Recurrent respiratory papillomatosis (RRP) is a chronic human papillomavirus (HPV)-related disease characterized by papillomatous lesions in the upper airway, usually caused by HPV-6 and/or HPV-11 infection in larynx. Both children and adults are affected by the disease, which shows a high tendency of recurrence and leads to repeated surgeries lifelong to avoid respiratory obstruction by the papillomatous lesions [1,2]. RRP with an aggressive clinical course can be described by multiple surgical interventions per year, tracheostomy need, papillomatous lesions detected in extra-laryngeal sites, and malignant transformation [[2], [3], [4]]. Molecular tests would be an important strategy to track RRP patients with active disease and to better stratify those at risk of an aggressive clinical course of the disease. Since RRP affects the upper airway, saliva can be a suitable and non-invasive sampling source for clinical monitoring of the patients. Furthermore, saliva can be used in several pathologies for viral detection, including screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Epstein-Barr virus (EBV), and HPV [[5], [6], [7], [8]].

Liquid biopsy is an emerging investigation area based on minimally invasive procedures with potential to track molecular alterations clinically useful through biological fluids (e.g., blood, saliva, and urine) [9,10]. One of the most investigated molecular category in liquid biopsy is represented by the cell-free DNA (cfDNA), usually released into biofluids from processes of cell death (e.g., apoptosis, necrosis, and autophagy) or active cellular secretions [11,12]. HPV cfDNA screening in biofluids has been reported as a diagnostic and prognostic biomarker useful for clinical surveillance and to describe the disease status of HPV-associated oropharyngeal cancer [13,14].

The HPV viral load dynamics can be assessed by polymerase chain reaction (PCR) assays through the cycle threshold (Ct) values. The Cobas® 4800 HPV Test (Roche Molecular Systems, Pleasanton, CA, USA) is an automated system clinically FDA-approved based on PCR technology to detect 14 high-risk HPV (HR-HPV) types. Cobas® test has been used to better risk stratification of cervical cancer and cervical precursor lesions through Ct values, in which smaller Ct values represent higher viral copies [15,16]. In this work, we investigated whether HPV DNA can be detected in saliva (both cellular pellets and circulating cfDNA) from RRP patients to evaluate the influence of the HPV viral load dynamics on clinical manifestation of the disease.