Expression profile of Bcl-2 family proteins in newly diagnosed multiple myeloma patients

Abstract

Antiapoptotic Bcl-2 family proteins are involved in myeloma cell survival. To date, their expression in multiple myeloma (MM) patients has mostly been analyzed at the RNA level. In the present study, we quantified for the first time the protein expression of the Bcl2-family members using a capillary electrophoresis immunoassay in 120 newly diagnosed MM patients, aged ≤65 years, treated in the context of the PETHEMA/GEM2012 study. We found that the pattern of expression of Bcl-2 family proteins was highly heterogeneous among patients. Although cases with t(11;14) had significantly higher levels of Bcl-2/Bcl-xL and Bcl-2+Bim+Bax/Bcl-xL ratios than those without t(11;14), the presence of this translocation was not synonymous with such high levels of expression. Conversely, some patients with other genetic alterations also showed higher levels of those ratios. Survival analysis revealed that the high expression of Bad and Puma proteins was associated with significantly longer overall survival (p = 0.001 and p < 0.001, respectively). Bcl-2 protein ratios predicting sensitivity to venetoclax in vitro were also able to distinguish patients with shorter time to progression after triplet-based induction therapy and ASCT. This is the first study to assess the expression of the most important Bcl-2 family proteins by a quantitative method in a large set of MM patients according to their cytogenetic abnormalities. We shed light on the impact of these proteins on MM prognosis, which could help to consider the levels of proteins involved in apoptosis in the development of new therapeutic strategies.

Figure 1

Bcl‐2 family protein expression in MM samples. (A) The expression of each protein was assessed by CNIA and normalized with respect to Gapdh expression in each case. (B) Percentage of patients with the presence of each protein. The antiapoptotic proteins (Mcl‐1, Bcl‐2, and Bcl‐xL) are represented in red, while the proapoptotic ones (Bak, Bax, Bad, Puma, and Bim) are shown in green. Only the patients with protein expression are shown.

Figure 2

Association between proteins and cytogenetic abnormalities. (A) Expression level of Bak‐1 and Bcl‐xL according to the presence of t(14;16) and t(11;14), respectively. (B) Expression level of the Bcl‐2/Mcl‐1, Bcl‐2/Bcl‐xL, and Bcl‐2+Bim+Bax/Bcl‐xL ratios, according to the presence or absence of t(11;14). The statistically significant differences between groups were determined by the Mann–Whitney U test. *p < 0.05; **p < 0.01: ns, non‐statistically significant.

Figure 3

Distribution of the Bcl‐2/Mcl‐1, Bcl‐2/Bcl‐xL, and Bcl‐2+Bim+Bax/Bcl‐xL protein expression ratios according to the cytogenetic abnormalities del(17p), t(4;14), t(11;14), 1q gain, TP53 mutation, del(1p), t(14;16). The first and third quartiles of the protein expression ratios in the group of patients with t(11;14) are indicated with a blue dashed line. Patients with more than one alteration are represented more than once.

Figure 4

Antimyeloma activity of venetoclax in in vitro studies on human multiple myeloma cell lines (HMCLs). The HMCLs were treated with increasing concentrations of venetoclax for 24 and 48 h. Cell viability was assessed by 3‐(4,5‐dimethylthiazol‐2‐yl)−2,5‐diphenyltetrazolium bromide (MTT) assay. The means ± standard deviation of three independent experiments are represented.

Figure 5

Association between Bcl‐2 protein expression analyzed at diagnosis and the response at three different time points in the treatment scheme (GEM2012 protocol). The response to treatment was assessed by IMWG response criteria (A) and minimal residual disease (MRD) by flow cytometry (B), at three points: after induction (in red), after autologous stem cell transplantation (ASCT) (in blue), and after consolidation (in green). The statistically significant differences between groups were determined by the Mann–Whitney U test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

Kaplan–Meier curves for Bad, Puma, Bim, and Bcl‐2. Probability of overall survival (OS) (A) and survival without progression (time to progression [TTP]) (B) of MM patients by protein expression. The log‐rank test p values are shown.

Figure 7

Multivariable analysis of OS and TTP. Forest plot of multivariable Cox proportional hazards regression models accounting for each potential risk factor associated with OS and TTP of MM: age, ISS III versus I/II, LDH high level, plasmacytoma, FISH risk (high cytogenetic risk, including del17p, t(4;14) and/or t(14;16), vs. standard risk), and the expression level of the studied proteins. For each factor, the hazard ratios and their 95% confidence intervals are shown. AIC, Akaike information criterion; FISH: fluorescence in situ hybridization; ISS, International Staging System; LDH, lactate dehydrogenase; OS, overall survival; TTP, time to progression.