AMMF’s Virtual 2021 European Cholangiocarcinoma Conference
Abstract No 4
Liquid biopsy is feasible in biliary tract cancer patients and opens new perspectives
- Vismara1, F. dell’Angelo1, M. Silvestri1, M. Niger2, F.Nichetti2, F. De Braud2,3, M.G. Daidone1, V. Cappelletti1
1Department of Applied Research and Technological Development, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Milan, Italy
2Department of Medical Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Milan, Italy
3University of Milan, Milan, Italy
Recent biological data support a molecular stratification of patients with biliary tract cancer (BTC) with direct therapeutic implications; however the limited access to tissue restrains its clinical applicability. Liquid biopsy represents a possible solution, but it has been so far poorly investigated in BTC. We addressed this gap in a series of 27 patients with metastatic and advanced BTC by evaluating circulating tumors cells (CTCs) and ctDNA fraction in longitudinally collected samples.
CTCs were enriched from peripheral blood samples with the Parsortix, labeled with DAPI and with antibodies against epithelial (EpCAM, panCK, EGFR) and leukocyte (CD45, CD14, CD16) markers, and analyzed in the DEPArray to collect single cells and evaluate copy number alterations to confirm their malignancy. Prior to treatment in 61% of patients at least 1 CTC/10 mL blood was detected.
Cell free DNA was extracted from plasma samples and used for ctDNA estimation. When DNA sequencing from tissue samples was available ctDNA (expressed as VAF-percentage) was evaluated by ddPCR using a custom mutation-specific ddPCR assays designed using the Thermo Fisher custom SNP genotyping assay tool or alternatively the Bio-Rad Mutation Detection Assay online design tool. Alternatively, ctDNA fraction was evaluated by direct sequencing using the Oncomine Pan‐Cancer Cell‐Free Assay, a target sequencing panel interrogating 272 amplicons from 52 cancer-related genes. All called variants were validated with the corresponding ddPCR assay. In the longitudinal study 41% of cases were positive for ctDNA at baseline.
We show that CTC and ctDNA evaluation for treatment-response monitoring is feasible in BTC patients, offers complementary information and contributes to classic imaging-based response. CTC represents an unbiased approach with limitations due to technical difficulties. On the contrary ctDNA is more straightforward, but its sensitivity may be limited by the number of tracked variants. Single cases will be discussed in the poster.
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