Development and Results of a Multiple Myeloma Specific Custom 77-Gene Mutation Panel for Clinical Targeted Sequencing
Authors: Kortuem K. Martin , Stewart A. Keith , Bruins Laura Ann , Ahmann Gregory , Vasmatzis George , Rajkumar S. Vincent , Kumar Shaji , Dispenzieri Angela , Lacy Martha Q , Gertz Morie A , Fonseca Rafael , Champion Mia , Bergsagel P. Leif , Braggio Esteban 
Published: October 2019

Abstract

Background:A goal of “post-genomic” cancer care is development of specific, affordable, reliable and rapid next-generation sequencing-based assays determining prognosis, therapeutic selection and minimal disease monitoring, replacing older technologies in the diagnostic and monitoring workup. In this regard, we designed and deployed a custom gene panel for identification of mutations, copy-number changes and clonal frequency in multiple myeloma (MM) samples.
Methods: The MM Mutation Panel Version 2.0 (M3P 2.0)1 includes 1271 amplicons from 77 genes that are either mutated in MM (genes with a published mutation incidence ≥3%), represent critical pathophysiological pathways, are targetable with small molecule-based therapies and/or are associated with drug resistance (eg. XBP-1, CRBN, IZKF, IRF4). The amplicons are spread across 21 chromosomes, allowing the identification of copy-number changes in these regions.
Results: We have sequenced 38 MM cases (150 will be presented) with paired tumor-germline DNA samples, using only 20ng of DNA for library preparation. Patients (Pts) included 29 untreated and 9 MM pts with multi drug refractory disease. Coverage depth averaged 964X. Hyperdiploid MM was successfully identified in 45% of cases, del(13q) in 34%, del(1p) in 18%, del(17p) in 8% and gain of 1q in 13%. Array CGH in representative cases confirmed concordance with semiconductor sequencing. Overall, 71 exonic missense and 3 nonsense mutations were detected, and 7 frameshift indels. In 89% of the pts a mutation in at least one M3P2.0 gene was identified (range 1-8). 92% of mutations were predicted damaging (Provean, Polyphene-2 or SIFT). Most commonly mutated were KRAS (37%), NRAS (21%), BRAF (13%), TP53, CDKN1B, DIS3 (each 11%), FAM46C, STAT3, and IRF4 (each 8%). The MEK-ERK pathway was mutated in 61% of cases and in 3 cases more than one gene in this pathway (NRAS, KRAS, BRAF) was simultaneously mutated. Six mutations in BRAF were identified in 5 untreated pts, 3 of them known to be activating and targetable (Val600E). Other commonly mutated pathways were the NFKB pathway in 24% of pts and the Cyclin D pathway, mutated in 16% of cases.
Genes of the cereblon pathway (CRBN, IRF4, IKZF3), essential for the action of lenalidomide (len) were mutated in 5 Pts. One newly diagnosed and len refractory Pt harbored a subclonal CRBN mutation (Asn316Lys, allelic fraction [AF] 20%) as well as 4 clonal IRF4 mutations within the DNA binding domain of the gene (Lys89Asn (AF 78%), Lys77Glu, Ser48Arg and Cys49Ser (AF 80% each). Another IRF4 mutated (Lys123Arg, AF 90%) hemodialysis dependent Pt was responsive to dose adapted treatment of len and dexamethasone (dex). Mutations in the C2H2-type 3 region of IKZF3 in a len resistant (Gly191Arg, AF 48%) pt occured as well as in a len and dex responsive (Arg242Gly, AF 52%) Pt. Furthermore, we identified a truncating XBP1 mutation (p.Leu232*, 97% AF) in a Pt refractory to bortezomib. One patient harbored a mutation in the steroid receptor NR3C1 (Pro255Leu, 22% variant reads). Finally, integrated mutation and copy-number data identified biallelic deletions of CDKN2C and FAM46C in a multi drug refractory Pt. Mutation and/or copy-number losses in both alleles were found in DIS3 in 4 Pts, in CYLD, TP53 and TRAF3 in 2 Pts and in CARD11, CDKN2A, CDKN1B, RB1and STAT3 in one Pt each.
Conclusion: In summary, we report the initial results from 38 MM Pts using a custom MM sequencing panel which employs small amounts of DNA requiring very few cells and providing deep coverage in clinically meaningful timeframes. Our findings frequently include prognostically significant information, actionable targets and mutations in genes related to drug resistance. Targeted mutation profiling will likely become part of the clinical workup in MM and our M3P2.0 mutation panel is a suitable tool to provide information needed to guide precision therapy and to set the basis for individually tailored treatment decisions.
1. Kortuem KM. et al. Development Of a 47 Gene Sequencing Panel For Common Mutations Present In Multiple Myeloma: Preliminary Results In 77 p53 Deleted Patients, ASH 2013.

Disclosures
Stewart: Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Array BioPharma: Consultancy; Sanofi: Consultancy; Takeda Pharmaceuticals International Co.: Research Funding; Novartis: Consultancy. Bruins:OncoSpire: Salary support Other. Vasmatzis:OncoSpire: Salary Support Other. Kumar:Celgene: Consultancy, Research Funding; Millennium: The Takeda Oncology Co.: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding; Sanofi-Aventis: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Skyline Diagnostics: Membership on an entity's Board of Directors or advisory committees. Fonseca:Medtronic, Otsuka, Celgene, Genzyme, BMS, Lilly, Onyx, Binding Site, Millennium, AMGEN: Consultancy, patent for the prognostication of MM based on genetic categorization of the disease. He also has sponsored research from Cylene and Onyx Other, Research Funding. Bergsagel:MundiPharma: Research Funding; OncoEthix: Research Funding; Constellation Pharmaceutical: Research Funding; Novartis: Research Funding.