Characterization of different CTC subpopulations in non-small cell lung cancer

Abstract

Circulating tumour cells (CTCs) serve as valuable biomarkers. However, EpCAM positive CTCs are less frequently detected in NSCLC patients compared to other epithelial tumours. First, EpCAM protein expression was analysed in primary and metastatic lung cancer tissue. In both groups 21% of the samples were EpCAM negative. Second, the CellSearch system identified 15% of patients (n = 48) as CTC positive whereas a multiplex RT-PCR for PIK3CA, AKT2, TWIST, and ALDH1 following EGFR, HER2 and EpCAM based enrichment detected CTCs in 29% of the patients. Interestingly, 86% of CTC positive patients were found to express ALDH1. Only 11% of the patients were CTC-positive by both techniques. CTC positivity was associated with patient disease state when assessed by the multiplex RT-PCR assay (p = 0.015). Patients harbouring tumours with an altered EGFR genotype were more frequently CTC-positive compared to patients with EGFR wildtype tumours. In subsets of patients, CTCs were found to express genes involved in resistance to therapy such as HER3 and MET. In conclusion, using multiple targets for CTC capture and identification increases the sensitivity of CTC detection in NSCLC patients, which can be explained by the presence of different CTC subtypes with distinct molecular features.

Critical issues in the clinical application of liquid biopsy in non-small cell lung cancer

“…In principle, the concordance of genomic alterations between tumor biopsies and CTCs/ctDNA has been demonstrated by several reports, both for EGFR mutations (62)(63)(64) and ALK translocations (50,54,65), leading to the inclusion of CTCs/ ctDNA in clinical trials as prognostic/predictive biomarkers. The results of these studies suggest that monitoring EGFR mutations or EML4-ALK rearrangements in the circulating compartment can reveal therapy resistance also prior to radiological progression evidence, thus guiding the therapeutic decision in the patient clinical course (62,65) (32,68). Notably, these studies also indicate the mandatory need to use methods that are more sensitive in the analysis of tumor biopsies at diagnosis, rather than Sanger sequencing.…”

“…Several studies associate CTC number to OS and/or PFS of metastatic NSCLC patients (32,33,48,57), meanwhile others do not find these associations (70,71). Many small trials have also demonstrated the association between CTC count and therapy efficacy, comparing the count before and after therapy (38,(72)(73)(74), or during target therapy, in order to detect the onset of resistance mutations (50,54,62,66,75).…”

“…Indeed, methods based on physical properties (48,49,53) or PCR-based methods (32), usually detect higher numbers of CTCs in lung cancer. However, so far, the FDA-cleared CellSearch ® assay, enriching and counting EpCAM+ CTCs (Table 1) has proved to identify 20-40% of CTC positive metastatic NSCLC patients (32,33,48,57). Moreover, a CTC positive status has been associated with a positive lymph node using this test (32,77).…”

“…However, so far, the FDA-cleared CellSearch ® assay, enriching and counting EpCAM+ CTCs (Table 1) has proved to identify 20-40% of CTC positive metastatic NSCLC patients (32,33,48,57). Moreover, a CTC positive status has been associated with a positive lymph node using this test (32,77). On the other side, the EpCAMlow/− fraction of CTCs alone has not been shown to correlate with patient outcome (53).…”

Sample types applied for molecular diagnosis of therapeutic management of advanced non-small cell lung cancer in the precision medicine

“…Single-cell molecular analysis based on CTCs can be applied as a research tool to reveal the mechanisms of metastases and resistance to targeted therapy [75,76]. Additionally, CTCs could be useful for the following purposes: as noninvasive surrogates for HE staining to definitively diagnose NSCLCs, for FISH testing to guide ALK-targeted therapy and for mRT-PCR testing to detect the MET and HER3 gene amplifications that are related to the resistance to EGFR-TKI therapy, when suitable tissue biopsies are unavailable [77,78].…”