Preoperative Circulating Tumor HPV DNA and Oropharyngeal Squamous Cell Disease

Key Points

Question

What is the association of preoperative circulating tumor human papillomavirus (HPV) DNA levels with disease burden in patients with HPV-associated oropharyngeal squamous cell carcinoma who have undergone transoral robotic surgery?

Findings

In this cross-sectional study of 70 patients, increasing levels of preoperative circulating tumor HPV DNA were not associated with increased pathologic tumor stage, the number of lymph nodes involved, or adverse surgical pathology characteristics.

Meaning

These findings suggest that preoperative circulating tumor HPV DNA levels may not be useful predictors of disease burden and adverse pathology characteristics in patients who have undergone primary transoral robotic surgery.

Abstract

Importance

The utility of preoperative circulating tumor tissue-modified viral human papillomavirus DNA (TTMV-HPV DNA) levels in predicting human papillomavirus (HPV)–associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC) disease burden is unknown.

Objective

To determine if preoperative circulating tumor HPV DNA (ctHPVDNA) is associated with disease burden in patients with HPV+ OPSCC who have undergone transoral robotic surgery (TORS).

Design, Setting, and Participants

This cross-sectional study comprised patients with HPV+ OPSCC who underwent primary TORS between September 2021 and April 2023 at one tertiary academic institution. Patients with treatment-naive HPV+ OPSCC (p16-positive) and preoperative ctHPVDNA levels were included, and those who underwent neck mass excision before ctHPVDNA collection were excluded.

Main Outcomes and Measures

The main outcome was the association of increasing preoperative ctHPVDNA levels with tumor size and lymph node involvement in surgical pathology. The secondary outcome was the association between preoperative ctHPVDNA levels and adverse pathology, which included lymphovascular invasion, perineural invasion, or extranodal extension.

Results

A total of 70 patients were included in the study (65 men [93%]; mean [SD] age, 61 [8] years). Baseline ctHPVDNA levels ranged from 0 fragments/milliliter of plasma (frag/mL) to 49 452 frag/mL (median [IQR], 272 [30-811] frag/mL). Overall, 58 patients (83%) had positive results for ctHPVDNA, 1 (1.4%) had indeterminate results, and 11 (15.6%) had negative results. The sensitivity of detectable ctHPVDNA for identifying patients with pathology-confirmed HPV+ OPSCC was 84%. Twenty-seven patients (39%) had pathologic tumor (pT) staging of pT0 or pT1, 34 (49%) had pT2 staging, and 9 patients (13%) had pT3 or pT4 staging. No clinically meaningful difference between detectable and undetectable preoperative ctHPVDNA cohorts was found for tumor size or adverse pathology. Although the median preoperative ctHPVDNA appeared to be higher in pT2 through pT4 stages and pN1 or pN2 stages, effect sizes were small (pT stage: η2, 0.002 [95% CI, −1.188 to 0.827]; pN stage: η2, 0.043 [95% CI, −0.188 to 2.600]). Median preoperative log(TTMV-HPV DNA) was higher in active smokers (8.79 [95% CI, 3.55-5.76]), compared with never smokers (5.92 [95% CI, −0.97 to 1.81]) and former smokers (4.99 [95% CI, 0.92-6.23]). Regression analysis did not show an association between tumor dimension or metastatic lymph node deposit size and preoperative log(TTMV-HPV DNA). After univariate analysis, no association was found between higher log(TTMV-HPV DNA) levels and adverse pathology.

Conclusions and Relevance

In this cross-sectional study, preoperative ctHPVDNA levels were not associated with disease burden in patients with HPV+ OPSCC who underwent TORS.

This cross-sectional study evaluates the association of preoperative circulating tumor HPV DNA levels with disease burden or adverse pathologic features among patients with HPV-associated oropharyngeal squamous cell carcinoma who have undergone primary transoral robotic surgery.

Introduction

Over the past few decades, human papillomavirus (HPV) has emerged as a leading factor co-occurring with oropharyngeal squamous cell carcinoma (OPSCC), with up to 80% to 90% of OPSCC cases associated with HPV.¹ Given the improved clinical prognosis of patients with HPV-positive OPSCC (HPV+ OPSCC) and the adverse effects associated with multimodal treatment, increased focus has been placed on the identification of prognostic and predictive markers to improve patient stratification and opportunities for treatment deintensification.^1,2,3

The recommended initial step to evaluate patients with suspected OPSCC is a fine-needle aspiration (FNA) of an enlarged cervical lymph node followed by an indirect or direct laryngoscopy with biopsy to identify a primary lesion.⁴ However, FNA as an initial screening method is limited by its intrinsic failure rate of 20% to 30% due to inadequate cellular material collection.^5,6 Additionally, the lack of standardization and low sensitivity of p16 immunohistochemistry staining, a surrogate marker for high-risk HPV infection, on FNA specimens limits its prognostic utility in patients with OPSCC.^7,8,9 Per the National Comprehensive Cancer Network, the modality of primary treatment in HPV+ OPSCC is currently determined by clinical stage based on pretreatment computed tomography and positron emission tomography–computed tomography findings.¹⁰ Recent efforts have been directed toward the evaluation of biomarkers that may complement clinical staging to improve diagnostic and prognostic capabilities.¹¹ Circulating tumor HPV DNA (ctHPVDNA) is a potential serum biomarker for HPV+ tumors. It was first investigated for use in the management of cervical cancers and has been an emerging focus of research in head and neck cancers.^12,13,14,15

Commercially available assays of plasma ctHPVDNA have been developed to quantify HPV DNA fragments shed by tumor cells and identify high-risk HPV genotypes.¹⁶ As a minimally invasive test, it has favorable characteristics to be used as a potential diagnostic and surveillance tool in a clinical setting.^17,18 Previous studies, including a report by Ferrandino et al¹⁸ published in 2023, have reported excellent sensitivity and specificity of ctHPVDNA assays for initial diagnosis (up to 91.5% and 100%, respectively) and identification of recurrence (up to 88.4% and 100%, respectively) in patients with OPSCC. These ctHPVDNA assays also have been validated to accurately reflect the patient’s response to systemic treatment.^{17,18,19,20,21,22} Although a study by Rettig et al²³ published in 2022 showed an association between pretreatment ctHPVDNA levels and clinical disease burden on pretreatment positron emission tomography–computed tomography imaging, this study population predominantly consisted of patients undergoing primary systemic therapy.^14,23 Notably, the utility of preoperative ctHPVDNA serum assays in predicting disease burden in patients who have undergone primary surgery has not yet been well evaluated.

For this study, a commercially available digital droplet polymerase chain reaction (PCR)–based assay was used to determine a normalized circulating tumor tissue-modified viral (TTMV)–HPV DNA level. The objective of this study was to determine whether pretreatment TTMV-HPV DNA levels are associated with disease burden in patients with HPV+ OPSCC who have undergone primary transoral robotic surgery (TORS). Our goal was to further the understanding of the role that this biomarker plays in guiding treatment discussions that weigh primary surgery against systemic or trimodality therapy.

Methods

Study Design and Population

This cross-sectional study of patients with pathology-confirmed HPV+ (p16 immunohistochemistry staining [p16+]) OPSCC treated with TORS and cervical lymphadenectomy was conducted at the University of Pennsylvania in Philadelphia between September 2021 and April 2023. Patients were included if they had a preoperative TTMV-HPV DNA level collected as part of routine clinical care. Patients who underwent mass excision (eg, excisional lymph node biopsy or tonsillectomy) before TTMV-HPV DNA collection or those who received salvage surgery were excluded. Tumors were classified as HPV+ OPSCC through p16 immunohistochemistry staining, per the American Society of Clinical Oncology and the College of American Pathologists recommendations.^24,25 This study was approved by the institutional review board at the University of Pennsylvania. Written informed consent was obtained from all participants.

Data Collection

Demographics were abstracted from the electronic medical record. Given reported differences in HPV+ OPSCC epidemiologic characteristics, racially and ethnically minoritized patients were combined into one category, inclusive of Asian, American Indian and/or Alaska Native, Black or African American, Native Hawaiian or other Pacific Islander, or unknown race. Smoking status at the time of clinical evaluation was self-reported by patients.

Surgical specimens were independently evaluated by a board-certified pathologist. Pathologic tumor (pT) and pathologic nodal (pN) staging were categorized according to the American Joint Committee on Cancer (AJCC) Staging Manual, eighth edition.²⁶ Tumor characteristics such as greatest dimension, perineural invasion (PNI), and lymphovascular invasion (LVI) were collected from the surgical pathology reports. Cervical lymph node specimens were also evaluated for features such as extranodal extension (ENE) and size of the largest lymph node metastatic deposit.

Circulating TTMV-HPV DNA level was determined by digital droplet PCR analysis using a commercially available assay called NavDx (Naveris). The assay is performed independently by the test developer in a College of Pathologists–accredited and Clinical Laboratory Amendments–certified laboratory. Furthermore, the assay measures tumor-derived HPV DNA through algorithmic analysis of digital droplet PCR data and tumor-specific fragmentation patterns.^19,27 The results are typically reported back to the clinical team within 14 days. The assay tests for the HPV-16 strain, and if the sample is negative, additional assays will be performed for other high-risk HPV subtypes (18, 31, 33, and 35). Normalized TTMV-HPV DNA levels are reported as fragments per milliliter of plasma (frag/mL), and results of testing are categorized as negative (<5 frag/mL), indeterminate (5-7 frag/mL for HPV-16; 5-12 frag/mL for HPV-18, HPV-31, HPV-33, and HPV-35), or positive (>7 frag/mL for HPV-16; >12 frag/mL for non–HPV-16). In this study, positive and indeterminate results of testing for circulating TTMV-HPV DNA levels were categorized as detectable, and negative results were considered undetectable.

Statistical Analysis

Descriptive statistics were used to describe the overall study population, as well as the cohort with detectable TTMV-HPV DNA. Categorical variables were reported as frequency (percentage), while continuous variables were reported as mean (SD) or median (IQR). To compare proportions between 2 groups, differences in proportions and medians were reported with 95% CIs. For the comparison of proportions between 3 or more groups, Kendall τ (95% CI) was used to estimate association strengths (<0.1 = very weak, 0.10-0.19 = weak, 0.20-0.29 = moderate, and ≥0.30 = strong). Effect size when comparing medians between 3 or more groups was estimated using η² (95% CI) (0.01 = small, 0.06 = medium, and 0.14 = large). Simple linear regression and univariate and multivariate logistic regression analyses were performed to evaluate factors associated with TTMV-HPV DNA levels. Logistic regression results are presented as an odds ratio (OR) with a 95% CI. An α level of .05 was used to determine statistical significance for all statistical tests.

Due to the skewed nature of the preoperative TTMV-HPV DNA level data and inclusion of zero levels, a Box-Cox transformation was applied (TTMV-HPV DNA(λ) = log(TTMV-HPV DNA⁺λ), λ = 0.14). Because the transformed TTMV-HPV DNA variable failed the Shapiro-Wilk test for normality, nonparametric tests and a generalized linear model using a logarithmic link function with a γ distribution were ultimately used for our analysis. Statistical analyses were performed using R Studio statistical software, version 2023.03.1 (R Project for Statistical Computing).

Results

Study Population

A total of 70 patients were included in the study with 65 (93%) men and a mean (SD) age of 61 (8) years. The sample included 61 White individuals (87%) and 9 racially and ethnically minoritized patients (13%) (Table 1). For the smoker status variable, 41 patients (59%) identified as never smokers, while 5 patients (7%) identified as active smokers at the time of clinical evaluation. Overall, baseline circulating TTMV-HPV DNA levels ranged widely from 0 frag/mL to 49 452 frag/mL, with a median (IQR) level of 272 (30-811) frag/mL. Overall, 58 patients (83%) had positive results for these levels, whereas 1 patient (1.4%) had indeterminate results, and 11 patients (15.6%) had negative results. In addition, 7 patients (10%) had circulating TTMV-HPV DNA levels of 0. The most common HPV genotype among the patients with nonzero TTMV-HPV DNA levels was HPV-16, consisting of 61 patients (98%), with 1 patient (2%) found to have HPV-18. The sensitivity of the detectable circulating TTMV-HPV DNA for the detection of pathology-confirmed HPV+ OPSCC was represented by 59 of the 70 patients (84%).

Table 1. Patient Demographics and Circulating Tumor Tissue-Modified Viral Human Papillomavirus DNA Characteristics.

Characteristic	Overall, No.(%) (N = 70)
Age, mean (SD), y	61 (8)
Sex
Female	5 (7)
Male	65 (93)
Race
Black	5 (7)
White	61 (87)
Othera	4 (6)
Smoking status
Never smoker	41 (59)
Former smoker	24 (34)
Active smoker	5 (7)
HPV subtype
HPV-16	61 (98)
HPV-18	1 (2)
Baseline TTMV-HPV DNA levels, median (IQR), frag/mL	272 (30-811)
log (baseline TTMV-HPV DNA) levels, median (IQR)	5.6 (3.4-6.7)

Abbreviations: frag/mL, fragments per milliliter of plasma; HPV, human papillomavirus; TTMV-HPV DNA, tumor tissue-modified viral human papillomavirus DNA.

^a Asian, American Indian and/or Alaska Native, Black or African American, Native Hawaiian or other Pacific Islander, or unknown race.

Clinicopathologic Characteristics Associated with Circulating TTMV-HPV DNA Detection

Overall, 27 patients (39%) had pT0 or pT1 tumor staging, and 34 (49%) had pT2 tumor staging. Nine patients (13%) had advanced pT3 or pT4 tumor staging (Table 2). No meaningful difference in pT or pN stage was found between patients with detectable and undetectable preoperative circulating TTMV-HPV DNA. Additionally, no clinically meaningful difference between patients with detectable and undetectable preoperative TTMV-HPV DNA was found regarding the tumor size, or evidence of LVI, PNI, or ENE.

Table 2. Clinicopathologic Characteristics by Preoperative Circulating Tumor Tissue-Modified Viral Human Papillomavirus DNA Detection.

Characteristic	No. (%)		Effect size (95% CI)		Preoperative circulating TTMV-HPV DNA, median (IQR), frag/mL	Effect size (95% CI)
	Overall	Detectable preoperative circulating TTMV-HPV DNAa	Proportion difference	Kendall τ		Difference in medians	η2
pT stage
0-1	27 (39)	24 (89)	NA	0.022 (−0.143 to 0.933)	157 (26 to 624)	NA	0.002 (−1.188 to 0.827)
2	34 (49)	27 (79)			447 (87 to 888)
3-4	9 (13)	7 (78)			209 (11 to 381)
pN stage
0	7 (10)	3 (43)	NA	0.151 (−0.097 to 0.926)	2 (0 to 432)	NA	0.043 (−0.188 to 2.600)
1	53 (77)	47 (89)			209 (44 to 883)
2	9 (13)	7 (78)			563 (43 to 729)
Lymphovascular invasion
None	48 (69)	38 (79)	0.12 (−0.088 to 0.269)	NA	188 (24 to 819)	205 (−217 to 551)	NA
Present	22 (31)	20 (91)			376 (87 to 742)
Perineural invasion
None	61 (87)	50 (82)	0.07 (−0.262 to 0.216)	NA	225 (25 to 811)	174 (−224 to 512)	NA
Present	9 (13)	8 (89)			399 (206 to 634)
Extranodal extension
None	43 (59)	31 (79)	0.14 (−0.045 to 0.292)	NA	382 (48 to 864)	−221 (−593 to 210)	NA
Present	27 (41)	25 (93)			161 (31 to 638)

Abbreviations: frag/mL, fragments per milliliter of plasma; NA, not applicable; TTMV-HPV DNA, tumor tissue-modified viral human papillomavirus DNA.

^a TTMV-HPV DNA levels were greater than 5 frag/mL.

Clinicopathologic Characteristics Associated with Increased Circulating TTMV-HPV DNA Level

Median preoperative log(TTMV-HPV DNA) is increased in active smokers (8.79 [95% CI, 3.55-5.76]) compared with never smokers (5.92 [95% CI, −0.97 to 1.81) and former smokers (4.99 [95% CI, 0.92-6.23]). The associations of pT and pN stage with detectable preoperative TTMV-HPV DNA levels were categorized as very weak and weak, respectively (Kendall τ, 0.022 [95% CI, −0.143 to 0.933]; Kendall τ, 0.151 [95% CI, −0.097 to 0.926]). Furthermore, small effect sizes were observed when assessing differences in medians between pT and pN stage subgroups (pT stage: η², 0.002 [95% CI, −1.188 to 0.827]; pN stage: η², 0.043 [95% CI, −0.188 to 2.600]) (Table 2).

When comparing the greatest tumor dimension between detectable and undetectable TTMV-HPV DNA cohorts, the difference in medians was 0.15 cm (95% CI, −0.35 to 0.65). When comparing the size of metastatic lymph node deposits, the difference in medians was 1.2 cm (95% CI, 0.0 to 2.8).

Regression analysis did not demonstrate any association of tumor dimension (r, 0.02 [95% CI, −0.22 to 0.25]) or size of metastatic lymph node deposit (r, 0.19 [95% CI, −0.07 to 0.43]) with preoperative log(TTMV-HPV DNA). Furthermore, after univariate analysis, no association was found between increasing log(TTMV-HPV DNA) levels and evidence of LVI (OR, 1.09 [95% CI, 0.91-1.33), PNI (OR, 1.04 [95% CI, 0.81-1.36), or ENE (OR, 0.99 [95% CI, 0.83-1.20]) on surgical pathology.

Discussion

This cross-sectional study evaluated the association between pretreatment ctHPVDNA and surgical pathology findings in patients with HPV+ OPSCC who underwent primary surgery. Among the 70 patients in this study who underwent primary TORS with cervical lymphadenectomy, no association between baseline circulating TTMV-HPV DNA levels and disease burden was found. Specifically, increased circulating TTMV-HPV DNA levels did not appear to predict tumor size, pT or pN stage, or adverse cervical lymph node characteristics, such as ENE greater than 2 mm. This finding has important implications as clinicians have sought to understand the utility of preoperative circulating TTMV-HPV DNA values as a diagnostic and prognostic marker of disease burden as valued guidance for stratifying patients according to primary treatment packages.²⁸

The findings of the present study supplement existing studies evaluating the association of pretreatment ctHPVDNA with clinical OPSCC staging. Rettig et al²³ found in a cohort of 110 patients, where 27 patients received primary surgery, increasing circulating TTMV-HPV DNA levels were associated with the clinical nodal burden on imaging. Similarly, in a study of 103 patients receiving definitive chemoradiation, Chera et al¹⁹ also identified a significantly higher ctHPVDNA level in patients with greater clinical nodal burden (cN2a or cN2b). However, Rettig et al²³ did not report any association between increasing TTMV-HPV DNA levels and pT characteristics, yet the results suggested that detectable TTMV-HPV DNA levels were associated with surgical pathology in confirmed nodal disease, as in the current study. In this study of 70 patients undergoing primary surgery, no association between increasing preoperative TTMV-HPV DNA level with pT or pN stage was identified. Although clinical staging is crucial to informing the primary treatment mode, pathologic staging and surgical pathology characteristics are underlying factors associated with postoperative treatment planning and inform clinical discussions around whether adjuvant therapy de-escalation would be appropriate in a patient undergoing primary surgery. In this setting, the findings of the present study suggest that pretreatment TTMV-HPV DNA levels do not appear to predict disease burden and may not be useful for prognostic purposes. However, previous studies have validated the use of positive results of TTMV-HPV DNA assays to diagnose disease with high sensitivity and specificity, highlighting its potential as an alternative to FNA, which can be nondiagnostic for the presence of malignant neoplasm or indeterminate for p16 status.¹⁸

Consistent with previous reports, the overall sensitivity of the commercially available serum-based TTMV-HPV DNA assay used in this study was 84%. Although this is higher than the pooled sensitivity of 65% of 10 studies totaling 457 patients included in a meta-analysis by Campo et al,²⁹ it remains within a 95% CI of 40% to 84%. In the patient population included in the present study, 11 patients had negative preoperative circulating TTMV-HPV DNA levels, of which 7 had levels of 0 despite positive p16 immunohistochemistry staining of surgical pathology. To date, no clear mechanism has been identified to explain these findings, though false-positive p16 results are possible, as p16 immunohistochemistry staining has an overall sensitivity of 94% and specificity of 83%.³⁰ The mechanisms behind undetectable circulating TTMV-HPV DNA levels and their association with p16 status warrant further investigation as HPV status crucially informs patient management. Interestingly, Chera et al¹⁹ identified 19 patients with undetectable ctHPV-16DNA, 8 of whom had positive test results for an alternative high-risk HPV strain (1 patient with HPV-31; 3 with HPV-33; 4 with HPV-35) via droplet PCR assays, and 2 were found to have a TP53 variant and negative test results for any high-risk HPV strain by droplet PCR assays, despite tumors being p16+. Given the known sensitivity of p16 immunohistochemistry staining and its use as a proxy for HPV status, undetectable ctHPVDNA may be an indicator of false-positive results for p16, which suggests methods such as preoperative RNA in situ hybridization could be used to clarify HPV status to confirm diagnoses. Two studies by Rettig et al^23,31 found that patients with undetectable levels appear to predominantly have cN0 disease, and ctHPVDNA was even detected in select patients several years before OPSCC diagnosis. Interestingly, in a study by Cao et al,³² 3 of 6 patients with stage III HPV+ OPSCC and undetectable TTMV-HPV DNA at pretreatment baseline failed primary chemoradiation therapy within 3 months, with 2 patients found to have persistent locoregional disease and 1 patient with distant failure of treatment. Previous research has suggested that undetectable baseline levels are associated with adverse tumor genomic characteristics, which have been linked to worse outcomes.^19,33,34,35 Continued investigation is warranted to identify mechanisms by which patients with biopsy-proven HPV+ tumors have undetectable TTMV-HPV DNA levels and to validate whether undetectable levels are a leading indicator of adverse biological tumor characteristics.

Lastly, active smoking was associated with increased baseline TTMV-HPV DNA in patients with HPV+ OPSCC tumors. A similar association was reported by Xi et al³⁶ in patients with HPV-16/HPV-18 cervical cancer, in which current smokers had a higher baseline viral load compared with never smokers. History of smoking was identified in 2010 by Ang et al³ as an independent risk factor for mortality and recurrence in patients with stage III/IV OPSCC regardless of HPV status. Interestingly, the ECOG 3311 trial did not find smoking status to be prognostic in patients undergoing primary surgery, although ENE and positive surgical margins were more likely to be found in the surgical pathology of current smokers.³⁷ It has been hypothesized that tobacco-related carcinogens can induce tumor damage, leading to increased serum levels of ctHPVDNA and genetic alterations that may affect tumor responsiveness to systemic treatment or radiotherapy. Additionally, vasoconstriction in active smokers and carbon monoxide exposure can contribute to tumor hypoxia, thereby limiting the efficacy of radiotherapy.^38,39,40,41 In 2021, Caliri et al⁴² published results of a study that suggest tumors growing in the presence of tobacco smoke appear better adapted to withstand oxidative stress induced by chemoradiation. A meta-analysis by Ference et al⁴⁰ concluded that though smoking history does not reduce disease-specific survival, decreased overall survival was observed in patients with any history of smoking. Smoking continues to be an important modifiable risk factor in patients with OPSCC, even in those with HPV+ OPSCC, for which smoking is not known to be a primary factor associated with outcomes of the disease.

Strengths and Limitations

Strengths of this study include the use of a clinically available commercial assay and the contemporary cohort of patients undergoing surgery at a high-volume institution. We believe that the findings reported in this study are informative for clinicians evaluating the utility of incorporating serum-based ctHPVDNA level testing in their practice and clarifying the relevance of pretreatment levels in assessing patient prognosis.

However, these findings should be considered in the context of several limitations. First, the participants were predominantly White and male, limiting the generalizability of the findings to diverse populations. Within our practice setting, clinicians also rely on p16 immunohistochemistry staining as a proxy for HPV+ head and neck tumors, and surgical specimens are not tested for HPV. Previously, p16 staining has been reported to have a sensitivity of 94% and specificity of 83%; therefore, misdiagnoses are possible.³⁰ The sample size for active smokers was small, consisting of 5 patients, so wide CIs limited the internal validity of our findings. Lastly, whether these findings are generalizable to most sensitive digital droplet-based PCR methods, similar to the commercially available ctHPVDNA assay used in this study, remains unclear. Of note, the true sensitivity for disease diagnosis of the assay used in the present study has been questioned, with Rettig et al²³ finding that the sensitivity may be as low as 50% in clinically N0 patients compared with more than 90% in those with nodal disease, in line with the association of increasing pathological tumor stage with detectable TTMV-HPV DNA levels categorized as weak strength in the present study. This highlights the potential shortcomings of the assay that we used, which relies on serum detection of circulating HPV DNA fragments and does not detect integrated or episomal high-risk HPV sequences within the tumor genome (as methods such as next-generation sequencing do).^{17,18,43,44,45} Further research to determine the true clinical validity of different assay methods for the diagnosis of HPV+ OPSCC is warranted.

Conclusions

In this cross-sectional study, the level of preoperative ctHPVDNA was not associated with disease burden in patients with p16+ OPSCC who underwent primary TORS. These findings inform the continuously evolving understanding of serum ctHPVDNA as a biomarker and how it may best be used to improve clinical decision-making related to the management of HPV+ OPSCC.

Supplement.

Data Sharing Statement