HPV-positive Oropharyngeal Squamous Cell Carcinoma Patients Research Articles

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.

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.

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.

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.

Abstract 1005: LINE-1 hypomethylation is associated with poor outcomes in locoregionally advanced oropharyngeal cancer

Abstract Background: Currently, human papillomavirus (HPV) positivity represents a strong prognostic factor for both reduced risk of relapse and improved survival in patients with oropharyngeal squamous cell carcinoma (OPSCC). However, a subset of HPV-positive OPSCC patients still experience poor outcomes. Furthermore, HPV-negative OPSCC patients, who have an even higher risk of relapse, are still lacking suitable prognostic biomarkers for clinical outcome. Here, we evaluated the prognostic value of LINE-1 methylation level in OPSCC patients, and further addressed the relationship between LINE-1 methylation status and p53 protein expression as well as genome-wide/gene-specific DNA methylation. Results: In this study, DNA was extracted from 163 formalin-fixed paraffin-embedded tissue samples retrospectively collected from stage III-IVB OPSCC patients managed with curative intent with up-front treatment. Quantitative methylation-specific PCR revealed that LINE-1 hypomethylation was directly associated with poor prognosis (5-year overall survival - OS: 28.1% for LINE-1 methylation&amp;lt;35% versus 69.1% for ≥55%; p&amp;lt;0.0001). When LINE-1 methylation was dichotomized as &amp;lt;55% vs. ≥55%, interaction with HPV16 emerged: compared with hypermethylated HPV-16 positive patients, subjects with hypomethylated HPV16-negative OPSCC reported an adjusted higher risk of death (HR: 4.83, 95% CI: 2.24-10.38) and progression (HR: 4.54, 95% CI: 2.18-9.48). Tumor protein p53 (TP53) gene is often mutated and overexpressed in HPV-negative OPSCC. Since p53 has been reported to repress LINE-1 promoter, we then analyzed the association between p53 protein expression and LINE-1 methylation levels. Following p53 immunohistochemistry, results indicated that among HPV16-negative patients with p53≥50%, LINE-1 methylation levels declined and remained stable at approximately 43%; any HPV16-positive patient reported p53≥50%. Finally, DNA methylation analysis demonstrated that genome-wide average methylation level at cytosine-phosphate-guanine sites was significantly lower in HPV16-negative OPSCC patients who relapsed within two years. The subsequent integrative analysis of gene expression and DNA methylation identified 20 up-regulated/hypomethylated genes in relapsed patients, and most of them contained LINE-1 elements in their promoter sequences. Conclusions: Evaluation of the methylation level of LINE-1 may help in identifying the subset of OPSCC patients with bad prognosis regardless of their HPV status. Aberrant LINE-1 hypomethylation might occur along with TP53 mutations, and lead to altered gene expression in OPSCC. Citation Format: Mariateresa Casarotto, Valentina Lupato, Giorgio Giurato, Roberto Guerrieri, Sandro Sulfaro, Annamaria Salvati, Elisa D'Angelo, Carlo Furlan, Anna Menegaldo, Lorena Baboci, Barbara Montico, Irene Turturici, Riccardo Dolcetti, Salvatore Romeo, Vittorio Baggio, Stefania Corrado, Gianluca Businello, Maria Guido, Alessandro Weisz, Vittorio Giacomarra, Giovanni Franchin, Agostino Steffan, Luca Sigalotti, Emanuela Vaccher, Paolo Boscolo-Rizzo, Jerry Polesel, Giuseppe Fanetti, Elisabetta Fratta. LINE-1 hypomethylation is associated with poor outcomes in locoregionally advanced oropharyngeal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1005.

LINE-1 hypomethylation is associated with poor outcomes in locoregionally advanced oropharyngeal cancer

Background and purposeCurrently, human papillomavirus (HPV) positivity represents a strong prognostic factor for both reduced risk of relapse and improved survival in patients with oropharyngeal squamous cell carcinoma (OPSCC). However, a subset of HPV-positive OPSCC patients still experience poor outcomes. Furthermore, HPV-negative OPSCC patients, who have an even higher risk of relapse, are still lacking suitable prognostic biomarkers for clinical outcome. Here, we evaluated the prognostic value of LINE-1 methylation level in OPSCC patients and further addressed the relationship between LINE-1 methylation status and p53 protein expression as well as genome-wide/gene-specific DNA methylation.ResultsIn this study, DNA was extracted from 163 formalin-fixed paraffin-embedded tissue samples retrospectively collected from stage III-IVB OPSCC patients managed with curative intent with up-front treatment. Quantitative methylation-specific PCR revealed that LINE-1 hypomethylation was directly associated with poor prognosis (5-year overall survival—OS: 28.1% for LINE-1 methylation < 35% vs. 69.1% for ≥ 55%; p < 0.0001). When LINE-1 methylation was dichotomized as < 55% versus ≥ 55%, interaction with HPV16 emerged: compared with hypermethylated HPV16-positive patients, subjects with hypomethylated HPV16-negative OPSCC reported an adjusted higher risk of death (HR 4.83, 95% CI 2.24–10.38) and progression (HR 4.54, 95% CI 2.18–9.48). Tumor protein p53 (TP53) gene is often mutated and overexpressed in HPV-negative OPSCC. Since p53 has been reported to repress LINE-1 promoter, we then analyzed the association between p53 protein expression and LINE-1 methylation levels. Following p53 immunohistochemistry, results indicated that among HPV16-negative patients with p53 ≥ 50%, LINE-1 methylation levels declined and remained stable at approximately 43%; any HPV16-positive patient reported p53 ≥ 50%. Finally, DNA methylation analysis demonstrated that genome-wide average methylation level at cytosine–phosphate–guanine sites was significantly lower in HPV16-negative OPSCC patients who relapsed within two years. The subsequent integrative analysis of gene expression and DNA methylation identified 20 up-regulated/hypomethylated genes in relapsed patients, and most of them contained LINE-1 elements in their promoter sequences.ConclusionsEvaluation of the methylation level of LINE-1 may help in identifying the subset of OPSCC patients with bad prognosis regardless of their HPV status. Aberrant LINE-1 hypomethylation might occur along with TP53 mutations and lead to altered gene expression in OPSCC.

Hypermethylation of nc886 in HPV-positive oropharyngeal cancer and its clinical implications: An epigenome-wide association study

The incidence of oropharyngeal squamous cell carcinoma (OPSCC) has increased rapidly in the United States, driven by rising human papillomavirus (HPV) infections in the U.S. population. HPV-positive OPSCC patients have a better prognosis than HPV-negative patients. To gain insights into the unique biology of HPV(+) OPSCC that may contribute to its clinical behaviors, we performed a multi-stage epigenome-wide methylation profiling of leukocyte and tumor DNA in OPSCC patients and compared the methylation levels of CpG sites between HPV(+) and HPV(-) OPSCC patients. We identified and validated a significantly differentially methylated region (DMR) of 1,355bp encompassing non-coding RNA 886 (nc886) gene and its promoter region. Nc886 is hypermethylated in both leukocytes and tumor DNA of HPV(+) OPSCC patients. Homozygous knockout of nc886 by CRISPR-Cas9 in head and neck cell lines was lethal, but nc886 could be knocked out on the background of protein kinase R (PKR) knockout. Our data suggest that HPV induces nc886 hypermethylation, and nc886 acts as both a viral sensor and a tumor sensor in OPSCC patients and contribute to the better prognosis of HPV(+) OPSCC patients. Nc886 may become a therapeutic target in OPSCC.

Impact of Alcohol and Smoking on Outcomes of HPV-Related Oropharyngeal Cancer.

The aim of this study was to evaluate the impact of adverse lifestyle factors on outcomes in patients with human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC). From 2010 to 2019, 150 consecutive non-metastatic OPSCC patients receiving curative treatment in our institution were retrospectively enrolled. HPV positivity was defined as p16 expression ≥75%. The effects of adverse lifestyle factors on overall survival (OS) and disease-free survival (DFS) on OPSCC patients were determined. The median follow-up duration was 3.6 years. Of the 150 OPSCCs, 51 (34%) patients were HPV-positive and 99 (66%) were HPV-negative. The adverse lifestyle exposure rates were 74.7% (n = 112) alcohol use, 57.3% (n = 86) betel grid chewing, and 78% (n = 117) cigarette smoking. Alcohol use strongly interacted with HPV positivity (HR, 6.00; 95% CI, 1.03-35.01), leading to an average 26.1% increased risk of disease relapse in patients with HPV-positive OPSCC. Heavy smoking age ≥30 pack-years was associated with increased risk of death (HR, 2.05; 95% CI, 1.05-4.00) and disease relapse (HR, 1.99; 95% CI, 1.06-3.75) in OPSCC patients. In stratified analyses, the 3-year absolute risk of disease relapse in HPV-positive OPSCC patients reached up to 50% when alcohol use and heavy smoking for ≥30 pack-years were combined. Alcohol acted as a significant treatment-effect modifier for DFS in HPV-positive OPSCC patients, diluting the favorable prognostic effect of HPV positivity. Heavy smoking age ≥30 pack-years was an independent adverse prognostic factor of OS and DFS in OPSCC patients. De-intensification treatment for HPV-related OPSCC may be avoided when these adverse lifestyle factors are present.

Genomic Adjusted Radiation Dose (GARD) Predicts Overall Survival and Outperforms AJCC 8th Edition in Prognostication of HPV-Positive Oropharyngeal Squamous Cell Carcinoma

<h3>Purpose/Objective(s)</h3> Treatment decision-making in oropharyngeal squamous cell carcinoma (OPSCC) includes clinical stage, HPV status, and smoking history. Despite improvements in staging with separation of HPV positive and negative OPSCC in AJCC 8^th edition (AJCC8), patients are largely treated with a uniform approach, with recent efforts on de-intensification in low-risk patients. We have previously shown, in a pooled analysis, that the genomic adjusted radiation dose (GARD) is predictive of radiation treatment benefit and can be used to guide dose selection. We hypothesize that GARD can be used to predict overall survival (OS) in HPV-positive OPSCC patients treated with radiotherapy (RT). <h3>Materials/Methods</h3> Gene expression profiles were analyzed for 234 formalin-fixed paraffin-embedded samples from HPV-positive OPSCC patients within an international, multi-institutional, prospective/retrospective observational study (Big Data and Models for Personalized Head and Neck Cancer Decision Support (BD2Decide, NCT02832102). GARD, a measure of the biological effect of RT, was calculated for each patient as previously described. In total, 203 patients received definitive treatment (chemoradiation or RT alone), and 31 patients received post-operative RT. Two RT dose fractionations were utilized for definitive cases (70 Gy in 35 fractions or 69.96 Gy in 33 fractions). Median RT dose was 70 Gy (mean 50.88-74) for definitive cases and 66 Gy (range 44-70) for post-operative cases. 29 patients received surgery and adjuvant chemoradiation, 14 received surgery and adjuvant RT, and 19 received RT alone. The median follow up was 46.2 months (95% CI, 41.2 to 50). Cox proportional hazards analyses were performed with GARD as a continuous variable and ROC analyses comparing the performance of GARD with AJCC8. <h3>Results</h3> Despite uniform radiation dose utilization, GARD showed significant heterogeneity (range 30-110), reflecting the underlying genomic differences in the cohort. On multivariate analysis, each unit increase in GARD was associated with an improvement in OS (HR= 0.97, 95% CI 0.94-1.0, p=0.040) compared to AJCC8 (HR=1.96, 95% CI 0.90-4.32, p=0.0952). ROC analysis for GARD at 24 months yielded an AUC of 75.6 (95% CI 61.8-89.4) compared with an AUC of 73.9 (95% CI 56.9-90.9) for AJCC8. GARD ≥64.97 was associated with improved OS (HR=0.29, p=0.005). <h3>Conclusion</h3> In this multi-institutional cohort of patients with HPV-positive OPSCC, GARD predicts OS as a continuous variable and outperforms AJCC8 in prognostication. We propose that GARD, which provides the first opportunity for genomic guided personalization of radiation dose, should be incorporated in the diagnostic workup of HPV-positive OPSCC patients and can be used to guide treatment decision making.

The clinical value of oral robotic surgery in the treatment of oropharyngeal squamous cell carcinoma

Objective: To explore the therapeutic effects of transoral robotic surgery (TORS) and traditional surgical modes in oropharyngeal squamous cell carcinoma (OPSCC). Methods: The clinicopathological data of patients with oropharyngeal squamous cell carcinoma treated at Sun Yat-sen University Cancer Center from 2010 to 2018 were retrospectively analyzed. 135 cases were treated with traditional surgery (non-TORS group), while 52 cases were treated with TORS (TORS group). The prognosis of the two groups of patients were analyzed by Kaplan-Meier method and Log rank test, the influencing factors were analyzed by Cox regression model. Results: The 2-year overall survival (OS, 94.2%) and 2-year progression-free survival (PFS, 93.8%) of patients in the TORS group were better than those in the non-TORS group (71.4% and 71.4%, respectively, P<0.05). The 2-year OS (93.3%) and 2-year PFS (92.8%) of TORS group patients in T1-2 stage were better than those of non-TORS group (73.1% and 72.8%, respectively, P<0.05). The 2-year OS (95.8%) and 2-year PFS (95.2%) of patients with stage Ⅰ to Ⅱ in the TORS group were not significantly different from those in the non-TORS group (84.1% and 83.9%, respectively, P>0.05). The 2-year OS (92.9%) and 2-year PFS rate (92.7%) of patients with stage Ⅲ to Ⅳ in the TORS group were better than those in the non-TORS group (64.7% and 63.9%, respectively, P<0.05). The 2-year OS (94.4%) of HPV-positive patients in the TORS group was not significantly different from that in the non-TORS group (83.3%, P=0.222). The 2-year OS of HPV-negative patients in the TORS group (94.1%) was significantly different from that in the non-TORS group (43.7%, P<0.001). HPV status was an independent prognostic factor (P=0.008). Conclusions: TORS has a better prognosis in the treatment of oropharyngeal squamous cell carcinoma compared with the traditional treatment methods. The patients with T1-T2 can achieve better survival benefits after TORS treatment. The HPV-positive OPSCC patients has a better prognosis than that of HPV-negative OPSCC patients, and regardless of HPV status, OPSCC patients in the TORS group could obtain a better survival prognosis.

The Impact of Human Papillomavirus (HPV) Associated Oropharyngeal Squamous Cell Carcinoma (OPSCC) on Nutritional Outcomes.

Background: Patients undergoing (chemo) radiotherapy for oropharyngeal squamous cell carcinoma (OPSCC) are at high risk of malnutrition during and after treatment. Malnutrition can lead to poor tolerance to treatment, treatment interruptions, poor quality of life (QOL) and potentially reduced survival rate. Human papillomavirus (HPV) is now known as the major cause of OPSCC. However, research regarding its effect on nutritional outcomes is limited. The aim of this study was to examine the relationship between HPV status and nutritional outcomes, including malnutrition and weight loss during and after patients’ (chemo) radiotherapy treatment for OPSCC. Methods: This was a longitudinal cohort study comparing the nutritional outcomes of HPV-positive and negative OPSCC patients undergoing (chemo) radiotherapy. The primary outcome was nutritional status as measured using the Patient Generated-Subjective Global Assessment (PG-SGA). Secondary outcomes included loss of weight, depression, QOL and adverse events. Results: Although HPV-positive were less likely to be malnourished according to PG-SGA at the beginning of treatment, we found that the difference between malnutrition rates in response to treatment was not significantly different over the course of radiotherapy and 3 months post treatment. HPV-positive participants had significantly higher odds of experiencing >10% weight loss at three months post-treatment than HPV-negative participants (OR = 49.68, 95% CI (2.7, 912.86) p ≤ 0.01). Conclusions: The nutritional status of HPV positive and negative patients were both negatively affected by treatment and require similarly intense nutritional intervention. In acute recovery, HPV positive patients may require more intense intervention. At 3- months post treatment, both groups still showed nutritional symptoms that require nutritional intervention so ongoing nutritional support is essential.

A prognostic gene expression signature for oropharyngeal squamous cell carcinoma.

BackgroundRobust prognostic stratification of patients with oropharyngeal squamous cell carcinoma (OPSCC) is important for developing individualized treatment plans. This study was conducted to develop and validate a clinically feasible prognostic classifier based on transcriptome-wide gene expression profiles.MethodsTumor tissues were collected from 208 OPSCC patients treated at Washington University in St. Louis and 130 OPSCC patients treated at Vanderbilt University, used for model training and validation, respectively. OPSCC patients (n = 70) from the TCGA cohort were also included for independent validation. Based on RNA-seq profiling data, Cox proportional hazards regression analysis was performed to identify genes associated with disease outcomes. Then, Lasso-penalized multivariate survival models were constructed to identify biomarker genes for developing a prognostic gene signature.FindingsA 60-gene signature was identified by RNA-seq profiling analysis. Computed risk score of the gene signature was significantly predictive of 5-year overall survival of the training cohort (Hazard ratio (HR) 28·32, P = 4·3E-41). Subgroup analysis stratified by HPV status revealed that the signature was prognostic in HPV-positive OPSCC patients (HR 30·55, P = 7·0E-37) and was independent of clinical features. Importantly, the gene signature was validated in two independent patient cohorts, including the TCGA cohort (HR 3·94, P = 0·0018) and the Vanderbilt cohort (HR 8·50, P = 5·7E-09) for overall survival.InterpretationThe prognostic gene signature is a robust tool for risk stratification of OPSCC patients. The signature remains prognostic among HPV-positive OPSCC patients.FundingNational Institutes of Health.

Prognostic Significance of Smoking in Human Papillomavirus-Positive Oropharyngeal Cancer Under American Joint Committee on Cancer Eighth Edition Stage.

To determine the prognostic significance of smoking in human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) when considering American Joint Committee on Cancer eighth edition (AJCC-8) stage. Retrospective cohort study. Three hundred seventeen HPV-positive OPSCC patients with known AJCC-8 stage and smoking status (<10 or ≥10 pack-years) seen at a tertiary center from 1997 to 2017 were studied. We used the Kaplan-Meier method to compare 5-year overall survival (OS) by smoking status and by clinical AJCC-8 stage and smoking status combined. Hazard ratios (HRs) were estimated with Cox proportional hazard regression for the independent effects of smoking and AJCC-8 stage. We also studied pathologic stage and estimated the combined effects of smoking and clinical stage. The ≥10 pack-years smokers had worse 5-year OS than <10 pack-years smokers (93.6%; 95% confidence interval (CI): 89.7-97.8 vs. 82.3%; 95% CI: 76.0%-89.1%). When stratified by AJCC-8 clinical stage, only stage I <10 pack-years smokers (98.7%; 95% CI: 96.3%-100.0%) had significantly better 5-year OS than their ≥10 pack-years (84.8%; 95% CI: 76.4%-94.1%) counterparts. In a multivariable analysis, ≥10 pack-years smoking was associated with increased hazard of death when adjusting for AJCC-8 clinical (HR: 2.52; 95% CI: 1.16-5.46) and pathologic (HR: 5.21; 95% CI: 1.47-18.5) stage. In both analyses, stage III patients demonstrated worse survival than stage I, and smoking had greater impact at lower stages. Smoking is a negative prognosticator in HPV-positive OPSCC and interacts with AJCC-8 clinical stage. It is important to understand the impact of smoking in HPV-positive disease when considering treatment plans and deintensification trials. 2b Laryngoscope, 130: 1961-1966, 2020.

Molecular mechanisms underlying increased radiosensitivity in human papillomavirus-associated oropharyngeal squamous cell carcinoma.

Oropharyngeal squamous cell carcinoma (OPSCC) is an important type of head and neck squamous cell carcinoma (HNSCC). The traditional risk factors for OPSCC include carcinogen intake, smoking, alcohol consumption, and lifestyle. In recent years, cases of human papillomavirus (HPV)-related OPSCC have gradually increased. At present, HPV-related OPSCC in developed Western countries comprise up to 90% of all OPSCC cases, while in other developing countries, the proportion of HPV-related OPSCC cases is also gradually increasing. Compared with HPV-negative OPSCC, HPV-positive OPSCC patients have better overall survival rates and local control rates and this improved prognosis may be related to the increased radiosensitivity of HPV-positive tumors. Due to this more favorable prognosis, many downgraded treatment schemes are gradually emerging, including simple radiotherapy instead of concurrent radiotherapy or reduced radiotherapy dose. However, there is insufficient theoretical basis for such schemes. Some studies have shown that delayed repair of DNA damage after radiation, G2/M arrest, increased hypoxia, and decreased proliferation capacity are the main reasons for the increased radiosensitivity of HPV-positive tumor cells. In this review, we discuss the four principles of tumor cell damage caused by radiation, including repair, reoxygenation, redistribution, and regeneration in order to reveal the mechanism whereby HPV increases the radiosensitivity of tumor cells. An attempt was made to provide sufficient information to facilitate more individualized treatment for HPV-positive OPSCC patients, under the premise of good tumor control.

Elemental Zn and its Binding Protein Zinc-\u03b12-Glycoprotein are Elevated in HPV-Positive Oropharyngeal Squamous Cell Carcinoma

Human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) is biologically distinct from HPV-negative HNSCC. Outside of HPV-status, few tumor-intrinsic variables have been identified that correlate to improved survival. As part of exploratory analysis into the trace elemental composition of oropharyngeal squamous cell carcinoma (OPSCC), we performed elemental quanitification by X-ray fluorescence microscopy (XFM) on a small cohort (n = 32) of patients with HPV-positive and -negative OPSCC and identified in HPV-positive cases increased zinc (Zn) concentrations in tumor tissue relative to normal tissue. Subsequent immunohistochemistry of six Zn-binding proteins—zinc-α2-glycoprotein (AZGP1), Lipocalin-1, Albumin, S100A7, S100A8 and S100A9—revealed that only AZGP1 expression significantly correlated to HPV-status (p < 0.001) and was also increased in tumor relative to normal tissue from HPV-positive OPSCC tumor samples. AZGP1 protein expression in our cohort significantly correlated to a prolonged recurrence-free survival (p = 0.029), similar to HNSCC cases from the TCGA (n = 499), where highest AZGP1 mRNA levels correlated to improved overall survival (p = 0.023). By showing for the first time that HPV-positive OPSCC patients have increased intratumoral Zn levels and AZGP1 expression, we identify possible positive prognostic biomarkers in HNSCC as well as possible mechanisms of increased sensitivity to chemoradiation in HPV-positive OPSCC.

Multiple imputation and clinico-serological models to predict human papillomavirus status in oropharyngeal carcinoma: An alternative when tissue is unavailable.

In cancer epidemiological studies, determination of human papillomavirus (HPV) in oropharyngeal squamous cell carcinoma (OPSCC) typically depends on the availability of tumor tissue testing, and/or tumor tissue access. Identifying alternative methods for estimating HPV status can improve the quality of such studies when tissue is unavailable. We developed multiple predictive models for tumor HPV status and prognosis by combining both clinico-epidemiological variables and either serological multiplex assays of HPV or multiple imputation of HPV status (HPVmi ). Sensitivity, specificity and accuracy of these methods compared to either p16 immunostaining (p16 IHC) or survival were assessed. When compared to a reference of tumor tissue p16 IHC in 783 OPSCC patients, the clinic-HPVsero model incorporating a composite of 20 HPV serological antibodies (HPVsero ) and 4 clinical factors (c-index: 0.96) performed better than using HPVsero (c-index: 0.92) or HPVmi (c-index: 0.76) alone. However, the model that contained a single HPV16 E6 antibody combined with four clinical variables, performed extremely well (clinic-s1-16E6; c-index: 0.95). When defining HPV status by HPVsero , s1-16E6, HPVmi or through p16 IHC, each of these definitions demonstrated improved overall and disease-free survival in HPV-positive OPSCC patients, when compared to HPV-negative patients (adjusted hazard ratios between 0.25 and 0.63). Our study demonstrates that when blood samples are available, a model that utilizes a single s1-16E6 antibody combined with several clinical features has excellent test performance characteristics to estimate HPV status and prognosis. When neither blood nor tumor tissue is available, multiple imputation, calibrated on local population characteristics, remains a viable, but suboptimal option.

Identification of prognostic molecular biomarkers in 157 HPV-positive and HPV-negative squamous cell carcinomas of the oropharynx.

The incidence of oropharyngeal squamous cell carcinoma (OPSCC) has been increasing due to high-risk HPV infection. We explored the significance of genetic alterations in HPV-positive (HPV-P) and HPV-negative (HPV-N) OPSCC patients on long-term outcome. A total of 157 cases of primary resected OPSCC diagnosed from 1978 to 2005 were subjected to a targeted exome sequencing by MSK-IMPACT™ interrogating somatic mutations in 410 cancer-related genes. Mutational profiles were correlated to recurrence and survival outcomes. OPSCC included 47% HPV-positive (HPV-P) and 53% HPV-negative (HPV-N) tumors arising in the base of tongue (BOT, 43%), palatine tonsil (30%) and soft palate (SP, 27%). HPV negative status, SP location and smoking were associated with poorer outcome. Poorer overall survival was found in NOTCH1-mutated HPV-P (p = 0.039), and in SOX2-amplified HPV-N cases (p = 0.036). Chromosomal arm gains in 8p and 8q, and 16q loss were more common in HPV-P (p = 0.005, 0.04 and 0.01, respectively), while 9p, 18q and 21q losses were more frequent in HPV-N OPSCC (p = 0.006, 0.002 and 0.01, respectively). Novel, potentially functional JAK3, MYC and EP300 intragenic deletions were found in HPV-P, and FOXP1, CDKN2A, CCND1 and RUNX1 intragenic deletions and one FGFR3 inversion were detected in HPV-N tumors. HPV-N/TP53-wild-type OPSCC harbored recurrent mutations in NOTCH1/3/4 (39%), PIK3CA, FAT1 and TERT. In comparison to their oral and laryngeal counterparts, HPV-N OPSCC were genetically distinct. In OPSCC, HPV status, tumor subsite and smoking determine outcome. Risk-stratification can be further refined based on the mutational signature, namely, NOTCH1 and SOX2 mutation status.

Different whole genome sequencing strategies for the clinical management of patients with oropharyngeal squamous cell carcinoma

Oropharyngeal squamous cell carcinoma (OPSCC) is a cancer of the base of the tongue and tonsils. This cancer was previously found in individuals with a smoking and drinking history but is increasingly being promoted by the presence of high risk types of human papillomavirus (HPV). In an attempt to determine the exact role that HPV plays in the development of this cancer we have been using mate-pair next generation sequencing (MP-Seq). Using MP-Seq we have found that HPV is only integrated into the human genome in 30% of HPV-positive OPSCCs, which is dramatically different than what is found in cervical cancer. MP-Seq not only can determine the physical status of HPV in a given sample, but can also ascertain genome-wide changes in any cancer. We describe how MP-Seq could be used in the clinical management of HPV-positive OPSCC patients. We have also explored another whole genome sequencing strategy that has been developed by the Beijing Genomics Institute. They can currently do whole genome sequencing for a total cost (and this includes analysis) of $600 and are developing methodologies to reduce this to $100. I will describe how these types of whole genome sequencing strategies could be used to direct therapies for these patients including facilitating the use of the liquid biopsy to monitor patients during their clinical treatment.

Magnitude of benefit for adjuvant radiotherapy following minimally invasive surgery in intermediate to high risk HPV-positive oropharyngeal squamous cell carcinoma

ObjectiveTo determine the outcomes and toxicities of minimally-invasive surgery with adjuvant intensity-modulated radiotherapy +/− chemotherapy (AT) compared to definitive surgical therapy (ST) in a contemporary cohort of HPV-positive oropharyngeal squamous cell carcinoma (OPSCC). MethodsFrom 2005 to 2013, a consecutive cohort of 190 HPV-positive OPSCC patients was retrospectively reviewed from multi-institutional databases maintained by the Departments of Otorhinolaryngology and Radiation Oncology. A total of 116 AT patients and 42 ST patients with intermediate or high risk pathologic features were included in the final analysis. All patients received minimally invasive surgery. Time to recurrence and time to death from the onset of surgery were evaluated. Toxicity data collected included dysphagia or xerostomia requiring feeding tube placement >6 months, or mandibular osteonecrosis requiring surgery or hyperbaric oxygen. ResultsAll AT patients received IMRT to a median dose of 60 Gy. Chemotherapy delivered to 67.2% of AT patients. AT group included more high-risk patients given higher nodal classification (p = 0.005) and extracapsular extension (p = 0.0005). AT improved disease-free survival (HR 2.77, CI 1.22–6.28; p = 0.02) and local-regional control (HR 14.83, CI 3.240–67.839; p = 0.001). Disease-free survival with AT and tumor extracapsular extension was improved when compared to ST (HR of 4.34, CI 1.540–12.213; p = 0.006). Dysphagia or mandibular osteonecrosis toxicity after AT vs. ST of 19.0% vs. 2.4%. ConclusionsAT improved local-regional control and disease-free survival but was associated with greater toxicity. The recurrence benefit was most pronounced in tumors with extracapsular extension.

MMP-7 expression may influence the rate of distant recurrences and disease-specific survival in HPV-positive oropharyngeal squamous cell carcinoma.

The objective of this study was to determine if matrix metalloproteinase-7 (MMP-7) expression is related to human papilloma virus (HPV) status, clinical parameters, and outcome in oropharyngeal squamous cell carcinoma (OPSCC). Tumor tissue specimens from 201 OPSCC patients treated with curative intent were available for immunohistochemistry, and the samples were stained with monoclonal MMP-7 antibody. All the patients were followed up at least 3years or until death. MMP-7 expression did not differ between HPV-positive and HPV-negative patients. MMP-7 was not prognostic among patients with HPV-negative OPSCC. In the HPV-positive subgroup, patients with moderate, high, or very high MMP-7 expression had significantly worse 5-year disease-specific survival (DSS) (56.6%) than patients with absent, or low MMP-7 expression (77.2%), and MMP-7 expression appeared as a prognostic factor in the multivariate analysis. In addition, among HPV-positive OPSCC with moderate, high, or very high MMP-7 expression, the 5-year distant recurrence-free survival was significantly lower (69.6%) than in those who had low or absent MMP-7 expression (97.5%). Our results suggest that among HPV-positive OPSCC patients, high MMP-7 expression is related to worse 5-year DSS and increased rate of distant recurrences.