Use of Droplet Digital Polymerase Chain Reaction to Identify Biomarkers for Differentiation of Benign and Malignant Renal Masses

Abstract

Several microRNAs (miRNAs) have been identified as cell-free biomarkers for detecting renal cell carcinoma (RCC). Droplet digital polymerase chain reaction (ddPCR) is a unique technology for nucleic acid quantification. It has the potential for superior precision, reproducibility, and diagnostic performance in identifying circulating miRNA biomarkers compared to conventional quantitative real-time PCR (qRT-PCR). This study aims to evaluate the performance of ddPCR compared to qRT- PCR in identifying miRNA biomarkers that differentiate malignant from benign renal masses. Potential biomarkers of RCC were identified from a literature review. RNA was extracted from the plasma of 56 patients. All the samples underwent analysis via ddPCR as well as qRT-PCR, and expression levels were recorded for the following miRNAs: miR-93, -144, -210, -221, and -222. Tumors were grouped into low-grade ccRCC, high-grade ccRCC, papillary RCC, and benign masses (primarily angiomyolipoma). The miRNA miR-210 (p = 0.034) and the combination of miRs-210 and miR-222 (p = 0.003) were expressed at significantly higher rates among those with RCC than those with benign masses, as measured by ddPCR. Using the combination of miR-210 and miR-222, ddPCR identified significant differences between the subgroups: papillary RCC versus benign (p = 0.03), low-grade ccRCC versus benign (p = 0.026), and high-grade ccRCC versus benign (p = 0.002). The only significant difference between these subgroups using qRT-PCR was between high-grade ccRCC and benign (p = 0.045). All the AUCs were significant when comparing each RCC subgroup with benign for both PCR technologies. Using a combination of miR-210 and miR-222, ddPCR identified significant differences between benign and malignant renal masses that were not identified as significant by conventional qRT-PCR.