Efficiency of in vivo purging with rituximab prior to autologous peripheral blood progenitor cell transplantation in B-cell non-Hodgkin’s lymphoma: a single institution study

K. Belhadj1,*, M.-H. Delfau-Larue2, T. Elgnaoui1, F. Beaujean3, J.-L. Beaumont3, C. Pautas1, I. Gaillard1, Y. Kirova4, A. Allain1, P. Gaulard5, J.-P. Farcet2, F. Reyes1 and C. Haioun1

1 CHU Henri Mondor, Service d’Hématologie Clinique; 2 CHU Henri Mondor, Laboratoire d’Immunologie; 3 CHU Henri Mondor, Unité de Thérapie cellulaire EFS; 4 CHU Henri Mondor, Service de Radiothérapie; 5 CHU Henri Mondor, Service d’Anatomo-pathologie, Créteil, France

Received 20 May 2003; revised 1 September 2003; accepted 7 November 2003;


    ABSTRACT
 Top
 ABSTRACT
 Introduction
 Patients and methods
 Results
 Discussion
 REFERENCES
 
Background:

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


    Introduction
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 ABSTRACT
 Introduction
 Patients and methods
 Results
 Discussion
 REFERENCES
 
Myeloablative therapy with bone marrow or peripheral blood progenitor cell transplantation may play an important role in the treatment of follicular lymphoma (FL) [1], mantle cell lymphoma (MCL) [2] and aggressive lymphoma [35]. A major concern in autografting programs is that cell harvest contains occult tumor cells that may contribute to disease relapse. This is particularly relevant in follicular, mantle cell or marginal zone lymphomas (MZL) in which bone marrow infiltration is a common feature.

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-Hodgkin’s 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.


    Patients and methods
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 ABSTRACT
 Introduction
 Patients and methods
 Results
 Discussion
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Patients
Between May 1998 and March 2001, 14 consecutive patients entered in a pilot unicentric study consisting of four courses of rituximab followed by HDT with autologous stem cell support. They included 11 patients with refractory/relapsed FL requiring salvage treatment and three patients in which consolidative HDT was delivered following first-line induction [two with transformed marginal zone lymphoma (tMZL) and one with MCL]. Eligibility criteria included histologically confirmed diagnosis, expression of CD20 by lymphoma cells, molecular marker detectable by PCR amplification of DNA in order to assess minimal residual disease (MRD), age ≤60 years, absence of severe organ dysfunction not related to tumor, and informed consent. As shown in Table 1, all the patients except one had bone marrow involvement and all had received an anthracyclin-containing regimen as first-line treatment.


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Table 1. Patient characteristics at diagnosis
 
Treatment plan
Figure 1 shows the treatment plan. Rituximab 375 mg/m2 was administered by slow intravenous infusion following standard guidelines at weekly intervals for 4 consecutive weeks. Premedication with an antipyretic (paracetamol) and antihistamine (dexchlorpheniramine) was given in association with corticosteroids. For the first course, rituximab was infused at an initial rate of 50 mg/h then escalated by regular increments of 50 mg/h every 30 min to a maximum of 400 mg/h. For subsequent infusions, rituximab was started at 100 mg/h and increased by steps of 100 mg every 30 min up to 400 mg/h. The infusion was interrupted if fever, chills, oedema, mucosa congestion, hypotension, or any other serious adverse event occurred.



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Figure 1. Treatment plan.

 
Mobilization therapy and graft collection
PBPC were collected during the hematological recovery phase following a specific mobilization regimen consisting of cyclophosphamide 4500 mg/m2 and etoposide 450 mg/m2 at day 1 (d1) in three divided doses, by 1 h infusion each. All patients received granulocyte colony-stimulating factor at a dosage of 5 µg/kg s.c. from day 6 after the start of the mobilization regimen until the completion of leukaphereses. The hematological recovery phase was assessed by daily CD34+ cell count and leukaphereses were started when they were >20/µl. Harvesting of PBPC from all patients was performed on a continuous flow cell processor (Cobe Spectra, Cobe BCT, Lakewood, CO). The median blood volume processed was 10745 ml (range 8624–14156) and the median number of CD34+ cells harvested was 5.3 x 106/kg (range 2.6–20.7).

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 phenol–chloroform 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].


    Results
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 ABSTRACT
 Introduction
 Patients and methods
 Results
 Discussion
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Patient characteristics
The main characteristics of the 14 enrolled patients are listed in Table 1. Histologic bone marrow involvement was documented in 13 cases and the presence of lymphoma cells on peripheral blood smears in five cases. A skin involvement was seen in one case of FL. The eleven patients with FL had reached either a partial (six cases) or a minor (five cases) response after first line treatment and, at the time of enrollment in the study, had disease progression requiring salvage treatment. The three remaining patients (two with tMZL, one with MCL) were enrolled after they had reached PR following first-line induction treatment.

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 3–21) x 106 CD34+ cells/kg were collected, with a median of one (range 1–3) 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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Table 2. Characteristics of the 14 patients before and after treatment
 
Molecular response to treatment
As indicated in Table 3, PCR status in peripheral blood after rituximab alone is available in only nine of our 14 patients. Conversion to negative PCR was observed in four patients, three of them remaining in molecular remission at last follow-up. Molecular detection of MRD after rituximab followed by the mobilization chemotherapy regimen was performed in harvests in 11 out of the 14 enrolled patients and revealed that no PCR-tumor-specific marker was amplifiable in nine (82%). After HDT, PCR analysis of peripheral blood samples showed that these nine patients were in molecular remission. It is noteworthy that the two remaining patients with FL who were reinfused with a PCR-positive harvest were, as expected, positive in peripheral blood after HDT. Finally, two of the latter three patients with no available molecular study in harvest were PCR negative in peripheral blood after HDT.


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Table 3. PCR analysis in peripheral blood before and after rituximab, in harvest after mobilization chemotherapy and follow-up of minimal residual disease in peripheral blood after high-dose therapy (HDT)
 
Rituximab-related toxicity
All patients tolerated the four, weekly scheduled infusions well. Non-hematological toxicity was observed during the first infusion, consisting of grade 2 or 3 pain in two patients and grade 2 perspiration in another. These side-effects were of brief duration and easily managed by adjustment of the infusion rate. Hematological toxicity occurred later and consisted of a grade 3 neutropenia after the second infusion in one patient and a grade 1 thrombocytopenia after the third infusion in another one who quickly recovered, and did not prevent the subsequent administration of scheduled infusions.

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 8–19). 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 16–55). Hematological engraftment was rapid with a median neutrophil recovery time of 14 days (range 12–17) and self-maintaining platelet count of 12 days (range 9–17). The median number of platelet and erythrocyte transfusions were two (range 1–7) and two (range 0–14), 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).


    Discussion
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 ABSTRACT
 Introduction
 Patients and methods
 Results
 Discussion
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Bone marrow involvement is difficult to eradicate in indolent lymphoma and concerns have been raised for more than a decade about the significance of persistent malignant cells in harvests of candidates for autologous hematopoietic rescue [21]. It is now generally accepted that contamination of peripheral blood stem cell collections by tumor cells may contribute to relapse, on the basis of studies showing that the presence of minimal residual disease in reinfused marrow is predictive for relapse in patients with FL [22, 23]. A study of 113 patients with informative relapsed follicular lymphoma [9] has shown that patients with autotransplanted bone marrow rendered PCR-negative for bcl2/IgH rearrangement after ex vivo purging experienced longer freedom from relapse than those with PCR-positive bone marrow. Recently, a multicenter Italian trial [24] that evaluated the efficacy of an intensified and prolonged high-dose sequential regimen—considered as an in vivo purging procedure—in untreated patients with follicular lymphoma showed that disease-free survival of the autotransplanted patients whose harvests were PCR negative was significantly superior to that of patients whose harvests were PCR positive.

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-Hodgkin’s 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.


    FOOTNOTES
 
* Correspondence to: Dr K. Belhadj, Hôpital H. Mondor, 51 Avenue de Lattre de Tassigny, 94010 Creteil, France. Tel/Fax: +33-1-49-81-21-71; E-mail: karim.belhadj{at}hmn.ap-hop-paris.fr Back


    REFERENCES
 Top
 ABSTRACT
 Introduction
 Patients and methods
 Results
 Discussion
 REFERENCES
 
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