Pulmonary venous circulating tumor cell dissemination before tumor resection and disease relapse

ABSTRACT Approximately 50% of patients with early-stage non-small-cell lung cancer (NSCLC) who undergo surgery with curative intent will relapse within 5 years1,2. Detection of circulating

tumor cells (CTCs) at the time of surgery may represent a tool to identify patients at higher risk of recurrence for whom more frequent monitoring is advised. Here we asked whether

CellSearch-detected pulmonary venous CTCs (PV-CTCs) at surgical resection of early-stage NSCLC represent subclones responsible for subsequent disease relapse. PV-CTCs were detected in 48% of

100 patients enrolled into the TRACERx study3, were associated with lung-cancer-specific relapse and remained an independent predictor of relapse in multivariate analysis adjusted for tumor

stage. In a case study, genomic profiling of single PV-CTCs collected at surgery revealed higher mutation overlap with metastasis detected 10 months later (91%) than with the primary tumor

(79%), suggesting that early-disseminating PV-CTCs were responsible for disease relapse. Together, PV-CTC enumeration and genomic profiling highlight the potential of PV-CTCs as early

predictors of NSCLC recurrence after surgery. However, the limited sensitivity of PV-CTCs in predicting relapse suggests that further studies using a larger, independent cohort are warranted

to confirm and better define the potential clinical utility of PV-CTCs in early-stage NSCLC. Access through your institution Buy or subscribe This is a preview of subscription content,

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institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS CIRCULATING TUMOUR DNA IN EARLY STAGE AND LOCALLY ADVANCED NSCLC: READY FOR

CLINICAL IMPLEMENTATION? Article 20 January 2025 PLASTICITY OF CIRCULATING TUMOR CELLS IN SMALL CELL LUNG CANCER Article Open access 21 July 2023 GENOMIC CHARACTERISTICS AND CLINICAL

SIGNIFICANCE OF CD56+ CIRCULATING TUMOR CELLS IN SMALL CELL LUNG CANCER Article Open access 03 March 2023 DATA AVAILABILITY Most data generated or analyzed during this study are included in

this published article. The sequencing data are available through the Cancer Research UK & University College London Cancer Trials Centre for non-commercial research purposes. Access

will be granted upon review of a project proposal that will be evaluated by a TRACERx data access committee, and an appropriate data access agreement will be entered into, subject to any

applicable ethical approvals. CHANGE HISTORY * _ 03 JUNE 2020 An amendment to this paper has been published and can be accessed via a link at the top of the paper. _ REFERENCES * Uramoto, H.

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Download references ACKNOWLEDGEMENTS We sincerely thank the patients and their families for donating blood samples for research. We thank E. Aidaros-Talbot for administrative assistance with

the manuscript. We also thank J. Shaw for kindly providing plasma (relapse time point) of patient CRUK0242. TRACERx is funded by Cancer Research UK (grant C11496/A17786). This research was

supported by Cancer Research UK–Core funding to the CRUK Manchester Institute (C5759/A27412) Centre, funding to the CRUK Manchester Centre (C5759/A25254) and funding of the CRUK Lung Cancer

Centre of Excellence. Support was also received from the Manchester Experimental Cancer Medicine Centre and Manchester NIHR Biomedical Research Centre. F.C. is funded by the CANCER-ID

Consortium (115749-Cancer-ID). B.M. is funded by Menarini Biomarkers Singapore PTE Ltd. C.S.K. is funded by The Manchester MRC Single Cell Research Centre (MR/M008908/1). C.S. is Royal

Society Napier Research Professor. This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001169, FC001202), the UK Medical

Research Council (FC001169, FC001202) and the Wellcome Trust (FC001169, FC001202). C.S. is funded by Cancer Research UK (TRACERx and CRUK Cancer Immunotherapy Catalyst Network), the CRUK

Lung Cancer Centre of Excellence, Stand Up 2 Cancer (SU2C), the Rosetrees Trust, the Butterfield and Stoneygate Trusts, NovoNordisk Foundation (ID16584), the Prostate Cancer Foundation and

the Breast Cancer Research Foundation (BCRF). The research leading to these results has received funding from the European Research Council (ERC) under the European Union’s Seventh Framework

Programme (FP7/2007-2013) Consolidator Grant (FP7-THESEUS-617844), European Commission ITN (FP7-PloidyNet 607722), an ERC Advanced Grant (PROTEUS) from the European Research Council under

the European Union’s Horizon 2020 research and innovation programme (grant agreement 835297). Support was also provided to C.S. by the National Institute for Health Research, the University

College London Hospitals Biomedical Research Centre and the Cancer Research UK University College London Experimental Cancer Medicine Centre. AUTHOR INFORMATION Author notes * These authors

contributed equally: Francesca Chemi, Dominic G. Rothwell, Nicholas McGranahan. * A list of members and affiliations appears online. AUTHORS AND AFFILIATIONS * Clinical and Experimental

Pharmacology Group, Cancer Research UK Manchester Institute, The University of Manchester, Alderley Park, UK Francesca Chemi, Dominic G. Rothwell, Sakshi Gulati, Simon P. Pearce, Cong Zhou, 

Jackie Pierce, Chang Sik Kim, Saba Ferdous, Deborah J. Burt, Daniel Slane-Tan, Barbara Mesquita, Jonathan Tugwood, Ged Brady, Caroline Dive, Jonathan Tugwood, Jackie Pierce, Dominic G.

Rothwell, Caroline Dive, Ged Brady & Francesca Chemi * Cancer Research UK Lung Cancer Centre of Excellence, The University of Manchester, Manchester, UK Francesca Chemi, Fabio Gomes, 

Philip Crosbie, Fiona Blackhall, Ged Brady, Caroline Dive, Caroline Dive, Ged Brady, Francesca Chemi, Fabio Gomes, Fiona Blackhall, Philip Crosbie, Elaine Kilgour, Lynsey Priest & 

Matthew G. Krebs * Cancer Research UK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK Nicholas McGranahan, Mariam Jamal-Hanjani, Crispin Hiley, 

Charles Swanton, Charles Swanton, Mariam Jamal-Hanjani, Crispin Hiley & Nicholas McGranahan * University College London Cancer Institute, London, UK Nicholas McGranahan, Chris Abbosh, 

Gareth A. Wilson, Mariam Jamal-Hanjani, Nicolai Birkbak, David Moore, Crispin Hiley, Selvaraju Veeriah, Allan Hackshaw, Charles Swanton, Charles Swanton, Mariam Jamal-Hanjani, Crispin Hiley,

 Nicholas McGranahan, Allan Hackshaw, Chris Abbosh, Selvaraju Veeriah, David Moore, Gareth A. Wilson, Nicolai Birkbak, Yin Wu, Marcin Skrzypski, Robert E. Hynds, Andrew Georgiou, Mariana

Werner Sunderland, James L. Reading, Sergio A. Quezada, Karl S. Peggs, Teresa Marafioti, John A. Hartley, Pat Gorman, Helen L. Lowe, Leah Ensell, Victoria Spanswick, Angeliki Karamani, 

Maryam Razaq, Stephan Beck, Ariana Huebner, Michelle Dietzen, Cristina Naceur-Lombardelli, Mita Afroza Akther, Haoran Zhai, Nnennaya Kannu, Elizabeth Manzano, Supreet Kaur Bola, Ehsan

Ghorani, Marc Robert de Massy, Elena Hoxha, Emine Hatipoglu, Stephanie Ogwuru & Benny Chain * Cancer Evolution and Genome Instability Laboratory, The Francis Crick Institute, London, UK

Nicholas McGranahan, Gareth A. Wilson, Nicolai Birkbak, Maise Al Bakir, Sophia Ward, Dhruva Biswas, Charles Swanton, Charles Swanton, Nicholas McGranahan, Gareth A. Wilson, Nicolai Birkbak, 

Sophia Ward, Maise Al Bakir & Dhruva Biswas * Department of Molecular Medicine, Aarhus University, Aarhus, Denmark Nicolai Birkbak & Nicolai Birkbak * Department of Thoracic Surgery,

Wythenshawe Hospital, Manchester University NHS Foundation Trust, Manchester, UK Rajesh Shah & Rajesh Shah * Division of Cancer Sciences, Faculty of Biology Medicine and Health,

University of Manchester, Manchester, UK Yvonne Summers, Philip Crosbie, Fiona Blackhall, Fiona Blackhall, Philip Crosbie & Yvonne Summers * North West Lung Centre, Wythenshawe Hospital,

Manchester University NHS Foundation Trust, Manchester, UK Philip Crosbie & Philip Crosbie * Scientific Computing Core Facility, Cancer Research UK Manchester Institute, The University

of Manchester, Alderley Park, UK Marek Dynowski * RNA Biology, Cancer Research UK Manchester Institute, The University of Manchester, Alderley Park, UK Crispin Miller * Cancer Research UK

& UCL Cancer Trials Centre, London, UK Yenting Ngai, Abigail Sharp, Cristina Rodrigues, Oliver Pressey, Sean Smith, Nicole Gower & Harjot Dhanda * Cancer Research UK Manchester

Institute, University of Manchester, Manchester, UK Elaine Kilgour * Christie NHS Foundation Trust, Manchester, UK Lynsey Priest, Matthew G. Krebs, Mathew Carter & Colin R. Lindsay * The

Francis Crick Institute, London, UK Thomas B. K. Watkins, Rachel Rosenthal, Mickael Escudero, Aengus Stewart, Andrew Rowan, Jacki Goldman, Peter Van Loo, Richard Kevin Stone, Tamara Denner,

 Emma Nye, Emilia Lim, Stefan Boeing, Maria Greco, Kevin Litchfield, Jerome Nicod, Clare Puttick, Katey Enfield, Emma Colliver & Brittany Campbell * Wythenshawe Hospital, Manchester

University NHS Foundation Trust, Manchester, UK Raffaele Califano, Paul Taylor, Piotr Krysiak, Kendadai Rammohan, Eustace Fontaine, Richard Booton, Matthew Evison, Stuart Moss, Juliette

Novasio, Leena Joseph, Paul Bishop, Anshuman Chaturvedi, Helen Doran, Felice Granato, Vijay Joshi, Elaine Smith & Angeles Montero * Aberdeen Royal Infirmary, Aberdeen, UK Gillian Price, 

Sylvie Dubois-Marshall, Keith Kerr, Shirley Palmer, Heather Cheyne, Joy Miller, Keith Buchan, Mahendran Chetty & Mohammed Khalil * Ashford and St Peter’s Hospitals NHS Foundation Trust,

Chertsey, UK Veni Ezhil & Vineet Prakash * Barnet Hospital and Chase Farm Hospital, London, UK Girija Anand & Sajid Khan * Barts Health NHS Trust, London, UK Kelvin Lau, Michael

Sheaff, Peter Schmid, Louise Lim & John Conibear * Berlin Institute for Medical Systems Biology, Max Delbrueck Center for Molecular Medicine, Berlin, Germany Roland Schwarz * German

Cancer Consortium (DKTK), partner site Berlin, Berlin, Germany Roland Schwarz * German Cancer Research Center (DKFZ), Heidelberg, Germany Roland Schwarz * Cancer Research Centre, University

of Leicester, Leicester, UK John Le Quesne, Joan Riley, Lindsay Primrose, Luke Martinson, Nicolas Carey, Jacqui A. Shaw & Dean Fennell * Leicester University Hospitals, Leicester, UK

Dean Fennell, Apostolos Nakas, Sridhar Rathinam, Louise Nelson, Kim Ryanna, Mohamad Tuffail, Amrita Bajaj & Jan Brozik * Cardiff & Vale University Health Board, Cardiff, UK Fiona

Morgan, Malgorzata Kornaszewska, Richard Attanoos, Haydn Adams & Helen Davies * Danish Cancer Society Research Center, Copenhagen, Denmark Zoltan Szallasi * Department of Pathology,

GZA-ZNA Antwerp, Antwerp, Belgium Roberto Salgado * Department of Physics of Complex Systems, ELTE Eötvös Loránd University, Budapest, Hungary Istvan Csabai & Miklos Diossy * Departments

of Radiation Oncology and Radiology, Dana-Farber Cancer Institute, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA Hugo Aerts * Department of Radiology, Netherlands

Cancer Institute, Amsterdam, The Netherlands Hugo Aerts * Golden Jubilee National Hospital, Clydebank, UK Alan Kirk, Mo Asif, John Butler, Rocco Bilanca & Nikos Kostoulas * Independent

Cancer Patients’ Voice, London, UK Mairead MacKenzie & Maggie Wilcox * University of Leicester, Leicester, UK Sara Busacca, Alan Dawson & Mark R. Lovett * Liverpool Heart and Chest

Hospital NHS Foundation Trust, Liverpool, UK Michael Shackcloth, Sarah Feeney & Julius Asante-Siaw * Royal Liverpool University Hospital, Liverpool, UK John Gosney * Manchester Cancer

Research Centre Biobank, Manchester, UK Angela Leek, Nicola Totten, Jack Davies Hodgkinson, Rachael Waddington, Jane Rogan & Katrina Moore * National Institute for Health Research

Leicester Respiratory Biomedical Research Unit, Leicester, UK William Monteiro & Hilary Marshall * NHS Greater Glasgow and Clyde, Glasgow, UK Kevin G. Blyth, Craig Dick & Andrew Kidd

* Royal Brompton and Harefield NHS Foundation Trust, London, UK Eric Lim, Paulo De Sousa, Simon Jordan, Alexandra Rice, Hilgardt Raubenheimer, Harshil Bhayani, Morag Hamilton, Lyn Ambrose, 

Anand Devaraj, Hema Chavan, Sofina Begum, Aleksander Mani, Daniel Kaniu, Mpho Malima, Sarah Booth, Andrew G. Nicholson, Nadia Fernandes, Jessica E. Wallen & Pratibha Shah * Sheffield

Teaching Hospitals NHS Foundation Trust, Sheffield, UK Sarah Danson, Jonathan Bury, John Edwards, Jennifer Hill, Sue Matthews, Yota Kitsanta, Jagan Rao, Sara Tenconi, Laura Socci, Kim

Suvarna, Faith Kibutu, Patricia Fisher, Robin Young, Joann Barker, Fiona Taylor & Kirsty Lloyd * The Princess Alexandra Hospital NHS Trust, Harlow, UK Teresa Light, Tracey Horey, 

Dionysis Papadatos-Pastos & Peter Russell * The Whittington Hospital NHS Trust, London, UK Sara Lock & Kayleigh Gilbert * University College London Hospitals, London, UK David

Lawrence, Martin Hayward, Nikolaos Panagiotopoulos, Robert George, Davide Patrini, Mary Falzon, Elaine Borg, Reena Khiroya, Asia Ahmed, Magali Taylor, Junaid Choudhary, Penny Shaw, Sam M.

Janes, Martin Forster, Tanya Ahmad, Siow Ming Lee, Javier Herrero, Dawn Carnell, Ruheena Mendes, Jeremy George, Neal Navani, Dionysis Papadatos-Pastos, Marco Scarci, Elisa Bertoja, Robert C.

M. Stephens, Emilie Martinoni Hoogenboom, James W. Holding & Steve Bandula * University Hospital Birmingham NHS Foundation Trust, Birmingham, UK Babu Naidu, Gerald Langman, Andrew

Robinson, Hollie Bancroft, Amy Kerr, Salma Kadiri, Charlotte Ferris, Gary Middleton, Madava Djearaman & Akshay Patel * University Hospital Southampton NHS Foundation Trust, Southampton,

UK Christian Ottensmeier, Serena Chee, Benjamin Johnson, Aiman Alzetani & Emily Shaw * Velindre Cancer Centre, Cardiff, UK Jason Lester Authors * Francesca Chemi View author publications

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Katrina Moore * , William Monteiro * , Hilary Marshall * , Kevin G. Blyth * , Craig Dick * , Andrew Kidd * , Eric Lim * , Paulo De Sousa * , Simon Jordan * , Alexandra Rice * , Hilgardt

Raubenheimer * , Harshil Bhayani * , Morag Hamilton * , Lyn Ambrose * , Anand Devaraj * , Hema Chavan * , Sofina Begum * , Aleksander Mani * , Daniel Kaniu * , Mpho Malima * , Sarah Booth *

, Andrew G. Nicholson * , Nadia Fernandes * , Jessica E. Wallen * , Pratibha Shah * , Sarah Danson * , Jonathan Bury * , John Edwards * , Jennifer Hill * , Sue Matthews * , Yota Kitsanta * ,

 Jagan Rao * , Sara Tenconi * , Laura Socci * , Kim Suvarna * , Faith Kibutu * , Patricia Fisher * , Robin Young * , Joann Barker * , Fiona Taylor * , Kirsty Lloyd * , Teresa Light * , 

Tracey Horey * , Dionysis Papadatos-Pastos * , Peter Russell * , Sara Lock * , Kayleigh Gilbert * , David Lawrence * , Martin Hayward * , Nikolaos Panagiotopoulos * , Robert George * , 

Davide Patrini * , Mary Falzon * , Elaine Borg * , Reena Khiroya * , Asia Ahmed * , Magali Taylor * , Junaid Choudhary * , Penny Shaw * , Sam M. Janes * , Martin Forster * , Tanya Ahmad * , 

Siow Ming Lee * , Javier Herrero * , Dawn Carnell * , Ruheena Mendes * , Jeremy George * , Neal Navani * , Dionysis Papadatos-Pastos * , Marco Scarci * , Elisa Bertoja * , Robert C. M.

Stephens * , Emilie Martinoni Hoogenboom * , James W. Holding * , Steve Bandula * , Babu Naidu * , Gerald Langman * , Andrew Robinson * , Hollie Bancroft * , Amy Kerr * , Salma Kadiri * , 

Charlotte Ferris * , Gary Middleton * , Madava Djearaman * , Akshay Patel * , Christian Ottensmeier * , Serena Chee * , Benjamin Johnson * , Aiman Alzetani * , Emily Shaw *  & Jason

Lester CONTRIBUTIONS C.S., C.D., P.C., D.G.R. and G.B. developed the clinical study, directed research and co-wrote the manuscript. F.C. designed and conducted experiments, analyzed data and

drafted the manuscript with the assistance of D.G.R. and N.M.G. S.G., S.P.P., G.W., N.B., N.M.G., C.S.K., S.F., C.M. and M.D. provided bioinformatic support for the study. C.A. provided

support for the clinical interpretation of the data. C.Z. performed statistical analysis. C.A. and D.M. performed central pathology review. D.B., D.S.T. and B.M. provided support for

single-cell isolation. M.J.-H., J.P., F.G., R.S., M.A.B., C.H., S.V., Y.S., P.C., S.W., D.B., J.T., F.B. and A.H. supported patient recruitment and sample management and provided clinical

support for the study. CORRESPONDING AUTHORS Correspondence to Ged Brady, Charles Swanton or Caroline Dive. ETHICS DECLARATIONS COMPETING INTERESTS C.D. receives research grants/support from

AstraZeneca, Astex Pharmaceuticals, Bioven, Amgen, Carrick Therapeutics, Merck, Taiho Oncology, GSK, Bayer, Boehringer Ingelheim, Roche, BMS, Novartis, Celgene and Epigene Therapeutics,

Menarini, Angle PLC and Clearbridge Biomedics, all of which are outside the scope of this paper. C.D. has received honoraria/consultancy fees from Biocartis, Merck and AstraZeneca and

Illumina, again outside the scope of this work. C.S. receives grant support from Pfizer, AstraZeneca, BMS and Roche-Ventana, Boehringer-Ingelheim. C.S. has consulted for Pfizer, Novartis,

GlaxoSmithKline, MSD, BMS, Celgene, AstraZeneca, Illumina, Genentech, Roche-Ventana, GRAIL, Medicxi and the Sarah Cannon Research Institute and is an adviser for Dynamo Therapeutics. C.S. is

a shareholder of Apogen Biotechnologies, Epic Bioscience and GRAIL, and has stock options in and is cofounder of Achilles Therapeutics. ADDITIONAL INFORMATION PEER REVIEW INFORMATION Joao

Monteiro was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. PUBLISHER’S NOTE Springer Nature

remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 PV-CTCS ARE ASSOCIATED WITH LUNG-CANCER-SPECIFIC

RELAPSE. A, tdROC curves showing true-positive and false-positive rates for the 65th, 75th and 85th PV-CTC quantiles (≥3, ≥7, and ≥39 PV-CTCs per 7.5 ml blood, respectively) alongside the

previously published threshold from our pilot study (≥18 PV-CTCs per 7.5 ml blood)11. All predictions were made at 720 d. Sensitivity and specificity of each category are shown along with

area under ROC value. B, Kaplan–Meier curve showing lung-cancer-specific relapse-free survival for 98 patients stratified as PV-CTC high or low according to the 75th quantile (≥7 PV-CTCs per

7.5 ml blood). The number of patients at risk for each time point is indicated below the time point and color coded according to the high or low group. _P_ values, HRs and relative 95% CIs

(two-sided log-rank test) are indicated. C, Forest plot showing the results of multivariate regression analysis for patients with PV-CTC high or low status (≥7 PV-CTCs per 7.5 ml blood). The

_x_-axis represents the HR with the reference line (dashed), and significance was calculated using a Cox proportional hazards model. The estimated HRs and their 95% CIs are presented as

error bars. The log-rank test used was two sided. EXTENDED DATA FIG. 2 TECHNICAL DETAILS OF SINGLE-CELL ANALYSIS OF PATIENT CRUK0242. A, Consort diagram describing samples used for

downstream analysis. Only patients with ≥5 PV-CTCs (_n_ = 29) were processed through single-cell isolation (DEPArray). Single cells were not isolated from 6 of the 29 samples owing to

failures during sample loading into the DEPArray machine. From the remaining 23 samples, 7 patients whose single CTCs isolated did not meet morphology criteria (Methods) were excluded.

Sixteen samples were processed for WGA, and two patients whose CTCs did not show good quality GII in quality control after WGA were removed (Methods). B, Table showing cases of relapse in

the patients with single PV-CTCs isolated. C, Agarose gel showing results of a quality-control PCR assay used to determine the genome integrity of each sample (_n_ = 1). 0–4 bands determine

the overall DNA integrity of each sample. DEPArray images of corresponding PV-CTC (green, cytokeratin; blue, CD45; purple, DAPI) are shown above. D, Examples of copy number profiles detected

in single PV-CTCs, CECs, and WBC controls. Blue and red indicate regions of copy number loss and gain, respectively. EXTENDED DATA FIG. 3 GENETIC RELATIONSHIP BETWEEN PV-CTCS, PRIMARY TUMOR

AND METASTATIC DISEASE. A, Venn diagram showing the overlap of somatic mutations detected among single PV-CTCs, primary tumor and metastatic tumor. B, Venn diagram showing the overlap of

somatic mutations detected among single PV-CTCs, metastatic tumor and cfDNA isolated at the time of relapse. EXTENDED DATA FIG. 4 SUMMARY OF ALL MUTATIONS DETECTED IN PATIENT CRUK0242. Heat

map showing the comparison of SNVs detected in primary tumor regions, metastasis, PV-CTCs, CECs, WBCs and cfDNA samples (cfDNA before surgery was isolated from peripheral blood; cfDNA

surgery was isolated from the pulmonary vein; and cfDNA relapse was isolated at the time of relapse). Mutations are ordered according to their clonality established by primary tumor

analysis. SUPPLEMENTARY INFORMATION REPORTING SUMMARY SUPPLEMENTARY TABLES 1–12 Supplementary Tables 1-12 RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE

Chemi, F., Rothwell, D.G., McGranahan, N. _et al._ Pulmonary venous circulating tumor cell dissemination before tumor resection and disease relapse. _Nat Med_ 25, 1534–1539 (2019).

https://doi.org/10.1038/s41591-019-0593-1 Download citation * Received: 09 July 2019 * Accepted: 20 August 2019 * Published: 07 October 2019 * Issue Date: October 2019 * DOI:

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