Abstract
Immunohistochemistry remains the overwhelming technique of choice for test biomarker evaluation in both clinical or research settings when using formalin-fixed, paraffin embedded tissue sections. However, validations can be complex with significant issues about specificity, sensitivity and reproducibility. The vast array of commercially available antibodies from many vendors may also lead to non-standard approaches which are difficult to cross-reference. In contrast mRNA detection, by in situ hybridization (ISH) with sequence specific probes, offers a realistic alternative, with less validation steps and more stringent and reproducible assessment criteria. In the present study mRNA ISH was evaluated in prospectively and retrospectively collected FFPE samples within a cancer biobank setting. Three positive control probes, POLR2A, PPIB and UBC were applied to FFPE sections from a range of tumour types in FFPE whole-face (prospective collection) or TMA (retrospective collection) formats and evaluated semi-quantitatively and by image analysis. Results indicate that mRNA can be robustly evaluated by ISH in prospectively and retrospectively collected tissue samples. Furthermore, for 2 important test biomarkers, PD-L1 and c-MET, we show that mRNA ISH is a technology that can be applied with confidence in the majority of tissue samples because there are quantifiable levels of control probes indicating overall mRNA integrity.
Highlights
Detection of mRNA in formalin-fixed, paraffin embedded (FFPE) tissue samples by chromogenic RNA in situ technology has become a reliable alternative for a wide range of biomarkers in many areas of research including cancer and neurosciences [1,2,3]
The purpose of the present study was twofold: Firstly, to assess, using 3 RNAscope control probes (POL2RA, PPIB and UBC), the suitability of RNAscope in four tumour types whereby FFPE tissue had been prospectively collected from patients consented to the Northern Ireland Biobank (NIB); secondly to assess the midrange control probe PPIB expression and correlate to test probe biomarkers (PD-L1 and c-MET) in a series of 353 colorectal cancer (CRC) FFPE samples collected over a 5-year period within routine pathology practice in the Belfast Health and Social Care Trust (BHSCT)
The goal of this study was to determine whether the suitability of FFPE blocks for mRNA detection could be confirmed using RNAscope is situ hybridization technology
Summary
Detection of mRNA in formalin-fixed, paraffin embedded (FFPE) tissue samples by chromogenic RNA in situ technology has become a reliable alternative for a wide range of biomarkers in many areas of research including cancer and neurosciences [1,2,3]. The technology should be robust enough to take account of acceptable variations in fixation parameters (time before fixation, time of fixation, time before dissection of gross resection specimen to optimal block size) which may present in large numbers of routine samples within a study cohort This was demonstrated recently by a review of the challenges and pitfalls in detecting PD-L1 expression in lung cancer, by IHC [6]. Comparison of PD-L1 expression in different NSCLC clinical trials showed a high variation in PD-L1 prevalence This variation can potentially be explained by differences in patient demographic, therapies and by variation in quality of assay antibodies or IHC methods used for detection. Alternative methods like RNAscope® ISH have been tested to complement IHC analysis and it has been suggested that “mRNA ISH may identify patients that would benefit from immunotherapy that would otherwise be negative for PD-L1 protein expression by IHC” [6]
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