Comportamento delle mutazioni driver e non driver nei pazienti con Mielofibrosi in trattamento con Ruxolitinib

Background: Introduction of ruxolitinib changed the outcome of patients with myelofibrosis (MF), offering longer survivals. Nevertheless, 50% of patients loss response; in some cases, this phenomenon has been ascribed to driver and non-driver mutations, but with conflicting results: Patel et al. (Blood 2015) reported that having >3 mutations well correlated with shorter time to discontinuation and overall survival, whereas in the COMFORT-II study the MF-associated mutations did not correlate with response, survival or discontinuation probability (Guglielmelli, Blood 2014). Aims: in order to investigate if ruxolitinib could play any role in changing the mutational landscape in MF, we assessed the 3 driver and 8 non-driver mutations in 36 MF patients; all were assessed a diagnosis, 19 also after 12 months of ruxolitinib, and other 4 after hydroxyurea. Methods: In addition to assessment of the driver mutations (JAK2, CALR, MPL), a PCR plate with pre-spotted primers able to detect 8 non-driver mutations was designed (Custom qBiomarker Somatic Mutation PCR Array® - Qiagen, Italy). ASXL1, EZH2, DNMT3A, IDH1, IDH2, SRSF2, TET2, TP53, for total 38 hot-spot sites, were assessed. Results: JAK2 was mutated in 70% of cases, CALR in 20%, whereas 10% of cases were triple-negative. The median OS was significantly longer for primary MF (160 months) vs post-ET (80 months) or post-PV MF (35 months)(p=0.03), and for CALR- vs JAK2-mutated patients. At the last follow-up, 4 patients (11%) progressed to AML, and 12 (33%) died. The non-driver mutations were found at diagnosis in 33% of cases receiving ruxolitinib and in one/4 patients treated with hydroxyurea. Considering both driver and non- driver mutations, 24 cases (67%) were mutated, with 16 cases carrying one, and 10 two mutations. The most frequently detected mutations belonged to the methylation pathway (DNMT3A, IDH, TET2 = 75%), followed by TP53 (17%), SRSF2 (8%), ASXL1 (8%), and EZH2 (8%). During treatment, JAK2 allele burden remained stable, whereas non-driver mutations changed in 13 cases: 9 acquired a new mutation (DNMT3A in 5, IDH2 in one, and TP53 in another one) while other 4 lost mutation. None of the CALR-mutated cases carried non-driver mutations. In the 4 cases treated with hydroxyurea, one acquired the TP53 and another one the DNMT3A mutation. On the other hand, 4 cases in the group of ruxolitinib, and none in the group of hydroxyurea lost mutations present at diagnosis (TP53, IDH2, ASXL1, DNMT3A). Presence/absence of non-driver mutations, their number (>1), the molecular subgroup (methylation, splicing, chromatin) did not significantly condition OS. Conclusions: In this work, even if on a small series of patients, we showed that during ruxolitinib about the half of cases developed non-driver mutations, a percentage overlapping to that observed in cases receiving hydroxyurea. Interestingly, ruxolitinib and not hydroxyurea allowed disappearance of mutations in one third of cases.