The Prognostic Value of MYC, BCL2 and BCL6 Overexpression Evaluated By Immunohistochemistry (IHC) in De-Novo Diffuse Large B Cell Lymphoma (DLBCL) Treated with Rituximab-CHOP
Authors: Botto Barbara , Novero Domenico , Chiappella Annalisa , Novo Mattia , Castellino Alessia , Ciochetto Chiara , Cametti Giovanni , Evangelista Andrea , Freilone Roberto , Ghione Paola , Ladetto Marco , Riccomagno Paola , Vittone Federico , Ciccone Giovannino , Vitolo Umberto 
Published: November 2019

Abstract
Introduction : MYC, BCL2 and BCL6 overexpression, assessed by IHC, with the latter conferring a better prognosis, have been reported to be a prognostic factor in DLBCL, but data are not consistent and sometimes contradictory.
The aim of the present study was to assess the prognostic impact of overexpression of MYC, BCL2, and BCL6 in a retrospective cohort of de-novo DLBCL, selected for an high proliferation index (MIB1 ≥70%), treated consecutively with R-CHOP regimen.
Methods: Patients with de-novo DLBCL diagnosed between January 2010 and December 2013 were included into the study. Inclusion criteria were: high proliferation index MIB1 ≥ 70% and a full course of R-CHOP regimen. Paraffin-embedded tumor samples were collected and investigated using immunohistochemistry (IHC) for MYC, BCL2 and BCL6. Fluorescence in situ hybridization (FISH) is ongoing. MYC/BCL2+ or MYC/BCL6+ double expression cases were identified if they had rearrangements of MYC and BCL2 or BCL6. MYC immunochemistry was done on TMA sections using the antibody clone Y69. BCL2 and BCL6 staining had been evaluated previously at diagnosis. Tumor cells were defined positive for MYC and BCL2 or BCL6 protein expression by immunostaining if >40%, >40% and >25% of cells showed positive expression, respectively. Progression free survival curves (PFS) were estimated using the Kaplan-Meier method and compared between groups using the log-rank test and Cox models.
Results : One hundred and sixty seven patients are evaluable for clinical characteristics and 69/167 had paraffin embedded tumor samples available for immunohistochemistry at the time of present analysis. Clinical characteristics of the 69 cases were: median age 66 years (IQR 57;73), 45 (65%) male, 47 (68%) stage III-IV, 35 (54%) with elevated LDH levels and 46 (67%) at International Prognostic Index (IPI) high intermediate or high risk. Overexpression of MYC was detected in 28 cases (41%), 50 (72%) and 38 (55%) showed BCL2 and BCL6 overexpression respectively. Nineteen (28%) cases showed MYC/BCL2+ and 17 (25%) MYC/BCL6+ double expression. With a median follow up of 26 months, the median 2-years PFS was 59%. Overexpression of MYC and BCL2 proteins and low expression of BCL6 were associated with an inferior 2-years PFS in univariate analysis: MYC- vs MYC+ 64% vs 55%; BCL2- vs BCL2+ 71% vs 56%; BCL6+ vs BCL6- 61% vs 54%. In a Cox multivariate regression model adjusted for IPI and age, MYC overexpression, BCL2 positivity and BCL6 negativity showed prognostic relevance as significant independent indicators with different risk (Hazard ratio 2.53 for MYC+, 2.08 for BCL2+ and 1.62 for BCL6-). Established that the three variable contributed with different risk in the multivariate analysis, an IHC sum additive score of 0-5 was calculated proportionally to the coefficient estimated (coefficient [Log hazard ratio] 0.92 for MYC+, 0.73 for BCL2+ and 0.48 for BCL6-), assigning an individual risk of 2 points for MYC or BCL2 positivity and 1 point for BCL6 negativity. Two years-PFS was significantly different between all separate groups (Hazard ratio for unit increase 1.57 95% CI 1.11-2.22, p=0.01). After pooling scores 0-1 (with or without BCL6), 2 (presence of MYC or BCL2 only), and 3-4-5 (MYC+/BCL6-, BCL2+/BCL6-, MYC+/BCL2+, MYC+/BCL2+/BCL6-) 2-yrs PFS rates were different across the three groups: 100% vs 64% vs 50% (log rank p= 0.04) (figure 1).
Conclusion: Our data showed, with the limits of a small sample size, that MYC overexpression alone or with high expression of BCL2 and/or low expression of BCL6 correlates with a worse prognosis independently by IPI score in a cohort of DLBCL selected for high proliferation index and treated with R-CHOP. Assessment of MYC, BCL2 and BCL6 expression by IHC represents a rapid, inexpensive, and reproducible technique. These results need to be confirmed in our complete series of 167 patients (analysis ongoing) and validated prospectively in a larger cohort, using standardized staining and scoring methodologies. Thus, MYC and BCL2 represent relevant biomarkers that should be tested in future clinical trials using novel effective and targeted agents in order to improve the prognosis of DLBCL.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.