Clinical Significance of Apoptosis Is Independent from Proliferation in Acute Myeloid Leukemia (AML).
Authors: Del Poeta Giovanni , Del Principe Maria Ilaria , Venditti Adriano , Maurillo Luca , Buccisano Francesco , Consalvo Maria Irno , Mazzone Carla , Cox Maria Christina , Bruno Antonio , Lo Coco Francesco , Amadori Sergio 
Published: October 2019

Abstract
Multidrug resistance and recurrent disease are key problems in the variable response of AML pts to treatment. Tumor cells in a high proliferative state have a high density of transferrin receptors, as demonstrated in breast cancer (Yang DC, 2001) and in adult T-cell leukemia/lymphoma (Moura IC, 2004). On the other hand, defects in apoptotic pathways such as higher levels of bcl-2 and Mcl-1 were reported in “poor risk” AML pts. Current availability of antisense oligonucleotides targeted both to the transferrin receptor genes and bcl-2 incited us to evaluate the impact of proliferative and/or apoptotic pathways on AML prognosis. Therefore, a large series of 325 pts, affected by de novo AML, except FAB M3, median age 55 years, treated with intensive chemotherapy regimens, were studied. The aims of our research were: 1) to correlate bax/bcl-2 ratio with the proliferation levels, determined by the transferrin receptor (CD71) and 2) to demonstrate that the clinical significance of spontaneous apoptosis is independent from proliferation. CD71, bcl-2 and bax proteins were determined by multicolor flow cytometry. CD71 was evaluated as mean fluorescence intensity (MFI) and bax/bcl-2 ratio was obtained by dividing MFI bax/MFI bcl-2. One hundred-seventy five pts (53.8%) were bax/bcl-2 ratio positive and 204/324 (63%) were CD71 positive, respectively. There was a close correlation between higher CD71 expression and Ki-67 positive staining by flow cytometry (r=0.86), confirming that transferrin receptor overexpression is really linked to increased cellular proliferation in AML. No significant correlation was found between a higher bax/bcl-2 ratio and a lower CD71 MFI (p=0.16), confirming that an apoptosis resistant protein profile may have variable proliferation levels. A significant lower complete remission (CR) rate was found in pts with lower bax/bcl-2 ratio (43% vs 72%, p<0.00001) or higher CD71 (47% vs 74%, p=0.00001). Overall survival (OS) was significantly shorter either in pts with lower bax/bcl-2 ratio (1% vs 15% at 4 years; p=0.00001) or higher CD71 MFI (5% vs 104% at 5 years; p=0.002).
Noteworthy, higher bax/bcl-2 ratio plus lower CD71 MFI identified an AML subset at better prognosis with regard to CR (91% vs 32%; p<0.00001) and OS (31% vs 0% at 2 years; p<0.00001). In order to confirm the independent prognostic value of bax/bcl-2 ratio from proliferation levels, we investigated the CD71 + (203 pts) and the CD71- (121 pts) AML subgroups. As a matter of fact, a lower CR rate was found in patients with lower bax/bcl-2 ratio either within CD71+ (34% vs 61%, p=0.0009) or CD71- (60% vs 90%, p=0.0006) pts. Also a lower bax/bcl-2 ratio was associated with a shorter OS both within CD71+ (2% vs 10% at 4 years, p=0.008) and within CD71- (0% vs 24% at 3.5 years, p=0.00008) pts. The independent prognostic value of bax/bcl-2 ratio was confirmed in multivariate analysis with regard to CR (p=0.00007) and OS (p=0.00003). The lack of significant correlation between bax/bcl-2 ratio and CD71 MFI confirms the independence of apoptosis from proliferation pathways. Furthermore, the capacity of bax/bcl-2 ratio of clearly identifying patients at different prognosis within the CD71+ and CD71- subgroups implies that apoptosis has an intrinsic more relevant clinical significance as compared to proliferation. That has to be taken in account when strategies are to be planned in order to resolve both chemoresistance and relapse in AML.