Spontaneous Apoptosis and Proliferation Predict Disease Progression within ZAP-70 Negative B-Cell Chronic Lymphocytic Leukemia (B-CLL).
Authors: Del Poeta Giovanni , Del Principe Maria Ilaria , Niscola Pasquale , Maurillo Luca , Venditti Adriano , Buccisano Francesco , Consalvo Maria Irno , Mazzone Carla , Panetta Paola , Degan Massimo , Pasqua Irene , Bruno Antonio , Gattei Valter , Coco Francesco Lo , Amadori Sergio 
Published: April 2020

Abstract
Inhibition of spontaneous apoptosis and upregulation of the anti-apoptotic protein bcl-2 define clinical prognosis in B-CLL. However, increasing evidence exists that disease progression (DP) relies upon a proliferating pool of cells in lymph nodes and bone marrow which might feed the accumulating pool in the blood. Moreover, unmutated immunoglobulin VH genes predict for a rapid DP and an inferior overall survival. Unmutated B-CLL cells express ZAP-70 protein kinase associated with an early DP risk. The availability both of rapamycin or proteasome inhibitors effective against proliferating cells and bcl-2 targeting drugs incited us to evaluate the impact of proliferation and apoptosis pathways on B-CLL prognosis. The primary aims of our study were: 1) to determine progression-free survival (PFS) upon apoptosis/proliferation and ZAP-70 expression; 2) whether apoptosis/proliferation could predict varied outcome within ZAP-70 subgroups; and finally 3) whether ZAP-70 and apoptosis/proliferation were independent prognostic factors. Therefore, we investigated 249 pts, median age 64 years, almost all belonging to the intermediate Rai stage. ZAP-70 was quantified by a multicolor flow cytometric method. Bcl-2 (Bcl-2B/T) was determined dividing mean fluorescence intensity (MFI) of CD19+B-CLL cells by MFI of T-cells. CD71 was used as a measure of proliferation in percentage. Combining Bcl-2B/T with CD71 (Bcl2CD71) we enucleated three subgroups: 1) Bcl2CD71- [low proliferation (CD71&lt;8%) and high apoptosis (Bcl-2B/T&lt;1.6)]; 2) Bcl2CD71+ [high proliferation (CD71&gt;8%) and low apoptosis (Bcl-2B/T&gt;1.6)]; and 3) Bcl2CD71+/− [low proliferation/low apoptosis or high proliferation/high apoptosis]. ZAP-70+ B-CLL pts were 87/249 (35%). We found significant associations either between lower ZAP-70 and lower Bcl-2B/T (p=0.0008) or lower ZAP-70 and Bcl2CD71- (p=0.0007), showing that low ZAP-70 levels were characterized by high apoptosis and low proliferation. With regard to clinical outcome, a significant shorter PFS was observed in ZAP-70+ pts (6% vs 58% at 12 years; p&lt;0.00001) and in Bcl2CD71+ pts (10% vs 56% at 12 years; p&lt;0.00001). The Bcl2CD71+/− subgroup showed an intermediate outcome (30% at 12 years). To further explore the prognostic impact of Bcl2CD71 index, we investigated its expression within ZAP-70+ (87) and ZAP-70 negative (162) pts. As a matter of fact, Bcl2CD71 index was not able to identify different prognosic subsets within ZAP-70+ pts, because all these cases presented a similar short PFS. On the contrary, Bcl2CD71 clearly identified two subsets at different PFS within the ZAP-70 negative subgroup (73% for Bcl2CD71- pts vs 29% for Bcl2CD71+ pts at 12 years, p=0.00007). In multivariate analysis of PFS, ZAP-70 resulted to be the strongest indipendent prognostic factor (p=0.00002). However, the apoptotic/proliferative index Bcl2CD71 was very useful to identify our pts at different PFS within the ZAP-70 negative subgroup. In conclusion, ZAP-70 negative B-CLL represents a large and clinically heterogeneous population with a variable DP and the determination of the amount of apoptosis combined with the proliferation, could allow us both to distinguish early progressive pts and to take timely therapeutic decisions.