ALBERT

Description: 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.

Description: Abstract 1820 Introduction: Since the introduction of thalidomide into the treatment of multiple myeloma (MM) immunomodulatory drugs (IMiDs) have become an essential treatment modality for the management of MM. Recently, cereblon (CRBN) has been identified as the thalidomide binding protein disrupting the E3 ubiquitin ligase complex composed by CRBN, DDB1 and Cul4 (Ito et al., Science 2010). Moreover, CRBN is essential for the antimyeloma activity of lenalidomide and pomalidomide (Zhu et al.; Blood 2011). However, the genetic and epigenetic mechanisms by which CRBN is regulated are not understood so far. Therefore, we aimed to determine if CRBN expression associates with clinically relevant subgroups and response to therapy with lenalidomide. In addition, we investigated additional regulatory layers by identifying key microRNAs (miRNAs) correlated with CRBN expression. Methods: CRBN expression was measured by real-time PCR (qPCR) using CD138 purified plasma cells from 17 patients with a monoclonal gammopathy of undetermined significance (MGUS) and 139 patients with MM. CRBN expression was normalized to GUSB (used as internal control) and to the expression levels of CRBN in normal plasma cells (n=4). MiRNA expression profiles were generated using Agilent mirRNA-arrays on patients with the highest and lowest CRBN mRNA expression levels, respectively (n=42). All patients were characterized by a comprehensive set of FISH probes for the presence of recurring cytogenetic abnormalities. Results: CRBN expression was variable in the investigated samples (median: 0.717; range: 0.078 – 5.285). In patients with MGUS (median: 0.833) CRBN expression was significantly (p=0.01) lower as compared to patients with a MM (median: 0.673). CRBN expression was associated with cytogenetic subgroups (p

Description: Abstract Introduction: Immunity and inflammatory response impact tumor microenvironment and progression of malignancies. Metabolic and inflammatory parameters of the peripheral blood, and ratios of the latter, correlate with outcome in cancer patients. There exist several established inflammation-based scores of prognostic significance including the Glasgow-prognostic-score (GPS; integrating serum-CRP (〉10 mg/L: 1pt) and albumin (〈 35g/L: 1 pt). Methods: In this retrospective multi-center study we investigated the prognostic capabilities of an integrated scoring system including GPS and information on cytogenetic high-risk aberrations as determined by FisH (CytoGPS) in transplant-eligible MM patients as a complementary resource for risk-stratification. Patients with MM admitted to our institutions between January 2000 and July 2017 were screened and established prognostic factors were assessed. CytoGPS was calculated as conventional GPS score plus one additional point for high-risk cytogenetics. Statistical evaluation resulted in three significantly divergent groups in terms of clinical outcome (Group I: 0-1 pts.; group II. 2 pts.; III: 3 pts.). Characteristics significantly associated with OS or PFS were included in a proportional-hazard-model. The study was approved by the local ethics committee. Results: Following initial assessment we identified 212 eligible and fully evaluable as well as transplant eligible patients. Centralized review of pathology and cytogenetic reports was conducted and central hematopathology assessment was performed in 163/212 (76.9%) cases. All patients included in the study proceeded to high-dose melphalan and subsequent autologous stem cell transplantation. Median age at diagnosis was 59 years (range 35 - 76 years) with a median follow-up of 76 months. Mean GPS was 0.849, with a mean CytoGPS of 0.472. Multivariate analysis revealed ISS (HR = 1.677, 95% CI = 1.035 - 2.716, p = 0.036) and CytoGPS but not R-ISS to be the only independent predictors of OS and CytoGPS constituted the only independent predictor of PFS (OS: HR = 2.172; 95% CI = 1.607 - 2.936 p = 0.001; PFS: HR = 1.517; 95% CI = 1.174 - 1.960, p = 0.001). The impact of CytoGPS on OS and PFS is presented in Figure 1. Discussion: There is growing evidence stating a drastic impact of systemic inflammatory scores and cytogenetic data on clinical outcome in various malignancies including lymphoma and solid tumors. Our data show that baseline CytoGPS, integrating both these aspects, correlates with rates of relapse and refractory disease across all primary stages of MM in transplant-eligible patients. Upon multivariate analysis these effects were preserved with prognostic impact beyond established prognosticators. CytoGPS constitutes a promising means of risk-stratification in MM requiring further validation. Acknowledgments The authors would like to thank Mr. Jan Kroenke (ASH-Member) for the sponsorship of this abstract. Figure 1: Overall and Progression-free Survival in transplant-eligible Multiple Myeloma patients according to CytoGPS (Log-rank: A: p 〈 0.0001, B: p = 0.0001). Disclosures No relevant conflicts of interest to declare.

Description: Telomerase represents an attractive target for a mechanism-based therapeutic approach because its activation has been associated with unlimited proliferation in most cancer cells. Recently, a nonnucleosidic small molecule inhibitor, BIBR1532 (2-[(E)-3-naphtalen-2-yl-but-2-enoylamino]-benzoic acid), has been identified that is highly selective for inhibition of telomerase, resulting in delayed growth arrest of tumor cells. Here we examined the effects of BIBR1532 in different leukemia cell lines as well as in primary cells from patients with acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) in short-term culture assays. We observed a dose-dependent direct cytotoxicity in concentrations ranging from 30 to 80 μM. Interestingly, cell death was not dependent on the catalytic activity of telomerase but was delayed in cells with very long telomeres. We observed time-dependent individual telomere erosion, which was associated with loss of telomeric repeat binding factor 2 (TRF2) and increased phosphorylation of p53. Importantly, the proliferative capacity of normal CD34+ cells from cord blood and leukapheresis samples was not affected by treatment with BIBR1532. We conclude that using this class of telomerase inhibitor at higher concentrations exerts a direct cytotoxic effect on malignant cells of the hematopoietic system, which appears to derive from direct damage of the structure of individual telomeres and must be dissected from telomerase-suppressed overall telomere shortening. (Blood. 2005; 105:1742-1749)

Description: Lenalidomide, bortezomib, melphalan and dexamethasone are standard drugs for the treatment of multiple myeloma (MM). Although many patients initially respond to treatment regimens including these drugs, the majority ultimately relapses due to the development of resistance of the MM cells, what may result from acquired genetic alterations. Here we performed fluorescence in situ hybridization (FISH) and whole exome sequencing (WES) on 16 paired pre-treatment/ progression MM samples followed by functional validation through CRISPR/Cas9-based screens to identify gene mutations that are associated with resistance. Treatment between two samples consisted of lenalidomide (n=16), bortezomib (n=14), dexamethasone (n=16) and melphalan (n=9). Cytogenetic analyses by FISH revealed that the majority of translocations (7 of 10) and chromosomal gains and deletions (22 of 28) were concordant between pre-treatment and relapse samples. In contrast, gene mutations assessed by WES were highly variable: of the total of 794 identified mutations 6% (n=46) were present only at diagnosis, 59% (n=474) at both time points and 35% (n=274) specifically at relapse with an increase of the median number of mutations from 29 (range 9-103) in pre-treatment to 47 (range 13-110) in progression samples (figure 1A). Recurrent mutations detected pretreatment were in general stable at progression: NRAS (3/3), KRAS (4/4), IGLL5 (3/3) and DIS3 (2/3). Only very few of the newly acquired gene mutations at progression were recurrent: TP53 (n=4), DNAH5 (n=4) and WSCD2 (n=3) while the remaining were non-recurrent. In order to investigate the functional impact of relapse-specific gene mutations on drug resistance we performed pooled CRISPR-Cas9-based knockout screens (figure 1B). We included 160 gene mutations that fulfilled the following criteria: 1) a variant allele fraction (VAF) of 〉20% at the time of progression, 2) found exclusively in progression samples or had a more than 2-fold increase in VAF at progression as compared to pre-treatment, 3) predicted to be loss-of-function. In addition, we included genes found recurrently mutated in relapsed MM in previously published studies. Resistance screens were performed in three different MM cell lines (MM1S, NCIH-929, KMS27) with 4 sgRNA per gene in the presence of lenalidomide, dexamethasone, melphalan, bortezomib or DMSO as a control. None of the sgRNAs included in the screen conferred resistance to all four drugs. In contrast, we identified several genes whose inactivation caused resistance to a specific drug. For lenalidomide, the top hits were members of the CRBN-CRL4 E3 ubiquitin ligase, the primary target of IMiDs, including CRBN, CUL4B and DDB1. CRISPR-mediated inactivation of these genes was specifically associated with lenalidomide resistance since sensitivity towards other drugs was not affected. In addition, we found sgRNAs targeting SYT5, a membrane protein involved in Ca2+-dependent exocytosis, to be enriched in lenalidomide-treated cells that so far has not been related to lenalidomide resistance. SgRNAs targeting TP53 were also weakly enriched after lenalidomide treatment in two of the three cell lines but conferred a high level of resistance to melphalan in all three cell lines (figure 1C). Consistently, three of the four TP53 mutations identified by WES were detected in samples obtained after cytotoxic chemotherapy and one after 3 years of treatment with lenalidomide/dexamethasone. Our screens also revealed an increased susceptibility to melphalan by inactivation of ATM, FANCA, BIRC3, and BRCC3, all involved in DNA damage repair. The top sgRNAs causing resistance to dexamethasone were directed against ANKMY2 and BIRC3 in two cell lines (MM1S and NCI-H929). For bortezomib, inactivation of only one gene, TMC2, encoding a transmembrane protein was associated with resistance in two cell lines whereas BIRC3 inactivation provided increased susceptibility to bortezomib. In conclusion, by combination of comprehensive genetic analyses of tumor samples before and after treatment with functional genetic screens we found mutations that are causally linked with drug-specific resistance and sensitivity. These results may help to personalize therapy in patients with multiple myeloma. Figure 1 Disclosures Bohl: Pfizer: Honoraria. Döhner:Celgene, Novartis, Sunesis: Honoraria, Research Funding; AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria; AROG, Bristol Myers Squibb, Pfizer: Research Funding. Bullinger:Astellas: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Daiichi Sankyo: Honoraria; Gilead: Honoraria; Hexal: Honoraria; Janssen: Honoraria; Jazz Pharmaceuticals: Honoraria; Menarini: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; Seattle Genetics: Honoraria; Bayer: Other: Financing of scientific research; Abbvie: Honoraria; Amgen: Honoraria. Krönke:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: Introduction: During the last decade, the outcome of patients (pts) with symptomatic multiple myeloma (MM) has markedly improved. However, there is still a significant proportion of pts who do not achieve a longtime control of their disease. In particular, pts presenting with a deletion 17p (del17p) still have dismal prognosis. In order to better stratify this important group of MM pts we sought to investigate the prognostic impact of the following parameters in a larger cohort of del17p pts: del17p clone size, concomitant genetic abnormalities, treatment modalities and the incorporation of the novel agents lenalidomide and bortezomib. Methods: We identified 54 MM pts diagnosed between 1998 – 2012 who had a del17p at diagnosis and were treated at the University Hospital of Ulm. The patients were screened for additional chromosomal aberrations by fluorescence in situ hybridization (FISH) performed on purified bone marrow plasma cells. Results: The median age at MM diagnosis was 59 years and the proportion of male pts was 52%. At presentation the median del17p clone size was 83% (range: 28%-98%). In the vast majority of cases (83%) the presence of a del17p was associated with the presence of a del13q14. Other concomitant genetic abnormalities detected by FISH were t(4;14) in 17%, t(11;14) in 30% and gain at 1q21 (+1q21) in 31% of cases (figure 1). The median overall survival (OS) was poor (18.9 months) and did not change substantially over time (similar median survival in pts diagnosed before 2006 versus pts diagnosed thereafter). The del17p clone size had no impact on OS, neither the presence of a t(4;14) or a t(11;14). In patients with an additional +1q21 OS was significantly shorter (15.2 versus (v) 32.4 months; p=0,032). The incorporation of one of the novel agents into first-line treatment did not change the outcome significantly. In contrast, pts receiving at least one autologous transplantation showed a significantly longer OS (33.1 v 12.7 months; figure 2). On univariate analysis there was an improved median OS for pts undergoing an allogeneic transplantation (n=15; 32.4 v 14.4 months; p=0.025). In multivariate analysis ISS stage and the implementation of an autologous transplantation remained significant prognostic factors for OS. Conclusions: The outcome of MM pts with a del17p remains poor, even after the introduction of lenalidomide and bortezomib into clinical practice. The development of novel therapeutic strategies therefore is urgently warranted. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Description: Background Previously, it has been reported, that AML with mutated NPM1 is associated with a distinctive immunophenotype. In particular, low or absent expression of CD34 accompanied by high expression of CD33, and – at least in part of the cases - absence of HLA-DR expression was reported. CD45/side scatter (SSC) gating is widely used for the identification of blasts by flow cytometry (FC). Blast cell gates typically are defined by a low SSC and moderate CD45 expression. However, in a number of patients with NPM1mutation this typical blast cell gate comprises significantly lower blast percentages when compared to the morphological evaluation. In these patient samples a second population is present, which is characterized by a higher expression of CD45 and a brighter SSC signal (myelomonocytic region). Here we provide evidence, that the implementation of an adapted gating procedure integrating cells with higher CD45 expression and moderate SSC improves diagnostic accuracy in these cases. Aim To evaluate differential gating strategies in diagnostic multicolor flow cytometry in NPM1mutated AML. Methods After informed consent diagnostic work-up of patient samples with newly diagnosed AML within the AMLSG BiO Study (clinicaltrials.gov NCT01252485) was initiated and included rapid molecular screening for NPM1 and FLT3 mutations, and CBFß-MYH11, RUNX1-RUNX1T1 and PML-RARA fusions, conventional karyotyping, multicolor flow cytometry and centralized morphological assessment. Multicolor flow cytometry was performed using a Becton Dickinson FACS Canto-II and a comprehensive antibody panel (cytoplasmic staining: TdT, MPO, CD3, CD34, CD45, CD79a; surface staining: HLA-DR, NG2, CD3, CD7, CD10, CD11b, CD11c, CD13, CD14, CD15, CD19, CD33, CD34, CD41, CD42b, CD45, CD56, CD61, CD64, CD117, CD235) according to ELN-recommendation (Döhner et al. Blood 2010). Results Between October 2011 and July 2013 n=2117 patients were included into the AMLSG BiO protocol. In the current study immunophenotypic data of a total of n=263 pretreatment bone marrow samples of patients with NPM1 mutated AML (age 18 to 60 years) were included for further analyses. In n=175 patients only one blast population was present, which was characterized by the CD45 low/SSC low blast cell gate (group-1), whereas in n=87 two populations were detected and gated (group-2). Concurrent activating FLT3 mutations were present in 48% and 39% in group-1 and group-2 (p=0.19), respectively. In a first attempt, the immunophenotypically determined blast infiltration rate was correlated with morphological assessment. In group-1 the morphological blast count correlated well with the immunophenotypically determined blast infiltration (r=0.62, p=0.0001), whereas in group-2 the two methods did not correlate (r=0.34, p=0.07), when only the blast gate was taken into account. However, by applying the two-gate strategy including the myelomonocytic window the correlation could be restored (r=0.67, p