Publication Only: Myeloproliferative neoplasms - Biology & translational research
Conventional genetic testing methods hardly can describe the wide molecular profile of myeloproliferative neoplasms (MPN), and in some cases, they are unable to confirm the clonal nature of the disease. Given the undoubted diagnostic and prognostic significance of molecular markers, it's of current interest to apply the next-generation sequencing (NGS) for analyzing a large array of genetic loci.
To analyze the molecular profile of MPN in patients with different course of the disease.
We evaluated whole blood DNA samples of 7 patients with MPN. Six patients were diagnosed with primary myelofibrosis (PMF), one-with essential thrombocythemia (ET). All patients were tested for the presence of clonality markers by conventional methods: driver mutations (DM) in JAK2 (allele-specific PCR), MPL (PCR-RFLP), CALR (Sanger sequencing) genes, as well as aberrations of ASXL1, EZH2, SRSF2 genes (Sanger sequencing). A self-designed myeloid neoplasm-relevant 55-gene panel was used for NGS with AmpliSeq tool kits on MiSeq/HiSeq 2500 (Illumina Inc.).
JAK2V617F was found in 3 patients, CALR mutations - in 3 patients. No DM was detected in 1 case. Aberrations of the ASXL1 gene were detected in 3 patients. One patient had ASXL1 and EZH2 mutations simultaneously. All identified mutations were confirmed by NGS.
A preliminary diagnosis of ET in Patient No.1 without markers of clonality was confirmed when 2 somatic mutations in IDH1 (Y183 C, COSM6494783) and KIT (I924 T, rs1000138811) genes were found using myeloid NGS panel. JAK2V617F+Patient No.2 with the prefibrotic stage of PMF and a favorable disease course had the only additional aberration in the TET2 gene (G355D, COSM6494926). Patient No.3 with possible transformation of PMF in chronic myelomonocytic leukemia (CMML) had the CALR mutation type 1 and ASXL1/EZH2/SRSF2-negative status. The NGS method revealed a mutation in the KRAS gene (G12A, COSM522) which is found in ˜20% patients with CMML.
In the remaining 4 patients a combination of a DM and unfavorable aberrations in the ASXL1 gene were previously identified. The adverse clinical course of the disease was noted in all these cases, 2 patients (No.5 and No.7) died.
In PMF Patient No.4 with a rapid progression of bone marrow (BM) fibrosis accompanied by an increase in leukocytosis and splenomegaly JAK2V617F and ASXL1 (Y591X, COSM1681609) mutations were complemented by aberrations in CBL (E366 K, COSM6840675; R246X, COSM4387952) and TET2 (P29R, COSM5020249) genes. PMF in Patient No.5 progressed with the development of grade IV neutropenia and severe thrombocytopenia in about one year from the diagnosis. Besides JAK2V617F and ASXL1 (R1068X, COSM41715) mutations the patient harbored Pro278Leu aberration with unclear clinical value in the GNAS gene. Clinical course of PMF in CALR-type-2+Patient No.6 characterized by steady growth of the blast percentage in the peripheral blood, marked BM fibrosis and osteosclerosis. Defects in ASXL1 (G646fsX12, COSM914346), EZH2 (D730fsX1), GNAS (R844H, COSM94388) and PDGFRA (I989 V, rs145019788) genes were also revealed. For Patient No.7 having CALR-type-1 and ASXL1 (L691fsX25) mutations, the leukemic transformation of PMF was ascertained. NGS identified anomalies in IDH1 (R132S, COSM28748) and DNMT3A (F734L) genes that are often found in AML.
Results obtained with NGS allow to broaden our understanding of MPN molecular profile. Clinical course of MPN varies depending on the set of detected mutations. The introduction of NGS using extended myeloid panel in clinical practice is expedient already at the time of diagnosis.