Concurrent BCL2 and MYC Protein Expression by Immunohistochemistry Determines Clinical Outcome In DLBCL Patients Treated with R-CHOP

Concurrent translocations in MYC and BCL2 determined by fluorescence in-situ hybridization (FISH), have been associated with a poor outcome in diffuse large B cell lymphoma (DLBCL) patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). However, unlike immunohistochemistry (IHC), FISH is expensive and is not routinely available in all clinical laboratories. The aim of this study was to determine if MYC protein expression or expression of the proliferation marker Ki-67 by IHC could be used to identify samples that harbour MYC translocations.

DLBCL samples, diagnosed by an expert panel of hematopathologists according to the WHO criteria of 2008 and derived from 167 patients treated with R-CHOP, have been subjected to gene expression profiling (GEP) and FISH using commercial break-apart probes for BCL2 and MYC. MYC mRNA expression was determined using log₂ normalized expression values of probe set 202431_s_at. The presence of MYC, BCL2 and Ki-67 protein expression was determined by IHC using commercially available antibodies (clone Y69 (Epitomics), clone 124 (Dako) and clone Ki-67 (Dako) respectively). MYC protein is not normally expressed in germinal centers (tonsils, negative control), thus the % of tumour cells staining for MYC was noted in each case. Thresholds for BCL2 protein (50%) and MYC mRNA (>9.4) were determined using the statistical software X-tile which determines the optimal threshold based on its association with clinical outcome. For MYC protein expression, no threshold was significant by X-tile thus a 40% threshold was used based on the bimodal distribution of the data, with a through occurring at 40%. Protein expression for MYC and Ki-67 was correlated to the presence of MYC translocations, MYC mRNA expression and outcome including overall survival (OS) and progression free survival (PFS).

Over-expression of MYC was present in 56/167 (34%) of DLBCL samples (18/167 translocations, 19/167 high mRNA and 47/167 high protein expression) and was not specific to the germinal center B cell (GCB) or activated B cell (ABC) molecular subtype. MYC protein expression (in ≥40% of cells) captured 13/18 MYC translocations and in 4/5 remaining cases, MYC staining was present in 20–39% of cells. MYC protein expression, alone, was not associated with OS but the presence of a MYC translocation was associated with an inferior OS in the ABC subtype only. BCL2 protein expression was also associated with an inferior OS in this cohort (p=0.002). Concurrent expression of MYC and BCL2 protein by IHC was associated with a markedly inferior OS compared to MYC protein- or MYC protein+/BCL2 protein- (median OS of 2 years, 7 years and > 7 years respectively, p<0.001). In a Cox-multivariate model, the co-expression of MYC and BCL2 proteins by IHC maintained prognostic significance independent of the IPI (OS p=0.007, PFS p=0.008). In contrast, Ki-67 staining (>90%) identified only 5/18 cases with MYC translocations and was not associated with outcome alone or in combination with BCL2.

MYC deregulation in DLBCL is more common than previously reported (34%) and can occur in the absence of a MYC translocation. MYC and BCL2 protein expression could be easily determined by routine IHC in most clinical laboratories and should be prospectively tested as potential predictive biomarkers in DLBCL patients treated with R-CHOP.

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