Usman Hassan

Determination of Frequency of Epstein-Barr Virus in Non-Hodgkin Lymphomas Using EBV Latent Membrane Protein 1 (EBV-LMP1) Immunohistochemical Staining

BACKGROUND The presence of Epstein-Barr virus (EBV) in Non-Hodgkins lymphoma can be identified by immunohistochemistry for detection of EBV latent membrane protein (LMP). The role of EBV as an etiologic agent in the development of non-Hodgkin lymphoma has been supported by detection of high levels of latent membrane protein 1 (LMP-1) expression in tumors. However, no study has been conducted in a Pakistani population up till now to determine the frequency of Epstein-Barr virus positivity. The objective of our study was to determine a value for non-Hodgkin lymphoma patients using EBV LMP-1 immunostaining in our institution. MATERIALS AND METHODS This study was carried out at the Department of Histopathology, Armed Forces Institute of Pathology (AFIP), Pakistan from December 2011 to December 2012. It was a cross sectional study. A total of 71 patients who were diagnosed with various subtypes of NHL after histological and EBV LMP-1 immunohistochemical evaluation were studied. Sampling technique was non-probability purposive. Statistical analysis was achieved using SPSS version 17.0. Mean and SD were calculated for quantitative variables like patient age. Frequencies and percentages were calculated for qualitative variables like subgroup of NHL, results outcome of IHC for EBV and gender distribution. RESULTS Mean age of the patients was 53.6 ± 16 years (Mean ± SD). A total of 50 (70.4%) were male and 21 (29.6%) were female. Some 9 (12.7%) out of 71 cases were positive for EBV-LMP-1 immunostaining, 2 (22.2%) follicular lymphoma cases, 1 (11.1%) case of T-cell lymphoblastic lymphoma, 4 (44.4%) cases of diffuse large B cell lymphomas, 1 (11.1%) mantle cell lymphoma and 1 (11.1%) angioimmunoblastic T cell lymphoma case. CONCLUSION In our study, frequency of EBV in NHL is 12.7% and is mostly seen in diffuse large B cell lymphoma. This requires further evaluation to find out whether this positivity is due to co-infection or has a role in pathogenesis.

Prognostic sub-grouping of diffuse large B-cell lymphomas into germinal centre and post germinal centre groups by immunohistochemistry after 6 cycles of chemotherapy.

INTRODUCTION Diffuse large B-cell lymphomas (DLBCL) can be divided into germinal centre (GC-DLBCL) and post germinal centre (post GC-DLBCL) groups by applying immunohistochemical antibodies. As these subgroups respond differently to chemotherapy, it is possible at diagnosis to select a poor prognostic subgroup for aggressive treatment. OBJECTIVE To determine the frequencies of GC-DLBCL and post GC-DLBCL in patients by immunohistochemistry (IHC) and the clinical response after six cycles of chemotherapy. SUBJECTS AND METHODS In this descriptive study conducted in AFIP and CMH, Rawalpindi and NORI, Islamabad, from September 2010 to September 2011, a total of 75 pretreatment cases of DLBCL diagnosed during the study period were included. Cases were segregated in to GC-DLBCL and post GC-DLBCL groups according to results of immunohistochemistry markers CD10, BCL6 and MUM1. Immediate clinical response was assessed after 6 cycles of chemotherapy. Response was divided into complete response, partial response, stable disease or relapse or progression. RESULTS The mean age was 54.2 ± 15. Males were 53 (70.7%). Forty (53.3%) cases comprised the GC-DLBCL group; 25(62.5%) of them showed a complete response. Most patients of the post GC-DLBCL 19(54%) showed relapse/progression. Results of immediate clinical response in both prognostic subgroups were significant (p<0.05). Results regarding positivity with immunohistochemical antibodies CD10 (p 0.011), BCL6 (p 0.013) and MUM1 (p 0.000) regarding immediate clinical response were also significant. CONCLUSION GC-DLBCL group shows better response to CHOP chemotherapy regimen. Immunohistochemistry should be used to further classify DLBCL as this can enable us to select aggressive group for aggressive treatment. This manuscript is important because the study is the first to becarried out exclusively in Pakistan or our part of the world.

Triple Gallbladder: A Rare Entity

To the Editor: Triple gallbladder is also called vesica fellae triplex (1). It is one of the very rare congenital anomaly of the biliary tract. According to literature, 11 cases have been reported till now (2). The very first case was reported in a human cadaver in 1752 by Huber (3). Most of the cases of congenital anomalies of gallbladder remain undiscovered and come to attention only when they are associated with symptoms due to cholelithiasis, cholecystitis, biliary sludge, obstructive symptoms, and carcinoma. We report a case of triple gallbladder, which was incidentally diagnosed in a 30-year-old female who presented with complaints of pain in the right hypochondrium. Her radiological investigation revealed a duplication of gallbladder and chronic cholecystitis. However, a third gallbladder was found during surgery.

Fluorescence in Situ Hybridization (FISH) for Differential Diagnosis of Soft Tissue Sarcomas

Introduction: Soft tissue sarcomas are rare tumors comprising 1 percent of solid malignancies. The latest edition of WHO soft tissue pathology lists 94 benign and malignant soft tissue tumors. Many of these show a large degree of morphological overlap. Immunohistochemistry has been shown to be reliable in many cases for differential diagnosis of lesions, although cytogenetic tests are considered the gold standard for many entities.Fluorescence in-situ hybridization (FISH) is a cytogenetic technique that uses fluorescent probes that bind to only those parts of the chromosome which have a high degree of sequence complementarity. Many soft tissue tumors show recurrent genetic mutations that are now being used as diagnostic markers. Knowledge of the molecular identity allows prediction of behavior, prognosis and treatment response. Objective: The aim of this study was to identify genetic mutations in soft tissue sarcomas using FISH testing and to assess correlations with histological diagnosis. Material and methods: A total of 25 cases of different soft tissue sarcomas diagnosed on histology with the help of immunohistochemical staining and for which FISH studies were requested were included in this study. Three pathologists with a special interest in soft tissue sarcomas reviewed the cases. FISH tests for EWS, the X:18 translocation, FOXO1 and MDM2 were respectively applied for 8 cases of Ewing sarcoma, 8 cases of synovial sarcoma, 2 cases of rhabdomyosarcoma and 7 cases of dedifferentiated liposarcoma and atypical lipomatous tumors/well differentiated liposarcomas. Results: EWS gene fusion was detected in 7 out of 8 cases of Ewing sarcoma and the X: 18 translocation was positive in 3 of the 8 cases of synovial sarcoma. FOXO1 was not detected in either of the two rhabdomyosarcomas. MDM2 by FISH was detected in only one out of 5 cases of atypical lipomatous tumors and 1 out of 2 dedifferentiated liposarcomas. Conclusion: FISH is a useful adjunct in the diagnostic assessment of different types of soft tissue sarcomas. It is easy to set up, is relatively inexpensive and has the ability to diagnose sarcomas with great accuracy, especially in cases which can not be accurately classified even after thorough histological and immunohistochemical evaluation. It may play a very important role in the accurate diagnosis and correct management of patients.

Mesenchymal Chondrosarcoma, Clinicopathological Characteristics of an Uncommon Tumor

Mesenchymal chondrosarcomas (MCS) are very rare malignant tumors. They comprise 2–10% of all chondrosarcomas. Approximately 600 cases of MCS have been published, as case reports and small series. In English language medical literature only three series include 20 or more cases have been described. This paper aims to document various histological findings, histological patterns that can be seen in MCS. In addition, demographic features, clinical and radiological findings and survival data are also incorporated to assess prognostic impact of various histological findings. This is a descriptive cross sectional study including cases of MCS diagnosed at Shaukat Khanum Memorial Cancer Hospital and Research Center, Lahore, Pakistan between 2002 and 2012. The slides were reviewed by two consultant pathologists independently and findings (pattern, cellularity, spindle cell component, necrosis, mitoses, hemangiopericytoma like vessels) were documented on a specially developed form. The clinical information was extracted from case files. Survival data was obtained by contacting the patient or their families in those cases for which contact numbers were available. Data analysis was done using SPSS 20.0. Of the total 18 cases, 10 were males (55.6%) and 8 were females (44.4%). Patients were most commonly in their second or third decades of life. Tumor locations included craniofacial bones (n=5, 28%), ribs and chest wall (n=3, 16%), spine (n=3, 16%), and the lower extremity bones (n=5, 28%). Extraskeletal sites included parapharyngeal region (n=1, 6%) and thigh (n=1, 6%). Histologically biphasic pattern was seen 16 cases (89%). Two cases showed sheets of round to spindle cells with very focal cartilaginous component (11%). Spindle cell component was seen in 14 cases. Necrosis was present in 7 cases (38.9%). Survival data was available for 11 patients (61%). Among these, n=7 (64%) patients developed metastatic disease. The mean survival for cases with metastatic disease was 14.2 months. In patients without metastatic disease, mean survival was 37 months. As the incidence of mesenchymal chondrosarcoma is extremely low, the limited number of patients discussed in most of the studies precludes statistically significant conclusions. MCS should be considered in the differential diagnosis, when pathologists encounter a bone and soft tissue tumor with biphasic pattern composed of round to spindle cell component and hyaline cartilage. Given the diagnostic pitfalls discussed, clinicians may consider obtaining larger biopsy specimens to limit sampling error or confirming the diagnosis with immunohistochemical stains or genetic analysis when a larger biopsy is not possible.

Effectiveness Of Vascular Markers (immunohistochemical Stains) In Soft Tissue Sarcomas

OBJECTIVE To ascertain the effectiveness of IHC markers of vascular origin like CD31, CD34, FLI1 and ERG in vascular soft tissue sarcomas including angiosarcomas, Kaposi sarcomas, epithelioid hemangioendothelioma and a non-vascular soft tissue sarcoma (Epithelioid sarcoma). STUDY DESIGN Descriptive study. PLACE AND DURATION OF STUDY Shaukat Khanum Memorial Cancer Hospital and Research Centre, Lahore, from 2011 to 2017. METHODOLOGY Diagnosed cases of angiosarcomas (n=48), epithelioid hemangioendothelioma (n=9), Kaposi sarcoma (n=9) and epithelioid sarcoma (n=20) were selected. Immunohistochemical staining as performed on formalin fixed paraffin embedded sections. The sections were stained for the following markers: CD34 (VENTANA clone Q Bend 10), CD31 (Leica clone 1 A 10), FLI1 (CELL MARQUE clone MRQ-1) and ERG (CELL MARQUE clone EP111). RESULTS A complete panel of CD34, CD31 and ERG was applied on 8/48 cases of angiosarcomas with triple positivity in 6 cases. Eight cases showed positivity for only CD31 and ERG and 2 cases showed positivity for only ERG. A complete panel of CD34, CD31 and ERG was applied on 3/9 cases of epithelioid hemangioendothelioma with positivity for all markers in 2 cases. Combined positivity for ERG and CD34 was seen in 2 cases and on 4 cases only CD31 immunohistochemical was solely applied with 100% positivity. FLI1 was not applied on any case. Among 9 cases of Kaposi sarcoma, ERG, CD34 and CD31 in combination were applied on only 1 case with triple positivity. Remaining cases show positivity for either CD34, CD31 or FLI1. Majority of cases of epithelioid sarcomas were diagnosed on the basis of cytokeratin and CD34 positivity with loss of INI1. The other vascular markers showed negativity in all cases. CONCLUSION Among these four markers, ERG immunohistochemical stain is highly effective for endothelial differentiation due to its specific nuclear staining pattern in normal blood vessel endothelial cells (internal control) as well as neoplastic cells of vascular tumors and lack of background staining.

Immunohistochemical Expression of CD-10, BCL-6 and MUM-1 Antibodies and Immediate Clinical Response in Patients of Diffuse Large B-Cell Lymphomas after Six Cycles of Chemotherapy

OBJECTIVE To determine the expression of CD-10, BCL-6 and MUM-1 in patients with diffuse large B-cell lymphoma (DLBCL) and its association with immediate clinical response after six cycles of CHOP chemotherapy. STUDY DESIGN Analytical study. PLACE AND DURATION OF STUDY Armed Forces Institute of Pathology (AFIP), Rawalpindi in collaboration with Nuclear medicine, Oncology and Radiotherapy Institute (NORI), Islamabad from September 2010 to September 2011. METHODOLOGY CD-10, BCL-6 and MUM-1 antibodies were applied on cases diagnosed as DLBCL. Immediate clinical response was noted after 6 cycles of chemotherapy with the help of oncologist and divided into complete response, partial response, stable disease and relapse/ progression. Patients age, results of expression of CD-10, BCL-6 and MUM-1 and results of immediate clinical response to chemotherapy were noted. Regarding analysis of prognostic markers (CD-10, BCL-6 and MUM-1), chi-square test was used for immediate clinical response to chemotherapy in DLBCL. RESULTS CD-10 was positive in 40% cases, BCL-6 in 58.7% cases and MUM-1 was positive in 46.7% cases. About 41.3% of patients showed complete response, 10.6% partial response, 17.3% stable disease and 30.8% showed relapse/progression. CD-10 expression in DLBCL was associated with better immediate clinical response (p=0.011) whereas MUM-1 expression in DLBCL was associated with poor immediate clinical response (p<0.0001). However, there was no statistically significant association of BCL-6 with immediate clinical response (p=0.22). CONCLUSION DLBCL shows expression of CD-10, BCL-6 and MUM-1 in nearly fifty percent of the cases. CD-10 is associated with good whereas MUM is associated with poor response. However, there was no association of BCL-6 with immediate clinical response.