Mar 4, 2018 - using findings from histology as the reference. The. 5-microRNA signature identified T1 cancers with lymph node invasion in cohort 1 with an ...
Gastroenterology 2018;154:844–848
BRIEF REPORTS
A MicroRNA Signature Associated With Metastasis of T1 Colorectal Cancers to Lymph Nodes Tsuyoshi Ozawa,1 Raju Kandimalla,1 Feng Gao,2 Hiroaki Nozawa,3 Keisuke Hata,3 Hiroshi Nagata,3 Satoshi Okada,3 Daisuke Izumi,1 Hideo Baba,4 James Fleshman,5 Xin Wang,2 Toshiaki Watanabe,3 and Ajay Goel1 1
Center for Gastrointestinal Research and Center for Translational Genomics and Oncology, Baylor Scott & White Research Institute, Charles A Sammons Cancer Center, Baylor University Medical Center, Dallas, Texas; 2Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China; 3Department of Surgical Oncology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; 4Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan; and 5Department of Colorectal Surgery, Baylor University Medical Center, Dallas, Texas Most T1 colorectal cancers treated by radical surgery can now be cured by endoscopic submucosal dissection. Although 70%L80% of T1 colorectal cancers are classified as high risk, 0.5, P < .01, Wilcoxon rank-sum test); expression level of the miRNA was relatively high (average expression >3). Lastly, we excluded miRNAs that overlapped or highly correlated with each other (mean absolute Pearson’s correlation >0.5), and established a panel of 5-miRNA signature. Subsequently, the detective power of this miRNA panel was evaluated using patients with T1 CRC (n ¼ 19; 2 LNMpositive and 17 LNM-negative, respectively) in the TCGA cohort, followed by subsequent validation in a clinical cohort. The study design is described in Figure 1A.
Patients and Sample Collection A total of 188 formalin-fixed paraffin-embedded (FFPE) specimens from patients with T1 CRCs were obtained from 2 cohorts; cohort 1 (n ¼ 60; 7 LNM-positive and 53 LNMnegative, respectively) from Kumamoto University, and cohort 2 (n ¼ 128; 20 LNM-positive and 108 LNM-negative, respectively) from the University of Tokyo. All of the patients underwent radical surgery between 2005 and 2014 in the cohort 1 patients, and between 2005 and 2016 in the cohort 2 patients. Eight patients (13%) and 1 patient (1%) underwent endoscopic resection before surgery in cohorts 1 and 2, respectively. Matched FFPE biopsy samples from 83 patients (15 LNM-positive and 68 LNM-negative, respectively), which were taken during colonoscopy before surgery, were also obtained from the cohort 2 subjects. The exclusion criteria were as follows: synchronous CRCs (T14), presence of distant metastases, presence of neoadjuvant chemo-/radiotherapy, hereditary or inflammationassociated CRC, non-adenocarcinoma, and nonavailability of FFPE specimens. All of the patients underwent standard surgical procedures (resection of affected segment of colon or rectum and regional lymphadenectomy), and all of the specimens were evaluated by pathologists of each participating institute according to the 7th edition of the American Joint Committee on Cancer TNM grading system and Japanese guidelines.1,2 Tumor budding was evaluated in 41 cases from cohort 2, and specimens with 10 mm, LNM was estimated to be positive. This study was approved by the ethical committee of each participating institution.
Total RNA Extraction and Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction Total RNA was extracted from 10-mm-thick FFPE specimens by macrodissection from cancer cellrich areas (>75% of tumor cells) and using AllPrep DNA/RNA FFPE kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Complementary DNA was synthesized from total RNA using Taqman MicroRNA Reverse Transcription Kit according to the manufacturer’s recommendations (ThermoFisher Scientific, Waltham, MA). Real-time quantitative reverse transcription polymerase chain reaction was performed using the QuantStudio 7 Flex Real Time PCR System (Applied Biosystems, Foster City, CA), and expression levels were evaluated with Applied Biosystems QuantStudio 7 Flex Real Time PCR System Software. The relative abundance of target transcripts was evaluated and normalized to the expression levels of miR-16 as an internal control using the 2-DCt method; DCt means the difference of Ct values between the miRNA of interest and the normalizer. Normalized values were further log10 transformed. All of the primers for miRNAs used in this study were purchased from ThermoFisher Scientific. Catalogue number of the primers of all miRNAs is 4427975, and assay IDs of each miRNA are as follows: Hsa-miR-32-5p: 2109, Hsa-miR-181b: 1098, Hsa-miR-193b-3p: 2367, Hsa-miR-195-5p: 494, Hsa-miR-411-5p: 1610, Hsa-miR-16: 391.
Evaluation of MicroRNA Regulatory Network Four integrated online databases (TarBaseV6.0, mir2Disease, miRecords, and miRTarBase) were used to evaluate the miRNA regulatory mRNAs, and pathway enrichment analysis. In these databases, the association between miRNA and mRNA was validated experimentally. Pathway enrichment analysis was performed using Kyoto Encyclopedia of Gene and Genomes pathway analysis and Gene Ontology analysis, and pathway with a P value of
