Molecular analysis of circulating tumor cells identifies distinct copy-number profiles in patients with chemosensitive and chemorefractory small-cell lung cancer

Abstract

In most patients with small-cell lung cancer (SCLC)-a metastatic, aggressive disease-the condition is initially chemosensitive but then relapses with acquired chemoresistance. In a minority of patients, however, relapse occurs within 3 months of initial treatment; in these cases, disease is defined as chemorefractory. The molecular mechanisms that differentiate chemosensitive from chemorefractory disease are currently unknown. To identify genetic features that distinguish chemosensitive from chemorefractory disease, we examined copy-number aberrations (CNAs) in circulating tumor cells (CTCs) from pretreatment SCLC blood samples. After analysis of 88 CTCs isolated from 13 patients (training set), we generated a CNA-based classifier that we validated in 18 additional patients (testing set, 112 CTC samples) and in six SCLC patient-derived CTC explant tumors. The classifier correctly assigned 83.3% of the cases as chemorefractory or chemosensitive. Furthermore, a significant difference was observed in progression-free survival (PFS) (Kaplan-Meier P value = 0.0166) between patients designated as chemorefractory or chemosensitive by using the baseline CNA classifier. Notably, CTC CNA profiles obtained at relapse from five patients with initially chemosensitive disease did not switch to a chemorefractory CNA profile, which suggests that the genetic basis for initial chemoresistance differs from that underlying acquired chemoresistance.

Molecular analysis of single circulating tumour cells following long‐term storage of clinical samples

“…We and others have previously demonstrated the potential clinical benefit of molecular characterization of CTCs following single‐cell isolation from patient blood samples (Carter et al ., 2017; Diaz and Bardelli, 2014; Fusi et al ., 2013; Rothwell et al ., 2016). To facilitate single CTC isolation and analysis, we have established a simple yet robust workflow, which allows banking of either the entire CellSearch ® sample prior to cell isolation, or individual CTCs after DEPArray™ single‐cell isolation (Fig.…”

“…Both CTCs and circulating free DNA (cfDNA) can be readily obtained from a simple peripheral blood draw and together represent a ‘liquid biopsy’ that holds considerable potential for providing biomarkers that can improve cancer patient management (Carter et al ., 2017; Diaz and Bardelli, 2014; Fusi et al ., 2013; Krebs et al ., 2014; Rothwell et al ., 2016). The isolation and characterization of single tumour cells from the circulation can give an insight into the heterogeneity and genetic status of tumours, which has been associated with prognosis (Carter et al ., 2017; Casasent et al ., 2017; Navin, 2015).…”

“…The molecular characterization of tumours also has the potential to give insight into mechanisms of resistance to therapies and tumour heterogeneity (Burrell and Swanton, 2014; Carter et al ., 2017; Casasent et al ., 2017; Jamal‐Hanjani et al ., 2015; Navin, 2015). Circulating tumour cells (CTCs) found in the blood of patients with cancer are being evaluated for clinical utility as a liquid biopsy that can facilitate molecular characterization of the patient's disease (Carter et al ., 2017; Diaz and Bardelli, 2014; Fusi et al ., 2013; Krebs et al ., 2014; Rothwell et al ., 2016). Use of a simple blood draw and CTC analysis has the added benefit of ease of sample collection as well as the possibility of repeat sampling.…”

“…To enable single‐cell analysis, enriched samples require further manipulation on platforms such as the DEPArray™ (Silicon Biosystems, Bologna, Italy), a microfluidic system that combines high‐resolution imaging with the dielectrophoretic movement of fluorochrome‐labelled cells enabling selection as well as isolation of individual cells. We and others demonstrated the utility of a combined CellSearch ® and DEPArray™ workflow to enable molecular analysis of CTCs (Carter et al ., 2017; De Luca et al ., 2016; Hodgkinson et al ., 2014). Although effective, the isolation of CTCs using the combined CellSearch ® and DEPArray™ approach is both time‐consuming and costly, making it impractical for routine analysis of all CellSearch ® ‐enriched samples.…”

Challenges and unanswered questions for the next decade of circulating tumour cell research in lung cancer

“…Over the last decade, advances in both CTC enrichment protocols and molecular methodologies has culminated in a range of successful strategies for genome wide amplification and analysis of single CTCs which have extended many of the observations made with enriched CTCs. There are several advantages of single CTC analysis including: (I) single CTC analysis is 100% pure (assuming a CTC was isolated) and avoids any complications or dilution of signal due to contaminating cells; (II) since a cell has on average only two DNA copies of each gene and around 100,000 mRNA molecules (41) the degree of next generation sequencing (NGS) required for generating a representative single cell snapshot is relatively low; (III) single cell genomic analysis can unambiguously identify co-existing genetic changes; (IV) the integration of RNA and DNA analysis from the same cell can establish linkage between the genetic alterations and alterations in RNA pathways (42); (V) single cell data provides an ideal means of establishing the degree of heterogeneity and estimating tumour evolution (43,44). For single CTC molecular analysis the main problems encountered are largely technical, financial and logistical.…”