Table 4.

Current available biomarkers for BTCs.

Biomarkers	Available data
PD-L1	Controversial data: - Keynote 15833 and Keynote 02833: no correlation between PD-L1 levels and response to ICIs; - NCT0282991837: patients with ≥1% of tumor cells expressing PD-L1 had a higher median PFS compared with patients with PD-L1-negative tumor tissue.
TMB	No sufficient data, given the low rate of TMB high BTCs: - Keynote 15833: tumors with TMB high showed better efficacy of Pembrolizumab. None of the patients with BTCs were TMB high.
MSI	MSI is a good predictor of response to ICIs; however, MSI-high BTCs are rare. - Keynote 15833: Pembrolizumab for treatment of BTC that had MSI or dMMR.
EpCAM-enriched CTCs	- Reduzzi et al.85 confirmed the prognostic role of eCTCs on survival in BTCs.
V-CTCs	- Han et al86: V-CTC > 50/mL blood is a significant factor affecting survival in patients with BTCs.
ctDNA	Potential complementary tool in the clinical practice to detect gene alterations, aiding in screening patients who may benefit from targeted therapies. - Chen et al93: for most genes, the mutation frequencies in ctDNA were similar with those detected in tissue samples. - Csoma et al93: positive correlation between the estimated tumor volume and cfDNA yield; Comparing tissue and LB results, similar tumor variant burden was observed.
ncRNA (eg: miRNA)	- Kishimoto et al103: increased level of miR-21 in patients with BTCs, making it a highly sensitive biomarker.

PD-L1: programmed death-ligand 1, ICIs: immune checkpoint inhibitors, PFS: progression free survival, TMB: tumor mutational burden, MSI: microsatellite instability, dMMR: mismatch repair deficiency, EpCAM: epithelial cell adhesion molecule, CTC: circulating tumor cells, V-CTC: vimentin-positive CTC, ctDNA: circulating tumor DNA, LB: liquid biopsy, cfDNA: circulating free DNA, ncRNA: non coding RNA, miRNA: micro-RNA.