Annals of Oncology Advance Access originally published online on April 11, 2008
Annals of Oncology 2008 19(8):1485-1487; doi:10.1093/annonc/mdn163
hematologic malignancies |
Neutrophil role in in vivo anti-lymphoma activity of rituximab: FCGR3B-NA1/NA2 polymorphism does not influence response and survival after rituximab treatment
1 EA3853, Immuno-Pharmaco-Génétique des Anticorps Thérapeutiques, Université François Rabelais, Tours
2 INSERM U847, Biothérapies des cellules souches normales et cancéreuses and Service d'Hématologie et Biothérapies, CHU Lapeyronie, Montpellier
3 Service d'Hématologie, Hospices Civiles de Lyon, Université Claude Bernard, Lyon
4 Department of Hématologie, Centre Jean-Bernard, Le Mans
5 Service d'Hématologie et Thérapie Cellulaire, CHU Bretonneau, Tours, France
* Correspondence to: Dr G. Cartron, Service d'Hématologie Clinique, CHU Lapeyronie, 191 avenue du doyen Gaston Giraud 34295 Montpellier, France. Tel: +33 4 67 33 83 62; Fax: +33 4 67 33 91 94; E-mail: guillaume.cartron{at}med.univ-tours.fr
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Abstract |
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Background: Neutrophils could play an important role in in vivo rituximab anti-lymphoma activity. Fc
RIIIb is expressed only by neutrophils and FcRIIIb-neutrophil antigen (NA)1/NA2 polymorphism influenced phagocytosis of immunoglobulin G1-opsonized particles. We formulated the hypothesis that if neutrophils are critical cells for in vivo rituximab activity, FcRIIIb-NA1/NA2 polymorphism could influence the response to rituximab. Patients and methods: FCGR3B-NA1/NA2 genotypes were determined in 46 patients having received rituximab for a previously untreated, follicular, non-Hodgkin's lymphoma. The clinical response and the disappearance of the BCL2-JH gene rearrangement in both peripheral blood and bone marrow were evaluated at 2 months (M2) and each year during 7 years.
Results: They were 13% homozygous for FCGR3B-NA1, 61% homozygous for FCGR3B-NA1/NA2 and 26% heterozygous. The objective response rates at M2 were 67% in homozygous FCGR3B-NA1 patients compared with 75% in homozygous FCGR3B-NA2 and 75% in heterozygous patients (not significant). We found no difference for progression-free and overall survival by FCGR3B-NA1/NA2 genotypes.
Conclusion: These results indicate no association between FCGR3B-NA1/NA2 polymorphism and response to rituximab indicating no significant role of phagocytosis mediated by neutrophils in in vivo mechanism of rituximab activity.
Key words:
FcRIIIb, follicular lymphoma, neutrophils, rituximab
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introduction |
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Although the clinical efficacy of rituximab (MabThera®, Roche Pharmaceuticals, Bâle, Switzerland, Rituxan® IDEC Pharmaceuticals, Sans Diego, CA) has no more to be demonstrated in B-cell lymphomas, its in vivo mechanisms of action have yet to be elucidated. In vitro data indicate that it induces apoptosis, complement-mediated lysis and cellular mechanisms including antibody-dependent cell-mediated cytotoxicity and phagocytosis [1]. These latter mechanisms could be exerted by a range of effector cells, each of them expressing a unique panel of receptors for the Fc portion of immunoglobulin (Ig) (Fc
R). It is, however, difficult to evaluate the respective contributions of each of these mechanisms, cells and receptors in clinical setting. Pharmacogenetic analyses appear as a fruitful approach to molecularly identify the crucial mechanisms operating in vivo. For example, we were the first to demonstrate that patients homozygous for the FCGR3A-158V allele (encoding the FcRIIIa allotype of highest affinity for IgG1) have a better response to rituximab [2], an observation found also by several other groups [3]. By contrast, there is a debate whether FCGR2A polymorphism actually influences the response to rituximab, despite the fact that its encoded receptor, FcRIIa, has been involved in the phagocytosis of rituximab-sensitized cells by macrophages [4]. Thus, a study reported the influence of FcRIIa-131H/R polymorphism on rituximab response in relapsed follicular non-Hodgkin's lymphoma (NHL) [3], whereas we did not find correlation in a previous study [2].
Murine models using rituximab have shown the role of neutrophils in the elimination of CD20-expressing tumors, raising the question of their actual role in patients [5]. Murine neutrophils mostly express FcRIV [6], an FcR--associated activatory receptor, whereas human neutrophils express both FcRIIa and FcRIIIb, two receptors without any murine counterpart. FcRIIa, encoded by FCGR2A, is an immunoreceptor tyrosine-based activatory motif expressed by neutrophils, macrophages and platelets. FcRIIIb, encoded by FCGR3B, is a glycosylphosphatidylinositol-linked FcR, thought to recruit lipid raft-associated transducing molecules [7]. FcRIIIb exists in two allotypic forms named neutrophil antigen (NA)1 and NA2 which differ by four amino acid residues [8]. FRIIIb-NA1 internalizes hu-IgG1-opsonized particles more efficiently than FRIIIb-NA2 [9, 10]. Since FRIIIb is expressed only by neutrophils [11], we hypothesized that if neutrophils are critical for in vivo rituximab activity, FCGR3B-NA1/NA2 polymorphism might influence the response to rituximab. Genotyping of FCGR3B was therefore carried out on patients with previously untreated follicular NHL who had received rituximab alone [12], and in which we have demonstrated the influence of FcRIII-158VF polymorphism [2].
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patients and treatment
Clinical trial design, eligibility criteria and end-point assessment have been previously reported [12]. Patients were eligible if they had previously untreated follicular CD20 positive NHL with stage II–IV disease and low tumor burden. A total of four 375 mg/m2 doses of rituximab (Roche, Neuilly, France) were administered by i.v. infusion (days 1, 8, 15, 22). Clinical response was evaluated at 2 months (M2) and progression each year until 7 years. Molecular analysis of BCL2-JH gene rearrangement was carried out by PCR [12], on both peripheral blood (PB) and bone marrow (BM) at diagnosis, M2 and each year. The study protocol was approved by an ethics committee, and all patients gave their informed consent
FCGR3B-NA1/NA2 genotyping
Out of the 49 patients included in the clinical trial, two patients refused to be followed and one patient died after 1 year. Forty-six patients were therefore available for genotype analysis. FCGR3B-NA1/NA2 was genotyped as previously described [13]. Both alleles were amplified in duplex reaction containing 0.5 mM of each forward (NA1: 5'-CAGTGGTTTCACAATGTGAA-3'; NA2: 5'-CAATGGTACAGCGTGCTT-3') and reverse (5'-ATGGACTTCTAGCTGCAC-3') primers in the 20 µ PCR reaction mixture with 50 ng of genomic DNA, 1 mM MgCl2, 250 µM of each dNTP (MBI Fermentas, Mundolsheim, France), and 0.16 U of EurobioTaq® DNA polymerase (Eurobio, Courtaboeuf, France) in supplied reaction buffer. PCR conditions consisted in 4 min at 94°C, then 30 cycles (94°C for 1 min, 49°C for 2 min, 72°C for 1 min) and 7 min at 94°C to achieve complete extension. Amplification products (141 bp and 219 bp for NA1 and NA2, respectively) were resolved using standard electrophoresis.
statistical analysis
Departure of genotype frequencies from Hardy–Weinberg equilibrium was tested by an exact test (GENOPOP® software) [14]. The clinical and laboratory characteristics and the clinical and molecular responses were compared using a Fisher's exact test. Progression-free survival (PFS) was calculated using the method of Kaplan and Meier, and comparison of PFS by FCGR3B genotype was carried out using the log-rank test.
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Out of the 46 patients tested for the FCGR3B-NA1/NA2 polymorphism, six (13%) and 28 (61%) were homozygous for FCGR3B-NA1 and FCGR3B-NA2, respectively, and 12 (26%) were heterozygous. Alleles and genotype frequencies were similar to those reported elsewhere [8, 15] and do not depart from those expected from Hardy–Weinberg equilibrium. The three groups were not significantly different in terms of sex, disease stage, BM involvement, number of extranodal sites involved or presence of BCL2-JH rearrangement in PB and BM at diagnosis (Table 1). Odds ratio (OR) rates and survival with an extended follow-up of 7 years have been already described for the entire cohort [12, 16]. OR rates at M2 were 67%, 75% and 75% in homozygous FCGR3B-NA1, FCGR3B-NA2 and heterozygous patients, respectively (Table 2). Median PFS were 18 months [95% confidence interval (CI) 4–57 months], 21 months (95% CI 14–37 months) and 30 months (95% CI 12–64 months) for homozygous FCGR3B-NA1, FCGR3B-NA2 and heterozygous patients, respectively (Figure 1). Seven-year estimated OS were 50%, 69% and 100% for homozygous FCGR3B-NA1, FCGR3B-NA2 and heterozygous patients, respectively.
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Considering that the number of patients in this cohort could show a trend between FCGR3A-158VF polymorphism and both clinical and molecular response to rituximab [2], our results indicate the lack of influence of FCGR3B-NA1/NA2, polymorphism on rituximab activity. Furthermore, because Fc
RIIIb is expressed only by neutrophils and FCGR3B-NA1/NA2 polymorphism modifies IgG1-mediated phagocytosis, these data could also indicate that rituximab-mediated phagocytosis by neutrophils is not crucial in vivo.
FcRIIa, expressed on neutrophils, macrophages and platelets, displays a polymorphism at amino acid position 131 (FcRIIa-131H/R) which is known to influence hu-IgG2, but not hu-IgG1 binding [7]. A study has shown, however, an influence of this polymorphism on clinical response indicating a role of this receptor in in vivo rituximab activity [3]. Since rituximab is an IgG1, these results remain unexplained and are probably related to a known genetic linkage between FCGR2A and FCGR3A [15] which are physically very close together [17]. In an NHL severe combined immunodefiency (SCID) mouse model, neutrophils depleted SCID mice did not respond to rituximab and had a decreased survival time compared with nondepleted mice [5]. Murine FcR cell expression and Ig specificity are, however, quite different from those of human FcRs [7]. Nimmerjahn et al. [6] have recently reported a novel murine-activating receptor named FcRIV which is expressed by neutrophils and binds to hu-IgG1. The role of neutrophils in rituximab-mediated anti-lymphoma activity observed in murine models could be therefore due to the ability of rituximab to recruit murine neutrophils through FcRIV. This could also explain why an enhanced survival is observed in SCID mouse lymphoma model with the association of granulocyte colony-stimulating factor and rituximab [18], whereas this association did not improve response to rituximab in humans [19]. This work underlines that the human FcR system has evolved differently compared with the murine system, questioning the relevance of murine models for the understanding of monoclonal antibody mechanisms of action in humans [20].
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Association pour la Recherche contre le Cancer (3229); Fondation Langlois; Cancéropôle Grand Ouest (MabIMPACT).
Received for publication January 5, 2008. Revision received February 9, 2008. Accepted for publication March 18, 2008.
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