Cell-free RNA urogentital cancer biomarker detection from urine

Background: Urogenital cancers encompass a variety of tumor types including kidney, bladder and prostate cancer, the most prevalent cancer in men. Current early detection methods rely on blood screening of prostate-specific antigen (PSA), however this method is associated with overdiagnosis and overtreatment, has a high rate of false positives, and the subsequent procedures carry risks. As a result, the search for alternative biomarkers is of much interest. Urine is an ultra-non-invasive analyte ideal for urogenital cancer detection, including prostate cancer. Use of cell free RNA (cfRNA) is ideal for biomarker identification for use in diagnostics, treatment monitoring, and tumor tissue of origin prediction. Methods: Here we describe urine cfRNA extraction and RNA-Seq analysis on a series of urogenital cancer affected urine samples and normal control urine samples. Several extraction kits were tested, including a novel cfRNA isolation technique that has highly efficient recovery rates of cfRNA with variable input amounts of 1-100 mL of sample. Following extraction, quantitative and qualitative QC were performed directly from the isolate, as well as functional downstream testing via RNA-Seq to assess recovery, transcriptome complexity, and detectability of known cancer biomarkers. Results: Preliminary testing of the cfRNA isolation methods showed that traditional extraction methods did not achieve the yield required for detection of low-abundance biomarkers. In contrast, the novel extraction method demonstrated high efficiency cfRNA recovery. Along with traditional quantitative and qualitative QC of the extracted cfRNA, functional testing was performed using RNA-Seq. RNA-Seq results demonstrated detection of rare transcripts and cancer-associated biomarkers, confirming this is a reliable method for biomarker detection. Conclusion: As these results show, cfRNA can be extracted from urine, with the detection of rare biomarkers associated with disease. The high-efficiency extraction method enables sensitive detection of low-frequency transcripts, addressing limitations of conventional techniques. This method is not limited to urine and cfRNA, with the potential for application in other biofluids including plasma and other nucleic acid types such as mitochondrial DNA.