Phase II Study of G3139, a Bcl-2 Antisense Oligonucleotide, in Combination With Dexamethasone and Thalidomide in Relapsed Multiple Myeloma P
http://www.100md.com
《临床肿瘤学》
the Greenebaum Cancer Center and Departments of Pathology and Epidemiology and Preventive Medicine, University of Maryland, Baltimore
Cancer Therapy Evaluation Program, National Cancer Institute, Bethesda, MD
ABSTRACT
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.
INTRODUCTION
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.
PATIENTS AND METHODS
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
G3139 and thalidomide were provided free of charge to all patients by the National Cancer Institute, Cancer Therapy Evaluation Program. Table 1 lists the study schedule. G3139 was delivered through a central line as a continuous intravenous infusion using a CADD ambulatory infusion pump (Sims Deltec, St Paul, MN) on days 1 to 7 at a dose of 5 mg/kg/d for the first three patients and at a dose of 7 mg/kg/d for subsequent patients. On day 4 of G3139, patients received dexamethasone at 40 mg orally for 4 days, and thalidomide was started at a dose of 200 mg and gradually increased to 400 mg daily, as tolerated, for the duration of the study. Cycles were repeated every 21 days for three cycles (induction phase). Responding and stable patients proceeded to a maintenance phase consisting of G3139 (days 1 to 7), dexamethasone 20 mg daily for 4 days, and thalidomide at the tolerated dose. Cycles were repeated every 35 days. Patients with continued clinical improvement were eligible to receive up to 2 years of therapy. All therapy was administered on an outpatient basis.
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
G3139 doses were adjusted at each cycle for creatinine as follows: 7 mg/kg/d for patients with creatinine less than 1.5 mg/dL, 5 mg/kg/d for creatinine of 1.5 to 2 mg/dL, and 3 mg/kg/d for creatinine of 2 to 3 mg/dL; the dose was held if the creatinine was more than 3 mg/dL. G3139 was restarted once the creatinine decreased below 2 mg/dL. Later in the study, entry was limited to creatinine less than 2 mg/dL. For G3139-related grade 3 to 4 adverse events, including thrombocytopenia with bleeding, prothrombin time partial thromboplastin time prolongation, and abnormalities of serum AST, ALT, or bilirubin, the infusion was delayed until toxicity resolved and was then restarted at 75% of the previous dose. If toxicity did not resolve to less than grade 2 within 2 weeks, the patient was removed from the protocol. Thalidomide dose was reduced to 75% for any grade 2 toxicity persisting more than 2 weeks, and for grade 3 to 4 toxicity, thalidomide was held until toxicity resolved to less than grade 2 and was then resumed at 50% of the previous dose. Patients unable to tolerate thalidomide were permitted to continue dexamethasone and G3139. Dexamethasone was held for grade 3 to 4 related toxicities including neuropsychiatric complications, GI bleeding, pancreatitis, and unmanageable hypertension or hyperglycemia. Once toxicity resolved, dexamethasone was restarted at 50% of the previous dose. If toxicity did not resolve to less than grade 2 within 2 weeks from the onset, the patient was removed from the study.
Criteria for Response and Toxicity
The response criteria as defined by Blade et al18 were used. Complete response (CR) required absence of monoclonal paraprotein in serum and urine by immunofixation, less than 5% plasma cells in the bone marrow, no increase in size or number of lytic bone lesions, and disappearance of soft tissue plasmacytoma. Near CR met all CR criteria except for a positive immunofixation in the serum and/or urine. Partial response (PR) required a more than 50% reduction in the level of the serum monoclonal paraprotein and reduction in 24-hour urine light-chain excretion either by more than 90% or to less than 200 mg. Minimal response (MR) required a 25% to 49% reduction in the level of the serum monoclonal paraprotein and a 50% to 89% reduction in 24-hour urine light-chain excretion that exceeds 200 mg/24 hours. Stable disease (SD) did not meet the criteria for either MR or progressive disease (PD). Relapse required reappearance of serum or urinary paraprotein on immunofixation for CR patients and more than 5% plasma cell in the bone marrow. PD was defined as any increase of more than 25% in the level of the serum monoclonal paraprotein (with a minimum absolute increase of 500 mg/dL in the serum or 200 mg/24 hours for light-chain in urine) and/or a more than 25% increase in the plasmacytosis the bone marrow (an absolute increase of 10%). Adverse events were graded according to the National Cancer Institute Common Toxicity Criteria (version 3.0; http://ctep.info.nih.gov). Any grade 3 or 4 nonhematologic systemic toxicity was reported to the institutional review board and National Cancer Institute.
Quantification of Bcl-2 Protein
Bone marrow aspirates (5 to 10 mL) were obtained from patients before starting G3139 infusion and on days 4 and 29. Day 4 samples were obtained on day 7 and, in some patients, on day 10. Multiple myeloma cells were isolated using anti-CD138–coated microbeads (Miltenyi Biotec, Auburn, CA) as described previously.16 CD138+ cells were lysed in a modified RIPA buffer (50 mm Tris-HCl, pH 7.4; 1% NP-40, 0.25% Na-deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L NaF, 1 mmol/L Na3VO4, 1 mmol/L phenylmethylsulfonylfluoride, 5 μg/mL aprotinin, and 5 μg/mL leupeptin), and protein extracts were analyzed by immunoblotting as described previously.16 The signal was detected by enhanced chemoluminescence (Amersham, Arlington Heights, IL). Equal amounts of protein were analyzed for each patient (10 to 20 μg per lane), and loading was controlled by analysis of tubulin levels. Antibodies used for Bcl-2 (anti–Bcl-2, clone 124; Upstate Biotechnology, Lake Placid, NY), Mcl-1 (S-19; Santa Cruz Biotechnology, Inc, Santa Cruz, CA), Bax (anti-Bax, NT; Upstate Biotechnology), Bcl-xl (Bcl-XL; BD Transduction Laboratories, Franklin Lakes, NJ), and tubulin (tubulin Ab-4; NeoMarkers, Fremont, CA). Relative levels of protein expression were determined by densitometric analysis.
Real-Time Polymerase Chain Reaction Analysis
RNA (1 μg) from each sample was reverse transcribed using an Omniscript RT kit (Qiagen, Inc, Valencia, CA) with random primers (Invitrogen Inc, Carlasbad, CA). After reverse transcription, polymerase chain reaction (PCR) was conducted using a CFD-3240 Chromo4 Detector with Opticon Monitor software version 2.03 (MJ Research Inc, Waltham, MA). The PCR reaction was performed in the 30-μL volume containing 0.3 μM of sequence-specific primers for Bcl-2 (5'-CGC CCT GTG GAT GAC TGA GT-3' and 5'-GGG CCG TAC AGT TCC ACA A-3'; 93 bp), as previously described.17 Primers specific for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) were used as an internal control (5'-GGA GCC AAA AGC GTA ATC ATC TC-3' and 5'-AGT GGG TGT CGC TGT TGA AGT C-3'; 531 bp; Sigma-Genosys Ltd, Woodlands, TX). Reaction buffer containing modified Tbr DNA polymerase, SYBR green, 2.5 mmol/L MgCl2, and deoxynucleotide triphosphates was included in the SYBR Green qPCR Kit (MJ Research Inc, Reno, NV). Amplification of cDNAs involved a 10-minute denaturation step followed by 40 cycles with a 95°C denaturation for 10 seconds, 60°C annealing for 10 seconds (20 seconds for GAPDH), and 72°C extension for 10 seconds (20 seconds for GAPDH). A data acquisition step was performed at an elevated temperature (76 to 78°C) to minimize the interference of primer dimers with quantification. The specificity of the amplification reactions was confirmed by melting curve analysis and, subsequently, by agarose gel electrophoresis. The negative control sample was prepared by replacing the DNA template with PCR grade water. A dilution series (1:1, 1:3, 1:10, 1:30, and 1:100) of the reference cDNA sample was used to generate a standard curve against which the experimental samples were quantified. GAPDH mRNA was measured in each sample as an internal control. Relative Bcl-2 mRNA levels were normalized to GAPDH mRNA levels and presented as an X-fold (percent difference) in the expression level after treatment, relative to that before treatment. For each sample, PCR amplification was performed in quadruplicates (each sample was run in duplicates in two independent reverse transcriptase PCR [RT-PCR] reactions).
Statistical Considerations
The analysis was performed using intent-to-treat strategy. Survival time for 33 patients on the study was estimated as the time from the enrollment date to the date of death or last known alive. Progression-free survival (PFS) was defined as the time from enrollment to the date of PD or death as a result of any cause. Patients who had stable disease as their best response were censored at the time last known to be stable. The duration of objective response (CR, near CR, PR, and MR) was defined as the time from achieving a response to the date of PD or last known remission without evidence of relapse. Time to event data was analyzed using the Kaplan-Meier and the log-rank test. To compare categoric data, patients were grouped as responders versus nonresponders. The Fisher's exact test for 2 x 2 tables and Fisher-Freeman-Halton test for 2 x C contingency tables were applied to asses whether the objective response was associated with plausible risk factors, such as sex, race, chromosomal abnormalities, isotype, platelets, hemoglobin, beta2-microglobulin, and creatinine.
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 .05.
RESULTS
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
Three patients were removed from the study after 1 week because of toxicity. One patient discontinued treatment because of development of renal failure not requiring dialysis; she had rapidly progressive renal failure before enrollment onto the study as a result of her disease. One patient had a transient ischemic attack 3 days after starting thalidomide and was found to have a near complete occlusion of internal carotids. This was surgically corrected, but the patient decided not to continue on the study. The third patient developed acute weakness that had also occurred on prior thalidomide; this resolved off therapy.
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
Thirty patients were assessable for response; 24 (73%) of 33 patients had documented responses, including two CRs, four near CRs, and 12 PRs; six patients had MR. Eighteen patients had achieved PR or better. Six patients had PD. The median duration of response was 13 months. The estimated median PFS was 12 months, and the median OS was 17.4 months (Fig 1). The upper limits of the 95% CIs for PFS and OS have not been reached. As of July 2004, seven patients (21%) had died, and 26 (79%) are alive, with 16 patients continuing on the study. Details of responses on intent-to-treat analysis are listed in Table 4.
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.
DISCUSSION
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.
Authors' Disclosures of Potential Conflicts of Interest
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.
Acknowledgment
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.
NOTES
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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Klasa RJ, Gillum AM, Klem RE, et al: Oblimersen Bcl-2 antisense: Facilitating apoptosis in anticancer treatment. Antisense Nucleic Acid Drug Dev 12:193-213, 2002
Van de Donk, Kamphuis M, Van Dijk M, et al: Evaluation of bcl-2 antisense oligonucleotide drugs in multiple myeloma. Blood 96:757a, 2000 (abstr)
Liu Q, Gazitt Y: Potentiation of dexamethasone-, paclitaxel-, and Ad-p53-induced apoptosis by Bcl-2 antisense oligodeoxynucleotides in drug-resistant multiple myeloma cells. Blood 101:4105-4114, 2003
Zhang B, Gojo I, Fenton RG: Myeloid cell factor-1 is a critical survival factor for multiple myeloma. Blood 99:1885-1893, 2002
van de Donk NW, Kamphuis MM, van Dijk M, et al: Chemosensitization of myeloma plasma cells by an antisense-mediated downregulation of Bcl-2 protein. Leukemia 17:211-219, 2003
Blade J, Samson D, Reece D, et al: Criteria for evaluating disease response and progression in patients with multiple myeloma treated by high-dose therapy and haemopoietic stem cell transplantation: Myeloma Subcommittee of the EBMT—European Group for Blood and Marrow Transplant. Br J Haematol 102:1115-1123, 1998
Lee CK, Barlogie B, Munshi N, et al: DTPACE: An effective, novel combination chemotherapy with thalidomide for previously treated patients with myeloma. J Clin Oncol 21:2732-2739, 2003
Morris MJ, Tong WP, Cordon-Cardo C, et al: Phase I trial of BCL-2 antisense oligonucleotide (G3139) administered by continuous intravenous infusion in patients with advanced cancer. Clin Cancer Res 8:679-683, 2002
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Marshall J, Chen H, Yang D, et al: A phase I trial of a Bcl-2 antisense (G3139) and weekly docetaxel in patients with advanced breast cancer and other solid tumors. Ann Oncol 15:1274-1283, 2004
Rudin CM, Kozloff M, Hoffman PC, et al: Phase I study of G3139, a bcl-2 antisense oligonucleotide, combined with carboplatin and etoposide in patients with small-cell lung cancer. J Clin Oncol 22:1110-1117, 2004
Tolcher AW, Kuhn J, Schwartz G, et al: A phase I pharmacokinetic and biological correlative study of oblimersen sodium (Genasense, G3139), an antisense oligonucleotide to the bcl-2 mRNA, and of docetaxel in patients with hormone-refractory prostate cancer. Clin Cancer Res 10:5048-5057, 2004
Raynaud FI, Orr RM, Goddard PM, et al: Pharmacokinetics of G3139, a phosphorothioate oligodeoxynucleotide antisense to bcl-2, after intravenous administration or continuous subcutaneous infusion to mice. J Pharmacol Exp Ther 281:420-427, 1997
Marcucci G, Byrd JC, Dai G, et al: Phase 1 and pharmacodynamic studies of G3139, a Bcl-2 antisense oligonucleotide, in combination with chemotherapy in refractory or relapsed acute leukemia. Blood 101:425-432, 2003
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Cancer Therapy Evaluation Program, National Cancer Institute, Bethesda, MD
ABSTRACT
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.
INTRODUCTION
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.
PATIENTS AND METHODS
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
G3139 and thalidomide were provided free of charge to all patients by the National Cancer Institute, Cancer Therapy Evaluation Program. Table 1 lists the study schedule. G3139 was delivered through a central line as a continuous intravenous infusion using a CADD ambulatory infusion pump (Sims Deltec, St Paul, MN) on days 1 to 7 at a dose of 5 mg/kg/d for the first three patients and at a dose of 7 mg/kg/d for subsequent patients. On day 4 of G3139, patients received dexamethasone at 40 mg orally for 4 days, and thalidomide was started at a dose of 200 mg and gradually increased to 400 mg daily, as tolerated, for the duration of the study. Cycles were repeated every 21 days for three cycles (induction phase). Responding and stable patients proceeded to a maintenance phase consisting of G3139 (days 1 to 7), dexamethasone 20 mg daily for 4 days, and thalidomide at the tolerated dose. Cycles were repeated every 35 days. Patients with continued clinical improvement were eligible to receive up to 2 years of therapy. All therapy was administered on an outpatient basis.
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
G3139 doses were adjusted at each cycle for creatinine as follows: 7 mg/kg/d for patients with creatinine less than 1.5 mg/dL, 5 mg/kg/d for creatinine of 1.5 to 2 mg/dL, and 3 mg/kg/d for creatinine of 2 to 3 mg/dL; the dose was held if the creatinine was more than 3 mg/dL. G3139 was restarted once the creatinine decreased below 2 mg/dL. Later in the study, entry was limited to creatinine less than 2 mg/dL. For G3139-related grade 3 to 4 adverse events, including thrombocytopenia with bleeding, prothrombin time partial thromboplastin time prolongation, and abnormalities of serum AST, ALT, or bilirubin, the infusion was delayed until toxicity resolved and was then restarted at 75% of the previous dose. If toxicity did not resolve to less than grade 2 within 2 weeks, the patient was removed from the protocol. Thalidomide dose was reduced to 75% for any grade 2 toxicity persisting more than 2 weeks, and for grade 3 to 4 toxicity, thalidomide was held until toxicity resolved to less than grade 2 and was then resumed at 50% of the previous dose. Patients unable to tolerate thalidomide were permitted to continue dexamethasone and G3139. Dexamethasone was held for grade 3 to 4 related toxicities including neuropsychiatric complications, GI bleeding, pancreatitis, and unmanageable hypertension or hyperglycemia. Once toxicity resolved, dexamethasone was restarted at 50% of the previous dose. If toxicity did not resolve to less than grade 2 within 2 weeks from the onset, the patient was removed from the study.
Criteria for Response and Toxicity
The response criteria as defined by Blade et al18 were used. Complete response (CR) required absence of monoclonal paraprotein in serum and urine by immunofixation, less than 5% plasma cells in the bone marrow, no increase in size or number of lytic bone lesions, and disappearance of soft tissue plasmacytoma. Near CR met all CR criteria except for a positive immunofixation in the serum and/or urine. Partial response (PR) required a more than 50% reduction in the level of the serum monoclonal paraprotein and reduction in 24-hour urine light-chain excretion either by more than 90% or to less than 200 mg. Minimal response (MR) required a 25% to 49% reduction in the level of the serum monoclonal paraprotein and a 50% to 89% reduction in 24-hour urine light-chain excretion that exceeds 200 mg/24 hours. Stable disease (SD) did not meet the criteria for either MR or progressive disease (PD). Relapse required reappearance of serum or urinary paraprotein on immunofixation for CR patients and more than 5% plasma cell in the bone marrow. PD was defined as any increase of more than 25% in the level of the serum monoclonal paraprotein (with a minimum absolute increase of 500 mg/dL in the serum or 200 mg/24 hours for light-chain in urine) and/or a more than 25% increase in the plasmacytosis the bone marrow (an absolute increase of 10%). Adverse events were graded according to the National Cancer Institute Common Toxicity Criteria (version 3.0; http://ctep.info.nih.gov). Any grade 3 or 4 nonhematologic systemic toxicity was reported to the institutional review board and National Cancer Institute.
Quantification of Bcl-2 Protein
Bone marrow aspirates (5 to 10 mL) were obtained from patients before starting G3139 infusion and on days 4 and 29. Day 4 samples were obtained on day 7 and, in some patients, on day 10. Multiple myeloma cells were isolated using anti-CD138–coated microbeads (Miltenyi Biotec, Auburn, CA) as described previously.16 CD138+ cells were lysed in a modified RIPA buffer (50 mm Tris-HCl, pH 7.4; 1% NP-40, 0.25% Na-deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L NaF, 1 mmol/L Na3VO4, 1 mmol/L phenylmethylsulfonylfluoride, 5 μg/mL aprotinin, and 5 μg/mL leupeptin), and protein extracts were analyzed by immunoblotting as described previously.16 The signal was detected by enhanced chemoluminescence (Amersham, Arlington Heights, IL). Equal amounts of protein were analyzed for each patient (10 to 20 μg per lane), and loading was controlled by analysis of tubulin levels. Antibodies used for Bcl-2 (anti–Bcl-2, clone 124; Upstate Biotechnology, Lake Placid, NY), Mcl-1 (S-19; Santa Cruz Biotechnology, Inc, Santa Cruz, CA), Bax (anti-Bax, NT; Upstate Biotechnology), Bcl-xl (Bcl-XL; BD Transduction Laboratories, Franklin Lakes, NJ), and tubulin (tubulin Ab-4; NeoMarkers, Fremont, CA). Relative levels of protein expression were determined by densitometric analysis.
Real-Time Polymerase Chain Reaction Analysis
RNA (1 μg) from each sample was reverse transcribed using an Omniscript RT kit (Qiagen, Inc, Valencia, CA) with random primers (Invitrogen Inc, Carlasbad, CA). After reverse transcription, polymerase chain reaction (PCR) was conducted using a CFD-3240 Chromo4 Detector with Opticon Monitor software version 2.03 (MJ Research Inc, Waltham, MA). The PCR reaction was performed in the 30-μL volume containing 0.3 μM of sequence-specific primers for Bcl-2 (5'-CGC CCT GTG GAT GAC TGA GT-3' and 5'-GGG CCG TAC AGT TCC ACA A-3'; 93 bp), as previously described.17 Primers specific for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) were used as an internal control (5'-GGA GCC AAA AGC GTA ATC ATC TC-3' and 5'-AGT GGG TGT CGC TGT TGA AGT C-3'; 531 bp; Sigma-Genosys Ltd, Woodlands, TX). Reaction buffer containing modified Tbr DNA polymerase, SYBR green, 2.5 mmol/L MgCl2, and deoxynucleotide triphosphates was included in the SYBR Green qPCR Kit (MJ Research Inc, Reno, NV). Amplification of cDNAs involved a 10-minute denaturation step followed by 40 cycles with a 95°C denaturation for 10 seconds, 60°C annealing for 10 seconds (20 seconds for GAPDH), and 72°C extension for 10 seconds (20 seconds for GAPDH). A data acquisition step was performed at an elevated temperature (76 to 78°C) to minimize the interference of primer dimers with quantification. The specificity of the amplification reactions was confirmed by melting curve analysis and, subsequently, by agarose gel electrophoresis. The negative control sample was prepared by replacing the DNA template with PCR grade water. A dilution series (1:1, 1:3, 1:10, 1:30, and 1:100) of the reference cDNA sample was used to generate a standard curve against which the experimental samples were quantified. GAPDH mRNA was measured in each sample as an internal control. Relative Bcl-2 mRNA levels were normalized to GAPDH mRNA levels and presented as an X-fold (percent difference) in the expression level after treatment, relative to that before treatment. For each sample, PCR amplification was performed in quadruplicates (each sample was run in duplicates in two independent reverse transcriptase PCR [RT-PCR] reactions).
Statistical Considerations
The analysis was performed using intent-to-treat strategy. Survival time for 33 patients on the study was estimated as the time from the enrollment date to the date of death or last known alive. Progression-free survival (PFS) was defined as the time from enrollment to the date of PD or death as a result of any cause. Patients who had stable disease as their best response were censored at the time last known to be stable. The duration of objective response (CR, near CR, PR, and MR) was defined as the time from achieving a response to the date of PD or last known remission without evidence of relapse. Time to event data was analyzed using the Kaplan-Meier and the log-rank test. To compare categoric data, patients were grouped as responders versus nonresponders. The Fisher's exact test for 2 x 2 tables and Fisher-Freeman-Halton test for 2 x C contingency tables were applied to asses whether the objective response was associated with plausible risk factors, such as sex, race, chromosomal abnormalities, isotype, platelets, hemoglobin, beta2-microglobulin, and creatinine.
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 .05.
RESULTS
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
Three patients were removed from the study after 1 week because of toxicity. One patient discontinued treatment because of development of renal failure not requiring dialysis; she had rapidly progressive renal failure before enrollment onto the study as a result of her disease. One patient had a transient ischemic attack 3 days after starting thalidomide and was found to have a near complete occlusion of internal carotids. This was surgically corrected, but the patient decided not to continue on the study. The third patient developed acute weakness that had also occurred on prior thalidomide; this resolved off therapy.
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
Thirty patients were assessable for response; 24 (73%) of 33 patients had documented responses, including two CRs, four near CRs, and 12 PRs; six patients had MR. Eighteen patients had achieved PR or better. Six patients had PD. The median duration of response was 13 months. The estimated median PFS was 12 months, and the median OS was 17.4 months (Fig 1). The upper limits of the 95% CIs for PFS and OS have not been reached. As of July 2004, seven patients (21%) had died, and 26 (79%) are alive, with 16 patients continuing on the study. Details of responses on intent-to-treat analysis are listed in Table 4.
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.
DISCUSSION
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.
Authors' Disclosures of Potential Conflicts of Interest
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.
Acknowledgment
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.
NOTES
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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