No abstract

Background - The domain of precision oncology includes improving patient treatment outcomes based on the application of advanced molecular profiling methodologies to pathological specimens. To this end, optimal utilization of tumor tissue from diagnostic biopsies is an unmet medical need. This is especially relevant today since precision oncology is a rapidly evolving field where timely tumor genotyping is essential for the indication of many advanced and targeted therapies. National Comprehensive Cancer Network (NCCN) guidelines now mandate molecular testing for clinically actionable targets in certain malignancies. Patients diagnosed with advanced non-small cell lung cancer (NSCLC) are commonly of an older age and have significant co-morbidities. This frequently causes clinical dilemmas regarding the ability to obtain adequate amounts of tissue for tumor genotyping. In these cases, the tumor tissue may have been obtained by an image-guided biopsy, and the diagnosis of NSCLC proper determined via cytology. In certain instances, adequate tissue for tumor genotyping and/or a more advanced mutational analysis to identify oncogenic drivers may not be available. Methods - Formalin fixed paraffin embedded (FFPE) specimens were examined using advanced immuno-based laser capture microdissection (LCM), following a formal pathology review. In preparation for droplet digital PCR (ddPCR), DNA was extracted from samples and run with a series of positive and negative controls. Results - Utilizing lung cancer as an example, an improved genotyping approach for NSCLC solid tumors was developed and tested. The strategy involves optimization of the microdissection process and analysis of a large number of identical target cells from FFPE specimens sharing similar characteristics, in other words, single-cell subtype analysis. Immunostaining status, cell phenotype, and spatial location within a histological section are examples of shared characteristics that can guide cell procurement. Conclusions - Synergy between microdissection and ddPCR enhances molecular analysis. Demonstrated is a methodology that illustrates genotyping of a solid tumor from a small tissue biopsy sample in a time and cost-efficient manner, using immunohistochemistry directed LCM and ddPCR detection. Citation Format: Donald J. Johann, Ikjae Shin, Erich Peterson, Mathew Steliga, Jason Muesse, Katy Marino, Sarah Laun, Adam Roberge, Robert Weigman, Michael Emmert-Buck, Michael Tangrea. Advancing precision oncology by synergizing ddPCR with microdissection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 373.

Immunohistochemical (IHC) staining is an established technique for visualizing proteins in tissue sections for research studies and clinical applications. IHC is increasingly used as a targeting strategy for procurement of labeled cells via tissue microdissection, including immunodissection, computer-aided laser dissection (CALD), expression microdissection (xMD), and other techniques. The initial antigen retrieval (AR) process increases epitope availability and improves staining characteristics; however, the procedure can damage DNA. To better understand the effects of AR on DNA quality and quantity in immunodissected samples, both clinical specimens ( KRAS gene mutation positive cases) and model system samples (lung cancer patient-derived xenograft tissue) were subjected to commonly employed AR methods (heat induced epitope retrieval [HIER], protease digestion) and the effects on DNA were assessed by Qubit, fragment analysis, quantitative PCR, digital droplet PCR (ddPCR), library preparation, and targeted sequencing. The data showed that HIER resulted in optimal IHC staining characteristics, but induced significant damage to DNA, producing extensive fragmentation and decreased overall yields. However, neither of the AR methods combined with IHC prevented ddPCR amplification of small amplicons and gene mutations were successfully identified from immunodissected clinical samples. The results indicate for the first time that DNA recovered from immunostained slides after standard AR and IHC processing can be successfully employed for genomic mutation analysis via ddPCR and next-generation sequencing (NGS) short-read methods.