Current challenges for detection of circulating tumor cells and cell-free circulating nucleic acids, and their characterization in non-small cell lung carcinoma patients. What is the best blood substrate for personalized medicine?

Abstract

The practice of "liquid biopsy" as a diagnostic, prognostic and theranostic tool in non-small cell lung cancer (NSCLC) patients is an appealing approach, at least in theory, since it is noninvasive and easily repeated. In particular, this approach allows patient monitoring during treatment, as well as the detection of different genomic alterations that are potentially accessible to targeted therapy or are associated with treatment resistance. However, clinical routine practice is slow to adopt the liquid biopsy. Several reasons may explain this: (I) the vast number of methods described for potential detection of circulating biomarkers, without a consensus on the ideal technical approach; (II) the multiplicity of potential biomarkers for evaluation, in particular, circulating tumor cells (CTCs) vs. circulating tumor DNA (ctDNA); (III) the difficulty in controlling the pre-analytical phase to obtain robust and reproducible results; (IV) the present cost of the currently available techniques, which limits accessibility to patients; (V) the turnaround time required to obtain results that are incompatible with the urgent need for delivery of treatment. The purpose of this review is to describe the main advances in the field of CTC and ctDNA detection in NSCLC patients and to compare the main advantages and disadvantages of these two approaches.

Keywords: Lung cancer; circulating tumor DNA (ctDNA); circulating tumor cells (CTCs); methods; personalized medicine; pre-analytical phase.

Figure 1

Circulating tumor cells detected in lung cancer patients using an indirect method, the CellSearch system. (A) Cell with a round morphology, a visible DAPI-positive nucleus, positive CK-PE staining in the cytoplasm and negative staining for CD45 is considered as typical intact CTC; (B) image of cell not included in the CTC count; (C) CD45-positive cell (arrows) is not considered as CTC. CTC, circulating tumor cells.

Figure 2

Circulating tumor cells detected in lung cancer patients using the ISET method (Rarecells company). Circulating tumor cells with malignant features in lung adenocarcinoma (A) and squamous cell carcinoma (B) patients (A and B, May Grunwald Giemsa ×1,000). Circulating tumor cells expressing cytokeratin (C) and vimentin (D) alone (C, anti-cytokeratin antibody, immunoperoxidase ×1,000; D, anti-vimentin antibody, immunoperoxidase ×1,000).

Figure 3

Circulating tumor cells detected in lung cancer using the system of ScreenCell company. Circulating tumor cells showing malignant feature (A, May Grunwald Giemsa ×400; B, May Grunwald Giemsa ×1,000).

Figure 4

The melting curve profile for KRAS exon 2 mutation (p.G12C, c.34G>T), as detected in ctDNA from one NSCLC patient. The wild-type allele is shown as a horizontal red line. ctDNA, circulating tumor DNA; NSCLC, non-small cell lung cancer.