Citations

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 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 Background: Lenalidomide with dexamethasone (Rd) is a standard of care used for pts with relapsed multiple myeloma (RMM). The randomized, open-label, multicenter, phase 3 study ASPIRE (NCT01080391) is comparing carfilzomib, lenalidomide, and dexamethasone (KRd) to Rd in pts with RMM. The primary end point is progression-free survival (PFS; assessed by an independent review committee). Secondary end points include overall survival (OS), overall response rate (ORR), duration of response (DOR), health-related quality of life (EORTC QLQ-C30 Global Health Status/QoL), and safety. Methods: Adults with RMM who received 1–3 prior regimens were eligible. Pts were randomized (1:1) to receive KRd or Rd and were stratified by β2-microglobulin levels (<2.5 vs ≥2.5 mg/L), prior bortezomib (no vs yes), and prior R (no vs yes). All pts received lenalidomide (25 mg) on days 1–21 and dexamethasone (40 mg) on days 1, 8, 15, and 22 of a 28-day cycle. In addition, KRd pts received K as a 10-min infusion on days 1, 2, 8, 9, 15, and 16 during cycles 1–12 (20 mg/m2 [days 1 and 2 of cycle 1]; 27 mg/m2 thereafter); K was omitted on days 8 and 9 during cycles 13–18 and was not administered beyond 18 cycles. Cycles were repeated until disease progression, unacceptable toxicity, or withdrawal of consent. The study had 90% power to detect a 25% reduction in risk for PFS events for KRd vs Rd (hazard ratio [HR]=0.75) at a 1-sided significance level of 0.025. A stratified log-rank test was used for the PFS comparison. Results: Data are presented for KRd followed by Rd throughout the abstract. Between July 2010 and March 2012, 792 pts from 20 countries were randomized. Baseline characteristics were balanced between the 2 groups. Median age was 64.0 years (range: 31.0‒91.0). Pts received a median of 2 prior regimens in each group. In both the KRd and Rd groups, 66% of pts received prior bortezomib; 20% of pts in each arm received prior R. Median treatment exposure was 22 cycles (KRd) and 14 cycles (Rd). At the time of the prespecified interim analysis, the study met the primary end point for PFS (HR=0.69; 95% confidence interval [CI]: 0.57–0.83; P<.0001), with a longer duration of median PFS in the KRd group (26.3 months [mo]; 95% CI: 23.3–30.5) compared with the Rd group (17.6 mo; 95% CI: 15.0–20.6). Median OS was not reached in either group; however, there was a trend toward longer OS with KRd compared with Rd (HR=0.79; 95% CI: 0.63–0.99; P=.018), which did not meet the prespecified statistical boundary at the interim analysis of survival (P=.005). The Kaplan-Meier OS event-free rates at 24 mo were 73.3% (95% CI: 68.6‒77.5) and 65.0% (95% CI: 59.9‒69.5). The ORR was 87.4% (95% CI: 83.7–90.5) with KRd and 66.9% (95% CI: 62.0–71.5) with Rd. Median DOR was 28.6 mo (95% CI: 24.9–31.3) and 21.2 mo (95% CI: 16.7–25.8). In the KRd and Rd groups, 31.8% vs 9.4% achieved a stringent complete response (sCR) or complete response (CR) (sCR: 14.1% vs 4.3%; CR: 17.7% vs 5.1%), and 70.4% and 40.7% achieved ≥very good partial response. KRd consistently improved Global Health Status/QoL compared with Rd over 18 cycles of treatment (P=.0001). Treatment discontinuation due to an adverse event (AE) occurred in 15.2% (KRd) and 17.4% (Rd) of pts. In each group, 7.7% and 8.5% of pts died while still on study treatment or within 30 days of receiving the last dose of study treatment. The most common hematologic treatment-emergent AEs (TEAEs) (≥grade 3) included neutropenia (29.6% vs 26.5%), anemia (17.9% vs 17.2%), and thrombocytopenia (16.6% vs 12.3%). The most common nonhematologic TEAEs (all grade) included diarrhea (42.3% vs 33.7%), fatigue (32.9% vs 30.6%), and cough (28.8% vs 17.2%). The most common nonhematologic TEAEs (≥grade 3) included pneumonia (12.5% vs 10.5%), hypokalemia (9.4% vs 4.9%), and hypophosphatemia (8.4% vs 4.6%). Other TEAEs of interest (grouped terms; all grade) included dyspnea (22.4% vs 18.0%), hypertension (preferred term; all grade: 14.3% vs 6.9%; grade 3: 4.3% vs 1.8%), acute renal failure (8.4% vs 7.2%), cardiac failure (6.4% vs 4.1%), and ischemic heart disease (5.9% vs 4.6%). Rates of peripheral neuropathy (PN) (grouped terms; all grade) were 17.1% and 17.0%; ≥grade 3 PN was infrequent (2.6% and 3.1%). Conclusion: The addition of carfilzomib to lenalidomide and dexamethasone in pts with RMM resulted in a statistically significant and clinically meaningful improvement in PFS. KRd had an acceptable safety and tolerability profile and represents a potential new standard of care. Disclosures Stewart: Novartis: Consultancy; Array BioPharma: Consultancy; BMS: Consultancy; Millenium: Research Funding; Celgene: Consultancy. Off Label Use: Carfilzomib is approved in the United States for the treatment of patients with multiple myeloma who have received at least two prior therapies including bortezomib and an immunomodulatory agent and have demonstrated disease progression on or within 60 days of completion of the last therapy. ASPIRE is a randomized, multi-center, phase 3 study investigating the use of carfilzomib in combination with lenalidomide and dexamethasone for the treatment of patients with relapsed multiple myeloma who have received 1–3 prior regimens.. Dimopoulos:Celgene: Consultancy, Honoraria; Onyx: Consultancy, Honoraria. Masszi:Janssen Cilag: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Spicka:Janssen-Cilag: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau. Oriol:Celgene: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau. Hájek:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees. Rosiñol:Celgene: Honoraria; Janssen: Honoraria. Siegel:Onyx: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Millennium: Honoraria, Speakers Bureau. Niesvizky:Onyx Pharmaceuticals: Consultancy, Research Funding, Speakers Bureau; Millennium: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy, Research Funding, Speakers Bureau. Jakubowiak:Onyx Pharmaceticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. San Miguel:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees; Millinnium: Membership on an entity's Board of Directors or advisory committees. Ludwig:Celgene: Research Funding, Speakers Bureau; Takeda Celgene: Research Funding, Speakers Bureau; Bristol-Myers: Research Funding, Speakers Bureau. Zojwalla:Onyx Pharmaceticals, an Amgen subsidiary: Employment, Equity Ownership. Tonda:Onyx Pharmaceticals, an Amgen subsidiary: Employment, Equity Ownership. Xing:Onyx Pharmaceticals, an Amgen subsidiary: Employment, Equity Ownership. Moreau:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; 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; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees. Palumbo:Onyx Pharmaceuticals: Consultancy, Honoraria; Millennium Pharmaceuticals: Consultancy, Honoraria; Janseen-Cilag: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Genmab A/S: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Amgen, Inc,: Consultancy, Honoraria; Array BioPharma: Honoraria.

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 1517 Purpose: Decitabine, a deoxycytidine nucleoside derivative, is an inhibitor of DNA-methyltransferases indicated for treatment of myelodysplastic syndrome (MDS) including previously treated and untreated, de novo or secondary MDS of all French-American-British (FAB) subtypes, Intermediate-1, Intermediate-2, and High-Risk International Prognostic Scoring System (IPSS) groups. Laboratory evidence shows that pretreatment of AML cell lines can sensitize leukemia cells to chemotherapy as well as inhibit clonogenic potential. The purpose of this study is to evaluate the safety, pharmacokinetics, and efficacy of decitabine when used as priming before induction therapy in children with newly diagnosed AML. Exploratory analyses of genomic methylation and RNA expression patterns and minimal residual disease (MRD) are embedded correlative studies. Methods: This multicenter, randomized, two-arm, open-label study enrolls pediatric pts ages 1–16 with newly diagnosed AML to either Arm A: daunorubicin, cytarabine, etoposide (D-ADE) preceded by a 5-day course of decitabine (experimental arm) or Arm B: daunorubicin, cytarabine, etoposide (ADE; control arm). Following completion of induction on study, post-induction therapy is at the treating physician's discretion. To date, 20 pts, 10 in each arm (1–16 yrs, median 9.4 yrs in both arms) have been enrolled. Results: At the protocol-specified bone marrow sampling time 3 weeks post-induction, there were 2 CRs, 6 CRi (CR with incomplete hematopoietic recovery), 1 leukemia not in remission and 1 pt who discontinued due to an AE in Arm A. In Arm B, 6 CRs, 2CRi, 1 PR and 1 pt with marrow aplasia have been confirmed. Decitabine PK in all arm A pts (100%) were characterized by mean+SD Cmax, 297+109 ng/mL, AUC0-t, 216+73.7 ng*h/mL, CL, 136+93.9L/h, Vdss 93.1+70.7 L, t1/2 0.456+0.073 h, and median tmax0.925 h. All pts had at least one AE. Grade 3 and 4 non-hematologic AEs in Arm A that were not seen in Arm B were caecitis in 2 (20%), anorexia in 3 (30%), and hypophosphatemia in 2 (20%) of pts. All pts experienced neutropenia and thrombocytopenia as expected. One pt in Arm A had appendicitis and colon perforation on Day 6 that led to study discontinuation, later determined to have been leukemic infiltrate. Two pts have died to date (both in Arm A), one of necrotic bowel, Pseudomonas sepsis and multisystem organ failure 6 months after completing study induction therapy. One pt died 5 months following study treatment from multisystem organ failure after stem cell transplant. Epigenetic changes associated with decitabine pretreatment were analyzed and will be presented. Conclusions: Decitabine in combination with ADE chemotherapy is well tolerated in children with newly diagnosed AML. There were no AEs that had not been previously identified in adults with MDS or AML. No differences have been observed between treatment arms in hematologic toxicities based on the current sample size. To date, the decitabine-treated pts in this study may have more gastrointestinal toxicity and hypophosphatemia. Decitabine-treated pts achieved PK exposures that were similar to those observed in adults treated with decitabine. Preliminary responses by CR/CRi were similar between treatment arms; expected adverse events of neutropenia and thrombocytopenia were similar between both Arms. Molecular changes associated with decitabine pretreatment may be important in the sensitization of clonogenic AML cells. Total accrual of 40 subjects is planned. Disclosures: Off Label Use: AC220 in relapsed/refractory pediatric acute leukemia. Tarassoff:Eisai, Inc.: Employment. Jones:Eisai Inc: Employment.

Abstract Abstract 1563 Intro: Optimal treatment for non-follicular low grade B-cell lymphoma (NFLGL) is not well defined. Clinical trial design for these subtypes would be enhanced by further understanding of early presentation and management. A large prospective observational study was used to identify presenting clinical features, describe patterns of care and compare the prognostic utility of International Prognostic Index (IPI) and the Follicular Lymphoma International Prognostic Index (FLIPI). Methods: The University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER) is a prospective, longitudinal observational study designed to collect information on patterns of care and outcomes for patients with newly diagnosed lymphoma. Patients seen at the Mayo Clinic, Rochester MN and the University of Iowa within 9 months of their initial diagnosis of lymphoma are offered enrollment. Baseline patient reported data, clinical data and initial management are collected using a standard protocol and a central pathology review is performed. After enrollment, patients are actively followed for events (disease progression, retreatment, and death) and disease related comorbidities. This analysis includes 198 patients diagnosed with extranodal marginal zone (EMZL), 22 with nodal marginal zone (NMZL), 47 with splenic marginal zone (SMZL), 48 with lymphoplasmacytic lymphoma (LPL), as well as 67 patients with unclassifiable low-grade B-cell lymphoma (B-NOS). Results: 382 newly diagnosed NFLGBL patients were enrolled in the MER from 2002–2009. The median age at enrollment was 63 years (range 22–95). 52% were male. The most common types of initial therapy after diagnosis were observation (41 %), alkylator- based chemotherapy +/− rituximab (R) (17%), radiation therapy alone (15%) and R monotherapy (13%). At median follow-up of 60 months (range 1–121), 51 patients were deceased and 155 had an event. The clinical characteristics that comprise the IPI and FLIPI were similar across the subtypes. Approximately half of the cases presented at low risk (55% IPI; 48% FLIPI). A majority had a normal LDH (81%), limited nodal involvement (91% had ≤ 4 groups), normal hemoglobin (71% ≥12 g/dL), good performance status (95% 0–1), and advanced stage (57% stages III-IV). There were notable differences among the NFLGBL subtypes. Patients with SMZL and LPL presented with high risk FLIPI of 3–5 (49% and 46% respectively) and high to high-intermediate IPI of 3–5 (21% and 13%). Abnormal LDH and anemia (< 12 g/dL) were more frequent in patients with SMZL (40%; 56%) and LPL (25%; 62%). EMZL presented with lower stages (70% stage I-II) and NMZL presented with increased nodal involvement (38% >4 sites). SMZL had the highest rates of surgical resection (21%) and EMZL was more frequently treated with radiation therapy (25%). LPL (30%) and NMZL (32%) more commonly received alkylator based chemotherapy +/− R. While both IPI and FLIPI differentiated outcome in these patients, FLIPI had higher c-statistics than IPI for both EFS (0.583 vs 0.568) and OS (0.685 vs 0.661), respectively. Conclusions: This large prospective collection of data on NFLGBL patients presenting to academic medical centers provides a modern picture of presentation and management patterns useful to investigators designing clinical trials. FLIPI may have more prognostic utility than IPI in this group of patients. Disclosures: Ansell: Seattle Genetics, Inc.: Research Funding.

Abstract Background: The incidence of secondary Acute Lymphoblastic leukemia (sALL) after a preceding first primary solid organ malignancy (1M) is not well defined. We undertook a Surveillance Epidemiology and End Results (SEER)-based analysis to describe the occurrence of sALL among adult patients with a history of common 1M's. We also evaluated differences in sALL survival on the basis of age, the site and extent of 1M, and for evidence of any underlying racial/ethnic disparity. Methods: The SEER-18 database (1973-2011) was interrogated for the current study. All confirmed cases of ALL (ICD-O-3 codes: 9811, 9812, 9814-18, 9826-28, 9835-37) in adult patients (age ≥18 years) were identified. De novo ALL vs. sALL was determined by using the SEER variable ‘First Malignant Primary Indicator'. 1M prior to the diagnosis of ALL was identified by merging all SEER databases together by patient ID number. Those with 1M stage of ‘in situ'; sALL cases reported <2 months after the diagnosis of 1M; and those with “unknown” information on 1M (n=471) were excluded. Standard race/ethnicity categories were used. Latency interval (time from diagnosis of 1M to sALL) was calculated for all patients. Overall survival (OS) was calculated as the interval from diagnosis to death, or censored at date of last contact, December 31st 2011, or 5 years after diagnosis, whichever came first. A proportional hazards Cox regression model was performed to evaluate the impact of sALL and latency on OS, adjusting for age at diagnosis, race/ethnicity, marital status, birthplace, SEER registry site, and year of diagnosis. All statistical tests were two-sided and utilized the SAS software (v9.4) with a two-sided significance level of 0.05. Results: A total of n=8,398 adult patients with primary ALL (1ALL) and n=414 with sALL were identified. Patient characteristics were significantly different between 1ALL & sALL (Table). Males comprised 49.8% of sALL patients as compared to 58% with 1ALL. Age at diagnosis of ALL was also significantly different, with 55.5% of sALL patients being ≥65 years as compared to 51.4% of 1ALL observed in the 18-44 years age group. Non-Whites comprised 27.3% of sALL and 40.4% of 1ALL, with Hispanics having the most differential distribution of 13.3% and 24.6%, respectively in the two groups. Distribution of sALL patients by latency from 1M is shown in Table. The majority of sALL patients had a diagnosis of local/regional (rather than advanced stage) 1M (Table). sALL patients had a significantly inferior OS when compared to 1ALL (HR 1.211, 95%CI 1.079-1.359) (Median OS 14 months vs. 18 months, Figure). sALL cases with longer latency interval (>5 year) appeared to have better median OS compared to shorter latency intervals, but this difference was not statistically significant (p=0.53). Conclusion: We have performed the largest population-based analysis identifying sALL after 1M, demonstrating that sALL patients are older and have significantly worse OS than 1ALL. We noted a lower relative incidence of sALL in Hispanics as compared with 1ALL, while the converse was true for Whites. Although detailed treatment data (specifically with respect to chemotherapeutic agents) is not available in SEER, it is possible that treatment for local/regional 1M - presumably adjuvant in nature - may be associated with the risk of sALL. Further analyses are required and are ongoing to confirm the relative incidence after specific 1M's, and the impact of both specific treatment modalities and the era of treatment for 1M on risk of developing sALL. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Abstract Background: Chronic lymphocytic leukemia (CLL) patients exhibit variable times from diagnosis to the need to initiate therapy. Previous studies have demonstrated that the rates of CLL cell proliferation (birth) and elimination (death plus recirculation to solid tissues) could be estimated using the incorporation of deuterium (2H), taken in the form of heavy water (2H2O), into the DNA of dividing cells. In a cross-sectional study of patients with early and late stage CLL, birth rates above 0.35% per day were associated with more aggressive disease. The NIH-sponsored CLL Research Consortium (CRC) conducted a prospective study on patients with early stage disease to assess the relationship between CLL-cell birth rate and the length of time to initiation of initial therapy. Methods: Eligible patients had untreated early stage (Rai 0, 1 or 2) disease, and were no more than 3 years from diagnosis. Additional clinical parameters included age at diagnosis, gender, mutational status of the immunoglobulin heavy chain variable region (IGHV), 70-kD zeta-associated protein (ZAP-70) (binary split at 20%) and CD38 (binary split at 30%) expression, and FISH cytogenetics by the Dohner hierarchy. Heavy water was administered daily for 6 weeks, and peripheral blood was obtained for kinetic measurements at the end of weeks 3, 6, 9, 12, and 16. CLL birth rates for blood samples were measured at the same facility (KineMed, Inc, Emeryville, CA) by quantifying deuterium incorporation into the deoxyribose moiety of DNA of tumor cells, using isotope ratio mass spectrometry (IRMS). Subjects were followed for time to administration of therapy; subjects still without treatment at time of analysis were censored, as were subjects who exited the study to receive treatment outside of the established criteria. To assess the impact of birth rate and other factors on time to first therapy, Kaplan-Meier curves were compared using the logrank test for univariable analyses; stepwise Cox proportional hazards regression models were used to build multivariable models. Results: Between July 2005 and February 2009, 119 subjects were enrolled through the CRC. 107 subjects were eligible. For 10 eligible patients, exit from the study prior to completion of protocol-mandated heavy water consumption (n=5) or technical issues (n=5) made it impossible to obtain estimates of birth rate. The 97 eligible subjects for whom birth rates were available form the basis of this report. 60% of subjects were male; median age 57 years (range 40-85). Median birth rate was 0.32%/day (range 0.07-1.31%/day). 43 subjects (44%) had birth rates higher than the previously reported threshold of 0.35%/day. With a median follow up of 4 years, 33 eligible subjects (34%) required treatment. IGHV status (31 subjects unmutated), elevated ZAP-70 (31 subjects), elevated CD38 (21 subjects), and hierarchical categorization of FISH anomalies (4 del(17p), 5 del(11q), 8 tri(12), 50 del(13q)) were examined for association with differential time to first therapy in univariable analyses. All were associated with earlier need for therapy (p <0.001) except for genomic aberrations defined by FISH (p = 0.96). Birth rate > 0.35%/day was also significantly associated with shorter time to first therapy (p < 0.001). In multivariable modeling, only birth rate and IGHV status met the criteria for inclusion (Wald p-value < 0.05). Hazard ratios were 3.51 (p = 0.002) for birth rate above 0.35%/day and 5.0 for unmutated IGHV status (p < 0.001). The association between elevated birth rate and shorter time to first treatment was observed within subjects with both unmutated and mutated IGHV (see Figure 1). Conclusion: An increased birth rate of CLL cells early in the disease course is a strong predictor of an earlier need for treatment in univariable analysis, as are unmutated IGHV, elevated ZAP-70 expression and elevated CD38 expression. However, only unmutated IGHV and an increased birth rate contribute significantly to the multivariable model. This adds a direct measure of B-cell kinetics to the array of biomarkers available to subjects with CLL. Figure 1 Time to treatment by birth rate and IGHV status Figure 1. Time to treatment by birth rate and IGHV status Disclosures Murphy: KineMed, Inc: Equity Ownership. Emson:KineMed, Inc.: Employment, Equity Ownership. Li:KineMed, Inc.: Employment, Equity Ownership. McConnel:KineMed, Inc.: Equity Ownership. Turner:KineMed, Inc.: Employment, Equity Ownership. Busch:KineMed, Inc.: Equity Ownership. Hellerstein:KineMed Inc: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Chiorazzi:KineMed, Inc.: Stock options Other.

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 Next-generation sequencing (NGS) has revealed new insight into the complexity of clonal and subclonal architecture of multiple myeloma (MM). The introduction of targeted studies allows the detection of mutations with very low allele frequencies at affordable price. However, there is few information about the prognostic impact of this mutational profile in series of homogeneously treated MM patients. In this study, we analyzed the most frequently mutated genes in MM from patients included in a randomized clinical trial applying the highest read depth to date. We performed a correlation study between our results and cytogenetics aberrations and clinical outcomes. We used Ampliseq Library Kit 2.0 to target 77 genes (M3Pv2.0 panel) related to critical pathophysiological pathways, associated to drug resistance or targetable with molecular drugs in MM. We sequenced DNA (15ng) from CD138+ purified plasma cells from 80 MM patients at diagnosis included in GEM2010MAS65 clinical trial. We sequenced at an average read depth of 1500X and using Ion Proton sequencer (Thermo Fisher, USA, PaloAlto, CA). We used Ion Reporter software applying in-house modifications in call variants process. We also sequenced DNA from CD138 negative population in 30% of patients in order to filter out potential artifacts and stablish conditions for excluding germline variants. We first fitted a cox-proportional hazard model to predict survival and a second approach using a penalized regression LASSO. We identified 137 gene mutations in the 80 patients analyzed, 54 mutations were predicted pathogenic by Shif and Poliphen and 65 have been described in COSMIC database. 85% of patients harbored at least 1 mutation. The most frequently mutated genes were KRAS (16%), DIS3 (15%), NRAS (13%), BRAF (6%), and TP53 (5%), accounting for the 54% of the total number of mutations. The most frequently mutated pathways were RAS and NFKB in 34% and 28% of patients respectively. Mutational allele frequency for KRAS, BRAF and TP53 was, in all cases, lower than 50% while DIS3 showed mutations in a broad range (from 2 to 85%). Only one patient presented one mutation in NRAS at 73 % of allele frequency. For patients with more than one mutation, two different scenarios have been found. Some patients showed several genes mutated in a similar allele frequency values. On the contrary, a complex subclonal structure was confirmed in 3 patients, with mutations at very different clonal frequency. CD138 negative fraction was sequenced to confirm that these mutations were exclusive of plasma cells. The first of them had two mutations in DIS3 at 8 and 53% of allele frequency, second in KRAS and DIS3 at 13 and 63% respectively and the third in NRAS and IFNGR2 at 5 and 37% respectively. We found no differences in the survival based on pathway, mutational allele frequency or number of mutations. We also investigated pairwise associations between events and we not found significant correlations (Figure 1). Cox-proportional hazard model only showed a negative impact in survival of patients with TP53 mutations. This ultra-deep targeted sequencing strategy is able to detect mutations in most of patients, at very low allele frequency and using a minimal amount of DNA. Despite of its huge capacity to detect mutations, we have not identified new prognostic factors in MM patients treated with highly efficient therapies. However, this study again confirms the complexity of the genomic landscape of MM and the great heterogeneity between patients. The role of theses mutations at relapse as well as in the subsequent treatment effectiveness remains unknown and should be preferably investigated in larger cohorts of homogeneously treated MM patients. Figure 1 Figure 1. Disclosures Martínez-López: Novartis: Honoraria, Speakers Bureau. Oriol:Janssen: 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. Stewart:celgene: Consultancy.