Clinical Utility and Limitations of Circulating HPV DNA in Tonsillar Squamous Cell Carcinoma: A Narrative Review

Abstract

The rising incidence of human papillomavirus (HPV)-positive tonsillar cancer, despite the availability of highly effective treatment modalities, presents an ongoing challenge in the area of disease surveillance. While routine clinical examinations, often supported by CT or even PET imaging, remain the standard approach for monitoring, they may lack sensitivity in detecting early or submucosal recurrences. Consequently, there is a growing need to integrate novel diagnostic tools—such as liquid biopsy—into routine follow-up strategies to enhance the accuracy of disease monitoring. The aim of this study is to provide a comprehensive overview of HPV-positive tonsillar cancer, encompassing its etiology, molecular pathogenesis, disease progression, diagnostic approaches, treatment strategies, and current surveillance practices. Special emphasis is placed on the emerging role of liquid biopsy in disease monitoring, particularly for assessing disease burden and enhancing early detection of local recurrence. The review encompassed studies from diverse sources, including experimental, observational, and clinical research published between 2000 and 2025. Data were collected through comprehensive literature searches using databases such as PubMed, Scopus, and the Cochrane Library with defined inclusion and exclusion criteria. Emerging evidence supports the clinical utility of liquid biopsy—particularly circulating tumor (ct) HPV DNA detection—for monitoring HPV-positive tonsillar cancer. Studies report high specificity (87-100%) and sensitivity (72-100%) for recurrence detection, often identifying disease earlier than imaging. Commercial ctHPV DNA assays have demonstrated strong performance in distinguishing true recurrence from benign HPV presence. However, technical variability, lack of standardization, and reduced sensitivity for non-HPV16 genotypes remain key limitations. Therefore, this review aims to evaluate the clinical application of circulating HPV DNA as a surveillance tool in tonsillar squamous cell carcinoma (TSCC), focusing on its diagnostic performance, current limitations, and integration into real-world practice.

Plain Language Summary

This review addresses the growing global health concern of HPV-positive tonsillar cancer, highlighting key molecular mechanisms, evolving epidemiological trends, and emerging diagnostic innovations. Particular focus is given to liquid biopsy as a promising tool for improving both early detection and post-treatment surveillance, especially in identifying disease recurrence.

Introduction

Human papillomavirus (HPV) -positive tonsillar cancer is currently on the rise, with incidence rates steadily increasing. ¹ While specific data on HPV-positive tonsillar carcinoma are often not reported separately, it is estimated that tonsillar cancers account for approximately 50% of HPV-related oropharyngeal cancers—suggesting an annual incidence of around 25 000 cases in the United States. ² Since it remains the most common presentation of HPV-positive oropharyngeal carcinoma,^2,3 we focused on tonsillar cancer location in our study.

Tonsillar squamous cell carcinoma (TSCC) constitutes a distinct anatomical and biological subtype within oropharyngeal cancers, with important implications for diagnosis, treatment, and post-treatment surveillance. Although HPV-positive TSCC is generally associated with a favorable prognosis, late recurrences and subclinical residual disease continue to pose significant challenges during long-term follow-up. ⁴ Standard treatment protocols—particularly radiotherapy combined with cisplatin-based chemotherapy—have markedly improved outcomes in HPV-related oropharyngeal carcinomas, with reported response rates ranging from 82% to 90%. ⁴ Nevertheless, locoregional recurrence remains a notable concern, with some studies reporting failure rates of 17.3% to 32% within 3 years post-treatment, ⁵ underscoring the need for improved surveillance tools and risk-adapted follow-up strategies.

Evaluation of the tonsillar fossa following radiotherapy is generally feasible using routine clinical examination, often supported by fiberoptic endoscopy. ⁶ Nevertheless, detecting recurrence can be challenging, as recurrent lesions frequently develop submucosally and may not be readily apparent on surface inspection. In addition, post-radiation mucosal and submucosal changes often present as contrast-enhancing areas on CT imaging, complicating the distinction between true recurrence and benign post-treatment effects. ⁷ Therefore, even advanced imaging modalities such as MRI may fail to provide definitive differentiation.

Although PET scans can enhance diagnostic sensitivity, ⁸ their limited availability and longer turnaround times may hinder timely decision-making. Given these limitations, there is an increasing need to incorporate alternative or adjunctive diagnostic strategies into the routine surveillance of patients with HPV-positive tonsillar cancer.

In the era of precision oncology, there is increasing emphasis on tools that support both early cancer detection and post-treatment surveillance. Liquid biopsy has emerged as a minimally invasive, versatile modality that aligns closely with this paradigm, offering real-time, molecular-level insights throughout the cancer care continuum.^9-11 In HPV-positive TSCC, the detection of circulating HPV DNA (ctHPV DNA) enables non-invasive assessment of tumor burden and facilitates early identification of recurrence risk.^12,13 Among the various liquid biopsy targets, circulating tumor DNA (ctDNA)—particularly ctHPV DNA—has shown considerable promise for monitoring disease dynamics in HPV-associated oropharyngeal squamous cell carcinomas (OPSCC). ¹⁴ However, the majority of published studies have aggregated data across all oropharyngeal subsites, without accounting for the anatomical and biological nuances specific to the tonsils.^15,16 The lymphoepithelial structure, deep crypt architecture, and subsite-specific shedding behavior in TSCC may influence the sensitivity, specificity, and overall kinetics of ctHPV DNA detection. ¹⁷ This creates a critical gap in our understanding of how liquid biopsy performs in tonsillar cancers compared to other regions such as the base of tongue or soft palate.

Therefore, this review aims to provide a comprehensive overview of HPV-positive TSCC, encompassing its diagnostic strategies, clinical features, and current treatment approaches. Particular emphasis is placed on the emerging role of liquid biopsy—specifically ctHPV DNA—as a non-invasive tool for disease monitoring, with a focus on its utility in detecting locoregional recurrence. By synthesizing the available evidence on ctHPV DNA in the context of TSCC, we evaluate current detection platforms, assess diagnostic performance, and explore clinical applications in post-treatment surveillance. Moreover, by narrowing the scope to this anatomically and biologically distinct subsite, we underscore the need for subsite-specific validation and highlight the translational potential of liquid biopsy in advancing personalized management for patients with TSCC.

Methodology

This narrative review focuses on HPV-positive tonsillar cancer, emphasizing not only its pathogenesis, etiology, clinical evaluation and treatment approach but particularly the challenges associated with recurrence monitoring. The aim of this study is to provide a comprehensive synthesis of existing literature—including observational, clinical, and experimental studies—to contextualize current knowledge and highlight areas requiring further research.

Inclusion Criteria

• Original research articles (observational, clinical trials, experimental studies)

• Systematic reviews and meta-analyses, relevant narrative reviews and expert consensus papers (for context)

• Human subjects diagnosed with HPV-positive tonsillar or HPV-positive oropharyngeal squamous cell carcinoma

• Studies addressing etiology, molecular mechanisms, pathogenesis, clinical presentation, treatment, or recurrence monitoring

• Research evaluating or discussing liquid biopsy, imaging, or molecular diagnostics and precision oncology

• Articles exploring novel therapies (eg, immunotherapy, targeted therapy) related to HPV-positive tonsillar cancer

• Preferably published between 2000 and 2025

Exclusion Criteria

• editorials, commentaries, or letters to the editor that did not include original data or peer-reviewed scientific content;

• case reports or case series with fewer than 5 patients;

• animal or in vitro studies, unless they provided significant mechanistic insights relevant to recurrence detection or molecular diagnostics;

• studies focusing exclusively on HPV-negative oropharyngeal cancers;

• research involving pediatric populations;

• articles limited to surgical techniques or rehabilitation without addressing monitoring, surveillance, or molecular profiling.

Search Strategy

A comprehensive literature search was conducted using PubMed, Scopus, and the Cochrane Library databases. The search strategy included combinations of the following keywords: “tonsillar cancer,” „oropharyngeal cancer”, “HPV,” “epidemiology,” “etiology,” “recurrence,” “disease monitoring”, „precision oncology”, „liquid biopsy”. In addition, current National Comprehensive Cancer Network (NCCN) guidelines were reviewed to supplement the evidence base.

Synthesis Approach

The narrative review was organized into thematic sections encompassing molecular pathogenesis, epidemiology, diagnostic strategies, treatment approaches, recurrence monitoring in HPV-positive tonsillar cancer and liquid biopsy description. While formal quality assessment tools such as GRADE were not applied due to the narrative nature of the review, priority was given to high-quality evidence, including large-scale studies, randomized controlled trials (RCTs), and meta-analyses. Observational studies and preclinical data were included where relevant, particularly to highlight emerging diagnostic tools such as liquid biopsy. Greater emphasis was placed on consensus guidelines from authoritative bodies such as the National Comprehensive Cancer Network (NCCN), as well as studies demonstrating methodological rigor and clinical relevance.

Organization and Synthesis of Information

Following data extraction, the results were organized into thematic sections reflecting the major domains of research in HPV-positive tonsillar cancer. These included epidemiology, etiology, molecular pathogenesis, diagnostic and imaging strategies, current treatment modalities and post-treatment surveillance methods, including liquid biopsy utensils. Within each thematic area, data from multiple studies were synthesized to highlight both consistent trends and areas of divergence. This integrative narrative approach emphasizes key connections—such as how molecular insights relate to diagnostic innovation, or how treatment effectiveness impacts surveillance protocols—and identifies critical gaps where further research is needed to improve clinical outcomes.

Tonsillar Cancer Epidemiology

Tonsillar cancer represents the most common subsite of oropharyngeal malignancies, a group that collectively ranks as the sixth most common cancer worldwide. ¹⁵ The rising incidence of tonsillar cancer is strongly associated with HPV infection, particularly in high-income countries. ¹⁸ This subtype accounts for approximately 23.1% of all oropharyngeal cancers, with an overall incidence rate of 8.4 cases per 100 000 individuals. ¹⁹ Importantly, while the incidence of HPV-negative tonsillar cancer has remained relatively stable over time, HPV-positive cases have increased significantly. ²⁰ In the United States alone, HPV-positive tonsillar cancer is estimated to account for 25 000 to 30 000 new cases annually, comprising approximately 50-60% of all HPV-associated oropharyngeal squamous cell carcinomas (OPSCCs). ²¹

Etiology

While HPV-negative tonsillar cancer is primarily associated with tobacco and alcohol use and typically affects older male patients, HPV-positive tonsillar cancer presents a distinctly different clinical profile. ²² Namely, HPV-positive tonsillar cancer is predominantly caused by infection with high-risk HPV genotypes, most notably HPV-16, which accounts for approximately 83% of HPV-associated OPSCCs. ²³ Other high-risk genotypes occur less frequently: HPV33 (3.3%), HPV26 (2.6%), HPV35 (2.2%), HPV18 (1.8%), with all others contributing to less than 1% of cases. ²⁴ HPV-positive tonsillar cancer tends to affect younger individuals, often without a history of significant tobacco or alcohol exposure. ²⁵ Instead, key risk factors include orogenital sexual practices, particularly oral sex and having multiple sexual partners. ²⁵ These behaviors contribute to the higher incidence of this cancer subtype, especially among male patients. ²⁵ Interestingly, one study assessing HPV-related cancer risk in a cohort of homosexual individuals reported a higher rate of oropharyngeal cancers among women than men, suggesting the complexity of transmission dynamics. ²⁵ However, the higher prevalence of HPV-positive tonsillar cancer among men is thought to be linked to the greater likelihood of acquiring oral HPV through oral sex with women compared to with men. ²⁶ Supporting this, studies show that oral HPV prevalence increases more sharply with the number of sexual partners in men than in women. ²⁷

Pathogenesis

When discussing the pathogenesis of tonsillar cancer, it is important to highlight several histologic features of tonsillar tissue that predispose it to HPV infection. Notably, the tonsillar crypts are lined with reticulated squamous epithelium, which is thin, frequently discontinuous, and poorly keratinized. ²⁸ These characteristics facilitate direct access of HPV to the basal epithelial layer, where viral infection is initiated. Furthermore, the high proliferative rate of basal epithelial cells within the crypts increases opportunities for viral replication and integration into the host genome, thereby promoting malignant transformation. Another contributing factor is the elevated expression of attachment molecules—such as heparan sulfate proteoglycans—on the epithelial surface, which enhances HPV binding and cellular entry. As components of the mucosa-associated lymphoid tissue (MALT), the tonsils also play an immunoregulatory role that may favor immune tolerance, enabling persistent HPV infection. ²⁹ Additionally, a low density of Langerhans cells in the crypt epithelium further diminishes local immune surveillance, facilitating viral persistence and potential oncogenesis. ²⁹ Figure 1 illustrates the key steps involved in HPV-mediated infection and transformation of tonsillar tissue. The molecular mechanism underlying HPV-driven carcinogenesis in tonsillar cancer centers on the actions of the viral oncoproteins E6 and E7, which play pivotal roles in disrupting normal cell cycle regulation. ³⁰ E6 promotes the degradation of the tumor suppressor protein p53 via the ubiquitin-proteasome pathway, thereby impairing apoptosis, DNA repair, and cell cycle arrest. ³¹ Concurrently, E7 inactivates the retinoblastoma protein (pRb), a key regulator of the G1/S phase transition. ³² Upon infection of epithelial cells, HPV DNA integrates into the host genome, frequently disrupting the E1/E2 open reading frame. This leads to the loss of E2 protein function, which normally represses E6 and E7 expression. ³³ The resulting deregulation leads to upregulated transcription of E6 and E7, intensifying their oncogenic effects. ³³ In the absence of functional pRb, E2F transcription factors are released, promoting the expression of genes necessary for DNA replication and allowing unchecked progression through the G1/S checkpoint (Figure 2). ³³ Moreover, the inactivation of pRb triggers a compensatory overexpression of p16, a cyclin-dependent kinase inhibitor. Elevated p16 levels serve as a widely accepted surrogate marker for HPV-associated malignancies, including HPV-positive tonsillar cancer. ³⁴

Figure 1.

Mechanism of HPV Infection and Carcinogenesis in Tonsillar Tissue.

Figure 2.

Schematic Representation of HPV-Driven Oncogenesis and Its Link to ctHPV DNA-Based Surveillance.

Clinical Presentation and Diagnostic Challenges in Tonsillar Cancer

While tonsillar asymmetry and palpable or visible lesions are typical findings in advanced stages (Figure 3) of tonsillar cancer, early-stage disease may present with subtle or even imperceptible clinical signs. ³⁵ In some cases, no overt tonsillar abnormalities are initially evident, and the first clinical indication may be cervical lymphadenopathy—occasionally on the contralateral side of the neck. ³⁶ Additionally, referred otalgia can serve as an early symptom in certain patients. ³⁷ In contrast, symptoms such as dysphagia, odynophagia, and significant weight loss are more commonly associated with advanced disease. ³⁸ Although routine oral examination in the clinical setting is often sufficient to identify advanced cases, it may fail to detect early or submucosal tumors. ³⁹ Oral examination is usually complemented by fiberoptic endoscopy, which improves visualization of the oropharynx but may still miss small or deeply located primary or recurrent tumors. ³⁹ Consequently, additional imaging modalities are often required to establish a definitive diagnosis. Contrast-enhanced CT is commonly used as a first-line diagnostic tool and is generally effective in evaluating the size and spread of the tumor. ⁴⁰ However, its sensitivity is limited in detecting early-stage or submucosal tumors. According to published data, CT demonstrates a sensitivity of approximately 80-90% for detecting tonsillar masses larger than 1 cm, with a specificity of around 70-85%. ⁴¹ In contrast, MRI offers superior soft tissue resolution and is more effective in identifying submucosal lesions and parapharyngeal space involvement. It demonstrates higher diagnostic performance, with a sensitivity of 85-95% and a specificity of 80-90%, making it a valuable tool in both the initial assessment and detailed local staging of tonsillar cancer. ⁴² PET scans are particularly valuable when other imaging modalities such as CT or MRI yield inconclusive results, or when the cancer presents with an unknown primary origin. However, limited availability and, in some cases, the prolonged time required to obtain results are significant drawbacks of this technique. ⁴³ Nevertheless, all patients with oropharyngeal carcinoma should be tested for HPV using p16 immunohistochemistry and/or direct HPV testing (PCR, ISH). The use of p16 as a prognostic marker is the standard of care according to NCCN guidelines. ⁴⁴

Figure 3.

Intraoperative Views of Transoral Surgical Resection of HPV-Positive Tonsillar carcinoma.

Treatment Strategies

Surgical management, including transoral robotic surgery (TORS) and transoral laser microsurgery (TLM), is primarily reserved for early-stage tumors, with both approaches demonstrating comparable clinical outcomes. ⁴⁵ TORS is gaining popularity due to its advantages, including reduced operating time, improved postoperative swallowing function, and shorter hospital stays compared to conventional tonsillectomy. ⁴⁶ In contrast, TLM is less commonly used because it involves piecemeal tumor resection, which can compromise accurate histopathological margin assessment. ⁴⁷ Regardless of the surgical technique selected, bilateral tonsillectomy is not routinely recommended, although it may be considered in rare cases due to the potential for synchronous bilateral tonsillar carcinoma. ⁴⁸ According to NCCN guidelines, in HPV-positive cases, radical tonsillectomy should be accompanied by unilateral or bilateral neck dissection—depending on tumor staging—to include levels II to IV. ⁴⁴ Nevertheless, the high radiosensitivity of HPV-positive tonsillar cancer—unlike its HPV-negative counterpart—has significantly improved patient outcomes and prompted ongoing research into treatment de-escalation strategies, including radiation dose reduction. ⁴⁹ Current evidence suggests no significant differences in overall survival (OS), progression-free survival (PFS), or local control (LC) between primary radiotherapy and surgical approaches in HPV-positive tonsillar carcinoma. ⁴⁹ However, advanced tumors (T4) are best managed with definitive chemoradiotherapy. For locally advanced head and neck squamous cell carcinoma, radiotherapy combined with concurrent platinum-based chemotherapy—most commonly cisplatin—remains the standard of care. ⁵⁰ Although the monoclonal antibody cetuximab is employed in cases where cisplatin is contraindicated, multiple studies, including the pivotal RTOG 1016 and De-ESCALaTE HPV trials, have demonstrated inferior overall survival and higher recurrence rates with cetuximab compared to cisplatin in HPV-positive oropharyngeal cancers. ⁵¹

Follow-up Assessment and Recurrence Monitoring

While the transoral approach allows direct access to the tonsils and tonsillar fossa without the need for advanced equipment, accurate assessment in post-treatment patients is often difficult due to anatomical changes and treatment-related tissue alterations. Routine follow-up evaluation includes a thorough oral examination usually supplemented by fiberoptic nasopharyngolaryngoscopy.^35,44 This should be performed every 1-3 months during the first year following treatment completion, every 2-6 months during the second year, and every 4-8 months during years 3 to 5. ⁴⁴ After the fifth year, annual follow-up is recommended. ⁴⁴ A PET-CT or other imaging study is performed once at 3-6 months after completion of treatment to assess the treatment response. ⁴⁴ Further imaging is justified only in the presence of clinical symptoms or a high risk of recurrence. However, current surveillance strategies have notable limitations. For instance, while PET scans demonstrate a high negative predictive value (NPV) exceeding 90%, their positive predictive value (PPV) is considerably lower—ranging from approximately 62% to 77%—and varies depending on the interval since treatment completion.^52,53 Frequent false-positive results can lead to unnecessary invasive procedures, including biopsies and surgeries. Moreover, the NPV of PET imaging, although generally strong, is reduced in cases of low-volume disease or tumors with low metabolic activity.^54,55 Although PET/CT performed at 3 months post-treatment is considered the standard of care for response assessment, its role in long-term surveillance is limited. Beyond this initial imaging window, routine follow-up primarily relies on clinical examination, including fiberoptic laryngoscopy. However, these examinations are often insufficient for detecting asymptomatic recurrences, particularly in previously irradiated tissues where visualization and palpation may be compromised.^56,57 In fact, studies have shown that in asymptomatic patients, standard surveillance protocols detect recurrences in only 0.3% to 2% of cases, highlighting the need for more sensitive and specific tools for post-treatment monitoring. ⁵⁸ Unfortunately, no specific recommendations for HPV testing in the context of recurrence monitoring have been outlined by the NCCN or other major clinical guidelines.

HPV Status Evaluation in Terms of Tonsillar Cancer Recurrence

To address the limitations posed by the lack of reliable diagnostic tools for disease monitoring, assessment of circulating HPV DNA status (ctHPV DNA) has emerged as a potentially valuable component in ongoing surveillance. Several prospective studies have demonstrated that ctHPV DNA can detect molecular recurrence with high sensitivity and specificity, often months before clinical or radiologic evidence becomes apparent. For instance, Chera et al. reported that 2 consecutive positive ctHPV DNA tests during post-treatment surveillance yielded a PPV of over 90% for recurrence. The rapid clearance of ctHPV DNA following definitive therapy has also been associated with favorable prognosis, whereas its persistence may indicate residual disease. ⁵⁹ Moreover, emerging evidence suggests that treatment adaptation guided by liquid biopsy can improve patient outcomes by minimizing unnecessary chemotherapy or radiotherapy, while maintaining therapeutic efficacy and preserving quality of care.^9,60,61 In a study by Jacobsen et al. involving 72 patients with HPV-positive/p16-positive OPSCC, 41 patients had undetectable levels of circulating cell-free HPV DNA (cfHPV-DNA) in all follow-up plasma samples, and none of these individuals experienced disease recurrence. In contrast, 13 patients were classified as cfHPV-DNA–positive during follow-up, of whom 5 developed recurrent disease and 3 had residual cancer. The baseline sensitivity of cfHPV-DNA detection was reported at 97.2% (95% confidence interval: 90.3%-99.6%). ⁶² Similarly, in a surveillance cohort study by Ferrandino et al., 290 patients were evaluated, of whom 23 had pathologically confirmed recurrences detected by molecular testing. The Tumor Tissue–Modified Viral Human Papillomavirus DNA (TTMV-HPV DNA) assay demonstrated a sensitivity of 88.4% and a specificity of 100% for identifying recurrent disease. The PPV was 100%, while the NPV reached 99.1%, underscoring the assay’s high diagnostic accuracy in post-treatment surveillance. ⁵⁶ A recent systematic review and meta-analysis combining data from 10 studies involving 457 patients reported that ctDNA-based assays achieved a pooled sensitivity of 65% and a specificity of 99% for disease detection. While specificity was consistently high across studies, sensitivity showed greater variability. ⁶³ As assay technologies continue to advance, with improvements in both sensitivity and specificity, their clinical utility in diagnosis, treatment response assessment, and surveillance is expected to expand significantly. Ongoing clinical trials are expected to further clarify its role in personalized follow-up strategies and early intervention protocols.

Detection Techniques for Assessing HPV Status

The primary method used to assess HPV status in patients with HPV-positive tonsillar carcinoma through liquid biopsy involves the detection of circulating tumor HPV DNA (ctHPV DNA) in plasma^64,65 (Figure 4). Liquid biopsy begins with collecting 10-20 mL of blood into EDTA or Streck tubes. EDTA samples must be processed within 6 hours, while Streck tubes preserve DNA for up to 7 days. Plasma is separated via 2 centrifugation steps and then used for nucleic acid extraction, typically taking 1-2 hours. ⁶⁶ ctDNA is analyzed using platforms like droplet digital polymerase chain reaction (ddPCR), quantitative PCR (qPCR), or next generation sequencing (NGS), with PCR methods yielding faster results (6 hours) and NGS taking up to 5 days. The timing of blood collection relative to treatment is critical.^10,67 Combining multiple analytes—ctDNA, CTCs, and EVs—with AI-enhanced interpretation improves accuracy. ⁶⁸ However, differences in lab protocols and lack of standardization remain major challenges to broader clinical adoption. Nevertheless, the concept of liquid biopsy is gaining increasing recognition, not only in cancer diagnosis but also as a valuable tool in precision oncology. Although liquid biopsy is an emerging non-invasive tool in the management of various cancers, including HPV-positive OPSCC, its reliability depends on both the analytical platform employed and the specific biomarker selected for detection. ⁶⁹ Current research efforts are primarily focused on ctHPV DNA, which can be identified in up to 90-95% of patients with advanced HPV + OPSCC. The viral etiology of these tumors contributes to elevated levels of circulating tumor DNA, which serves as a tumor-specific biomarker. ⁷⁰ Unlike traditional tissue biopsy, liquid biopsy allows for serial monitoring of tumor-derived nucleic acids via a simple blood draw, providing real-time insights into tumor burden and dynamics. ⁷¹ In HPV + OPSCC, tumor cells shed viral DNA—most commonly HPV16—into the bloodstream, which can be detected using highly sensitive methods. ⁷² In addition, HPV-driven tumors release extracellular vesicles (EVs) containing both viral and tumor-derived components, which may influence immune responses and represent additional liquid biopsy targets. ⁷³ Thus, the choice of liquid biopsy method should be tailored to the specific goals of disease assessment and monitoring. Although not yet implemented in standard clinical practice, liquid biopsy represents a promising adjunct to conventional surveillance strategies, with ongoing clinical trials aiming to validate its role in risk-adapted follow-up and early therapeutic intervention.

Figure 4.

Schematic Representation of Liquid Biopsy in HPV-Positive Tonsillar Cancer.

Rationale and Biomarker Biology

TSCC exhibits unique anatomical and biological features that have important implications for the performance and interpretation of liquid biopsy assays, particularly those targeting ctHPV DNA. The palatine tonsils are composed of lymphoid tissue with deep crypts, which can hinder the consistent release of tumor-derived nucleic acids into the bloodstream. ⁷⁴ This complex microarchitecture may result in heterogeneous shedding patterns, reducing the sensitivity of ctHPV DNA detection—especially in early-stage disease or when tumor burden is low. Although salivary sampling offers a practical, non-invasive alternative to blood-based assays, its diagnostic utility in TSCC is limited by variable tumor accessibility and dilution of analytes in oral fluids. ⁷⁵ These subsite-specific factors introduce a risk of sampling bias and false-negative results, underscoring the need for tailored biomarker strategies and standardized protocols in TSCC surveillance. ⁷⁶ Comparatively, HPV-positive tumors of the base of tongue are often deeper and less accessible, complicating both diagnostic and follow-up evaluations. ⁷⁷ The soft palate, though more amenable to salivary diagnostics due to its superficial location, represents only a small fraction of HPV-driven oropharyngeal cancers. ¹⁵ Recognizing these anatomical and functional distinctions is essential for interpreting ctHPV DNA results and for designing subsite-specific surveillance strategies that maximize diagnostic yield and clinical utility.

CTCs and ctDNA Assessment

CTCs are epithelial-derived cells shed from the primary tumor into the bloodstream. ⁷⁸ They exhibit genetic heterogeneity and often undergo epithelial–mesenchymal transition (EMT), a process that enhances their migratory capacity and invasive potential. ⁷⁹ The detection of CTCs in the bloodstream serves as a valuable prognostic marker for disease severity, metastatic potential, and treatment response. However, their presence is typically scarce—estimated at around one CTC per billion blood cells—making detection technically challenging. ⁸⁰ In HPV-related tonsillar carcinoma, this difficulty may be further compounded by the tumor’s relatively low degree of EMT, which limits CTC release into circulation. ⁸¹ As a result, CTC analysis is often more feasible in advanced disease stages, where their numbers may be elevated. ⁸² Additionally, CTCs offer complementary information on tumor heterogeneity and metastatic potential. ⁸³ Emerging CTC-based diagnostic approaches include the assessment of expressed viral oncoproteins, enabling phenotypic profiling associated with treatment resistance and dissemination. ⁸⁴ On the other hand, ctDNA consists of short fragments of tumor-derived DNA, typically 150-200 base pairs in length, detectable in the bloodstream of cancer patients. ⁸⁵ It originates primarily from apoptotic and necrotic tumor cells and carries somatic mutations associated with both primary and metastatic lesions. ⁸⁶ In addition to genetic alterations, ctDNA may also reflect epigenetic changes, such as DNA methylation patterns. ⁸⁷ These characteristics make ctDNA a valuable biomarker for cancer diagnosis, prognosis, and monitoring of therapeutic response as it is used already in monitoring non-small cell lung cancer and as a screening marker for colorectal cancer.^88,89 ctDNA represents a cost-effective diagnostic tool, useful not only for assessing treatment response but also for ongoing clinical monitoring. While NGS allows for in-depth analysis of tumor heterogeneity and potential resistance mechanisms, it remains a high-cost approach that requires advanced instrumentation and specialized personnel.^90,91 As a result, NGS is primarily suited for comprehensive molecular tumor profiling, whereas ddPCR is more practical and feasible for real-time disease surveillance. ⁹² Therefore, several important distinctions exist among the technical platforms used for ctHPV DNA detection that warrant detailed discussion. Quantitative PCR (qPCR) remains widely accessible and cost-effective but may lack the sensitivity required for detecting low levels of circulating HPV DNA, particularly in early or minimal residual disease. ⁹³ Droplet digital PCR (ddPCR) offers superior analytical sensitivity and precision by enabling absolute quantification of low-copy HPV DNA fragments, making it particularly well-suited for post-treatment monitoring and early recurrence detection. ⁹⁴ In contrast, NGS allows for broader genomic profiling and the detection of co-existing mutations but generally exhibits lower sensitivity for low-abundance targets and incurs higher costs and longer turnaround times. ⁹⁵ These distinctions are especially relevant in TSCC, where biomarker concentrations may vary due to subsite-specific shedding patterns. Table 1 summarizes key performance attributes of each method. Moreover, multiple studies have demonstrated that HPV16 ctDNA levels strongly correlate with tumor burden and therapeutic response.^96-98 Although HPV16 is the predominant genotype in HPV-positive head and neck squamous cell carcinoma (HNSCC), less common high-risk types such as HPV18, 31, and 33 differ in the structure of E6 and E7 oncogenes, which may affect detection accuracy. ⁹⁹ However, a new clinically validated molecular assay - NavDx quantifies tumor tissue–modified HPV DNA (TTMV-HPV DNA or ctHPV DNA) in the bloodstream. ¹⁰⁰ It accurately distinguishes tumor-derived HPV recurrence from benign HPV-related lesions, demonstrating high sensitivity and specificity.^63,100 It has been validated in several independent clinical studies, demonstrating high sensitivity and specificity for identifying minimal residual disease and predicting recurrence in HPV-associated oropharyngeal cancers.^99,101 For example, independent validation by Chera et al. and others has shown that NavDx can detect recurrence several months before clinical or radiographic evidence of disease. ¹⁰² However, we emphasize that comparable sensitivity has also been reported with high-quality, in-house ddPCR-based assays, particularly when well-optimized and applied within experienced molecular laboratories. Importantly, while NavDx offers standardization and regulatory oversight, including CLIA certification, laboratory-developed tests (LDTs) provide greater flexibility in assay design, target selection, and cost control, which may be advantageous in research settings or resource-limited environments. ¹⁰³ Currently used ctDNA detection methods, such as ddPCR and NGS, are primarily optimized for HPV16, which may lead to reduced sensitivity and specificity when applied to other high-risk HPV genotypes. ¹⁰⁴ Furthermore, the ability to detect both viral and somatic mutations in ctDNA provides valuable insight into disease status, with some studies reporting lead times of several months over conventional imaging in identifying recurrence. ¹⁰⁵ Although the majority of published studies have focused on ctHPV DNA in HPV + OPSCC, ctDNA has also been detected in other head and neck cancers, including those of the larynx, hypopharynx, and oral cavity—even in tumors not associated with HPV.^106,107 Importantly, the clinical behavior and molecular characteristics of non-oropharyngeal (non-OP) HPV-positive head and neck squamous cell carcinomas (HNSCCs) differ from those of oropharyngeal cases. ¹⁰⁸ These differences may affect both the dynamics of ctDNA shedding and the overall detectability of ctHPV DNA. While ctHPV DNA assays have shown excellent sensitivity (90-95%) and specificity (>98%) in detecting oropharyngeal tumors, their performance in non-OP HPV-positive tumors remains less well defined.^56,109 This distinction is critical, as biomarker dynamics in non-OP cases may not mirror those observed in OPSCC, thereby limiting the generalizability of current findings. Nonetheless, multiple studies have demonstrated that ctHPV DNA–based liquid biopsy can effectively detect minimal residual disease and early recurrence, often preceding radiographic or clinical findings by several months. ¹¹⁰ Furthermore, post-treatment clearance of ctHPV DNA is associated with improved clinical outcomes, whereas persistent or rising levels may signal impending relapse. ¹¹¹ In a systematic review by Chennareddy et al., 23 studies were analyzed—10 conducted retrospectively and 13 prospectively. Diagnostic approaches included plasma-based ddPCR, n = 13, qPCR, n = 4, digital PCR (dPCR, n = 3), NGS, n = 3, and one study utilizing a commercial ctDNA detection kit. Test sensitivities during disease surveillance ranged from 72% to 100%, while specificities ranged from 87.2% to 100%. ¹¹² These findings support the utility of circulating tumor DNA as a highly effective biomarker for disease monitoring.

Table 1.

Comparison of Liquid Biopsy Modalities in Tonsillar Cancer

Method	Analyte	Sample type	Sensitivity	Specificity	Turnaround time	Cost	Clinical availability
Circulating tumor DNA (ctDNA)	Mutated tumor DNA fragments	Blood (plasma)	High in advanced stages	High (when targeted)	1-2 weeks	High	Limited to specialized centers
Cell-free DNA (cfDNA)	Total extracellular DNA	Blood (plasma/serum)	Moderate	Moderate	3-5 days	Moderate	Increasingly available
Extracellular vesicles (EVs)	Proteins, mRNA, miRNA	Blood, saliva	Variable (depends on biomarker)	Variable	1-2 weeks	High	Mostly research setting
Exosomes	RNA, DNA, proteins	Blood, saliva	Moderate to high	High (targeted)	5-10 days	High	Limited, mainly experimental
Salivary diagnostics	cfDNA, HPV DNA, Exosomal RNA	Saliva	Moderate (site-dependent)	Moderate to high	1-3 days	Low	High (non-invasive, point-of-care)

Tumor-Derived Extracellular Vesicles (TD-EVs)

Tumor-derived extracellular vesicles (TD-EVs) are a heterogeneous population of membrane-bound particles released by tumor cells that play a pivotal role in intercellular communication. ¹¹³ They mediate the transfer of proteins, lipids, and genetic material between cells, influencing key processes such as tumor progression, immune modulation, and metastasis. ¹¹⁴ Notably, EV-associated microRNA signatures and viral transcripts are being actively investigated as promising diagnostic biomarkers and potential therapeutic targets. ¹¹⁵ Subtypes of TD-EVs include exosomes, microvesicles, nanovesicles, exomeres, apoptotic bodies, and large oncosomes. ¹¹⁶ Similar to ctDNA, TD-EVs are present at low concentrations in peripheral blood, necessitating sample enrichment for reliable detection. ¹¹⁷ Furthermore, their isolation is technically challenging due to the risk of contamination with similarly sized particles, such as lipoproteins.^118,119 Their diverse molecular composition—which includes membrane-bound proteins and lipids, cytosolic proteins, and nucleic acids—provides a valuable reservoir of biomarker candidates, yet also demands thorough analytical validation to confirm disease specificity and biological relevance. Despite these challenges, overcoming the technical barriers associated with TD-EV analysis can unlock their full potential as a robust, minimally invasive biomarker platform—particularly useful for evaluating tumor stage, metastatic potential, and treatment response in various cancers.

Epigenetic Biomarkers

Epigenetic biomarkers, particularly DNA methylation signatures and circulating RNA species such as microRNAs (miRNAs) and messenger RNAs (mRNAs), are gaining traction as valuable tools in the liquid biopsy landscape. ¹²⁰ These molecular alterations often occur early in carcinogenesis and can persist throughout disease progression, offering a window into tumor biology that is complementary to genetic alterations. In the context of TSCC, where anatomical features like deep crypts and lymphoid-rich tissue may hinder consistent ctHPV DNA release, epigenetic biomarkers provide an alternative means of detection, especially for MRD or early recurrence. ¹²¹ Methylation-based assays have demonstrated the ability to discriminate between benign HPV infections and malignant transformation, addressing a key challenge in HPV-driven cancers. ¹²² Specific promoter hypermethylation patterns—such as in the genes CADM1, MAL, or PAX1—have been correlated with HPV-associated malignancies and may enhance diagnostic precision. ¹²³ Likewise, circulating miRNAs have emerged as promising biomarkers due to their stability in body fluids and their regulatory roles in oncogenic pathways, including proliferation, immune evasion, and apoptosis. ¹²⁴ For instance, dysregulation of miR-21 and miR-155 has been linked to poor outcomes in head and neck squamous cell carcinomas. ¹²⁵ While mRNA-based signatures remain more technically challenging due to lower stability, advances in exosome isolation and RNA protection technologies are improving their feasibility for clinical use. ¹²⁶ Although these epigenetic biomarkers are still in early-stage clinical investigation, they hold significant promise for improving the sensitivity and specificity of liquid biopsy assays, especially when combined with ctHPV DNA in multimodal biomarker panels. Future research should focus on validating these markers in TSCC-specific cohorts, establishing reproducible assay protocols, and integrating them into risk-stratified surveillance frameworks.

Implications for Precision Oncology

The fundamental goal of precision oncology is to tailor medical management to the specific molecular and clinical characteristics of a patient’s malignancy, thereby enabling more effective treatment while minimizing adverse effects. ¹²⁷ A central focus of precision oncology is the optimization of treatment outcomes through the identification of germline and somatic variants that drive cancer initiation and progression, thereby informing the most effective therapeutic strategies. ¹²⁸ However, precision oncology extends beyond molecularly guided therapy selection; it also encompasses early cancer detection, precision prevention, and the identification of molecular markers predictive of recurrence, enabling proactive disease management. ¹²⁹ Published research underscores the significant potential of precision oncology to enhance patient care; however, persistent challenges remain—particularly global disparities in access, implementation, and the equitable adoption of these advanced approaches. ¹³⁰ Nevertheless, precision oncology offers a promising avenue for improving patient prognosis by enabling accurate prediction of cancer recurrence. It has transformed recurrence risk assessment across multiple cancer types through the adoption of highly individualized, molecularly guided surveillance strategies. In a study conducted by Keogen et al., the authors employed precision oncology approaches to model long-term recurrence risk in breast cancer. The study leveraged emerging label-free digital technologies that retain spatial tissue context. Specifically, Fourier-transform infrared (FTIR) chemical imaging was combined with deep learning models—utilizing 2D and 2D-separable convolutional neural networks (CNNs)—to predict breast cancer recurrence. The models achieved an area under the ROC curve (AUC) of approximately 0.64, comparable to established clinical prognostic assays. ¹³¹ This work underscores the potential of fully digital, spatially informed, and non-invasive tools to enhance individualized recurrence risk assessment in precision oncology. Similarly, in colorectal cancer, ctDNA-based assays have demonstrated the ability to detect minimal residual disease (MRD) several months before radiologic evidence of recurrence. Postoperative ctDNA positivity has been strongly correlated with an increased risk of disease relapse, highlighting its potential as an early and reliable biomarker for recurrence surveillance. ¹³² In a TRACERx study dealing with non-small cell lung cancer (NSCLC) it was confirmed that ctDNA variations could predict recurrence and clonal evolution, guiding adjuvant treatment decisions. ¹³³ Similarly, in bladder cancer, urinary tumor DNA analysis offers a non-invasive tool for recurrence surveillance, reflecting tumor heterogeneity and early molecular relapse. ¹³⁴ These applications illustrate how precision oncology—through liquid biopsy, genomic profiling, and personalized surveillance—enables early recurrence detection, risk stratification, and treatment adaptation across various cancer types. Therefore, incorporation of ctDNA assessment in tonsillar cancer disease surveillance holds promise for facilitation of early recurrence cancer detection.

Clinical Applications and Limitations

Although circulating HPV DNA represents a promising biomarker for surveillance in HPV-related tonsillar cancer, several critical aspects of its clinical implementation remain insufficiently standardized. These include the lack of consensus on assay selection and validation, with considerable variability in analytical sensitivity and reporting formats across platforms. Moreover, there are no established guideline regarding the optimal timing and frequency of sample collection during follow-up, which limits comparability across studies and hinders clinical decision-making. Cutoff thresholds that define clinically meaningful changes in ctHPV DNA levels are also inconsistent, further complicating the interpretation of results. Finally, integration of ctHPV DNA testing into established treatment and surveillance pathways has yet to be formally defined, underscoring the need for prospective studies and consensus frameworks to ensure its effective translation into routine practice. ¹³⁵ In addition, while many published studies group all HPV-associated oropharyngeal cancers together, such aggregation may obscure important subsite-specific differences in biology, anatomy, and clinical behavior.^136,137 In particular, the palatine tonsils are encapsulated lymphoid tissues with deep crypts, which can influence both tumor growth patterns and the kinetics of viral DNA release into circulation. These crypts may create a barrier to consistent shedding of tumor-derived nucleic acids, potentially leading to lower ctHPV DNA detectability compared to tumors located at non-cryptic sites, such as the soft palate or lateral pharyngeal wall. ¹³⁸ Conversely, the base of tongue—while also lymphoid-rich—differs in surface architecture and is more commonly associated with occult primaries, which may lead to distinct ctDNA kinetics. ¹³⁹ These anatomical and histological distinctions underscore the importance of considering tumor location when interpreting ctHPV DNA results, particularly in surveillance settings. Furthermore, we note that many meta-analyses and pooled studies have not stratified results by subsite, limiting their applicability to TSCC.^140,141 Therefore, future research should prioritize subsite-specific validation studies to refine biomarker utility and better tailor clinical decision-making in HPV-related head and neck cancers. Moreover, despite promising evidence supporting ctHPV DNA as a biomarker for surveillance in HPV-associated oropharyngeal cancers, including TSCC, its integration into routine clinical practice remains limited. A key barrier is the lack of standardized thresholds for ctDNA positivity, which complicates interpretation and clinical decision-making across institutions. ¹⁴² Additionally, current clinical guidelines do not yet include recommendations for ctHPV DNA testing, reflecting the need for further validation and consensus-building. ¹⁴³ Practical challenges also persist, including the high cost of advanced molecular assays, which may not be covered by insurance or accessible in resource-limited settings. Furthermore, there is considerable variability in the timing and frequency of ctDNA sampling, with no established protocols guiding longitudinal monitoring. These issues collectively highlight the gap between research and real-world implementation, underscoring the importance of multicenter trials and expert consensus to facilitate broader clinical adoption of liquid biopsy in TSCC surveillance pathways. Furthermore, it appears that among the most pressing challenges are the high cost and uncertain reimbursement pathways for advanced molecular assays, which limit accessibility, particularly outside academic centers. Additionally, high-sensitivity platforms such as ddPCR and NGS are not universally available, contributing to disparities in diagnostic capability. A further impediment is the lack of formal guideline endorsement, which hampers standardization and clinician confidence in incorporating ctHPV DNA into surveillance protocols. Compounding these issues is the variability in assay design, analytical thresholds, and result interpretation, making cross-study comparisons difficult and slowing clinical integration. Addressing these barriers will be essential for transitioning liquid biopsy from a promising research tool to a reliable component of post-treatment monitoring in HPV-related tonsillar cancer.

Future Directions

As ctHPV DNA continues to gain traction as a non-invasive biomarker in HPV-related head and neck cancers, future research must focus on validating its performance specifically in TSCC. Given the unique anatomical and histological characteristics of the palatine tonsils, subsite-specific studies are needed to establish reliable sensitivity thresholds and clarify biomarker kinetics. Prospective, multicenter trials with standardized sampling protocols and long-term follow-up will be essential to assess the clinical utility of ctHPV DNA in detecting minimal residual disease, monitoring treatment response, and guiding recurrence surveillance. In parallel, emerging epigenetic biomarkers, such as DNA methylation patterns and circulating RNA species, warrant further investigation as potential complementary tools to enhance diagnostic accuracy. The development of cost-effective, scalable assays and integration into evidence-based clinical guidelines remain key challenges. Ultimately, a more personalized approach to TSCC surveillance, guided by liquid biopsy and molecular profiling, may significantly improve patient outcomes and reduce unnecessary interventions.

Conclusions

Despite its generally favorable prognosis and high radiosensitivity, HPV-positive tonsillar cancer is affecting a growing number of patients worldwide. A persistent clinical challenge lies in accurately identifying recurrence, particularly in early or subclinical stages, where conventional surveillance methods such as imaging and physical examination may lack sufficient sensitivity. Precision oncology offers a transformative approach by integrating molecular profiling with individualized patient care. In this context, liquid biopsy has emerged as a powerful, non-invasive tool capable of detecting tumor-derived biomarkers, such as circulating cell-free HPV DNA, which can signal recurrence well before clinical or radiologic evidence becomes apparent. When combined with traditional diagnostics—including imaging and histopathology—liquid biopsy enables a more personalized and dynamic evaluation of disease status. However, widespread clinical implementation remains limited by the lack of standardized protocols, assay variability, and inconsistent validation across care settings. Moving forward, a precision oncology–driven strategy that unifies clinical, radiologic, and molecular data will be essential for establishing optimized follow-up pathways and improving outcomes in patients with HPV-associated head and neck cancers.