Abstract 450: A targeted capture panel to identify translocations, copy number abnormalities, and mutations in multiple myeloma
Authors: Walker Brian Andrew , Ahsan Aarif , Sudha Parvathi , Kausar Tasneem , Rane Sushmita , Flynt Erin , Thakurta Anjan 
Published: July 2021

Abstract
Multiple myeloma (MM) is a genetically heterogeneous disease where risk stratification and outcomes are associated with translocations of the immunoglobulin (Ig) and MYC loci, copy number abnormalities and mutations. Therefore, we designed a comprehensive MM targeted sequencing panel (MM panel) to investigate the common genomic abnormalities and validated it against known standards. The MM panel includes targeted mutation and translocation panels encompassing ~ 990 kb and ~ 4.7 Mb respectively. The mutation panel includes exons of 228 genes and the translocation panel investigates the structural and copy number changes. The two panels are manufactured separately and combined in a 5:1 ratio to prevent over-sequencing of the larger translocation panel. To validate the MM panel, we performed targeted sequencing on 110 MM patient samples and compared the results to different orthogonal technologies. Libraries were prepared from the DNA extracted from CD138+ bone marrow cells and non-tumor tissue (peripheral blood or saliva) using the KAPA workflow. Following hybridization and amplification the libraries were sequenced using 75 bp paired end sequencing. Sequences were aligned to hg19 and mutations and translocations were identified using Strelka and Manta. Copy number was determined using the ratio of non-tumor to tumor reads in each targeted region. Data was further validated using clinical FISH (copy number and translocations), MLPA (copy number), known standards (mutations), or ddPCR (mutations). Canonical IgH translocations were detected in 33.6% of patients by sequencing which agreed with FISH except for one patient in which a t(11;14) was detected by FISH only. The overall sensitivity and specificity for translocation detection was 97.4% and 100%, respectively. Non-canonical translocations were detected in 14.5% of samples, 43% of which were to the MYC locus. MYC abnormalities were detected in 46.4% of samples. Copy number was determined by sequencing and MLPA for 22 regions that were directly comparable in 103 patient samples and the concordance between the technologies was R2=0.969. For the important prognostic regions, the concordance was R2=0.962 (CDKN2C), R2=0.986 (CKS1B), and R2=0.973 (TP53). Mutation detection validation was performed using samples with known variant allele frequencies (VAF) for common mutations. We were able to determine the sequencing VAF for 74 mutations across 5 samples with a concordance of R2=0.9849 between the expected and observed frequencies. The minimum detected VAF was 1.3% at an average depth of 891x. We also performed ddPCR on 6 patient samples with the common KRAS, NRAS and BRAF mutations with a VAF concordance of R2=0.9983. In summary, we have developed and validated a sequencing panel which is being deployed to clinical laboratories that is relevant to MM patient prognosis, risk stratification, and treatment.
Citation Format: Brian Andrew Walker, Aarif Ahsan, Parvathi Sudha, Tasneem Kausar, Sushmita Rane, Erin Flynt, Anjan Thakurta. A targeted capture panel to identify translocations, copy number abnormalities, and mutations in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 450.