ALBERT

Description: Key Points CYR61/CCN1 is a bone marrow microenvironmental biomarker for myeloma progression and for transformation of MGUS and asymptomatic disease to overt myeloma. CCN1 reduces myeloma bone disease and tumor growth and is a potential therapeutic target for myeloma.

Description: Key Points Jumping translocations of 1q12 (JT1q12) provide a mechanism for the deletion of 17p in cytogenetically defined high-risk myeloma. Sequential JT1q12s introduce unexpected copy number gains and losses in receptor chromosomes during subclonal evolution.

Description: Multiple myeloma (MM) is a B-cell malignancy stratified in part by cytogenetic aberrations including the high-risk copy number alterations (CNAs) of 17p- and +1q21. CNAs of 1q21 are known to occur as a result of jumping translocations of 1q (JT1q12) which increase the copy number (CN) of 1q21, while at the same time causing collateral genomic damage including deletions and amplifications in some receptor chromosomes (RC). We have previously reported the decondensation of the 1q12 region and triradial chromosomes in the bone marrow specimens of patients with aggressive disease, and have speculated that hypomethylation of the pericentromeric region of 1q12 may play a role in the origin of 1q21 CNAs. To test the hypothesis that 1q12 hypomethylation induces CN gains of 1q21, we used the hypomethylating agent 5-azacytidine to treat blood lymphocyte cultures of five myeloma patients with balanced constitutional 1q12 aberrations as well as five normal control subjects. The patients with constitutional aberrations of 1q12 were selected based on their potential to show either novel CN gains of regions translocated distal to 1q12 or CN gains of 1q21 when 1q12 was translocated to a non-homologous chromosome. The constitutional aberrations included two patients with pericentromeric inversions of chromosome 1 inv(1)(p11q12), and one patient each with a balanced translocation of t(1;2)(q12;p11), t(1;2)(q12;q34), or t(1;9)(q12;q11). These particular aberrations should provide a unique model for testing the association between the hypomethylation of 1q12 and the origin of CN gains of 1q21. In vitro blood lymphocyte cultures of patients and controls were treated for 72 hours with 5-azacytidine at a final concentration of 10uM. 5-azacytidine is known to cause site-specific hypomethylation and chromatin decondensation in the 1q12 region. Utilizing FISH probes for 1q12 (satII/III) and 1q21(CKS1B), we analyzed 100 metaphase cells from each patient and each control for CN gains for 1q21 and any novel region translocated distal to 1q12. To fully characterize the induced rearrangements of 1q12, we also applied inverse G-banding and spectral karyotyping in selected cells. Remarkably, all patients with the balanced constitutional 1q12 rearrangements showed CN gains of chromosome regions on the derivative chromosomes distal to the inverted or translocated 1q12 regions. Recurring aberrations of 1q12 included pericentromeric decondensation and triradials of chromosome arms 1q, 1p, and 2p, in addition to localized pulverizations distal to 1q12 of these same arms. Specifically, in both patients with inv(1), whole-arm CN gains of 1p were identified in 2-3% of cells, while in the patient with t(1;2)(q12;p11), whole-arm gains of 2p (MYCN) were identified in 3% of cells. In these same three patients, on the derivative chromosomes where the 1q12 region was separated from 1q21 by an inversion or translocation, no CN gains of the 1q21 occurred. In the patients with 1q12 translocated to a non-homologous chromosome, 1q21 was amplified on the RC in 4% of the cells in the patient with t(1;2)(q12;q34), and in 3% in the patient with t(1;9)(q12;q11). Control subjects showed CN gains of 1q21on both chromosomes 1 in the range of 3-13%. Whole-arm JT1q12s to RC16q were identified in three patients and three controls. This JT1q12 results in a CN gain of 1q21, and a whole-arm CN loss in RC16q, identical to those found in the bone marrow specimens of MM patients. Interestingly, one control showed JT1q12s to two different RCs, 9q and 12q, causing whole-arm deletions in the long arms of both. The key structural aberration responsible for the generation of CN gains of 1q21 and subsequent JT1q12s is the formation of a triradial involving the 1q12 region. The novel finding in this study is the induction of triradials and CN gains for chromosome arms of 1p and 2p, which demonstrates that epigenetic modifications to 1q12 can amplify non-homologous chromosome regions juxtaposed to it. Furthermore, the CN gains induced here occurred only distal to 1q12 on all normal and derivative chromosomes and mimic the triradials and JT1q12s reported in the bone marrow of patients with aggressive disease. These findings demonstrate for the first time that the hypomethylation of the 1q12 region can potentially amplify any genomic region distal to it, and provide evidence for an epigenetic origin of the high-risk 1q21 CNAs found in the progression of MM. Disclosures No relevant conflicts of interest to declare.

Description: Background: The definition of high risk smoldering multiple myeloma (HR-SMM) is in flux. There are several models using serologic, bone marrow and radiologic data that predict for time to progression (TTP) to clinical myeloma (CMM). Lenalidomide and Dexamethasone in HR-SMM is reported to delay onset of end organ damage and improve overall survival, stressing the clinical utility of early intervention. We previously reported a GEP70 based score cutoff (-0.26, serum M spike ≥3g/dL, and involved SFLC 〉25 mg/dL identified a subset of patients with 67% risk of progression at 2 years. With longer follow up, we now examine whether unique gene probe sets can be identified at the AMM stage that portend an earlier time to therapy (TTT). Patients and Methods: We identified 105 patients with AMM who had baseline GEP data on our S0120 protocol, after IRB approval for retrospective data review, and evaluated each of 54,675 Affymetrix gene probes for their potential to predict TTT. Probes were ranked by their q-values; we found 40 probes with q-value 〈 0.05 and 7 probes with q-value 〈 0.01; the top probe had a q-value of 0.00066. Scores based on the number of significant probes at these cut-points were computed by subtracting the sum of the expressions of the up-regulated probes from the sum of the expressions of the down-regulated probes, then dividing by the total number of probes. Results: In the GEP40 model, an optimal cut-point for risk of progression was identified at 7.05. The 3-year TTT probability was 83% with scores 〉=7.05 and only 11% for patients with values under this threshold (Figure 1A; p65 (HR: 2.3), Albumin3g/dl (HR: 4.99), BM plasmacytosis〉=10% (HR: 12.2), GEP70〉-0.26 (HR: 3.4), GEP40〉=7.05 (HR: 16.41), GEP proliferation index 〉 -0.26 (HR: 2.8), GEP PR subgroup (HR: 9.4) and GEP PolyPC 〉11.6 (HR: 0.22) to be significant. In the multivariate model, GEP40〉=7.05 was the most significant (HR: 13.7), followed by SFLC〉10mg/dl and M-protein〉3g/dl. GEP40 score positively correlated with proliferation index (R: 0.804), and showed no correlation with GEP polyPC score (R: -0.156). Next, we used recursive partitioning on data from 72 patients and identified 23 patients with GEP40 score 〉=7.05 of whom 22 suffered TTT by 3 years (87%). Among the remaining 49 patients with GEP40 59 years identified 24 patients, of whom 11 suffered progression with a 3 year TTT estimate of 25%. In the 25 patients with GEP40

Description: Intra-tumoral heterogeneity is a hindrance to curing malignant disease. By employing all MM-active drugs upfront, TT was designed to overcome such obstacle by targeting all potential MM sub-clones broadly. We are reporting on long-term results of phase-2 TT1, phase-3 TT2 and phase-2 TT3a trials, with median follow-up times of 21yr, 12yr and 9yr, respectively. Five year estimates of OS, PFS and CR duration (CRD) increased from 58%, 28% and 40% with TT1 to 65%, 42% and 50% with TT2’s control arm (TT2-), to 68%, 56% and 58% with TT2’s thalidomide arm (TT2+), and to 74%, 65% and 74% with TT3a (all p

Description: Background: Marital status, social support, and socioeconomic status (SES) have been long identified as factors that have a role in outcomes in patient care and health. In patients with solid malignancies, both marital status and socioeconomic status influence the timing and stage of disease presentation. In such malignancies, late presentations are often consistent with incurable metastatic disease. In contrast, patients with hematological malignancies are often considered to have curable disease independent of their stage or timing of presentation. Given this discrepancy, we set out to determine the impact of marital status and SES on outcomes in patients with highly curable hematological malignancies such as acute promyelocytic leukemia (APML). Methods: We used the Surveillance, Epidemiology, and End Results (SEER) program to identify patients diagnosed with APML between 1999 and 2010. Linkage of SEER to Area Health Resources Files (AHRF) allowed county-level evaluation of socioeconomic factors. The association of individual patient factors on both 30-day mortality and long-term survival were analyzed to evaluate for differing influence on early-versus-delayed APML mortality. Results: A total of 2,635 individuals had baseline and follow-up information available for multivariable logistic regression and Cox regression analysis. The models included a standardized socioeconomic status (SES) index along with measures of county-level uninsurance rates and urban-rural stratification. In addition to increased early mortality with rising age, the likelihood of death during the first 30 days was higher in men (OR 1.22, 95% CI 1.01-1.44; p = 0.04). Marital status was not a significant predictor of death at 30 days, but there was a 2% increase in the odds of early death with every 1% increase of county-level uninsurance (p = 0.02). Conversely, gender and census-level uninsurance rates did not predict survival beyond the first 30 days, however, marriage and SES index above the median were associated with improved long-term survival (OR 0.70, 95% CI 0.57-0.81; OR 0.64, 95% CI 0.46 – 0.84 respectively, p ≤ 0.001). Conclusion: The impact of marital status, gender, and socioeconomic factors on clinical outcomes of patients with newly diagnosed APML appears to differ between the early acute and late clinical settings. Male sex and uninsurance rates were associated with early APML mortality and may suggest delay in seeking acute medical care. Marital and SES status appear to have greater influence on late survival of patients with APML and may be related to improvements in long-term medical adherence. Overall, our analysis suggests marital status, insurance coverage, and SES factors affect the outcomes of patients with APML, a highly curable malignancy. Future studies investigating the impact of social support on outcomes of other highly curable hematological malignancies may allow identification of important patient factors that affect clinical course. Table 1: 30-day mortality, multivariable logistic regression Variable Unadjusted OR (95% CI) Adjusted OR (95% CI) p Age at diagnosis (years) 1.03 (1.02-1.04) 1..03 (1.02-1.04)

Description: Progression of Asymptomatic Monoclonal Gammopathies to Myeloma requiring treatment -Clinical Multiple Myeloma (CMM)- is an important issue in current clinical investigation toward secondary prevention, i.e. treating high-risk AMG. Several predictive models have been published, including one that incorporated gene expression profiling (GEP) of plasma cells (PC) where a GEP70 score ≥0.26 was linked to higher AMG-CMM progression in a multivariate model (Dhodapkar, Blood 2014). We have applied 2-parameter flow cytometry of DNA and cytoplasmic immunoglobulin (FDC) of bone marrow aspirates as part of baseline staging of all patients with plasma cell dyscrasia. A modification introduced in August 2006 on the doublet discrimination method increased accuracy and reproducibility of FDC results considerably and allowed for the detection of a plasma cell population with a low CI17% (HR: 6.76, P

Description: Multiple myeloma (MM) is a malignancy of terminally differentiated clonal plasma cells displaying significant molecular heterogeneity with 7 subgroups defined by gene expression profiling (GEP). Our previous work showed that both the MS and MF subgroups associated with inferior survival (Zhan et al, Blood 2006). Furthermore, clinical studies have demonstrated that the addition of the proteasome inhibitor (PI) bortezomib (Bzb) to high dose melphalan based regimens provided a major advantage to patients in MS subgroup while patients in the MF subgroups (including C-MAF and MAFb) did not benefit from Bzb (Nair, Blood 2010). We have previously demonstrated that Bzb prevents c-MAF protein degradation leading to primary drug resistance (Abstract # 281, ASH 2013). In the present study, we assessed the ability of MAFb, another MAF family member, to influence the innate resistance to proteasome inhibitors (PI) and identify the molecular mechanism underlying the resistance of proteasome inhibitors in high MAFb-expressing patients. To investigate the association of the limited therapeutic effect of proteasome inhibitors in the different molecular subgroup of myeloma, we compared the IC50 of Bzb and carfilzomib (CFZ) in 29 myeloma cell lines (MMCL) belonging to different GEP-based molecular subgroups. IC50 concentrations of Bzb were higher (〉25 nM) in all 4 MAFb MMCL and 〉60 nM in one MAFb MMCL, which expressed the highest level of MAFb protein, as determined by immunoblot analysis. In contrast, Bzb IC50 levels were lower (7.5-20 nM) for the MMCL belonging to the other molecular subgroups. For CFZ, IC50 concentrations were higher (〉30nM) in all 11 c-MAF cell lines, while the IC50 levels were lower (2-20 nM) for the MMCL belonging to other molecular subgroup. One MMCL harboring t (14; 20) with high IC50 of Bzb (35 nM) showed low IC50 (10 nM) for CFZ. These results indicate that high MAFb expression in myeloma cells may contribute to primary resistance to Bzb and MM cells that express high MAFb protein although resistant to Bzb are sensitive to CFZ. Mechanistically, immunoblotting analysis demonstrated that exposure to Bzb resulted in increased MAFb protein levels in a dose-dependent manner, suggesting that Bzb prevents the degradation of MAFb protein in myeloma cells. To further confirm that drug-induced stabilization of MAFb protein confers resistance to Bzb and partially to CFZ, we generated loss of functional MAFb cells by silencing MAFb expression in a t (14;20) positive myeloma cell line using lentiviral shRNA specific to MAFb mRNA (shMAFb). shMAFb infected myeloma cells had 85% lower levels of MAFb mRNA and protein level compared with the cells infected with scrambled shRNA. Additionally, significantly decreased ITGB (9.1 fold), E-cadherin (2.5-fold), CCND2 (5.5-fold), and CCR1 (25-fold) levels were observed in these cells, compared with the cells infected with control viral vector. Silencing MAFb expression significantly decreased proliferation of myeloma cells (65.1% decrease, p=2.1E-6). Moreover, Bzb treatment of the cells infected with shMAFb led to 50.2%, inhibition (P=2.9E-8) of proliferation compared with control cells. Similarly, CFZ treatment of cells with silenced MAFb resulted in 54.3% (P=3.5E-8) inhibition of proliferation compared with control cells. Taken together, our results indicate that high expression of MAFb protein, similar to c-MAF, confers primary resistance to Bzb as well as to CFZ. In addition Bzb induces stabilization of MAFb protein, further increasing resistance to Bzb. These data provide the molecular rational for adopting an alternative therapeutic strategy for high-MAFb expressing myeloma patients. Disclosures Morgan: Celgene Corp: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Myeloma UK: Membership on an entity's Board of Directors or advisory committees; International Myeloma Foundation: Membership on an entity's Board of Directors or advisory committees; The Binding Site: Membership on an entity's Board of Directors or advisory committees; MMRF: Membership on an entity's Board of Directors or advisory committees.

Description: Prolonged survival of myeloma patients on Total Therapy regimens including IMIDS and novel agents has been associated with increased incidence of treatment-related myelodysplastic syndrome and acute leukemia (t-MDS/AL). MDS cytogenetic abnormalities (MDS-CAs) are often observed prior to t-MDS/AL development. Among 1,080 patients on TT2 and TT3 protocols, MDS-CA occurred in 11% and t-MDS/AML in 3%. Risk features of MDS-CA included TT3b treatment, age ³65 yr, male sex, elevated B2M, and MM relapse. Lower doses of CD34 HSCs applied with first transplants raised the probability of MDS-CAs, and lower CD34 HSCs dosing was an independent contributor to MDS-CAs and clinical t-MDS (Usmani et al., 2013). Although patients with MDS-CAs do not always develop t-MDS/AML, the phenomenon, also recognized in other tumors, requires understanding and development of preventive measures. We analyzed gene expression profiling (GEP) of bone marrow core biopsies at diagnosis from patients enrolled in our TT2 (n=88) and TT3 (n=263; training set) trials to identify genes associated with time to MDS-CA. Univariate Cox regression identified BCL11A as the only gene with expression associated with the time to development of MDS-CA (q 5.5 mg/L, GEP70-defined high risk and low BCL11A expression, while female sex was linked with longer time to MDS-CA development. In multivariate analysis, females retained independent prognostic significance for time to MDS-CA (HR-0.41, p 0.006), as did B2M 〉 5.5 mg/L (HR-1.95, p=0.038) and BCL11A expression

Description: Does the dogma that multiple myeloma is incurable still hold?. The genomic chaos and resulting resistance to apoptosis of myeloma, long considered an obstacle to cure, formed the basis of Total Therapy (TT) program. The TT approach uses all myeloma-active drugs upfront to target drug-resistant subclones during initial treatment to prevent later relapse. Long-term follow-up of 1202 patients (TT1: n = 231, median follow-up: 21 years; TT2: 668, median follow-up: 12 years; TT3a: n = 303, median follow-up: 9 years) permitted investigation of whether progression-free survival (PFS) and complete response (CR) duration were consistent with curability, ie observation of plateaus in Kaplan-Meier plots for PFS and CR duration. In the subset of 627 patients with plasma cell gene expression profiling data, cure plateaus were apparent at 5 years in the 14% with high-risk myeloma compared with 10 years in the remainder with low-risk disease. A parametric model based on PFS and CR duration supported an increase in curability: 10-year PFS and CR estimates increased from 8.8%/17.9% in TT1 to 15.5%/28.2% in TT2’s control arm to 25.1%/35.6% in TT2’s thalidomide arm and to 32.9%/48.8% in TT3a. Toward developing novel therapies, we recommend a concerted focus on patients with high-risk myeloma whose outcome has not been advanced.