Henriett Butz1, Roy Nofech-Mozes2, Qiang Ding2, Heba W Z Khella2, Peter M Szabó3, Michael Jewett4, Antonio Finelli4, Jason Lee5, Michael Ordon5, Robert Stewart5, Sergey Krylov6, George M Yousef7. 1. Department of Laboratory Medicine and the Keenan Research Centre for Biomedical Science of St. Michael's Hospital, Toronto, Ontario, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. 2. Department of Laboratory Medicine and the Keenan Research Centre for Biomedical Science of St. Michael's Hospital, Toronto, Ontario, Canada. 3. Biometric Research Branch, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. 4. Department of Surgery, Princess Margaret Hospital, Toronto, Ontario, Canada. 5. Department of Surgery, St. Michael's Hospital, Toronto, Ontario, Canada. 6. Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Ontario, Canada. 7. Department of Laboratory Medicine and the Keenan Research Centre for Biomedical Science of St. Michael's Hospital, Toronto, Ontario, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. Electronic address: yousefg@smh.ca.
Abstract
BACKGROUND: Apart from an invasive biopsy, currently no tools are available to confirm the diagnosis of clear cell renal cell carcinoma (ccRCC); this resulted in approximately 30% of patients being diagnosed with metastatic disease. OBJECTIVE: To determine whether urinary microRNAs (miRNAs) can serve as biomarkers to confirm the diagnosis of ccRCC. DESIGN, SETTING, AND PARTICIPANTS: Global miRNA expression was assessed in 28 preoperative urine samples from patients with ccRCC and 18 healthy participants. The independent validation set consisted of 81 ccRCC patients, 24 patients with benign lesions, and 33 healthy participants. We extracted both cell-free and exosomal RNA for miRNA expression analysis using miRNA-specific polymerase chain reaction assays. We also investigated exosomal miRNA secretion in cell line models and performed exosome transfer between RCC and endothelial cell types. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Receiver operating characteristic analysis was applied to identify the discrimination power of miRNAs. RESULTS AND LIMITATIONS: Overall, miR-126-3p combined with miR-449a or with miR-34b-5p could significantly distinguish ccRCC patients from healthy participants (miR-126-3p-miR-449a: area under the curve [AUC]: 0.84; 95% confidence interval [CI], 0.7620-0.9151; p<0.001; miR-126-3p-miR-34b-5p: AUC: 0.79; 95% CI, 0.7013-0.8815; p<0.001). The combination of miR-126-3p and miR-34b-5p was also able to distinguish small renal masses (pT1a, ≤4cm) from healthy controls (AUC: 0.79; 95% CI, 0.6848-0.8980; p<0.001). Using miR-126-3p and miR-486-5p in combination, we were able to differentiate between benign lesions and ccRCC (AUC: 0.85; 95% CI, 0.7295-0.9615; p<0.01). The expression of a number of miRNAs returned to a level comparable with health after surgery. Kidney cancer cell lines were found to secrete exosomal miR-126-3p, miR-17-5p, miR-21-3p, and miR-25-3p, and these miRNAs were found to be internalized by other cell types. CONCLUSIONS: We identified exosomal miRNAs as potential noninvasive diagnostic urinary biomarkers for ccRCC and provided evidence that miRNAs are secreted by the tumor and can function as a tool for intercellular communication. PATIENT SUMMARY: We identified urinary microRNAs that can serve as diagnostic biomarkers for clear cell renal cell carcinoma.
BACKGROUND: Apart from an invasive biopsy, currently no tools are available to confirm the diagnosis of clear cell renal cell carcinoma (ccRCC); this resulted in approximately 30% of patients being diagnosed with metastatic disease. OBJECTIVE: To determine whether urinary microRNAs (miRNAs) can serve as biomarkers to confirm the diagnosis of ccRCC. DESIGN, SETTING, AND PARTICIPANTS: Global miRNA expression was assessed in 28 preoperative urine samples from patients with ccRCC and 18 healthy participants. The independent validation set consisted of 81 ccRCC patients, 24 patients with benign lesions, and 33 healthy participants. We extracted both cell-free and exosomal RNA for miRNA expression analysis using miRNA-specific polymerase chain reaction assays. We also investigated exosomal miRNA secretion in cell line models and performed exosome transfer between RCC and endothelial cell types. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Receiver operating characteristic analysis was applied to identify the discrimination power of miRNAs. RESULTS AND LIMITATIONS: Overall, miR-126-3p combined with miR-449a or with miR-34b-5p could significantly distinguish ccRCC patients from healthy participants (miR-126-3p-miR-449a: area under the curve [AUC]: 0.84; 95% confidence interval [CI], 0.7620-0.9151; p<0.001; miR-126-3p-miR-34b-5p: AUC: 0.79; 95% CI, 0.7013-0.8815; p<0.001). The combination of miR-126-3p and miR-34b-5p was also able to distinguish small renal masses (pT1a, ≤4cm) from healthy controls (AUC: 0.79; 95% CI, 0.6848-0.8980; p<0.001). Using miR-126-3p and miR-486-5p in combination, we were able to differentiate between benign lesions and ccRCC (AUC: 0.85; 95% CI, 0.7295-0.9615; p<0.01). The expression of a number of miRNAs returned to a level comparable with health after surgery. Kidney cancer cell lines were found to secrete exosomal miR-126-3p, miR-17-5p, miR-21-3p, and miR-25-3p, and these miRNAs were found to be internalized by other cell types. CONCLUSIONS: We identified exosomal miRNAs as potential noninvasive diagnostic urinary biomarkers for ccRCC and provided evidence that miRNAs are secreted by the tumor and can function as a tool for intercellular communication. PATIENT SUMMARY: We identified urinary microRNAs that can serve as diagnostic biomarkers for clear cell renal cell carcinoma.
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