Abstract 1373: IHC and RT-PCR assays for detection of cancer antigen NY-ESO-1 in human tissues
Authors: Halim Abdel , Bagley Rebecca G. , Dauffenbach Lisa , Olsen Eric P. , Keler Tibor 
Published: July 2016

Abstract
Introduction: NY-ESO-1 is a cancer testis antigen frequently expressed in a broad range of tumors: lung, breast, ovarian, bladder, liver cancer, melanoma, sarcoma and myeloma. CDX-1401, a fully human monoclonal antibody linked to full length NY-ESO-1, binds to dendritic cell (DC) receptor DEC-205 to stimulate NY-ESO-1 specific CD4 and CD8 responses. Immunohistochemistry (IHC) and quantitative RT-PCR (qRT-PCR) assays were developed to determine NY-ESO-1 expression in human tumors and normal adjacent tissues (NAT). A qRT-PCR assay for the detection of LAGE-1, a cancer testis antigen with significant DNA homology to NY-ESO-1, was also developed but no commercially specific antibody was available for IHC.
Methods: Formalin fixed, paraffin embedded tumors and NAT were procured from a commercial source or obtained from Mosaic Laboratories; split anonymized samples were analyzed for NY-ESO-1 by IHC at Mosaic and for NY-ESO-1 and LAGE-1 by qRT-PCR at MolecularMD (OR) under IRB-approved protocols allowing in vitro analysis of remnant human samples. The study included: 18 each melanoma and ovarian cancers; 13 each colorectal (CRC), head & neck (H&N), and non-small cell lung cancers (NSCLC); and 38 NAT. HT1080 fibrosarcoma and SKOV3 ovarian cancer cell lines (ATCC, VA) and normal testis were used as controls. The qRT-PCR assays were validated using in vitro transcribed RNA on NY-ESO-1 and LAGE-1 kits provided by Life Technologies and the NY-ESO-1 IHC was developed using a mouse monoclonal antibody (Sigma, MO).
Results: The NY-ESO-1 and LAGE-1 qRT-PCR assays had a linear range of 100-1,000,000 copies/reaction with limit of detection between 1-10 copies. An excess of LAGE-1 copies did not impact the specificity of the NY-ESO-1 assay and vice-versa. NY-ESO-1 protein was detected by IHC and NY-ESO-1 transcripts and LAGE-1 transcripts were detected in 9 (12%), 12 (16%) and 16 (21.3%), respectively, of the 75 tumor tissues while only 2 of the 38 normal samples were positive for LAGE-1. When stromal staining was considered in addition to epithelial staining, 16 (21.3%) tumor samples were positive by IHC. NY-ESO-1 protein was more prevalent in NSCLC (38%), ovarian cancer (11%) and melanoma (11%) vs. other tumor types. Positive staining by IHC was achieved in tumor tissues shown positive for NY-ESO-1 and negative for LAGE-1 transcripts. In general, tumor tissues obtained from Mosaic were of better quality, as assessed by the pathologist, and more positive than commercially procured samples.
Conclusions: Two qRT-PCR assays were developed to specifically quantify NY-ESO-1 or LAGE-1 mRNA without cross reaction. The antibody used in IHC assay seemed specific to NY-ESO-1. Inadequate sample quality impacted target prevalence. Implementing these assays in clinical trials may aid in prospectively identifying patients with NY-ESO-1 positive malignancies who could derive benefit from investigational NY-ESO-1 vaccines designed to induce anti-tumor immunity, such as CDX-1401.
Citation Format: Abdel Halim, Rebecca G. Bagley, Lisa Dauffenbach, Eric P. Olsen, Tibor Keler. IHC and RT-PCR assays for detection of cancer antigen NY-ESO-1 in human tissues. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1373.