The evaluation of prediction machines is an important step for a successful implementation of genomic-enabled selection in plant breeding. Computation time and predictive ability constitute key metrics to determine the methodology utilized for the consolidation of genomic prediction pipeline. This study introduces two methods designed to couple high prediction accuracy with efficient computational performance: 1) a non-MCMC method to estimate marker effects with a Laplace prior; and 2) an iterative framework that allows solving whole-genome regression within mixed models with replicated observations in a single-stage. The investigation provides insights on predictive ability and marker effect estimates. Various genomic prediction techniques are compared based on cross-validation, assessing predictions across and within family. Properties of quantitative trait loci detection and single-stage method were evaluated on simulated plot-level data from unbalanced data structures. Estimation of marker effects by the new model is compared to a genome-wide association analysis and whole-genome regression methods. The single-stage approach is compared to a GBLUP fitted via restricted maximum likelihood, and a two-stages approaches where genetic values fit a whole-genome regression. The proposed framework provided high computational efficiency, robust prediction across datasets, and accurate estimation of marker effects. Genome-wide markers are utilized in plant and animal breeding to capture quantitative trait loci (QTL) and relationship among individuals for prediction and selection . Most inEvaluating the predictive performance of these various methodologies has become an important step for a successful implementation of genomic-enabled selection . Hence the method of choice must have two desirable features: computational feasibility and accurate prediction across various scenarios  Estimate the genetic values from phenotypes of replicated trials; 2) Calibrate marker effects upon the genetic values to estimate the breeding values and enable prediction. This approach is referred to as a ”two-stage” approach. However, single-stage analysis can benefit genomic evaluation by jointly modeling genotypes and replicated phenotypes , an iterative method to fit a WGR using a Laplace prior. Laplace priors are popular in genetic analysis for QTL detection and genomic prediction  estimates of regression coefficients alongside their associated parameters, updating one parameter at a time . The term ”single-stage” has been used to define the joint modeling of replicated observations at the phenotypic level  represents genetic signal not captured by the markers. Genotypes may have variable number of observations and weights can be assigned to the genotypes to account for the unbalancedness. The WGR step can be solved assuming unweighted observations for computational convenience, or weighted according to the number of observations of each genotype, thus b, a, and β until convergence  must be updated conditional to g.Conditional to other model terms, the variance component associated to this random effect is estimated as :σg2=g′gnmated as :σg2≅ of grain yield collected in as many as 18 environments under a modified augmented design . BLUPs wThe BLUPs are not optimal for conducting second stage analyses  the predictive ability measured as the correlation between predicted and observed genetic values, computed within-family from fivefold cross-validations with individuals were sampled at random  in each round and this procedure was repeated 20 times, and leave-family-out cross-validation by using 39 families to predict the family left out, and repeating this procedure for all 40 families; and 2) the elapsed time for calibrating the model using the whole data.π and BayesDπ implemented in the bWGR; GBLUP with REML variance components implemented in rrBLUP (ν and ϵ support vector machines (SVM) implemented in kernlab  and reproducing kernel Hilbert spaces (RKHS) implemented in BGLR ; BayesCπn rrBLUP ; boostinn rrBLUP ; L1L2 man glmnet ; random n glmnet ; ν and ϵ kernlab ; and the kernlab . The latδ is the average value of Methods above were deployed with default settings. Tuning parameters for ridge, LASSO and elastic-net were computed through 10-fold cross validation in the training set. To mitigate the computational burden necessary to tune parameters, PLS used 5 components and the empirical Bayes Lasso hyperparameters a-b were set to 0.5. The Gaussian kernel employed for RKHS was computed as An experimental population was generated through simulation to evaluate FLM estimates of large effect parameters. From an F2 bi-parental cross with 1000 individuals, 250 individuals were randomly selected and randomly mated to generate a new population of 1000 individuals. This bottle-necking with subsequent random mating was repeated 5 times. The resulting allele frequency ranged from 0.32 to 0.63. The simulated genome had 10 chromosomes of length 100 cM. The genotyping density was 0.5 marker/cM. A causative marker was assigned to the center of each chromosome with alternating values of positive and negative one.The response variable was evaluated under heritability of 0.25 and 0.50. The ability of FLM to detect major genes was compared to the Bayesian ridge regression and Bayesian LASSO implemented in the R package BGLR , and a mBreeding data are inherently unbalanced. Genotypes are often unreplicated or not equally distributed across environments, and observations from different environments present a variable degree of noise. The single-stage approach was evaluated on simulated datasets that recreated such condition.The simulations were based on assigning the simulated individuals described in the previous section, a genetic pool with 1000 genotypes, to a random set of environments. Each simulated scenario was performed with a combination of number of observations across trials  and genetic architectures . The number of environments for each simulation was sampled from a uniform distribution between 4 and 10 with effect For the simulated scenarios with less than 1000 observations, most genotypes were unreplicated since the observed individuals were sampled from a pool of 1000 genotypes. Selection across unreplicated trials are not unusual when genomic prediction is deployed  Iterative single-stage with Laplace (FLM-SS) and Gaussian prior , implemented in RcppEigen , carryinThe criteria for comparison was the computation time necessary to fit the model as the elapsed time, and the prediction accuracy as the correlation between estimated breeding values and true breeding values.y). GBLUP and Single-Stage fit environment (u) as function of intercept (μ) and marker effects :Two-stage approach:GBLUP:github.com/alenxav/FLM). The FLM-SS code can be made available for research purposes.The soybean data are available in the R package SoyNAM. The R code for FLM is in the appendix. Cross-validation scripts are available on GitHub  is presented in The accuracy of Gaussian prior models (GBLUP and RR-SS) was sensitive to the number of observations when the trait was controlled by a small number of QTL. As the number of QTL increased, the predictive advantage of FLM-SS and two-stages over GBLUP and RR-SS decreased, and GBLUP outperformed the other methods under the scenario with the lowest number of observations.The computation time of GBLUP was considerably larger than iterative single-stage, and two-stage approach yielded intermediate computational performance. The discrepancy in computation time between iterative single-stage and two-stages can be attributed to the MCMC sampling in the second step and the estimation of variance components in the first step.The discussion section frames FLM as a potential method of choice for genomic prediction in plant breeding. The proposed methodology provided accurate prediction across datasets, as well as computational efficiency. Besides the predictive and computational performance, the FLM is an easy-to-implement regression method (Algorithm 1) without the need for tuning or matrix inversion.Most methods provide comparable predictive performance  build the kernel and 2) compute its inversion or Eigendecomposition. The time needed to build the kernel depends on the number of both individuals and parameters. Many kernels require the computation of distance matrices, which is more computationally demanding.For the prediction of new observations, kernels must be augmented with the genotypes of observed and unobserved individuals, making the inversion or spectral decomposition more challenging. This is particularly cumbersome in plant breeding where the size of the offspring being predicted and selected can be much larger than the training set, whereas the parameter estimates required for prediction from regression and tree models can be stored and easily employed for prediction of new observations.The two-stage and iterative single-stage approaches were faster than GBLUP by an order of magnitude. Such difference can be attributed to the sparse nature of the algorithm and the complexity associated to the estimation of variance components. GBLUP was fit using AI-REML, a general-purpose algorithm, whereas FLM-SS was specifically designed to provide efficient computation of breeding models. The two-stages model provided an intermediate outcome.The lower accuracy of the GBLUP and RR-SS in scenarios with few QTL can be attributed to the statistical nature based an infinitesimal model. Gaussian priors work by capturing the relationship among individuals  where thFrameworks where marker effects are estimate alongside all other parameters are not new, but rarely utilized (Multi-stage procedures have been proposed to address the computational burden of single-stage without compromising in the quality of the results . HoweverFuture directions for this research include the comparison of breeding values estimated from GBLUP, multi-stage and RR-SS to understand how the proposed algorithm reproduces standard procedures on real data. An evaluation of the estimated variance components is also a desirable target. Further studies must contrast scenarios with genotype-environment interaction, spatial trends, dominance and epistasis.A robust prediction methodology is a key component for a successful genomic-assisted breeding pipeline. This study introduced a fast and accurate algorithm for solving a WGR with Laplace prior, alongside a single-stage methodology that allows to connect WGR into mixed models with replicated observations.The proposed framework provided more accurate predictions and higher computational efficiency than other methods based on a cross-validation evaluation on real datasets. With a simulated dataset, it was shown that the fast Laplace model provided reasonably accurate estimation of QTL effects, being less biased than Bayesian LASSO and ridge regression, and proving less spurious signals than genome-wide association analysis. The algorithm extension to single-stage also presented promising properties, benefiting both computation and prediction.


This study evaluated an innovative wearable sensor based platform  to quickly quantify frailty phenotypes, without the need of walking test. 61 older adults  were recruited and assessed by Fried Frailty Criteria to determine frailty phenotypes. All subjects participated the iTMT test by standing in front of a computer, wearing a wearable sensor on the front lower shin. The sensor tracked the subject’s ankle movement and projected it on a computer-screen as a moving cursor at real-time. The subject rotated the ankle joint to navigate the cursor to reach 5 indexed circles (including numbers 1-to-3 and letters A&B placed in random orders) in the alternative order of numbers and letters. The iTMT required coordination of brain and body movement, testing subject’s cognitive-motor function. The sensor quantified ankle-rotation biomechanics during the test. All subjects completed the iTMT with average time less than 3 minutes. The ankle-rotation velocity generated during the test distinguished between the presence and absence of Slowness phenotype . The decline of ankle-rotation velocity determined the presence of Exhaustion phenotype . The ankle-rotation power generated during the test determined the presence of Weakness phenotype . The ankle-velocity variability determined the presence of Inactivity phenotype . This study demonstrated the feasibility and validity of the iTMT to quantify frailty phenotypes. This new platform is time-efficient and doesn’t require walking test. It’s more practical for routine assessment in small and busy clinics among patients with mobility limitation.


Predictive ability, unbiasedness and accuracy of predictions from conventional multi-trait G-BLUP were further optimized by using a combined pedigree-genomic relationship matrix. Results of this study suggest that multi-trait genomic evaluation combining routinely measured traits may be used to improve prediction of crop productivity and drought adaptability in grain sorghum.Grain yield and stay-green drought adaptation trait are important targets of selection in grain sorghum breeding for broad adaptation to a range of environments. Genomic prediction for these traits may be enhanced by joint multi-trait analysis. The objectives of this study were to assess the capacity of multi-trait models to improve genomic prediction of parental breeding values for grain yield and stay-green in sorghum by using information from correlated auxiliary traits, and to determine the combinations of traits that optimize predictive results in specific scenarios. The dataset included phenotypic performance of 2645 testcross hybrids across 26 environments as well as genomic and pedigree information on their female parental lines. The traits considered were grain yield (GY), stay-green (SG), plant height (PH), and flowering time (FT). We evaluated the improvement in predictive performance of multi-trait G-BLUP models relative to single-trait G-BLUP. The use of a blended kinship matrix exploiting pedigree and genomic information was also explored to optimize multi-trait predictions. Predictive ability for GY increased up to 16% when PH information on the training population was exploited through multi-trait genomic analysis. For SG prediction, full advantage from multi-trait G-BLUP was obtained only when GY information was also available on the predicted lines  Sorghum bicolor L. Moench) is a cereal crop that can play an important role for sustainable farming, as it is particularly resilient to stress conditions caused by drought and erratic rainfall  can increase the accuracy of genetic evaluations relative to single-trait (ST) analysis by exploiting information from correlated characters . The potGenome-based MT analysis has been applied for breeding in hybrid crops of other major cereals like maize, rice and wheat e.g., . In sorgThe aims of this study were to investigate if multi-trait analysis improves across-environment genomic predictions for grain yield and stay-green in sorghum, and to identify the combinations of traits that optimize results in different prediction scenarios. To attain these objectives, the optimal combination of traits was empirically determined for each scenario by evaluating the gain in prediction quality of alternative MT models relative to the ST model. In addition, we explored if the performance of best-predictive MT genomic models can be further enhanced by incorporating pedigree information.The dataset used in this study is part of the sorghum breeding program of female parental lines conducted by the University of Queensland and the Department of Agriculture and Fisheries in Queensland, Australia. The phenotypic records consisted of 26 testcross performance trials where a total of 646 female lines were tested in hybrid combinations across 12 locations between 2008 and 2014. Phenotypes of 2645 testcross hybrids were used to assess female lines performance across a target population of environments (TPE) covering the main sorghum cropping region of Australia. The series of trials belongs to an advanced stage of yield testing (AYT), where measurements are taken from relatively large plots. More details on field layout and structure of the dataset are given in 1. The sequence data generated were then aligned to the most recent version (v3.1.1) of the sorghum reference genome sequence  methodology involving complexity reduction of the genomic DNA to remove repetitive sequences using methylation sensitive restriction enzymes prior to sequencing on Next Generation sequencing platforms (DArT)sequence  to identsequence  in R  of the i-th female line crossed with the j-th male tester in the k-th environment, which was fitted by a random line effect (Li), a fixed male tester effect (Mj), a fixed environmental effect (Ek), and all possible interactions between these factors. Since line effects were considered random, all the interactions involving Li were random, while MEjk was fixed. Note that LMEijk includes the error of genotype mean. All random effects were assumed independent homoscedastic and normally distributed with zero mean.where i. Moreover, using BLUP(Li) as response variable in the genomic prediction model is problematic because it would result in double shrinkage of predicted breeding values. Therefore, to eliminate shrinkage in line BLUPs before the genomic prediction stage, we applied the deregression procedure of i), as obtained by inverting the coefficient matrix of the mixed model equation  to genomic effects; g is the vector of additive genomic effects with distribution g ∼ N (where the vector BLUP(Li) .h2) of line means were obtained as: Given the variance components from the ST prediction model, the narrow-sense heritabilities  and (2) were extended using multivariate mixed models. We present a general formulation for any combination among traits T = . The multi-trait case of model (1) can be represented in vector notation as:yijk is a vector collecting spatially adjusted BLUEs of multiple traits T from separate univariate analyses in stage one; and Li, LMij, LEik, LMEijk are the vectors of multi-trait random effects with respective assumed distributions Li ∼ MVN, LMij ∼ MVN, LEik ∼ MVN, LMEijk ∼ MVN, where Σq, for q = L, LM, LE, LME, is a covariance matrix among traits and ⊗ is the Kronecker product operator. For all random effects, the matrix Σq was modeled as unstructured, allowing for unequal variances across traits and specific covariances for each pair of traits.where in this case The multi-trait G-BLUP model was defined as:yT is now a multi-trait vector of deregressed BLUP(Li), ordered as lines within traits, obtained from joint multivariate analysis using model (3); gT is the vector of multi-trait additive genomic effects with distribution gT ∼ MVN; and the multivariate residual effects were assumed eT ∼ MVN, where matrix DT has diagonal elements containing weights based on individual reliabilities of deregressed BLUP(Li) for each trait, as obtained from the second stage of multi-trait analysis (model 3). The covariance matrices among traits for additive genomic effects (Σg) and residuals effects (Σe) were assumed unstructured.where the subscript refers to traits T; K = wA + (1 − w)sG, where A is the numerator relationship matrix among lines computed from the full pedigree data and sG is a scaled matrix G that is compatible with A in reference to the base breeding population , and collects additive genomic effects as well as residual polygenic effects. Under our approach, the optimal w is empirically determined in order to maximize predictive ability after evaluating a sequence of candidate weights within the range 0 < w < 1. To assess the benefits of including additional pedigree information, the best-predictive multi-trait model using conventional G-BLUP  was compared to the equivalent optimized multi-trait K-BLUP model.We also considered multi-trait prediction models fitting a kinship matrix that combines pedigree and genomic information. For this, we extended the BLUP method advocated by pulation . The wei2. Uni- and multivariate models in the second and the genomic prediction stages were fitted by REML using the average information algorithm as implemented in ASReml-R  and other auxiliary characters are used to potentially improve predictions of the target trait. Predictive performance of MT models was evaluated through fivefold cross-validation, where 80% of lines were randomly assigned to the training lines set (TLS) and the remaining 20% formed the validation lines set (VLS). We explored two potential scenarios for MT prediction . One sceThe first MT prediction scenario was studied for two cases: when GY or SG were the traits of interest. However, the Aux@TL + VL scenario was considered only for SG as target trait. Given that one of the main purposes of the trial series used in this study is to obtain representative measurements of GY productivity, the fact that lines are field-tested for other traits, but not for GY, would imply atypical circumstances seriously affecting GY during an experiment. Moreover, extremely reduced or aborted grain production affects normal canopy development; hence, data of traits such as PH or SG should be discarded or considered with extreme caution. For these reasons, the Aux@TL + VL scenario was not considered when GY was the target of prediction, as it would represent an unrealistic or exceptional situation in the context of an advanced stage of sorghum testing.yT and y were used to validate rPA) between yT (y). Unbiasedness of genomic predictions was measured by the regression coefficient (b) of yT (y) on b = 1 indicates an empirically unbiased predictor. Accuracy of predictions was assessed by computing the mean squared error of prediction (MSEP) from the linear regression. The evaluation of prediction models was based on average values over 20 replicates of each cross-validation scenario using the same random seed for all models.In each prediction scenario, the performances of all possible MT models combining two, three, and the four traits were compared to that of the ST model, with the latter taken as benchmark. The response variables used for validation of genomic predictions were consistent with the models applied for analysis: h2. The heritability of SG was higher than that of GY, but lower than those of PH and FT. Additive genetic correlations were significant only for GY with SG and PH, while FT was statistically uncorrelated with the other traits. Significant residual correlations were estimated between GY and the rest of the traits as well as between PH and FT.The narrow-sense heritability estimates using ST G-BLUP are given in b) for all models were below 1 for GY, which indicates overestimation of genomic predictions. The average empirical bias of GY predictions from the ST model was slightly reduced only by the MT model including PH alone as auxiliary trait. The relative decrease (%) in MSEP by MT models was consistent with their increases in predictive ability; MT G-BLUP exploiting PH information had a greater impact on improving accuracy of genomic prediction for GY. When SG was the target of prediction, ST G-BLUP outperformed MT G-BLUP models in predictive ability. Even though prediction models incorporating GY data in the TLS reduced the absolute deviation of b from 1, these models tended to over-predict additive genetic values for SG (b < 1). The inclusion of auxiliary traits in TLS produced higher MSEP relative to using only SG data for prediction.Different results were found for SG predictions when MT G-BLUP models used auxiliary trait records on both TLS and VLS . Under tK for optimization of multi-trait genomic prediction. K using w = 0.25) for all evaluation criteria to a marginal extent. Under the prediction scenario for SG, bias and MSEP from multi-trait models were slightly higher when K (using w = 0.30) was used instead of G. However, an additional 10% gain in predictive ability was obtained by MT K-BLUP in this scenario.After evaluating predictive performance of G-BLUP method, we explored the use of a combined pedigree-genomic matrix per se. Multi-trait BLUP can be seen as a generalized (linear) selection index, with the additional abilities to account for unbalanced information from any set of relatives while properly adjusting for fixed effects in the data . This is in line with the idea that genetic (genomic) similarities between relatives are actually trait-specific (G are derived from posterior trait-specific (co)variance estimates of SNP effects, which are obtained from a previous Bayesian analysis. The authors found, based on simulations, that the benefits of their weighting method were generally not significant for the low-heritability trait that more closely fitted an infinitesimal model (using 500 QTLs). Alternatively, our multi-trait K-BLUP uses a more straightforward approach that assumes a common weight across the genome, while still relaxing the assumptions of conventional multi-trait G-BLUP by allowing the similarity matrix to vary across traits in order to optimize prediction of the target. Given the complexity of traits and the highly structured breeding population used for this study, genomic predictions are likely to rely more on familial relationships and less on information from specific SNPs in LD with QTL (Here, we used a different optimal weight specific , which cwith QTL . In thiswith QTL .While genotyping costs are being constantly reduced, the efficient use of phenotypic data becomes more relevant for plant breeding programs, since field phenotyping is still costly and labor-intensive. This work has shown for the first time how phenotypes of several traits routinely measured in grain sorghum breeding can be efficiently utilized through multi-trait analysis to assist genome-based selection of a target trait. We demonstrated empirically that genomic prediction of parental breeding values for GY benefits mainly from using PH as auxiliary trait. This trait is particularly promising in practice, since PH phenotype can be potentially measured in all trials, as opposed to SG, which can only be phenotyped under specific environmental conditions. In addition, high throughput field phenotyping technologies are expected to increase accuracy of PH measurement in sorghum , which cBased on findings from this study, when SG is the target of prediction, most advantage from multi-trait genomic analysis can be obtained only if predicted lines have been phenotyped for GY. This may be beneficial when the interest is to predict the genetic aptitude for drought-adaptation in lines that have been field-tested for GY performance but could not yet experience post-flowering water stress. Such scenario is compatible with selection for broad adaptation across the TPE, which is generally the best approach to deal with the largely unpredictable occurrence of drought in the Australian sorghum region . Expressper se since, in this case, direct information from SG-GY genetic correlations is exploited predictively through multi-trait analysis. This article also shows, for the first time in plant breeding, how a similarity matrix using trait-specific combinations of pedigree and marker-based relatedness can further enhance multi-trait genomic prediction. Collectively, our results imply useful properties of multi-trait BLUP to evaluate alternative prediction schemes for genetic improvement of crop productivity and drought adaptability in grain sorghum. Given that the traits considered in this study are commonly measured in cereal breeding programs, findings presented here can be also relevant for practical implementation in other major crops.This study demonstrates, based on extensive breeding data, that there is potential to improve genome-based predictions of grain yield and stay-green traits in grain sorghum by using multi-trait genomic analysis. Results suggest that better predictive abilities and accuracies for GY prediction are obtained when PH information on the training lines is included in multi-trait genomic models. When SG is the target, the quality of predictions is likely to improve only if GY performance data is available for both the training and the predicted lines The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.JV, DJ, MM, and FvE designed the research. JV performed the statistical analyses and wrote the manuscript. DJ and FvE edited the manuscript. DJ coordinated the experiments and data collection. CH and EM processed and prepared the dataset. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Prostate cancer is the most common male cancer with one in four developing non-curable metastatic disease. Initial treatment responses to hormonal therapies are transient and further management options lie between (1) further hormone therapy or (2) a non-hormonal approach involving additional chemotherapy or molecular radiotherapy (radium-223). There is no clear rationale for choosing between these mechanistically different treatment approaches. The biology of hormone resistance is driven through abnormal androgen receptor activity and we can assay this through a blood test measuring androgen receptor variant 7 (AR-V7) expression in circulating tumour cells. Despite increasing evidence supporting AR-V7’s role as a prognostic marker, the clinical utility of such measures remains unknown in helping personalise treatment decisions.The VARIANT feasibility trial is a pragmatic design, to be run over 18 months with participants randomised into the intervention arm receiving biomarker (AR-V7) guided clinical treatment and participants randomised into the control arm with conventional standard management (no biomarker guidance). AR-V7 positive participants (likely to be insensitive to further hormone treatment) will receive chemotherapy or in other cases radium-223 (where routinely available). Seventy male ≥18 years old participants with metastatic castrate resistant prostate cancer clinically indicated to proceed to further hormone therapy or chemotherapy, will be recruited from three National Health Service Trusts based in England, Scotland and Wales. The feasibility primary outcome is willingness of patients to be randomised and clinicians to recruit to a biomarker-based treatment strategy, with trial data informing the basis of a definitive and appropriately powered randomised control trial.Formal ethics review was undertaken with a favourable opinion, through Wales NRES Committee 2 18/WA/0419. Findings to be disseminated through patient and professional organisations that have expressed their support, media outlets and peer-reviewed journal publication.ISRCTN10246848; pre-results Focuses on a priority area of need in advanced prostate cancer clinical practice.To date, the feasibility of delivering a randomised biomarker guided-treatment trial in prostate cancer to formally assess clinical utility is not established and will be addressed in this study.As a feasibility study, the planned sample size (70 participants) does not have sufficient power or precision to compare the ‘event’ rate between treatment arms, but will allow informed planning for a definitive randomised controlled trial with prominent clinicians from non-recruiting centres involved in feasibility, to aid with follow-on trial.Emerging evidence points to additional androgen receptor biology driving hormone resistance, such as other variant expression and mutations — these along with alternative biomarkers can be explored in the associated biobanked samples (including cell-free tumour DNA).Strong patient and public involvement (PPI) to inform study design with a clear commitment to informing participants of project outcomes, setting a clear new gold standard for PPI.Prostate cancer (PC) is the most common male cancer in the UK and the second highest cause of male cancer death.Medical castration ), blocks production of the hormone testosterone and/or targets the androgen receptor (AR) signalling axis that drives cancer cell growth. Although a good response to hormonal treatment seen often initially, disease progression to a lethal metastatic castration-resistant prostate cancer (mCRPC) is common.Management pathways for mCRPC are still evolving in response to emerging new treatments; however, it broadly follows one of two standard care approaches;A breakthrough in understanding the biology of PC revealed that hormonal treatments generate a selective pressure at the cellular level inducing complex molecular mechanisms characterised by an adaptation of the AR signalling axis. This results in tumour resistance mediated by the induced expression of alternative types of androgen receptor. These AR messenger RNA (mRNA) splice variants lack the important hormone-binding domain, resulting in a constitutively active cellular receptor, despite castration. The most widely studied variant is androgen receptor variant 7 (AR-V7).Published clinical data demonstrates a strong link between AR-V7 expression and mCRPC progression and highlights the potential for AR-V7 to be utilised as a treatment stratification biomarker to identify those men likely to be sensitive to further hormonal treatment (AR-V7 negative patients) and avoid futile treatments in those predicted to be insensitive (AR-V7 positive patients).Encouragingly, a cost saving analysis of performing AdnaTest ProstateCancerPanel AR-V7 biomarker testing in mCRPC demonstrated use of the biomarker would result in a substantial cost saving as long as the true prevalence of AR-V7 was >5% .The National Comprehensive Cancer Network Task ForceThe treatment landscape of hormone sensitive (PC) is evolving, altering treatment pathways for mCRPC. Recent data from the USA based PROPHECY trial reporting on a prospective observational cohort showed mRNA AR-V7  and protein AR-V7  biomarker positivity associated with worse progression free survival and OS in poor prognosis patients with advanced prostate cancer who receive abiraterone or enzalutamide treatment.We argue irrespective of the evolving treatment landscape, the opportunity to generate feasibility data for a biological (biomarker) informed approach to treatment selection over standard care protocol-based approaches, tests a highly relevant clinical question in these high risk CRPC patients . This would provide an appealing long-term strategy (for patients and service providers) to ultimately improve on clinical outcome .To determine the feasibility of conducting a definitive randomised control trial to evaluate the clinical utility of an AR-V7 blood biomarker assay in personalising treatment for men with mCRPC in UK NHS clinical practice.Feasibility study.To establish if it is feasible to conduct a definitive trial comparing AR-V7 biomarker-driven management with the current standard care in patients with mCRPC.To estimate AR-V7 biomarker prevalence in the trial population to inform sample size calculations for a definitive randomised control trial.To assess recruitment, compliance and retention rates.To confirm outcome measures for a future definitive trial and establish trial data response rates, variability and data quality.To establish a blood sample biorepository to include baseline, 12 and 24 week blood samples for future translational studies.To establish a complete serial blood tissue archive to include potential measures of cell-free DNA (cfDNA) and additional AR variants in CTCs and cfDNA biomarker measures to complement AR-V7 reads , depending on the ultimate biomarker performance characteristic established in this trial population. Blood will be collected, processed and archived at 0 weeks (baseline), 12 weeks and 24 weeks following the first treatment.To explore thresholds of the magnitude of AR-V7 positivity to investigate relationships with outcomes and estimate AR-V7 positivity rate assumptions regarding a cut-off point.To undertake cross-site validation of biomarker reads between two GCP laboratories (Newcastle University and Cardiff University).This feasibility study is a multicentre, two-arm, randomised control trial (RCT). All patients who consent to take part in the trial and who are eligible, will have a blood test to assess prevalence rate of the AR-V7 biomarker. Participants will be randomised in the ratio 1:1 to receive personalised standard treatment (intervention) guided by AR-V7 biomarker status or standard care (control) without biomarker-guided treatment. Those in the control group will not receive blood biomarker test results.The treatment for each patient is expected to be dependent on various factors , as well as randomised allocation and AR-V7 status in the personalised treatment arm. All treatments are part of standard care for these participants. Treatment options for participants randomised to the personalised standard treatment arm will be recommended, but not mandated within this feasibility trial, with reasons for not following the recommendation recorded and reported as outcome. A Consolidated Standards of Reporting Trials diagram of study protocol  is shown in Seventy patients with mCRPC who require a change in treatment will be recruited in three secondary care NHS Trusts in the UK spread across England , Scotland (NHS Greater Glasgow and Clyde) and Wales (Velindre University NHS Trust). We aim to recruit mCRPC patients with a predicted poor overall survival. We anticipate this group of mCRPC patients have the most to gain from a biological-based treatment approach as their disease is more likely to progress during a period of treatment with an inactive agent. Multivariate analysis from the metastatic population of STAMPEDEPatients will be aged ≥18 years old with metastatic castrate resistant prostate cancer (high risk features) clinically indicated to proceed to further hormone therapy or chemotherapy and fulfil all of the following criteria:Histologically or cytologically proven diagnosis of adenocarcinoma of the prostate.Radiographic and/or histological and/or cytological evidence of metastatic disease.Castrate levels of testosterone and documented ongoing medical or surgical castration. Testosterone level ≤50 ng/dL/1.73 nmol/L and maintaining on androgen suppression therapy.Disease progression since the last change in therapy defined by one or more of the following: (i) PSA progression as defined by the prostate cancer working group 3 criteria ≥2 ng/mL; (ii) bone disease progression as determined by the local radiology/multidisciplinary team; (iii) radiographic progression of nodal or visceral metastases as determined by the local radiology/multidisciplinary team.Suitable for treatment with at least one novel hormonal treatment  and one non-hormonal therapy .At least two high risk features: (i) age <60 years at time of diagnosis of metastatic disease; (ii) bone metastases present at time of initial metastatic prostate cancer diagnosis ; (iii) Gleason grade group 4 or 5 (Gleason score 8 to 10); (iv) presence of visceral metastases  at any time point. This does not include lymph node metastases; (v) PSA doubling time <3 months; (vi) elevated alkaline phosphatase above institutional upper limit of normal; (vii) Eastern Cooperative Oncology Group (ECOG) Performance Status worse than or equal to 1; (viii) previous treatment for castration resistant prostate cancer with docetaxel chemotherapy; (ix) previous treatment for castration resistant prostate cancer with abiraterone and/or enzalutamide or equivalent agent.Estimated life expectancy >6 months.Provision of written informed consent, including consent for biobanking of blood samples.Histological variants of prostate cancers with small cell or neuroendocrine features.Prior or current malignancy (except adenocarcinoma of the prostate) with an estimated ≥30% chance of relapse/progression within next 2 years.Previously identified brain metastases or spinal cord compression unless treated with full functional recovery.Administration of an investigational agent within 30 days of first dose of trial medication.Patients will be randomised to receive either personalised standard treatment (guided by AR-V7 biomarker status) or standard care (not guided by biomarker status) on a 1:1 basis using a method of random permuted blocks of concealed variable block size and stratified by site.This three-centre randomised feasibility study incorporates a control and an intervention arm. All patients will undergo AR-V7 biomarker assessment with results only made known to the patients and clinical team in the intervention arm.Treatment will be given as per standard care with recommendations guided by biomarker status; [1] if the participant is found to be AR-V7 positive, then non-hormonal treatment is recommended  or; [2] if the participant is found to be AR-V7 negative, then next generation hormonal treatment is recommended .The results of the AR-V7 biomarker assessment will be provided securely to the clinical team to enable tailored treatments based on AR-V7 expression from the biomarker result. Where a decision is made that the participant will receive a non-recommended therapy (either by the clinician or patient) this therapy, and the reasons for giving this, will be documented.Participants with their clinical care team will make an informed and preference-based decision to receive standard care, including either next generation hormone treatments abiraterone or enzalutamide or non-hormonal approaches including docetaxel or cabazitaxel chemotherapy or radium-223. Details of all treatment administered, including doses, will be recorded as part of the trial.The research team at sites will not receive the participants AR-V7 biomarker results.Standardised clinical assessment tools used in monitoring CRPC disease and progression on treatment will be reported .Clinical progression and survival within 6 months; (i) Number of patients who have progressed clinically at 6 months (includes change of systemic anti-cancer therapy and death from prostate cancer); (ii) Cancer specific survival at 6 months and (iii) overall survival at 6 months.Quality of life for patients with cancer (EORTC QLQ-C30 Questionnaire).Additional quality of life items patients with prostate cancer (EORTC QLQ-PR25 Questionnaire).Participant costs questionnaire .The proportion of prostate cancer patients identified through clinics who meet the eligibility criteria.The number of patients accrued per site per month over the course of the trial.Baseline prevalence of AR-V7 expression in the participant cohort .The willingness of patients to be randomised .Compliance rate (this will be defined as the number of patients who start randomised treatment as a proportion of the number randomised).The proportion of patients who: start AR-V7 recommended treatment; start treatment other than the recommended treatment; change treatment before disease progression or withdraw. (This measure will capture information regarding patients who choose not to take recommended treatment because of strong preferences and patients who progress rapidly while waiting for treatment with a change in eligibility for treatment options).The proportion of trial participants with assessable blood samples for biomarker status (which would affect treatment targeting).The median time from the blood sample being drawn to; (i) AR-V7 result being sent back to the site and (ii) patient starting treatment (and compared with standard of care treatment).The proportion of randomised patients for whom data is collected on each clinical and health economic outcome at baseline, 12 and 24 weeks.Further information on recruitment, screening, the patient consent procedure and informed consent literature, can be found in the 10.1136/bmjopen-2019-034708.supp1Supplementary dataA validated two-centre pipeline  to measure AR-V7 biomarker using the commercially available AdnaTest ProstateCancerPanel AR-V7 circulating tumour cell quantitative RT-PCR assay , has been set up according to assay manufacturers recommendations, analytical methods and sponsor agreed Standard Operating Procedures (SOP's). Following biomarker data analysis and data verification, for participants randomised to the intervention standard treatment arm (AR-V7 biomarker guided), the baseline biomarker result and biomarker treatment recommendation will be sent securely within 10 working days to the local principal investigator and delegated research staff. Further information on the specifics of AR-V7 biomarker driven personalised treatment , can be found in the Analyses will be conducted on an intention-to-treat basis, with sensitivity analyses used to investigate the impact of non-compliance to allocated arm. Given the feasibility status of this study, all statistical analyses will be descriptive. The majority of the outcome data will be presented in simple descriptive tables presenting percentages, means and SD or five-number summary (as appropriate), for each arm of the study. Analysis of clinical and biomarker measures will be assessed by; [1] clinical progression and survival within 6 months; [2] PSA response/progression (confirmed rising PSA more than 12 weeks after randomisation); [3] clinical progression and survival  within 6 months (includes change of cancer therapy for progression) and; [4] survival  estimates will be derived using the Kaplan-Meier method and presented as 6 month rates with CIs. The relationship between survival estimates and continuous AR-V7 biomarker expression will be modelled considering non-linear transformations in a univariate Cox model, or parametric alternative, presented as parameter estimates (HR) with CIs.Compliance with quality of life and health economic measures will be assessed by; [1] number of patients completing measures as a proportion of the number randomised and; [2] degree of completeness of each domain of the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC-QLQ) and economic questionnaire measures. These scores will be presented graphically and with numeric descriptive statistics.et al.This trial is designed as feasibility trial according to definition of Eldridge The performance of potential outcome measures for a definitive trial will be assessed by estimating data completeness of the instruments and any potential bias in the completion of follow-up data. This information will be used to inform the design, choice of outcomes, necessary sample size and approach to the analysis, of a future definitive trial.This is a low risk trial and no specific safety reporting is required. Should an Investigator have any concern regarding participant safety as an outcome of their participation in the trial, they will contact the Trial Management Group (TMG) and Chief Investigator as soon as possible. The Trial Oversight Committee (TOC) will monitor concerns as required.The trial will be conducted in accordance with the UK Policy Framework for Health and Social Care and as applicable, the Guidelines for Good Clinical Practice. The TMG is responsible for the day-to-day management of the trial, overseeing all aspects of the conduct of trial to ensure that the protocol is adhered to and take appropriate actions to ensure patient and data safety. The TOC will review trial conduct and accumulating clinical trial data and provide overall supervision for the trial on behalf of the Sponsor and the Funder. The constitution of the TMG and TOC including roles and responsibilities delegation for this trial can be found in the The design, planning and management of this trial has been supported by two prostate cancer patient representatives (co-applicant on the funding grant and TMG member). Both have advocated the dissemination of trial findings to patients and ensured that the public was adequately considered during trial design. PPI has been embedded into the study, with the patient’s voice a strong theme to inform and influence the ongoing research and development of participant information resources in collaboration with the ‘Cancer Perspectives’ patient representative group . A strong commitment is to inform the participants of the outcome of this project, a clear new gold standard for PPI.All parties will conduct the trial in accordance with ethical opinion. No amendment to protocol will be made without consideration and approval by the Trial Management Committee.Feasibility data will be published as a peer-reviewed article and if successful, these findings will contribute to gaining further funding for a HTA full trial. In addition, assessing clinical data and blood derivatives from the participant cohort will provide valuable material (circulating tumour cells transcript and plasma circulating tumour DNA), to validate translational studies of other AR aberrations and hormone targeting resistance pathways (or the emergence of biomarkers for chemosensitivity), to inform and contribute further to the rapidly evolving treatment developments for CRPC. Participants will remain anonymised in all publications.We will also use dissemination through patient and professional organisations that have expressed their support for this trial  and through media outlets including web resources, lay press, academic national and international conferences and peer-reviewed journal publication.


In a multifrequency phase-shifting (MFPS) algorithm, the temporal phase unwrapping algorithm can extend the unambiguous phase range by transforming the measurement range from a short fringe pitch into an extended synthetic pitch of two different frequencies. However, this undesirably amplifies the uncertainty of measurement, with each single-frequency phase map retaining its measurement uncertainty, which is carried over to the final unwrapped phase maps in fringe-order calculations. This article analyzes possible causes and proposes a new absolute depth measurement algorithm to minimize the propagation of measurement uncertainty. Developed from normalized cross-correlation (NCC), the proposed algorithm can minimize wrong fringe-order calculations in the MFPS algorithm. The experimental results demonstrated that the proposed measurement method could effectively calibrate the wrong fringe order. Moreover, some extremely low signal-to-noise ratio (SNR) regions of a captured image could be correctly reconstructed (for surface profiles). The present findings confirmed measurement precision at one standard deviation below 5.4 µm, with an absolute distance measurement of 16 mm. The measurement accuracy of the absolute depth could be significantly improved from an unacceptable level of measured errors down to 0.5% of the overall measuring range. Additionally, the proposed algorithm was capable of extracting the absolute phase map in other optical measurement applications, such as distance measurements using interferometry. Precise absolute depth measurement remains one of the most important challenges in 3D surface profilometry because most 3D shape-measuring systems trade accuracy for depth range. With a significant increase in surface discontinuity, such as step height, measurement accuracy and precision cannot be guaranteed to satisfy engineering requirements. One of the most popular 3D measurement methods in industries is structured light projection, in which a structured light is projected onto an object’s surface to obtain the optical path difference (OPD) of the detected object point. The OPD is then captured by an imaging sensor in a triangular optical configuration. The deformed structured pattern can be further decoded to extract the OPD using phase-shifting or other spatial wrapping algorithms. Among the existing fringe projection techniques, phase-shifting profilometry  and FourTo resolve the problem of phase ambiguity, temporal phase unwrapping was developed by Saldner and Huntley . This apThis article focuses on the multifrequency phase-shifting (MFPS) technique, which is the most widely used in industrial applications. Although this technique can avoid phase ambiguity, it does have a critical drawback. The MFPS technique uses a combination of several phase maps with different fringe pitches; hence, measurement uncertainty is amplified and affects the final result. Each phase map of a single frequency has phase noises, which will lead to 3D measurement errors. An example of measurement uncertainty propagation after fringe-order calculation is illustrated in In the MFPS technique, fringe order is calculated using the synthetic fringe pitch. The measurable range is extended after a combination of different pitches. The uncertainty of the synthetic pitch is also amplified in fringe-order calculations. This amplified uncertainty affects the 3D measurement results from fringe-order calculations. To minimize the amplification of measurement uncertainty, a new absolute height calibration technique is proposed. The calibrated absolute height will be employed to extract the fringe order with minimized uncertainty.The remainder of this article is organized as follows. In fringe projection profilometry (FPP), the distortion of fringe is estimated using a phase distribution map. Many techniques can extract the phase distribution of distorted fringes: the best-known ones are phase-shifting profilometry (PSP), wavelet-transform profilometry (WTP) ,17, and The wrapped phase map can be extracted by resolving at least three different phase-shifted equations (Equation (1)). The wrapped phase map is calculated using Equation (2):h” and “l” denote “high frequency” and “low frequency”, respectively). The relation between the unwrapped phase map and the wrapped phase map of high frequency can be expressed by Equation (4):When the multifrequency fringe projection is employed, the wrapped phase can be unwrapped from the coarsest to the finest fringe pitch . In the Similarly, the relation between the unwrapped phase map and the low-frequency wrapped phase map can be formulated as Equation (5):The relationship between the low- and high-frequency fringe pitches can be expressed as Equation (6):The ratio, K-frequencies is unique [There are four main strategies for temporal phase unwrapping ,23,24,25s unique . A lookud phases . Furtherd phases , the synThe synthetic phase map is generated from the wrapped phase difference between low- and high-frequency phase maps and can be expressed as Equation (8):N patterns can only encode The final unwrapped phase map will be extracted by applying Equations (4)–(6) when  speckle ,29. In tTo understand how various measurement errors encountered in PSP impact phase measurement accuracy, the measurement uncertainties in PSP were analyzed and modeled. In the analysis, the sensor noise variation, ariation ,30. In gariation ,33,34,35INT is the integer operation (because fringe order must be an integer number).Phase shifting is independently evaluated on a single pixel; hence, image coordinates hase map  and can INT operation in Equation (12) fails. Consequently, fringe-order error can be quantified using Equations (11) and (12):It is important to note that a fringe-order error will occur if the From the above derivation of the fringe-order calculation error, it can be concluded that the measurement uncertainty of multifrequency fringe projection is excessive and cannot be ignored. This study analyzes the measurement uncertainty and proposes a normalized cross-correlation (NCC)-based calibration method to effectively reduce the uncertainty of fringe order, thus enhancing the measurement accuracy of multifrequency fringe projection.The unambiguous phase range is extended by transferring the measurement range from the short to the synthetic fringe pitch. However, each single-frequency phase map retains measurement uncertainty, which is seen in unwrapped high-frequency phase maps with fringe-order calculations. In addition, the synthetic fringe pitch is amplified after the combination of different fringe frequencies. This process also amplifies the measurement uncertainty of involved single-frequency phase maps. In this paper, the phase noise model was derived by following the phase noise model of temporal phase-unwrapping algorithms, which was proposed by Zuo et al. in 2016 . MeasureThe proposed method aims to eliminate the amplified ratio Assuming that the stored reference phase K different frequencies to the matching score after NCC calculation. The final propagated measurement uncertainty is expressed asSimilarly, the uncertainty is propagated from the wrapped phase of Equation (17) can be further expressed asWith the inequality of Cauchy–Bunyakovsky–Schwarz , EquatioFrom Equations (15) and (16), the limits of From the above equations, the following inequality can be expressedBy substituting Equation (22) into the first element in the bracket of Equation (19), Equation (23) can be formed:In addition, the righthand side of Equation (19) can reach its maximum when By further substituting Equations (24) and (25) into Equation (19), the final propagated phase uncertainty can be expressed asK-frequency phase maps before being saved to the reference database. To realize this, a median filter with a 3 × 3 mask  can be employed to effectively minimize the random noise of K-frequency phase maps.Equation (26) indicates clearly that the distorted phase uncertainty varies with the uncertainty of the wrapped phase H when the reference plane is shifted perpendicularly to the optical axis of the sensor lens for Q layers with a height pitch N-step phase-shifting image bucket is performed in a time-averaging manner multiple times (M) to minimize random phase errors (in which M is set at 30 or more). In general, phase noises may be introduced from lighting illumination, sensor noises, and other possible environmental sources. The phase maps of K-frequencies are calibrated and obtained from each individual fringe projection and are stored in the reference database, as shown in K-frequency sinusoidal fringes are sequentially projected onto a tested object with an arbitrary surface profile, PSP is performed for each individual fringe to measure and determine the K-frequency phase maps.The proposed method is generally applicable to any multifrequency fringe projection with two or more different frequencies. In a general FPP-based system, the reference plane is aligned perpendicularly to the optical axis of the imaging lens in front of the sensor and can be vertically shifted to determine a precise depth to be calibrated. To ensure good quality of the captured fringe image, it is important to keep the measurable range strictly within the overlapped depth-of-field (DOF) between the sensor lens and the projection lens in the FPP system. In the experimental system setup, the maximum measurable range is defined as The proposed method works only when the uniqueness of the set of all wrapped phases in the multifrequency fringe projection has a phase value from 0 to Equation (27) presents a one-to-one relation between eference .To demonstrate that each wrapped phase of the synthetic fringe is unique, an example with a fringe pitch of two different frequencies is considered, in which d unique . The conK-frequency fringe projection is digitally correlated with the corresponding reference phases stored in the database, K-frequency phase-shifting images is acquired, in which the phase maps of every single frequency are calculated. The wrapped phase maps are employed to calibrate the absolute height using the proposed NCC-based method. The final unwrapped phase map can be reconstructed using the relationship in Equation (14), in which the absolute phase can be reconstructed accurately with the extracted fringe order When an absolute height is determined using the above method, an absolute phase map can be further reconstructed using the calculated fringe order and the wrapped phase map. The absolute height is employed to compute the fringe order for phase unwrapping. To achieve this, a phase-to-height calibration process is performed to accurately determine the mapping relationship between the absolute phase difference and the surface absolute depth. Equation (3) presents the relationship between the absolute phase and the surface absolute depth. Before transferring the absolute phase to the height, the phase discontinuity existing in the wrapped phase map has to be recovered by compensating for the missing fringe order, The fringe order can then be expressed:The final absolute phase map is transferred to the object’s height using phase-to-height calibration coefficients, which are estimated in the system calibration process.Using Equations (28) and (29), the fringe-order uncertainty of the proposed method can be further expressed asH = 10 mm, and one shifted step of the reference plane was Q = 100 layers stored in the database. A high accurate ceramic plate (flatness and roughness <5 µm) was chosen to build the database for absolute height calculations.During the experimental measurements, a projector projected sinusoidal patterns onto an object surface, and distorted fringe patterns were acquired by a sensor see a. To buiTo ensure the accuracy of this database, the reference plane was mounted onto a precalibrated accurate linear stage with a minimum incremental motion of 0.1 µm and an accuracy of 0.8 µm. The travel range of the linear stage was 25 mm, which covered the entire measurable range of the system. On the imaging side, a telecentric lens with a DOF of 16.7 mm was chosen. A step-height target was measured to evaluate the precision and accuracy of the proposed absolute height calculation technique. A step-height target was made of stainless steel through a strictly controlled grinding process. The parallelism, flatness, and step-heights of the target were measured using a Mitutoyo CMM for accurate estimation of the optical probe. The final absolute phase map was extracted by combining the calculated fringe order and the wrapped phase of the finest fringe projection, thus achieving high depth resolution and absolute depth measurement simultaneously. To determine the measurement accuracy of the proposed method, tests were performed on a step-height target and on industrial parts, including a cell phone button, screws (shiny and black), and an electrical connector. The measured results obtained using MFPS and NCCMFPS were analyzed and compared.To estimate the accuracy of the proposed method, the average step heights between The absolute distances between the centers of the three different spheres were extracted using their measured point cloud. When testing the feasibility of the proposed method, a cell phone button was chosen to represent an arbitrary shape. The most challenging problem using the FPP technique was nonuniform reflectivity and the high curvature of the measured object’s surface, which appears in In this article, new absolute height and fringe-order calculations developed from multiphase NCC, which effectively minimizes fringe-order error in MFPS, was proposed. Measurement uncertainty was successfully minimized by eliminating amplified ratios in the fringe pitch synchronization. The experimental results showed that the proposed measurement method could effectively calibrate fringe-order errors caused by the MFPS technique. The accuracy of the proposed approach was estimated using absolute distances in the center space of three spheres. The measurement results showed that the measurement accuracy of absolute depth could be significantly improved from an unacceptable level of measured errors down to 0.5% of the overall measuring range. Additionally, the proposed algorithm was also capable of extracting the absolute phase map in many different applications using multiphase maps for measurements, such as structured light projection, interferometry, and optical inspection.


Thus, this approach solves the requirement for mutated proteins to render the tumor cell visible to T cells. Although the choice of potential microbial antigens that can be inserted in AgAbs is practically unlimited, antigens derived from herpes viruses have multiple advantages. Epstein-Barr virus in particular latently infects 90% of the population, which guarantees the presence of EBV-specific T cells in a large proportion of cancer patients. Furthermore, the lifelong persistence of the virus in infected individuals guarantees that these T cells are present in substantial numbers at any age.Tremendous progress has been made in the field of cancer immunotherapy in the past few decades. The introduction of rituximab in 1997 marked a major milestone in the treatment of B-cell lymphomas and initiated the era of recombined anti-cancer antibodies. More recently, antibodies have been armed with cytotoxic drugs or radionuclides to increase their therapeutic potential or have been modified to enhance the immune response against tumor cells. The latter approach includes the development of bi-specific antibodies and of CAR-T cells that have since proven their efficacy in the clinic. Antibody-antigen conjugates represent another class of modified antibodies that were initially designed for vaccination purposes in various types of cells, including dendritic cells . Anotherin vitro. These AgAbs were efficiently internalized and processed, leading to the effective presentation of epitopes on the surface of tumor cells and resulted in efficient T-cell mediated killing. The recognition and killing rates were far superior to the response elicited by the same EBV peptides in stoichiometric proportions [ex vivo. The versatility of the AgAb system allows the incorporation of multiple EBV epitopes that elicit a T-cell response in the majority of EBV-infected individuals. We have recently shown that this approach is feasible and efficient for patients with chronic lymphocytic leukemia (CLL). AgAbs carrying multiple epitopes from the strongly immunogenic EBNA3C protein successfully expanded EBNA3C-specific CD4+ T cells in 12 blood samples from CLL patients [We previously demonstrated that AgAbs directed against CD19/CD20/CD21/CD22 combined to HLA II-restricted epitopes encoded by the Epstein-Barr virus genome can eliminate multiple Burkitt's lymphoma cell lines portions . As expepatients . NonetheThese results offer another example of the cytotoxic potential of CD4+ cells that share typical gene expression characteristics with CD8+ T cells and whose differentiation is dependent on CRTAM expression . CD4+ CTIn summary, antigen-armed antibodies represent a new class of immunostimulatory molecules with a great potential in cancer immunotherapy. A great advantage of the strategy is its flexibility as antibodies recognizing a broad spectrum of targets can be easily created, tailoring the therapy to changes and differences in antigens expression. The application of AgAb strategy can extend beyond lymphoid malignancies, provided that they can be tagged by antibodies and express MHC class II molecules. The potential of these molecules is currently being tested in animal models before the inception of clinical trials.


Chronic suppurative otitis media is a global middle ear disease with quality of life as economic implications, which are worse felt in low and middle income (LAMI) countries; thus the need for myringoplasty. This study aimed to explore audiological function in a group of adults following myringoplasty in South Africa, with an exploration of the possible influence of factors such as HIV/AIDS and type of surgical technique on hearing outcomes.Within a retrospective chart review research design, 41 participant files for a six-year period from two academic hospitals in Johannesburg, South Africa, were reviewed. Data were analysed using both descriptive and inferential statistics.Participant files comprised of 16 males and 25 females between 18-63 years. Findings revealed that clinically, overall hearing improved post-operatively, as indicated by improved tympanometry findings, pure tone air-conduction and speech reception thresholds. Descriptively, the predictors of improved hearing outcomes post-operatively appeared to be HIV negative status and butterfly cartilage inlay surgery as a surgical technique adopted. Although clinically, hearing outcomes improved post-operatively at all air-conduction frequencies tested; these clinical improvements were only statistically significant at specific frequencies.Current findings provide useful initial evidence on the benefits of myringoplasty from the South African context; particularly because of the HIV/AIDS prevalence and its potential influence on middle ear disease and its management. Prospective efficacy studies with bigger sample sizes are recommended, with early identification strategies for middle ear disease to reduce the need for myringoplasty seriously considered bearing in mind the resource constraints. Globally, hearing loss is the most common sensory deficit across the human race . Eighty Factors influencing health care in developing countries are numerous. South Africa has the largest number of people living with HIV/AIDS . DisordeSouth Africa has the highest income inequality globally and the gap between public and private health care, with regards to affordability and quality of service remains a great concern . As a reClinical studies comparing outcomes of tympanic membrane reconstruction with fascia and cartilage show contradictory results , due to A retrospective chart review design was adopted , where m(n) in the data analysis. For participants who had bilateral surgery each ear was considered separately. So, the total sample size (N) comprised of 41 participant files, of these 8 participants had undergone bilateral surgery with each ear being considered separately; thus a total of 49 ears were investigated.Through non-probability purposive sampling technique where participant files meeting specific inclusion criteria were included in the study ; a totalParticipants in the files selected had to meet the following criteria in order to be included in the study: 1) Participants needed to have undergone type 1 tympanoplasty primary surgery- myringoplasty at one of the two chosen tertiary academic hospitals. 2) Participants needed to be between 18-63 years of age as the study was focusing on adults; but the influence of presbycusis needed to be minimised as well. 3) HIV status of the participants needed to be known. 4) Each participant file needed to contain audiology testing results. The pre-operative results needed to have been within 3 months preceding surgery and post-operative results within 3-6 months following surgery. 5) Information on the type of surgery needed to be recorded in the files. Following a pre-study discussion with a senior oto-rhino-laryngologist on common surgical techniques at the research sites, two types of surgical techniques were included in the study i.e. butterfly cartilage inlay technique and fascia underlay technique.The researcher documented the following biographical information and pre-post medical and audiological findings on a spreadsheet .Biographical and medical information: 1) Age and gender of participant; 2) Ear in which surgery was performed; 3) Technique of surgery; 4) HIV status.Audiological information: 1) Tympanometry results: tympanometry results were classified according to the existing Jerger system for classification of types A-As, Ad, B, C and D with the type A regarded as normal middle ear functioning and all other types as abnormal, including Eustachian tube function findings where available [Air-conduction and bone-conduction thresholds: air-bone gaps (ABGs) at 500Hz, 1000Hz, 2000Hz and 4000Hz were calculated. According to Valente [Speech reception threshold (SRT): Speech reception threshold results for each ear were documented pre and post operatively.vailable . 2) Air- Valente , normal  Valente . 3) SpeeData were analysed using both descriptive and inferential statistics through the use of SPSS software. Descriptive analysis entailed a description of the demographical and medical presentation of the participants; and a clinical description of audiological data pre and post-operatively. Inferential statistics in the form of a paired t-test (two-tailed) to assess the changes in hearing function pre and post operatively as well as the influence of HIV status and type of surgical technique on hearing outcomes were used, set at a significance level of 0.05 ; with surgery and HIV being independent variables ad post op outcomes dependent variable.The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guidelines on human experimentation. Ethical approval from the University’s Medical Ethics Committee (Protocol number M121012) was obtained.The sample comprised 16 males (39%) and 25 females (61%), with ages ranging from 18-63years with a mean age of 39.4 years were recruited. All participants of the selected files had undergone type 1 tympanoplasty, i.e. myringoplasty surgery in the last six years of the study being conducted. The sample was divided according to variables that would potentially affect hearing outcomes such as HIV status and type of surgical technique. Of the 41 participant files, 30 (73%) of the participants were HIV negative and 11 (27%) were HIV positive. Two types of surgical techniques were investigated. Of the 49 ears, 11 (22%) underwent the fascia underlay technique (F) while 38 (78%) underwent the butterfly cartilage inlay technique (C).Descriptively, the type of hearing loss was analysed pre and post operatively . ResultsAs depicted in Pre and post-operative air-bone gaps (ABGs) at 500Hz, 1000Hz and 2000Hz were calculated. The air-bone gap is the difference between the air-conduction threshold and the bone-conduction threshold. Results in Results presented in n=27) predictors for improved speech reception threshold results were HIV negative status and having undergone butterfly cartilage inlay surgery.Out of 49 ears only 27 ears (55%) had complete speech reception thresholds (SRTs) both pre and post operatively. Current findings indicate that there was an overall decrease in speech reception threshold (SRT) means post-surgery, suggesting improved speech reception thresholds post operatively. Twenty-three (85%) of the ears were of HIV negative individuals and four (15%) were from HIV positive individuals. Seven (26%) of the ears underwent fascia underlay (F) surgery, while 20 (74%) underwent butterfly cartilage inlay (C) surgery. Thus it can be suggested that in the current sub-sample ; which implies improvement in functional hearing. These frequencies are the most important frequencies when evaluating hearing function as they are used to establish the degrees of hearing loss when the average threshold at three frequencies  is calculated. The fact that in the current study, bone conduction deteriorated post-surgery could possibly be due to the type of surgical technique used in the majority of the participants; the butterfly cartilage inlay surgery; as opposed to the temporalis fascia underlay surgery adopted in Shaikh et al. [In the current study, the mean hearing gain for air-conduction and bone-conduction thresholds showed that air-conduction thresholds remained the same or improved and bone-conduction thresholds deteriorated overall. The improvements found were both clinically and statistically significant changes; which strengthens current findings. The statistically significant changes were at 250Hz, 500Hz, 1000Hz and 2000Hz where , therefore, suggesting that butterfly cartilage inlay surgery had an influence on air-conduction thresholds at specific frequencies. This correlates well with findings by Chhapola and Matta [When assessing the influence of butterfly cartilage inlay surgery on hearing outcomes post-operatively; statistically significant differences with regard to air-conduction mean change in hearing function were observed at 250Hz, 500Hz, 1000Hz and 2000Hz where .On the other hand, no evidence of statistically significant difference in air-conduction mean change in hearing function was found in the ears that underwent fascia underlay surgery at all air-conduction thresholds; suggesting that this type of surgical technique had limited influence on hearing outcomes post-operatively; although this is a preliminary finding since the sample size was small for this type of surgery. Although clinically, there was a difference in the post-operative findings between the two surgeries, these were not statistically significant at all air-conduction thresholds. This suggests that, statistically, the type of surgical technique has no influence on the hearing outcomes post-operatively. These findings highlight the importance of clinical evaluation and indicate that more detailed clinical conclusions must be drawn from a descriptive clinical evaluation rather than just on statistical analysis. Gerber et al.  compared [et al.  found th [et al.  looked aIt must be noted that many of the studies reviewed did not use the same criteria to assess hearing outcomes when compared to that of the current study. There are several different kinds of myringoplasty techniques which are often adapted by surgeons according to their resources and clinical presentation of the patients; which differ from country to country. These variables make comparing findings from the current study to other similar studies challenging; hence the importance of interpreting current findings with these limitations in mind is recommended. Current findings should be interpreted within the identified methodological limitations. Firstly, the retrospective chart review design presented potential challenges related to data collection procedures and accuracy in data capturing. Secondly, the sample size as well as sample distribution limits generalization of current findings. The unequal distribution of participants between the two surgical techniques makes current findings preliminary in nature. Despite these limitations, current findings do raise important research, clinical management, policy, and planning future directions for this population within the South African context.The current study which aimed to explore the changes in hearing outcomes post myringoplasty and investigate the possible influence of HIV status and type of surgical technique on hearing outcomes post-operatively has implications for audiologists and otorhinolaryngologists working in South Africa. These findings can be used to guide audiologists in the management of patients’ pre and post myringoplasty surgery in terms of pre-testing and monitoring hearing function post-surgery; as well as in providing audiologists with possible variables that may influence hearing outcomes so that they can anticipate management protocols. This is particularly important in a resource constrained context where careful planning around health priorities is critical. This is also important because health budget allocation and spending is influenced by professionals proving the value of their service; and this is easily done with such efficacy studies. Current findings can also assist oto-rhino-laryngologists in anticipating hearing outcomes post myringoplasty surgery, which affects their decision making process in terms of type of surgical technique to be adopted. A study of this nature specifically investigating HIV status as a variable and its possible influence on hearing outcomes post myringoplasty surgery has never been conducted in the current context. Therefore, current findings may serve as a baseline for future studies which could specifically focus on HIV as an influencing factor; with strict controls of possible confounding variables which were not controlled for in the current study; since this was an exploratory study.Extensive evidence exists on the success rate of myringoplasty in relation to functional gain in hearing post-surgery worldwide;International evidence exists on the efficacy of myringoplasty surgery in terms of audiological improvement worldwide;It is known that there is paucity of evidence within the developing world context, particularly from Africa.Findings from this study indicating clinical improvement in hearing outcomes post-myringoplasty from a LAMI country, consistent with international studies, add to the surgical efficacy evidence from this context;The fact that HIV status was found to be a possible variable that may have an influence on hearing outcomes post myringoplasty is an important addition to the body of evidence;The importance of efficacy studies looking at both clinical and statistical significance of findings in their analysis was highlighted by the current study.The authors declare no competing interests.


To evaluate biomechanical differences between two surgical procedures for cervical open-door laminoplasty using human cadaveric spines.Twenty-four cervical vertebrae (C4-C6) from eight fresh-frozen human cervical spines were subjected to mechanical testing after being instrumented for open-door laminoplasty using a newly designed plate-spacer device with a monocoque structure  or by conventional miniplate-alone fixation . Cantilever bending testing was performed by applying a compressive load in the cranio-caudal direction to the base of the spinous process of the reconstructed laminar arch constructs until failure and strength and stiffness of the laminar arch were determined. The results are presented as mean ± standard deviation.p < 0.05). Stiffness in the plate-spacer group exhibited the same trend .The plate-spacer group was approximately twice as strong as the miniplate group (27.6 ± 16.5 N vs. 13.5 ± 7.3 N, The fixation with the monocoque plate-spacer construct for open-door laminoplasty provided higher structural properties when compared against the plate-alone fixation. The spacer in the plate-spacer construct appears to contribute by preventing large deformations of the laminar arch caused by bending in cranio-caudal direction. Future studies will be required to investigate stress/strain distribution in the laminar arch constructs. Cervical laminoplasty has gradually become a well-established surgical intervention since first introduction in 1983 . In the fixation .Spacers have been applied to laminoplasty to prevent lamina closure. Bone struts and ceramic and hydroxyapatite blocks have been used as a spacer element. However, dislodgement of the spacer without stable fixation is a concern, which could lead to subsequent premature laminoplasty closure and even root or cord compression if the spacer dislodges into the canal , 7.et al. first described the use of miniplates adapted from cranial fixation systems to secure cervical laminoplasty [As another approach to prevent laminar closure after the open-door procedures, plate fixation of the lamina has been introduced in laminoplasty. Frank noplasty . The usenoplasty , 10. Desnoplasty  and highnoplasty .et al. found that screw loosening was more common in cases without the spacers in the laminoplasty using plate fixation [et al. reported on open-door laminoplasty fixed by titanium miniplate and allograft bone, emphasizing that the combination of titanium miniplates for immediate stabilization and allograft bone for long-term fusion led to no loss of canal decompression [Plate fixation has been used in conjunction with bone struts connected to the plate by screws, which provides immediate stabilization and prevents bone strut dislodgement. The bone struts are expected to recreate a continuous bony laminar arch spanning from one lateral mass to the contralateral one which cannot be achieved by laminoplasty using the plate alone. Goto fixation . Shaffrepression . Despitepression –19.A new device for laminoplasty consisting of a titanium box-shaped spacer with two arms for plate fixation has been developed in response to the challenge of poor/unstable fixation by providing immediate stabilization and long term biological fixation by bony reconstruction of the laminar arch via bone induction of autogenous local bone chips inserted in the titanium spacer during surgery . ClinicaThe aims of the present study were to investigate biomechanical behavior of the laminar arch after cervical laminoplasty using the monocoque plate-spacer construct compared to the conventional miniplate alone as a standard using human cadaveric cervical spines.This cadaveric study was approved by IRB at Rush University Medical Center (ORA#13103105-IRB01). A total of 24 cervical vertebrae (C4-C6) were prepared from fresh frozen 8 human cervical spines obtained from a tissue bank . The donor sample consisted of 4 men and 4 women with an average age of 56 ± 12 years (mean ± standard deviation). Computed tomographic (CT) scans were obtained to exclude samples that had anatomical abnormalities or pathological changes such as fractures, severe deformity, and metastatic disease. The specimens were wrapped in saline-soaked gauze and stored frozen at -20°C. The day before testing, the specimens were thawed overnight at room temperature.The specimens were separated to individual vertebrae  and laminoplasty surgery was performed for each vertebra by a single board-certified spine surgeon to keep consistency in the surgical procedure. A 3.0 mm wide gutter was made by high-speed cutting burr and a unicortical hinge was created. After creating a bicortical defect on the opposite side of the hinge, the laminar arch was reconstructed by two types of titanium-made devices: either a monocoque plate-spacer device  running in displacement control with a cross-head speed of 1.0 mm/min until failure Figs  and 4. Ap < 0.05. The results are presented as mean and standard deviation.The load to failure process was recorded by video during testing and macroscopic failure mode was evaluated. Load-displacement curves were recorded at 10 Hz. The fixation strength was determined by the initial peak of the load-displacement curve. Structural stiffness of the laminoplasty construct was determined by the slope of the initial linear portion of the load-displacement curve. Spinal level effects of the fixation strength and stiffness were studied by one way ANOVA with a Tukey’s post-hoc test. The fixation strength and stiffness were compared between the plate-spacer and the miniplate groups by an unpaired t-test. A significance level was set at No screw loosening was observed in any of the specimens. Failure was initiated at the upper corner of the hinge with an audible cracking sound in all specimens. In all miniplate group specimens, the miniplate was rotated as deflection of the elevated lamina increased during loading, which caused displacement of the cut-edge of the lamina towards the caudal direction. This phenomenon caused rotation of the lamina around the anteroposterior axis and, in turn, the hinge was twisted in the coronal plane in addition to the rotation in the sagittal plane. Conversely, these phenomena were only observed in a single specimen in the plate-spacer group, which showed gaps between the box-shaped spacer and the lateral mass or the cut-surface of the lamina. In the remaining specimens from the plate-spacer group, the spacer contacted to the lateral mass and cut-surface of the lamina during loading, which protected rotation of the lamina in the coronal plane .p < 0.05). The stiffness in the plate-spacer group exhibited the same trend   .The present study demonstrated that the failure mechanism of the laminar arch reconstructed by either the plate-spacer or miniplate alone was hinge fracture under cantilever bending produced by compressive load in the cranio-caudal direction. However, the fixation strength and stiffness of the laminar arch with plate-spacer device were both approximately twice as high than those with the miniplate alone. Shear movement at the laminectomy gap under loading as presented by macroscopic deformation of the laminar cut-edge might have initiated hinge fracture starting at the gutter upper margin. In this study, single-screw fixation at the lateral mass and lamina ends was used in both the plate-spacer and miniplate groups based on clinical studies using one-screw fixation , 20. HowThe higher strength and stiffness in the plate-spacer group appears to be brought by a more uniform load sharing provided by the box-like spacer, which is, effectively a stiffer structure than just the plate. A finite element analysis (FEA) was performed to investigate stress distribution and construct stability in cervical open-door laminoplasty models using the plate-spacer device used in the present study . In thisBiomechanical analyses of the laminoplasty construct under compressive loading in a postero-anterior direction have been reported in the literature , 25. MaxThe present study only used middle cervical levels of C4—C6 and the presented results did not include the level effect. A larger sample size may demonstrate the differences of the biomechanical parameters of the laminoplasty reconstruct. Since extensive multilevel laminoplasty including upper and/or lower cervical levels has been often performed recently, biomechanical studies including C2, C3 and C7 levels are also important. Future studies including C2—C7 levels with a sufficient sample size will be required to provide relevant information for extensive multilevel laminoplasty.The biomechanical parameters studied in this study are limited to the overall structural characteristics and fracture mechanisms of the hinge and load transmission through the spacer were speculated by qualitative observation. Stress analysis of the hinge and the device-bone interfaces should be conducted to elucidate the failure mechanism of the laminar construct after laminoplasty. The present study used single vertebra experimental specimens to investigate individual vertebra fixation strength and stiffness of individual vertebra. However, multi-motion segments should be used to investigate structural properties of the cervical spine under different fixation techniques for multiple laminoplasty. Although the present study aimed to investigate initial fixation strength of the laminoplasty reconstruct after surgery, fatigue behavior of the construct should be investigated because early failure of the construct, such as screw loosening, could occur before biological fixation has been accomplished. The present study reported only the biomechanical evaluation of the open-door laminoplasty constructs using the newly-developed monocoque plate-spacer device and plate fixation techniques, even though a variety of other reconstruction techniques for laminoplasty is available. Nonetheless, the results of the present study would also be helpful to understand mechanical benefits of the other forms of plate-spacer constructs, i.e. bone graft strut or bone substitute such as hydroxyapatite and ceramic rigidly fixed to the plate, on the initial structural strength of the open-door laminoplasty constructs.Cervical open-door laminoplasty using a monocoque plate-spacer device provided approximately twice as much strength and stiffness as compared with plate-alone fixation under compressive loading applied to the base of the spinous process in cranio-caudal direction. Under this loading condition representing bending of the reconstructed laminar arch, all specimens failed from the upper corner of the hinge. While reconstructed laminar arch by plate-alone fixation exhibited coronal rotation under loading which caused twisting of the hinge, the plate-spacer device prevented this rotation by contact between the spacer and lamina or lateral mass during loading. Although further stress analysis will be required, load transmission through the spacer integrated in one structure appears to contribute to higher structural properties of the laminar arch after open-door laminoplasty.