DOI: 10.5061/dryad.4xgxd25fz
title: Study group charasteristics and sequencing data of patients with essential thrombocythemia and polycythemia vera

description: Polycythemia vera (PV) and essential thrombocythemia (ET) are diseases
 driven by canonical mutations in JAK2, CALR, or MPL gene. Previous studies
 revealed that in addition to driver mutations, patients with PV and ET can
 harbor other mutations in various genes, with no established impact on
 disease phenotype. We hypothesized that the molecular profile of patients
 with PV and ET is dynamic throughout the disease. In this study we
 performed 37-gene targeted next-generation sequencing panel on the DNA
 samples collected from 49 study participants in two time points, separated
 by 78-141 months. We identified 78 variants across 37 analyzed genes in
 the study population. By analyzing the change in variant allele
 frequencies (VAFs) and revealing the acquisition of new mutations during
 the disease, we confirmed the dynamic nature of molecular profile of
 patients with PV and ET. We found connections of specific variants with
 the development of secondary myelofibrosis, thrombotic events, and
 response to treatment. We confronted our results with existing
 conventional and mutation-enhanced prognostic systems, showing the limited
 utility of available prognostic tools. Results of this study underline the
 significance of repeated molecular testing in patients with PV and ET and
 indicate the need for further research within this field to better
 understand the disease and improve available prognostic tools.
description: Study participants have been
 selected from the population of patients of the Outpatient Ward,
 Department of Hematology and Transplantology, Medical University of
 Gdansk, Poland. Initially, all alive patients diagnosed with PV or ET were
 identified. Next, individuals in whom the diagnosis was made at least 5
 years prior to the study were selected. Among those patients, we
 identified individuals from whom DNA samples of sufficient quality and
 quantity were available from the time of diagnosis. Finally, 49 patients
 consented to the participate in the study.
 Diagnoses were verified with
 MPN 2016 WHO criteria[6]. Two patients with apparent features of
 myeloproliferative neoplasms but undetected driver mutation were labeled
 triple negative (TN). Each patient’s documentation was analyzed to collect
 the data regarding treatment, thrombotic complications, and disease
 progression. A peripheral blood sample was collected from
 each participant for molecular testing and comparison with corresponding
 archival samples. The median time between historical sample collection and
 a present sample was 104 months (range 78-141
 months) Genomic DNA was extracted from peripheral blood
 samples using QIAamp DNA mini kit (Qiagen) according to the
 manufacturer's instructions. DNA was quantified by spectrophotometric
 method and stored at –20°C for further analysis.
 Next-generation sequencing was
 performed using the Archer VariantPlex Core Myeloid kit (ArcherDX), Mid
 Output Kit (300-cycles), and MiniSeq (Illumina). It allowed performing a
 comprehensive analysis of 37 genes: <em>ABL1, ANKRD26, ASXL1, BCOR,
 BRAF, CALR, CBL, CABPA, CSF3R, DDX41, DNMT3A, ETNK1, ETV6, EZH2, FLT3,
 GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PHF6, PTPN11,
 RUNX1, SETBP1, SF3B1, SRSF2, STAG2, TET2, TP53, U2AF1, WT1,
 ZRSR2</em>. The results were analyzed using Archer Analysis
 v.6.0.3.2 software (ArcherDX), and 2,7% allele frequency (VAF) as a cutoff
 was applied. Collected data was used to stratify patients
 into the risk groups at the time of sample collection. Conventional risk
 stratification was made according to the revised IPSET-T system in ET and
 based on age and thrombotic complications in PV.  Acquired sequencing data was used to
 attribute patients into the low- intermediate- or high-risk groups
 according to MIPSS-ET or MIPSS-PV[5]. Since cytogenetic data were incomplete in the
 study population, all PV patients were considered to have normal
 karyotypes.                                                    
                                                           
 An
 additional exploratory risk stratification, including 5- and 10-year event
 free survival (EFS), risk of secondary myelofibrosis (sMF) or acute
 myeloid leukemia (AML) were assigned based on prognostication model
 developed by Grinfeld et al.. This stratification was made to assess the
 utility of the proposed MPN Personalized Risk calculator (available online
 at https://www.sanger.ac.uk/science/tools/progmod/progmod/). For this
 evaluation, all patients were considered as unknown karyotype.
description: Microsoft Excel