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Abstract Background: Primary systemic amyloidosis (AL) is an incurable plasma cell disorder. For selected patients high dose chemotherapy with peripheral blood stem cell support is effective, but even in those patients, only 50% derive organ responses. Other patients are too sick to undergo that therapy. Thalidomide has limited utility in this disease, largely because of its toxicity profile in patients with AL. Lenalidomide, Celgene’s lead clinical compound in a new group of drugs called IMiDs®, is highly active in patients with multiple myeloma, especially in conjunction with dexamethasone. We sought to determine the toxicity and efficacy of lenalidomide in patients with AL. Methods: Patients with symptomatic AL who had a measurable plasma cell disorder (defined as serum M-spike ≥ g/dL urine M-spike >200 mg/24 hours; or involved immunoglobulin free light chain (FLC) ≥10 mg/dL and an abnormal FLC ratio) and adequate organ reserve defined as a creatinine ≤3 mg/dL, absolute neutrophil count ≥1000, platelet count ≥75000, and a hemoglobin ≥8 g/dL were eligible. Patients were started on lenalidomide 25 mg/day for 21 days followed by a 7 day rest (1 cycle). Dose modifications were made based on toxicity. If there was evidence of progression before 3 months or no evidence of hematologic response after 3 cycles, dexamethasone 40 mg p.o. days 1–4 and 15–18 was added. Results: Twenty-three patients were enrolled between 10/28/04 and 4/7/05; 14 were previously treated. Patient characteristics are shown in Table. Organ involvement was as follows: cardiac (61%); renal (70%), hepatic (22%); nerve (13%). Nine patients withdrew from study before completing 3 months of therapy. The reasons were: cancel (1); death (4); adverse events (2); and progression (2). At the time of our preliminary analysis in July 2005, an additional 3 patients have withdrawn from study: death (1) and patient refusal (2). Of the 5 patients who died, 4 had severe cardiac involvement and at least 3 organs involved by amyloid. The median follow-up for the eleven patients remaining on study is 6.2 months. Of the 12 patients who have crossed the 3 month treatment landmark, there are 7 hematologic partial responses (4 confirmed and 3 unconfirmed), two renal responses and one liver response. Of these same 12 patients, all but one has had dexamethasone added to their treatment program. The most common grade 3–4 adverse advents at least possibly attributable to lenalidomide were neutropenia (43%), thrombocytopenia (26%), rash (17%), dyspnea (9%), fatigue (9%), and edema (4%). Expansion of the trial is planned to accrue an additional 10 evaluable patients. Conclusions: Early results suggest that lenalidomide ± dexamethasone has activity in patients with primary systemic amyloidosis. Supported in part by the Caliguiri Fund for Amyloidosis Research, Robert A. Kyle Hematologic Malignancies Program, and Celgene. Patient Characteristics Median Range Age 64 44–88 Major Organs 2 0–3 Involved FLC, mg/dl 23 4.1–278 Serum alb, g/dl 2.8 1.2–3.7 Urine protein, g/24 hrs 3.7 0.02–14.3 Creatinine, mg/dl 1.3 0.7–2.6 Alkaline phos IU/l (nml <115) 99 57–1729 NY Heart Class 1 1–3 Troponin T, mcg/l 0.03 0.01–0.55 NT-proBNP, ng/l 1419 109–42844 LV Septum, mm 13 9–24 LV EF, % 63 22–72

Abstract Background: High dose therapy with autologous stem cell rescue has been shown to prolong survival in patients with multiple myeloma in randomized controlled trials. However, most of the prospective studies have included younger patients, usually 65 or less. It is important to have a better understanding of the outcome of transplantation in the older patients given the median age of onset of myeloma of 65 years. We retrospectively reviewed our institutions experience with high dose therapy for patients over 70 years. Methods: We identified 35 patients with multiple myeloma, from the transplant database, who were at or over the age of 70 at the time of their high dose therapy. We matched these patients to 70 patients (two matches for each patient), based on stage at transplant (primary refractory, plateau phase, relapse off therapy, or relapse on therapy), Durie Salmon stage, high or low labeling index, conventional cytogenetics (abnormal vs normal), presence or absence of circulating plasma cells at time of transplant, and whether cyclophosphamide was used as part of mobilization in that order of priority. Results: The median age of the two groups were 55.3 (Range 37.3–64.8) and 71.7 (Range 70–75.8) years at the time of transplant. The median time to transplant from diagnosis was similar (6.4 for the older patients compared to 6.9 months for the other, P = NS). Ten of the 35 older patients received reduced dose melphalan (140 mg/2)) compared to 3 patients in the control group; P < 0.01. The median follow up from transplant was 10.1 months for the older patients compared to 18 months for the control group. The overall response rate was similar for the two groups (97.1% for the older patients compared to 95.5 for the control group). Eleven (31%) of the older patients and 17 (24%) of the control patients achieved a CR (P = NS). The post transplant progression free survival estimate at 1 year post transplant was 65.3% for the older patients compared to 66% for the control group (P = 0.3)The two year estimated overall survival from transplant was similar in the two groups; 58% for the older patients compared to 67% for the control group. The overall survival from diagnosis was similar for the two groups (P = 0.6). The median number of days hospitalized was 9 days for the older population compared to 5 days for the control group (P = 0.37). Four patients died within the first one hundred days, one (3%) among the older patient group and 3 (4.3%) in the control group. Conclusions: High dose therapy and autologous stem cell transplantation is feasible in selected patients with multiple myeloma over 70 years. It is likely that these older patients were selected based on their overall performance status, a factor that is difficult to analyze in this retrospective review. Nearly 70% of the elderly patients received full dose melphalan for conditioning (200 mg/m2). The toxicity of transplant as well as the outcome appears to be very similar to the younger patients. Patients with multiple myeloma should not be excluded from high dose therapy solely on the basis of their chronological age.

Abstract Background: Multiple myeloma (MM) is a plasma cell malignancy that is incurable with current approaches. The median survival for patients with MM is around four years and a significant proportion of patients experience a course characterized by multiple relapses treated with different therapies. The median survival for patients relapsing after the initial therapy is nearly 18 months and successive treatment strategies result in decreasing response durations, likely reflecting acquired drug resistance. In order to better understand the biological changes associated with advanced, relapsed, refractory MM, we compared gene expression profiles (GEP) of malignant plasma cells isolated from patients with relapsed refractory MM and compared them to plasma cells from patients with newly diagnosed MM. Methods: In order to obtain two relatively homogenous group of patients, we compared samples from 44 patient with newly diagnosed MM enrolled in the ECOG E1A00 clinical trial (comparing thalidomide and dexamethasone to dexamethasone alone) to 44 patients with relapsed refractory MM enrolled in a phase II trial of Velcade (SUMMIT), where most patients had four or more previous relapses. Plasma cells from bone marrow aspirates were separated by magnetic bead selection of CD138 positive cells and studied using Affymetrix HG-U133A chips using standard methodology. The arrays were analyzed using Genespring 7.2 software following GCRMA normalization and genes with differential expression between the two datasets were examined. Differentially expressed genes were further analyzed using Ingenuity Pathways Analysis program. Results: A total of 864 genes were identified which were at least two fold and significantly different between the newly diagnosed and relapsed patients. Using Ingenuity software, 437 of these genes were mapped to different biological networks. Examination of the canonical pathways demonstrated several important cellular pathways differentially regulated between the two groups. Several important mediators of the cytokines, receptors and respective signaling pathways appear to be down regulated in the relapsed group and included IGF-1, HGF, SDF-1 alpha, gp130 and importantly the MEK/ERK pathway. Additionally expression of adhesion molecules such as VCAM1 and PECAM was decreased in the relapsed group compared to newly diagnosed pts. There appear to increased tissue hypoxia in the relapsed marrow as indicated by up regulation of HIF-1 alpha as well increased levels of Placental growth factor. Myeloma cells from relapsed disease were characterized by decreased expression of mcl1, FLIP1, and bcl-xL and increased caspase 8 relative to newly diagnosed group. Also seen was decreased expression of the glucocorticoid and interferon receptors in the relapsed setting. Conclusion: Comparison of the GEP between MM cells from newly diagnosed and relapsed pts demonstrates important differences that have potential biological relevance. The plasma cell in the relapsed setting appears to be more independent of the tumor microenvironment. Additionally, differential expression of some of the genes provides clues to mechanisms of drug resistance commonly observed in the relapsed pts. We are in the process of validating some of the key findings from these analyses.

Abstract Background: Multiple Myeloma (MM) is characterized by accumulation of malignant plasma cells with relatively low proliferative and apoptotic rates. The proliferative rate of plasma cells, measured in terms of % plasma cells labeled by bromodeoxyuridine (BrdU) using slide based fluorescence microscopy (plasma cell labeling index or PCLI) has been shown to be a powerful prognostic factor in MM. We have previously shown that the % bone marrow plasma cells in apoptosis (PCAP) determined by a flow cytometry method that uses Annexin V staining or 7-amino Actinomycin D (7-AAD) correlates with disease stage in MM. The goal of this study was to examine the prognostic value of the PCAP. Methods: We studied 145 patients with MM including 74 with newly diagnosed MM, 40 with relapsed MM, and 31 with MM on treatment in whom simultaneous measurements of % plasma cell apoptosis and PCLI were available. The PCLI is expressed as the percentage of cytoplasmic immunoglobulin positive plasma cells that have taken up BrdU. The PCLI was characterized as high when >= 1%. Mononuclear cells isolated from bone marrow aspirate, following red cell lysis with ACK solution, are incubated with CD38-PE and either CD138-FITC or CD45-FITC. This is followed by incubation with 7-AAD and the plasma cells are gated using the CD38/CD45 staining pattern. The Annexin method used a similar strategy, with Annexin V in place of 7-AAD. The percentage of plasma cells in apoptosis (PCAP) was categorized as high when >5%, the median value. Results: The median follow up for the entire group was 34 months (range, 0.1 mo to 9 years) and 112 (77%) had died at the time of analysis. Forty seven patients (32%) had a high PCLI (>=1%). The median overall survival for the patients with high PCLI was 14.6 months versus 42.3 months for those with a low PCLI; P = 0.0005 (log rank test). Seventy-five (51.7%) patients had a high PCAP. The median overall survival for those patients was 28.9 months versus 40.2 months for those with a low PCAP; P = 0.078. We next examined if there was any correlation between a high PCAP and high PCLI and found none; P = NS by (Fisher’s exact test). Additionally in a multivariate Cox model using both PCLI and PCAP, both were independently prognostic for overall survival. We then developed an index by adding PCLI to log transformed PCAP (PCLI + log PCAP). Using a cutoff of 1.5 for the new index we were able identify a group of patients with poor survival. The 59 patients with a high value for the new index had a median survival of 19.3 months vs 46.1 months for the rest (P < 0.0001, log rank test). Conclusion: The measurement of plasma cell apoptosis has prognostic value in patients with MM. More importantly, we have shown that by combining measures of two important biological characteristics of the plasma cell, namely proliferation and apoptosis, patients with a poor prognosis can be identified. Such a prognostication strategy can help stratify patients in clinical trials as well as shed light on the disease biology.

Abstract Background: Multiple myeloma (MM) is an incurable hematological malignancy characterized by the expansion of a plasma cell clone that localizes to the bone marrow. Stromal cells residing in the bone marrow respond to signals from MM cells and other cell types by producing cytokines and other proteins that stimulate tumor cell growth, survival, adhesion, migration, and drug resistance. We have examined the proteins produced by stromal cells in response to stimulation by bone marrow from patients diagnosed with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and MM. Furthermore, we have begun analyzing the properties of one of these proteins, the pro-angiogenic chemokine IL-8, in MM. Methods: Bone marrow aspirates from patients with MGUS (n=3), SMM (n=7), and MM (n=6) were cultured for 48 hours, and the culture supernatants were incubated with stromal cells for an additional 48 hours. Protein levels were analyzed using antibody array and ELISA. Microvessel density (MVD) was determined as a measure of angiogenesis in patient bone marrow samples using CD34 staining. Flow cytometry analysis of MM cell lines and patient bone marrow samples was performed using monoclonal antibodies against IL-8 receptors CXCR1 and CXCR2. Results and Conclusion: We observed a significant increase in stromal cell IL-8 production stimulated by bone marrow cells from patients with active myeloma and a subset of SMM patients (16.67 ± 9.82 ng/ml) in comparison to bone marrow of patients with MGUS and all other SMM patients (0.55 ± 0.17 ng/ml; P=0.0004). Use of an IL-1 inhibitor and recombinant IL-1β demonstrated that IL-8 production was dependent upon IL-1β signaling. Increased BM microvessel density correlated with stimulation of stromal cell IL-8 production (P=0.0005). Furthermore, the majority of MM cell lines (7/9) and MM patient plasma cells were found to express IL-8 receptors CXCR1 and CXCR2. In addition to its function as a pro-angiogenic factor, IL-8 may directly influence MM cells through its CXCR1 and CXCR2 receptors. We conclude that stromal cell IL-8 production parallels MM disease activity, is IL-1β induced, correlates with bone marrow angiogenesis, and may influence MM disease via impact upon both the microenvironment and tumor cells.

Abstract Background: Primary systemic amyloidosis (AL) is an incurable plasma cell disorder. Lenalidomide, especially in conjunction with dexamethasone, has been shown to be highly active in patients with multiple myeloma. Methods: We studied the toxicity and efficacy of lenalidomide in patients with symptomatic AL. Patients received single agent lenalidomide. If progression by 3 months or no evidence of hematologic response after 3 cycles, dexamethasone was added. Originally, twenty-three patients (Cohort 1) were enrolled according to study design. Because of a significant early drop out rate and notable activity of the regimen, the trial was modified to include an additional 15 patients (Cohort 2). Baseline characteristics and adverse events are available for all enrolled patients, but at the time of this writing, response data are available for Cohort 1 patients due to short follow-up of Cohort 2, but will be updated by the time of the meeting. Results: Median age was 64 years, with 69% male. Twenty-three were previously treated. Organ involvement was cardiac (67%), renal (64%), hepatic (17%), nerve (17%). Thirty-three, twenty-two, and forty-four percent of patients were cardiac biomarker stage 1, 2, and, 3 respectively. Of the 37 patients, one was a cancel, and 6 have not yet made it through 3 months of protocol treatment and event monitoring. The respective median follow-ups for Cohorts 1 and 2 are 17 and 3.4 months. Of the remaining, 30 patients, within the first 3 cycles of therapy fifteen patients discontinued treatment: 7 early deaths and 8 adverse events or other causes. Three additional patients died 0.5 to 2 months after stopping treatment. The best predictor for early withdrawal and/or death was baseline NT-proBNP and cardiac biomarker staging system (cut-offs for serum troponin T <0.035 ng/ml and NT-proBNP <332 pg/ml--Stage I neither above cut-off; Stage III, both above cut-off; and Stage II, one above cut-off). Figure1 Figure1. Figure 2 Figure 2. Of the twenty 22 patients assessed for response ten patients responded to treatment for an overall response rate of 45%, including 23% organ responders. Among the patients with organ responses, there were four renal responses, two cardiac responses and two liver responses. All but one of the responders had dexamethasone added to their treatment program. The most common grade 3−4 adverse events at least possibly attributable to lenalidomide were neutropenia (38%), thrombocytopenia (21%), fatigue (15%), and rash (12%). Conclusions: Lenalidomide and dexamethasone has significant activity in patients with AL with an overall response rate of 45%. Baseline NT−proBNP may be an effective eligibility screening strategy for subsequent trials.

Abstract Background: The serum free light chain (FLC) assay is increasingly used to monitor patients (pts) with oligo-secretory or non-secretory multiple myeloma (MM) and pts with primary amyloidosis lacking measurable monoclonal protein in the serum or urine. Criteria to use this assay to assess response to therapy have recently been proposed (Rajkumar SV, Kyle RA. Best Pr Clin Haematol2005;18:585–601) but have not been validated. The goal of this study was to validate the response criteria for the FLC assay in a prospective trial of lenalidomide plus dexamethasone in newly diagnosed MM. Methods: 34 pts were enrolled in the trial; 27 pts who had serial FLC assessments were studied. FLC estimation was carried out using the serum FLC assay (FreeliteH, The Binding Site Limited, UK) performed on a Dade-Behring Nephelometer. Pts with κ /λ FLC ratio <0.26 were defined as having monoclonal λ FLC and those with ratios >1.65 as having a monoclonal κ FLC. The monoclonal light chain isotype was considered the “involved” FLC isotype, and the opposite light chain type as the “uninvolved” FLC type. Partial response (PR) required an abnormal baseline FLC ratio and any one of the two following criteria: 1) a 50% decrease in the level of the involved FLC plus a 50% decrease (or normalization) in the ratio of involved/uninvolved FLC or 2) 50% decrease in the difference between involved and uninvolved FLC levels. Complete response (CR) required normalization of FLC ratio and negative serum and urine immunofixation. Response at 4 months or earlier by the Bladé criteria was compared to FLC response criteria from the same evaluation. Results: Three pts had normal FLC levels and ratio and were not included in the analysis. 23 of the remaining 24 achieved a PR or better by Bladé criteria. A 50% decrease in the level of the involved FLC plus a 50% decrease (or normalization) in the ratio of involved/uninvolved FLC correctly classified 20 of the 22 responding pts (sensitivity 91%); 2 pts could not be evaluated since baseline FLC ratio could not be calculated. On the other hand, a 50% decrease in the mathematical difference between involved and uninvolved FLC levels correctly classified all 24 responding pts (sensitivity 100%). The one non-responding pt by Blade criteria was correctly classified by both FLC criteria. All pts were correctly classified by both criteria when only those with a baseline “involved” FLC level of at least ≥10mg/dL (≥100mg/L) were considered (15 pts). Conclusions: This study demonstrates that the serum FLC assay can be used to assess response to therapy. A 50% decrease in the difference between “involved” and “uninvolved” FLC levels will suffice as FLC criteria for PR, eliminating the need for the alternative criteria based on the involved FLC level and the ratio. We recommend that this FLC response criteria be used only in pts not having measurable levels of serum and /or urine M protein.The FLC response criteria will now enable most patients with oligo-secretory and non-secretory MM to enter trials for which they are currently ineligible due to “lack of measurable serum or urine M protein.” This study is limited by the lack of adequate non-responders to calculate specificity and lack pts who progressed to validate progression criteria and needs further validation.

Abstract Introduction: High dose melphalan followed by autologous stem cell transplantation (ASCT) is an effective treatment for patients with light chain associated (AL) amyloidosis. Longer patient survival and higher rates of organ response have now been documented by multiple studies. However, predictors of organ response remain unknown. Previously, we have reported the benefits of renal response after ASCT in this population. This study was conducted to investigate the characteristics that would predict renal response after ASCT. Methods: The study was performed retrospectively on consecutive patients that underwent ASCT at our institution from March of 1996 to December of 2004. Exclusion criteria include baseline proteinuria < 1 g/d, dialysis prior to ASCT and lack of laboratory data at follow up to determine renal response. Renal response was defined by > 50% reduction in baseline proteinuria with < 25% decline in renal function as measured by serum creatinine. Treatment related mortality and dialysis dependence prior to meeting criteria of response were viewed as treatment failures. Hematologic response was determined by 50% reduction of monoclonal protein (free light chain) or complete eradication if the monoclonal protein was too small to be quantified. Results: A total of 135 patients met criteria for study. Median age was 56.2 years at the time of transplant, 53.7% were male. Median baseline proteinuria and GFR were 6.4 g/d and 70 ml/min/1.73m2 respectively. Renal response was achieved in 35.6% of the patients while hematologic response was 71.1% in the 128 patients evaluated. Patient’s age, sex, albumin, GFR, proteinuria, conditioning regimen, and hematologic response were evaluated and the following were found to be associated with renal response: albumin (p = 0.001), proteinuria (p = 0.008), and hematologic response (p = 0.0002). The cutoff for albumin was found to be 1.6 mg/dl and proteinuria was 3.5 g/d. Multivariate analysis using a logistic regression model showed hematologic response and proteinuria to be independent predictors of renal response. The impact of proteinuria and hypoalbuminemia was then investigated together (Table 1). When combined, they were a better predictor then either one alone (Hazard ratio = 6.34 for combined, 3.43 for proteinuria, 3.32 for hypoalbuminemia). The combination was also a better independent predictor of renal response in the multivariate analysis. In this group of patients, renal response was associated with longer survival but hematologic response was not (p = 0.02). Discussion: Our study showed that besides hematologic response, baseline serum albumin and proteinuria are independent predictors of renal response in AL patients after ASCT. Hypoalbuminemia and nephrotic range proteinuria, both markers of the severity of renal disease, have strong negative impact on response. This implies that there may be a limit to the reversibility of organ damage even when hematologic response is achieved. This study also points out the importance of organ (renal) response in this disease as hematologic response alone did not predict long term outcome. Our results suggest ASCT should be done early for AL to insure optimal organ response and patient outcome. Table 1 The Effects of Hypoalbuminemia and Proteinuria on Renal Response after ASCT Hypoalbuminemia & Proteinuria No Renal Response Renal Response None 39.3% 60.7% One 66.2% 33.8% Both 81.8% 18.2%

Abstract Introduction: Molecular cytogenetic studies have revealed that, to a great extent, the heterogeneity of myeloma is largely dictated by the underlying genetic and cytogenetic aberrations present in the clonal plasma cells. Most recently the group from the University of Arkansas identified strong prognostic associations with an increased level of gene expression of a cell cycle associated gene, CKS1B. CKS1B favors cell cycle progression by promoting degradation of p27 with release of the cyclin dependent kinases and entry into mitosis. Patients and methods: To further test this hypothesis we studied: a) via FISH for CKS1B amplification in a cohort of patients treated at the Mayo Clinic with high dose chemotherapy and stem cell support, as well as a group of patients with cytogenetically defined hypodiploidy, and b) a cohort of myeloma patients that were studied by gene expression profiling. Gene expression analysis was performed on CD138-enriched plasma-cell RNA using Affymetrix U133A chips (Affymetrix, Santa Clara, CA). Results: Of 159 patients studied 46 exhibited FISH abnormalities consistent with increase number of signals for CKS1B (30%): amplification was marginal or low in 44 cases, and in only two cases the ratio between CKS1B and control probe was greater than 2.0. Therefore, the predominant pattern is one of gene duplication rather than amplification where gene copy usually exceeds 10 and ratio with control probe exceeds 5 (e.g. HER-2/neu amplification). Patients with CKS1B duplication had a higher prevalence of chromosome 13 deletion (72%), and t(4;14) (29%). The presence of CKS1B duplication was more frequent (53%) among 19 patients with hypodiploidy. Using gene expression data for CKS1B, we found a positive association with t(14;16)(q32;q23). The level of CKS1B gene expression positively correlated with the PCLI (p<0.0001) but there was heterogeneity in this relationship (r2 = 0.34). We found no correlation between the expression level of CKS1B and p27 (r2 = 0.008, p = 0.37). While CKS1B gene duplication had a negative effect on survival this effect was weak (29.9 versus 38 months, p = 0.124. Median follow-up is 55 months) and disappeared when the variable was entered into the multivariate model including PCLI, B2-microglobulin and all the major genetic abnormalities by FISH. Likewise the level of expression of CKS1B determined by gene expression only carried prognostic significance when extreme levels of expression were utilized as a prognostic variable. Discussion: In this study we show that increase copy number of CKS1B is present in one third of patients with MM (majority of these being gene duplication) and seem to be associated with a shorter overall survival, but the net effect of this is rather weak in our series. Further study is needed to understand its potential role as a progression factor in the plasma cell neoplasms.

Abstract Background: Multiple myeloma is a plasma cell malignancy that remains incurable with current approaches and newer therapies are needed to improve the outcome of patients with MM. While monoclonal antibody base therapies have been successful in some of the hematological malignancies, especially lymphoma, such approaches have not been very useful in the setting of myeloma. Targeting of antigens like CD138 on the myeloma cell surface has been hampered by the ubiquitous nature of this protein in the body. Thymoglobulin (polyclonal rabbit antithymocyte globulin, Genzyme) has been extensively evaluated in the setting of allogeneic blood and marrow transplantation and is currently in clinical use. Given the polyclonal nature of this product, it has antibodies against several B cell antigens and forms the rationale for its evaluation in B cell malignancies like 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. Drug induced apoptosis in the cell lines was measured by flow cytometry after staining with Annexin V-FITC and propidium iodide (PI). Apoptosis in primary patient derived plasma cells following treatment was measured after staining for Apo 2.7. 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. The LC50 in most of the cytotoxicity assays was around 1 mg/mL. Additionally, rATG was cytotoxic MM cell lines resistant to conventional agents such as doxorubicin (Dox40), melphalan (LR5) and dexamethasone (MM1R). The drug retained its cytotoxicity when myeloma cells were grown in the presence of various cytokines like IL-6, IGF-1 and VEGF. rATG treatment resulted in a time and dose dependent induction of apoptosis in MM cell lines. rATG was also able to induce apoptosis of freshly isolated myeloma cells from patient marrows. When tested in combination with other anti-myeloma agents an additive effect was seen with doxorubicin, PS341 and melphalan. Conclusions: Thymoglobulin appear to have in vitro activity against various myeloma cell lines as well as patient derived primary myeloma cells. Ability of the drug to overcome resistance to conventional drugs as well as the effect of combining rATG with these agents points towards non-overlapping mechanisms of action. Ongoing studies are trying to identify the particular B-cell antigens that are targeted by correlating response to expression of various B cell antigens expressed by these cell lines. These studies will provide the rational for future clinical development of this agent in myeloma alone or in combination with other agents.