Application of Cobas 6800 HPV testing and qPCR utilizing three genomically-optimized primer sets for detecting HPV DNA in the blood of HPV Positive dysplasia and cancer patients

Sep 22, 2026

Quantitative polymerase chain reaction (qPCR) measuring viral DNA in plasma can enable detection and/or monitoring of high-risk human papillomavirus (hrHPV)–associated cancers. The study evaluated Roche cobas 6800 HPV testing for hrHPV detection in plasma or peripheral blood mononuclear cells (PBMC) from patients with HPV-associated dysplasia or cancer. The assay detected hrHPV DNA in pre-treatment plasma of advanced cervical cancer patients, but not early-stage or dysplasia cases, and triplex qPCR increased sensitivity by detecting HPV16 DNA. Serial post-treatment sampling showed cobas HPV produced no reproducible signal, while triplex qPCR sporadically detected HPV DNA in a subset.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages14

Reading Time35 min

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Introduction

HPV infection and risk across cancer types

Screening strategies and the role of HPV testing

OPEN ACCESS Citation: Mostafa HH, Fall A, Gaillard S, Fakhry C, Khan Z, Seiwert T, et al. (2026) Application of Cobas 6800 HPV testing and qPCR utilizing three genomically-optimized primer sets for detecting HPV DNA in the blood of HPV Positive dysplasia and cancer patients. PLoSOne 21(8): e0354671. [https://doi.org/10.1371/](https://doi.org/10.1371/)[ ](https://doi.org/10.1371/)[journal.pone.0354671](journal.pone.0354671) Editor: Maria Lina Tornesello, Istituto Nazionale Tumori IRCCS Fondazione Pascale, ITALY Received: April 28, 2026 Accepted: July 7, 2026 Published: August 25, 2026 Copyright: © 2026 Mostafa 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 statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by National Institutes of Health ([https://www.nih](https://www.nih). RESEARCH ARTICLE Application of Cobas 6800 HPV testing and qPCR utilizing three genomically-optimized primer sets for detecting HPV DNA in the blood of HPV Positive dysplasia and cancer patients Heba H. Mostafa1, Amary Fall1, Stéphanie Gaillard2,3, Carole Fakhry4, Zubair Khan4, Tanguy Seiwert4, Hao Wang2, Hua-Ling Tsai2,5, Amanda N. Fader3, Charles A. Leath, III6, Warner K. Huh6, Rebecca C. Arend6, Ya-chea Tsai1, Joseph A. Califano7, Deyin Xing1, T.-C. Wu1,2,3, Richard B. S. Roden1,2,3* 1 Department of Pathology, Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America, 2 Department of Oncology, Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America, 3 Department of Gynecology/Obstetrics, Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America, 4 Department of Department of Otolaryngology Head and Neck Surgery, Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America, 5 Department of Biostatistics, Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America, 6 Division of Gynecologic Oncology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America, 7 Moores Cancer Center, University of California San Diego, San Diego, California, United States of America * [roden@jhmi.edu](roden@jhmi.edu) Abstract Use of quantitative polymerase chain reaction (qPCR) to measure viral DNA in plasma has promise for the detection and/or monitoring of high-risk human papillomavirus (hrHPV)-associated cancers. We examined Roche’s cobas 6800 HPV test for detection of hrHPV in plasma or peripheral blood mononuclear cells (PBMC) of patients with HPV-associated dysplasia or cancer. In our pilot study, this assay detected hrHPV DNA in 1 mL pre-treatment plasma of patients with advanced (stage II-IV) cervical cancer, but not stage I or dysplasia cases, or in PBMC. Plasma of oropharyngeal cancer patients also tested positive in most cases. Seeking to enhance sensitivity, we re-tested these pilot samples with qPCR employing three published primer sets (E7, BR E1-5, HR E5L2-4) selected for improved detection of HPV16 in cancer. This triplex assay detected quantifiable levels of HPV16 in pre-treatment plasma of advanced (stage II-IV) cervical cancer cases, and lower levels in some stage I and dysplasia cases. In serial sampling of HPV16 +cervical cancer patients post-treatment with no evidence of disease over ~2 years, no reproducible signal was detected by cobas HPV testing, whereas the triplex qPCR sporadically detected HPV DNA in plasma and PBMC samples of a subset of these patients. PLOS One | [https://doi.org/10.1371/journal.pone.0354671](https://doi.org/10.1371/journal.pone.0354671) August 25, 2026 1 / 14 gov/) grant P50CA098252 to T.-C.W. , grant R01CA237067 to T.-C.W. and R. B.S. R. , and grant P30CA006973 to R. B.S. R. The

FAQs

What was the purpose of using qPCR and the cobas 6800 HPV test in this study?

The study aimed to detect and/or monitor hrHPV associated disease by measuring HPV DNA in blood (plasma and PBMC), comparing cobas 6800 HPV testing with a more sensitive qPCR approach.

How did cobas 6800 perform for different patient groups in pre-treatment samples?

In the pilot study, cobas 6800 detected hrHPV DNA in 1 mL pre-treatment plasma from patients with advanced (stage II–IV) cervical cancer, while it did not detect in stage I or dysplasia cases, nor in PBMC.

What changed when the researchers retested samples with triplex qPCR primer sets?

To enhance sensitivity, the pilot samples were retested using qPCR with three genomically optimized primer sets (E7, BR E1-5, HR E5L2-4), which allowed detection of quantifiable HPV16 DNA in advanced cervical cancer plasma and lower levels in some stage I and dysplasia cases.

Sep 22, 2026

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