Published OnlineFirst October 14, 2016; DOI: 10.1158/1078-0432.CCR-16-1874 Cancer Therapy: Clinical Clinical Cancer Research FCGR Polymorphisms Influence Response to IL2 in Metastatic Renal Cell Carcinoma Amy K. Erbe1,WeiWang1, Jacob Goldberg1, Mikayla Gallenberger1, KyungMann Kim2, Lakeesha Carmichael2,DustinHess1,EneidaA.Mendonca2,3, Yiqiang Song2, Jacquelyn A. Hank1,Su-ChunCheng4, Sabina Signoretti5, Michael Atkins6,7, Alexander Carlson8,JamesW.Mier6,8, David J. Panka8, David F. McDermott6,8,and Paul M. Sondel1,3 Abstract Purpose: Fc-gamma receptors (FCGRs) are expressed on Results: When higher-affinity genotypes for FCGR2A, FCGR3A, immune cells, bind to antibodies, and trigger antibody-induced and FCGR2C were considered together, they were associated with cell-mediated antitumor responses when tumor-reactive antibo- significantly increased tumor shrinkage and prolonged survival in dies are present. The affinity of the FCGR/antibody interaction is response to HD-IL2. variable and dependent upon FCGR polymorphisms. Prior stud- Conclusions: Although associations of higher-affinity FCGR ies of patients with cancer treated with immunotherapy indicate genotype with clinical outcome have been demonstrated that FCGR polymorphisms can influence antitumor response for with mAb therapy and with idiotype vaccines, to our knowl- certain immunotherapies that act via therapeutically administered edge,thisisthefirst study to show associations of FCGR mAbs or via endogenous tumor-reactive antibodies induced from genotypes with outcome following HD-IL2 treatment. We tumor antigen vaccines. The previously published "SELECT" trial hypothesize that endogenous antitumor antibodies may of high-dose aldesleukin (HD-IL2) for metastatic renal cell carci- engage immune cells through their FCGRs, and HD-IL2 may noma resulted in an objective response rate of 25%. We evaluated enhance antibody-induced tumor destruction, or antibody- the patients in this SELECT trial to determine whether higher- enhanced tumor antigen presentation, via augmented acti- affinity FCGR polymorphisms are associated with outcome. vation of innate or adaptive immune responses; this FCGR- Experimental Design: SNPs in FCGR2A, FCGR3A, and mediated immune activity would be augmented through FCGR2C were analyzed, individually and in combination, for immunologically favorable FCGRs. Clin Cancer Res; 1–10. associations between genotype and clinical outcome. Ó2016 AACR. Introduction "SELECT" clinical trial of high-dose IL2 (HD-IL2) to prospectively determine whether certain clinical and pathologic criteria [the Patients with metastatic renal cell carcinoma (mRCC) show a type of mRCC tumor (clear cell vs. non–clear cell), carbonic 14% to 22% response to the standard high-dose regimen of anhydrase 9 (CA-9) tumor staining and CA-9 polymorphism aldesleukin (IL2). On the basis of retrospective analyses of status, programmed death ligand 1 (PD-L1) tumor staining, patients with mRCC that had been treated with IL2, potential B7-H3 tumor staining, as well as other criteria] are associated biomarkers that may be useful for response predictions were with response to IL2. As noted in the clinical report, correlations assessed in the "SELECT" clinical trial conducted by the Cytokine were observed between the immune cell modulatory ligand, Working Group. Patients with mRCC were entered into the PD-L1, and immune response, and the study produced a response rate of 25% (1). In an effort to further identify genetic markers that might associate with efficacy of the HD-IL2 treatment for patients 1 Department of Human Oncology, University of Wisconsin, Madison, Wisconsin. with mRCC, and potentially identify immunologic mechanisms 2Department of Biostatistics and Medical Informatics, University of Wisconsin, 3 involved in the response, we sought to identify genotypic factors Madison, Wisconsin. Department of Pediatrics, University of Wisconsin, Madi- fl son, Wisconsin. 4Department of Biostatistics, Dana Farber Cancer Institute, that may in uence the immune activity of HD-IL2 therapy. In this Boston, Massachusetts. 5Department of Pathology, Brigham and Women's study, we genotyped SNPs found in certain activating Fc-gamma Hospital, Boston, Massachusetts. 6The Cytokine Working Group. 7Department receptor (FCGR) genes (FCGR2A, FCGR3A, and FCGR2C). of Medicine, Georgetown University, Washington DC. 8Division of Hematology/ Variably expressed on immune cells, FCGRs bind the Fc frag- Oncology, Beth Israel Deaconess Medical Center, Boston, Massachusetts. ment of IgG antibodies (2–4). Upon engagement and cross- Note: Supplementary data for this article are available at Clinical Cancer linking, activating FCGRs transmit signaling within the immune Research Online (http://clincancerres.aacrjournals.org/). cell and initiate immune activation (5–7). FCGR2A (expressed on Corresponding Author: Paul M. Sondel, University of Wisconsin School of dendritic cells, macrophages, monocytes, neutrophils, and eosi- Medicine and Public Health, 1111 Highland Avenue, 4159 WIMR, Madison, WI nophils), FCGR3A [expressed on natural killer (NK) cells and 53705. Phone: 608-263-9069; Fax: 608-263-4226; E-mail: macrophages], and FCGR2C (also expressed on NK cells) are all [email protected] activating FCGRs (4, 8). The SNPs found in both FCGR2A and doi: 10.1158/1078-0432.CCR-16-1874 FCGR3A genes convey differential binding affinities for the Fc Ó2016 American Association for Cancer Research. portion of antibody. The FCGR2A SNP encodes amino acids of www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst October 14, 2016; DOI: 10.1158/1078-0432.CCR-16-1874 Erbe et al. Translational Relevance Materials and Methods DNA Associations with clinical outcome were found in this study A total of 106 patients from the SELECT trial had DNA available in individuals that have a "favorable" FCGR genotype (higher- for genotyping, along with clinical data for correlative analyses. affinity alleles of FCGR2A and FCGR3A with the expression of DNA was isolated from peripheral blood mononuclear cells FCGR2C), suggesting that greater functionality of FCGRs plays following the manufacturer's protocol of the DNeasy Blood and a role in the antitumor activity of high-dose IL2 for patients Tissue Kit (Qiagen). DNA was kept at 4C during the time of with metastatic renal cell cancer (mRCC). The data presented analyses, and later was transferred to À80C for long-term storage in this report suggest that FCGRs may play a role in the in vivo after completion of the analyses. antitumor effect seen in patients with mRCC receiving high- fi dose IL2. These ndings raise important hypotheses for future Genotyping research that may focus on the potential role of endogenous All SNP genotyping was performed on a StepOnePlus quanti- antitumor antibody and indicate that future work should be tative PCR machine (ABI/Life Technologies). The FCGR2A SNP pursued to test whether the combined analyses of FCGR3A/ was determined using TaqMan primer/probes available from ABI/ 2A/2C genotypes may become a useful biomarker for pro- Life Technologies and used as per the manufacturer's protocol. For spective clinical planning and retrospective outcome analyses both FCGR3A and FCGR2C, Rnase H primers and probes for each for other clinical trials of cancer immunotherapy that may gene were developed in our laboratory to allow for specific involve NK cells or other FCGR-bearing immune cells. amplification of each gene. For genotyping the FCGR3A SNP, Rnase H primers were developed to specifically amplify FCGR3A while not coamplifying FCGR3B. These primers were paired with specific probes to determine the SNP (31). For genotyping the either histidine (H) or arginine (R) at position 131 of the FCGR2A FCGR2C-C/T SNP, Rnase H primers were developed to specifically protein (FCGR2A-H131R, rs1801274), and the FCGR3A SNP amplify this gene while not coamplifying FCGR2B. Primers and encodes either valine (V) or phenylaline (F) at amino acid 158 probes for both FCGR3A and FCGR2C were designed through of FCGR3A (FCGR3A-V158R, rs396991; refs. 9–12). The Integrated DNA Technologies (IDTDNA). Specific method details FCGR2A-H and FCGR3A-V receptors each have higher binding can be found in Erbe AK and colleagues, 2016 (31). Genotyping affinities to human IgG than do the FCGR2A-R and FCGR3A-F was conducted in a blinded manner, where those individuals that receptors, respectively (2, 4, 11). This stronger binding affinity determined the genotype of the patients did not have access to the results in more potent in vitro antibody-dependent cell-mediated clinical outcome data. Genotypes were verified for Hardy–Wein- cytotoxicity (ADCC) and tumor cell death (13, 14). In some berg equilibrium agreement (32); specific genotype results can be clinical trials involving various chimeric or humanized mAbs found in Supplementary Table S1. specific for head and neck, colorectal, or B-cell malignancies, both FCGR2A-H and FCGR3A-V SNPs are associated with improved Clinical data – clinical response (13 16). Similarly, in a trial of an idiotypic The clinical results of the SELECT trial have been published (1). vaccine for B-cell lymphoma, designed to induce endogenous As noted in the clinical report, patients received 600,000 IU/kg/ anti-idiotypic antibody, better outcome was seen for patients with dose of IL2 (Prometheus Laboratories Inc., San Diego, CA) IV every fi the higher-af nity FCGR2A-H and FCGR3A-V SNPs (16). Alter- 8 hours for 5