Originally published as JCO Early Release 10.1200/JCO.2005.04.6011 on May 8 2006

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FCGR2A Polymorphism Is Correlated With Clinical Outcome After Immunotherapy of Neuroblastoma With Anti-GD2 Antibody and Granulocyte Macrophage Colony-Stimulating Factor

Nai-Kong V. Cheung, Rebecca Sowers, Andrew J. Vickers, Irene Y. Cheung, Brian H. Kushner, Richard Gorlick

From the Departments of Pediatrics and Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center; and the Department of Pediatrics, Children's Hospital at Montefiore, Albert Einstein College of Medicine, New York, NY

Address reprint requests to Nai-Kong V. Cheung, MD, PhD, Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021; e-mail: cheungn{at}mskcc.org

	ABSTRACT

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PURPOSE: Anti-GD2 murine IgG3 antibody 3F8 kills neuroblastoma cells by antibody-dependent cell-mediated cytotoxicity (ADCC). Granulocyte macrophage colony-stimulating factor (GM-CSF) enhances phagocyte-mediated ADCC. The differential affinity of the human FCGR polymorphic alleles for 3F8 may influence the effectiveness of antibody immunotherapy.

PATIENTS AND METHODS: The entire cohort of high risk neuroblastoma patients (N = 136) treated on protocol using 3F8 and GM-CSF were the subjects of this analysis. Tumor response was measured by standard clinical tools plus sensitive molecular monitoring using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Polymorphic alleles of FCGR2A and FCGR3A were determined by PCR plus direct sequencing using genomic DNA samples obtained from marrow or blood of patients.

RESULTS: FCGR2A (R/R) genotype correlated with progression-free survival for the entire cohort (P = .049) and for the subset of patients with no history of prior relapse (P = .023). FCGR2A (R/R) also correlated with marrow remission 2.5 months after treatment initiation: by histology (P = .021 and P = .036, for the entire cohort and the subset, respectively) and by qRT-PCR (P = .052 and P = .033, respectively).

CONCLUSION: The favorable outcome associated with FCGR2A (R/R) genotype is consistent with the proposed role of FCGR2A and phagocyte-mediated ADCC in 3F8 plus GM-CSF immunotherapy.

	INTRODUCTION

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Monoclonal antibodies (MoAbs) show promise in cancer therapy. They can kill tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC), complement-mediated cytotoxicity (CMC), and apoptosis. ADCC and CMC are mediated by Fc receptors (FcR), where individual FcRs are suited for unique effector functions as well as unique effector cell types.^1,2 Since natural antibody responses to specific antigens or pathogens are polyclonal, their interaction with FcR can be quite complex. When MoAb is used for disease therapy, its preferential interaction with specific FcR alleles translates into distinct biologic effects. Thus, it is not surprising that polymorphic FcR alleles can influence and correlate with clinical efficacy of MoAb immunotherapy.

FcR2A has two polymorphic alleles at amino acid position 131. The FcR2A-H131 allelotype (histidine) has higher binding efficiency for human IgG2 and IgG3 antibodies when compared with FcR2A-R131 (arginine). On the other hand, FcR2A-V158 (valine) has higher affinity than FcR3A-F158 (phenylalanine) for human IgG1.³ Greater binding affinity of FcR2A-H131 is expected to trigger stronger inflammatory responses predisposing patients to disease like Guillain-Barre syndrome.⁴ However, ineffective FcR function may impair immune complex clearance, precipitating autoimmune processes (eg, FcR2A-R131 in lupus nephritis⁵). Impaired FcR function in clearing opsonized bacteria (eg, FcR2A-R131) has also been associated with infections, bacteremic pneumonia, periodontitis, and fulminant meningococcal septic shock.⁶ The interaction of IgG with the inhibitory FcR2B on B-cells, macrophages, and monocytes adds another dimension to antibody regulation of inflammatory responses.⁷ Whether the polymorphic forms of this FCGR2B will translate into allele-specific outcomes remains to be elucidated.⁸

To date, almost all MoAbs in clinical trials are either chimeric or humanized antibodies bearing the human 1 Fc domain. Antibodies of this subclass are known to interact preferentially with FcR3A-V158. Several reports have found a favorable association between this allelotype with clinical outcome following treatment with rituximab.^9,10 In contrast, there have been limited studies on the interaction of murine antibodies with human FcR. Mouse anti-CD3 MoAbs are used extensively as immunosuppressive agents for graft rejection; FcR-dependent mitogenic properties and cytokine release are believed to be responsible for the systemic adverse effects. Unlike murine IgG2a anti-CD3 antibodies, which bind both FcR2A allelotypes equally well, IgG1 subclass antibodies prefer the FcR2A-R131 over the -H131 allelotype, resulting in differential mitogenic potency and cytokine release,¹¹ believed to be responsible for the first-dose effect of anti-CD3 MoAb in patients,¹² characterized by systemic release of cytokines, chills, fever, nausea, headache, and diarrhea. Similar observations of FcR2A allelotype preference were made for the murine IgG1 antibody Leu2a specific for human CD8,¹³ and platelet-activating murine IgG1 antibodies,¹⁴ and cellular aggregation in the presence of IgG1 in flow cytometry.¹⁵ The murine IgG3 anti-GD2 antibody 3F8 is being used in the clinic for its antitumor activity in high-risk neuroblastoma patients. In vitro studies using 3F8 have shown potent ADCC.¹⁶ In the presence of granulocyte macrophage-colony-stimulating factor (GM-CSF), antitumor activity can be markedly increased. By enzyme-linked immunosorbent assay, 3F8 has preferential binding to FcR2A-R131 over FcR2A-H131. In this report, we correlate FCGR genotypes with antitumor response and survival among the entire cohort of 136 high-risk neuroblastoma patients who were treated with an immunotherapy regimen using murine MoAb 3F8 and GM-CSF.

	PATIENTS AND METHODS

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Patients
One hundred and thirty-six consecutive patients with neuroblastoma (38 with MYCN amplification of more than 10 MYCN copies per diploid human genome) treated on protocol IRB 9418 at Memorial Sloan-Kettering Cancer Center (MSKCC; New York, NY) were the subjects of this study. One hundred and thirty of 136 patients had metastatic stage 4 neuroblastoma, and six patients had high-risk stage 3 (four of six with MYCN amplification). Except for one infant with MYCN-amplified stage 4 neuroblastoma, 135 of 136 patients were diagnosed at older than 12 months of age, and 126 of 136 patients (93%) were diagnosed at older than 18 months of age, generally regarded as the highest-risk age group. This protocol utilized anti-GD2 MoAb 3F8 plus GM-CSF in children after chemotherapy.¹⁷ Written informed consent was obtained from the patients and/or their parents/guardians. Their disease status at protocol entry was stratified into four categories:¹⁸ complete remission/very good partial remission, primary refractory, second refractory, and progressive disease.¹⁷ Of 136 patients, 124 had follow-up bone marrow (BM) samples after treatment cycle two, at a median of 2.5 months from protocol entry. There were 69 deaths and 90 relapses during follow-up. Median follow-up for survivors was 34 months and 36 months for overall survival (OS) and progression-free survival (PFS), respectively. Outcome at 2 years was correlated with disease status at the time of protocol entry. Patients who had prior relapse (either progressive disease or secondary refractory) fared worse; 97% progressed before 2 years compared with 62% of those with no prior relapse (ie, either complete remission/very good partial remission or primary refractory disease status) at protocol entry. Two of the 136 patients chose to go off protocol to enroll on other treatment studies.

Histologic Examinations of BM Samples
All 136 patients had marrow studies under general anesthesia, before and after the second cycle of therapy. Each study consisted of six histopathologic examinations of two biopsy specimens and four aspirates from different sites, with a fresh needle for each site. After clearance of contaminating skin, bone, or endothelial cells, 2 mL to 2.5 mL of heparinized marrow from each aspiration site was pooled, mononuclear cells were then isolated and cryopreserved.

Real-time quantitative polymerase chain reaction (qRT-PCR) was performed on cryopreserved BM using the primers and probes for GD2 synthase and the endogenous reference glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as previously described.¹⁹ The transcript levels of GD2 synthase and GAPDH were determined from their respective standard curves using serially diluted cDNA from neuroblastoma cell line NMB7. Normalized transcript levels were expressed as multiples of GAPDH expression.

Fc Polymorphism Genotyping
Genomic DNA was purified from peripheral blood or marrow mononuclear cells using DNA extraction kit (Qiagen, Valencia, CA). Genotyping of FCGR2A 131-H/R and FCGR3A 158-V/F polymorphism was performed by PCR, followed by allele-specific restriction enzyme digestion.^20,21 One hundred and fifty ng genomic DNA was amplified using final concentrations of 0.5 µmol/L each of forward and reverse primer (Table 1) , 0.2 mmol/L dNTP Mix (Promega, Madison WI), 1.5 mmol/L MgCl₂, 1X AmpliTaq Gold Buffer II (Applied Biosystems [ABI], Foster City, CA), and 1 U AmpliTaq Gold (ABI). All reactions included one 8-minute 94°C hot-start before cycling followed by 40 cycles of 30-second denaturation (94°C), 1-minute annealing (52°C or 60°C), 45-second extension (72°C), and one final 8-minute extension (72°C). FCGR2A polymorphism was detected by a subsequent restriction digest of PCR product with BstUI (New England Biolabs, Beverly, MA) where 15 µL of FCGR2A PCR product was incubated overnight at 60°C with 5 units of BstUI. Digested products were run on a 2% agarose gel. Genotypes were represented by 322 base pairs (bp) alone (G/G), 343bp and 322 bp together (A/G), or 343 bp alone (A/A). FCGR3A polymorphisms were identified by direct sequencing of the PCR product. PCR products were cut from the agarose gel and purified using QIAquick PCR Purification Kit (Qiagen). Five µL of purified PCR product was sequenced using 0.625 µmol/L of the forward PCR primer, 1x BigDye Terminator sequencing buffer and 2 µL BigDye Terminator v3.1 cycle sequencing mix (ABI) in a total volume of 20 µL. After completion of sequencing reaction, products were ethanol-precipitated as follows: 16 µL sterile water and 64 µL 95% ethanol were added to each 20 µL sample, incubated for 15 minutes at room temperature, then centrifuged at maximum speed (3,200 x g) at 4°C for 45 minutes. Supernatant was decanted and pellet was washed in 70% ethanol followed by a 10-minute centrifugation at 3200 x g at 4°C. Seventy percent ethanol supernatant was then poured off; to eliminate any residual ethanol, samples were inverted and centrifuged for 1 minute at 300 x g. Dried pellet was resuspended in 20 µL Hi-Di Formamide (ABI). Samples were denatured at 94°C for 2 minutes and immediately placed on ice for 10 minutes and then loaded onto an ABI 3100-Avant Genetic Analyzer per manufacturer's instructions. This analysis was carried out in accordance with the guidelines of the institutional review board of MSKCC.

	RESULTS

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Fc Polymorphism Among Patients With Neuroblastoma
The genotype distribution of FCGR2A and FCGR3A among the 135 patients is detailed in Table 2. Samples from one patient were not assessable. The ethnic breakdown for the 135 patients was as follows: 78.5% white non-Hispanic, 7.4% black non-Hispanic, 5.2% Asian/Pacific Islander, 5.2% white-Hispanic, 3% South Asian, and 0.7% black-Hispanic. The allele frequency was 0.47 for FCGR2A-R131, 0.53 for FCGR2A-H131, 0.27 for FCGR3A-V158, and 0.73 for FCGR3A-F158. The genotype distributions for both FCGR2A and FCGR3A were as expected and they were in Hardy-Weinberg Equilibrium.²⁴

View larger version (7K): [in this window] [in a new window]   Fig 1. Kaplan-Meier plot of progression-free survival with respect to FCGR2A genotype among 135 high-risk neuroblastoma patients treated with an immunotherapy protocol using anti-GD2 antibody 3F8 plus granulocyte macrophage-colony-stimulating factor. Progression-free survival (PFS) of two patients were censored when they chose to go off protocol. PFS in the FCGR2A-131R/R group was superior to that in the R/H or H/H group (P = .049).

View larger version (7K): [in this window] [in a new window]   Fig 2. Kaplan-Meier plot of progression-free survival with respect to FCGR2A genotype among patients with no history of prior relapse (N = 106). Progression-free survival in the FCGR2A-131R/R group was superior to that in the R/H or H/H group (P = .023).

We also tested the influence of FCGR2A and FCGR3A on PFS in a cohort of 39 patients similarly treated with intensive chemotherapy, where 23 of 39 also received 3F8 but without GM-CSF. There were 25 progression events, with a median follow-up for survivors of nearly 14 years. We found no statistically significant association between PFS and allelotypes. The hazard ratio for FCGR2A R/R compared with FCGR2A H/H and H/R was 1.62 (95% CI, 0.48 to 5.43). The lower bound of the 95% CI was only slightly lower than our central estimate (0.57; Table 3) if GM-CSF was added to the treatment. This suggests that the effects of FCGR2A on outcome are likely to be GM-CSF treatment-dependent.

Fc Polymorphism and Marrow Remission
The prognostic impact of Fc polymorphism on marrow remission by histology and molecular marker GD2 synthase was also analyzed. Post-treatment marrow histology data were available for 122 patients and GD2 synthase qRT-PCR data for 123 patients. As previously described,²⁵ patients were considered to be in molecular remission if the GD2 synthase transcript level of the post-treatment BM was 5 units. The relationships between FCGR2A and histologic plus molecular remission are shown in Table 4. FCGR2A-R/R genotype predicted histologic remission of post-treatment marrow for the entire cohort and for the subgroup of patients without prior relapse (P = .021 and P = .036, respectively), and with molecular remission (P = .052 and P = .033, respectively).

	DISCUSSION

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Treatment success following immunotherapy with 3F8 plus GM-CSF is highly correlated with patients having the FCGR2A-R/R genotype. Since FcR2A is expressed on human neutrophils, macrophages, and antigen-presenting cells, these findings are consistent with a critical role of phagocyte-mediated antitumor response. Whether the effect is direct tumor cytotoxicity,^16,27 or immune activation through enhanced antigen presentation, will require a more detailed analysis.

There are well known pitfalls encountered in molecular discovery,^28,29 which include reproducibility, adequate case delineations, false discovery, and adherence to the general guidelines for biomarker studies.³⁰ We chose the entire cohort of patients treated on the protocol MSKCC IRB 9418, in which 135 of 136 patients had samples assessable for polymorphism. No patient was excluded from analysis for other reasons. In terms of reproducibility of the assay, the entire 135 samples were reanalyzed using the ABI Taqman SNP allelic discrimination assay. There was 100% agreement between restriction digest (used in this report) and the Taqman single-nucleotide polymorphism findings. Although there were only two principal analyses, the potential risk for false discovery still exists. Clearly, further validation in prospectively collected patient samples will be needed. A preliminary analysis of the first 45 patients in a follow-up study using 3F8 plus GM-CSF again showed that FCGR2A-R/R was highly correlated with PFS (data not shown).

The ability of phagocytes to mediate efficient ADCC against human tumors in vitro is well known.^16,31-34 However, defects in neutrophil ADCC have been found in cancer patients,³⁵ and reversal with cytokines, such as GM-CSF and G-CSF, is possible.^16,36,37 In vivo tumor models have also demonstrated the antitumor effects of neutrophil-mediated ADCC, especially when activated by cytokines.^38,39 In vitro studies with neuroblastoma indicated that neutrophil ADCC was particularly effective^16,40,41 in the presence of anti-GD2 MoAb 3F8. Similar observations were reported for another murine IgG3 anti-GD2 MoAb 7A4,⁴² and the human-mouse chimeric anti-GD2 MoAb ch14.18.^42-44 The exact tumoricidal mechanism is still unclear, but probably involves granular proteases⁴³ and independent of oxidative intermediates.⁴¹

Despite the compelling evidence in preclinical models, the clinical role of phagocyte mediated ADCC in human cancer is controversial. Since FcR2A is preferentially expressed on neutrophils/macrophages, and not on lymphocytes, dependence of clinical outcome on FCGR2A genotype provides indirect evidence of a pivotal role of phagocyte ADCC. A recent report described improved clinical outcome for patients with the FCGR2A-H/H genotype after rituxan (chimeric IgG1)⁹; although, an earlier study failed to find such an association.¹⁰ Furthermore, this association was unexpected since the H/H genotype was not known to have preferential affinity for human IgG1.⁴⁵ The preferential binding of FcR2A-R131 ectodomain to mouse IgG and the correlation with patient outcome strongly suggests that FCGR2A is mediating an antitumor effect in vivo. However, we should be cautious not to over interpret the published literature or our results since they are relatively small studies.

In addition to its expression on phagocytes and platelets, FcR2A is also found on dendritic cells and langerhans cells.^1,2 Although neutrophil is a major myeloid population activated in patients receiving GM-CSF, and is a likely cell type through which FcR2A polymorphism exerts its biologic effects, it remains possible that 3F8 helps target tumors to macrophages or antigen-presenting cells. In neuroblastoma, BM metastasis is a common event. In addition to being the site of minimal residual disease after induction therapy, marrow is also an organ conducive to phagocyte ADCC since the trafficking of MoAb or effector cells is not a limiting factor. Here, FcR2A may exert its effect by enhancing ADCC and/or adaptive immunity through positive antibody feedback.^46,47An in depth analysis of induced tumor specific immunity will be needed to understand the underlying mechanisms.

In vitro binding studies of individual MoAb to recombinant FcR may help define the importance of different polymorphic alleles in the antitumor mechanism of MoAb in vivo. An accurate prediction of outcome based on FCGR allelotype can help identify patients most likely to benefit from a specific form of MoAb. Historically, FCGR2A polymorphism is particularly relevant among patients with complement deficiency where phagocytosis is the only intact defense for clearing infections. Thus, one might expect FCGR2A genotype to be most critical if the antitumor mechanism is complement dependent. Identification of the clinically important FcR for each MoAb will help focus various strategies on Fc to improve their affinities for specific FcR. Since FcR is only one of the many receptors important in ADCC, CMC, and APC function, a better understanding of gene polymorphism in antibody-based therapies may provide insight in making this modality even more effective.⁴⁸

	Authors' Disclosures of Potential Conflicts of Interest

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The authors indicated no potential conflicts of interest.

	Author Contributions

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... Author Contributions REFERENCES

 

Conception and design: Nai-Kong V. Cheung Financial support: Nai-Kong V. Cheung, Richard Gorlick Administrative support: Nai-Kong V. Cheung, Brian H. Kushner Provision of study materials or patients: Nai-Kong V. Cheung, Brian H. Kushner Collection and assembly of data: Nai-Kong V. Cheung, Rebecca Sowers, Richard Gorlick Data analysis and interpretation: Nai-Kong V. Cheung, Rebecca Sowers, Andrew J. Vickers, Irene Y. Cheung, Richard Gorlick Manuscript writing: Nai-Kong V. Cheung, Irene Y. Cheung Final approval of manuscript: Nai-Kong V. Cheung, Rebecca Sowers, Andrew J. Vickers, Irene Y. Cheung, Brian H. Kushner, Richard Gorlick

 

	ACKNOWLEDGMENTS

 
We thank Kim Kramer, MD, and Shakeel Modak, MD, and neuroblastoma nurses Ester Dantis, Ursula Tomlinson, Yi Chih Lin, Linda D’Andrea, Latisha Jones, and Catherine Enero; Hongfen Guo and Yi Feng for technical assistance; and Karen Danis, Neha Dalal, and Elizabeth Leon for data management.

	NOTES

 
Supported by in part by Grants No. CA106450 and CA095742 from the National Institutes of Health, and by grants from the Robert Steel Foundation, Pediatric Cancer Foundation, The Aubrey Fund, Margaux's Miracle Foundation, Hope Street Kids, and Katie-Find-A-Cure Fund.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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Submitted October 22, 2005; accepted March 13, 2006.

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