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Supplementary Figures 1 - 8 from miRNA Biomarkers in Cyst Fluid Augment the Diagnosis and Management of Pancreatic Cysts

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posted on 2023-03-31, 17:50 authored by Hanno Matthaei, Dennis Wylie, Maura B. Lloyd, Marco Dal Molin, Jon Kemppainen, Skye C. Mayo, Christopher L. Wolfgang, Richard D. Schulick, Laura Langfield, Bernard F. Andruss, Alex T. Adai, Ralph H. Hruban, Anna E. Szafranska-Schwarzbach, Anirban Maitra

PDF file, 917K, Supplemental Figure 1: (A) Laser microdissection in an IPMN (hematoxylin and eosin; original magnification: 10x); (B) After precise marking and microdissection the neoplastic epithelium is catapulted into a sterile tube for subsequent RNA extraction. (C) The section after dissection. Supplemental Figure 2: Detailed diagram of the study design, describing experimental setup of FFPE (FTS) and cyst fluid (CFS) studies, including number of specimens used and number of miRNA candidates identified in the course of each study. Supplemental Figure 3: Mean Cts for all FTS and CFS specimens. (A) Megaplex RT-qPCR data for FTS. (B) and (C) Singleplex RT-qPCR data for FTS. (D) Megaplex RT-qPCR data for CFS. (E) and (F) Singleplex RT-qPCR data for CFS. Supplemental Figure 4: Boxplots showing raw Ct values for Megaplex (A) and singleplex (B) RT-qPCR expression analyses of FTS1 and FTS1 plus FTS2, respectively. The diagnosis associated with each sample is indicated by color, while the FTS1 and FTS2 specimens are depicted as separate panels in the singleplex boxplot (B). Supplemental Figure 5: Boxplots showing raw Ct values for Megaplex (A) and singleplex (B) RT-qPCR expression analyses of cyst fluid specimens from the CFS1 and CFS1 plus CFS2, respectively. Diagnosis is indicated by color, the CFS1 and CFS2 specimens by panel. Supplemental Figure 6: Boxplots of raw Ct values for singleplex RT-qPCR expression analysis of the CFS1 and CFS2 specimens with reassignment to test or training set indicated by separation of panels. Supplemental Figure 7: Raw Ct values of miRNAs involved in DiffPairs comprising the logistic regression model in the CFS1 and CFS2 specimens. Raw Ct for miR-21, which is not a part of the logistic regression model, are also shown. Supplemental Figure 8: PCA applied to raw Cts (A) and restricted mean-center normalized Cts. (B) CFS1 and CFS2 singleplex RT-qPCR data. Note that CFS1 and CFS2 specimens do not separate in either plot.

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ARTICLE ABSTRACT

Purpose: The diagnosis of pancreatic cystic lesions has increased dramatically. Most are benign, whereas some, such as intraductal papillary mucinous neoplasms (IPMN), represent precursors of pancreatic adenocarcinoma. Therapeutic stratification of IPMNs is challenging without precise information on dysplasia grade and presence of invasion. We assessed the diagnostic benefit of using miRNAs as biomarkers in pancreatic cyst fluid, focusing on IPMNs because of their frequency and malignant potential.Experimental Design: RNA was extracted from 55 microdissected formalin-fixed, paraffin-embedded (FFPE) IPMN specimens, and 65 cyst fluid specimens aspirated following surgical resection. Expression of 750 miRNAs was evaluated with TaqMan miRNA Arrays using 22 FFPE and 15 cyst fluid specimens. Differential expression of selected miRNA candidates was validated in 33 FFPE and 50 cyst fluid specimens using TaqMan miRNA Assays.Results: We identified 26 and 37 candidate miRNAs that distinguish low-grade from high-grade IPMNs using FFPE and cyst fluid specimens, respectively. A subset of 18 miRNAs, selected from FFPE and cyst fluid data, separated high-grade IPMNs from low-grade IPMNs, serous cystadenomas (SCA) and uncommon cysts, such as solid pseudopapillary neoplasms (SPN) and cystic pancreatic neuroendocrine tumors (PanNET). A logistic regression model using nine miRNAs allowed prediction of cyst pathology implying resection (high-grade IPMNs, PanNETs, and SPNs) versus conservative management (low-grade IPMNs, SCAs), with a sensitivity of 89%, a specificity of 100%, and area under the curve of 1.Conclusions: We found candidate miRNAs that helped identify patients with high-grade IPMN and exclude nonmucinous cysts. These classifiers will require validation in a prospective setting to ultimately confirm their clinical usefulness. Clin Cancer Res; 18(17); 4713–24. ©2012 AACR.

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