To investigate effects of the preautografting administration of rituximab on the mobilization and engraftment of peripheral blood stem cells (PBSC), we retrospectively analyzed the outcomes of 43 newly diagnosed diffuse-large B-cell lymphoma patients who received CHOP chemotherapy with or without rituximab as a first-line treatment before autologous PBSC transplantation (PBSCT). There was no difference in the number of CD34+ cells among PBSC between the non-rituximab and the rituximab groups. Although B-cells were completely depleted from PBSC in the rituximab group, we found no difference in the expression of CXCR-4, VLA-4 and c-Kit on PBSC, indicating that rituximab did not affect the expression of these adhesion molecules, which might be involved in the mechanism of mobilization. There was no significant difference in the recovery of neutrophils and platelets, transplant-related toxicity and post-transplant complications between the two groups. Despite the short follow-up, there was no significant difference in progression-free survival between the two groups. These results indicated no adverse effect of rituximab on the mobilization and engraftment of PBSC. Larger studies are required to determine the impact of rituximab on the mobilization and function of PBSC as well as whether a survival advantage exists in patients who undergo auto-PBSCT with rituximab.
The emergence of new, more effective therapies has benefited patients with aggressive non-Hodgkin's lymphoma (NHL). As high-dose chemotherapy with autologous stem cell rescue has been shown to provide a survival advantage over salvage chemotherapy in relapsed patients with NHL,1, 2 many investigators have attempted to extend the use of auto-SCT approaches for the treatment of aggressive NHL. Recent reports have revealed that high-dose chemotherapy followed by autologous peripheral blood stem cell transplantation (auto-PBSCT) is superior to standard chemotherapy as a primary treatment for newly diagnosed patients with aggressive NHL.3, 4 Moreover, rituximab has changed the treatment paradigm of CD20-positive lymphoma, and has improved response and survival rates in combination with chemotherapy,5, 6, 7 and rituximab-containing chemotherapy is increasingly becoming the primary standard for patients with diffuse-large B-cell lymphoma (DLBCL). Recent trials have focused on how to incorporate rituximab into high-dose chemotherapy followed by auto-PBSCT as a first-line treatment,8, 9 including the concept of in vivo purging of lymphoma cells from the circulation before the collection of auto-PBSC.10, 11 However, there is little evidence to support an effect of rituximab on the mobilization and engraftment of auto-PBSC,12, 13 though rituximab might be associated with a poor mobilization and impaired engraftment of PBSC. In an attempt to clarify this issue, we retrospectively compared characteristics of collection and transplantation of auto-PBSC in DLBCL patients treated with a protocol consisting of six courses of the CHOP regimen and high-dose conditioning chemotherapy followed by auto-PBSCT with or without rituximab as a primary treatment. In addition, we also tested the expression of adhesion molecules such as VLA-4, CXCR-4, and c-Kit on mobilized PBSC in the two groups to elucidate whether rituximab affects the expression of these molecules, as degradation of adhesion molecules could lead to the release of stem/progenitor cells from bone marrow into peripheral blood.14, 15
Patients and methods
Patients aged 15–65 years with newly and histologically diagnosed DLBCL, were enrolled in this study. CD20 expression was determined at each participating institution, and further immunostaining was performed on central review if the lineage assignment was ambiguous. Eligible patients had an ECOG performance status of 0–3, high-intermediate to high risk according to the International Prognostic Index,16 and at least one objective, measurable disease parameter. Exclusion criteria included transformed follicular lymphoma, central nervous system involvement, inadequate organ function, concomitant malignancy and an active viral infection such as hepatitis B, hepatitis C and human immunodeficiency virus. This study was conducted in accordance with the ethical guidelines mandated by the Declaration of Helsinki. All patients signed informed consent forms approved by the institutional review board at each participating hospital.
Between May 2001 and January 2006, a total of 43 patients were enrolled into this retrospective analysis: 20 patients were treated with CHOP chemotherapy and autologous PBSCT from May 2001 to May 2003 before the era of rituximab treatment (non-rituximab group). After September 2003 when rituximab was approved for the treatment of DLBCL in Japan, 23 patients enrolled in our study were treated with exactly the same regimen mentioned above plus rituximab (rituximab group) as a first option. Patient characteristics were well balanced and not statistically different between the two groups with regard to sex, age and clinical conditions (Table 1).
All patients were treated with a standard CHOP regimen consisting of cyclophosphamide 750 mg/m2 on day 1, doxorubicin 50 mg/m2 on day 1, vincristine 1.4 mg/m2 to a maximum of 2 mg on day 1 and prednisone 100 mg/m2 on days 1 through 5, every 21 days for six courses. Patients in complete remission (CR), CR of undetermined significance (CRu) or partial remission (PR) after three cycles of CHOP received high dose of etoposide 500 mg/m2 for 3 days followed by granulocyte colony-stimulating factor (G-CSF) to mobilize PBSC as described previously.17 PBSC were collected using a COBE Spectra (Gambro JAPAN Inc., Tokyo, Japan) blood cell separator.17 The target cell dose was 2 × 106 CD34+ cells/kg. Harvested PBSC were cryopreserved until used as described previously.18
After the collection of PBSC, all patients received additional three courses of CHOP. Thereafter, the patients in CR, CRu or PR underwent autologous PBSCT: the conditioning regimen consisted of ranimustine 200 mg/m2 on day −8 and day −3, carboplatin 300 mg/m2 on day −7 through −4, etoposide 500 mg/m2 on day −6 through −4 and cyclophosphamide 50 mg/kg on day −3 and day −2. On day 0, unpurged PBSC were reinfused followed by administration of G-CSF 5 μg/kg. Engraftment was confirmed by a granulocyte count >0.5 × 109/l and platelet counts >20 × 109/l or independence of platelet transfusion.
On the other hand, 23 patients in the rituximab group received rituximab (Chugai Pharmaceutical Co., Tokyo, Japan) 375 mg/m2 one day before the 2nd, 3rd, 5th and 6th CHOP regimen, on day −9 and day +1 after autologous PBSCT. Rituximab was also administered one day before the high-dose etoposide regimen, and one day before PBSC collection again for in vivo purging of circulating lymphoma cells to avoid contamination from lymphoma cells in the PBSC harvest. In total, patients in the rituximab group received eight courses of rituximab 3000 mg/m2 in this protocol.
Cell staining and fluorescence activated cell sorter (FACS) analysis
Peripheral blood mononuclear cells (PBMNC) were prepared by thawing the frozen PBSC harvest samples, which were stored at −80°C.18 PBMNC were stained with a Cy5-PE-conjugated lineage (Lin) cocktail (anti-CD3, CD4, CD7, CD8, CD11b, CD16, CD56 and glycophorin A; Caltag, Burlingame, CA, USA), fluorescein isothiocyanate-conjugated anti-CD19 (Becton Dickinson (BD) Pharmingen, San Jose, CA, USA), PE-conjugated anti-CXCR-4, VLA-4 and c-Kit (BD Pharmingen), allophycocyanin-conjugated anti-CD34 (BD Pharmingen) and PE-Cy7-conjugated anti-CD38 (Caltag) antibodies. Nonviable cells were excluded by propidium iodide staining. Expression of adhesion molecules was detected on progenitors using a highly modified triple laser (488 nm argon laser, 633 nm helium-neon laser and 407 nm crypton laser) FACS (FACS Aria; BD) as described previously.15
The test of independence between the rituximab and non-rituximab groups was made with the χ2 test, Fisher’s exact test, or the Kruskal–Wallis test, where appropriate. Distribution of time to progression-free survival (PFS) was summarized with Kaplan–Meier product limit estimators and compared by log-rank test.
Stem cell mobilization
One course of apheresis for the collection of PBSC was sufficient to obtain the number of CD34+ cells needed for transplantation in all patients of both groups. There was no significant difference in white blood cell count (WBC) at collection (R vs NR group, mean WBC 9.49 × 109/l vs 8.72 × 109/l, P=0.55), platelet counts at collection (mean, 168.1 vs 188.7 × 109/l, P=0.29), duration from administration of etoposide to collection of PBSC (median day, 20 vs 20, P=0.36), percentage of CD34+ cells at harvest (mean, 3.66 vs 3.98%, P=0.85), or CD34+ cell dose collected (mean, 11.10 vs 13.00 × 106/kg, P=0.64) between the two groups (Table 2).
Engraftment of PBSC
The characteristics of auto-PBSCT are shown in Table 3. Engraftment was rapid and documented in all patients. There was no significant difference in the CD34+ cell dose reinfused (R vs NR group, mean CD34+ cell dose 9.82 vs 10.99 × 106/kg, P=0.46), days taken to achieve a granulocyte count of 0.5 × 109/l (median day, 9 vs 9, P=0.39), and a platelet count of 20 × 109/l in platelet counts (median day, 10 vs 10, P=0.57), or independence of platelet transfusion (median day, 8 vs 9, P=0.15) (Table 3). There were no treatment-related deaths, and no significant difference in the frequency of more than grade 3 adverse events scored using the National Cancer Institute Common Toxicity Criteria between two groups (17 vs 15%, P=0.45). Neutropenic fever occurred in the majority of patients in both groups; however, we found no difference in the prevalence of documented infections (26 vs 30%, P=0.42).
The median follow-up period was 51 months in the non-rituximab group and 31 months in the rituximab group. At the time of reporting, there was no significant difference in 1-year PFS (R vs NR groups, 80.2 vs 78.3%, P=0.59) (Table 3).
Expression of adhesion molecules on PBSC
Four courses of rituximab were administered before the collection of PBSC to eliminate DLBCL cells. Therefore, we compared the contents of B-cells in the harvest products to evaluate the efficacy of B-cell purging by rituximab. PBSC harvests contained a small fraction of CD19+ mature-B cells (0.89±0.39% of MNC, n=7) in the non-rituximab group, whereas only a few B cells were circulating (0.06±0.05% of MNC, n=7) in the rituximab group (P<0.001) (Table 2).
We next tested for the expression of c-Kit and adhesion molecules such as VLA-4 and CXCR-4 on immature CD34+ stem/progenitor cells in the PBSC harvest, as downregulation of these molecules resulted in release of stem/progenitor cells from marrow and mobilization into the circulation.15 Figure 1 shows the mean fluorescence intensity (MFI) for these molecules in seven patients in the non-rituxiamb group and seven in the rituximab group. There was no significant difference in MFI of c-Kit (NR vs R groups, 206.4±35.9 vs 182.0±22.0, P=0.15), CXCR-4 (176.6±17.9 vs 161.5±27.2, P=0.25) and VLA-4 (457.7±18.0 vs 454.0±16.9, P=0.69) between the two groups.
Promising results have been obtained for an initial treatment for NHL patients with high-dose chemotherapy followed by auto-PBSCT3 as well as the addition of rituximab to standard chemotherapy.6, 7 Emerging new treatment strategies have focused on a combination of rituximab and high-dose chemotherapy with auto-PBSCT. Several trials have been conducted to design the optimal timing of rituximab administration during the treatment course of chemotherapy and auto-PBSCT.9 A large number of protocols have incorporated rituximab administration preceding chemotherapy and pretransplant conditioning to enhance the chemosensitivity of lymphoma cells by exposure to rituximab.19 In addition, to reduce or eliminate circulating lymphoma cells contaminating the PBSC harvest, concurrent administration of rituximab with cytotoxic chemotherapy for PBSC mobilization has achieved in vivo purging in most patients whose residual lymphoma cells were undetectable in the harvest products by polymerase chain reaction assay.11, 20 However, few studies have examined the effect of rituximab on mobilization and engraftment of auto-PBSC.12, 13 In general, factors associated with poor mobilization include extensive treatment before mobilization,21 and rituximab administration may impair the efficiency of PBSC mobilization compared to that in the non-rituximab group. Hoerr et al.13 reported that patients in the rituximab group had delays in platelet recovery post-transplant, but rituximab did not affect PBSC mobilization, and post-transplant neutrophil recovery, early complications, and mortality rates. In contrast, Benekli et al.12 have shown the detrimental effect of rituximab on the mobilization and engraftment of PBSC because there was a significantly lower number in collected PBSC and a prolonged neutrophil recovery in the rituximab group. However, both studies were performed in patients with relapsed or refractory NHL who had been heavily treated previously, and the exact effect of rituximab on PBSC mobilization still remains unclear. Therefore, in the newly diagnosed DLBCL patients who were treated with the same treatment protocol consisting of chemotherapy with or without rituximab and auto-PBSCT as a primary treatment, we evaluated the characteristics of mobilization and transplantation of auto-PBSC. In our study, we found no disadvantage in the number of CD34+ cells collected, recovery of neutrophils and platelets or post-transplant complications. As none of our patients were previously treated and this study was conducted as a primary therapy, mobilization potential might not have been impaired, resulting in no adverse effect of rituximab on the mobilization and engraftment of PBSC.
Mechanisms of PBSC mobilization may involve chemotherapy/G-CSF-induced modulation of chemokines, adhesion molecules and proteolytic enzymes.14 Proteolytic enzymes such as neutrophil elastase, cathepsin G and matrix metalloproteinase-9 released from the activated neutrophils and monocytes can degrade and/or inactivate adhesion molecules such as VCAM-1/VLA-4, chemokines such as stromal-derived factor (SDF)-1/CXCR-4 and soluble Kit ligand, resulting in the disruption of contact between stem/progenitor cells and the bone marrow microenvironment, and then stem/progenitor cells would be released to migrate into peripheral blood.14, 15 However, recently late-onset neutropenia has been reported following rituximab-based chemotherapy,22, 23 and Dunleavy et al.24 have suggested that rituximab may induce perturbations of SDF-1/CXCR-4 interaction, which could retard the egress of neutrophils from bone marrow. Therefore, we investigated expression levels of adhesion molecules on PBSC in the two groups. Low levels were documented in both groups, but we did not find any difference in expression levels of VLA-4, CXCR-4 and c-Kit on PBSC. Moreover, there was no difference in neutrophil counts, which might partially contribute to mobilization, at the time of PBSC collection, and the recovery in neutrophil and platelet counts was equally rapid following auto-PBSCT in the two groups. These results indicated that rituximab might not impair the mobilization as well as homing, engraftment and repopulation of PBSC, without altering the expression of adhesion molecules including at least VLA-4, CXCR-4 and c-Kit.
In summary, our data provide evidence that rituximab has no adverse effect on the mobilization and engraftment of PBSC, when rituximab is employed in the first-line treatment for newly diagnosed DLBCL patients. As rituximab received approval in September 2003 for the treatment of DLBCL in Japan, the median follow-up is too short to evaluate its effect on survival. Moreover, a high incidence of late-onset neutropenia following rituximab-containing chemotherapy and/or autologous stem cell transplantation has been reported, however, its mechanism still remains to be solved.25, 26 Larger studies and longer follow-ups are necessary to confirm these findings and to determine more optimal combinations of rituximab and auto-PBSCT as well as the impact of rituximab on disease-free survival in the treatment of NHL.
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We thank the medical and nursing staff working on the Fukuoka Blood and Marrow Transplantation Group for providing patients samples and information. We also are grateful to Chugai Pharmaceutical Co. (Tokyo, Japan) for information about rituximab. This work was supported in part by a Grant-in-Aid from Daiwa Securities Health Foundation, Tokyo, Japan to TM.
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Kamezaki, K., Kikushige, Y., Numata, A. et al. Rituximab does not compromise the mobilization and engraftment of autologous peripheral blood stem cells in diffuse-large B-cell lymphoma. Bone Marrow Transplant 39, 523–527 (2007) doi:10.1038/sj.bmt.1705649
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