Jenny Antonello

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

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

The CellSearch® semiautomated CTC enrichment and staining system has been established as the ‘gold standard’ for CTC enumeration with CellSearch® CTC counts recognized by the FDA as prognostic for a number of cancers. We and others have gone on to show that molecular analysis of CellSearch® CTCs isolated shortly after CellSearch® enrichment provides another valuable layer of information that has potential clinical utility including predicting response to treatment. Although CellSearch® CTCs can be readily isolated after enrichment, the process of analysing a single CellSearch® patient sample, which may contain many CTCs, is both time‐consuming and costly. Here, we describe a simple process that will allow storage of all CellSearch®‐enriched cells in glycerol at −20 °C for up to 2 years without any measurable loss in the ability to retrieve single cells or in the genome integrity of the isolated cells. To establish the suitability of long‐term glycerol storage for single‐cell molecular analysis, we isolated individual CellSearch®‐enriched cells by DEPArray™ either shortly after CellSearch® enrichment or following storage of matched enriched cells in glycerol at −20 °C. All isolated cells were subjected to whole‐genome amplification (WGA), and the efficacy of single‐cell WGA was evaluated by multiplex PCR to generate a Genome Integrity Index (GII). The GII results from 409 single cells obtained from both ‘spike‐in’ controls and clinical samples showed no statistical difference between values obtained pre‐ and postglycerol storage and that there is no further loss in integrity when DEPArray™‐isolated cells are then stored at −80 °C for up to 2 years. In summary, we have established simple yet effective ‘stop‐off’ points along the CTC workflow enabling CTC banking and facilitating selection of suitable samples for intensive analysis once patient outcomes are known.

Abstract 3371: Genetic, phenotypic and functional characterisation of vasculogenic mimicry in small-cell lung cancer

Background: Despite a good initial response to chemotherapy, most small cell lung cancer (SCLC) patients relapse with drug resistant disease. Targeting tumor vasculature in SCLC with anti-angiogenic drugs produced disappointing results, therefore angiogenesis-independent tumor vascularisation pathways warrant further investigation. Vasculogenic mimicry (VM) is the ability of tumor cells to mimic endothelial cells by trans-endothelial differentiation, characterised by increased expression of vascular markers including VE-Cadherin. We previously demonstrated that VM correlates with poor Overall Survival in Limited Stage SCLC patients and sought to phenotypically and genetically characterise VM vessels using SCLC Circulating Tumour Cells (CTCs) and CTC-Derived eXplant (CDX) models1 and to explore the functional significance of VE-Cadherin for VM formation in vitro and in vivo.Methods: VM was evaluated using CD31/periodic acid-Schiff (PAS) staining in tumors from 10 CDX models. Laser Capture Microdissection (LCM) and Copy Number Alteration (CNA) analysis was performed on CDX regions with high and low levels of VM. VE-Cadherin expression in SCLC CTCs was evaluated following ISET microfiltration of patients’ blood and Immunofluorescence staining for DAPI, CD45, pan Cytokeratin and VE-Cadherin. VE-Cadherin function was evaluated in vitro by Matrigel network assay using H446 SCLC cells and H446 shRNA VE-Cadherin knockdown (KD) cell lines, and in vivo by growth as xenografts, further treated with Cisplatin. Results: VM was present in CDX models and LCM followed by CNA analysis of VM vessels confirmed their SCLC origin. ISET microfiltration and immunofluorescent staining of CTCs from 37/38 SCLC patients revealed VE-Cadherin as a putative VM biomarker in SCLC CTCs. VE-Cadherin shRNA KD in VM competent H446 SCLC cells abrogated their ability to form VM networks in vitro and in vivo. Cisplatin treatment of mice bearing H446 VE-Cadherin KD tumors resulted in reduced cisplatin binding compared to parental H446 tumors. Conclusions: VM is present in CDX models and co-localises with VE-Cadherin expression. CNA confirms that VM vessels originate from tumour, and these VM-enriched regions bear a unique chromosomal signature compared to the low-VM regions. VE-Cadherin is required for network formation in vitro and VE-Cadherin levels and VM vessel numbers are positively correlated in vivo. Moreover, levels of VM in tumors had significant impact on both tumour growth kinetics and cisplatin delivery which has implications for drug senstivity. Our future research will interrogate the VM signaling pathway in SCLC and its role in chemosensitivity and establish feasibility for therapeutic targeting. Citation Format: Francesca Trapani, Stuart Williamson, Robert L. Metcalf, Hui Sun Leong, Benjamin Abbott, Jenny Antonello, Cassandra Hodgkinson, Lynsey Franklin, Mary J. Hendrix, Richard E b Seftor, Elisabeth Seftor, Dominic Rothwell, Ged Brady, Crispin Miller, Fiona H. Blackhall, Kathryn L. Simpson, Caroline Dive. Genetic, phenotypic and functional characterisation of vasculogenic mimicry in 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 3371.

Abstract 3155: Investigating chemoresistance in small cell lung cancer through the molecular profiling of single circulating tumour cells

Background Chemoresistance, both intrinsic and acquired, represents one of the greatest challenges in the management of Small Cell Lung Cancer (SCLC), contributing to the very poor survival seen in this disease. The investigation of SCLC, and particularly serially monitoring the development of changes associated with resistance, is hampered by the limited availability of tumour tissue for research. Circulating tumour cells (CTCs) in contrast, are abundant in this disease and represent a potential minimally invasive ‘liquid biopsy’ to study the molecular landscape of SCLC. Methods To investigate tumour genetics in SCLC CTCs were isolated from chemorefractory patients and chemoresponsive patients’ blood samples using the DEPArray©. Blood samples were collected prior to chemotherapy and again at progression with relapsed disease. Following single-cell whole genome amplification low coverage whole genome sequencing and whole exome sequencing (WES) of CTCs were used to investigate mutations and to generate genome-wide patterns of copy number alterations (CNA). Results Hallmark SCLC molecular abnormalities such as TP53 mutations and copy number loss in tumour suppressor genes such as RB1 (previously identified in bulk tumour profiling), were noted in the isolated SCLC CTCs. Distinct CNA profiles were found in the CTCs isolated from patients with chemorefractory disease compared to those isolated from patients with chemoresponsive disease. A potential signature based on differential copy number changes in 2183 loci between the two groups of patients’ CTCs was identified. When tested in an independent set of CTC-derived explants this signature achieved 100% accuracy in classifying patients with chemoresponsive disease. There were no profound differences in CNA pattern in the CTCs isolated from initially chemoresponsive patients when they had relapsed post chemotherapy when compared to the baseline samples. However, changes in the proportion of C>A transversions were identified by WES between baseline and relapse CTCs. Conclusions Our study highlights the utility of molecular profiling CTCs in the research of SCLC. The data presented confirm CTCs represent a novel medium for the investigation of chemoresistance. Citation Format: Louise R. Carter, Dominic G. Rothwell, HuiSun Leong, Yaoyong Li, Deborah J. Burt, Jenny Antonello, Cassandra Hodgkinson, Karen Morris, Lynsey Franklin, Crispin J. Miller, Fiona Blackhall, Caroline Dive, Ged Brady. Investigating chemoresistance in small cell lung cancer through the molecular profiling of single circulating tumour cells. [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 3155.

Abstract 3060: Circulating tumor cells from small cell lung cancer patients are tumorigenic

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Small cell lung cancer (SCLC), is a highly aggressive and metastatic disease with a 5 year survival of 5%. SCLC represents 15-20% of all lung cancers and causes >160,000 deaths a year. The majority of patients present with metastatic disease and consequently resections are rare, while biopsies are small and only for diagnostic purposes thus hampering the study of SCLC. However, circulating tumour cells (CTCs) are highly prevalent in SCLC patients and may represent an avenue for the better understanding this disease. Our aim was to determine whether CTCs isolated from SCLC patients were able to form tumours in immunocompromised mice. This was accomplished by erythrocyte and leukocyte depletion and implantation of the remaining cells. Of the 6 initial patients whose CTCs were implanted, 4 gave rise to tumours in less than 5 months. Immuno-histochemical analysis of the tumours revealed them to be human in nature and express markers consistent with SCLC. Whole exome sequencing demonstrated that the tumours had mutations (e.g. TP53 and RB1) and CNV (e.g. loss of 3p and 13q) commonly observed in SCLC samples. Furthermore, single cell analysis of CTCs isolated from the corresponding patient revealed that genetic abnormalities detected in the tumour were also present in the patients CTCs. This confirmed that the tumours (termed CDX for CTC derived explant), were indeed derived from CTCs. In all 4 successful cases, analysis of parallel blood samples by CellSearch demonstrated that the patients had more than 400 CTCs/7.5 ml blood. Two of these patients were initially sensitive to platinum/etoposide therapy, while 2 were refractory. The doubling times of CDX derived from refractory and sensitive patients were 7.2 and 5.0 days compared to 14.2 and 13.4 days, consistent with refractory SCLC being more aggressive than sensitive SCLC. We have been able to successfully passage, freeze and resurrect all the CDX models and aim to report whether the patient response to therapy is mirrored in their CDX. These data demonstrate that SCLC CTCs are tumorigenic and we are investigating whether the CTC derived tumours represent a faithful model of the clinical disease. Citation Format: Christopher J. Morrow, Cassandra L. Hodgkinson, Yaoyong Li, Robert Metcalf, Dominic Rothwell, Francesca Trapani, Radoslaw Polanski, Debbie Burt, Kathryn Simpson, Karen Morris, Stuart Pepper, Daisuke Nonaka, Alastair Greystole, Paul Kelly, Matthew Krebs, Jenny Antonello, Mahmood Ayub, Suzanne Faulkner, Lynsey Priest, Louise Carter, Catriona Tate, Crispin J. Miller, Fiona Blackhall, Ged Brady, Caroline Dive. Circulating tumor cells from small cell lung cancer patients are tumorigenic. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3060. doi:10.1158/1538-7445.AM2014-3060