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Using DNA sequencing data to quantify T cell fraction and therapy response

Nature volume 597, pages 555–560 (2021)Cite this article

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Abstract

The immune microenvironment influences tumour evolution and can be both prognostic and predict response to immunotherapy1,2. However, measurements of tumour infiltrating lymphocytes (TILs) are limited by a shortage of appropriate data. Whole-exome sequencing (WES) of DNA is frequently performed to calculate tumour mutational burden and identify actionable mutations. Here we develop T cell exome TREC tool (T cell ExTRECT), a method for estimation of T cell fraction from WES samples using a signal from T cell receptor excision circle (TREC) loss during V(D)J recombination of the T cell receptor-α gene (TCRA (also known as TRA)). TCRA T cell fraction correlates with orthogonal TIL estimates and is agnostic to sample type. Blood TCRA T cell fraction is higher in females than in males and correlates with both tumour immune infiltrate and presence of bacterial sequencing reads. Tumour TCRA T cell fraction is prognostic in lung adenocarcinoma. Using a meta-analysis of tumours treated with immunotherapy, we show that tumour TCRA T cell fraction predicts immunotherapy response, providing value beyond measuring tumour mutational burden. Applying T cell ExTRECT to a multi-sample pan-cancer cohort reveals a high diversity of the degree of immune infiltration within tumours. Subclonal loss of 12q24.31–32, encompassing SPPL3, is associated with reduced TCRA T cell fraction. T cell ExTRECT provides a cost-effective technique to characterize immune infiltrate alongside somatic changes.

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Fig. 1: Overview and validation of T cell ExTRECT.
Fig. 2: Determinants of T cell fraction.
Fig. 3: Prognostic value of TCRA T cell fraction for LUAD but not for LUSC.
Fig. 4: TCRA T cell fraction is predictive of survival and response to immunotherapy.

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Data availability

The RNA-seq data, WES data and histopathology-derived TIL scores (in each case from the TRACERx study) generated, used or analysed during this study are not publicly available and restrictions apply to the availability of these data. Such RNA-seq, WES data and histopathology-derived TIL scores are available through the Cancer Research UK and University College London Cancer Trials Centre (ctc.tracerx@ucl.ac.uk) for academic non-commercial research purposes upon reasonable request, and subject to review of a project proposal that will be evaluated by a TRACERx data access committee, entering into an appropriate data access agreement and subject to any applicable ethical approvals. Details of all other datasets obtained from third parties used in this study can be found in Extended Data Table 1. Clinical trial information (if applicable) is also available in the associated publications described in Extended Data Table 1.

Code availability

The code used to produce TCRA T cell fraction scores is available for academic non-commercial research purposes at https://github.com/McGranahanLab/TcellExTRECT. All other code used in the analysis and to produce figures is available at https://github.com/McGranahanLab/T-cell-ExTRECT-figure-code-2021.

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Acknowledgements

R.B. is supported by the NIHR BRC at University College London Hospitals. K.L. is funded by the UK Medical Research Council (MR/P014712/1 and MR/V033077/1), Rosetrees Trust and Cotswold Trust (A2437) and Cancer Research UK (C69256/A30194). T.B.K.W. is supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001169), the UK Medical Research Council (FC001169) and the Wellcome Trust (FC001169) as well as the Marie Curie ITN Project PLOIDYNET (FP7-PEOPLE-2013, 607722), Breast Cancer Research Foundation (BCRF), Royal Society Research Professorships Enhancement Award (RP/EA/180007) and the Foulkes Foundation. E.L.L. receives funding from NovoNordisk Foundation (ID 16584). R.R. is supported by Royal Society Research Professorships Enhancement Award (RP/EA/180007). C.M.-R. is supported by Rosetrees. C.T.H. is supported by the NIHR BRC at University College London Hospitals. M.J.-H. has received funding from Cancer Research UK, National Institute for Health Research, Rosetrees Trust, UKI NETs and NIHR University College London Hospitals Biomedical Research Centre. N.M. is a Sir Henry Dale Fellow, jointly funded by the Wellcome Trust and the Royal Society (Grant Number 211179/Z/18/Z) and also receives funding from Cancer Research UK, Rosetrees and the NIHR BRC at University College London Hospitals and the CRUK University College London Experimental Cancer Medicine Centre. C.S. is a Royal Society Napier Research Professor. His work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001169), the UK Medical Research Council (FC001169) and the Wellcome Trust (FC001169). C.S. is funded by Cancer Research UK (TRACERx, PEACE and CRUK Cancer Immunotherapy Catalyst Network), Cancer Research UK Lung Cancer Centre of Excellence, the Rosetrees Trust, Butterfield and Stoneygate Trusts, NovoNordisk Foundation (ID16584), Royal Society Research Professorships Enhancement Award (RP/EA/180007), the NIHR BRC at University College London Hospitals, the CRUK-UCL Centre, Experimental Cancer Medicine Centre and the Breast Cancer Research Foundation (BCRF). This research is supported by a Stand Up To Cancer-LUNGevity-American Lung Association Lung Cancer Interception Dream Team Translational Research Grant (SU2C-AACR-DT23-17). Stand Up To Cancer is a program of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the Scientific Partner of SU2C. C.S. also receives 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 (835297) and Chromavision from the European Union’s Horizon 2020 research and innovation programme (665233). The TRACERx study (Clinicaltrials.gov no: 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 (C11496/A17786) and coordinated through the Cancer Research UK and UCL Cancer Trials Centre. The results published here are based in part on data generated by The Cancer Genome Atlas pilot project established by the NCI and the National Human Genome Research Institute. The data were retrieved through the database of Genotypes and Phenotypes (dbGaP) authorization (accession number phs000178.v9.p8). Information about TCGA and the constituent investigators and institutions of the TCGA research network can be found at http://cancergenome.nih.gov/. This project was enabled through access to the MRC eMedLab Medical Bioinformatics infrastructure, supported by the Medical Research Council (MR/L016311/1). In particular, we acknowledge the support of the High-Performance Computing at the Francis Crick Institute as well as the UCL Department of Computer Science Cluster and the support team. In addition, this work was supported by the CRUK City of London Centre Award (C7893/A26233). We thank all investigators, funders and industry partners that supported the generation of the data within this study, as well as patients for their participation. Specifically, we thank Merck & Co, Genentech and Bristol-Myers Squibb for generating the industry datasets used in this study, and E. Van Allen, L. Diaz, T. A. Chan, L. A. Garraway, R. S. Lo, D. F. Bajorin, D. Schadendorf, T. Powles, S.-H. Lee, A. Ribas and S. Ogawa for academic datasets. This work has been funded by Merck and Co. We gratefully acknowledge the patients and their families, the investigators and site personnel who participated in the KEYNOTE-001, -006, -012 and -028 studies.

Author information

Author notes

  1. These authors contributed equally: Robert Bentham, Kevin Litchfield, Thomas B. K. Watkins

Authors and Affiliations

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

    Robert Bentham, Carlos Martínez-Ruiz & Nicholas McGranahan

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

    Robert Bentham, Kevin Litchfield, Emilia L. Lim, Carlos Martínez-Ruiz, Crispin T. Hiley, David A. Moore, Mariam Jamal-Hanjani, Charles Swanton & Nicholas McGranahan

  3. The Tumour Immunogenomics and Immunosurveillance Lab, Cancer Research UK Lung Cancer Centre of Excellence, University College London Cancer Institute, London, UK

    Kevin Litchfield

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

    Thomas B. K. Watkins, Emilia L. Lim, Rachel Rosenthal, Crispin T. Hiley, Maise Al Bakir & Charles Swanton

  5. Department of Pathology, GZA-ZNA, Antwerp, Belgium

    Roberto Salgado

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

    Roberto Salgado

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

    David A. Moore

  8. Department of Medical Oncology, University College London Hospitals, London, UK

    David A. Moore, Mariam Jamal-Hanjani & Charles Swanton

  9. Cancer Metastasis Lab, University College London Cancer Institute, London, UK

    Mariam Jamal-Hanjani

  10. The Francis Crick Institute, London, UK

    Nicolai J. Birkbak, Mickael Escudero, Aengus Stewart, Andrew Rowan, Jacki Goldman, Peter Van Loo, Richard Kevin Stone, Tamara Denner, Emma Nye, Sophia Ward, Stefan Boeing, Maria Greco, Jerome Nicod, Clare Puttick, Katey Enfield, Emma Colliver, Brittany Campbell, Alexander M. Frankell, Daniel Cook, Mihaela Angelova, Alastair Magness, Chris Bailey, Antonia Toncheva, Krijn Dijkstra, Judit Kisistok, Mateo Sokac, Oriol Pich, Jonas Demeulemeester, Elizabeth Larose Cadieux, Carla Castignani, Krupa Thakkar, Hongchang Fu, Takahiro Karasaki, Othman Al-Sawaf & Mark S. Hill

  11. University College London Cancer Institute, London, UK

    Takahiro Karasaki, Othman Al-Sawaf, Christopher Abbosh, Yin Wu, Selvaraju Veeriah, Robert E. Hynds, Andrew Georgiou, Mariana Werner Sunderland, James L. Reading, Sergio A. Quezada, Karl S. Peggs, Teresa Marafioti, John A. Hartley, Helen L. Lowe, Leah Ensell, Victoria Spanswick, Angeliki Karamani, Dhruva Biswas, Stephan Beck, Olga Chervova, Miljana Tanic, Ariana Huebner, Michelle Dietzen, James R. M. Black, Cristina Naceur-Lombardelli, Mita Afroza Akther, Haoran Zhai, Nnennaya Kanu, Simranpreet Summan, Francisco Gimeno-Valiente, Kezhong Chen, Elizabeth Manzano, Supreet Kaur Bola, Ehsan Ghorani, Marc Robert de Massy, Elena Hoxha, Emine Hatipoglu, Benny Chain, David R. Pearce, Javier Herrero & Simone Zaccaria

  12. University College London Hospitals, London, UK

    Mark S. Hill, David Lawrence, Martin Hayward, Nikolaos Panagiotopoulos, Robert George, Davide Patrini, Mary Falzon, Elaine Borg, Reena Khiroya, Asia Ahmed, Magali Taylor, Junaid Choudhary, Sam M. Janes, Martin Forster, Tanya Ahmad, Siow Ming Lee, Neal Navani, Dionysis Papadatos-Pastos, Marco Scarci, Pat Gorman, Elisa Bertoja, Robert C. M. Stephens, Emilie Martinoni Hoogenboom, James W. Holding, Steve Bandula, Ricky Thakrar, Radhi Anand, Kayalvizhi Selvaraju, James Wilson, Sonya Hessey, Paul Ashford, Mansi Shah & Marcos Vasquez Duran

  13. Swansea Bay University Health Board, Swansea, UK

    Jason Lester

  14. Cardiff and Vale University Health Board, Cardiff, UK

    Fiona Morgan, Malgorzata Kornaszewska, Richard Attanoos, Haydn Adams & Helen Davies

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

    Jacqui A. Shaw, Joan Riley, Lindsay Primrose & Dean Fennell

  16. Leicester University Hospitals, Leicester, UK

    Dean Fennell, Apostolos Nakas, Sridhar Rathinam, Rachel Plummer, Rebecca Boyles, Mohamad Tufail, Amrita Bajaj, Jan Brozik, Keng Ang & Mohammed Fiyaz Chowdhry

  17. National Institute for Health Research Leicester Respiratory Biomedical Research Unit, Leicester, UK

    William Monteiro & Hilary Marshall

  18. University of Leicester, Leicester, UK

    Alan Dawson, Sara Busacca, Domenic Marrone & Claire Smith

  19. Barnet & Chase Farm Hospitals, Barnet, UK

    Girija Anand & Sajid Khan

  20. Aberdeen Royal Infirmary, Aberdeen, UK

    Gillian Price, Mohammed Khalil, Keith Kerr, Shirley Richardson, Heather Cheyne, Joy Miller, Keith Buchan, Mahendran Chetty & Sylvie Dubois-Marshall

  21. The Whittington Hospital NHS Trust, London, UK

    Sara Lock & Kayleigh Gilbert

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

    Babu Naidu, Gerald Langman, Hollie Bancroft, Salma Kadiri, Gary Middleton, Madava Djearaman, Aya Osman, Helen Shackleford & Akshay Patel

  23. Manchester Cancer Research Centre Biobank, Manchester, UK

    Angela Leek, Nicola Totten, Jack Davies Hodgkinson, Jane Rogan, Katrina Moore & Rachael Waddington

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

    Raffaele Califano, Rajesh Shah, 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 & Philip Crosbie

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

    Philip Crosbie

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

    Philip Crosbie, Fiona Blackhall, Lynsey Priest, Matthew G. Krebs, Caroline Dive, Dominic G. Rothwell, Alastair Kerr & Elaine Kilgour

  27. Christie NHS Foundation Trust, Manchester, United Kingdom

    Fiona Blackhall, Lynsey Priest, Matthew G. Krebs, Katie Baker, Mathew Carter, Colin R. Lindsay & Fabio Gomes

  28. Cancer Research UK Manchester Institute, University of Manchester, Manchester, UK

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

  29. Berlin Institute for Medical Systems Biology, Max Delbrueck Center for Molecular Medicine, Berlin, Germany

    Roland Schwarz & Matthew Huska

  30. German Cancer Consortium (DKTK), partner site Berlin, Berlin, Germany

    Roland Schwarz

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

    Tom L. Kaufmann

  32. BIFOLD, Berlin Institute for the Foundations of Learning and Data, Berlin, Germany

    Tom L. Kaufmann

  33. Danish Cancer Society Research Center, Copenhagen, Denmark

    Zoltan Szallasi

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

    Istvan Csabai & Miklos Diossy

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

    Hugo Aerts

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

    Hugo Aerts & Charles Fekete

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

    Gary Royle & Catarina Veiga

  38. Department of Oncology and Radiotherapy, Medical University of Gdańsk, Gdańsk, Poland

    Marcin Skrzypski

  39. Independent Cancer Patients Voice, London, UK

    Mairead MacKenzie & Maggie Wilcox

  40. Cancer Research UK and UCL Cancer Trials Centre, London, UK

    Allan Hackshaw, Yenting Ngai, Abigail Sharp, Cristina Rodrigues, Oliver Pressey, Sean Smith, Nicole Gower, Harjot Kaur Dhanda, Kitty Chan & Sonal Chakraborty

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

    Christian Ottensmeier, Serena Chee, Benjamin Johnson, Aiman Alzetani, Judith Cave, Lydia Scarlett & Emily Shaw

  42. 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, Silviu I. Buderi, Daniel Kaniu, Mpho Malima, Sarah Booth, Andrew G. Nicholson, Nadia Fernandes, Christopher Deeley, Pratibha Shah & Chiara Proli

  43. Barts Health NHS Trust, London, UK

    Kelvin Lau, Michael Sheaff, Peter Schmid, Louise Lim & John Conibear

  44. Ashford and St Peter’s Hospitals NHS Foundation Trust, Chertsey, UK

    Madeleine Hewish

  45. 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

  46. Liverpool Heart and Chest Hospital NHS Foundation Trust, Liverpool, UK

    Michael Shackcloth & Julius Asante-Siaw

  47. Royal Liverpool University Hospital, Liverpool, UK

    John Gosney

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

    Teresa Light, Tracey Horey, Peter Russell & Dionysis Papadatos-Pastos

  49. NHS Greater Glasgow and Clyde, Glasgow, UK

    Kevin G. Blyth, Craig Dick & Andrew Kidd

  50. Golden Jubilee National Hospital, Clydebank, UK

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

  51. Achilles Therapeutics UK Limited, London, UK

    Gareth A. Wilson

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  •  & Gareth A. Wilson

Contributions

R.B. helped conceive the study, designed and conducted the bioinformatic analysis, and wrote the manuscript. K.L. curated the CPI1000+ cohort used in the study and provided considerable bioinformatic support on its analysis. T.B.K.W. provided considerable bioinformatic support on the analysis of the multi-sample pan-cancer cohort and helped conceive the study and write the manuscript. T.B.K.W. and E.L.L jointly curated the multi-sample pan-cancer cohort used in the study. R.R. and C.M.-R. provided considerable bioinformatic support in the transcriptomic analysis performed in the study, providing RNA-seq immune score metrics and assisting with the RNA-seq gene-expression analysis respectively. R.S., M.A.B., D.A.M. and C.T.H. jointly analysed histopathology-derived TIL estimates. M.J.-H. designed study protocols and helped to analyse patient clinical characteristics. C.S. helped provide study supervision and helped direct the avenues of bioinformatics analysis and also gave feedback on the manuscript. N.M. conceived and supervised the study and helped write the manuscript.

Corresponding author

Correspondence to Nicholas McGranahan.

Ethics declarations

Competing interests

D.A.M. reports speaker fees from AstraZeneca. M.A.B. has consulted for Achilles Therapeutics. R.R. has consulted for and has stock options in Achilles Therapeutics. 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, and a consulting role with Monopteros Therapeutics. C.T.H. has received speaker fees from AstraZeneca. M.J.-H. is a member of the Scientific Advisory Board and Steering Committee for Achilles Therapeutics. N.M. has stock options in and has consulted for Achilles Therapeutics and holds a European patent in determining HLA LOH (PCT/GB2018/052004). C.S. acknowledges grant support from Pfizer, AstraZeneca, Bristol Myers Squibb, Roche-Ventana, Boehringer-Ingelheim, Archer Dx Inc. (collaboration in minimal residual disease sequencing technologies) and Ono Pharmaceutical; is an AstraZeneca Advisory Board Member and Chief Investigator for the MeRmaiD1 clinical trial; has consulted for Amgen, Pfizer, Novartis, GlaxoSmithKline, MSD, Bristol Myers Squibb, AstraZeneca, Illumina, Genentech, Roche-Ventana, GRAIL, Medicxi, Bicycle Therapeutics, Metabomed and the Sarah Cannon Research Institute; has stock options in Apogen Biotechnologies, Epic Bioscience and GRAIL; and has stock options and is co-founder of Achilles Therapeutics. C.S. holds patents relating to assay technology to detect tumour recurrence (PCT/GB2017/053289); to targeting neoantigens (PCT/EP2016/059401), identifying patent response to immune checkpoint blockade (PCT/EP2016/071471), determining HLA LOH (PCT/GB2018/052004), predicting survival rates of patients with cancer (PCT/GB2020/050221), to treating cancer by targeting Insertion/deletion mutations (PCT/GB2018/051893); identifying insertion/deletion mutation targets (PCT/GB2018/051892); methods for lung cancer detection (PCT/US2017/028013); and identifying responders to cancer treatment (PCT/GB2018/051912).

Additional information

Peer review information Nature thanks Florian Markowetz and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Overview and validation of T cell ExTRECT.

a, Outline of quantification of the TCRA T cell fraction utilising V(D)J recombination and TRECs. top: Schematic demonstrating how RDR signals are used to detect SCNA gain or loss events in a standard tumour and matched control sample analysis. In this analysis cells consist of three distinct cell types: tumour cells, T cells and all other stromal cells. bottom: Schematic of how this same process works when focussing on the TCRA gene in relation to V(D)J recombination and TRECs, the lower right panel indicates an increased number of breakpoints detected in the TRACERx100 dataset within the TCRA gene relative to surrounding areas of 14q, suggesting that the TREC signal is captured. b, c, Plots showing examples of RDR in two TRACERx100 samples demonstrating either increased levels of T cell content in blood compared to matched tumour (b) or increased levels of T cell content in tumour compared to matched blood (c). VDV segments refer to variable segments in both the TCRα and TCRδ locus. d, TCRA T cell fraction (non-GC corrected) value for FFPE and fresh frozen samples for bladder and melanoma tumours within the CPI1000+ cohort (bladder: n = 228, melanoma: n = 297, two sided Wilcoxon rank-sum (Mann-Whitney U) test used, boxplot shows lower quartile, median and upper quartile values). e, Summary of linear model for prediction of non-GC corrected TCRA T cell fraction from histology and FFPE sample status within the CPI cohort. f, Pie charts of calculated TCRA T cell fraction from WES of either T cell-derived cell lines or non-T cell derived cell lines, all HCT116 cell lines had calculated fractions < 1 e-15. g, Overview of samples in the TRACERx100 cohort. e, Association of the CDR3 V(D)J read score based on the iDNA method to TCRA T cell fraction in TRACERx100, error bands represent the 95% confidence interval of the fitted linear model.

Extended Data Fig. 2 Accuracy of TCRA T cell fraction by copy number and depth.

a, Simulated log RDR from a sample consisting of 24% T cells, 75% tumour, and 1% non-T cell stroma (TCRA copy number = 1). b, Calculated TCRA T cell fraction versus actual T cell fraction value for simulated data c, Difference between calculated naive T cell fraction and actual fraction for range of tumour purities and local tumour copy number states at the TCRA locus. d, Difference between TCRA T cell fraction and actual fraction for a range of local tumour copy number to the TCRA locus and tumour purities. e,. Downsampling of 5 TRACERx100 samples to different depths. f, Downsampling of simulated data to different depth levels. g, Downsampling of the 5 TRACERx100 samples that with the highest CDR3 read counts to different depths and the resulting CDR3 read counts.

Extended Data Fig. 3 Extended analysis on determinants of TCRA T cell fraction.

a, Association of blood TCRA T cell fraction to histology in TRACERx100 (n = 93 LUAD and LUSC patients). b, Predictors of blood TCRA T cell fraction in TCGA LUAD and LUSC cohort (left panel: n = 1017, middle panel: n = 976, right panel: n = 714). c, Overview of samples in the TCGA LUAD and LUSC cohort. d, Summary of mean TCRA T cell fraction in PNE cohort. e, Overview plot of PNE cohort containing multi-sample microdissected tissue paired with normal blood samples. f, Summary of linear model for predicting blood TCRA T cell fraction, PNE infiltration defined as TCRA T cell fraction > 0.001, ESCC = Oesophageal squamous cell carcinoma, HGD = high grade dysplasia. g, Linear model for TCRA T cell fraction in PNE samples from genomic factors. h, Association of microbial reads from Kraken with TCRA T cell fraction in tumour samples (n = 880). i, -Log10 p-values for 59 microbial species tested for association with TCRA T cell fraction in blood and tumour sample in LUAD and LUSC. Red line represents the significance threshold at P = 0.000423. j, The significant hit Willamsia in LUAD tumours, red dots represent samples where reads were detected while blue represent samples with no reads detected (n = 501). k, The significant hit Paeniclostridium in LUSC tumours (n = 379). All Wilcoxon tests refer to Wilcoxon rank-sum (Mann-Whitney U) tests and are two sided. Boxplots represent lower quartile, median and upper quartile.

Extended Data Fig. 4 Subclonal SCNAs and T cell infiltration.

a, Overview of immune heterogeneity across multi-sample pan-cancer cohort with tumour samples ranked by TCRA T cell fraction, upper panel: histogram of entire cohort, lower panel: tumour sample grouped by patients with solid horizontal lines joining regions from the same patient, each line includes 2 or more tumour region and dashed red line is at the mean TCRA T cell fraction in the cohort (0.11). b, Overview of patients in the multi-sample pan-cancer cohort. c, Lower panel: number of tumours in pan-cancer multi-sample cohort with subclonal gains (dark red) or losses (dark blue) across the genome, horizontal lines signify the samples which have more than 30 tumours (Methods) with subclonal gains or losses. Upper panel: - log10(p-value) of the 160 cytoband regions tested for association between TCRA T cell fraction and subclonal gains (dark red points) or losses (dark blue points). Red horizontal line marks significance threshold, only one region is significant, a loss event on chromosome 12q24.31-32. d, Volcano plot for the RNA-seq analysis in the TRACERx100 cohort between samples with 12q24.31-32 loss and samples without, genes within the locus are labeled, dotted lines at fold change of 0.25 and adjusted P = 0.05.

Extended Data Fig 5 Association of TCRA T cell fraction with prognosis.

a, Kaplan-Meier curves for the multi-sample TRACERx100 cohort for LUAD (top) and LUSC (bottom) divided by the number of cold samples in the tumour. Immune-hot and immune-cold samples were defined by using the median of all the tumour samples (0.0736) as a threshold. In each Kaplan-Meier curve the included patients were restricted to those with total samples greater than the number of immune-cold samples used in defining the threshold. b, Kaplan-Meier curves for overall and progression free survival in the TCGA LUAD cohort, dividing the cohort into immune-hot and immune-cold groups using the mean of the TCGA LUAD cohort (0.109) as a threshold. c, Kaplan-Meier curves for the TCGA LUSC, and TCGA LUAD & LUSC cohorts for overall and progression free survival using the mean of the TCGA LUAD cohort (0.109) as a threshold for distinguishing hot and cold tumours. d, Log2(Hazard ratios) from Kaplan-Meier plots for the TCGA separating the tumour samples into immune-hot and immune-cold based on different thresholds from 0 to 0.16 in steps of 0.0025 for overall and progression free survival. e, Hazard ratios of separate Cox regression models relating disease free survival to different multi-sample measures related to the TCRA T cell fraction in the entire TRACERx100 cohort as well as the LUAD and LUSC patients separately. TCRA divergence score is defined as the maximum divided by the upper 95% confidence interval of the minimum. f, Hazard ratios of separate Cox regression models for TCRA T cell fraction for the TCGA LUAD and LUSC cohort for both overall survival (OS) and progression free survival (PFS).

Extended Data Fig 6 Overview of CPI1000+ cohort.

a, Cohort overview of the CPI1000+ dataset. b, Overview of samples in the CPI1000+ cohort excluding Snyder et al.49 and those with prior CPI treatment. c, ROC plot of GLM models for predicting CPI response (blue: clonal TMB, red: clonal TMB + TCRA T cell fraction, green: clonal TMB + CD8A expression). d, Cohort overview of the CPI lung dataset, red lines in upper panel reflect the median TCRA T cell fraction in patients with (0.10) or without (0.0070) a response to CPI, note that Tumour TCRA T cell fraction particularly in non-responders is often zero. e, Overview of patients in the CPI Lung cohort.

Extended Data Table 1 Original source publications
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Bentham, R., Litchfield, K., Watkins, T.B.K. et al. Using DNA sequencing data to quantify T cell fraction and therapy response. Nature 597, 555–560 (2021). https://doi.org/10.1038/s41586-021-03894-5

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