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Phase I-II Study of Oxaliplatin, Fludarabine, Cytarabine, and Rituximab Combination Therapy in Patients With Richter's Syndrome or Fludarabine-Refractory Chronic Lymphocytic Leukemia

Apostolia M. Tsimberidou, William G. Wierda, William Plunkett, Razelle Kurzrock, Susan O'Brien, Sijin Wen, Alessandra Ferrajoli, Farhad Ravandi-Kashani, Guillermo Garcia-Manero, Zeev Estrov, Thomas J. Kipps, Jennifer R. Brown, Albert Fiorentino, Susan Lerner, Hagop M. Kantarjian, Michael J. Keating

From the Departments of Leukemia, Experimental Therapeutics, Phase I Program, and Biostatistics, The University of Texas M.D. Anderson Cancer Center, Houston, TX; University of California San Diego Moore's Cancer Center, San Diego, CA; and Dana-Farber Cancer Institute, Boston, MA

Corresponding author: Apostolia M. Tsimberidou, MD, PhD, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Unit 455, Houston, TX 77030; e-mail: atsimber{at}mdanderson.org

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
Purpose Richter's syndrome (RS) and fludarabine-refractory chronic lymphocytic leukemia (CLL) are associated with poor clinical outcomes. We conducted a phase I-II trial of oxaliplatin, fludarabine, cytarabine, and rituximab (OFAR) in these diseases.

Patients and Methods The OFAR regimen consisted of increasing doses of oxaliplatin (17.5, 20, or 25 mg/m²/d) on days 1 to 4 (phase I), fludarabine 30 mg/m² on days 2 to 3, cytarabine 1 g/m² on days 2 to 3, rituximab 375 mg/m² on day 3 of cycle 1 and day 1 of subsequent cycles, and pegfilgrastim 6 mg on day 6, every 4 weeks for a maximum of six courses. Dose-limiting toxicity (DLT) was defined as any nonhematologic, treatment-related toxicity grade 3.

Results Fifty patients were treated (20 patients had RS, and 30 had CLL). The highest tolerated oxaliplatin dose was 25 mg/m², which was the highest dose tested. DLT was not observed. Pharmacodynamic analyses demonstrated enhanced leukemia cell killing by oxaliplatin in the presence of fludarabine and cytarabine. The overall response rates were 50% in RS and 33% in fludarabine-refractory CLL. The overall response rate in 14 patients with age 70 years was 50%. Responses were achieved in seven (35%) of 20 patients with 17p deletion, two (29%) of seven patients with 11q deletion, all four patients with trisomy 12, and two (40%) of five patients with 13q deletion. The median response duration was 10 months. Toxicities were mainly hematologic; prolonged myelosuppression was not observed.

Conclusion The OFAR regimen is highly active in RS and has activity in fludarabine-refractory patients with CLL. This regimen warrants further investigation in the treatment of these disorders.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
Richter's syndrome (RS) refers to the development of high-grade non-Hodgkin's lymphoma (NHL) in patients with chronic lymphocytic leukemia (CLL).¹ RS may be triggered by viral infections, such as Epstein-Barr virus (EBV), or by genetic defects acquired within the malignant clone and occurs in approximately 4% of CLL patients.^2,3 Various regimens, including fractionated cyclophosphamide, vincristine, liposomal daunorubicin, and dexamethasone plus rituximab⁴ and rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP),⁵ have been used in RS.^6,7 Response rates with these regimens range from 7% to 41%. Our analysis of 130 patients treated over a 30-year period demonstrated that the median survival time of patients with RS treated with chemotherapy with or without rituximab was 8 months.³

Fludarabine has shown marked activity in CLL, inducing response in 70% to 80% and 45% to 55% of patients with untreated and relapsed CLL, respectively.^8,9 Fludarabine combined with cyclophosphamide induced an overall response in 88% of untreated patients (complete response, 35%) and in 39% of patients with fludarabine-refractory disease (complete response, 3%).^10-12 Fludarabine, cyclophosphamide, and rituximab induced an overall response in 95% of untreated patients (complete response, 70%)¹³ and in 73% of patients with relapsed CLL (complete response, 25%).¹⁴

Oxaliplatin is a third-generation platinum compound in which the platinum atom is complexed with 1,2-diaminocyclohexan carrier ligand and an oxalate ligand.^15,16 It has a different spectrum of activity and low cross-resistance with cisplatin¹⁷ and a favorable toxicity profile.^16,18 Oxaliplatin had shown activity in relapsed/refractory NHL.^19-22 It has been substituted for cisplatin in the dexamethasone, high-dose cytarabine, and cisplatin regimen^23,24 and combined with CHOP²⁵ for the treatment of large-cell NHL.^19,20,22,26

In 2004, we initiated a phase I-II study of oxaliplatin, fludarabine, cytarabine, and rituximab (OFAR) for patients with RS and fludarabine-refractory CLL. The rationale for developing this regimen was based on preclinical data demonstrating synergistic cytotoxicity between cisplatin and the nucleoside analogs cytarabine²⁷ and fludarabine.^28-30 Additionally, the administration of fludarabine before cytarabine increases the cellular concentration of the active metabolite, cytarabine triphosphate.³¹ We hypothesized that the fludarabine-cytarabine combination might increase the sensitivity of leukemia cells to oxaliplatin by inhibiting DNA excision repair of the oxaliplatin adducts. The objectives of this trial were to determine whether oxaliplatin up to 25 mg/m² daily for 4 days could be administered in the OFAR regimen without unacceptable toxicity, to identify dose-limiting toxicities (DLTs) of oxaliplatin, to determine pharmacodynamic end points (phase I), and to assess the efficacy and toxicity of the OFAR regimen (phase II).

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
Patients
Signed informed consent explaining the investigational nature of the trial was obtained in accordance with institutional policy. Eligibility criteria included histologically or cytologically confirmed Richter's transformation or fludarabine-refractory CLL, age 18 years, Zubrod performance status of 0 to 2, and absence of uncontrolled life-threatening infections. Fludarabine-refractory CLL was defined as disease that failed to respond to fludarabine-based treatment or progressive disease within 6 months of response to a fludarabine-based regimen. Platelet counts 20,000/µL were required, unless lower counts were a result of disease involvement or autoimmune disorders. Adequate hepatic function (bilirubin 2 mg/dL, AST or ALT 3x the upper limit of normal) and renal function (serum creatinine 2 mg/dL or creatinine clearance > 30 mL/min) were also required unless abnormalities were a result of disease involvement. Patients were excluded if they were pregnant; had a history of oxaliplatin, fludarabine, cytarabine, or rituximab intolerance; had received chemotherapy and/or radiation therapy within the previous 4 weeks; or had any other medical condition deemed by the investigator to be likely to interfere with a patient's ability to give informed consent or cooperate/participate in the study or interfere with the interpretation of the results.

Patients with RS underwent gallium scanning plus computed tomography (CT) or positron emission tomography-CT scanning³² of the chest, abdomen, and pelvis. Pretreatment EBV testing of peripheral blood by polymerase chain reaction at a sensitivity level of 10^–4 to 10^–5 was also performed (see Appendix, online only).

Treatment Plan
In the phase I portion of this study, there were three dose cohorts for oxaliplatin (17.5, 20, or 25 mg/m²/d intravenously [IV]), which was administered on days 1 through 4 and infused over 2 hours. On days 2 through 3, fludarabine 30 mg/m² IV was administered within 30 minutes of oxaliplatin completion, and cytarabine 1 g/m² IV was administered as a 2-hour infusion approximately 4 hours after fludarabine was started. Rituximab 375 mg/m² IV was administered on day 3 of course 1 and on day 1 of subsequent courses in a 4- to 6-hour infusion, and pegfilgrastim 6 mg subcutaneously was administered on day 6. No DLT of oxaliplatin was noted; therefore, oxaliplatin was administered at 25 mg/m²/d IV for 4 days in the phase II study (Table 1). OFAR therapy was repeated every 4 weeks for a maximum of six courses. Prophylaxis for Pneumocystis carinii pneumonia and herpes zoster virus was administered.

End Points and Statistical Methods
For patients with RS, the response and end point assessments conformed to the published International Workshop response criteria for NHL.³⁴ In fludarabine-refractory CLL, the response criteria were those defined by the National Cancer Institute–Sponsored Working Group.³⁵ CT scans were not required for evaluation of response. Response was calculated on an intent-to-treat basis. Survival was measured from start of treatment until death from any cause or last follow-up. Failure-free survival (FFS) was defined as the time from treatment until progression, relapse, or toxic death. The ² test was used to investigate the independence between two categoric variables. Survival curves were estimated using the Kaplan-Meier method. To test the association between variables and survival or FFS, the two-sided log-rank test (or the Breslow-Gehan-Wilcoxon test, if the survival curves overlapped) was used. A multivariate Cox regression model was used to assess simultaneously the effect of two or more variables on survival and to examine the association between treatment and survival after adjusting for other factors. P values were derived from two-sided tests, and P < .05 was considered to be statistically significant. Statistical analyses were carried out using SAS 8.0 (SAS Institute, Cary, NC) and S-Plus 2000 (Statistical Sciences, Seattle, WA).

In the phase I part of the study, a conventional 3 + 3 design was used to assess doses of oxaliplatin up to 25 mg/m² daily for 4 days in the OFAR regimen (see Appendix). Toxicities were graded according to the National Cancer Institute Common Terminology Criteria of Adverse Events, version 3.0. DLT was defined as any grade 3 or higher nonhematologic, treatment-related toxicity.

The phase I portion of the study was conducted at M.D. Anderson Cancer Center. The phase II portion was conducted as a multicenter trial of the CLL Research Consortium, which in addition to M.D. Anderson, included the University of California San Diego Moore's Cancer Center (San Diego, CA) and the Dana-Farber Cancer Institute (Boston, MA).

	RESULTS

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Patient Characteristics
From November 2004 to August 2006, 21 patients with RS and 30 patients with fludarabine-refractory CLL were enrolled onto this phase I-II clinical trial. One otherwise eligible patient with RS was excluded because his insurance would not pay for the treatment. The pretreatment characteristics with respect to diagnosis are listed in Table 2. The median ages were 66 years (range, 41 to 78 years) for patients with RS and 59 years (range, 34 to 77 years) for patients with fludarabine-refractory CLL. Ten men and 10 women with RS and 25 men and five women with fludarabine-refractory CLL were treated. Two patients with RS were previously untreated. The remaining patients were heavily pretreated (Table 3). The median number of prior therapy courses was two (range, zero to 10 courses) for patients with RS and four (range, one to 11 courses) for patients with fludarabine-refractory CLL.

Response to Therapy
Two patients with fludarabine-refractory CLL who were not assessable for response are still reported in this analysis on an intent-to-treat basis. One patient withdrew consent on completion of chemotherapy infusion, and the other patient, who was responding to the first course of OFAR, was referred for SCT and, therefore, removed from the protocol.

The overall response rate (CR+PR) was 50% in RS patients and 33% in fludarabine-refractory CLL patients (Table 4). When only the phase II dose (25 mg/m²/d) of oxaliplatin was considered, the overall response rates were 46% and 38% for RS and CLL patients, respectively. Twenty percent of patients with RS and 6% of patients with fludarabine-refractory CLL achieved CR, and an additional 30% of patients with RS and 27% of patients with fludarabine-refractory CLL achieved PR. The response rate in 14 patients with age 70 years was 50% (one CR and six PRs). OFAR was active in patients with genomic aberrations (Table 5). Notably, among patients with a 17p deletion, two (40%) of five patients with RS and five (33%) of 15 patients with fludarabine-refractory CLL responded to OFAR. Among RS patients who had polymerase chain reaction testing for EBV in peripheral blood, the overall response rates were 50% (two of four patients) and 50% (seven of 14 patients) for EBV-positive and -negative patients, respectively. In patients with fludarabine-refractory CLL, the overall response rates were 33% (three of nine patients) and 25% (four of 16 patients) for EBV-positive and -negative patients, respectively. The median response duration of all responders was 10 months. In univariate analysis, the only factors predicting response were albumin levels and the CLL/small lymphocytic lymphoma score³⁷ (Table 6 and Appendix).

View larger version (13K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. (A) Survival by diagnosis. (B) Failure-free survival by diagnosis. (C) Pharmacokinetic studies in patients with Richter's syndrome or fludarabine-refractory chronic lymphocytic leukemia (CLL) treated with oxaliplatin, fludarabine, cytarabine, and rituximab. Apoptotic cell death was measured by annexin V binding. Arrows indicate timing of drug infusion. Each box, triangle, and so on, represents a different patient. O, oxaliplatin, F, fludarabine, A, cytarabine.

In univariate analysis (Table 6), factors predicting shorter survival were albumin level less than 3.5 g/dL (P < .0001), tumor size 5 cm (P = .008), platelet count less than 100 x 10⁹/L (P = .03), and high CLL/small lymphocytic lymphoma score (score of 3 to 5; P = .02).³⁷ As expected, patients with RS tended to have shorter survival than patients with fludarabine-refractory CLL (P = .07). In multivariate analysis, factors predicting shorter survival were albumin levels less than 3.5 g/dL (relative risk = 12.4, P = .0008) and tumor size less than 5 cm (relative risk = 3.6, P = .009).

FFS
Treatment failure occurred in 28 patients. At 6 months, the FFS rates were 47% for patients with RS and 48% for patients with fludarabine-refractory CLL (P = .97; Fig 1B). Among patients with 17p deletion, the 6-month FFS rates were 36% for patients with fludarabine-refractory CLL and 27% for patients with RS (log-rank, P = .72; Appendix Fig A1C). In univariate analysis, the only factor predicting shorter FFS was albumin level less than 3.5 g/dL (Table 6).

Subsequent Therapy
Seventeen patients received salvage therapy, including seven patients who received two or more subsequent therapies. Fifteen patients underwent SCT after OFAR (RS, n = 3; fludarabine-refractory CLL, n = 12; Appendix Tables A1 and A2, online only). Seven of these patients were in PR, and eight patients had refractory or progressive disease at the time of transplantation. Eleven patients received SCT from a matched unrelated donor, and four received SCT from a matched sibling. At 1 year, 70% of the 15 patients who underwent SCT remained alive compared with 39% of the remaining 35 patients who did not undergo SCT (P = .057).

Toxicity
The toxicity from this chemoimmunotherapeutic regimen was mainly hematologic (Table 7). OFAR caused grade 3 to 4 neutropenia and/or thrombocytopenia in the majority of patients. The regimen was well tolerated in patients with age 70 years (n = 14). The median times to grade 3 neutropenia, thrombocytopenia, and anemia were 12, 9, and 13 days, respectively. The median time to platelet recovery 50 x 10⁹/L was 33 days (range, 24 to 46 days), and the median time to leukocyte recovery 10⁹/L was 28 days (range, 5 to 65 days). Subsequent cycles of OFAR were administered after a median of 28 days (range, 21 to 54 days). The number of patients who required transfusion of RBCs, by oxaliplatin dose, was as follows: 17.5 mg/m², two (67%) of three patients; 20 mg/m², five (63%) of eight patients; and 25 mg/m², 26 (67%) of 39 patients. No RBC transfusions were required as a result of autoimmune hemolytic anemia. In addition, the number of patients who required platelet transfusion, by oxaliplatin dose, was as follows: 17.5 mg/m², one (33%) of three patients; 20 mg/m², seven (88%) of eight patients; and 25 mg/m², 23 (59%) of 39 patients. The most common nonhematologic adverse events were fatigue (34%) and nausea (26%). Six patients developed grade 3 to 4 infections or neutropenic fever, including one patient with cytomegalovirus reactivation. Four patients developed grade 1 to 2 sensory neuropathy.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
The rationale for designing the OFAR regimen was based on the activity of oxaliplatin in colorectal cancer,^38,39 the minimal renal and auditory oxaliplatin-associated toxicity,^16,18 the activity of oxaliplatin-based therapies in lymphomas,^19,20,22,26 and our prior experience with cisplatin-based therapies in RS and progressive CLL.^7,40 On the basis of preclinical data demonstrating synergistic cytotoxicity between cisplatin and cytarabine²⁷ or fludarabine,^28-30 we also hypothesized that the fludarabine-cytarabine combination might modulate oxaliplatin sensitivity in leukemia cells by inhibiting DNA excision repair of the oxaliplatin adducts, thereby resulting in synergistic cytotoxicity in RS and CLL.⁴¹

The results of this study demonstrate that OFAR in RS results in response rates that are at least as high as those seen with other therapies, such as fractionated cyclophosphamide, vincristine, liposomal daunorubicin, and dexamethasone plus rituximab or rituximab plus CHOP therapies, and that OFAR is clinically active in CLL.^3,4 Fifty percent of patients with RS and 33% of patients with fludarabine-refractory CLL attained a CR or PR with OFAR therapy. Notably, among patients with age greater than 70 years, the overall response rate was 50%. OFAR was generally well tolerated, and grade 3 to 4 nonhematologic toxicities were rare. As expected with antileukemic cytotoxic regimens in heavily pretreated patients, OFAR induced neutropenia, thrombocytopenia, and anemia in most patients.

The superiority of OFAR compared with prior cisplatin-based therapies (see Appendix)^7,40 is attributed to the substitution of oxaliplatin for cisplatin, the addition of rituximab, and the considerable increase in the cytarabine dose. The antileukemic activity of the OFAR regimen is elucidated by the pharmacokinetic studies, which showed enhancement of apoptotic death when fludarabine and cytarabine followed oxaliplatin. Pharmacologic data demonstrated that fludarabine interferes with the removal of interstrand DNA cross-links caused by oxaliplatin in primary CLL lymphocytes and that fludarabine and oxaliplatin in combination cause greater-than-additive cell death.³² This is similar to the action of fludarabine on the cross-linking caused by cisplatin, which has been shown in cell lines.²⁷

The efficacy of OFAR in our patient population is of particular interest, considering the high proportion of patients in our study with poor prognostic features. For instance, 90% of patients with fludarabine-refractory CLL and 75% of patients with RS had received two or more prior therapies (Table 3); more importantly, 52% and 25%, respectively, carried a 17p deletion, and higher proportions of patients had multiple deletions (Table 2). Whereas purine analogs have minimal antitumor activity in patients with a 17p deletion,⁴² OFAR induced responses in five (33%) of 15 patients with fludarabine-refractory CLL and two (40%) of five patients with RS, demonstrating activity comparable to that of alemtuzumab in patients with CLL carrying 17p deletions.⁴³ Another observation was that responses occurred regardless of the EBV status. We have reported that EBV-encoded RNAs are more frequently detected in patients with more advanced RAI stage CLL and that they are associated with a trend toward shorter survival.⁴⁴

Others' experiences with oxaliplatin-based therapy in lymphoid malignancies have been promising.^19-22,45,46 Single-agent oxaliplatin has activity, inducing remission (all PRs) in 26% of 30 patients with relapsed/refractory NHL.⁴⁵ With oxaliplatin combination therapies, such as dexamethasone, high-dose cytarabine, and oxaliplatin with or without rituximab, results were encouraging. Although the numbers of patients in those studies were small (< 25 patients), the overall response rates ranged from 50% to 73% (CR, 10% to 53%), and the main toxicity was myelosuppression.^19,20,22 Oxaliplatin combined with CHOP in untreated, diffuse, large B-cell lymphoma induced response in eight of 10 patients (one CR).²⁶

In conclusion, the current study demonstrates the efficacy and tolerability of the OFAR regimen in heavily pretreated patients with RS and fludarabine-refractory CLL. Given the activity of OFAR, particularly in patients with 17p deletions and in patients older than 70 years, a larger study of OFAR is currently being pursued by our group.

	AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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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 or Leadership Position: None Consultant or Advisory Role: None Stock Ownership: None Honoraria: Michael J. Keating, Berlex Laboratories, Genentech Research Funding: Apostolia M. Tsimberidou, Sanofi-Aventis; William G. Wierda, Berlex Laboratories, Sanofi-Aventis, Memgen, Genitope, Genmab; William Plunkett, Sanofi-Aventis; Susan O'Brien, Berlex Laboratories, Genentech, Biogen Idec; Farhad Ravandi-Kashani, Genentech, Biocryst, Berlex Laboratories, Genzyme, Abiogen, MGI Pharma, Bristol-Myers Squibb Co; Jennifer R. Brown, Berlex Laboratories, Genentech; Hagop M. Kantarjian, Novartis, Bristol-Myers Squibb Co, MGI Pharma Expert Testimony: None Other Remuneration: None

	AUTHOR CONTRIBUTIONS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
Conception and design: Apostolia M. Tsimberidou, Michael J. Keating

Financial support: Apostolia M. Tsimberidou, Michael J. Keating

Administrative support: Apostolia M. Tsimberidou, Michael J. Keating

Provision of study materials or patients: Apostolia M. Tsimberidou, William G. Wierda, William Plunkett, Razelle Kurzrock, Susan O'Brien, Alessandra Ferrajoli, Farhad Ravandi-Kashani, Guillermo Garcia-Manero, Zeev Estrov, Thomas J. Kipps, Jennifer R. Brown, Hagop M. Kantarjian, Michael J. Keating

Collection and assembly of data: Apostolia M. Tsimberidou, William Plunkett, Razelle Kurzrock, Albert Fiorentino, Susan Lerner

Data analysis and interpretation: Apostolia M. Tsimberidou, William G. Wierda, William Plunkett, Razelle Kurzrock, Sijin Wen, Susan Lerner, Michael J. Keating

Manuscript writing: Apostolia M. Tsimberidou, William Plunkett

Final approval of manuscript: Apostolia M. Tsimberidou, William G. Wierda, William Plunkett, Razelle Kurzrock, Susan O'Brien, Alessandra Ferrajoli, Farhad Ravandi-Kashani, Guillermo Garcia-Manero, Zeev Estrov, Thomas J. Kipps, Jennifer R. Brown, Albert Fiorentino, Susan Lerner, Hagop M. Kantarjian, Michael J. Keating

	Appendix

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 

View larger version (14K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig A1. (A) Survival in patients with 17p deletion by diagnosis. (B) Survival by response to oxaliplatin, fludarabine, cytarabine, and rituximab. (C) Failure-free survival in patients with 17p deletion by diagnosis. CLL, chronic lymphocytic leukemia. CR, complete remission; PR, partial remission; NR, no response.

Pretreatment evaluation. Pretreatment evaluation included medical history, physical examination, CBCs, differential and platelet counts, a biochemical assay panel (including liver and renal function studies), serum β₂-microglobulin and serum immunoglobulin measurements, bone marrow aspiration and biopsy, and acquisition of marrow samples for immunophenotyping.

Pretreatment Epstein-Barr virus (EBV) testing of peripheral blood by polymerase chain reaction (PCR) was also performed. Briefly, DNA was isolated from peripheral-blood mononuclear cells before treatment using a Qiagen kit (Qiagen, Valencia, CA). EBV was detected by a real-time PCR assay using an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA) at a sensitivity level of 10^–4 to 10^–5 and an EBV R-gene (BXLF1) quantification kit (Ref. 69-002B; Argene, Varilhes, France), according to the manufacturers' instructions.

Patient monitoring during therapy. Staging evaluations, including a review of patient history, physical examination, blood counts, and bone marrow biopsy, were performed after three and six courses. Patients with Richter's syndrome (RS) were additionally assessed with computed tomography (CT) or positron emission tomography-CT scans of the chest, abdomen, and/or pelvis at the same time intervals.

End points and statistical methods. For patients with RS, complete remission (CR) was defined as the complete disappearance of all detectable clinical and radiographic evidence of disease and all disease-related symptoms and the normalization of biochemical abnormalities definitely assignable to lymphoma. Partial remission (PR) was defined as a reduction by 50% or more of the sum of the products of the greatest diameters of bidimensionally measurable disease. Any other response was considered a treatment failure.

For patients with fludarabine-refractory chronic lymphocytic leukemia (CLL), a CR required the disappearance of all palpable disease; the normalization of blood counts, with neutrophil counts 1.5 x 10⁹/L, platelet counts more than 100 x 10⁹/L, hemoglobin levels more than 11 g/dL, and bone marrow aspirate lymphocytes less than 30%; and no evidence of disease on bone marrow biopsy. A nodular PR met the same criteria as a CR, with the exception that lymphoid nodules could be seen on bone marrow biopsy. A PR required a reduction 50% in palpable disease as well as at least one of the following: neutrophil counts more than 1.5 x 10⁹/L or a 50% improvement over baseline; a 50% improvement in platelet counts over baseline; or a 50% improvement in hemoglobin levels over baseline without transfusions. No bone marrow evaluation was required for the determination of PR.

A conventional 3 + 3 design was used to determine the maximum-tolerated dose from three dose levels in the phase I part of the study. A dose-limiting toxicity was defined as any oxaliplatin-related nonhematologic toxicity grade 3 involving a major organ system (brain, heart, kidney, liver, or lung) in the National Cancer Institute Common Terminology Criteria of Adverse Events, version 3.0. However, the institutional review board required the maximum cumulative dose of oxaliplatin not to exceed a total of 600 mg/m². Therefore, the maximum allowed oxaliplatin dose was 100 mg/m² per cycle or 25 mg/m² daily for 4 days. A maximum of 34 patients were to be accrued in the phase II part of the study and a 90% credible interval of the response rate was estimated. The objective of the current study was to increase the response rate to 40%. This trial would be regarded as successful if the addition of oxaliplatin resulted in a 10% increase in response rate over the previously used regimen for RS (ie, fractionated cyclophosphamide, vincristine, liposomal daunorubicin, and dexamethasone [hyper-CVXD] plus rituximab combination therapy). If the trial proceeded to maximum accrual, the CR+PR rate could be estimated with a 90% credible interval width of 0.25, under the assumption of a 50% response rate. The trial was to be regarded as successful (ie, achieving a 10% improvement in response rate over previous therapy) if at least 14 responses (CR and/or PR) were observed among 40 patients (six patients at the maximum-tolerated dose of the phase I trial plus 34 patients in the phase II part of the study). The study would be terminated prematurely if there were two responses among the first 10 patients, five responses among the first 20 patients, or nine responses among the first 30 patients.

Results
Histologic diagnosis. The diagnosis of RS was based on biopsy in 15 patients. In particular, the diagnosis of RS was based on an excisional lymph node biopsy in eight patients and on a bone marrow biopsy in seven patients, with concurrent involvement of other tissue in four of the seven patients (lung, n = 1; lymph node, n = 2; and vertebrae, n = 1). In three additional patients, a fine-needle aspiration was performed, which demonstrated large cells, including retroperitoneal lymph node involvement in one patient and a core biopsy of an arm mass in another patient. In two additional patients with symptoms suggestive of RS, a positron emission tomography-CT scan demonstrated bulky lymphadenopathy associated with high standardized uptake value, which was consistent with Richter's transformation of CLL; in these two patients, no biopsy or fine-needle aspiration was performed because of the urgency to begin treatment.

Overall, 47 patients were diagnosed at M.D. Anderson Cancer Center, and three patients were enrolled by collaborating institutions of the Chronic Lymphocytic Leukemia Research Consortium (University of California San Diego Moore's Cancer Center, n = 2; and Dana-Farber Cancer Institute, n = 1). Histopathology was reviewed at M.D. Anderson Cancer Center for the 47 patients seen there.

Patients. Three, eight, and 39 patients were treated at the oxaliplatin dose levels of 17.5, 20, and 25 mg/m², respectively (Table 7). The highest dose level was 25 mg/m². Eight patients were treated at the 20-mg dose level because two patients presented during accrual at that level with Richter's transformation, which needed treatment. Because four patients had been treated safely at the 20-mg/m² dose level at the time and safety information was not available at the time for patients 5 and 6, we decided to include these two additional patients in the 20-mg/m² dose level group. There were six patients at the 20-mg/m² dose level because there were concerns about myelosuppression. This dose was determined to be safe with regard to nonhematologic and hematologic toxicities, so the oxaliplatin dose was escalated to 25 mg/m². Theoretically, the oxaliplatin dose could be higher than 25 mg/m² daily for 4 days per cycle for six cycles. However, the institutional review board restriction for oxaliplatin not to exceed a cumulative dose of 600 mg/m² together with our observation of cytopenias led us to use 25 mg/m² as the highest tested dose.

Response to prior alemtuzumab in fludarabine-refractory CLL. Fourteen of the fludarabine-refractory patients had received alemtuzumab-containing therapy before oxaliplatin, fludarabine, cytarabine, and rituximab (OFAR). Their responses were as follows: cyclophosphamide, fludarabine, alemtuzumab, and rituximab: five PRs and three failures; alemtuzumab plus rituximab: two PRs and three failures; and alemtuzumab alone: one PR. In these patients, OFAR induced only one PR, which occurred in a patient whose disease had failed to respond to cyclophosphamide, fludarabine, alemtuzumab, and rituximab.

Clinical outcomes by pretreatment characteristics. Twenty (47%) of 43 patients with albumin levels 3.5 g/dL and none of seven patients with albumin levels less than 3.5 g/dL responded to OFAR therapy (P = .02; Table 6). Similarly, 11 of 17 patients with a low CLL/small lymphocytic lymphoma score (score, 0 to 2) and nine of 29 patients with a high CLL/small lymphocytic lymphoma score responded to treatment (P = .03).

Factors other than those presented in Table 6, such as age, performance status, levels of creatinine, lactate dehydrogenase, or β₂-microglobulin, 17p deletion, EBV status by PCR, and number of prior therapies, were not associated with rates of response, survival, or failure-free survival.

Subsequent therapies. Salvage therapies included hyper-CVXD with rituximab (10 patients; one PR and nine treatment failures); rituximab with or without methylprednisolone (two patients; both treatment failures); alemtuzumab (two patients; both treatment failures); rituximab and alemtuzumab combination therapy (two patients; both treatment failures); rituximab, ifosfamide, carboplatin, and etoposide therapy (one patient; treatment failure); and lenalidomide (one patient; treatment failure).

Historical comparison of OFAR with other regimens. We compared OFAR in RS with hyper-CVXD plus rituximab and rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) in RS. In the OFAR group, 18 patients treated with oxaliplatin 20 mg/m² or higher were included in this analysis. Patients treated with OFAR were older compared with other patients, whereas the R-CHOP group had fewer prior therapies. Despite the fact that OFAR was administered in a poorer prognosis group, the response rate was 56% compared with 46% with hyper-CVXD + rituximab and 50% with R-CHOP (Appendix Table A3).

Toxicity. The median number of RBC units per OFAR cycle administered, by oxaliplatin dose, was as follows: 17.5 mg/m², 2 units (range, 1 to 2 units); 20 mg/m², 2 units (range, 1 to 10 units); and 25 mg/m², 2 units (range, 1 to 8 units). In addition, the median number of platelet transfusion events per OFAR cycle administered, by oxaliplatin dose, was as follows: 17.5 mg/m², one event (range, one to five events); 20 mg/m², two events (range, one to five events); and 25 mg/m², three events (range, one to six events).

The median number of OFAR cycles administered was two (range, one to six cycles). To address whether OFAR was a feasible regimen, we compared these data with those of our prior best treatment in RS or fludarabine-refractory CLL (ie, hyper-CVXD plus rituximab alternating with methotrexate and cytarabine plus rituximab and granulocyte-macrophage colony-stimulating factor therapy; Appendix Table A4; Tsimberidou AM, Kantarjian HM, Cortes J, et al. Cancer 97:1711-1720, 2003).

The majority of these patients were heavily pretreated, as evidenced by the large number of prior therapies. It is well established that the bone marrow reserve in heavily pretreated patients is poor, and therefore, administration of intensive chemotherapy is not commonly feasible.

Discussion
In RS or refractory CLL, the response rate with fludarabine, cytarabine, cyclophosphamide, cisplatin, and granulocyte-macrophage colony-stimulating factor was 5% (one of 19 assessable patients), and the response duration was 10 months (Tsimberidou AM, O'Brien SM, Cortes JE, et al. Leuk Lymphoma 43:767-772, 2002). In patients with CLL after fludarabine failure, the response rate with cisplatin and fludarabine, with or without cytarabine, was 19% (all PRs), and the median overall survival duration was 6 months (Giles FJ, O'Brien SM, Santini V, et al. Leuk Lymphoma 36:57-65, 1999). Other regimens, such as doxorubicin, methylprednisolone, high-dose cytarabine, and cisplatin, have not been systematically studied in CLL or RS. In mucosa-associated lymphoid tissue lymphoma, single-agent oxaliplatin is highly active, inducing response in 94% (CR, 56%) of 16 patients (nine of whom were therapy naïve) (Raderer M, Wohrer S, Bartsch R, et al. J Clin Oncol 23:8442-8446, 2005).

	NOTES

 
Supported in part by a Career Development Award from the American Society of Clinical Oncology (A.M.T.).

Presented in part at the 48th Annual Meeting of the American Society of Hematology, December 9-12, 2006, Orlando, FL, and at the 42nd Annual Meeting of the American Society of Clinical Oncology, June 2-6, 2006, Atlanta, GA.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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Submitted March 23, 2007; accepted October 1, 2007.

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