P53 and pRB induction improves response to radiation therapy in HPV-positive laryngeal squamous cell carcinoma.

INTRODUCTION: Patients with Human Papillomavirus (HPV+)-associated Laryngeal Squamous Cell Carcinoma (LSCC) exhibit dramatically improved survival relative to those with HPV-Negative (HPV-) tumors. In this study, the authors aimed to investigate the radiosensitivity of all available confirmed HPV+ and HPV-LSCC cells in vitro and in vivo. METHODS: Primary LSCC cells were generated from tumor specimens obtained from patients. Real-time PCR was performed to confirm HPV infection and the expression of HPV-related genes (E6 and E7), p53, and pRB. Clonogenic survival assays, western blotting, and flow cytometry were used to assess radiation sensitivity, apoptosis, and the expression of p53 and pRB. p53 and pRB knockout cells were generated using CRISPR/Cas9 technology. RESULTS: HPV+ LSCC cells displayed enhanced radiation sensitivity compared to HPV- cells. Radiation-induced apoptosis in HPV+ LSCC cells, accompanied by increased levels of p53 and pRB. Knockout of p53 or pRB led to radiation resistance and attenuated radiation-induced apoptosis in HPV+ LSCC cells. In vivo experiments showed similar results, where knockout of p53 or pRB decreased radiosensitivity in tumor-bearing mice. CONCLUSION: The present findings demonstrated that HPV+ LSCC cells displayed obvious inherent radiation sensitivity, corresponding to increased apoptosis following radiation exposure. Mechanism study showed that the expression of p53 and pRB in HPV+ cells are required for radiation sensitivity. These findings highlight a novel mechanism by which p53 and pRB play key roles in the radiation sensitivity of HPV+ LSCC compared to HPV-LSCC.

Translational risk-adapted approaches to de-escalated radiation for human papillomavirus-positive oropharyngeal cancer: Past, present, and future.

Interest in the use of de-escalated radiation to treat patients with newly diagnosed human papillomavirus (HPV)-positive oropharyngeal cancer has grown dramatically with the publication of prospective trials demonstrating the efficacy of such an approach. While the rationale for de-escalation--- namely to decrease treatment-related toxicity while maintaining the excellent rates of disease control historically observed in patients with this disease-is inherently obvious, uncertainty exists regarding how to best select patients for de-escalation. Consequently, risk-adapted strategies using a variety of translational and clinical platforms have been increasingly popularized to better refine treatment. These have integrated contemporary methods of mid-treatment response assessment using advanced technologies and molecular assays to customize the radiation dose. By monitoring the response as patients actively proceed through treatment, risk-adapted protocols have the potential to provide insight into the biological behavior of tumors and make individualized therapy possible. The purpose of this review is to summarize the evidence to date on risk-adapted approaches to de-escalated radiation-- highlighting the clinical, radiological, and biological data which may ultimately help usher the principles of precision medicine into practice for patients with HPV-positive oropharyngeal cancer.

Upfront surgery versus definitive radiotherapy: competing risk analyses for cancer-specific and noncancer mortality in oropharyngeal cancer.

PURPOSE: The optimal treatment strategy for oropharyngeal cancer (OPC) is undetermined. We aim to compare the survival outcomes of OPC patients treated with upfront surgery versus definitive radiotherapy (RT). METHODS: A total of 8057 cases were retrieved from the Surveillance, Epidemiology, and End Results database. Primary endpoints were cancer-specific and noncancer mortalities, which were estimated using cumulative incidence function and compared by Gray's test. Univariate and multivariate Fine-Gray subdistribution hazard models were used to estimate the effects of treatment modality on mortality. Subgroup analyses were performed in propensity-score-matched cohorts. All the analyses were conducted separately in human papillomavirus (HPV)-negative and HPV-positive cohorts. RESULTS: In the HPV-negative cohort, definitive RT was independently associated with increased risk of cancer-specific mortality (adjusted subdistribution hazard ratio [SHR], 1.31; 95% confidence interval [CI], 1.05-1.64; P = 0.017) and noncancer mortality (adjusted SHR, 1.59; 95% CI 1.13-2.25; P = 0.008). In the HPV-positive cohort, definitive RT was independently associated with increased risk of cancer-specific mortality (adjusted SHR, 1.51; 95% CI 1.23-1.85; P < 0.001) and noncancer mortality (adjusted SHR, 1.53; 95% CI 1.11-2.12; P = 0.009). CONCLUSION: Upfront surgery is a superior treatment modality compared with definitive RT in terms of lowering cancer-specific and noncancer mortality in OPC patients, regardless of HPV status. Further prospective clinical trials are needed to confirm our findings.

Association of follow-up imaging frequency with temporal incidence and patterns of distant failure following (chemo) radiotherapy for HPV related oropharyngeal cancer.

OBJECTIVES: Emerging data supports radical intent therapy for oligometastatic (OM) relapsed human papilloma virus (HPV+) related oropharyngeal cancer (OPC). We assess the association of follow-up imaging frequency amongst HPV + OPC, with temporal and spatial patterns of distant relapse, to inform rationalisation of routine post-treatment imaging. MATERIALS AND METHODS: A retrospective single centre cohort study was carried out of consecutive HPV + OPC patients treated with radical intent (chemo)radiotherapy ((CT)RT) between 2011 and 2019. OM state was defined as ≤ 5 metastasis, none larger than 3 cm (OMs) or, if interval from last negative surveillance imaging > 6-months, then ≤ 10 metastasis, none larger than 5 cm, (OMp). Patients not meeting OMs / OMp criteria were deemed to have incurable diffuse metastatic disease (DMdiffuse). RESULTS: 793 HPV-OPC patients were identified with median follow-up 3.15years (range 0.2-8.9). 52 (6.6 %) patients had radiologically identified DM at first failure and were considered for analysis. The median time to recurrence was 15.1 months (range: 2.6-63 months). 87 % of distant metastasis (DM) occurred in the first two years after treatment. Twenty-seven (52 %) patients had OM (OMs or OMp) at time of failure, with 31 % having OMs. The median time from completion of treatment to diagnosis of DMdiffuse vs OM was 22.2 months (range: 2.6-63.1 months) vs 11.6 months (range: 3.5-32.5 months). The probability of being diagnosed with OM vs DMdiffuse increased with reducing interval from last negative surveillance scan to imaging identifying DM (≤6 months 88.9 %, 7-12 months 71.4 %, 13-24 months 35 %, > 24 months 22.2 %). CONCLUSION: We demonstrate that a reduced interval between last negative imaging and subsequent radiological diagnosis of DM is associated with increased likelihood of identification of OM disease. Consideration of increased frequency of surveillance imaging during the first two years of follow up is supported, particularly for patients at high risk of distant failure.

The Effects of Human Papillomavirus Status and Treatment on the Positive Predictive Value of Post-radiotherapy 18F-Fluorodeoxyglucose Positron Emission Tomography-Computed Tomography in Advanced Head and Neck Squamous Cell Carcinoma.

AIMS: The high negative predictive value of post-chemoradiation (CRT) positron emission tomography-computed tomography (PET-CT) is well established in head and neck squamous cell cancers (HNSCC). The positive predictive value (PPV) remains under scrutiny, with increasing evidence that it is affected by several factors. The aim of this study was to assess the PPV of post-treatment PET-CT for residual nodal disease when stratified by treatment modality and tumour human papillomavirus (HPV) status. MATERIALS AND METHODS: This was a retrospective cohort study in a tertiary oncology centre carried out between January 2013 and December 2019. Patients were radically treated with radiotherapy only/CRT for node-positive HNSCC. PET-CT nodal responses were categorised as complete, equivocal (EQR) or incomplete (ICR), and outcomes extracted from electronic records. RESULTS: In total, 480 patients were evaluated, all had a minimum potential follow-up of 2 years, with a median of 39.2 months. The PPV of 12-week PET-CT was significantly different between HPV-positive (22.5%) and HPV-unrelated (52.7%) disease, P < 0.001. It was also significantly different between the CRT (24.8%) and radiotherapy-only (51.1%) groups, P = 0.001. The PPV of an EQR was significantly less than an ICR, irrespective of HPV status and primary treatment modality. In HPV-positive disease, the PPV of an EQR was 9.0% for the CRT group compared with 21.4% for radiotherapy only, P = 0.278. The PPV in those who achieved an ICR was 34.2% in the CRT group, significantly lower than 70.0% in the radiotherapy-only group, P = 0.03. CONCLUSION: The PPV of 12-week PET-CT is significantly lower for HPV-positive compared with HPV-unrelated HNSCC. It is poorer in patients with HPV-positive disease treated with CRT compared with radiotherapy alone.

Human papillomavirus E6/E7 oncoproteins promote radiotherapy-mediated tumor suppression by globally hijacking host DNA damage repair.

Rationale: Whatever the mucosa primary infected, HPV-positive cancers are traditionally associated with a favorable outcome, attributable to a high sensitivity to radiation therapy. However, the direct impact of viral E6/E7 oncoproteins on the intrinsic cellular radiosensitivity (and, globally, on host DNA repair) remains mostly speculative. Methods: Using several isogenic cell models expressing HPV16 E6 and/or E7, the effect of viral oncoproteins on global DNA damage response was first investigated by in vitro/in vivo approaches. The binary interactome of each individual HPV oncoprotein with factors involved in the various host DNA damage/repair mechanisms was then precisely mapped by Gaussia princeps luciferase complementation assay (and validated by co-immunoprecipitation). The stability/half-life of protein targets for HPV E6 and/or E7 as well as their subcellular localizations were determined. At last, the host genome integrity following E6/E7 expression and the synergy between radiotherapy and compounds targeting DNA repair were analyzed. Results: We first showed that the sole expression of one viral oncoprotein from HPV16 was able to significantly increase the sensitivity to irradiation of cells without affecting their basal viability parameters. In total, 10 novel targets (CHEK2, CLK2, CLK2/3, ERCC3, MNAT1, PER1, RMI1, RPA1, UVSSA and XRCC6) for E6 and 11 (ALKBH2, CHEK2, DNA2, DUT, ENDOV, ERCC3, PARP3, PMS1, PNKP, POLDIP2 and RBBP8) for E7 were identified. Importantly, not degraded following their interaction with E6 or E7, these proteins have been shown to be less linked to host DNA and to colocalize with HPV replication foci, denoting their crucial implication in viral life cycle. Finally, we found that E6/E7 oncoproteins globally jeopardize host genome integrity, increase the cellular sensitivity to DNA repair inhibitors and enhance their synergy with radiotherapy. Conclusion: Taken together, our findings provide a molecular insight into the direct hijacking of host DNA damage/repair responses by HPV oncoproteins, demonstrate the significant impact of this phenomenon on both intrinsic cellular radiosensitivity and host DNA integrity and suggest novel connected therapeutic vulnerabilities.

Clearance Profile of Circulating Tumor Human Papillomavirus DNA During Radiotherapy Predicts Clinical Outcomes in Human Papillomavirus-Related Oropharyngeal Cancer.

PURPOSE: This study aimed to examine whether circulating tumor human papillomavirus type 16 (HPV16) DNA (ctHPV16DNA) can help identify patients with locally advanced HPV16-related oropharyngeal squamous cell carcinoma who may benefit from deintensified treatment. MATERIALS AND METHODS: We serially collected blood samples before, during, and after treatment from 22 patients who received 70 Gy radiotherapy alone and longitudinally quantified ctHPV16DNA using droplet digital polymerase chain reaction. We correlated the clearance profile of ctHPV16DNA with clinical outcomes. RESULTS: The percentage of patients with detectable ctHPV16DNA decreased after every 10 Gy of radiotherapy. By contrast, the percentage of patients who later developed treatment failure among patients with detectable ctHPV16DNA gradually increased as radiotherapy proceeded, reaching 100% after 60 Gy of radiotherapy. We defined patients with and without detectable ctHPV16DNA after receiving 40 Gy as having slow and rapid clearance profiles, respectively. All 12 patients with a rapid clearance profile remained disease-free after radiotherapy. Of the 10 patients with a slow clearance profile, three had persistent or progressive disease at response evaluation after radiotherapy and one developed distant metastasis during follow-up (ie, four patients experienced treatment failure). The median follow-up for surviving patients was 38.6 months, and the 3-year failure-free survival rates of patients with rapid and slow clearance profiles were 100% and 58%, respectively (P = .02). Neither baseline ctHPV16DNA levels nor metabolic tumor volume was an independent predictor of the pattern of the clearance profile. CONCLUSION: In patients with HPV16-related oropharyngeal squamous cell carcinoma receiving radiotherapy, a slow ctHPV16DNA clearance profile could prelude unfavorable outcomes. Monitoring ctHPV16DNA is essential for determining the clearance profile, which might help optimize treatment intensity individually.

CENPM upregulation by E5 oncoprotein of human papillomavirus promotes radiosensitivity in head and neck squamous cell carcinoma.

OBJECTIVES: This study aims to investigate how human papillomavirus (HPV) affects the key gene in the biological behaviors of head and neck squamous cell carcinoma (HNSCC) that leads to better response to radiotherapy. MATERIALS AND METHODS: The expression of key gene CENPM was analyzed using The Cancer Genome Atlas (TCGA) HNSCC data and HPV positive and HPV negative HNSCC tumors and cells. Assays with siRNAs, CRISPR/Cas9-based models, Western blot, qRT-PCR, ChIP, etc., were used to explore how HPV affects CENPM and response to radiotherapy for HNSCC. RESULTS: CENPM occupies the hub in the HPV-related gene network. HPV-positive HNSCC showed higher level of CENPM expression comparing with HPV-negative HNSCC. HPV E5 has the most pronounced impact on CENPM (R = 0.44, p = 0.00081). This might result from the binding of transcription factor E2F1 to CENPM. We further found that inhibition of CENPM expression in HPV-positive HNSCC cell line SCC47 increased resistance to X-ray radiation by approximately 59% under 2 Gy irradiation, which may be resulted from a reduced proportion of mitotic cells. CONCLUSION: HPV E5 enhances CENPM expression by transcription factor E2F1 in HNSCC, which results in a radiosensitive profile in cell cycle redistribution of HNSCC. Thus, HPV infection in HNSCC provides profound evidence that underscores the magnitude of E2F1 control of CENPM expression illustrating the potential clinical benefit of CENPM examination for difficult-to-treat HPV-negative cancers.

Increasing global accessibility to high-level treatments for cervical cancers.

Human papillomaviruses (HPV)-related gynecological cancers are a major health care issue, and a leading cause of cancer death in low- and middle-income countries (LMIC). In 2020, the World Health Organization launched a program aimed at cervical cancer elimination, by screening and vaccination strategies. Offering the best possible care to women diagnosed with invasive cancer is a complementary objective. Treatment of cervical cancer as per modern standards is complex and multimodal, mainly relying on surgery, external-beam radiotherapy (+/-chemotherapy) and brachytherapy. In parallel with the pivotal role of multidisciplinary discussion, international societies provide guidance to define the most effective and least toxic anti-cancer strategy, homogenize treatment protocols and provide benchmark quality indicators as a basis for accreditation processes. The challenge is to offer the appropriate diagnostic workup and treatment upfront and to avoid non- evidence-based treatment that consumes resources, impairs quality of life (QoL), and compromises oncological outcome. Various strategies may be applied for improving treatment quality: development of surgical mentorship, companion-training programs and international cooperation. The lack of radiotherapy/brachytherapy facilities is a major concern in LMIC. Reinforcing international support in terms of education, training, research and development and technical cooperation with national projects is required to increase access to minimum requirements but also introduce modern techniques, upgrade radiotherapy/brachytherapy services, and expand access to modern systemic treatments. In countries with robust economies, compliance to standards should also be increased. Integrative cancer care and multidisciplinary approaches are needed to tackle the dual challenge of increasing cure rates while minimizing QoL impairment. Appropriate dimensioning of the resources to avoid harmful treatment delays and access to expert referral centers is also a priority.

Current considerations for radiotherapy in HPV-associated head and neck cancer.

Human papillomavirus (HPV)-mediated oncogenesis confers increased sensitivity to radiotherapy and HPV head and neck cancer is associated with improved patient outcomes. As such, management of HPV-related head and neck cancer requires a multidisciplinary approach that balances maximizing locoregional control with minimizing treatment-related toxicity. We highlight considerations in radiation dose and target delineation, as well as considerations for chemoradiation, postoperative radiotherapy, and single modality, definitive radiotherapy.

Radiation Treatment Deintensification for HPV-Associated Oropharyngeal Cancer.

Human papillomavirus-associated oropharyngeal cancer (HPV-OPC) is a distinct clinical entity with a favorable prognosis compared with non-HPV-OPC. Surgery and radiotherapy (RT) result in adverse effects, and negative quality of life or functional outcomes, which impact a significant proportion of HPV-OPC survivors. Ongoing studies aim to reduce these negative treatment effects while maintaining high cure rates through deintensified therapy typically use either a primary surgical or RT approach. A single-day curative surgery will remain relevant for many patients with early-stage disease. However, the average patient with HPV-OPC will have indications for adjuvant therapy. A primary RT approach to deintensified therapy has more available data from patients on prospective multi-institutional trials, provides broader patient selection, and may be more cost-effective. Anticipated results from an active phase II/III NCTN trial will help guide the standard of care using primary RT. Next generation trials will help further refine patient selection and/or radical deintensification (30-50 Gy).

HPV transcript expression affects cervical cancer response to chemoradiation.

Persistent HPV infection is causative for the majority of cervical cancer cases; however, current guidelines do not require HPV testing for newly diagnosed cervical cancer. Using an institutional cohort of 88 patients with cervical cancer treated uniformly with standard-of-care chemoradiation treatment (CRT) with prospectively collected clinical outcome data, we observed that patients with cervical tumors containing HPV genotypes other than HPV 16 have worse survival outcomes after CRT compared with patients with HPV 16+ tumors, consistent with previously published studies. Using RNA sequencing analysis, we quantified viral transcription efficiency and found higher levels of E6 and the alternative transcript E6*I in cervical tumors with HPV genotypes other than HPV 16. These findings were validated using whole transcriptome data from The Cancer Genome Atlas (n = 304). For the first time to our knowledge, transcript expression level of HPV E6*I was identified as a predictive biomarker of CRT outcome in our complete institutional data set (n = 88) and within the HPV 16+ subset (n = 36). In vitro characterization of HPV E6*I and E6 overexpression revealed that both induce CRT resistance through distinct mechanisms dependent upon p53-p21. Our findings suggest that high expression of E6*I and E6 may represent novel biomarkers of CRT efficacy, and these patients may benefit from alternative treatment strategies.

Radiation Response of Cervical Cancer Stem Cells Is Associated with Pretreatment Proportion of These Cells and Physical Status of HPV DNA.

Radio- and chemoresistance of cancer stem cells (CSCs) is considered as one of the possible causes of adverse results of chemoradiotherapy for various malignancies, including cervical cancer. However, little is known about quantitative changes in the CSC subpopulation in the course of treatment and mechanisms for individual response of CSCs to therapy. The purpose of the study was to evaluate the association of radiation response of cervical CSCs with clinical and morphological parameters of disease and features of human papillomavirus (HPV) infection. The proportion of CD44+CD24low CSCs was determined by flow cytometry in cervical scrapings from 55 patients with squamous cell carcinoma of uterine cervix before treatment and after fractionated irradiation at a total dose of 10 Gy. Real-time PCR assay was used to evaluate molecular parameters of HPV DNA. Post-radiation increase in the CSC proportion was found in 47.3% of patients. Clinical and morphological parameters (stage, status of lymph node involvement, and histological type) were not significantly correlated with radiation changes in the CSC proportion. Single- and multifactor analyses revealed two independent indicators affecting the radiation response of CSCs: initial proportion of CSCs and physical status of HPV DNA (R = 0.86, p = 0.001 for the multiple regression model in the whole).

Pre- and Post-Radiotherapy Radiologic Nodal Features and Oropharyngeal Cancer Outcomes.

OBJECTIVES: To assess the prognostic value of pre-/post-radiotherapy (pre-/post-RT) radiologic lymph node (LN) features in human papillomavirus (HPV)-positive and HPV-negative oropharyngeal carcinoma (OPC) patients treated with definitive (chemo-)RT. METHODS: Clinical node-positive OPCs treated from 2011 to 2015 were reviewed. Nodal features were reviewed by a radiologist on pre-/post-RT computed tomography (CTs). Univariable analysis calculated hazard ratio (HR) for regional failure (RF), distant metastasis (DM), and deaths. Multivariable analysis estimated adjusted HR (aHR) of significant nodal features identified in univariable analysis adjusting for confounders. RESULTS: Pre-RT CT was undertaken in 344 HPV-positive and 94 HPV-negative OPC patients, of whom 242 (70%) HPV-positive and 67 (71%) HPV-negative also had a post-RT CT. Median follow-up was 4.9 years. Pre-RT LN calcification (pre-RT_LN-cal) increased the risk of RF in HPV-negative (aHR: 5.3, P = .007) but not HPV-positive patients (P = .110). Pre-RT radiologic extranodal extension (pre-RT_rENE+) increased the risk of DM and death in both HPV-negative (DM: aHR 6.6, P < .001; death: aHR 2.1, both P = .019) and HPV-positive patients (DM: aHR 4.9; death: aHR 3.0, both P < .001). Increased risk of RF occured with < 20% post-RT LN size reduction in both HPV-negative (HR 6.0, P = .002) and HPV-positive cases (HR 3.0, P = .049). Post-RT_LN-cal did not affect RF, DM, or death regardless of tumor HPV status (all P > .05). CONCLUSION: Pre-RT_LN-cal is associated with higher RF risk in HPV-negative but not in HPV-positive patients. Pre-RT_rENE increases risk of DM and death regardless of tumor HPV status. Minimal post-RT LN size reduction (< 20%) increases risk of RF in both diseases. Post-RT_LN-cal + has no apparent influence on outcomes in either disease. LEVEL OF EVIDENCE: 4 (a single institution case-control series) Laryngoscope, 131:E1162-E1171, 2021.

Sensitivity of human papillomavirus­positive and ­negative oropharyngeal cancer cell lines to ionizing irradiation.

Human papillomavirus­positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) has increased in incidence and has a much better prognosis than HPV­negative (HPV­) OPSCC with radiotherapy alone, but exactly why is unknown. The present study therefore aimed to further examine the sensitivity and possible changes in gene expression of several HPV+ and HPV­ OPSCC, including various novel cell lines, upon ionizing irradiation (IR). Previously established HPV+ UM­SCC­47, UPCI­SCC­90, CU­OP­2, CU­OP­3 and HPV­ UM­SCC­4, UM­SCC­6, UM­SCC­74a, UM­SCC­19 and newly established CU­OP­17 and CU­OP­20, characterised here, were subjected to 0­6 Gy. Surviving fractions of each cell line were tested by clonogenic assays, and irregularities in cell cycle responses were examined by flow cytometry, while changes in gene expression were followed by mRNA sequencing. HPV+ OPSCC cell lines showed greater variation in sensitivity to ionizing irradiation (IR) and tended to be more sensitive than HPV­ OPSCC cell lines. However, their IR sensitivity was not correlated to the proportion of cells in G2 arrest, and HPV­ cell lines generally showed lower increases in G2 after IR. Upon IR with 2 Gy, mRNA sequencing revealed an increase in minor HPV integration sites in HPV+ cell lines, and some changes in gene expression in OPSCC cell lines, but not primarily those associated with DNA repair. To conclude, HPV+ OPSCC cell lines showed greater variation in their sensitivity to IR, with some that were radioresistant, but overall the HPV+ OPSCC group still tended to be more sensitive to IR than the HPV­ OPSCC group. In addition, HPV+ OPSCC lines were more frequently in G2 as compared to HPV­ cell lines, but the increase in G2 arrest upon IR in HPV+ OPSCC was not correlated to sensitivity to IR. Increases in minor HPV integration sites and changes in gene expression were also demonstrated after irradiation with 2 Gy.

Intrinsic Radiosensitivity Is Not the Determining Factor in Treatment Response Differences between HPV Negative and HPV Positive Head and Neck Cancers.

Head and neck squamous cell carcinomas (HNSCC) resulting from human papillomavirus (HPV) are increasing in incidence but demonstrate significantly better treatment response than HNSCC from other causes such as tobacco and alcohol. This study sought to identify differences in HNSCC, intrinsic to HPV status, in their response to radiation dose. Previously unexamined changes in radio-responsiveness following fractionated X-ray irradiation were compared between HPV positive and negative statuses of HNSCC. Six HNSCC cell lines, 3 of each HPV status, were investigated for radiosensitivity by clonogenic assay and modelled by response as a function of dose. Generational cultures of each cell line were developed to follow changes in radiosensitivity after repeated irradiations simulating fractionated radiation therapy. As a group, the HPV positive cell lines were more radiosensitive, but with changes following repeated fractions of dose, and modelling of response as a function of dose, both statuses displayed large radiobiological heterogeneity. These findings challenge current radiobiological assumptions of head and neck cancers as early responding tissue to radiation and may go some way in explaining difficulties reaching consensus in stratification of treatment by HPV status. Consequently, results from this study do not support stratifying radiation therapy by HPV status.

M2 macrophages reduce the radiosensitivity of head and neck cancer by releasing HB­EGF.

The aim of the present study was to examine the potential role of human heparin­binding epidermal growth factor (HB­EGF) secreted by M2 macrophages in the development of radioresistance in head and neck squamous cell carcinoma (HNSCC). Immunohistochemistry was used to detect radiosensitivity in human papilloma virus (HPV)­positive and HPV­negative HNSCC tissues and immunohistochemical staining with specific antibodies for macrophage surface markers was used to assess the infiltration of M1 and M2 macrophages in HPV­positive and ­negative HNSCC tissues. The expression of HB­EGF in HPV­positive and ­negative HNSCC tissues was determined by multi­cytokine detection in order to determine the relationship between HB­EGF and radiosensitivity. M1 and M2 macrophages were co­cultured with the HNSCC cell line CAL27 and treated with HB­EGF and its neutralizing antibodies to assess radiation sensitivity. Finally, the major DNA double­strand break repair pathways required for the activation of HB­EGF and promotion of epidermal growth factor receptor (EGFR) were identified. The results revealed that radiosensitivity was higher in HPV­positive HNSCC compared with HPV­negative. There was a higher infiltration of M2 macrophages in HPV­negative HNSCC, which were revealed as the main source of HB­EGF secretion. Furthermore, it was determined that overexpression of HB­EGF induced radioresistance in HPV­negative HNSCC. HB­EGF promoted the activation of the non­homologous end­joining pathway by activating EGFR. To the best of our knowledge, this is the first study to demonstrate the association between HB­EGF and radiosensitivity in HNSCC. These results indicated that the secretion of HB­EGF by M2 macrophages could induce radioresistance of HPV­negative HNSCC.

HPV + HNSCC-derived exosomal miR-9 induces macrophage M1 polarization and increases tumor radiosensitivity.

Human papillomavirus (HPV) is an etiological risk factor for a subset of head and neck squamous cell carcinoma (HNSCC). HPV + HNSCC is more radiosensitive than HPV- HNSCC, however, the mechanism underlying this observation remains unknown. Tumor microenvironment can regulate tumor response to radiation therapy. Secretory exosomes are emerging as crosstalk mediators between tumor cells and the tumor microenvironment. In this study, we attempted to determine the role of HPV + HNSCC exosomes in increased radiation sensitivity. We found that HPV + HNSCC exosomes were able to transform macrophages into the M1 phenotype, which subsequently increased the radiosensitivity of HNSCC. miR-9 was found enriched in HPV + HNSCC exosomes and it could be transported into macrophages, inducing M1 macrophage polarization via downregulation of PPARδ. After incubating with M1 macrophages or macrophages treated with miR-9 mimics, HNSCC had strikingly increased radiosensitivity. The clinical significance of miR-9 in HNSCC was confirmed by using profiling data from The Cancer Genome Atlas. Our data suggest that miR-9-enriched exosomes from HPV + HNSCC can polarize macrophages into M1 phenotype and increase the radiosensitivity of HPV + HNSCC. Hence, miR-9 may be used as a potential treatment for HNSCC.

Influence of the human papillomavirus on the radio-responsiveness of cancer stem cells in head and neck cancers.

A growing proportion of head and neck cancers (HNC) result from HPV infection. Between HNC aetiological groups (HPV positive and HPV negative) clinical evidence demonstrates significantly better treatment response among HPV positive cancers. Cancer stem cells (CSCs) are identified in HNC tumour populations as agents of treatment resistance and a target for tumour control. This study examines dynamic responses in populations of a CSC phenotype in HNC cell lines following X-irradiation at therapeutic levels, and comparing between HPV statuses. Variations in CSC density between HPV groups showed no correlation with better clinical outcomes seen in the HPV positive status. CSC populations in HPV positive cell lines ranged from 1.9 to 4.8%, and 2.6 to 9.9% for HPV negative. Following 4 Gy X- irradiation however, HPV negative cell lines demonstrated more frequent and significantly greater escalation in CSC proportions, being 3-fold that of the HPV positive group at 72 hours post irradiation. CSC proportions of tumour populations are not fixed but subject to change in response to radiation at therapeutic dose levels. These findings imply a potential effect of aetiology on radio-responsiveness in CSCs, illustrating that clonogen treatment response may be more informative of therapy outcomes than inherent population density alone.