Pharma Focus Asia

Isolation of Salivary Cell-free DNA for Cancer Detection

Patricia J. Brooks, Ethan Z. Malkin, Steven De Michino, Scott V. Bratman


Saliva is an emerging source of disease biomarkers, particularly for cancers of the head and neck. Although analysis of cell-free DNA (cfDNA) in saliva holds promise as a liquid biopsy for cancer detection, currently there are no standardized methodologies for the collection and isolation of saliva for the purposes of studying DNA. Here, we evaluated various saliva collection receptacles and DNA purification techniques, comparing DNA quantity, fragment size, source, and stability.


Head and neck cancers, of which head and neck squamous cell carcinomas (HNSCCs) make up the majority, are the 7th most common cancer worldwide. Generally, HNSCCs are associated with a poor 5-year overall survival of less than 50%, a number that has remained unchanged for many years due to diagnoses being made most frequently at locoregionally advanced disease stage.


Materials and Methods:

Sample collection

All work was approved by the Research Ethics Board at University Health Network (21–5592.0). Written informed consent was obtained from all participants for inclusion in the study. Samples were collected from healthy donors and from patients with newly diagnosed HPV-positive oropharyngeal carcinoma as confirmed by tissue biopsy with >70% p16-positivity by immunohistochemistry.

DNA purification, quantification, and size selection

DNA purification from saliva samples was performed utilizing three different approaches. First, cellular debris was removed from the samples by centrifuging at 300g for 20 minutes followed by 10,000g for 20 minutes.



As the known clinical utility of blood-based ctDNA testing expands, there is increasing interest in evaluating alternative biofluids as sources of ctDNA for complementary diagnostic applications. Saliva presents distinct advantages over blood particularly for localized HNSCC due to the proximity to primary tumors and ease of collection.



The authors would like to thank the healthy donors and patients for their contributions. We thank Katrina Rey-McIntyre and Kezia Willie-Ramharack for assisting with clinical protocols, patient enrolment, and provision of samples.

Citation: Brooks PJ, Malkin EZ, De Michino S, Bratman SV (2023) Isolation of salivary cell-free DNA for cancer detection. PLoS ONE 18(5): e0285214.

Editor: Elingarami Sauli, Nelson Mandela African Institute of Science and Technology, UNITED REPUBLIC OF TANZANIA

Received: January 29, 2023; Accepted: April 17, 2023; Published: May 2, 2023.

Copyright: © 2023 Brooks et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper.

Funding: The Cancer Research Society (Grant 843299) was received by SVB. The Catalyst Grant from Princess Margaret Cancer Centre was received by SVB. SVB was supported by the Gattuso-Slaight Personalized Cancer Medicine Fund at the Princess Margaret Cancer Centre and the Mariano Elia Chair in Head & Neck Cancer Research at University Health Network and University of Toronto. PJB was supported by a Princess Margaret Cancer Research Institute Fellowship as well as a Strategic Training in Transdisciplinary Radiation Science for the 21st Century (STARS21) training award. EZM was supported by the Frederick Banting and Charles Best Canada Graduate Scholarship (FRN: FBD-175873) through the Canadian Institutes for Health Research, with additional support from the Government of Ontario and the Temerty Faculty of Medicine, University of Toronto. SDM was supported by the Frederick Banting and Charles Best Canada Graduate Scholarship (FRN: FBD-457244) through the Canadian Institutes for Health Research. We would like to acknowledge the Princess Margaret Cancer Foundation and the Princess Margaret Head & Neck Translational Program, supported by philanthropic funds from the Wharton Family, Joe’s Team, Gordon Tozer, and the Reed Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: SVB is inventor on patents related to cell-free DNA mutation and methylation analysis technologies that have been licensed to Roche Molecular Diagnostics and Adela, respectively. SVB is a cofounder of, has ownership in, and serves in a leadership role at Adela. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

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