- Email: [email protected]
60: Selection and adaptation during metastatic cancer progression
S15
EACR-23 Oral Presentations, Monday 7 July 2014 / European Journal of Cancer 50, Suppl. 5 (2014) S12–S20 cases discordance was accompanied by increasing plasma fractions of other potential drivers, for example mutations in TP53, suggesting selection of predominant tumour clones as a result of the targeting agents. The EGFR resistance-conferring T790M mutation was detected in plasma in over half of the patients with previous EGFR-TKI resistance, as well as a subset of TKIna¨ıve patients months before radiological disease progression. One patient who progressed on gefitinib had a ctDNA resistance-conferring/activating mutation ratio of over 50% during first-line treatment. After switching to chemotherapy, upon completion of treatment the ratio fell below 2%. On rechallenge with gefitinib the disease showed renewed sensitivity. Serial quantitative mutational analysis in plasma can be used for monitoring of the changes in relative abundance of activating and resistance-conferring mutations, as well as to detect the emergence of alternative actionable targets. It provides a rational framework for adaptive sequential therapy to improve the management of lung cancer. No conflict of interest. 59 CTCs and cfDNA, will they be useful for early detection of cancer? C. Dive1 , P. Crosbie2 , R. Shah3 , R. Metcalf4 , F. Blackhall5 . 1 CRUK Manchester Institute, Clinical and Experimental Pharmacology, Manchester, United Kingdom, 2 University of Manchester and University Hospital of South Manchester, Respiratory Medicine, Manchester, United Kingdom, 3 University Hospital of South Manchester, Thoracic Surgery, Manchester, United Kingdom, 4 CRUK Manchester Institute University of Manchester, Clinical and Experimental Pharmacology, Manchester, United Kingdom, 5 University of Manchester, Institute of Cancer Sciences, Manchester, United Kingdom Background: ‘Liquid biopsies’ for the detection of early cancers present signiicant challenges regarding assay sensitivity. Certainly the prevalence of circulating tumour cells (CTCs) in the peripheral blood of early non small lung cancer (NSCLC) patients is low. We are evaluating sereval CTC technologies to increase sensitivity and testing the hypothesis that number, phenotype or genotype of CTCs in the pulmonary vein of patients with early stage, resectable NSCLC may predict disease recurrence. Surgical resection of NSCLC can produce long-term survival (5 year survival: Stage I 58−73%, Stage II 36−46%, Stage IIIA 24%). However, recurrence occurs in 50% of cases, most commonly at distant sites when it is almost universally fatal. Standard staging methods do not reliably detect micrometastatic disease therefore CTC analysis may identify patients most at risk of recurrence. We initiated a study to explore CTC prevalence in pulmonary venous blood at the time of resection of early stage NSCLC. As the pulmonary veins directly drain blood from the lungs we postulated that CTC number would be higher than in matched peripheral blood. Aim: To compare CTC number in peripheral and pulmonary vein blood at the time of surgical resection of NSCLC by cellSearch and marker independent technology platforms. Methods: Ethically approved surgical case series of patients undergoing resection of NSCLC at the Thoracic Surgical Department, University Hospital of South Manchester. Pre-operative peripheral blood (10ml) was compared to intra-operative pulmonary vein blood (10ml), taken prior to vessel ligation or tumour manipulation. Sample (7.5ml) processing and analysis was undertaken using CellSearch and by filtration. Results: 34 patients were recruited (20M/14F). CTC count ranged from 0−4 in peripheral and 0–3093 in pulmonary vein blood, with CTC count significantly higher in pulmonary vein blood (p = 0.002). CTCs were detected more frequently in pulmonary compared to peripheral vein samples (1 CTC: 16/34, 47% vs. 7/32, 22%; p = 0.041) (2 CTCs: 14/34, 41% vs. 2/32, 6%; p = 0.001) and circulating microemboli (CTM) only in pulmonary vein blood (6/34, 18%). Pulmonary vein CTC count was positive (2 CTCs) in 4/12 (33%) patients with stage I and 10/22 (46%) patients with stage II−IV disease (p = 0.49). Mesenchymal and epithelial CTCs were observed by filtration. Pulmonary vein sampling was safe with no significant adverse events and was possible irrespective of surgical approach. Conclusion: CTC prevalence is significantly higher in pulmonary vein blood compared to matched peripheral vain samples in patients undergoing surgical resection of early stage NSCLC. Ongoing studies and future Goals: A workflow from CellSearch enrichment of CTCs to their isolation by DEPArray has been optimised. Single cell molecular analysis of early CTCs is underway and matched plasma is being collected for ctDNA analysis. The ability of pulmonary CTCs to generate explant tumour models in immune-compromised mice will also be evaluated. No conflict of interest.
Monday 7 July 2014
10:45−12:30
Symposium
AEK Supported Symposium: Invasion and Metastasis 60 Selection and adaptation during metastatic cancer progression C. Klein1 . 1 Abteilung fur ¨ Onkogenomik, Regensburg, Germany Cancer is often regarded as a process of asexual evolution driven by genomic and genetic instability. Mutation, selection and adaptation are by convention thought to occur primarily within, and to a lesser degree, outside the primary tumour. However, we previously noted in several cancers, including breast and prostate cancer, that metastatic dissemination occurs often early, suggesting that metastatic founder cells may lodge and evolve considerable periods of time outside the primary tumour. The evidence for this was derived from comparative genetic studies (primary tumour versus disseminated cancer cells, DCC), analysis of DCIS patients and from transgenic animal models. We now investigated a large cohort of 1027 melanoma patients and determined the thickness of the primary melanoma at dissemination. We then investigated melanoma DCCs and corroborated the breast cancer-derived concept of early dissemination of immature cancer cells. Strikingly, upon earliest evidence of metastatic colonization, early DCCs acquire critical somatic mutations, which appear to drive the lethal metastatic process. Acquisition of these properties enhanced the ability to engraft in immunodeficient NSG mice, providing strong evidence that colonization in addition to dissemination is a fundamentally critical step of metastasis. Abrogation of ectopic progression may provide new opportunities for therapeutic intervention, which may change diagnostic procedures and adjuvant therapies. No conflict of interest. 61 Role of the Hippo transducers YAP and TAZ in mechanotransduction and cancer stem cells S. Piccolo1 . 1 University of Padova, Department of Medical Biotechnologies Section of Histology and Embryology, Padova, Italy Cells perceive their microenvironment not only through soluble signals but also in term of physical and mechanical cues, such as extracellular matrix (ECM) stiffness or cell shape. By mechanotransduction systems, cells translate these stimuli into biochemical signals controlling multiple aspects of cell behavior, including growth, differentiation and malignant progression; but how rigidity mechanosensing is ultimately linked to activity of nuclear transcription factors remains poorly understood. Here we report evidence that the Yorkiehomologues YAP and TAZ as nuclear relays of mechanical signals exerted by ECM rigidity and cell-shape. Crucially, YAP/TAZ emerge as universal mediators of the biological effects of these structural and unsoluble cues. YAP and TAZ are also regulated by EMT and disturbed cell polarity, a hallmark of cancer. We found that TAZ regulates biological traits operationally associated with breast Cancer Stem Cells (CSCs): TAZ is required to sustain self-renewal and tumor-initiation capacities in cellular models of breast cancer progression. Gain-of-TAZ in non-CSCs induces them to adopt CSCs-like behaviors and promotes the transition from low- to high-grade tumors. TAZ is downstream of EMT. Central to this regulation is the formation of an endogenous complex between TAZ and the cell polarity determinant Scribble: induction of EMT, or loss-of-Scribble, disrupts the association of TAZ with the core Hippo kinases MST and LATS, leading to TAZ activation. This links breast CSCs to the Hippo pathway, and reveals a mechanistic basis of the control of Hippo kinases by cell polarity. No conflict of interest. 62 Proffered Paper: Cell-specific proteomic analysis of contact-initiated tumour-endothelial signalling identifies novel regulators of transendothelial migration M. Locard-Paulet1 , L. Lim1 , J. Sinclair1 , C. Jorgensen1,2 . 1 The Institute of Cancer Research, London, United Kingdom, 2 Cancer Research UK Manchester Institute, The University of Manchester, United Kingdom Background: Most cancer related deaths are caused by metastatic disease, presenting formidable challenges to improved treatment. Haematological spread rely on the ability of cancer cells to both intra- and extravasate. Although these two events are essential for the spread of tumour cells, little is known of the molecular networks underpinning these processes. Here we present a novel approach to interrogate key processes underlying metastatic spread. Critically, using a novel mass spectrometry technique, we describe the cellspecific analysis of contact-initiated signalling between tumour and endothelial cells, and identify EPHA2 as a novel regulator in extravasation. Methods: To identify regulators of tumour-endothelial cell adhesion (TEA) and transendothelial cell migration (TEM) we conducted a phospho-proteomics
EACR-23 Oral Presentations, Monday 7 July 2014 / European Journal of Cancer 50, Suppl. 5 (2014) S12–S20 cases discordance was accompanied by increasing plasma fractions of other potential drivers, for example mutations in TP53, suggesting selection of predominant tumour clones as a result of the targeting agents. The EGFR resistance-conferring T790M mutation was detected in plasma in over half of the patients with previous EGFR-TKI resistance, as well as a subset of TKIna¨ıve patients months before radiological disease progression. One patient who progressed on gefitinib had a ctDNA resistance-conferring/activating mutation ratio of over 50% during first-line treatment. After switching to chemotherapy, upon completion of treatment the ratio fell below 2%. On rechallenge with gefitinib the disease showed renewed sensitivity. Serial quantitative mutational analysis in plasma can be used for monitoring of the changes in relative abundance of activating and resistance-conferring mutations, as well as to detect the emergence of alternative actionable targets. It provides a rational framework for adaptive sequential therapy to improve the management of lung cancer. No conflict of interest. 59 CTCs and cfDNA, will they be useful for early detection of cancer? C. Dive1 , P. Crosbie2 , R. Shah3 , R. Metcalf4 , F. Blackhall5 . 1 CRUK Manchester Institute, Clinical and Experimental Pharmacology, Manchester, United Kingdom, 2 University of Manchester and University Hospital of South Manchester, Respiratory Medicine, Manchester, United Kingdom, 3 University Hospital of South Manchester, Thoracic Surgery, Manchester, United Kingdom, 4 CRUK Manchester Institute University of Manchester, Clinical and Experimental Pharmacology, Manchester, United Kingdom, 5 University of Manchester, Institute of Cancer Sciences, Manchester, United Kingdom Background: ‘Liquid biopsies’ for the detection of early cancers present signiicant challenges regarding assay sensitivity. Certainly the prevalence of circulating tumour cells (CTCs) in the peripheral blood of early non small lung cancer (NSCLC) patients is low. We are evaluating sereval CTC technologies to increase sensitivity and testing the hypothesis that number, phenotype or genotype of CTCs in the pulmonary vein of patients with early stage, resectable NSCLC may predict disease recurrence. Surgical resection of NSCLC can produce long-term survival (5 year survival: Stage I 58−73%, Stage II 36−46%, Stage IIIA 24%). However, recurrence occurs in 50% of cases, most commonly at distant sites when it is almost universally fatal. Standard staging methods do not reliably detect micrometastatic disease therefore CTC analysis may identify patients most at risk of recurrence. We initiated a study to explore CTC prevalence in pulmonary venous blood at the time of resection of early stage NSCLC. As the pulmonary veins directly drain blood from the lungs we postulated that CTC number would be higher than in matched peripheral blood. Aim: To compare CTC number in peripheral and pulmonary vein blood at the time of surgical resection of NSCLC by cellSearch and marker independent technology platforms. Methods: Ethically approved surgical case series of patients undergoing resection of NSCLC at the Thoracic Surgical Department, University Hospital of South Manchester. Pre-operative peripheral blood (10ml) was compared to intra-operative pulmonary vein blood (10ml), taken prior to vessel ligation or tumour manipulation. Sample (7.5ml) processing and analysis was undertaken using CellSearch and by filtration. Results: 34 patients were recruited (20M/14F). CTC count ranged from 0−4 in peripheral and 0–3093 in pulmonary vein blood, with CTC count significantly higher in pulmonary vein blood (p = 0.002). CTCs were detected more frequently in pulmonary compared to peripheral vein samples (1 CTC: 16/34, 47% vs. 7/32, 22%; p = 0.041) (2 CTCs: 14/34, 41% vs. 2/32, 6%; p = 0.001) and circulating microemboli (CTM) only in pulmonary vein blood (6/34, 18%). Pulmonary vein CTC count was positive (2 CTCs) in 4/12 (33%) patients with stage I and 10/22 (46%) patients with stage II−IV disease (p = 0.49). Mesenchymal and epithelial CTCs were observed by filtration. Pulmonary vein sampling was safe with no significant adverse events and was possible irrespective of surgical approach. Conclusion: CTC prevalence is significantly higher in pulmonary vein blood compared to matched peripheral vain samples in patients undergoing surgical resection of early stage NSCLC. Ongoing studies and future Goals: A workflow from CellSearch enrichment of CTCs to their isolation by DEPArray has been optimised. Single cell molecular analysis of early CTCs is underway and matched plasma is being collected for ctDNA analysis. The ability of pulmonary CTCs to generate explant tumour models in immune-compromised mice will also be evaluated. No conflict of interest.
Monday 7 July 2014
10:45−12:30
Symposium
AEK Supported Symposium: Invasion and Metastasis 60 Selection and adaptation during metastatic cancer progression C. Klein1 . 1 Abteilung fur ¨ Onkogenomik, Regensburg, Germany Cancer is often regarded as a process of asexual evolution driven by genomic and genetic instability. Mutation, selection and adaptation are by convention thought to occur primarily within, and to a lesser degree, outside the primary tumour. However, we previously noted in several cancers, including breast and prostate cancer, that metastatic dissemination occurs often early, suggesting that metastatic founder cells may lodge and evolve considerable periods of time outside the primary tumour. The evidence for this was derived from comparative genetic studies (primary tumour versus disseminated cancer cells, DCC), analysis of DCIS patients and from transgenic animal models. We now investigated a large cohort of 1027 melanoma patients and determined the thickness of the primary melanoma at dissemination. We then investigated melanoma DCCs and corroborated the breast cancer-derived concept of early dissemination of immature cancer cells. Strikingly, upon earliest evidence of metastatic colonization, early DCCs acquire critical somatic mutations, which appear to drive the lethal metastatic process. Acquisition of these properties enhanced the ability to engraft in immunodeficient NSG mice, providing strong evidence that colonization in addition to dissemination is a fundamentally critical step of metastasis. Abrogation of ectopic progression may provide new opportunities for therapeutic intervention, which may change diagnostic procedures and adjuvant therapies. No conflict of interest. 61 Role of the Hippo transducers YAP and TAZ in mechanotransduction and cancer stem cells S. Piccolo1 . 1 University of Padova, Department of Medical Biotechnologies Section of Histology and Embryology, Padova, Italy Cells perceive their microenvironment not only through soluble signals but also in term of physical and mechanical cues, such as extracellular matrix (ECM) stiffness or cell shape. By mechanotransduction systems, cells translate these stimuli into biochemical signals controlling multiple aspects of cell behavior, including growth, differentiation and malignant progression; but how rigidity mechanosensing is ultimately linked to activity of nuclear transcription factors remains poorly understood. Here we report evidence that the Yorkiehomologues YAP and TAZ as nuclear relays of mechanical signals exerted by ECM rigidity and cell-shape. Crucially, YAP/TAZ emerge as universal mediators of the biological effects of these structural and unsoluble cues. YAP and TAZ are also regulated by EMT and disturbed cell polarity, a hallmark of cancer. We found that TAZ regulates biological traits operationally associated with breast Cancer Stem Cells (CSCs): TAZ is required to sustain self-renewal and tumor-initiation capacities in cellular models of breast cancer progression. Gain-of-TAZ in non-CSCs induces them to adopt CSCs-like behaviors and promotes the transition from low- to high-grade tumors. TAZ is downstream of EMT. Central to this regulation is the formation of an endogenous complex between TAZ and the cell polarity determinant Scribble: induction of EMT, or loss-of-Scribble, disrupts the association of TAZ with the core Hippo kinases MST and LATS, leading to TAZ activation. This links breast CSCs to the Hippo pathway, and reveals a mechanistic basis of the control of Hippo kinases by cell polarity. No conflict of interest. 62 Proffered Paper: Cell-specific proteomic analysis of contact-initiated tumour-endothelial signalling identifies novel regulators of transendothelial migration M. Locard-Paulet1 , L. Lim1 , J. Sinclair1 , C. Jorgensen1,2 . 1 The Institute of Cancer Research, London, United Kingdom, 2 Cancer Research UK Manchester Institute, The University of Manchester, United Kingdom Background: Most cancer related deaths are caused by metastatic disease, presenting formidable challenges to improved treatment. Haematological spread rely on the ability of cancer cells to both intra- and extravasate. Although these two events are essential for the spread of tumour cells, little is known of the molecular networks underpinning these processes. Here we present a novel approach to interrogate key processes underlying metastatic spread. Critically, using a novel mass spectrometry technique, we describe the cellspecific analysis of contact-initiated signalling between tumour and endothelial cells, and identify EPHA2 as a novel regulator in extravasation. Methods: To identify regulators of tumour-endothelial cell adhesion (TEA) and transendothelial cell migration (TEM) we conducted a phospho-proteomics