Pb2175: next-generation sequencing in the diagnosis, prognosis and features of the course of the disease in patients with ph-negative myeloproliferative neoplasms

Topic: 15. Myeloproliferative neoplasms - Biology & Translational Research Background: Determination of “driver” mutations in the JAK2, CALR and MPL genes in patients with myeloproliferative neoplasms not associated with the Philadelphia chromosome (Ph-MPN) is the gold standard in diagnosis. However, the genomic landscape of such patients is very complex and is characterized by the presence of mutations not only in the driver genes. Such mutations can cause a more aggressive course of the disease. The next generation sequencing (NGS) method allows to perform simultaneous comprehensive analysis of an extensive panel of genes, which can significantly reduce the execution time and cost of the study. In addition, an important advantage of NGS is the ability to read the entire gene, including areas not studied by the standard Sanger sequencing method. This fact makes NGS an important tool in detecting pathogenic mutations and predicting the course of the disease. Aims: To evaluate the possibilities of using NGS technology in the diagnosis and determination of prognostic features of the course of the disease in Ph-MPN patients. Methods: The study included 31 patients (12 men and 19 women) aged from 27 to 85 years (Me=55 years). The Ph-MPN diagnosis was previously established in all patients: PMF (18/31), PV (3/31), ET (7/31), unspecified MPN – in 3/31 patients. All patients were analyzed for the presence of mutations in the driver genes – in 19/31 (61.2%) cases a mutation was detected in the JAK2 (V617F), 5/31 (16.2%) CALR, 3/31 (9.7%) MPL, 4/31 (12.9%) had no mutations in any of the driver genes (“triple-negative status”). In all patients, sequencing was performed using a myeloid panel of 121 genes with an average reading depth of 200x or 1000x on a MiSeq (Illumina) device. When analyzing the data obtained, a 3% threshold of allele frequency (VAF) was used. The clinical significance of mutations was established using COSMIC and ClinVar databases. To analyze the survival rate, the Kaplan–Meyer method was used with an assessment of statistical significance using the Cox-Mantel test. Results: During the NGS analysis, genetic abnormalities were detected in all patients. At the same time, mutations of somatic nature were detected in 90% of cases (28/31), on average 5 mutations in one patient (1-18 in one patient). In 94% of the studied samples (29/31), from 1 to 5 pathogenic mutations were detected (Me=2). Somatic mutations were found for 3 out of 4 patients with triple-negative status, which made it possible to confirm the clonality of the disease with the help of NGS and establish a diagnosis. Pathogenic mutations in “non-driver” genes were found in 16/27 patients. These genes perform various functions – epigenetic regulation (ASXL1 (7/31), TET2 (4/31), IDH1 (2/31), EZH2 (1/31), SETBP1 (1/31)), RNA splicing (SRSF2 (2/31), U2AF1 (2/31), DDX3X (1/31)), signal transmission (CBL (1/31), EP300 (1/31), KRAS (1/31), APC (2/31)) and chromatin remodeling (ATRX (2/31). Mutant variants of ASXL1 (7/31) were found in more than 10% of patients. The presence of mutations in the ASXL1 gene is associated with a deterioration in the median overall survival compared to patients without mutation (7,6 years and 12 years, respectively, p=0.043) (Fig. 1).Figure 1. Survival of patients with Ph-MPN depending on the presence of mutations in the ASXL1 gene, p=0.043 Summary/Conclusion: The study of the mutational profile of patients with Ph-MPN by NGS allows us to confirm the clonal nature of the disease. The use of the NGS method makes it possible to predict the course of the disease and determine indications for allogeneic HSCT, including Ph-MPN patients without driver mutations. Keywords: Thrombocythemia, Idiopathic myelofibrosis, Polycythemia vera, Myeloproliferative disorder