Citations

Abstract Abstract 1246 Poster Board I-268 B-cell chronic lymphocytic leukemia (B-CLL) is characterized by a variable clinical course. Genomic aberrations as detected by FISH analysis and the mutational status of the IgVH genes have been found to have prognostic clinical significance with unmutated IgVH genes associated with clinically more aggressive disease. Previous studies have suggested that unmutated IgVH B-CLL is associated with increased chromosomal instability and increased chromosomal aberrations leading to accelerated disease progression. We recently performed a high-throughput, high-resolution array-based comparative genomic hybridization (aCGH) in a cohort of B-CLL patients. Fifty-eight patients have been analyzed by using the Sureprint G3 microarray (one million probes, Agilent). Our working hypothesis was that we would see increased genomic aberrations and instability in the unmutated IgVH group. The total number of copy number aberrations and total megabases (Mb) deleted or gained per genome were used as a surrogate marker for genomic instability. Variations in chromosomal aberrations in various VH families were also evaluated. B-CLL with unmutated IgVH genes utilized members of the VH1, VH3, VH4 and VH5 families with VH1-69 the most prevalent. B-CLL with mutated IgVH genes utilized VH families VH1, VH2, VH3, VH4, and VH6. Considerable variation was seen in the number of genomic abnormalities between cases. Overall, 315 abnormalities have been identified (236 deletions and 79 gains), with a median of 4 copy-number abnormalities per patient (range 0-32). In patients with IgVH mutational status available the median number of chromosomal aberrations in the mutated group was 5 (range 1-17) and unmutated group was 4 (range 0-20). Copy number deletions were significantly more common than chromosomal gains for the total group and in both the unmutated and mutated IgVH gene groups. Mean number of megabases (Mb) of DNA deleted in the unmutated IgVH group was 17.80Mb (standard deviation 24.17 Mb, median 6.63 Mb, range 0 - 90.55Mb) and 40.65 Mb in the mutated IgVH group (standard deviation 86.04 Mb, median 15.35Mb, range 0 – 307.57Mb). Mean number of Mb gained in the unmutated group was 37.74Mb (standard deviation 57.58Mb, median 0.44Mb, range 0 – 132.47Mb) and 32.99Mb (standard deviation 59.76Mb, median 0.14Mb, range 0 – 172.50Mb) in the mutated group. Nine patients had B-CLL with VH1-69 genes and 8 of them were unmutated. Median number of chromosomal deletions was 2 and gains also 2 (ranges 0 – 8 deletions and 0 – 4 gains) for this group. Similar findings were seen for the other VH families. These results initially suggest that total copy number variations do not account for the observed clinical differences between mutated and unmutated IgVH B-CLL. The catalogue of potentially clinically relevant chromosomal abnormalities found in this aCGH study is being currently evaluated. Disclosures Zent: Genentech, Bayer, Genzyme, Novartis: Research Funding. Shanafelt:Genentech: Research Funding; Hospira: Membership on an entity's Board of Directors or advisory committees, Research Funding; Polyphenon E International: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Bayer Health Care Pharmaceuticals: Research Funding. Kay:Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding; Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees. Fonseca:BMS: Consultancy; Amgen: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Medtronic: Consultancy; Genzyme: Consultancy.

Abstract Abstract 1999 Recurrent chromosomal translocations involving the IGH locus are characteristic of B-cell non-Hodgkin lymphoma (NHL) and MALT lymphoma in particular has been pathologically characterized by specific IGH translocations. However, these translocations are present in only a subset of cases, suggesting that additional uncharacterized translocations might exist. In previous studies we characterized the novel t(X;14)(p11.4;q32) translocation in a patient with MALT lymphoma and found that GPR34, an orphan G-protein coupled receptor (GPCR), was highly expressed due to its juxtaposition to the IGHSA2 switch region. Although the t(X;14)(p11;q32) translocation was only identified in one patient, we wanted to determine its expression across a spectrum or normal and malignant lymphoma tissue, as other mechanisms may contribute to overexpression of genes. We measured GPR34 mRNA expression by quantitative PCR in tissue biopsies from a panel of lymphomas. As controls, we also measured GPR34 mRNA expression in normal resting or activated CD19+ B cells, resting and activated CD3+ T cells, and CD14+ monocytes. Expression of GPR34 was significantly increased in MALT (37-fold, n=35), LPL (23-fold, n=23), nodal marginal zone B cell lymphoma (18-fold, n=21), and splenic marginal zone B cell lymphoma (21 fold, n=33) compared to resting CD19+ B cells (n=11). In a gastric MALT lymphoma specimen we detected GPR34 expression at levels similar to the t(X;14)(p11;q32) patient and interphase FISH studies showed an extra intact GPR34 signal, but no translocation involving IGH or GPR34, suggesting that other mechanisms, including gene dosage effect, can upregulate GPR34. The receptor encoded by GPR34 is most similar to the PY2 receptor subfamily of GPCRs and signaling studies suggest that overexpression of GPR34 results in an accumulation of inositol phosphates and MAP-kinase activation. To further characterize the impact of GPR34 overexpression on cell signaling, HeLa cells were transduced with WT GPR34, a signaling deficient GPR34 (GPR34 DRY), or a vector control. To determine if MAPK activity was upregulated, the phosphorylation status of ERK1/2 in GPR34 WT, GPR34 DRY, and vector control cells was analyzed and we found that ERK1/2 was constitutively phosphorylated at higher levels (1.6-fold) in GRP34 WT cells compared to GRR34 DRY or vector control cells. To further explore which signaling pathways were affected by GPR34 overexpression, GPR34 WT or vector control cells were transfected with an AP-1, CRE, NF-kB, E2F, SRE, NFAT, or ISRE- luciferase reporter plasmid. GPR34 expressing cells had increased luciferase activity driven by AP-1 (5.35-fold), CRE (4.7-fold), NF-kB (2.6-fold), and E2F (2.1) when compared to vector control cells. GPR34 DRY was unable to activate these pathways. AP-1 and CRE have been implicated in a large variety of cellular processes, including transformation, and both AP-1 and CRE activity is induced upon activation of MAP kinases. In untreated cells, both AP-1 and CRE activity were significantly higher in GPR34 WT cells compared to GPR34 DRY cells and both AP-1 and CRE luciferase activity was inhibited by the MEK1 inhibitor PD98059, suggesting that activation of these pathways is MAP-kinase dependent. To determine the biologic impact of GRP34 overexpression, the proliferation rates of vector control, GPR34 WT, and GPR34 DRY cells were compared and we found that proliferation of GPR34 WT cells was significantly higher (5.6-fold) than that seen in GPR34 DRY cells. We next tested the effect of the MEK inhibitor on proliferation and saw a dose dependent decrease in proliferation of GPR34 WT expressing cells. These results suggest that GPR34-mediated proliferation is dependent on MAPK. The presence of elevated GPR34 expression in tumors and the increased proliferation mediated by GPR34 suggests that it may have oncogenic potential. We therefore tested the ability of GPR34 WT cells to generate tumors in a tumor colony-forming assay. Only the GRP34 WT cells had the ability to form tumors in soft agar suggesting that overexpression of GPR34 can result in tumor formation. Taken together, our results identify a novel translocation in MALT lymphoma, emphasize a novel role for GPR34 in tumor cell growth, and suggest that MEK inhibitors may be useful in a subset of translocation-harboring or GRP34 high-expressing tumors. Disclosures: Fonseca: Genzyme: Consultancy; Medtronic: Consultancy; BMS: Consultancy; AMGEN: Consultancy; Otsuka: Consultancy; Celgene: Consultancy, Research Funding; Intellikine: Consultancy; Cylene: Research Funding; Onyx: Research Funding; FISH probes prognostication in myeloma: Patents & Royalties.

Abstract Abstract 2406 The outcome for a given CLL patient is difficult to predict. While there are promising models, they require collation of multiple clinical and laboratory parameters, and it remains to be seen whether they will apply to typical CLL patients in the community. To further dissect out explanations for this dramatic clinical heterogeneity, we sought to understand genomic complexity of clonal B-cells as a possible explanation of clinical variability with specific application to genomic complexity as a predictor of therapeutic response and clinical outcome in CLL. Thus we wished to identified global gains and losses of genetic material in order to define copy-number abnormalities (CNA) in 48 clinically progressive CLL patients who were about to be treated on a chemoimmunotherapy protocol. This protocol was previously reported by us (Blood. 109:2007) and had an induction phase with pentostatin (2 mg/m2), cyclophosphamide (600 mg/m2) and rituximab (375 mg/m2) given every 3 weeks for 6 cycles and then responding patients were followed ever three months until relapse. In order to estimate CNA, we employed array-based comparative genomic hybridization (aCGH) using a one-million oligonucleotide probe array format on the leukemic B-cells from the 48 patients entering this trial. In those same patients, the aCGH data were compared to a) FISH detecxtable data using a panel for the common recurring genetic defects seen in CLL and b) to their clinical outcome on this trial. With aCGH we found that 288 CNA were identified (median of 4 per patient; range 0–32) of which 215 were deletions and 73 were gains. The aCGH method identified most of the FISH detected abnormalities with a complete concordance for 17p13.1- deletion (17p-) between aCGH and FISH. We also identified chromosomal gain or loss in ≥6% of the patients on chromosomes 3, 8, 9, 10, 11, 12, 13, 14 and 17. We found that CLL patients with ≥15 CNA had a significantly worse progression free survival (PFS) than patients with <15 CNA (p=0.004)(figure). Patients with ≥15 CNA also had a shorter duration of response than those with <15 CNA (p=0.0726). Of interest, more complex genomic features were found both in patients with a 17p13.1 deletion and in more favorable genetic subtypes such as 13q14.1. Thus, for 5 patients with >15 CNAs the following FISH patterns were seen: +12/13q14.1-x1/13q14.1 -x2, 13q14.1 ×1 (n=2), and 17p13.1 (n=2). In addition, a 17p- by FISH was positively associated with the number of CNA and total deletion size. The odds of having an overall response decreased by 28% (95% CI: 5–55%; p=0.015) with each additional CNA for the 17p13.1- patients. In addition to defining genomic complexity as the total number of CNA for each patient, we also defined complexity as the sum of the lengths of all interstitial chromosomal gains and losses. When defined as the total size of chromosomal gains or losses, genomic complexity was significantly associated with 17p13.1 and worse overall clinical response. In summary, this analysis utilized the global assessment of copy number abnormalities using a high-resolution aCGH platform for clinically progressive CLL patients prior to initiation of their treatment. One outcome was that we found higher genomic complexity was associated with shorter progression-free survival, reduced duration of response and predicted a poor response to treatment. In addition since we did find genomic complexity in more traditionally favorable FISH categories, such as 13q14.1 type defects, this may explain why some of the latter patients do not fare as well as might be expected even with aggressive chemoimmunotherapy approaches. This study adds information on the association between inferior trial response and increasing genetic complexity as CLL progresses. Disclosures: Off Label Use: Pentostatin. Kipps: GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding; Memgen: Research Funding; Igenica: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Research Funding; Abbott Laboratories: Research Funding.

Abstract Abstract 1807 We have recently demonstrated that cereblon (CRBN) mediates the direct anti-myeloma activity of immunomodulatory drugs (IMiDs). However, the genes/pathways downstream of CRBN associated with anti-myeloma activity remain unclear. We, and others, identified interferon regulatory factor 4 (IRF4) as one of the downstream targets of CRBN-associated signaling. Both lenalidomide treatment and CRBN knockdown downregulate IRF4. IRF4 levels return to baseline in IMiD resistant cells surviving CRBN silencing. To determine whether IMiD-induced IRF4 downregulation is critical to anti-MM activity, we overexpressed IRF4 in two IMiD-sensitive human MM cell lines (HMCLs), KMS11 and MM1.S, followed by lenalidomide treatment. Lenalidomide-induced cytotoxicity was greatly impaired in both HMCLs overexpressing IRF4 compared with the control virus infected cells. Further analysis indicated that IRF4 over-expression does not completely prevent lenalidomide-induced growth arrest, but reduces cell death by 70% after lenalidomide treatment. Immunoblotting analysis of KMS11 cells indicated that IRF4 over-expression blocks lenalidomide-induced activation of caspase 8, reduces up-regulation of p21waf and increases CDK6 expression but does not significantly affect lenalidomide-induced MYC down-regulation. Although cereblon and IRF4 are broadly expressed in MM, baseline levels of expression are only weakly correlated (r=0.22) in primary MM patient gene expression analysis. Gene expression studies revealed statistical changes in 1,368 genes when comparing high versus low CRBN expression in primary myeloma samples. Interestingly genes associated with high CRBN expression included cyclin D2, SOCS3 and IL4 while genes associated with low cereblon expression included cyclin D1, FRZB and CD200. In order to understand how CRBN is connected with downstream anti-myeloma signaling, a structure-function study was performed to determine which CRBN domain is required for lenalidomide-induced IRF4 down-regulation and cytotoxicity. Lentiviral constructs expressing wild-type CRBN and a series of mutated CRBN were generated, including mutations at thalidomide binding site (Y384A/W386A), deletion of DDB1 binding region (ΔMid) and truncations at N-terminal and C-terminal. Lentiviruses from these constructs were used to infect IMiDs resistant HMCLs, OCI-MY5 and MM1.S res. Both of these cell lines have very low endogenous CRBN expression and they became sensitive to lenalidomide after introduction of wild-type CRBN. Conversely, introduction of CRBN with mutated thalidomide-binding site or with DDB1 binding region depletion failed to mediate lenalidomide toxicity and down-regulation of IRF4. OCI-MY5 cells expressing either N-terminal or C-terminal truncated CRBN showed substantial reduced responses (more than 50%) to lenalidomide compared with wild-type CRBN expressing cells. Deletion of only 20–30 amino acids at either ends of CRBN greatly impaired the protein function, suggesting that protein folding might be important for CRBN-mediated IMiD response. Our data indicate that IMiD induced myeloma cytotoxicity is largely mediated by modifying CRBN associated E3 ubiqutin ligase and subsequent IRF4 downregulation, suggesting the CRBN-IRF4 axis is a potential target for development of new anti-myeloma drugs. Disclosures: Schmidt: Karyopharm: Research Funding. Stewart:Millenium: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy.

Abstract Introduction A unique biological characteristic of MM plasma cells (PCs) is the substantial production of immunoglobulin proteins. This process requires disulfide bond formations that in turn generate large amounts of H2O2. The Trx-TrxR reductase system exists to combat this oxidative stress; however, cytotoxicity can occur when the cells' antioxidative capacity is overwhelmed. Recently, we demonstrated that IMiDs inhibit peroxidase-mediated decomposition of H2O2, and that lenalidomide sensitivity in MM correlates with the degree of antioxidative capacity (Sebastian et al. 2015 ASH). In this study we sought to better understand this inhibitory process. Methods We used various human MM cell lines with different sensitivity to lenalidomide. Cell viability was assessed by the MTT assay. Antioxidative capacity was measured via the total cellular oxidation of reduced flavin adenine dinucleotide (FADH2) and reduced nicotinamide adenine dinucleotide (NAD(P)H) after H2O2treatment. A CRBN CRISPR/Cas9 KO plasmid was used to generate CRBN knockout cells and lentivirus system was used to over-express CRBN in MM cells. Results We found that IMiDs inhibit TrxR mediated intracellular H₂O₂ decomposition in a CRBN dependent fashion. Unlike lenalidomide, the direct TrxR inhibitor auranofin was very effective in inducing cell death in MM cell lines, in a CRBN independent fashion. By using MM.1R isogeneic cells with and without CRBN, and the CRBN-overexpressing OCIMY-5 cell line (transfected with wt-CRBN) we showed that auranofin treatment caused cytotoxicity irrespective of CRBN level of expression, while lenalidomide was only effective in CRBN expressing cells. Clinically achievable concentrations of auranofin treatment induced cell signals similar to lenalidomide, including degradation of IKZF1, IKZF3. As with lenalidomide, the sensitivity of CRBN expressing cell lines to auranofin correlated with their basal antioxidative capacity (MM.1S cells are sensitive and have with lower anti-oxidative capacity while RPMI-8226 is resistant due to its high anti-oxidative capacity). We also found that targeting Trx (downstream effector of TrxR) with the direct inhibitor PX12 overcame this cellular anti-oxidative capacity and inhibited cell proliferation in both RPMI-8226 and MM.1S in a CRBN independent fashion. Combination of auranofin with bortezomib showed a synergistic effect, but only in cells with lower anti-oxidative capacity. In contrast, PX-12, showed a synergistic effect with bortezomib in all analyzed MM cells lines, irrespective of their anti-oxidative capacity and CRBN expression. Conclusions IMiDs inhibit TrxR-mediated intracellular H2O2decomposition in CRBN expressing MM. While the direct TrxR inhibitor, auranofin, bypassed the CRBN requirement for inducing cytotoxicity in MM, its cytotoxic effect could be overcome by the cells antioxidative capacity. Direct Trx inhibition (such as PX-12) seems like a potent strategy for MM therapy to overcome cellular CRBN requirements and higher anti-oxidative capacity cells. Disclosures Bergsagel: Novartis: Research Funding; Amgen, BMS, Novartis, Incyte: Consultancy. Stewart:celgene: Consultancy. Fonseca:Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Janssen: Consultancy; Bayer: Consultancy; Millennium, a Takeda Company: Consultancy; Millennium, a Takeda Company: Consultancy; BMS: Consultancy; Millennium, a Takeda Company: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Celgene: Consultancy; AMGEN: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Millennium, a Takeda Company: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Janssen: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Bayer: Consultancy; AMGEN: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Novartis: Consultancy; AMGEN: Consultancy; Sanofi: Consultancy; AMGEN: Consultancy; Sanofi: Consultancy; BMS: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms.

Abstract Sequencing studies have significantly improved our understanding of the CLL genetic landscape and allowed the identification of the first associations between specific mutations and clinical outcome. Hence, mutations in TP53, NOTCH1, SF3B1, BIRC3 and MYD88 are known to be associated with differential prognosis and have been incorporated in CLL risk stratification models. Despite the work performed on these 5 genes, several additional putative tumor-driver genes are recurrently mutated in CLL, but their clinical impact remains unclear. In order to gain further understanding, we performed targeted sequencing in baseline samples of 308 CLL patients (pts) participating in the North American Intergroup study (NAIG) designated as E1912 and led by ECOG. This trial is a randomized Phase III of Ibrutinib (Ibr) and Rituximab (R) vs FCR in previously untreated CLL. Besides the 5 genes aforementioned, we screened another 19 genes recurrently mutated in CLL, and 2 genes (BTK and PLCy2) recently associated with Ibr resistance. The CD5+/CD19+ B lymphocyte population of these pts was flow-sorted. All coding regions were amplified using customized oligos. Libraries were templated and enriched using IonOneTouch2 and IonOneTouch ES systems, respectively. Samples were run on an IonTorrent sequencer using 318TM chips. Variants were called using IonTorrent Somatic VariantCaller. VCF files were annotated using BioR, which provides annotation from NCBI/Ensembl, UCSC, dbSNP, HapMap, 1000Genomes, COSMIC, SIFT, and PolyPhen-2. Somatic variants with a Mapping Quality <20 or read depth <5X were removed. Variants of interest were inspected using Integrative Genomics Viewer. The mutational status of genes and pathways was analyzed for association with chromosomal abnormalities detected by FISH, expression of CD38, CD49d and ZAP70, Rai stage, age and gender. We were not able to evaluate associations to clinical responses for the different arms as this trial is still underway and remains blinded for clinical outcome. We found 511 SNVs (442 missense, 45 nonsense and 24 splice sites) and 115 indels. An average coverage of 770X depth was obtained, allowing the detection of minor subclonal mutations (<10% of reads) in almost 35% (218/626 mutations). Overall, 215 pts (70%) carried at least one mutated gene, 102 (33%) carried 2 or more and 32 (10%) 3 or more mutated genes. The most commonly mutated gene was SF3B1 (22%), followed by NOTCH1 (19%), ATM (12%), XPO1 (11%), BIRC3 (9%), KRAS (7%), BRAF, DDX3X, EGR2, POT1 and TP53 (6% each). No BTK or PLCy2 mutations were found in progressive but untreated CLLs. In contrast to a recent report (Nature 2015;526:525-30), we found BRAF mutations located in the canonical hotspot p.V600E (25% of total mutations). Another 13% were located in amino acid K601 (p.K601E/N) and the remaining in the kinase domain (p.G469A, p.D594G, p.F595L, p.G596R, p.L597Q).Nearly 27% of cases with activating mutations in the RAS pathway (BRAF, KRAS, NRAS), showed >1 mutation affecting one or more genes of the pathway. Similar observations were found in mutational hotspots of BIRC3 (30% of mutated cases showed >1 mutation), MED12 (18%), SF3B1 (7%), XPO1 (6%), and NOTCH1 (5%), suggesting the existence of genetic subclones showing convergent evolution in a substantial number of CLL. We found multiple associations between mutations and known prognostic and clinical parameters. These included mutations in NOTCH1 associated with +12 (p=0.005), and expression of ZAP70 (p=0.01), CD38 (p=0.001) and CD49d (p=0.004). Mutant BIRC3 was associated with +12 (p<0.001) and CD38 expression (p=0.01). Mutations in RAS pathway, with +12 (<0.001), and expression of CD38 (p=0.002) and CD49d (p=0.05). ATM (p=0.007) and EGR2 (p=0.002) with -11q; POT1 with CD49d expression (p=0.01), and MYD88 with -13q (p=0.04) and lack of CD38 (p=0.01) and CD49d (p=0.01) expression. This study, performed on a large cohort of untreated but progressive CLLs, provides novel data about the mutation status of driver genes and their associations with clinical and prognostic parameters. Our data indicate that convergent evolution is a common event in CLL, affecting several driver genes. Furthermore, it reinforces that BTK and PLCy2 mutations are not found in Ibr-naive pts. Finally, this study provides the genetic groundwork for subsequent prospective comparative analyses of the clinical outcome for CLL cohorts treated with Ibr-R vs FCR in this phase 3 NAIG trial. Disclosures O'Brien: Janssen: Consultancy, Honoraria; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding. Fonseca:Novartis: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Novartis: Consultancy; Janssen: Consultancy; BMS: Consultancy; AMGEN: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; AMGEN: Consultancy; Millennium, a Takeda Company: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Sanofi: Consultancy; Celgene: Consultancy; Celgene: Consultancy; Bayer: Consultancy; Janssen: Consultancy; Millennium, a Takeda Company: Consultancy; Millennium, a Takeda Company: Consultancy; AMGEN: Consultancy; AMGEN: Consultancy; Bayer: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Millennium, a Takeda Company: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms. Shanafelt:Genentech: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding; GlaxoSmithkKine: Research Funding; Janssen: Research Funding; Cephalon: Research Funding; Hospira: Research Funding.

Abstract Abstract 1846 Background The low frequency of TP53 (p53) alterations (<10%) in MM makes this tumor an ideal candidate for p53-activation therapies. Unfortunately, DNA alkylating agents (cyclophosphamide and melphalan), when used alone, cannot fully exploit p53 mediated apoptosis. It is currently unknown the extent to which DNA alkylating agents and p53 activating small molecules are activating functional p53 signaling to induce apoptosis in primary MM cells, because, p53 mainly induces cell cycle arrest/DNA repair or apoptosis in the DNA damage response. Emerging studies demonstrate that PARP activation and p53 poly(ADP-ribosyl)ation can negatively influence apoptosis induction but the exact molecular mechanisms are unknown. Methods For apoptosis and gene expression analysis 05 × 106 cells were incubated with 2.5 μM of melphalan, 10 μM of olaparib and 2.5 μM of nutlin-3a alone and/or combinations of melphalan with olparib and/or nutlin3a with olaprib for 24 to 72 hours before they were examined for cell death by the annexin-PI and FACS analysis. Gene and protein expression were measured by RT-PCR, western blot, and immunohistochemistry. For combination studies, cells were incubated with 0.194–50 μM of melphalan and 0.625–40 μM of either olaparib or ABT-888 for 72 hours. p53 knock-down performed by lentiviral mediated shRNA. Vk*MYC mice with significant gammopathy (>20g/l on SPEP) used for in-vivo drug combination studies. Results Preliminary results support our principle hypothesis that, a PARP inhibitor (olaparib) enhanced Melphalan sensitivity in wild type p53 MM cell lines H929 and MM1S but not in p53 mutated or homozygously deleted cell lines U266 and KMS11, respectively. Moreover, we demonstrate that p53 knock-down decreased the synergistic effect of combining a PARP inhibitor with melphalan and Nutlin-3a. Again, combining a PARP inhibitor with the DNA alkylating agent melphalan induced clear PARP cleavage, a signature of apoptosis in wild type p53 MM cell lines H929 and MM1S. No significant PARP cleavage was observed after melphalan or olaparib treatment alone in H929 and MMIS. As expected, the p53 mutant cell line U266 did not show any PARP cleavage and p21 up-regulation after both drug treatments. Here we propose that the hyper-activated p53 from PARP inhibition along with DNA alkylating agents (melphalan) and/or p53 activating agents (nutlin-3a) result in better responses. Finally, in a murine xenograft model of human MM, olaparib potentiated melphalan activity in vivo, with significant reduction in M spike level. Conclusion Our studies indicate that DNA alkylating agents activate wild type p53, but leads to DNA repair and cell survival and this can be abolished by PARP inhibition in wild type p53 expressing MM cases. Moreover, our study also found that olaparib, and ABT-888 - PARP1 inhibitors currently in clinical trials - display different dose responses in MM cell lines and in the abilities of individual PARP inhibitors to sensitize MM cell lines to melphalan varied to a great extent in a cell context- and cell line specific manner. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: CLL patients (pts) treated with novel targeted therapies continue to experience disease progression. Approximately 7-15% of relapsed CLL treated with ibrutinib or venetoclax developed Richter's syndrome (RS), an aggressive high grade lymphoma transformation. Standard chemotherapy has limited efficacy in RS. We hypothesized that checkpoint blockade would re-establish anti-tumor immune response in progressive CLL/RS and here we aim to update the clinical data and correlative analysis of MC1485: a phase 2 study of PD-1 blocking antibody pembrolizumab in relapsed/refractory CLL and RS. Methods: Relapsed/refractory CLL including RS enrolled in the CLL arm of MC1485 trial was reported here. The primary end point of this study is overall response rate (ORR). Pts with prior allogeneic stem cell transplant were excluded. Pembrolizumab 200 mg was administered intravenously every 3 weeks. NCI CTCAE v4.0 and IWCLL 2008 criteria were used for non-hematological adverse events (AE) and for CLL grade hematological AE. Tumor expression of PD-1 and PD-L1 were assessed with standard immunohistochemical (IHC) staining. Percentage expressions of individual antigens were analyzed with whole-slide scanning followed by image analysis with Image-Pro software (Media Cybernetics). Fluorescence in situ hybridization (FISH) was performed on available CLL/RS tissue sections to assess copy number of chromosome 9p region that contains PD-L1 and PD-L2. Results: 25 pts including 16 CLL and 9 RS (biopsy-proven large cell lymphoma) were enrolled in CLL arm. The median age was 69 years (46-81). Twelve (48%) pts had del(17p) or monosomy 17 or TP53 mutation. The median number of prior therapies was 4 (1-10). 96%, 72% or 48% of pts had received alkylator and anti-CD20 antibody, purine analog or anthracycline. Fifteen (60%) pts had prior ibrutinib and 12 (48%) progressed clinically while receiving ibrutinib, including 6 who progressed to RS and 6 who developed progressive CLL. The median number of pembrolizumab doses that pts received was 3 (1 to 21), administered over a median treatment duration of 11 weeks (1 to 56). Drug-related AE occurred in 21 pts (88%) with the most common ones being neutropenia in 9 (37%), cough in 7 (29%) and dyspnea in 6 (25%) pts. Drug-related grade 3 or above AE occurred in 9 (38%) pts with most common ones being thrombocytopenia (5, 21%), dyspnea (2, 8%), and fatigue (2, 8%). The most common immune-related AE was liver enzyme elevation (12%, 8% G3) and was reversible with therapy interruption or steroid therapy. Based on investigator assessment using the Revised Response Criteria for Lymphomas and IWCLL 2008 criteria, one RS pt had complete response (CR, 4%), 3 RS had partial responses (PR, 12%). Among 9 RS pts, 1 CR (11%), 3 PR (33%), 3 SD (33%) and 2 PD (22%) were observed. Thus the ORR in RS was 44%. No CLL pts achieved a CR/PR, 3 had SD and 9 had progressive disease (PD). The ORR of all pts was 16%. Of the 4 RS who had CR/PR, responses occurred early after 2 cycles of therapy and median duration of therapy for CR/PR pts was 8.3 months with data cut-off by June 30th 2016 (Figure 1A). The causes for therapy discontinuation in RS were PD (3, 33%), alternate therapy (2, 22%), and thrombocytopenia due to increased marrow CLL (1, 11%). Increased marrow CLL was observed in 2 RS pts with PR to RS phase of disease assessed by PET, thus the protocol was amended to allow addition of idelalisib/ibrutinib to control the underlying marrow CLL. For RS pts who had a CR/PR or SD, variable nodal responses were observed (Figure 1B). In particular, 6 RS who had prior ibrutinib experienced CR or PR or SD with nodal responses. After a median follow-up of 10.2 (1.9 to 16.1) months, the median overall survival (OS) for all pts was 10.7 months (95% confidence interval [CI], 5.4 to not reached). The 6 month OS rate for RS and CLL pts were 73% and 59%, respectively. Biomarker assessment using IHC analysis on 10 pts with available tumor/nodal tissues (6 RS and 4 CLL) showed an increased expression of PD-L1 (p = 0.02) and a trend for an increased expression of PD-1 (p = 0.1) in the group of pts with CR/ PR versus pts in the group with no clinical response. One out of 10 tested showed polysomic for chromosome 9p. Conclusion: Pembrolizumab had an acceptable safety profile in CLL and RS patients. We confirmed that pembrolizumab has substantial therapeutic activity in RS. Single-agent pembrolizumab does not appear to have clear activity in CLL. Disclosures Ding: Merck: Research Funding. Parikh:Pharmacyclics: Honoraria, Research Funding. Shanafelt:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Hospira: Research Funding.

Abstract Introduction Tumor specific somatic mutations can potentially influence patient response to targeted immunotherapies. Since somatic mutations generate neoantigens, novel tumor-specific peptides can be processed, loaded into MHC class I molecules, and presented to T-cells as potentially immunogenic targets. Indeed, both somatic mutation burden and neoantigen burden have been correlated with responsiveness to checkpoint inhibitor blockade in highly mutagenic advanced melanoma and lung cancer. However, correlation of response with mutation and neoantigen burden has not been reported for cancers of lower characteristic mutational burden such as multiple myeloma (MM). Indeed early results of checkpoint inhibitors in MM have been underwhelming. We therefore wished to better understand the mutation and neoantigen landscape of MM patients as it relates to responsiveness to current and future therapies. Materials/Methods Somatic mutation burden, neoantigen load, and response to therapy was obtained using interim data from theCoMMpassstudy (NCT01454297), a study by the Multiple Myeloma Research Foundation that collects longitudinal clinical and molecular data on 1,000 newly diagnosed MM patients from baseline through treatment and disease progression. Next-generation exome sequencing data was used to call somatic mutations andbioinformaticallydetermine patient specific HLA type. Neoantigen binding affinity was predicted for all unique patient specific somatic missense mutation and HLA allele combinations using a well validated neural network and machine learning based algorithm. Binding affinity of the corresponding non-mutated peptide was also predicted to identify mutated neoantigens witha high binding affinity while the wild type counterpart has low predicted binding affinity. Results For 627 patients with available sequencing and clinical data, mutation and neoantigen profiles were created. Within this population, the mean overall canonical somatic mutation load was 405.84 (s=608.55) mutations per patient. These mutations included 38,222 unique missense mutations in 12,994 genes. The mean somatic missense mutation load was 64.51(s=95.29) mutations per patient including immunoglobulin genes (52.70, s=94.85 excluding immunoglobulins). There was a positive linear correlation between mutation burden and neoantigen burden. The mean neoantigen load using thresholds of mutant peptide binding affinity (half maximal inhibitory concentration, IC50) less than 500nM was 69.57 (s=124.76) neoantigens (58.53, s=123.58 excluding immunoglobulins). Limiting binding affinity of wild type peptide sequence to greater than 500nM decreased the mean neoantigen load to 23.84 (s=53.55) neoantigens (20.74, s=53.28 excluding immunoglobulins). Progression free survival (PFS) following induction therapy was used to determine correlates of mutation burden excluding immunoglobulin mutations with response. Kaplan-Meier survival analysis revealed significantly shorter PFS time in patients with greater than average tumor somatic missense mutation load (N=126) relative to those with less than average mutation load (49.1% vs. 73.0% PFS at 2yrs, p=0.0055, Figure 1) Similarly, patients with greater than average neoantigen load (N=164) had significantly shorter PFS relative to those with less than average neoantigen load (48.0% vs. 74.2% PFS at 2yrs, p=0.00259, Figure 2). Moreover, patients with both high mutation and neoantigen load (N=85) had significantly shorter PFS relative to those with less than average mutation and neoantigen load (45.3% vs. 72% PFS at 2yrs, p= 0.0168). Conclusion While mutation and neoantigen load has been correlated with response to targeted immunotherapies in highly mutated solid tumors, we report here a negative correlation between mutation, neoantigen load and MM patient survival. This indicates that a high mutation andneoantigen load negatively impacts survival of myeloma patients under current standard of care. It is hypothesized that this is because current treatments fail to recruit T cell immunity necessary to exploit tumor neoantigens fundamental to immune-mediated killing of cancer cells. This presents an encouraging basis for pursuing targeted and personalized immunotherapies for multiple myeloma, especially for patients with a higher mutational burden. PFS and mutation load. PFS and mutation load. PFS and neoantigen load. PFS and neoantigen load. Disclosures Lonial: BMS: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Onyx: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Janssen: Consultancy; Onyx: Consultancy; Novartis: Consultancy; BMS: Consultancy; Millenium: Consultancy; Merck: Consultancy.

Abstract Introduction: Proteasome inhibition is the backbone of various Multiple Myeloma (MM) treatment regimens, leading to durable responses and high quality remissions. However, under prolonged therapy patients eventually develop drug resistance and the underlying mechanisms have been poorly understood. Proteasome inhibitor resistant cell lines were generated by continuous exposure to proteasome inhibiting drugs. In these cell line models a number of variants within the PSMB5 gene were observed. This includes single point mutations, leading to conformational changes of the _5 subunit within the 20S proteolytic core of the proteasome, impairing its chymotryptic catalytic function and the binding of inhibitory drugs. However, no PSMB5 mutations could, so far, be identified in human disease, leaving the functional relevance of such mutations to be determined. Methods: We applied the MM Mutation Panel (M3P) and used the Personal Genome Machine (Life Technologies) to sequence CD138+ purified bone marrow plasma cells and peripheral blood germ line control from a MM patient in third relapse that had been previously treated by various proteasome inhibitor containing therapies (VTD-PACE, VCD, PAD-Rev). This patient was subsequently treated with a pomalidomide, bortezomib, adriamycin and dexamethasone combination therapy (Pom-PAD) and achieved a partial remission after 4 cycles of therapy. When being exposed to VCD at earlier relapse a complete remission was induced within 6 cycles of therapy, demonstrating excellent response to proteasome inhibition at this earlier disease stage. Results: We identified clonal missense mutations in this patient at known NRAS hotspot location (p.Gln61Arg) and in MAX (p.Arg35His). Most notably we found and validated four subclonal single point mutations in PSMB5, each of them in subclonal frequencies with variant reads (VR) ranging from 1.9%- 5.9% (average read depth 750X). All PSMB5 mutations occurred in a highly conserved region in exon 2 of the gene (p.Cys122Tyr, p.Met104Ile, p.Ala86Pro, p.Ala79Thr), with three of them being located within the proteasome inhibitor drug binding site. Our 400bp amplicon design allowed us to observe that each mutation identified in PSMB5 is exclusively present on a different sequencing read and no reads are shared between the mutations. This implies that the mutations are present on different subclones of the tumor, which means that, despite low VR frequencies of the single mutation, in sum more than a quarter of the whole tumor might be affected by mutated PSMB5. At a disease stage when the patient was well responsive to proteasome inhibitor treatment (at diagnosis and at first relapse), of note, none of the mutations in PSMB5 is detectable. Conclusion: Here we report the first in human PSMB5 mutation in a MM patient. These mutations evolved in parallel within different subclones of the disease under the selective pressure of bortezomib- containing treatment regimens, representing branching evolution. It is to speculate whether previous investigations negating the existence of PSMB5 mutations in proteasome inhibitor treated patients might not have sequenced deep enough or did set up a sensitivity cut-off too rigid to detect such subclonal mutations. Our finding suggests that the mutations identified may contribute to the development of proteasome inhibitor resistance, emphasizing the need for more detailed genomic characterization of tumors, including minor subclonal mutation detection. Functional analysis of the mutations is ongoing and results will be presented at the meeting. Figure Figure. Disclosures No relevant conflicts of interest to declare.