Droplet digital PCR quantification of miR-1290 as a circulating biomarker for pancreatic cancer

Abstract

Droplet digital PCR was used to validate miR-1290 as circulating biomarker for pancreatic cancer (PC). The diagnostic performance of miR-1290 was evaluate in 167 PC patients and 267 healthy subjects at clinical risk of developing the disease (HS). MiR-1290 plasma levels were compared to CA 19-9 determinations, and the combination of the two biomarkers was also taken into account. Plasma levels of miR-1290 were higher in PC patients compared to HS (p = 2.55 × 10^-16). A similar trend was observed for CA 19-9 determinations (p = 1.03 × 10^-47). ROC curve analysis revealed that miR-1290 in combination with CA 19-9 was effective for discriminating between PC patients and HS (AUC = 0.956, 95% CI = 0.933-0.979) than the two biomarkers tested alone (miR-1290: AUC = 0.734, 0.678-0.789; CA 19-9: AUC = 0.914, 0.877-0.951). The discriminating ability was higher when only PC patients with low or slightly increased CA 19-9 levels were compared with HS. MiR-1290 concentrations were not able to differentiate between PC patients with single or multiple risk factors for developing PC. Our data suggest that the absolute quantification of circulating miR-1290 levels does not allow to select patients at clinical risk of PC for entry into a surveillance program, and underline the methodological challenges still existing in utilizing circulating miRNAs as new promising biomarkers for PC.

Figure 1

Absolute quantification of circulating levels of CA 19-9 and miR-1290 in healthy subjects (HS) and in patients with pancreatic cancer (PC). Each blot indicates the levels of CA 19-9 and miR-1290 tested using the ELISA assay and droplet digital PCR, respectively. The median levels in each HS and PC subgroup are indicated by the horizontal black bars. Values are reported in log scale (y-axis). HC: green dots; PC: blue dots

Figure 2

Receiver operating characteristics (ROC) curve analysis for discriminating healthy subjects (HS) from patients with pancreatic cancer (PC). The diagnostic performance of miR-1290 and CA 19-9 was tested independently and then in combination using a logistic regression model.

Figure 3

Receiver operating characteristics (ROC) curve analysis for discriminating healthy subjects (HS) from pancreatic cancer (PC) patients with slightly increased CA 19-9 levels. The diagnostic performance of miR-1290 and CA 19-9 was tested independently and then in combination using a logistic regression model.

Figure 4

Scatter plotting of miR-1290 plasma levels against CA 19-9 serum value for healthy subjects (HS) and pancreatic cancer (PC) patients with slightly increased CA 19-9 levels. White circles: PC; green circles: HS; horizontal black line: cut-off value for CA 19-9 (11.6 U/ml) for differentiating HC from PC patients with slightly increased CA 19-9 levels; vertical black line: cut-off value for miR-1290 (662 no. copies/μl) for differentiating HC from PC patients with slightly increased CA 19-9 levels. Values are reported in log scale.

Figure 5

Absolute quantification of circulating levels of miR-1290 plasma and CA 19-9 in subjects with intraductal papillary mucinous neoplasm (IPMN) of the pancreas and chronic pancreatitis (CP) compared with healthy subjects (HS) and pancreatic cancer (PC) patients. Each blot indicates the levels of miR-1290 and CA 19-9 tested by droplet digital PCR and ELISA assay, respectively. The median levels in each subgroup of patients and in HS are indicated by the horizontal black bars. Values are reported in log scale (y-axis).

Figure 6

Association between miR-1290 plasma levels and pre-operative classification of pancreatic cancer (A). The box indicates median, interquartile range (Q1–Q3) and lower and upper adjacent values (vertical bars) for each subject group. miR-1290 values are reported in log scale (y-axis). Kaplan–Meier curve for survival (OS) and disease-free survival (DFS) in patients with pancreatic cancer according to the cut-off value of 662 n.copies/μl derived from the ROC curve for circulating miR-1290 levels (B). Cum.: cumulative.