Triple Negative Myelofibrosis and Myelodysplastic Syndrome with Fibrosis: Clinico-Biological Characterization and Correlation with Gene Mutations
Authors: Correa Juan Gonzalo , Alvarez-Larrán Alberto , Lopez-Guerra Monica , Hernandez Boluda Juan Carlos , Tormo Mar , Rozman María , Martínez Daniel , Colomer Dolors , Esteve Jordi , Cervantes Francisco 
Published: October 2019

Abstract
Introduction: Triple negative primary myelofibrosis (TN-PMF) and myelodysplastic syndromes with fibrosis (F-MDS) are rare entities, often difficult to distinguish each other. Currently, no specific molecular markers allowing a precise differential diagnosis are available. In this sense, next generation techniques (NGS) might be useful to distinguish between both entities and to refine prognosis.
Methods: Thirty-nine patients with TN-PMF (n=16) or F-MDS (n=23) were analyzed, Targeted NGS was performed in 28 cases (10 TN-PMF and 18 F-MDS) using the Sophia Genetics Myeloid Tumor Solution Panel including the following genes: ABL1, ASXL1, BRAF, CALR, CBL,CEBPA, CSF3R,CSNK1A1,DNMT3A, ETV6, EZH2, FLT3,HRAS, IDH1, IDH2, JAK2, KIT, KMT2A, KRAS, MPL, NPM1, NRAS, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1, U2AF2, WT1 and ZRSR2. High molecular risk (HMR) was defined by the presence of any mutation in ASXL1, SRSF2, EZH2 or IDH1/2 genes. Clinical and biological data at diagnosis were compared among both groups of patients, including bone marrow cytogenetics, bone marrow histology, and the results from NGS analysis. The probabilities of survival and progression to acute leukemia were estimated by the Kaplan Meier method using the log rank test for comparisons.
Results: Median age at diagnosis for the whole series was 63 years (range 42-87). Cytogenetic abnormalities were detected in 16 (57%) out of 28 assessable patients, being classified as low risk (n=17), intermediate risk (n=5), and high risk (n=6) There were no significant differences among TN-PMF and F-MDS regarding age, sex, hematological values, spleen size or cytogenetic risk or the number of blasts in peripheral blood or bone marrow.
Mutations were detected by NGS in 26 out of 28 (93%) patients. Median number of mutations was 3 (Range: 1-7). Most frequent mutated genes were: ASXL1 n=10 (36%), TET2 n=7 (25%), SRSF2 n=6 (21%), DNMT3A n=6 (21%), U2AF1 n=5 (18%), ETV6 n=4 (14%), SETBP1 n=3 (11%), RUNX1 n=3 (11%), EZH2 n=2 (7%), TP53 n=2 (7%), ZRSR2 n=2 (7%) and KMT2A n=2 (7%). Mutations in either TET2, U2AF1, SETBP1, TP53 or RUNX1 genes were observed in only 1 patient with TN-PMF in contrast with 12 out of 18 (67%) F-MDS cases (p=0.006). HMR mutations were detected in 12 cases (43%) with 7 of them (21%) carrying two or more HMR mutations. There were no significant differences among TN-PMF and F-MDS regarding the total number of mutations per case or in the presence of HMR mutations.
Median survival was 1.6 and 3.7 years for patients with TN-PMF and F-MDS, respectively (p=0.4). Leukocyte count > 25x109/L and presence of blasts ≥ 1% in peripheral blood were associated with a significantly lower survival. High risk cytogenetics and ≥ 2 HMR mutations were associated with a tendency towards lower survival. Median survival was 1.6 years in patients with ≥ 3 mutations in comparison with 8.3 years in those with < 3 mutations, respectively (p=0.03). Progression to acute myeloid leukemia was observed in 11 patients, resulting in a probability of 32% at 3 years. There were no significant differences in the probability of progression to acute leukemia among TN-PMF and F-MFDS. Time to acute leukemia was significantly shorter in patients with ≥ 3 mutations (3-year probability 45% versus 16%, p=0.039).
Conclusions: TN-PMF and F-MDS showed a high rate of mutations in myeloid genes with TET2, U2AF1, SETBP1, TP53 or RUNX1 being more frequently mutated in F-MDS. Despite survival being short in both entities, NGS allowed the identification of a subgroup of patients with especially poor prognosis characterized by the presence of ≥ 3 mutations.

Disclosures
Hernandez Boluda: Novartis: Consultancy; Incyte: Consultancy. Cervantes:Hospital Clinic Barcelona: Employment; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees.