Analysis of Single Circulating Tumor Cells (CTCs) to Infer Phenotype and Genome Changes in Response to Therapeutic Pressures in Biliary Tract Cancer

Reduzzi, Carolina (2020). Analysis of Single Circulating Tumor Cells (CTCs) to Infer Phenotype and Genome Changes in Response to Therapeutic Pressures in Biliary Tract Cancer. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0001179d

Abstract

Biliary tract cancer (BTC) is a highly fatal disease mainly treated with standard chemotherapy, albeit with limited efficacy. New therapeutic options are greatly needed, but the use of targeted treatments is often prevented by the impossibility of obtaining tissue biopsies for molecular characterization. Circulating tumor cells (CTCs) could represent a valuable alternative to tissue biopsies, also allowing a real-time monitoring of disease evolution and therapeutic resistance. Unfortunately, by using conventional CTC detection methods, which are based on the expression of epithelial markers, CTCs have been detected in only a small proportion of BTC patients, thus limiting their implementation in BTC clinical management.

Here, we developed a protocol for the identification of not only the classic epithelial CTCs (eCTCs), but also of non-conventional CTCs (ncCTCs) lacking epithelial and leukocyte markers, but presenting aberrant genomes, and therefore representing bona fide CTCs. CTCs were analyzed in 41 blood samples longitudinally collected from 21 patients with BTC. The detection of ncCTCs in addition to eCTCs resulted in an increase in CTC-positivity from 19% to 83%. The presence of at least 1 eCTC/10 mL of blood at baseline was associated with a significantly shorter median disease-specific survival (9 months vs. 17 months, p=0.03). Conversely, ncCTCs were not prognostic but variations in their number during treatment mirrored patient response, supporting their role for treatment monitoring.

The developed workflow also allowed the molecular characterization of single-CTCs. Copy number alteration profiling was performed for 88 single-CTCs collected from 23 BTC patients. Unsupervised clustering analysis revealed a segregation of CTCs according to patient best response and allowed the identification of genomic regions possibly involved in mechanisms of therapeutic resistance.

Overall, our results demonstrate the presence of a novel subpopulation of CTCs in BTC, paving the way for the use of liquid biopsy to improve clinical management of BTC patients.

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