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Originally published as JCO Early Release 10.1200/JCO.2006.07.1191 on February 12 2007 © 2007 American Society of Clinical Oncology. Randomized Phase III Trial of Fludarabine Plus Cyclophosphamide With or Without Oblimersen Sodium (Bcl-2 antisense) in Patients With Relapsed or Refractory Chronic Lymphocytic Leukemia
From The University of Texas M. D. Anderson Cancer Center, Houston, TX; Duke University Medical Center, Durham, NC; Yakima Regional Cancer Care Center, Yakima, WA; Cross Cancer Institute, Edmonton, AB, Canada; Klinika Hematologii Collegium Medicum Uniwersytetu Jagiellonskiego, Cracow, Poland; Instituto Argentino de Diagnostico y Tratamiento SA, Buenos Aires, Argentina; Roswell Park Cancer Institute, Buffalo; Long Island Jewish Medical Center, New Hyde Park, NY; Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia; and University of Nebraska Medical Center, Omaha, NE Address reprint requests to Susan O'Brien, MD, Department of Leukemia, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030; e-mail: sobrien{at}mdanderson.org
Purpose: Expression of Bcl-2 protein is associated with chemotherapy resistance and decreased survival in chronic lymphocytic leukemia (CLL). We evaluated whether oblimersen would improve response to chemotherapy in patients with relapsed or refractory CLL. Patients and Methods: Patients had received at least one prior fludarabine-containing regimen and were stratified on the basis of prior fludarabine response, number of prior regimens, and duration of response to last prior therapy. Patients were randomly assigned to 28-day cycles of fludarabine 25 mg/m2/d plus cyclophosphamide 250 mg/m2/d administered intravenously for 3 days with or without oblimersen 3 mg/kg/d as a 7-day continuous intravenous infusion (beginning 4 days before chemotherapy) for up to six cycles. The primary end point was the proportion of patients who achieved complete response (CR) or nodular partial response (nPR). Results: Of 241 patients randomly assigned, CR/nPR was achieved in 20 (17%) of 120 patients in the oblimersen group and eight (7%) of 121 patients in the chemotherapy-only group (P = .025). Achievement of CR/nPR was correlated with both an extended time to progression and survival (P < .0001). In patients who remained sensitive to fludarabine, oblimersen was associated with a four-fold increase in the CR/nPR rate and a significant survival benefit (P = .05). Oblimersen was frequently associated with thrombocytopenia and, rarely, tumor lysis syndrome and cytokine release reactions; the incidence of opportunistic infections and second malignancies was similar in both groups. Conclusion: The addition of oblimersen to fludarabine plus cyclophosphamide significantly increases the CR/nPR rate in patients with relapsed or refractory CLL (particularly fludarabine-sensitive patients), as well as response duration among patients who achieve CR/nPR.
Chronic lymphocytic leukemia (CLL) therapy has improved in the last 15 years. Purine nucleosides such as fludarabine are the treatment of choice for previously untreated patients; monoclonal antibodies such as rituximab are commonly used in combination therapy. Response rates have raised expectations that many patients can achieve complete remission.1-3 Various factors associated with poor prognosis in newly diagnosed CLL include cytogenetic abnormalities, mutations in p53,4 lack of immunoglobulin VH mutations,4 and expression of CD385-7 and ZAP-70.7-9 Expression of the antiapoptotic protein Bcl-2 is associated with the pathogenesis of CLL.4,10-13 As a negative regulator of the intrinsic apoptotic pathway, overexpression of Bcl-2 confers chemoresistance in a number of hematologic cancers14,15 and solid tumors.15 Although protein levels vary among cells and patients, Bcl-2 is expressed in virtually all patients with CLL,11,13 and Bcl-2 upregulation plays a critical role in CLL. Deletion of miRNA regulators of Bcl-2 expression is frequently found in cells of patients with CLL and in association with Bcl-2 upregulation. Notably, reintroduction of micro-RNAs (miRNA) in a lymphoid cell line downregulated Bcl-2 and induced apoptosis.16,17 New CLL models confirm dependence of tumorigenicity on Bcl-2 expression and the positive correlation of Bcl-2 imbalance with tumor aggressiveness.18 Antisense oligonucleotides that bind mRNA can target specific proteins, induce enzymatic cleavage of the message, and thereby prevent protein translation.19 Bcl-2 downregulation with antisense phosphorothioate oligonucleotides has been confirmed in primary CLL cells, and enhanced chlorambucil-induced apoptosis documented.20,21 Other effects (eg, immunostimulation) have been associated with CpG- and non–CpG-containing oligonucleotides in B-cell lines and primary CLL cells. Results are highly inconsistent20-23; no firm conclusion can be reached about nonantisense effects. The antisense oligonucleotide oblimersen downregulates bcl-2 mRNA and Bcl-2 protein in a concentration- and time-dependent manner.15 Preclinically, oblimersen demonstrated single-agent activity against lymphoma cells 24-26 and CLL cells,27,28 and potentiated cytotoxic activity of commonly used CLL agents, including fludarabine,27 alemtuzumab,29 and rituximab.27 Bcl-2 downregulation correlated with oblimersen uptake in leukemia cell lines and acute myeloid leukemia cells from oblimersen-treated patients30 and resulted in 10-fold enhancement of sensitivity to daunorubicin.31 Clinically, oblimersen displayed modest single-agent activity in a phase I/II trial in relapsed/refractory CLL.32,33 We evaluated whether adding oblimersen would increase the rate of complete responses (CR) plus nodular partial responses (nPRs) with fludarabine/cyclophosphamide in patients who had relapsed after or become refractory to fludarabine-based therapy.
Patients Eligibility requirements included age at least 18 years; measurable and active disease (National Cancer Institute–sponsored Working Group [NCI-WG] Guidelines)34; intermediate- or high-risk stage (modified Rai criteria)34; requiring therapy; relapse from or no response to at least one prior chemotherapy regimen, at least one of which must have included two or more cycles of fludarabine; Eastern Cooperative Oncology Group (ECOG) performance status of 2 or lower; adequate organ function; platelet count at least 50,000/mm3 without hematopoietic growth factor or transfusion support; and negative direct and indirect Coombs’; tests.
Exclusion criteria were major surgery/therapy for CLL within 3 weeks before enrollment; prior organ allograft or stem-cell transplantation; serious comorbidity; concurrent investigational or corticosteroid therapy; pregnancy/lactation; hypersensitivity to phosphorothioate-containing oligonucleotides, fludarabine, or cyclophosphamide; and secondary leukemia or history of other cancer (except for adequately treated basal or squamous cell skin cancer, in situ cervical cancer, or other cancer from which the patient was disease-free for The study was conducted in accordance with applicable requirements regarding ethical review and informed consent, including those in the Declaration of Helsinki.
Random Assignment and Treatment In the chemotherapy-only group, patients received fludarabine 25 mg/m2/d intravenously over 20 to 30 minutes, followed by cyclophosphamide 250 mg/m2/d intravenously over 30 to 60 minutes on cycle days 1, 2, and 3. In the oblimersen group, oblimersen 3 mg/kg/d was administered on days 1 through 7 by continuous intravenous infusion via central or peripheral catheter and ambulatory pump, and fludarabine and cyclophosphamide were administered at the doses described on days 5, 6, and 7. Treatment was repeated every 28 days for up to six cycles. If toxicity occurred, oblimersen could be delayed; fludarabine and cyclophosphamide doses could be reduced or delayed. Before patients resumed treatment, recovery of hemoglobin and platelet count to at least 10.0 g/dL and at least 75,000/mm3, respectively, or to within 10% of baseline values was required, and absolute neutrophil count (ANC) was to be at least 1,000/mm3 in patients with ANC more than 2,000/mm3 and WBC count less than 20,000/mm3. During cycle 1 (weeks 1 to 2), patients received allopurinol. They started sulfamethoxazole-trimethoprim and acyclovir (or similar agents) when study treatment began, continuing for up to 6 months after last dose of study medication. Filgrastim was initiated within 72 hours after completion of study medication in each cycle and was continued for at least 1 week or until postnadir ANC was more than 1,000/mm3 for 2 days or ANC was more than 10,000/mm3; administration could be extended, but was to be stopped 48 hours before the next cycle. Erythropoietin was used at the investigator's discretion. It was recommended that patients receive (5-HT)3 antiemetics (or equivalent) before fludarabine/cyclophosphamide.
Evaluations on Study
Response Assessment and Criteria
End Points and Statistical Analysis On the basis of the literature, we assumed that a combined CR/nPR rate of 24% was achievable with fludarabine and cyclophosphamide alone,35 and that oblimersen would increase the CR/nPR rate from 24% to 44%. Thus, a total of approximately 200 assessable patients would provide 80% power to detect a significant difference at an overall alpha of .05 (two-sided). A single interim analysis was planned using a Lan-DeMets alpha-spending approach and an O'Brien-Fleming stopping rule after 50% of patients had been observed for 6 months from randomization. The interim alpha for the primary analysis was .001. That analysis determined that sample size would not be increased; the final primary analysis used an adjusted nominal alpha of .049.
Efficacy was analyzed in the intent-to-treat population. Response rates were compared using a continuity-corrected Pearson
Response duration was analyzed using the log-rank test. TTP was analyzed using the log-rank test and Cox proportional hazards model (using variables included in logistic regression analyses of the primary end point). Clinical benefit parameters were analyzed based on best response using descriptive statistics, a
Patient Characteristics and Treatment A total of 241 patients were randomly assigned at 100 sites in eight countries between August 2001 and June 2003 (oblimersen group, n = 120; chemotherapy-only group, n = 121). Groups were well balanced at baseline (Table 1). Median time from CLL diagnosis was 70 months and 58 months in the oblimersen and chemotherapy-only groups, respectively.
In both groups, 115 patients initiated treatment, and the median number of cycles completed was four. The most common reason for early discontinuation was adverse event/toxicity in 36% and 35% of patients in the oblimersen and chemotherapy-only groups, respectively. Fludarabine and cyclophosphamide were administered at or near protocol-specified doses in both groups. Reductions in fludarabine and/or cyclophosphamide doses were recorded for 34% and 25% of patients in the oblimersen and chemotherapy-only groups, respectively. Delays with fludarabine and/or cyclophosphamide occurred in 42% and 35% of patients in the oblimersen group and chemotherapy-only groups, respectively. In both groups, adverse event/toxicity was the primary reason for dose modifications, and thrombocytopenia was most often associated with dose reduction. Associated most often with administration delays or interruptions were thrombocytopenia, fever, and rigors in the oblimersen group, and fever in the chemotherapy-only group.
Efficacy
Among patients with CR/nPR, baseline cytogenetic data were available for nine of the 20 patients in the oblimersen group (including six with normal cytogenetic factors, one with trisomy 12, and two with 17p or 11q) and two of the eight patients in the chemotherapy-only group (both with 17p or 11q). Multivariate analysis of response using a logistic regression model that adjusted for all prespecified baseline factors (including the three stratification factors, age, sex, Rai stage, hemoglobin, platelet count, lactate dehydrogenase, and beta-2 microglobulin) confirmed a significant difference in the CR/nPR rate favoring the oblimersen group (P = .016). The oblimersen regimen was superior across all prospectively defined strata (Table 2). Maximum benefit was observed in fludarabine-sensitive (relapsed) patients (ie, those who had a partial response or better for at least 6 months after fludarabine therapy and then relapsed). Among these patients (oblimersen group, n = 51; chemotherapy-only group, n = 50), the CR/nPR rate was 25% in the oblimersen group and 6% in the chemotherapy-only group (P = .016). Overall response rate, which included PR, was not significantly different between groups.
Response duration.
Among patients with CR/nPR in both groups, all responses were durable (ie, lasting
TTP. There was no significant difference in TTP with 24 months of minimum follow-up (Fig 2A). However, TTP was correlated with response (P < .0001; Fig 2B). In patients with CR/nPR, TTP was 2 or more years in 70% of patients in the oblimersen group compared with 50% in the chemotherapy-alone group.
Clinical benefit. Achievement of a CR/nPR was associated with the disappearance of all predefined CLL symptoms (fever, night sweats, fatigue, abdominal discomfort or early satiety due to hepatosplenomegaly, impaired cosmesis or impaired mobility due to lymphadenopathy, and "other") for at least 180 days (Pearson  2 test P < .0001). Among patients in both groups who were symptomatic at baseline, 17 patients (94%) who had a CR/nPR compared with 36 (59%) who had a PR were symptom free for at least 180 days before disease progression or initiation of new therapy. Patients with a CR/nPR had a minimum of approximately 11 more symptom-free months than did those with a PR. There were no significant between-group differences in clinical benefit parameters assessed as secondary end points. Survival. With protocol-specified follow-up for 36 months, estimated median survival was 33.8 months in the oblimersen group and 32.9 months in the chemotherapy-only group (Fig 3A), and the estimated 3-year survival rate was 46% and 37.5%, respectively. As observed with TTP, survival was correlated with response (P < .0001; Fig 3B). In patients with CR/nPR, survival was 3 or more years in 70% of patients in the oblimersen group compared with 38% in the chemotherapy-alone group.
Among fludarabine-sensitive patients, the addition of oblimersen to standard chemotherapy resulted in a statistically significant benefit in survival (P = .05; Fig 3C).
Safety
The most common grade 4 event in both groups was neutropenia (oblimersen group, 7%; chemotherapy-only group, 11%). Among remaining grade 4 events occurring in at least 2% of patients in either group, thrombocytopenia, febrile neutropenia, and hypotension were more common in the oblimersen group. Anemia and leukopenia were more common in the chemotherapy-only group, in which more patients received filgrastim and erythropoietin. More patients in the oblimersen group than in the chemotherapy-only group received platelet transfusions. The oblimersen group had a greater incidence of bleeding events (all grades) than did the chemotherapy-only group (19% v 8%, respectively); however, the incidence of grade 3 to 4 bleeding events was low in both groups (4% and 2%, respectively). The incidence of opportunistic infections (oblimersen group, 4%; chemotherapy-only group, 7%) and second malignancies (1% v 4%, respectively) was similar in the groups. Within the oblimersen group during cycle 1, two patients (2%) developed tumor lysis syndrome and one (1%) developed a cytokine release–type reaction; these events were characterized by fever, dehydration, weakness, back pain, and/or hypotension. A small number of patients had a treatment-related adverse event with an outcome of death (oblimersen group, five patients [4%]: septic shock,2 tumor lysis syndrome,1 cytokine release syndrome,1 and increased blood creatinine1; chemotherapy-only group, two patients [2%]: neutropenic sepsis1 and pseudomonal pneumonia1). Several of these patients had underlying disease and comorbidities. Causes of death were generally similar except for tumor lysis/cytokine release syndrome in two patients with underlying cardiac disease.
This study demonstrates that oblimersen can improve the outcomes of standard CLL chemotherapy. As previously shown,2 we confirmed that patients with CLL who achieve CR/nPR enjoy superior survival compared with patients whose best response is partial remission or stable disease. These responses were durable and significantly longer than were those achieved with chemotherapy alone. We also confirmed these patients experienced an extended TTP and prolonged symptom-free time. Results of this trial suggest that oblimersen therapy is most beneficial in patients who remain fludarabine sensitive. The CR/nPR rate with fludarabine/cyclophosphamide was lower than anticipated in this study. CR rates alone in patients with relapsed or refractory CLL have ranged from 3% to 12% after fludarabine/cyclophosphamide.35 Thus, CR and nPR rates of 17% and 7% with fludarabine/cyclophosphamide with and without oblimersen, respectively, in heavily pretreated relapsed or refractory patients seem consistent, especially in a multicenter setting. Differences exist between this trial and others in which higher response rates have been reported.35-37 Patients were more heavily pretreated than in other studies; assessment of disease status included CT/ultrasound at the investigator's discretion, which has been reported to reduce response rate by approximately one third38; and hematopathologic/clinical responses were determined centrally by experts blinded to study treatment. Oblimersen did not increase fludarabine-/cyclophosphamide-associated myelosuppression/immunosuppression. Although the incidence of severe neutropenia was slightly higher in the control arm, the major adverse hematologic effect of oblimersen was thrombocytopenia. Oblimersen was associated with first-cycle reactions (tumor lysis syndrome, cytokine release–type reaction), as reported with single-agent oblimersen in CLL.32 Precautions to minimize risks of dehydration and tumor lysis are warranted. Other routes of administration (eg, subcutaneous injection) for oblimersen under evaluation should reduce the incidence of catheter-related complications. Overall, oblimersen plus fludarabine/cyclophosphamide significantly increases CR/nPR rate, compared with fludarabine/cyclophosphamide alone. These responses are durable and significantly longer than those achieved with chemotherapy alone.
Although all authors completed the disclosure declaration, the following authors or their immediate family members 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. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors. Employment: N/A Leadership: N/A Consultant: Benjamin Koziner, Genta; Asher A. Chanan-Khan, Genta; John F. Seymour, Schering A.G., AMGEN; Kanti R. Rai, Genta Stock: N/A Honoraria: Joseph O. Moore, Amgen, Novartis, Biogen IDEC; Loree M. Larratt, Amgen, Novartis, Celgene, Alexion; Benjamin Koziner, Genta; Asher A. Chanan-Khan, Genta Research Funds: Susan O'Brien, Berlex, Genentech, Biogen IDEC; Joseph O. Moore, Novartis, Amgen; Loree M. Larratt, Genta; Aleksander Skotnicki, Genta; Benjamin Koziner, Genta; Asher A. Chanan-Khan, Genta; John F. Seymour, Schering A.G., AMGEN Testimony: Kanti R. Rai, Genta Other: John F. Seymour, Schering, AMGEN
Conception and design: Susan O'Brien Provision of study materials or patients: Susan O'Brien, Joseph O. Moore, Thomas E. Boyd, Loree M. Larratt, Benjamin Koziner, Asher A. Chanan-Khan, John F. Seymour, R. Gregory Bociek, Steve Pavletic, Kanti R. Rai Collection and assembly of data: Joseph O. Moore, Thomas E. Boyd, Aleksander Skotnicki, Benjamin Koziner, Asher A. Chanan-Khan, R. Gregory Bociek, Steve Pavletic Data analysis and interpretation: Susan O'Brien, Joseph O. Moore, Kanti R. Rai Manuscript writing: Joseph O. Moore, John F. Seymour, Kanti R. Rai Final approval of manuscript: Susan O'Brien, Joseph O. Moore, Loree M. Larratt, Benjamin Koziner, John F. Seymour, R. Gregory Bociek, Steve Pavletic, Kanti R. Rai
We thank the patients who participated in this study and their families. We thank the following individuals for their expertise: hematopathologic review—Gregory Threatte, MD (University Pathologists Laboratories, LLP, Syracuse, NY), John M. Bennett, MD (University of Rochester Medical Center, Rochester, NY), and Randy D. Gascoyne, MD (British Columbia Cancer Agency, Vancouver, BC); radiologic review—James F. Caravelli, MD (Memorial Sloan-Kettering Cancer Center, New York, NY); and biostatistical guidance—Janet Wittes, PhD (Statistics Collaborative, Washington, DC), and Gary Koch, PhD (Department of Biostatistics, University of North Carolina at Chapel Hill, Chapel Hill, NC). The Oblimersen CLL Study group includes: B. Afanassiev, F. Ahmed, S. Allen, R. Anderson, T. Anderson, S. Anthony, N. Bartlett, B. Bernhardt, R. Bezares, R. Bociek, T. Boyd, K. Bradstock, W. Breyer, J. Brown, A. Chakrabarti, A. Chanan-Khan, C. Chen, W.-M. Chuu, J. Cicco, P. Cobb, G. Cohen, T. Cosgriff, S. Del Prete, N. DiBella, A. Dmoszynska, S. Durrant, C. Emmanouilides, L. Fernandez, J. Fleagle, C. Freter, N. Gabrail, A. Golenkov, M.F. Gonzalez, D. Gravenor, J. Gribben, M. Guarino, V. Gupta, K. Guter, W.G. Harker, G. Harrer, L. Hellerstein, R. Herrmann, N. Horvath, W. Jedrzejczak, S. Johnson, N. Kay, L. Kessler, J. Kloczko, K. Kolibaba, M. Komarnicki, B. Koziner, F. Kruter, K. Kuliczkowski, L. Larratt, J. Leonard, A. Levine, J. Liesveld, B. Link, J. Lister, D. Loesch, S. Maj, P. Marlton, B. Mavromatis, R. McCroskey, A. Melnyk, J. Moore, A. Morrison, V. Morrison, J. Muscato, R. Nakamura, M. Neubauer, S. O'Brien, T. Odenike, M. Olsen, L. Palmer, N. Patton, Z.S. Pavletic, V. Pavlov, L. Piro, T. Pluard, E. Podoltseva, A. Prentice, K. Rai, M. Rarick, T. Rearden, D. Richards, M. Saleh, F. Schnell, J. Seymour, S. Shao, C. Shustik, A. Skotnicki, R. Sobecks, M. Tallman, K. Taylor, A. Turkina, P. Venugopal, M. Volkova, P. Vongkovit, M. Wax, M. Yoffe, B. Zanger, and J. Zonder.
published online ahead of print at www.jco.org on February 12, 2007. Presented in part at the annual meeting of the American Society of Hematology, San Diego, CA, December 4-7, 2004, and at the XI International Workshop on CLL, New York, NY, September 16-18, 2005. Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Faderl S, Keating MJ, Do K-A, et al: Expression profile of 11 proteins and their prognostic significance in patients with chronic lymphocytic leukemia. Leukemia (CLL) 16:1045-1052, 2002 11. Hanada M, Delia D, Aiello A, et al: bcl-2 gene hypomethylation and high-level expression in B-cell chronic lymphocytic leukemia. Blood 82:1820-1828, 1993 12. Robertson LE, Plunkett W, McConnell K, et al: Bcl-2 expression in chronic lymphocytic leukemia and its correlation with the induction of apoptosis and clinical outcome. Leukemia 10:456-459, 1996[Medline] 13. Schena M, Larsson L-G, Gottardi D, et al: Growth- and differentiation-associated expression of bcl-2 in B-chronic lymphocytic leukemia cells. Blood 79:2981-2989, 1992 14. Reed JC, Kitada S, Takayama S, et al: Regulation of chemoresistance by the bcl-2 oncoprotein in non-Hodgkin's lymphoma and lymphocytic leukemia cell lines. Ann Oncol 5:S61-S65, 1994 (suppl 1) 15. 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Leukemia 18:1093-1101, 2004[CrossRef][Medline] 38. Eichhorst BF, Busch R, Hopfinger, et al: Fludarabine plus cyclophosphamide versus fludarabine alone in first-line therapy of younger patients with chronic lymphocytic leukemia. Blood 107:885-891, 2006 Submitted April 27, 2006; accepted December 15, 2006. This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
5 years) or hematologic disorder before CLL. 
6 months). A patient was considered refractory if he/she had failed to achieve at least a partial response (PR) or if disease recurred within 6 months of treatment, and was considered relapsed if disease recurred after achievement of at least a PR lasting more than 6 months. Patients were centrally randomly assigned to treatment groups using a permuted block method. 
