Dataset Information

Comparison of next generation sequencing, droplet digital PCR, and quantitative real-time PCR for the earlier detection and quantification of HPV in HPV-positive oropharyngeal cancer.

ABSTRACT:

Background

Human papillomavirus (HPV) causes nearly 80% of oropharynx cancers diagnosed in the United States, with incidence increasing each year. Analysis of cfDNA in plasma and oral rinse has the potential to detect these cases earlier than their typical presentation, but their utility and the best method to detect HPV in plasma and oral rinse samples is unknown.

Materials and methods

We directly compared next generation sequencing (NGS), droplet digital PCR (ddPCR), and quantitative real-time PCR (qPCR) for their ability to detect HPV16 DNA in plasma and oral rinse from 66 patients diagnosed with HPV16-positive oropharyngeal cancer (HPV16-OPC).

Results

HPV DNA detection by NGS and ddPCR in plasma samples both had good sensitivity (70%) for HPV16-OPC compared to 20.6% sensitivity by qPCR (p < 0.001). In oral rinse, NGS demonstrated a superior sensitivity of 75.0% as compared to both ddPCR (8.3%, p < 0.001) and qPCR (2.1%, p < 0.001). In a limited cohort of follow up patients, HPV levels detected in plasma by NGS but not ddPCR or qPCR reflected disease remission or progression.

Conclusions

These results suggest that NGS has the best sensitivity for detecting HPV in both plasma and oral rinse and may play a role in monitoring patients for disease recurrence. Additional studies are needed to define the specificity of NGS for similar patient cohorts.

SUBMITTER: Mattox AK

PROVIDER: S-EPMC9058207 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Publications

Comparison of next generation sequencing, droplet digital PCR, and quantitative real-time PCR for the earlier detection and quantification of HPV in HPV-positive oropharyngeal cancer.

Mattox Austin K AK D'Souza Gypsyamber G Khan Zubair Z Allen Hailey H Henson Stephanie S Seiwert Tanguy Y TY Koch Wayne W Pardoll Drew M DM Fakhry Carole C

Oral oncology 20220322

<h4>Background</h4>Human papillomavirus (HPV) causes nearly 80% of oropharynx cancers diagnosed in the United States, with incidence increasing each year. Analysis of cfDNA in plasma and oral rinse has the potential to detect these cases earlier than their typical presentation, but their utility and the best method to detect HPV in plasma and oral rinse samples is unknown.<h4>Materials and methods</h4>We directly compared next generation sequencing (NGS), droplet digital PCR (ddPCR), and quantit ...[more]

PMID: 35334415

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Project description:Cell-associated (CA) HIV-1 RNA is considered a potential marker for assessment of viral reservoir dynamics and antiretroviral therapy (ART) response in HIV-infected patients. Recent studies employed sensitive seminested real-time quantitative (q)PCR to quantify CA HIV-1 RNA. Digital PCR has been recently described as an alternative PCR-based technique for absolute quantification with higher accuracy compared to qPCR. Here, a comparison was made between the droplet digital PCR (ddPCR) and the seminested qPCR for quantification of unspliced (us) and multiply spliced (ms) CA HIV-1 RNA. Synthetic RNA standards and CA HIV-1 RNA from infected patients on and off ART (N = 34) were quantified with both methods. Correlations were observed between the methods both for serially diluted synthetic standards (usRNA: R2 = 0.97, msRNA: R2 = 0.92) and patient-derived samples (usRNA: R2 = 0.51, msRNA: R2 = 0.87). Seminested qPCR showed better quantitative linearity, accuracy and sensitivity in the quantification of synthetic standards than ddPCR, especially in the lower quantification ranges. Both methods demonstrated equally high detection rate of usRNA in patient samples on and off ART (91%), whereas ddPCR detected msRNA in larger proportion of samples from ART-treated patients (p = 0.13). We observed an average agreement between the methods for usRNA quantification in patient samples, albeit with a large standard deviation (bias = 0.05±0.75 log10). However, a bias of 0.94±0.36 log10 was observed for msRNA. No-template controls were consistently negative in the seminested qPCR, but yielded a positive ddPCR signal for some wells. Therefore, the false positive signals may have affected the detection power of ddPCR in this study. Digital PCR is promising for HIV nucleic acid quantification, but the false positive signals need further attention. Quantitative assays for CA HIV RNA have the potential to improve monitoring of patients on ART and to be used in clinical studies aimed at HIV eradication, but should be cross-validated by multiple laboratories prior to wider use.

Dataset Information

Comparison of next generation sequencing, droplet digital PCR, and quantitative real-time PCR for the earlier detection and quantification of HPV in HPV-positive oropharyngeal cancer.

Background

Materials and methods

Results

Conclusions

Publications

Comparison of next generation sequencing, droplet digital PCR, and quantitative real-time PCR for the earlier detection and quantification of HPV in HPV-positive oropharyngeal cancer.

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