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Originally published as JCO Early Release 10.1200/JCO.2005.14.381 on May 2 2005 © 2005 American Society of Clinical Oncology. Phase II Study of G3139, a Bcl-2 Antisense Oligonucleotide, in Combination With Dexamethasone and Thalidomide in Relapsed Multiple Myeloma PatientsFrom the Greenebaum Cancer Center and Departments of Pathology and Epidemiology and Preventive Medicine, University of Maryland, Baltimore; and Cancer Therapy Evaluation Program, National Cancer Institute, Bethesda, MD Address reprint requests to Ashraf Badros, MB, ChB, University of Maryland, Greenebaum Cancer Center, 22 S Greene St, Baltimore, MD 21201; e-mail: abadros{at}umm.edu
PURPOSE: Bcl-2 regulates the mitochondrial apoptosis pathway that promotes chemotherapy resistance. Bcl-2 antisense oligonucleotide, G3139, targets Bcl-2 mRNA. PATIENTS AND METHODS: G3139 was administered (3 to 7 mg/kg/d for 7 days) by continuous intravenous infusion. On day 4, patients started thalidomide (100 to 400 mg as tolerated) and dexamethasone (40 mg daily for 4 days) on 21-day cycles for three cycles. Stable and responding patients continued on 35-day cycles for 2 years. RESULTS: Thirty-three patients (median age, 60 years; range, 28 to 76 years) received 220 cycles. Patients received a median of three prior regimens including thalidomide (n = 15) and stem-cell transplantation (n = 31). The regimen was well tolerated; the median number of cycles per patient was eight (range, one to 16+ cycles). Toxicities included reversible increase in creatinine, thrombocytopenia, neutropenia, fatigue, anorexia, constipation, fever, neuropathy, edema, electrolyte disturbances, and hyperglycemia. Fifty-five percent of patients had objective responses, including two complete responses (CRs), four near CRs (positive immunofixation), and 12 partial responses; six patients had minimal responses (MRs). Of patients who received prior thalidomide, seven had objective responses, and three had MRs. The median duration of response was 13 months, and estimated progression-free and overall survival times were 12 and 17.4 months, respectively. Responding patients had significant increase in polyclonal immunoglobulin M (P = .005), indicating innate immune system activation. Western blot analysis of Bcl-2 protein isolated from myeloma cells before and after G3139 demonstrated a decrease of Bcl-2 levels in three of seven patients compared with six of nine patients using reverse transcriptase polymerase chain reaction. CONCLUSION: G3139, dexamethasone, and thalidomide are well tolerated and result in encouraging clinical responses in relapsed multiple myeloma patients.
Multiple myeloma remains an incurable malignancy despite responses to conventional and high-dose chemotherapy.1 On relapse, deregulation of signaling pathways and impairment of apoptosis contribute to resistance to chemotherapy and radiotherapy. The recent developments of novel therapeutics, such as thalidomide and bortezomib, have markedly improved outcomes.2,3 However, these salvage regimens have not resulted in sustained remissions, and relapse remains unavoidable. Bcl-2 overexpression is a major determinant of tumor resistance and predicts poor survival in many cancers including multiple myeloma.4,5 Physiologically, Bcl-2 expression is low during early stages of B-cell development and increases among more mature long-lived memory B cells.6,7 Upregulation of Bcl-2 expression is seen in myeloma cells after exposure to doxorubicin and etoposide.8 Several in vitro studies have shown that overexpression of Bcl-2 renders myeloma cells resistant to apoptosis by agents that traditionally work through the intrinsic apoptotic pathway, and in some studies, overexpression is associated with unfavorable outcome.9-12 Bcl-2 antisense, G3139 (oblimersen, Genasense; Genta, Berkeley Heights, NJ), is an 18-base phosphorothioate oligonucleotide complementary (antisense) to the first six codons of Bcl-2 mRNA, which leads to selective decrease in concentrations of Bcl-2 mRNA and protein levels.13 Several studies both in vitro and in vivo using tumor xenografted mice demonstrated that G3139 alone could induce apoptosis in low Bcl-2–expressing myeloma cells.14,15 However downregulation of Bcl-2 alone did not induce apoptosis in cells with high levels of Bcl-2 expression, possibly because other Bcl-2 family members (eg, Mcl-1 and Bcl-XL) substitute as survival factors.16 However, downregulation of Bcl-2 resulted in significantly enhanced susceptibility to dexamethasone- or daunorubicin-induced apoptosis.17 We hypothesized that the administration of G3139 to downregulate Bcl-2 mRNA and protein expression would increase the efficacy of thalidomide and dexamethasone in relapsed myeloma patients. Considering the toxicity profile of each agent, we did not anticipate significant myelosuppression, which is a major concern in heavily pretreated and relapsed patients. The primary objectives of this trial were to determine the safety and tolerability of the G3139, dexamethasone, and thalidomide regimen and to correlate changes in Bcl-2 mRNA and protein levels with the clinical outcome.
Eligibility and Enrollment All patients provided written informed consent that was approved by the University of Maryland School of Medicine Institutional Review Board (Baltimore, MD). Patients had relapsed multiple myeloma after chemotherapy or transplantation (allogeneic transplantation patients were eligible if they had no evidence of active graft-versus-host disease). Patients had measurable disease at study entry, which was defined by increasing levels of quantitative immunoglobulin levels in serum and/or urine and bone marrow plasmacytosis. Patients with nonsecretory disease were eligible if imaging studies could accurately measure at least one plasmacytoma. Patients had a Karnofsky performance score of at least 60 and had a life expectancy of greater than 3 months. Patients were excluded if they had received more than four prior regimens for treatment of their myeloma, had inadequate marrow function as defined by an absolute neutrophil count less than 1,000/µL or a platelet count less than 50,000/µL (except if myelosuppression was secondary to marrow plasmacytosis > 80%), or had abnormal liver function (bilirubin, AST, or ALT more than twice the upper limit of normal). In the initial phase of the study, patients with creatinine levels less than 3 mg/dL were treated with dose modification (outlined in Dose Modification), but once renal toxicity of G3139 was observed, the creatinine threshold for enrollment was changed to 2 mg/dL. Women of childbearing potential and all men agreed to use two methods of adequate contraception for the duration of study participation. Patients were excluded from the study if they had demonstrated grade 2 or higher skin reaction to thalidomide or grade 2 or higher sensory or motor neuropathy. The study opened in November 2002; the analysis was carried through July 2004 for 33 patients.
Study Protocol
Supportive measures included antimicrobial prophylaxis with sulfamethoxazole/trimethoprim or dapsone for patients with known sulfa allergy; during the week of dexamethasone treatment, patients received famciclovir, levofloxacin, and fluconazole. To decrease risk of catheter-related thrombosis, warfarin sodium was administered at 1 mg daily. Granulocyte colony-stimulating factor was administered for absolute neutrophil counts less than 1,000/µL; erythropoietin was administered to patients with hemoglobin less than 10 mg/dL. Bisphosphonates were withheld during the induction phase of the study to avoid possible synergistic renal toxicity but were restarted during the maintenance phase; bisphosphonates were not administered during the week of G3139 infusion.
Dose Modification
Criteria for Response and Toxicity
Quantification of Bcl-2 Protein
Real-Time Polymerase Chain Reaction Analysis
Statistical Considerations
RT-PCR data were analyzed using SPSS statistical software (SPSS, Inc, Chicago, IL) and expressed as mean ± SEM. Differences between means were evaluated by independent t tests, with statistical significance assigned at P
Patient Characteristics Thirty-three patients were enrolled onto this study (11 women and 22 men; Table 2). The median age was 60 years. All patients had relapsed after a median of three prior treatments (range, two to four treatments); none had primary refractory disease. Of the 33 patients, 30 had received autologous stem-cell transplantation, and one had a matched related allogeneic transplantation. All patients had received dexamethasone.
Fifteen patients had received prior thalidomide for a median duration of 6.5 months (range, 2 to 8 months). Four patients received dexamethasone and thalidomide in combination with chemotherapy (platinum, doxorubicin, cyclophosphamide, and etoposide [DT-PACE]) for primary refractory disease.19 Two patients achieved PR to DT-PACE, and two patients had PD. Subsequently, all four patients received high-dose chemotherapy and stem-cell support; two of the patients continued dexamethasone and thalidomide maintenance after transplantation until progression. Ten patients received dexamethasone and thalidomide either as maintenance after stem-cell transplantation (n = 3) or salvage for relapsed disease (n = 7). One patient received single-agent thalidomide for relapsed disease. The median time to study entry from the initial diagnosis was 40 months (range, 9 to 170 months). Three patients with creatinine more than 2 mg/dL were treated before the protocol was amended to limit enrollment to patients with creatinine less than 2 mg/dL. Other high-risk features for relapsed myeloma included chromosome 13 deletions in 16 patients, beta2-microglobulin more than 2 mg/L in 15 patients, and high lactate dehydrogenase (x 1.5 normal) in seven patients.
Toxicity A total of 220 cycles of G3139 have been administered as of July 2004, with a median of eight cycles per patient (range, one to 16 cycles). Table 3 lists the cumulative incidence of toxicity for all cycles by grade. Myelosuppression was the main hematologic toxicity in this study. Although grade 3 and 4 neutropenia was observed, patients responded well to granulocyte colony-stimulating factor, and none had a life-threatening infection. Reversible thrombocytopenia was seen; the median platelet counts were not different between cycles 1, 3, and 6 (cycle 1: median, 165,000/µL; range, 73,000 to 499,000/µL; cycle 3: median, 192,000/µL; range, 59,000 to 395,000/µL; and cycle 6: median, 169,000/µL; range, 68,000 to 237,000/µL; P = .054 and .08, respectively).
There was an increase in creatinine level from a median of 1.2 mg/dL (range, 0.6 to 2.5 mg/dL) at baseline to 1.5 mg/dL (range, 0.9 to 2 mg/dL) at cycle 6 (P < .001). Of 17 patients with increased creatinine, 13 eventually returned to baseline creatinine levels; in four patients, renal function worsened with evidence of progressive myeloma. Other toxicities included fatigue, electrolyte disturbance that affected calcium, phosphorus, and magnesium levels, fluid retention and edema, muscle cramps, rash, hypotension, and nausea. Fever during G3139 infusion began on day 2 to 3 of the infusion and was mostly low grade with no evidence of infection. This pyrexia was treated and prevented in subsequent cycles with acetaminophen. Most patients required thalidomide dose reduction to 50 to 200 mg/d because of neuropathy, and only three patients maintained the 400-mg dose. Peripheral neuropathy was observed in most patients on the study, with symptom relief provided by gabapentin (300 to 1,000 mg/d). Constipation was manageable in most patients using prophylactic laxatives. Three patients had thyroid stimulating hormone levels more than 5 mU/L, and one patient was symptomatic, with thyroid stimulating hormone increasing from 3.5 to 75 mU/L in 3 months; he responded well to supplemental levothyroxine and was continued on thalidomide. Two episodes of deep venous thrombosis were reported despite the prophylactic use of low-dose warfarin; one episode was catheter related.
Response and Survival
Of the 12 patients who received prior thalidomide and dexamethasone, two achieved near CR, four achieved PR, three had MR, one had PD, and two were not assessable. The single patient who progressed on prior thalidomide achieved PR on study. Neither of the two patients who progressed on prior DT-PACE responded. The only variable predictive of response was an early and statistically significant (P = .005) increase in polyclonal immunoglobulin M (IgM) from a median of 35.5 mg/dL (range, 8 to 75 mg/dL) to 94 mg/dL (range, 45 to 111 mg/dL) for responders after cycle 3 versus 26 mg/dL (range, 9 to 52 mg/dL) to 35 mg/dL (range, 6 to 58 mg/dL) in nonresponders (Fig 2).
Pharmacodynamics Of the 18 patients in whom protein isolation was attempted for immune blotting, only 11 had sufficient numbers of CD138+ cells isolated both on the pretreatment and the day 4 to 10 bone marrow aspirate to permit analysis. In four patients, undetectable tubulin levels precluded densitometric analysis. Therefore, only seven patients were assessable; whenever possible, the levels of other Bcl-2-family proteins were also determined. The data is listed in Table 5. Examples of the patients' analyses are shown in Figure 3. Of the seven assessable patients, three patients exhibited a decrease in Bcl-2 levels (one patient on day 7, one on day 10, and one on day 29); clinically, this corresponded to nonassessable (patient 12), PD (patient 6), and PD (patient 10), respectively. In patient 6, although the Bcl-2:tubulin ratio indicated a drop in Bcl-2 levels, comparison with Mcl-1 and Bcl-XL (which are proteins that had levels that did not change in any patients after G3139 treatment) suggested that the Bcl-2 levels may have actually increased (Fig 3). Patient 12 had a significant decrease in Bcl-2 protein level on day 7, had rapid progression of her disease resulting in renal failure, and was not assessable for response. Of the two patients with increased Bcl-2 levels, one had PR (patient 2), and one had PD (patient 10). After this series of patients, it became clear that immunoblotting using the low levels of protein obtained from CD138+ multiple myeloma cells present in marrow aspirates obtained in these studies was challenging and, for most patients, was not possible. Nonetheless, even our limited experience with Bcl-2 protein does not suggest a correlation with clinical outcome. Therefore, we shifted our approach to determining G3139 effects on Bcl-2 expression by examining mRNA levels using RT-PCR.
RNA isolation was attempted in the subsequent eight patients, in addition to four patients from the group tested for Bcl-2 protein that had enough CD138+ cells to isolate RNA. Of the 12 patients evaluated by RT-PCR, nine had successful amplification of Bcl-2 and the control GAPDH, as summarized in Table 5. Six of the nine patients demonstrated a significant decrease in Bcl-2 mRNA levels, and five patients had PRs. In three patients (patients 1, 7, and 8), Bcl-2 mRNA levels were already significantly decreased in cells isolated from day 4 to 10 aspirate, suggesting a fairly rapid effect that is likely attributable to the antisense oligonucleotide. In two patients (patients 3 and 4), there was a downward trend in Bcl-2 mRNA levels on days 4 to 10 that became significant by day 29. One patient (patient 6) did not have a day 4 to 10 specimen but demonstrated a significant decrease on day 29. One patient (patient 2) had a statistically significant increase in Bcl-2 mRNA, although this patient clinically achieved a PR. The other two patients (patients 5 and 9) with essentially unchanged levels had PD and MR, respectively. In four patients with enough cells to measure both Bcl-2 protein and mRNA, there was concordance in two patients. One patient (patient 2) had increased levels, although he achieved a PR clinically, and patient 6 had decreased levels and achieved a PD clinically. Therefore, a tentative conclusion of our study is that clinical response did not correlate with Bcl-2 protein expression in contrast to RT-PCR analysis, where five of six patients with significant decrease in Bcl-2 mRNA had achieved PR.
In this study, G3139 was safely combined with thalidomide and dexamethasone, with clinical responses observed in 73% of patients on an intent-to-treat analysis and in 80% of assessable patients completing the induction phase. Several phase I trials of G3139 have shown that the maximum-tolerated dose varied with the type of malignancy.20-24 In solid tumors, the dose-limiting toxicities reported were fatigue, fever, thrombocytopenia, and hypotension. Patients with lymphoid malignancies are more sensitive to G3139; a dose of 3 mg/kg/d in chronic lymphocytic leukemia patients was associated with hypotension, fever, thrombocytopenia, and grade 3 to 4 cytokine release syndrome. In this study, G3139 was started at 5 mg/kg/d for 1 week (first three patients) and subsequently increased to 7 mg/kg/d. No attempt was made to establish a maximum-tolerated dose for G3139 in combination with thalidomide and dexamethasone because previous pharmacokinetic studies had shown that a steady-state plasma concentration of more than 1 µg/mL is achieved with 3 mg/kg/d of G3139 with adequate downregulation of Bcl-2, at least when examined in peripheral-blood lymphocytes.25,26 G3139 toxicities were reversible, especially fever and fatigue, which resolved once the infusion was stopped. The observed reversible renal insufficiency in our study was also reported in patients with solid tumors but seems to be more frequent in myeloma patients.27 Several reports had suggested that the phosphorothioate DNA backbone could induce activation of the coagulation pathway as well as the complement system, although the contribution of these factors to renal toxicity is speculative.28,29 Through careful monitoring of the creatinine and dose reductions for each cycle as outlined earlier, most patients were able to continue treatment on study. It is worth noting that, in a randomized phase III trial of dexamethasone with and without G3139, there was no increased renal toxicity in the G3139 arm.30 The development of neutropenia and thrombocytopenia during G3139 infusion is possibly related to cytokine-mediated inhibition of early progenitors perhaps through Bcl-2 regulation of platelet production and release from megakaryocytes.31,32 Thrombocytopenia occurred mostly in the first few cycles of therapy with nadir counts on day 7 of G3139 infusion; all patients recovered to normal by the time of the next infusion, and no case of thrombocytopenia was associated with bleeding. Thalidomide toxicities were predictable and not aggravated by the addition of G3139. The incidence of deep venous thrombosis was lower than that reported with thalidomide and dexamethasone (15% to 25%), despite the use of central venous access.33 The most frequent side effect for thalidomide was peripheral neuropathy, which was the main reason for thalidomide dose reductions.34 There was no evidence that G3139 enhanced neuropathy in these patients. Several large studies have shown that thalidomide as a single agent can induce objective responses (PR or better) in approximately 30% of patients; an additional 10% of patients achieved MR. The addition of dexamethasone to thalidomide increases the response rate by 20%, possibly through a synergistic mechanism because approximately 30% of patients who did not respond to single-agent thalidomide achieved a response when dexamethasone was added35-38 Although comparison of these clinical results to our data is not feasible because of the variability in patient selections, we have achieved comparable, if not better, responses. Ninety percent of the patients on the study had received prior transplantation, and more than 45% had received prior thalidomide (n = 15), including one third of patients (n = 12) who had relapsed or progressed after prior thalidomide and dexamethasone. An encouraging objective response rate of 60% and overall response rate of 80% (including MR) was observed. In most patients, the maximum dose of thalidomide was 200 mg daily and less; this may suggest that Bcl-2 antisense could sensitize myeloma cells to thalidomide and dexamethasone effects. One other trial had shown that G3139 could overcome resistance and restore sensitivity to vincristine, doxorubicin, and dexamethasone chemotherapy in refractory myeloma patients. In that trial, the addition of G31339 to vincristine, doxorubicin, and dexamethasone resulted in responses in seven of eight patients, with a median PFS of 6 months (range, 2 to 7+ months).39 A randomized phase III trial presented in abstract form has shown that the addition of G3139 to high-dose dexamethasone did not increase the time to progression or overall response rate in patients who have received extensive prior therapy. The objective response rate was 16% v 20% (P = .6) with a median time to progression of 3.1 months v 3.5 months (P = .26) for the G3139-dexamethasone versus dexamethasone alone arms, respectively.30 It is also interesting to note that, unlike patients with chronic lymphocytic lymphoma, the use of G3139 was not associated with cytokine release reactions, possibly because of the corticosteroid use, which may have suppressed the immune modulatory effects of the molecule. We feel that the results are encouraging and call for a randomized trial to compare G3139 plus thalidomide rather than dexamethasone. The best end point would be time to progression, and there would be a cross-over design to G3139 arm in nonresponding patients to establish the potential value of G3139 in overcoming resistance. The pharmacodynamic activity of G3139 was an intended primary end point of this study. In this study, the low number of plasma cells isolated from bone marrow aspirates significantly affected our ability to analyze Bcl-2 expression in every patient. Bcl-2 mRNA levels decreased in six of the nine patients when tested by RT-PCR, whereas Western blotting showed mixed results with three of seven assessable patients showing decreases and four patients showing significant increases in Bcl-2 levels. Given the limited number of multiple myeloma cells in the aspirates, it is clear that the analysis of Bcl-2 mRNA expression is more efficient because of the small amount of RNA required for cDNA synthesis. Furthermore, RT-PCR studies were performed in quadruplicate, imparting greater statistical validity compared with immunoblot analysis. Although the numbers are too small to make any firm conclusions, our data suggest that a decrease in Bcl-2 mRNA levels may correlate with clinical response. Decreasing Bcl-2 expression may render multiple myeloma cells more sensitive to apoptosis via the intrinsic pathway that is activated by dexamethasone and thalidomide. These findings are similar to what was reported in acute myeloid leukemia and in various solid tumors.23,24,26 The increase in IgM observed early after G1339 infusion on the current protocol, as well as its correlation with clinical response, merits special consideration. This polyclonal IgM activation occurred in myeloma patients, who normally have suppressed capacity to produce normal immunoglobulins, suggesting that alternative pathways that do not involve stimulation of the B-cell receptor are involved. One possibility is that G3139, which encodes two CpG motifs (cytosine and guanine dinucleotides in particular base context) in a phosphorothioate backbone, may stimulate the innate immune system to secrete polyclonal IgM. It is now clear that unmethylated CpG motifs in bacterial DNA can stimulate Toll-like receptor-9 expressed on subsets of human B cells and lymphoid dendritic cells and that this can induce the production of polyclonal IgM and the maturation of the dendritic cell with secretion of multiple cytokines including interleukin (IL) -6, IL-10, and interferon alpha.40-43 In an elegant experiment, 5'-methylation of cytosines in CpG motifs of G3139 to create an immune-silent molecule resulted in a similar downregulation of Bcl-2 and increase in apoptosis in a human melanoma SCID mouse xenotransplantation model, as did G3139. However, mice treated with G3139 had a pronounced increase in spleen weight and IL-12 plasma levels relative to mice treated with the methylated form, suggesting that G3139 can be immunostimulatory.44 It is unlikely that the production of IgM is secondary to treatment/response to thalidomide plus dexamethasone because most patients treated with these agents in the absence of G3139 demonstrated persistent suppression of IgM levels. This is the first clinical report, to our knowledge, to suggest that an important biologic effect of G3139 is stimulation of the immune system. The measured increase in IgM may be a surrogate for other G3139 immune modulatory effects, such as changes in cytokine secretion by monocytes and dendritic cells. This is supported by the febrile responses seen in patients while on G3139 infusion. The question of whether this immune response can be used to improve vaccine efficiency using CpG motifs would be supported by this observation and outcome.45,46 Given the results of the current study, we conclude that the combination of G3139 with thalidomide and dexamethasone is feasible and effective in refractory and relapsed multiple myeloma. The responses are promising and should be further investigated in the refractory and in the newly diagnosed setting. The biologic data derived from this study suggest that G3139 is active against its target Bcl-2 mRNA, at least in some patients. The observed activation of the innate immune system is worth further investigation and should be explored in combination of G3139 with drugs that do not inhibit the immune system.
The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Consultant/Advisory Role: Ashraf Z. Badros, Celgene. Honoraria: Jodi A. Flaws, Wyeth. For a detailed description of these categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and Disclosures of Potential Conflicts of Interest found in Information for Contributors in the front of each issue.
We thank the patients who participated in the clinical trial. We also thank Edward Sausville, MD, PhD, for the thoughtful critical review of the manuscript and Judy Karp, MD, for the advice and support in starting the trial.
Supported in part by the National Cancer Institute grant No. UO1 CA69854. Authors' disclosures of potential conflicts of interest are found at the end of this article.
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ABSTRACT
INTRODUCTION
.05. 
