Aligning tumor mutational burden (TMB) quantification across diagnostic platforms: phase II of the Friends of Cancer Research TMB Harmonization Project.

BACKGROUND: Tumor mutational burden (TMB) measurements aid in identifying patients who are likely to benefit from immunotherapy; however, there is empirical variability across panel assays and factors contributing to this variability have not been comprehensively investigated. Identifying sources of variability can help facilitate comparability across different panel assays, which may aid in broader adoption of panel assays and development of clinical applications. MATERIALS AND METHODS: Twenty-nine tumor samples and 10 human-derived cell lines were processed and distributed to 16 laboratories; each used their own bioinformatics pipelines to calculate TMB and compare to whole exome results. Additionally, theoretical positive percent agreement (PPA) and negative percent agreement (NPA) of TMB were estimated. The impact of filtering pathogenic and germline variants on TMB estimates was assessed. Calibration curves specific to each panel assay were developed to facilitate translation of panel TMB values to whole exome sequencing (WES) TMB values. RESULTS: Panel sizes >667 Kb are necessary to maintain adequate PPA and NPA for calling TMB high versus TMB low across the range of cut-offs used in practice. Failure to filter out pathogenic variants when estimating panel TMB resulted in overestimating TMB relative to WES for all assays. Filtering out potential germline variants at >0% population minor allele frequency resulted in the strongest correlation to WES TMB. Application of a calibration approach derived from The Cancer Genome Atlas data, tailored to each panel assay, reduced the spread of panel TMB values around the WES TMB as reflected in lower root mean squared error (RMSE) for 26/29 (90%) of the clinical samples. CONCLUSIONS: Estimation of TMB varies across different panels, with panel size, gene content, and bioinformatics pipelines contributing to empirical variability. Statistical calibration can achieve more consistent results across panels and allows for comparison of TMB values across various panel assays. To promote reproducibility and comparability across assays, a software tool was developed and made publicly available.

Skill sets required for the management of military head, face and neck trauma: a multidisciplinary consensus statement.

INTRODUCTION: The evolution of medical practice is resulting in increasing subspecialisation, with head, face and neck (HFN) trauma in a civilian environment usually managed by a combination of surgical specialties working as a team. However, the full combination of HFN specialties commonly available in the NHS may not be available in future UK military-led operations, necessitating the identification of a group of skill sets that could be delivered by one or more deployed surgeons. METHOD: A systematic review was undertaken to identify those surgical procedures performed to treat acute military head, face, neck and eye trauma. A multidisciplinary consensus group was convened following this with military HFN trauma expertise to define those procedures commonly required to conduct deployed, in-theatre HFN surgical combat trauma management. RESULTS: Head, face, neck and eye damage control surgical procedures were identified as comprising surgical cricothyroidotomy, cervico-facial haemorrhage control and decompression of orbital haemorrhage through lateral canthotomy. Acute in-theatre surgical skills required within 24 hours consist of wound debridement, surgical tracheostomy, decompressive craniectomy, intracranial pressure monitor placement, temporary facial fracture stabilisation for airway management or haemorrhage control and primary globe repair. Delayed in-theatre procedures required within 5 days prior to predicted evacuation encompass facial fracture fixation, delayed lateral canthotomy, evisceration, enucleation and eyelid repair. CONCLUSIONS: The identification of those skill sets required for deployment is in keeping with the General Medical Council's current drive towards credentialing consultants, by which a consultant surgeon's capabilities in particular practice areas would be defined. Limited opportunities currently exist for trainees and consultants to gain experience in the management of traumatic head, face, neck and eye injuries seen in a kinetic combat environment. Predeployment training requires that the surgical techniques described in this paper are covered and should form the curriculum of future military-specific surgical fellowships. Relevant continued professional development will be necessary to maintain required clinical competency.

A urine-based methylation signature for risk stratification within low-risk prostate cancer.

BACKGROUND: Prostate cancer overdiagnosis and overtreatment represents a major problem. Many men with low-grade disease on biopsy are undergraded and they harbour high-grade disease at prostatectomy with no reliable way to identify these men. We used a novel urine-based 2-gene methylation test to identify prostate cancers with aggressive features. METHODS: Following a proof of concept study in 100 post-radical prostatectomy tissue samples, urine samples were tested from 665 men at multiple U.S. centers undergoing prostate needle biopsy for elevated prostate-specific antigen (2-10 ng ml(-1)). A prediction model was then developed from a combination of clinical factors and the urine-based markers. It was then prospectively tested for accurate prediction of adverse disease (surgical Gleason score ⩾7 and/or a pathological stage ⩾T3a) using urine from a separate cohort of 96 men before radical prostatectomy. RESULTS: Among pre-prostatectomy men with a biopsy Gleason score <7, 41% had adverse disease of which 100% were correctly identified by the test with a negative predictive value of 100% (95% confidence interval, 86-100%). CONCLUSIONS: This urine-based test accurately identifies men with clinical low-risk disease who do not have adverse pathology in their prostates and would be excellent candidates for active surveillance.

Acquisition and retention of military surgical competencies: a survey of surgeons' experiences in the UK Defence Medical Services.

INTRODUCTION: The acquisition and retention of militarily relevant surgical knowledge and skills are vital to enable expert management of combat casualties on operations. Opportunities for skill sustainment have reduced due to the cessation of combat operations in Iraq and Afghanistan and lack of military-relevant trauma in UK civilian practice. METHODS: A voluntary, anonymous online survey study was sent to all UK Defence Medical Services (DMS) surgical consultants and higher surgical trainees in Trauma and Orthopaedics, Plastic and Reconstructive, and General and Vascular surgical specialties (three largest surgical specialties in the DMS in terms of numbers). The online questionnaire tool included 20 questions using multiple choice and free text to assess respondents' subjective feelings of preparedness for deployment as surgeons for trauma patients. RESULTS: There were 71 of 108 (66%) responses. Sixty-four (90%) respondents were regular armed forces, and 46 (65%) worked in a Major Trauma Centre (MTC). Thirty-three (47%) had never deployed on operations in a surgical role. Nineteen (27%) felt they had sufficient exposure to penetrating trauma. When asked 'How well do you feel your training and clinical practice prepares you for a surgical deployment?' on a scale of 1-10, trainees scored significantly lower than consultants (6 (IQR 4-7) vs 8 (IQR 7-9), respectively; p<0.001). There was no significant difference in scores between regular and reservists, or between those working at an MTC versus non-MTC. Respondents suggested high-volume trauma training and overseas trauma centre fellowships, simulation, cadaveric and live-tissue training would help their preparedness. CONCLUSIONS: There was a feeling among a sample of UK DMS consultants and trainees that better preparedness is required for them to deploy confidently as a surgeon for combat casualties. The responses suggest that UK DMS surgical training requires urgent attention if current surgeons are to be ready for their role on deployed operations.

Forklift-related lower limb injuries: a retrospective case series study with patient-reported outcome measures (PROMs).

AIM: Forklift trucks can cause serious lower limb trauma with long-lasting sequelae to patients. The aim of this study was to analyse a case series of patients with forklift-related injuries over 7 years at a level 1 major trauma centre in the UK and present their patient-reported outcome measures (PROMs) with long-term follow-up. To the best of the authors' knowledge, this is the largest case series study in the UK describing forklift injuries. METHODS: Retrospective case note analysis of 19 patients over 7 years. Data including demographics, injury mechanism, pattern of injury, management, length of hospital stay, number of operations and complications were extracted from the notes. We used 'Enneking score' as a validated tool for PROMs. RESULTS: Seventeen men and two women with mean age of 47 years; 20% had bilateral injuries and 34% had multi-level fractures. The mean number of theatre sessions was 5.21, while the mean length of hospital stay was 30.10 days. There was one mortality. Twelve patients (63%) required reconstruction with free tissue transfer, with one flap failure. The mean long-term Enneking percentage score was 57.33%. The mean Enneking score for patients in this study is lower than our institute's score for Gustilo 3B, highlighting the gravity of these injuries. CONCLUSION: Forklifts can cause grave injuries with massive energy transfer. This study highlights the seriousness of those injuries, thus guiding patient counselling and optimising planning of management.

Planning for UK terror attacks: Analysis of blast and ballistic injuries.

Terrorist attacks have become more acute, less predictable and frequently involve use of explosives and gunfire to inflict mass casualty to civilians. Resource demand has been reported in Role 3 Medical Facilities but the continued resource required to manage blast and ballistic injuries has not been quantified. This study aimed to assess the resource required for blast and ballistic injuries at the United Kingdom's Role 4 Medical Facility. Military patients admitted to the Queen Elizabeth Hospital (Role 4 Medical Facility) from Afghanistan with blast or ballistic injuries during the 2012 calendar year were retrospectively reviewed. Injury pattern, theatre resource, length of stay and cost analysis were performed. This study included 99 blast and 53 gunshot wound (GSW) patients. Blast patients were more likely to suffer polytrauma than GSW (53% vs 23%), underwent more surgical procedures and utilized double the theatre time. Blast injury patients had a longer length of stay in hospital. The average cost per patient for blast patients was double that of the GSW injury cohort. The Queen Elizabeth experience represents a continuous flow of severely injured military casualties whilst managing concurrent civilian trauma over a long period. This workload has encouraged systematic advancements in managing high numbers of injured patients from point of wounding to rehabilitation. Distribution of resource, theatre planning and multi-disciplinary team working are critical in effectively managing Major Incidents such as terror attacks. Drawing on previous Role 4 Medical Facility experience can aid UK hospitals in terms of strategy and resource distribution.

From chaos to a new norm: The Birmingham experience of restructuring the largest plastics department in the UK in response to the COVID-19 pandemic.

The coronavirus disease 2019 (COVID-19) pandemic presented unprecedented challenges for healthcare systems worldwide. The Queen Elizabeth Hospital, Birmingham, has one of the largest burns, hands and plastics department in the UK, totalling 83 doctors. Our response to the COVID-19 response was uniquely far reaching, with our department being given responsibility of an entire 36 bed medical COVID-19 ward in addition to our commitment to specialty-specific work, and saw half of our work force re-deployed to Intensive Treatment Unit (ITU). Our aim was to exploit the high calibre of doctors found in plastic surgery, and to demonstrate, we were able to support the COVID-19 effort beyond our normal scope of practice. In order to achieve this aim, the department underwent significant structural and leadership changes. Factors considered included: rota and shift pattern changes to implement depth and resilience to sudden fluctuations in staffing levels; a preparatory phase for focussed upskilling and relevant training packages to be delivered; managing the COVID-19 ward cover and ITU deployment; adjustments to our front of house and elective specialty-specific service, including developing alternative and streamlined patient pathways; mitigating the effects on plastic surgical training during the pandemic; the importance of communications for patient care and physician wellbeing; and leadership techniques and styles we considered important. By sharing our experience during this pandemic, we hope to reflect on and share lessons learned, as well as to demonstrate that it is possible to rapidly mobilise and retrain plastic surgeons at all levels to contribute safely and productively beyond a specialty-specific scope of care.

Optimising ballistic facial coverage from military fragmenting munitions: a consensus statement.

VIRTUS is the first United Kingdom (UK) military personal armour system to provide components that are capable of protecting the whole face from low velocity ballistic projectiles. Protection is modular, using a helmet worn with ballistic eyewear, a visor, and a mandibular guard. When all four components are worn together the face is completely covered, but the heat, discomfort, and weight may not be optimal in all types of combat. We organized a Delphi consensus group analysis with 29 military consultant surgeons from the UK, United States, Canada, Australia, and New Zealand to identify a potential hierarchy of functional facial units in order of importance that require protection. We identified the causes of those facial injuries that are hardest to reconstruct, and the most effective combinations of facial protection. Protection is required from both penetrating projectiles and burns. There was strong consensus that blunt injury to the facial skeleton was currently not a military priority. Functional units that should be prioritised are eyes and eyelids, followed consecutively by the nose, lips, and ears. Twenty-nine respondents felt that the visor was more important than the mandibular guard if only one piece was to be worn. Essential cover of the brain and eyes is achieved from all directions using a combination of helmet and visor. Nasal cover currently requires the mandibular guard unless the visor can be modified to cover it as well. Any such prototype would need extensive ergonomics and assessment of integration, as any changes would have to be acceptable to the people who wear them in the long term.

Mutagenicity and toxicity studies with high pressure nitrous oxide.

The mutagenic and toxic potential of nitrous oxide were assessed in vitro by microbial assay using two histidine dependent strains of Salmonella typhimurium, TA98 and TA100. Bacteria on plates and in liquid suspension in the presence or absence of enzymes prepared from rat liver, were exposed in a pressure chamber to partial pressures of nitrous oxide ranging from 0.5 to 6 atmospheres. Nitrous oxide decreased viability of both strains of bacteria at 4 and 6 atmospheres but was not mutagenic at any pressure tested.

Toxicity and teratogenicity of inhaled anesthetics in Drosophila melanogaster.

The toxic and teratogenic effects of inhaled anesthetics were assessed in an in vivo assay using the fruit fly Drosophila melanogaster. Eggs were exposed during development (metamorphosis) to enflurane, isoflurane or halothane at a vapor concentration of 0.1 or 0.2% (v/v), to fluroxene at 0.025 or 0.05% (v/v), or to nitrous oxide at 20 or 40% (v/v). Flies produced in each group were counted and were examined for morphological abnormalities within one day of hatching. All the anesthetics except nitrous oxide produced a dose-dependent increase in the duration of metamorphosis and a decrease in the number of flies. Despite these effects on development, no morphological abnormalities were observed in any fly.

Effect of high helium and hydrostatic pressure on bacterial mutation and growth.

The mutagenic and toxic effect of high helium and hydrostatic pressure were assessed in an in vitro microbial assay using two histidine-dependent strains of Salmonella typhimurium. TA98 and TA100. Bacteria in the presence or absence of mammalian enzyme system were subjected to either 50 or 100 atmospheres absolute (ATA) helium or 300 ATA hydrostatic pressure. The number of revertant colonies, colony forming units and cell growth rates were measured. There was no mutagenic effect of decrease in number of colony forming units at any of the pressures tested. However, a statistically significant decrease in growth rate of exponentially dividing cells was seen at 300 ATA hydrostatic pressure and 100 ATA helium, but not at 50 ATA helium.

Mutagenic activity of vinyl compounds and derived epoxides.

Many vinyl compounds, such as vinyl chloride and some inhalational anesthetics, are known to be mutagens. In the present study, 10 vinyl compounds or derived epoxides, widely used in industry, were assayed in the Salmonella typhimurium/mammalian microsome system. 3 strains of histidine-dependent S. typhimurium, TA1535, TA98 and TA100 were used. Of the 10 compounds, 4 were mutagens. They were 9-vinylanthracene, vinylcarbazole, 3-vinyl-7-oxabicyclo[4.1.0]heptane and 3-epoxyethyl-7-oxabicyclo[4.1.0]-heptane. The study confirmed the overall genotoxicity of vinyl compounds and epoxides and the need to carefully screen them for mutagenic/carcinogenic effects.

Fluroxene mutagenicity.

The commercially available volatile anesthetic fluroxene (2,2,2-trifluoroethyl vinyl ether) which contains the stabilizer N-phenyl-1-napthylamine, was tested for mutagenicity using four strains of S. typhimurium, TA1535, TA1537, TA98 and TA100, and one strain of E. coli, WP2. In addition, purified fluroxene; N-phenyl-1-napthylamine; trifluoroethanol, a major metabolite of fluoroxene; and urine from rats anesthetized with fluroxene were tested. Several procedures were utilized including exposure of bacteria to vapor in desiccators and in liquid suspension. Results indicate that fluroxene, but not its stabilizer, was mutagenic to strains TA1535, TA100 and WP2 only in liquid suspension and only in the presence of a rat-liver enzyme system. Trifluoroethanol and urine from fluroxene-treated rat were not mutagenic to any strain of bacteria. These findings indicate that fluroxene is a promutagen which requires preincubation before it is recognized. Further experiments were performed with enzymes prepared from mouse, hamster and human liver. Fluroxene was mutagenic only in the presence of enzymes prepared from Aroclor 1254 pretreated rodents. Since fluroxene was not mutagenic in the presence of enzymes prepared from three human livers, the significance of these findings to man are unclear.

Effects of subchronic intermittent exposure to isoflurane in Swiss Webster mice.

Swiss Webster mice were treated to determine if subchronic intermittent exposure to the inhalation anesthetic isoflurane causes organ toxicity or enhances its own metabolism or that of other anesthetics. One-hundred twenty, four-week-old male and female mice were exposed to compressed air or to 0.02%, 0.1% or 0.5% of isoflurane for four hours per day, five days per week for nine weeks. Body weights among the groups were the same prior to exposure. Overall, there were no significant differences in body weights among exposure groups (ANOVA with day as a repeated measure: females - F = 2.12, P = 0.1085; males - F = 1.80, P = 0.1583). There was, however, a significant interaction of group and days; differences were isolated to the start of exposure (weeks 1 through 3 for females; week 2 for males). At all times, differences remained within 10% of the control body weights. Organ weights (liver, spleen, kidney, testis and uterus), hematocrits, and SGOT levels were similar among exposure groups. Histologic evaluation of organs revealed no anesthetic-related organ toxicity. The concentration of hepatic cytochromes, b5 and P-450, per mg of microsomal protein were similar among exposure groups and between sexes. The rates of hepatic microsomal metabolism (defluorination) of three volatile halogenated ether anesthetics (methoxyflurane, enflurane, and isoflurane) were not different among groups following nine weeks of exposure. Isoflurane exposures of 0.5% or less for four hours per day for five days per week would appear to be the maximum tolerated concentration for any chronic study. Since there was no evidence of organ toxicity or of enhanced or inhibited hepatic microsomal enzyme activity, isoflurane seems to be relatively non-toxic inhalation anesthetic under the conditions of this study.

Effects of subchronic intermittent exposure to nitrous oxide in Swiss Webster mice.

Swiss Webster mice were treated to determine whether the inhalational anesthetic, nitrous oxide (N2O), causes organ toxicity and enhances anesthetic defluorination. Two-hundred and sixteen young adult male and female mice were exposed to room air or to 5,000 (.5%), 50,000 (5%) or 500,000 (50%) N2O for four hours per day, five days per week for periods up to fourteen weeks. Body weight was measured twice weekly throughout the experiment. Liver, kidney, spleen and testis were weighed and examined histologically along with brain, stomach, seminal vesicle, and ovary for evidence of drug induced damage. Blood smears were examined microscopically and complete blood count, differential white cell count, and reticulocyte and platelet counts were performed. In addition, liver microsomal cytochrome P-450 content and the rates of defluorination of enflurane and methoxyflurane were determined. The maximum tolerated concentration of N2O was approximately 5000,000 ppm. Even at this high dose, there was no evidence of organ damage. Following N2O exposure, neither the hepatic microsomal cytochrome P-450 content nor the rates of anesthetic defluorination were increased; the rate of in vitro inorganic fluoride production was greater for methoxyflurane than for enflurane. Since there was no evidence of specific organ toxicity or of enzyme induction or inhibition, it was concluded that N2O is a comparatively nontoxic inhalational anesthetic under the conditions of this study.

Expression of mRNA for alpha1-adrenoceptor subtypes at the beginning of neurulation in rat embryos.

We recently reported that alpha1-adrenoceptor agonists, administered at the beginning of neurulation (Stage 11a) in rat embryos grown in culture, interfere with normal development of the left/right body axis leading to situs inversus. Despite these pharmacological findings, expression of alpha1-adrenoceptor genes at such an early stage of development has not been demonstrated. In the present study, we examined the expression of mRNAs for cloned alpha1-adrenoceptor subtypes in rat embryos at Stage 11a. Timed-pregnant Sprague-Dawley rats were killed in the morning of gestational day 9 (vaginal plug day = day 0), and the implantation sites were removed. The implantation sites were separated into embryo, ectoplacental cone and decidua, only those at Stage 11a were selected, and these were immediately frozen on dry ice, and subsequently their total RNA was isolated. RNase protection assays were then performed for cloned alpha1a-, alpha 1b- and alpha1d-adrenocepter subtypes using 20-30 mug of total RNA for each hybridization. In all tissues, strong and weak signals were detected for alpha1b- and alpha1a-adrenoceptor subtype mRNAs, respectively. In contrast, a signal for alpha1d mRNA was not detected in any tissues. These results, together with previous pharmacological findings, suggest the existence of alpha1a- and alpha1b-adrenoceptor subtypes in rat embryos at Stage 11a.