Analysis of cooperating pathogenic gene variants in patients with myelofibrosis

Abstract

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm derived from a clonal hematopoietic stem-cell associated with bone marrow fibrosis. Different prognostic schemes to predict survival are available, among them, the Dynamic International Prognostic Scoring System (DIPSS) considers the age, hemoglobin level, leukocyte count, circulating blasts and constitutional symptoms. Most of patient present one driver mutations within JAK2, CALR or MPL genes, and the majority may also acquire others affecting epigenetic (ASXL1, IDH1/2) and splicing (SRSF2) genes, often in multiple combinations.In the current study, we screened hot-spot regions of ASXL1, IDH1/IDH2 and SRSF2 to identify pathogenic variants and to describe their prevalence in the context of the DIPSS classification.The series included 67 patients with PMF diagnosed according to the 2016 WHO criteria with a median age of 65 years old (range 20?88) and 61% of females. At the moment of gene testing, laboratory characteristics included a median hemoglobin level of 10 g/dL (3-16), leukocyte counts of 11x109/L (16-124) and circulating blast of 0% (0-15). And, the distribution according to the DIPSS was: 18% low, 18% intermediate-1, 30% intermediate-2, and 34% high risk. Driver mutational status revealed 49% JAK2, 30% CALR, 9% MPL and 12% triple-negatives.For the present purpose, genomic DNA was amplified using allele-specific-primers for IDH1/IDH2 (exon 4), high-resolution melting/ Sanger sequencing for SRSF2 (exon 1) and Sanger sequencing for ASXL1 (exon 12-13). Fifteen patients (22%) presented pathogenic variants in ASXL1, 2 (3%) in IDH2, 1 (1.5%) in SRSF2 and 6 (9%) combined two of them. Overall mutational frequencies according to the DIPSS were 3% for low, 6% intermediate-1, 9% intermediate-2 and 18% high risk patients.Our data show a duplication of subclonal cooperating pathogenic variants as the DIPSS risk increases associated with a more aggressive disease with clinical and therapeutic implications.