MicroRNAs in stool samples as potential screening biomarkers for pancreatic ductal adenocarcinoma cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) accounts for approximately 90-95% exocrine malignant tumors of the pancreas. The high prevalence of metastasis and the difficulty of early diagnosis lead to a dismal prognosis. MicroRNAs (miRNAs) play a critical role in extensive biological processes. The purpose of this study was to evaluate the feasibility of stool miRNAs as novel biomarker for PDAC screening. MiRNAs were extracted from clinical specimens which included cancer and matched adjacent benign pancreatic tissues of 30 PDAC patients, pancreatic juice of 20 from the 30 PDAC patients and 10 chronic pancreatitis (CP) patients, stool samples of the 30 PDAC patients, the 10 CP patients and 15 healthy volunteers. Relative expression of a panel of 5 dysregulated miRNAs (miR-21, miR-155, miR-196a, miR-216 and miR-217) was analyzed with qRT-PCR. Receiver operating characteristic curve (ROC) analysis was performed to assess the diagnosing value of stool miRNAs in PDAC patients. The study showed that our methods of extracting and detecting miRNAs from pancreatic juice and stool specimens had high reproducibility. Compared to matched adjacent benign pancreatic tissues and pancreatic juice of CP patients, the expression of miR-21 (P = 0.0021 and P = 0.0027) as well as miR-155 (P = 0.0087 and P = 0.0067) was significantly higher and the expression of miR-216 (P < 0.0001 and P = 0.0044) was significantly lower in primary tumor tissues and pancreatic juice of PDAC patients. PDAC patients had a significantly higher stool miR-21 and miR-155 (P = 0.0049 and P = 0.0112) and lower miR-216 level (P = 0.0002) compared to normal controls. The same results were obtained in the expression levels of stool miR-21, miR-155 and miR-216 between PDAC and CP patients (P = 0.0337, P = 0.0388 and P = 0.0117, respectively). Receiver operating characteristic (ROC) analysis by using stool miRNAs expression indicated that combination of miR-21 and miR-155 had best sensitivity of 93.33% while the combination of miR-21, miR-155 and miR-216 would be best for detecting and screening PDAC with area under the curve (AUC) of 0.8667 (95% CI: 0.7722-0.9612) and a better balance of sensitivity and specificity (83.33% vs. 83.33%). Our data indicate that miRNAs could be extracted and detected from pancreatic juice and stool efficiently and reproducibly. MiR-21, miR-155 and miR-216 in stool have the potential of becoming biomarkers for screening PDAC.

Keywords: PDAC; diagnosis; microRNA; pancreatic juice; stool.

Figure 1

Detectability of miRNAs and RNU6B. To evaluate the detectability of selected miRNAs (miR-21, miR-155, miR-196a, miR-216, miR-217 and RNU6B), we analyze all Ct value of miRNAs and RNU6B. A. Shows the expression of all miRNAs and RNU6B in all primary cancer tissue of PDAC patients. B. Hows the expression of all miRNAs and RNU6B in all matched adjacent benign pancreatic tissues of PDAC patients. C. Hows the expression of all miRNAs and RNU6B in all pancreatic juice of PDAC patients. D. Hows the expression of all miRNAs and RNU6B in all stools of PDAC patients. The Figure shows the detectability miRNAs and RNU6B in tissue, pancreatic juice and stool.

Figure 2

Expression levels of miR-155 and RNU6B in cancer tissue (C), pancreatic juice (J) and stool (S) of all PDAC patients.

Figure 3

Reproducibility of miRNAs extraction and detection in pancreatic juice and stool samples. A. Reproducibility of our methodology for extraction of miRNAs in pancreatic juice shows a high linear correlation (R² = 0.9609, P < 0.0001). B. Reproducibility of our methodology for extraction of miRNAs in stool shows a high linear correlation (R² = 0.9882, P < 0.0001). C. Reproducibility of miRNAs detection in pancreatic juice shows a high linear correlation (R² = 0.9357, P < 0.0001). D. Reproducibility of miRNAs detection in stool shows a high linear correlation (R² = 0.9068, P < 0.0001).

Figure 4

MiRNA expression patterns in primary cancer tissue and matched adjacent benign pancreatic tissues. A-E. Represent the difference expression of different miRNAs between primary cancer tissue and matched adjacent benign pancreatic tissues. Abbreviations: N- adjacent benign pancreatic tissues, PDAC- primary cancer tissue. The data are present as box-and-whiskers plot: the upper and lower limits of the boxes indicate the 75th and 25th percentiles, the lines inside the boxes-the medians, and the upper and lower horizontal bars denote the 90th and 10th percentiles, respectively.

Figure 5

MiRNA expression patterns in pancreatic juice of CP and pancreatic juice of PDAC. A-D. Represent the difference expression of different miRNAs between pancreatic juice of CP and pancreatic juice of PDAC. Abbreviations: CP- chronic pancreatitis, PDAC- pancreatic ductal adenocarcinoma.

Figure 6

MiRNA expression patterns in stool of normal controls and stool of PDAC. A-C. Represent the difference expression of different miRNAs between stool of normal controls and stool of PDAC. Abbreviations: N- normal volunteers, PDAC- pancreatic ductal adenocarcinoma.

Figure 7

Diagnostic performance of the expression data of fecal miR-21, miR-155 and miR-216. A-C. Represents the AUC-ROC of different single fecal miRNAs of PDAC patients from controls. D. Shows the difference among the AUC-ROC of miR-21, miR-155 and miR-216. ROC = receiver operating characteristics.

Figure 8

Diagnostic performance of combination of two or three miRNAs (miR-21, miR-155 and miR-216). A-D. Represents the AUC-ROC of combination of miR-21 and miR-155, combination of miR-21 and miR-216, combination of miR-155 and miR-216 and combination of miR-21, miR-155 and miR-216 of PDAC from controls. The data of combination of miRNAs obtained through processing the original expression data of miR-21, miR-155 and miR-216 by logistic regression analysis. ROC = receiver operating characteristics.