Clinical Validation Of A Dickkopf-1 (Dkk1) Chromogenic In-Situ Hybridization (Cish) Assay For Manual And Image-Analysis Assisted Pathologist Interpretation.

Abstract 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 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 image analysis (IA) software (Flagship Biosciences) to unbiasedly and reproducibly quantify DKK1 staining in the same set of samples. The DKK1 CISH assay for manual scoring passed all pre-determined criteria of sensitivity, specificity, accuracy, and precision. 100% of the 40 evaluated G/GEJ tissues demonstrated acceptable staining in target tumor cells and absence in non-target cells. 100% of the evaluated tissues passed sensitivity with a broad dynamic range of signal expression across target cells, and negligible background staining. Reproducibility was measured by blinded pathology scoring of a serial subset of 12 cases, resulting in 11/12 (92%) with concordant DKK1 H-scores. Accuracy was assessed with a DKK1 qPCR assay on a 20-sample subset and a significant correlation with the H-score data was observed. The IA algorithm also passed all pre-determined criteria of sensitivity, specificity, accuracy, and precision. 36/40 samples (90%) passed analytical specificity with the IA algorithm correctly classifying staining on true cells belonging to the tumor. Failed samples were attributed to non-specific alkaline phosphatase activity, which in practice would be disregarded by the reviewing pathologist. 100% of samples passed the sensitivity criteria of appropriate cell identification and classification. IA precision compared H-scores across 3 days of staining, with 11/12 (92%) cases having concordant DKK1 H-scores and an ICC of 0.9009 (95% CI: 0.7117-0.9601). Digital H-scores were highly correlated to the validated manual H-scores of the 40-sample set (r = 0.72, p <0.0001). Taken together, these data demonstrate a clinically validated DKK1 RNAscope CISH laboratory-derived test (LDT) for manual and IA-assisted pathologist interpretation. The DKK1 RNAscope LDT is currently being applied as part of a phase 2 clinical study of DKN-01 in combination with tislelizumab to prospectively identify previously treated G/GEJ adenocarcinoma patients with elevated DKK1 tumor expression (NCT04363801). Citation Format: Charles Caldwell, Mike Kagey, Sofia Reitsma, Will Paces, Elizabeth Bueche, Vitria Adisetiyo, Roberto Gianani. Clinical validation of a Dickkopf-1 (DKK1) chromogenic in-situ hybridization (CISH) assay for manual and image-analysis assisted pathologist interpretation [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 433.