HPV-negative Oropharyngeal Squamous Cell Carcinoma Research Articles

Outcomes for potentially Resectable patients undergoing primary chemoradiation treatment for T1-T2 HPV Negative oropharyngeal squamous cell carcinoma.

Transoral surgical resectability (TOS) is a prognostic factor for patients with HPV+ T1-2 oropharyngeal squamous cell carcinoma (OPSCC) disease undergoing radiotherapy (RT), but it is unclear whether this holds for HPV-negative (HPV-) patients. We aimed to compare outcomes of potential TOS-candidates vs. non-TOS candidates, among patients who underwent RT/CRT for early T-stage HPV- OPSCC. For patients treated with RT/CRT for early T-stage HPV-negative OPSCC between 2014 and 2021, pretreatment imaging was reviewed by four head-and-neck surgeons, masked to clinical outcomes, to assess primary-site suitability for TOS. Extracapsular extension (ECE) was assessed by a head-and-neck neuroradiologist. We compared outcomes based on surgical resectability relating to: (1) the primary site tumor alone, and (2) the primary site plus the absence/presence of ECE (overall assessment). Kaplan-Meier curves for overall survival (OS), disease-specific survival (DSS), and progression-free survival (PFS) were compared using the log-rank test. Seventy patients were included in the analysis. The primary site was TOS-favorable in 46/70 (66%). Based on the overall assessment, 41/70 (58.6%) were TOS-favorable. The 3-year OS, DSS and PFS for primary site TOS-favorable versus unfavorable were OS: 76.9% versus 37.4%; DSS: 78.1% versus 46.2%, PFS: 69.9% versus 41.3%, (log-rank test = 0.01, 0.03, 0.04; respectively). Additionally, patients with an overall assessment of TOS favorability demonstrated better survival outcomes compared with TOS-unfavorable patients (OS: 77.3% vs. 46.2%; DSS: 78.2% vs. 56.5%, PFS: 72.3% vs. 42.1%, log-rank test = 0.01, 0.04, 0.01; respectively). Patients with TOS-favorable HPV-negative early T-stage OPSCC have superior survival outcomes than TOS-unfavorable patients.

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.

Curcumin Alone and Combined With PI3K Inhibitors Elicits Positive Effects on Oropharyngeal Cancer Cell Lines Regardless of HPV Status.

Human papillomavirus positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) is rising in incidence. Compared to HPV-negative (HPV-) OPSCC, HPV+ cases have a better 5-year survival. With its severe side-effects, today's chemoradiotherapy has not improved outcome compared to radiotherapy alone, so new therapies are needed. Mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), fibroblast growth factor receptor 3 (FGFR3) and cell division cycle 27 (CDC27) are found in HPV+ OPSCC, and in vitro targeted therapy combining PI3K and FGFR inhibitors showed synergistic effects. Here the effects of targeting CDC27 with curcumin with/without various inhibitors or cisplatin on OPSCC cell lines were examined. Curcumin was administered to HPV+ OPSCC cell lines CU-OP-2, CU-OP-3 and CU-OP-20, and HPV- CU-OP-17 with/without PI3K, cyclin-dependent kinase 4/6, FGFR, poly (ADP-ribose) polymerase or WEE1 inhibitors (BYL719, PD-0332991, JNJ-42756493, BMN-673 and MK-1775, respectively), or cisplatin. The cell lines were then assessed for 72 h after treatment for viability, proliferation and cytotoxicity. Curcumin led to dose-dependent responses with reduced viability and proliferation; upon combining it with BYL719, additional positive effects were found for most OPSCC lines grown as monolayers, and these effects were validated in CU-OP-2 cells grown as spheroids. Curcumin with MK-1775 or PD-0332991 also elicited some positive effects on CU-OP-2 and CU-OP-17 cells. Curcumin alone led to dose-dependent responses and when combined with BYL719, positive effects were revealed, as they were when it was combined with MK-1775 or PD-0332991, suggesting a potential use of some of these combinations for HPV+ OPSCC.

Relating pre-treatment non-Gaussian intravoxel incoherent motion diffusion-weighted imaging to human papillomavirus status and response in oropharyngeal carcinoma

Background and purposeDiffusion-weighted imaging (DWI) is a promising technique for response assessment in head-and-neck cancer. Recently, we optimized Non-Gaussian Intravoxel Incoherent Motion Imaging (NG-IVIM), an extension of the conventional apparent diffusion coefficient (ADC) model, for the head and neck. In the current study, we describe the first application in a group of patients with human papillomavirus (HPV)-positive and HPV-negative oropharyngeal squamous cell carcinoma. The aim of this study was to relate ADC and NG-IVIM DWI parameters to HPV status and clinical treatment response. Materials and methodsThirty-six patients (18 HPV-positive, 18 HPV-negative) were prospectively included. Presence of progressive disease was scored within one year. The mean pre-treatment ADC and NG-IVIM parameters in the gross tumor volume were compared between HPV-positive and HPV-negative patients. In HPV-negative patients, ADC and NG-IVIM parameters were compared between patients with and without progressive disease. ResultsADC, the NG-IVIM diffusion coefficient D, and perfusion fraction f were significantly higher, while pseudo-diffusion coefficient D* and kurtosis K were significantly lower in the HPV-negative compared to HPV-positive patients. In the HPV-negative group, a significantly lower D was found for patients with progressive disease compared to complete responders. No relation with ADC was observed. ConclusionThe results of our single-center study suggest that ADC is related to HPV status, but not an independent response predictor. The NG-IVIM parameter D, however, was independently associated to response in the HPV-negative group. Noteworthy in the opposite direction as previously thought based on ADC.

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.

INHBA is Enriched in HPV-negative Oropharyngeal Squamous Cell Carcinoma and Promotes Cancer Progression.

Patients with HPV-negative OPSCC have a poorer prognosis due to distinct molecular pathways. This study reveals significant transcriptomic differences between HPV-negative and HPV-positive OPSCC, identifying INHBA as a key upregulated gene in HPV-negative OPSCC's oncogenic pathways. INHBA is crucial in promoting EMT, cell proliferation, and an immunosuppressive tumor environment, suggesting its potential as a therapeutic target for HPV-negative OPSCC.

575 - Differences in mitochondrial gene expression between white and african americans with HPV-negative oropharyngeal squamous cell carcinoma

575 - Differences in mitochondrial gene expression between white and african americans with HPV-negative oropharyngeal squamous cell carcinoma

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.

Cause of Death in Patients with Oropharyngeal Carcinoma by Human Papillomavirus Status: Comparative Data Analysis.

The incidence of oropharyngeal squamous cell carcinomas (OPSCC) has increased in recent decades, and human papillomavirus (HPV) infection is the main cause of OPSCC. The data regarding causes of death (CODs) are vitally important in informing follow-up strategies and revising treatment strategies to deal with any possible preventable treatment-related COD. However, limited studies have assessed the competing COD by HPV status in patients with OPSCC. We aimed to analyze the distribution of the competing COD according to HPV status in OPSCC. We retrospectively included stage I-IVB patients with OPSCC from the Surveillance, Epidemiology, and End Results database between 2010 and 2015. The association between HPV status and head and neck cancer-specific mortality (HNCSM), second primary cancer mortality (SPCM), and noncancer-caused mortality (NCCM) were analyzed. The chi-square test, Kaplan-Meier analysis, and Fine and Gray model were used for statistical analysis. We included 5852 patients in this study and 73.2% (n=4283) of them had HPV-related tumors. A total of 1537 (26.3%) patients died, including 789 (51.3%), 333 (21.7%), and 415 (27%) patients who died from head and neck cancer, second cancer, and noncancer causes, respectively. The 5-year HNCSM, SPCM, NCCM, and overall mortality were 14.7%, 6.5%, 7.7%, and 26.4%, respectively. Those with HPV-positive disease had a lower cumulative incidence of HNCSM (subdistribution hazard ratio [sHR] 0.362, 95% CI 0.315-0.417; P<.001), SPCM (sHR 0.400, 95% CI 0.321-0.496; P<.001), and NCCM (sHR 0.460, 95% CI 0.378-0.560; P<.001) than those with HPV-negative disease. The 5-year risk of HNCSM was 26.9% and 10.7% in those with HPV-negative and HPV-positive disease, respectively (P<.001). The 5-year risk of SPCM was 12.4% and 4.6% in those with HPV-negative and HPV-positive disease, respectively (P<.001). The 5-year risk of NCCM of death was 13.7% and 5.8% in those with HPV-negative and HPV-positive disease, respectively (P<.001). Using the Fine and Gray competing-risks model, our results show that those with HPV-negative tumors had a significantly higher risk of HNCSM (P<.001), SPCM (P<.001), and NCCM (P<.001) than those with HPV-negative tumors. HPV-positive OPSCC has a lower NCSM, SPCM, and NCCM as compared to those with HPV-negative OPSCC. HPV positivity is a favorable prognostic factor in the context of overcoming cancer as well as in terms of reducing the risk of other CODs in OPSCC. Our finding supports the need to tailor patient follow-up based on the HPV status of patients with OPSCC.

HPV16 Impacts NHERF2 Expression in Oropharyngeal Cancers.

Infection with human papillomaviruses (HPVs), in particular with HPV type 16, is now considered to be a key risk factor for the development of a subset of oropharyngeal squamous cell carcinomas (OPSCC) that show different epidemiological, clinical, and prognostic characteristics from HPV-negative (HPV-) OPSCCs. So far, extensive research efforts aiming to distinguish these two distinct entities have not identified specific biomarkers, nor led to different therapies. Previous research has shown that HPV16 E6 oncoprotein binds NHERF2, inducing its proteasomal degradation, and consequently increasing cell proliferation; we therefore aimed to investigate how this might be reflected in human histological samples. We analyzed NHERF2 expression patterns in HPV16-positive (HPV16+) and HPV- OPSCC samples, to investigate any potential differences in NHERF2 pattern. Interestingly, we observed a statistically significant decrease in NHERF2 levels in HPV16+ and poorly differentiated HPV- OPSCCs, compared with healthy tissue. Furthermore, we observed a significant reduction in the percentage of NHERF2 immunoreactive cancer cells in HPV16+ tumors, compared with well and moderately differentiated HPV- OPSCCs, suggesting the importance of 16E6's targeting of NHERF2 in HPV-driven oncogenesis in the head and neck area.

Performance of Liquid Biopsy for Diagnosis and Surveillance of Human Papillomavirus–Associated Oropharyngeal Cancer

There is growing interest in the use of circulating plasma tumor human papillomavirus (HPV) DNA for diagnosis and surveillance of patients with HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). Recent advances in the assays, combining the identification of circulating HPV tumor DNA and tumor DNA fragment analysis (tumor tissue-modified viral [TTMV]-HPV DNA), have been shown to be highly accurate. However, use of these newer techniques has been limited to small cohort studies and clinical trials. To establish the clinical efficacy of plasma TTMV-HPV DNA testing in the diagnosis and surveillance of HPV-associated OPSCC in a contemporary clinical setting. This retrospective observational cohort study included patients with OPSCC who underwent TTMV-HPV DNA testing between April 2020 and September 2022 during the course of routine clinical care. For the diagnosis cohort, patients with at least 1 TTMV-HPV DNA measurement prior to initiation of primary therapy were included. Patients were included in the surveillance cohort if they had at least 1 TTMV-HPV DNA test performed after completion of definitive or salvage therapy. Per-test performance metrics, including sensitivity, specificity, positive predictive value, and negative predictive value, for TTMV-HPV DNA testing. Of 399 patients included in the analysis, 163 were in the diagnostic cohort (median [IQR] age, 63 [56-68.5] years; 142 [87.1%] male), and 290 were in the surveillance cohort (median [IQR] age, 63 [57-70] years; 237 [81.7%] male). Of the 163 patients in the diagnostic cohort, 152 (93.3%) had HPV-associated OPSCC while 11 (6.7%) had HPV-negative OPSCC. The TTMV-HPV DNA sensitivity in pretreatment diagnosis was 91.5% (95% CI, 85.8%-95.4% [139 of 152 tests]), and the specificity was 100% (95% CI, 71.5%-100% [11 of 11 tests]). In the surveillance cohort, 591 tests conducted in 290 patients were evaluated. A total of 23 patients had molecularly confirmed pathologic recurrences. The TTMV-HPV DNA test demonstrated sensitivity of 88.4% (95% CI, 74.9%-96.1% [38 of 43 tests]) and specificity of 100% (95% CI, 99.3%-100% [548 of 548 tests]) in detecting the recurrences. Positive predictive value was 100% (95% CI, 90.7%-100% [38 of 38 tests]), and negative predictive value was 99.1% (95% CI, 97.9%-99.7% [548 of 553 tests]). The median (range) lead time from positive TTMV-HPV DNA test to pathologic confirmation was 47 (0-507) days. This cohort study demonstrated that when evaluated in a clinical setting, the TTMV-HPV DNA assay demonstrated 100% specificity in both diagnosis and surveillance. However, the sensitivity was 91.5% for the diagnosis cohort and 88.4% for the surveillance cohort, signifying that nearly 1 in 10 negative tests among patients with HPV-associated OPSCC was a false negative. Additional research is required to validate the assay's performance and, if validated, then further research into the implementation of this assay into standard clinical practice guidelines will be required.

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.

Abstract 1212: Horizontal transfer of functional human papillomavirus genes in head and neck squamous cell carcinoma

Abstract Human papillomavirus (HPV) infections are currently associated with more than 70% of oropharyngeal squamous cell carcinomas (OPSCCs). HPV-positive OPSCCs display a distinct biologic behavior and treatment response and thus are now considered a distinct entity than HPV-negative OPSCCs. However, the underlying process of HPV-related OPSCC carcinogenesis is not entirely clear. HPV as a driver of cervical carcinogenesis is well-characterized with identification of a canonical mechanism dependent on HPV DNA integration, loss of HPV E2 gene expression, and increased expression of HPV E6 and E7 oncogenes with no active infection present in malignant lesions. However, whole genome sequencing found no HPV DNA integration in ~25% of HPV-associated OPSCC, suggesting an alternative mechanism of HPV carcinogenesis. Expression of all HPV genes, including self-assembling capsid proteins, in a substantial portion of HPV+ OPSCC, led us to hypothesize that tumors with episomal HPV are capable of transferring HPV genes to surrounding cells and that HPV gene transfer contributes to carcinogenesis in this subset of OPSCC. Our data support this hypothesis, showing that growth media from HPV+ OPSCC cell lines, as well as isolates from HPV+ tumors or nodal metastases, effectively transferred functional HPV genes to cells lacking HPV. The HPV DNA Transferring Agent (HDTA) derived from HPV+ OPSCC contained HPV L1 capsid protein and intact HPV genome protected from DNAase degradation. Initial characterization revealed that anti-L1 antibody blocked gene transfer and that exposure of cells to the HDTA initiated a robust innate immune response in recipient cells. Negative staining of HDTA isolate from metastatic lymph nodes revealed many pleomorphic, electron-dense particles with diameter ranging between 20 and 80nM, with approximately 40% of particles being between 50 and 60nM, consistent with the size of HPV. We propose that development and/or maintenance of OPSCCs depend on continuous production of HDTA capable of transferring HPV genes into neighboring cells. This paradigm of tumorigenesis/maintenance in OPSCC is most likely to apply to cancers lacking HPV integration, but may also apply to tumors with integration, since recent data suggested that integrated HPV may excise from the genome to recreate a transient episomal like structure, even containing cellular DNA. Our findings offer novel insights into oropharyngeal carcinogenesis and suggest targeting HDTA as a new therapeutic strategy for HPV-positive OPSCCs. Citation Format: Jason Tasoulas, Wendell Gray Yarbrough, Natalia Issaeva. Horizontal transfer of functional human papillomavirus genes in head and neck squamous cell carcinoma [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 1212.

Novel Immunotherapeutic Approaches to Treating HPV-Related Head and Neck Cancer.

Head and neck cancer (HNC) is the seventh most common malignancy, with oropharyngeal squamous cell carcinoma (OPSCC) accounting for a majority of cases in the western world. While HNC accounts for only 5% of all cancers in the United States, the incidence of a subset of OPSCC caused by human papillomavirus (HPV) is increasing rapidly. The treatment for OPSCC is multifaceted, with a recently emerging focus on immunotherapeutic approaches. With the increased incidence of HPV-related OPSCC and the approval of immunotherapy in the management of recurrent and metastatic HNC, there has been rising interest in exploring the role of immunotherapy in the treatment of HPV-related OPSCC specifically. The immune microenvironment in HPV-related disease is distinct from that in HPV-negative OPSCC, which has prompted further research into various immunotherapeutics. This review focuses on HPV-related OPSCC, its immune characteristics, and current challenges and future opportunities for immunotherapeutic applications in this virus-driven cancer.

Investigation of somatic mutation profiles and tumor evolution of primary oropharyngeal cancer and sequential lymph node metastases using multiregional whole-exome sequencing.

Lymph node (LN) metastasis is an important factor in determining the treatment and prognosis of oropharyngeal squamous cell carcinoma (OPSCC). Here, we compared the somatic mutational profiles and clonal evolution of primary and metastatic LNs using multiregion sequencing of human papilloma virus (HPV)-positive OPSCC and HPV-negative OPSCC. We performed high-depth whole-exome sequencing (200×) of 76 samples from 18 patients with OPSCC (10 HPV-positive and 8 HPV-negative), including 18 primary tumor samples, 40 metastatic LN samples, and 18 normal tissue samples. Among 40 metastatic LNs, 22 showed extranodal extension (ENE). Mutation profiles of HPV-positive OPSCC and HPV-negative OPSCC were similar to those reported previously. Somatic mutations in CDKN2A and TP53 were frequently detected in HPV-negative OPSCC. Somatic mutations in HPV-positive OPSCC samples showed APOBEC-related signatures. Somatic mutations from metastatic LNs showed a different pattern than the primary tumor. Somatic mutations acquired in the WNT pathway during metastasis showed a significant relationship with ENE. Clonal evolution analysis of primary and metastatic LNs showed that, in some cases, each metastatic LN originated from a different primary tumor sub-clone.

Correlation of immune makers with HPV 16 infections and the prognosis in oropharyngeal squamous cell carcinoma

ObjectivesThis study aims to investigate the association of immune markers with high risk human papillomavirus 16 (HPV 16) infection status and to evaluate the prognostic value of programmed death ligand-1 (PD-L1) in patients with oropharyngeal squamous cell carcinoma (OPSCC).Materials and methodsThis retrospective study collected 50 cases of HPV positive and HPV negative OPSCC from January 2011 to December 2015. The correlation of CD8 + tumor infiltrating lymphocytes (TILs), programmed death-1 (PD-1), and PD-L1 expression with HPV 16 infection status was analyzed via immunofluorescent staining and quantitative real-time PCR.ResultsThere was no significant difference in the baseline data between the two groups. Patients with HPV + OPSCC had better prognosis compared to HPV − patients (5-year overall survival [OS], 66% vs. 40%, P = 0.003; 5-year disease specific survival [DSS], 73% vs. 44%, P = 0.001). The expressions of immunity related makers were significantly higher in the HPV + group than the HPV − group (CD8 + TIL: P = 0.039; PD-L1: P = 0.005; PD-1: P = 0.044). Positive CD8 + TIL and PD-L1 were independent factors for better prognosis of OPSCC (DSS, P < 0.001; OS, P < 0.001, respectively). Kaplan–Meier survival analysis indicated that patients with TILs of high HPV + /CD8 + expression were more likely to have better prognosis than those with TILs of low HPV + /CD8 + expression (DSS, P < 0.001; OS, P < 0.001), TILs of high expression of HPV − /CD8 + (DSS, P = 0.010; OS, P = 0.032), and TILs of low expression of HPV − /CD8 + (DSS, P < 0.001; OS, P < 0.001). Furthermore, HPV + /PD-L1 + OPSCC patients had significant better prognosis compared to patients with HPV + /PD-L1 − (DSS, P < 0.001; OS, P = 0.004), HPV − /PD-L1 + (DSS, P = 0.010; OS, P = 0.048) and HPV − /PD-L1 − (DSS, P < 0.001; OS, P < 0.001).ConclusionsHPV + OPSCC had a significantly better prognosis, and PD-L1 expression was elevated in HPV + OPSCC. PD-L1 positivity might be related to the better prognosis of HPV + OPSCC.Clinical relevanceThis study provides a theoretical basis and baseline data for the application of immune checkpoint inhibitors in head and neck tumors.

Race and socioeconomic status interact with HPV to influence survival disparities in oropharyngeal squamous cell carcinoma.

HPV-related oropharyngeal squamous cell carcinoma (OPSCC) is associated with a favorable prognosis, yet patients of color and low socioeconomic status (SES) continue to experience inferior outcomes. We aim to understand how the emergence of HPV has impacted race and SES survival disparities in OPSCC. A retrospective cohort of 18,362 OPSCC cases from 2010 to 2017 was assembled using the SEER (Surveillance, Epidemiology, and End Results) database. Cox proportional regressionand Fine and Gray regression models were used to calculate hazard ratios (HRs) adjusting for race, SES, age, subsite, stage, and treatment. Black patients had lower overall survival than patients of other races in HPV-positive and HPV-negative OPSCC (HR 1.31, 95% CI 1.13-1.53 and HR 1.23, 95% CI 1.09-1.39, respectively). Higher SES was associated with improved survival in all patients. Race had a diminished association with survival among high SES patients. Low SES Black patients had considerably worse survival than low SES patients of other races. Race and SES interact variably across cohorts. High SES was protective of the negative effects of race, although there remains a disparity in outcomes among Black and non-Black patients, even in high SES populations. The persistence of survival disparities suggests that the HPV epidemic has not improved outcomes equally across all demographic groups.

Magnetic resonance imaging based radiomics prediction of Human Papillomavirus infection status and overall survival in oropharyngeal squamous cell carcinoma

ObjectivesHuman papillomavirus- (HPV) positive oropharyngeal squamous cell carcinoma (OPSCC) differs biologically and clinically from HPV-negative OPSCC and has a better prognosis. This study aims to analyze the value of magnetic resonance imaging (MRI)-based radiomics in predicting HPV status in OPSCC and aims to develop a prognostic model in OPSCC including HPV status and MRI-based radiomics. Materials and methodsManual delineation of 249 primary OPSCCs (91 HPV-positive and 159 HPV-negative) on pretreatment native T1-weighted MRIs was performed and used to extract 498 radiomic features per delineation. A logistic regression (LR) and random forest (RF) model were developed using univariate feature selection. Additionally, factor analysis was performed, and the derived factors were combined with clinical data in a predictive model to assess the performance on predicting HPV status. Additionally, factors were combined with clinical parameters in a multivariable survival regression analysis. ResultsBoth feature-based LR and RF models performed with an AUC of 0.79 in prediction of HPV status. Fourteen of the twenty most significant features were similar in both models, mainly concerning tumor sphericity, intensity variation, compactness, and tumor diameter. The model combining clinical data and radiomic factors (AUC = 0.89) outperformed the radiomics-only model in predicting OPSCC HPV status. Overall survival prediction was most accurate using the combination of clinical parameters and radiomic factors (C-index = 0.72). ConclusionPredictive models based on MR-radiomic features were able to predict HPV status with sufficient performance, supporting the role of MRI-based radiomics as potential imaging biomarker. Survival prediction improved by combining clinical features with MRI-based radiomics.

Analysis of T1-T2 stage oropharyngeal squamous cell carcinoma treated with transoral robotic surgery.

Transoral robotic surgery (TORS) has become an effective treatment for early-stage oropharyngeal squamous cell carcinomas (OPSCCs). We aimed to analyze the clinical safety and efficacy of TORS for human papilloma virus (HPV)-positive and HPV-negative OPSCC in China. Patients with OPSCC of pT1-T2 stage who underwent TORS from March 2017 to December 2021 were analyzed. A total of 83 patients (HPV-positive, n=25; HPV-negative, n=58) were included. The median age of the patients was 57.0 years and 71 were men. The majority of primary tumor sites were palatine tonsils (52, 62.7%) and base of tongues (20, 24.1%). Three patients have a positive margin. A total of 12 (14.5%) patients received tracheotomies, the average duration of tracheostomy tube use was 9.4 days, and nasogastric tube was 14.5 days. No patient had a long-term tracheotomy. The 3-year overall survival (OS), disease-free survival (DFS), and recurrence-free survival (RFS) for all 83 patients were 89.5%, 80.1%, and 83.3%, respectively. The OS at 3 years between the HPV-positive group and HPV-negative group were 100% versus 84.3% (P=.07), while the DFS and RFS between two groups also showed no significant difference. Among multivariate cox regression analysis of all potential risk factors, smoking was the significant risk factors for disease recurrence (P< .05). Transoral robotic surgery achieved encouraging oncologic outcomes and safety in T1-T2 stage OPSCC treatment, regardless of HPV status. 4.