LiKidMiRs: A ddPCR-Based Panel of 4 Circulating miRNAs for Detection of Renal Cell Carcinoma

Abstract

Background: Decreased renal cell cancer-related mortality is an important societal goal, embodied by efforts to develop effective biomarkers enabling early detection and increasing the likelihood of curative treatment. Herein, we sought to develop a new biomarker for early and minimally invasive detection of renal cell carcinoma (RCC) based on a microRNA panel assessed by ddPCR.

Methods: Plasma samples from patients with RCC (n = 124) or oncocytomas (n = 15), and 64 healthy donors, were selected. Hsa-miR-21-5p, hsa-miR-126-3p, hsa-miR-155-5p and hsa-miR-200b-3p levels were evaluated using a ddPCR protocol.

Results: RCC patients disclosed significantly higher circulating levels of hsa-miR-155-5p compared to healthy donors, whereas the opposite was observed for hsa-miR-21-5p levels. Furthermore, hsa-miR-21-5p and hsa-miR-155-5p panels detected RCC with high sensitivity (82.66%) and accuracy (71.89%). The hsa-miR-126-3p/hsa-miR-200b-3p panel identified the most common RCC subtype (clear cell, ccRCC) with 74.78% sensitivity.

Conclusion: Variable combinations of plasma miR levels assessed by ddPCR enable accurate detection of RCC in general, and of ccRCC. These findings, if confirmed in larger studies, provide evidence for a novel ancillary tool which might aid in early detection of RCC.

Keywords: circulating miRNA; ddPCR; diagnosis; malignancy; renal cell carcinoma.

Figure 1

Violin plots with miRNA levels in Healthy Donors (HD) and Renal Cell Carcinoma (RCC) samples of hsa-miR-21-5p (A) and hsa-miR-155-5p (B), and respective Receiver-Operating Characteristic Curve (without resampling analysis) (C,D). In violin plots, dashed lines indicate the interquartile range and horizontal line the median of miR levels. In ROC curves, red line indicates the reference line and blue line the identity line for each miRNA. Abbreviations: AUC—Area Under the Curve; CI—Confidence Interval, HD—Healthy Donors, RCC—Renal Cell Carcinoma, *—p-value < 0.05, ***—p-value < 0.0001.

Figure 2

Violin plots of miRNAs levels in Healthy Donor (HD) and early stages of Renal Cell Carcinoma (Stage I and II) samples of hsa-miR-21-5p (A) and hsa-miR-155-5p (B), and respective Receiver-Operating Characteristic Curve (without resampling analysis) (C,D). In violin plots, dashed lines indicate the interquartile range and horizontal line the median of miR levels. In ROC curves, red line indicates the reference line and blue line the identity line for each miRNA. Abbreviations: AUC—Area Under the Curve; CI—Confidence Interval; HD—Healthy Donors, **—p-value < 0.001.

Figure 3

Violin plots of hsa-miR-21-5p (A), hsa-miR-126-3p (B), hsa-miR-155-5p (C) and hsa-miR-200b-3p (D) levels in the malignant subtypes (ccRCC, pRCC and chRCC). Dashed lines indicate the interquartile range and horizontal line the median of miR levels. Abbreviations: ccRCC—Clear-Cell Renal Cell Carcinoma; chRCC—Chromophobe Renal Cell Carcinoma; pRCC—Papillary Renal Cell Carcinoma; n.s.—not significant, *—p-value < 0.05, **—p-value < 0.001, ***—p-value < 0.0001.

Figure 4

Violin plots of hsa-miR-21-5p (A), hsa-miR-126-3p (B), hsa-miR-155-5p (C) and hsa-miR-200b-3p (D) levels in ccRCC and other RCCs (pRCC and chRCC). Dashed lines indicate the interquartile range and horizontal line the median of miR levels. Abbreviations: ccRCC—Clear-Cell Renal Cell Carcinoma; RCC—Renal Cell Carcinomas; n.s.—not significant, *—p-value < 0.05, **—p-value < 0.001.

Figure 5

Receiver-Operating Characteristic Curves (without resampling analysis) of hsa-miR-21-5p (A), hsa-miR-126-3p (B), hsa-miR-155-5p (C) and hsa-miR-200b-3p (D) in ccRCC and other RCCs (pRCC and chRCC). Red line indicates the reference line and blue line the identity line for each miRNA. Abbreviations: AUC—Area Under the Curve; CI—Confidence Interval.

Figure 6

Graphical representation of the potential clinical impact of LiKidMiRs. Created with BioRender.com.