Twice-Yearly Depemokimab in Severe Asthma with an Eosinophilic Phenotype.

BACKGROUND: Depemokimab is an ultra-long-acting biologic therapy with enhanced binding affinity for interleukin-5 that may enable effective 6-month dosing intervals. METHODS: In these phase 3A, randomized, placebo-controlled replicate trials, we evaluated the efficacy and safety of depemokimab in patients with severe asthma and an eosinophilic phenotype characterized by a high eosinophil count (≥300 cells per microliter in the previous 12 months or ≥150 cells per microliter at screening) and a history of exacerbations despite the receipt of medium- or high-dose inhaled glucocorticoids. Patients were randomly assigned in a 2:1 ratio to receive either depemokimab (at a dose of 100 mg subcutaneously) or placebo at weeks 0 and 26, plus standard care. The primary end point was the annualized rate of exacerbations at 52 weeks. Secondary end points, which were analyzed in a hierarchical manner to adjust for multiplicity, included the change from baseline in the score on the St. George's Respiratory Questionnaire (SGRQ), the forced expiratory volume in 1 second, and asthma symptom reports at 52 weeks. RESULTS: Across the two trials, 792 patients underwent randomization and 762 were included in the full analysis; 502 were assigned to receive depemokimab and 260 to receive placebo. The annualized rate of exacerbations was 0.46 (95% confidence interval [CI]), 0.36 to 0.58) with depemokimab and 1.11 (95% CI, 0.86 to 1.43) with placebo (rate ratio, 0.42; 95% CI, 0.30 to 0.59; P<0.001) in SWIFT-1 and 0.56 (95% CI, 0.44 to 0.70) with depemokimab and 1.08 (95% CI, 0.83 to 1.41) with placebo (rate ratio, 0.52; 95% CI, 0.36 to 0.73; P<0.001) in SWIFT-2. No significant between-group difference in the change from baseline in the SGRQ score was observed in either trial, so no statistical inference was drawn on subsequent secondary end points. The proportion of patients with any adverse event was similar in the two groups in both trials. CONCLUSIONS: Depemokimab reduced the annualized rate of exacerbations among patients with severe asthma with an eosinophilic phenotype. (Funded by GSK; SWIFT-1 and SWIFT-2 ClinicalTrials.gov numbers, NCT04719832 and NCT04718103.).

Integration of patient and public involvement in a doctoral research study using the research cycle.

BACKGROUND: Patient and public involvement (PPI) in research is widely acknowledged as essential to achieving successful and impactful research. Despite this acknowledgement, there are limited reports on how to approach and apply meaningful PPI throughout the research cycle and how to address challenges for researchers such as doctoral students, particularly when undertaking research on sensitive topics. This paper provides insights and examples for researchers new to PPI, on the impact of active PPI and recommendations for building and developing a PPI group in a paediatric focused doctoral research study with bereaved parents and carers. METHODS: PPI was informed by the research cycle. The GRIPP2 short-form checklist was used to report PPI. The research was funded by the National Institute for Health and Care Research. RESULTS: PPI enhanced the research through input into the study design, recruitment, co-design of the study website and branding; and ethics amendments to increase participation in response to the COVID-19 pandemic. The literature review was extended to incorporate a PPI consultation phase and members contributed to data analysis. A flexible approach enabled involvement to develop iteratively throughout the research study, resulting in changes being made to enhance the study design and outcomes. CONCLUSION: This paper contributes to the limited knowledge base on embedding PPI into a doctoral research study and within the paediatric setting specifically working in partnership with bereaved parents and carers. Employing an adaptive approach to meet individual PPI needs, building a trusting and respectful partnership, creating shared ownership and investment in the research, are essential components to successful PPI.

Involving patients and the public in research provides the opportunity to develop meaningful outcomes that are relevant to the population being studied. Despite the benefits of patient and public involvement in research, guidelines that support researchers in doing so, lack detail on how to do this effectively. This is particularly important for those new to research such as doctoral students, who have so much to learn in developing a research study. Different approaches and applications to involvement are also likely to be needed depending on the population being studied. There are limited published papers on examples of how doctoral students have engaged and involved patients and the public in the context of their studies, and specifically within the children's setting, working in partnership with bereaved parents and carers, or those with seriously ill children. This paper offers examples and insights for those new to research in how to involve patients and the public throughout the research cycle. Specifically undertaking research in a sensitive subject of a particular childhood cancer which has poor outcomes and how to incorporate and evaluate successful patient and public involvement in their research activities such as study design and analysis of the results. Parent and carer reflections on their experiences of being involved are also reported and researcher recommendations for approaching and working with a patient and public group are described.

[11C]metomidate PET-CT versus adrenal vein sampling for diagnosing surgically curable primary aldosteronism: a prospective, within-patient trial.

Primary aldosteronism (PA) due to a unilateral aldosterone-producing adenoma is a common cause of hypertension. This can be cured, or greatly improved, by adrenal surgery. However, the invasive nature of the standard pre-surgical investigation contributes to fewer than 1% of patients with PA being offered the chance of a cure. The primary objective of our prospective study of 143 patients with PA ( NCT02945904 ) was to compare the accuracy of a non-invasive test, [11C]metomidate positron emission tomography computed tomography (MTO) scanning, with adrenal vein sampling (AVS) in predicting the biochemical remission of PA and the resolution of hypertension after surgery. A total of 128 patients reached 6- to 9-month follow-up, with 78 (61%) treated surgically and 50 (39%) managed medically. Of the 78 patients receiving surgery, 77 achieved one or more PA surgical outcome criterion for success. The accuracies of MTO at predicting biochemical and clinical success following adrenalectomy were, respectively, 72.7 and 65.4%. For AVS, the accuracies were 63.6 and 61.5%. MTO was not significantly superior, but the differences of 9.1% (95% confidence interval = -6.5 to 24.1%) and 3.8% (95% confidence interval = -11.9 to 9.4) lay within the pre-specified -17% margin for non-inferiority (P = 0.00055 and P = 0.0077, respectively). Of 24 serious adverse events, none was considered related to either investigation and 22 were fully resolved. MTO enables non-invasive diagnosis of unilateral PA.

Long-term morbidity after surgery for perihilar cholangiocarcinoma: A cohort study.

BACKGROUND: Surgery for perihilar cholangiocarcinoma (pCCA) offers the only possibility of long-term survival, but remains a formidable undertaking. Traditionally, 90-day post-operative complications and death are used to define operative risk. However, there is concern that this metric may not accurately capture long-term morbidity after such complex surgery. METHODS: A retrospective review of a prospective database of patients undergoing surgery for pCCA at a Western centre between January 2009-2020. RESULTS: Eighty-five patients underwent surgical resection for pCCA with a median overall survival of 36.3 months. Post-op (<90day) morbidity rates were high with 46% of patients developing a major complication (Clavien-Dindo grade 3-4). Post-op mortality rate was 13%. In total 38% (28/74) of patients experienced at least 1 episode of delayed morbidity (>90-days of surgery) resulting in 53 separate admissions with a median LOS of 7 days (IQR 2-15). These episodes were predominately secondary to biliary obstruction with the majority requiring radiological intervention (Clavien-Dindo grade 3). The development of long-term morbidity was associated with increased recurrence rates and correlated with poorer OS (27.6 months vs. 65.7 months HR 2.2 CI 1.63-2.77). CONCLUSIONS: Routinely cited 90-day morbidity and mortality does not accurately capture the patient morbidity experienced following surgery for pCCA. Surgery clearly offers a survival benefit and should be pursued in selected patients, but they must be fully counselled on the potential for long-term morbidity before embarking on this strategy.

A scalable phenotyping approach for female floral organ development and senescence in the absence of pollination in wheat.

In the absence of pollination, female reproductive organs senesce, leading to an irrevocable loss in the reproductive potential of the flower, which directly affects seed set. In self-pollinating crops like wheat (Triticum aestivum), the post-anthesis viability of unpollinated carpels has been overlooked, despite its importance for hybrid seed production systems. To advance our knowledge of carpel development in the absence of pollination, we created a high-throughput phenotyping approach to quantify stigma and ovary morphology. We demonstrate the suitability of the approach, which uses light-microscopy imaging and machine learning, for the analysis of floral organ traits in field-grown plants using fresh and fixed samples. We show that the unpollinated carpel undergoes a well-defined initial growth phase, followed by a peak phase in which stigma area reaches its maximum and the radial expansion of the ovary slows, and a final deterioration phase. These developmental dynamics were consistent across years and could be used to classify male-sterile cultivars. This phenotyping approach provides a new tool for examining carpel development, which we hope will advance research into female fertility of wheat.

To transplant, or not to transplant? That is the question. A patient advocate evaluation of autologous stem cell transplant in neuroblastoma.

High-dose chemotherapy with autologous stem cell transplant (ASCT) has been a mainstay of high-risk neuroblastoma treatment for several decades, demonstrating improvements in event-free survival but with risks of serious or even life-threatening acute toxicities, severe long-term adverse health effects for survivors, and ongoing contention regarding overall survival benefit. The merits of ASCT in the modern era of immunotherapy are a source of debate among parents, advocates, and some physicians. Here we examine evidence for and against ASCT, explore parent attitudes and their turmoil over decision-making, and strongly encourage international research consortia to develop a coordinated strategy to accelerate progress toward a future that avoids the routine use of ASCT in high-risk neuroblastoma.

A Phase 1 study of the long-acting anti-IL-5 monoclonal antibody GSK3511294 in patients with asthma.

AIMS: GSK3511294 is a humanized anti-interleukin (IL)-5 monoclonal antibody (mAb) engineered for extended half-life and improved IL-5 affinity versus other anti-IL-5 mAbs. This study examined its safety, tolerability, pharmacokinetics (PK) and effect on blood eosinophil counts. METHODS: This was a double-blind, parallel-group, single-ascending-dose, multicenter, Phase 1 study (205 722;NCT03287310) in patients with asthma and a blood eosinophil count ≥200 cells µL-1 . Patients were randomized 3:1 within dose cohorts to receive a single subcutaneous dose of GSK3511294 (2, 10, 30, 100 or 300 mg) or placebo and followed for up to 40 weeks to assess safety (primary endpoint), ratio to baseline in blood eosinophil count, plasma PK parameters and frequency/titers of binding antidrug antibodies (all secondary). RESULTS: Forty-eight patients received the study drug and completed the study. Adverse events (AEs) occurred in 92% of placebo-treated and 81% of GSK3511294-treated patients. There were no AEs leading to study withdrawal or serious AEs; hypersensitivity (one event in one patient) and injection-site reaction (three events in two patients) occurred infrequently. Marked reductions (>48%) in blood eosinophil count were seen from 24 hours post-dose with all GSK3511294 doses but not placebo; suppression was maintained for longer with increasing dose (82% and 83% adjusted reductions vs placebo with 100 and 300 mg, respectively, at week 26). PK were linear and dose proportional over the dose range; terminal half-life was 38-53 days. CONCLUSIONS: GSK3511294 was well tolerated, with linear and dose proportional PK, extended half-life and blood eosinophil count reduction, supporting less frequent dosing versus other anti-IL-5 mAbs.

Surgical management of suspected gallbladder cancer: The role of intraoperative frozen section for diagnostic confirmation.

BACKGROUND: Preoperative diagnosis for suspected gallbladder cancers is challenging, with a risk of overtreating benign disease, for example, xanthogranulomatous cholecystitis, with radical cholecystectomies. We retrospectively evaluated the surgeon's intraoperative assessment alone, and with the addition of intraoperative frozen sections, for suspected gallbladder cancers from a tertiary hepatobiliary multidisciplinary team (MDT). METHODS: MDT patients with complex gallbladder disease were included. Collated data included demographics, MDT discussion, operative details, and patient outcomes. RESULTS: A total of 454 patients with complex gallbladder disease were reviewed, 48 (10.6%) were offered radical surgery for suspected cancer. Twenty-five underwent frozen section that led to radical surgery in 6 (25%). All frozen sections were congruent with final histopathology but doubled the operating time (p < 0.0001). Both the surgeon's subjective and additional frozen section's objective assessment, allowed for de-escalation of unnecessary radical surgery, comparing favourably to a 13.0% cancer diagnosis among radical surgery historically. CONCLUSIONS: The MDT process was highly sensitive in identifying gallbladder cancers but lacked specificity. The surgeon's intraoperative assessment is paramount in suspected cancers, and deescalated unnecessary radical surgery. Intraoperative frozen section was a safe and viable adjunct at a cost of resources and operative time.

New gender-specific formulae for estimating extracellular fluid volume from height and weight in adults.

AIMS: First, to derive gender-specific formulae for estimation of extracellular fluid volume (eECV) and second, compare eECV as a scaling metric for slope-intercept glomerular filtration rate (GFR) with estimated body surface area (eBSA), lean body mass (eLBM) and total body water (eTBW). METHODS: GFR and 'slope-only' GFR (GFR/ECV), both single compartment-corrected, were measured in a previously published multicentre database of healthy potential kidney transplant donors. Measured ECV (mECV) was obtained as ratio GFR-to-GFR/ECV. Formulae for eECV in men and women were derived from the relationship of mECV with height and weight and expressed as eECV = a.weight.height. In a population of prospective kidney transplant donors from a single centre, eECV was compared with mECV. GFR was scaled to eECV, eBSA, eLBM and eTBW, estimated from previously published formulae. RESULTS: In men and women, respectively, a was 0.0755 and 0.0399, x was 0.6185 and 0.6065 and y was 0.4982 and 0.6217. In the single centre, biases (±precisions) of eECV against mECV in men and women were 0.26 (±1.68) and 0.31 (±1.67) l. Mean GFR/eBSA was higher in men but mean GFR/eLBM and GFR/eTBW were higher in women. Mean GFR/ECV and mean GFR/eECV were very similar between the two genders. GFR/ECV and GFR/eECV showed correlations with each other that were almost identical between men and women. CONCLUSIONS: New formulae are described for estimating eECV. Scaling GFR to eECV is more physiological than scaling to eBSA and accounts for gender. eECV used for measuring GFR from a single blood sample should be gender-specific.

CamGFR v2: A New Model for Estimating the Glomerular Filtration Rate from Standardized or Non-standardized Creatinine in Patients with Cancer.

PURPOSE: Management of patients with cancer, specifically carboplatin dosing, requires accurate knowledge of glomerular filtration rate (GFR). Direct measurement of GFR is resource limited. Available models for estimated GFR (eGFR) are optimized for patients without cancer and either isotope dilution mass spectrometry (IDMS)- or non-IDMS-standardized creatinine measurements. We present an eGFR model for patients with cancer compatible with both creatinine measurement methods. EXPERIMENTAL DESIGN: GFR measurements, biometrics, and IDMS- or non-IDMS-standardized creatinine values were collected for adult patients from three cancer centers. Using statistical modeling, an IDMS and non-IDMS creatinine-compatible eGFR model (CamGFR v2) was developed. Its performance was compared with that of the existing models Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI), Modification of Diet in Renal Disease (MDRD), Full Age Spectrum (FAS), Lund-Malmö revised, and CamGFR v1, using statistics for bias, precision, accuracy, and clinical robustness. RESULTS: A total of 3,083 IDMS- and 4,612 non-IDMS-standardized creatinine measurements were obtained from 7,240 patients. IDMS-standardized creatinine values were lower than non-IDMS-standardized values in within-center comparisons (13.8% lower in Cambridge; P < 0.0001 and 19.3% lower in Manchester; P < 0.0001), and more consistent between centers. CamGFR v2 was the most accurate [root-mean-squared error for IDMS, 14.97 mL/minute (95% confidence interval, 13.84-16.13) and non-IDMS, 15.74 mL/minute (14.86-16.63)], most clinically robust [proportion with >20% error of calculated carboplatin dose for IDMS, 0.12 (0.09-0.14) and non-IDMS, 0.17 (0.15-0.2)], and least biased [median residual for IDMS, 0.73 mL/minute (-0.68 to 2.2) and non-IDMS, -0.43 mL/minute (-1.48 to 0.91)] eGFR model, particularly when eGFR was larger than 60 ml/minute. CONCLUSIONS: CamGFR v2 can utilize IDMS- and non-IDMS-standardized creatinine measurements and outperforms previous models. CamGFR v2 should be examined prospectively as a practice-changing standard of care for eGFR-based carboplatin dosing.

Multicenter Validation of the CamGFR Model for Estimated Glomerular Filtration Rate.

Important oncological management decisions rely on kidney function assessed by serum creatinine-based estimated glomerular filtration rate (eGFR). However, no large-scale multicenter comparisons of methods to determine eGFR in patients with cancer are available. To compare the performance of formulas for eGFR based on routine clinical parameters and serum creatinine not calibrated with isotope dilution mass spectrometry, we studied 3620 patients with cancer and 166 without cancer who had their glomerular filtration rate (GFR) measured with an exogenous nuclear tracer at one of seven clinical centers. The mean measured GFR was 86 mL/min. Accuracy of all models was center dependent, reflecting intercenter variability of isotope dilution mass spectrometry-creatinine measurements. CamGFR was the most accurate model for eGFR (root-mean-squared error 17.3 mL/min) followed by the Chronic Kidney Disease Epidemiology Collaboration model (root-mean-squared error 18.2 mL/min).

Evaluation of the utility of prognostic models for patients with resected hilar cholangiocarcinoma.

BACKGROUND: Several prognostic systems have been proposed to guide management strategies post-resection for patients with hilar cholangiocarcinoma. The objective of this study was to evaluate the efficacy of these conventional prognostic models, with respect to Overall Survival (OS), on patients in a modern single-centre resectional cohort. METHOD: Patients diagnosed with hilar cholangiocarcinoma, referred to a supra-regional tertiary referral centre between February 2009 and February 2016, were retrospectively analysed from a prospectively held database linked to Hospital Episode Statistics and Somerset Cancer Registry data. RESULTS: Two-hundred and one patients were assessed for suitability for surgery. Eighty-three (41%) patients considered to have potentially resectable disease underwent surgical assessment of resectability. Fifty-six (68%) patients proceeded to resection. Multivariate analysis demonstrated that pre-operative Serum CA 19-9 (p = 0.007), Radiological Arterial Involvement (p = 0.005) and Amsterdam Medical Centre (AMC) prognostic model score (p = 0.032) retained significance in association with OS. Multivariate models developed from this cohort out-performed the conventional prognostic systems for OS. CONCLUSION: The cohort-derived multivariate models demonstrated significantly improved prognostic capability compared to conventional systems in explaining OS.

Role of a pre-operative radiological scoring system in determining resectability for potentially resectable hilar cholangiocarcinoma.

AIMS: Cholangiocarcinoma is a rare cancer arising from the biliary tree. Case series indicate that 25-40% of all borderline resectable primary tumours are potentially resectable. The Memorial Sloane Kettering System (MSKCC) stratifies patients for resectability by longitudinal and radial extension of the hilar tumour. The Bismuth-Corlette system describes the longitudinal extension of the tumour within the biliary duct system. We sought to validate and, if possible, augment these two scores within an independent validation cohort. METHODS: Patients diagnosed with hilar cholangiocarcinoma between January 2009 and December 2016 were analysed from a prospectively held database. Patients with distal cholangiocarcinoma, peripheral cholangiocarcinoma and gallbladder cancer were excluded. Comparison of surgical findings to pre-operative radiological imaging was undertaken at the time of surgery. RESULTS: The validation cohort was formed of 198 patients, of which, 55 (27.8%) patients underwent resection. Logistic regression analyses identified that BC score, MSKCC score, age at diagnosis and left artery involvement were all significant independent predictor's univariately. BC score explained 28% of the variability in resectability compared to 26% explained by MSKCC. In combination, the model consisting of BC score, age at diagnosis and left artery involvement explained 39% of variability in resectability compared to the 34% explained same model including MSKCC score instead of BC score. CONCLUSION: In this cohort an augmented BC score, incorporating left hepatic artery involvement, is more discriminative in predicting resectability than the current MSKCC system.

A systematic review of 3251 emergency department thoracotomies: is it time for a national database?

PURPOSE: Emergency department thoracotomy (EDT) is a potentially life-saving procedure, performed on patients suffering traumatic cardiac arrest. Multiple indications have been reported, but overall survival remains unclear for each indication. The objective of this systematic review is to determine overall survival, survival stratified by indication, and survival stratified by geographical location for patients undergoing EDT across the world. METHODS: Articles published between 2000 and 2016 were identified which detailed outcomes from EDT. All articles referring to pre-hospital, delayed, or operating room thoracotomy were excluded. Pooled odds ratios (OR) were calculated comparing differing indications. RESULTS: Thirty-seven articles, containing 3251 patients who underwent EDT, were identified. There were 277 (8.5%) survivors. OR demonstrate improved survival for; penetrating vs blunt trauma (OR 2.10; p 0.0028); stab vs gun-shot (OR 5.45; p < 0.0001); signs of life (SOL) on admission vs no SOL (OR 5.36; p < 0.0001); and SOL in the field vs no SOL (OR 19.39; p < 0.0001). Equivalence of survival was demonstrated between cardiothoracic vs non-cardiothoracic injury (OR 1.038; p 1.000). Survival was worse for USA vs non-USA cohorts (OR 1.59; p 0.0012). CONCLUSIONS: Penetrating injury remains a robust indication for EDT. Non-cardiothoracic cause of cardiac arrest should not preclude EDT. In the absence of on scene SOL, survival following EDT is extremely unlikely. Survival is significantly higher in the non-USA publications; reasons for this are highly complex. A UK multicentre prospective study which collects standardised data on all EDTs could provide robust evidence for better patient stratification.

Should pre-hospital resuscitative thoracotomy be reserved only for penetrating chest trauma?

PURPOSE: The indications for pre-hospital resuscitative thoracotomy (PHRT) remain undefined. The aim of this paper is to explore the variation in practice for PHRT in the UK, and review the published literature. METHODS: MEDLINE and PUBMED search engines were used to identify all relevant articles and 22 UK Air Ambulance Services were sent an electronic questionnaire to assess their PHRT practice. RESULTS: Four European publications report PHRT survival rates of 9.7, 18.3, 10.3 and 3.0% in 31, 71, 39 and 33 patients, respectively. All patients sustained penetrating chest injury. Six case reports also detail survivors of PHRT, again all had sustained penetrating thoracic injury. One Japanese paper presents 34 cases of PHRT following blunt trauma, of which 26.4% survived to the intensive therapy unit but none survived to discharge. A UK population reports a single survivor of PHRT following blunt trauma but the case details remain unpublished. Ten (45%) air ambulance services responded, each service reported different indications for PHRT. All perform PHRT for penetrating chest trauma, however, length of allowed pre-procedure down time varied, ranging from 10 to 20 min. Seventy percent perform PHRT for blunt traumatic cardiac arrest, a procedure which is likely to require aggressive concurrent circulatory support, despite this only 5/10 services carry pre-hospital blood products. CONCLUSIONS: Current indications for PHRT vary amongst different geographical locations, across the UK, and worldwide. Survivors are likely to have sustained penetrating chest injury with short down time. There is only one published survivor of PHRT following blunt trauma, despite this, PHRT is still being performed in the UK for this indication.

Optimization of PET/CT image quality using the GE 'Sharp IR' point-spread function reconstruction algorithm.

OBJECTIVE: The objective of this study was to quantify any improvement with the GE 'Sharp IR' point-spread function (PSF) reconstruction algorithm in addition to ordered subsets expectation maximum (OSEM) and time-of-flight (TOF) reconstruction algorithms and establish the optimum parameters to be used in clinical studies. MATERIALS AND METHODS: We conducted a range of experiments using the National Electrical Manufacturers Association image quality phantom filled with a 4 : 1 signal-to-background ratio. We scanned the phantom using the GE Discovery 690 PET/CT scanner. We varied iteration number and Gaussian filtration. Results were compared for OSEM, OSEM+TOF and OSEM+TOF+PSF reconstructions. A sample of 15 whole-body fluorine-18-fluorodeoxyglucose were reconstructed with OSEM+TOF and OSEM+TOF+PSF using a selection of optimum reconstruction parameters determined in phantom studies. Clinicians qualitatively ranked their preferred images to choose optimum parameters. RESULTS: The addition of PSF improved signal-to-noise ratios (SNRs), contrast, hot contrast recovery coefficients and noise over OSEM and OSEM+TOF reconstruction algorithms. SNRs were the highest at two iterations and with 0 or 2 mm filters with OSEM+TOF+PSF reconstruction in all phantom studies. Clinicians generally favoured OSEM+TOF+PSF reconstruction with three iterations and a 2 mm filter. CONCLUSION: PSF reconstruction significantly improved image quality for both clinical and phantom studies. We recommended the optimum reconstruction parameters using three iterations, 24 subsets and a 2 mm filter, which improved SNRs by up to 28.8% for small lesions (P<0.05).

The wheat Phs-A1 pre-harvest sprouting resistance locus delays the rate of seed dormancy loss and maps 0.3 cM distal to the PM19 genes in UK germplasm.

The precocious germination of cereal grains before harvest, also known as pre-harvest sprouting, is an important source of yield and quality loss in cereal production. Pre-harvest sprouting is a complex grain defect and is becoming an increasing challenge due to changing climate patterns. Resistance to sprouting is multi-genic, although a significant proportion of the sprouting variation in modern wheat cultivars is controlled by a few major quantitative trait loci, including Phs-A1 in chromosome arm 4AL. Despite its importance, little is known about the physiological basis and the gene(s) underlying this important locus. In this study, we characterized Phs-A1 and show that it confers resistance to sprouting damage by affecting the rate of dormancy loss during dry seed after-ripening. We show Phs-A1 to be effective even when seeds develop at low temperature (13 °C). Comparative analysis of syntenic Phs-A1 intervals in wheat and Brachypodium uncovered ten orthologous genes, including the Plasma Membrane 19 genes (PM19-A1 and PM19-A2) previously proposed as the main candidates for this locus. However, high-resolution fine-mapping in two bi-parental UK mapping populations delimited Phs-A1 to an interval 0.3 cM distal to the PM19 genes. This study suggests the possibility that more than one causal gene underlies this major pre-harvest sprouting locus. The information and resources reported in this study will help test this hypothesis across a wider set of germplasm and will be of importance for breeding more sprouting resilient wheat varieties.

Mutation Scanning in Wheat by Exon Capture and Next-Generation Sequencing.

Targeted Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach to identify novel sequence variation in genomes, with the aims of investigating gene function and/or developing useful alleles for breeding. Despite recent advances in wheat genomics, most current TILLING methods are low to medium in throughput, being based on PCR amplification of the target genes. We performed a pilot-scale evaluation of TILLING in wheat by next-generation sequencing through exon capture. An oligonucleotide-based enrichment array covering ~2 Mbp of wheat coding sequence was used to carry out exon capture and sequencing on three mutagenised lines of wheat containing previously-identified mutations in the TaGA20ox1 homoeologous genes. After testing different mapping algorithms and settings, candidate SNPs were identified by mapping to the IWGSC wheat Chromosome Survey Sequences. Where sequence data for all three homoeologues were found in the reference, mutant calls were unambiguous; however, where the reference lacked one or two of the homoeologues, captured reads from these genes were mis-mapped to other homoeologues, resulting either in dilution of the variant allele frequency or assignment of mutations to the wrong homoeologue. Competitive PCR assays were used to validate the putative SNPs and estimate cut-off levels for SNP filtering. At least 464 high-confidence SNPs were detected across the three mutagenized lines, including the three known alleles in TaGA20ox1, indicating a mutation rate of ~35 SNPs per Mb, similar to that estimated by PCR-based TILLING. This demonstrates the feasibility of using exon capture for genome re-sequencing as a method of mutation detection in polyploid wheat, but accurate mutation calling will require an improved genomic reference with more comprehensive coverage of homoeologues.

Prognostic molecular markers in resected extrahepatic biliary tract cancers; a systematic review and meta-analysis of immunohistochemically detected biomarkers.

UNLABELLED: Better prognostic information for resected extrahepatic cholangiocarcinoma could guide treatment strategies and potentially improve outcome. This study performed a systematic review and meta-analysis to identify prognostic biomarkers for further investigation. METHODS: Relevant literature was identified using Medline, EMBASE and Web of Science. Primary end point was overall survival assessed on univariate analysis. Log hazard ratio and variance were calculated and pooled using a random effects inverse variance approach. Hazard ratio and 95% confidence intervals were calculated. RESULTS: Thirty-seven studies, including 2371 patients, met the inclusion criteria. Subsequently nine biomarkers predictive of OS were identified (HR, 95% CI): VEGF (2.32, 1.57-3.44), COX-2 (1.94, 1.01-3.71), GLUT-1 (2.09, 1.52-2.89), Cyclin D1 (1.96, 1.02-3.76), p16 (0.68, 0.47-0.98), p27 (0.48, 0.3-0.78), E-Cadherin (0.47, 0.35-0.63), Fascin (2.19, 1.35-3.55), and Ki-67 (1.69, 1.02-2.79). CONCLUSION: Meta-analysis has identified a number of prognostic biomarkers for resected extrahepatic cholangiocarcinoma. These markers warrant further investigation as potential therapeutic targets and validation in a prospective setting.