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Clinical Trials |
1 Department of Hematology and Internal Medicine IV, Kitasato University School of Medicine, Kanagawa, Japan; 2 Department of Internal Medicine, Fujita Health University School of Medicine, Toyoake, Japan; and 3 Department of Pathology, Saitama Medical Center, Saitama Medical School, Kawagoe, Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design: We administered rituximab with CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) treatment with G-CSF to 15 patients with follicular lymphoma, and we investigated the safety and efficacy of this regimen. We investigated ADCC activity in neutrophils and the expression of cell surface antigens including Fc
receptor type I [FcRI (CD64)] on neutrophils to determine the optimal dose of G-CSF.
Results: Adverse reactions occurred in 14 of 15 patients and consisted mainly of grade 3/4 hematological toxicity. The response rate was 100%, with complete remission in 12 patients (80%) and partial remission in 3 patients (20%). At 14 months, the median length of the observation period, 2 of 12 patients had relapsed. G-CSF administration increased both FcRI expression and ADCC activity. There were no significant differences in the levels of FcRI expression or ADCC activity between the 2 µg/kg G-CSF and 5 µg/kg G-CSF groups, indicating that the optimal dose of G-CSF was 2 µg/kg.
Conclusions: We conclude that the combination of rituximab-CHOP and G-CSF is well tolerated. We plan to carry out a randomized trial to compare efficacy between rituximab-CHOP treatment and treatment with a combination of rituximab-CHOP and G-CSF.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rituximab is a chimeric IgG1
mAb with a mouse variable region and human constant regions, and it recognizes the CD20 antigen on B lymphocytes. It has been shown that rituximab exerts cytotoxic effects through complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC; Ref. 5
) and that it directly induces apoptosis (6)
. Clynes et al. (7)
demonstrated that ADCC through Fc receptor (FcR) substantially contributes to the cytotoxic action of rituximab. More recently, an association between the FCGR3A-158V genotype and a better response to rituximab treatment was found, suggesting that FcRIIIa is involved in the antitumor mechanism of rituximab (8
, 9)
. Taken together, these studies support that FcR-dependent mechanisms play a role in the effects of rituximab treatment. In vitro studies have confirmed that granulocyte colony-stimulating factor (G-CSF) up-regulates the expression of FcRI (CD64) on polymorphonuclear (PMN) cells, which play an important role in exerting ADCC activities (10
, 11) , and it has been considered that G-CSF augments the effects of rituximab. In addition, experiments using a bispecific antibody (Fc
RI x CD20) have confirmed that G-CSF enhances cytotoxic activities regulated by FcRI by increasing the number of effector cells, although it does not up-regulate FcRI (CD89) expression (12)
. Meanwhile, rituximab inhibits interleukin (IL)-10 expression and down-regulates Bcl-2 expression, inducing apoptosis of tumor cells (13)
. However, G-CSF increases IL-4 and IL-10 levels in the peripheral blood, thereby predominantly activating Th-2 cells (14)
. Therefore, it is important to investigate whether the addition of G-CSF to rituximab in chemotherapy regimens can enhance the antitumor effects. In this study, we investigated the optimal dose of G-CSF when used in combination with R-CHOP, we investigated the expression of cell surface antigens including FcRI (CD64) and ADCC activity to assess the enhanced antitumor effects of G-CSF in combination therapy with R-CHOP for untreated follicular lymphoma, and we report the safety of this combination regimen.
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PATIENTS AND METHODS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RI (CD64) and ADCC activity. In addition, the safety of these regimens was investigated. Patients with follicular lymphoma grade 1/2 who were being treated at Kitasato University School of Medicine or Fujita Health University School of Medicine were enrolled. This study was approved by the ethics committee at each center and performed in accordance with the guidelines of the Declaration of Helsinki.
Eligibility Criteria.
Eligible patients had histologically documented follicular lymphoma of grade 1 or grade 2 as defined by the WHO lymphoma classification (15)
. It was confirmed that CD20 antigen was expressed on the surface of lymphoma cells by either immunohistochemical analysis or flow cytometry using B1 or L26 anti-CD20 mAb. All patients were being treated for follicular lymphoma for the first time. Patients who were between the ages of 20 and 80 years at the time of the study with an expected survival of >4 months and a performance status of 0–2 using the Eastern Cooperative Oncology Group scale were included. Patients with stage III or IV disease, as assessed by the Ann Arbor classification, were included (16)
. Pretreatment laboratory examination was performed within 1 month of study entry, and patients with the following range of laboratory results were included: absolute neutrophil count > 1.5 x 109/liter; platelets > 75 x 109/liter; creatinine < 1.5x the upper limit of normal; bilirubin < 2.0x the upper limit of normal; and aspartate transaminase < 5x the upper limit of normal. Patients with uncontrolled infection, concomitant malignancy (excluding carcinoma in situ of the cervix and skin basal cell carcinoma), unstable angina pectoris, symptomatic cardiac arrhythmia, clinical heart failure, or symptomatic pleural effusions were excluded. Pregnant or lactating women as well as patients who had clinically apparent central nervous system lymphoma were also excluded from the study. All patients gave informed consent for both treatment and sample collection in accordance with institutional policy.
Treatment.
Patients were treated with R-CHOP and G-CSF (lenograstim). The CHOP regimen consisted of 750 mg/m2 cyclophosphamide, 50 mg/m2 doxorubicin, and 1.4 mg/m2 vincristine, up to a maximal dose of 2 mg, i.v. on day 1, with 100 mg of prednisone orally per day for 5 days. Patients were given six cycles of CHOP, once every 3 weeks. Rituximab, at a dose of 375 mg/m2, was given on day –2 of cycles 1, 2, 4, and 6. Between 30 and 60 min before the start of rituximab infusion, the patient was given oral acetaminophen (650 mg) and diphenhydramine hydrochloride (50 mg). Corticosteroids were never given as premedication. In the first infusion of rituximab, the rituximab solution was administered i.v. at an initial rate of 50 mg/h, and the rate was escalated in 50 mg/h increments every 30 min, to a maximum rate of 400 mg/h. If the patient developed hypersensitivity or an infusion-related event, the infusion was temporarily slowed or stopped and then resumed at one-half the previous rate on improvement of the patient’s symptoms. Subsequent rituximab infusions were administered at an initial rate of 100 mg/h, and the rate was increased in 100 mg/h increments at 30-min intervals to a maximum rate of 400 mg/h as tolerated. G-CSF was administered on days –4, –3, and –2 in cycles 1, 2, 4, and 6, and it was administered 3–6 h before rituximab infusion on day –2. G-CSF was administered s.c. The patients in group 1 initially received 0 µg/kg G-CSF with the R-CHOP regimen, the patients in group 2 initially received 2 µg/kg G-CSF with the R-CHOP regimen, and the patients in group 3 received 5 µg/kg G-CSF with the R-CHOP regimen. If there were no safety problems in a group 1 or group 2 patient, the dosage of G-CSF was increased to the next level.
Response Criteria.
The tumor response was assessed after the six cycles of treatment or at the end of treatment. Disease assessment included the following evaluations: physical examination and assessment of performance status and B symptoms (baseline; weeks 6, 12, and 18; and every 3 months through 2 years); chest X-ray (baseline); bone marrow aspiration and biopsy (baseline, and to confirm a CR, if they were positive at baseline); and computed tomography or magnetic resonance imaging (baseline; weeks 6, 12, and 18; and every 3 months through 2 years). The tumor responses were classified as CR, partial remission, stable disease, or progressive disease according to the international workshop for non-Hodgkin’s lymphoma response criteria (17)
. These classifications were defined as follows: CR, the disappearance of all lesions and of radiological or biological abnormalities observed at diagnosis and the absence of new lesions; partial remission, regression of all measurable lesions by >50%, the disappearance of nonmeasurable lesions, and the absence of new lesions; stable disease, regression of measurable lesions by 
50% or no change in the nonmeasurable lesions, and no growth of existing lesions or no appearance of new lesions; progressive disease, the appearance of new lesions, growth of the initial lesions by >25%, or growth of measurable lesions that had regressed during treatment by >50% of their smallest dimensions.
ADCC Assay.
The ADCC assay was performed as described previously (12)
. In brief, target cells were labeled with 51Cr (100 µCi/106 cells) at 37°C for 1 h. Cells were washed three times and resuspended in culture medium at a concentration of 5 x 106 cells/ml. Cells were then sensitized with antibodies (final concentration, 5 µg/ml). The neutrophils of a patient and target cells were added to 96-well, flat-bottomed microtiter plates at an E:T ratio of 10:1 and adjusted to a final volume of 200 µl. The plates were centrifuged at 200 x g for 1 min and incubated at 37°C under 5% CO2 for 20 h. 51Cr release was measured in triplicate and expressed as cpm. The percentage of specific lysis was calculated using the following formula: percentage of specific lysis = (experimental cpm – spontaneous cpm)/(maximal cpm – spontaneous cpm) x 100. The maximal 51Cr release was determined by adding saponin [5% (m/v), 100 µl] to target cells; spontaneous 51Cr release was determined by measuring 51Cr release from unsensitized target cells in the absence of effector cells. ADCC experiments were performed in duplicate.
Immunophenotyping.
After cells were washed once in PBS and resuspended in PBS containing 1% BSA (w/v), immunophenotyping was performed as described previously (18)
, using phycoerythrin R-conjugated mAbs (Beckman-Coulter, Hialeah, FL) directed against CD3, CD19, CD20, CD64 (FcRI), or CD89 (FcRI). An irrelevant isotype-matched control mAb was used in all experiments. Flow cytometry was carried out using a FACScan (Becton Dickinson, San Jose, CA). The mean fluorescence intensity was measured by flow cytometry, and data were always corrected for the mean fluorescence intensity in the presence of the irrelevant control antibody.
Cytokine Production Assay.
The levels of IL-4 and IL-10 in serum samples were analyzed by ELISA. ELISA kits for human IL-4 and IL-10 were purchased from BD PharMingen (San Diego, CA). The lower limit of detection was 2.0 pg/ml for IL-10 and 2.0 pg/ml for IL-4.
Statistical Analyses.
All statistical analyses were performed with SAS software (version 6.10; SAS Institute, Cary, NC). Data are expressed as means ± SD unless otherwise indicated. Serum concentrations were compared using the Wilcoxon signed rank test. Correlations were assessed using Pearson’s correlation coefficient. The duration of the response and survival were assessed using the method of Kaplan and Meier (19)
. P < 0.05 was considered significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. There were 10 males and 5 females with a median age of 56 years (range, 43–63 years). The histological subtype was follicular lymphoma grade 1 in 5 patients and follicular lymphoma grade 2 in 10 patients. Eight patients had stage III disease, and seven patients had stage IV disease.
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Analysis of Cell Surface Antigen Expression on Neutrophils on G-CSF Administration.
The rationale for adding G-CSF to the treatment regimen containing rituximab was to induce expression of FcRI on the circulating neutrophils and, at the same time, increase the number of neutrophils. As shown in Fig. 1
, the number of neutrophils did not change at the time of rituximab infusion in patients who did not receive G-CSF, whereas the number of neutrophils increased remarkably just before the first rituximab infusion in patients who received 2 or 5 µg/kg G-CSF [2 µg/kg G-CSF, number of neutrophils increased from 4.4 ± 0.7 to 15 ± 2.4 (x 109/liter), P < 0.01; 5 µg/kg G-CSF, number of neutrophils increased from 4.8 ± 0.6 to 13.4 ± 1.3 (x 109/liter), P < 0.01]. Moreover, the level of FcR1 (CD64) expression on neutrophils increased from 62.4 ± 4.1 (mean fluorescence intensity) at baseline to 331.7 ± 23.2 (P = 0.0005) after 3 days of G-CSF (2 µg/kg) administration. Similarly, in group 3 patients who received 5 µg/kg G-CSF, the level of FcRI (CD64) expression on neutrophils increased from 64.0 ± 5.8 at baseline to 320.4 ± 15.5 three days after the start of G-CSF treatment (P = 0.0005). At 3 days after the start of G-CSF treatment, the mean expression level of FcRI (CD64) on circulating neutrophils was still elevated. The increase in neutrophil count and induction of FcRI expression by G-CSF showed similar patterns during the second course and during the sixth course of treatment (data not shown). The expression of FcRI (CD89) on neutrophils was examined in a similar fashion. There was no remarkable change in the level of FcRI expression before and after G-CSF administration, irrespective of the dose of G-CSF. The level of ADCC activity increased significantly from 27.7 ± 5.1% at baseline to 68.4 ± 3.8% 3 days after the start of administration of 2 µg/kg G-CSF (P = 0.0004). The level of ADCC activity in the 5 µg/kg G-CSF group also increased significantly from 22.9 ± 5.1% to 70.2 ± 4.5% (P = 0.0004). On G-CSF treatment, the level of ADCC activity changed in a manner similar to the level of FcRI (CD64) expression on neutrophils, and both the level of ADCC activity and FcRI (CD64) expression were elevated on the third day of G-CSF treatment. On comparison of the 2 and 5 µg/kg G-CSF groups, there were no significant differences in the number of PMN cells, the level of FcRI (CD64) expression, or the level of ADCC activity 3 days after the start of G-CSF treatment.
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Response to the R-CHOP and G-CSF Regimen.
As shown in Table 2
, the response rate to the R-CHOP regimen with or without G-CSF was 100% (CR, 12 patients, 80%; partial remission, 3 patients, 20%). CR was achieved in three of the five patients (60%) who received 0 µg/kg G-CSF, four of the five patients (80%) who received 2 µg/kg G-CSF, and all five patients (100%) who received 5 µg/kg G-CSF. The median length of the observation period was 14 months, and 2 of the 12 patients relapsed. The 2-year survival rate was 90.9%.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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each increase CD20 expression on the surface of tumor cells, enhance the binding of rituximab to tumor cells, and significantly augment apoptosis (20)
. On the other hand, clinical studies have shown that rituximab in combination with cytokines such as G-CSF (21)
, IFN- (22)
, IL-2 (23)
, or IL-12 (24)
may have higher efficacy than rituximab alone in the treatment of relapsed low-grade lymphoma, and the time to progression with the combined treatment was reported to be a little longer than that with rituximab alone. However, additional studies on whether this leads to improved survival are needed.
The adverse reactions to G-CSF among the patients in the current study were mild and consisted mainly of bone pain and flu-like symptoms, and this is in agreement with a previous study on G-CSF administration (25)
. Currently, G-CSF is administered to patients who develop neutropenia after R-CHOP therapy, and no enhanced adverse reaction was reported. van der Kolk et al. (21)
reported that among patients with relapsed low-grade lymphoma who were treated with rituximab and G-CSF, most of the adverse reactions were of grade 1/2; 29% of patients developed fever, and 19% of patients developed an allergic reaction. The response rate was 42%, which is comparable with that of rituximab monotherapy, but the duration of the response to the combination of G-CSF and rituximab was longer than that to rituximab alone, indicating the usefulness of the combination of G-CSF and rituximab. In the current study, we examined the antitumor effect of G-CSF in combination with the R-CHOP regimen for the treatment of follicular lymphoma. We determined the optimal dose of G-CSF and used the levels of FcRI (CD64) expression on neutrophils and ADCC activity to assess the antitumor effect. We also investigated the safety of this regimen. We found that the adverse reactions to R-CHOP with G-CSF were comparable with the adverse reactions to R-CHOP, and no new adverse reaction or aggravation of a side effect was observed. A previous study showed that the combination of granulocyte macrophage colony-stimulating factor and R-CHOP yielded an overall response of 100%, but CR was seen in only 21% of the patients, which was lower than the CR rate in the current study, and that the regimen caused heart toxicity in two patients, indicating that G-CSF with R-CHOP is less likely to cause severe adverse reactions (26)
.
Three classes of FcR are expressed in humans, and PMN cells normally express the two low-affinity receptor classes, i.e., FcRII (CD32) and FcRIII (CD16). During G-CSF therapy, the neutrophils of patients with various malignancies and different hematological disorders were found to additionally express a high level of a receptor with a high affinity for FcRI (CD64; Ref. 10
). The high-affinity receptor for FcRI is an early myeloid differentiation marker that is lost at the metamyelocyte stage during normal final maturation of PMN cells and is therefore not expressed on mature cells. It has been demonstrated that G-CSF acts on committed myeloid progenitor cells rather than on mature cells and that G-CSF strongly induces FcRI expression in the precursor cells, resulting in FcRI-positive mature PMN cells (11)
. FcRI appeared to be the main FcR involved in neutrophil-mediated ADCC assays. Furthermore, G-CSF administration induces a large increase in the number of circulating FcRI-positive neutrophils. Therefore, adding G-CSF to rituximab therapy could theoretically enhance the efficacy of rituximab by exploiting the mechanism of ADCC. van der Kolk et al. (27)
examined the neutrophil-mediated CD20-dependent cellular cytotoxicity in B-cell lines and found that G-CSF-primed PMN cells were capable of functioning as effector cells in CD20-dependent cellular cytotoxicity. In addition, they analyzed the involvement of FcR and reported that although CD20-induced ADCC was mediated mainly via FcRI, FcRI and FcRII were both required for optimal lysis. In the present study, G-CSF administration for 3 days in combination with R-CHOP significantly increased FcRI expression on PMN cells, and the level of FcRI expression did not differ between the 2 µg/kg G-CSF group and the 5 µg/kg G-CSF group. Therefore, the optimal dose of G-CSF was considered to be 2 µg/kg. Alas and Bonavida (13)
reported that rituximab down-regulated bcl-2 expression in some B-cell lymphoma cell lines through an IL-10-dependent autocrine loop, rendering the resistant cells susceptible to chemotherapeutic drugs, and that the signal transducer and activator of transcription 3 protein was involved. It has also been reported that IL-4 significantly up-regulates CD20 expression on peripheral blood mononuclear cells (20)
. In addition, because G-CSF stimulates IL-4 and IL-10 release from Th-2 cells, G-CSF-induced IL-10 expression may attenuate the effect of rituximab, whereas G-CSF-induced IL-4 expression may enhance it. Hence, we measured the serum levels of IL-4 and IL-10 in serum samples obtained before and after G-CSF administration. We found that there were no significant differences in the IL-4 and IL-10 levels before and after administration, and it is unlikely that IL-4 and IL-10 modified the effect of rituximab in this study.
In conclusion, R-CHOP with G-CSF was safe for the treatment of untreated follicular lymphoma. We are planning to carry out a randomized controlled trial comparing this regimen and R-CHOP therapy.
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FOOTNOTES |
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Requests for reprints: Nozomi Niitsu, Department of Internal Medicine IV, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara-shi, Kanagawa 228-8555, Japan. Phone: 81-42-778-8111; Fax: 81-42-778-9978; E-mail nniitsu{at}med.kitasato-u.ac.jp
Received 11/30/03; revised 2/17/04; accepted 3/30/04.
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REFERENCES |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RIIIa polymorphism in relapsed follicular non-Hodgkin’s lymphoma patients treated with rituximab. Blood, 100: 1368a 2002.
-receptor (CD89) recruits neutrophils as effecter cells for CD20-directed antibody therapy. J Immunol, 165: 5954-61, 2000.This article has been cited by other articles:
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  F. Buchegger, O. W. Press, A. B. Delaloye, and N. Ketterer Radiolabeled and Native Antibodies and the Prospect of Cure of Follicular Lymphoma Oncologist, June 1, 2008; 13(6): 657 - 667. [Abstract] [Full Text] [PDF]
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 W. Y. Lin, Q. Gong, D. Seshasayee, Z. Lin, Q. Ou, S. Ye, E. Suto, J. Shu, W. Pun Lee, C.-W. V. Lee, et al. Anti-BR3 antibodies: a new class of B-cell immunotherapy combining cellular depletion and survival blockade Blood, December 1, 2007; 110(12): 3959 - 3967. [Abstract] [Full Text] [PDF]
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 Y.-T. Tai, X. Li, X. Tong, D. Santos, T. Otsuki, L. Catley, O. Tournilhac, K. Podar, T. Hideshima, R. Schlossman, et al. Human Anti-CD40 Antagonist Antibody Triggers Significant Antitumor Activity against Human Multiple Myeloma Cancer Res., July 1, 2005; 65(13): 5898 - 5906. [Abstract] [Full Text] [PDF]
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 N. Niitsu, M. Khori, M. Hayama, K. Kajiwara, M. Higashihara, and J.-i. Tamaru Phase I/II Study of the Rituximab-EPOCT Regimen in Combination with Granulocyte Colony-Stimulating Factor in Patients with Relapsed or Refractory Follicular Lymphoma Including Evaluation of Its Cardiotoxicity Using B-Type Natriuretic Peptide and Troponin T Levels Clin. Cancer Res., January 15, 2005; 11(2): 697 - 702. [Abstract] [Full Text] [PDF]
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