Hiroko Sudo

High-Throughput MicroRNA (miRNAs) Arrays Unravel the Prognostic Role of MiR-211 in Pancreatic Cancer

Background Only a subset of radically resected pancreatic ductal adenocarcinoma (PDAC) patients benefit from chemotherapy, and identification of prognostic factors is warranted. Recently miRNAs emerged as diagnostic biomarkers and innovative therapeutic targets, while high-throughput arrays are opening new opportunities to evaluate whether they can predict clinical outcome. The present study evaluated whether comprehensive miRNA expression profiling correlated with overall survival (OS) in resected PDAC patients. Methodology/Principal Findings High-resolution miRNA profiles were obtained with the Torays 3D-Gene™-miRNA-chip, detecting more than 1200 human miRNAs. RNA was successfully isolated from paraffin-embedded primary tumors of 19 out of 26 stage-pT3N1 homogeneously treated patients (adjuvant gemcitabine 1000 mg/m2/day, days-1/8/15, every 28days), carefully selected according to their outcome (OS<12 (N = 13) vs. OS>30 months (N = 6), i.e. short/long-OS). Highly stringent statistics included t-test, distance matrix with Spearman-ranked correlation, and iterative approaches. Unsupervised hierarchical analysis revealed that PDACs clustered according to their short/long-OS classification, while the feature selection algorithm RELIEF identified the top 4 discriminating miRNAs between the two groups. These miRNAs target more than 1500 transcripts, including 169 targeted by two or more. MiR-211 emerged as the best discriminating miRNA, with significantly higher expression in long- vs. short-OS patients. The expression of this miRNA was subsequently assessed by quantitative-PCR in an independent cohort of laser-microdissected PDACs from 60 resected patients treated with the same gemcitabine regimen. Patients with low miR-211 expression according to median value had a significantly shorter median OS (14.8, 95%CI = 13.1–16.5, vs. 25.7 months, 95%CI = 16.2–35.1, log-rank-P = 0.004). Multivariate analysis demonstrated that low miR-211 expression was an independent factor of poor prognosis (hazard ratio 2.3, P = 0.03) after adjusting for all the factors influencing outcome. Conclusions/Significance Through comprehensive microarray analysis and PCR validation we identified miR-211 as a prognostic factor in resected PDAC. These results prompt further prospective studies and research on the biological role of miR-211 in PDAC.

MicroRNA Markers for the Diagnosis of Pancreatic and Biliary-Tract Cancers

It is difficult to detect pancreatic cancer or biliary-tract cancer at an early stage using current diagnostic technology. Utilizing microRNA (miRNA) markers that are stably present in peripheral blood, we aimed to identify pancreatic and biliary-tract cancers in patients. With “3D-Gene”, a highly sensitive microarray, we examined comprehensive miRNA expression profiles in 571 serum samples obtained from healthy patients, patients with pancreatic, biliary-tract, or other digestive cancers, and patients with non-malignant abnormalities in the pancreas or biliary tract. The samples were randomly divided into training and test cohorts, and candidate miRNA markers were independently evaluated. We found 81 miRNAs for pancreatic cancer and 66 miRNAs for biliary-tract cancer that showed statistically different expression compared with healthy controls. Among those markers, 55 miRNAs were common in both the pancreatic and biliary-tract cancer samples. The previously reported miR-125a-3p was one of the common markers; however, it was also expressed in other types of digestive-tract cancers, suggesting that it is not specific to cancer types. In order to discriminate the pancreato-biliary cancers from all other clinical conditions including the healthy controls, non-malignant abnormalities, and other types of cancers, we developed a diagnostic index using expression profiles of the 10 most significant miRNAs. A combination of eight miRNAs (miR-6075, miR-4294, miR-6880-5p, miR-6799-5p, miR-125a-3p, miR-4530, miR-6836-3p, and miR-4476) achieved a sensitivity, specificity, accuracy and AUC of 80.3%, 97.6%, 91.6% and 0.953, respectively. In contrast, CA19-9 and CEA gave sensitivities of 65.6% and 40.0%, specificities of 92.9% and 88.6%, and accuracies of 82.1% and 71.8%, respectively, in the same test cohort. This diagnostic index identified 18/21 operable pancreatic cancers and 38/48 operable biliary-tract cancers in the entire cohort. Our results suggest that the assessment of these miRNA markers is clinically valuable to identify patients with pancreato-biliary cancers who could benefit from surgical intervention.

Use of Non-Amplified RNA Samples for Microarray Analysis of Gene Expression

Demand for high quality gene expression data has driven the development of revolutionary microarray technologies. The quality of the data is affected by the performance of the microarray platform as well as how the nucleic acid targets are prepared. The most common method for target nucleic acid preparation includes in vitro transcription amplification of the sample RNA. Although this method requires a small amount of starting material and is reported to have high reproducibility, there are also technical disadvantages such as amplification bias and the long, laborious protocol. Using RNA derived from human brain, breast and colon, we demonstrate that a non-amplification method, which was previously shown to be inferior, could be transformed to a highly quantitative method with a dynamic range of five orders of magnitude. Furthermore, the correlation coefficient calculated by comparing microarray assays using non-amplified samples with qRT-PCR assays was approximately 0.9, a value much higher than when samples were prepared using amplification methods. Our results were also compared with data from various microarray platforms studied in the MicroArray Quality Control (MAQC) project. In combination with micro-columnar 3D-Gene™ microarray, this non-amplification method is applicable to a variety of genetic analyses, including biomarker screening and diagnostic tests for cancer.

Sa1940 microRNA Markers for the Diagnosis of Pancreatic and Bile Duct Cancers

It is recently reported that microRNAs (miRNAs) are stably present in serum and potentially useful in the diagnosis of cancer. Using highly sensitive microarray, 3D-Gene®, we examined comprehensive miRNA expression profiles in serum obtained from 66 pancreatic cancer patients, 66 bile duct cancer patients and 100 matched healthy volunteer as the study set, and found that 122 miRNA markers for pancreatic cancer and 125 miRNA markers for bile duct cancer differentiated pancreatic / bile duct cancer patients from healthy control with a statistical significance. We then validated the diagnostic performance of these markers using serum obtained from 33 pancreatic cancer patients, 33 bile duct cancer patients and 50 healthy controls. One of the single miRNA markers for pancreatic cancer yielded more than 90% diagnostic sensitivity and specificity, and combined use of two markers perfectly differentiated cancer and healthy samples. We also found that over 100 miRNA markers of pancreatic cancer overlapped with miRNA markers of bile duct cancer. Our results suggest that these miRNA markers have clinical values to screen out pancreatic and bile duct cancer patients.

Abstract 5034: Healthy and cancerous serum RNA profiling by the novel RNA extraction reagent and highly sensitive DNA chip

Proteins, metabolites and DNA are already known as components of serum or plasma biomarkers, however RNA has not been a strong biomarker candidate because of its instability. Exosomes that are small vesicles secreted by various cells are recently reported to play important roles in intercellular communications by transferring proteins, DNA and also RNA to distant cells through circulatory system. Surprisingly, the exosomal RNA in serum preserves its integrity and thus holds a potential to be a new blood biomarkers. In this report, we show the exhaustive analysis of miRNA and mRNA in serum by DNA chip for the highly purified RNA extracted with a novel reagent. Serum contains various types of nucleic acids, mainly small RNA, mRNA, and also short DNA fragment. We suppose that the contamination of DNA to the extracted RNA often causes a discrepancy between the DNA chip analysis and qRT-PCR validation. The novel reagent was able to extract RNA from serum without contamination of short DNA fragment, resulting in better RNA quantification and decreasing DNA-related noise outputs. Using this novel RNA extraction reagent and the highly sensitive DNA chip “3D-Gene,” we analyzed healthy and cancerous serum miRNA profiles. Over 500 miRNAs are detected in healthy and cancerous sera reproducibly, and some miRNAs were detected specifically in cancerous sera, such as breast, gastric, cervix cancer. In addition, we detected over 19,000 mRNAs from amplified RNA which were similarly extracted from healthy serum and detected by the DNA chip. This indicates that not only serum miRNA but also serum mRNA have a potential capability to be a biomarker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5034. doi:1538-7445.AM2012-5034

Abstract C143: Expression profiles of miRNA, FFPE, and nonamplified samples for diagnostic tests using a supersensitive microarray

We have developed a novel gene expression microarray system, 3D-Gene™, featuring micro‐columnar structure on the platform made of black resin and micro‐bead agitation for efficient hybridization. With these technological characteristics, 3D‐Gene™ achieves dramatic reduction of background noise, exceptionally high sensitivity and reproducibility, and high correlation with qRT‐PCR methodology particularly for genes with low expression. In addition to the general high performance, however, the medical devices in clinical settings must be versatile for a variety of biomarker types as well as sample types. Employing this microarray, we further developed a highly sensitive system to detect attomole‐level miRNA which is reported to be a novel biomarker for some cancers. Moreover, we were able to detect gene profiles reproducibly and accurately from formalin‐fixed paraffin‐embedded (FFPE) tissue which is known to be stored worldwide in a large number for retrospective studies. Finally, we developed a RNA sample preparation protocol with no amplification process, resulting in a quick and cost‐effective protocol with high accuracy. Taken together, we developed systems for gene expression detection using a high performance microarray suitable for a wide range of clinical applications. 3D‐Gene™ could be a powerful tool for drug target discovery and clinical diagnostic for various diseases. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C143.