Disovery of Novel Mutations Exclusively Shared By Accelerated and Blast Crisis Phase CML Patients Using Whole Exome Sequencing:Implication in Hunt for Common Biomarker /Drug Target of CML Progression

Abstract

Introduction:Chronic Myeloid Leukemia (CML) is resulted due t (9;22) which leads to fusion oncogene BCR-ABL. Although chronic phase (CP) CML is treatable, accelerated (AP) and blast phases (BP) have high degrees of drug resistance and subsequent treat failures 1. Mechanism of this disease progression in CML is poorly understood and few markers studied in this regard are found only in a subset of advanced phase CML patients 2,3. Therefore, this study was designed to find out common gene mutations (trunk mutations) associated with all advanced phase CML patients.Material and Methods:We investigated imatinib responder AP (N=13), BC (10) and CP (5) CML patients along with age-sex matched healthy controls using whole exome sequencing (WES). Illumina NGS instrument (HiSeq) generated bcl files which were converted to fastq files by using bcl2fastqtool 4. Raw reads were aligned to genome using BWA tools while whole exome variants were annotated using Illumina Variant Studio 4. R package was employed to align specific gene mutants to disease phases 5. In order find a common CML progression biomarkers, gene mutations shared by all patients at a CML phase (so called trunk mutations) were selected and confirmed using Sanger sequencing.Results:Whole exome sequencing revealed novel gene mutations associated with different disease phases in CML. CP CML patients with long term CML response harbored mutations in TTN, PDLIM4, CELF2, ANO5, DOK2, MYO16, RAI1, LTBP3, CNNM3. On the other hand, specific genes were found mutated in AP (EV15L, FAM120B, ZNF208, PML, TMEM145, ASXL1, ABL1) and BC (ATXN3)CML patients.Discussion & Conclusions:All the genes mutated in AP and BC CML were either involved in other cancers or cellular processes like DNA repair, regulation of gene expression (for instance ATXN3 gene mutated in all BC-CML is involved in DNA repair and in repressing expression of some other genes6 which indicates role of damaged DNA repair in CML blast crisis). Mutations in long term CML responders indicate role of all are some of these genes in relation to sensitivity to tyrosine kinase inhibitors (TKIs). Very recently, it has been reported that CML patients with rs460089-GC (SLC22A4) genotype had stable major molecular response 7. Biomarkers of stable response can help identify patients who can be the candidates of cessation of TKIs which is one of the major focus of many CML trials. Identification of biomarkers of early CML progression can help identify and clinically intervene patients at high risk of transformation to advanced and blast phases of CML which can considerably minimize morbidities and mortalities associated with CML patients 2,3.