Robust Computational Tools for Multiple Testing with Genetic Association Studies

Resolving the interplay of the genetic components of a complex disease is a challenging endeavor. Over the past several years, genome-wide association studies (GWAS) have emerged as a popular approach at locating common genetic variation within the human genome associated with disease risk. Assessing genetic-phenotype associations upon hundreds of thousands of genetic markers using the GWAS approach, introduces the potentially high number of false positive signals and requires statistical correction for multiple hypothesis testing. Permutation tests are considered the gold standard for multiple testing correction in GWAS, because they simultaneously provide unbiased Type I error control and high power. However, they demand heavy computational effort, especially with large-scale data sets of modern GWAS. In recent years, the computational problem has been circumvented by using approximations to permutation tests, but several studies have posed sampling conditions in which these approximations are suggestive to be biased. We have developed an optimized parallel algorithm for the permutation testing approach to multiple testing correction in GWAS, whose implementation essentially abates the computational problem. When introduced to GWAS data, our algorithm yields rapid, precise, and powerful multiplicity adjustment, many orders of magnitude faster than existing employed GWAS statistical software. Although GWAS have identified many potentially important genetic associations which will advance our understanding of human disease, the common variants with modest effects on disease risk discovered through this approach likely account for a small proportion of the heritability in complex disease. On the other hand, interactions between genetic and environmental factors could account for a substantial proportion of the heritability in a complex disease and are overlooked within the GWAS approach. We have developed an efficient and easily implemented tool for genetic association studies, whose aim is identifying genes involved in a gene-environment interaction. Our approach is amenable to a wide range of association studies and assorted densities in sampled genetic marker panels, and incorporates resampling for multiple testing correction. Within the context of a case-control study design we demonstrate by way of simulation that our proposed method offers greater statistical power to detect gene-environment interaction, when compared to several competing approaches to assess this type of interaction

Use of the cell cycle progression (CCP) score for predicting systemic disease and response to radiation of biochemical recurrence

BACKGROUND: Determining the optimal treatment for biochemical recurrence (BCR) after radical prostatectomy (RP) is challenging. OBJECTIVE: We evaluated the ability of CCP score (a prognostic RNA expression signature) to discriminate between systemic disease and local recurrence in patients with BCR after RP. METHODS: Sixty patients with BCR after RP were selected for analysis based on: 1) metastatic disease, 2) non-response to salvage external beam radiotherapy (EBRT), and 3) durable response to salvage EBRT. CCP scores were generated from the RNA expression of 46 genes. Logistic regression assessed the association between CCP score and patient group. RESULTS: Passing CCP scores were generated for 47 patients with complete clinical and pathologic data. CCP score predicted clinical status when comparing patients with metastatic disease or non-responders to salvage therapy to patients with durable response (p = 0.006). CCP score remained significantly predictive of clinical status after accounting for time to BCR, PSA level at BCR, and Gleason score (p = 0.0031). CONCLUSIONS: Elevated CCP score was associated with increased risk of systemic disease, indicating that CCP score may be useful in identifying patients with BCR who are most likely to benefit from salvage radiation therapy

The development and validation of a scoring tool to predict the operative duration of elective laparoscopic cholecystectomy

Background: The ability to accurately predict operative duration has the potential to optimise theatre efficiency and utilisation, thus reducing costs and increasing staff and patient satisfaction. With laparoscopic cholecystectomy being one of the most commonly performed procedures worldwide, a tool to predict operative duration could be extremely beneficial to healthcare organisations. Methods: Data collected from the CholeS study on patients undergoing cholecystectomy in UK and Irish hospitals between 04/2014 and 05/2014 were used to study operative duration. A multivariable binary logistic regression model was produced in order to identify significant independent predictors of long (> 90 min) operations. The resulting model was converted to a risk score, which was subsequently validated on second cohort of patients using ROC curves. Results: After exclusions, data were available for 7227 patients in the derivation (CholeS) cohort. The median operative duration was 60 min (interquartile range 45–85), with 17.7% of operations lasting longer than 90 min. Ten factors were found to be significant independent predictors of operative durations > 90 min, including ASA, age, previous surgical admissions, BMI, gallbladder wall thickness and CBD diameter. A risk score was then produced from these factors, and applied to a cohort of 2405 patients from a tertiary centre for external validation. This returned an area under the ROC curve of 0.708 (SE = 0.013, p  90 min increasing more than eightfold from 5.1 to 41.8% in the extremes of the score. Conclusion: The scoring tool produced in this study was found to be significantly predictive of long operative durations on validation in an external cohort. As such, the tool may have the potential to enable organisations to better organise theatre lists and deliver greater efficiencies in care

The QuinteT Recruitment Intervention supported five randomized trials to recruit to target: a mixed-methods evaluation

ObjectiveTo evaluate the impact of the Quintet Recruitment Intervention (QRI) on recruitment in challenging randomized controlled trials (RCTs) that have applied the intervention. The QRI aims to understand recruitment difficulties, and then implements ‘QRI-actions’ to address these as recruitment proceeds.Study Design and SettingA mixed-methods study, comprising: a) before-and-after comparisons of recruitment rates and numbers of patients approached, and b) qualitative case studies, including documentary analysis and interviews with RCT investigators.ResultsFive UK-based publicly-funded RCTs were included in the evaluation. All recruited to target. RCT2 and RCT5 both received up-front pre-recruitment training before the intervention was applied. RCT2 did not encounter recruitment issues and recruited above target from its outset. Recruitment difficulties, particularly communication issues, were identified and addressed through QRI-actions in RCTs 1, 3, 4 and 5. Randomization rates significantly improved post-QRI-action in RCTs 1,3, and 4. QRI-actions addressed issues with approaching eligible patients in RCTs 3 and 5, which both saw significant increases in patients approached. Trial investigators reported that the QRI had unearthed issues they had been unaware of, and reportedly changed their practices post QRI-action.ConclusionThere is promising evidence to suggest the QRI can support recruitment to difficult RCTs. This needs to be substantiated with future controlled evaluations

Body composition of long-term survivors of acute lymphoblastic leukaemia

Background Long-term quality of life is of growing importance in children previously treated for malignancy. Obesity defined indirectly from indices of height and weight, has been described in long-term survivors of acute lymphoblastic leukaemia (ALL) and hypothesised to be a consequence of previous cranial irradiation. Procedure In this study, measures of whole and regional body composition using skinfold and dual energy X-ray absorptiometry (DEXA) measurements have been made in 35 long-term survivors of ALL who had received cranial irradiation and chemotherapy. To assess the influence of cranial irradiation, results were compared with those obtained in 21 children treated for other malignancies, who received chemotherapy alone and with 31 healthy sibling controls. Results Girls treated for ALL were significantly fatter than those treated for other malignancies or healthy control siblings whether measured by skinfold thickness (median (range) 37.4% (17.9–41.3) vs. 24.6% (19.1–35.0) and 28.8% (19.6–43.1), respectively, P < 0.01) or DEXA (33.5% (20.5–42.8) vs. 25.5% (16.5–31.0) and 24.5% (18.8–53.6), respectively, P < 0.01). Boys treated for ALL were not significantly fatter than boys in the other two groups. Measures of whole body percent fat derived from DEXA were persistently less than those derived from skinfold measurements with a mean (95% CI) difference of 2.4% (1.7–3.1, P < 0.001) for all groups combined. In ALL survivors, using regression equations for skinfold thicknesses derived from controls with DEXA as the ‘gold standard’ method, fat mass was significantly overestimated. Conclusion Female survivors of ALL are significantly fatter than those of other malignancies and healthy sibling controls. Caution should be observed in the application of published equations, derived from the normal population, for the calculation of body composition in children treated for ALL. The mechanism of onset of obesity remains unclear, but is probably multifactorial and related to previous cranial irradiation

Body composition in children with type 1 diabetes in the first year after diagnosis: relationship to glycaemic control and cardiovascular risk

INTRODUCTION: Rapid weight gain is often observed following initiation of insulin therapy in children with type 1 diabetes mellitus (T1DM) and girls are particularly at risk of becoming overweight. The authors evaluated body composition changes in children during the first year after diagnosis and related this to markers of cardiovascular risk. METHODS: Body mass index (BMI) and body composition measured by whole body dual energy x-ray absorptiometry (DEXA) were assessed in 30 patients (18 boys) with T1DM 3-10 days after diagnosis, 6 weeks later and at 1 year, and on two occasions 1 year apart in 14 controls (8 boys). Cardiovascular risk markers were assessed in T1DM subjects at 1 year. RESULTS: T1DM subjects had lower BMI SD scores (SDS) at diagnosis than controls (mean (SD) BMI SDS -0.67 (1.34) vs 0.20 (1.14), p<0.05) and reduced percentage body fat (20.3% (4.6) vs 24.5% (7.7), p<0.05). T1DM subjects normalised their body composition at 6 weeks and this was maintained 1 year later. Girls with diabetes were thinner than boys at diagnosis (BMI SDS -1.64 (1.02) vs -0.02 (1.17), p<0.05) and at 1 year (BMI SDS -0.58(0.9) vs 0.65 (0.98), p<0.05). Girls had higher glycated haemoglobin (HbA1c) (8.8% (1.2) vs 7.8% (1.0), p<0.05), insulin dose (1.01 (0.30) vs 0.82 (0.18) U/kg/day, p=0.04), total cholesterol (4.30 (0.45) vs 3.79 (0.50) mmol/l, p<0.05) and high-density lipoprotein (2.62 (0.53) vs 2.02 (0.37) mmol/l). High sensitivity C reactive protein and fibrinogen were in the normal range and there were no differences between genders. DISCUSSION: Insulin deficiency at diagnosis of diabetes causes a catabolic state that is predominantly lipolytic. Body composition normalises within 6 weeks of treatment, though girls remain thinner than boys both at diagnosis and 1 year thereafter, in contrast to published findings. Despite girls being prescribed a larger insulin dose, their HbA1c and cholesterol levels are higher at 1 year suggesting increased insulin resistance and cardiovascular risk

Osteopenia, excess adiposity and hyperleptinaemia during 2 years of treatment for childhood acute lymphoblastic leukaemia without cranial irradiation

objective Osteopenia and excess adiposity occur following treatment of childhood acute lymphoblastic leukaemia (ALL) and the use of cranial irradiation is thought to be a significant contributory factor. Hyperleptinaemia has also been demonstrated following cessation of treatment for childhood ALL. Therefore a prospective study was undertaken to evaluate serial changes in percentage bone mineral content (BMC), adiposity and serum leptin concentrations during 2 years of treatment of children with ALL with chemotherapy but without cranial irradiation. design and patient Only patients treated using the MRC ALL 97/ALL 97 (modified 99) protocols for childhood ALL were eligible for entry into the study. A total of 14 patients (seven male, with a median age of 7·5 years (range 3·4–16·7 years) were recruited. Serial dual energy X-ray absorptiometry (DEXA) scanning was undertaken at diagnosis and during two years of treatment. Serum leptin concentrations were determined at the same time as the scans. results Reductions in %BMC were observed at the hip and lumbar spine by 12 months (P < 0·01) and remained low after 24 months of treatment. Subanalysis of %BMC measurements at the hip demonstrated a greater reduction in %BMC at the trochanteric region compared to the femoral neck. The percentage corrected fat mass increased from 6 months whereas the body mass index (BMI) standard deviation score (SDS) was increased after 24 months of treatment (P < 0·05). Serum leptin concentrations increased following 24 months of therapy (P < 0·05). conclusions Children treated for ALL with contemporary regimens have a predisposition to osteopenia, excess adiposity and hyperleptinaemia during treatment without cranial irradiation administration. We speculate that in addition to glucocorticoid administration, leptin resistance may account in part for these observations

PTEN loss in biopsy tissue predicts poor clinical outcomes in prostate cancer

OBJECTIVES: To determine whether PTEN status in prostate biopsy represents a predictor of intermediate and long-term oncological outcomes after radical prostatectomy, and whether PTEN status predicts response to androgen deprivation therapy. METHODS: In a retrospective analysis of 77 men treated by radical prostatectomy who underwent diagnostic biopsy between 1992-2006, biopsy samples were stained for PTEN expression by the PREZEON assay with \u3e 10% staining reported as positive. Cox proportional hazards and log-rank models were used to assess the correlation between PTEN loss and clinical outcomes. RESULTS: During a median follow-up period after radical prostatectomy of 8.8 years, 39 men (51%) developed biochemical recurrence, four (5%) had castration-resistant prostate cancer, two (3%) had metastasis and two (3%) died from prostate cancer. PTEN loss was not significantly associated with biochemical recurrence (hazard ratio 2.1, 95% confidence interval 0.9-5.1, P = 0.10), but significantly predicted increased risk of castration-resistant prostate cancer, metastasis and prostate cancer-specific mortality (all log-rank, P \u3c 0.0001), and time from androgen deprivation therapy to castration-resistant prostate cancer (log-rank, P = 0.003). No patient without PTEN loss developed metastases or died from prostate cancer. CONCLUSIONS: PTEN loss at the time of biopsy seems to predict time to development of metastasis, prostate cancer-specific mortality and, for the first time, castration-resistant prostate cancer and response to androgen deprivation therapy after radical prostatectomy. If confirmed by larger studies, this would support the use of PTEN loss as an early marker of aggressive prostate cancer

High throughput analysis of B cell dynamics and neutralizing antibody development during immunization with a novel clade C HIV-1 envelope.

A protective HIV-1 vaccine has been hampered by a limited understanding of how B cells acquire neutralizing activity. Our previous vaccines expressing two different HIV-1 envelopes elicited robust antigen specific serum IgG titers in 20 rhesus macaques; yet serum from only two animals neutralized the autologous virus. Here, we used high throughput immunoglobulin receptor and single cell RNA sequencing to characterize the overall expansion, recall, and maturation of antigen specific B cells longitudinally over 90 weeks. Diversification and expansion of many B cell clonotypes occurred broadly in the absence of serum neutralization. However, in one animal that developed neutralization, two neutralizing B cell clonotypes arose from the same immunoglobulin germline and were tracked longitudinally. Early antibody variants with high identity to germline neutralized the autologous virus while later variants acquired somatic hypermutation and increased neutralization potency. The early engagement of precursors capable of neutralization with little to no SHM followed by prolonged affinity maturation allowed the two neutralizing lineages to successfully persist despite many other antigen specific B cells. The findings provide new insight into B cells responding to HIV-1 envelope during heterologous prime and boost immunization in rhesus macaques and the development of selected autologous neutralizing antibody lineages