Nucleophosmin Gene Mutations Exhibit High Spontaneous Apoptosis and Favorable Prognosis in Acute Myeloid Leukemia.

Nucleophosmin gene mutations (NPM1-Mt) are the hallmark of a large adult acute myeloid leukemia (AML) subgroup with normal karyotype and interact with p53 and its regulatory molecules (Arf, Hdm2/Mdm2), thus lowering cell proliferation and increasing apoptosis (Falini, 2007). Moreover, AML pts show co-existing NPM1-Mt and internal tandem duplications of FLT3 (FLT3-ITD) which increase potential for cell proliferation. Furthermore, genes and proteins involved in apoptosis, such as bcl-2 and bax, have been demonstrated to be relevant in response to treatment and outcome (Del Poeta, 2003). Therefore, we analysed NPM1-Mt, FLT3-ITD and apoptosis proteins (bcl-2 and bax) in 222 pts, affected by de novo non-M3 AML, median age 60 years, treated with intensive chemotherapy regimens according to GIMEMA-EORTC protocols. The aims of our study were:

1. to correlate NPM1-Mt or FLT3-ITD with bax/bcl-2 ratio levels, as a measure of spontaneous apoptosis;

2. to assess the independent prognostic significance of NPM1-Mt and FLT3-ITD.

Bcl-2 and bax proteins were determined by multicolor flow cytometry and bax/bcl-2 ratio was obtained by dividing mean fluorescence intensity (MFI) of bax/MFI bcl-2. The threshold of positivity was set at the median value >0.35 (range 0.01–9.1). NPM1 mutations and FLT3-ITD were detected by multiplex PCR and capillary gel electrophoresis. One hundred-twenty-one/222 (54.5%) pts were bax/bcl-2 ratio positive, 54/222 (24.3%) were NPM1-Mt and 52/222 (23.4%) presented FLT3-ITD; 17/222 (7.6%) pts carried both FLT3-ITD and NPM1-Mt. There was a strong correlation between higher WBC counts (>100x10⁹/L) and FLT3-ITD (P<0.00001), confirming their high proliferative potential. On the contrary, a higher apoptosis (bax/bcl-2 ratio>0.35) and NPM1-Mt without FLT3-ITD were significantly associated (30/37; P=0.0001), demonstrating that NPM1-Mt alone express high amount of spontaneous apoptosis. Moreover, NPM1-Mt cases were significantly related to FAB M4 or M5 AML (P=0.03). A normal karyotype was found in 37/45 (82%) NPM1-Mt pts (P=0.00001) and almost all NPM1-Mt cases were CD34 negative (47/54; P<0.00001). With regard to clinical outcome, a significant higher complete remission (CR) rate was found in NPM1-Mt/FLT3-ITD negative pts (90%) vs NPM1-Mt/FLT3-ITD+ (35%) or only FLT3-ITD+ cases (47%) [P=0.0002]. Equally, overall survival (OS) was significantly longer in NPM1-Mt/FLT3-ITD negative pts in comparison with NPM1-Mt/FLT3-ITD+ or only FLT3-ITD+ cases (35% vs 0% vs 6% at 2 years; P=0.00007). Furthermore, NPM1-Mt/FLT3-ITD negative subset showed a disease free survival longer than only FLT3-ITD+ cases (44% vs 0% at 1.2 years; P=0.008). Finally, NPM1-Mt/FLT3-ITD negative pts showed a better outcome than the large subgroup negative both for NPM1-Mt and FLT3-ITD, with regard to CR (90% vs 66%) and OS (35% vs 15% at 2 years). In multivariate analysis, bax/bcl-2 ratio (P<0.00001), age (P=0.0003) and FLT3-ITD (P=0.01) were significant for CR, while bax/bcl-2 ratio (P<0.00001), WBC count (P=0.01) and FLT3-ITD (P=0.01) resulted to be independent prognostic factors for OS. In conclusion, we demonstrated that NPM1 mutations exhibit high levels of spontaneous apoptosis, which strenghten, in the absence of FLT3-ITD, their favorable prognosis. On the contrary, FLT3-ITD dominate the myeloid leukemic phenotype conferring a poor outcome to pts with NPM1 mutations.