Prevalence of Double MYC/BCL2 Expression and the Cell of Origin in Diffuse Large B-Cell Lymphomas Diagnosed at a Tertiary Level Referral Laboratory

Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most commonly diagnosed non-Hodgkin’s lymphoma in adults worldwide. In Kenya DLBCL accounts for 64% of all non-Hodgkin’s lymphomas diagnosed in adults. It is a heterogeneous disease and patients show varying outcomes despite standard treatment. This variability is attributed to the differences in the biology and molecular pathogenesis of DLBCL. The five most important prognostic factors for DLBCL include scores for the revised international prognostic index (R-IPI), the cell of origin (COO), presence of myelocytomatosis (MYC) and B-cell lymphoma 2 (BCL2) gene rearrangements by fluorescent in situ hybridisation (FISH) or standard cytogenetics, the absolute lymphocyte and monocyte count, and imaging with positron emission tomography (PET scan). Current data shows that the use of immunohistochemistry in identifying cases of DLBCL with MYC and BCL2 protein over-expression (double-expressing lymphomas) yields important prognostic information since these patients form a higher proportion of cases compared to those with concurrent MYC and BCL2 translocations (double-hit lymphomas) using FISH/cytogenetics . The double expressing DLBCL have a poorer prognosis compared to cases of DLBCL lacking MYC/BCL2 double-expression. With advances in targeted therapy for DLBCL, it is important to also identify patients who may benefit from new regimens by determining their cell of origin. Gene expression profiling (GEP), the technique for determining the COO is not available to the routine diagnostic laboratory. Consequently, robust immunohistochemistry surrogates have been developed in place of GEP for this purpose. Hans’ algorithm which uses three antibodies in sequence (CD10, BCL6 and MUM1) is the most widely applied for determining COO. The World Health Organization (WHO) guidelines currently recommend definition of the cell of origin and testing for double MYC/BCL2 expression for every patient diagnosed with diffuse large B cell lymphoma. Furthermore, in order to establish a diagnosis of DLBCL, NOS with certainty, other high-grade B-cell lymphomas, such as Burkitt lymphoma and plasmablastic lymphoma, need to be excluded. This can be achieved by a comprehensive antibody panel that includes MYC and BCL2. These antibodies need to be optimised and standardised for the settings in which a particular laboratory operates. Objectives: To determine the prevalence of MYC/BCL2 double-expression among diffuse large B cell lymphomas diagnosed at The Aga Khan University Hospital, Nairobi (AKUH, N) Laboratory. To define the cell of origin of DLBCL diagnosed at AKUH, N laboratory. Methods: Formalin fixed Paraffin embedded (FFPE) blocks of DLBCL cases diagnosed between January 1st 2012 and December 31st 2015 were retrieved from the archives at the pathology Department of AKUHN. Hematoxylin /Eosin sections were reviewed for adequacy and Tissue Microarrays (TMA) constructed and stained with CD3, CD5, CD10, CD20, CD30, MUM1, BCL6, BCL2, MYC and Ki67 for determination of COO and BCL2/MYC double expression Additional cyclin D1 immuno-staining was performed on all CD5 positive cases to exclude pleomorphic mantle cell lymphoma. Data Analysis: Data analysis utilized SPSS® version 23 (IBM corporation, Armonk, New York, USA). Descriptive statistics for median age, sex preponderance, tumour site (whether nodal or extranodal) were analysed. The prevalence of cases expressing both MYC and BCL2 was calculated as a proportion of the total number of cases included in the study. The cell of origin for each case was determined as either germinal center origin (GCB) or non-germinal center (non-GCB) with their respective proportions calculated from the total number of cases included in the study. The proportion of CD5 and CD30 positive cases was also determined. Fisher’s exact test was performed on 2 by 2 tables for correlation between double expression and sex, cell of origin and site with a p value of Results: A total of 165 cases of DLBCL were included in the study for which two TMA blocks were constructed and stained for cell of origin and double expression. The median age for the study group was 50 years. There was an even distribution by gender with males accounting for 54.5% of cases. Majority of the cases were from nodal sites (57.9%). The mean Ki67 index was 30% (Range: 0 to 100%). Only 18 cases were found to be positive for both MYC and BCL2 accounting for 10.9% of all DLBCLs. The double expressing tumors showed a higher mean Ki67 index of 45% compared to the non-double expressing group (29%). No difference was noted in median age, sex preponderance, cell of origin or site of occurrence between the two groups. A total of 67 cases accounting for 40.6% of the cases were classified as GCB. Of the 97 non-GCB cases, 64 (70%) occurred in extranodal sites, a statistically significant correlation (p=0.016). The non-GCB group also showed mean higher Ki67 index (36%) compared to GCB group (26%) (p=0.008). No differences were detected in sex or median ages between these two groups. A total of 121 cases were analysed for CD5 immunoreactivity with 11(9.1%) cases showing positivity. All but one of the positive cases occurred in males. No differences were detected in median ages, mean Ki67 indices, cell of origin or site of occurrence between the CD5 positive and negative groups. None of the CD5 positive cases expressed cyclin D1. Only 3 (2.5%) cases out of the 122 stained expressed CD30. Conclusion: We report a lower prevalence of MYC/BCL2 double expression as compared to studies conducted in the West. This understates the reasons for this as an area for future studies. However, we report a higher prevalence of the poor prognosis non-GCB subtype of DLBCL understating the need for routine analysis of COO for all DLBCL cases to include rituximab as an adjuvant to CHOP. Classification for cell of origin at diagnosis is also crucial to prognosticate and predict outcomes in our patients. Prevalence of CD5 positive cases is similar to reported literature. The effect of higher prevalence of HIV in our region was not studied due to lack of clinical information and certainly warrants further exploration in future prospective studies. The value of routine CD30 staining remains to be confirmed given the low prevalence of this sub-group in the study