Deciphering Discordance between MYC mRNA and MYC IHC in DLBCL: The Role of MYC Exon 2 Mutations and N11S Polymorphism

Introduction

Diffuse large B-cell lymphoma (DLBCL) is a molecularly diverse group of lymphoid cancers that display heterogeneous clinical behaviour and treatment outcomes. Development of an antibody (Y69) that can detect MYC protein using immunohistochemistry (IHC) has facilitated the evaluation of the impact of MYC protein expression in DLBCL. Various thresholds have been used to define positivity, with the goal of either identifying cases that harbor rearrangements of MYC or defining high-risk patient populations. The prognostic impact of MYC expression alone remains controversial; however, dual protein expression of MYC and BCL2 has consistently defined a group with an inferior outcome.

Rearrangement of the MYC locus is observed in ~12 % of DLBCL and has been associated with poor prognosis, particularly in the context of concurrent rearrangement of BCL2 . This has contributed to the establishment of a new entity in the 2016 revision of the WHO: high grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements, which includes cases with DLBCL morphology. At a threshold of ≥40%, 60-80% of tumors with a MYC rearrangement are reported as positive for MYC protein expression using the Y69 antibody. Uniform implementation of the WHO classification requires universal FISH testing for MYC rearrangement in tumors with DLBCL morphology. It has been suggested that resources could be saved if MYC IHC was used to screen cases for FISH testing and that MYC rearrangement without detectable MYC protein expression may not confer the same poor prognosis. Herein, we examine DLBCL tumors that harbor a MYC rearrangement and express high levels of MYC mRNA, but are negative by MYC IHC, to examine potential mechanisms for this disparity.

Methods

The following analyses were performed on diagnostic biopsies from 322 patients with de novo DLBCL drawn from a population-based registry: FISH for MYC rearrangement (VYSIS and DAKO breakapart probes), targeted sequencing of MYC , RNAseq, and MYC IHC using the Y69 antibody (positivity defined as staining in ≥40% of tumor cells). Western blots using the MYC Y69 (Epitomics) and 9E10 (Abcam) antibodies were performed on HEK cells that had been transfected with vectors expressing wild type (WT) or variant MYC.

Results

MYC IHC was performed in 304 cases: 127 (42%) were positive, including 34/45 (76%) of the MYC rearranged tumors. Interestingly, the 3 IHC negative MYC rearranged tumors with the highest mRNA expression (>80^th percentile) all harbored mutations in exon 2. The Y69 antibody is known to bind within the N-terminal 100 amino acids of MYC, encoded for by a region within exon 2. Therefore, it was hypothesized that these mutations could interfere with Y69 binding, conferring false negative protein staining. Interestingly, two of these cases carried the germline N11S variant, while the third case had mutations affecting nearby residues. We observed N11S in 19 (6%) tumors in our cohort, within which 17/18 (94%) with evaluable IHC were negative for MYC. Of note, 7 of these MYC IHC negative cases had MYC mRNA expression levels above the cohort median.

Xu-Monette et al ( Clin Cancer Res 2016) reported the functional implications of the N11S polymorphism in a fibroblast cell line giving rise to reduced MYC protein expression (Y69 antibody), but having equivalent impact on proliferation and induction of apoptosis as the wild type. Intriguingly, the N11S fibroblasts gave rise to slower growing tumors in a xenograft model. To explore whether the N11S polymorphism gives rise to lower protein expression, as suggested by Xu-Monette et al , or alternatively disrupts the Y69 target epitope, western blots were performed on HEK cells transfected with vectors expressing WT or N11S MYC. Weaker expression in the N11S transfected cells was observed with the Y69 antibody while equivalent expression was seen with the 9E10 antibody, which binds to the C-terminal region of MYC. This is consistent with the hypothesis that epitope disruption mediates the apparent decreased expression.

Conclusions

Mutations in exon 2 and the N11S polymorphism lead to disruption of the relationship between MYC mRNA levels and MYC IHC results. This raises potential issues for using MYC IHC to screen DLBCL cases for FISH testing as well as determining whether detected rearrangements lead to MYC protein expression. The functional implications of exon 2 mutations and the N11S polymorphism require further study in the B-cell context.