Mutational Landscape of Patients with Myelofibrosis That Do Not Harbor Mutations in JAK2, MPL and Calreticulin Driver Genes
Authors: Rotunno Giada , Guglielmelli Paola , Pacilli Annalisa , Fanelli Tiziana , Mannarelli Carmela , Pancrazzi Alessandro , Vannucchi Alessandro M. 
Published: October 2019

Abstract
Background. In primary myelofibrosis (PMF), mutations of JAK2, CALR and MPL driver genes can be detected in about 60%, 20% and 5% of the patients (pts), respectively. Therefore, about 10% of the pts lack any of the 3 driver mutations and are operationally called "triple negative" (TN). TN pts present a higher risk of developing anemia and thrombocytopenia, suffer from reduced overall survival compared to other genotypes, particularly to CALR type1/type1-like mutations, and may be at higher risk of leukemic transformation (Blood 2014; 124:1062; Leukemia 2014; 28:1472; Blood 2014;124:2465).
Aims The aim of the study was to analyse the molecular landscape of TN PMF pts by genotyping a set of myeloproliferative-neoplasms associated genes whose mutated status was shown to be prognostically relevant in previous studies (Leukemia 2013;27:1861; Blood 2014;123:2220; Leukemia 2014;28:1804;Leukemia 2014;28:1494).
Methods. Diagnosis ofPMF was made according to WHO2008 criteria. All pts provided informed written consent. Previously published methods were used to screen mutations involving JAK2, MPL and CALR. A deep sequencing custom panel was designed to genotype the following genes: EZH2, ASXL1, IDH1/2, SRSF2, TP53, TET2, RUNX1, CBL, NRAS, KRAS, DNMT3A, SF3B1, IKZF1, NFE2, SH2B3. Analysis was performed using the Ion torrent PGM platform. Comparisons of quantitative variables between groups were carried out by the nonparametric Wilcoxon rank-sum test. The prognostic value of the molecular variables with regard to OS was estimated by the Kaplan-Meier method and Cox regression.
Results. We analysed 28 TN PMF collected at the time of diagnosis. Median age was 66.7y, 57% were male. Median follow up was 2.1y (0.3-14.5). Overall, 8 patients (28.6%) progressed to AML. Death occurred in 20 pts (71.4%) after a median follow up of 2.3y (1.4-3.3y); progression to leukemia was the cause of death in 5 pts (25%).
Overall, 22 pts (78.6%) presented at least one mutation in any of the 15 genes of the panel; 14 pts (50%) presented at least 2 mutations in different genes. The frequency of mutated genes was: SRSF2 39.3%, ASXL1 28.6%, EZH2 21.4%, NRAS 21.4%, TET2 10.7%, CBL 10.7%, IDH 3.6%, DNMT3A 3.6%, SH2B3 3.6%, U2AF1 3.6%. Twenty pts (71.4%) were classified as high molecular risk (HMR: ie, any mutated gene of ASXL1, EZH2, SRSF2, IDH1/2), a proportion significantly higher (P<0.01) than among JAK2 V617F (31.5%), CALR Type1/1-like (22.0%) and CALR Type2-type2-like (5.0%) (Leukemia 2013; 27:1861). Mutated genes were grouped into 3 different pathways: epigenetic regulation (ASXL1, EZH2, TET2, IDH), splicing machinery (SRSF2, U2AF1) and leukemic transformation (NRAS, DNMT3A, SH2B3, CBL). The most frequently mutated pathway was the epigenetic one with mutations in 14 pts (63.6%) of which 3 pts (21.4%) had 2 or more mutated genes of the pathway; 12 pts (54.5%) presented mutations in the splicing machinery and of these 8 pts (66.7%) had 2 or more mutated genes of the pathway; genes involved in leukemic transformation were mutated in 11 pts (50%) and 10 of 11 (90.9%) had 2 or more mutated genes. In three cases (13.6%) all 3 pathways were concurrently involved.
Among the mutated genes, SRSF2 was associated with shorter survival [1.9y (1.6-2.2)] compared to pts with un-mutated SRSF2 [3.2y (0.9-5.4y)] (HR 2.3, 95%CI 0.9-6.4). SRSF2 mutations were also associated with shorter leukemia free survival (LFS): LFS not reached in un-mutated pts compared to 2.2y (1.8-2.7y) for mutated pts, with a HR=4.5 (95%CI 10.3-19.9). We also found that in pts with grade 1 bone marrow fibrosis the splicing and the leukemic pathway were more frequently mutated compared to grade 2-3 fibrosis (57% vs 28.5% and 50% vs 28.5% respectively).
Conclusions. "Triple-negative" pts with PMF present high rate of mutations of MPN-associated genes, most of them are classified as "high molecular risk" and harbor >2 mutations. Mutated SRSF2 was particularly associated with shorter LFS. Such complex molecular landscape might help to explain the negative outcome of TN PMF pts.

Disclosures
Vannucchi: Baxalta: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shire: Speakers Bureau.