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Identification et caractérisation des cellules tumorales circulantes dans le cancer rénal à cellules claires

Abstract : Dissemination in the circulating tumor cells (CTC) from the primary tumor is an early sign of tumor invasion and risk of metastases. Therefore, the ability to detect CTC through a very sensitive and specific test is expected to be clinically important for cancer prognosis, patient monitoring and customization of therapy. CTCs are rare cells, and several methods have been proposed for their detection. The ISET technique (Isolation by Size of Epithelial /Tumor cells) is based on the difference in size of CTC as compared to leucocytes and provides high sensitivity of CTC isolation and high specificity of CTC identification. This methods also allows cytopathological, immunological and molecular analyses of the isolated cellsKidney cancer accounts for 3% of adult cancers and is a clear cell renal cell carcinoma (RCC) in 75% of cases. RCC is one of very rare cancers characterized by a DNA mutations. IRCC tumor cells are in fact characterized by by mutations in the VHL gene. This research project aims at a comparative molecular and cytopathological analysis of, CTCs isolated from patients with RCC in order to evaluate, through a molecular approach, the diagnostic criteria used for cytopathological identification of CTC. Our study included 29 patients tested by the ISET technique before surgery.The cytopathological analysis was performed using the criteria described by the group of P. Hofman to define CTC (CNHC-MF) and Circulating Atypical Cells "CAC" (UMF-CNHC). Genetic analysis of the VHL gene was successfully performed by sequencing on DNA from 205 individual cells isolated by ISET, on DNA from tumor tissue and on genomic DNA from each patient. Of the 29 tumors studied, 25 were characterized by mutations in the VHL gene. One hundred and sixty-one cells, CTC and CAC, isolated from the blood of the 25 patients, with the tumor having VHL mutation, showed genetic variations in the VHL gene identical to those found in the DNA from the tumor tissue. We found 18 different mutations affecting the three exons of this gene.We found CTC/CAC 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/CAC, we obtained VHL-specific results in 205 including 64 CTC and 141 CAC, according to the cytopathological analysis. VHL mutations were blindly detected in 57/64 CTC and in 125/141 CAC. However, we then observed that the 8 and 16 residual CTC and CAC, respectively, had been isolated from patients without detectable VHL mutations in the tumor tissue. Conclusion: This is the first study comparing genetic and cytopathological diagnosis of CTC/CAC in patients with a solid cancer, CRCC. Our results suggest that broaden cytopathological criteria could be applied to the diagnosis of CTC in patients with CCRC. Although further and larger studies are now needed, this approach opens the way to a genetic approach for the accurate diagnosis of Circulating Tumor Cells.
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Basma Gloulou. Identification et caractérisation des cellules tumorales circulantes dans le cancer rénal à cellules claires. Médecine humaine et pathologie. Université René Descartes - Paris V, 2012. Français. ⟨NNT : 2012PA05T077⟩. ⟨tel-00806775⟩

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