## 4 Discussion  
Approved circulating biomarkers for the prognostication of patients with pancreatic cancer are currently lacking. Clinical follow-up of pancreatic cancer patients in the Netherlands is not standardized [27] and currently consists of physical examination and at times radiographical imaging by CT scans which have a low performance in detecting early metastatic disease. Carbohydrate antigen 19-9 (CA19-9) is a sialylated Lewis blood group antigen associated with different cancers, including PDAC [28] and could serve as biomarker for progressive disease [29]. However, the sensitivity is low and up to 20% of the population is Lewis negative and cannot synthesize CA19-9 [30]. We sought to identify circulating biomarkers that can indicate high risk of fast recurrence shortly after resection, before metastases are visible on CT scan. Repeated blood draws followed by miR expression analysis that could stratify patients at higher risk for progressive disease and could prompt treatment adjustment would improve patient care. MiRs are suitable candidates for the prediction of cancer progression due to their altered expression during tumorigenesis and their stability in the circulation [22]. Numerous studies have shown that circulating miRs are altered in early PDAC as well as in metastatic PDAC [31]. [PERSON] et al. showed that patients with early PDAC stage could be distinguished from healthy controls by the upregulation of circulating miR-122-5p; miR-125b-5p; miR-192-5p; miR-193b-3p; miR-122-3p; and miR-27b-3p [14]. In contrast, they found that among the PDAC patients, decreased miR-125b-5p levels before treatment are associated with worse overall survival. Also, increased serum expression of the miR-99 family is recently found associated with the  
Figure 4: MiR-99a-5p detection by in situ hybridization in pancreatic cancer tissues. Serial FFPE tissue sections were stained with H&E a DIG labeled miR-99a-5p probe, or the B-lymphocyte lineage marker CD79A antibody by immunohistochemistry (brown). Red arrows indicate miR-99a-5p expression, detected with silver staining resulting in brown/black coloring, whereas cell nuclei are stained with hematoxylin in blue. Scale bars are 250 μm in the first two columns; scale bar is 100 μm in the right-most column. diagnosis of pancreatic cancer [32]. Studies similar to these, which assess circulating miR levels for the diagnosis of PDAC, are difficult to corroborate. Inconsistent results are due to interpatient diversity as well as technical differences in quantitation and data normalization [33]. Unfortunately, a subset of patients with PDAC that undergo primary tumor resection rapidly succumb to disease recurrence, whereas other patients have longer progression free survival after surgery. Identifying the patients at risk for early disease recurrence could prompt adjustments in adjuvant treatment decisions and thus improve patient outcome. In the current study, we performed a prognostic miR biomarker analysis using serum samples from treatment-naive patients with surgically resectable PDAC. We profiled serum miR expression before and after surgery and compared the changes to the PFS of the patients. Our comparison shows the changes in serum miR levels after removal of the primary cancer. The aim was to select biomarkers that could identify patients at risk, shortly after resection, before metastases are visible on CT scan. Therefore, no serum from the patients included here was collected at time of radiographically visible metastatic disease. Genetically engineered mouse models of pancreatic cancer have led to major improvements in the understanding of PDAC development. Specifically, the _LSL-Kras_G12D/+; _LSL-Trp53R172H/+; P48-Cre mouse model indicated by the acronym 'KPC' succumbs to progressive PDAC that mimics the features of the human disease [23]. KPC mice initially develop preinvasive acinar to ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) lesions before widespread PDAC, all in the presence of an intact immune system.