The diagnostic approach and management of neck mass have changed drastically during the last century. Hayes Martin was the first to recognize that cervical metastasis was a common presentation of squamous cell carcinomas (SCCs) of the aerodigestive tract in 1944 (1). This realization led Martin to introduce a new approach to the workup of neck masses, advocating for physical examination with a headlamp and direct laryngoscopy with biopsy of the primary instead of excisional biopsy (2).

Much has changed since the time of Hayes Martin. Squamous cell carcinoma of unknown primary (SCCUP) is now rarely due to smoking and drinking-related laryngeal and hypopharyngeal cancers (3–5). Instead, human papillomavirus (HPV) oropharyngeal and Epstein–Barr virus (EBV) nasopharyngeal carcinomas are the most likely sources of SCCUP, which is reflected in the recently published American Society of Clinical Oncology (ASCO) guideline for diagnosis and management of SCCUP (6).

The current workup begins with a physical examination, fiberoptic flexible in-office nasopharyngoscopy, and fine-needle aspiration (FNA) biopsy followed by immunohistochemical (IHC) p16 nuclear staining and/or DNA/RNA in situ hybridization (ISH) for high-risk HPV DNA/RNA, and/or EBV-encoded small RNAs. FNA of the neck mass can both be diagnostic and narrow the location of the primary to the oropharynx or nasopharynx. However, FNA biopsy in the workup of HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) has limitations. HPV-related cervical metastases are often cystic with significant necrosis, which can lead to indeterminate p16 staining patterns and a lack of nuclear material for HPV DNA/RNA ISH studies in as many as 49.5% of patients (7). For this reason, direct laryngoscopy with biopsy (DLB) of suspicious lesions is still needed but will only identify the primary only 20% of the time (8). Localization of the primary has been associated with improved overall survival (9). PET-CT can be of little use in the oropharynx, particularly for primaries smaller than 1 cm. Therefore, in cases where there remains no obvious tumor, transoral robotic surgery (TORS) is currently recommended to find microcarcinomas in the oropharynx and help target radiation therapy (6). Using this algorithm, 80% of SCCUPs should be found (9).

Recently, circulating tumor HPV DNA (ctHPVDNA) detectable in cell-free plasma has been developed as a biomarker for active HPV-positive cancers (10). Thus far, it has been used as a marker for treatment response and recurrence after chemoradiotherapy (11) and as a prognostic marker for recurrence after surgical treatment of OPSCC (12). Research examining the use of ctHPVDNA for diagnostic purposes has recently been conducted in non-smoking populations and has found a positive predictive value of 98.4% (13). To date, the diagnostic accuracy in SCCUP has not been specifically assessed. Given that delay in diagnosis is common for SCCUP, we suspect that ctHPVDNA may offer an improvement compared to current diagnostic methods. The present study aims a) to compare the diagnostic accuracy of ctHPVDNA in known vs. unknown primaries (5) and b) to describe how this test may fit into the diagnostic algorithm for SCCUP.

This pilot study is a retrospective review of prospectively collected data from a tertiary referral cancer center in West Virginia. Institutional Review Board approval was obtained for the purposes of the study (protocol: #2201496852). Due to the retrospective, observational nature of the study, the need for individual informed consent was waived. Twenty-seven consecutive patients with OPSCC presenting as a neck mass from January 2021 to October 2022 were included. All patients presenting for other reasons (incidentally found mass on imaging, hemoptysis, otalgia, dysphagia, and odynophagia) were excluded. All patients underwent routine diagnostic tests and procedures to obtain a diagnosis, determine HPV status, and stage the patient. The diagnostic workup flowchart is presented in Figure 1 .

The sample consisted of 27 patients and was predominantly male (77.8%) with a mean age of 62.3 (SD = 10.3; range 37–79). Of these patients, 70.4% were current or former smokers with a >10 pack-year history. The remaining 29.6% had no smoking history. After routine physical examination and flexible laryngoscopy, 48.1% (N = 13) had an identifiable primary, while the remainder (N = 14) had SCCUP. The tumor characteristics and diagnostic procedures for the cohort are presented in Table 1 .

This study is the first to compare the diagnostic accuracy of ctHPVDNA in known vs. SCCUP of the oropharynx. For patients with identifiable oropharyngeal primaries after physical examination and flexible laryngoscopy, 100% of patients with HPV-positive tumors had positive ctHPVDNA. Using the standard workup, 30.7% (4/13) of patients with known primaries required DLB solely in order to obtain HPV status. ctHPVDNA added to the diagnostic algorithm could reduce the number of operative biopsies for patients with identified primaries. For SCCUP, FNA biopsy with p16 staining and HPV-ISH demonstrated high rates of indeterminate findings in our patient sample, for a diagnostic yield of 71.4% in patients with SCCUP. For patients with unknown primary tumors, ctHPVDNA had 90.9% sensitivity, 100% specificity, 100% PPV, and 75% NPV. In this way, ctHPVDNA provided a more accurate diagnosis of HPV-related SCCUP than the standard workup.

Incorporating the use of ctHPVDNA into the ASCO diagnostic algorithm guidelines may help refine the application of TORS in SCCUP ( Figure 2 ). First, used in this way, ctHPVDNA may help reduce the total number of procedures required to obtain HPV status in unknown primaries since multiple FNAs and sometimes operative biopsies are often required. Second, for patients with SCCUP and non-operable neck disease, the presence of ctHPVDNA may help encourage the use of diagnostic TORS when FNA biopsy is non-diagnostic or indeterminate per the ASCO guidelines in the hope of finding the primary, targeting radiation, and improving survival (8).

Finally, in select patients with unidentified primaries, ctHPVDNA use may reduce the number of diagnostic TORS procedures in favor of therapeutic TORS procedures. Diagnostic TORS comes with significant pain and a 5% risk of oropharyngeal hemorrhage (9). Therefore, reducing the total number of diagnostic TORS procedures may reduce this morbidity. In this study, only 14.3% (2/14 patients with SCCUP) of patients underwent purely diagnostic TORS procedures, favoring therapeutic TORS with neck dissection when HPV-positive disease was confirmed. Using ctHPVDNA to determine HPV status, SCCUP patients with a single metastasis who were treated with definitive TORS and neck dissection were able to avoid adjuvant therapy 50% of the time. Since there is often a delay in diagnosis for SCCUP, ctHPVDNA used to obtain HPV status could potentially reduce the time from diagnosis to completion of treatment for SCCUP.

This is the first study to investigate the diagnostic utility of ctHPVDNA in determining the HPV status in SCCUP. For the unidentified primary tumors, ctHPVDNA had 90.9% sensitivity, 100% specificity, 100% PPV, and 75% NPV. Incorporation of ctHPVDNA into the diagnostic algorithm for SCCUP may reduce the need for multiple procedures to establish HPV status and may facilitate definitive treatment.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by West Virginia University Institutional Review Board. The studies were conducted in accordance with the local legislation and institutional requirements. The ethics committee/institutional review board waived the requirement of written informed consent for participation from the participants or the participants’ legal guardians/next of kin because this was a retrospective chart review performed without contact with patients and involving no risk to patients.

AK: Writing – original draft, Data curation. SS: Data curation, Formal Analysis, Writing – review & editing. PH: Data curation, Writing – review & editing, Formal Analysis. DC: Writing – review & editing. WS: Writing – review & editing. TF: Writing – review & editing. RC: Formal Analysis, Validation, Writing – original draft. EM: Formal Analysis, Writing – original draft. MT: Conceptualization, Formal Analysis, Methodology, Supervision, Writing – original draft, Writing – review & editing.