Development of a high-performance multi-probe droplet digital PCR assay for high-sensitivity detection of human papillomavirus circulating tumor DNA from plasma

Chandan Bhambhani; Erin Sandford; Catherine T Haring; Collin Brummel; Kirsten L Tuck; Mary Olesnavich; Apurva D Bhangale; Heather M Walline; Sarah M Dermody; Matthew E Spector; Steven B Chinn; Keith Casper; Michelle Mierzwa; Paul L Swiecicki; J Chad Brenner; Muneesh Tewari

doi:10.1016/j.oraloncology.2023.106436

Development of a high-performance multi-probe droplet digital PCR assay for high-sensitivity detection of human papillomavirus circulating tumor DNA from plasma

Oral Oncol. 2023 Aug:143:106436. doi: 10.1016/j.oraloncology.2023.106436. Epub 2023 Jun 1.

Authors

Chandan Bhambhani¹, Erin Sandford¹, Catherine T Haring², Collin Brummel², Kirsten L Tuck¹, Mary Olesnavich¹, Apurva D Bhangale², Heather M Walline², Sarah M Dermody², Matthew E Spector³, Steven B Chinn³, Keith Casper², Michelle Mierzwa⁴, Paul L Swiecicki⁵, J Chad Brenner⁶, Muneesh Tewari⁷

Affiliations

¹ Department of Internal Medicine, Division of Hematology and Oncology, University of Michigan, USA.
² Department of Otolaryngology- Head and Neck Surgery, University of Michigan, USA.
³ Department of Otolaryngology- Head and Neck Surgery, University of Michigan, USA; Rogel Cancer Center, University of Michigan, USA.
⁴ Rogel Cancer Center, University of Michigan, USA; Department of Radiation Oncology, University of Michigan, USA.
⁵ Department of Internal Medicine, Division of Hematology and Oncology, University of Michigan, USA; Rogel Cancer Center, University of Michigan, USA; VA Ann Arbor Healthcare System, Ann Arbor, MI, USA.
⁶ Department of Otolaryngology- Head and Neck Surgery, University of Michigan, USA; Rogel Cancer Center, University of Michigan, USA; Department of Pharmacology, University of Michigan, USA.
⁷ Department of Internal Medicine, Division of Hematology and Oncology, University of Michigan, USA; Rogel Cancer Center, University of Michigan, USA; VA Ann Arbor Healthcare System, Ann Arbor, MI, USA; Department of Biomedical Engineering, University of Michigan, USA; Center for Computational Medicine and Bioinformatics, University of Michigan, USA. Electronic address: mtewari@med.umich.edu.

PMID: 37269557
DOI: 10.1016/j.oraloncology.2023.106436

Abstract

Objectives: To develop a high-performance droplet digital PCR (ddPCR) assay capable of enhancing the detection of human papillomavirus (HPV) circulating tumor DNA (ctDNA) in plasma from patients with HPV-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC).

Materials and methods: Plasma samples from subjects with HPV+ OPSCC were collected. We developed a high-performance ddPCR assay designed to simultaneously target nine regions of the HPV16 genome.

Results: The new assay termed 'ctDNA HPV16 Assessment using Multiple Probes' (CHAMP- 16) yielded significantly higher HPV16 counts compared to our previously validated 'Single-Probe' (SP) assay and a commercially available NavDx® assay. Analytical validation demonstrated that the CHAMP-16 assay had a limit of detection (LoD) of 4.1 copies per reaction, corresponding to < 1 genome equivalent (GE) of HPV16. When tested on plasma ctDNA from 21 patients with early-stage HPV+ OPSCC and known HPV16 ctDNA using the SP assay, all patients were positive for HPV16 ctDNA in both assays and the CHAMP-16 assay displayed 6.6-fold higher HPV16 signal on average. Finally, in a longitudinal analysis of samples from a patient with recurrent disease, the CHAMP-16 assay detected HPV16 ctDNA signal ∼ 20 months prior to the conventional SP assay.

Conclusion: Increased HPV16 signal detection using the CHAMP-16 assay suggests the potential for detection of recurrences significantly earlier than with conventional ddPCR assays in patients with HPV16+ OPSCC. Critically, this multi-probe approach maintains the cost-benefit advantage of ddPCR over next generation sequencing (NGS) approaches, supporting the cost-effectiveness of this assay for both large population screening and routine post-treatment surveillance.

Keywords: Anal cancer; Biomarker; Cell-free DNA; Cervical cancer; Circulating tumor DNA; HPV; Head and neck cancer; Liquid biopsy; Oropharyngeal cancer; ctDNA.

Published by Elsevier Ltd.

MeSH terms

Carcinoma, Squamous Cell* / pathology
Circulating Tumor DNA*
Head and Neck Neoplasms*
Human Papillomavirus Viruses
Human papillomavirus 16 / genetics
Humans
Oropharyngeal Neoplasms*
Papillomavirus Infections*
Polymerase Chain Reaction

Substances

Circulating Tumor DNA