Morphological Typing of Lymphomas with Immunohistochemistry

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1 Indian Medical Gazette APRIL Original Article Morphological Typing of Lymphomas with Immunohistochemistry Aparna Bhardwaj, Assosciate Professor, Sanjeev Kishore, Professor Department of Pathology, SGRR Institute of Medical & Health Sciences, Patel Nagar, Dehradun, India. Anuradha Kusum, Professor, Department of Pathology, Himalyan Institute of Medical Sciences, Jollygrant, Dehradun, India. Abstract Lymphoid malignancies (LM) are a heterogeneous group of disorders that are broadly divided into Hodgkin disease (HD) and Non-Hodgkin Lymphoma (NHL). Diagnosing lymphoid malignancies based on morphology in conjunction with immunohistochemistry (IHC) forms the basis of WHO classification and this has prognostic implications. With this background this study was designed thus including all the lymphoid malignancies both NHL and HD. Materials and Methods: This study was conducted at a tertiary centre in Uttarakhand and included a total of 116 cases of lymphoid malignancies. Of these 41 cases were of Hodgkin disease and 75 cases were of NHL. These cases were initially diagnosed on morphology employing Hematoxylin and Eosin (H&E) and special stains like Reticulin. Subsequently, a preliminary panel of monoclonal antibodies using CD3, CD15, CD20, CD30, and CD45 were employed. All the cases were then classified using WHO classification. Results: HD- Of the 41 cases of Hodgkin s disease the commonest subtype was Nodular Sclerosis seen in 26 cases (48.78%). Reed Sternberg in reactive milieu is diagnostic of Hodgkin disease. In all cases except one Reed Sternberg cells exhibited positivity for both CD15 and CD30. NHL Of the 75 cases of NHL an initial classification based on morphology was done. All the cases were classified according to International Working Formulation initially. Subsequently, IHC was employed using CD3, CD15, CD20 and CD45. The disease was then classified according to WHO classification and broadly divided into B or T cell types. B cell expression was seen in 60 cases (80%) and T cell expression in 15 cases (20%). The commonest B cell subtype was Diffuse Large B cell Lymphoma (26.4%). Keywords lymphoid malignancies, hodgkin disease, non- hodgkin lymphoma, immunohistochemistry Introduction Malignant lymphoma is the generic term given to the tumors of lymphoid system and their precursor cells whether T, B or null phenotype. The malignant lymphomas can be divided into two major categories, Hodgkin lymphomas characterized by Reed Sternberg (R S cells) and all others collectively known as Non Hodgkin s lymphoma. The diagnosis of malignant lymphomas until recently was primarily based on morphology. However, the advent of immunohistochemistry has revolutionized the diagnosis of lymphoma and serves an important diagnostic tool in the current WHO classification 1. In the present scenario interpretation of morphological features together with immunohistochemistry has become Address for correspondence: Dr Aparna Bhardwaj, Assosciate Professor, Department of Pathology, SGRR Institute of Medical & Health Sciences, Patel Nagar, Dehradun, India aparnapande1977@gmail.com

2 128 Indian Medical Gazette APRIL 2015 the bedrock for diagnosis and therapy of lymphoid malignancies. These studies have not only provided the objective classification but have identified the antigens that can be targeted for therapy 2. This study was conducted in a tertiary care centre in Uttarakhand with an aim to study malignant lymphomas by morphology and then further subtype them according to Immunophenotype. Thus all the cases diagnosed as Hodgkin s Lymphoma morphologically were further subjected for immunophenotyping using CD15 and CD30 to study immunoreactivity of Reed-Sternberg cells. Likewise all cases of NHL that were initially diagnosed on morphology were divided into B or T cell type employing pan B cell marker CD20 and pan T cell marker CD3 respectively. Thus, diagnosing Lymphoid Malignancies morphologically and subsequently immunophenotyping them using a carefully selected preliminary panel serves an important role in making diagnostic and therapeutic decisions. Material and Methods A total of 116 consecutive cases of malignant lymphomas diagnosed over a period of 18 months at a tertiary centre in Uttarakhand were studied retrospectively. A detailed history and clinical information related to age, sex was retrieved from medical records. Tissue sections were obtained from paraffin blocks and stained with Hematoxylin and Eosin (H&E) and Reticulin stains. After recording diagnosis based on morphology, the relevant cases were subjected to immunohistochemical staining. Pretreatment employed microwave technique for antigen retrieval and detection was done using labeled strepavidin biotin peroxidase complex. The panel of monoclonal antibodies routinely used included CD45, CD20, CD3, CD15 and CD30 (BIOGENEX). In specific cases antibodies against epithelial membrane antigen (EMA), cytokeratin and S 100 were used. The cases that were diagnosed as Round cell malignancy on morphology but after special stain and immunostaining did not fall into category of lymphoma were excluded from the study. The cases that were delineated from study were Plasma cell myeloma, Metastatic poorly differentiated carcinoma, Metastatic small cell carcinoma, Ewing s sarcoma/pnet. Each reaction set included external and internal positive control. Reactive hyperplasia served as external positive control. In addition, coexisting small lymphocytes served as internal positive and negative control in each slide. Slides were considered assessable if all the concurrent internal and external control stained appropriately. All lymphomas were classified either as HD (Hodgkin s disease) or NHL (Non Hodgkin s lymphoma) and further categorization was done using WHO classification. [3] Also, International Working Formulation was employed and NHL s were subdivided into high, intermediate and low grade. Results Of the 116 cases, 41 cases (33%) were of Hodgkin s disease and 75 cases (67%) were of NHL. Hodgkin Disease (HD) Among the 41 cases of HD, 30 patients (73.17%) were males and 11 patients were females (26.83%). The male to female ratio was 2.7:1. The age of patients ranged from 5 to 56 years, with a median age of 21 years. Thirty eight of 41 patients had localized lymphadenopathy at the time of diagnosis. The most common presentation amongst patients of HD was enlargement of cervical and axillary lymph nodes. Only 3 cases of HD presented with generalized lymphadenopathy and hepatosplenomegaly. On morphological examination of HL using H&E stain, it was classified into four subtypes. There were 26 cases of NS HD (48.78%), 10 cases of MCHD (24.39%), 4 cases of LPHD (9.75%) and 1 case of LDHD. All the cases of Hodgkin Lymphoma were further subjected to immunohistochemistry using LCA, CD15 and CD30. Reed Sternberg cells (RS cells) makes the diagnostic hallmark of HD. Classical RS cells show immunoreactivity for CD15 and CD30 on IHC and are negative for LCA. In our study R S cells in all the cases except one showed strong immunorectivity to CD30 and CD15 irrespective of the histopathologic subtype. In one case diagnosed morphologically as Mixed Cellularity R S cell exhibited CD30 Positive and CD15 Negative phenotype (Table 1). Non Hodgkin s lymphoma Of the 75 cases of NHL, 45 patients (66.67%) were males and 15 patients (33.33%) were females. The male to female ratio was 3: 1. The age of the patients ranged between 7 70 years and maximum number of cases were in the age group of years (29%). All the 75 cases

3 Indian Medical Gazette APRIL Table 1 Shows histopathologic distribution of 41 cases of HD HD type Lymphocyte predominance No. of cases (%) CD 15+ CD30+ 4 (9.75%) 4 4 Nodular sclerosis 26 (48.78%) Mixed Cellularity 10 (24.39%) 9 10 Lymphocyte depleted 1 (2.43%) 1 1 of NHL were examined morphologically and classified according to International Working Formulation classification (Table 2). All the cases of NHL were subjected to detailed immunochemistry employing CD3, CD20, and CD45. Among the 75 NHL, 60 cases (80%) were of B cell phenotype and 15 cases (20%) were of T cell phenotype. B cell NHL expressed CD45 and CD20 positivity on immunostaining, while T cell NHL expressed CD15 and CD3 positivity. A significant number of cases of anaplastic lymphoma (n = 11) were observed. Of these on immunostaining, 2 were of B cell type and according to WHO classification were classified as B cell anaplastic lymphoma and remaining 9 cases were classified as Anaplastic Ki - 1 type. All the 75 cases of NHL were classified according to WHO classification (Table 3). Out of 75 cases, 61 cases (67.3%) were nodal and 14 cases (36.58%) were extra nodal. Amongst the extra nodal cases, 5 cases (35.71%) were seen as intra abdominal lump followed by involvement of central nervous system and testis in 2 cases each (14.28%). Also, there were 2 cases presenting as ulcer in axillary and inguinal region, which were probably nodal originally but has presented as an ulcer. The remaining 3 cases (7.14% each) presented as tonsillar growth, parapharyngeal mass and swelling at an angle of mouth respectively (Table 4). Discussion Lymphoid malignancies are a heterogeneous group of disorder that may arise in lymph node or extra-nodal sites. Table 2 Shows the distribution of 75 cases of NHL according to Working Formulation Low grade n Percentage SLL/CLL % Follicular predominantly small cleaved cell % Follicular lymphoma mixed small cleaved and large cell % Intermediate grade Follicular predominantly large cell 3 4.0% Diffuse small cleaved cell 3 4.0% Diffuse mixed small and large cell % High grade Misc. Diffuse large cell % ML, lymphoblastic 6 8 % Burkitt lymphoma % Anaplastic % MALT oma %

4 130 Indian Medical Gazette APRIL 2015 Table 3 Distribution of 75 patients with NHL according to WHO classification No. of cases Age groups (%) < > 20 M F Extra noda; B Cell neoplasm CLL/Small lymphocytic follicle centre 1 (1.33) 1 1 Follicular 13 (17.33) Diffuse, predominantly small cells 03 (4%) Extranodal marginal zone B cell (Low grade B cell of MALT type) 01 (1.33) Diffuse large B cell 41 (54.66) Burkitt s and Burkitt like 1/60 (1.33) T Cell neoplasm Precursor T lymphoblastic 6 (8%) T Cell prolymphocytic Peripheral T cell Anaplastic 09 (12%) Mycosis fungoides Extranodal NK/T cell Angioimmunoblastic Table 4 The location and phenotypes of 14 primary extranodal NHL cases Location Number (%) (14) B cell phenotype T cell phenotype Parapharyngal mass 1 (7.14%) 1 Tonsillar growth 1 (7.14%) 1 Swelling angle of mouth 1 (7.14%) 1 Abdominal lump 5 (35.71%) 4 1 Testicular sw. 2 (14.28%) 2 S.O.L. in CNS 2 (14.28%) 2 Ulcer 2 (14.28%) 2 Morphological identification and classification of lymphomas based on immunophenotyping is of paramount importance for the management of patients and determining the prognosis because each type and subtype exhibits distinct clinicopatholgic features. The pathological classification of lymphoid malignancies has been a controversial subject. Numerous classifications were proposed used and then went out of favor because there were always lacunae that defied

5 Indian Medical Gazette APRIL comprehension. Repeated attempts for a unified classification were complicated by extra-ordinary confusion of terminology 4. Application of monoclonal antibodies and subsequent immunophenotyping is now accepted worldwide. It serves a necessary adjunct to the morphological diagnosis of Leukemia and lymphomas. The recent REAL classification and WHO classification are primarily based on morphological and immunohistochemical characteristics 5, 6. In the present study, 116 cases of lymphoma were studied for their morphology and immunophentype. Diagnosis of HD is made by the identification of RS cells in an appropriate reactive milieu comprising small lymphocytes, histiocytes, epithelioid histiocytes, neutrophils, eosinophils, plasma cells and fibroblasts in different proportions. In most of the cases, HD can be diagnosed morphologically. However, application of immunophenotyping has enabled the recognition of subgroup that has led to the modification of previous Rye classification with the recent WHO classification 7, 8. The ratio of incidence of NHL- HD is variable among European and Asian countries. The ratio of NHL-HD is 4.47 in European Union and 3.69 in USA 9, 10. In the present study, the ratio is The ratios stated by Ramani et al and Naresh et al were 2.32 and 2.19 respectively for different studies that were done in our country and are similar to our study 11, 12. On morphological examination of HD, the most frequently diagnosed entity was nodular sclerosis (48.78%) followed by mixed cellularity (24.39%). These results are in concordance with various studies conducted world wide and in India and the most frequent subtype recognized was HD NS 13. Immunophenotyping, in case of HD provides an important insight into classification and also are important in differential diagnosis that may mimic HD on routine histological sections 14. The immunophenotype of HD in most of the cases are CD15 and CD30 positive 15. The German Hodgkin s study group found that CD15 negative cases had an increased chance of relapse and a poor survival than CD15 positive cases. In our study all the cases except one were CD15 and CD30 positive. Ongoing studies suggest that there is prognostic significance of CD20 expression in classical HD. CD20 bears resemblance to Calcium ion channel and thus might decrease apoptotic resistance or even activate programmed cell death hence improving failure free survival and overall survival 16. NHL, are known to occur in lymph nodes as well as extra nodal sites. In our study, the ratio of nodal to extranodal site was 4.3: 1. This is in agreement with studies that have shown only about one third of Non Hodgkin s occur at extra nodal sites 17, 18. Among the NHL, immunophenotypic analysis revealed 80% NHL to be of B cell type while only 20% were of T cell type, and B cell: T cell ratio being 4. Thus, these findings were in agreement with studies conducted by various authors in India and world wide 13, 18. Diffuse large B cell lymphoma was the commonest subtype accounting for 54.66% of all the cases in our study. Studies from India and other parts of world show a slightly lower percentage of this subtype varying from 28.2% % 12, 17, 18, 19, 20. In our study 17.33% cases were of follicular lymphoma, which is slightly higher to frequency from entire country (12.5%) 21. This is similar to studies from west which shows a significantly high percentage of this subtype (33-40%) 22, 23. When the Working Formulation was used, low grade lymphomas (Follicular lymphomas and Small lymphocytic lymphomas) contributed 11 cases (14.8%). This finding is in agreement with general trends found in NHL series from most Asian countries where the proportions of low grade lymphomas range from 6% - 13%. This is in contrast to western countries where low grade lymphomas are seen at an unexpectedly high proportion ranging from 26% - 46% 17, 18. A significant finding in our study was relatively higher number of cases of Anaplastic lymphoma 11 cases. After immunostaining, 2 cases exhibited B cell phenotype and were classified as B cell Anaplastic lymphoma and 9 cases (12%) were classified as Anaplastic Ki 1 type. In our study T cell lymphoblastic leukemia contributed 6 cases (8%). This proportion is slightly lower when compared with studies in other parts of country where T ALL ranges from 9-12% 18, 21.

6 132 Indian Medical Gazette APRIL 2015 Thus, application of preliminary panel of antibodies CD3, CD45, CD20 in conjunction with morphological diagnosis enabled us to primarily classify NHL into B or T cell type and further subtype each type. Also Immunophenotyping provided to be useful in cases where a final diagnosis could not be made on morphology along. Besides, classifying NHL as B cell NHL has a better prognosis and disease free survival. Conclusion Though, lymphomas can be confidently diagnosed on morphology, application of monoclonal antibodies and identification of immunophenotypic profile has enhanced the diagnostic accuracy and reproducibility. Further, the immunophenotyping of lymphoid tumors is now considered to be vital for better management, prognosis and for routine pathological evaluation of lymphoproliferative disorders. References 1. D Ardenn A.J. Immunohistology.Stanfeld A.G., D. Ardenne A.J. Lymph node biopsy interpretation 2nd ed. Philadelphia; Churchill Livingstone; , Zola H., Swart B., Nicholson I., et al. CD molecules 2005: human cell differentiation molecules. Blood. 106: , Jaffe E.S., Harris N.L., Diebold J., Fandrin G., Muller Hermelink H.K. World Health Organisation Classification of Neoplastic Diseases of Hematopoietic and Lymphoid tissues. Am J Clin Pathol. 111:8-12, Gall E.A., Mallory T.B. Malignant lymphoma. A clinicopathologic survey of 618 cases. Am J Pathol. 18: , Chan J.K.C., Banks P.M., Cleary M.L., et al. A Revised European American classification of lymphoid neoplasms proposed by the International Lymphoma Study Group summary version. Am J Clin Pathol. 103: , Harris NL, Jaffe ES, Diebold J, et al. The WHO classification of neoplastic diseases of the hematopoietic and lymphoid tissue. Report of the Clinical Advisory Commitee Meeting, Virginia, Nov Histopathology. 36:69-87, Lukes R.J., Butler J.J. The patholgy and nomenclature of Hodgkin s disease. Cancer Res. 26: , Harris N.L., Jaffe E.S, Stein H., et al. A Revised European American classification of lymphoid neoplasms: a proposal from the international lymphoma study group. Blood. 84: , Greiner T.C., Mederios L.J., Jaffe E.S. Non Hodgkin s Lymphoma. Cancer. 75: , Greiner T.C., Mederios L.J., Jaffe E.S. Hodgkin s disease. Cancer. 75: , Ramani A., Kumar K.A., Rao K.K., Vidyasagar M.S., Kundage G.N. Clinico- pathological profile of lymphomas in south India: a prospective rural referral hospital study of 103 cases. J Assoc Physicians India. 39: , Naresh K.N., Agarwal B., Nathwani B.N., Diebold J., Muller-Hermelink, et al. Use of The World Health Organisation (WHO) classification of Non- Hodgkin s Lymphoma in Mumbai, India: A review of 200 Consecutive Cases by a panel of Five Expert Hematopathologists. Leukaemia & Lymphoma. 45: , Naresh K.N., Agarwal B., Sangal B.C., Basu D.D., et al. Regional Variation in the Distribution of Subtypes of Lymphoid Neoplasms in India. Leukemia and Lymphoma. 43: , Harris N.L. The many faces of Hodgkin s disease around the world: What have we learned from its pathology? Annals of Oncology. 9:45-56, Zukerberg L., Collins A., Ferry J., Harris N. Coexpression of CD15 and CD20 by Reed- Sternberg cells in Hodgkin s disease. Am J Pathol. 139: , Tzankov A., Krugmann J., Fend F., et al. Prognostic significance of CD20 expression in Classical Hodgkin Lymphoma: A Clinicopathologic Study of 119 cases. Clin Cancer Res. 9: , 2003.

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