The Amount of Mitochondrial Apoptosis Exerts Prognostic Impact on Normal Karyotype Acute Myeloid Leukemia.

The unbalance between anti-apoptotic (bcl-2) and pro-apoptotic mitochondrial proteins (bax) represents a critical mechanism explaining the high rate of resistance and treatment failure in acute myeloid leukemia (AML) (Del Poeta, 2003). Moreover, the availability of novel and effective pro-apoptotic compounds such as bortezomib (Attar, 2008) and gemtuzumab (Del Poeta, 2008) moved us to widely investigate the impact of mitochondrial proteins such as bcl-2, bax and 7A6 on AML prognosis. 7A6 antigen has been found to be exposed on the mitochondrial membrane during the early stages of apoptosis and is recognized by monoclonal antibody APO2.7. For this purpose, a large series of 420 non M3 AML patients (pts), median age 63 years, treated with intensive chemotherapy regimens, were tested. The aims of our research were:

1. to correlate bax/bcl-2 and APO2.7/bcl-2 ratios, as measures of mitochondrial apoptosis, with other well-known prognostic factors such as age, cytogenetics, FLT3-ITD, P-glycoprotein;

2. to evaluate whether mitochondrial apoptosis was able to dissect normal karyotype AML with regard to prognosis, and finally

3. to confirm that mitochondrial apoptosis is an independent prognostic factor.

Bcl-2, bax and 7A6 proteins were assessed by multicolor flow cytometry and bax/bcl-2 or APO2.7/bcl-2 ratios were obtained by dividing mean fluorescence intensity (MFI) of bax/MFI bcl-2 or MFI of APO2.7/MFI bcl-2. The thresholds of positivity were set at the median values >0.35 and >0.60, respectively. Fifty-nine percent of pts were bax/bcl-2 positive and 50% were APO2.7/bcl-2 positive. There were strict correlations between higher bax/bcl-2 or higher APO2.7/bcl-2 and FAB M2 or M4/M5 AML subgroups (p<0.0001). Bax/bcl-2>0.35 or APO2.7/bcl-2>0.60 and CD34 negativity (p<0.0001) or normal karyotype (p<0.0001 and p=0.003) were closely associated. On the other hand, no significant relationships were found between bax/bcl-2 or APO2.7/bcl-2 and age, white blood cell count, P-glycoprotein or FLT3-ITD. A higher complete remission (CR) rate was found in pts either with higher bax/bcl-2 or higher APO2.7/bcl-2 (68% vs 32% and 60% vs 40%, p<0.0001). Moreover, a shorter time to relapse was observed in pts with bax/bcl-2 <0.35 (3.3 months vs 6 months, p=0.011). Overall survival (OS) and disease-free survival (DFS) were longer in pts with higher bax/bcl-2 (18% vs 0% at 3.5 years, p<0.0001 and 19 vs 0% at 2.7 years, p=0.00007) or higher APO2.7/bcl-2 (14% vs 2% at 4 years, p<0.0001 and 8% vs 0% at 4 years, p=0.02). Furthermore, in order to demonstrate the independent prognostic impact of the mitochondrial proteins, we investigated bax/bcl-2 and APO2.7/bcl-2 within the normal karyotype subgroup (243 pts). As a matter of fact, within this subset, higher bax/bcl-2 or higher APO2.7/bcl-2 were associated with higher CR rate (76% vs 24% and 64% vs 36%, p<0.0001) and longer OS (16% vs 0% at 3.5 years, p=0.00001 and 14% vs 4 at 4 years, p=0.002). The independent prognostic value of bax/bcl-2 and APO2.7/bcl-2 was confirmed in multivariate analysis with regard to CR (p=0.00007 and p=0.0005) and OS (p=0.0009 and p=0.03). Therefore, the significant and independent prognostic role of mitochondrial apoptosis implies that therapeutic strategies for improving outcome in AML should be focused on apoptosis-inducing compounds combined with conventional chemotherapy.