ALBERT

Description: INTRODUCTION: Multiple myeloma (MM) is a biologically and clinically heterogeneous disease. Different recurrent driver genomic events have been reported, but to date no unifying feature has been identified in MM evolution. The recent interest in signatures of mutational processes through analysis of whole-exome sequencing data has led to initial insights into what generates MM mutational repertoire (Bolli et al, Nat Com 2014). Here, taking advantage of the increased power provided by whole genome sequencing (WGS), we analyzed 22 paired samples from 11 patients first at the smoldering (SMM)/MGUS stage and subsequently at the time of progression to symptomatic MM to gain a deeper understanding of the full landscape of mutational processes operative in MM, especially during their evolution over time. MATERIAL AND METHODS: DNA from bone marrow CD138+ cells underwent WGS along with a matched normal sample using HiSeq X Ten machines (Illumina, Inc.). Mutational signature extraction was performed running non-negative matrix factorization (NMF) as previously described (Alexandrov et al, Nature 2013). RESULTS: We have observed and utilized a median number of 5780 (range 2599-7760) substitutions per patient at the asymptomatic stage and 5954 (ranges 2824-8227) at progression to MM stage to extract mutational signatures. NMF extracted 5 main signatures (http://cancer.sanger.ac.uk/cosmic/signatures). Specifically, APOBEC- (signature #2) and age-related signatures (signatures #1 and #5) accounted for 13% (1-21%) and 23% (3.2-40%) of all substitutions, respectively. In addition, we found two known signatures that were not implicated in MM so far: non-canonical AID (Signature #9), contributing to 28% of all substitutions (17-55%); and signature #8, accounting for 28% of all substitutions (13-45%) and pertaining to a yet unknown mutational process. Finally, the fifth signature did not match any of the previously described ones, representing a potential novel process which we defined as MM-1 (7%, range (1-16%). Interestingly, we found a differential contribution of processes in non-coding and intronic regions where AID was more prevalent, while exonic regions where APOBEC and age signatures were more prevalent. In intronic regions we found widespread regions of kataegis (9/11 patients), reflective of localized hypermutation. In our patients, kataegis was associated with rearrangements in 60% of cases, and was present in both the SMM and MM sample in 84% of cases, suggestive of an early event during tumor development. Contrary to what is observed in solid cancers, APOBEC signature was only responsible for 25% of kataegis variants, vs 70% for AID, suggesting a causative role of aberrant AID activity in shaping the early mutational repertoire of neoplastic plasma cells. To confirm this, we looked at serial samples in our cohort. While the percent contribution of each signature varied in each patient, confirming genomic heterogeneity of MM, it did not change when paired SMM and MM samples from the same patient were compared. This shows that mutational processes required for the development of symptomatic MM act early, and have been already operative at the SMM stage. However, by clustering substitutions as clonal (early variants present at the time of tumor initiation) or subclonal (late variants arisen closer to the time of sampling) using a Bayesian hierarchical Dirichlet process (Bolli et al, Nature Comms 2014), we could analyze processes operative before SMM was diagnosed. NMF analysis of these clusters reported striking differences. Specifically, AID and age were the predominant mutational processes in early substitutions in all patients, contributing to a median of 35% (25-54%) and 30% (15-43%) of variants respectively. Conversely the contribution of AID was minimal among late substitutions (5%, 1-22%), where instead APOBEC, Signature #8 and MM-1 activity was prominent [19% (1-43), 38% (8-73%), 16% (2-50%) respectively]. CONCLUSION: WGS data allowed the identification of mutational processes operative well before MM becomes clinically evident. Our observation that all samples have signs of aberrant AID at the time of tumor initiation supports a unifying model where MM precursors are initially transformed with the contribution of AID, providing a fertile ground for other later processes (i.e APOBEC and signature #8) to act and shape the final genomic landscape of overt multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Description: Rearrangements of the c-myc oncogene have been found in most plasmacytomas induced in mice and human myeloma cell lines (HMCLs) analyzed so far. However, neither induced mouse plasmacytomas nor HMCLs represent relevant models for human multiple myeloma (MM). To evaluate the incidence of c-mycrearrangements in human plasma cell dyscrasias, sets of probes were generated to allow direct assessment of c-myctranslocations on interphase plasma cells by using fluorescence in situ hybridization. After validation of these probes, a large cohort of patients with either newly diagnosed MM (n = 529), relapsed MM (n = 58), primary plasma cell leukemia (PCL; n = 23), monoclonal gammopathy of undetermined significance (n = 65), or smoldering MM (n = 24) were analyzed. C-myc rearrangements were identified in 15% of patients with MM or primary PCL, independently of the stage of the disease (ie, diagnosis or relapse and MM or primary PCL). Analysis of the 2 main translocations observed on karyotyping, ie, t(8;14) and t(8;22), revealed that these specific translocations represented only 25% (23 of 91) of c-mycrearrangements. c-myc rearrangements were then correlated with several other patients' characteristics: illegitimateIgH recombinations, chromosome 13 deletions, and serum β2-microglobulin levels. The only significant correlation was with a high β2-microglobulin level (P = .002), although a trend for association with t(4;14) was observed (P = .08). Thus, c-myc rearrangement analysis in patients with MM revealed a strikingly lower incidence than that in HMCLs and plasmacytomas induced in mice, indicating that data obtained with these models cannot be directly extrapolated to human MM.

Description: Epigenomic changes have become an important component of cellular regulation and ultimately, of our understanding of oncogenomics in Multiple Myeloma (MM) as well as in other cancers. In recent years, both clinical and preclinical studies have confirmed that MM is vulnerable to epigenetic intervention, with histone deacetylases (HDACs) emerging as the most promising epigenetic targets. Although Pan-HDAC inhibitors are effective as therapeutic agents, there is increasing emphasis on understanding the biological and molecular roles of individual HDACs. Here we have evaluated the role of HDAC8, a member of Class I HDAC isoenzymes in MM. First, we evaluated the expression of HDAC8 in 172 newly-diagnosed MM patients from the IFM myeloma dataset and observed HDAC8 overexpression as well as its significant correlation with poor survival outcome (p

Description: Although most multiple myeloma (MM) cases are characterized by the detection of a monoclonal immunoglobulin in the serum, about 15% of the patients present only immunoglobulin light chains, detected either in the urine or serum or both. These patients are designated as having light-chain (LC) MM. Using fiber-fluorescent in situ hybridization, and in contrast to patients and myeloma cell lines secreting heavy chains (who presented a legitimate functional IgH rearrangement in every case), LC MM never displayed a functional IgH recombination. Interestingly, most LC MM cases presented one IgH allele with a germline configuration (including the DJ region), the second allele being usually involved in an illegitimate recombination. Of note, most of these translocations occurred close to (or at) switch regions, even though in some cases, breakpoints involving nonswitch regions were observed. Thus, this study clearly showed that LC MM is due to the absence of legitimate IgH rearrangement at the DNA level, reflecting possible abnormalities in the IgH gene recombinations during B-cell maturation. Furthermore, it showed that this defect did not prevent the activation of the switch process because most of 14q32 translocations observed in LC MM occurred at switch regions.

Description: Pharmacogenomic profiles of genes involved in bortezomib - dexamethasone response may help to understand resistance and could provide new therapeutic targets as well as contributing to novel prognostic markers in multiple myeloma. We have used gene expression profiling to analyze the complex signaling pathways regulating the response to bortezomib - dexamethasone. Gene expression profiles were established in 9 cell lines, derived from 9 myeloma patients, incubated or not with a combination of bortezomib 10 nM and dexamethasone 1 μM. These concentrations correspond to the ones used for patients in the IFM 2005-01. Cells were collected after 6 hours of treatment. We focused our interest in early response genes, making the hypothesis that the comprehension of early effects would help to better understand the mechanisms of resistance that take place in at least two third of myeloma patients. Supervised analysis with permutations identified significantly up regulated genes involved in stress responses (heat shocks proteins, RTP801/dig2/REDD1/DDIT4), endoplasmic reticulum stress (HERP/HERPUD1, gadd145/CHOP/DDIT3), ubiquitin/proteasome pathway (proteasome 26S subunits PSMB7, PSMC4, PSMD3 and PSMD13), unfolded protein response (such as SQSTM1, ATF4) or redox equilibrium (PLRX, PRDX1). We assumed that these genes might represent a molecular signature of response to bortezomib and provide important insight into the complex mechanisms of action of these drugs. We focused on REDD1 a gene cloned in 2002 that is known to be rapidly induced by a wide variety of stress conditions (arsenic, hypoxia, dexamethasone, thapsigargin, tunimycin and heat shock) and DNA damages (ionizing radiation, ultraviolet radiation, DNA alkylant). We found that both REDD1 gene and protein expression were early and highly induced after bortezomib exposure alone or in combinaison with dexamethasone. This effect was dependent upon cell line: REDD1 was overexpressed within two hours in resistant cell lines in association with a cell size decrease while in sensitive cell lines, neither REDD1 induction nor morphological changes occured. REDD1 induction was associated with the dephosphorylation of S6K1, a key substrat of mTOR, a protein kinase which controls cell growth and cell size in response to various signals. SiRNA studies confirmed that bortezomib lead to a negative regulation of mRTor activity mediated by REDD1: disruption of REDD1 abrogates both S6K1 phosphorylation and early transitory cell size reduction. Our results are in accordance with data obtained in mouse showing an early regulation of mTOR pathway and cellular proliferation induced by REDD1 expression in response to stress. Our study suggests that mTOR regulation could be a resistance mechanism mediated by REDD1 expression. As we found that REDD1 was differentially induced in primary plasma cells from patients, this gene expression could help to predict response to bortezomib. Our objective is now to clarify the pathway that links bortezomib to REDD1 in multiple myeloma and to investigate REDD1 expression in patients enrolled in IFM 2005-01 clinical trial.

Description: Key Points The majority of mutations are found in genes that have low or no detectable biological expression. Mutated genes often show differential allelic expression in multiple myeloma patient samples.

Description: Progress in the treatment of multiple myeloma (MM) has increased extent and frequency of response, as well as prolonged progression-free (PFS) and overall survival (OS). Today complete remission (CR) rates up to 70% are achieved with new drug combinations. This has lead to development of sensitive next generation sequencing (NGS) -based methods to predict deeper responses that may more accurately predict survival outcomes in MM. Our large recent study has confirmed the clinical impact of achieving MRD- status in MM. Here we are evaluating the genomic alterations that may predict attainment of MRD negative status in MM. MRD status was evaluted in 279 patients from IFM/DFCI 2009 trial. We obtained gene expression by RNA-seq, and copy number profile by cytoScan HD array to evaluate genomic differences between MRD negative and MRD positive groups. We generated copy number data for 175 / 279 patients (72 MRD- and 103 MRD+) with Affymetrix Cytoscan HD array and compared genome wide copy number alterations. We observed statistically significant copy number alterations in chromosome 1p, 2, 4q, 11q, 13, 14 and 20 between MRD- and MRD+ patients. However, the extent of alterations in these regions is limited. The largest difference was on chromosome 11q arm where MRD- patients had 2.2 copies on average and MRD+ had 2.4 (p value 〈 0.001). Similarly, we generated gene expression profiles with RNAseq for 69 MRD- patients and 92 MRD+ patients to study gene expression alterations that may predict attainment of MRD negative status and to examine possible biological pathways. Although first two component of principle component analysis (PCA) showed that two groups have similar expression profile, we were able to identify 586 differentially expressed genes; 333 of those were up and 253 were down regulated in MRD+ compared to MRD- groups. We found that seven oncogenes (CCND1, CD79B, IDH1, PATZ1, PAX5, POU2AF1, RUNX1) were significantly high in MRD+ and two (CCND2 and MYCN) were high in MRD-. Additional genes that were high in MRD+ samples were enriched in genes regulated by NF-kB in response to TNF, P53 pathway, KRAS signaling and genes down-regulated in response to ultraviolet (UV) radiation. Genes that were high in MRD- compared to MRD+ were also enriched in genes up-regulated by STAT5 in response to IL2 stimulation, p53 pathways and networks, and genes up-regulated in response to ultraviolet (UV) radiation pathways. Finally, we have created a signature to predict MRD+ and MRD- in MM samples from differentially expressed genes. We used 40 genes that has at least 2 fold change difference between MRD+ and MRD- groups as a predictor and we randomly separated 161 RNAseq samples into train (n=99) and test group (n=62). We developed our classifiers with diagonal discriminant analysis and we achieved 0.79 classifier performance on test dataset. Then we tested our signature against 1000 random signature and it was significantly different than random signatures (Figure). In conclusion, we here report a first genomic landscape predictive of minimal residual disease (MRD) in Multiple Myeloma (MM). This analysis will help understand genomic and molecular correlates of achieving minimal residula disease and confirms feasibility of using RNAseq data from diagnosis sample to predict MRD status. The ongoing integration of other genomic correlates such as copy number status as well as alternate splicing may allow further improvement in the performance of prediction. Figure 1. Figure 1. Disclosures Anderson: Gilead: Consultancy; acetylon pharmaceuticals: Equity Ownership; Oncocorp: Equity Ownership; Celgene Corporation: Consultancy; BMS: Consultancy; Millennium: Consultancy. Attal:jansen: Honoraria; celgene: Membership on an entity's Board of Directors or advisory committees. Munshi:onyx: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; novartis: Membership on an entity's Board of Directors or advisory committees.

Description: Long intergenic non-coding RNA (lincRNA) are transcripts longer than 200 nucleotides which have a diverse sets of regulatory functions but do not get translated into protein. lincRNAs are located between the protein coding genes and do not overlap exons of either protein-coding or other non-lincRNA. However precise role of individual lincRNA in disease biology remains unclear. Here, we have evaluated the lincRNA expression and their potential biological functions in MM. We performed RNA-seq on CD138+ MM cells from 296 newly diagnosed patients and 16 normal bone marrow plasma cells (NBM) and analyzed for lincRNA expression. Data from paired-end RNAseq reads were mapped to the latest human genome, differentially expressed lincRNAs were identified and for each expressed lincRNA event free survival was examined with univariate cox regression model and support vector machine. Finally, we identified protein coding genes that are strongly correlated (cor 〉 0.5) with lincRNAs with significant altered expression in MM and impact on EFS to identify their biological role. lincRNA and protein coding genes that have more than 10 reads/million reads for at least 15 normal samples or 62 MM samples (20% all MM samples) were included in the analysis. We identified 60 differentially expressed lincRNA (adj p value 0.5), affecting immune system pathways and pathways in cancer including Jak-STAT signaling pathway. We also found that these lincRNAs are also highly correlated with tumor development genes such as TNFRSF1B,FGR,TP53BP2,TNF and T or B cells related genes PIK3CD, BCL6. In addition, two of these lincRNAs (LINC00936 and CTB-61M7.2) were found highly correlated with their protein coding neighbor genes ATP2B1(cor = 0.45) and FCAR (cor = 0.95) respectively and MIR22HG was host gene for mir22 which may indicate lincRNAs are using different machinery in MM to regulate protein coding genes. In summary, we report that lincRNA is differentially expressed and prognostically significant in myeloma and may function through their impact on immune system and tumor progression. Our ongoing integrative approach will provide further evidence of their regulatory role in MM with potential therapeutic application. Figure 1. Figure 1. Disclosures Anderson: acetylon pharmaceuticals: Equity Ownership; Celgene Corporation: Consultancy; Gilead: Consultancy; Oncocorp: Equity Ownership; Millennium: Consultancy; BMS: Consultancy. Munshi:onyx: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; novartis: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 2960 Despite advances and significant improvement in survival, multiple myeloma (MM) remains incurable and nearly all patients relapse after treatment. In order to gain insights into the genomic lesions associated with acquisition of drug resistance and progression of disease, we performed high resolution genome-wide single nucleotide polymorphism and copy number analyses on matched diagnostic and relapse samples from 24 MM patients either treated with proteasome inhibitor (bortezomib)-based induction regimen (n=12) or conventional chemotherapeutic agents (n= 12). All relapse samples have a clear relationship to the diagnosis clone. The vast majority of patients (92%) acquired additional copy number abnormalities (CNAs) or uniparental disomy (UPD) at relapse or exhibited change in the pattern of lesions present at diagnosis. Of these, 45% acquired new lesions and 41% both acquired new lesions and lost lesions present at diagnosis. Remarkably, loss of lesions at relapse was significantly associated with initial bortezomib treatment (8 out of 12 versus 1 out of 12; P = 0.009). Moreover, in 75% of the bortezomib-treated MM, the lesions lost at relapse included either UPD or deletion providing direct evidences that the relapse clone arose from a common prediagnosis clone present as a minor population at diagnosis that acquired additional abnormalities before emerging as the relapse clone. These results suggest that resistance to novel therapeutic agent known to target MM cells in the bone marrow milieu preferentially goes through with selection of minor prediagnosis clone while escape to conventional chemotherapeutic agents is almost exclusively associated with clonal evolution from diagnosis clone. These data support the proposal to combine several anti-myeloma drug upfront in order to obtain long-term remissions. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau.