1 CHU Henri Mondor, Service dHématologie Clinique; 2 CHU Henri Mondor, Laboratoire dImmunologie; 3 CHU Henri Mondor, Unité de Thérapie cellulaire EFS; 4 CHU Henri Mondor, Service de Radiothérapie; 5 CHU Henri Mondor, Service dAnatomo-pathologie, Créteil, France
Received 20 May 2003; revised 1 September 2003; accepted 7 November 2003;
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ABSTRACT |
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Rituximab induces clinical response in advanced B-cell lymphoma and is efficient in removing circulating B-cell from peripheral blood. We therefore postulated that rituximab might be a useful in vivo purging agent before high-dose therapy in this setting.
Patients and methods:
Fourteen patients with relapsed follicular, marginal zone and mantle cell lymphomas (11, two and one cases, respectively) and a PCR-detectable molecular marker were treated first with rituximab, then a mobilization chemotherapeutic regimen, followed by high-dose therapy with peripheral blood stem cell transplantation. PCR analyses were performed in peripheral blood before rituximab and during follow-up, and in harvest.
Results:
Harvests were free of PCR-detectable molecular marker in nine of the 11 studied cases (82%). After high-dose therapy, clinical complete remission was obtained in 13 (93%) patients and molecular remission in 11 (79%). With a median follow-up of 3 years, the 14 transplanted patients were alive, 11 of them remaining in clinical complete remission and eight in molecular remission at last follow-up.
Conclusion:
Rituximab treatment followed by high dose therapy appears to be effective in achieving complete clinical and molecular response. In vivo harvest purging is predictive of prolonged clinical and molecular remission.
Key words: B-NHL, high-dose therapy, PCR, purging, rituximab
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Introduction |
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With the aim of eliminating malignant cells from the graft, various ex vivo techniques involving the use of monoclonal antibodies or chemotherapeutic drugs have been developed [610]. While generally effective, the purging procedures are labor intensive, delay hematopoietic recovery after transplantation and substantially increase the costs of treatments [11].
CD20 is a B-cell-surface protein expressed on >95% of normal and neoplastic B cells. Treatment with the humanized CD20 antibody rituximab (IDEC-C2BS) has an effective anti-tumor activity in patients with relapsed B-cell non-Hodgkins lymphoma (NHL) [12, 13]; moreover it has been reported that some patients could achieve a molecular response, suggesting that this treatment may also clear circulating lymphoma cells [14].
In the present study, we investigated the ability of rituximab followed by high-dose therapy (HDT) with peripheral blood progenitor cell (PBPC) transplantation to induce durable clinical and molecular complete remission in patients with refractory/relapsed FL, or as a consolidative treatment after anthracyclin containing regimen in MCL or transformed MZL.
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Patients and methods |
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High-dose therapy and supportive care
The conditioning regimen in the 14 patients consisted of cyclophosphamide 60 mg/kg on days 6 and 5, etoposide 300 mg/m2 on days 6, 5 and 4, and 1000 cGy total body irradiation in a single fraction dose on day 1, followed by PBPC infusion on day 0. All patients had an indwelling central venous catheter and were housed in laminar air-flow rooms for the duration of aplasia. No hematopoietic growth factors were administered systematically after PBPC infusion. No prophylactic antiviral or oral antibiotic therapy was given, but patients were treated with intravenous broad spectrum antibiotics if they developed fever and had an absolute neutrophil count (ANC) <0.5 x 109/l. All patients received irradiated packed red blood cells and platelet products to maintain a hemoglobin level >8 g/dl and a platelet count >20 x 109/l. Time to hematological reconstitution was defined as the median time to reach an ANC of 0.5 x 109/l and an unsubstituted platelet count of 20 x 109/l.
Molecular marker identification
Characterization of tumoral marker was performed on involved lymph node, blood, marrow or skin. When no tumoral tissue was available, a marker was considered as tumoral marker when it was detected on two consecutive blood samples.
DNA was obtained by a standard proteinase K digestion and a phenolchloroform extraction. For immunoglobulin gene rearrangement analysis, two PCRs were performed as described previously [15]. Briefly, in one PCR, the 5' sense oligonucleotide matched homologous sequences within framework 2 region [FR2: GC(C/T) (C/T)CC GG(A/G) AA(A/G) (A/G)GT CTG GAG TGG], and in the second PCR it matched homologous sequences within framework 3 region [FR3: ACA CGG C(C/T)(G/C) TGT ATT ACT GT]. In the two PCRs, the 3' antisense oligonucleotide hybridized the JH region (ACC TGA GGA GAC GGT GAC C). After 40 cycles, 1% of the amplified products were run on a ABI prism 310 genetic Analyzer (Applied Biosystems). Results were analyzed using Genscan analysis software (Applied Biosystems). Samples with a unique dominant peak within the polyclonal background were considered clonal and the clonal population was characterized by its CDR3 size.
For t(14,18) analysis, upstream primer matched the Bcl2 gene, either in the MBR (TAT GGT GGT TTG ACC TTT A) or mcr (GAC TCC TTT ACG TGC TGG TAC C) region, and downstream primer matched the JH segments (ACC TGA GGA GAC GGT GAC CAG GGT). Major translocation cluster (MTC) t(11,14) was analyzed as described previously [16]. After 40 PCR cycles, translocation-derived PCR products were run on a 1.5% agarose gel, stained with ethidium bromide and visualized under UV light.
PCR analysis of residual disease
Molecular monitoring of residual disease was performed on harvests and peripheral blood mononuclear cells (PBMCs). Immunoglobulin heavy chain gene rearrangement was used as tumoral marker when no translocation-derived DNA could be amplified. Sensitivity was assessed by serial dilution of RL cell line (kindly provided by C. Bastard) for MBR t(14,18) or massively invaded tumoral tissue for mcr t(14,18), MTC t(11,14) and IgH rearrangement in healthy donor PBMC, and was 1/105, 1/104, 1/104 and 1/102, respectively.
Monitoring
At baseline, before rituximab treatment, patients were staged by means of physical examination, serum chemistries, full blood counts, bone marrow biopsy, body scan and PCR analysis on blood samples. Clinical evaluation of response was performed 1 month after the last cycle of rituximab. Restaging studies, including MRD assessment in peripheral blood, were repeated 2 months after completion of HDT and then at 6-month intervals during the first 2 years of follow-up, with restaging including bone marrow biopsy once a year, and then yearly or in the case of new symptoms. Complete response (CR) was defined as the disappearance of all sites of disease including bone marrow biopsy in the case of initial involvement. Undetermined complete response (CRu) was defined as clinical complete response when bone marrow biopsy was not performed. Partial response (PR) was defined as 50% to 75% reduction in tumor volume. Stable disease was defined as less than PR and progressive disease required 50% increase in tumor volume and/or appearance of any new lesion during or at the end of therapy.
Toxicity
The severity of side-effects that occurred during rituximab treatment, mobilization regimen and HDT were assessed according to the World Health Organization scoring system [17].
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Results |
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Stem cell mobilization and harvesting of CD34-positive cells
The mobilization regimen was well tolerated. Three out of the 14 patients developed neutropenic fever, which resolved after antibiotic therapy. A median number of 6 (range 321) x 106 CD34+ cells/kg were collected, with a median of one (range 13) leukapheresis.
Clinical response to treatment
Table 2 shows that after four courses of rituximab, one patient was in CR, two in CRu (bone marrow biopsy not available), nine in partial response and one failed to respond. The latter patient (case 5) was subsequently treated with two courses of fludarabine leading to a partial response. After completion of HDT, 12 patients achieved a complete response, one patient was in CRu and the last one was in partial response.
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High-dose therapy-related toxicity
All 14 patients developed neutropenic fever. One patient (case 12) had bacterial documented infection (Klebsiella oxytoca). Fever and/or infections were successfully managed in all patients with broad-spectrum antibiotics. The median duration of intravenous antibiotics administration after HDT was 14 days (range 819). One patient (case 5), treated with two courses of fludarabine after rituximab failure, developed a serious adverse event (SAE) consisting of herpes simplex pneumonia and encephalitis, which recovered under treatment with aciclovir. Two other reversible SAEs consisted of cardiac arrhythmia (case 5) and acute pulmonary embolism (case 6). Grade 3 mucositis requiring morphine occurred in two patients. The median duration of hospitalization for HDT was 25 days (range 1655). Hematological engraftment was rapid with a median neutrophil recovery time of 14 days (range 1217) and self-maintaining platelet count of 12 days (range 917). The median number of platelet and erythrocyte transfusions were two (range 17) and two (range 014), respectively.
Clinical and molecular follow-up and outcome
With a median follow-up of 3 years, all 14 enrolled patients are alive. Eight of them remained in clinical and molecular remission after HDT without additional treatment. Three patients with FL progressed to 5 months (case 3) and 6 months (cases 8 and 11) after HDT. Two of them never achieved molecular remission (cases 3 and 8) and one was PCR-negative in peripheral blood after HDT, but converted to a PCR-positive status while in disease progression; these patients were salvaged by an anthracycline-based chemotherapy regimen combined with rituximab and are considered in clinical complete remission at the time of analysis. The third patient (case 11) was treated by rituximab alone and is also in clinical complete remission. The three remaining patients are in clinical complete remission 54, 41 and 38 months after enrollment in the study; two of them (cases 1 and 6) were in molecular remission after HDT and converted to a PCR-positive status 36 and 12 months after HDT, respectively, and the third patient (case 5) never achieved a PCR-negative status (Table 3).
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Discussion |
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However, it must be recognized that on one hand, ex vivo purging techniques have limitations since they are labor intensive, expensive and may delay bone marrow recovery and immune reconstitution, and that on the other hand, in vivo purging by chemotherapy may hamper the subsequent collection of progenitors [2528].
Rituximab immunotherapy offers an active, non-toxic treatment for relapsed and refractory low-grade non-Hodgkins lymphomas [12] and is efficient in removing B-cells from peripheral blood [14]. This has suggested that rituximab administration used for in vivo purging before mobilization chemotherapy might constitute an alternative to ex vivo techniques [29, 30].
In the present study, we evaluated the feasibility, toxicity and efficacy of four courses of rituximab followed by HDT with autologous stem cell support for the treatment of patients with B-cell lymphoma.
Rituximab can be safely associated with conventional chemotherapy such as CHOP [3133], but little data are available to date regarding the safety of rituximab given before, or in combination with, intensified chemotherapeutic regimens [29, 30, 34]. Our results indicate that rituximab followed by HDT is a feasible treatment option since all patients successfully completed the procedure. Rituximab treatment did not adversely affect either yield of PBPC collection or time of engraftment.
Toxicity was another potential concern in this setting, due to the risk of delivering HDT to patients with profound and durable B-cell depletion. In this series, only one serious viral infection was observed in a patient who had received fludarabine after rituximab failure, thus resulting from major cellular immunity impairment. No other severe infectious complications were seen in the remaining 13 patients.
Three recent pilot studies performed in patients with low grade lymphoma or MCL have emphasized that the use of rituximab in addition to chemotherapy provides efficient depletion of tumor cells in leukapheresis products. Flinn et al. [35] reported on the efficacy of rituximab delivered just before cyclophosphamide as an in vivo purging agent, since six of seven stem-cell products tested were free of tumor contamination. On the basis of historical comparisons, Voso et al. [30] added four doses of rituximab to a sequential HDT program and found that harvests of all informative patients were PCR-negative, whereas this was observed in only 26% of patients previously treated with the same HDT program without rituximab [36]. Similar results were obtained by Magni et al. [29], who found that adding rituximab to a high-dose inductive sequential regimen based on cyclophosphamide and cytarabine significantly increased PCR-negativity of harvests (93% versus 40%; P = 0.007). Although there is no uniform modality of treatment or timing, our results obtained from a homogeneously treated series of patients are in line with such observations. Indeed, following rituximab and mobilization chemo-therapy, in vivo purging was obtained in nine (82%) out of 11 patients whose harvest could be studied, and such a PCR-negative status appears to be predictive of prolonged clinical and molecular remission.
We conclude that rituximab, by increasing the complete response rate and by ensuring in vivo tumor depletion of harvests, may improve the results of a consolidative high-dose therapy in poor-risk patients. Longer follow-up will help to clarify the potential long-term benefit of such an approach over rituximab-free autografting programs.
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FOOTNOTES |
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