MM-356 Deep Coverage Targeted Next Generation Sequencing Approach to Determine Somatic Mutational Spectrum of Recurrently Mutated Genes in Newly Diagnosed Multiple Myeloma in a Large Indian Cohort

Abstract

To determine the mutational spectrum of recurrently mutated genes in a large cohort of Indian patients presenting with multiple myeloma (MM) in our center by performing deep coverage-targeted NGS. Samples of 151 multiple myeloma patients (149 NDMM and 6 follow-up) were sequenced. Patients were diagnosed with MM per IMWG criteria 2014. The study was initiated following approval from the institute's ethical committee, and written informed consent was obtained from all patients. BMA was subjected to plasma cell isolation using CD138+ MACS. DNA underwent automated extraction using the Maxwell System. Mutations were tested on a commercially available 47-gene custom panel (Illumina) specifically designed for MM. Libraries were prepared using a TruSeq Custom Amplicon Low Input Kit (Illumina). DNA (25 ng) was hybridized with oligos specific for targeted regions of interest. Libraries were pooled. Paired-end sequencing was carried out on Illumina's MiSeq sequencer using micro flow cell with V3 chemistry and MS600 V3 series reagent cartridge. Sequenced reads were mapped to GRCh37/Hg19 reference and data obtained as FASTQ files and processed with MiSeq Reporter. As output, .vcf files were generated. Genomic variants were annotated by ANNOVAR. Sequencing coverage of 4771 X (Range 2063X-10980X) revealed different types of variants (exonic, intronic, splice region, 3' and 5' UTR, and upstream gene). Pathogenicity was checked by COSMIC. At least one pathogenic mutation was found in 77% (116/151) of patients (median 1 mutation per patient, range 0-7) and 51% (24/47) of genes. Forty-five percent (68/151) of patients had mutations in more than one gene. The most frequently mutated genes were KRAS, NRAS, BRAF, TP53, DIS3, FAT4, FAT3, and DNAH5. Genomic aberrations found were compared with the frequency of mutations as per the MMRF and TCGA datasets and related literature; good concordance was found (especially in MAPK pathway genes). BRAF and RAS mutations were mutually exclusive. Few mutations were validated by Sanger sequencing. The high frequency of mutations in MAPK pathway genes (KRAS, NRAS, BRAF) suggested the importance of this pathway in myelomagenesis and therapeutics. Follow-up sample sequencing may help deduce clonal evolution during disease course. Identification of clinically relevant target driver mutations may aid in prognosis and therapy personification.