Classification of Highly Proliferative B Cell Lymphomas Using Differential Expression of cMYT Target Genes.
Authors: Barrans S. L. , O’Connor S. J.M. , Rawstron A. C. , Allan J. , Owen R. G. , Mead G. M. , Jack A. S. 
Published: October 2019

Abstract
B cell lymphomas with 100% Ki67 expression, represent a spectrum of disease incorporating Burkitt Lymphoma (BL). The currently recruiting MRC (UK) LY10 trial aims to determine the efficacy of CODOX-M/IVAC in adult BL. Due to the unfeasibility of demonstrating cMYC rearrangement prior to trial entry, 100% Ki67 is being used to define trial eligibility. The purpose of this study is to evaluate the heterogeneity of tumors selected on this basis, and to determine the effectiveness of 100% Ki67 as a surrogate marker for cMYC deregulation. To date we have immunophenotypic and FISH data on tumors from 145 patients. 93 cases have 100% Ki67 and are eligible for the trial. 62/93 (66.67%) cases were found to have a BL-specific phenotype (defined as CD20+, CD10+, BCL6+, BCL2−, P53+P21- and 100% cell cycle fraction) and of these 44 (71%) have rearrangement of cMYC. Rearrangement of cMYC was exclusive to cases with a BL phenotype, however 18/62 cases express the phenotype with no evidence of cMYC rearrangement. The remaining cases were GC- or activated-DLBCL. To test the hypothesis that cMYC may be deregulated by a mechanism other than gene rearrangement in cases with a BL-Phenotype, we have compared the gene expression profiles of samples expressing a BL-phenotype, but lacking a cMYC rearrangement (BL-Phen), with typical BL cases with rearranged cMYC (BLt), using Affymetrix Human U133 plus 2.0 chips. Due to the high levels of apoptosis in these tumors, cases with fresh material to extract sufficient RNA are rare, and only 8 cases were suitable for analysis. These were split into 3 groups: BLt (3); BL-Phen (3) and 2 DLBCL with 100% Ki67 but no other features of BL. A condition tree clustering algorithm was produced using GeneSpring v6.1 and correlation of the related samples was determined by Pearson correlation (P). In addition, a sub-analysis was performed on 33 genes reported to be downstream targets of cMYC. Using this approach, the 3 BLt cases clustered, as did the 3 BL-Phen cases, however comparison between groups showed a negative association (P= −0.3), suggesting that, although the phenotype is identical to BL, the molecular pathogenesis of BL-Phen cases does not involve deregulation of cMYC. A >3 fold increase in cMYC expression was found in the BLt compared to the BL-Phen group, and several genes, including nucleolin, ornithine decarboxylase 1, eukaryotic translation initiation factor 4E, carbamoyl-phosphate synthetase 2, high mobility group AT-hook 1, transferrin receptor and branched chain aminotransferase 1, were differentially expressed in BLt compared to BL-Phen cases. Interestingly, one of the non-BL-Phen samples had similar expression of cMYC and downstream genes as the BLt cases, suggesting cMYC activation by an alternative mechanism in this case. In conclusion, B-cell lymphomas with 100% Ki67 are a heterogeneous group of tumors, in which 47% are typical BL as defined by phenotype and cMYC rearrangement. Cases with a BL-Phenotype that lack cMYC rearrangement have an expression profile suggesting that cMYC is not deregulated, and that these patients may not be suitable for treatment with high intensity chemotherapy regimens. The data presented suggests that altered expression of the cMYC targets, as described, may be a more appropriate surrogate marker for rearrangement of cMYC, and it is likely that this approach will more accurately determine which patients will benefit from high dose therapy.