Mutational Analysis of Tumor Samples From Patients with Relapsed or Refractory Multiple Myeloma (MM) Highlights the Prevalence of RAS/RAF Pathway Activation and Demonstrates Previously Unreported Mutations in Known Cancer Genes
Authors: Mulligan George , Lichter David I , Di Bacco Alessandra , Blakemore Stephen J , Berger Allison , Koenig Erik , Bernard Hugues , Trepicchio William L. , Li Bin , Lonial Sagar , Richardson Paul G. , Anderson Kenneth C. , Sonneveld Pieter , San Miguel Jesús F , Esseltine Dixie-Lee , Schu Matthew 
Published: October 2019

Abstract
Abstract 1377

Background:
MM is a plasma cell malignancy that remains generally incurable; however, there is significant inter-patient variation in the clinical course of MM and in survival, which is thought to relate, at least in part, to the biological heterogeneity of patients' tumors. At the molecular level, MM is sub-classified by chromosomal translocations, genetic mutations, and risk classifiers that integrate gene expression. Some of these genetic abnormalities have established associations with disease outcome in MM; for example, hyperdiploid patients have a better disease prognosis compared with non-hyperdiploid patients, and patients with a deletion of the p53 locus on chromosome 17 exhibit very poor clinical outcomes compared with patients who do not have this deletion. A better understanding of the molecular diversity of MM will likely inform therapeutic decision making and also identify additional intracellular targets for future drug development. A recent whole genome sequencing study of 38 MM patients described several somatic mutations that had not previously been described in MM or other cancers (Chapman et al, Nature 2011), as well as mutations in known cancer genes that had either not previously been reported in MM (BRAF), or that were observed at a higher frequency in MM than previously reported (KRAS, NRAS). The present study was conducted to confirm the prevalence of mutations in a panel of established cancer genes in tumor samples from patients with relapsed or refractory MM.

Methods:
Bone marrow aspirates were collected from 133 patients who participated in phase 2 (SUMMIT, CREST) and 3 (APEX) clinical studies of bortezomib for relapsed or refractory MM. Tumor DNA was amplified and screened for mutations in a panel of cancer genes using the MassARRAY®/Sequenom mass spectrometry-based methodology. This custom panel evaluates 514 known mutations in 43 distinct oncogenes and tumor suppressor genes. Mutations were identified with standard software and subsequently verified via manual inspection.

Results:
The most common mutations observed in MM tumor samples were in KRAS (n=32 [24.1%], 95% CI: 17.0–31.3) and NRAS (n=26 [19.5%], 95% CI: 12.8–26.3); for both genes, mutations in codon 61 were more common (47% and 85% for KRAS and NRAS, respectively) than mutations in codon 13, as previously described for MM (Chng et al, Leukemia 2008). Mutations in BRAF were detected in three patient samples (2.3%, 95% CI: 0–4.8). For all three genes, the mutation rate in this patient population was similar to that reported by Chapman et al (26.3%, 23.7%, and 4% for KRAS, NRAS, and BRAF, respectively). The frequency of mutations in KRAS and NRAS was similar in patients who had received one or more than one prior line of therapy, suggesting that these mutations arise early in the pathogenesis of MM and are not driven by treatment-exerted selection pressures. KRAS, NRAS, and BRAF mutations were mutually exclusive, consistent with their described biological roles in modulating a common signaling pathway. These data suggest that the RAS/RAF pathway is activated by mutation in 45.9% (95% CI: 37.0–54.3) of patients with relapsed or refractory MM. Mutations in a number of other genes, including PIK3CA, TP53, MET, PDGFRA, and JAK3 were detected in at least two patient tumor samples; several of these genes have not previously been reported to be mutated in MM.

Conclusions:
These data confirm the high prevalence of activating mutations in genes of the RAS/RAF pathway in patients with relapsed or refractory MM, and begin to shed light on additional mutations that may co-operate with or act independently of this key cellular signaling axis. Data on the co-occurrence of these mutations in patients with MM, and their association with clinical outcomes, will be presented.

Disclosures:
Mulligan: Millennium Pharmaceuticals, Inc.: Employment. Lichter:Millennium Pharmaceuticals, Inc: Employment. Di Bacco:Millennium Pharmaceuticals, Inc: Employment. Blakemore:Millennium Pharmaceuticals, Inc: Employment; Takeda Pharmaceuticals: Equity Ownership. Berger:Millennium Pharmaceuticals, Inc: Employment. Koenig:Millennium Pharmaceuticals, Inc: Employment. Bernard:Millennium Pharmaceuticals, Inc: Employment. Trepicchio:Millennium Pharmaceuticals, Inc: Employment. Li:Millennium Pharmaceuticals, Inc: Employment. Lonial:Bristol-Myers Squibb: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Millennium Pharmaceuticals, Inc.: Consultancy; Onyx: Consultancy; Merck: Consultancy. Richardson:Millennium Pharmaceuticals, Inc.: Consultancy; Johnson & Johnson: Consultancy; Celgene: Consultancy. Anderson:Merck: Consultancy; Onyx: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Millennium Pharmaceuticals, Inc.: Consultancy; Bristol-Myers Squibb: Consultancy; Actelion: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Sonneveld:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding. San Miguel:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals, Inc: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Esseltine:Millennium Pharmaceuticals, Inc.: Employment; Johnson & Johnson: Equity Ownership. Schu:Millennium Pharmaceuticals, Inc.: Employment.