ALBERT

Description: This study analyzed the frequency and clinical significance of t(4;14)(p16;q32) in multiple myeloma (MM) among 208 patients with MM and 52 patients with monoclonal gammopathy of undetermined significance (MGUS); diagnosed between 1994 and 2001. Patients with the translocation were identified using reverse transcription–polymerase chain reaction (RT-PCR) to detect hybrid immunoglobulin heavy chain (IgH)–MMSET transcripts from the der(4) chromosome. We found 31 (14.9%) t(4;14)+ MM patients and 1 (1.9%) t(4;14)+ MGUS patient. IgH-MMSET hybrid transcripts were detected in bone marrow (BM) and blood. Breakpoint analysis revealed that 67.7% of t(4;14)+ patients expressed hybrid transcripts potentially encoding full-length MMSET, whereas the remainder lacked one or more amino terminal exons. Expression of fibroblast growth factor receptor 3 (FGFR3), presumptively dysregulated on der(14), was detected by RT-PCR in only 23 of 31 (74%) patients with t(4;14)+ MM. Patients lacking FGFR3 expression also lacked detectable der(14) products. Longitudinal analysis of 53 MM patients with multiple BM and blood samples showed that, over time, BM from t(4;14)+ patients remained positive and that t(4;14)− patients did not acquire the translocation. IgH-MMSET hybrid transcripts and FGFR3 transcripts disappeared from blood during response to therapy. No correlation was observed between the occurrence of t(4;14) and known prognostic indicators. However, we find the t(4;14) translocation predicts for poor survival (P = .006; median, 644 days vs 1288 days; hazard ratio [HR], 2.0), even in FGFR3 nonexpressors (P = .003). The presence of t(4;14) is also predictive of poor response to first-line chemotherapy (P = .05). These results indicate a significant clinical impact of the t(4;14) translocation in MM that is independent of FGFR3 expression.

Description: Abstract 2842 Poster Board II-818 In addition to full length (FL) transcripts, clinically significant HAS 1 splice variants (Va, Vb and Vc) have been previously identified in multiple myeloma (MM) and Waldenstrom's macroglobulinemia. Increased HAS1Vb expression correlates with poor survival in a cohort of MM patients. Here, we show that directed mutation of HAS1 intron 3 alters HAS1 splicing and generates a pattern of HAS1 variant expression that mimics patterns detected in MM patients. This suggests that hypermutation of HAS1 and consequent expression of HAS1 splice variants may contribute to oncogenesis in MM. HAS1FL comprises of 5 exons (2089 bp); Va skips exon 4 (133 bp); Vb skips exon 4 and partially retains 59 bp of intron upstream of exon 5 (+59); Vc has all 5 exons and partially retains 26 bp of intron downstream of exon 4. In MM, frequent intronic mutations have been observed in introns 3 and 4, suggesting possible contributions to HAS1 alternative splicing. We have utilized a mammalian expression system to analyse HAS1 splicing by fusing a minigene extending from exon 3 to exon 5 (g345) with the upstream cDNA sequences. HAS1 expression is determined by transfection and RT-PCR using appropriated primer sets. This study focuses on identification of intronic mutations that may affect HAS1 splicing. We target mutations on (A/U)GGG motif because of its high abundance in HAS1 intron 3. The (A/U)GGG repeat was also shown to enhance the splicing of alternative intron in chicken β-tropomyosin (Sirand-Pugnet, P, et al, NAR, 1995, 23, 3501) and intronic G runs could work in a combinatorial way to control the selection of the proper 3' splice site in human thrombopoietin (Marcucci, R, et al, NAR, 2006, 35, 132). A 580 bp long human HAS1 intron 3 is GC-rich and comprises of 28 (A/U)GGG motifs (sequentially identified as G1, G2.., G28). HeLa cells transfected with an unmutated intron 3 construct mainly produce FL with a small amount of HAS1Va, a profile that is similar to CD40L/IL-4 activated normal B cells. Site directed mutagenesis of all 28 (A/U)GGG motifs (G1-28) abolished FL expression, but not HAS1Va, suggesting that these sequence alterations are highly unfavorable for constitutive splicing. It may be due to the loss of essential cis-acting element(s) and/or undesirable conformational changes that prevent spliceosome formation. Mutagenesis of G1-18 is shown to eliminate constitutive expression by increasing the usage of multiple alternative donor sites. Mutagenesis of G19-28 produces more HAS1Va than FL, presumably due to increased exon 4 skipping events. An increased Va/FL ratio could also be achieved by mutagenesis of G25-28 or G27-28, suggesting that this subregion is important for pathway selection. Mutagenesis was also studied in del1 construct, a unique derivative of HAS1 minigene that partially deletes intron 4. Similar to g345, del1 produces FL and HAS1Va as well as promotes expression of novel HAS1Vd, an isoform that includes +59 bp (like Vb) and exon 4. Alteration of HAS1 splicing profile caused by mutagenesis shown in g345 series is also observed in del1 series. Additionally, there is a shift from Vd to Vb expression in all constructs analysed (del1/G1-28, del1/G1-18, del1/G19-28, del1/G25-28 and del1/G27-28), a pattern of aberrant splicing that found in MM patients. Thus, in del1, increased exon 4 skipping events promote both Va and Vb expression. Sequencing of HAS1 intron 3 in a cohort of 50 MM patients indicates that recurrent mutations are found in the G repeat regions and that new repeats are generated by recurrent MM-specific HAS1 mutations. This suggests that mutation of the HAS1 construct mimics HAS1 mutation events that occur in MM patients themselves, and contributes to the clinically significant aberrant HAS1 splicing we have reported in MM (Adamia et al. Blood, 2008, 112, 5111; Blood, 2005, 105, 4836). Overall, critical mutations that could alter HAS1 expression and the ratio of HAS1 variants to FL were identified in intron 3. In intron 4, critical mutations that increase the usage of alternative splice site (+59) remain to be studied. We speculate that cumulative mutations within these two intronic sequences could bring the two events together to promote HAS1Vb splicing. While trans-acting elements are likely to regulate RNA splicing and its pathway, our studies clearly suggest that intronic mutations play an important role in the aberrant splicing of human HAS1, with probable contributions to disease progression in MM. Disclosures: No relevant conflicts of interest to declare.

Description: BACKGROUND Outcomes in multiple myeloma have improved dramatically over the last decade however, optimal sequencing of therapy remains unknown. Specifically, in an era where post-transplant lenalidomide (L) maintenance is now as established standard of care, questions remain around the utility of full dose L-based regimens in second line therapy. In this series, we sought to evaluate the impact of different regimens used at first relapse in patients who received autologous stem cell transplant (ASCT) in the frontline setting treated with and without lenalidomide maintenance (LM). We focused on the impact of L-based therapies in patients relapsing on LM. METHODS Using our prospectively maintained institutional MM database we retrospectively analyzed patients treated at the Cross Cancer Institute from January, 2005 to January, 2016 to ensure 2 years of follow-up for surviving patients. 4 categories were identified based on 2 variables: receipt of LM following 1st line therapy (yes or no) and receipt of L-based 2nd line therapy (yes or no). The primary endpoint was 2nd PFS defined as time of initiation of second line therapy to relapse, death or last follow-up. OS was defined as time of initiation of first line induction therapy to death or last follow-up. Second OS was defined as time of initiation of second line therapy to death or last follow-up. Survival statistics were determined using the Kaplan-Meier method with SPSS software. A p - value of

Description: Multiple myeloma (MM) plasma cells (PCs) express receptor for hyaluronan-mediated motility (RHAMM), a hyaluronan-binding, cytoskeleton and centrosome protein. The most abundant RHAMM isoforms in MM are full-length RHAMM (RHAMMFL) and the splice variant RHAMM-exon4. We separately examined the significance of RHAMM expression, and isoform balance, in 2 groups of MM patients. In oligonucleotide microarray experiments (n = 210, Arkansas), increasing RHAMM mRNA expression in MM PCs is strongly associated with osteolytic bone lesions (P 〈 .001), and event-free (P = .05) and overall (P = .04) survival. Semiquantitative determination of RHAMM isoform expression (Alberta, Canada) used capillary electrophoretic detection and measurement of RHAMM-exon4/RHAMMFL reverse-transcriptase-polymerase chain reaction (RT-PCR) products. RHAMM isoforms are rarely expressed concurrently in single MM PCs; the pattern of isoform expression, at the single-cell level, is approximated in larger numbers of cells by the RHAMM-exon4/RHAMMFL ratio. Absolute RHAMM expression and the RHAMM-exon4/RHAMMFL ratio are only partially correlated in MM PCs; in cell lines, absolute RHAMM expression is elevated in mitosis, while RHAMM ratios remain stable. Temporal examination of MM patients' peripheral blood reveals that the RHAMM-exon4/RHAMMFL ratio increases with disease burden. The RHAMM-exon4/RHAMMFL ratio in diagnostic bone marrow samples (n = 101, Alberta) is an independent prognostic factor. Thus, expression and splicing of RHAMM are important molecular determinants of disease severity in MM. (Blood. 2004;104:1151-1158)

Description: Abstract 813FN2 Introduction: Carfilzomib is a next-generation proteasome inhibitor that selectively and irreversibly binds to its target, resulting in sustained inhibition absent of off-target effects relative to bortezomib. Carfilzomib has demonstrated durable anti-tumor activity and an acceptable tolerability profile in patients with multiple myeloma (MM). Final results will be presented for the bortezomib-naïve group of PX-171-004, a phase 2 study of single-agent carfilzomib in patients with relapsed and/or refractory MM. Herein we report the most recent data analysis available at time of abstract submission. Updated final results for the bortezomib-naïve group of PX-171-004, including OS data, will be presented at the meeting. Methods: Patients received either 20 mg/m2 for all treatment cycles (Cohort 1) or a stepped-up dose-escalating regimen of 20 mg/m2 for Cycle 1 and 27 mg/m2 for all treatment cycles thereafter (Cohort 2). Carfilzomib was administered over 2–10 minutes on Days 1, 2, 8, 9, 15, and 16 of every 28-day cycle, for a maximum of 12 cycles. The primary endpoint was the best overall response rate (ORR; [CR + VGPR + PR]) determined according to the IMWG Uniform Response Criteria. Secondary endpoints included the clinical benefit response rate (CBR; [ORR + MR per EBMT criteria]), progression-free survival (PFS), time to progression (TTP), duration of response (DOR), OS, and safety. The result of efficacy analysis from disease assessment by the Independent Review Committee is presented in this abstract. Results: 127 of 129 enrolled bortezomib-naïve patients were evaluable for response. Prior therapies included thalidomide (59%), lenalidomide (59%), alkylating agents (81%), and stem cell transplant (73%). Patients had received a median of 2 prior regimens (1 in 54 patients, 2 in 40 patients, 3 in 28 patients, and ≥4 in 7 patients). 84 patients (65%) were disease refractory to their most recent therapy, defined as ≤25% response or progression during or within 60 days after completion of therapy. The median duration of carfilzomib treatment is 7 cycles (range 1−12) in Cohort 1; 8 patients were receiving drug as of February 2011 in Cohort 2 with a median treatment of 6.5 cycles (range 1−13) at that time. Best ORR was 42% in Cohort 1 and 52% in Cohort 2. Median TTP was 8.3 months and median DOR was 13.1 months in Cohort 1. The median TTP and DOR for Cohort 2 have not been reached at the time of this interim analysis; the lower bound of the 95% CI for the median TTP was 10.2 months, and 84% were estimated to have DOR ≥1 year at the time of data cutoff. Higher response rates for Cohort 2 compared with Cohort 1 do not appear to be associated with higher toxicities. Patients with unfavorable cytogenetic characteristics (≥1 abnormality) per mSMART criteria had an ORR of 37% and CBR of 42% compared with 50% and 65%, respectively, for patients with no abnormality. The most common treatment-emergent adverse events (AEs), regardless of relationship to carfilzomib in Cohorts 1 and 2, respectively, were fatigue (71%, 54%), nausea (54%, 43%), anemia (46%, 37%), and dyspnea (49%, 33%). These were primarily ≤Grade 2 in severity. The most common Grade 3/4 AEs were anemia (15%), lymphopenia (15%), thrombocytopenia (13%), pneumonia (12%) and neutropenia (12%). Treatment-emergent peripheral neuropathy (PN) was mild and infrequent (16%). Only 1 case of Grade 3 PN (0.8%) was observed. Overall, 38 patients (30%) completed 12 cycles and 22 of these patients continued to receive carfilzomib therapy under a safety extension protocol (PX-171-010), an ongoing, multicenter, open-label phase 2 study to monitor long-term use of single-agent carfilzomib. Conclusions: To date we have seen robust and durable single-agent activity for carfilzomib in bortezomib-naïve patients with relapsed and often refractory MM with an ORR of 42−52% and a CBR of 59−63% from 2 separate dose cohorts in a population wherein 92% received an immunomodulatry drug and 73% had undergone an autologous stem cell transplant previously. These data are suggestive of a dose–response relationship and are being further evaluated in the exploratory phase 1b/2 study PX-171-007. Carfilzomib was associated with minimal PN and excellent long-term tolerability, with nearly one-third of patients completing 12 cycles and 22 of these continuing treatment beyond 1 year in the extension protocol PX-171-010. Disclosures: Vij: Onyx Pharmaceuticals: Consultancy, Research Funding; Celgene: Research Funding, Speakers Bureau; Millennium: Speakers Bureau. Kaufman:Millenium: Consultancy; Onyx Pharmaceuticals: Consultancy; Novartis: Consultancy; Keryx: Consultancy; Merck: Research Funding; Celgene: Research Funding. Jakubowiak:Ortho Biotech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Exelixis: Consultancy, Honoraria. Wang:Onyx Pharmaceuticals: Research Funding. Jagannath:Millennium: Honoraria; Celgene: Honoraria; Onyx Pharmaceuticals: Honoraria; Merck: Honoraria; OrthoBiotec.Imedex: Membership on an entity's Board of Directors or advisory committees; Medicom World Wide: Membership on an entity's Board of Directors or advisory committees; Optum Health Education: Membership on an entity's Board of Directors or advisory committees; PER Group: Membership on an entity's Board of Directors or advisory committees. Kukreti:Celgene: Honoraria. Alsina:Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Allergan: Research Funding. Belch:Celgene: Research Funding; Onyx: Research Funding. Gabrail:Millennium: Research Funding. Matous:Celgene: Speakers Bureau; Millenium: Speakers Bureau; Cephalon: Speakers Bureau. Vesole:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Speakers Bureau. Orlowski:Onyx Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kunkel:VLST biothech: Consultancy; Threshold: Consultancy; Onyx Pharmaceuticals: Consultancy. Wong:Onyx Pharmaceuticals: Employment, Equity Ownership. Stewart:Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding.

Description: Abstract 289 Introduction: Chronic lymphocytic leukemia (CLL) is a disease mainly of older adults, with a median age of 72 at diagnosis, yet landmark trials that have established chemoimmunotherapy as standard initial therapy for this disease include patients (pts) with median ages between 58–64 years. Outcomes in other types of leukemia have been shown to be influenced by age, and one randomized phase III study (German CLL5) demonstrated a lack of benefit of fludarabine (F) over chlorambucil (Ch) in CLL pts over age 64. To help determine the current optimal standard therapy for older pts with CLL we reviewed the data on all pts enrolled on successive phase II and III CALGB CLL trials for previously untreated pts to determine if the efficacy varied by age. Particular interest was paid to ideal chemotherapy choice and the benefit of rituximab or alemtuzumab in older pts. Methods: 663 pts with untreated CLL enrolled on CALGB first-line studies were evaluated (515 pts 70 vs

Description: Specific tyrosine kinase inhibition (TKI) of the bcr-abl fusion protein in Philadelphia chromosome positive chronic myelogenous leukemia (CML) was introduced into general use in 2000 after a plenary abstract presentation to ASH in December 1999. Here we report the impact of the use of TKI on survival of all patients with CML in a total population based registry in the province of Alberta in Canada where BMT has been in use since 1980, α interferon +/− cytarabine since 1990 and TKI since 2000. All therapies have been fully funded by the public health care system in Alberta. Alberta’s population in 2000 was 2.97 million. Prior to 2000, allogeneic stem cell transplant (BMT) was the only curative therapy available but its application was limited to a minority of patients with CML. Alpha interferon induced a cytogenetic remission in some CML and alpha interferon plus cytarabine prolonged survival in CML. CML AML n 5 yr survival % BMT n 5 yr survival % BMT *59% of deaths occurred in the first year after diagnosis in the 2000–06 cohort 1980–89 303 40% NA 540 13% NA 1990–99 276 48% 25 692 15% 13 2000–06 216 83%* 8 680 25% 16 p

Description: Cytarabine (araC) is converted to araC 5′-triphosphate after entering leukemia cells as a substrate for nucleoside transport processes. This study tested the relationship between araC cytotoxicity, measured in an in vitro tetrazolium dye reduction assay of cell viability, and the cellular abundance of es nucleoside transport elements, assayed by a flow cytometric method that used the es-specific stain, 5-(SAENTA-x8)-fluorescein (5-(Sx8)-F), in cultured leukemia cells and in myeloblasts and lymphoblasts (blasts) from leukemia patients. Cellular es site abundance (Bmax value for 5-(Sx8)-F binding) varied sixfold among nine leukemic myeloblast samples from patients. In cultured OCI/AML-2 myeloblasts and CCRF-CEM T-lymphoblasts, and in fresh leukemic blasts, es sites were fractionally blocked by treatment with graded concentrations of nitrobenzylthioinosine (NBMPR), an inhibitory es site ligand, to simulate the variation in es expression found in leukemic blasts from patients with acute myeloid leukemia. When the cytotoxicity of a single concentration of araC was determined in NBMPR-treated leukemia cells, cell kill correlated closely with the intensity of 5-(Sx8)-F fluorescence (r = .92 to .99), a measure of the cell surface abundance of functional es nucleoside transporter sites. Concentrations of NBMPR that achieved half-maximal reduction (4.3 to 12 nmol/L) of cellular 5-(Sx8)-F fluorescence (measured by flow cytometry) approximated IC50 values (1 to 10 nmol/L) previously found for inhibition by NBMPR of es-mediated nucleoside fluxes in several cell types, supporting the view that 5-(Sx8)-F interacted with the es transporter. The correlation of araC cytotoxicity and the Bmax for 5-(Sx8)-F binding to es sites in cultured leukemia cells and in leukemic blasts from acute leukemia patients (r = .95) suggests that the flow cytometry assay of es capacity may be useful in predicting clinical response to araC.