Abstract 740: A robust rapid mRNA based test to profile simultaneously ER, AR, PI3K and MAPK functional signaling pathway activity for precision oncology
Authors: den Biezen Eveline , Vermeer Saskia , Keizer Diederick , Akse Martijn , van Zelst Martijn , van Strijp Dianne , Park Hannah , Magliocco Anthony 
Published: July 2021

Abstract
Introduction: OncoSignal test was developed to determine and quantify functional signal transduction pathway activity levels of 4 key oncogenic signaling pathways (ER, AR, PI3K and MAPK), based on the quantitative measurement of mRNA expression levels of the direct target genes. Here we present data of a 4 pathway qPCR test, that can be used in external labs and is able to measure simultaneously the 4 pathway activities in tumor tissue samples possibly providing important information for optimal therapy selection for treatment of breast cancer.
Materials and methods: OncoSignal 4 pathway test was compared between 2 reference laboratories; the Philips service lab in The Netherlands and at Protean BioDiagnostics, a US CAP-certified CLIA service lab. mRNA was isolated from unstained FFPE tissue sections of well characterized breast tissues obtained from 65 patients (retrospective). Annotated areas with at least 50% tumor cells were used (total volume about 0.25mm3). The OncoSignal test was used to measure the signal transduction pathway activity of AR, ER, PI3K and MAPK pathways. IHC staining for ER, PR, Ki67 and HER2 was also available for all samples enabling tumor categorization into conventional breast cancer subtypes.
Results: Excellent inter-lab concordance was found with correlation coefficients of 0.99, 0.99, 0.98 and 0.99 for ER, AR, PI3K and MAPK, respectively. Average absolute difference between pathway activities was 3.6 activity points on a 0-100 activity score scale. All 32 cases passed the criteria set for reproducibility. Of the 65 tested breast cancer samples, 58 could be subtyped based on IHC. Five of the 58 samples failed QC. Of interest, ER IHC positive luminal A (41%) and B (28%) breast cancer showed high variation in ER pathway activity between cases, suggesting that the ER signaling pathway is not the tumor driving pathway in a significant subset of IHC ER positive tumors. As expected the Her2-enriched (17%) and TNBC (17%) sub-groups predominantly had a very low ER pathway activity score. AR pathway activity was most prominent in the HER2 enriched tumor groups, while in TNBC the MAPK pathway was most active.
Conclusion: The OncoSignal 4 pathway test is robust and can be implemented in external (local) labs as shown by the excellent correlation results. The results of the signal pathway activity analysis correlate well with breast subtype classification, however, the assay has the potential to uncover specific pathway activation independent of subtype which may have significant value for precision oncology and targeted therapy selection. The test has potential for wide utilization as it can be performed on conventional FFPE prepared breast specimens, and may be complementary to conventional mutational and IHC analysis for classifying breast cancer and selecting appropriate therapy.
Citation Format: Eveline den Biezen, Saskia Vermeer, Diederick Keizer, Martijn Akse, Martijn van Zelst, Dianne van Strijp, Hannah Park, Anthony Magliocco. A robust rapid mRNA based test to profile simultaneously ER, AR, PI3K and MAPK functional signaling pathway activity for precision oncology [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 740.