Rituximab in Chronic Lymphocytic Leukemia

Rituximab in Chronic Lymphocytic Leukemia Samantha M. Jaglowskia and John C. Byrdb Rituximab is a class I chimeric anti-CD20 antibody that has shown efficacy in chronic lymphocytic leukemia (CLL), both as a single agent and in combination with traditional chemotherapies. The modest activity demonstrated in early studies evaluating rituximab in relapsed CLL was improved with higher doses or more dose-intensive regimens that overcame the unfavorable pharmacokinetic features commonly found in CLL. These studies led to a variety of combination trials of rituximab with chemotherapy, where both phase II and later phase III studies have shown great promise for the advancement of CLL therapy. Despite the therapeutic success of rituximab in CLL, studies demonstrating the definitive relative mechanism of tumor clearance are still lacking and this requires further investigation. In addition to being used as a therapy for CLL, rituximab is an effective treatment for autoimmune CLL complications such as hemolytic anemia and immune thrombocytopenia (ITP). Patients with CLL may experience early infusion-related side effects that can be diminished with corticosteroid pretreatment and stepped-up dosing. Risk factors for infusion-related toxicity may relate to atypical CLL expressing bright CD20 antigen expression, although several different studies have not clearly implicated elevated white blood cell count as a risk factor. Other adverse events, including delayed cytopenias, reactivation of hepatitis B, and development of progressive multifocal leukoencephalopathy, are rare. Future efforts focusing on novel combination-based strategies will be required to fully appreciate the benefit of this therapy in CLL. Semin Hematol 47:156 –169. © 2010 Published by Elsevier Inc.

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hronic lymphocytic leukemia (CLL)is one of the most common types of adult leukemia and is characterized immunophenotypically by expression of the B-cell markers CD19, CD20, CD22, and CD23, with co-expression of the T-cell marker CD5. The majority of patients present with asymptomatic disease, and based on the absence of a survival benefit with early treatment, they are generally observed until symptoms of enlarged nodes, organomegaly, or cytopenias develop. Genetically, CLL has several common chromosomal aberrations, including del(13q14), trisomy 12, del(11q22.3), and del(17p13.1); the latter two aberrations are associated with earlier development of symptomatic disease requiring therapy.1 Sentinel observations have demonstrated that subtypes of CLL can be biologically distinguished by IgVH mutational status.2,3 Patients with IgVH unmutated disease aDivision

of Hematology-Oncology, Department of Medicine, The Ohio State University, Columbus, OH. bDivision of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH. This work was supported by grants from Leukemia and Lymphoma Society and The D. Warren Brown Foundation. Address correspondence to John C. Byrd, MD, 455B, OSUCCC, 410 W 12th Ave, Columbus, OH 43210. E-mail: [email protected] 0037-1963/10/$ - see front matter © 2010 Published by Elsevier Inc. doi:10.1053/j.seminhematol.2010.01.005

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often have overexpression of ZAP70 and enhanced B-cell receptor signaling.4 – 6 This subgroup has a higher frequency of genomic instability and high-risk interphase cytogenetic abnormalities and rapidly progresses to requiring therapy. In contrast, CLL patients with IgVH mutated disease have a more indolent disease course with extended time from diagnosis to treatment. A role of disordered apoptosis in CLL has been suggested with constitutive activation of several antiapoptotic transcription factors, including nuclear factor-␬B (NF-␬B), nuclear factor of activated T cells (NFAT), and signal transducer and activator of transcription 3 (STAT3), that enhance overexpression of several anti-apoptotic proteins, including bcl-2, mcl-1, XIAP, and A1.7–9 CLL cells also derive significant survival signals from both direct contact with stromal cells and cytokines (stromal cell– derived factor 1 [SDF1], CD40-ligand, B-cell–activating factor [BAFF], interleukin-4 [IL-4], and vascular endothelial growth factor [VEGF]).10 Treatment of CLL for many years relied on palliative alkylator-based therapy (chlorambucil or cyclophosphamide), which promotes response in 40% to 60% of patients.11 The nucleoside analog fludarabine was demonstrated to be superior to alkylator-based therapy in several phase III studies with respect to overall response rate (ORR), complete response (CR) rate, and progression-free survival (PFS).12–15 Subsequent combiSeminars in Hematology, Vol 47, No 2, April 2010, pp 156 –169

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Table 1. Selected Phase II Trials of Weekly Rituximab in CLL/SLL

Reference (Authors, year) McLaughlin et al, 199824 Nguyen et al, 199926 Winkler et al, 199927 Ladetto et al, 200028 Huhn et al, 200129 Itala et al, 200230 Hainsworth et al, 200340 Thomas et al, 200141

Doses

Prior Therapy

Evaluable Patients

Response Rate (ORR)

4 4 4 4 4 4 4 8

Yes Yes Yes Yes Yes Yes No No

30 10 9 7 28 25 44 21

13% 10% 11% 0% 25% 35% 58% 90%

nation studies with fludarabine and cyclophosphamide have demonstrated superior CR rate, ORR, and PFS when compared to fludarabine monotherapy.12,16,17 However, none of these cytotoxic treatment strategies have attained the high CR rates observed in aggressive lymphoma and acute leukemia, where cure is appreciated. Attaining this goal in CLL would require application of therapies with different mechanisms of action. While the benefit of rituximab is now clearly seen as an important contributor to improving outcomes in CLL patients, the historical review of development clearly documents a less promising potential during the early days of rituximab clinical development.

EARLY DEVELOPMENT OF RITUXIMAB AS A SINGLE AGENT IN CLL Rituximab is a chimeric murine/human antibody directed at the CD20 antigen. CD20 is expressed almost exclusively on B cells from beyond pre–B-cell stage but is lost as post-germinal cells differentiate to become plasma cells. The function of CD20 is germane to B-cell development as CD20 knock-out mice demonstrate normal B-cell number and localization with a very modest phenotype of immature and mature B cells where IgM expression is moderately lower and CD19induced intracellular calcium response is significantly reduced.18 The CD20 antigen is neither shed nor internalized in resting B cells, making it an ideal antigen for targeting with therapeutic antibodies.19 Early preclinical studies with rituximab demonstrated this antibody could mediate both complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) against CD20⫹ tumor targets.20 Preclinical primate studies demonstrated depletion of normal B cells in vivo20 with rituximab, providing sufficient validation for this antibody to be tested in a phase I study in CD20⫹ non-Hodgkin lymphoma (NHL). The initial phase I dose-escalation study of rituximab demonstrated a dose-dependent and specific depletion of B cells in patients with relapsed NHL, specifically at doses greater than 100 mg/m2, and in spite of the fact

that this was a study of a single infusion of rituximab, some partial responses (PRs) and minor responses were noted.21 The subsequent phase I multidose trial also demonstrated the rapid depletion of circulating normal B cells, as well as malignant cells in patients with relapsed B-cell malignancies.22 Patients received 4 weekly doses of 125 mg/m2, 250 mg/m2, or 375 mg/m2 of rituximab; 18 patients received all four doses. Three of nine patients who received 375 mg/mg2 had PRs; the majority of adverse events were infusion-related and all were reversible.22 An early communication of the subsequent phase II study reported on 37 patients with relapsed NHL.23 Three of these patients had CRs, and another 14 had PRs. In those patients who responded, the median time to progression was 10.2 months. The most common adverse event noted was, again, infusion-related toxicity.23 The best responses were observed in patients with follicular histology, and the phase III study of 166 patients was focused on those patients with low-grade or follicular lymphoma.24 Forty-eight percent of patients had a response, and 6% had a CR. Of the 75 patients who did not meet criteria for a CR or PR, 56 had a measurable disease response. The projected median time to progression was 13 months.24 However, when response was broken down according to International Working Group criteria, the response among the 33 patients with small lymphocytic lymphoma (SLL) was only 12%.25 Similarly disappointing results were obtained in several other small studies enrolling 10 or fewer SLL/ CLL patients each.26 –28 Studies performed by the German CLL Study Group (GCLLSG) (n ⫽ 28 patients) and Nordic study group (n ⫽ 25 patients) demonstrated ORRs of 25% and 35%, respectively, with short remission duration.29,30 Responses in each of these studies were predominately in the blood and nodal compartment, with little improvement in marrow disease. Relapse of disease was generally noted within 2 to 4 months following completion of therapy. The results of selected phase II studies are listed in Table 1. The very modest response to rituximab as compared to other types of B-cell lymphomas provided some

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pause for developing this agent in CLL/SLL. Potential reasons for lower response in this disease included diminished CD20 expression on CLL cells,31 altered innate immune function,32,33 and different pharmacokinetic features25 compared to lymphoma, due to the often large tumor load in the blood. Two trials performed by the M.D. Anderson Cancer Center and our own group administered either higher doses of rituximab weekly (up to 2,250 mg/m2 per dose) or thriceweekly doses to relapsed CLL patients and showed improved response.34 –36 Benefit in these two trials was again seen predominately in the blood and nodal compartment, although response duration approached that achieved in follicular B-cell NHL in previous trials. Unfortunately, no patients with del(17p13.1) responded to thrice-weekly rituximab, indicating that single-agent rituximab is ineffective in this high-risk population.37,38 Nonetheless, both dose escalation and thrice-weekly dosing improved response rate, established a role for single-agent rituximab in relapsed CLL, and provided support for the study of rituximab in combination strategies in previously untreated CLL. Such studies were also supported by early investigations demonstrating sensitization of CD20⫹ tumor cells to the effects of cytotoxic therapy with rituximab.39 Subsequent studies demonstrated that weekly single-agent rituximab at the lower 375 mg/m2 dose had greater clinical efficacy in previously untreated CLL patients.40 Forty-four previously untreated patients with SLL/CLL received 4 weekly doses of rituximab at 375 mg/m2; the ORR after the first course of rituximab was 51% (CR 4%). Twenty-eight patients with stable or responsive disease received additional 4-week courses of rituximab every 6 months for up to four cycles. However, there was only a modest increase in ORR (58%) and CR rate (9%), and the median PFS of 19 months was shorter than the 36- to 40-month median PFS obtained by the same investigators using the same regimen in previously untreated patients with follicle center B-cell NHL.41 Nonetheless, this response duration compared favorably with the response duration to fludarabine in the upfront setting, suggesting that rituximab is active and may have a role in the upfront therapy of SLL/CLL.

EARLY STUDIES EXAMINING MECHANISM OF ACTION OF RITUXIMAB IN CLL Therapeutic antibodies such as rituximab can mediate CDC, ADCC, and direct apoptosis.42 Extensive investigation of each mechanism of action in CLL has been pursued. While CDC is relevant to rituximabmediated cytotoxicity in some B-cell lines, CLL cells express dim CD20 with only a small subset of cells being susceptible to CDC by rituximab.43,44 Very elegant preclinical in vitro and in vivo studies have demonstrated that CLL cells are prone to CD20 shaving

S.M. Jaglowski and J.C. Byrd

following treatment, and this diminishes the ability of CDC to occur. B lymphocytes are rapidly cleared from the bloodstream following initiation of infusion of rituximab. However, at high blood concentrations of rituximab (⬎100 ␮g/mL), there is a substantial resurgence of B cells. Interestingly, these B cells have very low levels of CD20. Rather than being internalized, rituximab-CD20 complexes are removed from opsonized cells by monocytes in an Fc␥RI-mediated process via the direct exchange of membrane fragments between effector and target cells, resulting in “shaving” of CD20 from B lymphocytes.45– 48 The transfer of CD20 from lymphocytes to natural killer (NK) cells and monocytes was again recently demonstrated and presented at the annual meeting of the American Society of Hematology (ASH).49 Attempts to abrogate the phenomenon of shaving have been undertaken clinically by administering very low doses of rituximab on a thrice-weekly schedule to subvert the recrudescence of these B cells with low levels of CD20 with very modest clinical activity.46,50 Thus, while a very strong hypothesis with supportive preclinical data supports shaving as a reason for modest rituximab clinical activity when administered at higher doses, it is not clear what CDC contributes to tumor elimination. Monocytes mediate antibody-dependent cellular phagocytosis (ADCP)51 and NK cells mediate ADCC51 against rituximab-labeled CLL cells in vitro. Despite this, the function of both monocytes52,53 and NK cells32,33 to mediate ADCP and ADCC respectively are compromised in vivo in CLL patients due to increased T-regulatory cells, chemotherapy administration, and marrow compromise promoted by active leukemia. Whereas single-nucleotide polymorphisms of Fc␥RIIIa and Fc␥RIIa enhance ADCC and response to therapy in other types of lymphoma,54,55 no added therapeutic benefit has been observed in CLL patients with high affinity Fc␥RIIIa or Fc␥RIIa polymorphisms in two separate studies of rituximab.38,56 Collectively, this suggests that ADCC may not play a major role in rituximab clearance of CLL cells. Finally, our group and several others have demonstrated that rituximab can mediate both caspase-dependent and -independent apoptosis in CLL through activation of distinct signaling pathways in vitro.57 In most cases, this requires co-incubation of rituximab with an anti-human Fc-directed antibody.57– 61 Type II anti-CD20 antibodies such as B1 and the humanized GA101 antibody promote direct apoptosis without a cross-linking antibody.62 Differences between type I (rituximab) and type II (GA101, B1) anti-CD20 antibodies lie predominately in their ability to redistribute CD20 into plasma membrane lipid rafts.63 Type II anti-CD20 antibodies do not segregate CD20 into lipid rafts and are very effective at activating a caspase-independent, lysosomal-dependent mechanism of death that is dependent upon homotypic adhesion.62 The relevance of this observation in vivo among CLL patients receiving type II CD20

Rituximab in chronic lymphocytic leukemia

antibody therapy remains unexplored. Our group was the first to note the occurrence of apoptosis with caspase activation in CLL cells in vivo following administration of rituximab.57 This was accompanied by serial down-modulation of mcl-1 in CLL cells derived from patients receiving this therapy.57 These studies, along with many other pre-clinical studies in lymphoma,39,64 provided justification to move forward with combination treatment strategies proposed below.

PHASE II CHEMOIMMUNOTHERAPY TRIALS Rituximab has synergistic effects with several chemotherapeutic agents traditionally used for CLL and NHL. Two early studies pursued the combination of fludarabine and rituximab (FR) in CLL. The first study performed by the GCLLSG included 31 patients, 20 of whom were previously untreated and 11 of whom had relapsed disease.65 Fludarabine was administered at 25 mg/m2 on days 1–5, 29 –33, 57– 61, and 85– 89, and rituximab was administered at 375 mg/m2 on days 57, 85, 113, and 151. Toxicity included predominately cytopenias and infection. The ORR was 87% with 10 (32%) patients achieving a CR. The median duration of response in this study was 75 weeks.65 A second study led by the Cancer and Leukemia Group B (CALGB) also examined this combination.66,67 In CALGB 9712 patients were randomized to receive sequential fludarabine at 25 mg/m2 on days 1–5 of a 28-day cycle that was repeated for a total of six cycles followed by 4 weekly cycles of 375 mg/m2 rituximab given to those patients with stable disease after 2 months or to receive fludarabine given concurrently with 375 mg/m2 rituximab on days 1 and 4 of cycle 1 then on day 1 of the subsequent cycles. This was followed by 4 weekly cycles of rituximab at 375 mg/m2 in those patients with stable disease after 2 months. Patients in the concurrent arm experienced more grade 3 or 4 hematologic toxicity and infusion-related toxicity, but the ORR was 90% with a CR rate of 47% compared with an ORR of 78% and CR rate of 28% in the sequential arm.66 Long-term follow-up data from CALGB 9712 were recently presented. Remissions appeared quite durable with extended follow-up. There also does not appear to be an increase in treatment-related myeloid neoplasms or second cancers.68 Made possible by similar patient characteristics, the results of CALGB 9712 were retrospectively compared to those of CALGB 9011, a study comparing fludarabine and chlorambucil as single agents and their combination.67 Only the fludarabine arm was used in the comparison. Multivariate analyses controlling the pretreatment characteristics demonstrated that patients receiving the FR combination had significantly better 2-year PFS, with 2-year PFS probabilities of 0.67 versus 0.45 and 2-year overall survival probabilities of 0.93 versus 0.81.67 A recent third Italian phase II study of sequential FR confirmed good re-

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sponse rates, with 78% of patients achieving a CR; Zap-70 –positive and CD38⫹ patients had significantly shorter PFS with 25% and 18% at 3 years, respectively.69 The combination of fludarabine, cyclophosphamide, and rituximab (FCR) has been observed to have significant activity in CLL. In a single-arm study, 300 previously untreated patients with progressive CLL were given 375 mg/m2 of rituximab on day 1 followed by 25 mg/m2 of fludarabine and 250 mg/m2 of cyclophosphamide on days 2, 3, and 4 of the first of six 28-day cycles.70,71 The rituximab was increased to 500 mg/m2 and the fludarabine and cyclophosphamide were given on days 1–3 of subsequent cycles. The ORR was 95%, with CR in 72%, nodular partial remission (nPR) in 10%, PR due to cytopenia in 7%, and PR due to residual disease in 6%.71 Two patients (⬍1%) died within 3 months of starting therapy. Six-year overall and failurefree survival rates were 77% and 51%, respectively. Median time to progression was 80 months.71 Pretreatment characteristics associated with inferior response in this trial were age (⬎70 years), beta-2-microglobulin (␤2M) elevated more than twice normal, white blood cell count greater than 150 x 109/L, abnormal chromosome 17 by standard karyotype, and lactate dehydrogenase greater than twice normal. A multivariate analysis of patients receiving serial fludarabine-based treatments at M.D. Anderson Cancer Center demonstrated that receiving FCR therapy was the strongest variable associated with improved survival.71 Toxicity in this study included predominately cytopenias and associated infection. Following completion of therapy, 19% patients had persistent cytopenias lasting more than 3 months. Recurrent late cytopenias were also noted in 28% of patients enrolled on this study. These cytopenias were predominantly noted during the first year of remission. The risk of opportunistic infection was 14% during the first 2 years post-therapy. After this time period, infections were much less frequent and typically bacterial in origin. Eight patients developed myelodysplasia with no patient having been exposed to other cytotoxic therapy. Collectively, this study demonstrates the remarkable activity of the FCR regimen as initial therapy for CLL and also suggests long-term toxicity is modest outside of a low frequency of secondary leukemia. A second study of 177 previously treated patients at this same institution was pursued using the same schedule of FCR.72 The results of this study demonstrated a CR rate of 25% with an ORR of 73%.72 The median time to progression was 28 months, which varied significantly based on response. Specifically, the median times to progression for patients achieving CR, nPR, and PR were 39, 33, and 15 months, respectively. Toxicities observed in this trial were predominately grade 3 or 4 neutropenia (81%) and grade 3 or greater infection (16%). Other adverse events were generally manageable. A retrospective study has recently demon-

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strated that previously untreated CLL patients with the del(11q22.3) appeared to benefit from FCR with loss of the adverse PFS observed in fludarabine monotherapy studies.73 In contrast, patients with IgVH unmutated disease appear to have an inferior outcome with FCR as compared to patients with IgVH mutated disease.74 CALGB 10404 is a randomized phase II study comparing FR, FCR, and FR followed by consolidation therapy with lenalidomide. While both FR and FCR have been demonstrated to improve CR rate, ORR, and PFS, FCR is potentially more myelosuppressive and immunosuppressive than FR. This study is therefore assigning patients with lower risk CLL [those without del(11q22.3)] who have been shown to benefit from cyclophosphamide therapy to these three different therapies. This study is currently ongoing.

PHASE III CHEMOIMMUNOTHERAPY TRIALS The large phase II studies by the M.D. Anderson group described above along with several other small studies by others prompted confirmatory phase III studies. These studies focused on previously untreated disease (CLL8 from the GCLLSG)75 and early relapsed disease (REACH trial)76 have recently been presented and confirm improved response rates and PFS. In CLL8, previously untreated patients were randomized to receive either fludarabine and cyclophosphamide (FC) at 25 mg/m2 and 250 mg/m2, respectively, on days 1–3 of a 28-cycle for six cycles or FCR with rituximab given at 375 mg/m2 during the first cycle of therapy and 500 mg/m2 on day 1 of the second through sixth courses. At a median observation time of 25.5 months, the ORR for FCR was 95% versus 88% for FC, and the CRR was 52% compared to 27% in the FC arm.75 An analysis of genomic subgroups demonstrated particularly poor outcomes in those patients with del(17p13.1); shorter overall survival was seen in the FC arm, as was a trend towards shorter overall survival in the FCR arm in patients with unmutated IgVH.77 This experience was recently updated with a median observation of 37 months. The median PFS for patients receiving FCR was 51.8 months versus 32.8 months for patients receiving FC. The ORRs were virtually identical after the longer observation period, and the CR rates were 44% versus 21.8% for patients receiving FCR and FC, respectively. The greatest benefit was seen in patients with Binet stage A or B disease. In spite of an increased number of hematologic toxicities including neutropenia, an increased infection rate was not observed in the FCR arm, and the number of deaths in the FC arm was greater than that in the FCR arm.78 The REACH trial also compared FC to FCR, this time in relapsed/refractory patients. The dosing schedule in the FC arm was identical to that of CLL8, but the FCR arm included a dose of 375 mg/m2 rituximab with the first cycle. Median observation time was 25 months.

S.M. Jaglowski and J.C. Byrd

Observed PFS in the FCR arm was 30.6 months compared with 20.6 months in the FC arm. An ORR of 70% for FCR was observed, versus 58% for FC alone, and the CR rates were 24% versus 13% for FCR and FC, respectively.76 As observed in earlier studies, hematologic toxicities were the most significant adverse effects in both studies. The French Collaborative Group on CLL and WM (FCGCLL/MW) and the Groupe Ouest-Est d’Etudes Des Leucemies Aigues et Autres Maladies du Sang (GOELAMS) conducted a multicenter French and Belgian trial to evaluate the efficacy of fludarabine and cyclophosphamide in combination with alemtuzumab (FCCam) versus FCR. The CLL2007FMP is a phase III study where 178 fit patients under the age of 65 years without the del(17p) with previously untreated disease were given oral FC with 40 mg/m2 fludarabine on days 1–3 and 250 mg/m2 cyclophosphamide on days 1–3 for six 28-day cycles in combination with either rituximab at 375 mg/m2 on day 0 of the first cycle then 500 mg/m2 on day 1 of subsequent cycles, or alemtuzumab 30 mg subcutaneous on days 1–3 for six cycles. Recruitment to the study was stopped early because of excess mortality in the FCCam arm. The ORR in the first 100 patients evaluated was 96% in the FCR arm versus 85% in the FCCam arm and the CR rates were 78% and 58%, respectively.79 Based on this study, the use of alemtuzumab in place of rituximab in combination with fludarabine and cyclophosphamide in the frontline setting has an unfavorable safety profile and does not represent a valid therapeutic option.

ALTERNATIVE PHASE II CHEMOIMMUNOTHERAPY REGIMENS WITH RITUXIMAB Due to both the delayed cytopenias and less optimal outcome of FCR in elderly CLL patients, alternative dosing strategies have been evaluated. FCR-Lite is one such strategy.80 This regimen was evaluated in previously untreated patients, many of whom were treated in a community setting. The dose of fludarabine was decreased to 20 mg/m2 and cyclophosphamide was decreased to 150 mg/m2, both given on days 1 through 3 of a 28-day cycle for six cycles. The dose of rituximab was increased to 500 mg/m2 every other week, and maintenance rituximab was given every 3 months until relapse. The ORR and CR rates were 100% and 79%, respectively, with a median duration of response of 22.3 months.80 Sequential administration of FCR has also been evaluated. In this trial, all patients received 25 mg/m2 fludarabine for 5 days every 4 weeks. If there was no response after three cycles, they went on to high-dose cyclophosphamide; otherwise, they completed six cycles. Four to 6 weeks after completing fludarabine, patients received cyclophosphamide at 3,000 mg/m2 every 3 weeks for three doses. Approxi-

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mately 4 weeks after this, patients received a second consolidation with 375 mg/m2 rituximab weekly for four doses. The ORR was 89% with a CR rate of 61%. Thirty-three percent of patients achieved a molecular CR. Myelosuppression remained the most significant toxicity, with 26% of patients unable to complete six courses of therapy due to cytopenias.81 Pentostatin is a purine analog that has been suggested to be less myelotoxic than fludarabine while still active in CLL. A study of patients with previously treated CLL82 or other low-grade B cell neoplasms evaluated patients given 4 mg/m2 pentostatin, 600 mg/m2 cyclophosphamide, and 375 mg/m2 rituximab on day 1 of each of six 21-day cycles (PCR combination), with rituximab omitted in the first cycle. Among CLL patients, the ORR was 75% with a CR rate of 25%. The major toxicities were grade 3/4 infections in 28% of patients and grade 3/4 myelosuppression in 53%. The PCR combination was evaluated in previously untreated patients using a dose of pentostatin of 2 mg/m2.83 Ninety-one percent of patients had responses, with a 41% CR rate. Nineteen patients with a CR had less than 1% CD5⫹ and CD19⫹ cells in their bone marrow. Similar to the study with FCR, patients with del(11q22.3) had similar PFS compared with those without this aberration.83 While individuals over the age of 70 were more likely to experience treatment delays with this regimen, no significant differences in ORR, CR rate, and PFS were observed, nor was there a significant difference in the number of cycles administered or grade 3/4 hematologic, infectious, or other toxicities.84 A community-based phase III study comparing FCR to PCR in untreated or minimally treated patients randomized patients to receive either 4 mg/m2 pentostatin with 600 mg/m2 cyclophosphamide and 375 mg/m2 rituximab on day 1 of six 21-day cycles or 20 mg/m2 fludarabine with 600 mg/m2 cyclophosphamide and 375 mg/m2 rituximab on day 1 of six 28-day cycles.85 CR rates were 17% in the FCR group compared with 7% in the PCR group, and the ORR was 57.5% versus 45%, respectively. The CR rate was significantly different, but ORR was not. Infection rates were statistically equivalent.85 The reasons for the extremely low response to both regimens in this study are uncertain but emphasize the importance of developing treatment regimens that can be effectively translated to the practicing community or alternatively directing CLL treatment to centers with significant experience in managing these treatment regimens. Bendamustine is a bifunctional agent consisting of a mechlorethamine group, a benzimidazole ring, and a butyric acid side chain. It has been demonstrated in vitro to induce DNA cross-links and strand breaks, as well as inhibit mitotic checkpoints and induce mitotic catastrophe. A phase II study of 67 patients with relapsed indolent NHL and mantle cell lymphoma, including 10 patients with SLL, was recently published. Patients received 375 mg/m2 rituximab on day 1 followed

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by 90 mg/m2 bendamustine on days 2 and 3 every 28 days for four cycles (BR combination). Additional doses of rituximab were given 7 days before the first cycle and 28 days after the last cycle. Six cycles were permitted if disease regression was evident between the second and fourth cycles. ORR was 92% with 41% CRs, 14% unconfirmed CRs, and 38% PRs.86 This has been further evaluated in a phase III study comparing BR with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) as frontline therapy in patients with follicular, mantle cell, or indolent lymphoma, including SLL. The ORR was similar between the groups, but the CR rate was significantly higher for the BR group at 40.1% compared with 30.8% for RCHOP. The event-free survival was 51 months in the BR group and 31 months with R-CHOP. BR had a more favorable toxicity profile with significantly fewer grade 3 and 4 neutropenic and leukocytopenic events.87 BR was evaluated as a frontline therapy in patients with CLL in the CLL2M study performed by the GCLLSG. Bendamustine was given to 117 patients at a dose of 90 mg/m2 on days 1 and 2 combined with rituximab at 375 mg/m2 on day 1 of the first cycle and 500 mg/m2 on day 1 of subsequent cycles for 6 cycles. At a median observation time of 15.4 months, an ORR of 90.9% was observed with 32.7% CRs, 2.7% nPRs, and 55.5% PRs. Fifty-six of 63 patients with unmutated IgVH had a response. Based on these results, the GCLLSG is initiating CLL10, a randomized phase III study comparing the efficacy of BR to FCR.88

DOUBLE-ANTIBODY CHEMOIMMUNOTHERAPY While this review focuses predominately on rituximab, other therapeutic antibodies are being developed in CLL, including alemtuzumab and lumiliximab, that have been combined with chemoimmunotherapy approaches. The approaches that are most promising are reviewed below. Alemtuzumab is a humanized CD52 antibody that has demonstrated evidence of clinical activity in both upfront and relapsed CLL, including patents with del(17p13.1). Because this antibody targets an alternative antigen than rituximab, investigators at M.D. Anderson Cancer Center combined this with their FCR backbone therapy in previously treated patients with CLL. Seventy-eight patients with relapsed CLL received the CFAR regimen: fludarabine 20 mg/m2 on days 2– 4, cyclophosphamide 200 mg/m2 on days 2– 4, rituximab 375 mg/m2 (cycle 1) or 500 mg/m2 (cycles 2– 6) on day 2, and alemtuzumab 30 mg intravenously on days 1, 3, and 5 every 28 days for up to six cycles.89 Patients received pegfilgrastrim, as well as prophylaxis for Pneumocystis carinii pneumonia (PCP) and cytomegalovirus (CMV). Grade 3– 4 neutropenia was seen in 89% of patients, and 59% developed grade 3– 4 thrombocytopenia. The incidences of major infections (11%),

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minor infections (28%), and fever of unknown origin (36%) were similar to the same institution’s experience with FCR in the relapsed setting. However, prophylactic valganciclovir was significantly more effective in preventing CMV reactivation (three of 30 patients, 10%) than was prophylactic valacyclovir (25 of 48 patients, 52%). The ORR was 65%, with 24% of patients achieving CR. Median PFS was 27 months for the 19 patients achieving CR, compared to only 10 months for the 32 patients attaining PR. Significantly better results were observed in patients who were sensitive to their last fludarabine regimen (ORR 74%, CR 36%), compared to patients refractory to fludarabine (ORR 49%, CR 6%). Given these promising results, a follow-up study in previously untreated, high risk patients (␤2M ⬎2x normal) was initiated.90 This trial enrolled 60 patients, of whom 48 were evaluable for response and follow-up at the time of the initial report. The ORR was 94% and the CR rate was 69%. As compared to FCR, the CFAR regimen had more myelosuppression and CMV reactivation. CFAR has been evaluated in the upfront setting as well. Sixty patients were enrolled on a phase II study and treated according to the regimen described above. CR was achieved in 70% of patients, nPR in 3%, PR in 18%, and no response in 7%, leading to an ORR of 92%. Patients with unmutated IgVH had an ORR of 73%, and those with del(17p) had an ORR of 57%, but the time to progression was significantly shorter in these groups. Infectious complications were similar to those reported with FCR.91 Lumiliximab is a primatized antibody directed at CD23 that mediates apoptosis against CLL cells in vitro and enhances the cytotoxicity of both fludarabine and rituximab in xenograft models of CD23⫹ lymphoma.92 A phase I study of monotherapy with lumiliximab was well tolerated but demonstrated no CRs or PRs.93 We therefore pursued a phase 1/2 dose-escalation study of lumiliximab added to FCR in previously treated CLL patients.94 Thirty-one patients received either 375 mg/m2 (n ⫽ 3) or 500 mg/m2 (n ⫽ 28) of lumiliximab in combination with FCR as administered by the M.D. Anderson Cancer Center group for six cycles. The toxicity profile was similar to that previously reported for FCR in treatment of relapsed CLL. The ORR was 65%, with 52% of patients achieving CR, which compares favorably with the CR rate previously reported for the FCR regimen alone in relapsed CLL. The estimated median PFS for all responders was 29 months. Based on this study, a randomized phase III trial comparing lumiliximab plus FCR with FCR alone is underway to define the benefit of this combination in relapsed CLL.

RITUXIMAB WITH CORTICOSTEROIDS Corticosteroids are active in lymphoproliferative diseases including CLL, with preclinical data suggesting they both disrupt stromal cell interaction and in combination with rituximab induce synergistic apo-

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ptosis.64 Additionally, higher doses of solumedrol have been shown to promote responses in patients with chemotherapy-refractory CLL independent of del(17p13.1).95,96 Based on these date, investigators from the University of California, San Diego CLL group have performed several small pilot studies of solumedrol (1 g/m2 on days 1–5) in combination with rituximab 375 mg/m2 weekly ⫻ 4 in both relapsed97 and previously untreated CLL.98 Among the 14 relapsed patients treated with this regimen,97 the ORR was 93% and the CR rate was 36%. Median time to progression in the relapsed patient population was 15 months. This same regimen in 28 previously untreated patients administered for up to three consecutive treatments was explored, with an ORR of 96% and a 32% CR rate.98 Toxicity with this treatment in both the relapsed and upfront treatment settings was acceptably tolerated provided aggressive infectious prophylaxis was provided. A retrospective analysis of 37 patients with CLL treated with this regimen at the Mayo Clinic found an ORR of 78% and a CR rate of 22%.99 Responses were observed in patients with del(17p13.1).96 Although well tolerated, 11 (29%) patients developed severe infectious complications during the first week of therapy. While this regimen is effective in controlling both del(17p13.1) CLL and disease with bulky lymphadenopathy, it requires significant attention to complications that are commonly observed with corticosteroid therapy: electrolyte abnormalities, hyperglycemia, infectious complications, psychosis, and myopathy.

MAINTENANCE RITUXIMAB The question has arisen as to whether there is a role for maintenance rituximab. Maintenance and consolidation strategies using alemtuzumab have been evaluated by several groups including the CALGB and GCLLSG. While there is an improvement in CR and minimal residual disease (MRD) with consolidation alemtuzumab, this strategy has been fraught with complications, and the GCLLSG phase III study had to be stopped prematurely when seven of the 11 patients randomized to alemtuzumab consolidation developed severe infections.100,101 This was recently reiterated when the final analysis of CALGB 10101 was presented at ASH; five patients in CR after receiving fludarabine and rituximab who were receiving alemtuzumab consolidation died of infectious complications.102 To date, no randomized trials have been performed to determine if benefit is derived from maintenance rituximab. Two separate phase II studies have suggested this approach is feasible. The first study performed by Hainsworth and colleagues treated 44 previously untreated CLL patients with 375 mg/m2 rituximab weekly ⫻ 4.40 Patients with objective responses or stable disease went on to receive repeated four dose cycles at 6-month intervals. ORRs were better than what had

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been previously reported in relapsed/refractory patients, but the median PFS was only 18.6 months.40 A second phase II study of 75 previously untreated patients with CLL evaluated the efficacy of rituximab maintenance following treatment with fludarabine for six cycles.103 All patients received 4 weekly doses of 375 mg/m2 rituximab following therapy, and then those who were MRD-positive went on to consolidation with 4 monthly cycles of 375 mg/m2 rituximab followed by 12 monthly cycles of 150 mg/m2. MRD-positive patients in CR or PR who received consolidation had a significantly longer response duration than a subset of patients who did not receive consolidation (87% v 32% at 5 years). This study identified that patients with Zap-70 positivity, CD38 overexpression, and IgVH unmutated status all had lower likelihoods of improved outcome with rituximab maintenance.103 The infectious complications seen with consolidation alemtuzumab have not been observed in studies evaluating consolidation rituximab.

RITUXIMAB IN THE TREATMENT OF RICHTER’S SYNDROME Richter’s syndrome (RS) is an uncommon complication of CLL, and its prognosis is poor. Two phase II studies have examined the use of riutximab as part of aggressive salvage regimens used to treat this disease. A study evaluating fractionated cyclophosphamide, vincristine, liposomal doxorubicin, and dexamethasone plus rituximab and granulocyte-macrophage colonystimulating factor (GM-CSF) alternating with methotrexate and cytarabine plus rituximab and GM-CSF did not produce better results than an earlier study evaluating hyper-CVAD alone, with an overall survival rate of 38% and a 12-month failure-free survival rate of 27%.104 A phase I-II trial of oxaliplatin, fludarabine, cytarabine, and rituximab (OFAR) was conducted in patients with RS or fludarabine-refractory disease. Patients were given increasing doses of oxaliplatin on days 1– 4 with fludarabine 30 mg/m2 on days 2 and 3, cytarabine 1 g/m2 on days 2 and 3, and rituximab 375 mg/m2 on day 3 of cycle 1 and day 1 on subsequent cycles. The highest tested and tolerated dose of oxaliplatin was 25 mg/m2. The ORR was 50% in RS and 33% in fludarabinerefractory CLL, and responses were seen in seven of 20 patients with del(17p13.1); median duration was 10 months.105 In an attempt to enhance the response rate while decreasing myelosuppression, a follow-up study was conducted where the dose of cytarabine was decreased to 500 mg/m2 and oxaliplatin was increased to 30 mg/m2. Fludarabine and cytarabine were given on days 2– 4 in dose level 2, which was the maximal tolerated dose. Preliminary results indicated an ORR of 63%. Six of 13 patients with del(17p13.1) responded.106 While these studies suggest rituximab can be effec-

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tively added to regimens commonly used to treat RS, it is unclear if this adds any benefit.

RITUXIMAB IN THE TREATMENT OF AUTOIMMUNE COMPLICATIONS Autoimmune complications of CLL are well recognized and are reported in about 10% to 25% of patients during their disease course. These complications are both an intrinsic characteristic of CLL and potentiated by its therapy, mainly fludarabine. Autoimmune hemolytic anemia (AIHA) is the most common complication, followed by immune thrombocytopenia (ITP).107 Rituximab was initially described for the treatment of corticosteroid-refractory pure red blood cell aplasia or AIHA108; subsequently, the successful treatment of two patients with CLL who developed red blood cell aplasia with 375 mg/m2 rituximab weekly for 2 weeks was described.109 Several case reports of patients with AIHA who have been successfully treated with rituximab have been reported.110 –112 A series of eight patients with CLL and corticosteroid-refractory AIHA was treated with a combination of rituximab and dexamethasone, and all patients achieved a remission of their AIHA, with five patients achieving a Coombs-negative status. Re-treatment was also found to be successful.113 In a series of 14 patients with CLL and AIHA treated with rituximab monotherapy, all but two patients had an increase in their hemoglobin levels after treatment.114 Rituximab is effective in patients with chronic refractory ITP as well. Twenty-five patients with ITP, including eight who had failed splenectomy, were treated with rituximab at 375 mg/m2 weekly ⫻ 4, and 52% of patients responded; five had a CR.115 Rituximab has been evaluated in CLL specifically, as fludarabinerelated ITP is not responsive to corticosteroids. In a small series of patients, it was shown to be helpful in quickly reversing fludarabine-associated ITP.116 While randomized data demonstrating the exact benefit of rituximab in autoimmune complications is lacking, these data provide evidence for the effectiveness of this treatment. There is no data at present to suggest that the use of rituximab in combination regimens has reduced the incidence of autoimmune cytopenias in CLL, particularly those that occur with fludarabine. In the authors’ opinion, rituximab represents one of the more active therapies for the treatment of autoimmune complications of CLL not responding to initial corticosteroid treatment.

COMPLICATIONS OF RITUXIMAB THERAPY Rituximab is generally safe and well tolerated, but it has been associated with significant adverse events. In the phase II studies, it was commonly associated with fever, chills, rigors, and rarely bronchospasm, all of which tended to be self-limited and confined to the first

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one or two doses of treatment.23 While early studies suggested high blood tumor load may be predictive of infusion toxicity, subsequent studies have shown no relationship to this in CLL. This suggests that physicians administering rituximab to CLL patients must consider every CLL patient at risk for this toxicity and monitor closely. In this author’s opinion, the frequency of serious infusion toxicity with rituximab can be greatly diminished by administration of dexamethasone (20 mg) prior to the first dose of rituximab and by splitting the first dose over 2 days. Fatal reactivation of hepatitis B (HBV) after rituximab has been described in a small subset of CLL patients receiving this treatment.117–122 Because of this complication, screening for evidence of hepatitis B and C prior to treatment is recommended. For patients with past exposure to hepatitis B, close monitoring during and after therapy should be performed, as well as consideration of prophylaxis against re-activation. Organizing pneumonia and interstitial pneumonitis have been reported with rituximab, both alone and in combination with chemotherapy.123–125 Recently, cases of progressive multifocal leukoencephalopathy (PML) following rituximab use have been described; PML has a case-fatality rate of 90%, although it is extremely rare.126 –129

NEW DIRECTIONS AND COMBINATION STRATEGIES WITH RITUXIMAB While rituximab has generated significant promise for the treatment of CLL when incorporated into combination-based regimens, its effect as a monotherapy is somewhat limited. As such, investigators have explored a variety of strategies to enhance the mechanisms of killing that rituximab uses to eliminate CLL cells. To enhance complement-mediated killing, investigators have altered the schedule and dose of administration to diminish the influence of CD20 shaving on CLL cells.46,50 In addition, investigators have infused fresh frozen plasma to replete complement and enhance the efficacy of rituximab.130,131 Relative to ADCC, administration of therapeutics that enhance ADCC (lenalidomide132,133, IL-21134) or ADCP (CpG oligonucleotides, TLR7 angonists) has been pursued preclinically and, in some cases, in early phase I clinical trials in combination with rituximab. In this regard, IL-21 represents one of the most promising therapeutics51 as it both enhances autologous NK cell ADCC and promotes apoptosis through a Bcl-2–interacting mediator of cell death (BIM)-dependent pathway in CLL cells. In contrast, other therapeutic agents such as lenalidomide that activate both CLL cells and the innate immune system under certain conditions can downregulate CD20 and antagonize both rituximab ADCC and direct apoptosis with cross-linking.133 Nonetheless, this activation-induced CD20 internalization promoted by lenalidomide might offer the opportunity to effectively de-

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liver RNA-based therapy to CLL cells, as our group has previously demonstrated. These collective noninclusive examples of future strategies to combine rituximab with other therapeutics exemplifies the significant amount of work that remains in order to fully appreciate the complete potential of rituximab in CLL therapy more than a decade after its approval for this disease.

CONCLUSION The chimeric antibody rituximab binds to CD20 and effectively mediates CDC, ADCC, and direct apoptosis in some settings. The contribution of each of these mechanisms to rituximab tumor clearance in CLL remains unknown and requires further study. Rituximab, when administered in its optimal schedule, has demonstrated monotherapy activity in both untreated and previously treated CLL. Rituximab’s greatest contribution to CLL therapy is in combination, both in symptomatic, previously untreated CLL patients and in those with relapsed disease. In both of these settings, phase III studies have demonstrated improved ORR, CR rate and PFS as compared to chemotherapy alone. Rituximab also represents a very helpful therapy in managing autoimmune complications of this disease. To this point, insufficient data exist to recommend maintenance rituximab in CLL and this should not be considered out of the context of a clinical trial.

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