Abstract A021: Analytical platform to validate microRNA cancer biomarkers

Abstract We developed and tested a novel assay to quantify and validate microRNA (miRNA) cancer biomarkers in liquid biopsies. The assay is amplification-free, exploits a commercially available nanopore-array device for detection (MinION from Oxford Nanopore Technologies), uses total RNA isolated from biospecimen as the miRNA source, and proprietary osmylated oligonucleotides, complementary to the target miRNA, as probes. The assay exploits a bracketing experimental protocol to quantify miRNA, and yields miRNA copy numbers (copies) per 1microLiter of isolated total RNA, with an unprecedented accuracy of approximately +/- 20% across all concentrations. For development purposes and as a control/reference the combined serum of healthy men (# H6914 from Sigma-Aldrich) was used. During a period of 4 years different lots of H6914 yielded, within experimental error, comparable miRNA copies for let-7b, miR-16, miR-21, miR-15b, miR-375 and miR-141. Serum samples from healthy individuals and cancer patients were also tested. Once data were normalized to the same total RNA content, miRNA copies appeared to belong to one of two groups. The first group included miR-21, miR-375 and miR-141 copies in H6914 and in healthy samples. The second group included miR-21, miR-375 and miR-141 copies in cancer samples exhibiting an approximate 1.8-fold overexpression compared to the first group. miR-15b copies in H6914, healthy and cancer samples were all in the first group. The two groups exhibited zero data overlap which is unprecedented in the miRNA/cancer literature. Zero data overlap was the result of (i) the assay’s bracketing approach with a protocol-defined accuracy of approximately +/-20%, and (ii) the normalization of miRNA copies to the same total RNA content. Notably, these two groups included both serum and urine samples from cancer patients and healthy individuals. All subjects were deselected regarding age, sex, and ethnicity, while patients were selected for breast, prostate or pancreatic cancer, Stage I or II, pretreatment. To the best of our knowledge, this is the first-time agreement between serum and urine miRNA copies is reported and leads to the suggestion that a urine sample may replace a blood draw for the tested miRNAs. Not only clear discrimination between healthy and cancer samples was possible, but this analytical platform may be used to validate tentative cancer/disease biomarkers when miRNA overexpression between cancer/disease and healthy is equal or higher than 1.8-fold. Non-Coding RNA 2024, 10(4), 42; https://doi.org/10.3390/ncrna10040042 Citation Format: Anastassia Kanavarioti, M. Hassaan Rehman, Salma Qureshi, Aleena Rafiq, Madiha Sultan. Analytical platform to validate microRNA cancer biomarkers [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A021.