The Amount of Spontaneous Apoptosis Is An Independent Strong Disease Progression Indicator in B-Cell Chronic Lymphocytic Leukemia (B-CLL)
Authors: Del Poeta Giovanni , Del Principe Maria Ilaria , Buccisano Francesco , Piccioni Daniela , Maurillo Luca , Sarlo Chiara , Consalvo Maria Irno , Bulian Pietro , Zucchetto Antonella , Giannì Laura , Simotti Cristina , de Fabritiis Paolo , Venditti Adriano , Gattei Valter , Amadori Sergio 
Published: October 2019

Abstract
Abstract 1252
Poster Board I-274
Bcl-2 levels has emerged as the most important protein in predicting survival between 11 proteins in CLL cells that are implicated in the control of apoptosis, proliferation and differentiation (Faderl, 2002). In fact, malignant cells are arrested in the G0/early G1 phase of the cell cycle, and inhibition of spontaneous apoptosis with upregulation of the anti-apoptotic protein bcl-2 may define clinical prognosis. The today availability both of bcl-2 antisense oligonucleotides and of novel pro-apoptotic BH3 peptidomimetic prompted us to evaluate the real impact of apoptosis pathways on B-CLL prognosis. The primary aims of our study were: 1) to determine progression-free survival (PFS) and overall survival (OS) upon bax/bcl-2 ratio, 2) whether bax/bcl-2 ratio and ZAP-70 show additive prognostic impact and finally 3) whether bax/bcl-2 is an independent prognostic factor. Therefore we investigated 356 patients (pts), median age 65 years (range 37-89), 164 males and 192 females. With regard to modified Rai stages, 115 pts had a low stage, 229 an intermediate stage and 12 a high stage. Bax/bcl-2 ratio was determined by flow cytometry, dividing mean fluorescence intensity (MFI) of bax by MFI of bcl-2 on CD19+CD5+ B-CLL cells. We obtained the bax/bcl-2 ratio and the threshold was set at the median value >1.45 (range 0.27-13.6). ZAP-70 was quantified by multicolor flow cytometry and the cut-off was fixed at >20%. Two hundred-three pts were bax/bcl-2 ratio positive (203/356; 57%). Higher bax/bcl-2 ratio was significantly associated with low Rai stage (80/115; P=0.003), lymphocyte doubling time >12 months (183/299; P=0.0003), beta-2 microglobulin (B-2M) <2.2 mg/dl (129/200; P=0.001) and soluble CD23 (sCD23) <70 U/ml (141/228; P=0.005). Moreover, there were significant correlations between higher bax/bcl-2 ratio and IgVH gene mutated status (216 cases, 96/154; P=0.015) or low risk (normal or 13q-) FISH cytogenetics (255 cases, 109/187; P=0.011). Noteworthy, a very strict association was found between higher bax/bcl-2 ratio and lower ZAP-70 (147/220; P<0.00001), suggesting that low ZAP-70 expression is characterized by high apoptosis levels. With regard to clinical outcome, significant shorter PFS and OS were observed in pts with lower bax/bcl-2 ratio (10% vs 60% at 14 years; P<0.00001 and 51% vs 74% at 16 years; P=0.005, respectively) as well as in ZAP-70+ pts (5% vs 57% at 12 years; P<0.00001 and 30% vs 85% at 16 years, respectively). To further explore the prognostic impact of bax/bcl-2 ratio, we investigated its expression associated with ZAP-70 protein. As a matter of fact, higher bax/bcl-2 ratio plus ZAP-70 <20% identified the pts subset with the longest PFS (70% vs 2% at 12 years; P <0.00001, Figure) and OS (92% vs 33%; P<0.00001). The discordant pts presented an intermediate outcome (Figure). In multivariate analysis of PFS, in which cytogenetics, IgVH status, ZAP-70, CD38, bax/bcl-2, sCD23 entered, bax/bcl-2 (P=0.02), cytogenetics (P=0.02) and ZAP-70 (P=0.04), resulted to be independent prognostic factors. In conclusion, our apoptotic index (bax/bcl-2 ratio), performed by flow cytometry, was very useful to identify pts at different progression rate and since the ZAP-70 negative subset represents a large and heterogeneous B-CLL population with a variable progression, other biological factors, such as the amount of apoptosis, have to be added in order both to identify early and treat timely progressive pts.

Disclosures
No relevant conflicts of interest to declare.