanty cytoplasm

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Orbital Lymphoid Lesions

along with interspersed larger cells having abundant pale cytoplasm. Very occasional plasma cells and infrequent mitosis was noted. No residual lacrimal gland identified. A diagnosis ? atypical orbital lymphoid hyperplasia/ pseudolymphoma was given supplemented by IHC which revealed a polyclonal population of lymphoid cells. The IHC panel comprised of the markers available in our set up and included both B and T cell markers. T cell markers included CD 3 and CD8 showing mainly interfollicular area positivity, CD4 positive cells were randomly distributed throughout the nodule. Amongst B cells CD 20 positivity was seen with in the nodule in germinal center and scattered positivity in the interfollicular area. BCL2 showed non germinal centre positivity, CD 10 was negative and Lambda/kappa showed polyclonal pattern with no light chain restriction. Pancytokeratin was negative indicating absence of any epithelial component and residual lacrimal gland while Ki 67 showed < 1% positivity. Post Operative follow up for 3 months with steroids was uneventful, however patient came back with a recurrence and was advised radiotherapy.

Discussion

criteria.[4] Lymphoid tumors are most commonly seen in the orbit (52%) followed by conjunctiva (29%).[4] These tumors also known as lymphoproliferative lesions are the most common primary orbital tumors in adults comprising of reactive lymphoid lesions and lymphoma which are often difficult to differentiate.[2,4,10,11] Subsquently these lymphoproliferative lesions have been categorized by Knowles and Gaag et al into : 1. Lymphoma - Malignant end of spectrum 2. Benign pseudo lymphoma

Orbital pseudotumor

Orbital lymphoid hyperplasia

3. Orbital inflammatory diseases (OID) An inflammatory presentation is not uncommon in orbital lymphoid tumors which accounts for up to 6% of orbital diseases.[12] Differential diagnosis of OID ranges from idiopathic inflammatory disease to systemic or local inflammatory conditions to other associated conditions such as neoplasm, infection, congenital malformation, or trauma.

Localised lymphocytic proliferation within the orbit is commonly seen as causing swelling/proptosis and poses a challenging problem as their benign or malignant nature cannot usually be determined by clinical and radiological

It is divided into primary and secondary . Primary lesions being infectious ( bacterial, fungal , parasitic ), non infectious ( thyroid associated ) and idiopathic ( which is the third most common orbital disease after thyroid associated

Fig. 1: (1)56 years female showing a firm, mobile right upper eyelid swelling measuring 2x1cm (Case 1). (2,3) Cytosmear showing polymorphous population comprising of mature and transformed lymphocytes along with occasional plasma cells. (Giemsa 10X, 40X )

Fig. 2: (1) 72 years female with a firm left upper eyelid swelling measuring 2.7x 2.1 cm (Case 2). (2) Cytosmears showing highly cellular smears comprising of singly lying pleomorphic cells larger than the mature lymphocytes. (Giemsa 40X) (3) CD 20 positivity seen mainly in the larger cells with no T cell positivity . (IHC 40X)

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A-75 ophthalmopathy and lymphoproliferative diseases) . Secondary lesions occur in response to inflammation due to some orbital condition like lymphoproliferative disease, foreign body , trauma , hemorrhages etc. [10,12] The last two groups in the classification are characterized morphologically by a relatively monomorphous, hypercellular, reactive lymphoid hyperplasia, or by a heterogenous cell population with small and large lymphocytes, plasma cells, eosinophils, histiocytes and by lymphfollicles with germinal centres and fibrosis. Excluding lymphoma all are interchangeable terms, used to describe orbital mass lesions in which mature lymphocytes are noted to infiltrate orbital structures.[14] On the other hand Ghasemi [2] et al have categorized them into :

Fig. 3: (1) 46 years female showing a firm, oval swelling measuring 2x1x1cm in size located in upper lateral angle of left orbit (Case 3). (2) Cytosmear showing a relatively monotonous population of lymphoid cells in a background of lymphoglandular bodies and very occasional plasma cells. Arrow showing a plasma cell. (Giemsa 40X) (3) Histopathology showing a nonencapsulated mass with nodular proliferation of lymphoid cells involving the surrounding adipose tissue with formation of primary and secondary follicles. Arrow shows a follicle with surrounding mantle zone. ( H&E 10X ).

Fig. 4: (1) CD 20 positivity with in the nodule in germinal center and scattered positivity in the interfollicular area (10X) (2) CD 3 showing interfollicular area positivity. (10X) (3) CD4 positive cells randomly distributed throughout the nodule. (10X) (4) CD8 showing mainly interfollicular area positivity. ( 10X )

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1. Reactive lymphoid hyperplasia ( RLH ) - Composed of dense polymorphous population of well differentiated lymphocytes, other inflammatory cells and occasional lymphoid follicles with germinal centers. Mitosis if present is restricted only to the germinal centers and tingible body macropahges can also be seen. However dutcher bodies are absent. Immunohistochemically the tumors consist of mixed population of T- and Blymphocytes and show polyclonality for Kappa/lambda. BCL-2 positivity is seen in interfollicular zones only. 2. Atypical lymphoid hyperplasia (indeterminate lesions) - Represent between 3-12% of the lymphoproliferative lesions in the ocular adnexa.[4] It does not reveal overt benign or malignant features and lie in a gray zone .This group consists of those borderline cases in which the diagnosis of the lesions cannot be determined with certainity using conventional histological techniques. These lesions can have a diffuse/follicular pattern and cells manifest borderline maturity or contain atypical cells with large hyperchromatic nuclei. Often requires IHC to categorize and gives polyclonal pattern. 3. Lymphoma â&#x20AC;&#x201C; Ocular lymphomas represent 8 % of all the extranodal lymphomas and show classic cellular atypia or immunophenotypically, express monoclonality. Most of them are primary extranodal lymphoma of the marginal zone of mucosa associated with lymphoid tissue (MALT type lymphoma). Important histomorphologic features of ocular adenexal lymphomas is cytologic atypia and dutcher bodies though immunohistochemistry is required in one third of the cases.[13] Germinal centre formation, polyclonality and lack of cellular atypia indicate benign lesions however some polyclonal tumours eISSN: 2349-6983; pISSN: 2394-6466

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Orbital Lymphoid Lesions

may become malignant and some monoclonal tumors may remain benign. 27-29% of patients with RLH and 45% with indeterminate lesions are known to develop disseminated disease.[4] Some authors have attempted to distinguish orbital pseudotumor from orbital lymphoid hyperplasia, but these distinctions are difficult and of uncertain prognostic or therapeutic importance.[14] Case 1 in our study was categorized as orbital lymphoid hyperplasia as per both the classifications. Case 2 occupies the extreme malignant end of the spectrum as per the classifications and immunologically being monoclonal. However case 3 was categorized as Benign pseudolymphoma and atypical lymphoid hyperplasia respectively , though polyclonal immunologically. Similar to the reported age/sex distribution; out of three cases, two of benign nature were seen in patients in their fourth decade while case no.2 being malignant was seen in elderly with all being females.[1,11] though few authors observed no significant gender and age correlation.[2,4] These patients presented with palpable mass/swelling, pain and diplopia as also seen in 65% of cases, with symptoms being less acute in lymphoma . [1,2,4, ] Time duration of complaints were much longer in cases with lymphoma versus pseudolymphoma /chronic inflammation, similar to as seen in our study . [1] Case no.3 was chosen to illustrate how difficult and dilemmatic the diagnosis and management of orbital lymphoid tumors can be . In view of recurrent nature of the swelling even after course of steroids in this case, a biopsy was advised to rule out lymphoma. Also because the patients origin was from north eastern india, where the incidence of lymphomas is more.[15]. Morphologically infiltration of lymphoid cells into the surrounding adipose tissue was insignificant, as the site is extranodal. Presence of primary and secondary follicles with germinal centres along with fibrosis favoured benignity in Case 3 however the peripheral diffuse pattern even in absence of atypia favoured indeterminate category, and therefore IHC was used as an adjunct to rule out lymphoma[2] , which showed both B and T cell positivity with no light chain restriction. Also no histological atypia was seen , and therefore a diagnosis of pseudolymphoma was given . The low ki 67 labelling index also suggested the diagnosis . Low grade B cell lymphomas were ruled out by absence of bcl-2 positivity as seen in follicular lymphoma and Kappa/ lambda being monoclonal while Extranodal marginal zone lymphomas (MALT ) were excluded due to the absence of atypical monocytoid B cells and CD 5 as also noted by other workers.[1,2,6,7,9]

All the authors had to face diagnostic challenges in the form of indeterminate cases. In our study the lesions were divided into two groups reactive lymphoid hyperplasia and lymphoma ; with a gray zone of so called histologically indeterminate tumor was observed in case 3, as also reported by others . [ 1,2,4 ] However immunohistochemical studies along with other molecular studies helped in decreasing the number of indeterminate cases. IHC was a helpful tool in our study used for confirmation, diagnosis and categorization. Also used to determine the clonality of indeterminate cases with evaluation of clonal immunoglobulin light chains ( kappa/ lambda ) expression to find neoplastic cells. [1,2,4] Panel of monoclonal antibodies like CD45, CD30, CD20, CD5, CD8, CD21, CD23, ki67, p53, bcl2, ki67, p53 and pan cyto keratin were used. Polyclonal antibodies were used to test the expression of CD 3 antigens and of the immunoglobulin chains kappa, lamda , gamma etc. Proliferative markers like p53, ki 67 also correlate with the prognosis and divide the tumor accordingly into low and high grade . [4 ] Other sophisticated methods which can be applied are electron microscopy, immunofluorescence and molecular diagnostic tests like PCR . [1,2,4] Recent development of these techniques have made it easier to differentiate benign from malignant orbital lymphoid disorders . [14] Few authors suggested that 27%-29% of RLH and upto 45% indeterminate lesions ultimately develop disseminated disease and therefore accurate and prompt diagnosis with a close follow up of the lymphoid proliferations in the orbit results in better adjusted treatment for each group of patients [1,4] Most cases of pseudotumors respond well to steroids which are used as first line therapy , with durable complete response in 50% of the cases . However radiation therapy gives a 75%100% response with almost rare relapse rates .[14] Rituximab which is a chimeric monoclonal antibody directed against CD20 receptors is being used as a latest targeted therapy. Since benign lymphoid hyperplasia of the orbit predominantly consists of B-cells bearing the CD20 surface marker, rituxirnab is a treatment altemative for refractory benign lymphoid hyperplasias along with other lymphoproliferative disorders and when used in combination with radioimmunotherapy or standard chemotherapy gives a more durable response with lesser side effects than radiation therapy. [16] BLH can be left untreated if the associated symptoms are rninimal . There is evidence, however. that BLH can

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undergo malignant transformation especially in lesions involving the lacrimal gland and therefore a careful follow up of the pseudolymphoma patients and patients with a chronic inflammatory disease is mandatory in order to study whether a neoplastic disorder can arise locally from a reactive process. [1]

4. Coupland SE, Krause L, Delecluse HJ, Anagnostopoulas I, Foss DH, Hummel M, Bornfels N et al. Lympho-proliferative lesions of the ocular adnexa. Analysis of 112 cases. Ophthalmology 1998;105:1430-1441.

5. Knowles DM, Jakobiec FA. Ocular adnexal lymphoid neoplasms. Hum Pathol 1982; 13: 148-162 6. Subramanian R, Solo S, Mishra MM, Murugan P, Siddaraju N, Basu D, Srinivasan R. Fine needle aspiration cytology of primary lymphoid lesions of the orbit: report of four cases. Acta Cytol. 2007;51(3):417-420. 7. Tani E, Seregard S, Rupp G, Söderlund V, Skoog L .Fineneedle aspiration cytology and immunocytochemistry of orbital masses. Diagn Cytopathol. 2006;34(1):1-5. 8. Singhal N, Mundi IK, Handa U, Punia RP, Mohan H. FNA in diagnosis of orbital lesions causing proptosis in adults. Diagnostic Cytopathology. 2012; 40(10) : 861-864. 9. Lowen MS, Saraiva VS, Martins MC, Burnier Jr MN. Immunohistochemical profile of lymphoid lesions of the orbit. Can J Ophthalmol 2005; 40(5): 634-639. 10. Choi VBL, Yuen HKL, Biswas J, Yanoff M. Update in pathological diagnosis of orbital infections and inflammations. Middle East Afr J Ophthalmol 2011; 18(4) : 268-276. 11. Mannami T, Yoshino T, Oshima K, Takase S, Kondo E, Ohara N et al. Clinical, Histopathological, and Immunogenetic Analysis of Ocular Adnexal Lymphoproliferative Disorders: Characterization of MALT Lymphoma and Reactive Lymphoid Hyperplasia.Mod Pathol 2001;14(7):641–649. 12. L K Gordon. Orbital inflammatory disease: a diagnostic and therapeutic challenge. Eye 2006; 20:1196–1206. 13. Medeiros LJ, Harris N. Lymphoid infiltrates of the orbit and conjunctiva. A morphologic and immunophenotypic study of 99 cases. Am J Surg Pathol. 1989 Jun;13(6):459-71. 14. Chesbro AC, Quivey JM. Cancer and benign diseases of eye and orbit.In: Leibel SA, Phillips TL, editors. Textbook of radiation oncology, 2nd ed.Philadelphia: Saunders; 2004: 1443-1461. 15. Das D, Deka P, Bhattacharjee k, DasJK, Kuri G, Deka AK, Bhattacharjee H. Ocular adnexal lymphoma in the Northeast Indian population. Indian J Ophthalmol 2008 ; 56(2): 153–155. 16. Ho HH, Savar A, Samaniego F, Manning J, Kasyan A, Pro B et al . Treatment of Benign Lymphoid Hyperplasia of the Orbit with Rituximab . Ophthal Plast Reconstruct surgery 2010; 26:11- l3.

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Summary

Differentiation between benign and malignant lymphoid proliferations of the ocular adnexa creates a diagnostic dilemma . We endorse that relying purely on FNAC is not recommended and often requires a good histomorphologic correlation keeping in mind the similarity in clinical presentation of indeterminate cases to lymphomas. Majority recommend a biopsy if this can be performed without significant morbidity due to the unusual nature of lymphoproliferative disorders. Though histopathology is considered confirmatory in diagnosis of these lesions, immunohistochemistry with other molecular techniques is required for a more refined diagnosis of lymphoid proliferations. Multidisciplinary cooperation and a promt follow up leads to further improvement of diagnosis and treatment of ocular adnexal lymphoproliferative disease with a need to work up all patients regardless of histopathologic diagnosis.

Bibliography

1. Gaag R, koornneef L, Heerde P, Vroom T, Pegels J, Feltkamp S et al. Lymphoid proliferations in the orbit: malignant or benign ? British Journal of Ophthalmology 1984; 68:892-900. 2. Ghasemi M, Amoli AF, Gransar A. A clinicopathologic study of orbital and ocular adnexal lymphoproliferative lesions with immunohistochemical staining of indeterminate cases . Acta Medica Iranica 2003;41(1):11-14. 3. Ulivieri S, Motolese PA, Motolese I, Motolese E, Menicacci F, Gennari P. Benign orbital pseudolymphoma. Case report. Giugno-Luglio 2009; 30: 299-301.

Case Report A Rare Case of Juvenile Fibromatosis Infiltrating Neck Subcutis in a 3-year Old Girl Amit Kumar1, Rakesh Mehra2, Tanushree Narain1, Neha Garg1, Pallavi Agrawal1* Department of Pathology, Mahavir Cancer Institute And Research Centre, Patna (Bihar), India Department of Radiology, Mahavir Cancer Institute And Research Centre, Patna (Bihar), India

1 2

Keywords: Juvenile, Fibromatosis, Neck, Submandibular

ABSTRACT Background: Fibromatosis is a rare benign tumor of fibro-myofibroblastic origin. This non-metastasizing tumor has high potential to locally invade and recur after surgical excision. Since 1950, only 99 cases of fibromatosis of head and neck in pediatric age-group are reported in the literature. We present the 100th case in this category. Case-report: A three year old girl presented with a right sided neck mass progressively increasing in size since one year. On imaging the tumor was found extending from submandibular to supraclavicular region, which on incisional biopsy showed features consistent with the diagnosis of Juvenile Fibromatosis. Histologically, it may be confused with other entities under broad diagnostic category â&#x20AC;&#x2DC;fibromatosesâ&#x20AC;&#x2122;, but immuno-histochemical analysis confirmed the diagnosis. Patient underwent wide local excision and was followed-up for two years with no features of recurrence. Conclusion: A rare and interesting tumor of pediatric age group with high tendency to recur after surgical excision. But surgical resection with tumor free margins prognostically decreases the chance of recurrence.

*Corresponding author: Dr. Pallavi Agrawal, (M.D., DNB, PDCC) Consultant Pathologist, Room no. 314, Department of Pathology, Mahavir Cancer Institute And Research Centre, Patna (Bihar) 801505, India Phone: +91 7739165410 E-mail: Dr.pallavimamc@gmail.com

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Kumar et al.

Introduction

Fibromatosis is an extremely rare disorder with an incidence of 2-4 cases per million a year, representing only 0.03% of all neoplasm [1]. It is an apparently non-neoplastic condition characterized by proliferation of fibroblastic and myofibroblastic cells resulting in formation of tumor-like masses. It is a benign and locally invasive disorder without the metastatic potential but with tendency to recur [2, 3]. Till date only 99 cases of fibromatosis of head and neck in pediatric age group are reported in literature world-wide. Majority of the cases are sporadic with unknown etiology but some cases show familial predisposition i.e., occur as a part of Gardner’s syndrome or Familial adenomatous polyposis [4, 5]. It is divided into the superficial and deep subtypes. Superficial fibromatosis include palmar and plantar fibromatosis while the deep fibromatosis usually involves muscles of trunk and limb. The deeper fibromatosis show tendency to locally invade and recur after surgical excision. We present the whole clinico-pathologic spectrum of a case of 3 year old girl diseased with this entity.

A-79 diagnosis of Juvenile Fibromatosis was considered which was confirmed on IHC. The tumoral cells were diffusely positive for SMA (Fig-2). The patient was followed-up for two years with no evidence of recurrence.

Case Report

A 3-year old girl presented with a large right-sided neck mass since one year which was progressively increasing in size (Fig-1). On physical examination a firm, non-tender, mobile swelling measuring 9×5cm was identified extending from submandibular to the supraclavicular region. The overlying skin appeared excoriated but wasn’t fixed to the underlying mass. There was no regional lymphadenopathy. The child was otherwise doing well. All the hematological and biochemical parameters were within the normal limits. Contrast enhanced computerized tomography (CECT) revealed a well-defined heterogeneously enhancing mass lesion measuring 9×6×4cms with infiltration into the surrounding muscles. An incisional biopsy was performed from the visible mass which on histopathological examination showed ill-defined nodule like structure composed of an admixture of spindle to plump fibroblast in vague fascicular arrangement. Nuclear atypia and necrosis was not seen. It was diagnosed as ‘benign spindle cell tumor with likely possibility of Juvenile Fibromatosis’.

Fig-1: (A) Clinical picture showing the extension of the right sided neck mass. (B) Gross photograph of the solid well circumscribed tumor. (C) CECT scan shows a welldefined heterogeneously enhancing lesion with central necrosis. (D) High-power view showing spindle to plump fibroblasts arranged in vaguely fasciculated pattern with no nuclear atypia or mitoses, H&E 400X.

Multidisciplinary team planned for the wide local excision of the lesion due to the progressively increasing size of mass and close approximation of mass with the neuro-vascular bundle in neck. Internal jugular vein and sternocleidomastoid muscle were preserved. Gross examination showed a well-circumscribed non-capsulated solid mass measuring 10×7×6cms. Cut-surface was firm, homogenous and grey-white. Histopathological examination showed plump fibroblast in vague fascicular arrangement. No nuclear atypia, mitoses, necrosis hemorrhage or calcification was identified. A provisional

Fig-2: Photomicrograph showing tumor cells diffuse immunoreactive with SMA (DAB, 400X)

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Juvenile Fibromatosis in a 3-Year Old Girl

Discussion

Fibromatosis is a benign but locally invasive neoplasm of fibro-myofibroblastic origin. It can occur at any age, but very rarely seen in infants and juvenile age group. This tumor is of unknown etiology though the role of localized trauma, endocrine and genetic factors are suggested by some authors. Sometimes fibromatosis regresses at menarche and an anti-estrogen tamoxifen has been shown to have an inhibitory effect on such lesions suggesting the role of endocrine factor in etiology. The most common site of involvement in pediatric age group is scalp and neck usually presenting as a nodular mass in subcutis as is reported in this case [6]. Fibromatosis can occur at other sites like arm, shoulder, axilla, mesentery and abdominal wall. These lesions are usually solitary but can be multiple. This tumor is highly predisposed to locally invade the surrounding tissue. CT and MRI can help in assessing the degree of local invasion. These lesions never metastasize, however 10% exhibit unpredictable biological behavior with extremely aggressive growth potential. Histopathological examination remains the goldstandard modality for diagnosis. The microscopic picture comprises of well-defined nodules of plump to spindle shaped fibroblasts arranged in vaguely fascicular pattern. These tumors lack nuclear atypia, mitoses and necrosis. Immunohistochemical analysis shows diffuse positivity with vimentin and SMA. Fibromatosis constitutes part of a spectrum of poorly understood proliferative lesions whose histologic features overlap to such an extent that it becomes very important for the pathologist to be aware of the anatomic location of lesion, sex and clinical behavior before characterizing the lesion. Juvenile Fibromatosis is an entity which comes under the broader diagnostic group â&#x20AC;&#x2DC;Fibromatosesâ&#x20AC;&#x2122; which includes other tumors of fibro-

myofibroblastic origin sharing similar histologic features [7] . The differential diagnoses of Juvenile Fibromatosis are discussed in Table-1. Wide local excision of the tumor with clear margins remains the treatment modality of choice with proper follow-up. This makes fibromatosis a very difficult condition to treat in young patients especially in head and neck region where excision becomes difficult without sacrificing vital structures and neurological structures. There is no consistently successful drug therapy available. Cytotoxic chemotherapy utilizing drugs such as methotrexate, vinblastine/ vinorelbine have been given to delay progression of the tumor until child is considered old enough for radiotherapy. Therapeutic responses also have been observed after non-cytotoxic drugs such as anti-estrogens or non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs stimulate the immune response and impair the proliferation of tumor cells. Radiation therapy is indicated in inoperable cases, after R2 resection (resection with gross positive margins/ incomplete margins) and R1 resection (surgery with microscopic positive margin) [8]. Prognosis of the Juvenile Fibromatosis is very good with no metastasis. Great care must be taken in the clinicoradiologic diagnosis of soft tissue tumors and this entity must be always kept in mind while formulating diagnosis of such cases.

Conclusion

Juvenile Fibromatosis is a benign locally invasive neoplasm of rarity. The biopsy and immuno-histochemical examination remains the gold-standard for diagnosis of this entity. Surgical excision of the tumor is the main-stay of treatment. This case is presented to emphasize on the challenges faced for diagnosing such lesions as they have

Table 1: Differential diagnosis for juvenile fibromatosis. Differential Diagnosis

Histopathological features and immuno-histochemical feature

Reactive fibrosis

Variable growth pattern with focal hemorrhage and hemosiderin deposition

Fibromatosis colli

Originate from sternocleidomastoid. Diffuse fibroblastic proliferation of varying cellularity with entrapped and atrophic muscle fibers.

Fibrous hamartoma of infancy

Organoid pattern composed of interlacing fibrous trabeculae, islands of loosely arranged spindle shaped cells and mature adipose tissue. Vimentin(+), Actin(+)

Infantile myofibroma

Nodular growth pattern. Plump myoid spindle cells, cigar shaped nuclei with eosinophilic cytoplasm. Occasional nuclear atypia. Vimentin(+), actin(+), S-100(-)

Infantile fibrosarcoma

Sheets of solid packed spindle cells arranged in herring bone pattern, hyperchromatic nuclei with predominant nucleoli. Moderate mitoses. Vimentin(+), SMA(+), Desmin(-), myoglobin (-), S-100(-).

Benign fibrous histiocytoma

Short intersecting fascicles of fibroblastic cells arranged in storiform pattern. Occasional histiocytic with giant cells. CD68(+)

Juvenile aponeuritic fibroma

Plump fibroblast with round or ovoid nuclei, indistinct cytoplasm separated by dense collagen. Occasional mitotic figures.

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Kumar et al. to be differentiated from other soft tissue tumors which display borderline pathological features regarding benign or malignant behavior. Immuno-histochemical analysis is generally helpful to reach a definitive diagnosis.

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1. Basha SM, Dutt SC, Murthy NS, Syed G. Juvenile fibromatosis of the temporomandibular joint: A rare case report. Dent Res J, 2014; 11(2): 284-87.

2. Wilkins SA, Waldron CA, Matthews WH et al. Aggressive fibromatosis of the head and neck. Am J Surg. 1975; 130: 412-16. 3. Seper L, Burger H, Vormoor J, Joos U, Kleinheinz J. Aggressive fibromatosis involving the mandible: Case report and review of the literature. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2005:99; 30-8. 4. Lee JC, Thomas JM, Phillips S et al. Aggressive fibromatosis: MRI features with pathologic correlation. Am J Roentgenol, 2006; 186(1): 247-54. 5. Carr RJ, Zaki GA, Leader MB et al. Infantile fibromatosis with involvement of the mandible. Brit J Oral Maxillofacial Surg. 1992: 30; 257-262. 6. Perez Cruet MJ, Burke JM. Weber R et al. Aggressive fibromatosis involving the cranial base in children. Neurosurgery, 1998;43: 1069-102. 7. Debner Louis P. Soft Tissue, Peritoneum and Retroperitoneum: In pediatric Surgical Pathology. 2nd Ed. Baltimore, USA. Williams and Wilkins 1987: 885-90. 8. Halperin E, Brady L,Â Perez C,Â Wazer D. Principles and practice of radiation oncology, 6th Ed. Lippincott, Williams and Wilkins, Philadelphia 2013;(78):1535.

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Acknowledgements

The author would like to record the appreciation for the thoughtful and skilled guidance of the Consultants of the Department of Pathology, Mahavir Cancer Institute and Research Centre, Patna. Without the willing assistance of these people, it would have been difficult to work on this topic.

Funding None

Competing interests None

Reference

Case Report Carcinomatosis Peritonei of Prostatic Adenocarcinoma: Incidental Finding in a Case of Obstructed Umbilical Hernia Rupesh Prakashrao Gundawar*, Umesh Sidheshwar Kanade, Shubhada Yadavrao Bansode, Rahul Suryakantrao Abhange, Shivaji Dadarao Birare Dept. of Patholoy, Government medical college and hospital, Latur, India Keywords: Carcinomatosis Peritonei, Umbilical Hernia, Prostatic Adenocarcinoma, Crystalloids,

ABSTRACT Peritoneal metastasis arising from prostate cancer is a very rare phenomenon. We present a rare case of a patient presenting with carcinomatosis peritonei from a prostate primary. This case report is important in that it is generally not widely recognized that prostate cancer metastasizes to the peritoneum. In the literature, peritoneal metastasis from prostate cancer, if present, are usually associated with other sites of metastases and found at very advanced stages of the disease. In our patient carcinomatosis peritonei was the first presentation which led to the diagnosis of prostate cancer and was the only site of metastasis.

*Corresponding author: Dr Rupesh Prakashrao Gundawar, Dept. of Pathology, 3rd floor, College building, Govt. Medical College, Latur, Maharashtra, India Phone: +91 9503376899 E-mail: rupeshgundawar@gmail.com,

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Gundawar et al.

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Introduction

diagnosed as peritoneal carcinomatosis (or carcinomatosis peritonei) due to prostatic adenocarcinoma.

Prostate cancer is the second cause of cancer related deaths in men.1,2 Haematogenous metastases are present in 35% of patients with prostate cancer, with most frequent involvement sites being bone (90%), lung (46%), liver (25%), pleura (21%), and adrenals (13%).3,4 The peritoneum is an extremely rare metastatic site for prostatic adenocarcinoma, with only a few cases published to date. We present a rare case of an otherwise asymptomatic patient who presented with obstructed umbilical hernia.

Case Report

An 80 year old male presented with an umbilical hernia of 5-6 months which became irreducible since 15 days associated with intermittent vomiting. No obvious urinary complaints were noted. Ultrasonography of abdomen and pelvis reported an abdominal wall defect at umbilicus containing mesentery with few subcentimetric sized lymph nodes. Ultrasonography of scrotum was unremarkable. Routine laboratory investigations were within normal limits. Patient was posted for hernia repair. Prostate was not examined during pelvic USG as patient was primarily managed for obstructed umbilical hernia To the histopathology section, we received peritoneal tissue measuring 7Ă&#x2014;4.8Ă&#x2014;0.3cm. It was studded with multiple (total 27) variable sized greyish nodules ranging from 0.2cm to 1.8cm diameter (Fig.1). Nodules were processed for histopathological studies. Microscopically, nodules were composed of tumour arranged in glands and acini arranged mainly in cribriform, tubulocystic and solid pattern (Fig.2). Glands were particularly perpendicular to each other. Glands were smaller with straight luminal borders (Fig.3-A). Cells lining the glands showed stratification with moderate nuclear pleomorphism, vesicular chromatin, prominent nucleoli and amphophilic cytoplasm (Fig.3-inset). Intra-luminal PAS positive dense, pink, acellular secretion (Fig.4-A) and blue tinged mucin were appreciated. Few glands showed presence of eosinophilic crystalloids (Fig.4-B). With above mentioned characteristic histomorphology, we primarily thought of prostatic metastasis with old age being the only suggestive finding. Since the patient was lost in the follow up and available clinical details were inadequate, large immunohistochemistry (IHC) panel was put. Tumour was immunopositive for, PSA (prostate specific antigen) (Fig.4-A), AMACR (Îą-MethylacylCoenzyme-A racemose) (Fig.4-B) and pan-cytokeratin (AE1/AE3) (Fig.4-C). Tumor was immunonegative for CK7, CK20, CDX-2, chromogranin, synaptophysin, CD10, CEA and PAX-8. With above IHC panel, case was www.pacificejournals.com/apalm

Discussion

Prostatic adenocarcinoma are known for their metastatic notoriety with metastatic manifestations arising before symptoms due to primary cancer. Involvement of the prostatic capsule, bladder base, and seminal vesicles can occur via direct extension. Prostatic cancer is metastatic in 35% of cases, with a marked predilection for bony spread. Growth factors immobilized on bony matrix and adhesive molecules expressed in marrow stromal cells as well as production of PSA and urokinase-type plasminogen activator (u-PA) are some of the factors implicated for preferential homing of prostate cancer cells to the bones in 90% of metastatic cases.5 Other less common sites are lung, liver, pleura and adrenals. Autopsy reports have suggested that prostate cancer metastasizes to the peritoneum in about 7% of the general population.3 Peritoneal metastasis is generally found in late stages of disease with either primary tumour is diagnosed or metastasis at other commoner sites have been found. Our case is unique in its own way that the peritoneal metastasis is the only manifestation with undiagnosed primary tumour. Till date, very few cases of prostate adenocarcinoma metastatic to the peritoneum, have been reported.6,7,8,9 In each case, the diagnosis of prostate adenocarcinoma in the prostate gland had been documented before discovery of the ascites or peritoneal metastasis. Our case stands a special note with peritoneal metastasis found incidentally before primary tumour as the only metastatic manifestation.

Conclusion

With this article, we present an additional case of an unusual manifestation of prostate cancer presented with

Fig. 1: Omental pad of fat is studded with varying sized multiple grey white nodules

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Carcinomatosis Peritonei of Prostatic Adenocarcinoma

Fig. 1 Predominantly tubulocystic pattern with fused glands forming cribriform pattern (inset) (H&E, 40X).

Fig. 2 Glands are showing straight luminal borders. Tumour cells showing stratification with vesicular chromatin and prominent nucleoli (inset) (H&E, 100X).

Fig. 3 Dense eosinophilic intraluminal secretion (A) and intraluminal crystalloids (B) (H&E, 100X).

Fig. 4 Tumor is positive for PSA (A), AMACR (B) and Pan CK (C) (100X).

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Gundawar et al. peritoneal metastases. Thus, surgeons and histopathologist should draw their attention to this rare clinical presentation of metastatic prostatic cancer in all aged patients.

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Acknowledgements

4. Conti G, La Torre G, Cicalese V, Michaletti G, Ludovico MG, Cottonaro G, et al. Prostate cancer metastases to bone: observational study for the evaluation of clinical presentation, course and treatment patterns. Presentation of the METAURO protocol and of patient baseline features. Arch Ital Urol Andro 2008;80(2):59–64.

Funding

5. Roodman GD. Mechanisms of bone metastasis. NEngl J Med 2004;350:1655–64.

Competing Interests

6. Saif MW, Figg WD, Hewitt S, Dahut W. Malignant ascites as only manifestation of metastatic prostate cancer. Prostate Cancer Dis 1999;2:290–3.

The authors appreciate Dr Shruthi Deshkulkarni and Dr Sankalp Deshmukh for assisting in literature collection and snapping the excellent pictures of the case. None

None declared

References

1. Turkbey B., Basaran C., Boge M., Karcaaltincaba M., Akata D.: Unusual presentation of prostate cancer with generalized lymphadenopathy and unilateral leg edema. JBR-BTR, 2008,91: 211-213. 2. De Visschere P, Oosterlinck W, De Meerleer G, Villeirs G.: Clinical and imaging tools in the early diagnosis of prostate cancer. JBR-BTR, 2010,93: 62-70. 3. Bubendorf L, Scho¨ pfer A, Wagner U, Sauter G, Moch H, Willi N, et al. Metastatic patterns of prostate cancer: an autopsy study of 1,589 patients. Hum Pathol 2000;31(5):578–83.

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7. Kehinde EO, Abdeen SM, Al-Hunayan A, Ali Y. Prostate cancer metastatic to the omentum. Scand J Urol Nephrol 2002;36(3):225–7. 8. Zagouri F, Papaefthimiou M, Chalazonitis AN, Antoniou N, Dimopoulos MA, Bamias A. Prostate cancer with metastasis to the omentum and massive ascites; a rare manifestation of a common disease. Onkologie 2009; 32:758–61. 9. Brehmer B, Makris A, Wellmann A, Jakse G. Solitary peritoneal carcinomatosis in prostate cancer (German). Aktuelle Urol 2007; 38:408–9.

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Case Report Role of Cytology and Radiology in Diagnosis of Lacrimal Gland Pleomorphic Adenoma Kamal Kant Gupta*, Swayam Prava Pradhan, Ashok Kumar Dash, Debi Prasad Mishra Department of pathology, M.KC.G. Medical College, Berhampur, India. Keywords: Lacrimal Gland, Cytology, Orbit, Pleomorphic Adenoma.

ABSTRACT Tumors of the lacrimal gland are rare in fine needle aspiration practice. Amongst all, the most common epithelial tumor in the lacrimal gland is pleomorphic adenoma, which is a benign indolent tumor that usually affects adults in the second to fifth decades of life. The most frequent symptom is a painless palpable mass in the upper external quadrant of the orbit, with slow inferonasal displacement of the globe. We present a case of pleomorphic adenoma of lacrimal gland diagnosed on fine needle aspiration cytology (FNAC) and CT scan demonstrated lobulated heterogeneous enhancing solid mass in the right orbit. We emphasize the importance of FNAC and CT scan in the diagnosis of lacrimal gland tumor.

*Corresponding author: Dr. Kamal Kant Gupta, Junior resident, Department of pathology, M.KC.G. Medical College, Berhampur-760004, India Phone: +91 8093970832 E-mail: Dr.kamalkantgupta@Gmail.com

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

Gupta et al.

Introduction

Tumors of the lacrimal gland are rare in fine needle aspiration practice[1]. Amongst all, the most common epithelial tumor in the lacrimal gland is pleomorphic adenoma[2]. Pleomorphic adenoma is the most common epithelial tumor of the lacrimal gland. Lacrimal gland pleomorphic adenoma (LGPA) occurs commonly in second to fifth decades of life. The most frequent symptom is a painless palpable mass in the upper external quadrant of the orbit, with slow inferonasal displacement of the globe[1]. The most frequent LGPA constitutes 3-5% of all orbital tumors, 25% of all lacrimal gland lesions and 50% of epithelial lacrimal gland tumors[3].

A-87 cells. Nuclei were round to oval, eccentric, and have a bland, finely granular chromatin and inconspicuous nucleoli. The cells with plasmacytoid appearance had eccentric nuclei with abundant cytoplasm. Spindle or rounded cells were present within the stromal fragments. The fibrillar chondromyxoid stroma stained intensely red to purple on MGG (Fig. 2,3). The differential diagnosis of other benign lacrimal gland tumor was ruled out and diagnosis of pleomorphic adenoma of lacrimal gland was made. Later on, En bloc excision of the mass was performed by lateral orbitotomy

We describe a case of LGPA in a 40 year-old male. This report emphasizes the importance of CT scan and fine needle aspiration cytology (FNAC) in precise localization and characterization of superotemporal mass lesions. FNAC helps to rule out pathologies like lymphoma, pseudotumor and malignancy, and thus helps formulate the appropriate treatment plan. Early diagnosis and treatment helps preserve vision and prevent future recurrences and malignant transformation.

Case Report

A 40 year-old male presented with proptosis of the right eye for the duration of 6 months. The proptosis was painless, progressive and non-pulsatile. No postural variation in the proptosis was observed. There was no history of diminution of vision in the right eye. There was no history of any constitutional symptoms or trauma. Investigations: Clinical Examination revealed a visual acuity of 6/6 on the snellen chart of both eyes. The right eye was displaced inferiorly and medially. The ocular movement of the right eye was restricted in upgaze. A CT scan of the right orbit demonstrated lobulated heterogeneous enhancing solid space occupying lesion was seen. Lesion was arising from superolateral angle of the right side orbit causing proptosis. No evidence of underlying bone erosion was noted. No evidence of cystic areas were seen within it. No evidence of intracranial extension was noted (Fig. 1). CT scan findings were suggestive of right side lacrimal gland mass may be neoplastic or inflammatory. The differential diagnosis of lymphoma, inflammatory pseudotumor or benign tumor of the lacrimal gland was considered. All other hematological and biochemical investigations were within normal limit. Subsequently, the patient underwent ultrasound-guided FNAC of the right orbital mass. FNAC was performed using 22-gauge needle, which revealed cellular smears mixed with stromal epithelial fragments dispersed singly and in sheets with fibrillar chondromyxoid stroma. The cellular component consists of relatively uniform oval, plasmacytoid or spindle www.pacificejournals.com/apalm

Figure 1: A CT scan of the right orbit showing lobulated heterogeneous enhancing solid space occupying lesion, arising from superolateral angle causing proptosis. No evidence of underlying bone erosion or cystic areas and intracranial extension is noted.

Fig. 2: Typical low-power pattern of poorly cohesive epithelial-like cells associated with fibrillar chondromyxoid stroma staining brightly red/magenta (MGG, LP).

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Lacrimal Gland Pleomorphic Adenoma

approach. Histopathological examination showed cystic structures lined by benign epithelial cells and surrounding myoepithelial cells melting in chondromyxoid stroma (Fig. 4). On histopathological examination, we confirmed the diagnosis of pleomorphic adenoma. No feature of malignant transformation was seen.

LGPA is a benign indolent tumor consisting of a very firm mass that leads to compression atrophy of the normal gland, displacement of residual lacrimal tissues, and is surrounded by a â&#x20AC;&#x2DC;pseudocapsuleâ&#x20AC;&#x2122; into which small sprouts of adenoma may be projected[6]. Most cases (90%) involve the orbital lobe of the lacrimal gland[4]. LGPA is most frequent in adults[4], with no gender preponderance[1]. The clinical presentation is usually characterized by a painless palpable mass in the upper external quadrant of the orbit, with slow growth and inferonasal displacement of the globe[1]. There may also be an increased lacrimation and intrabulbar pressure, visual impairment and diplopia[2]. Radiological investigations may be done either by CT or MRI, as in our case. Both are similar in terms of providing information regarding extent, configuration, margins, and angulation features of a lacrimal gland fossa mass. However, CT scan provides more details about bone destruction and presence of calcification, while MRI provides better intralesional features and intracranial extension[2].

Fig. 3 : High-power view showing epithelial cells present in sheets and singly with plasmacytoid appearance (MGG, HP).

Fig. 4: Histopathological examination showed cystic structures lined by benign epithelial cells and surrounding myoepithelial cells melting in chondromyxoid stroma (H&E, HP).

Disscusion

Tumors of the lacrimal gland are a rare condition in FNA practice, constituting 7-9% of all orbital tumors[1]. Amongst all, the most frequent LGPA constitutes 3-5% of all orbital tumors, 25% of all lacrimal gland lesions and 50% of epithelial lacrimal gland tumors[3].

The safety of FNAC for the diagnosis of lacrimal gland tumors has been questioned due to potential risk of tumor recurrence caused by disruption of the lacrimal gland tumor pseudocapsule which leads to tumor seeding along the needle track[5]. However, there have been no reports of recurrence due to the use of FNAC[6] or evidence of tumor seeding upon serially sectioning the needle track. Lai et al [7] demonstrated that FNAC differentiates between various lacrimal gland pathologies and helps in definitive management; it prevents incomplete excision of a malignant lesion and future recurrence. Verma and kapila[8] demonstrated FNAC to have a specificity of 98.2% and positive predictive value of 96.7%. Vagefi et al[9] recommended a complete surgical excision of the tumor to prevent future recurrence and malignant transformation. Thus, a preoperative FNAC is prudent in diagnosing and tailoring the management of each individual case. The recurrence can further be prevented by excision of the biopsy track. On the basis of the cytological features, we differentiated pleomorphic adenoma from adenoid cystic carcinoma, monomorphic adenoma, mucoepidermoid carcinoma. In others tumors, there were no chondromyxoid ground substance present.

Conclusion

LGPA is a common differential of superotemporal mass, especially in adults. LGPA should be kept in mind when dealing with superotemporal masses in adults. FNAC and CT scan helps in ruling out other possible causes, each having diverse treatment approaches, for example, lymphoma validates an early institution of chemotherapy.

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Gupta et al. This highlights the importance of FNAC and CT scan in achieving the accurate preoperative diagnosis and inducting the appropriate treatment without delay.

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2. Chandrasekhar J, Farr DR, Whear NM. Pleomorphic adenoma of the lacrimal gland: case report. British Journal of Oral and Maxillofacial Surgery. 2001;39.5: 390.

3. Fenton S, DMDS Sie Go, MPh Mourits. Pleomorphic adenoma of the lacrimal gland in a teenager, a case report. Eye. 2004;18.1:77. 4. Rose GE. To crash or not to crash & quest; Probability in the management of benign lacrimal gland tumours. Eye. 2009;23.8:1625. 5. Wright JE, Stewart WB, Krohel GB. Clinical presentation and management of lacrimal gland tumours. British Journal of Ophthalmology. 1979;63.9:600. 6. Amy T, Tartter PI, Zappetti D. Breast cancer diagnosis by fine needle aspiration and excisional biopsy. Acta cytological. 1997;41.2:302. 7. Lai T., et al. Pleomorphic adenoma of the lacrimal gland: is there a role for biopsy & quest. Eye. 2009;23.1:2. 8. Verma K, Kapila K. Role of fine needle aspiration cytology in diagnosis of pleomorphic adenomas. Cytopathology. 2002;13.2: 121. 9. Vagefi, M. Reza, et al. Atypical presentations of pleomorphic adenoma of the lacrimal gland. Ophthalmic Plastic & Reconstructive Surgery. 2007;23.4: 272.

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Acknowledgements Nil

Funding None.

Competing Interests None declared.

References

1. Santos, Rodrigo Ribeiro, et al. Ten-year follow-up of a case series of primary epithelial neoplasms of the lacrimal gland: clinical features, surgical treatment and histopathological findings. Arquivos brasileiros de oftalmologia. 2010; 73.1: 33.

Case Report Congenital Hepatic Fibrosis : Report on Two Cases And its Clinicopathological Correlation Neeraj Dhameja1, Varnika Rai1*, Rajeev Singh2, Vineeta Gupta3, O.P Mishra3 Department of Pathology, Institute of Medical Sciences BHU, India Department of Radiodiagnosis and Imaging Medicine, Institute of Medical Sciences BHU, India 3 Department of Pediatrics, Institute of medical sciences, BHU, India 1

Keywords: Congenital Hepatic Fibrosis, Caroliâ&#x20AC;&#x2122;s Syndrome, Autosomal Recessive Polycystic Kidney Disease.

ABSTRACT Introduction : Congenital hepatic fibrosis (CHF) is a relatively rare disease belonging to ductal plate malformations and considered as variant of autosomal recessive polycystic kidney disease with juvenile and young adult presentation. Case report : Case 1 was a 13 year male, presented with complaints of abdominal pain and distension with multiple episodes of fever. Case 2 was a two year male patient, presented with complaints of abdominal swelling since birth, fever and nausea, along with palpable liver and non-palpable spleen. Histopathological examinations in both cases showed dense portal fibrosis with preserved hepatocytes. Within the fibrotic portal tract dilated biliary channels were seen with neutrophilic infiltration in first case which was suggestive of superimposed cholangitis. Hepatic parenchyma showed normal arrangement of hepatocytes. Based on the findings diagnosis of CHF was made. Conclusion : Congenital hepatic fibrosis though a rare disease should be considered in the differential diagnosis while dealing with pediatric liver biopsies.

*Corresponding author: Dr Varnika Rai , Sadhana Sadan , N4/35C- 92, Mahamana Nagar Colony, Karaundi, Suswahi, BHU, Varanasi- 221005, Uttar Pradesh. India Phone: +91 9956827228 E-mail: vrachievers@gmail.com

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

Dhameja et al.

Introduction

Congenital hepatic fibrosis (CHF) is considered as variant of autosomal recessive polycystic kidney disease with juvenile and young adult presentation.[1] It is a relatively rare disease pertaining to ductal plate malformations.[1] The disease appears in both sporadic (in many as 56% cases) and familial patterns. Congenital hepatic fibrosis-ARPKD is estimated to occur in 1 in 20,000 live births.[2] Arrest of maturation and the lack of remodeling of the ductal plate engenders persistence of an excess number of immature embryonic duct structures which in turn stimulates the formation of portal fibrous tissue conferring the clinical picture of recurrent cholangitis or portal hypertension and associated symptoms.[3] The disease appears in both sporadic and familial patterns. Clinically it is characterized by hepatic fibrosis, portal hypertension, and renal cystic disease. However clinical manifestations of CHF can be nonspecific. Here we are presenting two cases of congenital hepatic fibrosis presented within one year duration in SS Hospital, IMS BHU.

Case Reports

Case 1: A 13 year old male patient presented in the department of pediatrics of Sir Sunder Lal Hospital, IMS, BHU. Patient was having complaints of pain and distension in abdomen and multiple episodes of on and off fever and jaundice, along with history of melena and hematemesis. On physical examination- pallor present, there was increase in abdominal girth. Spleen and liver both were palpable. On ultrasound features of portal hypertension was present. On Investigations, hemoglobin was 5.1 g/dl, Total leucocyte count was 12.6 ×103 /μl, Platelet count was 64000 / μl. Liver function tests revealed total bilirubin was 2.5 mg/dl, ALT was 69 U/L, AST was 46 U/L. Total protein was 6.3 g/dl. Serum urea 27 mg/dl and serum creatinine 0.5 mg/dl. CT scan findings (fig 1A) showed hepatosplenomegaly with dilated central and peripheral intrahepatic biliary radicals with multiple round hypodense cystic areas showing central dot sign. Extra hepatic biliary channels were not dilated. CT findings are suggestive of Caroli’s disease. Bilateral kidneys were mildly bulky, however cortico-medullary differentiation was within normal limits. Histopathological examinations (fig 1B, C and D) showed dense portal fibrosis with preserved lobules of hepatocytes. Within the fibrotic portal tract dilated biliary channels were seen with neutrophilic infiltration which was suggestive of superimposed cholangitis. Hepatic parenchyma showed normal arrangement of hepatocytes. No granuloma or malignant cells were seen. Based on the clinical, radiological and histopathological findings, diagnosis of CHF was made. www.pacificejournals.com/apalm

A-91 Case 2: A 2 year old male patient was admitted in the pediatric ward of Sir Sunder Lal Hospital IMS BHU. Patient was having complaints of abdominal swelling since birth, fever and nausea since 15 days. On examination, pallor was present. Per abdomen, there was distension of abdomen with umbilicus central everted. Liver was palpable and spleen was non palpable. Bilateral renal lump palpable. Rest systemic examination were within normal limit. On Investigations, hemoglobin was 7.4 g/dl, Total leucocyte count was 12.82 ×103 /μl, Platelet count was 6.5lakh/ μl. Liver function tests revealed total bilirubin was 0.3 mg/ dl, AST was 152 U/L, ALT was 88 U/L. Total protein was 7.4 g/dl. serum urea 122 mg/dl and serum creatinine 2.9 mg / dl. CT findings (fig 2A & B) were mild dilatation of central intrahepatic biliary radicals with focally dilated peripheral biliary radicals with central dot sign, suggestive of Caroli’s disease. Bilateral kidneys were enlarged, hypodense with loss of cortico-medullary differentiation. Few focally scattered hyperdensities also noted likely calcifications. In view of findings of Caroli’s disease, CT differentials were ARPKD and medullary sponge kidney. Histopathological findings (fig 2C &D) were same as case no 1 however histopathological features of cholangitis were lacking. Liver biopsy showed dense portal fibrosis with several dilated ducts embedded in it. Lobules of hepatocytes were also noted with normal arrangement of hepatocytes thus further reinforcing our diagonosis of CHF. Family history of both patients were negative for any fibropolycystic disease.

Discussion

Congenital hepatic fibrosis is considered as an autosomal recessive disorder.[4] CHF is one of the fibropolycystic diseases, which also include Caroli’s disease, autosomal dominant polycystic kidney disease (ADPKD), and autosomal recessive polycystic kidney disease (ARPKD). [1]

Numerous cases of CHF coexists with autosomal recessive polycystic kidney disease (ARPKD) or dilatation of the intrahepatic or extrahepatic biliary tree, characteristic of Caroli’s syndrome.[5,6] On the basis of age of presentation and the degree of renal involvement, ARPKD has been divided into four types by Blyth and Ockenden[3] – perinatal, neonatal, infantile and juvenile. It predominantly affect children and adolscents.[7] Infants present with abdominal distension from enlarged organs, respiratory distress and systemic hypertension. Older eISSN: 2349-6983; pISSN: 2394-6466

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Congenital Hepatic Fibrosis

Fig. 1(A). NCCT Abdomen showing hepatosplenomegaly, dilated intrahepatic biliary radicals with hypodense cystic areas with “central dot sign” (arrow) suggestive of Caroli’s disease.(B)Liver biopsy with diagnostic dense fibrosis of portal tracts with numerous marginal dilated bile ductular profiles. (H& E 100 X). (C) Bile in few ducts with periductular neutrophilic infiltration (H& E, 200 X). (D) Portal area with extensive fibrosis and normal hepatocytes arrangement within nodule. (reticulin stain 100x)

Fig. 2(A). NCCT abdomen axial sections of Case 2 showing mild central intrahepatic biliary duct dilatation. (B) NCCT abdomen coronal sections of Case 2 showing bilateral enlarged hypodense kidneys-ARPKD. (C) Portal area with extensive fibrosis and several bile ducts with cuboidal epithelium, arrested at different stages of the maturation process. (H & E , 200X). (D) Portal area with extensive fibrosis and normal hepatocytes arrangement within nodule. (reticulin stain 100x)

patients present with hepatosplenomegaly or bleeding from oesophageal varices but can be asymptomatic.[8] Renal involvement is maximal in perinatal group and minimal in juvenile group of ARPKD.[3] In the infantile group the clinical picture is either of chronic renal failure or of increasing portal hypertension. The juvenile group classically incorporates children (mostly 1–5 years old) who present with portal hypertension. In liver histopathological changes are marked.[3]

cholangitic and latent forms.[10] The pure cholangitic form is rare. In the mixed form patients suffer from recurrent bouts of cholangitis, with or without jaundice, in addition to the manifestations of portal hypertension. Our first case is also showing feature of mixed type presentation with portal hypertension characterized by melena, hemetemsis and splenomegaly along with cholangitis characterized by fever, jaundice and pain in abdomen.

Patients of CHF with ARPKD typically present as neonates or infants with severe renal impairment or pulmonary insufficiency,[4] likewise our second case presented with features of renal insufficiency however pulmonary insuffiency features were absent. Caroli’s syndrome comprises a clinical syndrome which is a combination of Caroli’s disease and those of congenital hepatic fibrosis. Correspondingly our first case was also showing features of CHF in histology besides features of Caroli’s disease on radiological findings.[9] Four clinical forms of CHF have been recognized – portal hypertensive, cholangitic, mixed portal hypertensive-

Grossly Liver is enlarged in CHF, has a firm to hard consistency, and shows a fine reticular pattern of fibrosis, no cysts are visible to the naked eye.[10, 11,12] According to Shorbhagi et al 2010 [1] laboratory workup may reveal mild elevations in liver enzymes. Concordantly in our both cases there was increased level of liver transaminases along with increased level of alkaline phosphatase. There was also elevation in total serum bilirubin due to associated cholangitis in our 1st case. Desmet et al[13] 1992 also described that abnormal renal functions tests were associated with extensive cystic renal disease, which might even progress to end-stage renal failure , this in agreement to our second case.

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Dhameja et al. A confirmed diagnosis of congenital hepatic fibrosis can only be made by a examination of a liver biopsy. Histological findings: portal tracts show expansion by connective tissue, with wide areas of septal bridging fibrosis connecting portal tracts to each other. Persistent ductal remnants are usually evident along the margins of portal tracts and fibrous septa. Interlobular bile ducts may be ectatic. Signs of cholestasis may be observed in the setting of associated cholangitis. Portal veins may be hypoplastic or completely absent.[14]A portal vein anomaly can be present as a component of the disorder, rather than a consequence, since bile ducts and portal veins share embryonic origins. Livers affected by congenital hepatic fibrosis usually contain numerus Von Meyenburg complexes.[4] Because of the similarities in the clinical picture, it is necessary to differentiate CHF from early liver cirrhosis, for which a liver biopsy is essential.[1] Congenital hepatic fibrosis can be distinguished from inflammatory bridging fibrosis or cirrhosis by lack of inflammation, lack of regenerative nodules, and presence of anastomosing biliary channels rather than proliferating bile ductules. Reticulin stain is useful because it may confirm the presence of regenerative activity as evidenced by cell plates greater than two cells thick in the cirrhotic liver. As there was no supporting family history present both our cases appeared to be having autosomal recessive sporadic pattern. While our first case seemed to be a Caroli’s syndrome (CHF with Caroli’s disease), our second case was more likely a case of Congenital Hepatic Fibrosis with associated ARPKD. Both our patients are alive till date and are clinically stable.

Conclusion

Congenital hepatic fibrosis is a rare disease with varying age of manifestations. It can lead to portal hypertension and fatal complications appertained to it. Therefore pediatric liver biopsies should always be carefully evaluated in order to rule out diagnosis of CHF despite presence or absence of contributive clinical background of patient.

Acknowledgements

We are thankful to Dr Mohan Kumar

Funding None

Competing Interests None declared

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Reference

1. Shorbagi A, Bayraktar Y. Experience of a single center with congenital hepatic fibrosis: a review of the literature. World journal of gastroenterology 2010;16: 683. 2. Patel J N et al. Congenital Hepatic Fibrosis associated with polycystic kidney disease. Annals of Pathology and Laboratory Medicine 2015;2:187-191. 3. Blyth M, Ockenden BG. Polycystic disease of kidneys and liver. J Med Genet 1971; 8:257-84. 4. Arroyo M, Crawford JM. Pediatric Liver Disease and Inherited, Metabolic, and Developmental Disorders of the Pediatric and Adult Liver. In Odze RD, Goldblum JR, eds. Surgical Pathology Of The GI Tract, Liver, Biliary Tract and Pancreas. 2nd ed. Philadelphia: Saunders Elsevier; 2009.p.1245-1290. 5. Nakanuma Y, Terada T, Ohta G. Caroli’s disease in congenital hepatic fibrosis and infantile polycystic disease. Liver 1982;2:346–54. 6. Summerfield JA, Nagafuchi Y, Sherlock S, et al. Hepatobiliary fibropolycystic diseases. A clinical and histological review of 51 patients. J Hepatol 1986;2:141–56. 7. Portman BC, Roberts EA. Developmental abnormalities and liver disease in childhood. In: Burt AD, Portman BC, Ferrell LD, eds. MacSween’s Pathology of the Liver. 6th ed. London, UK: Churchill Livingstone Elsevier;2012.p.102-156 8. Averback P. Congenital hepatic fibrosis:asymptomatic adults without renal anomaly. Arch Pathol Lab Med 1977;101:260–1. 9. Yonem O, Bayraktar Y. Clinical characteristics of Caroli’s syndrome. World J Gastroenterol 2007; 13: 1934-1937 10. Fauvert R, Benhamou JP. Congenital hepatic fibrosis. In: Schaffner F, Sherlock S, Leevy CM, editors. The liver and its diseases. New York: Intercontinental Medical Book; 1974. p. 283–8. 11. Witzleben CL. Cystic diseases of the liver. In: Zakim D, Boyer TD, editors. Hepatology A textbook of liver disease. Philadelphia: WB Saunders; 1990.p. 1395–411. 12. De Vos BF, Cuvelier C. Congenital hepatic fibrosis. J Hepatol 1988;6:222–8. 13. Desmet VJ. What is congenital hepatic fibrosis? Histopathology 1992; 20: 465-477 14. Gocmen R, Akhan O, Talim B. Congenital absence of the portal vein associated with congenital hepatic fibrosis. Pediatr Radiol 2007; 37: 920-924.

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Case Report Squamous Cell Carcinoma of Gall Bladder: A Rare Presentation Chhanda Das1*, Koushik Sarkar1, Madhumita Mukhopadhyay1, Bedabrata Mukhopadhyay2 Dept of Pathology, Institute of Post Graduate Medical Education & Research, Kolkata, India 2 Department of Biochemistry, Institute of Medical Sciences BHU, India

Keywords: Gall Bladder, Squamous Cell Carcinoma , Biliary Colic

ABSTRACT Pure Squamous cell carcinoma of the gall bladder is a rare entity. Here we present a case of 50-yrs-female presented to our hospital with intermittent upper abdominal pain, jaundice, anorexia, weight loss for last 6 months. On ultrasonography it was found that, there was presence of mass measuring (8x7) cm in gall bladder region. Subsequently, exploratory laparotomy was done and gall bladder along with liver bed and adjacent lymph node surgically removed. Histopathologically the case was diagnosed as well differentiated squamous cell carcinoma infiltrating upto serosa without involving liver bed or lymph node.

*Corresponding author: Dr Chhanda Das, 31 Eastern park , First Road , Santoshpur .Kolkata 75, India Phone: +91 9433116223 E-mail: chhhdas@gmail.com

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

Das et al.

A-95

Introduction

Gallbladder carcinoma is rare; however, within the biliary tract, the gallbladder is the most common site for malignancy and is the fifth most common site among gastrointestinal tract-related organs [1]. Squamous cell carcinoma of the gall bladder is rare and accounts for 12.7% of all cases of gall bladder cancer. Pure squamous cell carcinoma is even less common with incidence of 3.3%[.2-5]Squamous cell carcinoma of the gall bladder usually presents with an ill defined clinical course and is frequently detected in an advanced stage because of its tendency to infiltrate adjacent organs and silent rapidly growth pattern[2,3].The overall prognosis of squamous cell carcinoma is better than adenocarcinoma[4].

Case Report

A 50yrs old female patient presented in the OPD in our hospital with history of pain abdomen, jaundice, anorexia and weight loss for last 6 months. On general examination there was presence of icterus. On local examination there was tenderness in right upper abdomen. Routine investigations revealed raised bilirubin level with elevated liver enzymes. On ultrasound it was found that there was presence of a mass in the gall bladder measuring (8x7) cm with mild ascites. (fig 1) Subsequently, open laparotomy done and gall bladder with mass along with liver bed and adjacent lymph node surgically removed and sent for histopathological examination.

is between 4th and 6th decade[5] . Our case presented at age of 50.Gall bladder cancers are asymptomatic at early at early stages. When symptomatic, the presentation is similar to biliary colic or chronic cholecystitis. If signs of biliary colic or chronic cholecystitis present in an elderly patient along with weight loss and anorexia, gall bladder carcinoma should be suspected.[7] The histogenesis of squamous cell carcinoma is not well understood. Epithelial tumor genesis is a multistep process resulting from sequential genetic alteration at different stages of evolution. Because a normal gall bladder has no squamous epithelium, the source of origin is questionable .Some researchers have stated that squamous cell carcinoma originates from pre-existing squamous metaplasia of gall bladder epithelium, while others concluded that it originates from squamous metaplasia of neoplastic cells of adenocarcinoma[.4,6.] Genetic changes also have been identified along with mutations that consist of decreased expression of 23nm and over-expression of c-erb B2 gene product.[ 8]

On gross examination there was a greyish mass measuring (9x8x2) cm3 containing, gall bladder and liver bed (fig 2). On cut section whitish areas were noted. Normal anatomy of gall bladder totally distorted. Multiple sections were taken. On histology the sections showed neoplastic cells were arranged in diffuse sheets of cells extending into the muscle layer upto serosa. Individual cells were large, polygonal containing abundant eosinophilic cytoplasm, pleomorphic vesicular nuclei and prominent nucleoli. There was also presence of intracellular bridges and numerous keratin pearls (fig 3 ,4 &5).Sections from liver bed and lymph node do not show any tumor deposit. Histologically the case was diagnosed as infiltrating moderately differentiated squamous cell carcinoma extending upto the serosa.

Discussion

Squamous cell carcinoma is a rare and aggressive form of gall bladder carcinoma. It usually presents as invasive growth and spreads by local invasion. Squamous cell carcinoma of the gallbladder is predominantly incident among females, in a proportion of 3:1 over males, The mean age for squamous cell carcinoma of gall bladder

Fig. 1: ultrasonography showing mass in gall bladder region.

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Squamous Cell ca GB

liver bed

Fig. 4: Histological examination: shows infiltrating well differentiated keratinized squamous cell carcinoma of gall bladder (H&E 100X)

Fig. 3 : Histological examination of gall bladder showing columnar epithelium (H&E 100X)

Fig 5 : Microscopic examination reveals tumor composed of well differentiated squamous cells characterized by keratin pearls and intercellular bridges (H&E 400X).

Fig. 2: Gross picture of showing the mass.

gall bladder

and

The etiology and pathogenesis of squamous cell carcinoma is not well understood; however, two important presumptive causative possibilities are gallstones and parasitic infestation[.6 ,9] .Other pathologies that have been associated with increased incidence of carcinoma of gall bladder are polypoid lesions, adenomas, calcified porcelain gall bladder, ulcerative colitis, adenomyomatosis, polyposis coli and anomalous connection between CBD and pancreatic duct. Traditionally it has been said that adenosquamous and squamous cell carcinoma have very poor prognosis. However, because of the better prognosis of pure squamous cell carcinoma it is important now to differentiate it from adenocarcinoma with squamous differentiation from. Pure squamous cell carcinoma of the gallbladder grows slowly, is usually localized and rarely metastasized. On the other hand adenosquamous carcinoma is aggressive and metastasizes widely[4] . Radical resection is the mainstay of treatment for patients with locally invasive squamous cell carcinoma and offers the only

chance for cure[.3] extent of tumor invasion at the time of diagnosis is the most important parameter in determining survival. Significant prognostic factors include residual tumor status, type of resection, patient age, and blood vessel invasion.

Conclusion

Pure squamous cell carcinoma of gall bladder is a rare entity. Radical resection is the mainstay of treatment for locally invasive squamous cell carcinoma. Early diagnosis is the most important parameter for improving the survival of the patient.

Acknowledgements None

FUNDING None

Competing Interests None Declared

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REFERENCE

1. Greenlee RT, Hill-Harmon MB, Murray T, et al. Cancer statistics, 2001. CA Cancer J Clin 2001;51:15-36. 2. Rekik W, Fadhel CB, Boufaroua AL, Mesteri H, Khalfallah MT, Buraroui S .et al “case report: Primary Pure Squamous Cell Carcinoma of Gall Bladder”, Journal of visceral surgery, 2011;148:e149-e151. 3. Soyama A, Tajima Y, Kuroki T, Tsuneoka N, Ohno S, Adachi T, et al “Radical Surgery for Advanced Pure Squamous Cell Carcinoma of the Gall Bladder: Report of a Case”, Hepatogastroenterology,2011:58:2118-20. 4. Roa JC, Tapia O, Cakir A, Basturk O, Dursun N, Akdemir D.et al “Squamous Cell and Adenosquamous Cell Caecinomas of the Gall Bladder: Clinicopathological Analysis of 34 cases Identified in 606 carcinomas” Modern Pathology 2011:24:1069-1078. 5. Waisberg J, Bromberg SH, Franco MIF, et al “Squamous Cell Carcinoma of Gall Bladder”Sao Paulo Medical Journal; 2001:119:.43.

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6. Roppongi T, Takeyoshi I, Ohwada S, Sato Y, Fujii T, Honma M et al. Minute squamous cell carcinoma of the gall bladder, a case report. jpn j clin oncol 2000;30;43-5;doi:10.1093/jjco/hyd 010.PubMed PMID:10770570. 7. Angelica MD, Jarnagin W. ”Tumors of the Gall Bladder”, In B L Saunders, Ed, Surgery of the Liver, Biliary Tract and Pancreas,4th edition , Elsevier, Amsterdam, 2006,p.746-781. 8. Rhodes PS, BS Gall Bladder Carcinoma with the unusual occurrence of squamous cell carcinoma. A case study, J Diagn Med Sonography, 2004,20;347-350. 9. Karasawa T, Itoh K, Komukai M, Ozawa U,Sakurai I, Shikata T. Squamous cell carcinoma of gallbladder-report of two cases and review of literature. Acta Pathol Jpn. 1981;31:299-308. PubMed PMID: 7257770.

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Case Report Interdigitating Dendritic Cell Sarcoma Arising in The Cervical Node S. Shifa Ibrahim*, N. Sharmila Thilagavthy, G. Meena Kumari Dept.of Pathology, Madurai Medical College, Madurai, India

Keywords: Immunohistochemistry, Peripheral Node, S100, dendritic Cell, Sarcoma

ABSTRACT Inter-digitating dendritic cell (IDC) sarcoma is a rare tumor presenting as an asymptomatic slow growing lesion. The median age of occurrence is around 56 years and has a male preponderance. Peripheral lymphadenopathy is the commonest presenting symptom. The clinical course of patients with IDC sarcoma varies from indolent to more aggressive with widespread metastases and death within a year. There is no standardized therapeutic approach for patients with IDC sarcoma. We are presenting a case of IDC sarcoma in a 40 year old male with cervical node and liver involvement. Histologically, there was complete effacement of nodal architecture with tumor cells arranged in sheets composed of spindle cells and epithelioid cells having convoluted nuclei. Immuno- reactivity for S100 and vimentin were observed. Other stains such as CD 1a, CD21, CD3 and CD45 were negative. Histology, Immuno histochemistry and differential diagnosis are discussed in our presentation which would help in arriving at the diagnosis of this rare tumor.

*Corresponding author: Dr. S. Shifa, 82, J.N.Nagar, Old Natham Road, Madurai-625017, India Phone: +91 9486669274 E-mail: shifaffrin@gmail.com

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Ibrahim et al.

Introduction

Inter-digitating dendritic cell (IDC) sarcoma is a rare tumor with a male preponderance. [1, 2] Mean age of occurrence is 56 years. [1, 2] Cervical lymph nodes, nasopharynx, tonsil, salivary glands, skin, chest wall, paraspinal region, liver, spleen, gastrointestinal tract, bladder, and testis and bone marrow are involved with this tumor. Of this peripheral lymphadenopathy[cervical node] is the commonest presenting symptom. [3] Along with the histological findings, Immunohistochemistry with S-100 is essential for the confirmation of diagnosis. We are presenting a case of IDC sarcoma in a forty year old male. He had an indolent course for about 11/2 years. The histological picture along with Immuno -histochemistry and differential diagnosis are discussed.

Case Report

40 year male with the complaints of swelling neck and fever was investigated elsewhere 11/2 years back. FNAC was done there and he was diagnosed as having tuberculous lymphadenitis. He had anti tuberculous treatment for three months. As the swelling did not subside, he was referred to our center. Further evaluation was done on him in our center. On examination the patient had multiple bilateral cervical nodes and supraclavicular nodes, [Fig: 1]. CT scan showed cervical node, supraclavicular nodes, para- aortic nodes and liver involvement. Radiologist reported it as lymphoma. FNAC of the node showed spindle and epithelioid cells in an inflammatory background composed of eosinophils, [Fig: 2]. Provisional diagnosis of spindle cell tumor was made and biopsy was advised. Excision biopsy of the nodes was taken and sent to the Department of Pathology, Madurai Medical College for evaluation. The specimen we received in our department was 1x1cm. All tissues were embedded and examined.

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Discussion

Dendritic cells[DCs] are the key antigen-presenting cells. Four types of DCs are seen in the lymph nodes. They are follicular, interdigitating, Langerhans, and fibroblastic.[4] Interdigitating cells are distributed in the paracortex and deep cortex of lymph nodes, periarteriolar lymphoid sheaths in spleen and interfollicular areas of mucosa associated lymphoid tissue.[1].Neoplasms arising from these DCs as such are rare and constitute about 1% of all lymph node tumors.[2] Of that Interdigitating dendritic cell sarcoma is a rare neoplasm with only around 100 cases reported in the literature.[1] Median age at diagnosis is 56years with a male predilection.[1,2,3]. An ethnic predilection among Asians was noted. [1] Peripheral nodes like cervical and axillary nodes are most commonly involved.[1] Extranodal sites involved by this tumor include nasopharynx, tonsil, salivary glands, skin, chest wall, paraspinal region, liver, spleen, gastrointestinal tract, bladder, and testis [5]. In 20% of the cases, bone marrow involvement is seen. [6]Usually asymptomatic, B-type symptoms like fever, weight loss, and night sweats can occur in about 20% of the cases. [6] Grossly the size of the tumor varies from 1-6cm. It is lobulated and has a solid cut surface. Foci of necrosis and haemorrhage can also be seen. Microscopically the tumor either partially or completely effaces the node architecture. In partial involvement the tumor occupies the paracortical areas. The neoplastic cells are arranged in sheets, whorled, storiform, or nests and a mixture of patterns. Among them, mixed patterns are more common. Individual cells have indistinct cell borders and are spindle-shaped or epithelioid. [7] They have abundant eosinophilic cytoplasm and elongated or oval nuclei with irregular or folded nuclear contours. Nucleoli can be inconspicuous or prominent. Multinucleated giant cells are common. [7] Atypia is mild

Microscopically, complete effacement of the lymph node architecture with a thick capsule was noted, [Fig 3]. The node was replaced by tumor cells arranged in fascicles, whorls and sheets. Individual cells were spindle to ovoid with abundant eosinophilic cytoplasm. The nucleus shows indentation, dispersed chromatin and prominent nucleoli. There were neither necrosis nor mitosis. Lymphocytes and eosinophilic infiltrates were seen among the tumor tissue, [Fig: 4 and 5]. He was diagnosed as dendritic cell neoplasm and immunohistochemical study was done for confirmation and categorization. The tumor cells showed diffuse S100 and vimentin positivity,[Fig: 6 and 7]. Other markers like CD45, CD1a, and CD21,CD3 were negative. That clinched the diagnosis of Inter digital dendritic cell sarcoma.

Fig. 1: 40 years male with multiple cervical and supraclavicular nodes

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Fig. 2: FNAC of the lesion show tumor cells arranged in fascicles(H&E 10x) .Inset shows tumor cells with eosinophilic cytoplasm and oval to indented nuclei and eosinophils in the background.(H&E 40x)

Fig. 3: Shows complete effacement of lymph node architecture with a thick capsule,[4x H&E]

Fig. 4and5: Show tumor cells arranged in fascicles and whorls. Individual cells show oval and indented nuclei in an inflammatory background composed of eosinophils.(H&E 40x).

Fig. 6: Shows diffuse S100 positivity

Fig. 7: Shows Vimentin positivity.

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Ibrahim et al. to marked. The mitotic rate is usually low in this tumor. It is associated with fibrosis and inflammatory cells like small lymphocytes, eosinophils and plasma cells. Usually erythrophagocytosis can be seen focally. Immunohistologically[IHC], strong S100 and vimentin positivity is characteristic of this lesion. CD11c, HLADR, and fascin are the other markers that are positive. Leukocyte common antigen CD45 is focally positive. [8] The histo-morphological differential diagnosis includes Histiocytic sarcoma, Langerhans cell sarcoma, Metastatic melanoma and Follicular dendritic cell sarcoma. In Histiocytic sarcoma, neoplastic cells have abundant eosinophilic to vacuolated cytoplasm admixed with giant cells having wreath-like nuclei leading on to a xanthomatous appearance. Immunohistochemically, CD11c, CD4, CD14, CD68 and lysozyme are strongly positive in Histiocytic sarcoma. Mixed spindle and epithelioid pattern seen in our case and IHC studies helped to rule out Histiocytic sarcoma. In Langerhans cell sarcoma [LCS], cells have eosinophilic cytoplasm, twisted and grooved nuclei. CD1a, S100 protein and langerin are positive. Grooved nucleus was not seen in our case and absent CD1a marker helped to rule out LCS. Metastatic melanoma cells are more epithelioid, have eosinophilic nuclei and show greater pleomorphism than IDC sarcoma. They also express S100 protein and CD68 like IDC. HMB45 and Melan A expression are more specific for melanoma. Both the cytological feature and the marker studies ruled out melanoma in our case. In Follicular dendritic cell [FDC] sarcoma, oval to spindle-shaped cells are arranged in sheets, fascicles, whorls, storiform, trabecular, or nodular arrangement. In differentiating it from IDC sarcoma Immunohistochemistry is more useful as there are morphological similarities between these two lesions. [9] Strong S100 positivity and vimentin positivity is seen in IDC sarcoma. In FDC sarcoma, clusterin, CD21, CD23, and CD35 are positive. [10]

A-101 The clinical course of patients with IDC sarcoma is variable. The course may be benign or aggressive with widespread metastases and death within a year, which is observed in 40% to 50% of patients. Patients with IDC sarcoma develop second neoplasms, like T- or B-cell lymphomas or leukemias or carcinomas. Young age, abdominal involvement, extranodal involvement combined with nodal involvement is associated with a grave prognosis. Our case is a relatively younger male with both nodal and extranodal involvement precluding a graver course. At present no treatment modality is available for IDC sarcoma. [11] Local disease is treated with surgery. Chemotherapy includes the regimen that is used for lymphoma. More research is needed in this arena.

Conclusion

In conclusion, Interdigitating dendritic cell sarcoma is a rare tumor of dendritic cells. The disease course can be indolent to aggressive. So far no therapeutic options are available. And there is a 15% risk of developing secondary tumors in these patients. That warrants a careful follow up in these patients. This case is presented to highlight the fact that more research is needed in the treatment aspect of this rare neoplasm.

Acknowledgements None

Funding None

Competing Interests None

References

The immunohistochemical algorithm that was followed by us: As our case was a primary spindle cell neoplasm of the node, marker S100 was tested on the node. S100 was positive, pointing out to four possibilities- IDC sarcoma, Langhans cell sarcoma, Follicular dendritic sarcoma and Melanoma. Negative CD1a ruled out LCS. CD21 and CD 35 were negative and FDC sarcoma was ruled out. Negative Melan A marker ruled out melanoma. Diffuse S100 and vimentin positivity, negative CD1a, CD21 and the histomorphological patterns were in favor of Interdigitating dendritic cell sarcoma, hence the final diagnosis of IDC sarcoma was made.

1. Saygin C, Uzunaslan D, Ozguroglu M, Senocak M, Tuzuner N. Dendritic cell sarcoma: A pooled analysis including 462 cases with presentation of our case series. Critical Reviews in Oncology/Hematology. 2013;88(2):253-271. 2. Venkata K. Pokuri, Mihai Merzianu,Shipra Gandhi, Junaid Baqai, Thom R. Loree, and Seema Bhat, Interdigitating Dendritic Cell Sarcoma, Journal of the National Comprehensive Cancer Network. 2015;13 (2) : 128-132. 3. Gaertner E, Tsokos M, Derringer G, Neuhauser T, Arciero C, Andriko J. Interdigitating Dendritic Cell Sarcoma. American Journal of Clinical Pathology. 2001;115(4):589-597. 4. Fonseca R, Yamakawa M, Nakamura S, van Heerde P, Miettinen M, Shek T et al. Follicular dendritic cell sarcoma and interdigitating reticulum cell sarcoma: A review. Am J Hematol. 1998;59(2):161-167.

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5. Kanaan H, Al-Maghrabi J, Linjawi A, Al-Abbassi A, Dandan A, Haider R A. Interdigitating Dendritic Cell Sarcoma of the Duodenum With Rapidly Fatal Course: A Case Report and Review of the Literature. Archives of Pathology & Laboratory Medicine.2006; 130(2):205-208. 6. Ohtake H, Yamakawa M. Interdigitating Dendritic Cell Sarcoma and Follicular Dendritic Cell Sarcoma: Histopathological Findings for Differential Diagnosis. Journal of Clinical and Experimental Hematopathology. 2013;53(3):179-184. 7. Lee E, Hyun D, Cho H, Lee J. A Rare Case of Interdigitating Dendritic Cell Sarcoma in the Nasal Cavity. Case Reports in Otolaryngology. 2013;2013:1-4. 8. Barwell N, Howatson R, Jackson R, Johnson A, Jarrett RF, Cook G. Interdigitating dendritic cell sarcoma of salivary gland associated lymphoid tissue

not associated with HHV-8 or EBV infection. J Clin Pathol 2004;57: 87-89. 9. Han HS, Lee OJ, Lim SN, An JY, Lee KM, Choe KH, Lee KH , Kim ST: Extranodal interdigitating dendritic cell sarcoma presenting in the pleura: a case report. J Korean Med Sci. 2011; 26: 304-307. 10. De Pas T, Spitaleri G, Pruneri G, Curigliano G, Noberasco C, Luini A, Andreoni B, Testori A, de Braud F. Dendritic cell sarcoma: An analytic overview of the literature and presentation of original five cases. Critical Reviews in Oncology / Hematology.2008; 65(1): 1 â&#x20AC;&#x201C; 7. 11. Zhang J, Liu B, Song N, Hua L, Wang Z, Yin C. Interdigitating dendritic cell sarcoma presenting in the kidney combined with retroperitoneal leiomyosarcoma: A case report and literature review. Oncology Letters. 2013; 7: 466-470.

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Case Report Fine Needle Aspiration as a Diagnostic Tool for Metastasis to Thyroid: A Dilemma Prashant Vijay Kumavat*, Anita Padmanabhan , Manisha S Khare, Chetan S Chaudhari, Nitin M Gadgil Dept. of Pathology, LTMMC and LTMGH Sion, Mumbai, India Keywords: Fine-Needle Aspiration, Immunohistochemistry, Lobular Breast Carcinoma, Thyroid Metastasis.

ABSTRACT Metastasis to the thyroid gland is very rare. It is very important to differentiate metastasis to thyroid from primary thyroid malignancy from the treatment point of view. A 48-years-old lady presented with weakness of lower limbs and backache since one and half months. External examinations reveal diffuse, nodular goitre. A fine-needle aspiration (FNA) of the thyroid nodule showed malignant epithelial cells with features characteristic of invasive lobular breast carcinoma. The diagnosis offered on FNAC was metastatic lobular breast carcinoma which was later confirmed on histopathological finding at autopsy. IHC performed on thyroid nodule was positive for: CK7, AE1AE3, ER and negative for: E-cadherin, confirming its lobular nature. TTF1 was negative. FNA could be useful for diagnosis of thyroid metastasis, but it should be confirmed by immunohistochemistry.

*Corresponding author: Dr. Prashant Vijay Kumavat, Room no 1303, floor no 13, building no. 27, Hawre City, Kasarvadavali, Ghodbander Road, Thane (W). India E-mail: drkumavat_83@rediffmail.com

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Cytolology of Metastasis to Thyroid

Introduction

Metastasis to thyroid is usually identified at autopsy. Metastasis to thyroid gland is quite rare and there are only sporadic clinical cases reported in the literature. The common metastases to thyroid arise from of carcinoma breast, liver, lungs, brain, and bone.[1] There is recent increase in the rate of diagnosing metastatic tumor in thyroid due to radiologically guided fine needle aspiration (FNA) of the thyroid gland. We report a case of metastasis to thyroid from lobular breast carcinoma which was picked up on FNA cytology and confirmed by histopathology findings at autopsy and immunohistochemistry.

Case Report

A 48-year-old female patient presented with weakness of lower limbs and backache since one month. On examination she was cachexic and had a nodular, firm thyroid swelling involving both lobes, right lobe measuring 3x3 cm and left lobe measures 2.5x2 cm . FNAC from thyroid showed few benign thyroid follicular epithelial cells and scattered in between were small clusters of cells with hyperchromatic nuclei with occasional prominent nucleoli and moderate amount of cytoplasm, which was vacuolated at places (Figure 1a). Diagnosis of suspicious for metastasis to thyroid was made and patient was advised further clinical workup. X ray Spine showed osteoporotic fracture of L2-L3 lumbar vertebral, suggestive of metastasis. Sonomammography of Right Breast revealed a large ill-defined, hypoechoic lesion measuring 3.8x 3.5x 3.2 cm in the retroalveolar region, suggestive of neoplastic etiology. The Patient was then referred back for FNAC of right breast. FNAC of breast showed scattered and occasional small loose cluster of ductal epithelial cells with hyperchromatic nuclei with occasional prominent nucleoli and moderate amount of cytoplasm which was vacuolated

at some places giving classic signet ring cell appearance (Figure 1b). Many of the cells showed cytoplasmic vacuolization, diagnosis offered on FNAC of thyroid and breast was infiltrating Lobular carcinoma of breast with metastasis to thyroid. One week later patient developed breathlessness and expired. At autopsy -Thyroid was enlarged and encapsulated. Right lobe measures 6x5x3 cm, left lobe measures 6.5x4x3 cm and isthmus measures 5x3x2 cm. Many enlarged lymph nodes were present. Cut section show multiple nodules with focal areas of haemorrhage (Figure 2). Histopathology of the breast mass revealed small to medium sized non cohesive cells arranged in linear pattern in between collagenous stroma, showing a Indian file pattern (Figure 3a). At many places malignant cells encircling breast ducts giving classic targetoid pattern (Figure 3b). Histopathological findings of thyroid showed similar type of cells as seen in breast which were small to medium sized cells, scattered in between thyroid follicular epithelial cells (Figure 3c). Cells showed moderate amount of cytoplasm, hyperchromatic nuclei. Focally cytoplasm showed vacuolization pushing the nucleus to periphery, giving signet ring cell appearance (Figure 3c). Some of the cells were Alcian blue- PAS positive (Figure 3d). To confirm the diagnosis, immunohistochemistry on the thyroid mass war performed. CK 7 and AE 1- AE3 was strongly positive which confirmed malignancy of epithelial origin. TTF 1 which was negative ruled out a primary carcinoma of thyroid. IHC for ER, PR and HER 2 neu was performed, of which, ER was weakly positive. PR and HER 2 were negative (Figure 4). E cadherin was negative, thus confirming the histopathological diagnosis of metastasis of lobular carcinoma of breast to Thyroid. Histopathology of the other organs did not reveal any significant pathology.

Fig. 1: (a) FNAC of thyroid show scattered malignant cell. Inset, Malignant cells with intracytoplasmic vocalization (Pap stain 40X). (1) FNAC of Breast show scattered malignant breast cells, Inset- Signet ring cells (Pap stain 40X).

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Discussion

Fig. 2: Gross specimen of thyroid showing metastatic lesion.

The thyroid gland is highly vascularized; however, metastasis of malignant tumours to the thyroid are relatively rare. The exact mechanism for decrease incidence of metastasis to the thyroid is unknown, but it is thought to be associated with immunological mechanisms. [2] The incidence of metastasis to the thyroid gland in autopsy series varies from 0.5 to 24.2%, [3, 4] the variation of which is likely due to the precision of investigation during the autopsy. In autopsy studies metastasis to thyroid gland is more common than primary thyroid malignancy. [5] In most autopsy series metastasis to thyroid were from breast and lung cancers, however in clinical studies renal cell carcinoma is the most common metastasis to thyroid. [5] In a clinical setting, metastasis of the thyroid gland is detected at a low frequency, and is commonly detected at the same time as the diagnosis of the primary site or shortly thereafter. Diagnosis of thyroid metastasis is difficult, and in general, most cases are asymptomatic and identified due to the appearance of solitary or multiple masses.[3] Liver and lung metastases are common in invasive ductal carcinoma, whereas gastrointestinal tract, peritoneum, and retroperitoneum metastases are common in invasive lobular carcinoma. [6] Thyroid metastasis is generally a part of advanced and disseminated disease and associated with poor prognosis. [4] Our patient had lumbar spine metastasis at the time of presentation.

Fig. 3: (a) Primary tumor in the breast showing infiltrating lobular carcinoma (H&E, 10x), Inset â&#x20AC;&#x201C; Indian file pattern (H&E, 40x). (b) Targeted pattern (10x), Inset (40x). (c) Metastatic cell in between thyroid follicles, Inset- Signet ring cell ( 40x ). (3) â&#x20AC;&#x201C; Alcian blue â&#x20AC;&#x201C; PAS positive cells (40x)

Fig. 4: Metastasis to thyroid from breast IHC strong positive staining for CK 7 and AE1 AE3, 2% weak positive ER and negative TTF -1 (40 x).

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FNAC is a useful tool to detect thyroid metastasis in clinically suspected cases.[7] FNAC is preferred as it is safe, inexpensive and simple procedure. [5] In most cases, the cytological features of the metastatic deposit are similar to those of the primary neoplasm, allowing a precise recognition. The patient described underwent FNAC of thyroid and breast, which suggested metastatic thyroid lesion from the breast cancer. Breast metastatic carcinoma to the thyroid is uncommon phenomena in cytology practice and may give a diagnostic dilemma, particularly in case of occult primary carcinoma in the presence of some morphologic similarities with primary thyroid lesions. So histopathology results should be confirmed by IHC examinations. IHC analysis on thyroid revealed strong reactivity for CK7, AE1-AE3. ER was weakly positive. PR and HER 2 were negative. To distinguish breast metastatic lesions from primary thyroid malignancies TTF1 is a ideal marker which was negative. E cadherin marker to distinguish ductal and lobular was negative. So diagnosis of infiltrating lobular carcinoma metastasing to thyroid was confirmed. eISSN: 2349-6983; pISSN: 2394-6466

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Cytolology of Metastasis to Thyroid

Estrogen receptor-positive cases tend to metastasize primarily to the thyroid and parathyroid glands, while progesterone receptor-positive cases tend to metastasize to the myocardium or the epithelium of the gastrointestinal and urinary tracts. [8]

Conclusion

We conclude that although the thyroid gland is an uncommon site of metastasis, both clinicians and pathologist should be aware of the possibility of metastatic disease in patients with history of malignancy. A patient’s complete clinical history is extremely useful, and FNAC as a diagnostic tool in such difficult cases with help of immunohistochemistry to confirm the diagnosis of metastatic malignancy is warranted.

Funding None

Competing Interests None declared

Reference

1. Rosen IB, Walfish PG, Bain J, Bedard YC: Secondary malignancy of the thyroid gland and its management. Ann Surg Oncol 1995;2:252–256.

2. Czech JM, Lichtor TR, Carney JA, van Heerden JA: Neoplasms metastatic to the thyroid gland. Surg Gynecol Obstet 1982;155:503–505. 3. Lam KY, Lo CY: Metastatic tumors of the thyroid gland: A study of 79 cases in Chinese patients. Arch Pathol Lab Med 1998;122:37–41. 4. Nakhjavani MK, Gharib H, Goellner JR, van Heerden JA: Metastasis to the thyroid gland. A report of 43 cases. Cancer 1997;79:574–578. 5. Gerges AS, Shehata SR, Gouda IA. Metastasis to the thyroid gland: unusual site of metastasis. J Egypt Natl Canc Inst 2006; 18: 67–72. 6. Harris M, Howell A, Chrissohou M, Swindell RI, Hudson M, Sellwood RA: A comparison of the metastatic pattern of infiltrating lobular carcinoma and infiltrating duct carcinoma of the breast. Br J Cancer. 1984;50:23–30. 7. Yu J, Seethala RR, Walls A, Cai G. Fine-needle aspiration of breast carcinoma metastatic to follicular variant of papillary thyroid carcinoma. Diagn Cytopathol 2009; 37: 665–6. 8. de la Monte SM, Hutchins GM, Moore GW: Estrogen and progesterone receptors in prediction of metastatic behavior of breast carcinoma. Am J Med 1984;76:11–17.

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Case Report Reactive Lymphocytosis: A Diagnostic Dilemma in Pleural Effusion Uma1, Shelly Sehgal2* 1

Department of Pathology, Veer Sawarkar Arogya Sansthan, Karawal Nagar, Delhi, India 2 Department of Pathology, Swami Dayanand Hospital, Dilshad Garden, Delhi, India Keywords: Effusion;Immunophenotyping; Lymphoma

ABSTRACT Effusions containing numerous lymphocytes may be due to inflammatory processes, to non-specific reactions complicating other diseases or tolymphoid malignancies. In many cases it is difficult to make a differential diagnosis on morphological criteria alone. We present a case of 25 year old male who presented with cough and fever for one month along with left sided pleural effusion. Pleural fluid differential count showedlymphocytosis. Cytology smears showed the presence of large mononuclear cells with high N:C ratio,convoluted nucleus and prominent nucleolus. On immunophenotyping it was proved to be a case of reactive T lymphocytosis. A single lymph node was also found in upper cervical region which showed epithelioid cell granulomas with ZN stain for AFB positive. To concludereactive lymphocytes are difficult to distinguish from lymphoma onmorphology alone. Diagnostic accuracy can be increased by history, clinical examination and the use of ancillary techniques like immunophenotyping or immunocytochemistry.

*Corresponding author: Dr. Shelly Sehgal, Specialist Pathologist, Department of Pathology, Swami Dayanand Hospital, Dilshad Garden, New Delhi- 110092 Phone: +91 9711196106 E-mail: shellysehg@gmail.com

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Reactive Lymphocytosis in Pleural Effusion

Introduction

Pleural effusions, the result of the accumulation of fluid in the pleural space, are a common medical problem. They can be caused by several mechanisms including increased permeability of the pleural membrane, increased pulmonary capillary pressure, decreased negative intrapleural pressure, decreased oncotic pressure, and obstructed lymphatic flow[1]. The cytological diagnosis of serous effusions containing numerous lymphoid cells may present difficulty in differentiating a reactive from a malignant process . Here, we present an interesting case of pleural effusion with lymphocytosis which posed a diagnostic problem.

Case Report

25 years old male, presented with sudden onset dyspnea and fever with non -productive cough for one month.On examination there were extensive decreased breath sounds on left side with stony dullness. Chest radiographshowed leftsided massivepleural effusion (fig.1). Pleural tap was done which revealed 400ml of straw coloured fluid and sent to cytopathology laboratory for evaluation. On examination cytospin smears showed highcellularity with predominance of large mononuclear cells with high N:C ratio,nuclear convolusions and 0-2 prominent nucleolus(fig. 2). Few scattered neutrophils, macrophages and occasional plasma also noted.Since by morphology no conclusive opinion could be made.

Fig. 1: Chest radiograph showing left sided massive effusion.

Flow cytometry was performed which showed 95% of the total acquired events had bright Cd45 expression and a low side scatter. Some of these cells (18%) of the total acquired events showed slightly less brighter CD45 expression [mature lymphocytes]. 83% of the total gated cells show positivity for T-celllineage like CD7,CD2,CD3, and CD5 (fig3).The brightest CD45 positive population [reactive lymphocytes] showed 64%CD8 and 30%CD4 (fig. 4).Only 11% of the mature lymphocytes were B-lymphocytes(CD19 positive) with no light chain restriction positive.(fig.5). These cells were negative for common acute lymphoblastic leukemia antigen CD10 and CD34 which is a stem cell marker.These immunophenotyping findings suggested reactive T cell proliferation.Also, on further evaluating the patient we found a tiny cervical lymph node. FNA from this showed epithelioid cell granulomas in alymphoid background and ZN stain showed acid fast bacilli. So, diagnosis of tuberculous effusion was given and antitubercular treatment was started. There was a rapid clinical response including resolution of fever and pleural effusion.

Fig. 2: Photomicrograph showing pleural fluid lymphocytosis. Cells have high N:C ratio with convoluted nuclei and prominent nucleoli.( Giemsa, 40X )

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Fig. 3: Flow cytometry dot plots showing bright Cd45 expression and a low side scatter in 95% of the total gated cells. 83% of these cells show positivity for T-celllineage like CD7 and CD3.

Fig. 4: Flow cytometry dot plots showing 64% CD8 and 30% CD4 lymphocytes.

Fig. 5: Flow cytometry dot plots showing 11% B-lymphocyteswith no light chain restriction.

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Reactive Lymphocytosis in Pleural Effusion

Discussion

Tuberculous pleuritis is a common manifestation of extrapulmonary tuberculosis (TB) and is the most common cause of pleural effusion in many countries.[2] Pleural TB occurs as a result of a TB antigen entering the pleural space, usually through the rupture of subpleural focus, followed by a local, delayed hypersensitivity reaction mediated by CD4+ cells.[3] This process may occur during primary or reactivation TB and may or may not involve viable bacilli entering the pleural space.[4] The presence of mycobacterial antigens in the pleural space elicits an intense immune response, initially by neutrophils and macrophages [5,6], followed by interferon (IFN)-c-producing T-helper cell (Th) type 1 lymphocytes [3, 7] , resulting in a lymphocyte-predominant exudative effusion. This cellular trafficking is facilitated by homing surface markers and chemokine gradients. [8,9] This intense but poorly understood local immune response by sensitised lymphocytes to TB antigen is synonymous with the Koch phenomenon. [10] Pleural fluid lymphocytosis is non-specific and may be found in malignancy, tuberculosis, collagen vascular diseases, sarcoidosis and lymphoma. [11] It is agreed that serous cavity involvement by alow grade lymphoma is notoriously difficult to differentiate from a reactive process on the basis of cytomorphology alone. [12] In our case also the cytomorphology alone was inconclusive.Therefore, we applied flow cytometry to come to conclusion. On immunophenotyping, it was proved to be a case of reactive T lymphocytosis. Also a thorough search for any peripheral lymph nodes revealed a single lymph node in upper cervical region.FNA from same showed epithelioid cell granulomas in alymphoid background. ZN stain for AFB was also positive. Immunophenotyping by flow cytometric analysis of body cavity fluids can be a very useful adjunct to conventional diagnostic cytopathology in the evaluation of serous effusions for lymphomatous involvement. [13] In a study of Czader and Ali, immunophenotyping by flow cytometry modified the provisional cytopathologic diagnosis in 16% of 115 consecutive serous cavity effusions and this included 10 benign and atypical or suspicious cases that became malignant. [14] Satouchi et al. could establish definite diagnosis of lymphoma in 2 cases, only by flow cytometric examination of pleural effusion. [15] Cytomorphology and immunophenotyping in combination can improve the diagnostic accuracy. Bangerter et al. in

a study of 33 lymphomatous and 21 reactive effusions found 7 false-negative cases, if only one method was used but the combined strategy of cytomorphology and immunophenotyping had 100% sensitivity and 100% specificity. [16]

Conclusion

To conclude reactive lymphocytes are difficult to distinguish from lymphoma on morphology alone. Diagnostic accuracy can be increased by meticulous clinical examination and the use of ancillarytechniques.

Acknowledgement None

Funding None

Conflict of interest No

Bibliography

1. Liam CK, Lim KH, Wong CM. Causes of pleural exudate in a region with a high incidence of tuberculosis. Respirology 2000; 5: 33-8. 2. Valde´s L, Pose A, San Jose´ E, Martinez Va´zquez JM. Tuberculous pleural effusions. Eur J Intern Med 2003; 14: 77-88. 3. Barnes PF, Mistry SD, Cooper CL, Pirmez C, Rea TH,Modlin RL. Compartmentalization of a CD4+ T lymphocytesubpopulation in tuberculous pleuritis. J Immunol 1989; 142:1114-9. 4. Kim HJ, Lee HJ, Kwon SY, Yoon HI, Chung HS, Lee CT, et al. The prevalence ofpulmonary parenchymal tuberculosis in patients withtuberculous pleuritis. Chest 2006; 129: 1253-8. 5. Antony VB, Repine JE, Harada RN, Good JT Jr, Sahn SA.Inflammatory responses in experimental tuberculosispleurisy. Acta Cytol 1983; 27: 355-61. 6. Antony VB, Sahn SA, Antony AC, Repine JE. Bacillus Calmette-Gue´rin-stimulated neutrophils release chemotaxins for monocytes in rabbit pleural spaces and in vitro. J Clin Invest 1985; 76: 1514–1521. 7. Sharma SK, Mitra DK, Balamurugan A, Pandey RM, Mehra NK. Cytokine polarization in miliary and pleural tuberculosis. J Clin Immunol 2002; 22: 345–352. 8. Gopi A, Madhavan SM, Sharma SK, Sahn SA. Diagnosis and treatment of tuberculous pleural effusion in 2006. Chest 2007; 131: 880–889. 9. Mitra DK, Sharma SK, Dinda AK, Bindra MS, Madan B, Ghosh B. Polarized helper T cells in tubercular

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pleural effusion: phenotypic identity and selective recruitment. Eur J Immunol 2005; 35: 2367–2375. Dheda K, Booth H, Huggett JF, Johnson MA, Zumla A, Rook GA. Lung remodeling in pulmonary tuberculosis. J Infect Dis 2005; 192: 1201–1209. Yam LT. Diagnostic significance of lymphocytes in pleural effusions. Ann Intern Med 1967,66:972-982. Santos GC, Longatto-FilhoA, de CarvalhoLV, Neves JI, Alves AC. Immunocytochemical study of malignant lymphomain serous effusions.Acta Cytol 2000; 44:539-542. Dunphy CH.Combined cytomorphologic and immunophenotypic approach to evaluation of effusion

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A-111 for lymphomatous involvement.Diagn Cytopathol 1996;15:427-430. 14. Czader M and Ali SZ. Flowcytometry as an adjunct to cytomorphologic analysis of serous effusions. Diagnostic cytology 2003;29(2):74-78. 15. Satouchi M, Urata Y, Ueda S, Kotani Y, Kado T, Adachi S,et al.Clinical evaluation of mediastinal hematologic malignancies.Nihon KokyukiGakkai Zasshi 2003;41:507-513 16. Bangerter M, Hildebrand A, Griesshammer M. Combined cytomorphologic and immunophenotypic analysis in diagnostic workup of lymphomatous effusions. Acta Cytol 2001;45:307-312.

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Letter to Editor Burkholderia Cepacia: An Unusual Cause of Post Surgical Endopthalmitis Vibha Bhargava*, Menka Kapil, Rohit Jain, G.N. Gupta Department of pathology, Santokba Durlabhji Memorial Hospital, Jaipur, Rajasthan. India

Dear Sir,

Postoperative acute endophthalmitis remains a serious and blinding complication even today. Microorganisms which reside in the skin around the eyelids, eyelashes, conjunctiva, or lacrimal sac are the usual source of infection. Staphylococcus epidermidis is the most common organism recovered from patients with cultureproven endophthalmitis. [1] Gram negative species are found in fewer cases. Burkholderia cepacia(previously known as Pseudomonas cepacia) is an opportunistic Gram Negative bacteria non‐fermentative bacilli widely distributed in the environment. Burkholderia cepacia does cause significant infection not only in cystic fibrosis, chronic granulomatous disease, and immunocompromised patients, but also in healthy individuals. [2] Organism able to survive on and in medical devices and disinfectants. Intrinsic resistance to multiple antimicrobials agents also contribute to the organism survival in hospitals. . [3] A 58 years old male presented in the outpatient department with pain, redness, swelling, fever, diminished vision in left eye just after a day of cataract surgery with posterior chamber IOL implantation. Examination revealed lid edema, conjunctival and circumcilliary congestion along with corneal edema. Intraocular pressure was high. At the same time only perception of light was present. Vitreous sample was sent for culture and sensitivity along with gram staining. Gram stain revealed presence of gram negative bacilli with morphology. Bacterial culture was done on blood agar and Mac Conkey agar media .Growth showed presence of gram negative, non fermenter, oxidase positive bacteria. The identification and sensitivity was done on automated VITEK 2c system, which revealed Burkholderia cepacia which was only sensitive to the

drug the ceftazidime. Treatment remains a big challenge as it can easily become resistant to antimicrobial agents like in this case, which was found to be resistant to all the antibiotics commonly used in ophthalmology. The poor penetration of antibiotics into the eye also results in it being a difficult infection to treat. [1] Treatment was started as injection moxifloxacin, amikacin and eye drop atropine and Predmet Opthalmic (Prednisolone Acetate), with no improvement of vision, pars plana vitrectomy was done. Two weeks after follow up patient improved clinically .The vision improved to 6/12 .The source of infection could not be traced. Despite the availability of modern surgical techniques and standard sterilization methods, the incidence of postoperative endophthalmitis is still seen in 0.05% to 0.15% cases of cataract surgery. [1]. Burkholderia cepacia can cause nosocomial infection and commonly acquired through contaminated medical source such as anesthetics, disinfectants, intravenous solutions, nebulizer solutions, mouth wash and medical devices. (4) Investigation revealed the presence of this organism in chlorhexidine (0.2 percent) mouthwash as well as other chlorhexidine antiseptic solutions used for routine urologic and obstetric procedures. [5] Better understanding of surgical techniques, instrumentation, prophylactic antibiotics, ambulatory surgery, and proper asepsis will significantly reduce its incidence. This will be helpful for reducing such incidences, otherwise outbreaks may occur in most vigilant setting and any sterile consumable may be a common link.

Acknowledgements None

Funding None

*Corresponding author: Dr .Vibha Bhargava, Consultant, Department of pathology, Sankokba Durlabhji Memorial Hospital, Jaipur, Rajasthan, India Phone: +91 - 9414498921 E-mail: drvibhaguptabhargava@gmail.com

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Burkholderia Cepacia Endopthalmitis

Competing Interests None

References

1. Khan T. Burkholderia cepacia: Rare cause of postsurgical acute endophthalmitis. JCRS Online Case Reports. 2013;1(1):e1-e2. . 2. I Eser, T Altan, J E Stahl, M D Aydin, N Inan, Z Kapran, O F Yilmaz. Two cases of Burkholderia cepacia endophthalmitis. Br J Ophthalmol. 2006 Sep; 90(9): 1211.

3. Forbes B, Sahm D, Weissfeld A. Bailey & Scottâ&#x20AC;&#x2122;s diagnostic microbiology. 11th ed. St. Louis, Mo.: Elsevier Mosby; 2002. 4. Winn W, Koneman E. Konemanâ&#x20AC;&#x2122;s color atlas and textbook of diagnostic microbiology. Philadelphia: Lippincott Williams & Wilkins; 2006 5. Sobel J, Hashman N, Reinherz G, Merzbach D. Nosocomial pseudomonas cepacia infection associated with chlorhexidine contamination. The American Journal of Medicine. 1982;73:183-6.

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Letter to Editor Intraparotid schwannoma on cytology Lakshmi Agarwal, Deepti Sukheeja*, Vinny Gupta, Swati Namdev, Naresh N Rai Department Of Pathology, Government Medical College & Associated Group of Hospitals, Kota, Rajasthan, India

Dear Sir,

Schwannomas are ectodermally derived benign, encapsulated tumours arising from the schwann cells of the neural sheath of motor and sensory peripheral nerves. It is difficult to diagnose preoperatively because of the low incidence rate, lack of classical sign of facial nerve palsy and clinically it mimics benign parotid parenchymal tumor. But fine needle aspiration cytology (FNAC) if done properly can helps in diagnosing this lesion which is further confirmed by histopathology. A 40 years old male presented with painless swelling of the right parotid region. The swelling was firm, and mobile measuring 1x1 cm. There was no lymphadenopathy. Oral cavity was normal. USG showed a small hypo-echogenic solid tumor in the deep part of parotid gland. Based on the above investigations, clinical diagnosis of pleomorphic adenoma was made. FNAC was performed. Cytosmears revealed large, cohesive fragments of wavy nuclei with pointed nuclear

ends. Verocay bodies, Antony A and Antoy B areas were evident. Background showed abundant fibrillary materials. Mitosis and necrosis were not seen. The diagnosis of benign nerve sheath tumor most probably schwannoma was made. Excision biopsy and histopathology was done which revealed the characteristic features of schwannoma and final diagnosis of intra parotid schwannoma of facial nerve was made. The intraparotid facial nerve schwannoma was first described by Ibraz in 1927. Most of the tumor arises from the intratemporal part of facial nerve. A few cases have been described originating from the intraparotid facial nerve (10%). [1,2,3] Most of these arise from the eighth nerve sheath but are relatively uncommon from seventh nerve. [4] The role of (FNAC) in diagnosis of parotid lesions is uncertain and often misleading. Inohara H et al., in 2008 had concluded that the accuracy of FNAC in diagnosis of parotid lesions was 80% and 62% for benign and malignant

Fig. 1: 1A: Cytology showing spindle cells alongwith myxoid background matrix.A cluster of parotid ductal cells also seen. 1B: Gross picture of schwannoma.1C: Tissue section showing spindle cells with palisading nuclei. *Corresponding author: Dr Deepti Sukheeja, House No 1,Vigyan Nagar Special, Kota-5, Rajasthan, India Phone: +91 - 8058010655 E-mail: deeptisukheeja2001@yahoo.co.in

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Cytology of intraparotid schwannoma

lesion, respectively. So FNAC of parotid lesion are rarely asked for. [5] So it becomes difficult to arrive at the diagnosis of intraparotid schwannoma pre operatively and can be easily misdiagnosed.The definite diagnosis of facial nerve schwannoma is usually confirmed by histopathology. Intra parotid schwannoma can be confused with spindle cell myoepithelioma on cytology. But it can be differentiated on the basis of strong positivity for S100, whereas negative reaction for P63, vimentin and cytokeratin. To conclude, intra parotid schwannoma can be diagnosed by FNAC if done properly. This will also help the surgeons in deciding the treatment of patient preoperatively.

Funding None

Competing Interests None Declared

Reference

1. Oncel S, Onal K, Ernete M. Schwannoma (neurilemoma) of the facial nerve as a parotid mass. J Laryngol Otol. 2002; 116: 642â&#x20AC;&#x201C;3. 2. Kumar BN, Walsh RM, Walter NM. Intraparotid facial nerve schwannoma in child. J Laryngol Otol.1996; 110: 1169â&#x20AC;&#x201C;70. 3. Liu R, Fagan P. Facial nerve Schwannoma: surgical excision versus conservative management. Ann OtolRhinolLaryngol 2001;110(11):1025-9. 4. Kyriakos M., Pathology of selected soft tissue tumors of the head & neck. In: Thawley S.E, Panje WR, Batsakis JG, and Lindberg RD, Eds. Comprehensive management of head and neck tumors .1st Ed. Philadelphia: WB Saunders, 1987:1254-1256. 5. Inohara H, Akahani S, Yamamoto Y, et al. The role of fine-needle aspiration cytology and magnetic resonance imaging in the management of parotid mass lesions. Acta Otolaryngol 2008;128(10):1152-8.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 02, April - June 2016

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