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brushings . An advantage of ERCP guided sampling is the lack of tumor seeding, a known risk described in EUS/FNA patients [20, 27].
There is a growing body of literature about the epigenetic molecular markers, microRNAs (miRs), which can be utilized in clinical applications based on the stability and the disease-specific expression of these small RNA molecules. MicroRNAs have been reported to have important functions in the regulation of carcinogenesis and cancer progression as well as homeostasis. Deregulated microRNAs can give information on transcriptional regulation and most importantly may serve as biomarkers for survival and early detection of pancreatobiliary cancers . The aim of the present study was to analyze whether determination of microRNA expression levels in intraductal brush BYL-719 specimens is a viable molecular technique and if the tumor-associated microRNA expression in brush cytology specimens is a valuable tool in the diagnosis of pancreatobiliary cancers.
The isolation of microRNAs was successful from brush cytology specimens, RT-PCR expression analyses of miR-16, miR-196a and miR-221 showed a clear and reproducible statistical significance between malignant and benign pancreatobiliary specimens, indicating approximately 11.3-fold, 23.6-fold and 4.3-fold increased microRNA levels in tumor tissue. Interestingly miR-21, a well-established diagnostic and prognostic marker in other sample sources , was not useful as a separation marker on brush cytology isolates in our cohort. Target microRNAs were significantly enriched in the subgroup analysis of malignant biliary specimens compared to normal samples (Figure 2). MiR-196a proved to be the best marker for pancreatobiliary brushings in general, and even more so for the biliary subgroup, with highly different expression values separating cholangiocellular carcinoma from normal
specimens (p=2.3x10-6). The prognostic value of miR-196a expression has not been tested in our study, however, it has been shown recently that high levels of miR-196 as a plasma biomarker is associated with dismal survival in pancreatic adenocarcinoma patients . ROC analyses for the miR-196a marker in combination with routine cytology showed that malignant and benign pancreatobiliary samples could be separated with a sensitivity of 84.6% and a specificity of 100% (Table 2). For the biliary subgroup miR-196a and cytology together reached a diagnostic sensitivity of 92.9% with no false positives (Table 4), the combination of several microRNA markers did not improve these statistics. Feldmann et al. analyzing 16 samples found that routine cytology combined with the detection of HoxB7 and HAAH messenger RNA increased the overall diagnostic sensitivity from 36% to 82 % in biliary strictures . In 2012 Nischalke et al. demonstrated that gene expression of a combination of messenger
RNA markers (IGF2BP3, HOXB7 and NEK2) from brush cytology specimens is a helpful adjunct to routine cytological readings resulting in sensitivities of 87.5% and specificities of 87.2% in the diagnosis malignant biliary strictures . Compared to messenger RNA (mRNA) markers, microRNA markers have several advantages (1) unlike screening for large numbers of mRNA expression (microarray approach of the past decade), a modest number of microRNAs might be sufficient to differentiate disease from normal (e.g. miR-196a above); and (2) unlike mRNAs, microRNAs in tissues and biofluids remain largely intact and have been proven more stable for detection even in archived paraffin-embedded samples .
ERCP procedures are performed in big numbers worldwide for therapeutic interventions mainly in the biliary system, with the possibility of sample acquisition in the same endoscopic session. Besides well-established indications in the neoadjuvant and palliative setting in jaundiced tumor patients, ERCP sampling might still have a role in pancreatic strictures with small (<1cm) or no detectable mass on imaging due to the shortcomings of EUS/FNA in these settings, especially in high-risk individuals for pancreatic adenocarcinoma. To generalize the use of brush cytology in the diagnostic work-up, supplementary techniques are needed to increase sensitivity to an acceptable level. The unique advantage of the RT-PCR based method to measure microRNAs in cytology brushings presented in our paper over other published techniques is that it is a cheap, fast and widely available tool in all centers to reliably separate tumors from benign samples based on microRNA molecular markers. In our routine, sample processing took 2 hours and the estimated cost of molecular analysis was 30 € per patient. We h ave analyzed microRNA expression on pancreatobiliary brush cytology specimens for the first time, and miR-196a as a single marker showed a sensitivity of 85.7% in biliary strictures, which corresponded to a specificity of 100% (Figure 3). Shortcomings of our pilot study are the small sample size, and the lack of a separate validation group for optimal cut-off values. A follow-up study on a big sample size is currently ongoing in our laboratory. In conclusion, if the good diagnostic performance can be replicated on a large set of samples, microRNAs have the potential of becoming biomarkers increasing the sensitivity of ERCP/brush cytology.