Bruinink, Davine Hofste op

Abstract Introduction Multiple myeloma (MM) is characterized by a highly variable disease course, which can be traced to initiating and acquired genomic events. Whole exome analysis of matched tumor and germline DNA from 287 MM patients identified recurrently somatically mutated genes (RSMGs) (Lohr et al. - Cancer Cell 2014, Bolli et al. - Nat Commun 2014). Despite the fact that these RSMGs affect pathways that are biologically important in MM, the clinical relevance of many of these genes in the context of conventional prognostic markers remains to be elucidated. Aims The aims of this pilot study were: (1) To validate the prevalence of RSMGs in our newly diagnosed MM patient cohort; (2) To assess the correlation between RSMGs, clinical parameters and outcome; (3) To thereby identify the potential clinical usefulness of introducing RSMG mutational profiling in larger MM trial cohorts. Material and Methods CD138+ enriched MM cells and peripheral blood were obtained with informed consent from chemotherapy-naive patients, participating in 3 clinical trials: HOVON-65/GMMG-HD4, HOVON-87/NMSG-18 and Carthadex (EudraCT number 2004-000944-26, 2007-004007-34 and 2009-014922-40, respectively). Matched tumor and germline DNA were sequenced on an Ion Torrent sequencing platform (PGM, Life Technologies), using the M3 P Mutational Panel v3.0, comprising 1327 customized oligos (Life Technologies), targeted at the coding sequences of 88 MM-relevant genes, including the RSMGs. Somatic mutations were considered positive when present in >=10% of tumor reads and <=10% germline reads, with a minimal coverage of 10x and being non-synonymous, or splice donor variants. All statistical analyses were performed in SPSS version 23, using the log-rank and Mann-Whitney U-test, with the Bonferroni test to correct for multiple comparisons. Results A total of 206 DNA samples were sequenced from 103 patients (HOVON-65/GMMG-HD4 (n=16), HOVON-87/NMSG-18 (n=67), Carthadex (n=20)) with an average coverage of 574x in tumor DNA, 451x in germline DNA and an overall coverage of 98%. We collected follow-up data from 102/103 patients, with a median follow-up time of 30 months. 168 somatic mutations were detected in 44/88 genes. 82% of patients had at least 1 somatic mutation. Genes most frequently mutated were: (1) NRAS (26%), (2) KRAS (22%), (3) DIS3 (14%), (4) FAM46C (9%), (5) TP53 (7%) and (6) BRAF (6%) (Figure 1). Of note, NRAS and KRAS mutations were mutually exclusive in our cohort. Moreover, all TP53 mutations were located in its DNA binding domain. Three out of 6 BRAF mutations were predicted to cause a V600E amino acid change. We focused on these 6 RSMGs in all further analyses. Correlating mutational status with Progression Free Survival (PFS) and Overall Survival (OS) showed that TP53 mutated patients had a significantly shorter PFS compared to those with wildtype TP53 (adj. p-value=0,018; n=7 versus n=95). Comparing the mutational status of the 6 RSMGs, transplant versus non-transplant protocol, number of mutated genes in the M3 P panel, del17p and t(4;14) status, EMC92 score and ISS stage between patients with a PFS <=1 year and >1 year (n=23 versus n=79), only showed a significant correlation with TP53 mutational status (adj. p-value=0,012). TP53 mutational status remained the only significant prognostic factor when comparing patients with an OS <=1 year and >1 year (adj. p-value=0,003; n=13 versus n=89). When comparing the number of mutated genes, del17p and t(4;14) status, EMC92 score, transplant versus non-transplant protocol and ISS stage between TP53 mutated and wildtype MM, TP53 mutated patients had a significantly higher number of mutated genes in the M3 P panel (adj. p-value=0,001). Conclusions (1) With the M3 P Mutational Panel, we confirm the published prevalence of RSMGs in MM in our cohort of chemotherapy-naive patients. NRAS, KRAS, DIS3, FAM46C, TP53 and BRAF are the most frequently mutated genes. (2) TP53 mutational status is the strongest unfavorable prognostic factor in our cohort and it seems to be associated with greater mutational burden. Validation in a more extensive population is planned. (3) This warrants further investigation of the mutational status of these genes in larger clinical trial cohorts, enabling a more robust comparison with conventional prognostic markers in a multivariate analysis. Disclosures Broijl: Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Zweegman:Takeda: Research Funding; Janssen: Research Funding; Celgene: Research Funding. Sonneveld:Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Karyopharm: Research Funding; SkylineDx: Membership on an entity's Board of Directors or advisory committees.

Abstract Introduction: Customized gene panel sequencing is an attractive approach to genomic tumor characterization in clinical care. Based on published MM exome data we developed a MM Mutation Panel (M3 P) that includes the most commonly mutated genes, actionable drug targets, genes targeted by current standard of care (SOC) therapies and which allows tracking of clonal evolution, copy number and sample purity. Methods and Material: M3 P (v3.0) covers 88 genes (1327 amplicons, 181kb). MM samples from 504 patients (pts) have been analyzed (corresponding germline in 81%) through collaborations between Mayo Clinic and Hospital-12-de-Octubre (Madrid, Spain), the DSMM and GMMG (Würzburg, Ulm, Freiburg and Heidelberg, Germany) and the HOVON trial groups (Rotterdam, The Netherlands). The investigated cohort includes 410 untreated pts (81%), which includes a high risk cohort of 72 pts with del17p, 25 paired samples with later follow up from the same cohort, and 94 relapsed patients, of which 50 were relapsed and refractory. Results: Overall coverage per mutation averaged >500x depth. We identified 945 variants (1.9 per pt) and in 83% of the pts a mutation was found. Clonal heterogeneity was assessed with mutations ranging from 3%-100% variant reads suggesting the presence of a significant number of subclones (e.g. 21% of mutations were in < 10% of reads). The mutation incidence was compared with and closely resembles the most recent MM comprehensive genomic data from the MMRF CoMMpass study: We compare here all pts sequenced by M3 P, untreated pts sequenced by M3 P and CoMMpass: KRAS (24%/23%/24%), NRAS (20%/20%/18%), DIS3 (13%/14%/10%), BRAF (9%/7%/6%), FAM46C (6%/6%/8%) and TRAF3 (6%/6%/7%). TP53 mutation incidence, however, was significantly increased in our cohort (14%/12%/4.2%), a difference explained by the inclusion of del17p (untreated) and relapsed refractory MM in panel sequenced pts, cohorts with elevated incidences of TP53 mutations (32% / 26% respectively). Potentially actionable targets include BRAF mutationsin 43 patients (9%), with druggable p.V600E in 19 or 5%, 8 pts (2%) with FGFR3 (p.R248C and p.G375C one patient each), p.R132 mutation in 4 out of 5 IDH1 (1%) and p.R172K IDH2 mutation in 1 of 3 (1%) pts. Mutations in the MAPK pathway (NRAS, KRAS, BRAF) were detected in 59% of pts, ranging from 36% untreated MM to 72% in refractory MM. Similarly, the CRBN/CUL4B/IKZF1/IKZF3/IRF4 pathway, important for IMiD function, harbored a significant enrichment of mutations in advanced disease (6% untreated vs 17% relapsed), including CRBN mutations (0.5% vs 7%). Nine of 17 IRF4 mutations were located at the p.K123R hotspot, with minor difference between early or late disease (1% vs 3%). Notably, in 8 of 9 pts with CRBN mutation and clinical information, all were unresponsive to IMiD therapy, supporting association of these mutations with resistance to IMiDs. Conversely, M3 P genes related to other SOC therapies, including NR3C1 (targeted by steroids) and 5 proteasome subunit genes (proteasome inhibitors), were rarely mutated across the cohorts not exceeding 1% mutation incidence for each gene. Significant differences in DIS3 and FAM46C mutation incidences were observed across cohorts: DIS3 mutations are more common in untreated pts with a 1.7 fold increased predominance (14% untreated and 8% treated). FAM46C has an expected incidence of 8% but was rarely mutated in untreated del17p high risk disease with only one of 100 patients harboring both mutations. The significance of this finding needs to be determined but implies a possible overlap in function. Finally we assessed impact on survival of the mutation variants identified in 142 untreated Mayo patients and found STAT3 mutations negatively impacting PFS (p=0.034) and OS (p=0.001). This gene is rarely mutated in MM (2% of the total cohort) thus the sample size was small and this finding needs further validation. Conclusion: We here describe 504 MM patients sequenced using the M3 P gene panel, which identified mutations in >80% of investigated patients, overlaps well with published whole exome sequence data and provides clinically relevant information. New findings were the high frequency of minor clones, the relative lack of overlap of del17 and FAM46C mutation, a higher frequency of DIS3 mutation at diagnosis compared to relapse, the prognostic significance of STAT3 mutation and the frequent presence of CRBN pathway mutation in drug resistant relapsed patients. Disclosures Sonneveld: Janssen-Cilag, Celgene, Onyx, Karyopharm: Honoraria, Research Funding; novartis: Honoraria. Mai:Janssen-Cilag: Other: Travel Grant; Onyx: Other: Travel Grant; Mundipharma: Other: Travel Grant; Celgene: Other: Travel Grant. Goldschmidt:Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millenium: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Chugai: Honoraria, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Knop:Celgene Corporation: Consultancy. Kull:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Martinez-Lopez:Novartis: Honoraria, Research Funding; Bristol-Meyer Squibb: Honoraria; Celgene: Honoraria; Janssen: Honoraria. Einsele:Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Amgen/Onyx: Consultancy, Honoraria, Speakers Bureau. Raab:Novartis: Research Funding. Stewart:Oncospire Inc.: Equity Ownership; Celgene: Consultancy; Novartis: Consultancy; BMS: Membership on an entity's Board of Directors or advisory committees.