Quantification of human papillomavirus cell-free DNA from low-volume blood plasma samples by digital PCR

ABSTRACT The incidence rate of human papillomavirus-driven oropharyngeal cancer (HPV-OPC) is increasing in countries with high human development index. HPV cell-free DNA (cfDNA) isolated from 3 to 4 mL blood plasma has been successfully used for therapy surveillance. A highly discussed application of HPV-cfDNA is early detection of HPV-OPC. This requires sensitive and specific cfDNA detection as cfDNA levels can be very low. To study the predictive power of pre-diagnostic HPV-cfDNA, archived samples from epidemiological cohorts with limited plasma volume are an important source. To establish a cfDNA detection workflow for low plasma volumes, we compared cfDNA purification methods [MagNA Pure 96 (MP96) and QIAamp ccfDNA/RNA] and digital PCR systems (Biorad QX200 and QIAGEN QIAcuity One). Final assay validation included 65 low-volume plasma samples from oropharyngeal cancer (OPC) patients with defined HPV status stored for 2–9 years. MP96 yielded a 28% higher cfDNA isolation efficiency in comparison to QIAamp. Both digital PCR systems showed comparable analytical sensitivity (6–17 copies for HPV16 and HPV33), but QIAcuity detected both types in the same assay. In the validation set, the assay had 80% sensitivity (n = 28/35) for HPV16 and HPV33 and a specificity of 97% (n = 29/30). In samples with ≥750 µL plasma, the sensitivity was 85% (n = 17/20), while in samples with <750 µL plasma, it was 73% (n = 11/15). Despite the expected drop in sensitivity with decreased plasma volume, the assay is sensitive and highly specific even in low-volume samples and thus suited for studies exploring HPV-cfDNA as an early HPV-OPC detection marker in low-volume archival material. IMPORTANCE HPV-OPC has a favorable prognosis compared to HPV-negative OPC. However, the majority of tumors is diagnosed after regional spread, thus making intensive treatment necessary. This can cause lasting morbidity with a large impact on quality of life. One potential method to decrease treatment-related morbidity is early detection of the cancer. HPV cfDNA has been successfully used for therapy surveillance and has also been detected in pre-diagnostic samples of HPV-OPC patients. These pre-diagnostic samples are only commonly available from biobanks, which usually only have small volumes of blood plasma available. Hence, we have developed a workflow optimized for small-volume archival samples. With this method, a high sensitivity can be achieved despite sample limitations, making it suitable to conduct further large-scale biobank studies of HPV-cfDNA as a prognostic biomarker for HPV-OPC.

1st Editorial Decision Re: Spectrum00024-24 (Quantification of Human Papillomavirus cell-free DNA from low volume blood plasma samples by digital PCR) Dear Dr. Daniela Höfler: Thank you for the privilege of reviewing your work.Below you will find my comments, instructions from the Spectrum editorial office, and the reviewer comments.
I have now received reviews of experts in the field, which are appended below.Reviewer #1 had two additional suggestions that should be addressed before publication.Thank you for submitting to Spectrum.
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Sincerely, Peter Pelka Editor Microbiology Spectrum
Reviewer #1 (Comments for the Author): The authors have established a dPCR assay and workflow for detecting HPV16 and HPV33 E6 in low volume plasma samples from OPC patients.This workflow was shown to detect sensitively and specifically HPV16 and HPV33.The article is very well written, and of a great interest for the scientific community as there is an urgent need to develop and validate body-fluids based screening methods for the early detection of HPV-driven OPCs, and screening.
Minor revisions: 1-The authors have used low volume plasma samples with defined HPV status stored for 2-9 years.Did the sensitivity vary with the storage time?Would it be possible to perform this correlation?2-Most of the HPV genotyping assays are based on L1 gene.Why did the authors target E6 or E7 genes for HPV16 and HPV33 detection?
Reviewer #2 (Comments for the Author): Some specific comments: A section in the discussion that talks about other potential methods for cfDNA purification; along with the merits and drawbacks would be useful -especially as it relates to the 2 methods tested in this study.Why did the authors pick these 2 methods to test and not others?This could be described better to give the readers more insight into the choices made for this study.
The volume of serum used to extract the cfDNA seems to be critical.Perhaps alluding to the importance of conducting an experiment where cfDNA isolation efficiency as a function of serum volume (spiked healthy donor) can be assessed to determine optimal volumes would be necessary here.
Overall the experiments are described well, and the conclusions are justified by the results.Reviewer #1: "The authors have used low volume plasma samples with defined HPV status stored for 2-9 years.Did the sensitivity vary with the storage time?Would it be possible to perform this correlation?"This is an interesting question which can be answered based on our data, so information regarding this question was added in section 3.4.In short, sensitivity does not follow a clear trend with age of the sample, so it does not seem to be influenced by storage times up to 9 years.

Reviewer #1: "Most of the HPV genotyping assays are based on L1 gene. Why did the authors target E6 or E7 genes for HPV16 and HPV33 detection?"
It is true that many HPV assays, especially those used for cervical cancer screening, are based on L1.Nevertheless, we compared the performance of three different L1 and E6/E7 based (q)PCR methods in cervical (pre-) cancerous swabs and did not find significant differences in detecting high grade lesions (unpublished data).In addition, other publications have found the E6 and E7 region of the HPV genome are highly conserved regions within the HPV genome and are often amplified in cancer (10.1101/gr.164806.113), which reduces the risk of a false negative result by primers and probes not binding due to sequence variations in the primer target regions or lack of target sequence.As E6 and E7 are necessary oncogenes that drive growth of HPVdriven carcinomas, these genes would very rarely be deleted in HPV-driven carcinoma cells and consequently not missed by a PCR targeting these genes.We now addressed your question in the introduction of the manuscript.
Reviewer #2: "A section in the discussion that talks about other potential methods for cfDNA purification; along with the merits and drawbacks would be useful -especially To Microbiology Spectrum as it relates to the 2 methods tested in this study.Why did the authors pick these 2 methods to test and not others?This could be described better to give the readers more insight into the choices made for this study." This is an important point that is now addressed in the results section 3.2 of the manuscript.The two methods chosen were chosen to represent the main two technologies used for DNA purificationcolumn-based methods (QIAmp kit) and magnetic bead-based methods (MagNA Pure).
Reviewer #2: "The volume of serum used to extract the cfDNA seems to be critical.Perhaps alluding to the importance of conducting an experiment where cfDNA isolation efficiency as a function of serum volume (spiked healthy donor) can be assessed to determine optimal volumes would be necessary here." The volume of blood plasma used is indeed highly important for the sensitivity of cfDNA analysis.Therefore, we included the comparison of sensitivity using different sample volumes in section 3.3 of the manuscript.As demonstrating the performance of DNA purification on clinical samples is closer to the intended application of the assay than using spike-in DNA we had decided not to include data about the effect of sample volume on isolation efficiency of spike-in DNA.
In the data included in the manuscript it is shown that isolation efficiency is virtually identical between the different volumes used as the number of HPV copies detected per volume was identical (see fig. 5).To emphasize this the result section 3.3 of the manuscript has been edited.
We hope that the edits made to the manuscript are sufficient to qualify it for publication.

Fabian Rosing
April 23, 2024 1st Revision -Editorial Decision Re: Spectrum00024-24R1 (Quantification of Human Papillomavirus cell-free DNA from low volume blood plasma samples by digital PCR) Dear Dr. Daniela Höfler: Your manuscript has been accepted, and I am forwarding it to the ASM production staff for publication.Your paper will first be checked to make sure all elements meet the technical requirements.ASM staff will contact you if anything needs to be revised before copyediting and production can begin.Otherwise, you will be notified when your proofs are ready to be viewed.
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. Dr. med.Dr. h. c. Michael Baumann Ursula Weyrich Deutsche Bank Heidelberg IBAN: DE09 6727 0003 0015 7008 00 BIC (SWIFT): DEUT DES M672 Deutsche Bundesbank Karlsruhe IBAN: DE39 6600 0000 0067 0019 02 BIC (SWIFT): MARK DEF 1660 Dear Reviewers, firstly, I would like to thank you for taking the time to review this manuscript, your feedback is much appreciated.I have carefully read your comments and have addressed any issues you have pointed out.Below you can find my pointby-point response to your comments.