Abstract: Smoldering myeloma (SMM) is associated with a high-risk of progression to myeloma (MM). We report the results of a study of 82 patients with both targeted sequencing that included a capture of the immunoglobulin and MYC regions. By comparing these results to newly diagnosed myeloma (MM) we show fewer NRAS and FAM46C mutations together with fewer adverse translocations, del(1p), del(14q), del(16q), and del(17p) in SMM consistent with their role as drivers of the transition to MM. KRAS mutations are associated with a shorter time to progression (HR 3.5 (1.5–8.1), p  = 0.001). In an analysis of change in clonal structure over time we studied 53 samples from nine patients at multiple time points. Branching evolutionary patterns, novel mutations, biallelic hits in crucial tumour suppressor genes, and segmental copy number changes are key mechanisms underlying the transition to MM, which can precede progression and be used to guide early intervention strategies.

Abstract: Deciphering Multiple Myeloma evolution in the whole bone marrow is key to inform curative strategies. Here, we perform spatial-longitudinal whole-exome sequencing, including 140 samples collected from 24 Multiple Myeloma patients during up to 14 years. Applying imaging-guided sampling we observe three evolutionary patterns, including relapse driven by a single-cell expansion, competing/co-existing sub-clones, and unique sub-clones at distinct locations. While we do not find the unique relapse sub-clone in the baseline focal lesion(s), we show a close phylogenetic relationship between baseline focal lesions and relapse disease, highlighting focal lesions as hotspots of tumor evolution. In patients with ≥3 focal lesions on positron-emission-tomography at diagnosis, relapse is driven by multiple distinct sub-clones, whereas in other patients, a single-cell expansion is typically seen ( p   〈  0.01). Notably, we observe resistant sub-clones that can be hidden over years, suggesting that a prerequisite for curative therapies would be to overcome not only tumor heterogeneity but also dormancy.

Abstract: Introduction Although fusion genes other than the immunoglobulin (Ig) translocation, t(4;14), which results in IGH-WHSC1fusions, are not frequently detected in multiple myeloma (MM), recent evidence suggests that kinase fusion gene fusions do occur relatively frequently and may inform on treatment algorithms. Here we use a hybrid-capture based, next-generation sequencing assay to survey fusion genes in patients with MM. Methods We report on 1421 samples from 958 individuals diagnosed with either monoclonal gammopathy of unknown significance (MGUS), smoldering multiple myeloma (SMM) or MM who underwent targeted sequencing with the FoundationOne Heme® (F1H) assay. Tumor samples were obtained from bone marrow aspirates, enriched by CD138+selection using magnetic beads (AutoMACs, Miltenyi Biotech, Cologne, Germany or RoboSep, StemCell Technologies, Vancouver, Canada). RNA and DNA were extracted using the AllPrep DNA/RNA mini kit (Qiagen, Hilden, Germany), RNeasy RNA extraction kit (Qiagen) or Puregene DNA extraction kit (Qiagen). ≥ 50 ng of extracted DNA or RNA was processed on the F1H assay. The current assay analyzes the complete coding DNA sequence of 405 genes, as well as selected introns of 31 genes involved in chromosomal rearrangements as well as the RNA sequence of 265 commonly rearranged genes resulting in gene fusions. Genes included in this assay encode known or likely targets of therapy, either approved or in clinical trials, or are otherwise known drivers of oncogenesis. Sequencing was to an average depth of 510x and was performed using the Illumina HiSeq 2500. Sequences were analyzed for selected gene rearrangements including fusion genes which were detected by a combination of DNA and RNA sequencing. Results Rearrangements into the Ig loci were detected and included the 5 main translocations: t(4;14), t(6;14), t(11;14), t(14;16), and t(14;20), as well as translocations involving MYC at 8q24. From a combination of DNA capture and RNA-seq expression values we used 107 samples in a training set with matching gene expression profiling data to determine cut-offs for FGFR3, WHSC1, CCND3, MAF, MAFB, CCND2 and CCND1to stratify patients into the 5 main translocation groups. We used these values to classify a further 391 samples with corresponding gene expression profiling (GEP) data, resulting in sensitivities and specificities of t(4;14), 98% and 100%; t(6;14), 100% and 99%; t(11;14), 99% and 95%; t(14;16), 77% and 100%; t(14;20), 100% and 100%, respectively. 40 non-Ig rearrangements were detected in 38 patients (4.2%), of which 21 in-frame fusion genes were predicted. Recurrent fusion-genes, identified in more than one patient, included EIF4E3-FOXP1, TXNDC5-MYC and SUB1-WHSC1. As well as TXNDC5, MYC was also partnered with FOXO3, both of which are known partners of the MYC translocation. 12 of the 21 in-frame fusion genes involved kinase domains, including fusions with BRAF (n=4), NTRK3 (n=2), ALK (n=1), ROS1 (n=1), MAPK14 (n=1), MAP3K14 (n=1), FGFR1 (n=1), and DLG2 (n=1). Fusions involving each of these genes have been documented in other cancers. BRAF fusions are thought to partner with genes encoding homodimerization domains, resulting in downstream activation of Ras signaling. Other kinase fusions result in receptor signaling and downstream activation of the Ras signaling pathway. Of the patients with kinase fusions, 2 had an activating KRAS, NRAS or BRAF mutation but only one was clonal (84% cancer clonal fraction). One patient with samples taken at different timepoints had a GTF2I-BRAF fusion and concomitant KRAS G13C mutation (16% allele frequency), both of which were not detectable 8 months later but an AGK-BRAF fusion was detected at that time suggesting clonal selection. Conclusion Non-Ig fusion genes are present in myeloma patients, but at a low frequency. Most of the fusions detected contained a kinase domain indicating activation of the Ras signaling pathway, which is also activated through KRAS, NRAS and BRAF mutations in 50% of patients. Although rare (1%), these kinase fusions are potential clinical targets in myeloma where kinase inhibitors, such as crizotinib, can be used which has shown to be effective against ALK and ROS1 fusions. Disclosures He: Foundation Medicine, Inc: Employment, Equity Ownership. Zhong:foundation medicine: Employment. Bailey:Foundation Medicine, Inc: Employment, Equity Ownership. Vergillo:Foundation Medicine, Inc: Employment. Ross:Foundation Medicine, Inc: Employment. Miller:Foundation Medicine: Employment, Equity Ownership. Stephens:Foundation Medicine: Employment, Equity Ownership. Mughal:Foundation Medicine: Employment, Equity Ownership. Davies:Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Morgan:Janssen: Research Funding; Univ of AR for Medical Sciences: Employment; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria.

Abstract: Introduction Achieving complete remission (CR) improves outcomes in multiple myeloma (MM). We have shown that 50% of patients enrolled in Total Therapy (TT) trials achieve CR within one year of enrollment irrespective of risk as defined by the GEP70 risk signature. Nevertheless, the majority of patients with high risk (HR) MM show an early relapse with grim prognosis, while most standard risk (SR) patients tend to relapse late, most often years after completion of maintenance therapy. Evaluation of minimal residual disease (MRD) has been shown to be of prognostic relevance in patients in CR with MRD negativity being associated with better outcomes. Here we report the impact of next generation sequencing (NGS)-based MRD assessment in TT patients who have achieved at least a very good partial response (VGPR) at two different time points during their treatment protocol: first after auto stem cell transplant (ASCT at 4-8 months of enrollment) and secondly during maintenance therapy (12-24 months). Materials and methods In brief, our TT protocols incorporate induction therapy consisting of Velcade and Thalidomide in conjunction with chemotherapy (Cisplatin, Doxorubicin, Cytoxan, Etoposide) followed by ASCT, consolidation and three years of maintenance with Velcade, Revlimid and Dexamethasone. HR and SR were assigned using the GEP70 risk signature. For MRD testing, we included 119 patients who were treated on our TT3b -TT6 protocols and who achieved at least VGPR and had bone marrow samples available at 4-8 months post ASCT and 12-24 months into maintenance. Thirty-eight patients had HR MM (32%), 75 patients had SR MM (63%) and no GEP70 data was available for 6 patients (5%). For NGS-based MRD assessment (Adaptive Biotechnologies Corp), genomic DNA was amplified using locus-specific primer sets for immunoglobulin heavy-chain complete (IGH-VDJH) and incomplete (IGH-DJ) as well as for immunoglobulin κ locus (IGκ). The amplified products underwent sequencing and clonal gene rearrangements were analyzed. MRD levels were calculated at a sensitivity level of 1 x 10-5. PET-CT and/or MRI were obtained at same time point as MRD assessment to evaluate presence of focal lesions. Results Using MRD status post-ASCT and during maintenance as a predictor, there were significant differences for PFS and OS in HR and SR MM. In HR disease, MRD positivity post ASCT was associated with significantly worse clinical outcomes with 2 year PFS/OS at 33% and 52% for MRD positive patients compared to 82%and 83% in MRD negative patients. Further 5 year PFS and OS for the same patient group were 11% and 24% for MRD positivity compared to 45% and 75% for HR patients that had achieved MRD negativity. In contrast to the HR group, there was no significant difference between MRD positivity and negativity in SR patients at the time point post ASCT we have investigated. In this latter patient group a significant difference only became obvious later during maintenance with 5 year PFS and OS at 55% and 63% for MRD positive patients compared to 83% and 93% for MRD negative patients. Of the 54 SR patients that were MRD positive post ASCT, 25 (46%) became negative later during maintenance, while in HR disease only 4 of 21 MRD positive patients post ASCT became negative during maintenance (19%). Importantly, a stratified analysis of our MRD data showed that even during maintenance the majority of cases in the favorable CD-2 subgroup were MRD positive, indicating that MRD results should further be interpreted in the context of patients' molecular subgroup. Conclusions Our data suggest that HR patients that do not achieve MRD negativity after their first ASCT have a very high likelihood of disease progression and death within 24 months. In contrast, SR patients that are still MRD positive post ASCT tend to have continuing response to treatment and MRD testing only becomes prognostic during maintenance. Of interest is that a high proportion of HR patients that achieve MRD negativity post ASCT and a smaller proportion of MRD negative SR patients still relapse within 5 years of enrollment suggesting that remaining residual MM cells are not detected by molecular MRD testing. Imaging studies with PET-CT and MRI could further stratify these patients as they detect any residual focal lesions that are not assessed by molecular MRD testing. We have combined PET-CT/MRI data for all of the 119 patients that were included in this study and analyzed data will be presented at ASH 2016. Disclosures Carlton: Adaptive Biotechnologies: Employment, Equity Ownership. Kong:Adaptive Biotechnologies Corp: Employment, Equity Ownership. Moorhead:Adaptive Biotechnologies: Employment. Barlogie:Signal Genetics: Patents & Royalties. Davies:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Morgan:Univ of AR for Medical Sciences: Employment; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria; Janssen: Research Funding.

Abstract: Introduction Achieving complete remission (CR) improves survival outcomes in multiple myeloma (MM). Applying more sensitive flow-cytometry or PCR based minimal residual disease (MRD) testing further improves prognostication and MRD negativity confers better outcomes among CR patients. In our Total Therapy (TT) trials, gene expression profiling based risk (GEP) identifies 15% patients with high risk (HR) MM whose clinical course is characterized by early relapsing disease and prognosis has remained grim despite advances in therapy. Of interest is whether high-risk (HR) patients fail to achieve MRD negativity leading to relapse. We have determined response rates across the total therapy (TT) trials and in this study we report the impact of molecular negativity by MRD assessment in patients who have achieved at least very good partial response (VGPR) or CR at 4-8 months and 12-24 months, respectively, after enrollment in TT3b-TT5 from 2005-2009. We use next-generation sequencing (NGS)-based MRD assessment (Adaptive Biotechnologies) which is sensitive to 1 MM cell in 106 normal cells. Furthermore, we evaluate MRD status in long term relapse-free survivors ( 〉 6 years) to determine the importance of MRD status in prolonged remission. Materials and methods Study subjects include 591 patients enrolled in TT3b-TT5 to determine VGPR/nCR/CR by EBMT/IMWG criteria and to analyze PFS and OS of HR and SR patients defined by our GEP model. For MRD testing, we identified 102 patients from the TT3b-TT6 protocols that achieved at least VGPR and had bone marrow (BM) sample available at 4-8 months and 12-24 months after enrollment. Of these 102 patients, 14 patients had HR disease and 88 had SR. Six of 14 HR patients already had clinical relapse between 12-24 months, but were included to determine MRD at 4-8 months. Of all 102 patients, 51 are currently still in CR 6-9 years after protocol enrollment and most recent flow-cytometric MRD status was evaluated. NGS-based MRD Assessment: Genomic DNA was amplified using locus-specific primer sets for immunoglobulin heavy-chain complete (IGH-VDJH) and incomplete (IGH-VDH) as well as for immunoglobulin κ locus (IGκ). The amplified products were subjected to sequencing and clonal gene rearrangements were analyzed. Final MRD measurement was calculated at a sensitivity level of 1 cancer cell per 1 million cell equivalents. Flow Cytometry based MRD assessment: Erythrocyte-lysed BM samples were immunophenotyped by use of 8-color (CD138/CD38/CD19/CD45/CD27/CD81/CD56/CD20) staining technique. Myelomatous Plasmacells (PCs) were separated from healthy PCs by different phenotype expression and MRD was deemed negative when no myelomatous PCs were detectable at a sensitivity of 〈 10-4 to 〈 10-5. Results Response by EBMT/IMWG criteria across our TT3b-TT5 (n=591) protocols showed no significant difference between HR and SR patients by year 1 of enrollment. In the SR group (n=502) 74.9%-77.9% of patients achieve at least nCR/VGPR and 40.3%-50.4% CR. For the HR patients (n=89) the results are similar with 75%-77.9% achieving at least VGPR and 50%-50.4% CR. However, the majority of HR patients start relapsing after one year of enrollment with a relapse rate of 59% for HR at 3 years compared to only 19% for SR at the same time point, indicating that MRD positive disease likely exists at 12-24 months for those patients with relapse. NGS-based MRD assessment will answer this question and data will be available by December 2015. Fifty one patients of the initial 102 patient group continue to be in complete remission to date 〉 6 years after enrollment. Of these, 47 are MRD negative at a MRD level of 〈 10-5 (3 HR and 44 SR), two are MRD negative at 〈 10-4 (SR), leaving only 2 patients (SR) with a detectable MRD burden at 〉 10-4. Conclusions Using conventional response methods, there is no difference between HR and SR MM at 1 year after initiation of treatment. However clinical outcome for HR disease is very poor and characterized by early relapsing disease, indicating that MRD persists even when patients have achieved CR. Sequencing is the most sensitive method for MRD detection and will be used in the present study to evaluate the importance of sequential MRD testing in HR and SR disease. We show that the majority of patients achieving long term remission at 6-8 years are MRD negative indicating the importance of molecular response for long term success. Disclosures Schinke: University of Arkansas for Medical Sciences: Employment. Mitchell:Cancer Research and Biostatistics: Employment. Faham:adaptive biotech: Employment, Other: stockholders. Patel:University of Arkansas for Medical Sciences: Employment. Thanendrarajan:University of Arkansas for Medical Sciences: Employment. Mohan:University of Arkansas for Medical Sciences: Employment. Mathur:University of Arkansas for Medical Sciences: Employment. Matin:University of Arkansas for Medical Sciences: Employment. Radhakrishnan:University of Arkansas for Medical Sciences: Employment. Stephens:University of Arkansas for Medical Sciences: Employment. van Rhee:University of Arkansa for Medical Sciences: Employment. Zangari:Novartis: Research Funding; Millennium: Research Funding; University of Arkansas for Medical Sciences: Employment; Onyx: Research Funding. Jethava:University of Arkansas for Medical Sciences: Employment. Petty:University of Arkansas for Medical Sciences: Employment. Alapat:University of Arkansas for Medical Sciences: Employment. Johnson:University of Arkansas for Medical Sciences: Employment. Epstein:University of Arkansas for Medical Sciences: Employment. Barlogie:University of Arkansas for Medical Sciences: Employment. Davies:Celgene: Consultancy; Onyx: Consultancy; Janssen: Consultancy; Millenium: Consultancy; University of Arkansas for Medical Sciences: Employment. Heuck:Millenium: Other: Advisory Board; Celgene: Consultancy; Janssen: Other: Advisory Board; Foundation Medicine: Honoraria; University of Arkansas for Medical Sciences: Employment. Hoering:Cancer Research and Biostatistics: Employment. Weinhold:University of Arkansas for Medical Sciences: Employment; Janssen Cilag: Other: Advisory Board. Morgan:Weismann Institute: Honoraria; MMRF: Honoraria; CancerNet: Honoraria; University of Arkansas for Medical Sciences: Employment; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract: Hits in driver genes and bi-allelic events affecting tumor suppressors increase apoptosis resistance and proliferation rate–driving relapse. Excessive biallelic inactivation of tumor suppressors in high-risk cases highlights the need for TP53-independent therapeutic approaches.

Abstract: The presence of ≥3 large focal lesions is associated with poor outcome in newly diagnosed myeloma patients. The prognostic impact of multiple large focal lesions is independent of R-ISS, GEP70, and extramedullary disease.

Abstract: Introduction Gene expression and comprehensive genomic profiling (CGP) underscore the importance of multiple myeloma (MM) being driven by diverse genomic abnormalities and are increasingly being integrated into personalized treatment algorithms to optimize clinical outcomes, in particular that of high risk disease. Furthermore, CGP allow for ultra-deep sequencing of various clinically relevant and targetable genomic alterations using a single assay, with an advantage of detection of low frequency variants. Methods Samples from 578 patients (monoclonal gammopathy of undetermined significance, MGUS, (n=19); smoldering multiple myeloma, SMM, (n=42); or multiple myeloma, MM, (n=517; 87 newly diagnosed (NDMM), 107after treatment (TRMM), and 323 at relapse (RLMM)) were analyzed using the FoundationOne® Heme (F1H) assay. 50 ng of DNA and RNA from CD138+ selected cells were analyzed for genomic alterations including base substitutions, indels, copy number alterations, and rearrangements. Sequencing was performed to a median depth of 468x in 405 genes, as well as selected introns of 31 genes involved in rearrangements. Additionally, matched Gene Expression Profiling (GEP) was performed using Affymetrix U133 Plus 2 array, and GEP70-defined risk status and molecular subgroups were calculated. Results Results of the F1H assay revealed the most common alterations in MM to be: KRAS (28.8%), NRAS (23.2%), TP53 (17.4%), BRAF (6.8%), CDKN2C (6.0%), RB1 (5.8%), TRAF3 (5.8%), DNMT3A (3.9%), TET2 (3.7%) and ATM (2.5%), including mutations, homozygous loss and rearrangements. When these frequencies were split across GEP70 risk groups, TP53, CDKN2C/FAF1, RB1, and the t(4;14) were significantly different (p 〈 0.05). As the disease progressed from MGUS to relapse, the number of mutations showed an increasing trend. Likewise, there were significant differences in the number of mutations between CCND1/CCND3 (CD-1) and low bone disease, CD-1 and hyperdiploid, and hyperdiploid and proliferation groups. In order to identify independent prognostic genomic alterations, we performed a multivariate Cox regression analysis on all the gene alterations that were present in at least 5% of the patient cohort, resulting in identification of four significant alterations: the t(4;14), mutation/loss of TP53, CDKN2C/FAF1 or RB1. Alterations in CDKN2C and RB1 were associated with the PR group. When the MM samples were split according to type (NDMM, TRMM, RLMM) the effect on survival of each of these alteration was more pronounced at relapse, but still present at diagnosis for CDKN2C and t(4;14). Bi-allelic events in CDKN2C, TP53 and RB1 were examined, by both homozygous deletion and monosomy with accompanying mutation, showing the rate of inactivation increased from 9.2% in NDMM to 17.9% at relapse, indicating that bi-allelic inactivation of these genes are correlated with relapse. CDKN2C and TP53 are known prognostic markers but the prognostic significance of RB1 has been debated. Previous data have shown that the association of t(4;14) with del(13q) results in insignificance of del(13q) as a prognostic marker in multivariate analyses. Here, we confirmed that the prognostic effect of RB1 is not due to association with t(4;14), and show that patients with either the t(4;14) or alteration of RB1 have a poor prognosis, which is worse when both lesions are present. Conclusions Using the F1H assay, we establish the mutational spectrum in MM, identifying lesions associated with high risk. This is the first study in MM to identify and confirm the poor prognostic effect of RB1 driven by bi-allelic inactivation, which is more prevalent at relapse. Furthermore, we determined the gene alterations that are independent prognostic markers in relapsed MM, thereby identifying novel therapeutic targets. Disclosures He: Foundation Medicine, Inc: Employment, Equity Ownership. Bailey:Foundation Medicine, Inc: Employment, Equity Ownership. Ashby:University of Arkansas for Medical Sciences: Employment. Zhong:foundation medicine: Employment. Nahas:Foundation medicine: Employment. Ali:Foundation Medicine: Employment, Equity Ownership. Vergillo:Foundation Medicine, Inc: Employment. Ross:Foundation Medicine, Inc: Employment. Miller:Foundation Medicine: Employment, Equity Ownership. Stephens:Foundation Medicine: Employment, Equity Ownership. Barlogie:Signal Genetics: Patents & Royalties. Mughal:Foundation Medicine: Employment, Equity Ownership. Davies:Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Morgan:Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Bristol Meyers: Consultancy, Honoraria; Janssen: Research Funding; Univ of AR for Medical Sciences: Employment.