Chronic Exposure to Cytoreductive Treatment Shapes Clonal Evolution in Myeloproliferative Neoplasms

Introduction: Myeloproliferative neoplasms (MPN) are a heterogeneous group of chronic myeloid malignancies resulting from the combination of a driver mutated gene (JAK2, MPL or CALR) and a variety of acquired additional somatic mutations. Although next-generation sequencing (NGS) has identified high molecular risk mutations associated with adverse prognosis (Vannucchi et al., Leukemia, 2013 , Guglielmelli et al., Leukemia, 2014 ), the clonal evolution of these mutations remains poorly described. Chronic exposure to cytoreductive treatment, especially genotoxic drugs such as hydroxyurea (HU), could impact clonal evolution. A previous study suggested that Interferon-α (IFN) could limit the accumulation of cytogenetic abnormalities compared to HU (Mondello et al., Blood, 2018). The objective of our study was to describe the long-term evolution of the mutational landscape in the era of NGS in a large cohort of MPN patients.

Methods: A total of 1538 consecutive patients were diagnosed with MPN according to WHO criteria and followed in our hospital between January 2011 and January 2021. This study included 1039 of them in whom a NGS molecular analysis targeting 36 myeloid genes with a sensitivity of 1% was performed at diagnosis and/or during follow-up. Patients with only one NGS (n=588), AML/MDS transformation at either the first (n=3) or the second NGS (n=2) were excluded from the analysis. Serial NGS data obtained in chronic MPN phase were thus analyzed for 446 patients. Clinical and biological characteristics at time of diagnosis and follow-up were collected from medical charts and electronic medical records. Mutation rates per year were calculated for each gene as the difference in the number of mutations between first and last NGS divided by the time interval (in years) between both NGS.

Results : Median age at MPN diagnosis in our whole cohort was 51 years [IQR 41-60]. Our cohort included 167 (37%), 205 (46%) and 64 (14%) patients with Polycythemia Vera (PV), Essential thrombocythemia (ET) and primary myelofibrosis (MF) respectively. 279 patients (63%) had at least one additional mutation at first NGS, and respectively 27 (6%) and 104 (23%) patients had TP53 and high molecular risk mutations. Median interval between MPN diagnosis and the first NGS was 6.5 years [IQR 1.7-13] while median time between the first and the last NGS was 2.5 years [IQR 1.6-4, range 0.3-14.3]. Overall, 178 patients (39.9%) acquired an additional mutation at last NGS evaluation, most frequently involving TET2, DNMT3A, ASXL1, TP53 and NFE2 genes . To study the impact of chronic MPN therapy on clonal evolution, we focused on patients who electively received HU (n=112) or IFN (n=92) as a monotherapy, or did not receive any cytoreductive treatment (n=119) between the first and the last NGS. The remaining patients received ruxolitinib (n=44), anagrelide (n=10), vercyte (n=7) or polytherapy (n=62). At last follow-up, 74 patients receiving IFN (80.4%) and 65 (58%) treated with HU had a complete hematological response. When combining all additional mutations, the global mutation rate per year did not significantly differ between treatment groups. When analyzing individual genes, TP53 mutation rate was higher in patients treated with HU compared to the patients receiving IFN (p=0.014) or not treated (p=0.008) (Figure). MDS/AML evolution occurred in 4 patients (3.6%) treated with HU, 2 (1.7%) without cytoreductive therapy versus none of the 92 patients treated with IFN (ns). In the whole cohort, MDS/AML evolution was significantly increased in patients harboring TP53 mutations (p= 0.004). In contrast, DNMT3A mutation rate was significantly increased in patients receiving IFN compared to patients treated with HU (p=0.045) (Figure). The latest result is in line with previous observations showing that loss of DNMT3A could confer resistance to IFN in a JAK2-V617F mouse model (Stetka et al., Blood, 2020).

Conclusion: Our results highlight the impact of chronic cytoreductive therapy on clonal evolution shaping in MPN. IFN limits the emergence of TP53 mutated clones compared to HU, thus potentially reducing the risk of leukemogenesis. Emergence of DNMT3A mutated clones under IFN therapy requires further exploration and could potentially play a role in therapeutic resistance. This study on a large clinically and biologically annotated cohort illustrates how serial NGS analysis may guide therapeutic options for MPN patients.

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