Citations

Abstract 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.

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that results in considerable morbidity and mortality. As it is incurable with the current therapeutic approaches, more effective therapies based on better understanding of the pathobiology of the disease are needed. In MM, malignant plasma cells are characterized by low proliferative and apoptotic rates compared to other malignancies. The tumor suppressor gene p53, responsible for induction of cellular apoptosis in response to genotoxic stimuli, is relatively intact in most cases of myeloma. However, p53 mutations or deletion can occur late in the course of disease. Here we evaluate a novel small molecule inhibitor of the interaction between p53 and its negative regulator, MDM2, in the setting of myeloma. Methods and Results: Mi-63 was cytotoxic to several different myeloma cell lines with a median effect observed at approximately 2.5 μM in cell lines including MM1.S that express wild type p53 and between 10–15 μM in cells with mutated p53 as measured using an MTT cell viability assay. Additionally, Mi63 induced cytotoxicity in myeloma cell lines resistant to conventional agents such as Melphalan (LR50), Doxorubicin (Dox40) and Dexamethasone (MM1.R), indicating non-overlapping mechanisms. To evaluate the ability of the drug to induce cell death in the tumor microenvironment, MM cells were co-cultured with marrow stromal cells or in the presence of VEGF or IL-6, two cytokines known to be important for myeloma growth and survival. Mi63 was cytotoxic to myeloma cells under these conditions as well, at doses similar to those seen with myeloma cells alone. Mi63 was able to inhibit proliferation and induce apoptosis in myeloma cells in a dose- and time-dependent fashion, as demonstrated by flow cytometry using Annexin/PI staining as well as cell cycle studies. Treatment of myeloma cells with Mi63 was associated with early mitochondrial membrane depolarization, inversion of Bax/Bcl-2 ratio, and down regulation of Mcl-1, indicating induction of mitochondrial mechanisms of cell death. Mi63 was also cytotoxic to freshly isolated primary patient myeloma cells, inducing apoptosis in a dose-dependent manner. In the patient cells the drug appears to have a differential effect on the CD45 positive and negative cells. Conclusion: Mi-63 has significant activity in vitro in the setting of myeloma as demonstrated by its effect on myeloma cell lines and primary patient cells. It clearly induces apoptosis in myeloma cells, with higher activity seen in cells with wild type p53. Given the lack of p53 abnormalities in most of the patients with myeloma, this drug alone or in combination is likely to have significant clinical activity. Studies combining this with various DNA damaging drugs are in progress. These studies will eventually form the framework for future clinical studies.

Abstract Background: An abnormal free light chain (FLC) indicating the presence of excess monoclonal FLC is associated with an increased risk of progression of monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) or related disorder. In addition, the risk of progression rises with increasingly abnormal FLC ratio, suggesting that the mechanism for markedly abnormal FLC ratios may be different compared to ratios closer to normal range. We hypothesized that as in light chain MM (in which IgH expression is completely lost), a more markedly abnormal FLC ratio is a marker for IgH translocations (TCs) involving 14q32. We tested this hypothesis using data obtained from gene expression profiling (GEP) to identify the presence of specific IgH TCs and correlating them to serum FLC ratio. Methods: We examined data from 128 GEPs from pts with MM (92), smoldering MM (13), MGUS (9) and primary amyloidosis (14). Presence of IgH translocations were identified using the specific probes for the partner chromosome TC sites, namely FGFR3/MMSET (4p16), cyclin D3 (6p21), cyclin D1 (11q13), c-maf (16q23), and mafB (20q12) as previously described. FLC estimation was carried out on stored serum samples from these patients using the serum FLC assay (FreeliteH, The Binding Site Limited, UK) performed on a Dade-Behring Nephelometer. Based on the established reference range, kappa/lambda FLC ratio <0.26 or >1.65 were defined as abnormal indicating the presence of monoclonal lambda and kappa FLC, respectively. The monoclonal LC isotype was considered the involved FLC isotype, and the opposite LC type as the uninvolved FLC type. Differential expression of genes was examined using Genespring 7.2 software. Results: Among the 92 pts with MM, 36 (42%) had evidence of one of the 5 primary IgH TCs: 20 pts with 11q13 (22%), 7 pts with 4p16 (8%), 4 pts with16q23 (4%), 3 pts with 20q12 (3%), and 2 patients with 6p21 (2%). Among the 11 pts with MM with an FLC ratio of > 1000, 8 (72%) had an IgH TC. The proportion of pts with an FLC ratio >1000 was significantly higher in pts with an IgH TCcompared to those without, 22% vs 5% respectively, P=0.02. The mean FLC ratio was 1728 (range; 1.6 to 31,008) in patients with an IgH TC vs. 412 (1–11340) for those without IgH TCs, P=0.09. Similarly in pts with MGUS, SMM or AL all three pts with a ratio > 50 had evidence of an IgH translocation. compared to 14/33 with lower ratio. The mean FLC ratio was 29 (range;1 to 209) in pts with an IgH TC vs. 9.1 (1–41) for those without IgH TCs, P=0.1. We correlated the results of FISH tests where available (122 of 128 pts) to determine the ability of GEP to identify IgH TCs. IgH translocation identified by the GEP could be detected on FISH in 42 of 47 patients (89%). Correspondingly, 61 of 75 patients (81%) without an IgH translocation on GEP had a similar result by FISH as well. We compared the GEP of MM pts with FLC ratio of >500 to the rest and identified 34 genes which were 2 fold differentially expressed between the two groups. Conclusion: By combining GEP data and serum FLC measurements, we show that very high FLC ratios is associated with IgH translocations. However, the correlation is not absolute, likely reflecting that markedly abnormal FLC ratios may be mediated through other mechanisms including increasing tumor load. To further explore this we analyzed differentially expressed genes between patients with high and low ratios and identified a set of 34 genes, which are currently being studied further.

Abstract Background: Autologous stem cell transplantation (SCT) improves survival in patients (pts) with multiple myeloma (MM). We have previously demonstrated that the degree of response at transplant does not impact on the outcome of transplant. However, newer induction regimens such as thalidomide and dexamethasone (Thal-Dex) result in higher response rates compared to previously used regimens such as single agent dexamethasone or vincristine, doxorubicin, and dexamethasone (VAD). We examined the outcome of SCT following three different induction therapies for newly diagnosed MM, namely VAD, single agent Dex, and Thal-Dex. Patients and Methods: 340 patients with MM who received their SCT within 12 mos of diagnosis (median 5.8, range 3–12) were studied. Patients receiving more than one induction therapy as well as those in whom thalidomide was added to dexamethasone for lack of response were excluded from the analysis. There were 105 pts in the VAD group, 140 in the Dex group and 95 in the Thal-Dex group. Responses were defined using standard criteria. Results: The study cohort consisted of 209 males (59%), with a median age of 57 years (range 30–76) at transplant. Baseline characteristics were similar in the 3 groups, except for lower age in the VAD group (median 55.8) compared to Dex (59.6) and Thal Dex (57.4) and shorter time to transplant in the Dex group (5.4 m) compared to VAD (6.4) and Thal Dex (5.9). Markers of disease activity pre-transplant, including B2M and marrow plasma cell percentage were higher in the Dex group compared to either VAD or Thal Dex. The proportion of patients with any response to induction therapy was lower in the Dex group compared to the other two. All pts in the Dex and the Thal-Dex groups received melphalan only conditioning compared to 70% in the VAD group, the rest receiving Melphalan/TBI. An objective response was achieved after SCT in 96%, 97%, and 98% of pts in the VAD, Dex and Thal-Dex groups respectively (P=0.8). A complete response to SCT was seen in 49% of patients in VAD group, 45% among those in the Dex group and 38% among those in the Thal Dex group (P=0.38). There was no difference in the median progression free survival after transplant (P=0.21) or overall survival from diagnosis (P=.34) between the three groups. The proportion free from progression at 2 years post transplant was 54%, 55% and 46% for Dex, VAD and Thal-Dex respectively. The proportion surviving at 4 years from diagnosis was 64%, 65.4% and 72% respectively for the three groups. Conclusion: We did not observe any difference in the response rates including complete responses to SCT in the three groups with nearly all pts in each group achieving a response. The progression free survival and overall survival appear to be comparable between the three groups. The results from initial therapy cannot be compared between the three regimens since the study population is restricted to patients reaching stem cell transplant. Within the limits of the study, there does not appear to be any long term impact of the initial therapy for the patients going onto an early stem cell transplant. Figure Figure

Abstract Purpose: Deep vein thrombosis (DVT) is increasingly recognized as a major complication of thalidomide (T; Thal) and lenalidomide (R; Rev) based therapy for multiple myeloma. Estimated incidence varies widely in the literature. There is no consensus on specific risk factors, indications for thromboprophylaxis or the agent of choice for DVT prophylaxis. The purpose of this study was to develop a consensus among members of the IMWG. Patients and Methods: A survey was electronically mailed to 67 IMWG members on July 21, 2006 and consisted of 24 multiple-choice questions. Results: Twenty-three IMWG members (34%) sent in their responses by the cut-off date. All (100%) felt that DVT was an important question, but most (95%) did not avoid Thal/Rev based therapy because of the DVT risk. Overall results are summarized in the Table below. A majority of physicians felt that concomitant therapy with high dose dexamethasone (90% of physicians), alkylators (55%), doxorubicin/liposomal doxorubicin (90%), and erythropoietin (75%) increase the risk of DVT associated with Thal or Rev based therapy. Aspirin (81 mg or 325 mg per day) was the preferred choice of prophylaxis when necessary. However, 65% of respondents preferred warfarin to a therapeutic INR or LMW heparin if either doxorubicin or liposomal doxorubicin was added to the regimens. Most (95–100%) felt that DVT prophylaxis was not indicated for: VAD, MP, bortezomib +/− dexamethasone regimens. Seventy-five percent felt it to be safe to resume therapy with Thal/Rev after therapeutic anticoagulation. Most (95%) believed randomized trials were needed. Conclusion: DVT is recognized as a major clinical problem associated with Thal/Rev containing regimens. Most respondents felt that the DVT risk did not warrant any prophylaxis when either Thal or Rev were used as single agents. Aspirin was the preferred form of DVT prophylaxis for most Thal/Rev containing regimens, except when doxorubicin/liposomal doxorubicin or melphalan were added. Additional responses will be analyzed both electronically and by conference call prior to the meeting. Therapy Estimated incidence of DVT (% respondents) Choice of thromboprophylaxis (% respondents) Single agent thal <5% (61%) None (65%) Single agent Rev <5% (80%) None (65%) Thal-Dex 10–20% (52%) Aspirin (60%) Rev-Dex 10–20% (48%) Aspirin (60%) MPT 10–20% (65%) Aspirin (40%) R-MP 5–10% (57%) Aspirin (70%)

Abstract Background: Isolation of malignant plasma cells from bone marrow of patients with monoclonal gammopathies is critical for studies into the disease biology. The isolation of plasma cells have generally been performed by positive selection using plasma cell markers such as CD138 or by negative selection by removing other marrow cells using a cocktail of antibodies. We have previously demonstrated differences in apoptotic rates in plasma cells with and without this enrichment step. Here we have examined the effect of CD138 magnetic bead selection on the surface phenotype of plasma cells by flow cytometry. Methods: Bone marrow aspirates from patients with myeloma (n=12) were first washed, and then lysed with ACK to eliminate red cells. The samples were split with a portion of the cells further processed for CD138 selection. ACK lysed only whole bone marrow (WBM) and sorted cells were stained with CD38, CD45, CD56, activation markers CD71 and CD69, adhesion markers CD49d, CD11b, and CD66, B cell markers CD19 and CD20, and clonality (kappa and lambda). Gates were drawn around the plasma cells and plasma cell subsets on the basis of CD38/45 expression for both sorted and unsorted samples. In addition, an aliquot of the sorted preparation was examined by immunohistochemistry to calculate the purity of the sorted sample. Results: Significant differences were observed in terms of the percentage of plasma cells expressing the different antigens when the cells were selected using CD138. This difference included a greater than 10% difference in expression between the two preparations as well as a change from positive to negative in several cases. There was a substantial loss in the expression of CD20, CD71 and CD11b on plasma cells following CD138 based sorting (Table). Among the other markers, CD49d remains unchanged and changes are variable for the other markers. In addition, in 8 of the 12 cases, there was a nearly complete loss of the CD45 positive subset with a loss of discrimination between CD45 negative and CD45 positive plasma cell subsets in the remaining CD138 sorted preparations (figure: unsorted left, sorted right). Conclusion: In conclusion, the process of CD138 sorting of plasma cells appears to change important markers on the plasma cells and may even eliminate a key subset from further analysis. This should be kept in mind when isolating plasma cells using CD138 positive selection for analysis such as gene expression profiling. Consideration should be given to negative selection using antibodies against other cell types to deplete them. Marker Unsorted % Sorted % CD56 74 67 CD69 11 2 CD71 23 8 CD49d 95 92 CD11b 33 4 CD66 5 1 CD20 16 6 Figure Figure

Translocations involving immunoglobulin (Ig) loci and chromosome 13 monosomy (Δ13) are frequent cytogenetic findings in multiple myeloma (MM). Similar chromosomal aberrations have been identified in the monoclonal gammopathy of undetermined significance (MGUS), but their prevalence and significance remain uncertain. Bone marrow from 72 patients with MGUS (n = 62) and smoldering MM (n = 10) was evaluated for translocations between the Ig heavy chain (IgH) and chromosomes 4, 11, and 16, translocations involving Ig light chain–lambda (IgL-λ, and Δ13. Fluorescence in situ hybridization (FISH) analysis was done on clonal plasma cells (PCs) detected by immunofluorescence (cIg-FISH) of the cytoplasmic light chain. We also studied cells for cyclin D1 and FGFR3 up-regulation by immunohistochemistry and immunofluorescence, respectively. Twenty-seven (46%) of 59 patients had IgH translocations, and 4 (11%) of 37 had an IgL-λ translocation. A t(11;14)(q13;q32) was found in 15 (25%) of 59 patients, a t(4;14)(p16.3;q32) in 9% of patients, and a t(14;16)(q32;q23) in 5% of patients. All patients with t(4;14)(p16.3;q32) tested (n = 3) had intense cytoplasmic fluorescence with an anti-FGFR3 antibody. PC nuclear staining of cyclin D1 was only observed in patients with t(11;14)(q13;q32); Δ13 was detected in the clonal PCs in 50% of patients. The percentage of abnormal PCs varied with any given abnormality. No obvious clinical or biologic correlations were associated with these chromosome abnormalities. Similar translocations are found in both MGUS and MM, including t(4;14)(p16.3;q32) and t(14;16)(q32;q23). Moreover, Δ13 is common in MGUS and unlikely to play a predominant role in the evolution of MGUS to MM.

Abstract Plasma cell leukemia (PCL) is an aggressive and rare hematological malignancy that can originate either as primary disease (pPCL) or as a leukemic transformation of multiple myeloma (MMLT). We report here on the clinical characteristics, survival and genetic studies of 80 patients with PCL treated at a single institution (including 41 with pPCL and 39 with MMLT) and make comparisons with 439 newly diagnosed cases of multiple myeloma (MM). From our results pPCL presents a decade earlier than MMLT (54.7 vs 65.3 years) and despite substantially poorer clinical and laboratory parameters at presentation (hepatomegaly 32% vs 11%, leukocytes 31.6 vs 20.9, β2M 8.5 vs 4.3) pPCL appears more responsive to conventional combination chemotherapy (VAD, VBMVP or VBAP) than MMLT and is associated with significantly longer survival (median 18.4 vs 3 months). Comparison of pPCL groups treated with combination chemotherapy (n=21) or with other regimens (40% MP, 10% Thal/Dex, 20% other, 30% none)(n=20) identifies no substantial pre-treatment differences between the groups although a higher proportion of patients who received combination chemotherapy subsequently proceeded to stem cell transplantation (30 vs 10%). Importantly, survival of pPCL patients treated initially with combination chemotherapy (median 13.9, mean 27.3 months) was significantly longer compared with patients who did not receive combination chemotherapy (median 5.2, mean 9.9 months)(p=0.035) with 2 long term survivors (> 7 years) in the chemotherapy group. By comparison, patients with MMLT received less active therapy for their leukemic disease than did patients with pPCL (21% VAD, VBMVP or VBAP; 18% MP). Treated MMLT patients showed selection bias for younger age (60.9 vs 66.4 years) and lack of bone disease (0 vs 65%)(p<0.01); however despite this, median survival was disappointingly short for MMLT patients in the treatment group and was not significantly improved compared with non-treated patients (2.3 vs 0.8 months)(p=0.34). Assessment of novel therapies in PCL is therefore urgently required. Karyotype analysis and targeted genetic studies demonstrate that IgH translocations are present with high frequency in both pPCL and MMLT (71 vs 86%) compared with MM (~50%). Notably, IgH translocation in pPCL is almost exclusively targeted at chromosome 11 (cyclin D1) belying its importance in establishing the phenotype. IgH translocation in MMLT is primarily to chromosome 11 [71%], but also targets chromosomes 4 (FGFR3/MMSET) [13%] and 16 (cMaf) [20%], recapitulating MM. Abnormalities of chromosomes 3,5,7,9,11,13,15 and 19 are present equally in all 3 disorders. Deletion of 17p13 (p53) shows strong selection pressure in the leukemias (pPCL 53.8%, MMLT 48.8%, MM 10.7%) and is complemented by p53 mutation (23%). Notably, pPCL tumors show lower rates of Ras mutation (12 vs 40%) and p14 (0 vs 31%) or p16 (25 vs 40%) promoter methylation than MMLT. Together these results therefore indicate that pPCL and MMLT are fundamentally different plasma cell disorders with distinctive biology, genetic abnormalities and treatment responses.

Abstract Background: Multiple myeloma remains incurable with current approaches and newer therapies are needed to improve the outcome of these patients. While monoclonal antibody based therapies have been successful in some of the hematological malignancies, such approaches have had limited efficacy in the setting of myeloma. Thymoglobulin (polyclonal rabbit antithymocyte globulin, Genzyme) (Thymo) has been extensively evaluated in the setting of allogeneic blood and marrow transplantation and solid organ transplants. Given the polyclonal nature of this product, with antibodies against different B cell antigens, we evaluated the in vitro and in vivo activity of Thymo in myeloma. Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity following drug treatment was measured using the MTT viability assay. Apoptosis was measured by flow cytometry using Annexin V/PI in cell lines and Apo 2.7 in primary patient plasma cells. Shifts in expression of a variety of different B cell and plasma cell antigens were examined on several different myeloma cell lines following Thymo treatment in order to identify the potential antigenic targets. In vivo activity of thymo was evaluated in a SCID plasmacytoma model injected with RPMI myeloma cell lines. Results: rATG was cytotoxic in vitro to several MM cell lines (RPMI 8226, U266, OPM1, OPM2) including the IL-6 dependent cell line Kas6/1 with LC50 of around 1 mg/mL. Additionally, thymo was cytotoxic MM cell lines resistant to conventional agents such as doxorubicin (Dox40), melphalan (LR5) and dexamethasone (MM1R). Thymo induced apoptosis in MM cell lines and in patient derived primary myeloma cells. When tested in combination with other anti-myeloma agents an additive effect was seen with doxorubicin, PS341 and melphalan. Using competitive flow cytometry, we identified CD138, CD38, Cd45, CD126, CD49d (VLA4), as well as CD20 as antigens likely to be targeted by Thymo. Tumor bearing mice injected with Thymo at two different doses (5 mg/kg and 10 mg/kg for five days) had significantly delayed tumor growth compared to non-injected mice, and this translated into a better survival for these mice. Mice receiving 10 mg/kg dose had a slower tumor growth compared to 5 mg/kg dose (Figure). Conclusions: Thymoglobulin has promising in vitro and in vivo activity in the setting of myeloma. These studies will provide the rational for future clinical development of this agent in myeloma alone or in combination with other agents. Based on these results, we are in the process of initiating a clinical trial combining Thymo with Melphalan. Figure Figure

Abstract Purpose: Updated results, including response rate data, from a randomized, double-blind, placebo-controlled study comparing thalidomide plus dexamethasone versus dexamethasone alone as primary therapy for newly diagnosed multiple myeloma (MM). Methods: Patients (pts) with previously untreated, symptomatic MM were eligible. Pts were randomized to Thal/Dex (Arm A) or placebo plus Dex (Arm B). Pts in Arm A received Thal 50 mg PO daily, escalated to 100 mg on day 15, and to 200 mg from day 1 of cycle 2; Dex 40 mg PO was given on days 1–4, 9–12, and 17–20. Pts in Arm B received placebo instead of Thal, and Dex as in Arm A. Each cycles was 28 days long. Treatment was continued until progression or undue toxicity. The primary endpoint was time to progression (TTP) defined using EBMT criteria. All analyses were conducted on an intent to treat basis. An independent data monitoring committee recommended release of results. Results: 470 pts were enrolled: 235 were randomized to Thal/Dex and 235 to placebo/Dex. Median follow up is 18 months. Median age was 65 yrs. The response rate (CR+PR) was significantly higher with Thal/Dex compared to placebo/Dex, 59% vs 42%, respectively, P<0.001. Eighty-two percent of patients have gone off-study. TTP was significantly superior with Thal/Dex vs placebo/Dex, median TTP not reached with Thal/Dex versus 8.1 months (95% CI: 6.5–12.8 months) with placebo/Dex, P<0.001. Major grade 3–4 events are summarized in the Table below. Deep vein thrombosis (DVT) was higher with Thal/Dex vs placebo/Dex, 15.4% vs 4.3%, respectively. Conclusions: Thal/Dex yields superior response rates and TTP compared to Dex alone when used as first-line therapy for multiple myeloma. Major Grade 3–4 adverse events Adverse Event Thal/Dex (n=234) Placebo/Dex (n=232) Venous thromboembolism (Grade >=3) 17.9% 4.3% Cerebral ischemia (Grade >=3) 3.0% 1.3% Myocardial ischemia (Grade >=3) 3.8% 2.2% Neuropathy (Grade >=3) 3.0% 0% Any toxicity (Grade >=4) 30.3% 22.8%