Dickkopf-1 (DKK1) is a secreted modulator of Wnt signaling that is frequently overexpressed in tumors and associated with a poor prognosis. In this study, we demonstrate an approach for clinically validating a RNAscope chromogenic in-situ hybridization (CISH) assay for determining the level of DKK1 RNA in Gastric (G) and Gastroesophageal (GEJ) tumor tissues according to CLIA guidelines. This two-step process validated first the performance of the wet chemistry assay along with the ability of a pathologist to manually score the CISH signal according to a dot-based H-score paradigm, and second, the ability of an image analysis (IA) solution (Flagship Biosciences) to unbiasedly and reproducibly quantify DKK1 staining in the same set of samples.
Results and Conclusions
The DKK1 RNAscope wet chemistry assay validation results (see poster Figs. 1 and 2) show that the DKK1 RNAscope assay yields high analytical sensitivity, specificity, accuracy, and precision in the determination of the manual pathology DKK1 H-score: > 90% of the tissue cohort met the passing criteria for sensitivity, specificity, and precision and the manual H-scores significantly correlated with the DKK1 qPCR assay for accuracy.
The DKK1 RNAscope IA solution validation results (see poster Figs. 4 and 5) show that the IA solution yields high analytical sensitivity, specificity, accuracy, and precision in the determination of the digital DKK1 H-score: ≥ 90% of the tissue cohort met the passing criteria for sensitivity, specificity, and precision and the digital and manual pathology H-scores were significantly correlated for accuracy.
These data demonstrate a clinically validated DKK1 RNAscope CISH laboratory-derived test (LDT) for manual and IA-assisted pathologist interpretation. This LDT is currently being used in a phase 2 clinical study of DKN-01, a DKK1 neutralizing antibody, in combination with tislelizumab to prospectively identify previously treated patients with elevated DKK1 tumor expression (Leap Therapeutics; NCT04363801).