Robert Metcalf

Molecular analysis of circulating tumour cells—biology and biomarkers

Growing evidence for intratumour heterogeneity informs us that single-site biopsies fall short of revealing the complete genomic landscape of a tumour. With an expanding repertoire of targeted agents entering the clinic, screening tumours for genomic aberrations is increasingly important, as is interrogating the tumours for resistance mechanisms upon disease progression. Multiple biopsies separated spatially and temporally are impractical, uncomfortable for the patient and not without risk. Here, we describe how circulating tumour cells (CTCs), captured from a minimally invasive blood test—and readily amenable to serial sampling—have the potential to inform intratumour heterogeneity and tumour evolution, although it remains to be determined how useful this will be in the clinic. Technologies for detecting and isolating CTCs include the validated CellSearch® system, but other technologies are gaining prominence. We also discuss how recent CTC discoveries map to mechanisms of haematological spread, previously described in preclinical models, including evidence for epithelial–mesenchymal transition, collective cell migration and cells with tumour-initiating capacity within the circulation. Advances in single-cell molecular analysis are enhancing our ability to explore mechanisms of metastasis, and the combination of CTC and cell-free DNA assays are anticipated to provide invaluable blood-borne biomarkers for real-time patient monitoring and treatment stratification.

Tumourigenic non-small-cell lung cancer mesenchymal circulating tumour cells: a clinical case study

An explant model derived from EpCam negative mesenchymal non-small-cell lung (NSCLC) cancer circulating tumour cells (a ‘liquid biopsy’) recapitulates the histology of the donor patients diagnostic specimen and chemoresistance to cisplatin and pemetrexed. This proof-of-principal landmark model opens a new avenue for study of advanced NSCLC biology when tissue biopsies unavailable.

Thrombosis in ovarian cancer: a case control study

Background:Thrombotic events are common in cancer patients and have been associated with an adverse prognosis in large registry-based studies.Methods:A retrospective cohort of 417 patients with ovarian cancer treated at a tertiary cancer centre between 2006 and 2009 was studied to identify the incidence and risk factors for thrombotic events and the prognostic impact of thrombosis. Patient outcomes were evaluated against a matched control group without thrombosis.Results:Ninety-nine thrombotic events occurred in 90 patients (21.6%) from 8 months before diagnosis to 56 months following diagnosis, peaking in the 4 months following diagnosis. Patients with thrombosis were older (mean 65 vs 61 years, P=0.007), had a worse performance status (PS ⩾2: 29.9% vs 9.5%, P<0.0001) and had a more advanced FIGO stage (FIGO III/IV 75.6% vs 56.9%, P<0.0001) than patients without thrombosis. Shorter overall survival was seen in patients with pulmonary embolism and pelvic/lower limb deep vein thrombosis than without thrombosis (P=0.001). When the control group was matched for stage and PS, no survival difference was seen (P=0.91).Conclusion:Ovarian cancer patients with thrombotic events had a shorter survival. However, when matched for prognostic factors (PS and FIGO stage), thrombosis did not impact upon prognosis.

Clinical Utility of Circulating Tumour Cell Detection in Non-Small-Cell Lung Cancer

Opinion statementRecent years have witnessed increased interest in the detection of circulating tumour cells (CTCs) for diagnosis, monitoring, and treatment decision making in patients with cancer. Factors that have led to accelerated research in this field include advances in technologies for examination of intact CTCs, personalised medicine with treatment selection according to molecular characteristics, and continued lack of understanding of the biology of treatment resistance and metastasis. CTCs offer promise as a surrogate for tissue where there is insufficient tissue for molecular analysis and where there is a requirement to serially monitor molecular changes in cancer cells through treatment or on progression. In patients with either small cell or non-small cell lung cancer (NSCLC), there is evidence that CTC number is prognostic and that CTCs counted before and after treatment mirror treatment response. In patients with molecularly defined subtypes of NSCLC, CTCs demonstrate the same molecular changes as the cancer cells of the tumour. However, CTCs are not quite ready for “primetime” in the lung cancer clinic. There are still more questions than answers with respect to the optimal technologies for their detection and analysis, their biological significance, and their clinical utility. Despite this the current pace of progress in CTC technology development seems set to make “liquid biopsies” a clinical reality within the next decade. For the everyday clinician and clinical trialist, it will be important to maintain knowledge of the strengths and weaknesses of the technologies and evolving evidence base for CTCs as a routinely used diagnostic tool.

Characterisation and risk assessment of venous thromboembolism in gastrointestinal cancers

AIM To characterise venous thromboembolism (VTE) in gastrointestinal cancer and assess the clinical utility of risk stratification scoring. METHODS We performed a retrospective analysis using electronic patient records of 910 gastro-oesophageal (GO) cancer and 1299 colorectal cancer (CRC) patients referred to a tertiary cancer centre to identify the incidence of VTE, its relationship to chemotherapy and impact on survival. VTE risk scores were calculated using the Khorana index. Patients were classified as low risk (0 points), intermediate risk (1 to 2 points) or high risk (3 points). Data was analysed to determine the sensitivity of the Khorana score to predict VTE. RESULTS The incidence of VTE was 8.9% for CRC patients and 9.7% for GO cancer patients. Pulmonary emboli (PE) were more common in advanced than in localised CRC (50% vs 21% of events respectively) and lower limb deep vein thrombosis (DVT) were more common in localised than in advanced CRC (62% vs 39% of events respectively). The median time to VTE from cancer diagnosis was 8.3 mo for CRC patients compared to 6.7 mo in GO cancer. In localised CRC median time to VTE was 7.1 mo compared with 10.1 mo in advanced CRC. In contrast in GO cancer, the median time to VTE was 12.5 mo in localised disease and 6.8 mo in advanced disease. No survival difference was seen between patients with and without VTE in this cohort. The majority of patients with CRC in whom VTE was diagnosed had low or intermediate Khorana risk score (94% for localised and 97% in advanced CRC). In GO cancer, all patients scored either intermediate or high risk due to the primary site demonstrating a limitation of the risk assessment score in discriminating high and low risk patients with GO cancers. Additional risk factors were identified in this cohort including surgery, chemotherapy or hospital admission. Overall, 81% of patients with CRC and 77% of patients with GO cancer had one or more of these factors within 4 wk prior to diagnosis VTE. These should be factored into clinical risk assessment scores. CONCLUSION The Khorana score has low sensitivity for thrombotic events in CRC and cannot discriminate low risk patients in high risk cancer sites such as GO cancer.

Biology and clinical relevance of circulating tumour cells

Assessment of circulating tumour cells (CTCs) provides a novel approach to interrogating cancer biology and CTCs have potential to serve as clinically useful biomarkers. The presence of CTCs and their role in metastasis has long been hypothesised but it is only in recent years that technology has allowed reliable isolation of CTCs leading to a renewed interest in this area. CTCs are commonly found in the blood of patients with cancer but are rare in the blood of healthy volunteers’ and those with benign disease (1). As technologies for CTC isolation have evolved, so too has the ability to characterise CTCs at the molecular level, paving the way to better understand metastasis biology; and CTCs could serve as a minimally invasive ‘liquid biopsy’ for longitudinal monitoring throughout the course of a patient’s treatment. This approach could be particularly useful to describe tumour evolution, assess pharmacodynamic biomarkers for drug development and reveal inherent or acquired treatment resistant mechanisms in a way that has been historically challenging due to difficulties in obtaining successive biopsies in lung cancer patients.

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 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

Abstract 1468: Circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) from patients with small cell lung cancer (SCLC) show heterogeneity in epithelial to mesenchymal transition (EMT) phenotype and evidence of vasculogenic mimicry (VM).

Background: Circulating tumour cells (CTCs) and circulating tumour microemboli (CTM) are prevalent in patients with aggressive small cell lung cancer (SCLC). Heterogeneity in epithelial and mesenchymal phenotypes in CTCs and within CTM was investigated given evidence in preclinical models of epithelial and mesenchymal cell co-operativity advantageous for hematogenous spread and the role of epithelial to mesenchymal transition (EMT) in invasion and metastasis. Methods: Four colour immunofluorescence assays were developed and applied to tumour cells enriched by filtration from peripheral blood of extensive stage SCLC patients prior to treatment. CTCs/CTM were assessed for expression of cytokeratins (CKs, epithelial marker) and vimentin (mesenchymal marker) with DAPI stained nuclei and negative selection of leukocytes and circulating endothelial cells (CECs) by CD45 and/or vascular endothelial-cadherin (VE-cadherin, CD144) expression respectively. Results and Conclusions: CTCs were identified in all patients that expressed epithelial and/or mesenchymal markers. Individual CTM were detected comprised of tumour cells of heterogeneous EMT status and CTM were also observed containing leukocytes and/or CECs. There was substantial heterogeneity in CK and vimentin expression in CTCs and within CTM consistent with EMT plasticity and with co-operativity between epithelial and mesenchymal tumour cells. A subpopulation of CTCs co-expressed CKs and VE-cadherin that intriguingly, raises the possibility that vascular mimicry accompanies and may facilitate hematogenous spread in this aggressive tumor type. Further studies are required to explore this new hypothesis. Citation Format: Robert Metcalf, Jessica Booth, Rajeeb Swain, Steven Bagley, Karen Morris, Matthew Krebs, Andrew Hughes, Gerard Brady, Fiona Blackhall, Caroline Dive. Circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) from patients with small cell lung cancer (SCLC) show heterogeneity in epithelial to mesenchymal transition (EMT) phenotype and evidence of vasculogenic mimicry (VM). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1468. doi:10.1158/1538-7445.AM2013-1468