Considerations for study design in the DAHANCA 35 trial of protons versus photons for head and neck cancer.

Proton radiotherapy offers a dosimetric advantage compared to photon therapy in sparing normal tissue, but the clinical evidence for toxicity reductions in the treatment of head and neck cancer is limited. The Danish Head and Neck Cancer Group (DAHANCA) has initiated the DAHANCA 35 randomised trial to clarify the value of proton therapy (NCT04607694). The DAHANCA 35 trial is performed in an enriched population of patients selected by an anticipated benefit of proton therapy to reduce the risk of late dysphagia or xerostomia based on normal tissue complication probability (NTCP) modelling. We present our considerations on the trial design and a test of the selection procedure conducted before initiating the randomised study.

Biological modeling in thermoradiotherapy: present status and ongoing developments toward routine clinical use.

Biological modeling for anti-cancer treatments using mathematical models can be very supportive in gaining more insight into dynamic processes responsible for cellular response to treatment, and predicting, evaluating and optimizing therapeutic effects of treatment. This review presents an overview of the current status of biological modeling for hyperthermia in combination with radiotherapy (thermoradiotherapy). Various distinct models have been proposed in the literature, with varying complexity; initially aiming to model the effect of hyperthermia alone, and later on to predict the effect of the combined thermoradiotherapy treatment. Most commonly used models are based on an extension of the linear-quadratic (LQ)-model enabling an easy translation to radiotherapy where the LQ model is widely used. Basic predictions of cell survival have further progressed toward 3 D equivalent dose predictions, i.e., the radiation dose that would be needed without hyperthermia to achieve the same biological effect as the combined thermoradiotherapy treatment. This approach, with the use of temperature-dependent model parameters, allows theoretical evaluation of the effectiveness of different treatment strategies in individual patients, as well as in patient cohorts. This review discusses the significant progress that has been made in biological modeling for hyperthermia combined with radiotherapy. In the future, when adequate temperature-dependent LQ-parameters will be available for a large number of tumor sites and normal tissues, biological modeling can be expected to be of great clinical importance to further optimize combined treatments, optimize clinical protocols and guide further clinical studies.

Independent external validation using the EORTC HNCG-ROG 1219 DAHANCA trial data of NTCP models for acute oral mucositis.

PURPOSE: To externally validate previously published Normal Tissue Complication Probability (NTCP) models developed by separate teams for grade 3 oral mucositis (g3OM). MATERIALS AND METHODS: Two models were validated: a logistic model, based on 144 head and neck cancer (HNC) patients receiving induction chemotherapy followed by chemo-IMRT; a multivariable logistic model for prediction of g3OM for 253 patients receiving radical treatment for the head and neck squamous cell carcinoma (HNSCC). The EORTC HNCG-ROG 1219 DAHANCA trial dataset, consisting of 169 patients was used as the validation cohort. This cohort was treated with accelerated fractionated chemo-IMRT, with/without the hypoxic radiosensitizer Nimorazole for HNSCC. External validity was assessed using the scaled Brier score. Calibration was assessed in terms of calibration curves as well as measures of mean and weak calibration. Hosmer-Lemeshow was used for goodness-of-fit test. Discrimination was calculated using the area under the receiver operating curve (AUC-ROC). RESULTS: The prevalence of g3OM in the validation cohort (35.5%) was similar to that of two development cohorts, i.e. 38.7% and 31.9% for Bhide logistic and Otter multivariable logistic models respectively. The scaled Brier scores showed good overall model performance. Perfect calibration was observed in the prevalence range of 20% to 40%. AUC-ROC was acceptable in external validation (0.67). The Hosmer-Lemeshow test showed good agreement between predicted and observed outcomes for two models. CONCLUSION: The NTCP models were validated and lead to valid predictions in a wide range of diverse treatment techniques and patient characteristics, also when Nimorazole is added as hypoxic radiosensitizer.

Early Mortality after Radical Radiotherapy in Head and Neck Cancer - A Nationwide Analysis from the Danish Head and Neck Cancer Group (DAHANCA) Database.

AIMS: Curative-intent radiotherapy (RT) or chemoradiation (CRT) of squamous cell carcinoma of the head and neck (HNSCC) produces high survival rates, but is associated with substantial toxicity. However, there are no commonly accepted quality metrics for early mortality in radiation oncology. To assess the applicability of early mortality as a clinical quality indicator, this study investigated the temporal distribution, risk factors and trends of 90- and 180-day overall and non-cancer mortality in a nationwide cohort of HNSCC patients treated with RT/CRT. MATERIALS AND METHODS: Information on all HNSCC patients treated with curative-intent RT/CRT in Denmark between 2000 and 2017 was obtained from the national Danish Head and Neck Cancer Group clinical database. Deaths in patients with residual or recurrent disease after RT/CRT were classified as cancer-related. Possible risk factors were investigated using logistic regression analysis. RESULTS: Data from 11 419 patients were extracted. In total, 90- and 180-day mortality risks were 3.1% and 7.1%, respectively. There was a uniform temporal distribution of 180-day mortality. In multivariable analysis, increasing age, stage, performance status, earlier treatment year and hypopharyngeal cancer were significantly associated with an increased risk (P < 0.05). Risk factor estimates were comparable for 90- versus 180-day mortality as well as for overall versus non-cancer mortality. Between 2000 and 2017 there was a significant decrease in 180-day mortality, which was driven by a reduction in cancer-related events. CONCLUSION: The distribution of 180-day overall and non-cancer mortality did not indicate a well-defined early high-risk period. Moreover, risk factor estimates were highly similar across risk periods and groups. Taken together, our findings question the applicability of early mortality as a standard metric for treatment-associated toxicity.

Dietary nutrient balance shapes phenotypic traits of Drosophila melanogaster in interaction with gut microbiota.

The dietary nutrient composition can affect insects' phenotypes by modulating their physiology. Furthermore, diet can affect gut microbiota composition and abundance, with indirect consequences for the host. In this study, we reared Drosophila melanogaster on five different diets; three with balanced sugar:yeast ratio, but with increasing caloric content (2:2, 8:8, 16:16, in weight %), and two with imbalanced sugar:yeast ratio, either with low sugar and high yeast content (2:16) or vice-versa (16:2). In each of these diets, we compared flies with conventional vs. artificially altered gut microbiota with antibiotics that reduced the bacterial load. The antibiotic treatment also had the surprising effect of increasing the amount of live yeast associated with the flies. We characterized flies from these ten treatments (5 diets × 2 microbiota) in terms of development, body mass, food preference, body reserves, metabolic rate and a range of stress tolerance traits (heat, cold, starvation and desiccation tolerance). Diets, and to a lesser extent antibiotic treatment, affected development rate, weight, and cold tolerance of adult flies. Other traits such as energy reserves, metabolic rate, food preference, or starvation tolerance were affected by diet alone. When detected, the effect of antibiotic treatment was stronger in yeast-poor diets, suggesting that gut bacterial community might help to counterbalance nutritional deficiencies. These results show that changes in dietary factors lead to a global re-organization of fly's physiology and development while the manipulation of gut microorganisms had minor effects that were mainly seen in case of protein restriction.

NTCP model validation method for DAHANCA patient selection of protons versus photons in head and neck cancer radiotherapy.

Introduction: Prediction models using logistic regression may perform poorly in external patient cohorts. However, there is a need to standardize and validate models for clinical use. The purpose of this project was to describe a method for validation of external NTCP models used for patient selection in the randomized trial of protons versus photons in head and neck cancer radiotherapy, DAHANCA 35. Material and methods: Organs at risk of 588 patients treated primarily with IMRT in the randomized controlled DAHANCA19 trial were retrospectively contoured according to recent international recommendations. Dose metrics were extracted using MatLab and all clinical parameters were retrieved from the DAHANCA database. The model proposed by Christianen et al. to predict physician-rated dysphagia was validated through the closed testing, where change of the model intercept, slope and individual beta's were tested for significant prediction improvements. Results: Six months prevalence of dysphagia in the validation cohort was 33%. The closed testing procedure for physician-rated dysphagia showed that the Christianen et al. model needed an intercept refitting for the best match for the Danish patients. The intercept update increased the risk of dysphagia for the validation cohort by 7.9 ± 2.5% point. For the raw model performance, the Brier score (mean squared residual) was 0.467, which improved significantly with a new intercept to 0.415. Conclusions: The previously published Dutch dysphagia model needed an intercept update to match the Danish patient cohort. The implementation of a closed testing procedure on the current validation cohort allows quick and efficient validation of external NTCP models for patient selection in the future.

IMRT - Biomarkers for dose escalation, dose de-escalation and personalized medicine in radiotherapy for head and neck cancer.

Radiotherapy (RT) is an integral component in the management of head and neck cancer. Despite progress in several respects, a noteworthy proportion of the treated patients do not achieve complete response after RT. Regardless of novel dose delivery technologies, RT for head and neck cancer is still associated with acute as well as late toxicity. These challenges could potentially be addressed by means of personalized treatment. In this paper, we discuss the possibilities for dose escalation, dose de-escalation and allocation to systemic concomitant treatment based on prognostic and predictive markers for tumor control as well as predictive markers for normal tissue radiosensitivity.

Laboratory maintenance does not alter ecological and physiological patterns among species: a Drosophila case study.

Large comparative studies in animal ecology, physiology and evolution often use animals reared in the laboratory for many generations; however, the relevance of these studies hinges on the assumption that laboratory populations are still representative for their wild living conspecifics. In this study, we investigate whether laboratory-maintained and freshly collected animal populations are fundamentally different and whether data from laboratory-maintained animals are valid to use in large comparative investigations of ecological and physiological patterns. Here, we obtained nine species of Drosophila with paired populations of laboratory-maintained and freshly collected flies. These species, representing a range of ecotypes, were assayed for four stress-tolerance, two body-size traits and six life-history traits. For all of these traits, we observed small differences in species-specific comparisons between field and laboratory populations; however, these differences were unsystematic and laboratory maintenance did not eclipse fundamental species characteristics. To investigate whether laboratory maintenance influence the general patterns in comparative studies, we correlated stress tolerance and life-history traits with environmental traits for the laboratory-maintained and freshly collected populations. Based on this analysis, we found that the comparative physiological and ecological trait correlations are similar irrespective of provenience. This finding is important for comparative biology in general because it validates comparative meta-analyses based on laboratory-maintained populations.

Failure pattern and survival after breast conserving therapy. Long-term results of the Danish Breast Cancer Group (DBCG) 89 TM cohort.

UNLABELLED: Based on the results from the DBCG 82 trial, breast conserving therapy (BCT) has been implemented as standard in Denmark since 1989, and today constitutes more than 70% of the primary treatment. Our aim was to evaluate the implementation of BCT as a routine procedure in patients treated according to the DBCG 89 program and compare recurrence pattern and survival both overall and when separated in age groups, with the results from the randomized DBCG 82 TM trial. MATERIAL AND METHODS: A total of 1847 patients treated between 1989 and 1999 were included in a retrospective population-based cohort study. Data from the DBCG database were completed via search through the Danish Pathology Data Bank and medical records. RESULTS: Median follow-up time was 17 years. At 20 years the cumulative incidences of local recurrence (LR) and disease-specific mortality (DSM) were 15.3% and 25.8%, respectively. Twenty-year overall survival (OS) and recurrence-free survival were 63.7% and 43.1%, respectively. Subdivided by age groups cumulative incidences at 20 years were LR: 18.9%, 10.5% and 12.4%, and DSM: 28.9%, 18.9% and 28.4% in young (≤45 years), middle-aged (46-55 years) and older (≥56 years) women, respectively. In an adjusted analysis age maintained a significant and independent effect on both LR and DSM. CONCLUSION: The DBCG 82 TM program was successfully implemented. The women treated with BCT in the DBCG 89 program displayed equal failure pattern and improved survival in comparison with women from the DBCG 82 TM protocol. Occurrence of first failure and mortality varied with age; demonstrated by increased risk of LR, DM and DSM in the young patients and increased risk of DM and DSM in the older patients, compared to the middle-aged patients.

High-pressure single crystal X-ray diffraction study of the linear metal chain compound Co3(dpa)4Br2·CH2Cl2.

The crystal structure of the linear metal chain compound Co3(dpa)4Br2·CH2Cl2 (1) has been investigated up to a pressure of 13.6(2) GPa in a diamond anvil cell (DAC) using single crystal X-ray diffraction. The structure remains orthorhombic as the unit cell volume is reduced by 30% at 12.8 GPa. At 13.6(2) GPa the diffraction pattern is of very poor quality and not even reliable unit cell parameters can be determined. Peak broadening resulting from non-hydrostatic conditions was avoided by annealing the loaded DAC prior to data collection, allowing reliable structural models to be refined up to a pressure of 11.8(2) GPa. On increasing pressure, the disordered CH2Cl2 crystal solvent molecule gradually becomes redistributed from one site to another. Hirshfeld surface analysis suggests that the redistribution is a result of repulsive HH interactions. Pressure also affects the molecular geometry, in particular the Co-Co and Co-Br bond lengths which decrease by 4% and 12%, respectively, at 11.8(2) GPa.

Study of the population pharmacokinetic characteristics of nimorazole in head and neck cancer patients treated in the DAHANCA-5 trial.

AIMS: To study the pharmacokinetic characteristics of the hypoxic radiosensitiser nimorazole in patients with head and neck squamous cell carcinoma. MATERIALS AND METHODS: The pharmacokinetics of the hypoxic radiosensitiser nimorazole were studied in 63 patients treated in the DAHANCA-5 trial. After the first day of treatment, serial venous blood samples were taken and plasma concentrations of nimorazole measured by high pressure liquid chromatography (HPLC). Plasma concentration profiles were subjected to non-compartmental pharmacokinetic analysis using validated PC-based software. The different pharmacokinetic parameters were calculated and correlated with the different patient- and treatment-related variables. RESULTS: HPLC measurements showed a linear relationship between peak plasma concentration and administered dose. The mean peak concentration adjusted for dose (in g/m(2)) was 32.2 ± 0.9 µg/ml. The time of peak concentration ranged between 30 and 180 min (median 60 min). Plasma elimination occurred with a mean half-life of 3.35 ± 0.09 h and was not significantly altered as a function of dose. There was a well-established linear-linear relationship between area under the concentration-time curve (AUC; mean 191 ± 6 µg·h/ml) and administered dose, especially when expressed as g/m(2). The mean apparent volume of distribution was 0.77 ± 0.02 l/kg. A statistically significant longer elimination half-life in men relative to women (mean difference 0.40 h; 95% confidence interval 0.77-0.03; P 0.03) was detected. Nimorazole was well tolerated; with 67% of patients reporting no toxicity; nausea/vomiting was the most reported toxicity in the remaining patients. CONCLUSION: The study supports the current nimorazole dose scheduling in patients.

No patterns in thermal plasticity along a latitudinal gradient in Drosophila simulans from eastern Australia.

Phenotypic plasticity may be an important initial mechanism to counter environmental change, yet we know relatively little about the evolution of plasticity in nature. Species with widespread distributions are expected to have evolved higher levels of plasticity compared with those with more restricted, tropical distributions. At the intraspecific level, temperate populations are expected to have evolved higher levels of plasticity than their tropical counterparts. However, empirical support for these expectations is limited. In addition, no studies have comprehensively examined the evolution of thermal plasticity across life stages. Using populations of Drosophila simulans collected from a latitudinal cline spanning the entire east coast of Australia, we assessed thermal plasticity, measured as hardening capacity (the difference between basal and hardened thermal tolerance) for multiple measures of heat and cold tolerance across both adult and larval stages of development. This allowed us to explicitly ask whether the evolution of thermal plasticity is favoured in more variable, temperate environments. We found no relationship between thermal plasticity and latitude, providing little support for the hypothesis that temperate populations have evolved higher levels of thermal plasticity than their tropical counterparts. With the exception of adult heat survival, we also found no association between plasticity and ten climatic variables, indicating that the evolution of thermal plasticity is not easily predicted by the type of environment that a particular population occupies. We discuss these results in the context of the role of plasticity in a warming climate.

Identifying microRNAs regulating B7-H3 in breast cancer: the clinical impact of microRNA-29c.

BACKGROUND: B7-H3, an immunoregulatory protein, is overexpressed in several cancers and is often associated with metastasis and poor prognosis. Here, our aim was to identify microRNAs (miRNAs) regulating B7-H3 and assess their potential prognostic implications in breast cancer. METHODS: MicroRNAs targeting B7-H3 were identified by transfecting two breast cancer cell lines with a library of 810 miRNA mimics and quantifying changes of B7-H3 protein levels using protein lysate microarrays. For validations we used western immunoblotting and 3'-UTR luciferase assays. Clinical significance of the miRNAs was assayed by analysing whether their expression levels correlated with outcome in two cohorts of breast cancer patients (142 and 81 patients). RESULTS: We identified nearly 50 miRNAs that downregulated B7-H3 protein levels. Western immunoblotting validated the impact of the 20 most effective miRNAs. Thirteen miRNAs (miR-214, miR-363*, miR-326, miR-940, miR-29c, miR-665, miR-34b*, miR-708, miR-601, miR-124a, miR-380-5p, miR-885-3p, and miR-593) targeted B7-H3 directly by binding to its 3'-UTR region. Finally, high expression of miR-29c was associated with a significant reduced risk of dying from breast cancer in both cohorts. CONCLUSIONS: We identified miRNAs efficiently downregulating B7-H3 expression. The expression of miR-29c correlated with survival in breast cancer patients, suggesting a tumour suppressive role for this miRNA.

Waiting times for diagnosis and treatment of head and neck cancer in Denmark in 2010 compared to 1992 and 2002.

BACKGROUND AND AIM: Significant tumour progression was observed during waiting time for treatment of head and neck cancer. To reduce waiting times, a Danish national policy of fast track accelerated clinical pathways was introduced in 2007. This study describes changes in waiting time and the potential influence of fast track by comparing waiting times in 2010 to 2002 and 1992. METHODS: Charts of all new patients diagnosed with squamous cell carcinoma of the oral cavity, pharynx and larynx at the five Danish head and neck oncology centres from January to April 2010 (n=253) were reviewed and compared to similar data from 2002 (n=211) and 1992 (n=168). RESULTS: The median time to diagnosis was 13 days (2010) versus 17 days (2002; p<0.001) and 20 days (1992; p<0.001). Median days from diagnosis to treatment start were 25 (2010) versus 47 (2002; p<0.001) and 31 (1992; p<0.001). Total pre-treatment time was median 41 days in 2010 versus 69 days (2002) (p<0.001) and 50 days (1992; p<0.001). Significantly more diagnostic imaging was done in 2010 compared to 2002 and 1992. When compared to current fast track standards the adherence to diagnosis improved slightly from 47% (1992) to 51% (2002) and 64% (2010); waiting time for radiotherapy was within standards for 7%, 1% and 22% of cases, respectively; waiting time for surgery was within standards for 17%, 22% and 48%, respectively. CONCLUSION: The study showed a significant reduction in delay of diagnosis and treatment of head and neck cancer in 2010, but still less than half of all patients start treatment within the current standards.

PET hypoxia imaging with FAZA: reproducibility at baseline and during fractionated radiotherapy in tumour-bearing mice.

PURPOSE: Tumour hypoxia is linked to treatment resistance. Positron emission tomography (PET) using hypoxia tracers such as fluoroazomycin arabinoside (FAZA) may allow identification of patients with hypoxic tumours and the monitoring of the efficacy of hypoxia-targeting treatment. Since hypoxia PET is characterized by poor image contrast, and tumour hypoxia undergoes spontaneous changes and is affected by therapy, it remains unclear to what extent PET scans are reproducible. Tumour-bearing mice are valuable in the validation of hypoxia PET, but identification of a reliable reference tissue value (blood sample or image-derived muscle value) for repeated scans may be difficult due to the small size of the animal or absence of anatomical information (pure PET). Here tumour hypoxia was monitored over time using repeated PET scans in individual tumour-bearing mice before and during fractionated radiotherapy. METHODS: Mice bearing human SiHa cervix tumour xenografts underwent a PET scan 3 h following injection of FAZA on two consecutive days before initiation of treatment (baseline) and again following irradiation with four and ten fractions of 2.5 Gy. On the last scan day, mice were given an intraperitoneal injection of pimonidazole (hypoxia marker), tumours were collected and the intratumoral distribution of FAZA (autoradiography) and hypoxia (pimonidazole immunohistology) were determined in cryosections. RESULTS: Tissue section analysis revealed that the intratumoral distribution of FAZA was strongly correlated with the regional density of hypoxic (pimonidazole-positive) cells, even when necrosis was present, suggesting that FAZA PET provides a reliable measure of tumour hypoxia at the time of the scan. PET-based quantification of tumour tracer uptake relative to injected dose showed excellent reproducibility at baseline, whereas normalization using an image-derived nonhypoxic reference tissue (muscle) proved highly unreliable since a valid and reliable reference value could not be determined. The intratumoral distribution of tracer was stable at baseline as shown by a voxel-by-voxel comparison of the two scans (R = 0.82, range 0.72-0.90). During treatment, overall tracer retention changed in individual mice, but there was no evidence of general reoxygenation. CONCLUSION: Hypoxia PET scans are quantitatively correct and highly reproducible in tumour-bearing mice. Preclinical hypoxia PET is therefore a valuable and reliable tool for the development of strategies that target or modify hypoxia.

A comprehensive assessment of geographic variation in heat tolerance and hardening capacity in populations of Drosophila melanogaster from eastern Australia.

We examined latitudinal variation in adult and larval heat tolerance in Drosophila melanogaster from eastern Australia. Adults were assessed using static and ramping assays. Basal and hardened static heat knockdown time showed significant linear clines; heat tolerance increased towards the tropics, particularly for hardened flies, suggesting that tropical populations have a greater hardening response. A similar pattern was evident for ramping heat knockdown time at 0.06°C min(-1) increase. There was no cline for ramping heat knockdown temperature (CT(max) ) at 0.1°C min(-1) increase. Acute (static) heat knockdown temperature increased towards temperate latitudes, probably reflecting a greater capacity of temperate flies to withstand sudden temperature increases during summer in temperate Australia. Larval viability showed a quadratic association with latitude under heat stress. Thus, patterns of heat resistance depend on assay methods. Genetic correlations in thermotolerance across life stages and evolutionary potential for critical thermal limits should be the focus of future studies.

Multipole electron-density modelling of synchrotron powder diffraction data: the case of diamond.

Accurate structure factors are extracted from synchrotron powder diffraction data measured on crystalline diamond based on a novel multipole model division of overlapping reflection intensities. The approach limits the spherical-atom bias in structure factors extracted from overlapping powder data using conventional spherical-atom Rietveld refinement. The structure factors are subsequently used for multipole electron-density modelling, and both the structure factors and the derived density are compared with results from ab initio theoretical calculations. Overall, excellent agreement is obtained between experiment and theory, and the study therefore demonstrates that synchrotron powder diffraction can indeed provide accurate structure-factor values based on data measured in minutes with limited sample preparation. Thus, potential systematic errors such as extinction and twinning commonly encountered in single-crystal studies of small-unit-cell inorganic structures can be overcome with synchrotron powder diffraction. It is shown that the standard Hansen-Coppens multipole model is not flexible enough to fit the static theoretical structure factors, whereas fitting of thermally smeared structure factors has much lower residuals. If thermally smeared structure factors (experimental or theoretical) are fitted with a slightly wrong radial model (s(2)p(2) instead of sp(3)) the radial scaling parameters (kappa' parameters) are found to be inadequate and the ;error' is absorbed into the atomic displacement parameter. This directly exposes a correlation between electron density and thermal parameters even for a light atom such as carbon, and it also underlines that in organic systems proper deconvolution of thermal motion is important for obtaining correct static electron densities.