P660: next-generation sequencing (ngs) for detecting bcr::abl-independent mutations in patients with chronic myeloid leukemia with resistance to tyrosine kinase inhibitor treatment

Background: Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm associated with the presence of the chimeric BCR::ABL gene, which is treated with tyrosine kinase inhibitors (TKIs). The presence of mutations in the kinase domain of the BCR::ABL causes the development of resistance to TKIs. As mutations are not detected in 100% of resistant patients, the question arises about the causes of resistance to therapy in these patients. Next generation sequencing (NGS) makes it possible to analyze mutations in a wide range of genes, evaluate their impact on resistance to TKI therapy, and select the appropriate therapy for each patient. Aims: To study BCR::ABL-independent pathways of resistance to TKI therapy using modern molecular genetic technologies. Methods: The study included two groups of patients. 32 patients with resistant CML (18 men and 14 women) with a median age of 44 (IQR 14-74 years). The control group was 11 patients who responded to TKI therapy (5 men and 6 women) with a median age of 58 (IQR 33-75 years). The absence of mutations in the kinase domain of the BCR::ABL gene in patients of both groups was confirmed by direct Sanger sequencing. The presence of resistance to TKI therapy was assessed according to the ELN 2021 criteria. For the NGS study, a myeloid panel of 118 genes was used with an average reading depth of 1000x on a MiSeq instrument (Illumina). The clinical significance of the mutations found was assessed using the COSMIC, ClinVar, and VarSome databases. Survival was analyzed using the Kaplan-Meier method with statistical significance assessed using the Mantel-Cox test. Results: During the NGS study in the group with resistance to TKI therapy, genetic abnormalities were found in all patients: an average of 5 mutations (1 to 10 per patient). 95% (144/152) mutations had unclear clinical significance: most often such mutations occur in the tumor suppressor NF1 (14), transcription regulator genes TET2 (14) and ATRX (11), and cohesin complex gene STAG2 (8). Pathogenic mutations were found in the gene involved in the activation of the Ras/MAPK cell signaling pathway PTPN11 (1), epigenetic regulation genes EZH2 (1) and ASXL1 (4), proto-oncogene RHOA (1), transcription factor RUNX1 (1), and a gene involved in DNA methylation DNMT3A (1). In the control group, genetic abnormalities were detected in 81% (9/11) of patients: an average of 3 mutations (0 to 4 per patient). All variants are nonpathogenic: the most frequent mutations were found in the NF1 (4) and TET2 (3) genes. Pathogenic mutations in RUNX1 and ASXL1 genes have been noted to occur with resistant CML and are associated with an unfavorable course of the disease. The presence of pathogenic mutations reduces the overall survival of therapy-resistant patients with CML (Fig. 1).Fig.1. Survival of patients with resistance to TKI therapy depending on the presence of pathogenic mutations In two patients with pathogenic mutations in ASXL1 gene, despite several lines of therapy, a large molecular response was not achieved. In the future, these patients showed progression of the disease to the blast phase. Summary/Conclusion: The latest generation sequencing method makes it possible to detect pathogenic mutations in the PTPN11, EZH2, ASXL1, RHOA, DNMT3A, RUNX1 genes in patients with resistant CML, which significantly worsen survival. Application of this method can reveal additional BCR::ABL-independent resistance pathways and evaluate the course of the disease. Keywords: Chronic myeloid leukemia, Resistance