A prospective cohort study of HPV-mediated oral and oropharyngeal cancer and efficient detection methods

A prospective cohort study of HPV-mediated oral and oropharyngeal cancer and efficient detection methods Zdravka Pashova-Tasseva1, Vessela Raykova2, Milena Ivanova-Shivarova3, Stanislav Yordanov4, Svetoslav Slavkov5, Daniel Markov5, Viktor Lenkov6, Elitsa Deliverska7 1 Department of Periodontology, Faculty of Dental Medicine, Medical University-Sofia, Sofia, Bulgaria 2 Medical University-Sofia, Sofia, Bulgaria 3 Department of Clinical immunology, University hospital Alexandrovska, Medical University-Sofia, Sofia, Bulgaria 4 Department of ENT diseases, University hospital St. Anna, Sofia, Sofia, Bulgaria 5 Department of Maxillofacial Surgery, Pirogov Hospital; Bulgaria., Sofia, Bulgaria 6 FDM, MU – Sofia, Sofia, Bulgaria 7 Department of Oral and Maxillofacial surgery, Medical University-Sofia, Sofia, Bulgaria Corresponding author: Elitsa Deliverska (elitsadeliver[email protected]m) Received 14 August 2025♦ Accepted 11 September 2025♦ Published 7 October 2025 Citation: Pashova-Tasseva Z, Raykova V, Ivanova-Shivarova M, Yordanov S, Slavkov S, Markov D, Lenkov V, Deliverska E (2025) A prospective cohort study of HPV-mediated oral and oropharyngeal cancer and efficient detection methods. Pharmacia 72: 1–10. https://doi.org/10.3897/pharmacia.72.e168813 Abstract The role of HPV is accepted in pathogenesis of oral and oropharyngeal squamous cell carcinoma (OPSCC) . The aim of this study is to investigate the association between HPV and oral and oropharyngeal carcinoma, to analyze the oncoanatomical localizations and effective detection methods. We investigated 89 patients-50 patients with histologically proven OPSCC and 39 people as a test group. Samples for HPV detection were taken with smear test and oral rinse of each patient. In 30% of cases with OPSSC we found an association with HPV in the test group. Strains were isolated from samples by PCR method. Combination of oral rinse and brush has the highest HPV detection rate (77.8%). Tumours in oropharynx have a higher risk of HPV infection. In the study, HPV16 was found to be predominant, as well as other strains 51,52,58,66,6,31,39, alone or in combination. Of important clinical importance is the accurate determination of the HPV status of tumor, which is important for management and prognosis. Keywords detection methods, Human papillomavirus, Oral carcinoma, Oropharyngeal Squamous cell carcinoma, prevalence Copyright Pashova-Tasseva Z et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Pharmacia 72: 1–10 DOI 10.3897/pharmacia.72.e168813 Research Article Pashova-Tasseva Z et al.: A prospective cohort study of hpv-mediated ora and oropharyngeal cancers2 Introduction Oral carcinoma ranks among the ten most common malignant diseases worldwide, which emphasizes its importance in the context of public health. Notably, the incidence of oropharyngeal squamous cell carcinoma (OSCC) in Western Europe and the United States has increased dramatically over the past two decades (Chaturvedi et al. 2011; Roman and Aragones 2021). In 2020, 94,412 new cases of (OSCC) and 48,143 deaths worldwide were registered (Sung et al. 2021; Khan et al. 2025). This increase in incidence is largely due to human papillomavirus (HPV) infection, as the incidence of HPV – mediated OPSCC (HPV + OPC) has increased but with clear geographic variations (Mehanna et al. 2013, 2016; Van Dyne et al. 2015). In Bulgaria for 2020, the incidence is 3.3/100,000, with a difference between the sexes, respectively 5.0/100,000 in men and 1.7/100,000 in women (Ferlay et al. 2020). There is also high variability in the incidence of infection by anatomical location (Yete et al. 2018). HPV-mediated oropharyngeal cancer is a distinct disease entity with distinct epidemiological and molecularfeatures and biological behaviour and is characterized by better response to treatment and survival than HPV-unrelated oropharyngeal cancer (Ang et al. 2010; Lechner et al. 2022; Jung et al. 2010). Oncogenesis in HPV-associated cancers is mediated by viral oncoproteins E6 and E7, which inhibit the tumor suppressor proteins p53 and retinoblastoma (Rb) protein, respectively (Hebner and Laimins 2006; Lechner et al. 2022). In contrast, HPV-negative HNSCC is due to DNA damage by carcinogens with subsequent mutations in tumor suppressor genes such as TP53 and CDKN2A (Khan et al. 2025). Given the prognostic advantage of HPV-related oropharyngeal cancer, the American Joint Committee on Cancer (AJCC) and the Union for International Cancer Control (UICC) (UICC) developed separate classifications for HPV-related and non-HPV-related diseases in the new TNM 8th edition staging system (TNM 8) for HPV-related oropharyngeal cancer (Craig et al. 2019). Data on the prevalence of HPV in the oral cavity are important for several reasons – to understand the profile of the tumor lesion – HPV positive or negative, as HPV positives have a better prognosis; to define risk groups; to be used as a measure of the results and efficacy of vaccination programs. Therefore, various effective and non-invasive methods of research are sought (Chaturvedi et al. 2011; Craig et al. 2019; De Martel et al. 2020). HPV mediation of oropharyngeal cancer can be established by testing for the presence of HPV DNA or mRNA in the tumor using PCR-based methods or in-situ hybridization(ISH). Overexpression of the p16 protein serves as an excellent surrogate biomarker for HPV causation in OPSSC (Smeets et al. 2007; Tang et al. 2020). The positive prognosis is also more pronounced in HPV-positive patients who are also p16 positive (Mehanna et al. 2023). The world literature discusses conventional methods for HPV detection (p16 immunohistochemistry, HPV DNA ISH, HPV DNA PCR, HPV E6/E7 mRNA RT-PCR, HPV RNA ISH, as well as emerging new approaches (HPV circulating tumor DNA, HPV16 E6 antibodies, oral HPV DNA/mRNA PCR, NGS). Currently, a combined testing approach using sequential screening with p16 immunohistochemistry and confirmation with HPV DNA PCR is preferred by many authors(Smeets et al. 2007; Tang et al. 2020; Mehanna et al. 2023). HPV RNA in-situ hybridization could potentially serve as a single test due to its good sensitivity and specificity, but is not routinely used (Jung et al. 2010; Mena et al. 2022; Khan et al. 2025). The use of liquid biopsies such as HPV circulating tumor DNA could be successfully used for early detection of HPV-associated oropharyngeal carcinomas. HPV16 E6 antibodies and oral HPV DNA PCR can be used as additional tests to aid diagnosis (Tang et al. 2020; Mehanna et al. 2023). However, there are limitations to the use of PCR for HPV DNA detection – the test can be very sensitive; it is impossible to distinguish whether HPV DNA detected by this method is from malignant tumor tissue or the virus is only present in adjacent, non-neoplastic tissues. Detection of HPV DNA by PCR, like DNA ISH, does not provide information on whether the virus is transcriptionally active. To address this question, several studies have shown that reverse transcription PCR can be used on formalin-fixed specimens to detect E6 and E7 mRNA transcripts, although this method is not currently available for clinical use and has not been compared with other methodologies (van Houten et al. 2001; Smeets et al. 2007; Palve et al. 2018; Tang et al. 2020; Mehanna et al. 2023; Hillier et al. 2025; Khan et al. 2025). It is assumed that to be biologically and clinically relevant, HPV must be transcriptionally active, and some investigators have suggested that detection of HPV RNA should be the gold standard for HPV testing. For example, patients with HPV DNA+/RNA+ have improved overall survival compared with those with HPV DNA+/RNA– (Jung et al. 2010). In the dynamic environment of new tests and developments in molecular biology, clinicians must have access to and choose cost-effective, accurate, and effective HPV tests, as there is still no standard for HPV virus detection. The aim of this study is to investigate the relationship between HPV and oral and oropharyngeal carcinoma, to analyze the oncoanatomical localizations, and to discuss effective detection methods. Design of the study This study represents a centralized analysis of individual data for patients diagnosed with oral and oropharyngeal carcinoma based on data from three hospitals in Sofia, Bulgaria. Material and methods A total of 89 subjects participated in the study – 50 cancer patients and 39 healthy controls. All participants agreed to participate in the study and signed an informed consent. Pharmacia 72: 1–10 3 The patients were recruited from Alexandrovska Hospital, Pirogov Hospital and St. Anna Hospital in Sofia, while healthy volunteers were randomly selected. A clinical questionnaire was prepared for the patient and the treating team of doctors to fill in information – information on age, gender, tobacco use and use of electronic cigarettes and alcohol; TNM 7th edition staging; comorbidity; local oral status in relation to the presence of periodontal disease and local irritants, location of the lesion, biological behaviour of lesion,clinical characteristic of lesion, and histopathological findings. Inclusion criteria were a patient with histologically verified primary oropharyngeal squamous cell carcinoma. Patients underwent imaging (CT or MRI) and histological confirmation by biopsy – formalin-fixed, paraffin-embedded tissue. Patients were treated with surgery, radiotherapy, chemotherapy or a combination of these, or were referred for palliative treatment. HPV testing was performed using oral rinse and oral brush samples for HPV DNA PCR detection. – oral rinse 15 – to 30-second rinsing/swishing, followed by 15 – to 30-second gargling. Brush samples are taken from the area of the tumor lesion. Samples were collected from all participants in the study using pre-defined protocols by the treating clinicians and stored and transported according to strict guidelines. In the collected samples, genotyping and quantification of HPV were performed by multiplex Real Time PCR using validated commercial kits HPV Quant 21 on the test group – 50 patients with oropharyngeal carcinoma and on the healthy controls – 39. Multiplex serologic testing was performed at Department of Microbiology – Faculty of Medicine – MU – Sofia. Kits for Real-Time PCR quantitative detection and genotyping of 21 HPV genotypes were used, including high-risk 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82 and low-risk 6, 11 and 44 . Molecular methods for HPV detection, specifically PCR-based viral DNA assays in our study were performed using oral rinse and brush biopsytest samples in our study is consistent with molecular methods for HPV detection, specifically PCR-based assays for viral DNA. These noninvasive approaches are suitable for identifying the presence of HPV in exfoliated oral epithelial cells and saliva, although they do not distinguish between transcriptionally active infections, but they allow us to compare the results of the two methods and determine which is more accurate. Database, ethics approval and consent to participate This study was performed in accordance with the Declaration of Helsinki. Informed consent was obtained for patients diagnosed in 2024–2025. A standardized Microsoft Excel form was used to collect and harmonize data. Ethics approval of the study was obtained by the ethics committee (KENIMUS) of the Medical University – Sofia. No. 03/23.02.2024., ethic approval date – №985-29/02/2024. Statistical analysis SPSS (Statistical Program for Statistics) was used to process the data from the study. Package for the Social Sciences ) was version 20.0. 1. Descriptive statistics – Quantitative variables are represented by summary statistical characteristics – arithmetic mean (Mean), standard deviation (SD); minimum and maximum value. – Categorical variables are represented by absolute (N) and relative (%) frequencies. 2. One-Sample Kolmogorov-Smirnov test to check the shape of frequency distributions for quantitative variables. 3. Chi-square test or Fisher’s exact test – when examining relationships between descriptive (categorical) data with two or more categories. 4. T-test – when comparing two independent groups when the distribution of the variable under study is normal. The adopted significance level is α=0.05. Statistical significance is accepted when the p value is less than α (p < 0.05). Results The results of our study show that the average age of patients with HPV positive (+) is 60 years, and HPV negative (-) is 62 years. No statistically significant relationship was observed (Table 1). Males are more affected, accounting for 80% of patients, with nearly 1/3 for HPV (+). In our study 70% of the test group – patients with OSSC – were HPV (-), while in the control group 95% were HPV (-). In 12% of the test group more than one strain was detected (Table 3). Table 1. Mean age of patients with OSCC carcinoma depending on HPV status. HPV status N Mean SD Min Max p Age No 35 61.97 10.93 38.00 83.00 0.783 Yes 15 60.17 10.15 44.00 78.00 Table 2. Distribution of patients by gender depending on HPV status. HPV status Total p No Yes Gender Male N 29 11 40 0.462 % 82.9% 73.3% 80.0% Female N 6 4 10 % 17.1% 26.7% 20.0% Pashova-Tasseva Z et al.: A prospective cohort study of hpv-mediated ora and oropharyngeal cancers4 In the conducted study, HPV16 was predominantly found both alone and in combination with other strains – 51, 52, 58, 66, 6, 31, 39 (Table 4) n=1.0717 close to statistical significance. It is interesting that other strains were also found both alone –HPV 6 and in combination –HPV 39 and 58. In the control group – 5.1% positive for HPV, only single strains were found – HPV16 and 51. The oncoanatomical localization of the studied patients (Fig. 1) from the test group shows that 20% of the patients have carcinoma of the tongue and 34% have carcinoma of the larynx, 10% have floor of the mouth, 8% have localization of the tongue and floor of the mouth, and 10% have pharyngeal involvement. In 22% of the patients, involvement of more than one oncoanatomical area was found. The results of our study show that pharynx carcinomas are HPV (+) in a higher percentage of cases, as well as pharynx and root of the tongue, without a statistically significant relationship. Regarding the oral cavity, the floor of the mouth and tongue are more affected, but again without a statistically significant relationship, but weakly expressed trends are visible. In the localization of carcinoma-larynx, over half of the cases are HPV (-). In 25 cases with the floor of the mouth involved, the carcinoma is negative for HPV in isolation or in various combinations , in 4 cases it is HPV (+). In all other localizations, 25 cases are HPV negative and 11 are HPV positive without a statistically significant relationship. In the 50 cases of malignant tumors in different areas of the oral cavity, larynx and pharynx studied, HPV was not detected in most cases (70%, 35/50), HPV positives were in a total 30% (15/50). Detection was found in:1 HPV strain in 9 cases (18%) and more than 1 HPV strain in 6 cases (12%)(Table 5, Fig. 2). This means that most tumors do not contain HPV, but in some patients the virus is present, which may be a factor in the development of the disease. Distribution by localization in the study as the most commonly affected area is the Larynx – 17 cases (34% of all), with 29% of them being HPV positive.The second most common is Tongue – 10 cases (20%), half of which are HPV positive. Other more common locations: Floor of the mouth – 5 cases (40% HPV positive), Pharynx – 5 cases (60% HPV positive). Locations with the highest HPV frequency. At 100% HPV positivity (all cases tested positive): it is found that by localization – Gingiva/Floor of mouth/ Tongue (1 case, >1 strain) , Floor of mouth/Tongue (2 cases, 1 strain) and Pharynx/Tongue (1 case, >1 strain). Although the number of cases in these groups is small, the complete HPV positivity suggests a strong association between this anatomical combination and HPV infection. In the oral cavity, HPV is probably not a major factor in tumor development. The conclusions that can be made are that localizations that involve the tongue or pharynx have a higher risk of HPV infection. This is supported by the high positivity rates. The larynx is the most commonly affected area, but Table 3. HPV status in test group and control group. Groups HPV status N % Patients with OSSC HPV (-) 35 70.0 1 strain 9 18.0 More than 1 strain 6 12.0 Total 50 100.0 Control group HPV (-) 37 94.9 strain 2 5.1 Total 39 100.0 Table 4. HPV strains in test group and control group. Group HPV status N % Test group No 35 70.0 16 8 16.0 16;51;66 1 2.0 16;52 1 2.0 16;6 1 2.0 16;6;31 1 2.0 39;58 2 4.0 6 1 2.0 Total 50 100.0 Control group No 37 94.9 16 1 2.6 51 1 2.6 Total 39 100.0 Figure 1. Oncoanatomical localization of OSSC. 2,0% 2,0% 2,0% 2,0% 2,0% 2,0% 2,0% 2,0% 2,0% 8,0% 10,0% 10,0% 20,0% 34,0% gingiva/bucal mucosa gingiva/palate gingiva/floor of the mouth gingiva/tongue/floor of the mouth hard palate floor of the mouth/gingiva/buccal mucosa floor of the mouth/tongue tongue/floor of the mouth/palate pharynx/tongue tongue/floor of the mouth floor of the mouth pharynx tongue larynx Oncoanatomical localization Pharmacia 72: 1–10 5 Table 5. Oncoanatomical localization of OSSC and HPV status. Oncoanatomical localization of OSSC HPV status Total No Yes Gingiva/buccal mucosa N 1 0 1 % 2.9% 0.0% 2.0% Gingiva/ hard palate N 1 0 1 % 2.9% 0.0% 2.0% Gingiva/floor of the mouth N 1 0 1 % 2.9% 0.0% 2.0% Gingiva/floor of the mouth /tongue N 0 1 1 % 0.0% 6.7% 2.0% Tongue N 8 2 10 % 22.9% 13.3% 20.0% Tongue/floor of the mouth N 4 0 4 % 11.4% 0.0% 8.0% Larynx N 12 5 17 % 34.3% 33.3% 34.0% Hard palate N 1 0 1 % 2.9% 0.0% 2.0% Floor of the mouth N 3 2 5 % 8.6% 13.3% 10.0% Floor of the mouth/buccal mucosa N 1 0 1 % 2.9% 0.0% 2.0% Floor of the mouth/tongue N 0 1 1 % 0.0% 6.7% 2.0% Floor of the mouth/tongue/palate N 1 0 1 % 2.9% 0.0% 2.0% Pharynx N 2 3 5 % 5.7% 20.0% 10.0% Pharynx/tongue N 0 1 1 % 0.0% 6.7% 2.0% Total N 35 15 50 % 100.0% 100.0% 100.0% Table 6. HPVstrains in different oncoanatomical locations. Oncoanatomical localization HPV finding Total No finding 1 strain More than 1 strain Gingiva/buccal mucosa N 1 0 0 1 % 2.9% 0.0% 0.0% 2.0% Gingiva/Palate N 1 0 0 1 % 2.9% 0.0% 0.0% 2.0% Gingiva/Floor of mouth N 1 0 0 1 % 2.9% 0.0% 0.0% 2.0% Gingiva/Floor of mouth/Tongue N 0 0 1 1 % 0.0% 0.0% 16.7% 2.0% Language N 8 2 0 10 % 22.9% 22.2% 0.0% 20.0% Tongue/Floor of mouth N 4 0 0 4 % 11.4% 0.0% 0.0% 8.0% Larynx N 12 4 1 17 % 34.3% 44.4% 16.7% 34.0% Palate N 1 0 0 1 % 2.9% 0.0% 0.0% 2.0% Floor of the mouth N 3 1 1 5 % 8.6% 11.1% 16.7% 10.0% Floor of mouth/ buccal mucosa N 1 0 0 1 % 2.9% 0.0% 0.0% 2.0% Floor of mouth/ Tongue N 0 1 0 1 % 0.0% 11.1% 0.0% 2.0% Floor of mouth/ Tongue/Palate N 1 0 0 1 % 2.9% 0.0% 0.0% 2.0% Pharynx N 2 1 2 5 % 5.7% 11.1% 33.3% 10.0% Pharynx/Tongue N 0 0 1 1 % 0.0% 0.0% 16.7% 2.0% Total N 35 9 6 50 % 100.0% 100.0% 100.0% 100.0% Figure 2. Oncoanatomical localization of OSSC and HPV status. 0,0% 5,0% 10,0% 15,0% 20,0% 25,0% 30,0% 35,0% 40,0% gingiva/buccal mucosa gingiva/hard palate gingiva/floor of the mouth gingiva/floor of the mouth/tongue tongue tongue/floor of the mouth larynx hard palаte floor of the mouth floor of the mouth/buccal mucosa floor of the mouth/tongue floor of the mouth/tongue/palate pharynx pharynx/tongue Oncoanatomical localization Total HPV finding - HPV finding - not always associated with HPV (most are HPV negative). In combined anatomical areas (e.g. Tongue + Pharynx) the incidence of HPV is higher. More than 1 HPV strain is less common (only 12%), but is often found in areas with a more aggressive tumor profile (pharynx, tongue) (Table 6, Fig. 3). Locations such as the gingiva and palate are less associated with HPV and other factors probably play a greater role there (smoking, alcohol, etc.). The oncoanatomic location as Gingiva/Floor of mouth/ Tongue, Floor of mouth/Tongue, and Pharynx/Tongue all have 100% HPV positivity, while several locations have 0%. Pashova-Tasseva Z et al.: A prospective cohort study of hpv-mediated ora and oropharyngeal cancers6 In our study, it was found that the predominant localization of the pharynx and larynx demonstrated a higher frequency of HPV (+) – in 11 patients HPV (+) carcinoma was detected , with 7 cases having one strain, and 4 cases having more than one strain (Table 7). Combination patients of oral rinse and brush has the highest detection rate of HPV (77.8%). And with statistical significance of the results (Table 8, Fig. 4). And oral rinse and brush test separately detect HPV in about 61% of cases, but the combined method increases the likelihood of detecting more than one strain. In the case of a pap smear in the oral cavity, 2 cases (40%) were diagnosed, and in the upper respiratory tract 3 cases (60%). The same ratio was observed in a mouthwash sample. When using both methods together, cases were identified only in the upper Table 8. Comparison of HPV detection methods in patients with OSSC. Group Sample HPV status Total p HPV(-) HPV(+) 1 strain HPV(+) More than 1 strain Test group Sample No N 35 0 0 35 <0.001 % 100.0% 0.0% 0.0% 70.0% N 0 4 1 5 % 0.0% 44.4% 16.7% 10.0% N 0 4 1 5 % 0.0% 44.4% 16.7% 10.0% Combined method (brush test and oral rinse) N 0 1 4 5 % 0.0% 11.1% 66.7% 10.0% Control group Sample No N 37 0 37 0.001 % 100.0% 0.0% 94.9% Brush test N 0 2 2 % 0.0% 100.0% 5.1% Figure 3. The percentage of HPV-positive cases for each tumor location. Table 7. HPV status in oral cavity carcinoma and pharyngeal and laryngeal carcinoma. HPV status Total p Negative 1 strain More than one strain Localization Oral mucosa N 21 2 2 25 0.104 % 60.0% 22.2% 33.3% 50.0% Pharyngeal/laryngeal cancer N 14 7 4 25 % 40.0% 77.8% 66.7% 50.0% Figure 4. Comparison of HPV detection methods in patients with OSSC. Pharmacia 72: 1–10 7 respiratory tract – 5 cases (100%). For greater accuracy, we recommend combining the two HPV detection tests. In summary, the results highlight the importance of HPV as an etiological factor in oropharyngeal and oral lesions, which may have serious and important diagnostic, clinical and therapeutic implications. Discussion Human papillomavirus (HPV)-positive oral and oropharyngeal squamous cell carcinomas (OSCCs) exhibit distinct molecular characteristics compared to their HPV-negative counterparts. A marked geographic heterogeneity in HPV-attributable fractions (HPV-AFs) across head and neck cancers (HNCs), particularly in oropharyngeal carcinoma (OPC), has been consistently reported (Mehanna et al. 2013, 2016; Anantharaman et al. 2017; Algudaibi et al. 2021). Within this context, our study provides an updated assessment of the epidemiological profile of HPV-associated OPC in the Bulgarian population. In our cohort, the mean age of HPV-positive patients was 60 years, compared to 62 years for HPV-negative patients. This difference was not statistically significant, and our findings align with those of other investigators who reported similar age distributions (Rettig et al. 2018a, b; Windon et al. 2018). These observations challenge the earlier perception that HPV-positive OPC predominantly affects younger individuals and instead highlight an increasing prevalence among older patients, a trend that has also been linked to less favorable survival outcomes. Conversely, several studies have documented a higher incidence of HPV-positive OPC in younger populations (Gillison et al. 2008; Ang et al. 2010; Mehanna et al. 2013; Mena et al. 2020), suggesting potential population – and region-specific differences. Regarding sex distribution, our findings confirm the predominance of males, with 80% of cases occurring in men, irrespective of HPV status. This male predominance has also been consistently documented in previous epidemiological studies (D‘Souza et al. 2007; Gillison et al. 2015). Globally, HPV is estimated to account for approximately 25–30% of OPCs, compared with only 2–4% of oral cavity carcinomas and 2–4% of laryngeal carcinomas (Alemany and Mena 2021; Algudaibi et al. 2021; Roman and Aragones 2021; Mena et al. 2022). Analysis of the oncoanatomical distribution of tumors in the study group revealed that 20% of patients presented with carcinoma of the tongue, 34% with carcinoma of the larynx, 10% with carcinoma of the floor of the mouth, 8% with combined involvement of the tongue and floor of the mouth, and 10% with pharyngeal carcinoma. In 22% of cases, more than one anatomical site was affected. These findings are consistent with previous reports showing that approximately 30% of new cases involve oropharyngeal carcinoma (OPC), compared with 2.1% of oral cavity carcinoma (OC) and 2.3% of laryngeal carcinoma (LC) (Chaturvedi et al. 2011; De Martel et al. 2020; Mena et al. 2022). Moreover, HPV-positive OPC remains substantially more common in males, a trend repeatedly confirmed across multiple largescale studies (Gillison et al. 2015; Mena et al. 2020; Lechner et al. 2022; Fonsêca et al. 2023; Mehanna et al. 2023). The present study further highlights the prevalence of HPV in lesions localized to the oral cavity, oropharynx, and larynx. These findings are consistent with international evidence supporting HPV as a key risk factor in the development of malignant and premalignant lesions across these anatomical regions. In our cohort, HPV positivity was most frequently observed in oropharyngeal carcinomas, in line with global trends. Within the oral cavity, the floor of the mouth and tongue were more commonly affected; however, these differences did not reach statistical significance, though weakly expressed trends could be observed. In contrast, more than half of laryngeal carcinomas were HPV negative. Specifically, among 25 cases involving the floor of the mouth, carcinoma – whether isolated or in combination with other localizations – was HPV negative in the majority, with only 4 cases testing HPV positive. The higher frequency of HPV in oropharyngeal lesions likely reflects a greater propensity for viral integration and malignant transformation in this region. This may be explained by distinct features of the immune microenvironment and site-specific tissue characteristics, which appear to facilitate viral attachment and persistence to a greater extent than in the oral epithelium. When comparing the localization of HPV-positive lesions between the oral cavity and oropharynx, our data indicate that, although HPV is less frequent in the oral cavity, its presence is clinically relevant and should not be overlooked. This underscores the importance of incorporating routine HPV testing into the diagnostic algorithm for patients with oral neoplasia, particularly in light of the rising incidence of HPV-associated OPC observed globally over the past two decades (Chaturvedi et al. 2011; Palve et al. 2018; Mena et al. 2022; Fonsêca et al. 2023). Another key consideration is the role of different HPV genotypes, as high-risk types such as HPV16 are strongly associated with more aggressive disease and poorer prognosis (Mehanna et al. 2019). In our study, 70% of patients in the test group with OSCC were HPV negative, compared with 95% in the control group. Interestingly, 12% of test group patients harbored multiple HPV strains. Although our sample size was limited, we identified multiple strains in cases of laryngeal carcinoma, floor of the mouth carcinoma, and pharyngeal carcinoma. HPV16 was the most commonly detected genotype, found both alone and in combination with other strains, including HPV51, HPV52, HPV66, HPV6, and HPV31. Additional strains such as HPV6, HPV39, and HPV58 were also identified, either individually or in combination. In contrast, only single strains – HPV16 and HPV51 – were detected in the control group. Notably, pharyngeal and laryngeal carcinomas demonstrated the highest rates of HPV positivity in our series: HPV-positive tumors were identified in 11 patients, of whom seven carried a single strain and four had multiple strains. Nevertheless, the laryngeal epithelium is generally regarded as less susceptible to HPV infection, owing to anatomical and immunological barriers (D’Souza et al. 2007; Roman and Aragones 2021; Mena et al. 2022). Pashova-Tasseva Z et al.: A prospective cohort study of hpv-mediated ora and oropharyngeal cancers8 The genotypic profile observed in our study largely parallels patterns reported internationally, where HPV31, HPV33, and HPV52 frequently emerge as additional oncogenic strains (van Houten et al. 2001; Gillison et al. 2008; Mehanna et al. 2016; Schache et al. 2016). While we did not detect HPV18 or HPV33 in our cohort, we identified other oncogenic types such as HPV51, HPV66, HPV6, HPV39, and HPV58. This diversity emphasizes the ubiquity of HPV and reinforces the necessity of effective preventive strategies, including vaccination, to reduce the burden of HPV-associated malignancies (Bhatia et al. 2015; Mehanna et al. 2019; Tsentemeidou et al. 2021). From a clinical standpoint, the identification of HPV-positive lesions has a direct impact on therapeutic decision-making and patient follow-up (Fakhry et al. 2008). HPV-positive oropharyngeal carcinomas, for instance, are characterized by an enhanced response to radiotherapy and chemotherapy, which translates into improved overall survival rates (Brenner et al. 2020). Accurate detection and genotyping of HPV strains are therefore crucial for diagnosis, prognosis, and long-term management of patients with HPV-related lesions. A variety of laboratory methods are currently employed in clinical practice, each with distinct advantages and limitations in terms of sensitivity, specificity, complexity, and applicability. Polymerase chain reaction (PCR) remains one of the most widely used approaches, offering high sensitivity and the capacity to detect both general HPV presence and individual genotypes. In our study, we employed PCR, including multiplex PCR, which enables the simultaneous detection of multiple HPV types – a valuable advantage in tumors with potential coinfection. Two types of samples were analyzed, tumor tissue obtained by brush sampling and oral rinse specimens. Our findings demonstrated a statistically significant advantage of using both approaches in combination, yielding the most reliable and accurate resultsл The choice of diagnostic method continues to be debated in the literature, with increasing interest in non-invasive, highly sensitive approaches. While PCR is rapid and sensitive, it does not provide information regarding viral integration. Immunohistochemistry (IHC) for p16 is cost-effective and simple yet lacks specificity. In situ hybridization (ISH) offers localization of viral DNA but is technically more demanding and costly. Next-generation sequencing (NGS) represents the most informative tool, capable of identifying integration events and viral variants, though it requires substantial resources and technical expertise (Gillison et al. 2008; Tang et al. 2020; Hillier et al. 2025). Based on our results, we conclude that combined brush and oral rinse sampling with PCR provides a practical, sensitive, and feasible strategy that could be integrated into diagnostic and screening protocols. Despite these promising findings, several limitations of our study must be acknowledged. The relatively small sample size and the diagnostic methods applied restrict the generalizability of our conclusions. Future research should focus on larger and more heterogeneous populations, employing more advanced molecular techniques to improve HPV detection and characterization. Given the sensitivity of HPV-driven tumors to immune-mediated mechanisms, novel therapeutic strategies such as immunotherapy and molecularly targeted therapies hold considerable promises for improving patient prognosis and survival (Wakeham et al. 2019). In conclusion, our study reinforces the pivotal role of HPV in the pathogenesis of lesions of the oral cavity and oropharynx and highlights the necessity of an integrated diagnostic and therapeutic approach that incorporates HPV status assessment. Such strategies will facilitate more effective prevention, provide a more accurate prognosis, and support the development of personalized treatment regimens. Conclusion HPV-mediated oral and oropharyngeal carcinomas have different pathogenesis and a better prognosis than HPV-negative or opharyngealcarcinomas, which affect staging andprognosis. Therefore, accurate determination of the HPV status of the tumor lesion is of important clinical importance, especially in oropharyngeal carcinomas. Acknowledgement We thank the medical and dental practitioners who participated in the study. Written consent form was considered as an agreement for participation in the study. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statements Clinical trials: An ethics statement was conducted in full accordance with the World Medical Association Declaration of Helsinki. The study was approved by The Institutional Ethic Committee of Medical University - Sofia, KENIMUS. No. 03/23.02.2024., ethic approval date- №985-29/02/2024. The authors declared that experiments on humans or human tissues were performed for the present study. Informed consent from the humans, donors or donors’ representatives: Medical UniversitySofia The authors declared that no experiments on animals were performed for the present study. The authors declared that no commercially available immortalised human and animal cell lines were used in the present study. Use of AI No use of AI was reported. Funding This research was funded by Scientific Research Fund, Ministry of Education and Science, Bulgaria, contract No. KP-06-N73/8 dated 5.12.23. Pharmacia 72: 1–10 9 Author contributions All authors had full access to the data in the study and took responsibility for the integrity of the data and the accuracy of the data analysis. Author ORCIDs Zdravka Pashova-Tasseva https://orcid.org/0000-0002-2635083X Vessela Raykova https://orcid.org/0000-0002-9199-9600 Milena Ivanova-Shivarova https://orcid.org/0000-0003-41289915 Stanislav Yordanov https://orcid.org/0009-0002-9145-7235 Svetoslav Slavkov https://orcid.org/0000-0001-9088-5532 Daniel Markov https://orcid.org/0000-0001-9169-6987 Viktor Lenkov https://orcid.org/0009-0000-8164-7559 Elitsa Deliverska https://orcid.org/0000-0002-2356-9932 Data availability All of the data that support the findings of this study are available in the main text. 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