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Abstract: Aim: EGFR and HPV-associated p16 are among the most investigated biomarkers in head and neck cancer. The aim was to investigate the correlation and interaction between these two markers and to evaluate their potential prognostic significance when combined. Materials and methods: 336 Oropharyngeal carcinomas treated with primary radiotherapy (66–68Gy, 2fx/day, 10–12Gy/week) and with known EGFR/p16-status estimated semiquantitatively by immunohistochemistry were included in the study. Data were evaluated by EGFR-expression (high/low) and p16-status (positive/negative) consequently dividing tumours into four groups by combination of the biomarkers. Patient/tumour characteristics and complete 5-year follow-up were available. Results: Low EGFR-expression was significantly more common in p16-positive tumours compared to p16-negative, p <0.0001. p16 positivity showed a strong prognostic impact (p <0.0001, HR=0.22 [0.13–0.38]), whereas EGFR was a weak prognostic marker when local control was used as endpoint (p =0.03, HR=0.53 [0.29–0.94]). Combination of EGFR/p16 did not add significant information to p16 alone and by multivariable analysis only p16 showed significant prognostic information for all evaluated endpoints. Conclusions: Both EGFR and p16 bear prognostic information in oropharyngeal cancer, although p16 is, by far, the strongest prognostic factor. The markers seem to be correlated and this might have influence when evaluating the effect of EGFR inhibition in oropharyngeal tumours. [Copyright &y& Elsevier]

Abstract: Background and purpose: Tumour HPV-positivity is a favourable prognostic factor in the radiotherapy of HNSCC, but the optimal radiotherapy regimen for HPV-positive HNSCC is not yet defined. Reducing overall treatment time is known to improve outcome in the radiotherapy of HNSCC as was also demonstrated in the randomised DAHANCA 6&7 trial. We aimed to assess the influence of tumour HPV-status, expressed by p16, on the response to accelerated fractionated radiotherapy in HNSCC through evaluation of the DAHANCA 6&7 trial. Materials and methods: Immunohistochemical detection of HPV-associated p16-expression was performed on FFPE-pre-treatment tumour-tissues from 794 patients enrolled in the DAHANCA 6&7 trial. The influence of tumour p16-status on loco-regional tumour control and survival as a function of fractionation schedule (5Fx/week vs 6Fx/week) was evaluated 5years after the completion of radiotherapy. Results: The significant and independent prognostic value of tumour p16-positivity in HNSCC radiotherapy was confirmed, with adjusted hazard ratios (HR) of 0.58 [0.43–0.78], 0.47 [0.33–0.67] and 0.54 [0.42–0.68] for loco-regional control, disease-specific and overall survival, respectively. Accelerated radiotherapy significantly improved loco-regional tumour control compared to conventional radiotherapy, adjusted HR: 0.73 [0.59–0.92] and the benefit of the 6Fx/week regimen was observed both in p16-positive (HR: 0.56 [0.33–0.96]) as well as in p16-negative tumours (HR: 0.77 [0.60–0.99]). Disease-specific survival was also significantly improved with accelerated radiotherapy in the group of p16-positive tumours (adjusted HR: 0.43 [0.22–0.82]). Conclusion: Accelerated radiotherapy significantly improves outcome in HNSCC compared to conventional fractionation. The observed benefit is independent of tumour p16-status and the use of a moderately accelerated radiotherapy regimen seems advantageous also for HPV/p16-positive HNSCC. [Copyright &y& Elsevier]

Abstract: The profound influence of Human papillomavirus (HPV) on the epidemiological pattern and clinical course of head and neck cancer (HNSCC) has led to a change in the traditional understanding of this disease entity. Separate therapeutic strategies based on tumour HPV status are under consideration and in this light provision of knowledge concerning the influence of tumour HPV on the radiation response in HNSCC appears highly relevant. This review provides a summary of the current understanding of the role of HPV in head and neck cancer with specific focus on the viral impact on radiotherapy outcome of HNSCC. [Copyright &y& Elsevier]

Abstract: Background: HPV/p16-positive head and neck cancers (HNSCC) show superior response to radiotherapy, compared with virus-negative tumours. Tumour hypoxia induces radioresistance and the randomised DAHANCA 5 trial found that the hypoxic cell radiosensitiser nimorazole significantly improved the outcome in HNSCC. Using p16-status as a retrospective stratification parameter, we aimed to assess the influence of p16-expression on the response to nimorazole in HNSCC. Materials and methods: Pre-treatment tumour blocks were available from 331 of the 414 patients in the DAHANCA 5 trial and evaluated by immunohistochemistry for p16-expression. The influence of p16-expression on outcome was analysed as a function of treatment group (nimorazole/placebo) 5years after radiotherapy. Results: Overall, patients treated with nimorazole had significantly better loco-regional control than did those given placebo: hazard ratio (HR) 0.70 [95% CI 0.52–0.93]. Positive expression of p16 also significantly improved outcome after radiotherapy (0.41 [0.28–0.61]). In the subgroup of patients with p16-negative tumours, loco-regional failure was more frequent in the placebo group than in the nimorazole group (0.69 [0.50–0.95]). However, in the p16-positive group, patients treated with nimorazole had a loco-regional control rate similar to patients given placebo (0.93 [0.45–1.91]). Conclusions: HPV/p16-expression significantly improved outcome after radiotherapy in HNSCC. Hypoxic modification improved outcome in HPV/p16-negative tumours but was of no significant benefit in HPV/p16-positive tumours, suggesting that hypoxic radioresistance may not be clinically relevant in these tumours. [Copyright &y& Elsevier]

Radiotherapy treatment planning hinges on a critical factor: the prescribed dose. Surprisingly, no consistent, standardised global approach to interpreting this prescription exists. This study aimed to examine and illustrate the variations in prescribed doses for the same treatment across North European and North American centres. The study analysed consecutively treated oropharynx cancer patients from six globally recognised radiotherapy departments. The criteria for inclusion encompassed curative IMRT or VMAT radiotherapy administered in 2017 or later. These centres were divided into three North American and three North European centres. Dose-volume histogram (DVH) data were extracted from the local treatment planning system for the Gross Tumour Volume (GTV), the high-dose Clinical Target Volume (CTV), and Planning Target Volume (PTV) for each patient. The DVH was sampled in 1 cGy dose bins across the 0 to 100 Gy range. All DVHs were scaled to a standard prescription of 70 Gy delivered in 35 fractions to facilitate straightforward comparisons across centres. No biological corrections were applied. For the three target volumes (GTV, CTV, PTV), we extracted and compared metrics such as D95% (Dose to 95% of the volume), D98%, D99%, V95% (Volume receiving 95% of the prescription dose), V105%, and V107%. We visually compared these metrics and conducted statistical testing using the Mann-Whitney U-test. Our study encompassed 1,375 patients treated across six centres, revealing a spectrum of 38 different dose prescriptions, ranging from 55 Gy in 20 to 70 Gy in 35 fractions. When normalised to 70 Gy, the median mean CTV dose exhibited a 4% difference, ranging from 70.12 Gy to 72.93 Gy across centres. Notably, the three European centres showed a high consistency, deviating by only 0.4%, while the three North American centres showed a slight variation within 2%. Figure 1 presents the mean CTV dose and the D98% boxplots. The interquartile range (IQR) for the mean CTV dose from 0.17 Gy to 1.22 Gy, with European centres showing the smallest IQR. [Display omitted] Examining the near-minimum CTV doses (D98% of CTV), we observed a range from 68.65 Gy to 70.96 Gy, with an IQR from 0.30 Gy to 1.15 Gy. A scatter plot of CTV mean dose against CTV D98% revealed distinct clusters for each of the six centres (Figure 2). Notably, the European centres cluster more densely compared to the North American centres. [Display omitted] The variation in prescribed doses for the same treatment regimen poses a significant challenge. Clinical interpretation of 70 Gy varies widely between centres and is influenced by each centre's individual experience, which, in turn, impacts the interpretation of published results. European centres primarily adhere to the ICRU dose prescription, targeting the median CTV dose. Conversely, North American centres tend to prescribe based on the minimum dose to the CTV or, in some cases, the PTV, as outlined in various RTOG protocols. Our study underscores that prescribing a dose of 70 Gy in 35 fractions for standard oropharynx cancer patients results in substantial variations in mean and near-minimum CTV doses. These dose prescription discrepancies significantly impact the interpretation of clinical trial outcomes comparison. Furthermore, this significant dosimetric variability has important implications for dose de-escalation strategies for HPV+ oropharynx cancer treatment. [ABSTRACT FROM AUTHOR]

Diverse post-translational modifications (PTMs) regulate protein function and interaction to fine-tune biological processes. Reversible phosphorylation, cysteines (Cys) modifications, and N-linked glycosylation are all essentially involved in cellular signaling pathways, such as those initiated by the action of pro-inflammatory cytokines, which can induce pancreatic β-cell death and diabetes. Here we have developed a novel strategy for the simultaneous and comprehensive characterization of the proteome and three PTMs including reversibly modified Cysteines (rmCys), phosphorylation, and sialylated N-linked glycosylation from low amount of sample material. This strategy, termed TiCPG, is based on a combination of chemical labeling and titanium dioxide (TiO 2) chromatography. We applied the TiCPG strategy to study the proteome and the three PTMs changes in β-cells subject to pro-inflammatory cytokines stimulation. It enabled quantitative analysis of 8346 rmCys sites, 10,321 phosphosites and 962 sialylated N-glycosites from 5496 proteins. Significant regulation was found on 100 proteins at the expression level, while 3020 PTM peptide isoforms from 1468 proteins were significantly regulated. The three PTMs were involved in cytokine mediated β-cell apoptosis, such as the NFκB and the inducible NO synthase signaling pathways. Overall, the TiCPG strategy is a cheap, straightforward, and powerful tool for studies targeting the three PTMs described above. The present study presents a fast and easy method for quantitative assessment of the proteome and three PTMs from minimal amount of sample material. This simple method provides comprehensive and significant knowledge on biological systems and cellular signaling with relatively low analysis time, suitable for younger researchers and researchers that do not have direct access to LC-MSMS in their laboratories. From sub-milligram amount of material, we were able to map known cellular signaling events of proinflammatory cytokine effect on beta-cells and to discover novel PTMs involved in several known signaling pathways. [Display omitted] • Presentation of a simple method to assess the proteome and three PTMs from low amount of biological material. • The TiCPG method is used to study changes in the three PTMs in INS-1E beta cells upon proinflammatory cytokine stimulation. • Using TiCPG to study the interplay between phosphorylation and reversibly modified cysteines in a biological system. [ABSTRACT FROM AUTHOR]

Abstract: Purpose: To validate the predictive impact of a hypoxia gene expression classifier in identifying patients with head and neck squamous cell carcinoma (HNSCC) having benefit from hypoxic modification of radiotherapy. Patients and methods: Gene expressions were quantified from formalin-fixed, paraffin-embedded tumour biopsies of 323 HNSCC patients randomized for placebo or nimorazole in conjunction with radiotherapy in the DAHANCA 5 study. Tumours were classified as either “more” or “less” hypoxic with a classifier constituting of 15 hypoxia responsive genes. The predictive impact was evaluated by analysing the response to nimorazole vs. placebo in terms of loco-regional tumour control (LRC) and disease-specific survival (DSS) in the two classified groups. Results: Hundred and fourteen patients (35%) were classified as having “more” hypoxic tumours. These patients had a significant benefit of hypoxic modification with nimorazole compared with placebo in terms of LRC (5-year actuarial values 49% vs. 18%; p =0.001) and DSS (48% vs. 30%; p =0.04). “Less” hypoxic tumours had no significant effect of hypoxic modification (LRC: 50% vs. 44%; p =0.39, DSS: 57% vs. 51%; p =0.49) and generally an outcome, which was similar to “more” hypoxic tumours treated with nimorazole. In contrast to HPV-negative tumours, HPV-positive tumours had a substantially better outcome in response to radiotherapy, which was irrespective of hypoxic modification. Conclusions: A predictive 15-gene hypoxia classifier could identify patients associated with improved outcome after combining radiotherapy with hypoxic modification and underlines the relevance of such therapy. The impact of the classifier was limited to HPV-negative tumours. [Copyright &y& Elsevier]

Abstract: Purpose: Hypoxia is a cause of resistance to radiotherapy, especially in patients with head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to evaluate 18F-fluoroazomycin arabinoside (FAZA) positron emission tomography (PET)/computed tomography (CT) hypoxia imaging as a prognostic factor in HNSCC patients receiving radiotherapy. Material and methods: Forty patients with HNSCC treated with radiotherapy (66–76Gy) were included. Static FAZA PET/CT imaging 2h post injection was conducted prior to irradiation. The hypoxic volume (HV) was delineated using a tumor-to-muscle value ⩾1.4. In 13 patients, a repetitive FAZA PET/CT scan was conducted during the radiotherapy treatment. Results: A hypoxic volume could be identified in 25 (63%) of the 40 tumors. FAZA PET HV varied considerably with a range from 0.0 to 30.9 (median: 0.3) cm3. The Tmax/Mmed ranged from 1.1 to 2.9 (median: 1.5). The distribution of hypoxia among the Human Papillomavirus (HPV) positive (12/16) and negative (13/24) tumors was not significant different. In the FAZA PET/CT scans performed during radiotherapy, hypoxia could be detected in six of the 13 patients. For these six patients the location of HV remained stable in location during radiotherapy treatment, though the size of the HV decreased. In 30 patients a positive correlation was detected between maximum FAZA uptake in the primary tumor and the lymph node. During a median follow up of 19months a significant difference in disease free survival rate with 93% for patients with non hypoxic tumors and 60% for patients with hypoxic tumors could be detected. Conclusion: This study emphasizes the role of FAZA PET/CT imaging as a suitable assay with prognostic potential for detection of hypoxia in HNSCC. [Copyright &y& Elsevier]