Published OnlineFirst September 10, 2013; DOI: 10.1158/1078-0432.CCR-13-1253 Clinical Cancer Human Cancer Biology Research Gene Expression Profiling using Nanostring Digital RNA Counting to Identify Potential Target Antigens for Melanoma Immunotherapy Rachel E. Beard, Daniel Abate-Daga, Shannon F. Rosati, Zhili Zheng, John R. Wunderlich, Steven A. Rosenberg, and Richard A. Morgan Abstract Purpose: The success of immunotherapy for the treatment of metastatic cancer is contingent on the identification of appropriate target antigens. Potential targets must be expressed on tumors but show restricted expression on normal tissues. To maximize patient eligibility, ideal target antigens should be expressed on a high percentage of tumors within a histology and, potentially, in multiple different malignancies. Design: A Nanostring probeset was designed containing 97 genes, 72 of which are considered potential candidate genes for immunotherapy. Five established melanoma cell lines, 59 resected metastatic mela- noma tumors, and 31 normal tissue samples were profiled and analyzed using Nanostring technology. Results: Of the 72 potential target genes, 33 were overexpressed in more than 20% of studied melanoma tumor samples. Twenty of those genes were identified as differentially expressed between normal tissues and tumor samples by ANOVA analysis. Analysis of normal tissue gene expression identified seven genes with limited normal tissue expression that warrant further consideration as potential immunotherapy target antigens: CSAG2, MAGEA3, MAGEC2, IL13RA2, PRAME, CSPG4, and SOX10. These genes were highly overexpressed on a large percentage of the studied tumor samples, with expression in a limited number of normal tissue samples at much lower levels. Conclusion: The application of Nanostring RNA counting technology was used to directly quantitate the gene expression levels of multiple potential tumor antigens. Analysis of cell lines, 59 tumors, and normal tissues identified seven potential immunotherapy targets for the treatment of melanoma that could increase the number of patients potentially eligible for adoptive immunotherapy. Clin Cancer Res; 1–10. Ó2013 AACR. Introduction and complete response rates of approximately 40% The development of successful immunotherapy for met- reported in trials treating patients with metastatic melano- astatic cancer requires the identification of appropriate ma (1). This strategy necessitates the acquisition of tumor target antigens. As immunotherapeutic strategies become specimens for generation of TIL and has primarily shown increasingly sophisticated and powerful, finding antigens success in treating melanoma. An alternative approach is that are overexpressed in malignancies but have restricted the infusion of lymphocytes that have been harvested from expression in normal tissue becomes challenging. To date, the patient and genetically engineered to recognize tumor- the most successful immunotherapeutic approach is the associated antigens (1, 2). adoptive cell transfer (ACT) of tumor-infiltrating lympho- Tumor antigen-reactive T-cell receptor (TCR) gene ther- cytes (TIL), with objective response rates of more than 70% apies have been used with success in multiple histologies; however, the number of patients that can be treated is somewhat limited as they must express a specific human leukocyte antigen (HLA; e.g. HLA-AÃ0201; refs. 2, 3). The Authors' Affiliation: Surgery Branch, Center for Cancer Research, Nation- al Cancer Institute, Bethesda, Maryland restricted expression of certain target antigens in tumor cohorts can also limit a therapy’s potential use. For example, Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). effective TCR therapies have been reported targeting the cancer-testis antigen (CTA) NY-ESO-1; however, only Corresponding Author: Richard A. Morgan, Surgery Branch, National Cancer Institute, 10 Center Drive, Building 10-Hatfield CRC, Rm 3-5942, around 20% to 30% of melanomas express this antigen Bethesda, MD 20891-1202. Phone: 301-594-9629; Fax: 301-435-5167; (4, 5). Clinical trials using non–MHC-restricted chimeric E-mail: [email protected] antigen receptor (CAR) therapy can potentially expand the doi: 10.1158/1078-0432.CCR-13-1253 number of patients eligible for ACT if the target antigen is Ó2013 American Association for Cancer Research. expressed on the cell surface (2). Recently, CAR therapies www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst September 10, 2013; DOI: 10.1158/1078-0432.CCR-13-1253 Beard et al. Immunohistochemistry Translational Relevance All tumors samples were confirmed to be metastatic Accurate quantitation of RNA levels is an essential step melanoma at the time of harvest by pathological evalu- in the identification of potential tumor antigens. Nano- ation including immunohistochemistry. Immunohisto- string is a solution-based gene expression profiling tech- chemical staining was carried out for expression of nology that accurately counts individual RNA molecules the antigen NY-ESO-1 (encoded by gene CTAG1B)with in the small amounts of total RNA (250 ng or less) that the specific anti-NY-ESO-1 monoclonal antibody E978 are often obtained from biopsy samples. We used this (Invitrogen; ref. 5). Immunohistochemical scores were technology to study potential tumor antigen gene assigned for intensity of staining and percentage of expression in 59 metastatic melanoma samples and tumors cells that stained positive. identified seven genes as potential targets for adoptive immunotherapy. Nanostring analysis Using the Nanostring nCounter Analysis System (Nano- string Technologies), gene expression analysis was con- ducted for each sample as previously described using a custom-designed codeset containing 97 genes (8). Each have shown success in treating non-melanoma and non- reaction contained 250 ng of total RNA in a 5 mL aliquot, solid organ cancers, specifically the ability of the anti-CD19 plus reporter and capture probes, and 6 pairs of positive CAR to effect regression of advanced B-cell malignancies control and 8 pairs of negative control probes. Analysis and (6, 7). normalization of the raw Nanostring data was conducted The most effective way to maximize the number of using nSolver Analysis Software v1.1 (Nanostring Technol- patients potentially eligible for a therapy would be to ogies). Raw counts were normalized to internal levels of target an antigen expressed on a high percentage of 7 reference genes: CNOT2, GAPDH, HPRT1, PHGDH, tumors within a given histology. This study uses Nano- SUMO2, SYS1, and WDR45L. A background count level string technology to achieve gene expression profiling of was estimated using the average count of the 8 negative melanoma cell lines, metastatic melanoma tumors, and control probes in every reaction plus 2 SDs. normal human tissue samples to identify potential target antigens for immunotherapy. This robust technology uses Gene expression analysis unique digital color-coded barcodes that hybridize direct- Principal component analysis (PCA) and ANOVA anal- ly to specific nucleic acid targets and allow for detection ysis were conducted using the Partek Genomic Suite (Partek and quantitation of hundreds of transcripts in a single Incorporated). PCA was used to characterize samples on the reaction. Unlike microarray approaches, it facilitates the basis of their gene expression profiles. ANOVA analysis was direct measurement of mRNA expression levels for sub- used to identify differentially expressed genes (significant sequent gene expression analysis, and has been shown to P value < 0.05) and samples were clustered by hierarchic be highly reproducible and as sensitive as real-time PCR clustering. assays while still allowing for the measurement of mul- tiple genes at one time (8). Flow cytometry and quantitative-PCR Flow cytometry (FACS) was conducted using conjugat- Materials and Methods ed monoclonal antibody (mAb) specific for human chon- Sample collection, RNA isolation, and cell lines droitin sulfate proteoglycan 4 (CSPG4) according to the Patients seen at the Surgery Branch, National Cancer manufacturers’ instructions (R&D Systems). Reverse tran- Institute (NCI; Bethesda, MD) for treatment of metastatic scription (RT) was conducted using High Capacity cDNA melanoma underwent surgical excision of metastatic Reverse Transcription Kit (Applied Biosystems). Quanti- lesions for harvest of TILs in protocols approved by the tative PCR was carried out with the TaqMan Fast Univer- Institutional Review Board and Food and Drug Adminis- sal PCR Master Mix (Applied Biosystems) by the use of a tration. Viable-appearing fragments of these tumors were 7500 Fast Real-Time PCR System (Applied Biosystems). freed from surrounding normal tissue, collected, and either Copy numbers were generated using a standard curve stored in RNAlater (Ambion) or flash-frozen and stored at from CSPG4 plasmid and results were normalized against À80 Celsius, until RNA isolation was conducted using a b-actin (ACTB). RNEasy Mini Kit (Qiagen). Analyzed tumor samples were collected between September 2007 and December 2012. Results RNA isolation was conducted in the same fashion for Samples and Nanostring probeset established human melanoma cell lines initiated at Memo- Gene expression profiling using Nanostring technology rial Sloan-Kettering (SKmel23) or the NCI Surgery Branch was conducted