High Grade B Cell Lymphoma with MYC and BCL2 and/or BCL6 Rearrangements Depict a High Complexity on the Cytogenetic, but Not on the Molecular Genetic Level and Show MYC Mutations As Prognostic Marker

Background: In the revised WHO classification of 2016, cases of large B cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements were categorized as "HGBL (high grade B cell lymphoma, with MYC and BCL2 and/or BCL6 rearrangements)", except for cases that fulfill the criteria for a follicular or lymphoblastic lymphoma. A detailed molecular analysis of these cases and comparison of accompanying aberrations has not been available to date.

Aims: Evaluation of associated genetic alterations and prognosis in HGBL cases with rearrangement of (i) MYC + BCL2 , (ii) MYC + BCL6 or (iii) MYC + BCL2+BCL6.

Patient cohorts and methods: The total cohort comprised 70 cases with MYC and BCL2 and/or BCL6 rearrangements (39 male, 31 female). Cases were selected by detection of the rearrangements by FISH (fluorescence in situ hybridization), cases diagnosed as CLL, follicular or lymphoblastic lymphoma were excluded. Median age was 67 years (range: 44-87 years). All patients were investigated using chromosome banding analysis (CBA) and FISH using probes for MYC , IGH-BCL2 and BCL6 . Additionally, mutation analyses of ATM , BCL2, BIRC3, BRAF, BTK, CBL, CSF3R, CSNK1A1, CXCR4, EGR2, ETNK1, FBXW7, FLT3, ID3, IDH1, IDH2, KIT, KLF2, KRAS, MAP2K1, MYC, MYD88, NFKBIE, NOTCH2, PHF6, PLCG2, POT1, PTPN11, SAMHD1, SETBP1, SF3B1, SRSF2, STAT3, STAT5B, TET2, TP53, U2AF1, UBR5, WT1, XPO1 and ZRSR2 was performed. Variants of unknown significance were excluded from statistical analysis.

Results: We identified 33/70 cases with rearrangement of MYC + BCL2 , 10/70 cases with MYC + BCL6 and 27/70 cases with MYC + BCL2 + BCL6 (47%, 14% and 39%, respectively). In the total cohort, CBA revealed a complex karyotype (>3 abnormalities) in 59/70 (84%) of patients, 10/70 (14%) harbored an aberrant but non-complex karyotype, whereas in 2/70 (3%) of cases, CBA analyses failed due to insufficient cell proliferation. Regarding the subgroups, a complex karyotype was observed in 82%, 80% and 89% of cases, respectively (for MYC + BCL2 , MYC + BCL6 and MYC + BCL2 + BCL6 rearrangement). If only aberrations additional to MYC / BCL2 / BCL6 rearrangements were calculated, >3 aberrations were detected in 64% of cases (67%, 70% and 60%, respectively). The most frequent additional aberrations included 13q deletions, +12, gain of 1q and 6q deletions. Median number of additional cytogenetic aberrations was n=8 (range: 0-36). Despite the high cytogenetic complexity, the frequency of TP53 deletions was relatively low (6% in the total cohort), three of them harboured a concomitant TP53 mutation in the other allele. Mutation analyses of 40 genes revealed a median number of n=1 mutations in the total cohort with no differences between the three subgroups. The range of the number of molecular mutations was 0-3 (0 mutations: 31%; 1 mutation: 44%; 2 mutations: 20%; 3 mutations: 5%). Interestingly, out of the 40 genes analysed, mutations were almost exclusively detected in BCL2 (40%), MYC (21%) and TP53 (21%) in the total cohort. Other recurrent mutations were observed for ID3 and PHF6 only (3%, respectively). Cases with MYC+BCL6 rearrangement showed a higher - although not statistically significant - frequency of MYC mutations compared to the other cases (40% vs. 18%), while BCL2 mutations were rarely detected in this subgroup (10% vs. 45%, p=0.033). Of note, mutations in TP53 were found to show a trend towards a lower frequency in cases with a "triple hit" ( MYC+BCL2+BCL6 ) in comparison to the "double hit" cases ( MYC+BCL2 and MYC+BCL6 ) (11% vs. 28%).

Median overall survival (OS) in the total cohort was 61 months. Cases with MYC + BCL2 showed a shorter OS compared to cases with MYC+BCL2+BCL6 rearrangements, although this was not statistically significant (37 vs.61 months). Unfortunately, OS could not be determined for cases with MYC+BCL6 due to the low number of cases. Mutations in MYC, but not in BCL2 or TP53 , showed a negative impact on OS in the total cohort (16 vs. 61 months, p=0.046).

Conclusion: 1) Cases with rearrangements in MYC , BCL2 and/or BCL6 depict a high complexity on the cytogenetic, but not on the molecular genetic level. 2) Molecular mutations are almost exclusively detected in BCL2 , MYC and TP53. 3) Cases with MYC+BCL6 rearrangements differ from the other subgroups by showing a higher frequency of MYC mutations and a lower frequency of mutations in BCL2 . 4) Mutations in MYC negatively impact on OS. 5) Thus, our data support the current WHO classification and complement it on a molecular level.