Driver Mutation Profile By MLPA in Myelofibrosis Patients
Authors: Juliana Minuncio , Nonino Alexandre , Mazzeu Juliana Forte , Mascarenhas Cintia do Couto 
Published: October 2019

Abstract
Myelofibrosis is the rarest and most severe Ph- myeloproliferative neoplasm and can present de novo or post Polycythemia Vera or Essential Thrombocythemia. It is characterized by bone marrow fibrosis, extramedullary hematopoiesis and abnormal expression of inflammatory cytokines resulting in several atypical events and may progress to Acute Leukemia. The disease arises from clonal expansion of a single hematopoietic stem cell (HSC), driven by a somatic mutation of JAK2, CALR or MPL genes combined with dysregulation of hematopoietic microenvironment, additional mutations and cytogenetic abnormalities.The aims of this study are to assess driver mutations status in primary or secondary myelofibrosis patients and to correlate their mutational profile with clinical outcomes. The search for JAK2V617F, exon 12 JAK2, calreticulin exon 9 c.1092_1143del52 and c.1154_1155insTTGT, MPLW515K and MPLW515L mutations was performed in 31 subjects using MLPA technique, a method of DNA analysis that allows simultaneous appraisal of different mutations in multiple samples. 48.4% of patients present the JAK2V617F mutation,indel CALR mutations in 38.7% of patients (of these, 66.7% with del52 bp, 33.3% harbored insTTGTC),MPL W515L in 3.2% of patients and 9.7% of patients were triple-negative. From the mutational profile information obtained by MLPA,the clinical-molecular risk score was calculated for each of the individuals in the sample, according Rumi et al.. Being at 12,9% (4) as very low risk, 9.7% (3) as low risk,35.4% (11) as intermediary risk, 29.1% (9) and of high risk, and 12.9%(4) as very high risk. Patients with mutated JAK2 were older, with minor degree of anemia and more leukocytosis, whereas those with CALR mutations had less frequency of leukocytosis and thrombocytopenia. Triple-negative subjects displayed the lowest median age at diagnosis (49.3 years), and bone marrow failure phenotype, similar to Myelodysplastic Syndrome. Risk stratification provided by DIPSS was similar to other centers.Individuals with PMF present constitutional symptoms significantly more often than those with post-ET MF(p = 0.0365).Mortality rate was 29%, and mean survival after diagnosis was 68.3 months. CALR mutated individuals presented higher average survival and median survival according to DIPSS was higher than predicted by the prognostic model, possibly due to the higher frequency of CALR mutations reported.Median follow-up time was 32 months (ranging from 10 months to 13 years).Thromboembolic phenomena were recorded in 19.3% of patients, and evolution to AML in 6.4% of patients and it was verified that 75% of the individuals with Myelofibrosis Post-ET presented thrombotic events at some point in the disease. The association between the DIPSS clinical-laboratory parameters and the demand of transfusion at diagnosis, with the occurrence of Acute Leukemia was assessed using Fisher's exact test but have no significant difference in these parameters between patients who evolved or not for Acute Leukemia. The JAK2 V617F mutation is expected to be present in 60 to 65% of individuals with Myelofibrosis, but this mutation has been identified in only 48% of patients. In contrast, the observed frequency of indel of the CALR, of 38%, was higher than the classically described, from 25 to 30%. The rate of mutation type 2 (ins 5-bp) was also higher than expected, 33.6%.Regarding the mutation subtypes of CALR, mutation type 1 (52-bp deletion) is observed in up to 80% of MFP cases, but has a similar frequency as type 2 (5-bp insertion) in patients with ET. The grouping of patients with primary MF and post-ET may have contributed to the higher incidence of type 2 CALR mutation observed in this sample, although much higher than the 13% frequency described in the literature in a mixed population. The type 1 mutation was observed in 66% of our patients with post-ET and mutated CALR, but the small number of individuals in the study does not allow to estimate the impact of this mutation in the evolution of the disease. Our fraction of CALR-mutated patients is much higher than those described in Asian, European, North American and Argentinean populations. The complex genetic landscape involved in initiation and progression of Myelofibrosis, instigates the adoption of integrative prognostic stratification models. In this scenario, MLPA is a powerful tool for molecular study, and a promising ally for MPN molecular characterization.

Disclosures
No relevant conflicts of interest to declare.