Association of Bax and Bcl-2 polymorphisms in Aggressiveness and Prognosis of Diffuse Large B-Cell Lymphoma

Background: Apoptosis, with participation of the pro-apoptotic BAX and the anti-apoptotic BCL-2 proteins, plays a key role in outcome of patients with diffuse large B cell lymphoma (DLBCL). The ability to induce apoptosis is variable in humans, since several proteins enrolled in the process are encoded by polymorphic genes. The G wild allele of the BAX G(-248)A and the variant A allele of the BCL2 C(-717)A single nucleotide polymorphisms (SNPs) are related to lower transcriptional activity and higher BCL-2 protein expression, respectively, compared to the A variant and the C wild alleles. Since the roles of these SNPs in clinical aspects and prognosis of DLBCL are still unknown, these were the aims of the present study.

Methods: Our analysis included 187 consecutive DLBCL patients at diagnosis seen at the University Hospital from December 2007 to June 2014. Genomic DNA from peripheral blood was analyzed by polymerase chain reaction followed by enzymatic digestion for discrimination of distinct genotypes of each SNP. Chi-Square test, Fisher's Exact test, and multivariate analysis, using the logistic regression model, served to assess associations between genotypes and clinical aspects. Kaplan-Meier analysis was used to evaluate the effect of clinical features and genotypes on the cumulative probability of overall survival (OS). OS was calculated from date of diagnosis until the date of death or last follow-up. We used the Cox proportional hazards regression model to evaluate the effect of B symptoms, Ki-67 labeling index, bone marrow involvement (BMI), Ann Arbor staging system, International Prognostic Index (IPI), BAX G(-248)A and BCL2 C(-717)A genotypes on OS, and the results of analysis were presented as hazard ratios (HRs) with their corresponding 95% confidence intervals (CIs). First, we examined these associations using univariate Cox proportional hazards regression. In a second step, all variables with P< 0.10 were included in a multivariate Cox regression. All reported P values were two-sided, and P< 0.05 was considered to indicate statistical significance.

Results: Ki-67 labeling index greater than or equal to 80% was more common in patients with extranodal disease than in those without this feature (55.2% vs. 32.2%, P=0.04). An excess of the BAX GG genotype was seen in patients with stage IV tumors compared to those with tumors of I+II+III stages (91.3% vs. 78.6%, P=0.04). The median time of observation of patients was 27 months (range: 1-79). Concerning OS, Kaplan-Meier curves relating to the presence of B symptoms (69.8% vs. 89.9%, P=0.007), BMI (51.6% vs. 80.2%, P=0.002), stage IV (64.2% vs. 82.1% P=0.02), and IPI 4 and 5 (52.3% vs. 78.4%, P=0.001) showed worse outcomes at 24 months of follow up. Moreover, at the same time, patients with BCL2 CA+AA genotype had worse outcome than those with the BCL2 CC genotype (70.62% vs. 88.9%, P=0.03). In univariate Cox analysis, the presence of B symptoms (P=0.01, HR= 3.79, 95% CI: 1.34-10.69), BMI (P=0.003, HR= 2.68, 95% CI: 1.38-5.16), stage IV (P=0.02, HR= 2.05, 95% CI: 1.08-3.89), IPI 4 and 5 (P=0.002, HR= 3.10, 95% CI: 1.52-6.31), and BCL2 CA+AA genotype (P=0.03, HR= 2.77, 95% CI: 1.06-7.22) were predictive of worse outcome. Since Ann Arbor staging system is included in the IPI, and in order to avoid redundant information, we selected the presence of B symptoms, Ki-67 labeling index greater than or equal to 80% (P=0.08, HR= 1.99, 95% CI: 0.91-4.35), BMI, IPI and BCL2 CA+AA genotype for multivariate Cox analysis; only BMI (P=0.01, HR= 2.66, 95% CI: 1.19-5.90), IPI 4 and 5 (P=0.002, HR= 3.57, 95% CI: 1.57-8.10) and BCL2 CA+AA genotype (P=0.03, HR= 3.92, 95% CI: 1.13-13.52) were found to be significant predictors of worse patient outcome.

Conclusions: Our data present, for the first time, preliminary evidence that inherited abnormalities in intrinsic apoptosis pathway, related to the BAX G(-238)A and BCL2 C(-717)A SNPs, influence aggressiveness and outcome of DLBCL patients.