Validation of a DKK1 RNAscope chromogenic in situ hybridization assay for gastric and gastroesophageal junction adenocarcinoma tumors

Abstract

Dickkopf-1 (DKK1) is a secreted modulator of Wnt signaling that is frequently overexpressed in tumors and associated with poor clinical outcomes. DKN-01 is a humanized monoclonal therapeutic antibody that binds DKK1 with high affinity and has demonstrated clinical activity in gastric/gastroesophageal junction (G/GEJ) patients with elevated tumoral expression of DKK1. Here we report on the validation of a DKK1 RNAscope chromogenic in situ hybridization assay to assess DKK1 expression in G/GEJ tumor tissue. To reduce pathologist time, potential pathologist variability from manual scoring and support pathologist decision making, a digital image analysis algorithm that identifies tumor cells and quantifies the DKK1 signal was developed. Following CLIA guidelines the DKK1 RNAscope chromogenic in situ hybridization assay and digital image analysis algorithm were successfully validated for sensitivity, specificity, accuracy, and precision. The DKK1 RNAscope assay in conjunction with the digital image analysis solution is acceptable for prospective screening of G/GEJ adenocarcinoma patients. The work described here will further advance the companion diagnostic development of our DKK1 RNAscope assay and could generally be used as a guide for the validation of RNAscope assays with digital image quantification.

Safety, Efficacy, and Biomarker Results from a Phase Ib Study of the Anti-DKK1 Antibody DKN-01 in Combination with Pembrolizumab in Advanced Esophagogastric Cancers

“…RNAscope is a highly sensitive and specific expression technique that overcomes antibody reagent limitations common with IHC. RNAscope was recently utilized to identify patients with elevated FGFR mRNA expression in a phase I dose-escalation trial for an orally available inhibitor of FGFR1–4 kinase activity, and the DKK1 RNAscope assay has been validated as a laboratory developed test for the prospective screening of patient tumoral tissue ( 32, 33 ). Our findings suggest that DKK1 expression assessed by RNAscope CISH potentially has substantial discriminatory ability in identifying patients more likely to benefit from DKN-01-based therapies.…”

Prostaglandin EP4 receptor mRNA expression in canine lymphoma

“…The following metrics were then calculated for ptger4 expression based upon the cumulative analysis results of the 10 digitised photomicrographs for the slide: average transcript copy number/cell, H‐score, and percent probe positive expression (percent of cells positive for EP4R mRNA). The H‐score is a weighted, semi‐quantitative image analysis/expression scale that is utilised for immunohistochemistry quantitative analysis to evaluate heterogeneity in marker expression and has also been validated for mRNA expression analysis by RNAscope 24–26 . These combined techniques allow for quantification of the gene expression within a specific cell type and tissue context, and comparison of expression across LSA and reactive lymph tissues.…”

“…The H-score is a weighted, semiquantitative image analysis/expression scale that is utilised for immunohistochemistry quantitative analysis to evaluate heterogeneity in marker expression and has also been validated for mRNA expression analysis by RNAscope. [24][25][26] These combined techniques allow for quantification of the gene expression within a specific cell type and tissue context, and comparison of expression across LSA and reactive lymph tissues.…”

European Society of Toxicologic Pathology (Pathology 2.0 Molecular Pathology Special Interest Group): Review of In Situ Hybridization Techniques for Drug Research and Development

“…In general, modern RNA ISH techniques are specific and sensitive that have been increasingly utilized in clinical studies, particularly in patient selection and/or stratification. [175][176][177][178] FGFR mRNA ISH was used to select patients with advanced cancers for treatment with FGFR inhibitor. 175,179 Similarly, DKK1 mRNA ISH was used to screen patients with gastric and gastroesophageal adenocarcinoma in clinical trials.…”