Feasibility of Fecal MicroRNAs as Novel Biomarkers for Pancreatic Cancer

<div><h3>Introduction</h3><p>Pancreatic cancer (PCA) is an aggressive tumor that associates with high mortality rates. Majority of PCA patients are diagnosed usually at late tumor stages when the therapeutic options are limited. MicroRNAs (miRNA) are involved in tumor development and are commonly dysregulated in PCA. As a proof-of-principle study, we aimed to evaluate the potential of fecal miRNAs as biomarkers for pancreatic cancer.</p> <h3>Materials and Methods</h3><p>Total RNA was extracted from feces using Qiagen's miRNA Mini Kit. For miRNA expression analyses we selected a subset of 7 miRNAs that are frequently dysregulated in PCA (miR-21, -143, -155, -196a, -210, -216a, -375). Subsequently, expression levels of these miRNAs were determined in fecal samples from controls (n = 15), chronic pancreatitis (n = 15) and PCA patients (n = 15) using quantitative TaqMan-PCR assays.</p> <h3>Results</h3><p>All selected miRNAs were detectable in fecal samples with high reproducibility. Four of seven miRNAs (miR-216a, -196a, -143 und -155) were detected at lower concentrations in feces of PCA patients when compared to controls (p<0.05). Analysis of fecal miRNA expression in controls and patients with chronic pancreatitis and PCA revealed that the expression of miR-216a, -196a, -143 und -155 were highest in controls and lowest in PCA. The expression of the remaining three miRNAs (miR-21, -210 and -375) remained unchanged among controls and the patients with either chronic pancreatitis or PCA.</p> <h3>Conclusion</h3><p>Our data provide novel evidence for the differential expression of miRNAs in feces of patients with PCA. If successfully validated in large-scale prospective studies, the fecal miRNA biomarkers may offer novel tools for PCA screening research.</p> </div

Clinico-pathological characteristics of patients included to the study.

*<p> <i>- at time point of sample collection;</i></p>#<p> <i>- endocrine pancreatic insufficiency is defined by impaired glucose tolerance test or by manifest diabetes mellitus;</i></p>§<p>- exocrine pancreatic insufficiency is defined by reduced elastase in stool.</p

Cumulative miRNA expression analyses improve the separation of PCA samples.

<p>Summation of the ΔCt-values of miR-196a, -216a, -143 and -155 was performed to calculate the Σ ΔCt-value. *- p<0.05, ***- p<0.001. Abbreviations: N-control subjects, CP- chronic pancreatitis, PCA- pancreatic cancer. The data are present as box-and-whiskers plots: the upper and lower limits of the boxes indicate the 75^th and 25^th percentiles, the lines inside the boxes - the medians, and the upper and lower horizontal bars denote the 90^th and 10^th percentiles, respectively.</p

Stability of fecal miRNAs.

<p>To evaluate the long-term stability of the samples, we performed miRNA analyses in the fecal samples collected at different time points. Samples 1–15 were collected between 2004 and 2006 and samples 16–30 between 2009 and 2010 from healthy subjects. All samples were stored and processed in similar conditions. Figure (A) shows variations in miR-16 and miR-196a expression among all feces samples. (B) miR-16 and (C) miR-196a expression in subgroup analyses showed similar expression (p>0.1). (D) Normalized miR-196a expression is comparable in long- and short-term stored samples (p = 0.441). (E) Since miR-216a was present in feces at lowest concentrations, and its expression was analyzed by two independent quantitative RT-PCR runs to evaluate the reproducibility of the analysis (p<0.0001). Normalization was performed with using miR-16 as internal normalizer.</p

The expression of analyzed fecal microRNAs in comparison to tumor tissues, pancreatic fluid and blood of patients with pancreatic cancer.

<p>The expression of analyzed fecal microRNAs in comparison to tumor tissues, pancreatic fluid and blood of patients with pancreatic cancer.</p

Detection of fecal miRNAs that are commonly dysregulated in pancreatic cancer tissues.

<p>(A) MiRNA microarray expression analyses were performed using Illumina microarray to evaluate the presence of selected miRNAs (miR-16, -375, -196a, -216a, -21, -143, -155 and -210) in a single stool sample of the healthy subject. Fecal miRNA expression was converted to log-expression values following Lumi Bioconductor normalization. (B & C) Expression of selected miRNA was confirmed in all 45 samples including controls, chronic pancreatitis and pancreatic cancer patients. Figure (B) represents the raw miRNA expression Ct-values for qualitative assessment. (C) Normalization was performed using standard ΔCt-method using miR-16 as internal fecal normalizer.</p

MiRNA expression patterns in patients with chronic pancreatitis and PCA.

<p>Figures (A) to (G) represent different miRNAs that were selected for the study based on the alterations in PCA tissues. *represents p<0.05, ***- p<0.001. Abbreviations: N-control subjects, CP- chronic pancreatitis, PCA- pancreatic cancer. The data are present as box-and-whiskers plots: the upper and lower limits of the boxes indicate the 75^th and 25^th percentiles, the lines inside the boxes - the medians, and the upper and lower horizontal bars denote the 90^th and 10^th percentiles, respectively.</p

Barcharts of <i>IL23A</i> median fold change by RT-qPCR in AGS and MKN-28 (n=4).

<p>BCM-300, P1, M1, and M2 are <i>H. pylori</i> strains. Both BCM-300 and P1 (wild-type) are CagA strains, whereas M1 and M2 are P1-derived isogenic mutants lacking CagA and VirB7, respectively. Generally, higher expression levels were seen in AGS more than MKN-28 cells. Significant upregulation of <i>IL23A</i> was seen in both cells with BCM-300, P1 (the highest), and M1 strain (P<.05), whereas no alteration was seen after M2 infection (non-functional T4SS) similar to that of the non-infected control samples (CO).</p

Correlation of <i>IL23A</i> with <i>IL8</i> and <i>EBI3</i> after infection with different <i>H. pylori</i> strains.

<p>Scatterplots of log-transformed fold changes of transcripts by RT-qPCR illustrate the relationship of <i>IL23A</i> with both <i>IL8</i> (panel A) and <i>EBI3</i> (panel B) transcripts after infection with different <i>H. pylori</i> strains using pair-wise Spearmen’s <i>rho</i> correlations. Robust regression line using an M estimator was plotted as well (red line). BCM-300, P1, M1, and M2 are <i>H. pylori</i> strains. Both BCM-300 and P1 (wild-type) are CagA strains, whereas M1 and M2 are P1-derived isogenic mutants lacking CagA and VirB7, respectively. BCM-300+Fi is infection with <i>H. pylori</i> BCM-300 bacteria after application of 0.22µm filter cap to prevent any physical contact with the cells, while permitting their secretory factors to pass through. Asterisks represent the significance levels (*P<.05, **<.01, ***<.001).</p

Boxplots of raw Cqs of the detected transcripts by RT-qPCR in non-infected AGS and MKN-28.

<p>Each box represents the lower quartile, the median, and the upper quartile score (vertical lines of the box). Means (small white squares) and outliers are also shown (black dots). Expression levels of detected transcripts were ordered in AGS cell line from highest (<i>ACTB</i>) to lowest (<i>IL23R</i>) based on their median values. Similar ranking order was seen in MKN-28 cells except for the least four abundant transcripts: <i>IL23A</i>, <i>IL12A</i>, <i>EBI3</i>, and <i>IL23R</i>.</p