Use of High-Plex Data Reveals Novel Insights into the Tumour Microenvironment of Clear Cell Renal Cell Carcinoma

SIMPLE SUMMARY: Cancer is a complex ensemble of morphological and molecular features whose role is still unclear. Moreover, their role may change in different areas of the same tumour. Artificial intelligence (AI) allows pathologists to go beyond human perception and bias and may help better understand how these features influence disease progression. Furthermore, by capturing variation intrinsic to the tumour, AI may improve the accuracy of current prognostic tools, such as Leibovich Score (LS), in predicting patient outcome and response to therapy. For these reasons, we studied clear cell renal cell carcinoma (ccRCC) tissue in which molecular features and their coexpression in the same cell were quantified and mapped using AI-based image analysis software. We demonstrated a novel approach for investigating ccRCC and revealed new potential biomarkers of prognosis which may also be able to direct patients towards the most appropriate personalised therapy. ABSTRACT: Although immune checkpoint inhibitors (ICIs) have significantly improved the oncological outcomes, about one-third of patients affected by clear cell renal cell carcinoma (ccRCC) still experience recurrence. Current prognostic algorithms, such as the Leibovich score (LS), rely on morphological features manually assessed by pathologists and are therefore subject to bias. Moreover, these tools do not consider the heterogeneous molecular milieu present in the Tumour Microenvironment (TME), which may have prognostic value. We systematically developed a semi-automated method to investigate 62 markers and their combinations in 150 primary ccRCCs using Multiplex Immunofluorescence (mIF), NanoString GeoMx(®) Digital Spatial Profiling (DSP) and Artificial Intelligence (AI)-assisted image analysis in order to find novel prognostic signatures and investigate their spatial relationship. We found that coexpression of cancer stem cell (CSC) and epithelial-to-mesenchymal transition (EMT) markers such as OCT4 and ZEB1 are indicative of poor outcome. OCT4 and the immune markers CD8, CD34, and CD163 significantly stratified patients at intermediate LS. Furthermore, augmenting the LS with OCT4 and CD34 improved patient stratification by outcome. Our results support the hypothesis that combining molecular markers has prognostic value and can be integrated with morphological features to improve risk stratification and personalised therapy. To conclude, GeoMx(®) DSP and AI image analysis are complementary tools providing high multiplexing capability required to investigate the TME of ccRCC, while reducing observer bias.