|Year : 2021 | Volume
| Issue : 2 | Page : 149-154
Immunohistochemical subtypes of non-hodgkin lymphomas with special emphasis on diffuse large B-cell lymphoma: An epidemiological study in a tertiary care center of Eastern India
Gaurav Pal1, Senjuti Dasgupta2, Uma Banerjee2
1 Truenat Laboratory, Barjora Superspeciality Hospital, Bankura, West Bengal, India
2 Department of Pathology, Medical College, Kolkata, West Bengal, India
|Date of Submission||07-Mar-2021|
|Date of Acceptance||27-Mar-2021|
|Date of Web Publication||16-Jun-2021|
Department of Pathology, Medical College, Kolkata, West Bengal
Source of Support: None, Conflict of Interest: None
Background: Non-Hodgkin lymphoma (NHL) is a heterogeneous disease with respect to etiology, morphology, immunohistochemistry, and prognosis. Diffuse large B-cell lymphoma (DLBCL) is categorized into two subtypes – germinal center B-cell like (GCB) and activated B-cell like, based on the stage of B-cell differentiation. The epidemiologic profile of NHL varies from one place to another. The aim of the present study was to delineate the frequencies of different types of NHL including subtypes of DLBCL in a tertiary care center. Another objective was to determine if there is any correlation between different subtypes of DLBCL and clinicopathological parameters. Materials and Methods: Immunohistochemical (IHC) examination was done on paraffin blocks of patients diagnosed as NHL by histopathological examination. The patient details, type of NHL including subtype of cases of DLBCL, diagnosed by IHC, were recorded. Results: The mean age of the study population of 62 patients was 53 ± 14.5 years. Forty patients (65%) were male and 22 (35%) were female. The majority (56, 90%) of the NHLs were of B-cell type. DLBCL was the most common B-cell lymphoma (23, 41%). GCB subtype (12, 21%) of DLBCL was slightly more frequent than non-GCB type (11, 20%). The correlation between subtypes of DLBCL and clinicopathological parameters was not statistically significant. Peripheral T-cell lymphoma was the most common type of T-cell lymphoma. Most of the NHLs presented with nodal involvement (38, 61%). Conclusion: The heterogeneity of NHLs in different parts of the country makes it necessary to undertake epidemiological studies so that patient care may be improved.
Keywords: Diffuse large B-cell lymphoma, immunohistochemistry, non-Hodgkin lymphoma
|How to cite this article:|
Pal G, Dasgupta S, Banerjee U. Immunohistochemical subtypes of non-hodgkin lymphomas with special emphasis on diffuse large B-cell lymphoma: An epidemiological study in a tertiary care center of Eastern India. Biomed Biotechnol Res J 2021;5:149-54
|How to cite this URL:|
Pal G, Dasgupta S, Banerjee U. Immunohistochemical subtypes of non-hodgkin lymphomas with special emphasis on diffuse large B-cell lymphoma: An epidemiological study in a tertiary care center of Eastern India. Biomed Biotechnol Res J [serial online] 2021 [cited 2021 Jul 23];5:149-54. Available from: https://www.bmbtrj.org/text.asp?2021/5/2/149/318427
| Introduction|| |
Non-Hodgkin lymphomas (NHLs) are malignancies of lymphoid origin which consist of many subtypes, each with its distinct etiology, morphology, immunohistochemistry, and prognosis. There is a wide variation in epidemiology and clinical presentation among its various subtypes.
NHL ranks as the tenth and twelfth most frequent cancer in males and females, respectively, worldwide. It is more common in the developed world, with the highest incidence rates found in Israeli Jews (age-standardized [world] rate of 17.6 per 100,000), followed by Australia, US Whites, Canada, and Portugal. The lowest rates are found in South Africa (1.6 per 100,000), followed by Vietnam and India. The subtype distribution also varies geographically. Follicular lymphoma and chronic lymphocytic leukemia are more frequent in the Western population, whereas diffuse large B-cell lymphoma (DLBCL) is the most prevalent in India., The most common clinical presentation is painless lymphadenopathy, and extranodal sites may also be involved, with gastrointestinal tract and head-and-neck region being the main sites of involvement, but NHL can affect virtually any part of the body.
DLBCL is a B-cell neoplasm and is the most prevalent subtype of NHL in India accounting for 55% of all NHLs. It is a heterogeneous group of lymphomas having the characteristic morphology of large neoplastic B-cells with nuclear size equal to or exceeding normal macrophage nuclei or more than twice the size of a normal lymphocyte exhibiting diffuse growth pattern. Gene expression profiling (GEP) study to identify the molecular heterogeneity of DLBCL has revealed that it could be divided into the two important subtypes of germinal center B-cell like (GCB) and activated B-cell like, each with different expression of genes affecting different stages of B-cell differentiation and activation causing different clinical outcomes and prognosis. Since the application of GEP technology and other molecular approaches in daily practice is not feasible, various algorithms have been proposed for distinguishing these subgroups, based on a panel of immunohistochemical (IHC) stains for the germinal center B-cell markers (CD10, BCL6, GCET1, and LMO2) and postgerminal center B-cell markers (MUM1/IRF4 and FOXP1). The results of the algorithms developed by Hans et al. and Choi et al. have correlated well with the corresponding GEP results using cDNA microarray and have also demonstrated prognosis and overall survival differences between the GCB and non-GCB DLBCL groups.
Most of these studies have been performed in the Western countries and a few countries of Asia, but there is a paucity of data, especially from the eastern region of India with respect to prevalence of subtypes of NHL and immunophenotypes of DLBCL. The present study was conducted to find out the frequencies of different subtypes of NHL and immunophenotypes of DLBCL, namely GCB and non-GCB, in a tertiary care center of eastern India. It was also done to determine if there is any correlation between the subtypes of DLBCL and various clinicopathological parameters.
| Methods|| |
The patients who had been diagnosed as NHL by histopathological examination and were undergoing IHC examination in the department of pathology of the institute between January 1, 2018, and June 30, 2019, were evaluated for the study. A total of 62 patients were classified into different subtypes of NHL, and the patients with DLBCL were further immunohistochemically subtyped into GCB and non-GCB groups.
The patients with recurrence of malignancy and those who had previously received radiotherapy or chemotherapy were excluded from the study. The clinical records were reviewed in all patients with particular reference to age at diagnosis, site of initial involvement, lymph node status at the time of presentation, and presence of B-symptoms, i.e., fever, night sweats, and loss of ≥10% of body weight over 6 months. Ethical clearance was obtained from the ethical committee of the institute. The reference number of the letter of ethical clearance is MC/Kol/IEC/Non-Spon/692/11-2017, dated 9th December, 2017.
Hematoxylin- and eosin-stained sections were prepared from the formalin-fixed paraffin-embedded tissue blocks to identify the diagnostic area. Sections of 2–3 μm thick were used for IHC staining. IHC examination was done with CD3, CD4, CD8, CD5, CD10, CD79a, CD30, CD20, CD23, Ki67, BCL2, BCL6, CyclinD1, Tdt, MUM1, and PAX5 markers to subtype NHL. Cases with DLBCL were further immunophenotyped using Hans algorithm for classification into two IHC subgroups of DLBCL.
According to Hans algorithm, cases were allocated to the GCB subgroup if CD10 alone was positive; CD10-negative cases were further tested for BCL6. If BCL6 was negative, then the case was assigned to the non-GCB subgroup. Cases with BCL6 positivity were further stained with MUM1; if MUM1 was negative, then the case was assigned to the GCB subgroup, and the positive cases were grouped under the non-GCB subgroup. During the evaluation of Hans algorithm, the staining for CD10, BCL6, and MUM1 antibodies was considered positive only when more than 30% of the tumor cells were stained with the respective antibody. For each case, the areas with the highest percentage of tumor cells stained (”hot spots”) were used for analysis. The morphology of the tumor cells was also evaluated. Staining of tissue sections for all the markers was done manually. The primary antibodies used were all monoclonal except CD3 and CD23.
The clones from which antibodies were prepared included:
- CD20 – L26, Dako
- CD79a – JCB117, Dako
- CD30 – Ber-H2, Dako
- CD8 – C8/144B, Dako
- CD3 – Polyclonal Rabbit Anti-Human, Dako
- BCL-2 – 124, Dako
- CD23 – Polyclonal Rabbit Anti-Human, BioGenex
- MUM1 – EP190, BioGenex
- Cyclin D1 – EP12, BioGenex
- CD5 – 4C7, BioGenex
- CD10 – 56C6, BioGenex
- CD4 – 4B12, BioGenex
- PAX5 – DAK-Pax5, Dako
- Ki-67 – MIB-1, Dako
- BCL-6 – GI191E/A8, Cell Marque
- Tdt – SEN28, Thermo Scientific.
Appropriate positive and negative controls were used for each of the antibodies. Result analysis was done using Microsoft Excel 2013 and SPSS 23 statistical software – UNICOM Global; Bengaluru, Karnataka, India. A study of significance was done with Chi-square test and P value. The results were considered significant if P < 0.05.
| Results|| |
A total of 62 cases were evaluated in the study. Eight cases were rejected due to 6 having recurrence of malignancy and 2 did not give consent for immunohistochemistry. The mean age of the study population was 53 ± 14.5 years, with a range between 3 and 86 years. Forty patients (65%) were male and 22 (35%) were female with a male-to-female ratio of 1.8:1.
The most frequent site of involvement among the lymph nodes was cervical, accounting for almost half of all the nodal cases followed by axillary lymph nodes (21%) and inguinal lymph nodes (18%). Extranodal cases accounted for 39% of the cases, with the most common sites being stomach and tonsils, each accounting for 17% of the extranodal cases. Approximately 55% of the patients presented with lymphadenopathy, but B-symptoms were present only in 10% of the patients.
The IHC examination revealed that the overwhelming majority of the lymphomas were B-cell type which accounted for 90% of all the cases [Figure 1]. DLBCL constituted most of the B-cell lymphomas at 41%, followed by follicular lymphoma, small lymphocytic lymphoma, nodal and extranodal marginal zone lymphoma, mantle cell lymphoma, and acute lymphoblastic lymphoma at 18%, 14%, 11%, 7%, 5%, and 4%, respectively. The subtypes of DLBCL were almost equally divided, with GCB being slightly more prevalent at 21% [Figure 2] and [Figure 3]. The most common type of T-cell lymphoma was peripheral T-cell lymphoma which accounted for half of all the T-cell lymphoma cases [Figure 4]. Other T-cell lymphomas found were angioimmunoblastic lymphoma and anaplastic large-cell lymphoma accounting for 33% and 17%, respectively.
|Figure 1: (a) Non-Hodgkin lymphoma with diffuse effacement of lymph node architecture (H and E, ×100). (b) High-power view of the same lymph node (H and E, ×400). (c) CD20 positivity in B-cell non-Hodgkin lymphoma (×400). (d) CD79a positivity in B-cell non-Hodgkin lymphoma (×400)|
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|Figure 2: (a) Mantle cell lymphoma: Cyclin D1 positive (×400). (b) CD23-positive cells in follicular lymphoma: (×400)|
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|Figure 3: (a) Diffuse large B-cell lymphoma, nongerminal center B-cell like (diffuse large B-cell lymphoma nongerminal center B-cell like type); BCL-2-positive cells (×400). (b) Diffuse large B-cell lymphoma, nongerminal center B-cell like (diffuse large B-cell lymphoma nongerminal center B-cell like type); MUM1 positivity (×400). (c) Diffuse large B-cell lymphoma, nongerminal center B-cell like (diffuse large B-cell lymphoma nongerminal center B-cell like type); Ki-67 stain showing 80% positivity (×100). (d) Diffuse large B-cell lymphoma, nongerminal center B-cell like (diffuse large B-cell lymphoma nongerminal center B-cell like type); High-power view of 80% Ki-67 positivity (×400)|
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|Figure 4: (a) Peripheral T-cell lymphoma; CD3-positive cells (×400). (b) Peripheral T-cell lymphoma; CD5-positive cells (×400)|
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Among the males, the most frequent diagnosis was DLBCL of non-GCB subtype at 22% followed closely by follicular lymphoma at 19%. Among the females, the most frequent diagnosis was DLBCL of GCB subtype at 32% followed by DLBCL of non-GCB subtype, follicular lymphoma, and small lymphocytic lymphoma each at 16%. The age and gender distribution of different subtypes of B- and T-cell lymphomas is shown in [Table 1].
Whereas B-cell lymphomas were found both in nodes and extranodal sites, T-cell lymphomas were exclusively found in the nodes. CD20 was positive in 94.6% of the cases of B-cell lymphomas, and CD3 was immunoreactive in 83.3% of the cases of T-cell lymphomas. In the extranodal sites, DLBCL of non-GCB subtype was the most prevalent accounting for almost a third of the cases. In the nodes, small lymphocytic lymphoma was the most prevalent as it accounted for almost a quarter of the cases. Both the subtypes of DLBCL were more frequent in the extranodal sites, with DLBCL of non-GCB subtype being more prevalent [Table 2].
|Table 2: Distribution of non-Hodgkin lymphoma according to site of involvement (lymph nodal and extranodal sites) and the presence of B-symptoms|
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Majority of the GCB cases, approximately 67%, were classified with CD10 positivity, whereas around 55% of the non-GCB cases were identified with CD10 and BCL6 negativity [Table 3]. The subtypes of DLBCL were correlated with age group distribution, gender, site of involvement, and Ki-67 scores. These associations were not found to be statistically significant [Table 4].
|Table 3: Distribution of diffuse large B-cell lymphoma cases into subtypes by immunophenotyping according to Hans algorithm|
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|Table 4: Clinicopathological correlations of diffuse large B-cell lymphoma subtypes|
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| Discussion|| |
NHL incidence rates showed increasing trends in the last decade of the earlier century but have gradually stabilized, especially in the developed world. A similar pattern has also been observed in some other parts of the world including India. The age-adjusted incidence rates for NHL in India are 2.9/100,000 and 1.5/100,000 for men and women, respectively. It is more prevalent in urban centers than the rural areas. The occurrence of NHL is higher in men than women. In the present study, males outnumbered females, as there were 40/62 cases (65%) of male patients and 22/62 cases (35%) of female patients. This male preponderance among patients suffering from NHL has been reported in other studies as well.,,,,,
The current study indicates that NHL is more common in older adults than younger patients, with the fifth and sixth decades being the most affected age groups. Sharma et al. and Padhi et al. found the most affected age group in the fourth decade, whereas Roy et al. showed the maximum incidence in the sixth decade. Other authors have shown similar incidences in the age groups, with the fourth to sixth decades being the most affected.,,
The overwhelming majority of the NHLs are B-cell lymphomas, which accounted for 90% of lymphomas in the present study. This is in agreement with the general consensus that T-cell lymphomas are relatively rare as shown by Sharma et al. who found 89% of NHLs to be B-cell lymphomas and Bhardwaj and Kishore who reported that B-cell lymphomas constitute 80% of NHLs. Roy et al. found only 54% of NHLs to be of B-cell type, but Padhi et al. showed 96% of NHLs to be of B-cell type. Mushtaq et al. also found 86% of NHLs to be of B-cell type and the rest 14% to be of T-cell type.
The individual B-cell neoplasms vary in their relative frequency in various parts of the world. Follicular lymphoma and chronic lymphocytic leukemia are more common in the USA, whereas Burkitt's lymphoma occurs more frequently in tropical Africa, immunoproliferative small intestinal disease in the Middle East, and adult T-cell leukemia/lymphoma in Southwest Japan and the Caribbean Basin. In the present study, the most common type of B-cell lymphoma was DLBCL, which accounted for 37% of all the cases of NHL. GCB subtype (19% of the cases) was more prevalent than non-GCB subtype (18% of the cases). These were followed by follicular lymphoma and small lymphocytic lymphoma accounting for 16% and 13% of the cases, respectively. Among T-cell lymphomas, peripheral T-cell lymphoma was the most common type which accounted for 5% of the total cases. Sharma et al. also found DLBCL to be the most common type which accounted for 46.8% of all the NHL cases. This was followed by small lymphocytic lymphoma causing 17% of the cases. Among the T-cell lymphomas, they found that the most common type was T-lymphoblastic lymphoma causing 6.4% of the cases. Only Shanmugasundaram et al. found small lymphocytic lymphoma to be the most common B-cell lymphoma otherwise Devi et al., Bhardwaj and Kishore, Roy et al., Padhi et al., Mushtaq et al., Mondal et al., and Nimmagadda et al. – all found DLBCL to be the most common B-cell lymphoma. Peripheral T-cell lymphoma was found to be the common T-cell lymphoma by Shanmugasundaram et al., Roy et al., and Mushtaq et al. On the other hand, Mondal et al. and Nimmagadda et al. found anaplastic large-cell lymphoma to be the common T-cell lymphoma.
Extranodal involvement of NHL is very well documented in the literature. The current study found 39% of the NHL cases having extranodal manifestations, with the most common sites being gastrointestinal region closely followed by head-and-neck region. Shanmugasundaram et al. found only 12% of the NHL cases to be extranodal, with gastrointestinal region being the most common site. Sandhu et al. found 44% of the NHL cases to be extranodal, but the most common site of involvement was the head-and-neck region. Devi et al. found 43% of the NHL cases to be extranodal, and the most common site of involvement was the gastrointestinal region. Mushtaq et al. and Mondal et al. also reported gastrointestinal region to be the most common site of extranodal involvement each accounting for 38% and 28% of the extranodal cases, respectively.
The present study showed that among the DLBCL cases, 52% were of GCB subtype, whereas the rest were of non-GCB subtype. A slightly different prevalence was reported by a study group from Malaysia which showed 33% of the cases to be of GCB subtype whereas the rest to be of non-GCB subtype using Hans algorithm. The correlation between different subtypes of DLBCL and clinicopathological parameters was not found to be statistically significant in the current study. Ki-67 is a nuclear stain which is used as a proliferative marker and has been suggested for a potential prognostic value in cases of DLBCL, but results have been inconclusive. In the present study, the correlation between subtypes of DLBCL and Ki-67 proliferation index was not statistically significant. On the other hand, a study from Romania has unequivocally suggested routine use of Ki-67 along with other markers in diagnostic algorithm of DLBCL.
| Conclusion|| |
The majority of the NHLs encountered in the present study were of B-cell type, among which the most common type was DLBCL. GCB subtype of DLBCL was slightly more frequent than the non-GCB type. The most common type of T-cell lymphoma found was peripheral T-cell lymphoma. Both nodal and extranodal sites were involved in B-cell lymphomas, whereas only nodes were affected in T-cell type. The different subtypes of DLBCL did not bear any statistically significant correlation with clinicopathological parameters. The delineation of different types of lymphoma in various regions of the country is essential for epidemiological purposes as well as to provide appropriate patient care.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Sharma M, Mannan R, Madhukar M, Navani S, Manjari M, Bhasin TS, et al
. Immunohistochemical (IHC) analysis of Non-Hodgkin's lymphoma (NHL) spectrum according to WHO/REAL classification: A Single Centre Experience from Punjab, India. J Clin Diagn Res 2014;8:46-9.
Miranda-Filho A, Piñeros M, Znaor A, Marcos-Gragera R, Steliarova-Foucher E, Bray F. Global patterns and trends in the incidence of non-Hodgkin lymphoma. Cancer Causes Control 2019;30:489-99.
Shanmugasundaram S, Balan K, Arumugam D. Immunohistochemical profile and distribution of non-Hodgkin and Hodgkin lymphoma - An experience in a medical college hospital in Tamil Nadu. Indian J Med Paediatr Oncol 2020;41:695-701. [Full text]
Sandhu DS, Sharma A, Kumar L. Non-Hodgkin's lymphoma in Northern India: An analysis of clinical features of 241 cases. Indian J Med Paediatr Oncol 2018;39:42-5. [Full text]
Devi AA, Sharma TD, Singh YI, Sonia H. Clinicopathological profile of patients with non-Hodgkin's lymphoma at a regional cancer center in Northeast India. J Sci Soc 2017;44:140-4. [Full text]
Dwivedi A, Mehta A, Solanki P. Evaluation of immunohistochemical subtypes in diffuse large B-cell lymphoma and its impact on survival. Indian J Pathol Microbiol 2015;58:453-8.
] [Full text]
Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, et al
. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 2000;403:503-11.
Hans CP, Weisenburger DD, Greiner TC, Gascoyne RD, Delabie J, Ott G, et al
. Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood 2004;103:275-82.
Choi WW, Weisenburger DD, Greiner TC, Piris MA, Banham AH, Delabie J, et al
. A new immunostain algorithm classifies diffuse large B-cell lymphoma into molecular subtypes with high accuracy. Clin Cancer Res 2009;15:5494-502.
Nair R, Arora N, Mallath MK. Epidemiology of non-Hodgkin's lymphoma in India. Oncology 2016;91 Suppl 1:18-25.
Bhardwaj A, Kishore S. Morphological typing of lymphomas with immunohistochemistry. Indian Medical Gazette 2015;149:127-33.
Roy A, Kar R, Basu D, Badhe BA. Spectrum of histopathologic diagnosis of lymph node biopsies: A descriptive study from a tertiary care center in South India over 5½ years. Indian J Pathol Microbiol 2013;56:103-8.
] [Full text]
Padhi S, Paul TR, Challa S, Prayaga AK, Rajappa S, Raghunadharao D, et al
. Primary extra nodal non Hodgkin lymphoma: A 5 year retrospective analysis. Asian Pac J Cancer Prev 2012;13:4889-95.
Mushtaq S, Akhtar N, Jamal S, Mamoon N, Khadim T, Sarfaraz T, et al
. Malignant lymphomas in Pakistan according to the WHO classification of lymphoid neoplasms. Asian Pac J Cancer Prev 2008;9:229-32.
Mondal SK, Mandal PK, Samanta TK, Chakaborty S, Roy SD, Roy S. Malignant lymphoma in Eastern India: A retrospective analysis of 455 cases according to World Health Organization classification. Indian J Med Paediatr Oncol 2013;34:242-6.
] [Full text]
Nimmagadda RB, Digumarti R, Nair R, Bhurani D, Raina V, Aggarwal S, et al
. Histopathological pattern of lymphomas and clinical presentation and outcomes of diffuse large B cell lymphoma: A multicenter registry based study from India. Indian J Med Paediatr Oncol 2013;34:299-304.
] [Full text]
Phang KC, Hussin NH, Abdul Rahman F, Tizen NM, Mansoor A, Masir N. Characterisation of immunogenotypes of diffuse large B-cell lymphoma. Malays J Pathol 2019;41:101-24.
Pătraşcu AM, Rotaru I, Olar L, Pătraşcu Ş, Ghiluşi MC, NeamŢu SD, et al
. The prognostic role of Bcl-2, Ki67, c-MYC and p53 in diffuse large B-cell lymphoma. Rom J Morphol Embryol 2017;58:837-43.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]