Recognizing the Continuous Nature of Expression Heterogeneity and Clinical Outcomes in Clear Cell Renal Cell Carcinoma

Clear cell renal cell carcinoma (ccRCC) has been previously classified into putative discrete prognostic subtypes by gene expression profiling. To investigate the robustness of these proposed subtype classifications, we evaluated 12 public datasets, together with a new dataset of 265 ccRCC gene expression profiles. Consensus clustering showed unstable subtype and principal component analysis (PCA) showed a continuous spectrum both within and between datasets. Considering the lack of discrete delineation and continuous spectrum observed, we developed a continuous quantitative prognosis score (Continuous Linear Enhanced Assessment of RCC, or CLEAR score). Prognostic performance was evaluated in independent cohorts from The Cancer Genome Atlas (TCGA) (n = 414) and EMBL-EBI (n = 53), CLEAR score demonstrated both superior prognostic estimates and inverse correlation with anti-angiogenic tyrosine-kinase inhibition in comparison to previously proposed discrete subtyping classifications. Inverse correlation with high-dose interleukin-2 outcomes was also observed for the CLEAR score. Multiple somatic mutations (VHL, PBRM1, SETD2, KDM5C, TP53, BAP1, PTEN, MTOR) were associated with the CLEAR score. Application of the CLEAR score to independent expression profiling of intratumoral ccRCC regions demonstrated that average intertumoral heterogeneity exceeded intratumoral expression heterogeneity. Wider investigation of cancer biology using continuous approaches may yield insights into tumor heterogeneity; single cell analysis may provide a key foundation for this approach.