ALBERT

Description: The occurrence of deep-vein thrombosis (DVT) in patients with newly diagnosed multiple myeloma, who were randomly assigned to receive identical induction chemotherapy with or without thalidomide, are reported in this study. The 2 study arms were comparable with respect to key myeloma prognostic factors and known risk factors for DVT. One hundred patients received induction chemotherapy including 4 cycles of continuous infusion of combinations of dexamethasone, vincristine, doxorubicin, cyclophosphamide, etoposide, and cisplatin, and each patient completed at least one induction cycle. DVT developed in 14 of 50 patients (28%) randomly assigned to receive thalidomide but in only 2 of 50 patients (4%) not given the agent (P = .002). All episodes of DVT occurred during the first 3 cycles of induction. Administration of thalidomide was resumed safely in 75% of patients receiving anticoagulation therapy. Thus, thalidomide given in combination with multiagent chemotherapy and dexamethasone is associated with a significantly increased risk of DVT, which appears to be safely treated with anticoagulation and does not necessarily warrant discontinuation of thalidomide.

Description: A correlation between increase in bone markers (alkaline phosphatase (ALP) and response to bortezomib in patients with multiple myeloma (MM) has been previously described. We now report results from a prospective study examining the relationship of serum PTH variation with skeletal effects and myeloma response to bortezomib treatment. Methods: Single agent bortezomib (1.3 mg/m2 patients 1–10; 1mg/m2 patient 11–20), was administered to patients with relapsed/refractory MM on days 1, 4, 8 and 11 on a 21 day interval for a total of 3 cycles; patients were not allowed to receive concurrent bisphosphonates or any other anti-myeloma drugs during the study period. Dynamic indices of bone turnover were prospectively evaluated by high-resolution microCT. Architectural parameters such as bone volume/total volume (BVTV), trabecular number (TbN), and thickness (Tb.Th) was obtained. PTH along with bone markers (osteocalcin, calcium, magnesium, phosphorus and serum creatinine) were measured on days 1, 4, 8, 11 before and after each bortezomib dose and every 4 hours thereafter, daily for the other days of the treatment cycle. Results: Seventeen patients were enrolled in the study with a median age of 63 years, 41 % were male and 3/4 of the patients previously failed high-dose chemotherapy. Histomorphometric microCT comparative analysis was completed (baseline and post-treatment) in 7 of the 17 patients enrolled in the trial. Baseline BV/TV values ranged from 13% to 90%. After 3 cycles of bortezomib treatment a statistically significant increase in BV/TV was recorded in 6 of 7 patients (P

Description: Background: Neurologic complications of multiple myeloma (MM) are numerous, however little is known about strokes occurring in the course of MM and its treatment. Methods: 2877 MM patients were seen for an initial evaluation at our institution over a 5-year period (1998–2002). Patients were identified using discharge summaries by combining ICD-9 codes for MM and stroke. They were included if they had clinical and radiological features of acute stroke. Ischemic stroke was defined as a new acute neurologic deficit lasting 〉 24 hours, irrespective of diffusion-weighted MRI results. Diagnosis of hemorrhagic stroke was made in patients with new acute neurologic deficit plus evidence of hemorrhage on CT scan. We retrospectively reviewed medical records for demographics, type of MM and treatment, stroke type, clinical features, relevant imaging and laboratory data, and outcomes. Results: The 11 patients that we identified included eight with ischemic strokes and three with subarachnoid hemorrhages (SAH). There were no patients with intraparenchymal hemorrhage. The overall incidence of stroke in this cohort was 76/100,000 per year, whereas the incidence of ischemic stroke was 56/100,000 per year. The mean age of patients with ischemic stroke was 59 years. Seven had one or more stroke risk factors and an equal number had received thalidomide. The latter were given small doses of coumadin as part of the treatment protocol; at the time of the stroke, INR range was 0.9–1.4. Based on clinical, radiological and other laboratory features, of the eight patients with ischemic strokes, two were presumed cardio-embolic (one had infective endocarditis), five were thrombotic, and one was caused by hypoperfusion. Two patients had findings of severe stenosis/occlusion in vessels corresponding to the infracted brain territory (internal carotid and basilar arteries). Two patients had documented normal plasma viscosity and three had evidence of extra-cranial thrombosis at the time of the stroke. In the three patients with SAH, hemorrhage occurred in the setting of trauma and thrombocytopenia, although one patient had an incidental anterior communicating artery aneurysm. Seven patients were left with minor or no deficits and four died, two in each of the groups. Conclusion: Overall, strokes did not appear to be more common in MM patients than in the general population, and the pathophysiology was likewise, not different than in patients without MM, with the exception of one patient with infective endocarditis, which is probably directly related to MM or its treatment. The highly selected referral population at our myeloma institute, and the retrospective nature of the study probably underestimate the incidence of stroke. The role of thalidomide in stroke remains unknown. Thrombocytopenia and trauma are probable risk factors for subarachnoid hemorrhage in MM patients. Fatal outcomes were frequent, but when death did not occur, the deficits were minor.

Description: Introduction - Multiple Myeloma (MM) is a hematologic malignancy characterized by clonal growth of differentiated plasma cells (PCs). Despite improvement in MM therapy, the disease remains mostly incurable and is characterized by recurrent relapses with development of resistant clones that eventually lead to patient death. The pathways that lead to resistant and aggressive MM are not fully understood highlighting the need to improve our understanding of MM biology to identify potential new pathways and therapeutical targets. PHD Finger Protein 19 (PHF19) is a regulator of Polycomb Repressive Complex 2 (PRC2), the sole methyltransferase complex capable of catalyzing H3K27me3 to induce and enforce gene repression. PRC2 employs enhancer of zeste homolog 1 and 2 (EZH1/EZH2) as enzymatic subunits to hypermethylate H3K27. While overexpression and gain of function mutations of EZH1/2 have been observed in many cancers the role of this particular pathway in MM remains poorly understood. In the present study, we report on PHF19 as a new candidate gene to play a potential crucial role in MM oncogenesis. Methods- Gene expression profiling (GEP; Affymetrix U133 Plus 2.0) was performed on 739 MM patients (from total therapy trials [TT] 3-5; low risk MM n=636, high risk MM n=103), 42 patients with monoclonal gammopathy of undetermined significance (MGUS), 73 smoldering MM patients, 42 patients with primary plasma cell leukemia and 34 healthy donors. Myeloma risk was determined by the GEP 70 signature as previously defined. To test the implications of functional PHF19 knock down (KD) we used TRIPZ inducible PHF19 shRNA vs. scrambled control (Dharmacon) in two MM cell lines (JJN3 and ARP1). Real time PCR as well as western blotting was used to confirm PHF19 KD as well as to elucidate the effect on H3K27me3 (Cell Signaling). Functional in vitro studies included proliferation (Promega), clonogenic assays (StemCell), cell cycle and apoptosis assays (both Invitrogen). In vivo studies were performed using SCID mice that were subjected to tail vain injection with PHF19 KD JJN3 cells (n=10) or scrambled shRNA control (n=10). Weekly ELISA (Bethyl) and in vivo imaging (Xenogen) were performed and survival was recorded. Results- GEP of the previously mentioned patient populations and healthy controls identified PHF19 (chr9q33.2) as a candidate gene that was consistently dysregulated in MM patients. Mean expression levels at different MM stages correlated with disease aggressiveness (ANOVA, p10.46) at diagnosis correlated significantly with adverse clinical parameters, including ISS III, anemia and elevated LDH, as well as worse overall survival (5 yr OS = 29% for patients with high PHF19 expression vs 77% for patients with low PHF19 expression [log275% reduction in both cell lines, p

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: Abstract 2839 Poster Board II-815 Multiple myeloma (MM) remains largely incurable. The existence of the putative myeloma stem cell population may account for drug-resistance and relapse. Previous investigation showed that the CD138-negative (CD138-) MM cell fraction contains the putative MM stem cells. However, little is known about the molecular characteristics of MM stem cells, which makes it difficult to specifically target such cells. In this study, by comparing gene expression profiles (GEP) of CD138+ and CD138- cells isolated from 10 MM cell lines, we discovered that RARα is the top one over-expressed gene in CD138- MM stem cells. RARα has two major isoforms, RARα1 and RARalpha2. Real-time PCR detected significantly higher expression of RARalpha2 but not of RARalpha1 in CD138- MM cells compared with CD138+ cells. We recently reported that increased RARalpha2 expression at diagnosis resulted in a significantly shorter overall-survival (P = 0.003); importantly, ATRA selectively killed RARalpha2-positive myeloma cells, but not RARalpha2-negative cells. These results suggested that ATRA could be used to eradicate specifically RARalpha2-over-expressing MM stem cells. Indeed, we found that ATRA selectively killed CD138- MM stem cells (P 〈 0.01) from ARP-1 and OCI-MY5 human myeloma cell lines and 5T33 murine myeloma cells but spared the CD138+ tumor cells from these cell lines. WNT and Hedgehog (HH) signaling pathways were activated in CD138- stem cells. To our surprise, ATRA treatment (10-6M) further increased WNT and HH signaling pathways in CD138- myeloma cells, based on increased protein levels of β-catenin and cleaved Shh/Ihh in ATRA-treated cells. Stimulation of these signaling pathways by LiCl (5 mM) and/or SHH(20 μg/mL) partially abrogated ATRA-induced cytotoxities on CD138- MM cells, demonstrating that stimulation of WNT and HH signaling induced partial ATRA-resistance in CD138- cells. A combinational treatment of ATRA (1 μM), a COX-2 inhibitor celecoxib (shown to inhibit WNT signaling, 50 μM) and a HH signaling inhibitor cyclopamine (10 μM) induced synergistic effects on cell death and growth inhibition of CD138- and RARalpha2-over-expressing MM cells in-vitro. In the 5T33 murine myeloma model, the combination of ATRA (20mg/Kg), celecoxib (10mg/Kg) and cyclopamine (20mg/Kg) induced synergistic inhibition of tumor growth in-vivo. Thus, our study provides novel approaches to specifically target MM stem cells. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Many prognostic models have been proposed for staging patients with MM, most recently the ISS system utilizing B2M and albumin. Patients and Methods: In the context of Total Therapy 2 (TT2) for newly diagnosed MM, we identified 220 patients in whom 4 standard prognostic factors (SPF: B2M, albumin, LDH, hemoglobin), imaging data (MRI-defined focal lesions [MRI-FL]), metaphase-derived cytogenetic abnormalities (CA), FISH-derived amp1q21 and del13q14, and gene expression profiling (GEP)-derived data are available. The baseline characteristics and clinical outcome of the 220 patients are similar to those of the entire population of 668 patients receiving TT2. The median follow-up of the 220 patients is 42mo compared to 52mo for all 668 patients. Five multivariate analysis-based prognostic models were derived, utilizing SPF only (model I), with progressive addition of CA (model II), MRI (model III), FISH (model IV) and GEP (model V). Results: In model I, B2M, LDH and hemoglobin, but not albumin were independently significantly associated with overall survival (OS) (R2=22.5%); upon introduction of CA (model II) (p=.008), only LDH remained significant (p=.033) (R2=28.3%); addition of MRI (model III) was not significant (R2=31.1%); addition of FISH (model IV) was significant for amp1q21 (p=.003) but not for del13q13, with CA remaining significant (p=.023) (R2=38.1%); when GEP was added (model V), the 70 gene-derived model (p

Description: Mobilization with chemotherapy (CT) plus hematopoietic growth factors (HGFs) is superior to mobilization with HGFs only. For stem cell mobilization after chemotherapy HGFs are typically given once or twice a day for approximately 14 days. The aim of this study was to evaluate whether mobilization of peripheral blood stem cells (PBSC) with DT-PACE plus pegfilgrastim was equivalent to mobilization with DT-PACE + filgrastim. Patients analyzed were enrolled in two consecutive studies 2001–12 (N=97) and 2003–41 (N=72) and included only patients who had received ≥ 2 cycles of prior therapy, but no prior transplant. Both protocols employed a single cycle of induction with DT-PACE (Lee et al. JCO2003;21:2732) followed by stem cell collection with either filgrastim 5μg/kg bid until completion of stem cell collection (2001–12) or pegfilgrastim 6mg on days 6 and 13 (2003–41). Patients then proceeded with tandem transplants, one consolidation cycle with DT-PACE and two years of maintenance with thalidomide (100mg daily) and dexamethasone (4 mg/day, days 1–4 q 3 weeks). Group characteristics were compared using the Kruskall-Wallis test. Time to hematopoietic recovery after transplantation was analyzed with Kaplan-Meier plots, and comparison was performed using the logrank statistic. The two studies had comparable characteristics, except that more patients ≥ 65 years were enrolled in 2003–41 (35% vs 13%; p=.001). The median number of collection days was 2 in both studies (p=.8). The median number of CD34 cells/kg (x106) collected per day was 9.7 (2001–12) vs 12.7 (2003–41) (p=.2). The total number of CD34 cell/kg (x106) collected was 20.4 (2001–12) versus 25.4 (2003–41) (p=.2). The median number of CD34 cells/kg (x106) infused after the first transplant was 4.1 (2001–12) and 4.5 (2003–41) (p=.5). The time to recover ANC 〉 500/μl and platelets 〉 20,000/μl without transfusion was shorter for 2003–41 patients (13 versus 15 days; p〈 .0001) (Figure 1). Mobilization of PBSC with pegfilgrastim is easier for patients because they only receive 2 injections and is as effective as filgrastim when combined with DT-PACE. Moreover, mobilization with pegfilgrastim may result in a more rapid hematologic recovery after transplantation. Cumulative incidence analysis Recovery of ANC to 500 and Platelets 〉 20K 2003-41 vs. 2001-12, Transplant 1 Cumulative incidence analysis Recovery of ANC to 500 and Platelets 〉 20K 2003-41 vs. 2001-12, Transplant 1

Description: Introduction Chromosome instability (CIN) is a driver of copy number aberrations (CNAs) in cancer, and is a major factor leading to tumor heterogeneity and resistance to therapy. By definition, CIN is an increased rate or ongoing acquisition and accumulation of CNAs and not simply the existence of structurally and numerically abnormal aneuploid clones. In multiple myeloma (MM), the most common whole-chromosome CNAs involve either hyperdiploid or non-hyperdiploid clones. Secondary segmental CNAs are associated with high-risk (HR) in MM and involve gains of 1q21 and deletions of 17p (del17p). These types of intra-chromosomal segmental CNAs are also found in the CIN phenotypes of the autosomal recessive (AR) chromosome instability syndromes. These syndromes include Fanconi anemia, Bloom's syndrome, and ICF syndrome (Immunodeficiency, Centromeric instability and Facial anomalies). These chromosome instability syndromes display a spectrum of aberrations characterized by higher rates of chromosomal breaks, chromatid exchanges, quadriradials, and pericentromeric aberrations. In particular, patients with ICF syndrome show a marked increase of 1q12 pericentromeric instability including 1q12 decondensation, triradials, multibranched chromosomes 1q, and 1q micronuclei. ICF patients also show transient 1q aberrations including isochromosome 1q (iso1q) and unbalanced translocations of 1q to 9q and 16q. In MM, we have previously reported increasing pericentromeric instability during tumor progression resulting in increasing CNAs of 1q21 by unbalanced jumping translocations of 1q12 (JT1q12). Strikingly, in a subset of MM patients with 1q21 CNAs of ≥ 5 a distinct cytogenetic phenotype emerges which demonstrates transient 1q12 aberrations including 1q12 decondensation, triradials, and multibranched chromosomes 1q morphologically identical to those seen in ICF patients. In MM this chromosome instability leads to a cascade of increasing clonal 1q21 duplications, iso 1qs, and unbalanced 1q translocations with 16q and 17p, resulting in losses in these receptor chromosomes (RC) and massive intra-clonal CNA heterogeneity. Methods To investigate the cytogenetic impact and progression of high CNAs of 1q21, we performed a comprehensive metaphase analysis of 50 patients showing segmental aneuploidies with 4 or more copies of 1q by G-banding. Locus specific FISH and spectral karyotyping were used to identify the key transient unstable and clonal structural aberrations of 1q12 resulting in segmental aneuploidies in the derivative RCs. Probe for 1q12 (Vysis) was used according to the manufacturer's protocol. Locus specific BAC clones for 1q21 (CKS1B) and 17p (TP53) were prepared and analyzed as previously described (Sawyer et al., Blood 123: 2014). IGH translocations were investigated with IGH break apart probes (Vysis). Results Data for 50 patients including CNAs of 1q21 of ≥ 4, IGH translocations, del(17p), derivative RCs, are presented. The t(4;14) was found in 15 patients, del(17p) in 23, and both aberrations were found in 8 patients. All patients showed unbalanced gains of 1q and deletions of RCs, the most frequent being 7 patients with der(1;16) and 6 with iso1q. In four of the 23 patients with del(17p), the deletion was due to a JT1q12 to 17p. Seven patients with 1q21 CNAs of ≥ 5 showed profound instability involving the 1q12 satellite DNA, demonstrating both transient and clonal aberrations driving the 1q21 CNAs. These aberrations included unstable 1q21 triplications, JT1q12s, iso1q formation with intra-arm 1q12 CNAs, and region specific breakage-fusion-bridge cycle amplifications. Conclusions Among patients with ≥ 5 CNAs of 1q21, a subset develop an acquired HR chromosome instability phenotype with an elevated rate of 1q12 pericentromeric instability characterized by concomitant deletions in 16q, iso1q, del(17p), and intra-arm segmental instability. These patients show pronounced instability in the 1q12 satellite DNA, morphologically identical to ICF syndrome, suggesting hypomethylation of this region as a driver of both 1q21 CNAs and deletions in RCs. We hypothesize that region specific hypomethylation of 1q12 provides the genomic background for the onset of an acquired 1q12 chromosome instability phenotype in MM similar to that found in ICF syndrome. For myeloma patients demonstrating this 1q12 chromosome instability phenotype we propose the term "jumping 1q syndrome." Disclosures Epstein: University of Arkansas for Medical Sciences: Employment. Davies:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; ASH: Honoraria; Abbvie: Consultancy; TRM Oncology: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; MMRF: Honoraria; Janssen: Consultancy, Honoraria. Morgan:Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria.

Description: Abstract 2914 Background: Myeloma (MM) - stromal cell-cell interactions play a crucial role in MM cell growth, survival, and drug resistance. Intercellular adhesion molecule-1 (ICAM-1) is up-regulated in different cancers, including MM, and represents one of the major adhesion molecules mediating tumor-stromal cell-cell contacts. Previous investigations have shown that ICAM antibodies induce antitumor activity in SCID mice xenografted with MM cell lines, likely involving Fc :Fc gamma receptor dependent anti-tumor mechanisms. We have examined the anti-MM activity in the SCID-hu mouse model of a specific humanized ICAM1 antibody (BI-505, Bio-Invent, Sweden) using primary MM cells. Materials and Methods: The expression of ICAM1 was examined in plasma cells from 22 donors, 351 newly diagnosed MMs, and 9 MM cell lines using Affymetrix U133 Plus2 chips. Human fetal femurs and tibias, obtained at 17 to 22 gestational weeks, were cut into fragments and implanted subcutaneously in 16 SCID mice (SCID-hu) at age 6 to 8 weeks. Four weeks after bone implantation, approximately 5 ×106 CD138+ plasma cells from MM patients were injected directly into the human bone of the SCID-hu mice in final volume of 30 to 40μl of phosphate-buffered saline(PBS). Human immunoglobin (hIg) levels by ELISA were used as an indicator of myeloma cell growth. When hIg level reached 10μg/mL or higher in 2 consecutive samples after 4 weeks of injection of the tumor cells, the mice were divided into four groups (n=4), Control group (not injected MM cells and no drug treatment); the other 3 groups were injected with MM cells: the isotype control group (isotype IgG 2mg/kg, i.p., twice weekly), BI-505 group (2mg/kg, i.p., twice weekly) and bortezomib group (1mg/kg, i.p., twice weekly). Bone remolding was detected by X-radiography and by measuring bone mineral density (BMD). Tumor cells were detected by immunohistochemistry using the CD138 antibody. Results: High expression of ICAM1 was observed in normal plasma cells, primary MM cells and MM cell lines. With a follow- up of 10 weeks, the tumor burden in the mice treated with BI-505 or bortezomib was significantly lower compared with the isotype control group (p: BI505 〈 0.01; p: bortezomib 〈 0.01). Also, the number of MM tumor cells stained with the CD138 antibody was significantly less in samples treated with either BI-505 or bortezomib than in the isotype control group (p 〈 0.01). In addition, 6 weeks after injection of tumor cells, X-rays showed severe bone resorption in the isotype-control group, while there was no obvious bone resorption in the fetal bones after treatment with BI-505 or bortezomib. The BMD (0.0715±0.006 g/cm2) of isotype control was significantly lower than that in other 3 groups including control: 0.1278±g/cm2, BI-505 group: 0.102±0.0064 g/cm2, and bortezomib group: 0.106±0.0059g/cm2. Importantly, the number of TRAP-positive cells was significantly higher in the isotype control group than in the other 3 groups (p 〈 0.01). Conclusion: ICAM1 expression presents in all subtypes of myeloma. The ICAM1 antibody BI-505 significantly inhibits primary MM cell growth and bone destruction in the SCID-hu mice to the same degree as bortezomib, indicating its clinical applicability in therapy of MM. Disclosures: No relevant conflicts of interest to declare.