Metastasis is the main cause of cancer-related death owing to the blood-borne dissemination of circulating tumor cells (CTCs) early in the process. A rare fraction of CTCs harboring a stem cell profile and tumor initiation capacities is thought to possess the clonogenic potential to seed new lesions. The highest plasticity has been generally attributed to CTCs with a partial epithelial-to-mesenchymal transition (EMT) phenotype, demonstrating a large heterogeneity among these cells. Therefore, detection and functional characterization of these subclones may offer insight into mechanisms underlying CTC tumorigenicity and inform on the complex biology behind metastatic spread. Although an in-depth mechanistic investigation is limited by the extremely low CTC count in circulation, significant progress has been made over the past few years to establish relevant systems from patient CTCs. CTC-derived xenograft (CDX) models and CTC-derived ex vivo cultures have emerged as tractable systems to explore tumor-initiating cells (TICs) and uncover new therapeutic targets. Here, we introduce basic knowledge of CTC biology, including CTC clusters and evidence for EMT/cancer stem cell (CSC) hybrid phenotypes. We report and evaluate the CTC-derived models generated to date in different types of cancer and shed a light on challenges and key findings associated with these novel assays.

Circulating tumor cell (CTC)-filtration methods capture high numbers of CTCs in non-small-cell lung cancer (NSCLC) and metastatic prostate cancer (mPCa) patients, and hold promise as a non-invasive technique for treatment selection and disease monitoring. However filters have drawbacks that make the automation of microscopy challenging. We report the semi-automated microscopy method we developed to analyze filtration-enriched CTCs from NSCLC and mPCa patients. Spiked cell lines in normal blood and CTCs were enriched by ISET (isolation by size of epithelial tumor cells). Fluorescent staining was carried out using epithelial (pan-cytokeratins, EpCAM), mesenchymal (vimentin, N-cadherin), leukocyte (CD45) markers and DAPI. Cytomorphological staining was carried out with Mayer-Hemalun or Diff-Quik. ALK-, ROS1-, ERG-rearrangement were detected by filter-adapted-FISH (FA-FISH). Microscopy was carried out using an Ariol scanner. Two combined assays were developed. The first assay sequentially combined four-color fluorescent staining, scanning, automated selection of CD45− cells, cytomorphological staining, then scanning and analysis of CD45− cell phenotypical and cytomorphological characteristics. CD45− cell selection was based on DAPI and CD45 intensity, and a nuclear area >55 μm2. The second assay sequentially combined fluorescent staining, automated selection of CD45− cells, FISH scanning on CD45− cells, then analysis of CD45− cell FISH signals. Specific scanning parameters were developed to deal with the uneven surface of filters and CTC characteristics. Thirty z-stacks spaced 0.6 μm apart were defined as the optimal setting, scanning 82 %, 91 %, and 95 % of CTCs in ALK-, ROS1-, and ERG-rearranged patients respectively. A multi-exposure protocol consisting of three separate exposure times for green and red fluorochromes was optimized to analyze the intensity, size and thickness of FISH signals. The semi-automated microscopy method reported here increases the feasibility and reliability of filtration-enriched CTC assays and can help progress towards their validation and translation to the clinic.

DNA damage and genomic instability contribute to non–small cell lung cancer (NSCLC) etiology and progression. However, their therapeutic exploitation is disappointing. CTC-derived explants (CDX) offer systems for mechanistic investigation of CTC metastatic potency and may provide rationale for biology-driven therapeutics. Four CDX models and 3 CDX-derived cell lines were established from NSCLC CTCs and recapitulated patient tumor histology and response to platinum-based chemotherapy. CDX (GR-CDXL1, GR-CDXL2, GR-CDXL3, GR-CDXL4) demonstrated considerable mutational landscape similarity with patient tumor biopsy and/or single CTCs. Truncal alterations in key DNA damage response (DDR) and genome integrity–related genes were prevalent across models and assessed as therapeutic targets in vitro, in ovo, and in vivo. GR-CDXL1 presented homologous recombination deficiency linked to biallelic BRCA2 mutation and FANCA deletion, unrepaired DNA lesions after mitosis, and olaparib sensitivity, despite resistance to chemotherapy. SLFN11 overexpression in GR-CDXL4 led to olaparib sensitivity and was in coherence with neuroendocrine marker expression in patient tumor biopsy, suggesting a predictive value of SLFN11 in NSCLC histological transformation into small cell lung cancer (SCLC). Centrosome clustering promoted targetable chromosomal instability in GR-CDXL3 cells. These CDX unravel DDR and genome integrity–related defects as a central mechanism underpinning metastatic potency of CTCs and provide rationale for their therapeutic targeting in metastatic NSCLC.

Circulating tumor cells (CTCs) provide an accessible tool for investigating tumor heterogeneity and cell populations with metastatic potential. Although an in-depth molecular investigation is limited by the extremely low CTC count in circulation, significant progress has been made recently in single-cell analytical processes. Indeed, CTC monitoring through molecular and functional characterization may provide an understanding of genomic instability (GI) molecular mechanisms, which contribute to tumor evolution and emergence of resistant clones. In this review, we discuss the sources and consequences of GI seen through single-cell analysis of CTCs in different types of tumors. We present a detailed overview of chromosomal instability (CIN) in CTCs assessed by fluorescence in situ hybridization (FISH), and we reveal utility of CTC single-cell sequencing in identifying copy number alterations (CNA) oncogenic drivers. We highlight the role of CIN in CTC-driven metastatic progression and acquired resistance, and we comment on the technical obstacles and challenges encountered during single CTC analysis. We focus on the DNA damage response and depict DNA-repair-related dynamic biomarkers reported to date in CTCs and their role in predicting response to genotoxic treatment. In summary, the suggested relationship between genomic aberrations in CTCs and prognosis strongly supports the potential utility of GI monitoring in CTCs in clinical risk assessment and therapeutic choice.

11515 Background: Duration of response in ALK-rearranged NSCLC patients treated by crizotinib is in the range of 10 months but all patients invariably acquire resistance. Using filter-adapted fluorescence in situ hybridization (FA-FISH) we previously reported ALK-rearrangement detection in circulating tumor cells (CTCs) from ALK-rearranged patients (Pailler et al., J Clin Oncol., 2013). Here we report the monitoring of ALK-rearranged and ALK-amplified CTCs under crizotinib therapy in an extended cohort of ALK-rearranged patients. The correlation between CTC subsets harboring distinct FISH patterns and clinical parameters including progression-free survival (PFS) and overall survival (OS) is presented. Methods: Forty ALK-rearranged patients were recruited of whom one was not evaluable. Blood samples were collected at baseline and at an early time point (1-3 months). Abnormal ALK-FISH patterns were examined in CTCs using immunofluorescence staining (DAPI/CD45) combined with FA-FISH after isolation by size of epithelial tumor cells (ISET) filtration. CTCs were defined into distinct subsets according to the number of FISH ‘break-apart’ signals (3’ and 5’) or isolated red signals (3’) and/or native copies (3’/5’). All ALK-abnormal cells were validated by a cytogeneticist. Clinical data was collected retrospectively. Results: ALK rearrangement was detected in CTCs: 32/39 patients (82%) had ≥4 ALK-rearranged CTCs per 1 ml of blood. Age ≥60 years (36%) and smoking status ≥15 pack years (33%) were associated with a higher mean number of ALK-amplified CTCs (p=0.0361 and p=0.0023 respectively). Using log-rank test, we observed a significant correlation (p=0.0255) between the evolution under crizotinib of the numbers of ALK-amplified CTCs and PFS. An increase in the numbers of ALK-amplified CTCs during treatment is associated with a PFS of 6 months, while a decrease in this subset is associated with a PFS of 13.9 months. There was no corresponding correlation with OS. Conclusions: Our data suggest that variation of ALK-amplified CTCs may be a surrogate biomarker for crizotinib benefit in ALK-rearranged NSCLC patients.

165 Background: In order to optimize molecular screening in advanced prostate cancer patients, we implemented a prospective molecular triage trial PETRUS (NCT01786031), based on molecular characterization of fresh tumor biopsies and circulating tumor cells (CTCs) in patients with mCRPC. The primary objective was to investigate the feasibility of prospectively performing molecular profiling of CRPC patients. Methods: A tumor biopsy from a metastatic and tumor primary site, along with blood samples specimen were collected from patients, and both frozen and FFPE samples were obtained. CTCs were detected using two methods, CellSearch and filtration (ISET, isolation by Size of Epithelial Tumor cells) combined to four-color immunofluorescent staining. Results: From December 2012 and March 2014, 51 CRPC patients with metastases accessible for biospy were included in 5 centres in France. Tumor biospy were successful (≥ 50% tumor cells) in 36/51 pts (72%). CTCs with different phenotypes were detected by CellSearch and ISET combined to four-color immunofluorescent staining in 28 (51%) pts. FISH AR and ERG was successful in only 54% and 32% of metastasis biopsies and primitive tumors respectively, most likely due to the absence of tumor cells or overfixation. The ISET platform was able to detect CTCs clusters, CTCs expressing epithelial and vimentin markers. AR amplification and ERG rearrangement analyses showed high level of heterogeneity with evidence of multiple CRPC iCTC subpopulation. A high level of heterogeneity between CTCs, metastasis biopsies and primitive tumors was observed for ERG rearrangement and AR amplification status. Conclusions: Our results demonstrate that CTCs could be an alternative source for surrogate molecular marker assessment in mCRPC, but neither approach could truly reflect the extreme heterogeneity observed in CTCs from mCRPC. Our preliminary results suggest also the operational feasibility of metastatic tumor biopsy: high interest for patients, significant yield of tumor biopsies and ad hoc molecular analysis. Clinical trial information: NCT01786031.