What information could the main actors of liquid biopsy provide? -a representative case of non-small cell lung cancer (NSCLC)

Abstract

In non-small cell lung cancer (NSCLC), there is a consensus regarding the use of liquid biopsy, generally, to detect "druggable" mutations and, in particular, to monitor tyrosine kinase inhibitor (TKI) treatments. However, whether circulating tumor cells (CTCs) are better tools than cell-free DNA (cfDNA), is still a matter of debate, mainly concerning which antigen(s) we should use to investigating simultaneously both epithelial and epithelial-to-mesenchymal transient (EMT) phenotype in the same sample of CTCs. To address this item, we exploited here a single-tube liquid biopsy, to detect both epithelial cell adhesion molecule (EpCAM)-positive CTCs and EpCAM-low/negative CTCs, because down-modulation of EpCAM is considered the first step in EMT. Furthermore, we analyzed the DNA from CTCs of four different phenotypes (ctcDNA), according to their EpCAM expression and cytokeratin pattern, and circulating tumor DNA (ctDNA) by droplet digital PCR (ddPCR), in order to disclose activating and resistance-driving mutations. Liquid biopsy reflected spatial and temporal heterogeneity of the tumor under treatment pressure. We provide the proof-of-concept that the complementary use of ctDNA and ctcDNA represents a reliable, minimally invasive and dynamic tool for a more comprehensive view of tumor evolution.

Keywords: Non-small cell lung cancer (NSCLC); circulating tumor DNA (ctDNA); circulating tumor cells (CTCs); epidermal growth factor receptor (EGFR); epithelial cell adhesion molecule-positive and epithelial cell adhesion molecule-low/negative (EpCAM pos and EpCAM low/neg); liquid biopsy.

Figure 1

Imaging and liquid biopsy monitoring. (A) CT scan of the initial evaluation, exhibiting lung mass located on the left upper lobe (5.5 cm × 3 cm, red arrow); (B) CT scan, performed immediately before surgery, showing a reduction in the lesion size (2.3 cm × 1.2 cm, red arrow); (C) PET-CT imaging showing relapse of tumor involving mediastinal lymph nodes; (D) PET-CT, carried out in November, showing a good response after radiotherapy; (E) longitudinal graphs of the four CTC subpopulations, namely EpCAM-positive SA, EpCAM-positive EA, EpCAM-low/negative SA, and EpCAM-low/negative EA. The ctDNA and CTC mutational status are also shown for L858R and T790M mutations at the different time points, namely: t0 = baseline, t1 = after one cycle of therapy, t2 = after two cycles of therapy, t3 = before lobectomy, t4 = after lobectomy, t5 = in conjunction with PET, after the end of radiotherapy. CTC, circulating tumour cells; PET, positron emission tomography; CT, computed tomography; SA, standard assay; EA, expanded assay; EpCAM, epithelial cell adhesion molecule; ctDNA, circulating tumour DNA; cfDNA, cell-free DNA; mut, mutant; wt, wild type.