An elevated expression of serum exosomal microRNA-191, - 21, -451a of pancreatic neoplasm is considered to be efficient diagnostic marker

Abstract

Background: Pancreatic cancer is associated with an extremely poor prognosis, so new biomarkers that can detect the initial stages are urgently needed. The significance of serum microRNA (miR) levels in pancreatic neoplasm such as pancreatic cancer and intraductal papillary mucinous neoplasm (IPMN) diagnosis remains unclear. We herein evaluated the usefulness of miRs enclosed in serum exosomes (ExmiRs) as diagnostic markers.

Methods: The ExmiRs from patients with pancreatic cancer (n = 32) or IPMN (n = 29), and patients without neoplasms (controls; n = 22) were enriched using ExoQuick-TC™. The expression of ExmiRs was evaluated using a next-generation sequencing analysis, and the selected three miRs through this analysis were confirmed by a quantitative real-time polymerase chain reaction.

Results: The expression of ExmiR-191, ExmiR-21 and ExmiR-451a was significantly up-regulated in patients with pancreatic cancer and IPMN compared to the controls (p < 0.05). A receiver operating characteristic curve analysis showed that the area under the curve and the diagnostic accuracy of ExmiRs were 5-20% superior to those of three serum bulky circulating miRs (e.g.; ExmiR-21: AUC 0.826, accuracy 80.8%. Circulating miR-21: AUC 0.653, accuracy 62.3%). In addition, high ExmiR-451a was associated with mural nodules in IPMN (p = 0.010), and high ExmiR-21 was identified as a candidate prognostic factor for the overall survival (p = 0.011, HR 4.071, median OS of high-ExmiR-21: 344 days, median OS of low-ExmiR-21: 846 days) and chemo-resistant markers (p = 0.022).

Conclusions: The level of three ExmiRs can thus serve as early diagnostic and progression markers of pancreatic cancer and IPMN, and considered more useful markers than the circulating miRs (limited to these three miRs).

Keywords: Exosome; Intraductal papillary mucinous neoplasm; Pancreatic cancer; Tumor marker; microRNA-21; microRNA-451a.

Fig. 1

ExmiR-191, − 21 and -451a were significantly up-regulated in PC and IPMN. The three candidate miRs extracted with next-generation sequencing analysis were further evaluated using a qRT-PCR targeting all cases. a, b The expressions of ExmiR-191 (a, left panel), ExmiR-21 (a, middle panel), ExmiR-451a (a, right panel), CmiR-191 (b, left panel), CmiR-21 (b, middle panel) and CmiR-451a (b, right panel) were plotted (median with interquartile range was also shown). The expression of ExmiR-191, ExmiR-21, and ExmiR-451a were significantly higher in PC (n = 32) and IPMN patients (n = 29) than in controls (n = 22), while the expressions of these CmiRs did not differ significantly among the groups

Fig. 2

Three ExmiRs were more sensitive markers for diagnosing IPMN and PC than CmiRs. a,b The ROC analysis between control and IPMN (a) or PC (b) was showed, as follows: miR-191 (left panel), miR-21 (middle panel), and miR-451a (right panel). The AUC, specificity and diagnostic accuracy of three ExmiRs were superior to those of three CmiRs. Among the three ExmiRs, ExmiR-21 showed the highest diagnostic accuracy (IPMN diagnostic accuracy = 78.0%, PC diagnostic accuracy = 80.8%)

Fig. 3

ExmiR-191, ExmiR-21, and ExmiR-451a were good diagnostic markers for IPMN and early-stage PC. a The levels of CEA (left) and CA19–9 (right) were plotted (median with interquartile range was also shown). CA19–9 was significantly higher in the PC group than in the control or IPMN groups. b The ROC analysis between control and IPMN was shown. The AUC and diagnostic accuracy of three ExmiRs were clearly superior to traditional markers. c The ROC analysis between control and early stage of PC including only patients with stage I or IIa showed. The diagnostic accuracy of three ExmiRs were superior to CEA, and tended to be better than CA19–9. d The ROC analysis between the control group and the advanced-stage PC group (stage ≥IIb). The AUC values and accuracy of the three ExmiRs were superior to CEA, but they were not as useful as CA19–9

Fig. 4

ExmiR-451a was correlated with the Clinical Features of IPMN, and might be able to diagnose high risk cases. a The correlation between the expression levels of the three ExmiRs and the Fukuoka criteria. There were no significant differences in the expression of ExmiR-191, ExmiR-21 or ExmiR-451a between FN, WF and HRS. However, the ExmiR-451a level appeared to be gradually increasing (p = 0.0602). b The association between the expressions of three ExmiRs and clinical features, gender, age, cyst diameter, mural nodules, main pancreatic dilatation and Progression of cyst diameter, in IPMN group was assessed. The ExmiR expressions were plotted and those median with interquartile range was also shown. The expression of ExmiR-451a was significantly higher in the patients with mural nodules. Although there were no other significant differences, ExmiR-451a also seemed to be high with a large cyst diameter and main pancreatic dilatation

Fig. 5

ExmiR-21 was a candidate prognostic factor for the survival of PC patients. The association between the three ExmiRs and the clinical outcomes in PC patients was evaluated. a, b PC patients were categorized into high- and low-ExmiR expression groups using the median miR value as the cut-off point. a The survival analysis by Kaplan-Meier method with a log-rank test was shown. The overall survival in the high-ExmiR-21 expression group was significantly shorter than that in the low-ExmiR-21 expression group (p = 0.0137, median OS of low-ExmiR-21 expression group: 846 days, median OS of high-ExmiR-21 expression group: 344 days), but ExmiR-191 and ExmiR-451a were not associated with the survival of PC patients. With regard to the other factors, UICC4 was a significant prognostic factor (p = 0.0232, median OS of UICC1,2,3: 1330 days, median OS of UICC4: 388.5 days). b Multivariate survival analysis using Cox’s proportional-hazards regression model was performed to assess the relationship between the overall survival and the following candidate prognostic factors that were found to be significant by the Kaplan–Meier method: UICC stage (stage IV) and ExmiR-21. UICC stage IV (hazard ratio: 3.902, 95% CI: 1.416–10.750) and the high expression of ExmiR-21 (hazard ratio: 4.071, 95%CI: 1.382–11.996) were identified as independent prognostic factors. c The association between the expressions of three ExmiRs and the clinical outcome of chemotherapy was also assessed. The ExmiR expressions were plotted and those median with interquartile range was also shown. The expression of ExmiR-21 and ExmiR-451a in the group with disease progression (PD) was significantly higher than in the group with complete response (CR), partial response (PR), or stable disease (SD) (p = 0.0221, p = 0.0429, respectively)