Vincent Faugeroux

Clinical Utility of Circulating Tumor Cells in ALK-Positive Non-Small-Cell Lung Cancer

The advent of rationally targeted therapies such as small-molecule tyrosine kinase inhibitors (TKIs) has considerably transformed the therapeutic management of a subset of patients with non-small-cell lung cancer (NSCLC) harboring defined molecular abnormalities. When such genetic molecular alterations are detected the use of specific TKI has demonstrated better results (overall response rate, progression free survival) compared to systemic therapy. However, the detection of such molecular abnormalities is complicated by the difficulty in obtaining sufficient tumor material, in terms of quantity and quality, from a biopsy. Here, we described how circulating tumor cells (CTCs) can have a clinical utility in anaplastic lymphoma kinase (ALK) positive NSCLC patients to diagnose ALK-EML4 gene rearrangement and to guide therapeutic management of these patients. The ability to detect genetic abnormalities such ALK rearrangement in CTCs shows that these cells could offer new perspectives both for the diagnosis and the monitoring of ALK-positive patients eligible for treatment with ALK inhibitors.

The potential diagnostic power of circulating tumor cell analysis for non-small-cell lung cancer.

In non-small-cell lung cancer (NSCLC), genotyping tumor biopsies for targetable somatic alterations has become routine practice. However, serial biopsies have limitations: they may be technically difficult or impossible and could incur serious risks to patients. Circulating tumor cells (CTCs) offer an alternative source for tumor analysis that is easily accessible and presents the potential to identify predictive biomarkers to tailor therapies on a personalized basis. Examined here is our current knowledge of CTC detection and characterization in NSCLC and their potential role in EGFR-mutant, ALK-rearranged and ROS1-rearranged patients. This is followed by discussion of the ongoing issues such as the question of CTC partnership as diagnostic tools in NSCLC.

Detection of Gene Rearrangements in Circulating Tumor Cells: Examples of ALK-, ROS1-, RET-Rearrangements in Non-Small-Cell Lung Cancer and ERG-Rearrangements in Prostate Cancer

Circulating tumor cells (CTCs) hold promise as biomarkers to aid in patient treatment stratification and disease monitoring. Because the number of cells is a critical parameter for exploiting CTCs for predictive biomarkers detection, we developed a FISH (fluorescent in situ hybridization) method for CTCs enriched on filters (filter-adapted FISH [FA-FISH]) that was optimized for high cell recovery. To increase the feasibility and reliability of the analyses, we combined fluorescent staining and FA-FISH and developed a semi-automated microscopy method for optimal FISH signal identification in filtration-enriched CTCs . Here we present these methods and their use for the detection and characterization of ALK-, ROS1-, RET-rearrangement in CTCs from non-small-cell lung cancer and ERG-rearrangements in CTCs from prostate cancer patients.

Toward a real liquid biopsy in metastatic breast and prostate cancer: Diagnostic LeukApheresis increases CTC yields in a European prospective multicenter study (CTCTrap): Toward a real liquid biopsy in metastatic breast and prostate cancer

Frequently, the number of circulating tumor cells (CTC) isolated in 7.5 mL of blood is too small to reliably determine tumor heterogeneity and to be representative as a “liquid biopsy”. In the EU FP7 program CTCTrap, we aimed to validate and optimize the recently introduced Diagnostic LeukApheresis (DLA) to screen liters of blood. Here we present the results obtained from 34 metastatic cancer patients subjected to DLA in the participating institutions. About 7.5 mL blood processed with CellSearch® was used as “gold standard” reference. DLAs were obtained from 22 metastatic prostate and 12 metastatic breast cancer patients at four different institutions without any noticeable side effects. DLA samples were prepared and processed with different analysis techniques. Processing DLA using CellSearch resulted in a 0–32 fold increase in CTC yield compared to processing 7.5 mL blood. Filtration of DLA through 5 μm pores microsieves was accompanied by large CTC losses. Leukocyte depletion of 18 mL followed by CellSearch yielded an increase of the number of CTC but a relative decrease in yield (37%) versus CellSearch DLA. In four out of seven patients with 0 CTC detected in 7.5 mL of blood, CTC were detected in DLA (range 1–4 CTC). The CTC obtained through DLA enables molecular characterization of the tumor. CTC enrichment technologies however still need to be improved to isolate all the CTC present in the DLA.

Routine clinical use of circulating tumor cells for diagnosis of mutations and chromosomal rearrangements in non-small cell lung cancer—ready for prime-time?

In non-small cell lung cancer (NSCLC), diagnosis of predictive biomarkers for targeted therapies is currently done in small tumor biopsies. However, tumor biopsies can be invasive, in some cases associated with risk, and tissue adequacy, both in terms of quantity and quality is often insufficient. The development of efficient and non-invasive methods to identify genetic alterations is a key challenge which circulating tumor cells (CTCs) have the potential to be exploited for. CTCs are extremely rare and phenotypically diverse, two characteristics that impose technical challenges and impact the success of robust molecular analysis. Here we introduce the clinical needs in this disease that mainly consist of the diagnosis of epidermal growth factor receptor (EGFR) activating alterations and anaplastic lymphoma kinase (ALK) rearrangement. We present the proof-of-concept studies that explore the detection of these genetic alterations in CTCs from NSCLC patients. Finally, we discuss steps that are still required before CTCs are routinely used for diagnosis of EGFR-mutations and ALK-rearrangements in this disease.

Filter-Adapted Fluorescent In Situ Hybridization (FA-FISH) for Filtration-Enriched Circulating Tumor Cells

Circulating tumor cells (CTCs) may represent an easily accessible source of tumor material to assess genetic aberrations such as gene-rearrangements or gene-amplifications and screen cancer patients eligible for targeted therapies. As the number of CTCs is a critical parameter to identify such biomarkers, we developed fluorescent in situ hybridization (FISH) for CTCs enriched on filters (filter-adapted-FISH, FA-FISH). Here, we describe the FA-FISH protocol, the combination of immunofluorescent staining (DAPI/CD45) and FA-FISH techniques, as well as the semi-automated microscopy method that we developed to improve the feasibility and reliability of FISH analyses in filtration-enriched CTC.

Abstract 4953: Whole-exome sequencing of single circulating tumor cells according to epithelial-mesenchymal marker expression in metastatic prostate cancer

Molecular characterization of metastatic castration-resistant prostate cancer (mCRPC) is limited by tumor tissue availability. The analysis of circulating tumor cells (CTCs) offers an attractive non invasive surrogate option to analyze molecular alterations. We report whole exome sequencing (WES) of CTCs at the single cell level in 11 mCRPC patients. We examined single somatic nucleotide variant (sSNV) shared between matched metastatic tumor sample and CTCs and sSNV specific to CTCs. Blood samples were drawn from 11 patients enrolled in the clinical program MOSCATO (2011-A00841-40). CTC enrichment, detection and single cell isolation were performed using three methods to obtain pools of 1-10 CTCs. The first method used ISET filtration, immunofluorescent staining (CD45, pan-cytokeratin, EpCAM, Vimentin and Hoechst 33342) on filters and laser microdissection of single CTCs; the second combined CellSearch and the VyCap puncher system; the third used RosetteSep enrichment, immunofluorescent staining and isolation by cell sorting. Whole Genome Amplification (WGA) was performed using the Ampli1 kit. WGA quality was assessed by qPCR of 7 genes located on different regions of the genome. WES was performed by preparation of a genomic DNA bank, Agilent capture and sequencing on the Illumina HiSeq 2000 platform. Data were aligned to the human genome reference hg19. GATK Haplotype Caller enabled identification of germline polymorphisms from each patient in normal DNA, metastatic sample and CTCs in order to consider WGA induced bias. The detection of sSNV in tumor biopsies and CTCs was assessed with Mutect and IndelGenotyper respectively. 189 WGAs of CTC pools were performed. A first round of WES showed that at least 3 well amplified genes were required to obtain a coverage of at least 50% at 10X depth sequencing. 34 pools of phenotypically different CTCs from 7 patients were selected and sequenced. Mean coverage of 51% was obtained at a sequencing depth of 10X. Allelic drop out was lower for CTC pools containing 5 to 10 cells. 17/34 (50%) CTC samples (4 patients) had shared sSNV with the paired tumor sample (range 0.35%-68%). Epithelial CTCs had more shared sSNV with metastatic biopsies than CTCs of other phenotypes but shared sSNV were also detected in large Cytokeratin-Vimentin- CTC. Shared sSNV in cancer genes between epithelial CTC pools, but not in the paired biopsy, were present in 2 patients. We report WES of CTC pools harboring distinct EMT marker phenotypes is possible with the use of 3 different approaches to enrich, detect and isolate CTCs. The detection of shared sSNV between CTC pools and corresponding biopsy could validate the use of CTCs as a liquid biopsy. The finding of sSNV specific to CTCs could offer additional data on tumor heterogeneity. Ongoing work examining if sSNV detected in phenotypically different CTCs converge to similar signaling pathways will be presented. Citation Format: Vincent Faugeroux, Celine Lefebvre, Emma Pailler, Valerie Pierron, Fanny Billiot, Charles Marcaillou, Philippe Vielh, Semih Dogan, Philippe Rameau, Maud Ngocamus, Jean Charles Soria, Karim Fizazi, Yohann Loriot, Sylvia Julien, Francoise Farace. Whole-exome sequencing of single circulating tumor cells according to epithelial-mesenchymal marker expression in metastatic prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4953.

Abstract 2256: Establishment and characterization of circulating tumor cell-derived xenografts in non-small cell lung cancer

Low numbers of circulating tumor cells (CTCs) have so far limited the establishment of CTC-derived xenografts (CDXs) to improve our understanding of tumor progression, drug resistance mechanisms, and their biological properties. We report the establishment and the phenotypic and molecular characterization of one NSCLC CDX. Blood samples (30 ml) were drawn from 49 NSCLC patients with advanced metastatic disease. CTCs were enriched by RosetteSep, embedded in matrigel, and implanted in the interscapular aspect of NSG mouse (Nod/Scid-IL2Rγ-/-). Mice were followed-up for one year according to ethical regulations. CDX tumours and CDX derived cell lines were phenotypically and molecularly characterized by immunofluorescence, immunohistochemistry, CGHarray, exome sequencing and transcriptome gene expression. CTCs from one NSCLC patient with 750 CTCs detected by CellSearch gave rise to a tumor 5 months after initial murine injection. Histological analysis confirmed the human origin of the tumor and the presence of a poorly differentiated adenocarcinoma consistent with the patient9s biopsy. Tumor was positive for EpCAM, EMA, CK8;18, and Ki67, and negative for vimentin. A fraction of cells (25%) from freshly dissociated tumors exhibited ALDH activity. CGH from CDX tumors at passage 1 and 2 shows multiple gene rearrangement, revealing a high degree of genomic instability. Transcriptome analysis of ALDH positive and negative cells is ongoing and should help of identifying a cancer stem cell gene expression signature. Whole-exome sequencing of CDX tumor is ongoing and will be compared to data obtained from single CTCs from the patient. A cell line established in vitro from the CDX model grows in 3D clusters and is tumorigenic in mice. Interestingly, this cell line is positive for cytokeratins, EpCAM, E-cadherin, N-cadherin, vimentin, and expresses multiple cancer stem cell markers including CD166, CD24, CD133 and, ALDH activity. The cell line is hypotetraploid (about 70 chromosomes) and its CGH profile was similar to that of the CDX tumour, revealing a high level of genome instability. By investigating DNA replication process in this cell line, we found that it exhibits a spontaneous enhanced DNA damage signaling associated to an accumulation of DNA double strand breaks mainly in S phase strongly suggesting that the CDX-derived cell line displays hallmarks on replication stress that could explain, at least partially, the genomic instability in the cells. We report a low success rate in the establishment of NSCLC CDX (2%). However one NSCLC CDX model harboring cancer stem cell properties and deficiency of DNA replication maintenance was established. Ongoing work to identify a cancer stem cell signature and characteristics replication stress markers in this CDX model will be presented. This NSCLC CDX model will be useful to test drugs targeting these alterations in vivo and improve our knowledge of drug resistance. Citation Format: Vincent Faugeroux, Olivier Deas, Judith Michels, Jean Gabriel Judde, Stefano Cairo, Philippe Vielh, Virginie Marty, Fanny Billiot, Maud Ngocamus, Benjamin Besse, Patricia Kannouche, Francoise Farace. Establishment and characterization of circulating tumor cell-derived xenografts in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2256.

Abstract 2254: Variations in the epithelial-mesenchymal transition (EMT) program by non-small cell lung cancer (NSCLC) circulating tumor cells (CTCs) do not influence survival

Background By definition, CTCs must undergo the EMT to enter the bloodstream where they can be isolated from cancer patients for translational and biological study. Here we examined survival patterns in relation to CTC EMT expression in different molecular subgroups of NSCLC. Methods 125 patients (pts) with advanced treatment-naive stage IIb-IV NSCLC were prospectively included for CellSearch CTC analysis as part of the Gustave-Roussy MSN study. Patients signed an informed consent for one CellSave tube prior to chemotherapy. Anti-vimentin (vim) antibody was added to the free channel in the CellSearch system for examination of EMT. Association of CTC number with clinical characteristics were assessed using Fisher9s exact, Mann-Whitney and chi-squared tests. Kaplan-Meier method and log-rank tests were used to analyse progression-free survival (PFS) and overall survival (OS) of vimentin-expression in molecular subgroups of NSCLC. Results 51/125 pts (40.8%) were CTC-positive by CellSearch (≥2 CTC), and 29/125 (23.2%) patient samples contained at least 1 vimentin-positive (+) CTC. 19/76 (25%) adenocarcinomas were KRAS mutated. In the KRAS subgroup, 0/19 patient samples (0%) from pts with mutated KRAS contained vim+ CTC, compared to 17/48 pts (35.4%) with wild-type (WT) KRAS (p = 0.0027). There was also a significantly higher overall number of vim+ CTCs in pts with KRAS WT cancer compared to KRAS mutated cancer (mean 0 vs 1.63, respectively, p = 0.0035). For KRAS WT pts, no survival difference was evident between vim+ and vim- subgroups in terms of PFS or OS. 21/89 adenocarcinomas were EGFR mutated (23.6%). In this subgroup, statistically higher numbers of EGFR mutated pts with both vim+ and total CTCs were observed compared to EGFR WT pts (vim+ CTC: 9/21 EGFR mutated vs 9/56 EGFR WT, p = 0.0134; total CTC: 12/21 EGFR mutated vs 18/56 EGFR WT, p = 0.0451). Similarly, there was a significantly higher overall number of vim+ CTCs in pts with EGFR mutated cancer compared to pts with EGFR WT cancer (mean 1.24 vs 0.91, respectively, p = 0.0189), but no PFS or OS difference was evident between vim+ and vim- subgroups in EGFR mutated pts. 14/71 (19.7%) adenocarcinomas were ALK rearranged, with further results pending. Conclusions At baseline stage IIIb-IV disease, there are statistically fewer vim+ CTCs (and pts with vim+ CTCs) in KRAS mutated NSCLC, while vim+ CTC (and vim+ CTC pts) are statistically higher in EGFR mutated NSCLC. Despite this differential CTC vimentin expression between molecular subgroups, no PFS or OS difference is evident between vim+ and vim- patients. This biological variation coupled to a lack of overall clinical impact favours the hypothesis that each individual CTC is a highly plastic cell that can cover a range of EMT expression. Citation Format: Colin R. Lindsay, Vincent Faugeroux, Emma Pailler, Maria-Virginia Bluthgen, Chloe Pannet, Maud Ngo-Camus, Guillaume Bescher, Fanny Billiot, Jordi Remon, Philippe Vielh, David Planchard, Jean-Charles Soria, Benjamin Besse, Francoise Farace. Variations in the epithelial-mesenchymal transition (EMT) program by non-small cell lung cancer (NSCLC) circulating tumor cells (CTCs) do not influence survival. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2254.