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Comparing the Diagnostic Performance of Quantitative PCR, Digital Droplet PCR, and Next-Generation Sequencing Liquid Biopsies for Human Papillomavirus–Associated Cancers

2023; Elsevier BV; Volume: 26; Issue: 3 Linguagem: Inglês

10.1016/j.jmoldx.2023.11.007

1943-7811

Saskia Naegele, Daniel A. Ruiz-Torres, Yan Zhao, Deborah Goss, Daniel L. Faden,

Head and Neck Cancer Studies

Human papillomavirus (HPV)-associated cancers, including oropharyngeal squamous cell carcinoma (HPV + OPSCC), cervical cancer, and squamous cell carcinoma of the anus (HPV + SCCA), release circulating tumor HPV DNA (ctHPVDNA) into the blood. The diagnostic performance of ctHPVDNA detection depends on the approaches used and the individual assay metrics. A comparison of these approaches has not been systematically performed to inform expected performance, which in turn affects clinical interpretation. A meta-analysis was performed using Ovid MEDLINE, Embase, and Web of Science Core Collection databases to assess the diagnostic accuracy of ctHPVDNA detection across cancer anatomic sites, detection platforms, and blood components. The population included patients with HPV + OPSCC, HPV-associated cervical cancer, and HPV + SCCA with pretreatment samples analyzed by quantitative PCR (qPCR), digital droplet PCR (ddPCR), or next-generation sequencing (NGS). Thirty-six studies involving 2986 patients met the inclusion criteria. The sensitivity, specificity, and quality of each study were assessed and pooled for each analysis. The sensitivity of ctHPVDNA detection was greatest with NGS, followed by ddPCR and then qPCR when pooling all studies, whereas specificity was similar (sensitivity: ddPCR > qPCR, P < 0.001; NGS > ddPCR, P = 0.014). ctHPVDNA from OPSCC was more easily detected compared with cervical cancer and SCCA, overall (P = 0.044). In conclusion, detection platform, anatomic site of the cancer, and blood component used affects ctHPVDNA detection and must be considered when interpreting results. Plasma NGS-based testing may be the most sensitive approach for ctHPVDNA overall. Human papillomavirus (HPV)-associated cancers, including oropharyngeal squamous cell carcinoma (HPV + OPSCC), cervical cancer, and squamous cell carcinoma of the anus (HPV + SCCA), release circulating tumor HPV DNA (ctHPVDNA) into the blood. The diagnostic performance of ctHPVDNA detection depends on the approaches used and the individual assay metrics. A comparison of these approaches has not been systematically performed to inform expected performance, which in turn affects clinical interpretation. A meta-analysis was performed using Ovid MEDLINE, Embase, and Web of Science Core Collection databases to assess the diagnostic accuracy of ctHPVDNA detection across cancer anatomic sites, detection platforms, and blood components. The population included patients with HPV + OPSCC, HPV-associated cervical cancer, and HPV + SCCA with pretreatment samples analyzed by quantitative PCR (qPCR), digital droplet PCR (ddPCR), or next-generation sequencing (NGS). Thirty-six studies involving 2986 patients met the inclusion criteria. The sensitivity, specificity, and quality of each study were assessed and pooled for each analysis. The sensitivity of ctHPVDNA detection was greatest with NGS, followed by ddPCR and then qPCR when pooling all studies, whereas specificity was similar (sensitivity: ddPCR > qPCR, P < 0.001; NGS > ddPCR, P = 0.014). ctHPVDNA from OPSCC was more easily detected compared with cervical cancer and SCCA, overall (P = 0.044). In conclusion, detection platform, anatomic site of the cancer, and blood component used affects ctHPVDNA detection and must be considered when interpreting results. Plasma NGS-based testing may be the most sensitive approach for ctHPVDNA overall. Human papillomaviruses (HPVs) are a family of DNA oncoviruses that cause benign and malignant lesions of the genital mucosa, upper respiratory tract, and skin. More than 200 distinct types of HPV have been identified, and at least 14 of them are classified as high risk, or capable of tumorigenesis, in specific anatomic sites.1Saraiya M. Unger E.R. Thompson T.D. Lynch C.F. Hernandez B.Y. Lyu C.W. Steinau M. Watson M. 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Cell-free HPV DNA provides an accurate and rapid diagnosis of HPV-associated head and neck cancer.Clin Cancer Res. 2022; 28: 719-727Crossref PubMed Scopus (36) Google Scholar In the past, conventional real-time quantitative PCR (qPCR) was the most common method used for the detection of ctDNA, but newer (and more costly) techniques, including droplet-digital PCR (ddPCR) and next-generation sequencing (NGS), have emerged.47Postel M. Roosen A. Laurent-Puig P. Taly V. Wang-Renault S.-F. Droplet-based digital PCR and next generation sequencing for monitoring circulating tumor DNA: a cancer diagnostic perspective.Expert Rev Mol Diagn. 2018; 18: 7-17Crossref PubMed Scopus (148) Google Scholar The diagnostic performance of ctHPVDNA detection depends on the approaches used and the individual assay metrics. A comparison of these approaches has not been systematically performed in the literature to inform expected performance, which in turn influences clinical interpretation. We tested the hypothesis that the sensitivity of NGS-based liquid biopsy is superior to that of qPCR and ddPCR at the time of diagnosis across HPV-associated cancers. A systematic review was performed by a medical librarian (D.G.) following the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses.48Moher D. Liberati A. Tetzlaff J. Altman D.G. PRISMA GroupPreferred reporting items for systematic reviews and meta-analyses: the PRISMA statement.Ann Intern Med. 2009; 151 (W64): 264-269Crossref PubMed Scopus (19877) Google Scholar A search of published studies in Ovid MEDLINE (Wolters-Kluwer, 1946–February 2022), Embase (Elsevier, 1947–February 2022), and Web of Science Core Collections (Clarivate, 1900–February 2022) was designed and conducted by a reference librarian (D.G.) on February 18 and February 23, 2022. (Registration may be required to access these databases.) Search strategies were customized for each database. Each search used a combination of controlled vocabulary and key word terms relating to the diagnosis of HPV-associated cancer (head and neck, anal, vulvar, cervical, vaginal, and penile) using qPCR, ddPCR, or NGS testing. The search was constructed to exclude non-human studies. No filters for language, study design, date of publication, or country of origin were used in the search, which produced 251 articles (Figure 1). All references were exported into EndNote X7.8 (Clarivate, Philadelphia, PA). Duplicates were removed first by the automated process in EndNote and then manually by the librarian; this left 153 articles, which were exported into Covidence (Melbourne, VIC, Australia) for study screening, selection, and data extraction. Nine subsequent articles were found through searching the references of included articles, thus yielding a total of 162 articles for screening. Studies that examined ctDNA in any HPV-associated cancer with qPCR, ddPCR, or NGS were considered eligible for inclusion. Extracted data comprised the following: anatomic subsite; HPV status; HPV assay detection method; number of patients tested on each assay; number of true-positive, false-positive, false-negative, and true-negative findings; source of ctHPVDNA (plasma or serum); probe target gene (if ddPCR was used); and amplicon or hybrid capture (if NGS was used). Only studies written in English were included. Titles and abstracts were screened independently by two authors (S.N. and D.A.R.-T.) for full-text review. The same two authors independently conducted the full-text review. Any disagreements in the screening process were settled by discussion and consensus between the two authors. All eligible studies were screened for duplicate data by comparing authors, timeframe of data collection, and outcomes. After full text screening, 36 studies remained for the quantitative synthesis. R 4.1.2 (R Foundation for Statistical Computing, Vienna, Austria; https://cran.r-project.org) was used to conduct the statistical analysis and the R packages "meta" and "metafor" were used for the meta-analysis. The studies missing one of the two values [true positive, false negative (TP, FN) or true negative, false positive (TN, FP)] were excluded. Using the random effects model, sensitivity, including 95% confidence intervals (CIs), was computed from TP and TP + FN, and specificity including 95% CI was computed from TN and TN + FP. Subgroups were defined differently for each model, and the pooled means were calculated respectively. Subsequently, separate meta-regressions were performed to test the association of each study characteristic with HPV sensitivities and specificities. Interstudy variability and between-study variance were assessed by using Cochran's Q statistic. The percentage of variation explained by true heterogeneity opposed to sampling error was calculated with the I2 statistic. A two-sided P < 0.05 was considered to be significant. Potential publication bias was evaluated by using the Quality Assessment of Diagnostic Accuracy Studies-2 tool (https://www.bristol.ac.uk/population-health-sciences/projects/quadas/quadas-2). The risk of bias was judged as high or low when the answers to all signaling questions in the four domains were yes or no, respectively. If the information was not sufficient, an unclear bias was used. Most studies were at unclear or low risk of bias for flow and timing and index test domains (Figure 2). Notably, for reference standard, three studies were at unclear risk of bias and for patient selection, four studies were at high risk of bias. Regarding applicability, 33 studies were at low risk of bias for reference standard and index test, but five studies were at high risk of bias for patient selection. 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