Diversity of virulence and antibiotic resistance genes expressed in Class A biosolids and biosolids-amended soil as revealed by metatranscriptomic analysis.

Class A biosolids is a treated sewage sludge, commonly applied to agricultural fields, home lawns/gardens, golf courses, forests, and remediation sites around the world. This practice is of public and agricultural concern due to the possibility that biosolids contain antibiotic-resistant bacteria and fungal pathogens that could persist for extended periods in soil. This possibility was determined by metatranscriptomic analysis of virulence, antibiotic resistance, and plasmid conjugation genes, a Class A biosolids, organically managed soil, and biosolids-amended soil under realistic conditions. Biosolids harbored numerous transcriptionally active pathogens, antibiotic resistance genes, and conjugative genes that annotated mostly to Gram-positive pathogens of animal hosts. Biosolids amendment to soil significantly increased the expression of virulence genes by numerous pathogens and antibiotic-resistant genes that were strongly associated with biosolids. Biosolids amendment also significantly increased the expression of virulence genes by native soil fungal pathogens of plant hosts, which suggests higher risks of crop damage by soil fungal pathogens in biosolids-amended soil. Although results are likely to be different in other soils, biosolids, and microbial growth conditions, they provide a more holistic, accurate view of potential health risks associated with biosolids and biosolids-amended soils than has been achievable with more selective cultivation and PCR-based techniques.

Nasopharyngeal cancer: the impact of guidelines and teaching on radiation target volume delineation.

Target volume delineation in the radiation treatment of nasopharyngeal cancer is challenging due to several reasons such as the complex anatomy of the site, the need for the elective coverage of definite anatomical regions, the curative intent of treatment and the rarity of the disease, especially in non-endemic areas. We aimed to analyze the impact of educational interactive teaching courses on target volume delineation accuracy between Italian radiation oncology centers. Only one contour dataset per center was admitted. The educational course consisted in three parts: (1) The completely anonymized image dataset of a T4N1 nasopharyngeal cancer patient was shared between centers before the course with the request of target volume and organs at risk delineation; (2) the course was held online with dedicated multidisciplinary sessions on nasopharyngeal anatomy, nasopharyngeal cancer pattern of diffusion and on the description and explanation of international contouring guidelines. At the end of the course, the participating centers were asked to resubmit the contours with appropriate corrections; (3) the pre- and post-course contours were analyzed and quantitatively and qualitatively compared with the benchmark contours delineated by the panel of experts. The analysis of the 19 pre- and post-contours submitted by the participating centers revealed a significant improvement in the Dice similarity index in all the clinical target volumes (CTV1, CTV2 and CTV3) passing from 0.67, 0.51 and 0.48 to 0.69, 0.65 and 0.52, respectively. The organs at risk delineation was also improved. The qualitative analysis consisted in the evaluation of the inclusion of the proper anatomical regions in the target volumes; it was conducted following internationally validated guidelines of contouring for nasopharyngeal radiation treatment. All the sites were properly included in target volume delineation by >50% of the centers after correction. A significant improvement was registered for the skull base, the sphenoid sinus and the nodal levels. These results demonstrated the important role that educational courses with interactive sessions could have in such a challenging task as target volume delineation in modern radiation oncology.

Oligorecurrent Non-Small-Cell Lung Cancer Treated by Chemo-Radiation Followed by Immunotherapy and Intracranial Radiosurgery: A Case Report and Mini Review of Literature.

Locally advanced non-small-cell lung cancer still represents a "grey zone" in terms of the best treatment choice and optimal clinical outcomes. Indeed, most patients may be suitable to receive different treatments with similar outcomes such as chemo-radiotherapy (CHT-RT) followed by immunotherapy (IO) or surgery followed by adjuvant local/systemic therapies. We report a clinical case of a patient submitted to primary thoracic surgery who developed a mediastinal nodal recurrence successfully treated by CHT-RT-IO. Subsequently, a single brain lesion was found to have been successfully treated by single fraction stereotactic ablative radiotherapy. The patient is still on follow-up and she is free from disease having a good quality of life. In this report, we also perform a mini review about the role of CHT-RT followed by IO in treating loco-regional relapse after surgery. The role of SABR after IO is also evaluated, finding that it is safe and well tolerated. More robust and larger clinical data are needed in this particular setting to better define the role of the combination of systemic and local treatments in the management of intrathoracic and intracranial relapse for patients already submitted to CHT-RT followed by immunotherapy.

Second primary tumors in head and neck cancer patients: The importance of a "tailored" surveillance.

OBJECTIVE: Head and neck cancer survivors have increased risk of developing second primary tumors compared to overall population. Because second primary represents a major cause of morbidity and mortality in this population, early detection is fundamental. MATERIALS AND METHODS: In this 10-year single-institution study, we investigated the following: incidence, clinical-pathological risk factors, and survival of patients with second primary tumor. We included all patients with diagnosis of squamous cell carcinoma of the head and neck seen at the Modena University Hospital from 2008 to 2018. RESULTS: Among 1,177 patients included, 222 (18.9%) developed second primary tumor; its survival probability at 5 years was 40.6%. Alcohol consumption (p = .0055) and index cancer in oropharynx (p = .0029), supraglottic larynx (p = .0000), glottic larynx (p = .0222) were associated with higher risk of second primary. The most common second primary sites were head and neck district and lung (70, 31.5%, and 67, 30.2%, respectively). Head and neck district were more common in oral cavity (18, 43%) and oropharynx index cancer (20, 31%); lung second primary in hypopharynx (4, 40%), supraglottic larynx (17, 43%), and glottic larynx index cancer (23, 35%). CONCLUSION: Head and neck cancer survivors developing a second primary tumor have dismal prognosis. Tailored surveillance is recommended.

A snapshot on radiotherapy for head and neck cancer patients during the COVID-19 pandemic: a survey of the Italian Association of Radiotherapy and Clinical Oncology (AIRO) head and neck working group.

OBJECTIVES: The objective of the paper was to assess real-life experience in the management of head and neck cancer (HNC) patients during the COVID-19 pandemic in radiotherapy departments and to evaluate the variability in terms of adherence to American Society of Radiation Oncology (ASTRO) and European Society for Radiotherapy and Oncology (ESTRO) recommendations. MATERIALS AND METHODS: In May 2020, an anonymous 30-question online survey, comparing acute phase of outbreak and pre-COVID-19 period, was conducted. Two sections exploited changes in general management of HNC patients and different HNC primary tumors, addressing specific statements from ASTRO ESTRO consensus statement as well. RESULTS: Eighty-eight questionnaires were included in the demographic/clinical workflow analysis, and 64 were analyzed for treatment management. Forty-eight percent of radiotherapy departments became part of oncologic hubs. First consultations reduced, and patients were addressed to other centers in 33.8 and 18.3% of cases, respectively. Telematic consultations were used in 50% of follow-up visits and 73.9% of multidisciplinary tumor board discussions. There were no practical changes in the management of patients affected by different primitive HNCs. Hypofractionation was not favored over conventional schedules. CONCLUSIONS: Compared to pre-COVID era, the clinical workflow was highly re-organized, whereas there were no consistent changes in RT indications and schedules.

A comparison of swallowing dysfunction after three-dimensional conformal and intensity-modulated radiotherapy : A systematic review by the Italian Head and Neck Radiotherapy Study Group.

PURPOSE: Dysphagia is one of the most important treatment-related side effects in head and neck cancer (HNC), as it can lead to severe life-threating complications such as aspiration pneumonia and malnutrition. Intensity-modulated radiotherapy (IMRT) could reduce swallowing dysfunction by producing a concave dose distribution and reducing doses to the swallowing-related organs at risk (SWOARs). The aim of this study was to review the current literature in order to compare swallowing outcomes between IMRT and three-dimensional conformal radiotherapy (3DCRT). METHODS: A search was conducted in the PubMed and Embase databases to identify studies on swallowing outcomes, both clinically and/or instrumentally assessed, after 3DCRT and IMRT. Dysphagia-specific quality of life and objective instrumental data are summarized and discussed. RESULTS: A total of 262 papers were retrieved from the searched databases. An additional 23 papers were retrieved by hand-searching the reference lists. Ultimately, 22 papers were identified which discussed swallowing outcomes after 3DCRT and IMRT for HNC. No outcomes from randomized trials were identified. CONCLUSION: Despite several methodological limitations, reports from the current literature seem to suggest better swallowing outcomes with IMRT compared to 3DCRT. Further improvements are likely to result from the increased use of IMRT plans optimized for SWOAR sparing.

Diverse Imaging Methods May Influence Long-Term Oncologic Outcomes in Oligorecurrent Prostate Cancer Patients Treated with Metastasis-Directed Therapy (the PRECISE-MDT Study).

Metastasis-directed therapy (MDT) has been tested in clinical trials as a treatment option for oligorecurrent prostate cancer (PCa). However, there is an ongoing debate regarding the impact of using different imaging techniques interchangeably for defining lesions and guiding MDT within clinical trials. Methods: We retrospectively identified oligorecurrent PCa patients who had 5 or fewer nodal, bone, or visceral metastases detected by choline or prostate-specific membrane antigen (PSMA) PET/CT and who underwent MDT stereotactic body radiotherapy with or without systemic therapy in 8 tertiary-level cancer centers. Imaging-guided MDT was assessed as progression-free survival (PFS), time to systemic treatment change due to polymetastatic conversion (PFS2), and overall survival predictor. Propensity score matching was performed to account for clinical differences between groups. Results: Of 402 patients, 232 (57.7%) and 170 (42.3%) underwent MDT guided by [18F]fluorocholine and PSMA PET/CT, respectively. After propensity score matching, patients treated with PSMA PET/CT-guided MDT demonstrated longer PFS (hazard ratio [HR], 0.49 [95% CI, 0.36-0.67]; P < 0.0001), PFS2 (HR, 0.42 [95% CI, 0.28-0.63]; P < 0.0001), and overall survival (HR, 0.39 [95% CI, 0.15-0.99]; P < 0.05) than those treated with choline PET/CT-guided MDT. Additionally, we matched patients who underwent [68Ga]Ga-PSMA-11 versus [18F]F-PSMA-1007 PET/CT, observing longer PFS and PFS2 in the former subgroup (PFS: HR, 0.51 [95% CI, 0.26-1.00]; P < 0.05; PFS2: HR, 0.24 [95% CI, 0.09-0.60]; P < 0.05). Conclusion: Diverse imaging methods may influence outcomes in oligorecurrent PCa patients undergoing MDT. However, prospective, head-to-head studies, ideally incorporating a randomized design, are necessary to provide definitive evidence and facilitate the practical application of these findings.

Lattice Radiation Therapy in clinical practice: A systematic review.

Purpose: Lattice radiation therapy (LRT) is an innovative type of spatially fractionated radiation therapy. It aims to increase large tumors control probability by administering ablative doses without an increased toxicity. Considering the rising number of positive clinical experiences, the objective of this work is to evaluate LRT safety and efficacy. Method: Reports about LRT clinical experience were identified with a systematic review conducted on four different databases (namely, Medline, Embase, Scopus, and Cochrane Library) through the August 2022. Only LRT clinical reports published in English and with the access to the full manuscript text were considered as eligible. The 2020 update version PRISMA statement was followed. Results: Data extraction was performed from 12 eligible records encompassing 7 case reports, 1 case series, and 4 clinical studies. 81 patients (84 lesions) with a large lesion ranging from 63.2 cc to 3713.5 cc were subjected to exclusive, hybrid, and metabolism guided LRT. Excluding two very severe toxicity with a questionable relation with LRT, available clinical experience seem to confirm LRT safety. When a complete response was not achieved 3-6 months after LRT, a median lesion reduction approximately ≥50 % was registered. Conclusion: This systematic review appear to suggest LRT safety, especially for exclusive LRT. The very low level of evidence and the studies heterogeneity preclude drawing definitive conclusions on LRT efficacy, even though an interesting trend in terms of lesions reduction has been described.

Validation of RayStation Monte Carlo dose calculation algorithm for multiple LINACs.

PURPOSE: A photon Monte Carlo (MC) model was commissioned for flattened (FF) and flattening filter free (FFF) 6 MV beam energy. The accuracy of this model, as a single model to be used for three beam matched LINACs, was evaluated. METHODS: Multiple models were created in RayStation v.10A for three linacs equipped with Elekta "Agility" collimator. A clinically commissioned collapsed cone (CC) algorithm (GoldCC), a MC model automatically created from the CC algorithm without further optimization (CCtoMC) and an optimized MC model (GoldMC) were compared with measurements. The validation of the model was performed by following the recommendations of IAEA TRS 430 and comprised of basic validation in a water tank, validation in a heterogeneous phantom and validation of complex IMRT/VMAT paradigms using gamma analysis of calculated and measured dose maps in a 2D-Array. RESULTS: Dose calculation with the GoldMC model resulted in a confidence level of 3% for point measurements in water tank and heterogeneous phantom for measurements performed in all three linacs. The same confidence level resulted for GoldCC model. Dose maps presented an agreement for all models on par to each other with γ criteria 2%/2mm. CONCLUSIONS: The GoldMC model showed a good agreement with measured data and is determined to be accurate for clinical use for all three linacs in this study.

Oropharyngeal squamous cell carcinoma: Prognostic factors for development of distant metastases and oncological outcomes.

BACKGROUND: Distant metastasis (DM) development in Oropharyngeal Squamous Cell Carcinoma (OPSCC) represents an important prognostic factor. The identification of a phenotype of metastatic patients may better define therapeutic and follow-up programs. METHODS: We included 408 patients with OPSCC, non-metastatic at the time of diagnosis, and treated with curative intent. The Overall Survival (OS) analyses were performed and the impact of developing DM on survival was analyzed through Cox proportional-hazard regression model. RESULTS: 57 (14%) patients develop DM. 302 (74%) were p16+ OPSCC and 35 of them experienced DM. Advanced clinical stage, smoking, p16-status, response to primary treatment, and loco-regional relapse influence the DM rate. Only in the p16+ group, DM onset results in a greater impact on OS (p < 0.0001). Lung metastases have a better OS compared to non-pulmonary ones (p = 0.049). CONCLUSION: This retrospective study shows a possible stratification of OPSCC patients based on the risk of the development of DMs.

Technological advancements and future perspectives in breast cancer radiation therapy.

INTRODUCTION: Breast cancer is still one of the most common tumors worldwide and radiation therapy has a central role in the oncological pathway. Several technological options are now available with the aim to improve therapeutic index, target definition, and patient selection. AREAS COVERED: In this review, we summarize current available technologies in the management of breast cancer. These advances can support the prescription of postoperative partial breast cancer treatment and preoperative stereotactic partial breast irradiation. Moreover, image-guided radiotherapy is crucial for high-quality radiation treatments. Additionally, the recent development of hybrid magnetic resonance linear accelerator can impact target volume outline procedure, adaptive planning and radiomics. Finally, artificial intelligence represents the new frontier in medicine, supporting clinicians in target definition, patient selection, and treatment planning. EXPERT OPINION: In patients with breast cancer the overall level of evidence about new technologies is still low even if some advances are potentially very interesting to further development.

Reirradiation on spine metastases: an Italian survey on behalf of palliative care and reirradiation study groups of Italian association of radiotherapy and clinical oncology (AIRO).

AIM: This survey derived from the collaboration between the Palliative Care and Reirradiation Study Groups of the Italian Association of Radiotherapy and Clinical Oncology (AIRO). Its aim was to obtain a real "snapshot" on the treatments of spinal metastases, focusing on reirradiation, among radiation oncologists in Italy. METHODS: The survey was elaborated on SurveyMonkey's online interface and was sent via e-mail to all Radiation Oncologists of AIRO that were invited to anonymously fill in the electronic form within 60 days. The questionnaire was prepared by the AIRO "Palliative care" and "Reirradiation" Study Groups and it consisted of 36 questions, 19 single-choice questions, 10 multiple-choice questions and 6 open questions. The data were analyzed and represented with tables and graphs. RESULTS: The survey shows that palliative radiotherapy remains a field of interest for most ROs in the Italian centers. 3D Conventional Radiation Therapy (3DCRT) alone or in combination with other techniques is the primary choice for patients with a life expectancy of less than 6 months. For patients with a life expectancy of more than six months, there is an increased use of new technologies, such as Volumetric Modulated Arc Therapy (VMAT). Factors considered for retreatment are time between first and second treatment, dose delivered to spine metastasis and spinal cord in the first treatment, vertebral stability, symptoms, and/or performance status. The most feared complication are myelopathy followed by vertebral fracture and local recurrence. This explain an increasing focus on patient selection and the use of high technology in the treatment of metastatic patients. CONCLUSION: Stereotactic body radiotherapy (SBRT) and image-guided radiotherapy allow the administration of ablative RT doses while sparing the constraints of healthy tissue in spinal metastases. However, there is still an unclear and heterogeneous reality in the reirradiation of spinal metastases. A national registry with the aim of clarifying the most controversial aspects of vertebral metastasis retreatments will enable better management of these patients and design more targeted study designs.

Treatment of loco-regional recurrence of nasopharyngeal carcinoma in a non-endemic area: oncologic outcomes, morbidity, and proposal of a prognostic nomogram.

Introduction: The study assessed outcomes and toxicities of different treatment modalities for local and/or regional recurrent nasopharyngeal carcinoma (NPC) in a non-endemic area. Methods: Patients treated with curative intent for recurrent NPC with salvage surgery, photon-based radiotherapy, proton therapy (PT), with or without chemotherapy, at different Italian referral centers between 1998 and 2020 were included. Adverse events and complications were classified according to the Common Terminology Criteria for Adverse Events. Characteristics of the patients, tumors, treatments, and complications are presented along with uni- and multivariate analysis of prognostic factors. A survival predictive nomogram is also provided. Results: A total of 140 patients treated from 1998 to 2020 were retrospectively assessed. Cases with lower age, comorbidity rate, stage, and shorter disease-free interval (DFI) preferentially underwent endoscopic surgery. More advanced cases underwent re-irradiation, fairly distributed between photon-based radiotherapy and PT. Age and DFI were independent factors influencing overall survival. No independent prognostic effect of treatment modality was observed. No significant difference in the morbidity profile of treatments was observed, with 40% of patients experiencing at least one adverse event classified as G3 or higher. Conclusion: Recurrent NPC in a non-endemic area has dissimilar aspects compared to its endemic counterpart, suggesting the need for further studies that can guide the choice of the best treatment modality.

A predictive model of polymetastatic disease from a multicenter large retrospectIve database on colorectal lung metastases treated with stereotactic ablative radiotherapy: The RED LaIT-SABR study.

Aim: Stereotactic ablative radiotherapy (SABR) showed increasing survival in oligometastatic patients. Few studies actually depicted oligometastatic disease (OMD) evolution and which patient will remain disease-free and which will rapidly develop a polymetastatic disease (PMD) after SABR. Therefore, apart from the number of active metastases, there are no clues on which proven factor should be considered for prescribing local treatment in OMD. The study aims to identify predictive factors of polymetastatic evolution in lung oligometastatic colorectal cancer patients. Methods: This international Ethical Committee approved trial (Prot. Negrar 2019-ZT) involved 23 Centers and 450 lung oligometastatic patients. Primary end-point was time to the polymetastatic conversion (tPMC). Additionally, oligometastases number and cumulative gross tumor volume (cumGTV) were used as combined predictive factors of tPMC. Oligometastases number was stratified as 1, 2-3, and 4-5; cumGTV was dichotomized to the value of 10 cc. Results: The median tPMC in the overall population was 26 months. Population was classified in the following tPMC risk classes: low-risk (1-3 oligometastases and cumGTV ≤ 10 cc) with median tPMC of 35.1 months; intermediate-risk (1-3 oligometastases and cumGTV > 10 cc), with median tPMC of 13.9 months, and high-risk (4-5 oligometastases, any cumGTV) with median tPMC of 9.4 months (p = 0.000). Conclusion: The present study identified predictive factors of polymetastatic evolution after SABR in lung oligometastatic colorectal cancer. The results demonstrated that the sole metastases number is not sufficient to define the OMD since patients defined oligometastatic from a numerical point of view might rapidly progress to PMD when the cumulative tumor volume is high. A tailored approach in SABR prescription should be pursued considering the expected disease evolution after SABR, with the aim to avoid unnecessary treatment and toxicity in those at high risk of polymetastatic spread, and maximize local treatment in those with a favorable disease evolution.

Malnutrition management in oncology: An expert view on controversial issues and future perspectives.

Cancer and anticancer treatments can lead to several negative side effects, including malnutrition. Despite the recognized need for adequate nutritional support in cancer patients, in daily clinical practice, nutrition is still not considered one of the first aspects to be considered. Malnutrition negatively affects the clinical outcomes, treatment response, and overall survival of cancer patients. In this study, three of the most controversial issues related to malnutrition, which emerged during an Italian Consensus Conference, were addressed specifically for patients with head and neck as well as gastrointestinal cancer. The timing of nutritional evaluation and intervention, extension of the Enhanced Recovery after Surgery (ERAS®) protocols, and cost-effectiveness of nutritional interventions have been considered. This study aimed to illustrate the state-of-the art of each issue and hypothesize future perspectives and actions to be taken, trying to suggest a new nutritional management model for cancer patients in Italy that overcomes the critical issues encountered. Of note, the timely diagnosis of nutritional issue appears to be essential to ensure the correct management of malnourished cancer patients as well as those who are at high risk of malnutrition. Standardized protocols, screening tests, and the inclusion of nutritional parameters in patient medical records would help to achieve good clinical outcomes. Finally, appropriate nutritional support is also associated with cost savings, and it seems necessary to promote its clinical and economic value to obtain improvements in both outcomes and management costs.

Adding Concomitant Chemotherapy to Postoperative Radiotherapy in Oral Cavity Carcinoma with Minor Risk Factors: Systematic Review of the Literature and Meta-Analysis.

When presenting with major pathological risk factors, adjuvant radio-chemotherapy for oral cavity cancers (OCC) is recommended, but the addition of chemotherapy to radiotherapy (POCRT) when only minor pathological risk factors are present is controversial. A systematic review following the PICO-PRISMA methodology (PROSPERO registration ID: CRD42021267498) was conducted using the PubMed, Embase, and Cochrane libraries. Studies assessing outcomes of POCRT in patients with solely minor risk factors (perineural invasion or lymph vascular invasion; pN1 single; DOI ≥ 5 mm; close margin < 2−5 mm; node-positive level IV or V; pT3 or pT4; multiple lymph nodes without ENE) were evaluated. A meta-analysis technique with a single-arm study was performed. Radiotherapy was combined with chemotherapy in all studies. One study only included patients treated with POCRT. In the other 12 studies, patients were treated with only PORT (12,883 patients) and with POCRT (10,663 patients). Among the patients treated with POCRT, the pooled 3 year OS rate was 72.9% (95%CI: 65.5−79.2%); the pooled 3 year DFS was 70.9% (95%CI: 48.8−86.2%); and the pooled LRFS was 69.8% (95%CI: 46.1−86.1%). Results are in favor of POCRT in terms of OS but not significant for DFS and LRFS, probably due to the heterogeneity of the included studies and a combination of different prognostic factors.

Development of a prognostic model of overall survival in oropharyngeal cancer from real-world data: PRO.M.E.THE.O.

Objective: The PRO.M.E.THE.O. study (PredictiOn Models in Ent cancer for anti-EGFR based THErapy Optimization) aimed to develop a predictive model (PM) of overall survival (OS) for patients with locally advanced oropharyngeal cancer (LAOC) treated with radiotherapy (RT) and cetuximab (Cet) from an Italian dataset. Methods: We enrolled patients with LAOC from 6 centres treated with RT-Cet. Clinical and treatment variables were collected. Patients were randomly divided into training (TS) (80%) and validation (VS) (20%) sets. A binary logistic regression model was used on the TS with stepwise feature selection and then on VS. Timepoints of 2, 3 and 5 years were considered. The area under the curve (AUC) of receiver operating characteristic of 2, 3 and 5 year and confusion matrix statistics at 5-threshold were used as performance criteria. Results: Overall, 218 patients were enrolled and 174 (79.8%) were analysed. Age at diagnosis, gender, ECOG performance, clinical stage, dose to high-risk volume, overall treatment time and day of RT interruption were considered in the final PMs. The PMs were developed and represented by nomograms with AUC of 0.75, 0.73 and 0.73 for TS and 0.713, 0.713, 0.775 for VS at 2, 3 and 5 years, respectively. Conclusions: PRO.M.E.THE.O. allows the creation of a PM for OS in patients with LAOC treated with RT-Cet.

AIRO GORTEC consensus on postoperative radiotherapy (PORT) in low-intermediate risk early stages oral squamous cell cancers (OSCC).

Evidence on the efficacy of postoperative radiotherapy (PORT) in low-intermediate risk squamous cell carcinoma of the oral cavity (OSCC) remains inconclusive. Members of a task force from two national radio-oncology Associations (AIRO and GORTEC) defined 14 clinically relevant questions to identify "gray areas" pertinent to the indication for PORT in this clinical setting. Consequently, a literature review was performed on the topic. The resulting statements were then rated by an Expert Panel (EP) using a modified Delphi method. Only radiation oncologists were part of the discussion and voting on the scenarios. There was agreement on the 14 statements at the first round of voting. The task force then decided to propose clinical cases for the two more controversial statements that had received a lower agreement to better capture the Experts' attitudes. The clinical cases highlighted a more significant decisional heterogeneity. However, the good level of consensus reached among the two Associations gives relevant support in informing clinical choices while acknowledging general indications cannot fit all clinical situations and do not replace multidisciplinary discussion.

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

BACKGROUND AND PURPOSE: 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 < 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. 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.