In vivo Purging

Rituximab in vivo purging is safe and effective in combination with CD34-positive selected autologous stem cell transplantation for salvage therapy in B-NHL


The purpose of this study was to evaluate feasibility and efficacy of Rituximab included into a sequential salvage protocol for CD20+ B-NHL in relapse or induction failure. Twenty-seven patients with CD20+ B-NHL in relapse or induction failure received Rituximab combined with DexaBEAM (R-DexaBEAM) for stem cell mobilization. Additional ex vivo selection of CD34-positive cells was performed using the CliniMacs device. Two doses of Rituximab were included in the high-dose therapy regimen (HDT). R-DexaBEAM was well tolerated and 26 of 27 patients mobilized sufficient numbers of CD34+ blood stem cells. Application of R-DexaBEAM resulted in significant depletion of peripheral B cells. No treatment-related deaths occurred after HDT and all patients showed stable engraftment of hematopoesis. Combined immunodeficiency was observed post HDT and eight patients developed CMV antigenemia. Remission rate post HDT was 96% (CR, 24/26; PR, 1/26). Overall and progression-free survival (PFS) at 16 months post HDT (range 6–27) is 95% and 77%, respectively. With regard to histology, PFS was 71% in aggressive lymphoma (n = 11), 74% in indolent FCL (n = 10) and 100% in MCL (n = 5). The treatment protocol has proven feasible, with high purging efficiency and encouraging remission rates.


High-dose cytotoxic therapy with autologous hematopoietic stem cell support has been proven effective therapy in B cell non-Hodgkin's lymphoma (NHL) in chemosensitive relapse or induction failure.1,2,3,4 However, relapse of lymphoma remains the major cause of treatment failure, and gene marking studies have established the contribution of residual tumor cells in blood stem cell products of patients with different hematologic malignancies, to recurrence of underlying disease.5,6 In addition, it has been shown, that effective ex vivo purging correlates with prolonged disease-free survival in patients with follicular lymphoma, who have undergone autologous bone marrow transplantation.7

The clinical development of the chimeric anti-CD20 monoclonal antibody Rituximab has added a new and effective agent for killing lymphoma B cells, and leads to rapid and sustained depletion of peripheral blood B cells.8,9 Czuzman et al10 first described a combination of Rituximab and chemotherapy to be feasible, resulting in high remission rates. This sparked a rapidly expanding number of trials using the antibody at different steps in lymphoma treatment. B cell depletion caused by Rituximab might improve the in vivo purging capacity of chemotherapeutic regimens used for stem cell mobilization, and recent reports indicate that this is true for a proportion of patients.11,12,13,14,15,16

Combining Rituximab, included into a sequential salvage therapy protocol, and additional ex vivo selection of peripheral blood stem cells, involves the risk of increasing transplant morbidity due to possibly delayed reconstitution of the cellular and humoral immune system. Accordingly, we included prospective monitoring of viral reactivation and reconstitution of the immune system, into our study protocol, which was mainly focused on purging efficiency and clinical outcome.

Patients and methods

Patient population

Between July 1998 and March 2000, 32 consecutive patients with CD20+ B-NHL in relapse or after failure of induction treatment were screened for possible inclusion into the study protocol. All patients received a first cycle of salvage chemotherapy (DexaBEAM) to prove chemosensitivity of their disease. A minimum response of stable disease was required for inclusion into the study protocol. Five patients with aggressive lymphoma who showed disease progression under therapy were therefore not included.

The study population consisted of 10 patients with indolent follicle center lymphoma (FCL) grade I/II, two with FCL grade III, five with mantle cell lymphoma (MCL), two with aggressive (transformed) marginal zone lymphoma (MZL) and eight with diffuse large cell lymphoma (DLCL). Fourteen patients had first or subsequent relapse after anthracycline-containing regimens (CHOP or MCP). Thirteen further patients failed conventional dose anthracycline-containing chemotherapy. Progressive disease (PD) on therapy was seen in two patients, four patients had stable disease or partial remission and rapidly progressed after cessation of therapy, seven patients displayed stable disease (n = 5), or PR (n = 2) with clinically relevant tumor masses after induction treatment. Median number of conventional anthracycline-based cycles per patient was six. Four patients had received previous radiation therapy.

The remission rate after one cycle of DexaBEAM in the remaining 27 patients was 55%, with 10 partial and five complete remissions, and 12 patients displaying stable disease.

All patients had given informed consent for the treatment protocol, which had local ethics committee approval. Summarized patient characteristics are shown in Table 1.

Table 1 Patient characteristics

Mobilization therapy, graft collection and cell processing

All patients received mobilization chemotherapy consisting of DexaBEAM (dexamethasone 8 mg three times a day days 1 to 10, BCNU 60 mg/m2 day 2, etoposide 75 mg/m2 days 4 to 7, cytarabine 2 × 100 mg/m2 days 4 to 7, melphalan 20 mg/m2 day 3) and Rituximab (Hoffmann-La Roche, Grenzach, Germany; 375 mg/m2 day 1). Starting on day 11, G-CSF 10 μg/kg bw sc. was given daily until completion of leukapheresis procedures. Leukaphereses were performed until a minimum of 6 × 106/kg bw CD34-positive cells were collected. Leukapheresis was performed on a continuous-flow blood cell separator (Cobe Spectra, Lakewood, CO, USA). Either single or pooled PBSC collections of at least 4 × 106/kg bw CD34-positive cells were processed using the CliniMACS device (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer's instructions.17 Enriched cells were cryopreserved with DMSO 7.5% and PBS/HSA 4%, heparin 10 U/ml. Cell suspensions were frozen at a controlled rate in a programmed freezer (Cryoson, Schöllkrippen, Germany) and were stored in liquid nitrogen until reinfusion. In addition, an unprocessed backup of at least 2 × 106/kg bw CD34-positive cells was cryopreserved. Grafts from patients in whom the target number of 6 × 106 CD34+ cells could not be reached, were cryopreserved without further in vitro manipulation, until reinfusion.

Conditioning regimen, PBSC reinfusion, supportive care

The pretransplant conditioning regimen in 18 patients consisted of fractionated total body irradiation (TBI) (14.4 Gy in 12 doses over 4 days) and cyclophosphamide (120 mg/kg). Six patients received high-dose BEAC chemotherapy consisting of carmustine (600 mg/m2), etoposide (1600 mg/m2), cytosine arabinoside (24 g/m2) and cyclophosphamide (90 mg/kg). Two further patients received high-dose BEAM consisting of carmustine 300 mg/m2, etoposide 400 mg/m2, cytosine arabinoside 800 mg/m2, melphalan 140 mg/m2. All patients received Rituximab (375 mg/m2) on day −10 and day −3.

Forty-eight hours after chemotherapy, the cryopreserved leukapheresis products were reinfused. Patients received prophylactic oral antibiotic therapy with ofloxacin. iv. antibiotic combination therapy was administered for fever exceeding 38.3°C and amphotericin B was added for documented fungal infection or persistent fever. Intravenous immunoglobulins were given to maintain IgG serum levels above 4 g/l until day +100. Patients were screened weekly until day +100 for CMV DNAemia or antigenemia (pp65). Pre-emptive antiviral therapy was started when pp65 antigen was detected in peripheral blood samples (CMV) or clinical signs of reactivation occurred (VZV). Supplements of packed red blood cells or platelets were administered for hemoglobin levels below 8 g/dl or platelet count below 10/nl.


Flow cytometric analyses for evaluation of CD34-positive cells, lymphoma cells and T, NK cell subsets in whole blood or leukapheresis products were performed by the direct immunofluorescence technique according to the slightly modified ISHAGE guidelines for CD34+ cell enumeration and CD4 quantitation.18,19 Cell sorting experiments were performed using the Coulter Epics XL-MCL flow cytometer (Krefeld, Germany). PE-or FITC-conjugated antibodies were purchased from Beckman-Coulter pharmaceuticals (Krefeld, Germany). Cell viability was evaluated by Trypan Blue and/or propidium iodide (PI) staining.

Monitoring of MRD

Nested PCR amplification at the major breakpoint region of the bcl-2/IgH rearrangement of t(14;18) was performed according to the method described by Gribben et al.20 Serial samples of blood and bone marrow were analyzed starting before salvage therapy and post HDT. Samples of unmanipulated and purged grafts were also analyzed.

Definitions, statistical methods

Response evaluation referred to the proposed response criteria of an International Workshop.21 Overall survival time was considered as the time between reinfusion of blood stem cells and death. Progression-free survival time (PFS) was considered the time between reinfusion of blood stem cells and occurrence of relapse or disease progression. Survival curves were estimated using the method described by Kaplan and Meier.22 Event-free (death or progression) subjects were censored at the time-point of their last follow-up visit.

To compare grouped samples Mann–Whitney U or Fisher's exact test were used whenever appropriate. An exact two-tailed P value <0.05 was considered significant.


Blood stem cell mobilization and processing

The mobilization regimen consisting of Rituximab and DexaBEAM chemotherapy was well tolerated. No Rituximab infusion-related severe adverse events and no non-hematopoetic grade III/IV (WHO) toxicity were observed. Twelve patients developed neutropenic fever which resolved on antibiotic therapy or with leukocyte regeneration, including one patient with interstitial pneumonia who tested positive for CMV-DNA on PCR analysis of broncho-alveolar fluid. In 26 of 27 patients, sufficient numbers of CD34-positive blood stem cells (>2 × 106/kg bw) were mobilized to proceed with the study protocol. One patient, in whom previous treatment included extensive radiation therapy, failed to mobilize the requisite number of CD34-positive cells, and was excluded from the study protocol.

With a median of 2 (range 1–4) leukapheresis procedures, a median of 9.3 (range 2.2–36) × 106/kg bw CD34-positive cells were collected. In 19 of 27 patients, sufficient numbers of CD34-positive cells were harvested to perform additional ex vivo purging with the Clini- MACS device.

The use of R-DexaBEAM resulted in a significant decrease in peripheral CD19+ B cells (P = 0.004). In three out of 26 subjects, CD19+ cells were detected in peripheral blood samples from the day of (first) leukapheresis or in the unmanipulated autologous grafts (Figure 1). Additional ex vivo selection of CD34+ cells cleared these three stem cell products of contaminating CD19+ B cells (Table 2). PCR analysis of the bcl-2/IgH rearrangement of samples from patients with FCL confirmed the additive B cell depletion capacity of both: the chemoimmunotherapy and the CD34-positive selection device. Of eight patients with FCL initially positive for bcl-2/IgH in peripheral blood or bone marrow samples, two were still positive at the day of graft collection and further ex vivo purging resulted in elimination of contaminating tumor cells detectable by PCR analysis in both manipulated leukapheresis products (Figure 2).

Figure 1

Effective depletion of peripheral B cells by Rituximab/DexaBEAM. Peripheral blood B cell counts (CD19+ cells/μl) of 26 patients comparing the following timepoints: (a) before Rituximab-DexaBEAM; (b) day of (first) leukapheresis; (c) before HDT. Symbols indicate individual values. Statistical analysis indicates significant decrease of peripheral B cells after R-DexaBEAM application (a vs b: P = 0.017; a vs c: P = 0.004).

Table 2 Characteristics of CD34-positive selection
Figure 2

Monitoring of MRD. PCR-based (bcl-2/IgH rearrangement) monitoring of MRD in blood, bone marrow and leukapheresis products (LP) from eight patients with FCL. (a) Before DexaBEAM; (b) before R-DexaBEAM; (c) before HDT; (d) LP before ex vivo purging; (e) LP after ex vivo purging. •, positive; , negative; ↓, relapse.

Blood stem cell transplantation

Conditioning therapy including TBI, high-dose chemotherapy and Rituximab infusion was well tolerated. No treatment-related deaths occurred.

Hematopoetic engraftment was rapid, with a neutrophil count >500/μl after a median of 11 days (range 9–15) and a self-maintaining platelet count >20/nl after a median of 12 days (range 9–39). Except for severe stomatitis, only two patients developed grade III/IV (WHO) non-hematopoetic organ toxicity. One 61-year-old female patient developed severe impairment of cognitive and behavioral functions approximately 1 month post transplantation, lasting approximately 6 months. Search for the underlying cause remained negative in spite of extensive microbiologic, neurologic and psychiatric diagnostic procedures. One further patient suffered from severe infection (WHO grade III) (septicemia with Staphylococcus aureus and Candida krusei) resolving with antibiotic and antifungal therapy.

Sixteen of 18 seropositive patients developed CMV-DNAemia at least once in weekly drawn blood samples. Eight of these 16 patients also tested positive for the pp65 antigen (Table 3). Patients with positive staining of CMV-pp65 or repeated positivity for CMV-DNA received ganciclovir therapy. Pre-emptive treatment effectively protected from CMV disease and led to clearance of pp65 and CMV-DNA from peripheral blood in all patients.

Table 3 Incidence of CMV infection and CMV disease

Herpes zoster occurred in four of 26 patients. The number of patients with documented episodes of fever, between 3 and 12 months post HDT was six of 26 (excluding episodes referring to herpes zoster infection).

Reconstitution of cellular and humoral immunity

Lymphocyte regeneration post HDT was studied in 25 patients with a median follow-up period of 16 months. B cells did not reappear in peripheral blood before day +180 and were present in all subjects when monitored at 1 year post transplantation. Of 14 patients monitored for serum immunoglobulin levels at 1 year post transplantation, six presented with IgA, IgM and IgG serum levels below the respective normal ranges. Mean immunoglobulin serum levels post HDT are shown in Figure 3.

Figure 3

Serum immunoglobulin levels before and post HDT. (a) Before R-DexaBEAM; (b) before HDT. Mean serum levels (g/l) ± s.e.m. of IgM, IgA (right y-axis), IgG (left y-axis) of 26 patients (median follow-up 16 months (6–27). Note: patients received i.v. Ig until day +100 post PBSCT to maintain IgG levels above 4 g/l. Normal ranges: IgA: 0.7–4 g/l; IgM: 0.4–2.3 g/l; IgG: 7–16 g/l.

Lymphocyte reconstitution post PBSC-T showed a pronounced proliferation of CD8+ T cells, leading to a striking inverse T4/T8 ratio for the entire study period. CD4+ T cell count only increased slowly over time, and half of the patients (9/19) presented with a T helper cell count still below 200/μl at 6 months post PBSCT. For further details see Figure 4.

Figure 4

Reconstitution of lymphocyte subsets post HDT. Absolute numbers of peripheral B cells, NK cells, CD4+ and CD8+ T cells in the post-HDT period expressed as mean values ± s.e.m. of 25 patients (median follow-up: 16 months. (a) pre-HDT values.

Treatment efficacy

At a median of 16 months post HDT (range 6–27) 25 of 26 patients who completed the entire study protocol were alive and 73% (19/26) remained alive without signs of clinical or molecular disease. One patient died at day +360 of disease progression (Figure 5). The patient excluded from the study protocol due to mobilization failure received autologous bone marrow transplantation and died in CR in the post-transplant period due to cardiac fibrillation.

Figure 5

Overall and progression-free survival post HDT. Kaplan–Meier estimation of overall survival (OS) and progression-free survival (PFS) for patients completing the entire treatment protocol (n = 26). Median time post HDT: 16 months (6–27). Symbols indicate censored subjects.

Response evaluation by CT scans and bone marrow biopsies before HDT revealed that use of R-DexaBEAM resulted in a remission rate of 85% (23/27) with 11 patients (40%) reaching complete remissions. Restaging 2 months post HDT showed further improvement in treatment response in 13 patients resulting in 24 complete remissions (92%) in subjects completing the entire treatment protocol (n = 26). Two patients received involved field irradiation at the site of initial bulky disease or residual tumor mass after recovery from HDT.

Disease progression was seen in a total of five patients (histology: 2 DLCL; 1 FCL grade III, 2 FCL grade I/II), resulting in a PFS of 77% in the entire study population (n = 26), 74% in patients with indolent FCL, 71% in patients with aggressive lymphoma (n = 11) and 100% in patients with MCL (n = 5).

Patients with follicular lymphoma were monitored for minimal residual disease (MRD) in blood or bone marrow samples. PCR analysis detected eight out of 12 patients positive for t(14;18) prior to the first cycle of salvage therapy. Conversion to PCR negativity was seen in all relevant patients with two patients still remaining positive at the day of graft collection. Three of the patients showed disease progression and molecular relapse preceded the occurrence of clinical relapse in one patient. The other patients (n = 5) remain in ongoing clinical and molecular remission with a duration of up to 25 months (Figure 3).


It has been established that residual disease and tumor cell contamination of autologous grafts can contribute to disease relapse post high-dose therapy in patients with B cell lymphoma.4 The primary aim of our study was to improve anti-lymphoma activity of pre-transplant salvage therapy with elimination of malignant B cells from harvested leukapheresis products.

We combined Rituximab with DexaBEAM and this resulted in a large proportion (88%) of subjects totally depleted of CD19+ B cells in peripheral blood on the day of graft collection. Furthermore, the use of R-DexaBEAM resulted in a substantial lymphoma response, even in an unfavorable group of patients who only reached stable disease status after the first cycle of DexaBEAM without Rituximab. Carrying out ex vivo CD34-positive selection eliminated graft-contaminating tumor cells, detectable by flow cytometric or PCR analysis in all subjects showing CD19+ B cells in the unmanipulated grafts. In addition, Rituximab had no detrimental effects on the well described stem cell-mobilizing capacity of DexaBEAM/G-CSF, and this favorably supports previously published trials, describing the characteristics of different Rituximab chemotherapy regimens used for stem cell mobilization.11,16,23,24

The combination of Rituximab with total body irradiation and/or high-dose chemotherapy was well tolerated and all patients showed immediate and sustained hematopoetic recovery post transplantation. There were no treatment-related deaths and only a minority of patients developed grade III/IV (WHO) organ toxicity during the immediate post-transplant period. These patterns are comparable to those obtained in subjects undergoing conventional HDT for the treatment of (recurrent) lymphoma.25,26

At the time we were starting our study, the first reports were published indicating that the use of ex vivo CD34-positive selected stem cells in autologous transplantation for hematologic malignancy might result in poor lymphocyte recovery and increased transplant morbidity secondary to infectious (viral) complications.27,28,29 We therefore prospectively monitored the reconstitution of lymphocyte subsets, serum immunoglobulins and signs of CMV infection. Our data indicate that inclusion of Rituximab into the pre-transplant salvage regimen and high-dose therapy results in a delay of peripheral B cell regeneration and restoration of serum immunoglobulin levels when compared to published data obtained from subjects receiving unmanipulated or ex vivo purged (B cell-depleted) grafts. T cell regeneration post HDT in our study population was characterized by late onset T helper cell regeneration and early expansion of CD8+ T cells, leading to a pronounced inverse T4/T8 ratio for the entire observation period. Despite the impairment of the immune system post HDT described, the number of bacterial infections between 3 and 12 months was not excessive, supporting recently published results by Horwitz et al.30

With a frequency of 89% (16/18) in CMV-seropositive patients we found CMV-DNAemia and 44% of seropositive patients showed positive staining results of CMV-pp65. This number of CMV reactivations seen in our prospective study population is higher than in the retrospective study published by Holmberg et al.31 The prolonged B cell deficiency seen in our study population may possibly contribute to the impaired ability of controlling virus replication.32

Pre-emptive antiviral therapy was started in cases of CMV-antigenemia and although the absence of clinical disease in the entire population is gratifying, the predictive value of CMV-antigenemia in patients receiving autologous blood stem cells remains unclear.33,34,35 Further controlled studies are required to clarify this clinically quite relevant question. Evaluation of treatment efficacy 2 months post HDT revealed an encouraging 92% complete remission rate. With a median follow-up period post HDT of 16 months (6–27) five patients showed progressive (relapsed) disease. Of note is that in the unfavorable groups of patients with histologies of aggressive lymphoma or mantle cell lymphoma, all remain alive with a PFS of 71% and 100%, respectively.

Taken together, we have shown that Rituximab included in salvage therapy of B cell NHL, combined with the use of ex vivo selected CD34-positive stem cells, is safe and highly efficient in producing lymphoma-free grafts and results in high remission rates. It also causes a prolonged, but transient combined immunodeficiency, which is associated with low morbidity, when appropriately monitored and treated. Based upon these promising results we have started a multicenter phase II clinical trial.


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We gratefully acknowledge the support by Gertrud Feldmann and the excellent technical assistance of Ursula Petrat-Benzaim, Brigitte Schuch, Sabine Mielke and Kristina Schäfer. This study was supported by the Deutsche Krebshilfe and the Forschungsverbund, grant numbers 70–2427 and 70–2428 and approved by the Deutsche Krebsgesellschaft.

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Correspondence to HG Derigs.

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Flohr, T., Hess, G., Kolbe, K. et al. Rituximab in vivo purging is safe and effective in combination with CD34-positive selected autologous stem cell transplantation for salvage therapy in B-NHL. Bone Marrow Transplant 29, 769–775 (2002).

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  • B-NHL
  • Rituximab
  • high-dose therapy
  • immune reconstitution
  • purging

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