Eighty-one individuals who later developed PC (cases), with a prediagnostic plasma sample available, were randomized to the prediagnostic training cohort (Fig. 1B). During miRNA isolation, 13 case samples and four controls were excluded due to sample hemolysis or insufficient sample volume. The four excluded controls were replaced with controls matched to the excluded cases. Two additional controls were excluded after RT qPCR analysis; one due to low miRNA yield and the other due to uncertain sample identity. The final cohort thus consisted of 67 PC patients and 132 matched controls. The clinical characteristics are summarized in Table 2.
Plasma miRNAs are Altered in Patients at Diagnosis
To identify candidate miRNAs that were altered at PC diagnosis, we analyzed a panel of 372 miRNAs by RT qPCR in the screening cohort. Patients and controls separated somewhat in principle component analysis based on all detectable plasma miRNAs (n = 233), although with a considerable overlap between the groups (Supplementary Figure 1, http://links.lww.com/SLA/B163). One hundred ninety miRNAs (51%) were expressed in ≥20 cases or controls and were thus included in the statistical analysis. No miRNA displayed an on/off pattern, meaning that none were solely detected in either cases or controls.
Our screening analysis revealed 15 miRNAs with significant abundance differences between cases and controls after correcting for false discovery rate. Of these 15 candidate miRNAs, 10 were increased and five were decreased in PC patients at diagnosis. miR-885-5p had the highest positive fold change among increased miRNAs, and miR-144-3p the highest negative fold change among decreased miRNAs (Table 3, Supplementary Table 2, http://links.lww.com/SLA/B163, Supplementary Figure 2, http://links.lww.com/SLA/B163). Among the significantly altered miRNAs, accuracy for patient/control discrimination (AUC) varied from 0.83 [95% confidence interval (95% CI) 0.70–0.95] for miR-574-3p down to 0.75 (95% CI 0.60–0.89) for miR-122-5p (Supplementary Figure 3, http://links.lww.com/SLA/B163).
Serum Bilirubin Does Not Correlate With miRNA Levels at Diagnosis
A majority (17/23) of the PC patients suffered from obstructive jaundice at disease presentation (Table 1). Bile duct obstruction can affect liver function,26,27which in turn is associated with plasma miRNA alterations.28 We reasoned that obstructive jaundice might affect levels of circulating miRNA and therefore investigated correlations between candidate miRNAs and SBR. SBR levels were available in-hospital charts from 21 out of 23 cases, with a median SBR level of 23 mmol/L (range 0 to 158 mmol/L). We found no correlation between candidate miRNAs and SBR levels after correcting for multiple testing (Supplementary Table 3, http://links.lww.com/SLA/B163).
Plasma miRNAs Are Not Altered Before Diagnosis
Having established that none of our 15 candidate miRNAs were significantly correlated with SBR, we investigated if they could be used to predict a future PC diagnosis. First, we assessed candidate miRNA alterations in the prediagnostic samples independent of time before diagnosis. This clearly showed that none of the 15 miRNAs that were found significantly altered at the time of diagnosis differed before diagnosis (Table 4). We reasoned that one explanation for this negative result could be the time differences in our prediagnostic cohort, where time of sampling varied from 3 months to 18 years before PC diagnosis. We therefore divided the prediagnostic cohort into three groups: 1) >10 years, 2) 5 to 10 years, and 3) <5 years before diagnosis. At <5 years before diagnosis (3 months to 4.9 years), the P value for miR-24-3p was <0.05 and miR-106b was close to being significant, although none passed false discovery rate testing. Of note, most miRNAs had case/control fold changes close to 1 (Table 4).
A Multivariate Statistical Model of Candidate miRNAs Separate Cases and Controls at Diagnosis but not Before
Multiple miRNAs have been shown to act synergistically in gene regulation29 and previous studies have combined several miRNAs to better separate PC patients from controls.8,30,31 We therefore hypothesized that a combination of our 15 candidate miRNAs might perform better to detect early alterations than single miRNAs alone. To test this, we generated a multivariate statistical model on the basis of candidate miRNAs. At diagnosis, the model clearly separated cases from controls, a separation that was consistent and significant after cross validation (Fig. 2B). However, the model failed to separate cases from controls at any time point before diagnosis, although a tendency to separation was noted <5 years before diagnosis (Fig. 2A). The poor group separation before diagnosis was independent of TNM stage and sex (Supplementary Figure 4, http://links.lww.com/SLA/B163). By comparison, Ca 19–9 levels were significantly altered <5 years before diagnosis, although only three of these cases presented with levels above 37 U/mL, which is the standard clinical cutoff for Ca 19–9.4 Interestingly, Ca 19–9 levels increased the closer the sampling date was to diagnosis (Figs. 2C, D, Supplementary Table 4, http://links.lww.com/SLA/B163).
To compare discriminative performance, we constructed ROC curves for the miRNA model and Ca 19–9. Our miRNA model outperformed Ca 19–9 at diagnosis, but at all time points before PC diagnosis, both the miRNA model and Ca 19–9 performed poorly in discriminating cases from controls (Fig. 3). In the light of the poor performance of miRNAs in the training set of the prediagnostic cohort, we refrained from further analysis in the validation set.
Circulating levels of miRNAs are altered in many different cancer forms and various miRNAs and miRNA-combinations have been suggested as potential biomarkers of disease.7 In PC, there is a pressing need for early detection biomarkers, as patients are generally asymptomatic until metastatic disease has developed.1 In the first study of its kind, we evaluated the potential of miRNAs in early PC detection by analyzing candidate miRNAs in plasma samples collected before a PC diagnosis. Although we did identify miRNAs that were altered at diagnosis, they were not suitable for early detection of PC. Early detection performance was unaffected by stratifications for both time to diagnosis and TNM stage. miR-24 was significant <5 years before diagnosis using a permissive significance level at 0.05, but the fold change was minimal and nonsignificant after correcting for false discovery rate, indicative of a false-positive finding.
Nonetheless, we identified 15 miRNAs that were associated with PC at the time of diagnosis and a multivariate model based on these miRNAs outperformed Ca 19–9 in ROC analysis. However, as the model was not validated in an independent PC patient cohort, we refrain from drawing conclusions regarding its clinical potential for PC diagnosis.
Several of the miRNAs we identified have previously been associated with PC, including increased let-7d, miR-22, -24, -34a, -122, -130b, -574, and -885 as well as decreased miR-106b-5p and -1448,9,32–35 (Supplementary Table 5, http://links.lww.com/SLA/B163). There are also previous reports of associations for miR-26a, -423, and -451a, although with fold changes in opposite directions9,30,33 (Supplementary Table 5, http://links.lww.com/SLA/B163). Of note, miR-451a changes should be interpreted with caution due to its enrichment in red blood cells and its concomitant association with sample hemolysis.17 But as all our screening samples passed hemolysis testing, the decreased levels of miR-451a are more likely to be disease-associated. Importantly, PC association of circulating miR-101 and miR-197 are novel findings. This is of particular interest, as both are implicated in epithelial-mesenchymal transition in PC cells,36,37 suggestive of involvement in tumor invasion and metastasis.
Several of the miRNAs that were altered at diagnosis have been identified in functional in vitro studies on PC cells or found at altered levels in PC tissue (Supplementary Table 5, http://links.lww.com/SLA/B163). miRNAs with supporting functional data include miR-24, -197, -26a, -101, -106b, and -144.36–42 Similarly, miRNAs with supporting tissue data include let-7d, miR-24, -26a, and -101.12,37,38,40,43 On the contrary, our results on miR-34a, -122, miR-130b, and -451a contradict previous tissue study findings.11,13,44,45 But the controversy in the latter should not be exaggerated, as it is known that miRNAs in PC serum do not readily correlate with tumor tissue expression.30 We also speculate that circulating miRNA levels could be affected by systemic changes associated with PC.
One contributor to systemic changes during PC is obstructive jaundice. Although often one of the first symptoms of disease, obstructive jaundice is a late complication during PC development and manifest close to diagnosis.1 In our study, some of the candidate miRNAs identified in diagnostic samples trended toward SBR correlation. Although correlations were insignificant after false discovery rate correction, obstructive jaundice cannot be completely ruled out as a potential confounder in samples close to diagnosis. We therefore strongly suggest that future studies should assess SBR correlations for candidate, circulating PC biomarkers.
Ca 19–9 was significantly elevated in PC patients <5 years before diagnosis. However, ROC analysis revealed a poor discriminative performance at this stage. Prediagnostic increases in Ca 19–9 levels have been reported previously in PC patients, with similar AUC at <12 months before diagnosis.46 The poor discriminative performance of Ca 19–9 in prediagnostic samples in that study and in ours may, in part, explain the low positive predictive value of Ca 19–9 evident from prospective cohort studies.4
Almost 90% of the cases in our early detection cohort had stage III or IV cancer at diagnosis. If the temporal development suggested by Yachida et al15 is correct, then a substantial proportion of these patients should have been at stage I or II at sample collection, and thus comparable to patients in the screening cohort. However, it is possible that the transition from stage I to stage IV in fact occurs more rapidly than suggested, which is supported by survival data from nonresected stage I-II patients, showing a median survival of <7 months.2
Although the present screen covered 372 miRNAs, over 2500 miRNA sequences have been annotated in the current miRBase version (www.mirbase.org). In an ideal setting, all known miRNAs should be tested in an unbiased manner using prediagnostic samples. Another powerful approach is to combine results from omics studies on diagnosed patients with a prediagnostic cohort or with animal models. One such metabolomics study demonstrated that branched-chain amino acids are elevated before diagnosis in both patient plasma and in a KRAS-driven mouse PC model.47 In a similar mouse model, early carcinogenic progression was found to correlate with miRNA changes.48 However, to our knowledge, the current study is the first to investigate levels of circulating miRNAs in samples collected before a PC diagnosis.
A panel of 15 circulating miRNAs can discriminate PC patients from controls at the time of diagnosis. These 15 miRNAs do not hold promise as early detection biomarkers, as the alterations appear late in the disease course.
We want to thank Anette Berglund for retrieving and keeping track of the frozen patient samples and the personnel at the endoscopy unit at the surgical ward at Umeå University Hospital and Marjo Andersson for helping out with sample collection.
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blood samples; early detection; micro-RNA; miRNA; pancreatic cancer
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