Hypoxia Is Associated with Increased Immune Infiltrates and Both Anti-Tumour and Immune Suppressive Signalling in Muscle-Invasive Bladder Cancer.

Hypoxia and a suppressive tumour microenvironment (TME) are both independent negative prognostic factors for muscle-invasive bladder cancer (MIBC) that contribute to treatment resistance. Hypoxia has been shown to induce an immune suppressive TME by recruiting myeloid cells that inhibit anti-tumour T cell responses. Recent transcriptomic analyses show hypoxia increases suppressive and anti-tumour immune signalling and infiltrates in bladder cancer. This study sought to investigate the relationship between hypoxia-inducible factor (HIF)-1 and -2, hypoxia, and immune signalling and infiltrates in MIBC. ChIP-seq was performed to identify HIF1α, HIF2α, and HIF1ß binding in the genome of the MIBC cell line T24 cultured in 1% and 0.1% oxygen for 24 h. Microarray data from four MIBC cell lines (T24, J82, UMUC3, and HT1376) cultured under 1%, 0.2%, and 0.1% oxygen for 24 h were used. Differences in the immune contexture between high- and low-hypoxia tumours were investigated using in silico analyses of two bladder cancer cohorts (BCON and TCGA) filtered to only include MIBC cases. GO and GSEA were used with the R packages "limma" and "fgsea". Immune deconvolution was performed using ImSig and TIMER algorithms. RStudio was used for all analyses. Under hypoxia, HIF1α and HIF2α bound to ~11.5-13.5% and ~4.5-7.5% of immune-related genes, respectively (1-0.1% O2). HIF1α and HIF2α both bound to genes associated with T cell activation and differentiation signalling pathways. HIF1α and HIF2α had distinct roles in immune-related signalling. HIF1 was associated with interferon production specifically, whilst HIF2 was associated with generic cytokine signalling as well as humoral and toll-like receptor immune responses. Neutrophil and myeloid cell signalling was enriched under hypoxia, alongside hallmark pathways associated with Tregs and macrophages. High-hypoxia MIBC tumours had increased expression of both suppressive and anti-tumour immune gene signatures and were associated with increased immune infiltrates. Overall, hypoxia is associated with increased inflammation for both suppressive and anti-tumour-related immune signalling and immune infiltrates, as seen in vitro and in situ using MIBC patient tumours.

Clinical Biomarkers of Tumour Radiosensitivity and Predicting Benefit from Radiotherapy: A Systematic Review.

Modern advanced radiotherapy techniques have improved the precision and accuracy of radiotherapy delivery, with resulting plans being highly personalised based on individual anatomy. Adaptation for individual tumour biology remains elusive. There is an unmet need for biomarkers of intrinsic radiosensitivity that can predict tumour response to radiation to facilitate individualised decision-making, dosing and treatment planning. Over the last few decades, the use of high throughput molecular biology technologies has led to an explosion of newly discovered cancer biomarkers. Gene expression signatures are now used routinely in clinic to aid decision-making regarding adjuvant systemic therapy. They have great potential as radiotherapy biomarkers. A previous systematic review published in 2015 reported only five studies of signatures evaluated for their ability to predict radiotherapy benefits in clinical cohorts. This updated systematic review encompasses the expanded number of studies reported in the last decade. An additional 27 studies were identified. In total, 22 distinct signatures were recognised (5 pre-2015, 17 post-2015). Seventeen signatures were 'radiosensitivity' signatures and five were breast cancer prognostic signatures aiming to identify patients at an increased risk of local recurrence and therefore were more likely to benefit from adjuvant radiation. Most signatures (15/22) had not progressed beyond the discovery phase of development, with no suitable validated clinical-grade assay for application. Very few signatures (4/17 'radiosensitivity' signatures) had undergone any laboratory-based biological validation of their ability to predict tumour radiosensitivity. No signatures have been assessed prospectively in a phase III biomarker-led trial to date and none are recommended for routine use in clinical guidelines. A phase III prospective evaluation is ongoing for two breast cancer prognostic signatures. The most promising radiosensitivity signature remains the radiosensitivity index (RSI), which is used to calculate a genomic adjusted radiation dose (GARD). There is an ongoing phase II prospective biomarker-led study of RSI/GARD in triple negative breast cancer. The results of these trials are eagerly anticipated over the coming years. Future work in this area should focus on (1) robust biological validation; (2) building biobanks alongside large radiotherapy randomised controlled trials with dose variance (to demonstrate an interaction between radiosensitivity signature and dose); (3) a validation of clinical-grade cost-effective assays that are deliverable within current healthcare infrastructure; and (4) an integration with biomarkers of other determinants of radiation response.

Implementation of Oxygen Enhanced Magnetic Resonance Imaging (OE-MRI) and a Pilot Genomic Study of Hypoxia in Bladder Cancer Xenografts.

BACKGROUND/AIM: Patients with hypoxic bladder cancer benefit from hypoxia modification added to radiotherapy, but no biomarkers exist to identify patients with hypoxic tumours. We, herein, aimed to implement oxygen-enhanced MRI (OE-MRI) in xenografts derived from muscle-invasive bladder cancer (MIBC) for future hypoxia biomarker discovery work; and generate gene expression data for future biomarker discovery. MATERIALS AND METHODS: The flanks of female CD-1 nude mice inoculated with HT1376 MIBC cells. Mice with small (300 mm3) or large (700 mm3) tumours were imaged, breathing air then 100% O2, 1 h post injection with pimonidazole in an Agilant 7T 16cm bore magnet interfaced to a Bruker Avance III console with a T2-TurboRARE sequence using a dynamic MPRAGE acquisition. Dynamic Spoiled Gradient Recalled Echo images were acquired for 5 min, with 0.1mmol/kg Gd-DOTA (Dotarem, Guerbet, UK) injected after 60 s (1 ml/min). Voxel size and field of view of dynamic contrast enhanced (DCE)-MRI and OE-MRI scans were matched. The voxels considered as perfused with significant post-contrast enhancement (p<0.05) in DCE-MRI scans and tissue were further split into pOxyE (normoxic) and pOxyR (hypoxic) regions. Tumours harvested in liquid N2, sectioned, RNA was extracted and transcriptomes analysed using Clariom S microarrays. RESULTS: Imaged hypoxic regions were greater in the larger versus smaller tumour. Expression of known hypoxia-inducible genes and a 24 gene bladder cancer hypoxia score were higher in pimonidazole-high versus -low regions: CA9 (p=0.012) and SLC2A1 (p=0.012) demonstrating expected transcriptomic behaviour. CONCLUSION: OE-MRI was successfully implemented in MIBC-derived xenografts. Transcriptomic data derived from hypoxic and non-hypoxic xenograft regions will be useful for future studies.

A hypoxia biomarker does not predict benefit from giving chemotherapy with radiotherapy in the BC2001 randomised controlled trial.

BACKGROUND: BC2001 showed combining chemotherapy (5-FU + mitomycin-C) with radiotherapy improves loco-regional disease-free survival in patients with muscle-invasive bladder cancer (MIBC). We previously showed a 24-gene hypoxia-associated signature predicted benefit from hypoxia-modifying radiosensitisation in BCON and hypothesised that only patients with low hypoxia scores (HSs) would benefit from chemotherapy in BC2001. BC2001 allowed conventional (64Gy/32 fractions) or hypofractionated (55Gy/20 fractions) radiotherapy. An exploratory analysis tested an additional hypothesis that hypofractionation reduces reoxygenation and would be detrimental for patients with hypoxic tumours. METHODS: RNA was extracted from pre-treatment biopsies (298 BC2001 patients), transcriptomic data generated (Affymetrix Clariom-S arrays), HSs calculated (median expression of 24-signature genes) and patients stratified as hypoxia-high or -low (cut-off: cohort median). PRIMARY ENDPOINT: invasive loco-regional control (ILRC); secondary overall survival. FINDINGS: Hypoxia affected overall survival (HR = 1.30; 95% CI 0.99-1.70; p = 0.062): more uncertainty for ILRC (HR = 1.29; 95% CI 0.82-2.03; p = 0.264). Benefit from chemotherapy was similar for patients with high or low HSs, with no interaction between HS and treatment arm. High HS associated with poor ILRC following hypofractionated (n = 90, HR 1.69; 95% CI 0.99-2.89 p = 0.057) but not conventional (n = 207, HR 0.70; 95% CI 0.28-1.80, p = 0.461) radiotherapy. The finding was confirmed in an independent cohort (BCON) where hypoxia associated with a poor prognosis for patients receiving hypofractionated (n = 51; HR 14.2; 95% CI 1.7-119; p = 0.015) but not conventional (n = 24, HR 1.04; 95% CI 0.07-15.5, p = 0.978) radiotherapy. INTERPRETATION: Tumour hypoxia status does not affect benefit from BC2001 chemotherapy. Hypoxia appears to affect fractionation sensitivity. Use of HSs to personalise treatment needs testing in a biomarker-stratified trial. FUNDING: Cancer Research UK, NIHR, MRC.

The Canadian C-spine rule versus the NEXUS low-risk criteria in patients with trauma.

BACKGROUND: The Canadian C-Spine (cervical-spine) Rule (CCR) and the National Emergency X-Radiography Utilization Study (NEXUS) Low-Risk Criteria (NLC) are decision rules to guide the use of cervical-spine radiography in patients with trauma. It is unclear how the two decision rules compare in terms of clinical performance. METHODS: We conducted a prospective cohort study in nine Canadian emergency departments comparing the CCR and NLC as applied to alert patients with trauma who were in stable condition. The CCR and NLC were interpreted by 394 physicians for patients before radiography. RESULTS: Among the 8283 patients, 169 (2.0 percent) had clinically important cervical-spine injuries. In 845 (10.2 percent) of the patients, physicians did not evaluate range of motion as required by the CCR algorithm. In analyses that excluded these indeterminate cases, the CCR was more sensitive than the NLC (99.4 percent vs. 90.7 percent, P<0.001) and more specific (45.1 percent vs. 36.8 percent, P<0.001) for injury, and its use would have resulted in lower radiography rates (55.9 percent vs. 66.6 percent, P<0.001). In secondary analyses that included all patients, the sensitivity and specificity of CCR, assuming that the indeterminate cases were all positive, were 99.4 percent and 40.4 percent, respectively (P<0.001 for both comparisons with the NLC). Assuming that the CCR was negative for all indeterminate cases, these rates were 95.3 percent (P=0.09 for the comparison with the NLC) and 50.7 percent (P=0.001). The CCR would have missed 1 patient and the NLC would have missed 16 patients with important injuries. CONCLUSIONS: For alert patients with trauma who are in stable condition, the CCR is superior to the NLC with respect to sensitivity and specificity for cervical-spine injury, and its use would result in reduced rates of radiography.

The bedside investigation of pulmonary embolism diagnosis study: a double-blind randomized controlled trial comparing combinations of 3 bedside tests vs ventilation-perfusion scan for the initial investigation of suspected pulmonary embolism.

BACKGROUND: We sought to determine whether using combinations of 3 bedside tests (7-variable clinical model, non-enzyme-linked immunosorbent assay D-dimer test, and alveolar dead-space fraction) to exclude pulmonary embolism (PE) before diagnostic imaging was as safe as a standard strategy of starting with ventilation-perfusion (V/Q) scan. METHODS: In this double-blind, randomized, controlled equivalency trial, patients were randomized to initial bedside tests or to initial V/Q scan without bedside tests. Patients assigned to the bedside test group had a sham V/Q scan performed if at least 2 of 3 bedside test results were negative; otherwise, they underwent an actual V/Q scan. Further diagnostic management was determined by a blinded physician after V/Q scan. The primary outcome measure was recurrent venous thromboembolic events during 3 months among patients who were not taking anticoagulant agents after the initial investigations were completed. RESULTS: Four hundred fifty-eight consecutive adults with suspected PE were eligible for the study; 398 of 399 consenting and randomized patients completed the study. The follow-up venous thromboembolic event rate was 2.4% in the bedside test group vs 3.0% in the V/Q scan group (P = .76). Pulmonary embolism was excluded in 34% (67/199) of the bedside test group patients with at least 2 negative results on 3 bedside tests vs 18% (35/199) excluded using only the 7-variable clinical model and the D-dimer test. CONCLUSION: Excluding PE with at least 2 negative results on 3 bedside tests safely eliminates the need for diagnostic imaging in 34% of patients with suspected PE.

Comparison of the Canadian CT Head Rule and the New Orleans Criteria in patients with minor head injury.

CONTEXT: Current use of cranial computed tomography (CT) for minor head injury is increasing rapidly, highly variable, and inefficient. The Canadian CT Head Rule (CCHR) and New Orleans Criteria (NOC) are previously developed clinical decision rules to guide CT use for patients with minor head injury and with Glasgow Coma Scale (GCS) scores of 13 to 15 for the CCHR and a score of 15 for the NOC. However, uncertainty about the clinical performance of these rules exists. OBJECTIVE: To compare the clinical performance of these 2 decision rules for detecting the need for neurosurgical intervention and clinically important brain injury. DESIGN, SETTING, AND PATIENTS: In a prospective cohort study (June 2000-December 2002) that included 9 emergency departments in large Canadian community and university hospitals, the CCHR was evaluated in a convenience sample of 2707 adults who presented to the emergency department with blunt head trauma resulting in witnessed loss of consciousness, disorientation, or definite amnesia and a GCS score of 13 to 15. The CCHR and NOC were compared in a subgroup of 1822 adults with minor head injury and GCS score of 15. MAIN OUTCOME MEASURES: Neurosurgical intervention and clinically important brain injury evaluated by CT and a structured follow-up telephone interview. RESULTS: Among 1822 patients with GCS score of 15, 8 (0.4%) required neurosurgical intervention and 97 (5.3%) had clinically important brain injury. The NOC and the CCHR both had 100% sensitivity but the CCHR was more specific (76.3% vs 12.1%, P<.001) for predicting need for neurosurgical intervention. For clinically important brain injury, the CCHR and the NOC had similar sensitivity (100% vs 100%; 95% confidence interval [CI], 96%-100%) but the CCHR was more specific (50.6% vs 12.7%, P<.001), and would result in lower CT rates (52.1% vs 88.0%, P<.001). The kappa values for physician interpretation of the rules, CCHR vs NOC, were 0.85 vs 0.47. Physicians misinterpreted the rules as not requiring imaging for 4.0% of patients according to CCHR and 5.5% according to NOC (P = .04). Among all 2707 patients with a GCS score of 13 to 15, the CCHR had sensitivities of 100% (95% CI, 91%-100%) for 41 patients requiring neurosurgical intervention and 100% (95% CI, 98%-100%) for 231 patients with clinically important brain injury. CONCLUSION: For patients with minor head injury and GCS score of 15, the CCHR and the NOC have equivalent high sensitivities for need for neurosurgical intervention and clinically important brain injury, but the CCHR has higher specificity for important clinical outcomes than does the NOC, and its use may result in reduced imaging rates.

Retrospective application of the NEXUS low-risk criteria for cervical spine radiography in Canadian emergency departments.

STUDY OBJECTIVE: We evaluate the accuracy, reliability, and potential impact of the National Emergency X-Radiography Utilization Study (NEXUS) low-risk criteria for cervical spine radiography, when applied in Canadian emergency departments (EDs). METHODS: The Canadian C-Spine Rule derivation study was a prospective cohort study conducted in 10 Canadian EDs that recruited alert and stable adult trauma patients. Physicians completed a 20-item data form for each patient and performed interobserver assessments when feasible. The prospective assessments included the 5 individual NEXUS criteria but not an explicit interpretation of the overall need for radiography according to the criteria. Patients underwent plain radiography, flexion-extension views, and computed tomography at the discretion of the treating physician. Patients who did not have radiography were followed up with a structured outcome assessment by telephone to determine clinically important cervical spine injury, a previously validated outcome measurement. Analyses included sensitivity and specificity with 95% confidence interval (CI), kappa coefficient, and potential radiography rates. RESULTS: Among 8,924 patients, 151 (1.7%) patients had an important cervical spine injury. The combined NEXUS criteria identified important cervical spine injury with a sensitivity of 92.7% (95% CI 87% to 96%) and a specificity of 37.8% (95% CI 37% to 39%). Application of the NEXUS criteria would have potentially reduced cervical spine radiography rates by 6.1% from the actual rate of 68.9% to 62.8%. Of 11 patients with important injuries not identified, 2 were treated with internal fixation and 3 with a halo. CONCLUSION: This retrospective validation found the NEXUS low-risk criteria to be less sensitive than previously reported. The NEXUS low-risk criteria should be further explicitly and prospectively evaluated for accuracy and reliability before widespread clinical use outside of the United States.

bFGF-containing electrospun gelatin scaffolds with controlled nano-architectural features for directed angiogenesis.

Current therapeutic angiogenesis strategies are focused on the development of biologically responsive scaffolds that can deliver multiple angiogenic cytokines and/or cells in ischemic regions. Herein, we report on a novel electrospinning approach to fabricate cytokine-containing nanofibrous scaffolds with tunable architecture to promote angiogenesis. Fiber diameter and uniformity were controlled by varying the concentration of the polymeric (i.e. gelatin) solution, the feed rate, needle to collector distance, and electric field potential between the collector plate and injection needle. Scaffold fiber orientation (random vs. aligned) was achieved by alternating the polarity of two parallel electrodes placed on the collector plate thus dictating fiber deposition patterns. Basic fibroblast growth factor (bFGF) was physically immobilized within the gelatin scaffolds at variable concentrations and human umbilical vein endothelial cells (HUVEC) were seeded on the top of the scaffolds. Cell proliferation and migration was assessed as a function of growth factor loading and scaffold architecture. HUVECs successfully adhered onto gelatin B scaffolds and cell proliferation was directly proportional to the loading concentrations of the growth factor (0-100 bFGF ng/mL). Fiber orientation had a pronounced effect on cell morphology and orientation. Cells were spread along the fibers of the electrospun scaffolds with the aligned orientation and developed a spindle-like morphology parallel to the scaffold's fibers. In contrast, cells seeded onto the scaffolds with random fiber orientation, did not demonstrate any directionality and appeared to have a rounder shape. Capillary formation (i.e. sprouts length and number of sprouts per bead), assessed in a 3-D in vitro angiogenesis assay, was a function of bFGF loading concentration (0 ng, 50 ng and 100 ng per scaffold) for both types of electrospun scaffolds (i.e. with aligned or random fiber orientation).

Postexercise hypotension causes a prolonged perturbation in esophageal and active muscle temperature recovery.

We examined the effect of two levels of exercise-induced hypotension on esophageal (Tes) and active and nonactive muscle temperatures during and following exercise. Seven males performed an incremental isotonic test on a Kin-Com isokinetic apparatus to determine their peak oxygen consumption during bilateral knee extensions (VO2sp). This was followed on separate days by 15-min of isolated bilateral knee extensions at moderate (60% VO2sp) (MEI) and high (80% VO2sp) (HEI) exercise intensities, followed by 90 min of recovery. Muscle temperature was measured with an intramuscular probe inserted in the left vastus medialis (Tvm) and triceps brachii (Ttb) muscles under ultrasound guidance. The deepest sensor (tip) was located approximately 10 mm from the femur and deep femoral artery and from the superior ulnar collateral artery and humerus for the Tvm and Ttb, respectively. Additional sensors were located 15 and 30 mm from the tip with an additional sensor located at 45 mm for the Tvm measurements only. Following exercise, mean arterial pressure (MAP) remained significantly below preexercise rest for the initial 60 min of recovery after MEI and for the duration of the postexercise recovery period after HEI (P< or =0.05). After HEI, significantly greater elevations from preexercise rest were recorded for Tes and all muscle temperatures paralleled a greater decrease in MAP compared with MEI (P< or =0.05). By the end of 90-min postexercise recovery, MAP, Tes, and all muscle temperatures remained significantly greater after HEI than MEI. Furthermore, a significantly shallower muscle temperature profile across Tvm, relative to preexercise rest, was observed at the end of exercise for both HEI and MEI (P< or=0.05), and for 30 min of recovery for MEI and throughout 90 min of recovery for HEI. No significant differences in muscle temperature profile were observed for Ttb. Thus we conclude that the increase in the postexercise hypotensive response, induced by exercise of increasing intensity, was paralleled by an increase in the magnitude and recovery time of the postexercise esophageal and active muscle temperatures.

Ultra-sound imaging for precision implantation of a multi sensor temperature probe in skeletal muscle tissue.

A technique for implanting multi sensor temperature probes in muscle tissue was developed to optimize the accuracy of the tissue temperature measurements and the internal localization of the probe. Real time ultra-sound imaging was used to (a) determine the best perpendicular insertion tract, (b) guide the insertion of the probe in order to avoid major blood vessels, and (c) verify the insertion point relative to discernable anatomic reference structures such as arteries and bone.

Tissue temperature transients in resting contra-lateral leg muscle tissue during isolated knee extension.

This study was designed to evaluate the role of non-active tissue in the retention and dissipation of heat during and following intense isolated muscle activity. Six subjects performed an incremental isotonic test (constant angular velocity, increases in force output) on a KIN-COM isokinetic apparatus to determine their maximal oxygen consumption during single knee extensions (VO2sp). In a subsequent session, a thin wire multi-sensor temperature probe was inserted into the left vastus medialis under ultrasound guidance at a specific internal marker. The deepest temperature sensor (tip, Tmu10) was located approximately 10 mm from the femur and deep femoral artery with 2 additional sensors located at 15 (Tmu25) and 30 (Tmu40) mm from the tip. Implant site was midway between and medial to a line joining the anterior superior iliac spine and base of patella. Esophageal temperature (Tes) temperature was measured as an index of core temperature. Subjects rested in a supine position for 60 min followed by 30 min of seated rest in an ambient condition of 22 degree C. Subjects then performed 15 min of isolated single right knee extensions against a dynamic resistance on a KIN COM corresponding to 60% of VO2sp at 60 degree x sec(-1). Exercise was followed by 60 min of seated rest. Resting Tes was 37 degree C while Tmu10, Tmu25, and Tmu40 were 36.58, 36.55 and 36.45 degree C, respectively. Exercise resulted in a Tes increase of 0.31 C above pre-exercise resting. Tmu of the non-exercising leg increased 0.23, 0.19 and 0.09 degree C for Tmu10, Tmu25, and Tmu40, respectively. While Tes decreased to baseline values within approximately 15 min of end-exercise, Tmu10 reached resting values following approximately 40 min of recovery. These results suggest that during isolated muscle activity, convective heat transfer by the blood to non-active muscle tissue may have a significant role in maintaining resting core temperature.

The Canadian C-spine rule performs better than unstructured physician judgment.

STUDY OBJECTIVES: We compare the predictive accuracy of emergency physicians' unstructured clinical judgment to the Canadian C-Spine rule. METHODS: This prospective multicenter cohort study was conducted at 10 Canadian urban academic emergency departments. Included in the study were alert, stable, adult patients with a Glasgow Coma Scale score of 15 and trauma to the head or neck. This was a substudy of the Canadian C-Spine and CT Head Study. Eligible patients were prospectively evaluated before radiography. Physicians estimated the probability of unstable cervical spine injury from 0% to 100% according to clinical judgment alone and filled out a data form. Interobserver assessments were done when feasible. Patients underwent cervical spine radiography or follow-up to determine clinically important cervical spine injuries. Analyses included comparison of areas under the receiver operating characteristic (ROC) curve with 95% confidence intervals (CIs) and the kappa coefficient. RESULTS: During 18 months, 6265 patients were enrolled. The mean age was 36.6 years (range 16 to 97 years), and 50.1% were men. Sixty-four (1%) patients had a clinically important injury. The physicians' kappa for a 0% predicted probability of injury was 0.46 (95% CI 0.28 to 0.65). The respective areas under the ROC curve for predicting cervical spine injury were 0.85 (95% CI 0.80 to 0.89) for physician judgment and 0.91 (95% CI 0.89 to 0.92) for the Canadian C-Spine rule (P <.05). With a threshold of 0% predicted probability of injury, the respective indices of accuracy for physicians and the Canadian C-Spine rule were sensitivity 92.2% versus 100% (P <.001) and specificity 53.9% versus 44.0% (P <.001). CONCLUSION: Interobserver agreement of unstructured clinical judgment for predicting clinically important cervical spine injury is only fair, and the sensitivity is unacceptably low. The Canadian C-Spine rule was better at detecting clinically important injuries with a sensitivity of 100%. Prospective validation has recently been completed and should permit widespread use of the Canadian C-Spine rule.