ALBERT

Description: We have taken a genetic strategy to define the mechanisms underlying the increased risk of VTE in myeloma patient treated with thalidomide using clinical trial data derived from the MRC, ECOG, Little Rock and HOVON trial groups. Baseline VTE rates in the MRC IX study, (CTD vs CVAD; n=748 no prophylactic anticoagulation), were 14%. Rates in both arms were equal but in the thalidomide arm, thrombotic events were all DVT’s or PE’s, whereas in the CVAD arm they split equally between line related and VTE’s. In the other comparison (CTD vs MP; n=542), the VTE rate was 17%, all in the thalidomide arm. Rates were similar or higher (30%) in the other trials depending on the intensity of treatment used. We carried out a nested case control on DNA from MRC IX on 90 VTE (45 thalidomide, 45 non-thalidomide related), matched to 180 controls. SNPlex analysis for 60 SNPs in the coagulation cascade, DNA repair, thalidomide, metabolism and myeloma specific pathways, identified 7 genes which affected risk (NBS1, MTHFR, Ku80, Cyp3A5, LGAL52 and IL12B). This suggests three major pathways contribute to the risk of thalidomide related VTE, metabolism of thalidomide, tumour responsiveness and some pro-thrombotic factors. These factors differ from non-thalidomide related risk where a strong pro-inflammatory response is most relevant. We tested this hypothesis further using an expanded panel of 3,404 SNPs (BOAC panel), using the Megalle system. Statistical analysis of this data defined the top 20 thalidomide and non-thalidomide associated SNPs, which were consistent with the initial hypothesis, the results of which exclude variation in the coagulation cascade as being a significant risk factor. We explored this hypothesis further using pathway-based analysis focussing on the MTHFR, DNA repair and WNT pathways on data from the ECOG, HOVON and Little Rock data sets. The biological relevance of the SNPs and genes identified was validated by correlating the level of expression using the Affymetrix U133 CHIP with genetic variation at the DNA level. To verify the relevance of DNA repair variability to tumour response further, we correlated gene expression profiling with response and again Ku80 was identified as one of a cluster of genes defining increased risk. The data suggests that the risk of thalidomide related VTE relates to rapid tumour lysis combined with a prothrombotic tendency, whereas in line related thrombosis, risk is dependent upon a strong proinflammatory response. We are building this genetic data into a clinical risk model in order to allow us to develop a rationale for intervention strategies.

Description: Background: Cytotoxic T-cell infiltrates are a nearly universal finding in the bone marrow of patients with multiple myeloma. It has been postulated that presence of T-cells in the bone marrow of multiple myeloma (MM) patients represents an immune response against the tumor and therefore, might be associated with an improved prognosis. However, the impact of bone marrow T-cells on the prognosis of multiple myeloma patients has not been studied systematically. Methods: Bone marrow biopsies of patients with newly diagnosed multiple myeloma were stained by immnohistochemistry for the CD8 antigen and reviewed by a blinded hematopathologist. Three high power fields are reviewed for each biopsy and the total number of CD8 positive cells counted and reported. For patients with more than 300 cells per 3 fields, results were reported as 〉300. The number of bone marrow CD8 positive cells was then correlated with patients’ clinical data, including other prognostic factors and overall survival. Results: Bone marrow biopsy specimens from 100 patients, performed within the week of a diagnosis of multiple myeloma and collected between May 1998 and January 2001 were evaluated. The median number of CD8 positive cells was 270 (33 – 〉300). Patients’ characteristics are shown in Table 1. Median follow up was 30 months (0–80). The number of cytotoxic T-cells as a continuous variable was a risk factor for shortened overall survival, HR 1.86 (95% CI 1.11–3.35). Using minimal p value approach, the cutoff of 270 cells (the median) risk stratified patients into two groups: the median survival of patients with 〉 270 CD8 positive cells was 16 months vs. 48 months in patients with ≤270 cells, p=0.005 (Figure). In multivariate analysis including age, B2M, albumin, CRP, bone marrow plasma cell percentage and plasma cell labeling index, the number of cytotoxic T-cells was an independent predictor of overall survival was HR 3.1, p=0.0017. Conclusion: We show that the number of cytotoxic T-cells in the bone marrow is a strong and independent prognostic factor in patients with newly diagnosed multiple myeloma. Our observation does not contradict the hypothesis that cytotoxic T-cells participate in an immune response against the tumor since our findings may represent a higher level of immune response associated with baseline aggressive disease biology. However, our study suggests for the first time that increased marrow cytotoxic T-cells have an adverse effect on outcome in myeloma, and suggest that these cells may have a direct facilitating effect on tumor growth and on the marrow microenvironment. Further studies of the biology of behind this observation are warranted. Characteristic N Median (range) Gender male 61 CRP 81 0.4mg/L (0.01–11.2) Albumin 99 3.6 g/dL (2.6–5.4) B2microglobulin 94 4.0 (0.9–28) μg/mL Marrow PC% 90 45% (11–99) PC labeling index 90 high (〉1%) 36 BM CD8 cells 100 270 (33 – 〉300) ISS 94 1 19 2 41 3 34 Figure Figure

Description: Background: In patients (pts) with newly diagnosed myeloma (MM) eligible for high dose therapy and autologous stem cell transplantation (ASCT), the standard approach is 4–6 months of induction therapy followed by ASCT. The combination of thalidomide and dexamethasone is one of the commonly used induction regimens, and has superior response rates compared to dexamethasone or VAD. Almost all patients undergoing an ASCT relapse at a median interval of 18–24 months. The choice of treatments at first relapse is variable, but re-application of the original induction regimen, if effective is often considered. It is not clear if the responses obtained at the time of induction therapy can be replicated at the time of relapse. We studied the response of thalidomide based regimens when used as first treatment after relapse from an ASCT in patients receiving thalidomide as induction treatment. Methods: Pts who received thalidomide and dexamethasone as induction therapy, and thalidomide ± dexamethasone at first relapse after ASCT were identified for the current study. A group of patients receiving dexamethasone as induction therapy and thalidomide ± dexamethasone at first relapse after ASCT was used as the control group. Results: There were 20 pts in the study group (group 1) and 14 patients in the control group (group 2) identified between 2002 and 2007. There were 15 (75%) males in group 1 and 10 (71%) in group 2. In group 1 there was 1(5%) complete response (CR), 5 (25%) very good partial response (VGPR) and 14 (70%) partial responses (PR) to induction therapy. In group 2 there was 1 (7%)VGPR, 12 (86%) PR and 1 (7%)stable disease following dexamethasone induction. Upon treatment with thalidomide based regimens at first relapse after ASCT, there was a high rate of treatment failures in group 1 as compared to group 2 (no response in 14 out of 20 patients (70%) versus 5 of 14 pts (35%), respectively, p = 0.04). Of the 10 patients with progressive disease in group 1 post transplant, 30% had previously achieved a VGPR and 70% had previously achieved a PR to thalidomide/dexamethasone pre-ASCT. Conclusions: Our study provides evidence toward the lack of efficacy of thalidomide based regimens on post ASCT relapse in patients who had thalidomide responsive disease prior to ASCT. It is possible that this applies to other induction regimens when used at post ASCT relapse and raises the possibility of selection of drug resistant clones with initial therapy. Response rates seen with drug regimens prior to ASCT may not be reproducible at the time of relapse after ASCT.

Description: Recombinant human erythropoietin (rHu-EPO) is used to treat anemia by activating the erythropoietin receptor (EPOR) in erythroid progenitor cells, leading to proliferation and differentiation into mature red blood cells. To allow less frequent dosing, a hyperglycosylated version of EPO has been developed with a longer half-life. In principle, an agonistic antibody targeting EPOR would offer an even longer half-life, support robust monthly dosing, and, unlike EPO products, reduce the risk of pure red cell aplasia. The efficiency of signaling and corresponding potency of previously reported antibody mimics are generally suboptimal compared with EPO and not suitable for clinical use. Here we describe a potent, fully human, agonistic antibody (ABT007) targeting EPOR that supports potent, more sustained, and less pulsatile elevation of hematocrit in a human EPOR–expressing transgenic mouse model compared with standard doses of rHu-EPO while requiring less frequent dosing. Resolution of the crystal structure of the EPOR extracellular domain (ECD) complexed to the ABT007 Fab fragment, determined at 0.32 nm, identifies a binding site that is consistent with a novel mechanism of receptor activation based on a unique antibody-imposed conformational change. These results demonstrate that a symmetric molecule can serve as a potent activator of the EPOR.

Description: Background: Solitary plasmacytoma of bone (SBP) is a localized collection of monoclonal plasma cells that is potentially curable with local radiation therapy but associated with a high risk of progression to multiple myeloma. We hypothesized that an abnormal immunoglobulin free light (FLC) ratio at diagnosis may be a prognostic indicator of transformation risk. Methods: We identified a cohort of 133 patients with SBP for whom stored serum taken at the time of diagnosis was available. The diagnosis was ascertained and serum FLC determined in 126 patients. Results: From this cohort, 48 patients have progressed to myeloma and the median time to progression among those who progressed was 1.9 years. On univariate analysis, age (p

Description: While there are certain common clinical features in myeloma, the disease shows significant heterogeneity with regard to disease progression, and responses to therapy, affecting both survival and toxicities. Heritable variations in a wide variety of genes and pathways affecting cellular functions and drug responses likely impact patient outcomes. In the Bank On A Cure (BOAC) program we have developed a custom chip that assesses 3,404 SNPs representing variations in cellular functions and pathways that may be involved in myeloma progression and response. The chip has gone through rigorous quality controls checks for high call rates, accuracy, and reproducibility that will be presented. Using the BOAC chip, we have conducted studies to look for SNPs that may identify biologic variations that are associated with good or poor response across a variety of treatments. In this study we looked for SNPs that may distinguish short term and long term survivors in two phase III clinical trials: ECOG E9486 and intergroup trial S9321. E9487 patients were treated with VBMCP followed by randomization to no further treatment, IFN-alpha, or cylcophosphamide; and, although there was variation in survival, no significant differences in survival were noted among the 3 arms of the trial. Patients included in this SNP study from S9321 received VAD induction followed by randomization to VBMCP or high dose melphalan + TBI. SNP profiles were obtained for patients with less than 1 year EFS (n=20 in E9487; n=50 in S9321) and patients showing greater than 3 years EFS (n=32 in E9486; n=41 in S9321). Statistical approaches were performed to identify single and groups of SNPs that best discriminated the survival groups. Previous studies have suggested genetic variations in drug metabolism genes, p-glycoprotein transport, and DNA repair genes may influence survival outcomes. Our results show significant survival associations of genetic variations in genes within these functional categories (eg. GST, XRCC, ABCB, and CYP genes). Although genetic variations were found that were uniquely associated with each clinical trial, several of these genetic variations show survival associations that increase in significance when the two trials were examined as a conglomerate data set. Grouping genetic variations through common pathway approaches using gene set enrichment analysis, as well as clustering or partitioning algorithms, further improve the value of the SNPs as potential prognostic markers of survival outcomes. These results and statistical approaches will be presented, and represent steps toward identifying patient variations in biologic mechanisms important in predicting therapeutic outcomes.

Description: Abstract 1175 Most steps in the blood coagulation cascade obligatorily take place on membrane surfaces and are dependent on the exposure of phosphatidylserine (PS). Previous studies from our lab and others have shown that phosphatidylethanolamine (PE) poorly supports clotting reactions by itself, but strongly synergizes with PS to promote several membrane-dependent steps in the blood clotting cascade, although the mechanism for PE-PS synergy has been unclear. We recently put forward a new mechanistic explanation – which we termed the ABC or Anything But Choline hypothesis – for how PS and PE synergize to enhance factor X (fX) activation by the factor VIIa-tissue factor complex (Tavoosi et al., J. Biol. Chem. 286:23247–53, 2011). The membrane contribution to this reaction is dominated by the affinity of fX for the membrane surface; since fX binds to membranes via its gamma-carboxyglutamate-rich (GLA) domain, the ABC hypothesis therefore focuses on the mechanisms by which GLA domains engage the phospholipid bilayer. We identified two main types of GLA domain-phospholipid interactions: a single phospho-L-serine-specific binding site in each GLA domain; and multiple ”phosphate-specific” interactions in which the phosphate groups of non-phosphatidylcholine phospholipids form coordination complexes with the tightly bound calcium ions in GLA domains. In the current study, we test the ABC hypothesis in the context of the prothrombinase complex – i.e., activation of prothrombin by the membrane-bound complex of fXa and factor Va (fVa). Using a variety of approaches including surface plasmon resonance analyses, we measured the contributions of varying phospholipid compositions to the membrane binding affinities of fXa, fVa and prothrombin, as well as to the enzymatic activity of prothrombinase. Our results suggest that phospholipid synergy in prothrombinase activity differs in certain respects from that observed for the factor VIIa-tissue factor complex. Not only did PS synergize with PE for enhancing the activity of prothrombinase, but phosphatidylglycerol (PG) and phosphatidylacid (PA) also synergized with PE, albeit more weakly than with PS (i.e., significantly higher levels of PG or PA in the presence of PE were required to achieve prothrombinase activities comparable to mixtures of PS and PE). In contrast, PE failed to synergize with either PG or PA to support fX activation by the factor VIIa-tissue factor complex. These differences primarily arise from differential membrane binding of the substrates for these two complexes (fX for factor VIIa-tissue factor and prothrombin for prothrombinase). The data suggest that the phospho-L-serine-specific binding site in the GLA domain of prothrombin may not be as stringent as that of fX, as high levels of PG or PA can substitute for PS in membrane binding of prothrombin but not for fX. This study provides further insights into the membrane's role in regulating blood clotting reactions, specifically the binding interactions between GLA domains and membrane surfaces. Disclosures: No relevant conflicts of interest to declare.

Description: Background: The presence of circulating myeloma cells (CMC) detected by flow cytometry at the time of diagnosis of multiple myeloma is associated with a shortened response to therapy and reduced overall survival (OS). We hypothesized that the presence of CMC at the time of stem cell collection prior to high dose therapy (HDT) and autologous stem cell transplantation (ASCT) would identifies a cohort of patients with a high risk of rapid progression. Methods: The Mayo Clinic myeloma transplant database was queried for patients who were mobilized using cyclophosphamide and hematopoietic growth factors. CMC was determined using flow cytometry by gating on a population of CD38 bright and CD45 negative cells. The impact of CMC on OS and time to progression (TTP) and its role in the context of established prognostic parameters was evaluated. Results: Of 246 patients with MM undergoing ASCT, 95 had CMC. Patients with CMC had significantly higher plasma cell labeling index, adverse cytogenetics, B2-M and resistant disease. Complete response (CR) rates post transplant were 32% and 36% for patients with and without CMC (p=0.5034). OS was 33.2 and 58.6 months (p=0.0052) while TTP was 14.1 and 22 months respectively (p=0.0005). Figure Figure On multivariate analysis, CMC remained an independent prognostic factor in a model that included cytogenetics and disease status at time of transplant (p=0.0314). A prognostic system based on the presence or absence of CMC and karyotype abnormalities was developed. Patients with neither, one or both parameters had a median, OS of 55, 48 and 21.5 months respectively (p