EP660 Novel approach to noninvasive assessment of lymph-nodes metastases with one-step isothermal quantification of mRNAs in primary tumor of endometrial cancer

Introduction/Background Lymphadenectomy in endometrial cancer should be considered depending on individual patients owing to its postoperative risks such as lymphedema. Although assessment of lymph node metastasis provides crucial information for appropriate adjuvant management, therapeutic importance of lymphadenectomy for low-intermediate risk patients is a matter of debate. Given that sentinel lymph node biopsy is a beneficial but complex method, noninvasive, simple and high-precision diagnosis method for lymph node metastatic state is highly demanded. In a previous study, we identified SEMA3D and a novel isoform of TACC2 as promising biomarkers to evaluate lymphatic metastasis based on gene expression patterns in the primary lesion. Here, we attempted to optimize sampling method considering tumor heterogeneity and to accelerate gene quantitative analysis for our biomarkers. Methodology Endometrial cancer tissues from new several patients were collected in addition to the previous 115 patients. We collected 5–13 pieces of primary tumor from each new patient. We verified gene expressions of each tissue piece by quantitative-PCR to optimize sampling method. Then we assessed whether RT-SmartAmp method, which can detect nucleic acids in one step consisting of a reverse transcription and an isothermal amplification of DNA, could quantify our biomarker RNA rapidly. We measured the speed of amplification and target specificity based on biomarker RNA as the template in one step containing reverse transcription step. Results We calculated the average expression levels by bootstrap method It suggested that even when the sampling number is two, each average expression level indicates correct diagnosis with 99%CI. Then we also succeeded to develop a promising candidate of primer set to detect the target mRNA by using SmartAmp. This candidate can detect RNA quantitatively within 30 minutes without non-specific amplification of negative control by using Eprimer as detection fluorescence. Conclusion Our findings pave the way for support clinical decisions that minimize irrelevant lymphadenectomy. Disclosure I have no COI with regard to our presentation. This work was supported by AMED under Grant Number 18cm0106433h0001 and 19cm0106452h0001, a Grant-in-Aid for Scientific Research (C) from the MEXT (15K10732, 17K11296, 18K09299), a research grant from MEXT through the RIKEN Preventive Medicine and Diagnosis Innovation Program, a research grant from MEXT to RIKEN Omics Science Center and a research grant from MEXT to RIKEN Center for Life Science Technologies (Division of Genomic Technologies).