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An immunohistochemical study of double-expressor lymphomas and its correlation with cell of origin

 Department of Pathology, St John's Medical College, Bengaluru, Karnataka, India

Date of Submission09-Apr-2021
Date of Decision18-May-2021
Date of Acceptance30-May-2021
Date of Web Publication28-Jan-2022

Correspondence Address:
Anuradha Ananthamurthy,
Department of Pathology, St John's Medical College, Bengaluru - 560 034, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.jcrt_587_21

 > Abstract 

Background: Diffuse large B cell Lymphomas (DLBCL) that co express C MYC and BCL 2 are known as 'double expressor lymphomas' and are believed to have a worse prognosis than other diffuse large B cell lymphomas. This was a study to assess the frequency of double expressor lymphomas in our cohort of DLBCL
Aims and Objectives: The aims of this study were to assess the frequency of double expression of C MYC and BCL 2 in cases of DLBCL and to correlate the same with clinicopathological parameters including cell of origin, namely germinal centre type versus non germinal centre type.
Materials and Methods: This was a retrospective observational study Immunostaining for MYC antibody and BCL2 were performed using the standard polymer/DAB technique. 40% for MYC and 50% for BCL2 were taken as cut off values.Chi square analysis was used to compare the variables, and a p value of less than 0.05 was taken as statistically significant
Results: Of 40 cases studied, 11 (27.5%) were double expressors. There was no significant correlation of double expression with gender, site (nodal versus extra nodal), cell of origin namely germinal centre/non germinal centre types and Ki67 index when compared to the other group which did not show double expression
Conclusions: Immunohistochemistry is a useful technique to detect double expressor lymphomas which are known to have an aggressive course. Cell of origin did not show significant correlation with double expression in our study.

Keywords: BCL2, C MYC, Double expression

How to cite this URL:
Ananthamurthy A. An immunohistochemical study of double-expressor lymphomas and its correlation with cell of origin. J Can Res Ther [Epub ahead of print] [cited 2022 Nov 29]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=336699

 > Introduction Top

Diffuse large B-cell Lymphomas (DLBCL) are recognized as a heterogeneous subtype with subtypes being recognized based on gene expression profiling, cytogenetic studies, and immunohistochemical findings. Standard of care in DLBCL involves the use of the R CHOP regime and cure and survival rates are influenced by clinicopathological factors such as age, IPI index, molecular subtype, and the presence of cytogenetic abnormalities. One of the first attempts to subclassify DLBCL in order to provide better prognostic information was based on gene expression profiling which identified two distinct subsets of patients, who differed in their prognoses and exhibited two distinct molecular profiles.[1] These subsets are known as germinal center type (GCB type and less aggressive) and nongerminal center (non-GCB type and more aggressive) are now also identified using immunohistochemical (IHC) algorithms.[2]

Cytogenetic studies further showed that some DLBCLs showed the presence of both MYC and BCL2 rearrangements and sometimes BCL6 also. These lymphomas behaved in a more aggressive fashion with a poor prognosis.[3],[4] Recognizing this subgroup, the revised WHO guidelines in 2016 documented the new subtype B cell lymphomas of, namely, the high-grade B-cell lymphomas based on the presence of MYC and BCL2 rearrangements as double-hit lymphomas and MYC, BCL2, and BCL6 rearrangements as triple-hit lymphomas.[5] Further, lymphomas that co-express MYC and BCL2 proteins on immunohistochemistry are termed as double expressor (DE) lymphomas. Although not a distinct entity under the WHO classification, this category is believed to have a poor outcome and may account for 20%–30% of DLBCL cases.

AS the DE category has been relatively recently described, there are very few studies that have systematically analyzed the occurrence of DE lymphomas, this study was undertaken to assess the frequency of these lymphomas among our subset of DLBCLs and also correlate the same with other clinic pathological parameters and cell of origin. This will also lead to a better understanding of the pathogenesis of the heterogeneous category of DLBCLs.

 > Materials And Methods Top

Ethical clearance was obtained from the institutional ethical board. All diagnosed cases of DLBCL from January 2017 to December 2019 (period of 3 years) were included in this study. Cases that had inadequate tissue in the blocks were excluded from the study. These cases were diagnosed as DLBCL based on morphology and immunohistochemistry, the routine markers being, CD20, CD3, CD10, BCL6, MUM1, CD30, and Ki67. They were further subclassified as GCB and non-GCB based on the Hans algorithm. Immunostaining for MYC antibody and BCL2 was performed using the standard polymer/DAB technique. Forty percent for MYC and 50% for BCL2 were taken as cut-off values in accordance with previous studies [Figure 1], [Figure 2], [Figure 3], [Figure 4].[5],[6],[7] The presence of MYC expression and double expression for BCL2 and MYC were then correlated with other parameters such as germinal center versus non GCB and other clinicopathological parameters. Paraffin blocks with scanty or no tissue as well as cases with prior treatment history were excluded from the study. The presence of double expression was correlated with clinic pathological factors using the Chi-square statistical analysis.
Figure 1: Stomach biopsy showing diffuse large B cell lymphomas, hematoxylin and eosin, ×400

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Figure 2: CD20 positivity in the atypical lymphoid infiltrate, ×400

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Figure 3: C-MYC nuclear positivity in about 60% of the lymphoid cells ×400

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Figure 4: BCL2 positivity in 70%–80% of cells in the same case as in Figures 1-3, ×400

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 > Results Top

A total of forty cases of DLBCL were included in this study. The age ranged from 32 to 84 years. There were 22 males and 18 females. Of these twenty were of the GCB and 20, of the non-GCB based on the Hans algorithm. There were 26 nodal cases and 14 extranodal cases, the extranodal sites being gastrointestinal tract, brain, and nasopharynx [Table 1].
Table 1: Clinicopathological and immunohistochemical findings in diffuse large B-cell lymphomas (n=40)

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All the cases satisfied the morphological and IHC criteria of DLBCL. The lymph nodes showed effacement of architecture with a diffuse proliferation of large lymphoid cells with oval-to-round vesicular nuclei with fine chromatin and some showing prominent nucleoli. The cells showed similar morphology in extranodal sites also [Figure 1].

Further, a panel of IHC markers was done which included CD20, CD3, CD10, BCL6, Mum1, and BCL2. CD30 and CD5 were also done in a subset of cases. Ki67 was performed in 37 cases and ranged from 50% to 90%. Cyclin D1 was additionally done in the subset that was positive for CD5 and those cases that had a blastoid morphology to exclude a Mantle cell lymphoma. A summary of the IHC findings is given in [Table 1].

Fourteen cases out of 40 were positive for C MYC, and 31 were positive for BCL2; of which 11 expressed both BCL2 and C MYC. Of these 11 cases, six were female and five were male. The ages ranged from 39 years to 84 years. Six were nodal and the rest extra nodal-stomach, nasopharynx, duodenum, colon, and spleen being the extranodal sites. The Ki 67 ranged from 50% to 90%.

There were no significant correlation of double expression with gender, site (nodal versus extranodal), and germinal center/non-GCBs when compared to the other group which did not show double expression [Table 2]. Furthermore, there was no significant difference between the two groups with regard to high Ki 67 (80% or more; P = 0.904).
Table 2: Correlation of double expression with other clinic pathological parameters

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Of the total number of cases that showed C MYC expression (n = 14), nine were GCB and five were nongerminal center (P = 0.228). Of the total number of cases that showed BCL2 expression (n = 31), 17 were GCB and 14 were non-GCB (P = 0.255).

 > Discussion Top

Cytogenetic studies have shown that a subset of cases of DLBCL is the “double-hit” lymphomas (DHLs), with MYC and BCL2 and/or BCL6 rearrangements.[8] There is also a growing interest now in the more common “double-expressor” lymphomas defined based on the IHC stains with MYC and BCL2 staining on more than a specified proportion of tumor cells. Most DE lymphomas do not carry the translocations. Although not as aggressive as the high-grade lymphomas that carry the translocations, they are known to be more aggressive than other DLBCL which are not DEs. These observations have suggested that double expression of MYC and BCL2 proteins without gene translocations should be considered a prognostic indicator in DLBCL.

In this study we found, double expression of C MYC and BCL2 in 27.5% of the cases of DLBCL which is somewhat lower than what was found in previous studies, when the cut off for C-MYC was taken as 40% and that of BCL2 taken as 50% based on previous studies.[9],[10] In their cohort of 109 cases of DLBCL, Hashmi et al. showed that 35% were DE phenotype.[9] Asati et al. found 43.9% to be DE phenotype of a total of 76 cases.[10]

The study by Hashmi et al. also showed that DE lymphomas were significantly more common in DLBCLs with nongerminal center phenotype and also in DLBCLs with a higher Ki 67 index.[9]]

In the present study, we did not see any significant correlation of double expression with germinal center versus nongerminal center subtype, being represented almost equally in both.

In the study from India by Asati et al. the authors attempted to predict DE lymphomas based on cell of origin and Ki67 index.[10] Although their study found a significant association of DE phenotype with high Ki67 and non GCB cell of origin, it lacked specificity and the authors conclude that this model cannot be used as a surrogate for DE phenotype and all patients with DLBCL should be further assessed by IHC to rule out DE phenotype. Their study also showed no significant correlation between CMYC or BCL2 expression and cell of origin, which is similar to our study.

Association of DE phenotype with a high Ki 67 index has prompted authors to recommend doing IHC for C MYC in DLBCLs that have Burkitt-like morphology and high Ki 67 index. Contrary to other studies, we did not see any significant correlation between high Ki67 and DE phenotype.[9],[10] This shows that high Ki67 may be an unreliable marker to predict DE phenotype in DLBCL cases.

Many studies have proposed that DE lymphomas may have a worse prognosis and aggressive clinical behavior when compared with non-DE lymphomas.[5] A recent clinicopathological study of double hit and DE lymphomas from Mehta et al. showed that these lymphomas have a worse prognosis warranting aggressive therapeutic interventions.[11] Their study showed that DE lymphomas were more common in the nongerminal center subtype. The authors also recommend testing all DE lymphomas for double hit translocations. In another study, Johnson et al. investigated the correlation between the presence of MYC and BCL2 proteins by immunohistochemistry (IHC) with survival in two independent cohorts of patients with DLBCL treated with R-CHOP. They found double expression in 21% of DLBCL, which is similar to our study, and also found that MYC protein expression was only associated with inferior overall and progression-free survival when BCL2 protein was co-expressed (P < 0.001).[12] One of the drawbacks of our study was that we did not have adequate follow-up of the patients in order to compare the prognosis and outcomes of patients with DE phenotype.

In conclusion, we have shown that C MYC and BCL 2 assessment by immunohistochemistry is an easy and robust method to risk stratify patients of DLBCL. We also found that there was no significant correlation between cell of origin and double expression. Further studies are warranted to assess prognosis and survival in these patients in the Indian cohort. Further, cytogenetic studies are need to be performed to find the prevalence of MYC and BCL2 translocations and to examine the relation between double expresser and double-hit lymphomas.


This study was supported by a grant from the “RGUHS Advanced Research Grant” of the Rajiv Gandhi University of Health Sciences, Bangalore.

 > References Top

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.  Back to cited text no. 1
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.  Back to cited text no. 2
Phuoc V, Sandoval-Sus J, Chavez JC. Drug therapy for double-hit lymphoma. Drugs in context 2019;8-1.  Back to cited text no. 3
Riedell PA, Smith SM. Double hit and double expressors in lymphoma: Definition and treatment. Cancer 2018;124:4622-32.  Back to cited text no. 4
Swerdlow SH, Campo E, Pileri SA, Harris NL, Stein H, Siebert R, et al. The 2016 revision of the World Health Organization classification of lymphoid neoplasms. Blood 2016;127:2375-90.  Back to cited text no. 5
Kawashima I, Inamoto Y, Maeshima AM, Nomoto J, Tajima K, Honda T, et al. Double-expressor lymphoma is associated with poor outcomes after allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 2018;24:294-300.  Back to cited text no. 6
Swerdlow SH. Diagnosis of 'double hit'diffuse large B-cell lymphoma and B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma: When and how, FISH versus IHC. Hematology 2014;2014:90-9.  Back to cited text no. 7
Said JW. Aggressive B-cell lymphomas: How many categories do we need? Mod Pathol 2013;26 Suppl 1:S42-56.  Back to cited text no. 8
Hashmi AA, Iftikhar SN, Nargus G, Ahmed O, Asghar IA, Shirazi UA, et al. Double-Expressor phenotype (BCL-2/c-MYC Co-expression) of diffuse large B-Cell lymphoma and its clinicopathological correlation. Cureus 2021;13:e13155.  Back to cited text no. 9
Asati V, Premalata CS, Babu KG, Lakshmaiah KC, Lokanath D, Jacob LA, et al. An attempt to predict double expresser DLBCL based on cell of origin and proliferative index (Ki-67): A prospective study from India. Ann Oncol 2018;29:ix90.  Back to cited text no. 10
Mehta A, Verma A, Gupta G, Tripathi R, Sharma A. Double hit and double expresser diffuse large b cell lymphoma subtypes: Discrete subtypes and major predictors of overall survival. Indian J Hematol Blood Transfus 2020;36:627-34.  Back to cited text no. 11
Johnson NA, Slack GW, Savage KJ, Connors JM, Ben-Neriah S, Rogic S, et al. Concurrent expression of MYC and BCL2 in diffuse large B-cell lymphoma treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone. J Clin Oncol 2012;30:3452-9.  Back to cited text no. 12


  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1], [Table 2]


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