Abstract 650: Effect of antigen retrieval on immuno-based microdissection methods

Abstract

Abstract Introduction - Immunohistochemical staining (IHC) of histological sections is a well-established and useful technique for a large number of research studies and clinical applications, and is increasingly used as a targeting strategy for procurement of labeled cells via tissue microdissection, including immuno-LCM, immuno/computer-LCM, expression microdissection, and other techniques. The antigen retrieval (AR) process employed at the beginning of most IHC protocols increases epitope availability and improves staining characteristics; however, the procedure can damage DNA to an unknown extent. Experimental Procedures - To better understand the effects of AR on DNA quality and quantity, model system samples (lung cancer PDX tissue) and clinical specimens (KRAS gene mutation positive cases) were subjected to commonly employed AR methods [heat induced epitope retrieval (HIER), protease digestion] and the effects on DNA were assessed by Qubit, Fragment Analyzer, agarose gels, kappa qPCR, digital droplet PCR, NGS library preparation, and targeted sequencing. Data Summary - The results showed that HIER resulted in optimal staining characteristics, but induced significant damage to DNA, producing extensive fragmentation and decreased yields. Variation in the HIER protocol mitigated but did not eliminate the negative effects. In contrast, the consequences of protease treatment on DNA were relatively minor and immunostaining quality was acceptable, although diminished in intensity compared to HIER treated sections. Apart from the AR procedure, the IHC process itself also resulted in a decreased yield of DNA. However, in spite of the observed deleterious effects on DNA, none of the AR methods combined with IHC negatively affected PCR amplification of small amplicons, and KRAS gene mutations were successfully identified in the clinical cases under all conditions. Conclusions - The data indicate that DNA recovered from histology slides after standard AR and IHC processing can be successfully employed for the majority genomic applications, but studies that require larger DNA fragments such as long-read sequencing should avoid the use of HIER. Citation Format: Michael Tangrea, Meeiyueh Liu, Adam Roberge, Kristen Noyes, Kennedy Sanders, Michael Emmert-Buck, Donald J. Johann. Effect of antigen retrieval on immuno-based microdissection methods [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 650.