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.

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.

Analyzing images of tissue sections stained with biomarker-specific indicators can generate quantitative, cell-specific expression data rather than relying on pathologist's ability to count cells and recapitulate confounding scoring paradigms. Beyond biomarker expression, numerous cell features can be measured such as the cell morphology, biomarker staining patterns and intensity, and localization of the cells within defined spatial compartments or relations to one another. One of the most studied immune cells in immuno-oncology (IO) is the cytotoxic T cell, whose biological function is to identify and destroy infected or dysfunctional cells; this function is complicated by numerous factors within the tumor microenvironment (TME). Tumor cells can aberrantly express immune checkpoint molecules designed to stop CD8 T cells from performing their tumor killing function. Additionally, CD8 T cell function may be perturbed by other immune modulating factors within the TME. IO drugs modulating tumor and immune cell interactions such as PD-L1 checkpoint inhibitors have shown that an inflammatory TME, represented by high CD8 presence in the tumors (inflamed tumors), is indicative of a better therapeutic response. Investigation of CD8 T cell status in biopsied tissues typically describes each tissue as one of three main phenotypes: Immune Desert, Immune Excluded, or Inflamed. Immune Desert tissues do not express appreciable levels of CD8 throughout the tissue. Immune Excluded tissues contain CD8, but the expression is almost exclusively localized to the stroma surrounding tumor nests. Inflamed tissues show higher percentages of CD8 within the tumor nests. While this phenotypic categorization is informative, CD8 expression is often calculated as a mean of expression through the tissue and does not take in to account the heterogeneous nature of tumor biology. This may result in a tumor containing one highly inflamed tumor nest being averaged out with multiple deserted tumor nests and a tissue categorized as excluded or deserted even though inflammation is present. To better represent the heterogeneity of inflammation within tumor tissues, we present an image analysis-based algorithm which not only separates out the tumor, stroma, and tumor/stroma margin, but identifies each tumor nest within the tissue as its own discrete object. This allows for the enumeration of number and size of all tumor nests within the tissue. Each tumor nest is given its own phenotypic classification (inflamed, excluded, or deserted) and the percentage of tumor nests displaying each phenotype. We demonstrate heterogeneity of inflammation assessment alongside standard mean phenotypic evaluations of CD8 expression in non-small cell lung cancer, bladder, and melanoma tumor samples. Practical use in clinical studies can help uncover response or resistance associated phenotypes related to tumor heterogeneity. Citation Format: Charles William Caldwell, Will Paces, Sofia Reitsma, Christopher Brueckner, Si T. Lee-Hoeflich, Roberto Gianani. Characterizing heterogeneity of CD8 inflammation in biopsied tumor tissues using novel image analysis techniques [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 3137.

TGFβ signaling appears to be a key mediator of primary resistance to programmed cell death protein (PD-1) pathway blockade. SRK-181 is an investigational stage, high-affinity, fully human antibody that selectively binds to latent TGFβ1 and inhibits its activation. Preclinical data demonstrate that combining SRK-181 with a PD-1 inhibitor modulates tumor microenvironment (TME), including an influx of CD8 positive T cells that correlates with anti-tumor responses. The ongoing DRAGON trial is a multi-center, open-label, Phase 1, first in-human (FIH), dose-escalation, and dose-expansion study. The goal of the trial is to evaluate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and efficacy of SRK-181 administered alone and in combination with an anti-PD-(L)-1 in adult patients with locally advanced or metastatic solid tumors. To support this ongoing DRAGON clinical trial and further explore the mechanism of action of SRK-181, a comprehensive biomarker strategy is being developed to assess the alternation of immune profile in TME and potential predictors of therapeutic response to SRK-181. Here we describe the development and refinement of several biomarker assays. First, an image analysis-based algorithm for CD8 immunohistochemistry (IHC) is established utilizing human cancer tissue in a pre-clinical study. This novel digital pathology analysis enables identification of CD8 positive T cells in discrete compartments, including the tumor nests, stroma and tumor/stromal margins, to better capture the heterogeneity of the CD8 signal within tissues. Second, we describe methods to evaluate the TGFβ pathway including quantitative analysis of tumor tissue phospho-Smad2 and circulatory levels of TGFβ1 ligand. A companion assay to exclude blood samples with nonspecific background signals has been characterized and will be performed in parallel when evaluating circulatory TGFβ1 in clinic. In summary, we present several novel, tailored biomarker readouts that are part of a broader biomarker strategy aimed at maximizing detection of relevant clinical data to both support the ongoing clinical trial and provide further insight into the mechanism of action of SRK-181. Citation Format: Christopher Brueckner, Ryan Faucette, Charles Caldwell, Jr., Sofia Reitsma, Stefan Wawersik, Ashish Kalra, Lu Gan, Si Tuen Lee-Hoeflich. Development of a comprehensive biomarker strategy to support phase 1 clinical trial of SRK-181 the latent TGFβ1 inhibitor [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 1801.