Chng, Wee Joo

Abstract Plasma cell leukemia (PCL) is an aggressive and rare hematological malignancy that can originate either as primary disease (pPCL) or as a leukemic transformation of multiple myeloma (MMLT). We report here on the clinical characteristics, survival and genetic studies of 80 patients with PCL treated at a single institution (including 41 with pPCL and 39 with MMLT) and make comparisons with 439 newly diagnosed cases of multiple myeloma (MM). From our results pPCL presents a decade earlier than MMLT (54.7 vs 65.3 years) and despite substantially poorer clinical and laboratory parameters at presentation (hepatomegaly 32% vs 11%, leukocytes 31.6 vs 20.9, β2M 8.5 vs 4.3) pPCL appears more responsive to conventional combination chemotherapy (VAD, VBMVP or VBAP) than MMLT and is associated with significantly longer survival (median 18.4 vs 3 months). Comparison of pPCL groups treated with combination chemotherapy (n=21) or with other regimens (40% MP, 10% Thal/Dex, 20% other, 30% none)(n=20) identifies no substantial pre-treatment differences between the groups although a higher proportion of patients who received combination chemotherapy subsequently proceeded to stem cell transplantation (30 vs 10%). Importantly, survival of pPCL patients treated initially with combination chemotherapy (median 13.9, mean 27.3 months) was significantly longer compared with patients who did not receive combination chemotherapy (median 5.2, mean 9.9 months)(p=0.035) with 2 long term survivors (> 7 years) in the chemotherapy group. By comparison, patients with MMLT received less active therapy for their leukemic disease than did patients with pPCL (21% VAD, VBMVP or VBAP; 18% MP). Treated MMLT patients showed selection bias for younger age (60.9 vs 66.4 years) and lack of bone disease (0 vs 65%)(p<0.01); however despite this, median survival was disappointingly short for MMLT patients in the treatment group and was not significantly improved compared with non-treated patients (2.3 vs 0.8 months)(p=0.34). Assessment of novel therapies in PCL is therefore urgently required. Karyotype analysis and targeted genetic studies demonstrate that IgH translocations are present with high frequency in both pPCL and MMLT (71 vs 86%) compared with MM (~50%). Notably, IgH translocation in pPCL is almost exclusively targeted at chromosome 11 (cyclin D1) belying its importance in establishing the phenotype. IgH translocation in MMLT is primarily to chromosome 11 [71%], but also targets chromosomes 4 (FGFR3/MMSET) [13%] and 16 (cMaf) [20%], recapitulating MM. Abnormalities of chromosomes 3,5,7,9,11,13,15 and 19 are present equally in all 3 disorders. Deletion of 17p13 (p53) shows strong selection pressure in the leukemias (pPCL 53.8%, MMLT 48.8%, MM 10.7%) and is complemented by p53 mutation (23%). Notably, pPCL tumors show lower rates of Ras mutation (12 vs 40%) and p14 (0 vs 31%) or p16 (25 vs 40%) promoter methylation than MMLT. Together these results therefore indicate that pPCL and MMLT are fundamentally different plasma cell disorders with distinctive biology, genetic abnormalities and treatment responses.

Abstract Background: Treatment of multiple myeloma has evolved considerably in the past few years with availability of several news drugs as well as increasing use of multidrug combinations. These changes have no doubt led to the improved survival seen among patients with MM. We have previously shown that outcomes of patients intolerant or refractory to one of the IMiDs and bortezomib had a poor outcome. Since that time, other drugs of the same class as well as new classes of drugs have been introduced for the treatment of MM. We designed this retrospective study to estimate the outcomes in patients with relapsed myeloma, who have become refractory to the current generation IMiDs and proteasome inhibitors. Patients and Methods: Patients with relapsed multiple myeloma who have received at least 3 prior lines of therapy, is refractory to both an IMiD (lenalidomide or pomalidomide) AND a proteasome inhibitor (bortezomib or carfilzomib), and has been exposed to an alkylating agent were identified from multiple centers. The time patients met the above criteria was defined as T0, and details of all treatment regimens before and after T0 were collected using electronic CRFs. The study was approved by the IRB at the respective centers. Results: 543 patients were enrolled in this study; median age was 62 years (31-87) and 61% were males. Patients were enrolled from centers in North America (n=181), Europe (n=318), and Asia Pacific (n=44). Patients were diagnosed between 2006 and 2014, the median duration between diagnosis of myeloma and study entry (T0) was 3.1 years (0.3 to 9). The median (95% CI) estimated follow up from diagnosis and from T0 were 61 (57, 66) months and 13 (11, 15) months respectively. The median number of lines of therapy prior to T0 was 4 (3-13), 48% had a prior transplant. The median OS from T0 for the entire cohort was 13 (11, 15) months. For these 462 patients, the median number of recorded regimens was 2 (1-9). The overall response and the depth of response to each line of treatment following T0 are as shown in the table. The median (95% CI) PFS and OS from T0 was 5 (4, 6), and 15.2 (13, 17), respectively. The overall survival for the 81 patients with no treatment post T0 was only 2.1 months. In a multivariate analysis, duration from diagnosis to T0, ISS stage III and number of lines of therapy were all associated with inferior PFS, as well as OS, and in addition, serum creatinine>2 mg/dL at T0 also predicted inferior OS. Conclusions: The study provides the expected outcome following development of myeloma that is refractory to a PI and an IMiD. The outcomes of these patients appear to be better than we had seen historically in patients refractory/ intolerant to bortezomib and IMiDs, highlighting the increased treatment options available for these patients. However, there is decreasing response rate to sequential regimens highlighting the development of drug resistance. The data provides a bench mark for comparison of new therapies that are being evaluated in this disease. Table Table. Disclosures Dimopoulos: Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Kastritis:Takeda: Consultancy, Honoraria; Genesis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Terpos:BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Celgene: Honoraria; Takeda: Consultancy, Honoraria; Genesis: Consultancy, Honoraria, Other: Travel expenses; Amgen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Novartis: Honoraria. Hillengass:Sanofi: Research Funding; Novartis: Research Funding; Amgen: Consultancy, Honoraria; BMS: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria. Leleu:TEVA: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; LeoPharma: Honoraria; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Pierre Fabre: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Takeda: Honoraria. Oriol:Janssen: Honoraria, Other: Expert board committee; Amgen: Honoraria, Other: Expert board committee. Cavo:Celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Mateos:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Vij:Shire: Consultancy; Takeda: Consultancy, Research Funding; Jazz: Consultancy; Karyopharma: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Bristol-Myers Squibb: Consultancy; Amgen: Consultancy, Research Funding. Lokhorst:Genmab: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. van de Donk:Amgen: Research Funding; Janssen: Research Funding; BMS: Research Funding; Celgene: Research Funding. Mark:Onyx: Research Funding, Speakers Bureau; Millenium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ludwig:Amgen: Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; BMS: Speakers Bureau; Janssen: Speakers Bureau. Jagannath:Novartis: Consultancy; Janssen: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Merck: Consultancy. Usmani:Array: Research Funding; Britsol-Myers Squibb: Consultancy, Research Funding; Skyline: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BioPharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; Millenium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Speakers Bureau. Dytfeld:Janssen Poland: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees. Moreau:Novartis: Honoraria; Amgen: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda: Honoraria; Janssen: Honoraria, Speakers Bureau. Lee:Amgen: Membership on an entity's Board of Directors or advisory committees. Shustik:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. de la Rubia:Celgene: Consultancy; Bristol Myers: Consultancy; Amgen,: Consultancy; Janssen: Consultancy. Durie:Takeda: Consultancy; Amgen: Consultancy; Janssen: Consultancy.

Abstract Multiple myeloma is a disease of the antibody producing plasma cells in the bone marrow. It has been characterized by frequent genomic alterations including gains and losses of chromosomes. Some of these alterations result in poor clinical prognosis. Bortezomib, a first in class proteasome inhibitor, is approved for the treatment of relapsed multiple myeloma. To better understand how the genome wide changes observed in myeloma relate to prognosis and treatment response, we have used single nucleotide polymorphism (SNP) array technology to assess genotypes and DNA copy number at up to 100,000 genomic loci. DNA from 147 bone marrow tumor biopsies collected in multi-center phase II and III clinical trials of relapsed multiple myeloma patients prior to treatment with Bortezomib were hybridized on SNP arrays to assess genomic aberrations. Commonly seen genomic alterations in myeloma were observed, including deletions of chromosome 13 (37%), 1p (25%), 6q (23%), and amplifications on 1q (35%). We found that 35% of this relapsed myeloma population exhibited a hyperdiploid genome characterized by gains of chromosomes 3, 5, 7, 9, 11, 15, and 19. Other notable genomic deletions included 17p (22%), 8p (27%) and 10p (16%). A correlation of response to bortezomib therapy in patients with differential gains and losses at the chromosomal level is ongoing. Consistent with previous reports, response to bortezomib was observed in patients with poor prognostic chr 13 deletions. A more in-depth analysis of specific loci associated with clinical outcome will be discussed. In summary, this data will help further define genomic aberrations in multiple myeloma and their association with treatment outcome.

Abstract Previous work in our laboratory has identified the importance of the NFB signaling pathway for plasma cell (PC) survival and accumulation in Multiple Myeloma (MM). Although up to 43% of patient PCs appear to have activated NFkB by gene expression profiling (GEP), the remainder must rely on an alternate signaling pathway for their survival. We examined our MM GEP datasets and supervised the clustering of patients according to an index of NFkB activity. The majority of patients not activating NFkB were associated with a high IL-6/Stat3 signature, which included the protein phosphatase of regenerating liver-3 (PRL-3). Interestingly, analysis of the PRL-3 promoter revealed competing binding sites for both NFkB and STAT3 within the first intron, suggesting that these play a role in the transcriptional regulation of this gene. PRL-3 has been previously demonstrated to be absent in most normal tissues while its expression in MM cells is quite elevated. As normal PCs have low levels of PRL-3 expression, we determined the upper limit of normal to be 3SD above the mean of these cells. Using this cutoff we report that elevated PRL-3 expression is observed in plasma cells from 1/15 normal individuals, 4/22 MGUS patients and 47/126 patients with either smoldering or overt MM (6.7%, 18.2% and 45.6% respectively). Less than 10% of patients were seen to have both an elevated NFkB index and high PRL-3 expression. Patients with higher expression of PRL-3 were found to have statistically significant higher mean values of creatinine, C-reactive protein, lower albumin levels and a lower overall survival in our Mayo Clinic MM dataset. To examine the role PRL-3 in MM biology we first stimulated MM cell lines with IL-6 and observed PRL-3 up-regulation in two cell lines (INA6, OPM1) but not in JK6L (an NFkB activated cell line). Using a luciferase reporter we have determined that STAT3 induction of PRL3 expression requires the first intron of the PRL3 gene which contains the putative STAT3 binding site. Finally we show that PRL-3 inhibition by shRNA leads to decreased cell growth and loss of IL-6 responsiveness in vitro in at least one cell line. Based on these results, we propose that the majority of MM patients depend on either the NFkB or STAT3 signaling pathways for their survival and that these are to a large degree mutually exclusive. Our experimental results suggest that PRL-3 plays a role in IL-6/STAT3 signaling and that it is a promising target for drug development.

Abstract It is well established that 17p13(del) and p53 mutations represent poor prognosis in MM patients. We have previously shown that p53 is a haploinsufficient tumor suppressor in myeloma and p53 expression level is correlated with functional status of the pathway. In addition, p53 expression levels do not always correlate with p53 genotype. The absence of p53 expression in myeloma cells severely abolishes the integrity of its downstream signaling response, thus, driving disease aggressiveness and drug resistance. There is a pressing need for a more effective treatment strategy for high-risk patients harbouring p53 abnormalities. PRIMA-1 is a first-in-class drug that was identified to specifically kill cancer cells harboring mutant p53. It has shown substantial efficacy in various solid tumors with mutant as well as non-mutant p53. However, the discovery of the anti-myeloma activity of PRIMA-1 is still at its infancy. By utilizing human myeloma cell lines (HMCLs) of different p53 status as our study model, we aim to identify the efficacy of PRIMA-1 in MM and to dissect the mechanisms behind PRIMA-1-induced toxicity. Despite its known ability to reactivate mutant-p53 function, we did not observe any restoration of mutant p53 transcriptional activities. The expression levels of the p53-direct-targets, p21, MDM2 and PUMA, were unchanged upon treatment in the p53-mutant-HMCLs. In fact, PRIMA-1 was found to be able to decrease the cell viability of all the HMCLs, irrespective of their p53 status (Mutant, WT, Null), strongly indicating a p53-independent response. Importantly, PRIMA-1 showed the highest cytotoxicity in a p53-deficient background (IC50 of 16uM in -/-, 55uM in WT/WT and 70uM in mutant HMCLs). This finding was validated when p53 silencing in a WT HMCL significantly enhanced its sensitivity to PRIMA-1. GEP analysis on the two most sensitive cell lines (JJN3 and KMS11) demonstrated significant enrichment of genes related to unfolded (p=1.90E-06 in JJN3 and 6.12E-08 in KMS11) and misfolded protein (p=2.99E-06 in JJN3 and 8.21E-08 in KMS11) responses. Subsequent validation by Western blot and qPCR confirmed the activation of ER stress pathway in response to PRIMA-1, evidenced by the up-regulation of ER stress markers such as GRP78, GADD34 and CHOP. Further investigations then revealed that the PERK/CHOP axis was most important as CHOP knockdown saw an attenuation of MM cells sensitivity to PRIMA-1-induced apoptosis. Recently, it was reported that cells with increased ER stress are more sensitive to bortezomib. Indeed, we showed that PRIMA-1 synergizes with bortezomib, and was able to rescue bortezomib resistance when both compounds were co-incubated in a bortezomib-resistant-HMCL. In conclusion, we identified a novel pathway by which PRIMA-1 exerts its toxicity in MM through induction of ER stress. This may be a novel therapeutic strategy to induce ER stress to sensitize cells to proteasome inhibitor. Owing to the versatility of the drug in targeting myeloma cells with or without p53 abnormalities; PRIMA-1 can potentially be used alone or in combination with other drugs such as bortezomib in a broad range of MM including those with high-risk disease. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 76 Background: We measured proliferation in MM cells transfected with 13,984 small interfering RNAs in the absence/presence of increasing concentrations of bortezomib (BTZ) and identified 37 non lethal genes as reproducible BTZ sensitizers. The strongest hit was CDK5 a gene expressed at high levels in MM and neural tissues with relatively low expression in other organs suggesting a large therapeutic window. The small molecule CDK5 inhibitor Dinaciclib is a novel, potent, small molecule inhibitor of cyclin dependent kinases (CDK). It selectively inhibits CDK1, CDK 2, CDK 5 and CDK9 with 50% inhibitory concentrations (IC50) in the low nanomolar concentration. Based on these data, we undertook the current trial to evaluate the maximally tolerated dose of Dinaciclib in this patient population as well as assess the efficacy of single agent Dinaciclib in relapsed MM. Patients and Methods: Patients with relapsed MM and measurable disease were enrolled on this phase 1/2 trial provided they had not more than 4 prior lines of therapy for MM, had adequate performance status and organ and hematological function. Patients receiving strong inhibitors/inducers of CYP3A4 were not allowed unless the drugs could be discontinued. The primary objectives were to (i) to determine the MTD of dinaciclib in subjects with relapsed MM (phase 1) and (ii) to evaluate the confirmed response rate with dinaciclib in patients with relapsed MM (phase 2). Dinaciclib was administered on day 1 of a 21-day cycle at doses of 30–50 mg/m2. Dose escalation was done using a 3+3 design and MTD was defined as one dose level below that resulted in >=2 DLTs among 6 patients. Results: Overall, twenty-nine patients were accrued to this study (19 phase I, 10 phase II) from July 2009 to December 2011. Two patients were replaced for study violations and are not included in analysis. The Phase II analysis included the 6 patients treated at the Phase I dose level 50mg/m2 as well as the first 9 patients accrued to the Phase II portion (15 evaluable patients). The median age of the 27 evaluable patients was 66 (range, 49–81); 52% were male. The median duration from diagnosis was 42 months (range, 7–207 months); median number of prior therapies was 4 (range, 1–5). Overall, 11 (41%) patients were considered high-risk by FISH and 56% were ISS stage III at study entry. Patients have received a median of 2 cycles (range: 1–12) and all patients have discontinued treatment for disease progression (18), alternative Treatment (2), adverse Event (4), or other reasons (3). One dose limiting toxicity was seen over all dose levels. One patient at 40 mg/m2 experienced grade 3 constipation possibly related to treatment. The dose level of 50 mg/m2 was determined to be the MTD for the Phase II portion. The overall confirmed response rate was 3 of 27 (11%); including two patients at 40 mg/m2 dose (1 VGPR, 1 PR) and one patient at 50 mg/m2 dose (1 VGPR) with a PR or better. In addition, two patients at 50 mg/mg2 dose have achieved an MR; translating to an overall response rate of 18.5% (5 of 27). Figure 1 shows a waterfall plot of the M protein responses among patients with a measurable M spike. Overall, 21 patients had disease progression and 14 patients have died. Median follow-up for patients still alive is 11.8 months (range: 6.5–19.3). Leukopenia and thrombocytopenia were the most common hematological AEs, and gastrointestinal symptoms, alopecia, and fatigue, were the most common non-hematological AEs seen in the study. Conclusions: The current study demonstrates potential single agent anti-myeloma activity of dinaciclib in a relapsed MM patient population with 2 patients achieving a deep response (VGPR) and many patients obtaining some degree of M protein stabilization or decrease. Ongoing studies are examining dinaciclib in combination with proteasome inhibitors, exploring both weekly and every three-week dosing schedules. Disclosures: Zonder: Millennium Pharmaceuticals, Inc: Consultancy, Research Funding; Celgene: Research Funding.

Abstract Abstract 834 Multiple myeloma (MM) is an incurable bone marrow cancer. Events mediating transformation from the pre-malignant monoclonal gammopathy of undetermined significance (MGUS) to MM is unknown. We analyzed 2 gene expression datasets generated on the Affymetrix U133 platform. The test set consisted of 22 MGUS and 101 MM (GEO Accession GSE6477) and the validation set 50 MGUS and 351 MM (GEO Accession GSE2658 and GSE5900). The gene expression profiles of MM were compared to MGUS using gene-set enrichment analysis. Genes over-expressed in MM were enriched for cell cycle, proliferation and MYC activation gene-sets. We dissected the relationship between MYC and cell cycle, and identified a MYC activation signature dissociated from proliferation. We validated our MYC signature in publicly available mouse and human cell line gene expression dataset (GEO Accession GSE3151 and GSE3158 respectively), showing specific expression of our MYC signature in cell lines forced to expressed MYC but not when over-expressing other oncogene such as E2F, HER2. In these analyses, we noted that tumors with RAS mutation also consistently express this signature. Applying this signature to the test dataset, we showed that MYC is activated in 60% of myeloma but none of MGUS. This pattern is reproduced in an independent validation dataset. In order to validate the hypothesis that RAS mutation may also lead to the MYC activation signature, we correlated RAS mutation with MYC activation as measured by a MYC index calculated from the median expression of genes that constitute the MYC activation signature, and showed that almost all cases with RAS mutation have a high MYC index. Together with samples with very high expression of MYC mRNA corresponding to those with MYC translocations, these 2 mechanisms account for 67% of cases with MYC activation. To further confirm MYC activation, we performed immunohistochemistry using a validated MYC antibody together with CD138 double-staining. We can clearly identify CD138 positive plasma cells with and without nuclear MYC expression and found that nuclear expression of MYC, a marker of MYC activation, correlated strongly with the MYC signature, therefore providing clear evidence that MYC activation is present in majority of newly diagnosed MM but not in MGUS. MYC activation is not very well correlated with proliferation as assessed by the plasma cell labeling index. Among newly diagnosed myeloma patients with plasma cell labeling index less than 1, those with nuclear MYC expression have significantly shorter survival than those without (Median survival 77.7 months versus 37.9 months, log-rank p-value 0.04). Multiple pathways converge on MYC activation, which is a common transforming event in MM associated with poor prognosis. MYC nuclear staining by IHC can be a useful clinical surrogate. Targeting MYC can be a chemoprevention strategy. Disclosures: Fonseca: Amgen: Consultancy; Medtronic: Consultancy; Celgene: Consultancy; Bristol Mayor Squibs: Consultancy; Genzyme: Consultancy; Otsuka: Consultancy.

Key PointsDUSP22-rearranged ALCLs belong to a distinct subset of ALCLs lacking activated STAT3. DUSP22-rearranged ALCLs have a unique molecular signature characterized by DNA hypomethylation and an immunogenic phenotype.