Head and neck squamous cell carcinomas are among the most common cancers worldwide (sixth) and rank as the eighth leading cause of global mortality [1]. In 60% of cases, the cancer is locally advanced, with a recurrence rate of 50%, which is most often incurable [2]. Tobacco and alcohol consumption are predisposing factors, as are infections with human papillomavirus (HPV) and Epstein-Barr virus. Hereditary genetic factors also predispose individuals to these cancers [3].

HPV-negative cancers, resulting from tobacco and alcohol exposure, are characterized by genetic instability and a mutation profile distinct from HPV-positive cancers. HPV-negative cancers are resistant to chemotherapy and radiotherapy and have a poorer prognosis than HPV-positive tumors. Indeed, patients with HPV-negative tumors have a lower 3-year overall survival compared with HPV-positive patients (3-year overall survival rates of 57.1% vs 82.4%). Three-year progression-free survival (PFS) rates are 43.4% and 73.7% in the HPV-negative and HPV-positive groups, respectively [3].

HPV-negative tumors also show a higher tendency to recur. In a prospective study of 124 patients with advanced oropharyngeal tumors (86% stage IV), HPV-negative patients were 3.5 times more likely to develop disease recurrence (hazard ratio 3.5, 95% CI 1.6-7.9; P=.002) than HPV-positive patients. Three-year recurrence rates were approximately 54% for HPV-negative patients compared with 29% for HPV-positive patients [4].

Most head and neck cancers are diagnosed at an advanced stage (70.3% stage III/IV). Early recurrence after treatment is difficult to detect with current imaging modalities and tumor markers due to the anatomical location and previous treatments of the primary tumor (radiotherapy, surgery, and reconstruction). In addition, the spatial, epidemiological, genetic, and epigenetic heterogeneity of these cancers further complicates the prediction of recurrence risk [5].

Currently, no effective biomarker exists to assess treatment response or early recurrence in HPV-negative patients. Circulating tumor DNA (ctDNA) may represent a promising biomarker for detecting minimal residual disease and monitoring treatment response. ctDNA consists of small DNA fragments derived from the tumor, 150 to 200 base pairs in length, that are present in the blood of patients with cancer [6]. This DNA is released into the bloodstream following apoptosis or necrosis of tumor cells and carries somatic mutations from primary and secondary tumors. The presence of ctDNA was first demonstrated in 1994 by Sorenson in patients with pancreatic carcinoma, showing that the same Kirsten rat sarcoma viral oncogene homolog gene mutation was present in both the tumor and circulating DNA [7].

Liquid biopsy, a technique developed in oncology over the past 50 years, allows the analysis of ctDNA in patients with cancer. This method is noninvasive, relatively inexpensive, and can provide multiple pieces of information, including genetic and epigenetic profiles, detection of minimal residual disease, and monitoring of treatment response [8-11]. Therefore, ctDNA detection could become a potential biomarker for HPV-negative head and neck cancers.

Longitudinal monitoring using Epstein-Barr virus and HPV DNA (E6/E7 oncogene segments) has been extensively studied, in contrast to the evaluation of ctDNA in HPV-negative patients [12-14]. A 2018 study of 7 HPV-positive and HPV-negative patients aimed to evaluate ctDNA as a biomarker for detecting recurrence or treatment response after surgery or chemoradiotherapy. ctDNA was detectable before treatment in all patients with stage IV cancer. Two patients with local recurrence had detectable ctDNA prior to clinical relapse, whereas no mutations were detected in 2 patients who developed distant metastases before clinical recurrence [15].

Several studies investigated the correlation between ctDNA, treatment response, and recurrence in 26 patients with head and neck squamous cell carcinoma (22 HPV-negative and 4 HPV-positive). Tumor mutations were analyzed along with peripheral blood mononuclear cells, and each patient’s mutations were tracked in the blood. Mutated ctDNA was detectable before treatment initiation in 15 of 18 analyzed patients (6 were excluded due to probe design failure). The 3 patients without detectable ctDNA before treatment included 2 with stage I tumors and 1 with external auditory canal carcinoma.

After curative treatment, ctDNA was detected in all patients with local recurrence or distant metastases (7/7), whereas the 11 patients with undetectable ctDNA remained alive and recurrence-free during follow-up.

This study had several limitations, including heterogeneous patient characteristics (stages I-IV, HPV-negative and HPV-positive) and curative treatments. Follow-up was also heterogeneous, with a median of 563 (range 96‐1033) days [16].

A recent prospective single-center study in 2022 included 17 patients with stage III or IVb HPV-negative squamous cell carcinoma of the oral cavity, pharynx, or larynx. All patients received primary curative surgery. The study primarily aimed to determine whether postoperative ctDNA detection could serve as a biomarker for minimal residual disease and recurrence. Patients were followed for a median of 12.2 (range 9.6‐17.5) months, and preoperative ctDNA was detected in all patients. During longitudinal follow-up, ctDNA was detected before clinical progression in all cases with clinically confirmed recurrence (5/17 patients, 29%), with lead times of 3.5 to 8.3 months. Among the 12 patients without evidence of recurrence at the last follow-up, all ctDNA samples were negative [17].

Finally, a clinical trial (NCT03712566) included 55 patients (37 HPV-negative and 15 HPV-positive) with recurrent or metastatic disease to assess longitudinal ctDNA kinetics during treatment. ctDNA was detected at baseline in 94.3% of patients. Early changes in ctDNA dynamics after 1 treatment cycle better predicted both progression-free and overall survival than baseline ctDNA levels. Moreover, a decrease in ctDNA from baseline to postcycle 1 identified patients with longer overall survival despite radiological progression [18].

These studies demonstrate that longitudinal ctDNA monitoring in patients with head and neck cancer is feasible and could serve as a useful biomarker for early recurrence detection, providing clinicians with additional guidance in therapeutic decision-making. However, given the small number of patients analyzed, heterogeneous populations, varied treatments, and inconsistent follow-up, larger prospective studies are required before this approach can be implemented in routine clinical practice.

This study is justified by the need to develop more precise tools for the early prediction of recurrence in patients with ENT (ear, nose, throat) cancers, particularly those who are HPV-negative, a population for which surveillance and treatment options are currently limited. By evaluating ctDNA concentrations postoperatively and during follow-up, we may be able to predict recurrence before it is detectable by conventional clinical or imaging methods. This innovative approach aims to identify a ctDNA threshold indicative of an increased risk of recurrence, enabling earlier and tailored therapeutic interventions. Given the limited number of studies conducted on HPV-negative patients, who have a poorer prognosis compared with HPV-positive patients, it is essential to focus efforts on this specific cohort. Furthermore, the ability to more rapidly predict recurrence using ctDNA could not only improve patient monitoring and management but also allow for earlier therapeutic adjustments, such as considering a second surgery or modifying treatment.

There will be no direct benefit for patients included in this study. Nevertheless, the CIRVAD study will contribute to advancing scientific knowledge on the correlation between ctDNA concentration and recurrence and provide additional insights into the emergence and monitoring of mutations in patients with HPV-negative ENT cancers undergoing treatment.

If positive results are obtained, this study could help establish an additional postoperative monitoring method (ctDNA concentration) that is low-cost, noninvasive, and minimally burdensome for patients with head and neck cancers. The study may also allow surgeons to assess the quality of surgical procedures. These findings could ultimately enable personalized follow-up to identify recurrence risk early through a simple blood test and optimize therapeutic strategies.

The risks associated with participation in the CIRVAD protocol are minimal, as patient management (treatments and examinations) follows standard care for patients with this pathology. Individual risks related to venipuncture are minimal and comparable to those associated with routine blood sampling. Patients may occasionally experience mild pain or bruising. Rarely, fainting, small blood clots, or infection may occur at the puncture site. These risks are negligible, as they are mitigated by proper skin asepsis and the expertise of experienced personnel performing the procedure.

A 28-month follow-up period was selected because most recurrences of head and neck squamous cell carcinoma occur within the first 2 years after curative treatment, corresponding to the period of highest recurrence risk and the most intensive postoperative surveillance recommended by international guidelines. Extending follow-up to 28 months accommodates study logistics while ensuring capture of most clinically relevant recurrence events. This timeframe is therefore appropriate for evaluating the ability of early postoperative and longitudinal tumor-informed ctDNA analysis to detect molecular residual disease and predict recurrence [3,4].

Secondary objectives include evaluating plasma ctDNA concentrations before surgery, in the early postoperative period, and during follow-up as predictive factors for clinical recurrence following therapy; assessing quantitative ctDNA concentrations and their longitudinal evolution as predictors of progression-free, overall, and disease-specific survival; describing the clinical characteristics of patients and the evolution of quantitative ctDNA concentrations during follow-up in patients with and without progression throughout the 28-month follow-up period; and describing the variation in quantitative ctDNA concentrations between the preoperative and postoperative periods and analyzing their relationship with tumor size and surgical quality. The study will also determine the proportion of patients with detectable preoperative plasma ctDNA and evaluate its feasibility as a biomarker for longitudinal disease monitoring.

This is a prospective, bicentric study. This protocol was developed in accordance with the SPIRIT (Standard Protocol Items: Recommendations for Interventional Trials) 2013 Statement [19].

Plasma ctDNA samples will be collected before surgery, in the early postoperative period (1‐2 weeks after surgery), and longitudinally during scheduled follow-up visits throughout the 28-month follow-up period. For patients receiving adjuvant radiotherapy and/or chemotherapy, ctDNA analysis will be performed after completion of adjuvant treatment according to the study schedule. Plasma concentrations of ctDNA will be quantitatively expressed in ng/mL of plasma.

PFS is defined as the time from surgery to disease progression or death (event: local or regional progression, distant metastases, or death from any cause).

Plasma ctDNA concentrations obtained at each time point will be used to evaluate the association between molecular residual disease and clinical outcomes, including recurrence, PFS, overall survival, and disease-specific survival.

Time to recurrence is defined as the interval between surgery and clinically confirmed recurrence (event: local or regional recurrence, distant metastases). Overall survival is defined as the time from surgery to death from any cause, and disease-specific survival as the time from surgery to cancer-related death.

The evolution of quantitative ctDNA concentrations during follow-up and the clinical characteristics of the study population will be described and compared between patients with and without progression throughout the 28-month follow-up period. Relative and absolute variations in plasma ctDNA concentrations between the preoperative and postoperative periods will be described. The quality of the surgical procedure will be evaluated according to resection margin classification (R0, R1, and R2).

The CIRVAD study was designed to address one of the major unmet clinical needs in HPV-negative head and neck squamous cell carcinoma, namely, the lack of reliable biomarkers capable of detecting minimal residual disease and predicting recurrence before clinical or radiological evidence becomes apparent. Based on previous studies suggesting the potential value of ctDNA monitoring, we hypothesize that postoperative and longitudinal ctDNA assessment may improve risk stratification and enable earlier identification of patients at high risk of relapse.

To address this need, the CIRVAD study was designed as a prospective bicentric study including a homogeneous population of treatment-naïve patients with HPV-negative, nonmetastatic head and neck squamous cell carcinoma treated by primary surgery. Compared with previous studies, which were generally limited by small sample sizes, heterogeneous patient populations, mixed HPV status, or different treatment strategies, this study aims to provide more robust prospective evidence regarding the clinical utility of longitudinal ctDNA monitoring.

Several methodological strengths support the design of the CIRVAD study. First, the prospective design minimizes selection and information biases compared with retrospective studies. Second, the bicentric recruitment strategy should improve the external validity of the findings. Third, serial blood sampling at predefined time points allows evaluation of ctDNA kinetics throughout patient follow-up rather than relying on a single measurement. Finally, standardized laboratory procedures and a predefined statistical analysis plan are expected to improve the reproducibility and robustness of the study.

This study also presents several anticipated limitations. First, as a pilot study including 60 patients, the statistical power may remain limited for some secondary analyses and subgroup comparisons. Second, although the study population has been restricted to HPV-negative patients treated by surgery, adjuvant treatment strategies may differ according to pathological findings. To reduce the impact of this heterogeneity, treatment modalities will be incorporated into multivariable statistical models whenever appropriate. Finally, as this study is conducted in 2 French tertiary centers, external validation in larger multicenter and international cohorts will be required before widespread clinical implementation.

If ctDNA monitoring demonstrates sufficient predictive performance, future interventional studies could evaluate ctDNA-guided surveillance strategies and personalized treatment adaptation. Such approaches may include intensified imaging follow-up, earlier salvage surgery, or treatment escalation in patients presenting with molecular evidence of residual disease before clinical recurrence. Moreover, future protocol amendments could consider extending this strategy to patients receiving neoadjuvant immunotherapy, considering recent therapeutic advances in head and neck cancer.

Longitudinal ctDNA monitoring in patients with HPV-negative head and neck squamous cell carcinoma. By evaluating postoperative molecular residual disease and its association with clinical outcomes, this study may contribute to the development of more personalized surveillance strategies and earlier therapeutic interventions. If the proposed biomarker is validated, ctDNA monitoring could become a valuable complement to conventional postoperative follow-up and support the implementation of precision oncology in head and neck cancer.

Dissemination of the study results will include presentations at national and international scientific meetings and publication in peer-reviewed journals. The findings will also contribute to the development of future multicenter studies evaluating ctDNA-guided management strategies in head and neck cancer.