Genetic analysis of uterine lavage fluids to identify women at high risk of endometrial cancer

The uterine lavage was done immediately before hysteroscopy or hysterectomy in the operating theatre. Cases were divided into three groups EC histologically confirmed endometrioid endometrial cancer EIN histologically confirmed endometrial intraepithelial neoplasia and control cases with benign histology.Fluids from uterine lavages were stored at 4 C and processed within 24 hours. Briefly samples were centrifuged at 3 200 g 4 C for 20 min. Pellets were resuspended in erythrocyte buffer and incubated for 15 min. The samples were then centrifuged again for 20 min at 3 200 g and the resulting pellets were frozen at minus 80 C until DNA isolation.DNA was isolated using DNeasy Blood and Tissue Kit Qiagen according to manufacturer instructions. Sequencing libraries were prepared using KAPA HyperPlus Library Preparation Kit. We designed a custom endometrial tumor amplicon panel to cover 22 genes with the highest mutation frequencies specific for EC list of genes POLE POLD1 PTEN PIK3CA TP53 CTNNB1 KRAS NRAS HRAS AKT1 EGFR FGFR2 FBXW7 RB1 ATM APC ARID1A ARID5B PIK3R1 CDKN2A PPP2R1A RPL22.