Distinct genetic changes reveal evolutionary history and heterogeneous molecular grade of DLBCL with MYC/BCL2 double-hit

Abstract

Using a Burkitt lymphoma-like gene expression signature, we recently defined a high-risk molecular high-grade (MHG) group mainly within germinal centre B-cell like diffuse large B-cell lymphomas (GCB-DLBCL), which was enriched for MYC/BCL2 double-hit (MYC/BCL2-DH). The genetic basis underlying MHG-DLBCL and their aggressive clinical behaviour remain unknown. We investigated 697 cases of DLBCL, particularly those with MYC/BCL2-DH (n = 62) by targeted sequencing and gene expression profiling. We showed that DLBCL with MYC/BCL2-DH, and those with BCL2 translocation, harbour the characteristic mutation signatures that are associated with follicular lymphoma and its high-grade transformation. We identified frequent MYC hotspot mutations that affect the phosphorylation site (T58) and its adjacent amino acids, which are important for MYC protein degradation. These MYC mutations were seen in a subset of cases with MYC translocation, but predominantly in those of MHG. The mutations were more frequent in double-hit lymphomas with IG as the MYC translocation partner, and were associated with higher MYC protein expression and poor patient survival. DLBCL with MYC/BCL2-DH and those with BCL2 translocation alone are most likely derived from follicular lymphoma or its precursor lesion, and acquisition of MYC pathogenic mutations may augment MYC function, resulting in aggressive clinical behaviour.

Fig. 1. Summary of cases of DLBCL included in the study.

A total of 697 cases were studied, including 400 from the REMoDL-B trial and 297 cases from population-based cohort, mainly from the Haematological Malignancy Research Network (HMRN). Laboratory data on chromosome translocations and molecular subtypes by gene expression profiling are indicated.

Fig. 2. DLBCL with BCL2 translocation harbour the cardinal mutation signature of follicular lymphoma.

a Heatmap illustration of mutation distribution according to chromosome translocation status; Where data available, evidence of previous or concurrent follicular lymphoma is indicated. b DLBCL with BCL2 translocation, particularly those with MYC/BCL2-DH, harbour the cardinal mutation signature of FL, and also the mutation profile associated with its high-grade transformation [–41]. Representative mutation data in FL and transformed FL are from the study by Kridel et al. [41], with EZH2 mutation considered as the core changes associated with FL [–41]. c Comparison of BCL2 mutation profile between BCL2 translocation positive DLBCL in the present study and FL in the study by Huet et al. [42]. d DLBCL with BCL2 translocation often have a previous or concurrent follicular lymphoma. MYC/BCL2-DH MYC/BCL2 double-hit, TH MYC/BCL2/BCL6 triple-hit, SH single-hit, tr +ve/−ve translocation positive/negative, FL follicular lymphoma.

Fig. 3

Molecular subtype of DLBCL according to translocation status. MHG molecular high-grade, GCB germinal centre B-cell like, ABC activated B-cell like, UNC unclassified.

Fig. 4. Mutation profile of MHG in comparison with the other molecular subtypes.

a Comparison of mutation profile among MHG, GCB and ABC subtypes. The panel includes only the genes (n = 57) that had a mutation frequency ≥5% in at least one molecular subtype to make the figure legible. b Mutation comparison between MHG and GCB within the MYC/BCL2-DH(TH) group. Only genes showing a significant or apparent difference are included in the figure panel, with BCL2 mutation included as a reference. Fisherʼs exact test was used to analyse the difference of mutation frequency between various groups with statistical significance indicated.

Fig. 5. MYC pathogenic mutations and their relationship to molecular subtype and genetic changes in DLBCL.

a Distribution of MYC variants with hotspot mutations clustered at the phosphorylation site (T58) and its neighbouring amino acids that are important for MYC degradation. *One case shows an in-frame deletion of codons 56–60 and all other mutations are missense changes. The codons 57, 58 and 59 hotspot mutations and the in-frame deletion are likely pathogenic and selected during lymphoma development. MYC variants are annotated according to transcript ENST00000377970.6 in keeping with the literature. b MYC hotspot mutations in codons 57, 58 and 59 are seen in a subset of cases with MYC translocation, more frequent in those with MYC/BCL2-DH, but are significantly enriched in MHG-DLBCL. c MYC hotspot mutations are more commonly seen in MYC/BCL2-DH DLBCL with IG gene as the MYC translocation partner, with a considerable overlap with MHG phenotype. d DLBCL with MYC mutation in codons 57–59 show a significantly higher MYC protein expression than those with MYC translocation, but lacking these pathogenic mutations. The MYC protein expression was investigated by immunohistochemistry, quantified using IHC Profiler Image-J software and presented as percentage of positive nuclear staining in lymphoma cells. Unpaired t-test was used to compare the two groups. MHG molecular high-grade, GCB germinal centre B-cell like, ABC activated B-cell like UNC, unclassified.

Fig. 6. Prognostic value of MYC codons 57–59 mutations in DLBCL.

a Differential impact on survival between MYC mutations in codons 57–59, and others. b MHG-DLBCL with MYC mutations in codons 57–59 show the worst overall survival in comparison with GCB-DLBCL. c Single variable Cox proportional hazards regression analysis of progression-free survival in GCB-DLBCL. *In multivariable analysis adjusting for MHG and MYC/BCL2-DH, MYC pathogenic mutations in codons 57–59 retain statistical significance in the REMoDL-B cohort, albeit not in the HMRN cohort.