Integrated Molecular Analysis for the Identification and Validation of Novel Biomarkers for Renal Cell Carcinoma and the Small Renal Mass

Renal cell carcinoma (RCC) constitutes an array of morphologically and genetically distinct tumors with varying clinical behavior, prognosis and response to therapy. RCC is frequently diagnosed incidentally as an early-stage small renal mass (SRM; pT1a, ≤ 4 cm). Overtreatment of patients with benign or clinically indolent SRMs is increasingly common and has resulted in a recent shift in treatment recommendations. There are currently no available biomarkers that can accurately predict risk of clinical progression. Additionally, there are no biomarkers that can accurately classify RCC. To that end, we developed an integrated label-free LC-MS/MS proteomics and peptidomics approach and targeted parallel-reaction monitoring (PRM) to identify early non-invasive biomarkers of RCC progression. In addition, we performed qRT-PCR to identify prognostic (predict early RCC progression) and diagnostic (RCC subtype classification) miRNAs. We identified biomarkers which displayed differential expression in clear cell RCC-SRMs (ccRCC-SRMs) compared to healthy controls and renal oncocytoma (≤ 4 cm). Additionally, we identified biomarkers which displayed differential expression in progressive relative to non-progressive ccRCC-SRMs. A two-protein signature (EPS8L2 and CCT6A) showed significant discriminatory ability in distinguishing progressive from non-progressive ccRCC-SRMs. Moreover, patients with EPS8L2 and CCT6A mRNA alterations showed significantly shorter overall survival compared to patients with no alterations in an independent cohort of ccRCC (stage I-IV) patients. Peptide SHTSDSDVPSGVTEVVVKL showed significant discriminatory ability in distinguishing progressive from non-progressive tumors. Additionally, patients with elevated SHTSDSDVPSGVTEVVVKL expression had significantly shorter overall survival compared to patients with low expression. miR-328-3p displayed significantly down-regulated expression in progressive relative to non-progressive ccRCC-SRMs. Patients with elevated miR-328-3p expression had significantly longer overall survival compared to patients with low miR-328-3p expression. In addition, we demonstrated that miR-221 and miR-222 can distinguish clear cell and papillary subtypes from chromophobe tumors and renal oncocytoma. We also showed that miR-126 can discriminate clear cell from papillary RCC and miR-200b can discriminate chromophobe RCC from renal oncocytoma. In summary, the research presented here adds to our current understanding of the natural history of clinically localized RCC-SRMs, and identifies early markers of clinical progression, which could potentially help assign patients to appropriate management programs.Ph.D