A Genome-Wide Investigation of MicroRNA Expression Identifies Biologically-Meaningful MicroRNAs That Distinguish between High-Risk and Low-Risk Intraductal Papillary Mucinous Neoplasms of the Pancreas

<div><p>Background</p><p>Intraductal papillary mucinous neoplasms (IPMNs) are pancreatic ductal adenocarcinoma (PDAC) precursors. Differentiating between high-risk IPMNs that warrant surgical resection and low-risk IPMNs that can be monitored is a significant clinical problem, and we sought to discover a panel of mi(cro)RNAs that accurately classify IPMN risk status.</p><p>Methodology/Principal Findings</p><p>In a discovery phase, genome-wide miRNA expression profiling was performed on 28 surgically-resected, pathologically-confirmed IPMNs (19 high-risk, 9 low-risk) using Taqman MicroRNA Arrays. A validation phase was performed in 21 independent IPMNs (13 high-risk, 8 low-risk). We also explored associations between miRNA expression level and various clinical and pathological factors and examined genes and pathways regulated by the identified miRNAs by integrating data from bioinformatic analyses and microarray analysis of miRNA gene targets. Six miRNAs (miR-100, miR-99b, miR-99a, miR-342-3p, miR-126, miR-130a) were down-regulated in high-risk versus low-risk IPMNs and distinguished between groups (<i>P</i><10⁻³, area underneath the curve (AUC) = 87%). The same trend was observed in the validation phase (AUC = 74%). Low miR-99b expression was associated with main pancreatic duct involvement (<i>P</i> = 0.021), and serum albumin levels were positively correlated with miR-99a (r = 0.52, <i>P</i> = 0.004) and miR-100 expression (r = 0.49, <i>P</i> = 0.008). Literature, validated miRNA:target gene interactions, and pathway enrichment analysis supported the candidate miRNAs as tumor suppressors and regulators of PDAC development. Microarray analysis revealed that oncogenic targets of miR-130a (<i>ATG2B, MEOX2</i>), miR-342-3p (<i>DNMT1</i>), and miR-126 (<i>IRS-1</i>) were up-regulated in high- versus low-risk IPMNs (<i>P</i><0.10).</p><p>Conclusions</p><p>This pilot study highlights miRNAs that may aid in preoperative risk stratification of IPMNs and provides novel insights into miRNA-mediated progression to pancreatic malignancy. The miRNAs identified here and in other recent investigations warrant evaluation in biofluids in a well-powered prospective cohort of individuals newly-diagnosed with IPMNs and other pancreatic cysts and those at increased genetic risk for these lesions.</p></div

Heatmap and unsupervised hierarchical clustering of low-risk (adenoma) and high-risk (carcinoma-in-situ) IPMN samples according to the expression of the most differentially expressed miRNAs.

<p>A) The heatmap is supervised, and is ordered by the type of IPMN, and shows the expression for the 25 most deregulated miRNAs. B) Unsupervised hierarchical clustering for the 6 most differentially expressed miRNAs. Expression values for the miRNAs are represented in a matrix format, with columns representing samples and rows representing miRNAs. Low expression values are colored green, and high expression values are colored red. Colored bars indicate the range of normalized log2-based signals. </p

Gene ontology categories of biological pathways overrepresented by miRNA-mediated changes in target gene expression that may differentiate between high- and low-risk IPMNs.

<p>Gene ontology categories of biological pathways overrepresented by miRNA-mediated changes in target gene expression that may differentiate between high- and low-risk IPMNs.</p

Laser capture microdissection (LCM) of epithelium from A) low- grade and B) high-grade IPMN tissue.

<p>Left Panel: Hematoxylin (H&E) stained slide (× 4). Middle Panel: H&E stained slide before LCM (× 4), with the red area representing cells of interest marked for capture. Right Panel: Cap showing adherent cells.</p

Clinical and Pathologic Characteristics of Patients with IPMNs (N = 49).

<p>Data represent counts (percentages) unless otherwise indicated. Counts may not add up to the total due to missing values, and percentages may not equal 100 due to rounding.</p><p>¹Low-risk IPMNs are represented by 12 low-grade and 5 moderate-grade IPMNs.</p><p>²High-risk IPMNs are represented by 30 high-grade and 2 invasive IPMNs.</p><p>³P-value for differences between low- and high-risk groups using chi-squared or Fisher’s exact tests and t-tests for categorical and continuous variables, respectively. Values in bold are statistically significant (P<0.05).</p><p>⁴Signs of malignant potential on endoscopic ultrasound (EUS) include main duct (MD) involvement, MD dilation (≥5 mm), mural nodules, septation, wall thickness, or cyst size ≥3 cm.</p><p>⁵Based on pathological review post-resection.</p><p>Clinical and Pathologic Characteristics of Patients with IPMNs (N = 49).</p