Unsupervised detection of fragment length signatures of circulating tumor DNA using non-negative matrix factorization

  1. Gabriel Renaud
  2. Maibritt Nørgaard
  3. Johan Lindberg
  4. Henrik Grönberg
  5. Bram De Laere
  6. Jørgen Bjerggaard Jensen
  7. Michael Borre
  8. Claus Lindbjerg Andersen
  9. Karina Dalsgaard Sørensen
  10. Lasse Maretty  Is a corresponding author
  11. Søren Besenbacher  Is a corresponding author
  1. Technical University of Denmark, Denmark
  2. Aarhus University, Denmark
  3. Karolinska Institute, Sweden
  4. Regional Hospital of West Jutland, Denmark
  5. Aarhus University Hospital, Denmark

Abstract

Sequencing of cell-free DNA (cfDNA) is currently being used to detect cancer by searching both for mutational and non-mutational alterations. Recent work has shown that the length distribution of cfDNA fragments from a cancer patient can inform tumor load and type. Here, we propose non-negative matrix factorization (NMF) of fragment length distributions as a novel and completely unsupervised method for studying fragment length patterns in cfDNA. Using shallow whole-genome sequencing (sWGS) of cfDNA from a cohort of patients with metastatic castration-resistant prostate cancer (mCRPC), we demonstrate how NMF accurately infers the true tumor fragment length distribution as an NMF component - and that the sample weights of this component correlate with ctDNA levels (r=0.75). We further demonstrate how using several NMF components enables accurate cancer detection on data from various early stage cancers (AUC=0.96). Finally, we show that NMF, when applied across genomic regions, can be used to discover fragment length signatures associated with open chromatin.

Data availability

Danish law requires ethical approval of any specific research aim and imposes restrictions on sharing of personal data. This means that the prostate cancer data used in this article cannot be uploaded to international databases. External researchers (academic or commercial) interested in analysing the prostate dataset (including any derivatives of it) will need to contact the Data Access Committee via email to kdso@clin.au.dk. The Data Access Committee is formed of co-authors Karina Dalsgaard Sørensen and Michael Borre, and Ole Halfdan Larsen (Department Head Consultant, Department of Clinical Medicine, Aarhus University). Due to Danish Law, for the authors to be allowed to share the data (pseudonymised) it will require prior approval from The Danish National Committee on Health Research Ethics (or similar) for the specific new research goal. The author (based in Denmark) has to submit the application for ethical approval, with the external researcher(s) as named collaborator(s)). In addition to ethical approval, a Collaboration Agreement and a Data Processing Agreement is required, both of which must be approved by the legal office of the institution of the author (data owner) and the legal office of the institution of the external researcher (data processor). Raw fragment length distributions along with ctDNA% estimates are available in Supplementary File 1.

The following previously published data sets were used

Article and author information

Author details

  1. Gabriel Renaud

    Department of Health Technology, Technical University of Denmark, Kongens Lyngby, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  2. Maibritt Nørgaard

    Department of Molecular Medicine, Aarhus University, Aarhus N, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  3. Johan Lindberg

    Department of Medical Epidemiology and Biostatistics, Karolinska Institute, Stockholm, Sweden
    Competing interests
    The authors declare that no competing interests exist.
  4. Henrik Grönberg

    Department of Medical Epidemiology and Biostatistics, Karolinska Institute, Stockholm, Sweden
    Competing interests
    The authors declare that no competing interests exist.
  5. Bram De Laere

    Department of Medical Epidemiology and Biostatistics, Karolinska Institute, Stockholm, Sweden
    Competing interests
    The authors declare that no competing interests exist.
  6. Jørgen Bjerggaard Jensen

    Department of Urology, Regional Hospital of West Jutland, Holstebro, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  7. Michael Borre

    Department of Urology, Aarhus University Hospital, Aarhus, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  8. Claus Lindbjerg Andersen

    Department of Molecular Medicine, Aarhus University, Aarhus N, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  9. Karina Dalsgaard Sørensen

    Department of Molecular Medicine, Aarhus University, Aarhus N, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  10. Lasse Maretty

    Department of Molecular Medicine, Aarhus University, Aarhus N, Denmark
    For correspondence
    lasse.maretty@clin.au.dk
    Competing interests
    The authors declare that no competing interests exist.
  11. Søren Besenbacher

    Department of Molecular Medicine, Aarhus University, Aarhus N, Denmark
    For correspondence
    besenbacher@clin.au.dk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1455-1738

Funding

The Independent Research Fund Denmark (Sapere Aude Research Leader)

  • Søren Besenbacher

The Danish Cancer Society

  • Karina Dalsgaard Sørensen

The Central Denmark Region Health Fund

  • Karina Dalsgaard Sørensen

Aarhus Universitet (Graduate School of Health)

  • Maibritt Nørgaard

Direktør Emil C. Hertz og Hustru Inger Hertz Fond

  • Karina Dalsgaard Sørensen

KV Fonden

  • Karina Dalsgaard Sørensen

Raimond og Dagmar Ringgård-Bohns Fond

  • Karina Dalsgaard Sørensen

Beckett Fonden

  • Karina Dalsgaard Sørensen

Snedkermester Sophus Jacobsen og Hustru Astrid Jacobsens Fond

  • Karina Dalsgaard Sørensen

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Human subjects: The prostate study was approved by The National Committee on Health Research Ethics (#1901101) and notified to The Danish Data Protection Agency (#1-16-02-366-15). All patients provided written informed consent.

Copyright

© 2022, Renaud et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Download links

Share this article

https://doi.org/10.7554/eLife.71569

Further reading

    1. Cancer Biology
    Han V Han, Richard Efem ... Richard Z Lin
    Research Article

    Most human pancreatic ductal adenocarcinoma (PDAC) are not infiltrated with cytotoxic T cells and are highly resistant to immunotherapy. Over 90% of PDAC have oncogenic KRAS mutations, and phosphoinositide 3-kinases (PI3Ks) are direct effectors of KRAS. Our previous study demonstrated that ablation of Pik3ca in KPC (KrasG12D; Trp53R172H; Pdx1-Cre) pancreatic cancer cells induced host T cells to infiltrate and completely eliminate the tumors in a syngeneic orthotopic implantation mouse model. Now, we show that implantation of Pik3ca−/− KPC (named αKO) cancer cells induces clonal enrichment of cytotoxic T cells infiltrating the pancreatic tumors. To identify potential molecules that can regulate the activity of these anti-tumor T cells, we conducted an in vivo genome-wide gene-deletion screen using αKO cells implanted in the mouse pancreas. The result shows that deletion of propionyl-CoA carboxylase subunit B gene (Pccb) in αKO cells (named p-αKO) leads to immune evasion, tumor progression, and death of host mice. Surprisingly, p-αKO tumors are still infiltrated with clonally enriched CD8+ T cells but they are inactive against tumor cells. However, blockade of PD-L1/PD1 interaction reactivated these clonally enriched T cells infiltrating p-αKO tumors, leading to slower tumor progression and improve survival of host mice. These results indicate that Pccb can modulate the activity of cytotoxic T cells infiltrating some pancreatic cancers and this understanding may lead to improvement in immunotherapy for this difficult-to-treat cancer.

    1. Cancer Biology
    2. Immunology and Inflammation
    Almudena Mendez-Perez, Andres M Acosta-Moreno ... Esteban Veiga
    Short Report

    In this study, we present a proof-of-concept classical vaccination experiment that validates the in silico identification of tumor neoantigens (TNAs) using a machine learning-based platform called NAP-CNB. Unlike other TNA predictors, NAP-CNB leverages RNA-seq data to consider the relative expression of neoantigens in tumors. Our experiments show the efficacy of NAP-CNB. Predicted TNAs elicited potent antitumor responses in mice following classical vaccination protocols. Notably, optimal antitumor activity was observed when targeting the antigen with higher expression in the tumor, which was not the most immunogenic. Additionally, the vaccination combining different neoantigens resulted in vastly improved responses compared to each one individually, showing the worth of multiantigen-based approaches. These findings validate NAP-CNB as an innovative TNA identification platform and make a substantial contribution to advancing the next generation of personalized immunotherapies.