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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.

Improving the utilization of tumor tissue from diagnostic biopsies is an unmet medical need. This is especially relevant today in the rapidly evolving precision oncology field where tumor genotyping is often 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. Utilizing advanced stage lung cancer as an example, an improved genotyping approach for solid tumors is possible. The strategy involves optimization of the microdissection process and analysis of a large number of identical target cells from formalin-fixed paraffin-embedded (FFPE) specimens sharing similar characteristics, in other words, single-cell subtype analysis. The shared characteristics can include immunostaining status, cell phenotype, and/or spatial location within a histological section. Synergy between microdissection and droplet digital PCR (ddPCR) enhances the molecular analysis. We demonstrate here a methodology that illustrates genotyping of a solid tumor from a small tissue biopsy sample in a time- and cost-efficient manner, using immunostain targeting as an example.

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.

e15083 Background: Improving patient treatment outcomes based on the application of advanced molecular profiling methodologies applied to pathological specimens is the purview of precision oncology. Optimal utilization of tumor tissue from diagnostic biopsies remains 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. Clinical dilemmas regarding the ability to obtain adequate amounts of tissue for tumor genotyping frequently occur. In these cases, the tumor tissue may have been obtained by an image-guided biopsy, and the diagnosis of NSCLC proper determined via cytology. Adequate tissue for tumor genotyping and/or a more advanced mutational analysis to identify oncogenic drivers may not be available, and the opportunity to explore more optimal therapy options (eg, TKI in lieu of chemotherapy) is lost. Methods: Following a formal pathology review formalin fixed paraffin embedded (FFPE) specimens were examined using advanced immuno-based laser capture microdissection (LCM). In preparation for droplet digital PCR (ddPCR), nucleic acids were 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: With this approach, tumor molecular analysis is enhanced through the synergy between microdissection and ddPCR. Here we demonstrate 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 along with the detection and absolute quantitation of mutations by ddPCR.

Introduction: Precision medicine promises to improve patient treatment outcomes based on the application of molecular profiling methodologies to pathological specimens. This approach enables the development of rationally derived therapy assignments for each individual patient's tumor (versus categorical treatment assignments). Since tumors are composed of heterogeneous cells forming clusters of subtypes, dissimilar molecular profiles may emerge depending on which region is sampled and analyzed. Additionally, a manual slide scrape may result in a reduced or nearly absent molecular signal (i.e. key driver). Clearly, for cancer there is a need for an efficient, upfront enrichment method to specifically isolate specific tumor cell populations. Immuno-Laser Capture Microdissection (immuno-LCM) is a technology based on immunohistochemistry (IHC) that can isolate specific cell types from a heterogeneous cellular population. We sought to determine the impact of various IHC chromogens on the efficacy of microdissection and downstream molecular analysis from immuno-LCM samples. Methods: Histological tissue specimens (clinical colorectal and lung cancer PDX) were processed using traditional IHC methods with a variety of chromogens, including: Vector Black, Impact Vector Red, 3-amino-9-ethylcarbazole (AEC), and 3,3'-diaminobenzidine (DAB). These chromogens were evaluated for staining quality, microdissection efficiency, biomolecular extraction, and evaluation of specific molecular targets. Results: Interestingly, DAB, the most commonly used chromogen, demonstrated a negative impact on the retrieval of biomolecules from immuno-LCM samples. However, chromogens such as Vector Black and Impact Vector Red showed much improved biomolecule extraction compared to DAB even though the yield of microdissected material was comparable. While other chromogens, such as AEC, demonstrated a greater efficiency of biomolecule extraction but showed incomplete immuno-LCM of specific IHC-stained cells. Conclusions: The data indicate that traditional IHC chromogens should be carefully considered when using immuno-LCM to analyze biomolecules. Citation Format: Sarah Laun, Adam Roberge, Robert Weigman, Aubrey Lewis, Valarie Greisman, Meeiyueh Liu, Owen Stephens, Erich Peterson, Ikjae Shin, Michael Emmert-Buck, Michael Tangrea, Donald J. Johann. Towards advanced precision oncology assays - Selection of immunohistochemistry chromogen impacts microdissection and downstream molecular analysis of archival specimens [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2947.

e15571 Background: Improving the utilization of tumor tissue from diagnostic biopsies is an unmet medical need. This is especially relevant today in the rapidly evolving precision oncology field where 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: Following pathology review, formalin fixed paraffin embedded (FFPE) specimens were subjected to advanced immuno-based laser capture microdissection (LCM), DNA was extracted and prepped for droplet digital PCR (ddPCR) and run with a series of positive and negative controls. Results: Utilizing advanced stage lung cancer as an example, an improved genotyping approach for solid tumors is possible. 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. The shared characteristics can include immunostaining status, cell phenotype, and/or spatial location within a histological section. Conclusions: Synergy between microdissection and ddPCR enhances the molecular analysis. We demonstrate a methodology that illustrates genotyping of a solid tumor from a small tissue biopsy sample in a time and cost-efficient manner, using immunostain targeting as an example.

Introduction: Clinical applications and research studies routinely utilize immunohistochemistry (IHC) approaches on tissue sections. The aim is to selectively target antigens (proteins) in the cells of a tissue section by exploiting the principle of antibody binding, thus enabling target specificity. 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 nucleic acids, in this case RNA, to an unknown extent. Experimental Procedures: To better understand the effects of AR on RNA quality and quantity, model system samples (lung cancer patient derived xenograft tissue) were subjected to a commonly employed AR method [heat induced epitope retrieval (HIER)] and the effects on RNA were assessed by Qubit, Fragment Analyzer, and digital droplet PCR (ddPCR). The ddPCR experiments were performed following reverse transcriptase (RT) product generation and construction of cDNA, which then served as input to the ddPCR assay. Absolute quantitation of gene expression levels and expressed mutations were evaluated by ddPCR. Data Summary: The results showed that HIER resulted in optimal staining characteristics, but induced significant damage to RNA, producing extensive fragmentation and decreased yields. In general, fragmentation showed an inflection point at ~200 nt transcript length. Variation in the HIER protocol mitigated but did not eliminate the negative effects. Apart from the AR procedure, the IHC process itself also resulted in a significant decreased yield of RNA. Of note, in these experiments DAB chromogen was used exclusively in the IHC process. However, in spite of the observed deleterious effects on RNA, none of the AR methods combined with IHC negatively affected RT product generation and PCR amplification of small amplicons. Regarding the detection of RT product under different conditions, the size of the amplicon was found to make a difference, with the detection of longer amplicons being more difficult. In addition to a number of successful human and mouse gene expression measurements, KRAS gene mutations were successfully identified in the clinical cases under all conditions. Conclusions: The data indicate that RNA recovered from histology slides after standard AR and IHC processing can be successfully employed for genomic applications utilizing RT product formation (cDNA) for gene expression and detection of expressed mutations, especially if the detection methods are based on relatively short length nucleic acids. Studies that require larger RNA fragments such as long-read sequencing should avoid the use of HIER. Citation Format: Donald J. Johann, Meeiyueh Liu, Adam Roberge, Sarah Laun, Michael Emmert-Buck, Michael Tangrea. Effect of antigen retrieval on immuno-based RNA microdissection methods [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1243.