HPV-positive Oropharyngeal Squamous Cell Carcinoma Research Articles

Risk factors associated with oral Human Papillomavirus (HPV) prevalence within a young adult population

BackgroundThe prevalence of, and risk factors for, genital Human Papillomavirus (HPV) infections within the young adult population are well-established; the same is not known for oral HPV. This observational study aimed to determine oral HPV prevalence and abundance within a UK young adult population, and examine if sexual practices and established risk factors of oropharyngeal squamous cell carcinomas (OPSCCs) (such as smoking and alcohol consumption) influenced HPV prevalence.MethodsConvenience sampling was used to recruit a small sample of 452 UK-based young adults studying at a higher education (HE) institution to the study; the study was not powered. A highly sensitive real-time PCR HPV screening method was developed for the detection of multiple HPV subtypes from oral swabs. HPV-positive samples were subsequently screened by qPCR for viral subtypes HPV-6, HPV-11, HPV-16, HPV-18. Results were analysed by univariate and multivariate methods and stratified for gender, with lifestyle behaviour data collected via questionnaire. Socio-economic status was not captured within the questionnaire.ResultsWe found a high oral HPV prevalence of 22.79%, with a dominance of high-risk viral type HPV-16 (prevalence 19.12%; abundance average 1.08 × 105 copies/million cells) detected within healthy young adults. Frequent smoking (p = .05), masturbation (p = .029), and engagement in multiple sexual activities (p = .057), were found to be associated with oral HPV prevalence, and HPV-16 prevalence, whilst behaviours traditionally associated with genital HPV were not.ConclusionsOur results strengthen the link between sexual practices and oral HPV transmission. We suggest that young adults should be considered high-risk for the contraction of oral HPV, although acknowledge that this sample of HE students may not be representative of the wider population. We show that high-risk HPV-16 is prevalent in the healthy population, as well as dominating within OPSCC; this study is one of the first to determine the dominance of oral HPV-16 prevalence and abundance within this population, presenting a clear need for greater awareness of oral HPV infections, and the risk factors for HPV-positive OPSCC within young adults.

MRI for Differentiation between HPV-Positive and HPV-Negative Oropharyngeal Squamous Cell Carcinoma: A Systematic Review.

Human papillomavirus (HPV) is an important risk factor for oropharyngeal squamous cell carcinoma (OPSCC). HPV-positive (HPV+) cases are associated with a different pathophysiology, microstructure, and prognosis compared to HPV-negative (HPV-) cases. This review aimed to investigate the potential of magnetic resonance imaging (MRI) to discriminate between HPV+ and HPV- tumours and predict HPV status in OPSCC patients. A systematic literature search was performed on 15 December 2022 on EMBASE, MEDLINE ALL, Web of Science, and Cochrane according to PRISMA guidelines. Twenty-eight studies (n = 2634 patients) were included. Five, nineteen, and seven studies investigated structural MRI (e.g., T1, T2-weighted), diffusion-weighted MRI, and other sequences, respectively. Three out of four studies found that HPV+ tumours were significantly smaller in size, and their lymph node metastases were more cystic in structure than HPV- ones. Eleven out of thirteen studies found that the mean apparent diffusion coefficient was significantly higher in HPV- than HPV+ primary tumours. Other sequences need further investigation. Fourteen studies used MRI to predict HPV status using clinical, radiological, and radiomics features. The reported areas under the curve (AUC) values ranged between 0.697 and 0.944. MRI can potentially be used to find differences between HPV+ and HPV- OPSCC patients and predict HPV status with reasonable accuracy. Larger studies with external model validation using independent datasets are needed before clinical implementation.

Role of TORS as De-Escalation Strategy in HPV-Related Oropharyngeal Cancer, What We Need to Know.

Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) presents unique challenges and opportunities for treatment, particularly regarding de-escalation strategies to reduce treatment morbidity without compromising oncological outcomes. This paper examines the role of Transoral Robotic Surgery (TORS) as a de-escalation strategy in managing HPV-related OPSCC. We conducted a comprehensive literature review from January 2010 to June 2023, focusing on studies exploring TORS outcomes in patients with HPV-positive OPSCC. These findings highlight TORS's potential to reduce the need for adjuvant therapy, thereby minimizing treatment-related side effects while maintaining high rates of oncological control. TORS offers advantages such as precise tumor resection and the ability to obtain accurate pathological staging, which can guide the tailoring of adjuvant treatments. Some clinical trials provide evidence supporting the use of TORS in specific patient populations. The MC1273 trial demonstrated promising outcomes with lower doses of adjuvant radiotherapy (RT) following TORS, showing high locoregional tumor control rates and favorable survival outcomes with minimal side effects. ECOG 3311 evaluated upfront TORS followed by histopathologically directed adjuvant therapy, revealing good oncological and functional outcomes, particularly in intermediate-risk patients. The SIRS trial emphasized the benefits of upfront surgery with neck dissection followed by de-escalated RT in patients with favorable survival and excellent functional outcomes. At the same time, the PATHOS trial examined the impact of risk-adapted adjuvant treatment on functional outcomes and survival. The ongoing ADEPT trial investigates reduced-dose adjuvant RT, and the DART-HPV study aims to compare standard adjuvant chemoradiotherapy (CRT) with a reduced dose of adjuvant RT in HPV-positive OPSCC patients. These trials collectively underscore the potential of TORS in facilitating treatment de-escalation while maintaining favorable oncological and functional outcomes in selected patients with HPV-related OPSCC. The aim of this scoping review is to discuss the challenges of risk stratification, the importance of HPV status determination, and the implications of smoking on treatment outcomes. It also explores the evolving criteria for adjuvant therapy following TORS, focusing on reducing radiation dosage and volume without compromising treatment efficacy. In conclusion, TORS emerges as a viable upfront treatment option for carefully selected patients with HPV-positive OPSCC, offering a pathway toward treatment de-escalation. However, selecting the optimal candidate for TORS-based de-escalation strategies is crucial to fully leverage the benefits of treatment de-intensification.

Fibroblasts from HPV-negative oropharynx squamous cell carcinomas stimulate the release of osteopontin from cancer cells via the release of IL-6.

HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) shows distinct biological and clinical behaviour when compared to HPV-negative OPSCC. The overall role of the tumour microenvironment (TME) in head and neck cancer progression and metastasis has been studied intensively, but differences in HPV-negative and HPV-positive OPSCCs are less understood. To investigate the role of cancer-associated fibroblasts (CAFs) and the functional interactions of normal tonsil fibroblasts (NTFs) and OP CAFs with HPV+ and HPV- OPSCC cells and explore novel candidates in tumour-fibroblast crosstalk. A retrospective cohort of 143 primary OPSCCs was characterised using HPV16/18 RNAScope assay, p16 IHC and ɑ-SMA. Four OPSCC, three NTF and 2 new OPSCC CAF cultures were used to assess the cytokine-based interactions using cytokine arrays on conditioned media (CM), followed by co-culture approaches to identify the role of individual cell types and the role of OPN (SPP1) and IL-6 in SCC/fibroblast communication. HPV status was associated with better overall survival. Although ɑ-SMA expression was observed in both OPSCC subtypes, it provided survival stratification only in the HPV-positive group (Log-Rank p = 0.02). Three normal tonsillar fibroblast cultures (NTFs) were characterised by induction of myofibroblastic and senescent phenotypes with similar reactivity to our published NOF phenotype. The OPSCC-derived CAF cultures were characterised and their baseline myofibroblastic and senescence phenotypes varied. Cytokine array analysis of CM to identify novel candidates in the crosstalk between OPSCC tumour cells and NTFs/CAFs identified differences in the cytokine profiles on comparison of HPV+ and HPV- OPSCC cells. Osteopontin (OPN/SPP1) was identified, particularly in HPV-negative OPSCC cell analyses. We have demonstrated that OPN was produced by the OPSCC cells and revealed an associated upregulation of IL-6 in fibroblasts. Treatment of NTFs with rOPN showed alteration in phenotype, including increased contraction and IL-6 production. Antibody-mediated inhibition of CD44v6 attenuated the production of IL-6 by OPN in NTFs. This investigation with OPSCC fibroblasts provides novel insights into the role of CAFs in OPSCC mediated by IL-6 stimulated release of OPN from HPV negative OPSCC cells. The details of HPV-positive SCC cell/fibroblast cytokine crosstalk remain elusive.

Quality of life outcomes comparing primary Transoral Robotic Surgery (TORS) with primary radiotherapy for early-stage oropharyngeal squamous cell carcinoma: A systematic review and meta-analysis

BackgroundTransoral Robotic Surgery (TORS) and radiotherapy are considered oncologically equivalent primary treatment options for early-stage HPV-positive oropharyngeal squamous cell carcinoma (OPSCC). Quality of Life (QoL) and Patient Reported Outcome Measures (PROMs) are therefore imperative in supporting clinical decision-making and optimising patient-centred care. The aim of this article is to evaluate how these primary treatment modalities compare in terms of QoL. Materials and methodsSystematic review and meta-analysis of studies comparing primary TORS and primary radiotherapy for OPSCC using validated QoL tools. Swallowing and global QoL were the primary endpoints with secondary endpoints including all other QoL domains. An inverse variance random-effects model was employed to calculate the weighted estimate of the treatment effects across trials. ResultsA total of six studies collectively reporting on 555 patients were included (n = 236 TORS and n = 319 radiotherapy). Meta-analysis showed no significant difference for swallowing (mean difference = −0.24, p = 0.89) and global QoL (mean difference = 4.55, p = 0.14). For the remaining QoL domains (neck/shoulder impairment, neurotoxicity, voice, xerostomia, speech, and distress), the scarcity of data did not permit meta-analysis. However, the existing data showed no significant difference for any except for xerostomia where TORS appears favourable in the sole study reporting on this. ConclusionsTORS and radiotherapy appear to be comparable primary treatment options for early stage OPSCC when it comes to QoL. However, a substantial proportion of patients in the TORS group received adjuvant (chemo)radiotherapy rendering it difficult to establish the ‘true’ QoL outcomes following surgery alone. There are also minimal studies reporting QoL outcomes beyond swallowing and global QoL. Further research is therefore needed, including more randomised trials adequately powered to detect differences in QoL outcomes.

The impact of distance to facility on treatment modality, short-term outcomes, and survival of patients with HPV-positive oropharyngeal squamous cell carcinoma

PurposeThis study compared treatment and outcomes for patients with HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) based on their travel distance to treatment facility. Materials and methodsPatients with cT1–4, N0–3, M0 HPV-positive OPSCC in the National Cancer Database from 2010 to 2019 were identified and split into four quartiles based on distance to facility, with quartile 4 representing patients with furthest travel distances. Multivariable-adjusted logistic regression and Cox proportional hazards modeling were used to analyze the primary outcome of treatment received, and secondary outcomes of clinical stage, overall survival, surgical approach (i.e., TORS versus other), and 30-day surgical readmissions. Results17,207 patients with HPV-positive OPSCC were evenly distributed into four quartiles. Compared to patients in quartile 1, patients in quartile 4 were 40 % less likely to receive radiation versus surgery (OR = 0.60; 95 % CI = 0.54–0.66). Among the patients who received surgery, quartile 4 had a higher odds of receiving TORS treatment compared to quartile 1 (4v1: OR = 2.38; 95 % CI = 2.05–2.77), quartile 2 (4v2: OR = 2.31, 95 % CI = 2.00–2.66), and quartile 3 (4v3: OR = 1.75; 95 % CI = 1.54–1.99). Quartile 4 had a decreased odds of mortality compared to Quartile 1 (4v1: OR = 0.87; 95 % CI = 0.79–0.97). There were no differences among the quartiles in presenting stage and 30-day readmissions. ConclusionsThis study found that patients with furthest travel distance to facility were more often treated surgically over non-surgical management, with TORS over open surgery, and had better overall survival. These findings highlight potential disparities in access to care for patients with HPV-positive OPSCC.

Global DNA Methylation Level in Tumour and Margin Samples in Relation to Human Papilloma Virus and Epstein-Barr Virus in Patients with Oropharyngeal and Oral Squamous Cell Carcinomas.

Aberrant DNA methylation is a common epigenetic modification in cancers, including oropharyngeal squamous cell carcinoma (OPSCC) and oral squamous cell carcinoma (OSCC). Therefore, the analysis of methylation levels appears necessary to improve cancer therapy and prognosis. The enzyme-linked immunosorbent assay (ELISA) was used to analyse global DNA methylation levels in OPSCC and OSCC tumours and the margin samples after DNA isolation. HPV detection was conducted by hybridisation using GenoFlow HPV Array Test Kits (DiagCor Bioscience Inc., Hong Kong, China). EBV detection was performed using real-time PCR with an EBV PCR Kit (EBV/ISEX/100, GeneProof, Brno, Czech Republic). OPSCC tumour samples obtained from women showed lower global DNA methylation levels than those from men (1.3% vs. 3.5%, p = 0.049). The margin samples from OPSCC patients with HPV and EBV coinfection showed global DNA methylation lower than those without coinfection (p = 0.042). G3 tumours from OSCC patients had significantly lower levels of global DNA methylation than G2 tumours (0.98% ± 0.74% vs. 3.77% ± 4.97%, p = 0.010). Additionally, tumours from HPV-positive OSCC patients had significantly lower global DNA methylation levels than those from HPV-negative patients (p = 0.013). In the margin samples, we observed a significant negative correlation between global DNA methylation and the N stage of OSCC patients (rS = -0.33, p = 0.039). HPV-positive OPSCC patients had higher global DNA methylation levels than HPV-positive OSCC patients (p = 0.015). We confirmed that methylation could be changed in relation to viral factors, such as HPV and EBV, as well as clinical and demographical parameters.

Human Papilloma Virus (HPV) driven oropharyngeal cancer in current or previous heavy smokers: should we look for a different treatment paradigm?

Human papillomavirus Virus (HPV)-associated oropharyngeal squamous cell carcinoma (OSCC) has increased in incidence in recent decades and represents a heterogeneous disease entity in the context of Head and Neck Squamous Cell Carcinoma (HNSCC), in terms of disease prognosis. Treatment of locoregionally advanced OSCC is mainly based on concurrent chemoradiotherapy. Given the younger age of patients, if compared with HPV-negative counterparts, and the high cure rates, the acute- and long-term toxicity in survivors represents a field of interest. However, patient selection for de-escalation trials remains a major challenge due to the lack of robust validated prognostic indicators within the HPV-associated OSCC. The impact of smoking status on HPV-associated OSCC prognosis has been demonstrated in the majority of studies. However, the magnitude of the association is unclear due to variability in smoking metrics and study outcomes. Smoking status has been identified as a potential confounding factor in HPV-positive de-escalation trials. Smokers with HPV-positive OSCC have a worse prognosis in most studies than non-smokers and may require different and more aggressive therapeutic strategies.

Human papillomavirus circulating tumor DNA: a diagnostic tool in squamous cell carcinoma of unknown primary-a pilot study.

Neck mass is the most common presentation of human papillomavirus-related (HPV-related) oropharyngeal squamous cell carcinoma (OPSCC). Recently, circulating tumor HPV-DNA (ctHPVDNA) assays have been developed to detect active OPSCC. This pilot study investigates the diagnostic accuracy of ctHPVDNA in establishing HPV status for known vs. unknown OPSCC presenting as a neck mass. A single-institution pilot study was conducted on all patients with OPSCC presenting as a neck mass between 2021 and 2022. The diagnostic accuracy of ctHPVDNA was compared to that of standard diagnostic procedures used to obtain HPV status according to the American Society of Clinical Oncology (ASCO) guideline for squamous cell carcinoma of unknown primary (SCCUP). Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of ctHPVDNA were calculated. A total of 27 patients were included; 70.4% were current or former smokers, 48.1% (N = 13) had identifiable primaries, and 51.9% (N = 14) had SCCUP. Four patients with known primaries required operative direct laryngoscopy with biopsy (DLB) to establish HPV status. Two patients with SCCUP underwent diagnostic transoral robotic surgery (TORS) to establish HPV status and localize the primary. Twelve patients underwent therapeutic TORS and neck dissection. The gold standard for HPV status was based on final histopathologic p16 or HPV in situ hybridization (ISH) staining during workup/treatment. ctHPVDNA had 95.8% sensitivity, 100% specificity, 100% PPV, and 75% NPV in predicting HPV-positive OPSCC in the whole sample. Binary logistic regression model using ctHPVDNA results to predict HPV-positive OPSCC was significant (-2 log likelihood = 5.55, χ2=8.70, p <.01, Nagelkerke's R squared = .67). Among patients with identifiable primaries, all patients had HPV-positive tumors on final pathology, and ctHPVDNA was positive in 100%. In the unknown primary patients, ctHPVDNA had 90.9% sensitivity, 100% specificity, 100% PPV, and 75% NPV. ctHPVDNA demonstrated good diagnostic accuracy for both known and unknown primaries. Incorporation of ctHPVDNA into the diagnostic algorithm for SCCUP may reduce the need for multiple procedures to establish HPV status.

Treatment Delay and HPV Status on OPSCC With Upfront Surgery: Analysis of National Cancer Database.

Evaluate the effect of treatment delay on survival in human papillomavirus (HPV)-positive and HPV-negative oropharyngeal squamous cell carcinoma (OPSCC) patients undergoing primary surgical resection. Retrospective cohort study using the 2010-2017 National Cancer Database. Multicenter database study. Patients >18 years old with OPSCC and known HPV status, treated surgically with or without postoperative radiation/chemotherapy were included. Two cohorts based on HPV status were grouped by time to treatment initiation (TD-TI, ≤30, 31-60, ≥61 days) and surgery to radiotherapy (TS-RT, ≤42, 43-66, ≥67 days). Univariate, Kaplan-Meier, and multivariate analyses assessed correlations between demographic and clinical factors with overall survival in treatment delay groups. Included were 1643 HPV-positive OPSCC patients and 391 HPV-negative OPSCC patients. No associations between survival and gender, age, race, insurance, or radiotherapy length were observed. Regardless of HPV status, larger tumor size (>2 cm) and lymphovascular invasion predicted worse survival. HPV negative patients with >4 lymph nodes involved had 2.5×greater mortality risk (P = .039). Robotic surgery was associated with improved survival only in HPV positive patients (hazard ratio [HR]: 0.41, P < .001). In HPV positive patients, higher TD-TI related to lower mean survival, although this was not significant on multivariate analysis. HPV negative patients with >42 days of TS-RT had decreased survival (43-66 days, HR 1.63, P = .049; ≥67 days, HR 2.10, P = .032). Longer TS-RT was associated with lower overall survival in HPV negative patients. Treatment delay was not associated with survival in HPV positive OPSCC according to multivariate analysis. These findings enhance knowledge about treatment delay effects in OPSCC, aiding providers in decisions and patient communication.

Abstract 3683: Prognostic value and biological correlates of LINE1 hypomethylation in oropharyngeal cancer

Abstract Background. We have recently demonstrated that LINE-1 hypomethylation is a negative prognostic factor in oropharyngeal squamous cell carcinoma (OPSCC). HPV-positive patients with LINE-1 methylation &amp;lt;55% showed an almost 5-fold higher risk of death than hypermethylated once, with an overall survival as poor as HPV-negative patients. Somatic TP53 mutations are a common feature in HPV-negative OPSCC, whereas their presence has been reported in only a small subset of HPV-related OPSCC. At present, little is known about the correlation between LINE-1 methylation and p53 expression status and/or TP53 mutation in OPSCC. Methods. Since 2019, an ongoing prospective study has been enrolling patients with OPSCC in nine cancer centers in Northern Italy. HPV16 E6 DNA was quantified using real-time quantitative PCR (qPCR). LINE-1 methylation was evaluated by methylation-specific qPCR. p53 expression was detected by immunohistochemical staining. TP53 exon mutations were analyzed by Next Generation Sequencing. Results. Up to date, 134 OPSCC patients have been enrolled, including cancer of the tonsil (n=74; 55.2%), base of tongue (n=47; 35.1%), and other subsites (n=13; 9.7%). HPV16 DNA was found in 54.3% of cases: 65.7% in base of tongue, 57.1% in tonsil, and 16.7% in other subsites (p&amp;lt;0.001). LINE-1 methylation was higher in HPV16-positive patients (median: 55.8%, interquartile rage [IQR]: 42.5-68.5) than HPV16-negative ones (median: 40.7; IQR: 23.9-59.1; p=0.003). Nevertheless, the median LINE-1 methylation was lower (50.7%) among HPV-16 OPSCC patients who relapsed than those who did not (56.0%), thus confirming the findings previously reported. However, data from the present investigation are presently not mature to purse the study aims (median follow-up: 12.3 months). The correlation between LINE-1 hypomethylation and p53 expression has also been evaluated in OPSCC patients. p53 expression was categorized into three groups (0%, 1-49%, and ≥50%) based on the overall intensity of nuclear staining of the tumor cells. p53 was considered null, overexpressed, or wild type when its nuclear staining was 0 %, ≥50%, or ranged between 1% and 49%, respectively. Of note, the highest level of LINE-1 methylation was observed in OPSCC tissues with nuclear p53 staining between 1% and 49% (57.8%), whereas OPSCC patients with p53 expression ≥50% showed a decline of LINE-1 methylation levels (40.6; p=0.026). Interestingly, LINE-1 methylation was lower in OPSCC patients harboring TP53 mutations in both HPV16-negative (24.8%) and HPV16-positive (24.0%) OPSCC patients, even if statistical significance was reached only in the HPV-negative group (p=0.015). Conclusion. Preliminary results confirm the potential of LINE-1 methylation to identify HPV16-positive patients with poor prognosis. In addition, this study suggest that TP53 mutations might influence LINE-1 methylation, irrespective of HPV status. Citation Format: Mariateresa Casarotto, Roberto Guerrieri, Paolo Boscolo-Rizzo, Monica Schiappacassi, Sara D'Andrea, Valentina Lupato, Susanna Chiocca, Marta Tagliabue, Rita De Berardinis, Anna Menegaldo, Alice Piccinato, Paola Stritoni, Mohssen Ansarin, Doriano Politi, Giuseppe Fanetti, Giorgio Giurato, Jerry Polesel, Elisabetta Fratta. Prognostic value and biological correlates of LINE1 hypomethylation in oropharyngeal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3683.

Preliminary Results from Retrospective Correlation of Circulating Tumor DNA (ct-DNA) with Imaging for HPV-positive Oropharyngeal Squamous Cell Carcinoma.

The role of molecular markers is increasingly being recognized for head and neck tumors ranging from benign lesions like paragangliomas to malignancies like squamous cell carcinomas (SCCa). Multiple studies have recently validated blood tests for circulating tumor tissue modified viral- human papillomavirus DNA (HPV ct-DNA) (NavDx, Naveris Laboratories) for posttreatment surveillance of HPV-driven oropharyngeal SCCa. This technology quantifies fragments of circulating DNA that are shed into the blood stream with very high (>95%) positive and negative predictive values and are also highly sensitive in distinguishing tumor HPV-DNA from a non-cancerous source. This study has a cohort of 34 patients with HPV-driven oropharyngeal SCCa, having at least three sequential imaging studies and ct-DNA values. The study showed a strong positive correlation between the imaging findings and ct-DNA level in recurrent HPV positive oropharyngeal SCCa. Findings also include 100% negative predictive value of HPV ct-DNA tests to rule out tumor recurrence. At our institution, we are now routinely performing the ct-DNA assay for surveillance of treated HPV-oropharyngeal SCCa. Correlation between clinical, radiological, and biomarker findings are now part of routine discussions during the multidisciplinary tumor boards.ABBREVIATIONS: ct-DNA=circulating tumor deoxyribonucleic acid; HPV=Human Papilloma virus;OPC=Oropharyngeal SCCa=Squamous cell carcinomas; PCR= Polymerase chain reaction.

Clinical and genomic characterization of chemoradiation-resistant HPV-positive oropharyngeal squamous cell carcinoma.

Most patients with HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) have an excellent response to chemoradiation, and trials are now investigating de-escalated treatment. However, up to 25% of patients with HPV-positive OPSCC will experience recurrence, and up to 5% will even progress through primary treatment. Currently, there are no molecular markers to identify patients with poor prognosis who would be harmed by de-escalation. Herein we report the clinical and genomic characteristics of persistent HPV-positive OPSCC after definitive platinum-based chemoradiation therapy. Patients with HPV-positive OPSCC treated with curative intent platinum-based chemoradiation between 2007 and 2017 at two institutions and with a persistent locoregional disease were included. We evaluated clinical characteristics, including smoking status, age, stage, treatment, and overall survival. A subset of five patients had tissue available for targeted exome DNA sequencing and RNA sequencing. Genomic analysis was compared to a previously published cohort of 47 treatment-responsive HPV+ OPSCC tumors after batch correction. Mutational landscape, pathway activation, and OncoGPS tumor states were employed to characterize these tumors. Ten patients met the inclusion criteria. The tumor and nodal stages ranged from T1 to T4 and N1 to N2 by AJCC 8th edition staging. All patients were p16-positive by immunohistochemistry, and eight with available in situ hybridization were confirmed to be HPV-positive. The 1-year overall survival from the time of diagnosis was 57%, and the 2-year overall survival was 17%. TP53 mutations were present in three of five (60%) persistent tumors compared to 2% (one of 47) of treatment-responsive HPV-positive tumors (p = 0.008). Other genes with recurrent mutations in persistent HPV-positive OPSCC tumors were NF1, KMT2D, PIK3C2B, and TFGBR2. Compared to treatment-responsive HPV-positive tumors, persistent tumors demonstrated activation of DNA Repair and p53, EMT, MYC, SRC, and TGF-beta signaling pathways, with post-treatment samples demonstrating significant activation of the PI3K-EMT-Stem pathways compared to pretreatment samples. Chemoradiation-resistant HPV-positive OPSCC occurs infrequently but portends a poor prognosis. These tumors demonstrate higher rates of p53 mutation and activation of MYC, SRC, and TGF-beta pathways. A comparison of tumors before and after treatment demonstrates PI3K-EMT-Stem pathways post-treatment in HPV-positive tumors with persistent disease after platinum-based chemoradiation.

Detection of antibody subclasses IgA, IgM and IgG against HPV L1 in HPV-positive oropharyngeal squamous cell carcinoma patients: a pilot study

PurposeDespite prognostic superiority of HPV-positive oropharyngeal squamous cell carcinoma (OPSCC), up to 25% of patients will suffer from recurrence within the first 5 years. Therefore, it is of great scientific interest to find relevant biomarkers to identify patients at risk. In this prospective observational study, we aimed to investigate the dynamics of HPV-L1 capsid protein specific antibody (AB) subclasses IgA, IgM, and IgG in HPV-positive OPSCC patients under therapy.MethodsSerum samples from HPV-positive OPSCC patients, identified by positive p16-immunohistochemistry, were collected before and during tumor-specific therapy and 3–6 months during follow-up. They were analyzed for the presence of HPV-L1 AB subclasses IgA, IgM, and IgG using an HPV-L1-specific immuno-assay. Additionally, a PCR-based HPV-DNA detection from the tumor tissue was performed.ResultsAltogether, 33 patients with a mean follow-up of 55 months were included. Analysis of a total of 226 serum samples revealed that the most common L1-AB-subclass pattern was characterized by IgG > > IgA > IgM without significant fluctuation during the course of disease. Patients with excessive IgG levels tended to higher tumor stages and three out of three patients with disease recurrence showed increasing IgG AB titers beforehand. Seven patients showed an IgA dominance at diagnosis, which was associated with a better disease-free survival.ConclusionDespite limited cases, our prospective pilot study revealed promising trends in HPV L1 AB subclasses and may contribute useful information for future risk stratification and post-treatment monitoring in HPV-positive OPSCC patients.

Clinical Predictors of Cisplatin Chemoradiation-Induced Ototoxicity in HPV-Positive Oropharyngeal Squamous Cell Carcinoma: A Case-Control Study.

Cisplatin-based chemoradiation is a standard treatment for many patients with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC), an etiologically distinct subset of head and neck cancer. Although associated with good long-term survival, clinical risk factors for ototoxicity have been understudied in this population. This study aimed to evaluate clinical predictors associated with ototoxicity in HPV-positive OPSCC patients treated with cisplatin chemoradiation. This retrospective case-control study included 201 adult patients (>18 years) with histologically confirmed HPV-positive OPSCC who received cisplatin chemoradiation as their primary treatment from 2001 and 2019 at a single tertiary cancer center. Ototoxicity was determined using baseline and follow-up audiometry and the Common Terminology Criteria for Adverse Events v5.0 grading criteria (Grade ≥2). Multivariable logistic regression [adjusted odds ratio (aOR)] identified significant predictors that increased the odds of ototoxicity. A total of 201 patients [165 males; median (IQR) age, 57 (11) years] were included in the study. The incidence of ototoxicity in the worst ear was 56.2%, with the greatest hearing loss occurring at high frequencies (4-8 kHz), resulting in a loss of 12.5 dB at 4 to 6 kHz and 20 dB at 6 to 8 kHz. High-dose cisplatin administration compared to weekly administration [aOR 4.93 (95% CI: 1.84-14.99), P = .003], a higher mean cochlear radiation dose [aOR 1.58 (95% CI: 1.12-2.30), P = .01], smoking history [aOR 2.89 (95% CI: 1.51-5.63), P = .001], and a 10 year increase in age [aOR 2.07 (95% CI: 1.25-3.52), P = .006] were each independently associated with increased odds of ototoxicity. Clinical predictors of ototoxicity in HPV-positive OPSCC patients treated with cisplatin-based chemoradiation include the use of a high-dose cisplatin regimen, higher cochlear radiation doses, a history of smoking, and older age. With the rising incidence of this malignancy in Western countries and overall improved survivorship, our research motivates future studies into risk stratification and earlier interventions to mitigate and reduce the risk of ototoxicity.

Mapping the landscape of HPV integration and characterising virus and host genome interactions in HPV-positive oropharyngeal squamous cell carcinoma.

Human papillomavirus (HPV) integration into the host genome is an important factor in HPV(+)OPSCC carcinogenesis, in conjunction with HPV oncoproteins E6/E7. However, a well-studied investigation about virus-host interaction still needs to be completed. Our objective is to characterise HPV integration to investigate potential mechanisms of tumourigenesis independent of E6/E7 oncoproteins. High-throughput viral integration detection was performed on 109 HPV(+)OPSCC tumours with relevant clinicopathological information. Of these tumours, 38 tumours underwent targeted gene sequencing, 29 underwent whole exome sequencing and 26 underwent RNA sequencing. HPV integration was detected in 94% of tumours (with a mean integration count of 337). Tumours occurring at the tonsil/oropharyngeal wall that exhibit higher PD-L1 expression demonstrated increased integration sites (p=.024). HPV exhibited a propensity for integration at genomic sites located within specific fragile sites (FRA19A) or genes associated with functional roles such as cell proliferation and differentiation (PTEN, AR), immune evasion (CD274) and glycoprotein biosynthesis process (FUT8). The viral oncogenes E2, E4, E6 and E7 tended to remain intact. HPV fragments displayed enrichment within host copy number variation (CNV) regions. However, insertions into genes related to altered homologous recombination repair were infrequent. Genes with integration had distinct expression levels. Fifty-nine genes whose expression level was affected by viral integration were identified, for example, EPHB1, which was reported to be involved in cellular protein metabolic process. HPV can promote oncogenesis through recurrent integration into functional host genome regions, leading to subsequent genomic aberrations and gene expression disruption. This study characterises viral integrations and virus-host interactions, enhancing our understanding of HPV-related carcinogenesis mechanisms.

Increasing Patients With HPV-positive Oropharyngeal Squamous Cell Carcinoma and its Oncologic Outcomes in the Rural Region in Northern Japan.

Despite the global rise in the incidence of human papillomavirus (HPV)-positive oropharyngeal carcinoma (OPC) in recent years, its prevalence and oncological outcomes in patients living in rural areas of Northern Japan has not been explored and should be investigated. A total of 105 patients with oropharyngeal squamous cell carcinoma who underwent HPV screening and received first-line treatment were included in this study. The annual changes in the number of patients, survival rates, and clinical factors affecting prognosis were examined. The HPV-positive rate in patients with OPC was low, with the lowest rate of 10.0% in 2013 and the highest rate of 46.7% in 2020. The number of HPV-negative cases remained almost unchanged, whereas the overall number of cases increased with the increasing number of HPV-positive cases. Additionally, HPV-positive cases exhibited a fairly good prognosis. The number of OPC cases increased not only in urban areas, but also in rural areas. HPV-positive cases had better outcomes than HPV-negative cases.

A novel ΔNp63-dependent immune mechanism improves prognosis of HPV-related head and neck cancer.

Deconvoluting the heterogenous prognosis of Human Papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OSCC) is crucial for enhancing patient care, given its rapidly increasing incidence in western countries and the adverse side effects of OSCC treatments. Transcriptomic data from HPV-positive OSCC samples were analyzed using unsupervised hierarchical clustering, and clinical relevance was evaluated using Kaplan-Meier analysis. HPV-positive OSCC cell line models were used in functional analyses and phenotypic assays to assess cell migration and invasion, response to cisplatin, and phagocytosis by macrophages in vitro. We found, by transcriptomic analysis of HPV-positive OSCC samples, a ΔNp63 dependent molecular signature that is associated with patient prognosis. ΔNp63 was found to act as a tumor suppressor in HPV-positive OSCC at multiple levels. It inhibits cell migration and invasion, and favors response to chemotherapy. RNA-Seq analysis uncovered an unexpected regulation of genes, such as DKK3, which are involved in immune response-signalling pathways. In agreement with these observations, we found that ΔNp63 expression levels correlate with an enhanced anti-tumor immune environment in OSCC, and ΔNp63 promotes cancer cell phagocytosis by macrophages through a DKK3/NF-κB-dependent pathway. Our findings are the first comprehensive identification of molecular mechanisms involved in the heterogeneous prognosis of HPV-positive OSCC, paving the way for much-needed biomarkers and targeted treatment.

Multiparametric MRI-based radiomics model for predicting human papillomavirus status in oropharyngeal squamous cell carcinoma: optimization using oversampling and machine learning techniques.

To develop and validate a multiparametric MRI-based radiomics model with optimal oversampling and machine learning techniques for predicting human papillomavirus (HPV) status in oropharyngeal squamous cell carcinoma (OPSCC). This retrospective, multicenter study included consecutive patients with newly diagnosed and pathologically confirmed OPSCC between January 2017 and December 2020 (110 patients in the training set, 44 patients in the external validation set). A total of 293 radiomics features were extracted from three sequences (T2-weighted images [T2WI], contrast-enhanced T1-weighted images [CE-T1WI], and ADC). Combinations of three feature selection, five oversampling, and 12 machine learning techniques were evaluated to optimize its diagnostic performance. The area under the receiver operating characteristic curve (AUC) of the top five models was validated in the external validation set. A total of 154 patients (59.2 ± 9.1years; 132 men [85.7%]) were included, and oversampling was employed to account for data imbalance between HPV-positive and HPV-negative OPSCC (86.4% [133/154] vs. 13.6% [21/154]). For the ADC radiomics model, the combination of random oversampling and ridge showed the highest diagnostic performance in the external validation set (AUC, 0.791; 95% CI, 0.775-0.808). The ADC radiomics model showed a higher trend in diagnostic performance compared to the radiomics model using CE-T1WI (AUC, 0.604; 95% CI, 0.590-0.618), T2WI (AUC, 0.695; 95% CI, 0.673-0.717), and a combination of both (AUC, 0.642; 95% CI, 0.626-0.657). The ADC radiomics model using random oversampling and ridge showed the highest diagnostic performance in predicting the HPV status of OPSCC in the external validation set. Among multiple sequences, the ADC radiomics model has a potential for generalizability and applicability in clinical practice. Exploring multiple oversampling and machine learning techniques was a valuable strategy for optimizing radiomics model performance. • Previous radiomics studies using multiparametric MRI were conducted at single centers without external validation and had unresolved data imbalances. • Among the ADC, CE-T1WI, and T2WI radiomics models and the ADC histogram models, the ADC radiomics model was the best-performing model for predicting human papillomavirus status in oropharyngeal squamous cell carcinoma. • The ADC radiomics model with the combination of random oversampling and ridge showed the highest diagnostic performance.

HPV testing versus p16 immunohistochemistry in oropharyngeal squamous cell carcinoma: results from the DAHANCA 19 study

Introduction The prognosis after primary (chemo-)radiotherapy for oropharyngeal squamous cell carcinoma (OPSCC) is affected by Human Papillomavirus (HPV) status, with a better prognosis in HPV-positive OPSCC. HPV-status is routinely assessed by p16 immunohistochemistry (IHC), but additional HPV DNA testing is debated. Also, there are numerous HPV genotypes, which prognostic role may need clarification. The purpose of this study was: (1) to test a custom-made targeted HPV next generation sequencing (NGS) panel in OPSCC, (2) to determine correlation with p16 IHC, and (3) to assess the impact of HPV DNA testing on outcome in the prospectively randomized clinical trial DAHANCA 19. Materials and methods We included 271 patients with OPSCC treated with primary (chemo-)radiotherapy in the DAHANCA 19 trial. Of these, 199 (73%) were p16-positive. HPV-status was determined by targeted HPV next generation sequencing (NGS), using a custom-made HPV genotyping panel. Results HPV was detected in 194 tumor samples. p16 IHC and NGS HPV status were concordant in 265 (98%) of 271 patients, whereas we did not detect HPV DNA in 5 p16-positive tumors. HPV16 accounted for 169 of 194 HPV-positive cases (87%). HPV genotypes 18, 31, 33, 35, and 59 were also detected. Loco-regional failure and overall survival were similar whether patients were separated by p16 IHC, or HPV DNA status (p < 0.0001 for all) and did not depend on HPV genotype (p = 0.9 and p = 0.7). Conclusion In the present study, HPV DNA testing or typing in a Danish OPSCC cohort did not add additional information to p16 IHC, the most widely used and accepted prognostic indicator.