Fonseca, Rafaël

Abstract Introduction: Monoclonal B cell lymphocytosis (MBL) is an asymptomatic expansion of clonal CD19+/CD5+ B cells with less than 5x109/L cells in the peripheral blood and without other manifestations of chronic lymphocytic leukemia (CLL). Approximately 1% of MBL evolves to CLL requiring therapy per year; thus it is critical to develop more precise tools to identify which MBL will progress to CLL and require treatment. Patients and Methods: In this study, we performed targeted deep sequencing (TDS) on 49 high-count MBL individuals (median B-cell count 3.7x109/L; range 0.8-4.9x109/L) and explored the mutation status of 20 driver genes recurrently mutated in CLL. We analyzed the clonal evolution in 45 of these 49 MBLs by screening 2-4 sequential samples (average time between samples 56 months, range 10-119 months). At last follow-up, 19 cases (39%) had progressed to Rai>0, and 10 cases (20%) required treatment. Tumor and germ line DNAs were isolated from sorted CD5+/CD19+ and CD5-/CD19- cell populations, respectively. Overall, 154 samples from 49 MBL cases (105 tumor and 49 germ line) were screened using semiconductor sequencing technology. The latter genetic information was integrated with relevant clinical and biological parameters, and we evaluated the effect of driver mutations and clonal expansion on time to CLL progression and time to treatment (TTT). Results and Discussion: Our cohort consisted in 17 women and 32 men, with a median age of 66 years (range: 44-80). Five cases presented secondary diseases, including melanoma, lung and bladder cancer. Clinical and biological parameters were collected, including IGHV mutation status (mutated 66%, unmutated 34%), ZAP70 and CD49 expression (25% each). At presentation, 46% of cases had del(13q), 27% trisomy 12, 6% del(11q), and 4% del(17p). Overall, we found somatic non-synonymous mutations in 23 of 49 MBLs (47%) at the initial time point including 22% of cases with more than one mutated driver gene. The average depth of coverage was 730x, thus allowing the identification of small subclonal mutations. Recurrent mutations were found in most of the drivers: CHD2, DDX3X (8% of cases), FBXW7, NOTCH1, SF3B1 (6% each), ATM, BCOR, BIRC3, BRAF, KRAS, MED12, MYD88 and ZMYM3 (4% each). Furthermore, ITPKB, POT1, SAMHD1 and XPO1 were mutated in only one case, whereas no mutations were found in HIST1H1E, RIPK1 and TP53. In 4 individuals, we found two mutations in the same gene (BRAF, DDX3X, KRAS and SAMHD1). Genes that are known to be associated with disease progression in CLL were either mutated with significantly lower incidence (NOTCH1, SF3B1) or not mutated (TP53). Mutations were detected on average 45 months (range 9-73) prior to progression to CLL Rai>0 indicating the early origin of most driver gene mutations in the MBL/CLL continuum. The presence of driver mutations in MBL was associated with shorter TTT (median TTT: present: 96 months vs. not present: not reached, HR: 5.52, 95% CI: 1.2-26.2, P =0.015). Next, we looked at clonal expansion of driver mutations over time (defined as >2-fold change in the allelic frequency of driver mutations between time points). Of 20 MBLs with mutations at baseline who had sequential samples available, 10 cases showed clonal expansion. Seven out of 10 MBLs who required therapy showed clonal expansion, which was detected on average 15 months (range 6-30 month) prior to treatment. Finally, the detection of clonal expansion was significantly associated with reduced TTT (median TTT: clonal expansion: 21 months vs. no clonal expansion: 84 months, HR: 7.79, 95% CI: 1.94-31.3, P <0.001). Conclusion: We have confirmed the existence of recurrent mutations in most CLL putative driver genes at the premalignant MBL stage many years before progression to CLL. Furthermore, the early identification of driver mutations and its clonal expansion predicts a shorter TTT. Of note, clonal evolution under selective pressure has recently been linked to the onset of CLL progression after therapy. In this study, we characterized the clonal dynamics in the pre-malignant stages of the disease and underlined its impact on clinical outcome. Despite the relatively small size of the cohort, these findings suggest that the sequential monitoring of MBL individuals with a simple and reliable technique, such as TDS, will be at least of prognostic use and thus its incorporation in the disease stratification and clinical management should be further tested. Disclosures Fonseca: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Applied Biosciences: Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Binding Site: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Onyx/Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Kay:Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Tolero Pharma: Research Funding; Genentech: Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hospira: Research Funding.

Introduction We have shown that in myeloma (MM) the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide, in a CRBN dependent fashion, inhibit thioredoxin reductase and thus increase intracellular oxidative stress as a consequence of peroxide accumulation (Sebastian et al. 2017). Accordingly, we have also shown that CRBN expression alone does not fully correlate with lenalidomide sensitivity, as the cells ability to decompose H2O2 is important - MM cells with lower H2O2 decomposition capacity are more sensitive to IMiDs. We hypothezised that the specific metabolic pathways used by cells for biomass production would influence IMiD sensitivity since they also influence this cellular oxidative capacity. Cellular proteins are key elements of this biomass, and required for the proliferation of MM cells and production of immunoglobulin proteins. Methods We cultured MM cells under various nutrients conditions to understand its effect on cell proliferation and drug responses. Cell viability was assessed by MTT assay, the Luminescent Assay, and direct cell counting. MM cells glycolytic rate was measured using the Seahorse XF analyzer. Western blots were performed to quantify various protein expression levels in MM cells cultured under different nutrients conditions. CRBN CRISPR/Cas9 KO plasmid was used to generate CRBN knockout cells and a lentivirus system was used to over-express CRBN in MM cells. Results MM cells resistant to lenalidomide, but still expressing functional CRBN, show a higher glycolytic pathway use than sensitive cells. We found that the lenalidomide sensitive cell line MM1.S is highly dependent on extracellular glutamine for cell proliferation. In contrast, JJN3, a lenalidomide resistant cells line with wild type CRBN, preferentially consumes glucose. We found that glutamine depletion from culture media completely abolished lenalidomide sensitivity in MM1.S cell line. Western blot analysis revealed that antibody production by MM1.S was primarily dependent on glutamine availability, and that JJN3 antibody production was dependent upon glucose availability. While glutamine depletion from culture medium completely abolished lenalidomide sensitivity in MM1.S cell line, IKZF1, and IKZF3 degradation was unchanged. We further studied the role of CRBN in glutamine dependent cellular biomass protein production using isogenic cell lines (+/- wtCRBN). We found that wtCRBN expressing cells proliferate more and show higher antibody production in the presence of glutamine, over CRBN negative isogenic cell lines. Lenalidomide treatment further increased glutamine dependent antibody production in wtCRBN expressing cells. One possibile explanation is that activation of glutamine catabolism can facilitate protein demethylation via the supplementation α-ketoglutarate (α-KG). Therefore, we tested whether lenalidomide treatment could induce protein demethylation in sensitive cell lines. Western blot probed with mono-methyl lysine antibody showed lenalidomide induced protein demethylation. We further confirmed that protein demethylation, and lenalidomide sensitivity is a consequence of elevated α-KG by treating cells with cell-permeable 5-octyl- α-ketoglutarate which act as a substrate of the lysine demethylases. 5-Octyl-α-ketoglutarate treatment inhibited cell proliferation preferentially in wtCRBN expressing cells, and also enhanced lenalidomide induced sensitivity. Protein demethylation is associated with protein ubiquitination and proteasomal degradation. We thus hypothesize that lenalidomide induced protein demethylation also likely increases proteasome inhibitors sensitivity in MM. Conclusion MM cells with preferential glutamine consumption are likely to be more sensitive to lenalidomide, and extracellular glutamine depletion can induce lenalidomide resistance. MM cells expressing CRBN, but not dependent on glutamine for protein biomass production are more likely to be resistant to lenalidomide. Our study postulates that CRBN protein requirement in lenalidomide sensitivity cells is restricted to high glutamine dependency, and that it is quite likely CRBN has a role in glutamine metabolism. Moreover, MM cells that consume more glutamine are under higher oxidative stress and exhibit less H2O2 decomposition capacity and therefore increasing sensitivity to IMiDs. References Sebastian , S., et al., Blood 2017 129:991-1007 Disclosures Stewart: Ono: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Takeda: Consultancy; Amgen: Consultancy, Research Funding; Bristol Myers-Squibb: Consultancy; Celgene: Consultancy, Research Funding; Ionis: Consultancy; Janssen: Consultancy, Research Funding; Oncopeptides: Consultancy. Bergsagel:Janssen Pharmaceuticals: Consultancy; Ionis Pharmaceuticals: Consultancy; Celgene: Consultancy. Fonseca:AbbVie, Amgen, Bayer, Celgene, Kite, Janssen, Juno, Merck, Pharmacylics, Sanofi, Takeda: Other: Consultant/Advisor; Prognosticatin of MM based on Genetic Categorization by FISH: Patents & Royalties; Adaptive Biotechnologies: Other: Scientific Advisory Board.

Background: Combinations of alkylating agents with proteasome inhibitors have demonstrated efficacy in newly diagnosed and relapsed multiple myeloma (MM), with melphalan or cyclophosphamide combinations being some of the commonly used regimens for initial treatment of MM. Ixazomib (Ixa) is an oral proteasome inhibitor that is approved for use in combination with lenalidomide for patients with relapsed MM. We examined if Ixa can be effectively combined with cyclophosphamide (Ctx) in order to develop a less expensive, all oral regimen for patients with relapsed MM. Patients and Methods: Patients with relapsed MM, who were proteasome inhibitor naïve OR have received less than 6 cycles of therapy with bortezomib and had a better than PR with no progression at the time of discontinuation, were enrolled. The primary objective was to determine overall response rate (ORR). Treatment consisted of Ixa 4mg PO days 1, 8, 15; Ctx 300 mg/m2 PO days 1, 8, 15, 22 and dexamethasone (Dex) 40 mg PO weekly in a 28-day cycle. Overall, 37 patients were accrued; data on 33 eligible patients were available for analysis as of July 18, 2019. Results: The median age was 71 (48-89), 61% were male and the median duration from diagnosis was 46 months (mos). Median number of prior lines of therapy was 4 (range 1-5), 76%, 42% and 67% respectively had a prior IMiD, proteasome inhibitor or stem cell transplant, respectively. At data cutoff, 22 (67%) had progressed, 4 (12%) had died and the median follow up of those alive was 21.3 mos. Fourteen patients are still receiving treatment, with median of 8.5 cycles. Most common reason for treatment discontinuation was disease progression (10 pts; 53%). The ORR was 60% including 6% CR and 24% VGPR. The median event free survival was 11.3 mos (95%CI: 9.0 - 26.8). Overall, 401 cycles have been administered across the study, with dose modifications/ hold required for Ixa, Ctx, and Dex in 9 (27%), 14 (42%), and 22 (67%) patients respectively, the most common reason being hematologic toxicity. A grade 3 or higher adverse event at least possibly attributed to the study drugs was seen in 77% of patients, hematologic in 67% and non-hematologic in 30%. (Table 1) The most commonly observed hematologic toxicities included thrombocytopenia, neutropenia, lymphopenia and anemia; for non-hematologic was nausea, diarrhea, peripheral neuropathy toxicity and fatigue. Conclusions: The combination of Ixa, Ctx and Dex (ICd) offers a convenient, all oral regimen for treatment of relapsed disease not refractory to proteasome inhibitors. The regimen has good efficacy in this group f heavily pretreated patients, with an acceptable toxicity profile. Disclosures Lacy: Celgene: Research Funding. Gertz:Ionis: Honoraria; Spectrum: Honoraria, Research Funding; Janssen: Honoraria; Celgene: Honoraria; Prothena: Honoraria; Alnylam: Honoraria. Ailawadhi:Takeda: Consultancy; Janssen: Consultancy, Research Funding; Cellectar: Research Funding; Pharmacyclics: Research Funding; Amgen: Consultancy, Research Funding; Celgene: Consultancy. Bergsagel:Janssen Pharmaceuticals: Consultancy; Celgene: Consultancy; Ionis Pharmaceuticals: Consultancy. Fonseca:AbbVie, Amgen, Bayer, Celgene, Kite, Janssen, Juno, Merck, Pharmacylics, Sanofi, Takeda: Other: Consultant/Advisor; Prognosticatin of MM based on Genetic Categorization by FISH: Patents & Royalties; Adaptive Biotechnologies: Other: Scientific Advisory Board. Dingli:alexion: Consultancy; Janssen: Consultancy; Millenium: Consultancy; Rigel: Consultancy; Karyopharm: Research Funding. Kapoor:Amgen: Research Funding; Takeda: Honoraria, Research Funding; Glaxo Smith Kline: Research Funding; Sanofi: Consultancy, Research Funding; Celgene: Honoraria; Cellectar: Consultancy; Janssen: Research Funding. Chanan-Khan:AbbVie: Research Funding; Pharmacyclics: Research Funding; Xencor: Research Funding; Merck: Research Funding; Jansen: Research Funding; Mayo Clinic: Employment; Ascentage: Research Funding; Millennium: Research Funding. Larsen:Janssen Oncology: 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. Russell:Imanis: Equity Ownership. Stewart:Roche: Consultancy; Seattle Genetics: Consultancy; Takeda: Consultancy; Ionis: Consultancy; Janssen: Consultancy, Research Funding; Oncopeptides: Consultancy; Ono: Consultancy; Amgen: Consultancy, Research Funding; Bristol Myers-Squibb: Consultancy; Celgene: Consultancy, Research Funding. Kumar:Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Research Funding.

Introduction Data on the economic burden of disease progression among multiple myeloma (MM) patients who have had a stem cell transplant (SCT) in the US are limited. This study evaluated the impact of disease progression on healthcare resource utilization (HRU) and costs among these patients in the US. Methods Adult patients (pts) with a confirmed MM diagnosis (Dx), defined as ≥1 inpatient or ≥2 non-diagnostic outpatient claims 30-365 days apart who initiated a course of MM therapy (Rx) during the study period (Jan 1, 2006 to Dec 31, 2016) and received SCT during the first year after the initial diagnosis of MM were identified from the Truven Analytics MedStat MarketScan® Commercial Claims and Encounters and Medicare and Coordination of Benefits databases. The first date with Dx for MM was defined as the "Dx date" and line of therapy (LOT) was identified based on an adaptation of a previously-developed algorithm. Pts were excluded if they had a gap in enrollment during the period beginning 6-month before the Dx date through 30-days after the first LOT (1L) initiation date, received MM Rx, unclassified antineoplastic medications, or SCT before the Dx date, or had missing information required to identify LOT or assess baseline characteristics or study outcomes. The 12 months after initiation of each LOT was designated the "event identification period" for progression. Disease progression was defined as advancement to next LOT, diagnosis or procedure codes indicative of bone metastasis, hypercalcemia, soft tissue plasmacytoma, skeletal related events, or acute kidney disease while off MM Rx, or death. The "event date" for progression was defined as date with evidence of progression. For each pt with progression, one pt without progression was randomly selected and assigned an event date to ensure that the distribution of time from LOT initiation to event date for pts without progression was the same as the distribution of time from LOT initiation to event date for pts with progression. For each LOT (L1-L3), annual HCRU and costs from index date, defined as 30-day before the event date, through disenrollment or end of the study period were compared for pts with vs without progression using inverse probability of treatment weighting (IPTW) to adjust for differences between groups in baseline characteristics including year of index date, age, gender, region, plan type, Medicare status, comorbidities, and pre-index HRU and healthcare costs. All costs were adjusted to 2016 US $. In a subgroup analysis, HCRU and costs were evaluated for LOTs initiated from 2013 to 2016 to reflect patterns of treatment in current era. Results Of the 28,184 adult MM pts who received ≥1 MM Rx during the study period, 3226 qualified for the study, including 837 pts with progression and 2389 without progression during 1L. The corresponding numbers for 2L, and 3L were 412 and 589, and 226 and 196, respectively. Mean age at index date was 57 years; 57% were male. After IPTW, all baseline characteristics were well balanced in all LOTs with standardized mean differences <0.1 for most baseline characteristics. The mean number of hospitalizations per year was greater for pts with progression in all LOTs, with differences ranging from 1.11 (95%CI 0.85, 1.37) for 2L to 0.34 (95%CI 0.21, 0.47) for 1L. In all LOTs, annual costs of inpatient and outpatient services were greater for pts with progression while costs of outpatient prescriptions were greater among patient without progression. Total healthcare costs were greater for pts with vs without progression in all LOTs with hospitalizations and outpatient visit costs being key drivers. Total incremental annual costs in those who progressed were higher by $13,386 (1L), $105,004 (2L), and $70,206 (3L). When the analysis was restricted to LOTs initiated between 2013 and 2016, the incremental costs in pts who progressed were generally greater in the most recent era: $43,381 (1L), $113,797 (2L), and $115,327 (3L). Conclusions For MM pts receiving drug Rx and SCT the economic burden of disease progression is substantial across all LOTs. Incremental costs of progression are largely attributable to hospitalizations and outpatient visits. Treatments that prevent or delay progression are likely to yield important reductions in downstream disease management costs. These savings should be considered in frameworks assessing the value of innovative treatments for MM. Disclosures Fonseca: AbbVie, Amgen, Bayer, Celgene, Kite, Janssen, Juno, Merck, Pharmacylics, Sanofi, Takeda: Other: Consultant/Advisor; Prognosticatin of MM based on Genetic Categorization by FISH: Patents & Royalties; Adaptive Biotechnologies: Other: Scientific Advisory Board. Hagiwara:Amgen Inc.: Research Funding. Panjabi:Amgen Inc.: Employment, Other: Owns Amgen stock, Research Funding. Yucel:Amgen Inc.: Employment, Other: Owns Amgen stock, Research Funding. Buchanan:Amgen Inc.: Employment, Other: Owns Amgen stock, Research Funding. Delea:Amgen Inc.: Research Funding.

PURPOSE Observation is the current standard of care for smoldering multiple myeloma. We hypothesized that early intervention with lenalidomide could delay progression to symptomatic multiple myeloma. METHODS We conducted a randomized trial that assessed the efficacy of single-agent lenalidomide compared with observation in patients with intermediate- or high-risk smoldering multiple myeloma. Lenalidomide was administered orally at a dose of 25 mg on days 1 to 21 of a 28-day cycle. The primary end point was progression-free survival, with disease progression requiring the development of end-organ damage attributable to multiple myeloma and biochemical progression. RESULTS One hundred eighty-two patients were randomly assigned—92 patients to the lenalidomide arm and 90 to the observation arm. Median follow-up is 35 months. Response to therapy was observed in 50% (95% CI, 39% to 61%) of patients in the lenalidomide arm, with no responses in the observation arm. Progression-free survival was significantly longer with lenalidomide compared with observation (hazard ratio, 0.28; 95% CI, 0.12 to 0.62; P = .002). One-, 2-, and 3-year progression-free survival was 98%, 93%, and 91% for the lenalidomide arm versus 89%, 76%, and 66% for the observation arm, respectively. Only six deaths have been reported, two in the lenalidomide arm versus four in the observation arm (hazard ratio for death, 0.46; 95% CI, 0.08 to 2.53). Grade 3 or 4 nonhematologic adverse events occurred in 25 patients (28%) on lenalidomide. CONCLUSION Early intervention with lenalidomide in smoldering multiple myeloma significantly delays progression to symptomatic multiple myeloma and the development of end-organ damage.

Abstract Introduction - IMIDs are critical in the treatment of multiple myeloma (MM). Unfortunately, many patients become resistant and this is a major challenge in MM therapy. IMID resistance can be caused by CRBN mutations (rarely) and by upregulation of specific antioxidative mechanisms. We hypothesized that measuring baseline antioxidative capacity might be a useful predictor of IMID sensitivity in the clinic. Methods - To develop a quantitative assay for determining cellular antioxidative capacity of MM cells we measured total cellular oxidized flavin adenine dinucleotide (FAD) after treatment with H2O2. FAD has auto-fluorescent properties that can be measured via flow cytometry. Cells with high antioxidative capacity generate more FAD, and cells with low anti-oxidative capacity generate less FAD. MM cells with an increase in FAD after H2O2 were considered IMID resistant and those with no change in FAD were considered IMID sensitive (Figure 1). Clinical IMID resistance/sensitivity was defined clinically during blinded retrospective review, with disease progression based on the IMWG criteria. After blinded review of clinical outcomes, the patient flow cytometry results were disclosed to evaluate the predictive value of the flow cytometry assay for detection of IMID resistance. Results - Of 40 patients who underwent testing, 26 were IMID resistant via flow and were considered as being clinical resistant 17 (65%). Conversely, 14 patients were IMID sensitive via flow and 12 (86%) were considered to be clinical sensitive. There was a significant correlation between clinical assessment and flow cytometry in determination of IMID resistance patterns (Table 1). The sensitivity and specificity of our assay was 89% and 57% with a positive and negative predictive value of 65% and 86% respectively. A limitation of our clinical assessment was that 16 patients (38%) were treated with combination therapy; IMIDs plus a proteasome inhibitor or a monoclonal antibody. Of the 11 patients with discordant results by flow and clinical outcome, 7 (64%) were not on therapy at the time of testing and 6 (55%) had flow testing on the bone marrow sample performed on day 100 following an autologous transplant. Two (7%) of the 29 patients with correlative results were off all therapy at the time of testing, 1 with testing on the day 100 marrow sample after transplant. Conclusions - This early testing of our assay shows promising results predicting clinical sensitivity via our flow based assay. The testing demonstrates a high sensitivity and negative predictive value, with less ability to assess for resistance. However, there were limitations to this study. The majority of patients with discordant results were off IMID therapy at the time of flow cytometry and many of these were assessed on transplant day 100 marrow samples. Depth of measureable residual disease was not readily available on all marrow samples tested. It is probable that flow testing for IMID resistance is inaccurate in patients with deeper responses to therapy, especially those who are minimum residual disease (MRD) negative. Additionally, several patients were on combination IMID, proteasome inhibitor, and monoclonal antibody. It is possible that clinical IMID resistance was not detected due to the anti-MM activity of the additional therapies. Additional larger scale prospective studies are needed to validate these findings as this method of testing could potentially provide providers with powerful information allowing them to personalize therapy for their patients. Disclosures Fonseca: Mayo Clinic: Employment; Amgen: Consultancy.

Abstract Background: Each additional line of therapy is associated with lower rates of deeper responses, shorter durations of treatment-free intervals, and increased rates of toxicities/comorbidities in patients with multiple myeloma (MM; Yong K, et al. Br J Haematol 2016. 175[2]:252-264), all of which are factors that contribute to treatment discontinuation. In order to characterize the newly diagnosed MM (NDMM) patient population in the United States not treated with a stem-cell transplant based on the number of subsequent lines of therapy (LOT) received, we examined baseline patient characteristics and pre-existing comorbidities, frontline treatment patterns, and attrition rates for these patients according to whether subsequent LOT was received or not received. Methods: NDMM patients were identified from 3 US patient-level data sources: the Surveillance, Epidemiology, and End Results (SEER)-Medicare Linked database (from January 2007 through December 2014), the OPTUM™ Commercial Claims database (from January 2000 through March 2017), and the OPTUM™ Electronic Medical Records (EMR) database (from January 2007 through March 2016). Patients included in the assessment had 1) an index diagnosis of MM on or after 1 January 2007, 2) known gender, 3) medical prescription coverage in place at diagnosis, 4) a 1-year look-back period prior to the index diagnosis, 5) no prior malignancies in the 1-year period prior to index diagnosis, and 6) received ≥1 LOT. The analysis was limited to patients who did not receive stem cell transplantation at any time during their follow-up. Descriptive and logistic regression analyses were conducted to characterize and compare patients who received or did not receive a subsequent LOT. Results: The analysis included a total of 7,724 patients (1 LOT only: n = 3,906; 2+ LOT: n = 3,818). A summary of baseline patient characteristics and pre-existing comorbidities by LOT is summarized in Table 1. Patients who received a subsequent LOT vs those that did not were significantly younger (median age 72.0 vs 74.0 years; P <0.0001) and had significantly lower mean Charleston Comorbidity Index (CCI) score due to lower occurrence of renal, cardiac, and hepatic comorbidities (Table 1). Older age at diagnosis and presence of renal, congestive heart failure (CHF), hepatic, and pulmonary comorbidities were associated with higher attrition. The regimens used in frontline treatment included bortezomib/dexamethasone (26%), lenalidomide/dexamethasone (21%), bortezomib/lenalidomide/dexamethasone (11%), a bortezomib/alkylator ± steroid combination (6%), bortezomib monotherapy (10%), lenalidomide monotherapy (6%), thalidomide monotherapy (4%), and other (17%). The proportions of patients progressing to each subsequent line of therapy are shown in Table 2. In total, 49% of patients went on to receive a second line of therapy, and 55% of patients receiving a second line of therapy went on to receive a third line of therapy. The incidence of patients not receiving subsequent lines of therapy due to death or being lost to follow up by LOT is summarized in Table 2. Conclusions: A majority of non-transplant patients with NDMM in the study did not progress to subsequent lines of therapy, with attrition as high as 50% per line of therapy and only <8% of patients reaching LOT 5. This high level of attrition is associated with older age and poor comorbidity status of non-transplant patients with NDMM. Therefore, utilizing the most optimal regimens upfront (rather than reserving them for later lines) is warranted. Disclosures Fonseca: Mayo Clinic: Employment; Amgen: Consultancy. Usmani:Abbvie, Amgen, Celgene, Genmab, Merck, MundiPharma, Janssen, Seattle Genetics: Consultancy; Amgen, BMS, Celgene, Janssen, Merck, Pharmacyclics,Sanofi, Seattle Genetics, Takeda: Research Funding. Mehra:Janssen Global Services, LLC: Employment. Slavcev:Janssen Global Services, LLC: Employment. Ukropec:Janssen Scientific Affairs, LLC: Employment. Lam:Janssen Global Services, LLC: Employment. Potluri:SmartAnalyst Inc.: Employment.

Abstract Despite recent advances and improvement in treatment options, chronic lymphocytic leukemia (CLL) is still an incurable disease. Recent advancement in parallel sequencing technology has provided better understanding of genetic landscape and clonal architecture underlying disease course. Furthermore, mutation status of TP53, SF3B1, NOTCH1, and BIRC3 has improved current CLL risk stratification. In order to better understand the genetic basis and clonal evolution underlying disease progression we analyzed a homogeneously treated cohort of CLL cases at multiple time points before and after therapy. We performed WES in 30 sequential samples from 12 CLL cases. All 12 cases received PCR (pentostatin, cyclophosphamide and rituximab) based chemoimmunotherapy as initial treatment. Of these 12 cases, two had samples available both >6 months prior to enrollment and at treatment initiation, seven patients who experienced relapse had samples available at both treatment initiation and at and the time of relapse, and three patients had samples >6 months prior to enrollment, at treatment initiation, and at relapse. These cases were previously characterized by aCGH/FISH. R function Mclust and kernel density estimate (KDE) plots were used to identify subclonal heterogeneity. An average of 102-fold depth coverage was obtained with an average of 80% of the targeted regions being covered by at least 30X. Overall, we detected a total of 219 nonsynonymous Single Nucleotide Variants (SNV) and indels (average 19, range 11-33). Del13q (42%) was identified as most prevalent copy number abnormality followed by trisomy 12 (33%), del11q and del17p (17% each). Recurrent mutations were identified in NOTCH1 (33%), DDX3X (25%), TP53, SF3B1 XPO1 and MED12 (17% each). Other tumor implicated genes such as CARD11, NOTCH2, NOTCH4, DIS3, TRAF2, NFKBID, CHK2 and RB1 were found mutated in individual cases. In all 12 cases we identified at least one relevant cytogenetic abnormalities (del11q32, del13q14, del17p13, trisomy 12) and/or a mutated driver gene. Interestingly we found NOTCH1, DDX3X and XPO1 mutated at a significantly higher prevalence than previous studies (10%, 2% and 4%, respectively). Since all cases in our cohort correspond to individuals with progressive disease, a plausible explanation for the higher prevalence of mutations in our cohort is that these mutations are associated with disease progression. By WES we identified multiple subclones in 58% of cases (7 of 12 cases), as compared to previously identified 33% (4 of 12 cases) by CGH/FISH. In 5 chemotherapy naïve cases, no significant increase in genomic complexity was detected with disease progression. In contrast, 5 out of 10 cases with samples analyzed before CIT and at the time of relapse had changes in clonal dynamics, with initially dominant subclones diminishing in response to therapy and the available empty niche being occupied by expansion of another “fitter” subclone at the time of relapse. No acquisition of tumor associated gene mutations was seen post therapy or associated with relapse in 9 of 10 cases. In the remaining case we identified an interesting example of convergent evolution. The pre-therapy sample was comprised of a unique clone characterized with del11q13, mutations in SF3B1 (G742D) and DDX3X (pE196fs). After therapy that clone was either eradicated or reduced to below detection level. Interestingly another clone emerged after relapse that harbored an independent del11q22 and mutations affecting different aminoacids of SF3B1 (K700E) and DDX3X (pL21fs). The strong selection of impairment in these genes during disease progression suggests a key role of these pathways/genes in the pathogenesis of this patient. In conclusion, these studies provide additional insight into the genomic landscape and clonal architecture in a cohort of homogeneously treated CLL cases before and after therapy. Stable clonal architecture was seen in prior to treatment, whereas clonal evolution leading to increased tumor heterogeneity occurred after the under selection selective pressure of therapy in relapsed cases. Early identification of tumoral heterogeneity and underlying genetic abnormalities might provide better targets for therapy to decrease the likelihood of relapse. Disclosures: Fonseca: Medtronic: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Genzyme: Consultancy; BMS: Consultancy; Lilly: Consultancy; Onyx: Consultancy, Research Funding; Binding Site: Consultancy; Millennium: Consultancy; AMGEN: Consultancy; Cylene: Research Funding; Prognostication of MM based on genetic categorization of the disease: Prognostication of MM based on genetic categorization of the disease, Prognostication of MM based on genetic categorization of the disease Patents & Royalties. Shanafelt:Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.

In multiple myeloma (MM) the interaction of plasma cells, bone marrow stromal cells and tumor associated macrophages (TAMs) plays a significant role in conferring resistance to therapy and in the maintenance of residual disease. Folate receptors (FR) are specifically expressed on metabolically active malignant cells and TAMs and their expression in normal tissues and resting macrophages is limited. FR mediates folate uptake by receptor-mediated endocytosis. This qualifies the receptor to be exploited for drug delivery of folate conjugated cancer therapeutics. Our primary goal in this study is to evaluate the expression of functional FR in MM cells and TAMs with a view to exploit this for folate conjugated targeted therapy for MM. First we evaluated the presence of TAMs in paraffin embedded bone marrow slides of newly diagnosed MM patients with CD68 (pan-Macrophage marker) and CD163 antibodies (specific marker of TAMs) and found extensive infiltration of macrophages in bone marrow from MM patients. Next to evaluate expression of FR in MM cells, we employed an FR antibody and evaluated MM cell lines with immunoblot, flow cytometry and confocal microscopy. In a panel of ten MM cell lines, we found that out of the three FR isoforms (α, β and γ), FR beta (FR-β) is expressed by all of them. Next to test whether this expressed receptor is indeed functional, we incubated the cells with folate deficient media, added different concentrations of EC17 (folate conjugated to FITC, Endocyte Inc.) to the medium and looked for the uptake by flow cytometry after washing off the drug at different time points (after 10, 20, 40 and 60 min).We observed that the uptake begins in 10 min and is saturated at 1 hour with 100nM Folate-FITC. With confocal imaging, Folate-FITC was found in the cytoplasm of MM cells suggesting internalization of Folate-FITC and localization in the cytoplasm. In addition to myeloma cell lines, we also confirmed the uptake of Folate-FITC in CD138+ plasma cells of a newly diagnosed myeloma patient by flow cytometry. This strongly suggests that MM cells express functional FR-β. To test the specificity of this FR mediated uptake, we pre-incubated cells with 0.1mM folic acid in medium for 30 min and then added EC17. This maneuver blocked the activity mediated by FR and no uptake was observed , which proves that the Folate-FITC is internalized only through the FR. To evaluate the expression of FR in-vivo samples, we stained paraffin embedded bone marrow slides of newly diagnosed MM patients with FR-β antibody and TAM specific markers. We observe that FR is expressed on both MM cells as well as TAMs. To assess the endurance of cytotoxic effect of folate conjugated chemotherapeutic agents, we treated MM cell lines with folate conjugated vinka alkaloids and compared them to unconjugated drug and found no significant difference in their action suggesting conjugation with folate does not alter its efficacy. To assess potential toxicity of folate conjugated therapeutics, we obtained CD34+ cells and looked for the uptake of Folate-FITC with flow cytometry. We found no uptake and this is in line with previous reports suggesting that CD34 positive cells express nonfunctional FR. So we propose that FR qualify as potential targets for cancer treatment. Folate targeted therapy using folate-conjugated drugs which can selectively act against both MM cells and supporting TAMs has the potential of specific anti-MM tumoricidal activity. This therapeutic approach would broaden the use of drugs that could be conjugated with folate for MM therapy. Additionally assessment of TAMs in bone marrow sections of MM patients would add another feature for grading, classifying and prognosticating MM. Disclosures: Fonseca: Cylene: Research Funding; AMGEN: Consultancy; Millennium: Consultancy; Binding Site: Consultancy; Onyx: Consultancy, Research Funding; Lilly: Consultancy; BMS: Consultancy; Genzyme: Consultancy; Celgene: Consultancy; Medtronic: Consultancy; Otsuka: Consultancy; Prognostication of MM based on genetic categorization of the disease: Prognostication of MM based on genetic categorization of the disease, Prognostication of MM based on genetic categorization of the disease Patents & Royalties.

Abstract Abstract 3527 Increasing evidence compiled from our lab and several others is illuminating the importance of epigenetic mechanisms in the pathogenesis of multiple myeloma (MM). The biochemical modifications that govern epigenetics are DNA methylation, and post-translational modifications of histone proteins on arginine and lysine residues. Modifications are generally associated with activation or repression of gene transcription depending upon the specific position of the modification. Histone methylation, catalyzed by histone methyltransferases (HMT) and histone demethylases (HDMT) are crucial for the proper programming of the genome during development. Deregulation of the methylation machinery can alter chromatin configuration and disrupt normal transcriptional programs, both features of cancer cells. Misregulated methylation may result from several genetic alterations in chromatin-modifying enzymes, which can include mutations, over-expression or chromosomal abnormalities. Indeed, genomic alterations of MLL, NSD1, CREBBP(CBP) MMSET and UTX, all encoding proteins involved in post-translational histone modifications, have been implicated in MM. The purpose of our study was to interrogate existing genomics datasets to identify additional HMT or HDMT genes that may be important in the pathogenesis of MM and, thus could become potential therapeutic targets. To identify novel HMT or HDMT in MM we mined existing expression, copy number and whole genome sequencing data generated as part of the Multiple Myeloma Research Consortium Genomics Initiative. Collectively, the datasets converged on numerous alterations involving histone methylation on lysine 9 (H3K9). One of these genes, a HDMT called JMJD1C, was over-expressed in approximately 15% of MM samples examined. We validated over-expression of JMJD1C expression by RT-PCR in over 50 clinical MM samples and 10 human myeloma cell lines (HMCLs). We also confirmed over-expression of JMJD1C by immunohistochemistry (IHC) of a tissue microarray (TMA) consisting of over 60 MM samples. Next we examined the levels of JMJD1C expression and H3K9 methylation by IHC, RT-PCR and western blot to determine the extent to which JMJD1C expression is associated with a decrease in H3K9 methylation (or vice versa). We demonstrated this negative correlation in 6/10 HMCL including OCIMY7 and KMS11 and in over 60% of clinical samples examined on the TMA. Furthermore, we demonstrated that H3K9 methylation, a repressive mark associated with heterochromatin, is low in a large percentage of samples. In addition, we generated double knockout isogenic cell line pairs using zinc finger nuclease technology to evaluate the direct role of JMJD1C on H3K9 methylation, cell function and chromatin and expression level changes using ChIP-seq and RNA-seq analyses. In summary our preliminary results demonstrate that JMJD1C is highly expressed in a significant percentage of MM patients. Our results demonstrate a noticeable inverse relationship of JMJD1C expression to H3K9. This reflects the importance of JMJD1C in modifying the H3K9 repressive mark and suggests that loss of repression due to JMJD1C-mediated histone demethylation disrupts the chromatin machinery and leads to neoplastic processes. Further studies are ongoing to realize JMJD1C as a potential and novel therapeutic target in MM. Disclosures: No relevant conflicts of interest to declare.