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Use of Droplet Digital Polymerase Chain Reaction to Identify Biomarkers for Differentiation of Benign and Malignant Renal Masses
Adam Wiggins, MD, Joshua P. Hayden, MD, Travis Sullivan, MS, Thomas Kalantzakos, BA, Kailey Hooper, BS, David Canes, MD, Alireza Moinzadeh, MD, Kimberly Rieger-Christ, PhD.
Lahey Hospital and Medical Center, Burlington, MA, USA.

Background: Several microRNAs (miRNAs) have been identified as biomarkers for differentiating clear cell renal carcinoma (ccRCC) from benign renal masses as well as predicting which malignant tumors progress to metastatic disease. Droplet digital polymerase chain reaction (ddPCR) is a relatively novel technology for nucleic acid quantification. It has the potential for superior precision, reproducibility, and diagnostic performance in identifying circulating miRNA biomarkers for cancer compared to conventional PCR. This study aims to evaluate the performance of ddPCR compared to conventional PCR in identifying miRNA biomarkers that differentiate malignant from benign renal masses. The ability to distinguish between malignant and benign renal masses using a minimally invasive, serum-based test could ultimately reduce the morbidity associated with renal mass biopsy, which itself is imperfect in identifying malignant versus benign renal masses.
Methods: Potential biomarkers of ccRCC were identified from literature review. RNA was extracted from the plasma of 56 patients with renal masses and was subsequently reverse transcribed. All samples then underwent nucleic acid amplification via ddPCR as well as traditional quantitative reverse transcription PCR (qRT-PCR) and expression levels were recorded for the following miRNAs: miR-93, miR-144, miR-210, miR-221, and miR-222. Tumors were grouped into low grade ccRCC, high grade ccRCC, papillary RCC and angiomyolipoma (AML). Absolute counts were compared between groups using Kruskal-Wallis tests. All statistical analysis were performed using Statistical Package for Social Sciences (SPSS) software version 27.0.
Results: The miRNAs miR-210 (p = .031), miR-224 (p = .04), and the combination of miR-210 and miR-222 (p = .003) were expressed at significantly higher rates among those with RCC compared to those with AML as measured by ddPCR. Using the combination of miR-210 and miR-222, ddPCR identified significant differences between papillary RCC versus AML (p = .034), low grade ccRCC versus AML (p = .014), and high grade ccRCC versus AML (p = .001). There were no significant differences between these groups using conventional PCR.
Conclusions:Droplet digital PCR was effective in identifying miR-210, miR-224, and the combination of miR-210 and miR-222 as biomarkers to differentiate between benign and malignant renal masses. Using a combination of miRNA biomarkers, ddPCR identified significant differences between benign and malignant renal masses that were not identified by conventional qRT-PCR. Future work should investigate the prognostic capabilities of serum-based miRNAs using ddPCR as it compares to renal mass biopsy in distinguishing between benign and malignant renal masses.
Table 1: Pairwise tumor group comparisons with Kruskal-Wallis test for the combination of miR-210 and miR-222 using ddPCR and conventional qRT-PCR

MethodTumor group comparisonTest statisticStandard errorStandard test statisticp-value
ddPCRAML vs. Papillary RCC-14.276.72-2.12.034
AML vs. Low-grade ccRCC-16.766.80-2.47.014
AML vs. High-grade ccRCC-23.306.97-3.34.001
Papillary RCC vs. Low-grade ccRCC-2.495.68-0.44.66
Papillary RCC vs. High-grade ccRCC-9.035.89-1.54.13
Low-grade RCC vs. High-grade ccRCC-6.555.97-1.10.27
qRT-PCRAML vs. Papillary RCC-11.856.87-1.73.085
AML vs. Low-grade ccRCC-11.506.94-1.66.097
AML vs. High-grade ccRCC-13.27.10-1.86.062
Papillary RCC vs. Low-grade ccRCC0.355.580.062.95
Papillary RCC vs. High-grade ccRCC-1.395.78-0.24.81
Low-grade RCC vs. High-grade ccRCC-1.735.86-0.30.77

ddPCR = droplet digital polymerase chain reaction; qRT-PCR = quantitative reverse transcription polymerase chain reaction; AML = angiomyolipoma; RCC = renal cell carcinoma; ccRCC = clear cell renal cell carcinoma

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