ALBERT

Beschreibung: The ataxia telangiectasia and RAD3-related (ATR) protein kinase is a component of the cellular DNA damage response pathway and promotes cell survival by signalling repair of collapsed replication forks generated by replication stress. We hypothesised that inhibition of ATR potentiates the anti-leukaemic activity of chain terminating nucleoside analogues used in the treatment of acute myeloid leukaemia (AML). We used VE-821 and its derivative VX-970 (Vertex Pharmaceuticals, Abingdon, UK) as potent and specific inhibitors of ATR kinase activity to examine the effects of ATR inhibition in AML cell lines, primary AML cells and AML xenografts. Co-treatment with 1mM VE-821 did not consistently potentiate the anti-proliferative effects of cytarabine, clofarabine or fludarabine in a panel of AML cell lines. However, there was consistent potentiation of hydroxyurea and gemcitabine in all 7 AML cell lines tested. Treatment with hydroxyurea, which induces replication stress via depletion of dNTPs, resulted in phosphorylation of CHK1, a downstream target of ATR. CHK1 phosphorylation was attenuated when 1mM VE-821 was co-administered with hydroxyurea. Exposure of cells to gemcitabine or hydroxyurea slowed transit through S phase, which was pronounced in combination with VE-821. HL-60 AML cell clones expressing either a constitutively active or inducible shRNA construct targeting ATR had reduced ATR protein expression compared to control cells and were significantly more sensitive to the anti-proliferative effects of gemcitabine and hydroxyurea, but not to cytarabine, clofarabine or fludarabine. The growth inhibitory effects of hydroxyurea and gemcitabine were also significantly potentiated by VE-821 in primary AML patient samples, which included three adult patients with de novo AML and a paediatric patient with therapy-related AML. In contrast, ATR inhibition did not potentiate the inhibitory effects of hydroxyurea or gemcitabine in primary bone marrow cells from healthy donors ex vivo. We next sought to determine whether ATR inhibition potentiated hydroxyurea and gemcitabine in an orthotopic mouse model of AML. MV4-11 AML cells engineered to express firefly luciferase (MV4-11 pSLIEW) were intrafemorally transplanted into immunodeficient Rag2-/- gc-/- mice. Bioluminescent imaging via IVIS Spectrum (PerkinElmer, Buckinghamshire, UK) demonstrated localised femoral engraftment first detectable 4-5 days post-injection, with luciferase signal developing in other parts of the body (liver, ovaries) between days 15 and 18 in untreated mice. Treatment was initiated 7 days post-injection when disease was localised to the femur and prior to emergence of disseminated luciferase signal. Single agent hydroxyurea (250mg per kg, IP days 0-4 and 7-11) conferred some early disease control compared to controls as determined by luciferase total body flux measured on day 14, but this was not statistically significant (p=0.18) and did not affect overall survival (mean 35 days for controls and 37 days for hydroxyurea, p=0.47). Monotherapy with VX-970 (60 mg per kg, orally on days 0-4 and 7-11) also conferred early disease control compared to vehicle-treated mice (p=0.18), and resulted in significantly longer overall survival (mean 40 days, p=0.017). Combination treatment with hydroxyurea and VX-970 did not result in more effective early disease control or improved overall survival compared to monotherapy with either agent. Treatment with gemcitabine monotherapy (100 mg per kg, intraperitoneal injection on days 0, 3, 7 and 10) conferred significant early disease control (p=0.002) and significantly improved overall survival compared to controls (mean survival 73 days, p

Beschreibung: Introduction A significant proportion of myeloma patients relapse early and show short survival with current therapies. Molecular diagnostic tools are needed to identify these high risk patients at diagnosis to stratify treatment and offer the prospect of improving outcomes. Two validated molecular approaches for risk prediction are widely used: 1) molecular genetic risk profiling [e.g. del(17p), t(4;14)] 2) gene expression (GEP) risk profiling, [e.g. EMC92 (Kuiper et al., Leukemia 2012)]. We profiled patients from a large multicentric UK National trial using both approaches for integrated risk stratification. Methods A representative group of 221 newly diagnosed, transplant eligible patients (median age 64 years) treated on the UK NCRI Myeloma XI trial were molecularly profiled. DNA and RNA were extracted from immunomagnetically CD138-sorted bone marrow plasma cells. Molecular genetic profiles, including t(4;14), t(14;16), Del(17p), Gain(1q) were generated using MLPA (MRC Holland) and a TC-classification based qRT-PCR assay (Boyle EM, et al., Gen Chrom Canc 2015, Kaiser MF, et al., Leukemia 2013). GEP risk status as per EMC92 was profiled on a diagnostic Affymetrix platform using the U133plus2.0-based, CE-marked MMprofiler (SkylineDx) which generates a standardised EMC92 risk score, called 'SKY92'. Progression-free (PFS) and overall survival (OS) were measured from initial randomization and median follow-up for the analysed group was 36 months. Statistical analyses were performed using R 3.3.0 and the 'survival' package. Results were confirmed in an independent dataset, MRC Myeloma IX, for which median follow-up was 82.7 months. Results Of the 221 analysed patients, 116 were found to carry an established genetic high risk lesion [t(4;14), t(14;16), del(17p) or gain(1q)]. We and others have recently demonstrated that adverse lesions have an additive effect and that co-occurrence of ≥2 high risk lesions is specifically associated with adverse outcome (Boyd KD et al, Leukemia 2011). 39/221 patients (17.6%) were identified as genetic high risk with ≥2 risk lesions (termed HR2). By GEP, 53/221 patients (24.0%) were identified as SKY92 high risk. Genetic and GEP high risk co-occurred in 22 patients (10.0%), 31 patients (14.0%) were high risk only by GEP and 17 patients (7.7%) by genetics only. SKY92 high risk status was associated with significantly shorter PFS (median 17.1 vs. 34.3 months; P

Beschreibung: Introduction. Minimal residual disease (MRD) is a powerful predictor of outcome in multiple myeloma (MM). We have previously demonstrated, in transplant eligible patients, that the level of MRD as a continuous variable independently predicts both PFS and OS, with approximately a one year median OS benefit per log depletion (J Clin Oncol 2013; 31:2540-7 and Blood 2015; 125:1932-5). The impact of MRD also appears to be independent of therapy received. There is more limited data on the applicability of MRD assessment in transplant ineligible patients, largely as a consequence of low rates of CR historically within this patient cohort. Patients and Methods. In this analysis we have assessed the impact of MRD on PFS amongst patients treated within the non-intensive arm of the NCRI Myeloma XI trial. Patients were randomised between thalidomide (CTDa) and lenalidomide (RCDa) based induction therapies with responding patients being subsequently randomised to maintenance with lenalidomide monotherapy, or no further therapy. Bone marrow aspirates were obtained at the end of induction and this analysis represents a subset of 297 patients (median age 74 years). MRD was assessed using flow cytometry (sensitivity 10-4) with a minimum of 500,000 cells evaluated with six-colour antibody combinations including CD138/CD38/CD45/CD19 with CD56/CD27 in all cases and CD81/CD117 in additional cases as required. Results. Overall MRD-negativity was demonstrated in 41/297 (13.8%). When considered according to induction therapy received 25/154 (16.0%) of patients randomized to RCDa were MRD-negative compared to 16/143 (10.8%) of those randomized to CTDa (p=0.24; Fisher's exact test). MRD-negativity was associated with a significant outcome advantage as the median PFS was 34 months versus 18 months for MRD-positive patients (p

Beschreibung: Introduction Segmenting multiple myeloma (MM) into subgroups with distinct pathogenesis and clinical behavior is important in order to move forward with advancements in therapy and implement a targeted therapy approach. Current technologies have elucidated five major translocation groups, which have a varying effect on prognosis: t(4;14), t(6;14), t(11;14), t(14;16) and t(14;20) along with recurrent copy number changes including deletion of CDKN2C (1p32.3) and TP53 (17p13.1) as well as gain or amplification of 1q21. However, minor translocation and mutational groups are poorly described because sample numbers are limited in small datasets. The availability of multiple sets of high quality mutation data associated with clinical outcomes has provided a unique opportunity in MM whereby clustering mutational data with chromosomal aberrations in the context of gene expression we can develop a molecular classification system to segment the disease into therapeutically meaningful subgroups. The Multiple Myeloma Genome Project (MGP) is a global collaborative initiative that aims to develop a molecular segmentation strategy for MM to develop clinically relevant tests that could improve diagnosis, prognosis, and treatment of patients with MM. Materials and methods We have established a set of 2161 patients for which whole exome sequencing (WES; n=1436), Whole Genome Sequencing (WGS; n=708), targeted panel sequencing (n=993) and expression data from RNA-Seq and Gene Expression arrays (n=1497) were available. These data were derived from the Myeloma XI trial (UK), Intergroupe Francophone du Myeloma/Dana-Faber Cancer Institute (MA), The Myeloma Institute (AR) and the Multiple Myeloma Research Foundation (IA1 - IA8). We assembled all data on a secure site and analyzed it using a streamlined and consistent pipeline using state of the art tools. First, BAM were converted to FASTQ using Picard tools v2.1.1 to extract read sequences and base quality scores. Next, all reads were realigned to the human genome assembly hg19 using BWA-mem. Duplicate marking and sorting was performed using Picard tools v2.1.1. For QAQC we use FASTQC and Picard tools. We identified somatic single nucleotide variants and indels with Mutect2 using default parameters. Translocations and large chromosomal aberrations were identified using MANTA and breakdancer and inferred copy number abnormalities and homozygous deletions using Sequenza v2.1.2 and ControlFreeC. Results We have begun to integrate these diverse large genomic datasets with various correlates. Samples were stratified by RNA-seq expression values and WES/WGS to identify the main cytogenetic groups with high concordance. In addition to the main translocation groups, translocations into MAFA, t(8;14), were detected in 1.2% of samples by both RNA-seq and WES/WGS. RNA-seq also detected fusion transcripts, including the known Ig-WHSC1 transcript in t(4;14). However, a proportion of identified in-frame fusion genes involved kinase domains consistent with activation of the Ras/MAPK pathway, which may be clinical targets for therapy. The main recurrent mutations included KRAS and NRAS, and negative regulators of the NF-κB pathway. In addition we identified recurrent copy number abnormalities and examined the interaction of these with mutations. This highlighted the interaction of the recurrent changes at 1p, 13q, and 17p with mutation of genes located within these regions, specifically indicating bi-allelic inactivation of CDKN2C, RB1 and TP53. Using WGS and RNA-Seq data we identified recurrent translocations and fusion genes that can be used to instruct therapy. Based on these data and the presence of homogeneous inactivation of key tumor expressed genes we will present clinically relevant clusters of MM that can form the basis of future risk and molecular targeted trials. Interaction of mutation with expression patterns has identified distinct expression signatures associated with mutational groups. Conclusions We have established the largest repository of molecular profiling data in MM along with associated clinical outcome data. Integrated analyses of these are enabling generation of clinically meaningful disease segments associated with differing risk. The MGP intends to build a global network by expanding collaboration with leading MM centers around the world and incorporating additional datasets through current and new collaborations. Disclosures Mavrommatis: Discitis DX: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Employment, Equity Ownership. Ashby:University of Arkansas for Medical Sciences: Employment. Ortiz:Celgene: Employment. Towfic:Celgene: Employment, Equity Ownership; Immuneering Corp: Equity Ownership. Amatangelo:Celgene: Employment, Equity Ownership. Yu:Celgene: Employment, Equity Ownership. Avet-Loiseau:celgene: Consultancy; janssen: Consultancy; sanofi: Consultancy; amgen: Consultancy. Jackson:Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau; MSD: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau. Thakurta:Celgene: Employment, Equity Ownership. Munshi:Takeda: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Merck: Consultancy; Pfizer: Consultancy; Oncopep: Patents & Royalties. Morgan:Univ of AR for Medical Sciences: Employment; Janssen: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria.

Beschreibung: Background Lenalidomide is an effective treatment for myeloma and has been studied in a range of combination regimens worldwide. The results of these studies have suggested that prolonged exposure to lenalidomide is important to improve outcomes both as a maintenance agent post-transplant (Attal M et al NEJM 2012, McCarthy et al NEJM 2012) and in the transplant ineligible population (Palumbo A et al NEJM 2012, Benboubker L et al NEJM 2014). In the Myeloma XI study, the largest of its kind, we explored the use of oral lenalidomide continued to disease progression compared to no therapy in both newly diagnosed transplant eligible (TE) and transplant non-eligible (TNE) populations. Here we present the results of this maintenance randomization, which demonstrate the efficacy and safety of maintenance lenalidomide. Methods The Myeloma XI study is a Phase III, UK-based, multicenter, open-label, parallel group, randomized controlled trial for newly diagnosed symptomatic myeloma patients of all ages and includes a maintenance comparison of lenalidomide versus no maintenance. Newly diagnosed symptomatic myeloma patients both TE and TNE were enrolled to the study. Induction treatment in both pathways was with thalidomide or lenalidomide plus cyclophosphamide and dexamethasone, with appropriate dose reductions for TNE patients. TE patients proceded to a standard melphalan 200mg/m2 transplant. Patients were randomized to either maintenance lenalidomide or observation after achieving maximum response (TNE) or at 100 days after transplant (TE). Lenalidomide was administered at a dose of 10mg daily in 21/28 day cycles until disease progression. Dose adjustments for renal impairment and following AEs were permitted. The primary endpoints for the maintenance randomization were progression-free (PFS) and overall survival. Secondary endpoints included response, toxicity and PFS2. Time-to-event endpoints were measured from maintenance randomization. This abstract summarizes a preliminary analysis, final data will be presented at the meeting. The median follow up in this analysis is 26 months [IQR 12-41]. Results A total of 1550 patients, 828 TE and 722 TNE, median age 61 and 74 years, respectively, were randomized between lenalidomide (n=857) and no maintenance (n=693). The arms were well-balanced for clinical features and response to induction therapy (e.g. ISS stage III: 27% vs 23%, VGPR/CR: 73% vs 73%). The maintenance randomization has met its primary endpoint demonstrating a 55% reduction in risk of progression or death for lenalidomide compared to no maintenance (HR 0.45 [95%CI 0.39-0.52], median PFS 37 vs 19 months, p

Beschreibung: Background The Myeloma XI study is the first randomized study to investigate a response-adapted approach to induction therapy for newly diagnosed myeloma (NDMM). The study addresses whether, for patients achieving less than optimum response to an initial immunomodulatory (IMiD) triplet combination, defined as at least VGPR, the use of a sequential proteasome inhibitor (PI) based triplet can improve outcomes. In total 581 patients were randomized into the study which confirms the clinically significant benefit of deepening responses by utilizing treatment with a different mode of action, and that this leads to better outcomes. Methods This phase III, UK-based, multicenter, open-label, parallel group, randomized controlled trial for NDMM patients of all ages, randomized patients initially between a thalidomide or lenalidomide triplet combination with cyclophosphamide and dexamethasone. This IMiD triplet was continued for a minimum of 4 cycles (transplant eligible, TE) or 6 cycles (transplant non-eligible, TNE) and to maximum response. At the end of this IMiD regimen response was assessed. Patients with a suboptimal response (MR/PR) were randomized between further induction therapy with bortezomib, cyclophosphamide and dexamethasone (CVD) or no further induction therapy. Patients with a good response (VGPR/CR) proceeded straight to ASCT (if TE), whilst refractory (SD/PD) patients all received the CVD regimen. For patients receiving CVD, treatment was planned to continue to maximum response, and eligible patients would proceed to ASCT. The primary endpoints of the adapted approach randomization were progression-free survival (PFS) and overall survival. Secondary endpoints included upgrading of response compared to baseline and the impact of the PI combination in a high-risk subgroup. This abstract contains a preliminary analysis, final data will be presented at the meeting. The median follow up in this analysis is 29 months [IQR 17-44]. Results 581 patients (366 TE, 215 TNE) with initial response to IMiD of MR/PR were entered into the CVD randomization. In total 292/581 patients were randomized to receive sequential treatment with CVD. The arms were well-balanced with respect to clinical features and response (e.g. ISS stage: III 21% vs 19%, PR: 88% vs 88%). This randomization has met its primary endpoint. Overall the sequential use of CVD significantly improved PFS from a median of 24 to 30 months (HR 0.67 [95%CI 0.53-0.85], p=0.0005). This was largely due to a significant improvement seen in the TE pathway, HR 0.56 [95%CI 0.40-0.77], median PFS no therapy 31 months vs CVD 55 months, p=0.0003. In the TNE pathway there was an early benefit with improved median PFS 14 months vs 20 months, but similar hazard after 2 years (HR 0.83 [95%CI 0.60-1.17], p=0.297). Importantly upgrading of response was seen with 118/289 (41% [95%CI 35-47]) of evaluable patients who received CVD moving from MR/PR to VGPR/CR. 115/289 remained in the same response category MR/PR, but these patients still had a mean reduction in paraprotein during CVD of 24% [95%CI 11-17]. The upgrade in response was seen in both pathways and was not affected by the IMiD received in the initial induction randomization. The impact of CVD in cases with molecularly defined high-risk disease compared to standard and multiparameter flow cytometry assessment of minimal residual disease status will be presented at the meeting. In the transplant eligible pathway an improved depth of response persisted in the 253 patients completing ASCT with VGPR/CR responses post ASCT of 65% for those who were randomized to CVD (VGPR n=133, CR n=86) compared to 38% for those who went straight to transplant (VGPR n=120, CR n=46). Sequential CVD was well tolerated with patients receiving a median of 4 cycles of therapy (range 1-8). Relevant grade 3/4 toxicities were: neutropenia 7.1%, thrombocytopenia 7.5%, anaemia 3.1%, peripheral neuropathy 5.1%. Conclusion For the first time we have shown that the use of a response-adapted therapy based on the use of chemotherapeutic agents with a different mode of action in myeloma can improve response rates, both pre- and post-transplant and that these translate into improved PFS. On behalf of the NCRI Haem-Onc CSG Disclosures Jackson: Roche: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; MSD: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau. Davies:Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Pawlyn:Celgene: Consultancy, Honoraria, Other: Travel Support; Takeda Oncology: Consultancy. Jones:Celgene: Honoraria, Research Funding. Kishore:celgene: Other: travel grant. Garg:Janssen: Other: Travel support, Research Funding, Speakers Bureau; Takeda: Other: Travel support; Novartis: Other: Travel support, Research Funding. Williams:Takeda: Honoraria, Other: Travel support, Speakers Bureau; Janssen: Honoraria, Other: Travel support, Speakers Bureau; Celgene: Honoraria, Other: Travel support, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Novartis: Honoraria. Karunanithi:Celgene: Other: Travel support, Research Funding; Janssen: Other: Travel support, Research Funding. Lindsay:Novartis: Other: Travel support; Janssen: Consultancy; Takeda: Other: Travel support; BMS: Consultancy, Other: Travel support; Celgene: Honoraria, Other: Travel support. Jenner:Novartis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel support, Research Funding; Takeda: Consultancy, Honoraria, Other: Travel support; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Other: Travel support. Cook:Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria; Glycomimetics: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau. Kaiser:BMS: Consultancy, Other: Travel Support; Takeda: Consultancy, Other: Travel Support; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Chugai: Consultancy. Drayson:Abingdon Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Owen:Takeda: Honoraria, Other: Travel support; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Other: Travel support. Morgan:Bristol Meyers: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Research Funding; Univ of AR for Medical Sciences: Employment; Celgene: Consultancy, Honoraria, Research Funding.

Beschreibung: Introduction Epigenetic dysregulation is a hallmark of cancer and has significant impact on disease biology. The epigenetic structure of myeloma is heterogeneous and we previously demonstrated that gene specific DNA methylation changes are associated with outcome, using low-resolution arrays. We now performed a high-resolution genome wide DNA methylation analysis of a larger group of patients from a UK national phase III study to further define the role of epigenetic modifications in disease behaviour and outcome. Patients and Methods Highly purified (〉95%) CD138+ myeloma bone marrow cells from 465 newly diagnosed patients enrolled in the UK NCRI Myeloma XI study were analysed. The extracted DNA was bisulfite-converted using the EZ DNA methylation kit (Zymo) and hybridized to Infinium HumanMethylation450 BeadChip arrays. Raw data was processed using the R Bioconductor package "minfi". SNP containing probes and probes on the sex chromosomes were removed. 464 samples and 441293 probes were retained following inspection of quality control metrics. Beta values were summarized across functional genomic units or differentially methylated regions (DMRs) that included: gene bodies, promoters, insulators, CpG-islands and enhancers. K-means was applied to each DMR to cluster patients into 2 groups (high or low methylation) per region. Filters were applied to define a clinically meaningful minimum group size and methylation differences between the groups. Overall survival (OS) and progression free survival (PFS) were assessed by a Cox proportional hazards regression model fitted to each DMR with a time-dependent covariate of the trial pathway. Pathway analyses were performed using GREAT (Stanford University) and GSEA (Broad Institute). Results We identified 589 differentially methylated regions that were significantly associated with PFS and OS when using a cut-off of P

Beschreibung: Background Deletion of chromosome 17p (del17p) is detected in 10% of multiple myeloma (MM) patients at diagnosis and is associated with both a dismal prognosis and increased prevalence after treatment. Even though this suggests that it might be a driver of disease progression, relatively little is known about the genomic landscape of these tumors. With this study, we aimed to identify recurrent aberrations in a large cohort of del17p MM patients and to assess their prognostic value within this patient group. Methods The study design consisted of a discovery phase and a validation phase. For the discovery phase, 44 newly diagnosed MM (NDMM) patients were included with a del17p in at least 50% of plasma cells, as detected with fluorescent in situ hybridization (FISH). From 12 del17p patients, 2 or 3 tumor samples were available to analyze clonal evolution during disease progression. DNA was isolated from peripheral blood and CD138+ enriched bone marrow mononuclear cells, followed by a custom capture of the whole exome, Chr17p and the IgH, Igk, IgL and MYC regions (SeqCap EZ Exome Plus, Nimblegen) and paired-end sequencing. Significantly mutated genes were determined with MutSigCV and significantly deleted or amplified genomic regions with GISTIC2. Single nucleotide variants (SNVs) in TP53, FAM46C, KRAS, NRAS, DIS3 and BRAF were validated using a custom amplicon panel (TruSeq Custom Amplicon Assay v1.5, Illumina), followed by deep sequencing on a MiSeq System. Findings were validated in a cohort of 463 NDMM patients of the UK Myeloma XI trial, from which whole exome sequencing data were available for paired tumor and germline DNA (Walker et al. - J Clin Oncol 2015). All samples in both the discovery and the validation cohort were reanalyzed with one bioinformatic pipeline, from alignment to variant calling. The copy number status of TP53 was determined with Sequenza, followed by selection of del17p samples after manual inspection of the results. From a third cohort of 233 MM patients with progressive disease (PDMM) treated at the University of Arkansas for Medical Sciences (UAMS), 406 cancer-related genes were paired-end sequenced from tumor DNA with the FoundationOne Heme assay (He et al. - Blood 2016). Results In the discovery cohort, we identified a commonly deleted region (CDR) on Chr17p of 235 kb, in which one or more somatic, nonsilent aberrations (SNSA) in TP53 were detected in 25/44 (57%) patients (adj. p

Beschreibung: Introduction With a multifactorial mechanism of action and excellent PFS associated with prolonged exposure, lenalidomide (len) is an attractive candidate for maintenance therapy. Len exerts its action by interaction with cereblon (CRBN) which forms a ubiquitin ligase complex with cullin-4A (CUL4), damaged DNA binding protein 1 (DDB1) and regulator of cullins 1 (ROC1). Downstream effects are mediated via Ikaros, Aiolos, MYC, IRF4, basigin (BSG) and solute carrier family 16 member 1 (SLC16A1). The impact of selective pressure on MM clonal architecture and mutational load has not been assessed. Although not a DNA damaging agent there is an apparent effect of maintenance len increasing the risk of second cancers and a suggestion that it could select for aggressive clones in high risk disease. We addressed the hypothesis that len may increase the rate of mutation at relapse by performing whole exome sequencing (WES) on 70 paired presentation/relapse samples from patients enrolled to the Myeloma XI trial (MXI), 35 of whom received maintenance Len and 35 not. Methods WES was performed to a median depth of 125x on 70 presentation/relapse pairs from patients enrolled to the MXI trial. MXI is a phase III study comparing thalidomide, len and bortezomib induction combinations and len vs observation maintenance treatment in both transplant eligible (TE) and transplant non-eligible (TNE) NDMM patients. We selected patients who had completed induction +/- ASCT and been randomised to receive maintenance therapy with len or observation. All patients had disease progression determined by IMWG criteria at the time of the relapse sample. Of the 70 patients, 30 were enrolled in the TE pathway and 40 in the TNE pathway. The median time to relapse following maintenance randomisation was 323 days (296 len vs 325 observation). 35 patients (50%) achieved a CR as their best response, 26 (37%) a VGPR and 9 (13%) a PR. The median age was 66 and 69 for those receiving len and those being observed respectively. High risk disease status was confirmed in 33 (47%) patients at presentation (≥ 1of t(4;14), t(14;16), t(14;20), +1q, -17p, -1p). Results The median number of non-silent mutations (NSM) found at presentation and relapse was 37 and 41 respectively (p=0.25). In patients receiving len maintenance the median number of NSM at presentation was 37 vs 34 at relapse (p=0.69). In those being observed the median number of NSM at presentation was 42 vs 52 at relapse (p=0.21). Mutations in genes important in myeloma pathogenesis seen in more than one patient at presentation included KRAS (16), NRAS (14), DIS3 (6), HIST1H1E (2), RB1 (2), EGR1 (2), TP53 (2) and FAM46C (2). These were seen in a total of 37 (53%) patients. One patient had both an NRAS and KRAS mutation. At relapse 7 patients lost mutations (NRAS (3), KRAS (3), DIS3 (1)) and 6 patients gained mutations (KRAS (2), NRAS (2), TP53 (1), FAM46C(1)). Paired presentation/relapse copy number (CN) data (MLPA) was available for 38 patients (54%). At relapse there was evidence of a change in CN status with 5 (13%) patients gaining CN changes associated with high risk (gain 1q (4), del 17p (1). Six patients (9%) were found to have mutations in genes associated with len action; CRBN (1), IRF4 (1), DDB1 (2), SLC16A1 (2). No mutations were found in Ikaros, Aiolos, ROC1, CUL4 or BSG. The CRBN mutation was found at relapse only, in a patient who had achieved a CR and undergone 232 days of len maintenance. The IRF4 mutation was seen at presentation and relapse in a patient who achieved CR and received 754 days of len prior to relapse. Both patients with DDB1 mutations received len induction, ASCT, achieved CR and were randomised to observation. In one patient the mutation was seen at presentation and relapse whilst in the other only at relapse. Both patients with mutations in SLC16A1 were treated with len induction and ASCT to CR. In one patient, randomised to observation the mutation was seen at both time points and they relapsed after 156 days. The other, with the mutation present at presentation only was randomised to len maintenance and relapsed after 256 days. Conclusions This is the largest study comparing the genetics of presentation/relapse myeloma in a len treated population. Overall, the number of mutations at presentation vs relapse remained stable. We show that len does not affect the mutational load at relapse but may select for mutations conferring len resistance although at present further analysis is required to confirm this. Disclosures Jones: Celgene: Honoraria, Research Funding. Pawlyn:Celgene: Consultancy, Honoraria, Other: Travel Support; Takeda Oncology: Consultancy. Cook:Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Glycomimetics: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau. Jenner:Amgen: Consultancy, Honoraria, Other: Travel support; Janssen: Consultancy, Honoraria, Other: Travel support, Research Funding; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Other: Travel support. Drayson:Abingdon Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Davies:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Kaiser:BMS: Consultancy, Other: Travel Support; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Other: Travel Support; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Chugai: Consultancy. Jackson:Takeda: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau; MSD: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau. Morgan:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria; Janssen: Research Funding; Univ of AR for Medical Sciences: Employment.

Beschreibung: Introduction High expression of the H3K27 histone methyltransferase EZH2 mRNA in myeloma (MM) patient samples is associated with molecular features of high risk disease, including increased proliferation, and adverse outcomes (1). Mutations or deletions in the H3K27 demethylase KDM6A are associated with similar findings (2) and would be expected to have the same epigenetic effect, increasing H3K27me3 levels, a mark associated with repression of gene expression. We, therefore, sought to identify the role EZH2 plays in controlling myeloma cell proliferation. Methods A panel of MM cell lines and primary patient samples (CD138 selected from bone marrow with consent) representing a variety of different MM molecular subgroups were used. Cell viability (WST-1), cell cycle (PI) and apoptosis (AnnexinV/PI, Caspase-Glo 3/7) assays were performed. Affymetrix gene expression arrays followed by validation with RT-PCR were used to identify patterns of gene expression change with EZH2i. Western blotting confirmed changes at the protein level and Chip-PCR was performed using a validated antibody and isotype control to identify H3K27me3 changes at the relevant gene promotors. Affymetrix gene expression data for 1213 patients enrolled in the Total Therapy studies were used to investigate the relevance of our findings in myeloma patient samples. Results We confirmed a reduction in viability following EZH2i using two chemically distinct, specific small molecule inhibitors (EPZ005687 and UNC1999) and the negative control compound UNC2400. There was a reduction in viability in 6/8 cell lines and 5/6 patient samples. Response to inhibition was not related to molecular subgroup or the presence of high-risk molecular features including del17p. Global levels of H3K27me3 measured by Western blot were reduced in all cell lines regardless of response to EZH2i. In responding cell lines EZH2i induced cell cycle arrest at G1/S followed by induction of apoptosis. Gene expression arrays performed using mRNA from KMS11 and KMM1 cell lines highlighted a change in expression of cell cycle control genes associated with EZH2i. This finding was validated using qRT-PCR, which demonstrated upregulation of the cyclin dependent kinase inhibitors CDKN2B, CDKN1A or both. These findings were confirmed at the protein level by Western blotting. Chip-PCR experiment using cell lysates from KMS11 cells following incubation with EZH2i over 6 days identified changes in H3K27me3 at the promoter and transcriptional start site (PROM/TSS) regions of the CDKN2B and CDKN1A genes. The most specific changes occurred at the CDKN1A PROM/TSS, which were more heavily marked with H3K27me3 at baseline compared to a region approx. 5KB upstream. Given these results, which suggest that CDKN1A expression may be controlled by changes in H3K27me3, we explored the effect of CDKN1A mRNA expression in our patient datasets. We found the expression of EZH2 and CDKN1A to be inversely correlated (R=-0.170, p