Potential of E-Learning Interventions and Artificial Intelligence-Assisted Contouring Skills in Radiotherapy: The ELAISA Study.

PURPOSE: Most research on artificial intelligence-based auto-contouring as template (AI-assisted contouring) for organs-at-risk (OARs) stem from high-income countries. The effect and safety are, however, likely to depend on local factors. This study aimed to investigate the effects of AI-assisted contouring and teaching on contouring time and contour quality among radiation oncologists (ROs) working in low- and middle-income countries (LMICs). MATERIALS AND METHODS: Ninety-seven ROs were randomly assigned to either manual or AI-assisted contouring of eight OARs for two head-and-neck cancer cases with an in-between teaching session on contouring guidelines. Thereby, the effect of teaching (yes/no) and AI-assisted contouring (yes/no) was quantified. Second, ROs completed short-term and long-term follow-up cases all using AI assistance. Contour quality was quantified with Dice Similarity Coefficient (DSC) between ROs' contours and expert consensus contours. Groups were compared using absolute differences in medians with 95% CIs. RESULTS: AI-assisted contouring without previous teaching increased absolute DSC for optic nerve (by 0.05 [0.01; 0.10]), oral cavity (0.10 [0.06; 0.13]), parotid (0.07 [0.05; 0.12]), spinal cord (0.04 [0.01; 0.06]), and mandible (0.02 [0.01; 0.03]). Contouring time decreased for brain stem (-1.41 [-2.44; -0.25]), mandible (-6.60 [-8.09; -3.35]), optic nerve (-0.19 [-0.47; -0.02]), parotid (-1.80 [-2.66; -0.32]), and thyroid (-1.03 [-2.18; -0.05]). Without AI-assisted contouring, teaching increased DSC for oral cavity (0.05 [0.01; 0.09]) and thyroid (0.04 [0.02; 0.07]), and contouring time increased for mandible (2.36 [-0.51; 5.14]), oral cavity (1.42 [-0.08; 4.14]), and thyroid (1.60 [-0.04; 2.22]). CONCLUSION: The study suggested that AI-assisted contouring is safe and beneficial to ROs working in LMICs. Prospective clinical trials on AI-assisted contouring should, however, be conducted upon clinical implementation to confirm the effects.

3-Dimensional versus standard 2-D laparoscopy for benign hysterectomy: A randomized clinical trial.

OBJECTIVE: Our aim was to evaluate possible short and long-term benefits of 3-dimensional (3-D) compared to 2-dimensional (2-D) laparoscopy for benign hysterectomy. Primary outcomes were long-term quality of life and postoperative pain. Secondary outcomes were operative time, surgical complications, time to return to work and length of hospitalization. STUDY DESIGN: A randomized controlled trial conducted at two Danish university hospitals. In each arm, 190 patients were needed for an alpha of 3.3 % and a power of 90 %. For various reasons, however, the study was prematurely terminated after including 97 patients. Patients were randomized to either 2-D (n = 48) or 3-D (n = 49) laparoscopy. A laparoscopic hysterectomy was performed. Quality of life was assessed by the Short Form Health Survey 36 (SF-36) questionnaire at the time of inclusion and 6 weeks postoperatively. Postoperative pain was assessed using a Numeric Rating Scale (NRS) and by monitoring the amount of analgesic consumption. RESULTS: Out of the 97 randomized patients, 77 patients completed both SF-36 questionnaires. No significant differences in mental (p = 0.5) and physical status (p = 0.9) were found. The 2-D group had significantly higher pain-score registered in the post anesthesia care unit (PACU) (p = 0.004) and higher consumption of oral morphine equivalent dose (MEqD) (p = 0.003) than the 3-D group. This regardless a higher rate of minilaparotomies in the 2D (n = 7) than in the 3D (n = 1) group (p < 0.03). The 2-D group had also higher rate of Clavien-Dindo 2 (CD2) (n = 2) and Clavien-Dindo 3 (CD3) complications (n = 3) (p = 0.03) than 3-D (n = 0). The other secondary outcome parameters did not vary between groups. CONCLUSIONS: The results are severely hampered by the premature termination of the study, as less than 25 percent of the patients were recruited. Thus, no firm conclusions can be drawn regarding the quality of life and many of the secondary outcomes, as the lack of difference may be attributed to a type 2 error. However, the significant differences in postoperative pain and in complication rates suggest a greater advantage of 3-D laparoscopy than originally expected. Despite the methodological problems, the current data deserve attention in a sparsely investigated field, emphasizing the urgent need for further studies.

HPV testing versus p16 immunohistochemistry in oropharyngeal squamous cell carcinoma: results from the DAHANCA 19 study.

INTRODUCTION: The prognosis after primary (chemo-)radiotherapy for oropharyngeal squamous cell carcinoma (OPSCC) is affected by Human Papillomavirus (HPV) status, with a better prognosis in HPV-positive OPSCC. HPV-status is routinely assessed by p16 immunohistochemistry (IHC), but additional HPV DNA testing is debated. Also, there are numerous HPV genotypes, which prognostic role may need clarification. The purpose of this study was: (1) to test a custom-made targeted HPV next generation sequencing (NGS) panel in OPSCC, (2) to determine correlation with p16 IHC, and (3) to assess the impact of HPV DNA testing on outcome in the prospectively randomized clinical trial DAHANCA 19. MATERIALS AND METHODS: We included 271 patients with OPSCC treated with primary (chemo-)radiotherapy in the DAHANCA 19 trial. Of these, 199 (73%) were p16-positive. HPV-status was determined by targeted HPV next generation sequencing (NGS), using a custom-made HPV genotyping panel. RESULTS: HPV was detected in 194 tumor samples. p16 IHC and NGS HPV status were concordant in 265 (98%) of 271 patients, whereas we did not detect HPV DNA in 5 p16-positive tumors. HPV16 accounted for 169 of 194 HPV-positive cases (87%). HPV genotypes 18, 31, 33, 35, and 59 were also detected.Loco-regional failure and overall survival were similar whether patients were separated by p16 IHC, or HPV DNA status (p < 0.0001 for all) and did not depend on HPV genotype (p = 0.9 and p = 0.7). CONCLUSION: In the present study, HPV DNA testing or typing in a Danish OPSCC cohort did not add additional information to p16 IHC, the most widely used and accepted prognostic indicator.

Socioeconomic position and the pre-diagnostic interval among patients diagnosed with head and neck squamous cell carcinoma - a population-based study from DAHANCA.

BACKGROUND: The socioeconomic differences in survival are pronounced for patients diagnosed with head and neck cancer; disease stage at diagnosis is suggested to be a main driver of this association. This nationwide, population-based study investigates socioeconomic differences in the pre-diagnostic interval and disease stage at diagnosis. MATERIAL AND METHODS: Information on patient-reported symptoms, symptom onset and disease-specific factors was obtained from the nationwide population-based Danish Head and Neck Cancer Group (DAHANCA) database for patients diagnosed with head and neck squamous cell carcinoma between 2008 and 2019 in Denmark. Socioeconomic position (SEP) was measured by individual-level education, income and cohabitation status obtained from administrative registers. Socioeconomic differences in the interval from symptom onset to diagnosis were investigated in general linear models with 95% confidence intervals (CIs); overall and by subsite, symptom and comorbidity score. Consultation patterns prior to diagnosis were examined using methods for change-point detection. Associations with advanced-stage disease were estimated in logistic regression models. RESULTS: Patients with low, medium and high SEP had a similar interval from patient-reported symptom onset to diagnosis of 10 weeks. Although this interval varied according to primary symptom and anatomical subsite, no apparent socioeconomic differences were observed within these subgroups. Aligned with the patient-reported symptom onset, a distinct increase in consultation rates was observed at 9 weeks (95% CI [7.3; 10.7]) for patients with low SEP and 7 weeks (95% CI [4.8; 9.2]) for patients with high SEP, with overlapping CIs. Patients with low compared to high SEP had increased odds for advanced-stage glottic and oral cavity squamous cell carcinoma. For the remaining subsites the association varied according to SEP-indicator and TNM-edition. CONCLUSION: The interval from symptom onset to diagnosis and consultation patterns were similar across SEP groups. Still, socioeconomic differences in stage at diagnosis were observed for some - but not all - subsites.

TiCPG - a strategy for the simultaneous enrichment of reversibly modified cysteine peptides, phosphopeptides, and sialylated N-Glycopeptides to study cytokines stimulated beta-cells.

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 ß-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 (TiO2) chromatography. We applied the TiCPG strategy to study the proteome and the three PTMs changes in ß-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 ß-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. SIGNIFICANCE: 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.

Association of Smoking, Comorbidity, Clinical Stage, and Treatment Intent With Socioeconomic Differences in Survival After Oropharyngeal Squamous Cell Carcinoma in Denmark.

Importance: The socioeconomic gap in survival after cancer is pronounced among patients with head and neck cancer. Understanding the mechanisms of this gap is crucial to target intervention strategies. Objective: To investigate socioeconomic differences in survival after oropharyngeal squamous cell carcinoma (OPSCC) according to human papillomavirus (HPV) status and the extent to which smoking, comorbidity, clinical stage, and treatment intent explain the survival gap. Design, Setting, and Participants: This nationwide, population-based cohort study was based on prospectively collected information on all patients with a diagnosis of OPSCC from the Danish Head and Neck Cancer Group database and administrative registries. The study included 4600 patients born in 1921 or later, aged 30 years or older, and residing in Denmark 1 year prior to OPSCC diagnosis. Patients with missing information (547 [12%]) were excluded. Patients were diagnosed between January 1, 2008, and December 31, 2019, and followed up until December 31, 2021. Data were analyzed from June 6 to October 4, 2022. Exposure: Socioeconomic position (educational level, disposable income, or cohabiting status). Main Outcomes and Measures: Socioeconomic differences in 5-year overall survival were estimated in Cox proportional hazards regression models by HPV status. The indirect effect and proportion mediated by smoking, comorbidity, clinical stage, and treatment intent were estimated based on a counterfactual approach. Results: The analyzed cohort comprised 4053 patients (1045 women [26%] and 3008 men [74%]). The median age was 61 years (IQR, 55-68 years), and 2563 patients (63%) had HPV-positive OPSCC while 1490 patients (37%) had HPV-negative OPSCC. The 5-year standardized overall survival was 10% to 15% lower among patients with a lower educational level, with low disposable income, or who were living alone (patients with HPV-positive OPSCC, 68%-71%; patients with HPV-negative OPSCC, 31%-34%) than patients with a higher educational level, high disposable income, or a cohabiting partner (patients with HPV-positive OPSCC, 81%-86%; patients with HPV-negative OPSCC, 43%-46%). Among patients with HPV-positive OPSCC, a considerable part of this survival gap was estimated to be associated with differences in smoking (27%-48%), comorbidity (10%-19%), clinical stage (8%-19%), and treatment intent (16%-28%). Among those with HPV-negative OPSCC, comorbidity (12%-22%) and treatment intent (16%-42%) were the primary potential mediators. Conclusions and Relevance: This cohort study suggests that, regardless of HPV status, patients with low socioeconomic position had 10% to 15% lower 5-year overall survival than patients with high socioeconomic position. A substantial part of this survival gap was associated with differences in smoking, comorbidity, clinical stage, or treatment intent at diagnosis.

Fever of Unknown Origin: A Validation Study of Danish ICD-10 Diagnosis Codes.

Background: Real-world data in form of routinely collected clinical data are a valuable resource for epidemiological research in infectious disease. We examined the validity of a discharge diagnosis of fever of unknown origin from hospital discharge registries. Methods: We identified patients with a first in- or outpatient diagnosis (primary or secondary) of fever of unknown origin (ICD-10 code R50.0; R50.8, R50.9) recorded in the Danish National Patient Registry (DNPR) between 2010 and 2017 in the North Denmark Region. We based the validation cohort on a mix of patients diagnosed at a highly specialized university department of infectious diseases (n=100), other internal medicine departments (n=50), and patients diagnosed at a regional non-university hospital (n=50). We estimate positive predictive value (PPV) of diagnosis for fever of unknown origin using medical records as reference. Results: The PPV of a diagnosis of fever of unknown origin for patients diagnosed at the infectious disease department was 61% (95% CI: 51-71%). For other internal medicine departments, it was 14% (95% CI: 6-27%), and for the non-university hospital it was 16% (95% CI: 7-29%). To achieve higher PPVs, we excluded immunocompromised patients, patients who were diagnosed with infection, cancer or rheumatic disease within 7 days after admission, and/or patients with a short hospital stay (maximum 3 days) and no subsequent hospital contact within 1 month. The PPV for diagnoses from the Department of Infectious Diseases improved to 82% (95% CI: 68-91%) for other internal medicine departments it improved to 31% (95% CI: 11-59%), and for the non-university hospital it improved to 36% (95% CI: 13-65%). Conclusion: We found that only diagnoses made in the Department of Infectious Diseases accurately identified fever of unknown origin, whereas diagnoses made in other units mainly covered infection-related fever, cancer-related fever, or short unspecific fever without further diagnostic work-up.

Treatment outcomes and survival following definitive (chemo)radiotherapy in HPV-positive oropharynx cancer: Large-scale comparison of DAHANCA vs PMH cohorts.

We compare outcomes in two large-scale contemporaneously treated HPV-positive (HPV+) oropharynx cancer (OPC) cohorts treated with definitive radiotherapy/chemoradiotherapy (RT/CRT). p16-confirmed HPV+ OPC treated between 2007 and 2015 at PMH and DAHANCA were identified. Locoregional failure (LRF), distant metastasis (DM), and overall survival (OS) were compared. Multivariable analysis (MVA) calculated adjusted-hazard-ratio (aHR) with 95% confidence interval (95% CI), adjusting for cohort, age, gender, performance status, smoking pack-years, T-category and N-category and chemotherapy. Compared to PMH (n = 701), DAHANCA (n = 1174) contained lower TNM-8T-categories (T1-T2: 77% vs 56%), N-categories (N0-N1: 77% vs 67%) and stages (stage I: 63% vs 44% (all P < .001). PMH used standard-fractionation CRT in 69% (481) while 31% (220) received hypofractionated or moderately accelerated RT-alone. All DAHANCA patients were treated with moderately accelerated RT; 96% (1129) received nimorazole (NIM) and 73% (856) concurrent weekly cisplatin. DAHANCA had shorter overall-treatment-time (P < .001), lower gross tumor (66-68 vs 70 Gy) and elective neck (50 vs 56 Gy) doses. Median follow-up was 4.8 years. DAHANCA had higher 5-year LRF (13% vs 7%, aHR = 0.47 [0.34-0.67]), comparable DM (7% vs 12%, aHR = 1.32 [0.95-1.82]), but better OS (85% vs 80%, aHR = 1.30 [1.01-1.68]). CRT patients had a lower risk of LRF (aHR 0.56 [0.39-0.82]), DM (aHR 0.70 [0.50-1.00]) and death (aHR 0.39 [0.29-0.52]) vs RT-alone. We observed exemplary outcomes for two large-scale trans-Atlantic HPV+ OPC cohorts treated in a similar manner. Concurrent chemotherapy was a strong, independent prognostic factor for all endpoints. Our findings underscore the need for a very careful approach to de-intensification of treatment for this disease.

Distant metastases in squamous cell carcinoma of the pharynx and larynx: a population-based DAHANCA study.

BACKGROUND: In head and neck cancer, distant metastases may be present at diagnosis (M1) or occur after treatment (DM). It is unknown whether M1 and DM follow the same clinical development and share prognosis, as population-based studies regarding outcomes are scarce. Therefore, we investigated the incidence, location of metastases and overall survival of patients with M1 and DM. MATERIALS AND METHODS: Patients diagnosed with squamous cell carcinoma of the pharynx and larynx in Denmark 2008-2017 were identified in the Danish Head and Neck Cancer Group (DAHANCA) database. We identified 7300 patients, of whom 197 (3%) had M1 and 498 (8%) developed DM during follow-up. RESULTS: The 5-year cumulative incidence of DM was 8%. 1- and 2-year overall survival for DM (27% and 13%) vs. M1 (28% and 9%) were equally poor. There was no significant difference in location of metastases for M1 and DM and the most frequently involved organs were lungs, bone, lymph nodes and liver, in descending order. In oropharyngeal squamous cell carcinomas, the location of metastases did not differ by p16-status. For p16-positive patients, 21% of DM occurred later than three years of follow-up compared to 7% of p16-negative patients. CONCLUSION: Incidence, location of metastases and prognosis of primary metastatic (M1) or post-treatment metastatic (DM) disease in pharyngeal and laryngeal squamous cell carcinoma are similar in this register-based study.

CSF proteome in multiple sclerosis subtypes related to brain lesion transcriptomes.

To identify markers in the CSF of multiple sclerosis (MS) subtypes, we used a two-step proteomic approach: (i) Discovery proteomics compared 169 pooled CSF from MS subtypes and inflammatory/degenerative CNS diseases (NMO spectrum and Alzheimer disease) and healthy controls. (ii) Next, 299 proteins selected by comprehensive statistics were quantified in 170 individual CSF samples. (iii) Genes of the identified proteins were also screened among transcripts in 73 MS brain lesions compared to 25 control brains. F-test based feature selection resulted in 8 proteins differentiating the MS subtypes, and secondary progressive (SP)MS was the most different also from controls. Genes of 7 out these 8 proteins were present in MS brain lesions: GOLM was significantly differentially expressed in active, chronic active, inactive and remyelinating lesions, FRZB in active and chronic active lesions, and SELENBP1 in inactive lesions. Volcano maps of normalized proteins in the different disease groups also indicated the highest amount of altered proteins in SPMS. Apolipoprotein C-I, apolipoprotein A-II, augurin, receptor-type tyrosine-protein phosphatase gamma, and trypsin-1 were upregulated in the CSF of MS subtypes compared to controls. This CSF profile and associated brain lesion spectrum highlight non-inflammatory mechanisms in differentiating CNS diseases and MS subtypes and the uniqueness of SPMS.

Prevalence of medication-related falls in 200 consecutive elderly patients with hip fractures: a cross-sectional study.

BACKGROUND: Hip fractures constitute a major health problem in elderly people and are often fall-related. Several factors can contribute to a fall episode leading to hip fracture, including fall-risk-increasing drugs (FRIDs), which are often used by elderly people. We aimed to investigate the prevalence of medication-related falls and to assess the role of FRIDs and potentially inappropriate medications (PIMs) in a population of elderly patients hospitalized for a hip fracture. METHODS: We reviewed the patient records of 200 consecutive patients, aged ≥65 years, who were admitted for a hip fracture and evaluated whether medications were likely to have contributed to the fall episode. PIMs were identified using the Screening Tool of Older Persons' Prescriptions version 2 (STOPP) and by evaluating indications, contra-indications and interactions of the prescribed medications for each patient. RESULTS: FRIDs were used by 175 patients (87.5%). Medications were considered a likely contributor to the fall in 82 patients (41%). These were most often psychotropic medications alone or in combination with antihypertensives and/or diuretics. The 82 patients with suspected medication-related falls used more medications, FRIDs and PIMs than the rest of the patients, and in 74 (90%) of the 82 patients, at least one medication considered to be a contributor to the fall was also a PIM. CONCLUSIONS: The prevalence of suspected medication-related falls was 41%. It seems likely that a medication review could have reduced, though not eliminated, the risk of falling in this group of patients.

Characterization and radiosensitivity of HPV-related oropharyngeal squamous cell carcinoma patient-derived xenografts.

Background: Oropharyngeal squamous cell carcinomas (OPSCC) are rising rapidly in incidence due to Human Papillomavirus (HPV) and/or tobacco smoking. Prognosis is better for patients with HPV-positive disease, but may also be influenced by tobacco smoking and other factors. There is a need to individualize treatment to minimize morbidity and improve prognosis. Patient-derived xenografts (PDX) is an emerging pre-clinical research model that may more accurately reflect the human disease, and is an attractive platform to study disease biology and develop treatments and biomarkers. In this study we describe the establishment of PDX models, compare PDX tumors to the human original, and assess the suitability of this model for radiotherapy research and biomarker development. Material and methods: Tumor biopsies from 34 patients with previously untreated OPSCC were implanted in immunodeficient mice, giving rise to 12 squamous cell carcinoma PDX models (7 HPV+, 5 HPV-). Primary and PDX tumors were characterized extensively, examining histology, immunohistochemistry, cancer gene sequencing and gene expression analysis. Radiosensitivity was assessed in vivo in a growth delay assay. Results: Established PDX models maintained histological and immunohistochemical characteristics as well as HPV-status of the primary tumor. Important cancer driver gene mutations, e.g., in TP53, PIK3CA and others, were preserved. Gene expression related to cancer stem cell markers and gene expression subtype were preserved, while gene expression related to hypoxia and immune response differed. Radiosensitivity studies showed high concordance with clinical observations. Conclusion: PDX from OPSCC preserves important molecular characteristics of the human primary tumor. Radiosensitivity were in accordance with clinically observed treatment response. The PDX model is a clinically relevant surrogate model of head and neck cancer. Perspectives include increased understanding of disease biology, which could lead to development of novel treatments and biomarkers.

DAHANCA 10 - Effect of darbepoetin alfa and radiotherapy in the treatment of squamous cell carcinoma of the head and neck. A multicenter, open-label, randomized, phase 3 trial by the Danish head and neck cancer group.

PURPOSE: To evaluate if correction of low hemoglobin (Hb) levels by means of darbepoetin alfa improves the outcomes of radiotherapy in patients with squamous cell carcinoma of the head and neck (HNSCC). PATIENTS AND METHODS: Patients eligible for primary radiotherapy and who had Hb values below 14.0 g/dl were randomized to receive accelerated fractionated radiotherapy with or without darbepoetin alfa. Patients also received the hypoxic radiosensitizer nimorazole. Darbepoetin alfa was given weekly during radiotherapy or until the Hb value exceeded 15.5 g/dl. RESULTS: Following a planned interim analysis which showed inferiority of the experimental treatment the trial was stopped after inclusion of 522 patients (of a planned intake of 600). Of these, 513 were eligible for analysis (254 patients treated with darbepoetin alfa and 259 patients in the control group). Overall, the patients were distributed according to the stratification parameters (gender, T and N staging, tumor site). Treatment with darbepoetin alfa increased the Hb level to the planned value in 81% of the patients. The compliance was good without excess serious adverse events. The results showed a poorer outcome with a 5-year cumulative loco-regional failure rate of 47% vs. 34%, Hazard Ratio (HR): 1.53 [1.16-2.02], for the darbepoetin alfa vs. control arm, respectively. This was also seen for the endpoints of event-free survival (HR: 1.36 [1.09-1.69]), disease-specific death (HR: 1.43 [1.08-1.90]), and overall survival (HR: 1.30 [1.02-1.64]). There was no enhanced risk of cardio-vascular events observed in the experimental arm or any significant differences in acute or late radiation related morbidity. All univariate analyses were confirmed in a multivariate setting. CONCLUSION: Correction of the Hb level with darbepoetin alfa during radiotherapy of patients with HNSCC resulted in a significantly poorer tumor control and survival.

Prognostic impact of HPV-associated p16-expression and smoking status on outcomes following radiotherapy for oropharyngeal cancer: The MARCH-HPV project.

BACKGROUND AND PURPOSE: Evaluate the prognostic and predictive impact of HPV-associated p16-expression and assess the combined prognostic impact of p16 and smoking on altered fractionated radiotherapy (AFRT) for oropharyngeal cancer (OPC) within the frames of the update of the Meta-Analysis of Radiotherapy in Carcinomas of Head and neck (MARCH). MATERIALS AND METHODS: Patients with OPC, known tumor p16-status and smoking history were identified from the MARCH update, resulting in a dataset of 815 patients from four randomized trials (RTOG9003, DAHANCA6&7, RTOG0129, ARTSCAN). Analysis was performed using a Cox model stratified by trial and adjusted on gender, age, T-stage, N-stage, type of radiotherapy fractionation, p16, smoking. Primary endpoint was progression-free survival (PFS). RESULTS: In total, 465 patients (57%) had p16-positive tumors and 350 (43%) p16-negative. Compared to p16-negative, p16-positive patients had significantly better PFS (HR = 0.42 [95% CI: 0.34-0.51], 28.9% absolute increase at 10 years) and OS (HR = 0.40 [0.32-0.49], 32.1% absolute increase at 10 years). No interaction between p16-status and fractionation schedule was detected. Smoking negatively impacted outcome; in the p16-positive subgroup, never smokers had significantly better PFS than former/current smokers (HR = 0.49 [0.33-0.75], 24.2% survival benefit at 10 years). CONCLUSIONS: No predictive impact of p16-status on response to AFRT could be detected but the strong prognostic impact of p16-status was confirmed and especially p16-positive never smoking patients have superior outcome after RT.

TNFα affects CREB-mediated neuroprotective signaling pathways of synaptic plasticity in neurons as revealed by proteomics and phospho-proteomics.

Neuroinflammation is a hallmark of Alzheimer's disease and TNFα as the main inducer of neuroinflammation has neurodegenerative but also pro-regenerative properties, however, the dose-dependent molecular changes on signaling pathway level are not fully understood. We performed quantitative proteomics and phospho-proteomics to target this point. In HT22 cells, we found that TNFα reduced mitochondrial signaling and inhibited mTOR protein translation signaling but also led to induction of neuroprotective MAPK-CREB signaling. Stimulation of human neurons with TNFα revealed similar cellular mechanisms. Moreover, a number of synaptic plasticity-associated genes were altered in their expression profile including CREB. SiRNA-mediated knockdown of CREB in human neurons prior to TNFα stimulation led to a reduced number of protein/phospho-protein hits compared to siRNA-mediated knockdown of CREB or TNFα stimulation alone and countermeasured the reduced CREB signaling. In vivo data of TNFα knockout mice showed that learning ability did not depend on TNFα per se but that TNFα was essential for preserving the learning ability after episodes of lipopolysaccharide-induced neuroinflammation. This may be based on modulation of CREB/CREB signaling as revealed by the in vitro / in vivo data. Our data show that several molecular targets and signaling pathways induced by TNFα in neurons resemble those seen in Alzheimer's disease pathology.

3-dimensional versus conventional laparoscopy for benign hysterectomy: protocol for a randomized clinical trial.

BACKGROUND: Hysterectomy is one of the most common surgical procedures for women of reproductive age. Laparoscopy was introduced in the 1990es and is today one of the recommended routes of surgery. A recent observational study showed that operative time for hysterectomy was significantly lower for 3-dimensional compared to conventional laparoscopy. Complication rates were similar for the two groups. No other observational studies or randomized clinical trials have compared 3-dimensional to conventional laparoscopy in patients undergoing total hysterectomy for benign disease. The objective of the study is to determine if 3D laparoscopy gives better quality of life, less postoperative pain, less per- and postoperative complications, shorter operative time, or a shorter stay in hospital and a faster return to work or normal life, compared to conventional laparoscopy for benign hysterectomy. METHODS/DESIGN: The design is a randomised multicentre clinical trial. Participants will be 400 women referred for laparoscopic hysterectomy for benign indications. Patients will be randomized to 3-dimensional or conventional laparoscopic hysterectomy. Operative procedures will follow the same principles and the same standard whether the surgeon's vision is 3-dimensional or conventional laparoscopy. Primary outcomes will be the impact of surgery on quality of life, assessed by the SF 36 questionnaire, and postoperative pain, assessed by a Visual Analogue scale for pain measurement. With a standard deviation of 12 points on SF 36 questionnaire, a risk of type I error of 3.3% and a risk of type II error of 10% a sample size of 190 patients in each arm of the trial is needed. Secondarily, we will investigate operative time, time to return to work, length of hospital stay, and - and postoperative complications. DISCUSSION: This trial will be the first randomized clinical trial investigating the potential clinical benefits and harms of 3-dimensional compared to conventional laparoscopy. The results may provide more evidence regarding the future place of 3-dimensional laparoscopy in the range of endoscopic approaches for benign hysterectomy. TRIAL REGISTRATION: This study is registered at ClinicalTrial.gov: NCT02610985 November 16th 2015. November 2015. The regional Ethical committee approved it on the 12. November 2015, approval number: SJ-498. Data handling was approved by the Danish Data Protection Agency: REG-109-2015 on the 13. November 2015.

Understanding Alzheimer's disease by global quantification of protein phosphorylation and sialylated N-linked glycosylation profiles: A chance for new biomarkers in neuroproteomics?

Phosphorylation and glycosylation are important protein modifications in the mammalian brain acting as drivers of neural development, neurotransmission signalling and neurite elongation as well as synaptic morphology. Despite their important functional roles in the brain, only a few studies have elucidated them in neurodegenerative diseases such as Alzheimer's disease. Here, we comprehensively review Alzheimer's pathology in relation to protein phosphorylation and glycosylation on synaptic plasticity from neuroproteomics data. Moreover, we highlight several mass spectrometry-based sample processing technologies including an in-house developed TiO2-SIMAC-TiO2-based enrichment protocol to isolate and enrich phosphorylated and glycosylated peptides enabling to elucidate hopefully new early disease biomarkers.

Human Papilloma Virus as a Biomarker for Personalized Head and Neck Cancer Radiotherapy.

A dramatic increase in the incidence of HPV-related oropharyngeal cancer has been reported in some parts of the western world over the past 30 years. They constitute a clinically distinct subgroup of cancers in terms of molecular biology, patient characteristics, and treatment outcome. This chapter describes the molecular characteristics, epidemiology, and demographics of the HPV-related head and neck cancers and discuss available methods to detect HPV-related tumours. The impact of HPV-related biomarkers in clinical studies on radiotherapy only, altered fractionation, modulation of hypoxia, and concurrent chemo- or bio-radiotherapy are reviewed as well as the perspectives of de-escalation and immune-modulation are discussed.

Simultaneous Enrichment of Cysteine-containing Peptides and Phosphopeptides Using a Cysteine-specific Phosphonate Adaptable Tag (CysPAT) in Combination with titanium dioxide (TiO2) Chromatography.

Cysteine is a rare and conserved amino acid involved in most cellular functions. The thiol group of cysteine can be subjected to diverse oxidative modifications that regulate many physio-pathological states. In the present work, a Cysteine-specific Phosphonate Adaptable Tag (CysPAT) was synthesized to selectively label cysteine-containing peptides (Cys peptides) followed by their enrichment with titanium dioxide (TiO2) and subsequent mass spectrometric analysis. The CysPAT strategy was developed using a synthetic peptide, a standard protein and subsequently the strategy was applied to protein lysates from Hela cells, achieving high specificity and enrichment efficiency. In particular, for Cys proteome analysis, the method led to the identification of 7509 unique Cys peptides from 500 µg of HeLa cell lysate starting material. Furthermore, the method was developed to simultaneously enrich Cys peptides and phosphorylated peptides. This strategy was applied to SILAC labeled Hela cells subjected to 5 min epidermal growth factor (EGF) stimulation. In total, 10440 unique reversibly modified Cys peptides (3855 proteins) and 7339 unique phosphopeptides (2234 proteins) were simultaneously identified from 250 µg starting material. Significant regulation was observed in both phosphorylation and reversible Cys modification of proteins involved in EGFR signaling. Our data indicates that EGF stimulation can activate the well-known phosphorylation of EGFR and downstream signaling molecules, such as mitogen-activated protein kinases (MAPK1 and MAPK3), however, it also leads to substantial modulation of reversible cysteine modifications in numerous proteins. Several protein tyrosine phosphatases (PTPs) showed a reduction of the catalytic Cys site in the conserved putative phosphatase HC(X)5R motif indicating an activation and subsequent de-phosphorylation of proteins involved in the EGF signaling pathway. Overall, the CysPAT strategy is a straight forward, easy and promising method for studying redox proteomics and the simultaneous enrichment strategy offers an excellent solution for characterization of cross-talk between phosphorylation and redox induced reversible cysteine modifications.