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

Introduction

Caldwell et al., present an approach to the targeted selection of suitable candidate patients into a phase 2 clinical study using a laboratory validated chromogenic RNAscope assay for DKK1.

Dickkopf-1 (DKK1), is a secreted modulator of Wnt signalling. It demonstrates elevated expression in a range of tumours and is associated with poor clinical prognosis. It has been demonstrated that DKK1 promotes tumour growth, can stimulate tumour angiogenesis and facilitates metastasis. However, The oncogenic activity of DKK1 can be blocked by a therapeutic IgG4 antibody, DKN-01.

DKK1 expression was retrospectively measured in selected tumour tissue from relapsed gastric / gastroesophageal junction patients that received a DKN-01 combination treatment as part of a phase 1 clinical trial. It was hypothesized that patients with a high expression level of DKK1 were more likely to benefit from the phase 2 DKN-01 + tislelizumab combination therapy – as a subgroup of patients in the phase 1 study who had not previously received an anti PD-1 / PD-L1 therapy showed good clinical response and increased progression free survival with high DKK1 expression. The DKK1 RNAscope assay was therefore developed and validated to assist with the confident selection of suitable patients with high DKK1 expression that may show a good response in the phase 2 trial.

Main Points

• RNAscope is an in situ hybridization technique in which probes are designed to selectively target mRNA. The assay can overcome sensitivity and specificity issues that can arise with routine IHC. RNAscope allows the detection of single mRNA transcripts of a target gene using a double z strategy that allows for amplification of the target signal with no background. It may be a useful alternative where suitable IHC reagents are not available for specific biomarkers. (As with DKK1 where the available range is limited)
• 4 cell lines with a wide range of DKK1 expression (PC3, A549, HeLa and Pfeiffer) were initially labelled with the DKK1 RNAscope. Corresponding RNA seq data from the 4 cell lines using qPCR and ELISA assays was also obtained. Positive and negative reference genes were also tested on the cell lines via RNAscope for use as controls.
• The resultant DKK1 RNAscope staining expression matched with the data from the qPCR and ELISA assays confirming specific DKK1 staining over a wide dynamic range. (PC3 cells had the greatest expression with a reduced signal in the A549 / HeLa cells. The Pfeiffer cells had no expression).
• Accuracy was further assessed by comparing the DKK1 RNAscope assay to a DKK1 IHC assay using the same cell line pellets. The RNAscope and IHC data were consistent across the cell lines however the RNAscope assay proved more sensitive and detected small amounts of DKK1 RNA in the HeLa pellet where no IHC signal was achieved.
• To test DKK1 specificity, other cell lines were labelled that expressed high levels of other dickkopf family members (DKK2, DKK3, DKK4 and DKKL1). The DKK1 RNAscope assay showed little to no labelling in any of the cell line pellets other than the DKK1 pellet demonstrating that the assay was not cross reacting with other close family members – further confirming DKK1 specificity. Taken together – these results indicate that the DKK1 RNAscope assay is highly specific and accurate.
• To analyse the stained resected tumour tissues, labelled slides were scanned at x40 and an image analysis algorithm was developed to identify tumour cells and to quantify the DKK1 signal by counting the number of dots per tumour cell and an H-score was subsequently applied. (The greater the number of DKK1 positive cells – the higher the H score). The algorithm was able to identify tumour cells from non-tumour cells and categorize cells as either positive or negative for DKK1. 6 tumour issues were initially assessed in duplicate by the algorithm and manually via 2 separate pathologists for comparison. The results demonstrated a much lower degree of variability when using the algorithm supporting the use of the digital image analysis solution.

Conclusion

Biomarkers have increasing importance in drug development and are often applied in trial design to target patients that are most likely to benefit from a specific treatment. Therefore it is essential that biomarker tests are robust and precise. The DKK1 RNAscope assay developed by Caldwell et al, has been demonstrated to be highly sensitive and specific with no cross-reactivity. As such, the assay has been applied as part of a phase 2 clinical study (of DKN-01 in combination with tislelizumab) to identify previously treated adenocarcinoma patients with elevated DKK1 tumoral expression for suitable inclusion in the study.

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