O1-5-3SINGLE CELL GENETIC ANALYSIS HELPS VALIDATING CYTOPATHOLOGICAL IDENTIFICATION OF CTCs IN PATIENTS WITH CCRC

Abstract Background and aim: Cytopathological identification of circulating tumor cells (CTC) is a critical issue in non-invasive predictive oncology. However, in very rare cases, such as kidney cancers, the diagnostic value of cytology is weak. We thus planned to study the impact of genetic molecular analysis in combination with cytology in the CTC field. To this aim, we have taken advantage from the model of Clear Cell Renal Carcinoma (CCRC), which is characterized by the VHL genetic marker, and used genetic analysis in addition to the cytopathological study of CTC. Methods: We have treated by ISET the blood of 30 patients with CCRC, collected before surgery, to isolate circulating rare cells. Cytopathological analysis was performed blindly by 3 pathologists on the isolated cells to identify CTC and distinguish them from circulating cells with uncertain malignant features (CC-UMF) and from cells with benign features (CC-BF). CTC, CC-UMF and CC-BF were then individually microdissected. Single cells DNA was preamplified, amplified by VHL-specific primers and analyzed by sequencing. VHL-specific genetic analysis was performed blindly in microdissected cells and in the corresponding tumorous tissue. Results: We found CTC/ CC-UMF in 29/30 analyzed patients with CCRC. VHL mutations were found in the tumor of 25 out of the corresponding 29 CCRC tumors. Among 327 microdissected CTC/ CC-UMF, we obtained VHL-specific results in 205 including 64 CTC and 141 CC-UMF, according to the cytopathological analysis. VHL mutations were blindly detected in 57/64 CTC and in 125/141 CC-UMF. These results were analyzed according to the presence or absence of VHL mutations in the tumor tissue. Conclusion: This is the first study using single cell genetic analysis and cytopathology to study CTC in patients with CRCC. Our results show that genetic analysis is a very interesting approach potentially complementing cytopathology for the diagnosis of CTC.