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Evolutionary characterization of lung adenocarcinoma morphology in TRACERx

Nature Medicine volume 29, pages 833–845 (2023)Cite this article

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Abstract

Lung adenocarcinomas (LUADs) display a broad histological spectrum from low-grade lepidic tumors through to mid-grade acinar and papillary and high-grade solid, cribriform and micropapillary tumors. How morphology reflects tumor evolution and disease progression is poorly understood. Whole-exome sequencing data generated from 805 primary tumor regions and 121 paired metastatic samples across 248 LUADs from the TRACERx 421 cohort, together with RNA-sequencing data from 463 primary tumor regions, were integrated with detailed whole-tumor and regional histopathological analysis. Tumors with predominantly high-grade patterns showed increased chromosomal complexity, with higher burden of loss of heterozygosity and subclonal somatic copy number alterations. Individual regions in predominantly high-grade pattern tumors exhibited higher proliferation and lower clonal diversity, potentially reflecting large recent subclonal expansions. Co-occurrence of truncal loss of chromosomes 3p and 3q was enriched in predominantly low-/mid-grade tumors, while purely undifferentiated solid-pattern tumors had a higher frequency of truncal arm or focal 3q gains and SMARCA4 gene alterations compared with mixed-pattern tumors with a solid component, suggesting distinct evolutionary trajectories. Clonal evolution analysis revealed that tumors tend to evolve toward higher-grade patterns. The presence of micropapillary pattern and ‘tumor spread through air spaces’ were associated with intrathoracic recurrence, in contrast to the presence of solid/cribriform patterns, necrosis and preoperative circulating tumor DNA detection, which were associated with extra-thoracic recurrence. These data provide insights into the relationship between LUAD morphology, the underlying evolutionary genomic landscape, and clinical and anatomical relapse risk.

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Fig. 1: Clonal evolutionary characteristics of LUAD growth patterns.
Fig. 2: Genomic determinants of predominant growth pattern.
Fig. 3: Morphological intra-tumoral heterogeneity reflects genomic heterogeneity.
Fig. 4: Growth pattern evolution from primary tumor to metastasis.
Fig. 5: Impact of tumor morphology upon site and risk of recurrence.

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Data availability

The WES data and RNA-seq data (in each case from the TRACERx study) used during this study have been deposited at the European Genome–phenome Archive (EGA), which is hosted by the European Bioinformatics Institute (EBI) and the Centre for Genomic Regulation (CRG) under the accession codes EGAS00001006494 (WES) and EGAS00001006517 (RNA-seq); access is controlled by the TRACERx data access committee and details on how to apply for access are available at the linked page. Hallmark gene sets58 were downloaded from Molecular Signatures Database (https://www.gsea-msigdb.org/gsea/msigdb/).

Code availability

Code used to process data and generate figures is available at https://doi.org/10.5281/zenodo.7683605.

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Acknowledgements

The TRACERx study (NCT01888601) is sponsored by University College London (UCL/12/0279) and has been approved by an independent Research Ethics Committee (13/LO/1546). TRACERx is funded by Cancer Research UK (CRUK; C11496/A17786) and coordinated through the CRUK & UCL Cancer Trials Centre, which has a core grant from CRUK (C444/A15953). We gratefully acknowledge the participants and relatives who participated in the TRACERx study. We thank all site personnel, investigators, funders and industry partners that supported the generation of the data within this study. In particular, we acknowledge the support of Scientific Computing, the Advanced Sequencing Facility and Experimental Histopathology departments at the Francis Crick Institute. This work was also supported by the CRUK Lung Cancer Centre of Excellence and the CRUK City of London Centre Award (C7893/A26233) and the UCL Experimental Cancer Medicine Centre. BioRender aided in the generation of Figs. 3a and 4c and Extended Data Figs. 1k and 6a. T.K. is supported by the JSPS Overseas Research Fellowships Program (202060447). D.A.M. is supported by the CRUK Lung Cancer Centre of Excellence (C11496/A30025). M.A.B. is supported by CRUK, the Rosetrees Trust and the Francis Crick Institute. T.B.K.W. is supported by the Francis Crick Institute, as well as the Marie Curie ITN Project PLOIDYNET (FP7-PEOPLE-2013, 607722), Breast Cancer Research Foundation, Royal Society Research Professorships Enhancement Award (RP/EA/180007) and the Foulkes Foundation. A. Huebner is supported by CRUK. A.M.F. is supported by Stand Up To Cancer (SU2C-AACR-DT23-17). H.Z. is supported by the China Scholarship Council for a 4-year PhD study and the Breast Cancer Research Foundation. F.G.-V. is supported by Generalitat Valenciana fellowships program (APOSTD/2021/168). M.D. is supported by CRUK and the Lung Cancer Centre of Excellence. C.M.-R. is supported by the Rosetrees Trust (M630) and Wellcome Trust. E.C. is supported by CRUK (TRACERx (C11496/A17786)) and the Francis Crick Institute. K.S.S.E. was supported by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement no. 838540 and the Royal Society (RF\ERE\210216). S.H. is supported by CRUK and the Rosetrees Trust. C.T.H. has received funding from NIHR University College London Hospitals Biomedical Research Centre. S.Z. is a CRUK Career Development Fellow (award ref. RCCCDF-Nov21\100005) and is supported by Rosetrees Trust (grant ref. M917) and CRUK UCL Centre Non-Clinical Training Award (CANTAC721\100022). K.L. is funded by the UK Medical Research Council (MR/P014712/1 and MR/V033077/1), Rosetrees Trust and Cotswold Trust (A2437) and CRUK (C69256/A30194). N.J.B. is a fellow of the Lundbeck Foundation (R272-2017-4040) and acknowledges funding from Aarhus University Research Foundation (AUFF-E-2018-7-14) and the Novo Nordisk Foundation (NNF21OC0071483).E.L.C., J.D. and P.V.L. are supported by the Francis Crick Institute, which receives its core funding from CRUK (CC2008), the UK Medical Research Council (CC2008) and the Wellcome Trust (CC2008). P.V.L. is a Winton Group Leader in recognition of the Winton Charitable Foundation’s support toward the establishment of the Francis Crick Institute. P.V.L. is a CPRIT Scholar in Cancer Research and acknowledges CPRIT grant support (RR210006). P.S.A.’s laboratory work is supported by grants from the National Institutes of Health (P30 CA008748, R01 CA236615, R01 CA235667 and U01 CA214195), the US Department of Defense (CA180889 and CA200437), the DallePezze Foundation and the Derfner Foundation. P.S.A.’s laboratory receives research support from ATARA Biotherapeutics. D.R.J. is supported by National Institutes of Health grants R01CA217169, R01CA240472 and P30 CA008748 and the Hamilton Family Foundation. T.M. is supported by the UK National Institute of Health Research University College London Hospital Biomedical Research Centre. N.M. is a Sir Henry Dale Fellow, jointly funded by the Wellcome Trust and the Royal Society (grant no. 211179/Z/18/Z) and also receives funding from CRUK, Rosetrees and the National Institute for Health Research (NIHR) Biomedical Research Centre at University College London Hospitals and the CRUK University College London Experimental Cancer Medicine Centre. M.J.-H. is a CRUK Career Establishment Awardee and has received funding from CRUK, IASLC International Lung Cancer Foundation, Lung Cancer Research Foundation, Rosetrees Trust, UKI NETs and NIHR University College London Hospitals Biomedical Research Centre. C.S. is a Royal Society Napier Research Professor (RSRP\R\210001). C.S. is supported by the Francis Crick Institute that receives its core funding from CRUK (CC2041), the UK Medical Research Council (CC2041), and the Wellcome Trust (CC2041). For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. C.S. is funded by CRUK (TRACERx (C11496/A17786), PEACE (C416/A21999) and CRUK Cancer Immunotherapy Catalyst Network); CRUK Lung Cancer Centre of Excellence (C11496/A30025); the Rosetrees Trust, Butterfield and Stoneygate Trusts; NovoNordisk Foundation (ID16584); Royal Society Professorship Enhancement Award (RP/EA/180007); NIHR University College London Hospitals Biomedical Research Centre; the CRUK University College London Centre; Experimental Cancer Medicine Centre; the Breast Cancer Research Foundation (US); and The Mark Foundation for Cancer Research Aspire Award (grant 21-029-ASP). This work was supported by a Stand Up To Cancer‐LUNGevity-American Lung Association Lung Cancer Interception Dream Team Translational Research Grant (grant no. SU2C-AACR-DT23-17 to S. M. Dubinett and A. E. Spira). Stand Up To Cancer is a division of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the Scientific Partner of SU2C. C.S. is in receipt of an ERC Advanced Grant (PROTEUS) from the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 835297).

Author information

Author notes

  1. These authors contributed equally: Takahiro Karasaki, David A Moore.

  2. These authors jointly supervised this work: Nicholas McGranahan, Charles Swanton, Mariam Jamal-Hanjani.

Authors and Affiliations

  1. Cancer Research UK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK

    Takahiro Karasaki, David A. Moore, Selvaraju Veeriah, Cristina Naceur-Lombardelli, Antonia Toncheva, Neil Magno, Sophia Ward, Maise Al Bakir, Kristiana Grigoriadis, Ariana Huebner, Alexander M. Frankell, Christopher Abbosh, Clare Puttick, Haoran Zhai, Francisco Gimeno-Valiente, Sadegh Saghafinia, Nnennaya Kanu, Michelle Dietzen, Emilia L. Lim, Carlos Martínez-Ruiz, James R. M. Black, Dhruva Biswas, Sonya Hessey, Crispin T. Hiley, Simone Zaccaria, Kevin Litchfield, Nicolai J. Birkbak, Abigail Bunkum, Corentin Richard, Foteini Athanasopoulou, Jie Min Lam, Kerstin Thol, Krupa Thakkar, Mariana Werner Sunderland, Michelle Leung, Monica Sivakumar, Olivia Lucas, Othman Al-Sawaf, Paulina Prymas, Robert Bentham, Sergio A. Quezada, Sharon Vanloo, Wing Kin Liu, Martin D. Forster, Siow Ming Lee, Nicholas McGranahan, Charles Swanton & Mariam Jamal-Hanjani

  2. Cancer Evolution and Genome Instability Laboratory, The Francis Crick Institute, London, UK

    Takahiro Karasaki, David A. Moore, Sophia Ward, Maise Al Bakir, Thomas B. K. Watkins, Kristiana Grigoriadis, Ariana Huebner, Mark S. Hill, Alexander M. Frankell, Clare Puttick, Haoran Zhai, Michelle Dietzen, Oriol Pich, Emilia L. Lim, Dhruva Biswas, Brittany B. Campbell, Claudia Lee, Emma Colliver, Katey S. S. Enfield, Crispin T. Hiley, Nicolai J. Birkbak, Gareth A. Wilson, Rachel Rosenthal, Andrew Rowan, Chris Bailey, Foteini Athanasopoulou, Jayant K. Rane, Michelle Leung, Mihaela Angelova, Olivia Lucas, Othman Al-Sawaf & Charles Swanton

  3. Cancer Metastasis Laboratory, University College London Cancer Institute, London, UK

    Takahiro Karasaki, Sonya Hessey, Abigail Bunkum, Jie Min Lam, Othman Al-Sawaf, Wing Kin Liu & Mariam Jamal-Hanjani

  4. Department of Cellular Pathology, University College London Hospitals, London, UK

    David A. Moore, Teresa Marafioti, Elaine Borg, Mary Falzon & Reena Khiroya

  5. Advanced Sequencing Facility, The Francis Crick Institute, London, UK

    Sophia Ward, Foteini Athanasopoulou & Jerome Nicod

  6. Cancer Genome Evolution Research Group, Cancer Research UK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK

    Kristiana Grigoriadis, Ariana Huebner, Clare Puttick, Michelle Dietzen, Carlos Martínez-Ruiz, James R. M. Black, Kerstin Thol, Michelle Leung, Robert Bentham & Nicholas McGranahan

  7. Bill Lyons Informatics Centre, University College London Cancer Institute, London, UK

    Dhruva Biswas & Javier Herrero

  8. Computational Cancer Genomics Research Group, University College London Cancer Institute, London, UK

    Sonya Hessey, Simone Zaccaria, Abigail Bunkum & Olivia Lucas

  9. Tumour Immunogenomics and Immunosurveillance Laboratory, University College London Cancer Institute, London, UK

    Kevin Litchfield

  10. Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark

    Nicolai J. Birkbak, Judit Kisistok & Mateo Sokac

  11. Department of Clinical Medicine, Aarhus University, Aarhus, Denmark

    Nicolai J. Birkbak, Judit Kisistok & Mateo Sokac

  12. Bioinformatics Research Centre, Aarhus University, Aarhus, Denmark

    Nicolai J. Birkbak, Judit Kisistok & Mateo Sokac

  13. Cancer Genomics Laboratory, The Francis Crick Institute, London, UK

    Elizabeth Larose Cadieux, Jonas Demeulemeester, Peter Van Loo, Peter Van Loo & Carla Castignani

  14. Medical Genomics, University College London Cancer Institute, London, UK

    Elizabeth Larose Cadieux, Carla Castignani, Miljana Tanić & Stephan Beck

  15. Integrative Cancer Genomics Laboratory, Department of Oncology, KU Leuven, Leuven, Belgium

    Jonas Demeulemeester

  16. VIB – KU Leuven Center for Cancer Biology, Leuven, Belgium

    Jonas Demeulemeester

  17. Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Peter Van Loo & Peter Van Loo

  18. Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Peter Van Loo & Peter Van Loo

  19. Thoracic Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Prasad S. Adusumilli, Waseem Cheema & David R. Jones

  20. Center for Cell Engineering, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Prasad S. Adusumilli

  21. Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Kay See Tan & Francisco Sanchez-Vega

  22. Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Natasha Rekhtman & William D. Travis

  23. Cancer Research UK & UCL Cancer Trials Centre, London, UK

    Allan Hackshaw, Abigail Sharp, Sean Smith, Nicole Gower, Harjot Kaur Dhanda, Kitty Chan, Camilla Pilotti & Rachel Leslie

  24. Department of Pathology, ZAS Hospitals, Antwerp, Belgium

    Roberto Salgado

  25. Division of Research, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia

    Roberto Salgado

  26. Cancer Research UK Beatson Institute, Glasgow, UK

    John Le Quesne, John Le Quesne & Kevin G. Blyth

  27. School of Cancer Sciences, University of Glasgow, Glasgow, UK

    John Le Quesne, John Le Quesne & Kevin G. Blyth

  28. Pathology Department, Queen Elizabeth University Hospital, NHS Greater Glasgow and Clyde, Glasgow, UK

    John Le Quesne & John Le Quesne

  29. Department of Histopathology, Royal Brompton and Harefield Hospitals, Guy’s and St Thomas’ NHS Foundation Trust, London, UK

    Andrew G. Nicholson

  30. National Heart and Lung Institute, Imperial College London, London, UK

    Andrew G. Nicholson

  31. Department of Oncology, University College London Hospitals, London, UK

    Sarah Benafif, Jie Min Lam, Olivia Lucas, Martin D. Forster, Siow Ming Lee, Dionysis Papadatos-Pastos, James Wilson, Tanya Ahmad, Charles Swanton & Mariam Jamal-Hanjani

  32. Singleton Hospital, Swansea Bay University Health Board, Swansea, UK

    Jason F. Lester

  33. University Hospitals of Leicester NHS Trust, Leicester, UK

    Amrita Bajaj, Apostolos Nakas, Azmina Sodha-Ramdeen, Keng Ang, Mohamad Tufail, Mohammed Fiyaz Chowdhry, Molly Scotland, Rebecca Boyles, Sridhar Rathinam & Dean A. Fennell

  34. University of Leicester, Leicester, UK

    Claire Wilson, Domenic Marrone, Sean Dulloo & Dean A. Fennell

  35. Cancer Research Centre, University of Leicester, Leicester, UK

    Gurdeep Matharu, Jacqui A. Shaw, Joan Riley & Lindsay Primrose

  36. Royal Free Hospital, Royal Free London NHS Foundation Trust, London, UK

    Ekaterini Boleti

  37. Aberdeen Royal Infirmary NHS Grampian, Aberdeen, UK

    Heather Cheyne, Mohammed Khalil, Shirley Richardson & Tracey Cruickshank

  38. Department of Medical Oncology, Aberdeen Royal Infirmary NHS Grampian, Aberdeen, UK

    Gillian Price

  39. University of Aberdeen, Aberdeen, UK

    Gillian Price & Keith M. Kerr

  40. Department of Pathology, Aberdeen Royal Infirmary NHS Grampian, Aberdeen, UK

    Keith M. Kerr

  41. The Whittington Hospital NHS Trust, London, UK

    Kayleigh Gilbert

  42. Birmingham Acute Care Research Group, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, UK

    Babu Naidu

  43. University Hospital Birmingham NHS Foundation Trust, Birmingham, UK

    Akshay J. Patel, Aya Osman, Christer Lacson, Gerald Langman, Helen Shackleford, Madava Djearaman, Salma Kadiri & Gary Middleton

  44. Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, UK

    Gary Middleton

  45. Manchester Cancer Research Centre Biobank, Manchester, UK

    Angela Leek, Jack Davies Hodgkinson & Nicola Totten

  46. Wythenshawe Hospital, Manchester University NHS Foundation Trust, Wythenshawe, UK

    Angeles Montero, Elaine Smith, Eustace Fontaine, Felice Granato, Helen Doran, Juliette Novasio, Kendadai Rammohan, Leena Joseph, Paul Bishop, Rajesh Shah, Stuart Moss, Vijay Joshi & Philip Crosbie

  47. Division of Infection, Immunity and Respiratory Medicine, University of Manchester, Manchester, UK

    Philip Crosbie

  48. Cancer Research UK Lung Cancer Centre of Excellence, University of Manchester, Manchester, UK

    Philip Crosbie, Anshuman Chaturvedi, Lynsey Priest, Pedro Oliveira, Alexandra Clipson, Jonathan Tugwood, Alastair Kerr, Dominic G. Rothwell, Elaine Kilgour & Caroline Dive

  49. The Christie NHS Foundation Trust, Manchester, UK

    Fabio Gomes, Kate Brown, Mathew Carter, Anshuman Chaturvedi, Lynsey Priest & Pedro Oliveira

  50. Division of Cancer Sciences, The University of Manchester and The Christie NHS Foundation Trust, Manchester, UK

    Colin R. Lindsay, Fiona H. Blackhall, Matthew G. Krebs & Yvonne Summers

  51. Cancer Research UK Manchester Institute Cancer Biomarker Centre, University of Manchester, Manchester, UK

    Alexandra Clipson, Jonathan Tugwood, Alastair Kerr, Dominic G. Rothwell, Elaine Kilgour & Caroline Dive

  52. Artificial Intelligence in Medicine (AIM) Program, Mass General Brigham, Harvard Medical School, Boston, MA, USA

    Hugo J. W. L. Aerts

  53. Department of Radiation Oncology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA

    Hugo J. W. L. Aerts

  54. Radiology and Nuclear Medicine, CARIM & GROW, Maastricht University, Maastricht, Netherlands

    Hugo J. W. L. Aerts

  55. Institute for Computational Cancer Biology, Center for Integrated Oncology (CIO), Cancer Research Center Cologne Essen (CCCE), Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany

    Roland F. Schwarz

  56. Berlin Institute for the Foundations of Learning and Data (BIFOLD), Berlin, Germany

    Roland F. Schwarz & Tom L. Kaufmann

  57. Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany

    Tom L. Kaufmann

  58. Danish Cancer Society Research Center, Copenhagen, Denmark

    Zoltan Szallasi & Miklos Diossy

  59. Computational Health Informatics Program, Boston Children’s Hospital, Boston, MA, USA

    Zoltan Szallasi & Miklos Diossy

  60. Department of Bioinformatics, Semmelweis University, Budapest, Hungary

    Zoltan Szallasi

  61. Department of Physics of Complex Systems, ELTE Eötvös Loránd University, Budapest, Hungary

    Miklos Diossy

  62. The Francis Crick Institute, London, UK

    Aengus Stewart, Alastair Magness, Clare E. Weeden, Dina Levi, Eva Grönroos, Jacki Goldman, Mickael Escudero, Philip Hobson, Roberto Vendramin, Stefan Boeing, Tamara Denner, Vittorio Barbè, Wei-Ting Lu, William Hill, Yutaka Naito & Zoe Ramsden

  63. University College London Cancer Institute, London, UK

    Angeliki Karamani, Benny Chain, David R. Pearce, Despoina Karagianni, Elena Hoxha, Felip Gálvez-Cancino, Georgia Stavrou, Gerasimos Mastrokalos, Helen L. Lowe, Ignacio Garcia Matos, James L. Reading, Jayant K. Rane, John A. Hartley, Kayalvizhi Selvaraju, Kezhong Chen, Leah Ensell, Mansi Shah, Marcos Vasquez Duran, Maria Litovchenko, Olga Chervova, Piotr Pawlik, Robert E. Hynds, Saioa López, Samuel Gamble, Seng Kuong Anakin Ung, Supreet Kaur Bola, Thanos P. Mourikis, Victoria Spanswick & Yin Wu

  64. Experimental Histopathology, The Francis Crick Institute, London, UK

    Emma Nye & Richard Kevin Stone

  65. Retroviral Immunology Group, The Francis Crick Institute, London, UK

    George Kassiotis & Kevin W. Ng

  66. Department of Infectious Disease, Faculty of Medicine, Imperial College London, London, UK

    George Kassiotis

  67. Department of Haematology, University College London Hospitals, London, UK

    Karl S. Peggs

  68. Cancer Immunology Unit, Research Department of Haematology, University College London Cancer Institute, London, UK

    Karl S. Peggs

  69. Department of Molecular Oncology and Immunology, the Netherlands Cancer Institute, Amsterdam, Netherlands

    Krijn Dijkstra

  70. Oncode Institute, Utrecht, Netherlands

    Krijn Dijkstra

  71. Experimental Oncology, Institute for Oncology and Radiology of Serbia, Belgrade, Serbia

    Miljana Tanić

  72. Immune Regulation and Tumour Immunotherapy Group, Cancer Immunology Unit, Research Department of Haematology, University College London Cancer Institute, London, UK

    Sergio A. Quezada

  73. Centre for Medical Image Computing, Department of Medical Physics and Biomedical Engineering, University College London, London, UK

    Catarina Veiga

  74. Department of Medical Physics and Bioengineering, University College London Cancer Institute, London, UK

    Gary Royle

  75. Department of Medical Physics and Biomedical Engineering, University College London, London, UK

    Charles-Antoine Collins-Fekete

  76. Institute of Nuclear Medicine, Division of Medicine, University College London, London, UK

    Francesco Fraioli

  77. Institute of Structural and Molecular Biology, University College London, London, UK

    Paul Ashford

  78. University College London, London, UK

    Tristan Clark

  79. Department of Radiology, University College London Hospitals, London, UK

    Alexander James Procter, Asia Ahmed, Magali N. Taylor & Arjun Nair

  80. UCL Respiratory, Department of Medicine, University College London, London, UK

    Arjun Nair

  81. Department of Thoracic Surgery, University College London Hospital NHS Trust, London, UK

    David Lawrence & Davide Patrini

  82. Lungs for Living Research Centre, UCL Respiratory, University College London, London, UK

    Neal Navani, Ricky M. Thakrar & Sam M. Janes

  83. Department of Thoracic Medicine, University College London Hospitals, London, UK

    Neal Navani & Ricky M. Thakrar

  84. University College London Hospitals, London, UK

    Emilie Martinoni Hoogenboom, Fleur Monk, James W. Holding, Junaid Choudhary, Kunal Bhakhri, Marco Scarci, Martin Hayward, Nikolaos Panagiotopoulos, Pat Gorman, Robert CM. Stephens, Yien Ning Sophia Wong & Steve Bandula

  85. The Institute of Cancer Research, London, UK

    Anca Grapa, Hanyun Zhang, Khalid AbdulJabbar & Xiaoxi Pan

  86. The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Yinyin Yuan

  87. Independent Cancer Patients’ Voice, London, UK

    David Chuter & Mairead MacKenzie

  88. University Hospital Southampton NHS Foundation Trust, Southampton, UK

    Serena Chee, Aiman Alzetani, Lydia Scarlett, Jennifer Richards, Papawadee Ingram & Silvia Austin

  89. Department of Oncology, University Hospital Southampton NHS Foundation Trust, Southampton, UK

    Judith Cave

  90. Academic Division of Thoracic Surgery, Imperial College London, London, UK

    Eric Lim

  91. Royal Brompton and Harefield Hospitals, Guy’s and St Thomas’ NHS Foundation Trust, London, UK

    Eric Lim, Paulo De Sousa, Simon Jordan, Alexandra Rice, Hilgardt Raubenheimer, Harshil Bhayani, Lyn Ambrose, Anand Devaraj, Hema Chavan, Sofina Begum, Silviu I. Buderi, Daniel Kaniu, Mpho Malima, Sarah Booth, Nadia Fernandes, Pratibha Shah & Chiara Proli

  92. Royal Surrey Hospital, Royal Surrey Hospitals NHS Foundation Trust, Guilford, UK

    Madeleine Hewish

  93. University of Surrey, Guilford, UK

    Madeleine Hewish

  94. Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, UK

    Sarah Danson

  95. Liverpool Heart and Chest Hospital, Liverpool, UK

    Michael J. Shackcloth

  96. Princess Alexandra Hospital, The Princess Alexandra Hospital NHS Trust, Harlow, UK

    Lily Robinson & Peter Russell

  97. Queen Elizabeth University Hospital, Glasgow, UK

    Kevin G. Blyth

  98. NHS Greater Glasgow and Clyde, Glasgow, UK

    Craig Dick

  99. Golden Jubilee National Hospital, Clydebank, UK

    Alan Kirk, Mo Asif, Rocco Bilancia, Nikos Kostoulas & Mathew Thomas

Authors
  1. Takahiro Karasaki
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  2. David A. Moore
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  3. Selvaraju Veeriah
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  4. Cristina Naceur-Lombardelli
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  5. Antonia Toncheva
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  6. Neil Magno
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  7. Sophia Ward
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  8. Maise Al Bakir
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  9. Thomas B. K. Watkins
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  10. Kristiana Grigoriadis
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  11. Ariana Huebner
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  12. Mark S. Hill
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  13. Alexander M. Frankell
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  14. Christopher Abbosh
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  15. Clare Puttick
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  16. Haoran Zhai
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  17. Francisco Gimeno-Valiente
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  18. Sadegh Saghafinia
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  19. Nnennaya Kanu
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  20. Michelle Dietzen
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  21. Oriol Pich
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  22. Emilia L. Lim
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  23. Carlos Martínez-Ruiz
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  24. James R. M. Black
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  25. Dhruva Biswas
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  26. Brittany B. Campbell
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  27. Claudia Lee
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  28. Emma Colliver
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  29. Katey S. S. Enfield
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  30. Sonya Hessey
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  31. Crispin T. Hiley
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  32. Simone Zaccaria
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  33. Kevin Litchfield
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  34. Nicolai J. Birkbak
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  35. Elizabeth Larose Cadieux
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  36. Jonas Demeulemeester
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  37. Peter Van Loo
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  38. Prasad S. Adusumilli
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  39. Kay See Tan
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  40. Waseem Cheema
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  41. Francisco Sanchez-Vega
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  42. David R. Jones
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  43. Natasha Rekhtman
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  44. William D. Travis
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  45. Allan Hackshaw
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  46. Teresa Marafioti
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  47. Roberto Salgado
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  48. John Le Quesne
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  49. Andrew G. Nicholson
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  50. Nicholas McGranahan
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  51. Charles Swanton
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  52. Mariam Jamal-Hanjani
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Consortia

TRACERx Consortium

  • Charles Swanton
  • , Mariam Jamal-Hanjani
  • , Nicholas McGranahan
  • , Takahiro Karasaki
  • , David A. Moore
  • , Selvaraju Veeriah
  • , Cristina Naceur-Lombardelli
  • , Antonia Toncheva
  • , Sophia Ward
  • , Maise Al Bakir
  • , Thomas B. K. Watkins
  • , Kristiana Grigoriadis
  • , Ariana Huebner
  • , Mark S. Hill
  • , Alexander M. Frankell
  • , Christopher Abbosh
  • , Clare Puttick
  • , Haoran Zhai
  • , Francisco Gimeno-Valiente
  • , Sadegh Saghafinia
  • , Nnennaya Kanu
  • , Michelle Dietzen
  • , Oriol Pich
  • , Emilia L. Lim
  • , Dhruva Biswas
  • , Brittany B. Campbell
  • , Emma Colliver
  • , Katey S. S. Enfield
  • , Sonya Hessey
  • , Crispin T. Hiley
  • , Simone Zaccaria
  • , Kevin Litchfield
  • , Nicolai J. Birkbak
  • , Elizabeth Larose Cadieux
  • , Jonas Demeulemeester
  • , Peter Van Loo
  • , Allan Hackshaw
  • , Teresa Marafioti
  • , Roberto Salgado
  • , John Le Quesne
  • , Andrew G. Nicholson
  • , Jason F. Lester
  • , Amrita Bajaj
  • , Apostolos Nakas
  • , Azmina Sodha-Ramdeen
  • , Keng Ang
  • , Mohamad Tufail
  • , Mohammed Fiyaz Chowdhry
  • , Molly Scotland
  • , Rebecca Boyles
  • , Sridhar Rathinam
  • , Claire Wilson
  • , Domenic Marrone
  • , Sean Dulloo
  • , Dean A. Fennell
  • , Gurdeep Matharu
  • , Jacqui A. Shaw
  • , Joan Riley
  • , Lindsay Primrose
  • , Ekaterini Boleti
  • , Heather Cheyne
  • , Mohammed Khalil
  • , Shirley Richardson
  • , Tracey Cruickshank
  • , Gillian Price
  • , Keith M. Kerr
  • , Sarah Benafif
  • , Kayleigh Gilbert
  • , Babu Naidu
  • , Akshay J. Patel
  • , Aya Osman
  • , Christer Lacson
  • , Gerald Langman
  • , Helen Shackleford
  • , Madava Djearaman
  • , Salma Kadiri
  • , Gary Middleton
  • , Angela Leek
  • , Jack Davies Hodgkinson
  • , Nicola Totten
  • , Angeles Montero
  • , Elaine Smith
  • , Eustace Fontaine
  • , Felice Granato
  • , Helen Doran
  • , Juliette Novasio
  • , Kendadai Rammohan
  • , Leena Joseph
  • , Paul Bishop
  • , Rajesh Shah
  • , Stuart Moss
  • , Vijay Joshi
  • , Philip Crosbie
  • , Fabio Gomes
  • , Kate Brown
  • , Mathew Carter
  • , Anshuman Chaturvedi
  • , Lynsey Priest
  • , Pedro Oliveira
  • , Colin R. Lindsay
  • , Fiona H. Blackhall
  • , Matthew G. Krebs
  • , Yvonne Summers
  • , Alexandra Clipson
  • , Jonathan Tugwood
  • , Alastair Kerr
  • , Dominic G. Rothwell
  • , Elaine Kilgour
  • , Caroline Dive
  • , Hugo J. W. L. Aerts
  • , Roland F. Schwarz
  • , Tom L. Kaufmann
  • , Gareth A. Wilson
  • , Rachel Rosenthal
  • , Zoltan Szallasi
  • , Judit Kisistok
  • , Mateo Sokac
  • , Miklos Diossy
  • , Abigail Bunkum
  • , Aengus Stewart
  • , Alastair Magness
  • , Andrew Rowan
  • , Angeliki Karamani
  • , Benny Chain
  • , Carla Castignani
  • , Carlos Martínez-Ruiz
  • , Chris Bailey
  • , Clare E. Weeden
  • , Claudia Lee
  • , Corentin Richard
  • , David R. Pearce
  • , Despoina Karagianni
  • , Dina Levi
  • , Elena Hoxha
  • , Emma Nye
  • , Eva Grönroos
  • , Felip Gálvez-Cancino
  • , Foteini Athanasopoulou
  • , George Kassiotis
  • , Georgia Stavrou
  • , Gerasimos Mastrokalos
  • , Helen L. Lowe
  • , Ignacio Garcia Matos
  • , Jacki Goldman
  • , James L. Reading
  • , James R. M. Black
  • , Javier Herrero
  • , Jayant K. Rane
  • , Jerome Nicod
  • , Jie Min Lam
  • , John A. Hartley
  • , Karl S. Peggs
  • , Kayalvizhi Selvaraju
  • , Kerstin Thol
  • , Kevin W. Ng
  • , Kezhong Chen
  • , Krijn Dijkstra
  • , Krupa Thakkar
  • , Leah Ensell
  • , Mansi Shah
  • , Marcos Vasquez Duran
  • , Maria Litovchenko
  • , Mariana Werner Sunderland
  • , Michelle Leung
  • , Mickael Escudero
  • , Mihaela Angelova
  • , Miljana Tanić
  • , Monica Sivakumar
  • , Olga Chervova
  • , Olivia Lucas
  • , Othman Al-Sawaf
  • , Paulina Prymas
  • , Philip Hobson
  • , Piotr Pawlik
  • , Richard Kevin Stone
  • , Robert Bentham
  • , Robert E. Hynds
  • , Roberto Vendramin
  • , Saioa López
  • , Samuel Gamble
  • , Seng Kuong Anakin Ung
  • , Sergio A. Quezada
  • , Sharon Vanloo
  • , Stefan Boeing
  • , Stephan Beck
  • , Supreet Kaur Bola
  • , Tamara Denner
  • , Thanos P. Mourikis
  • , Victoria Spanswick
  • , Vittorio Barbè
  • , Wei-Ting Lu
  • , William Hill
  • , Wing Kin Liu
  • , Yin Wu
  • , Yutaka Naito
  • , Zoe Ramsden
  • , Catarina Veiga
  • , Gary Royle
  • , Charles-Antoine Collins-Fekete
  • , Francesco Fraioli
  • , Paul Ashford
  • , Tristan Clark
  • , Martin D. Forster
  • , Siow Ming Lee
  • , Elaine Borg
  • , Mary Falzon
  • , Dionysis Papadatos-Pastos
  • , James Wilson
  • , Tanya Ahmad
  • , Alexander James Procter
  • , Asia Ahmed
  • , Magali N. Taylor
  • , Arjun Nair
  • , David Lawrence
  • , Davide Patrini
  • , Neal Navani
  • , Ricky M. Thakrar
  • , Sam M. Janes
  • , Emilie Martinoni Hoogenboom
  • , Fleur Monk
  • , James W. Holding
  • , Junaid Choudhary
  • , Kunal Bhakhri
  • , Marco Scarci
  • , Martin Hayward
  • , Nikolaos Panagiotopoulos
  • , Pat Gorman
  • , Reena Khiroya
  • , Robert CM. Stephens
  • , Yien Ning Sophia Wong
  • , Steve Bandula
  • , Abigail Sharp
  • , Sean Smith
  • , Nicole Gower
  • , Harjot Kaur Dhanda
  • , Kitty Chan
  • , Camilla Pilotti
  • , Rachel Leslie
  • , Anca Grapa
  • , Hanyun Zhang
  • , Khalid AbdulJabbar
  • , Xiaoxi Pan
  • , Yinyin Yuan
  • , David Chuter
  • , Mairead MacKenzie
  • , Serena Chee
  • , Aiman Alzetani
  • , Judith Cave
  • , Lydia Scarlett
  • , Jennifer Richards
  • , Papawadee Ingram
  • , Silvia Austin
  • , Eric Lim
  • , Paulo De Sousa
  • , Simon Jordan
  • , Alexandra Rice
  • , Hilgardt Raubenheimer
  • , Harshil Bhayani
  • , Lyn Ambrose
  • , Anand Devaraj
  • , Hema Chavan
  • , Sofina Begum
  • , Silviu I. Buderi
  • , Daniel Kaniu
  • , Mpho Malima
  • , Sarah Booth
  • , Nadia Fernandes
  • , Pratibha Shah
  • , Chiara Proli
  • , Madeleine Hewish
  • , Sarah Danson
  • , Michael J. Shackcloth
  • , Lily Robinson
  • , Peter Russell
  • , Kevin G. Blyth
  • , Craig Dick
  • , Alan Kirk
  • , Mo Asif
  • , Rocco Bilancia
  • , Nikos Kostoulas
  •  & Mathew Thomas

Contributions

T.K. collated the cohort and clinicopathological data, conducted bioinformatics analyses and wrote the manuscript. D.A.M. led the central pathology review, collated the cohort and pathological data and wrote the manuscript. S.V. led the sample extraction and management and was supported by C.N.-L., A.T. and N.M. S.W. led the genomic sequencing. M.A.B. collated the cohort and clinicopathological data, processed the genomic data and helped write the manuscript. T.B.K.W., K.G., A. Huebner, E.C. and K.S.S.E. processed the genomic data, assisted with bioinformatics analyses and helped write the manuscript. M.S.H., C.P., H.Z., F.G.-V., S.S., N.K., M.D., O.P., E.L.L., C.M.-R., J.R.M.B., D.B., B.B.C. and C.L. processed the genomic data and assisted with bioinformatics analyses. S.H. assisted with clinical annotation and helped write the manuscript. C.T.H. assisted with clinical annotation and data processing. A.M.F. and C.A. processed the genomic and ctDNA data, assisted with bioinformatics analyses and helped write the manuscript. N.J.B., K.L. and S.Z. helped direct bioinformatics and statistical analyses. P.S.A., K.S.T., W.C., F.S.-V., D.R.J., N.R. and W.D.T. analyzed external cohort data and contributed to writing the manuscript. E.L.C., J.D., P.V.L. and T.M. contributed to writing the manuscript. A. Hackshaw helped oversee the running of the TRACERx study. R.S., J.L.Q. and A.G.N. supported central pathology review and helped write the manuscript. M.J.-H., N.M. and C.S. jointly designed and supervised the study and helped write the manuscript. Study design, conduct and clinical and laboratory oversight: M.J.-H., N.M. and C.S. (jointly led by M.J.-H., N.M. and C.S). Informatics supervision: N.M., N.J.B., K.L., S.Z. and C.S (led by N.M.). Cohort and clinical annotation: T.K., D.A.M., M.A.B., C.A., S.H., C.T.H., M.J.-H., C.S., A. Hackshaw and UCL Cancer Trials Centre (jointly led by T.K. and M.A.B.). Central pathology review: D.A.M., A.G.N., J.L.Q., R.S., M.A.B. and T.K. (led by D.A.M.). Sample extraction and management: S.V., C.N.-L., A.T., N.M. and the EHP STP (Experimental Histopathology Science Technology Platform) (led by S.V.). Genomic sequencing: S.W. and the ASF STP (Advanced Sequencing Facility Science Technology Platform) (led by S.W.). Cohort overview: T.K., D.A.M. and M.D. (led by T.K.). Genetic alteration and evolutionary dependency: T.K., K.G., A.M.F., T.B.K.W., E.L.L., M.S.H., C.P., H.Z., B.B.C., S.Z. and N.M. (led by T.K.). Transcriptomic data analysis, T.K., C.M.-R., J.R.M.B., C.P., D.B., F.G.-V., S.S., N.K. and N.M. (led by T.K.). Metastasis data analysis, T.K., A. Huebner and M.A.B. (jointly led by T.K. and A. Huebner). ctDNA data analysis: C.A., A.M.F., T.K., N.J.B. and C.S. (jointly led by C.A., A.M.F. and T.K.). Tumor immune microenvironment analysis: T.K., C.P., O.P., C.P., C.L., K.G., E.C., K.S.S.E., C.T.H., N.M. and C.S. (led by T.K.). Regression analysis, survival analysis and statistics: T.K., M.S.H., A.M.F., K.L. and A. Hackshaw (led by T.K.). External cohort data analysis: P.S.A., K.S.T., W.C., F.S.-V., D.R.J., N.R. and W.D.T. (jointly led by P.S.A., K.S.T. and W.C.). Manuscript writing: D.A.M., T.K., M.A.B., T.B.K.W., A.M.F., K.G., A. Huebner, E.C., K.S.S.E., E.L.C., J.D., P.V.L., F.S.-V., P.S.A., K.S.T., W.C., D.R.J., N.R., W.D.T., T.M., R.S., J.L.Q., A.G.N., N.M., M.J.-H. and C.S. (jointly led by D.A.M. and T.K.).

Corresponding authors

Correspondence to Nicholas McGranahan, Charles Swanton or Mariam Jamal-Hanjani.

Ethics declarations

Competing interests

D.A.M. reports speaker fees from AstraZeneca, Eli Lilly and Takeda, consultancy fees from AstraZeneca, Thermo Fisher, Takeda, Amgen, Janssen, MIM Software, Bristol Myers Squibb (BMS) and Eli Lilly and has received educational support from Takeda and Amgen. S.V. is a co-inventor to a patent for detecting molecules in a sample (US patent no. 10578620). M.A.B. has consulted for Achilles Therapeutics. A.M.F. is co-inventor to a patent application to determine methods and systems for tumor monitoring (PCT/EP2022/077987). C.A. has received speaking honoraria or expenses from Novartis, Roche, AstraZeneca and Bristol Myers Squibb and reports employment at AstraZeneca. C.A. is an inventor on a European patent application relating to assay technology to detect tumor recurrence (PCT/GB2017/053289), the patent has been licensed to commercial entities and under their terms of employment C.A. is due a revenue share of any revenue generated from such license(s). C.A. declares a patent application (PCT/US2017/028013) for methods to detect lung cancer. C.A. is a named inventor on a patent application to determine methods and systems for tumor monitoring (PCT/EP2022/077987) and is a named inventor on a provisional patent protection related to a ctDNA detection algorithm. D.B. reports personal fees from NanoString and AstraZeneca, and has a patent PCT/GB2020/050221 issued on methods for cancer prognostication. K.S.S.E. received research grant funding from BMS. C.T.H. has received speaker fees from AstraZeneca. K.L. has a patent on indel burden and CPI response pending and speaker fees from Roche tissue diagnostics, research funding from CRUK TDL/Ono/LifeArc alliance, Genesis Therapeutics, and consulting roles with Monopteros Therapeutics and Kynos Therapeutics. N.J.B. is a co-inventor to a patent for identification of responders to cancer treatment (PCT/GB2018/051912), has a patent application (PCT/GB2020/050221) on methods for cancer prognostication and a patent on methods for predicting anti-cancer response (US14/466,208). E.L.C. is employed by and holds shares in Achilles Therapeutics. P.S.A. is a Scientific Advisory Board Member and Consultant for ATARA Biotherapeutics, Bayer, Carisma Therapeutics, Imugene, ImmPactBio, Johnston & Johnston, Orion and OutpaceBio; declares patents, royalties and intellectual property on mesothelin-targeted CARs and other T cell therapies, which have been licensed to ATARA Biotherapeutics, issued patent method for detection of cancer cells using virus, and pending patent applications on PD-1 dominant negative receptor, wireless pulse-oximetry device, and on an ex vivo malignant pleural effusion culture system. Memorial Sloan Kettering Cancer Center (MSK) has licensed intellectual property related to mesothelin-targeted CARs and T-cell therapies to ATARA Biotherapeutics, and has associated royalties. D.R.J. has consulted for AstraZeneca, is a member of Clinical Trial Steering Committee for Merck, is a speaker for DAVA Oncology and is a member of the NCI Board of Scientific Advisors. N.R. has served on the Advisory Board for Merck, and is a member of the NCI Thoracic Malignancies Steering Committee. W.D.T. is a non-paid consultant for the LCMC3 and LCMC4 neoadjuvant clinical trials. A. Hackshaw has received fees for being a member of Independent Data Monitoring Committees for Roche-sponsored clinical trials, and academic projects coordinated by Roche. R.S. reports nonfinancial support from Merck and BMS, research support from Merck, Puma Biotechnology and Roche, and personal fees from Roche, BMS and Exact Sciences for advisory boards. A.G.N. reports personal fees from Merck, Boehringer Ingelheim, Novartis, AstraZeneca, BMS, Roche, Abbvie, Oncologica, Uptodate, European Society of Oncology, Takeda UK and Liberium, and personal fees and grants from Pfizer. N.M. has stock options in and has consulted for Achilles Therapeutics and holds European patents relating to targeting neoantigens (PCT/EP2016/ 059401), identifying patient response to immune checkpoint blockade (PCT/ EP2016/071471), determining HLA LOH (PCT/GB2018/052004) and predicting survival rates of patients with cancer (PCT/GB2020/050221). M.J.-H. has consulted for, and is a member of, the Achilles Therapeutics Scientific Advisory Board and Steering Committee, has received speaker honoraria from Pfizer, Astex Pharmaceuticals and Oslo Cancer Cluster, and is co-inventor on a European patent application relating to methods to detect lung cancer (PCT/US2017/028013). This patent has been licensed to commercial entities and under terms of employment M.J.-H. is due a share of any revenue generated from such license(s). C.S. acknowledges grant support from AstraZeneca, Boehringer Ingelheim, BMS, Pfizer, Roche-Ventana, Invitae (previously Archer Dx—collaboration in minimal residual disease sequencing technologies) and Ono Pharmaceutical. C.S. is an AstraZeneca Advisory Board member and Chief Investigator for the AZ MeRmaiD 1 and 2 clinical trials and is also co-chief investigator of the NHS Galleri trial funded by GRAIL and a paid member of GRAIL’s Scientific Advisory Board. C.S. receives consultant fees from Achilles Therapeutics (also SAB member), Bicycle Therapeutics (also a SAB member), Genentech, Medicxi, Roche Innovation Centre – Shanghai, Metabomed (until July 2022) and the Sarah Cannon Research Institute. C.S. had stock options in Apogen Biotechnologies and GRAIL until June 2021, and currently has stock options in Epic Bioscience, Bicycle Therapeutics, and has stock options in and is co-founder of Achilles Therapeutics. C.S. is an inventor on a European patent application relating to assay technology to detect tumor recurrence (PCT/GB2017/053289); the patent has been licensed to commercial entities and under terms of employment C.S. is due a revenue share of any revenue generated from such license(s). C.S. holds patents relating to targeting neoantigens (PCT/EP2016/059401), identifying patient response to immune checkpoint blockade (PCT/EP2016/071471), determining HLA LOH (PCT/GB2018/052004), predicting survival rates of patients with cancer (PCT/GB2020/050221), identifying patients who respond to cancer treatment (PCT/GB2018/051912), US patent relating to detecting tumor mutations (PCT/US2017/28013), methods for lung cancer detection (US20190106751A1) and both a European and US patent related to identifying insertion/deletion mutation targets (PCT/GB2018/051892). C.S. is a co-inventor to a patent application to determine methods and systems for tumor monitoring (PCT/EP2022/077987) and is a named inventor on a provisional patent protection related to a ctDNA detection algorithm. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Histopathological assessment of the TRACERx 421 LUAD cohort.

a. Definition and categorisation of LUAD growth patterns. b. Representative haematoxylin and eosin (H&E) image of invasive mucinous adenocarcinoma (IMA). c-h. Representative H&E images of lepidic (c), papillary (d), acinar (e), cribriform (f), micropapillary(g), and solid (h)pattern observed in LUAD. Scale bars, 250 μm. i. Number of tumors by histological entity in the TRACERX 421 cohort and further annotation of the number of LUAD tumors by predominant subtype. j. Number of regions with growth pattern assessment. k. Schematic of histological assessment in the TRACERx study. Proportions of each subtype in the diagnostic slides were reported, and the predominant subtype was used to label each tumor. Multiregional sampling specimens were processed for whole exome sequencing and a subset was subjected to RNA-sequencing. Each region was annotated with the representative growth pattern. l. Overview of TRACERx 421 LUAD cohort. Fetal adenocarcinoma, colloid adenocarcinoma, and two tumors from a collision tumor, determined by genomic analysis, were not included in the plot (n = 244 tumors from 240 participants are shown). Each column represents one tumor. The proportion of each growth pattern (subtype) based on diagnostic sectional area, genomic variables, and Ki-67 fraction by immunohistochemical staining are summarised. Missing data are shown in white. WGD, whole genome doubling; TMB, tumor mutational burden; wGII, weighted genome instability index; FLOH, fraction of the genome subject to loss of heterozygosity; SCNA, somatic copy number alteration; ITH, intra-tumoral heterogeneity.

Extended Data Fig. 2 Genomic correlates of LUAD predominant subtypes.

a, b. Comparison of intra-tumoral heterogeneity (ITH) metrics calculated using TRACERx analytical pipeline vs orthogonal methods. (a) Comparison of the mean fraction of the genome subject to loss of heterozygosity (FLOH), mean weighted genome instability index (wGII), and somatic copy number alteration ITH (SCNA ITH) by SCNA profiles generated by the TRACERx pipeline (based on ASCAT43 with additional multi-sample SCNA estimation approach9,44) against SCNA profiles generated by Sequenza12 (n = 224 tumors) and (b) a comparison of truncal tumor mutational burden (TMB) using the TRACERx pipeline (clonality inferred by the modified version of PyClone45) vs ubiquitous TMB (n = 224 tumors). Blue lines and shaded areas represent linear regression lines with 95% confidence intervals (CIs). Pearson’s correlation coefficient and P value are shown. c. Correlation of genomic variables calculated using orthogonal methods and the proportion of high-grade patterns within each tumor. The color scale reflects Spearman’s rank correlation coefficient (rho). Correlation P values were corrected for multiple testing according to the Benjamini-Hochberg (BH) method and asterisks indicate q value ranges * q < 0.05, ** q < 0.01, *** q < 0.001, **** q < 0.0001. d–f. Frequency of truncal driver mutations (d), truncal driver gene amplifications (AMP; e), and truncal chromosomal arm level SCNAs (gain or loss/LOH; f) in LUAD predominant subtypes. Recurrent truncal alterations observed in more than 5% of the tumors in the cohort are shown. Asterisks represent the alterations observed in fewer than 10 tumors in both predominantly high- and low-/mid-grade predominant tumors. The color scale represents the frequency of the alteration observed within each subtype. Tumors with multi-regional SCNA data were included in the analysis (n = 224). g-i. Adjusted odds ratios with 95% CIs of truncal genomic alterations associated with the predominance of high-grade (n = 79 tumors) or low-/mid-grade patterns (n = 116 tumors). Genomic alterations selected by the model simplification are shown when (g) truncal alterations observed in more than 10% of the tumors in the cohort are included in the analysis, or when (h) SCNA profiles generated by Sequenza12 are used, or when (i) wGII is added to the model shown in Fig. 2a. Asterisks indicate type II ANOVA P value ranges * P < 0.05, ** P < 0.01, *** P < 0.001. The color represents the type of genomic alteration. Statistically significant alterations (P < 0.05) are indicated in bold. j, k. Mutual exclusivity and co-occurrence of truncal driver gene alterations and chromosome arm SCNAs specific to either predominantly high-grade or low-/mid-grade tumors when (j) truncal alterations observed in more than 10% of the tumors in the cohort are included in the analysis, or when (k) SCNA profiles generated by Sequenza12 are used. Truncal alterations with unadjusted P < 0.05 for both predominantly high- and low-/mid-grade tumors are filtered out. Color of the edge represents the relationship (mutual exclusivity vs co-occurrence) and the negative log of the q value (BH method) is represented in blue color scale in predominantly low-/mid-grade tumors and red color scale in predominantly high-grade tumors. Relationships with q < 0.1 are shown and asterisks indicate q value ranges * q < 0.05, ** q < 0.01. Covariates in statistically significant relationships (q < 0.05) are indicated in bold. l. Comparison of mean wGII between tumors with (n = 20) and without (n = 96) co-occurrence of truncal loss/LOH of chromosomes 3p and 3q in predominantly low-/mid-grade tumors. P value was calculated using a two-sided Wilcoxon rank-sum test.

Extended Data Fig. 3 SCNA and immune characteristics of LUAD predominant subtypes.

a. Across-genome plots showing the frequency of truncal and subclonal SCNAs of low-/mid-grade predominant tumors (top) and high-grade predominant tumors (bottom). Within each tumor type, the proportion of patients with gains or amplifications (top) and loss/LOH events (bottom) for each chromosome are described. The black line indicates the total (namely the sum of truncal and subclonal) proportion of tumours with SCNAs; the yellow and grey lines or shades indicate the proportion of tumors with subclonal and truncal gains, respectively. b, c. Comparison of ploidy adjusted mean copy number of chromosomal arm and driver genes between high-grade and low-/mid-grade predominant tumors, using SCNA profiles generated by Sequenza12 (b), and adding wGII to the regression model (c). Fixed effect coefficients of the linear mixed-effects model with tumor as a random effect are displayed on the x-axis, and the negative log of the q value (BH method) is displayed on the y-axis. The color represents the sign or the mean ploidy adjusted copy number, stratified with high-grade and low-/mid-grade predominance. Data points with q value ≥ 0.05 are colored in gray. Horizontal red dashed line represents q = 0.05. d. Number of genes with differential SCNA between high-grade and low-/mid-grade predominant tumors. Significantly increased copy number in high-grade predominant tumors compared with low/mid-grade predominant tumors: G2M checkpoint genes, 13/186; all genes, 2169/23657 (P = 0.36, chi-square test). e. Comparison of (left) PD-L1 expression on cancer cells measured by immunohistochemistry (left; n = 197 tumors) and stromal tumor infiltrating lymphocyte (TIL) scores (right; n = 228 tumors) across LUAD predominant subtypes. Each predominant subtype was compared against all other subtype tumors. P values were calculated using a two-sided Wilcoxon rank-sum test and corrected for multiple testing according to the BH method. Asterisks indicate q value ranges ** q < 0.01, **** q < 0.0001. Centre line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range. f. Adjusted odds ratios with 95% confidence intervals (CIs) for cancer cell PD-L1 positivity (≥ 1%) per tumor estimated by multivariable logistic regression model (PD-L1 positive, n = 75 tumors; PD-L1 negative, n = 102 tumors). Asterisks indicate type II ANOVA P value ranges * P < 0.05, *** P < 0.001. Statistically significant (P < 0.05) covariates are indicated in bold.

Extended Data Fig. 4 Morphological intra-tumoral heterogeneity reflects genomic heterogeneity.

a-c. Genomic distance between regions calculated by (a) LOH detected by Sequenza (n = 53 tumors) and genomic distance calculated by (b) mutation and (c) LOH only including tumor regions with purity ≥ 0.4 (n = 30 tumors). Each point represents a distance between a pair of regions in a tumor. Tumors containing both different subtype pair(s) and same subtype pair(s) are included in the analysis. Centre line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range. P values were calculated using a linear mixed-effects model, with tumor as a random effect. d. Comparison of tumor mutational burden (TMB) between ancestor-like and descendant-like regions (151 regional pairs in 54 tumors). Each line represents an ancestor-descendant-like regional pair. Each point represents one region and the plotted points were duplicated for regions associated with multiple ancestor-descendant-like pairs within a tumor. To assess the mutational burden shared in the majority of the cancer cells in the region, mutations with estimated cancer cell fraction (CCF) ≥ 95% were counted. Enrichment of higher TMB in descendant-like regions compared with the paired ancestor-like regions was evaluated by permutation test (1000 permutations, randomising TMB within each tumor, Monte Carlo procedure). e. Comparison of growth pattern by grades (left) and by the six growth patterns (right) between inferred ancestral-like and descendant-like regions. Tumors with single grades are included in the analysis. The color represents the transition of grade from ancestral-like to descendant-like region. Enrichment of lower-to-higher grade transition (upward transition) was evaluated using a permutation test (1000 permutations, randomising growth patterns within each tumor, Monte Carlo procedure). f. Comparison of regional growth pattern grade in ancestor-descendant-like pairs, inferred by various cutoffs of private LOH branch length proportion. All combinations of cutoff for ancestor-like and descendant-like inference shown in the figure yielded empirical P value < 0.05 (1000 permutations, Monte Carlo procedure) when the enrichment of lower-to-higher grade transition (upward transition) was tested. P values were not adjusted for the multiple comparisons shown in this panel. g-h. Comparison of regional pattern grade in ancestor-descendant-like pairs, inferred by (g) LOH profile generated by Sequenza12 and (h) both LOH profile and mutational profile (CCF ≥ 95%). Enrichment of lower-to-higher grade transition (upward transition) was evaluated using a permutation test (1000 permutations, randomising growth patterns within each tumor, Monte Carlo procedure).

Extended Data Fig. 5 Characterization of purely (homogeneously) solid tumors.

a. Comparison of G2M checkpoint gene expression in solid-pattern regions within purely solid tumors and mixed pattern tumors as defined by both diagnostic and regional growth pattern assessment. Centre line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range. P value was calculated using a linear mixed-effects model, with tumor as a random effect. b-e. Proportion of tumors which are purely solid, mixed pattern with solid component, and without any solid component, compared between tumors with and without truncal gain of chromosome arm 3q (b), truncal gain of arm or focal 3q (3q21.3-3q29) (c), truncal SMARCA4 mutation and/or LOH (d), and compared across the tumors stratified by truncal SMARCA4 mutation and LOH status (e). P value was calculated using a two-sided Fisher’s exact test. f. Comparison of the frequency of truncal copy number gain of chromosome arm 3q between mixed pattern tumors with solid component and purely solid tumors. A two-sided Fisher’s exact test was used. g-i. Comparison of the frequency of (g) copy number gain of chromosome arm 3q, (h) gain of arm or focal 3q (3q21.3-3q29), and (i) SMARCA4 mutation and/or LOH between mixed pattern tumors with solid component and purely solid tumors using SCNA profiles generated by Sequenza12. A two-sided Fisher’s exact test was used.

Extended Data Fig. 6 Analysis of morphology and genomics in metastasis samples.

a. Schematic of primary and secondary lung tumors in CRUK0296. Phylogenetic analysis confirmed the contralateral lung lesion to be a metastasis from the primary tumor resected three years earlier. STAS was positive in the primary tumor. b. Phylogenetic tree of a case having lung metastasis with pure lepidic appearance (CRUK0296). Blue nodes are mutation clusters found only in metastasis and gray nodes represent mutation clusters found in the primary tumor. Regional growth pattern is indicated in brackets; R, region. c. Representative H&E slide of a primary tumor of CRUK0296 showing tumor border (arrowheads) and STAS (arrow). d. Representative H&E slide of metastasis tumor in the contralateral lung of CRUK0296, which showed a pure lepidic pattern. Scale bars, 250 μm. e. Characteristics of five patients having lung recurrence samples sequenced and one patient having an intrapulmonary metastasis resected and sequenced at the time of primary surgery. All six patients showed positive STAS in the primary tumors and phylogenetic analysis revealed late metastatic divergence. f. Proportion of the timing of seeding clone divergence across predominant subtypes of primary tumors. g. Frequency of late or early divergence of the metastatic clone compared between tumors with and without STAS. A two-sided Fisher’s exact test was used.

Extended Data Fig. 7 Characterization of tumors with STAS and preoperative ctDNA shedding.

a. Overview of the TRACERx 421 LUAD cohort, ordered by the positivity of STAS, pre-operative ctDNA detection, and the site of the relapse (n = 223 patients). Participants with synchronous primary lung cancers were excluded. Colloid and fetal adenocarcinomas are included (predominant sutbype = Other). Each column represents a participant. IMA, invasive mucinous adenocarcinoma; LVI, lymphovascular invasion; PL, pleural invasion. Tumors that did not relapse before death or the development of a new primary cancer are treated as no recurrence (No rec). b. Kaplan-Meier (KM) curve of disease-free survival (DFS), comparing STAS present vs absent. Numbers at risk are described at the bottom. Unadjusted and adjusted hazard ratio (HR) (adjusted for age, pathological stage, smoking pack-years, surgery type, and receipt of adjuvant therapy) are shown. c. STAS positivity across predominant subtypes of the primary tumor. d. Histopathological features associated with STAS positivity (left) and pre-operative ctDNA detection (right). Negative log of the q values (BH method) obtained by multiple univariable logistic regression analyses are presented. Vertical dotted lines represent q = 0.05, and variables with q < 0.05 are presented in points with colors which represent the direction of the correlation. e. Frequency of driver mutations in 10 canonical oncogenic signalling pathways63 in STAS present and absent tumors. P values calculated by two-sided Fisher’s exact test were corrected for multiple testing according to the BH method and the asterisk indicates q value range * q < 0.05. f. Comparison of CTNNB1 gene expression (variance stabilisation normalised count) between STAS absent (n = 72 tumors) and present tumors (n = 114 tumors). Each point represents a tumor region (STAS absent, n = 169 regions; present, n = 289 regions). Centre line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range. P value was calculated using a linear mixed-effects model, with tumor as a random effect. g. Gene set enrichment analysis (GSEA) of Hallmark gene sets between STAS absent (n = 72 tumors) and present tumors (n = 112 tumors). Normalised enrichment score is displayed on the x-axis and indicates the enrichment for a given gene set. Gene sets with q < 0.25 (BH method) are described in gray. h. Pre-operative ctDNA positivity across predominant subtypes of the primary tumor.

Extended Data Fig. 8 Impact of predominant subtype upon site and risk of recurrence.

a. KM curve of DFS, comparing participants with predominantly high-grade tumors vs low-/mid-grade tumors. Numbers at risk are described at the bottom. Unadjusted and adjusted HRs (adjusted for age, stage, pack-years, surgery type, and adjuvant therapy) are shown. b. Frequency of the relapse site (intra- and/or extra-thoracic) across (left) predominant subtypes and (right) grades of the predominant subtype of the primary tumor. Tumors that did not relapse before death or the development of a new primary cancer are treated as no recurrence (No rec). c. Relapse-site specific (sub-distribution) HRs for predominantly high-grade tumors compared with low-/mid-grade tumors, adjusted for age, stage, pack-years, surgery type, and adjuvant therapy, are presented with 95% CIs on a logarithmic scale (n = 185 participants). Participants with synchronous primary lung cancers or uncertain sites of relapse were excluded from the analysis. P < 0.05 are described in red. No corrections were made for multiple comparisons.

Extended Data Fig. 9 Impact of STAS, pre-operative ctDNA positivity, and necrosis upon site and risk of recurrence.

a, b. Frequency of the relapse site (intra- and/or extra-thoracic), stratified by the positivity of STAS and pre-operative ctDNA detection. Pre-operative ctDNA data were based on (a) the assay previously reported by Abbosh et. al.8 (TRACERx100 cohort) and (b) the assay reported in our companion manuscript28 (TRACERx421 cohort), including 7 participants who underwent both assays in each cohort. Tumors that did not relapse before death or the development of a new primary cancer are treated as no recurrence (No rec). c. KM curves of DFS, stratified by the positivity of STAS and pre-operative ctDNA detection in participants with (top) pathological stage I disease and (bottom) stage II/III disease. HRs were adjusted for age, pathological stage, smoking pack-years, and receipt of adjuvant therapy. Surgery type was also added as a covariate for stage I disease but not for stage II & III disease, because only 1 participant underwent sublobar resection for stage II/III disease. Numbers at risk are described below the KM curves. d. Positivity of STAS and pre-operative ctDNA detection are incorporated with other tumor and clinical characteristics in a multivariable Cox proportional hazards model (DFS). HRs of each variable with 95% confidence intervals (CIs) are shown on the horizontal axis. e. Positivity of necrosis across predominant subtypes of the primary tumor. f. Frequency of the relapse site (intra- and/or extra-thoracic), stratified by the presence of STAS and necrosis in all LUADs. g. Relapse-site specific (sub-distribution) HR for positivity of necrosis, adjusted for age, stage, pack-years, surgery type, and adjuvant therapy, are presented with 95% CIs on a logarithmic scale (n = 211 participantsts). Participants with synchronous primary lung cancers or uncertain site of relapse were excluded from the analysis. P < 0.05 are shown in red. No corrections were made for multiple comparisons. h. Positivity of STAS and necrosis are incorporated with other tumor and clinical characteristics in a multivariable Cox proportional hazards model for DFS. HRs of each variable with 95% CIs are shown. i. KM curve of DFS, stratified by the positivity of STAS and the presence of necrosis. HRs were adjusted for age, stage, pack-years, surgery type, and adjuvant therapy. Numbers at risk are described at the bottom.

Extended Data Fig. 10 External validation of the impact of STAS and necrosis on disease-free survival.

a. Summary of patient demographics and clinical characteristics of the Memorial Sloan Kettering Cancer Center cohort (n = 712 patients). b. Positivity of STAS and necrosis are incorporated with other tumor and clinical characteristics in a multivariable Cox proportional hazards model of DFS. c. KM curve of DFS, stratified by the positivity of STAS and the presence of necrosis (n = 712 patients). Hazard ratios were adjusted for age, pathological stage, smoking pack-years, surgery type, and receipt of adjuvant therapy. Numbers at risk are described at the bottom.

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Karasaki, T., Moore, D.A., Veeriah, S. et al. Evolutionary characterization of lung adenocarcinoma morphology in TRACERx. Nat Med 29, 833–845 (2023). https://doi.org/10.1038/s41591-023-02230-w

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