BACKGROUND MicroRNAs (miRNAs) are RNA molecules that are involved in the regulation of many cellular processes, including those related to human cancers. The aim of this study was to determine, as a proof of principle, whether specific candidate miRNAs could be detected in fine-needle aspirate (FNA) biopsies of pancreatic ductal adenocarcinoma (PDAC) and could accurately differentiate malignant from benign pancreatic tissues. METHODS We used TaqMan® assays to quantify miRNA levels in FNA samples collected in RNARetain (n = 16) and compared the results with a training set consisting of frozen macrodissected pancreatic samples (n = 20). RESULTS Quantitative reverse-transcription PCR analysis confirmed that miRNA levels are affected in PDAC FNAs and correlate well with the changes observed in the training set of frozen pancreatic samples. Analysis of the amounts produced for a few specific miRNAs enabled identification of PDAC samples. The combination of miR-196a and miR-217 biomarkers further improved the ability to distinguish between healthy tissue, PDAC, and chronic pancreatitis in the training set (P = 8.2 × 10−10), as well as segregate PDAC FNA samples from other FNA samples (P = 1.1 × 10−5). Furthermore, we showed that miR-196a production is likely specific to PDAC cells and that its incidence paralleled the progression of PDAC. CONCLUSIONS To the best of our knowledge, this study is the first to evaluate the diagnostic potential of miRNAs in a clinical setting and has shown that miRNA analysis of pancreatic FNA biopsy samples can aid in the pathologic evaluation of suspicious cases and may provide a new strategy for improving the diagnosis of pancreatic diseases.

The microRNA encoding genes miR-34a and miR-34b/c represent direct p53 target genes and possess tumor suppressive properties as they mediate apoptosis, cell cycle arrest, and senescence. We previously reported that the miR-34a gene is subject to epigenetic inactivation by CpG methylation of its promoter region in primary prostate cancer and melanomas, and in 110 different cancer cell lines of diverse origin. Here we analyzed the methylation status of miR-34a and miR-34b/c in additional primary tumors of divergent sites. We found methylation of miR-34a or miR-34b/c in formalin-fixed, paraffin-embedded (FFPE) tumor samples from 178 patients with the following frequencies: colorectal cancer (74% miR-34a, 99% miR-34b/c; n = 114), pancreatic cancer (64%, 100%; n = 11), mammary cancer (60%, 90%; n = 10), ovarian cancer (62%, 69%; n = 13), urothelial cancer (71%, 57%; n = 7), and renal cell cancer (58%, 100%; n = 12). Furthermore, soft tissue sarcomas showed methylation of miR-34 gene promoters in FFPE samples (64%, 45%; n = 11), in explanted, cultured cells (53%, 40%; n = 40), and in frozen tissue samples (75%, 75%, n = 8). In the colorectal cancer samples a statistically significant correlation of miR-34a methylation and the absence of p53 mutation was detected. With the exception of sarcoma cell lines, the inactivation of miR-34a and miR-34b/c was concomitant in most cases. These results show that miR-34 inactivation is a common event in tumor formation, and suggest that CpG methylation of miR-34a and miR-34-b/c may have diagnostic value. The mutual exclusiveness of miR-34a methylation and p53 mutation indicates that miR-34a inactivation may substitute for loss of p53 function in cancer.

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in different biological processes by suppressing target gene expression. Altered expression of miR-30a-5p has been reported in colon carcinoma. To elucidate its potential biological role in colon cancer, miR-30a-5p was overexpressed via a lentiviral vector system in two different colon cancer cell lines. This induced in both lines miR-30a-5p-mediated growth inhibition, attributable to a cell cycle arrest at the G(1) phase and an induction of apoptosis. Combining global gene expression analyses of miR-30a-5p transgenic line HCT116 with in silico miRNA target prediction, we identified the denticleless protein homolog (DTL) as a potential miRNA-30a-5p target. Subsequent reporter gene assays confirmed the predicted miR-30a-5p binding site in the 3'untranslated region of DTL. Importantly, overexpression of DTL in HCT116 cells partially rescued these cells from miR-30a-5p-mediated growth suppression. In addition, TP53 and CDKN1A expression were increased in miR-30a-5p-overexpressing HCT116 cells, suggesting that miR-30a-5p is able to modulate the cell cycle via a DTL-TP53-CDKN1A regulatory circuit. Finally, 379 colorectal cancer tissues were screened for DTL expression and DTL was found to be overexpressed in 95.8% of human colorectal cancers compared with normal colon mucosa. In conclusion, our data identified miR-30a-5p as a tumor-suppressing miRNA in colon cancer cells exerting its function via modulation of DTL expression, which is frequently overexpressed in colorectal cancer. Thus, our data suggest that restoring miR-30a-5p function may prove useful as therapeutic strategy for tumors with reduced miR-30a-5p expression.

Improved non-invasive strategies for early cancer detection are urgently needed to reduce morbidity and mortality. Non-coding RNAs, such as microRNAs and small nucleolar RNAs, have been proposed as biomarkers for non-invasive cancer diagnosis. Analyzing serum derived from nude mice implanted with primary human pancreatic ductal adenocarcinoma (PDAC), we identified 15 diagnostic microRNA candidates. Of those miR-1246 was selected based on its high abundance in serum of tumor carrying mice. Subsequently, we noted a cross reactivity of the established miR-1246 assays with RNA fragments derived from U2 small nuclear RNA (RNU2-1). Importantly, we found that the assay signal discriminating tumor from controls was derived from U2 small nuclear RNA (snRNA) fragments (RNU2-1f) and not from miR-1246. In addition, we observed a remarkable stability of RNU2-1f in serum and provide experimental evidence that hsa-miR-1246 is likely a pseudo microRNA. In a next step, RNU2-1f was measured by qRT-PCR and normalized to cel-54 in 191 serum/plasma samples from PDAC and colorectal carcinoma (CRC) patients. In comparison to 129 controls, we were able to classify samples as cancerous with a sensitivity and specificity of 97.7% [95% CI 5 (87.7, 99.9)] and 90.6% [95% CI 5 (80.7, 96.5)], respectively [area under the ROC curve 0.972]. Of note, patients with CRC were detected with our assay as early as UICC Stage II with a sensitivity of 81%. In conclusion, this is the first report showing that fragments of U2 snRNA are highly stable in serum and plasma and may serve as novel diagnostic biomarker for PDAC and CRC for future prospective screening studies.

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Pancreatic ductal adenocarcinoma (PDAC) is known for its poor prognosis resulting from being diagnosed at an advanced stage. Accurate early diagnosis and new therapeutic modalities are therefore urgently needed. MicroRNAs (miRNAs), considered a new class of biomarkers and therapeutic targets, may be able to fulfill those needs. Combining tissue microdissection with global miRNA array analyses, cell type-specific miRNA expression profiles were generated for normal pancreatic ductal cells, acinar cells, PDAC cells derived from xenografts and also from macrodissected chronic pancreatitis (CP) tissues. We identified 78 miRNAs differentially expressed between ND and PDAC cells providing new insights into the miRNA-driven pathophysiological mechanisms involved in PDAC development. Having filtered miRNAs which are upregulated in the three pairwise comparisons of PDAC vs. ND, PDAC vs. AZ and PDAC vs. CP, we identified 15 miRNA biomarker candidates including miR-135b. Using relative qRT-PCR to measure miR-135b normalized to miR-24 in 75 FFPE specimens (42 PDAC and 33 CP) covering a broad range of tumor content, we discriminated CP from PDAC with a sensitivity and specificity of 92.9% [95% CI5(80.5, 98.5)] and 93.4% [95% CI5(79.8, 99.3)], respectively. Furthermore, the area under the curve (AUC) value reached of 0.97 was accompanied by positive and negative predictive values of 95% and 91%, respectively. In conclusion, we report pancreatic cell-specific global miRNA profiles, which offer new candidate miRNAs to be exploited for functional studies in PDAC. Furthermore, we provide evidence that miRNAs are well-suited analytes for development of sensitive and specific aid-in-diagnosis tests for PDAC.Pancreatic ductal adenocarcinoma (PDAC) is characterized by its late clinical presentation, early and aggressive local invasion and high metastatic potential. The lack of sensitive early detection strategies and its strong resistance to chemotherapy and radiation therapy compounds the overall very poor prognosis of PDAC, which has a median survival time following diagnosis of 3-5 months. Surgery is still the only effective treatment option, improving the median survival time to 10-20 months; however, at the time of diagnosis, only 20% of PDACs are amenable to surgery and cure is rarely achieved. 1 Thus, improved early diagnosis modalities as well as new therapeutic targets for the development of effective treatment strategies are urgently needed to improve the dismal prognosis of PDAC. MicroRNAs (miRNAs), a class of 18-23 nt noncoding RNAs, have gained much attention as a new family of molecules involved in cancer development 2,3 and thus are under investigation for their suitability as diagnostic biomarkers and therapeutic targets.miRNAs as biomarkers offer a number of advantages. First, compared with mRNAs, which are very sensitive to degradation, miRNAs are more stable in compromised human specimens (e.g., formalin-fixed paraffin-embedded; FFPE). 4,5 Second, their expression levels can be measured reliably in FFPE tissue sampl...

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CCR7 expression on tumor cells promotes lymphatic spread in several malignant tumors. However, a comprehensive characterization of the CCL19/CCL21-CCR7 axis in pancreatic ductal adenocarcinoma (PDAC), which is known for its high rates of lymph-node metastases, is still lacking. CCR7 mRNA and CCR7 protein were found to be expressed in spheroid cultures of all six examined PDAC cell lines. In migration assays, CCR7 expressing PDAC cells showed enhanced migration toward CCL19 and CCL21, the two ligands of CCR7. In an orthotopic nude mouse model, CCR7-transfected PT45P1 cells gave rise to significantly larger tumors and showed a higher frequency of lymph vessel invasion and lymph-node metastases than mocktransfected cells. In an analysis using quantitative real-time PCR, CCR7 showed fourfold overexpression in microdissected PDAC cells compared to normal duct cells. Moderate-to-strong immunohistochemical CCR7 expression, found in 58 of 121 well-characterized human PDACs, correlated with high rates of lymph vessel invasion. Conversely, PDACs completely lacking CCR7 expression showed only low rates of lymph vessel invasion and lymph-node metastases. The evaluation of CCL21 expression by immunofluorescence staining revealed a significant upregulation of CCL21 in peritumoral and intratumoral lymph vessels compared to lymph vessels in disease-free pancreata. In conclusion, our study revealed strong evidence that lack of CCR7 impairs the metastatic potential of PDAC. Lymph vessel invasion by CCR7 expressing PDAC cells may be additionally enhanced by upregulation of CCL21 in tumor-associated lymph vessels, representing a previously unknown factor of lymphatic spread.Early spread to lymph nodes is a characteristic feature of human pancreatic ductal adenocarcinomas (PDAC). These lymph-node metastases and disseminated tumor cells in the lymphatic vessels of peripancreatic tissues could be the source of local tumor recurrence, which frequently occurs in patients after PDAC resection. 1The mechanisms determining lymphatic tumor spread are poorly understood. One possible mechanism is the induction of new lymph vessels by neoplastic or inflammatory cells, facilitating lymphangic invasion. Yet, we were able to show that there was no relevant increase in lymphatic vessel density (LVD) in PDAC. In contrast, the number of intratumoral lymph vessels was dramatically decreased compared to the normal pancreas. In line with these data, LVD proved not to be related to indicators of tumor progression including lymphatic spread in PDAC. 2 The results of Schneider et al. 3 also suggested that lymphangiogenesis is not required for lymphovascular spread of PDAC. This was the rationale of our strategy to search for other mechanisms of lymphatic dissemination in PDAC.There is an increasing body of evidence that the dissemination of tumor cells in lymphatics and visceral organs is a directed process mediated by interactions between tumor and normal cells rather than a random event. In several tumor