DNA methylation-based age estimation for adults and minors: considering sex-specific differences and non-linear correlations.

DNA methylation patterns change during human lifetime; thus, they can be used to estimate an individual's age. It is known, however, that correlation between DNA methylation and aging might not be linear and that the sex might influence the methylation status. In this study, we conducted a comparative evaluation of linear and several non-linear regressions, as well as sex-specific versus unisex models. Buccal swab samples from 230 donors aged 1 to 88 years were analyzed using a minisequencing multiplex array. Samples were divided into a training set (n = 161) and a validation set (n = 69). The training set was used for a sequential replacement regression and a simultaneous 10-fold cross-validation. The resulting model was improved by including a cut-off of 20 years, dividing the younger individuals with non-linear from the older individuals with linear dependence between age and methylation status. Sex-specific models were developed and improved prediction accuracy in females but not in males, which might be explained by a small sample set. We finally established a non-linear, unisex model combining the markers EDARADD, KLF14, ELOVL2, FHL2, C1orf132, and TRIM59. While age- and sex-adjustments did not generally improve the performance of our model, we discuss how other models and large cohorts might benefit from such adjustments. Our model showed a cross-validated MAD and RMSE of 4.680 and 6.436 years in the training set and of 4.695 and 6.602 years in the validation set, respectively. We briefly explain how to apply the model for age prediction.

Confirmatory adaptive group sequential designs for single-arm phase II studies with multiple time-to-event endpoints.

Existing methods concerning the assessment of long-term survival outcomes in one-armed trials are commonly restricted to one primary endpoint. Corresponding adaptive designs suffer from limitations regarding the use of information from other endpoints in interim design changes. Here we provide adaptive group sequential one-sample tests for testing hypotheses on the multivariate survival distribution derived from multi-state models, while making provision for data-dependent design modifications based on all involved time-to-event endpoints. We explicitly elaborate application of the methodology to one-sample tests for the joint distribution of (i) progression-free survival (PFS) and overall survival (OS) in the context of an illness-death model, and (ii) time to toxicity and time to progression while accounting for death as a competing event. Large sample distributions are derived using a counting process approach. Small sample properties are studied by simulation. An already established multi-state model for non-small cell lung cancer is used to illustrate the adaptive procedure.

A statistical framework for planning and analysing test-retest studies of repeatability.

There is an increasing number of potential quantitative biomarkers that could allow for early assessment of treatment response or disease progression. However, measurements of such biomarkers are subject to random variability. Hence, differences of a biomarker in longitudinal measurements do not necessarily represent real change but might be caused by this random measurement variability. Before utilizing a quantitative biomarker in longitudinal studies, it is therefore essential to assess the measurement repeatability. Measurement repeatability obtained from test-retest studies can be quantified by the repeatability coefficient, which is then used in the subsequent longitudinal study to determine if a measured difference represents real change or is within the range of expected random measurement variability. The quality of the point estimate of the repeatability coefficient, therefore, directly governs the assessment quality of the longitudinal study. Repeatability coefficient estimation accuracy depends on the case number in the test-retest study, but despite its pivotal role, no comprehensive framework for sample size calculation of test-retest studies exists. To address this issue, we have established such a framework, which allows for flexible sample size calculation of test-retest studies, based upon newly introduced criteria concerning assessment quality in the longitudinal study. This also permits retrospective assessment of prior test-retest studies.

Two-sample survival tests based on control arm summary statistics.

The one-sample log-rank test is the preferred method for analysing the outcome of single-arm survival trials. It compares the survival distribution of patients with a prefixed reference survival curve that usually represents the expected outcome under standard of care. However, classical one-sample log-rank tests assume that the reference curve is known, ignoring that it is frequently estimated from historical data and therefore susceptible to sampling error. Neglecting the variability of the reference curve can lead to an inflated type I error rate, as shown in a previous paper. Here, we propose a new survival test that allows to account for the sampling error of the reference curve without knowledge of the full underlying historical survival time data. Our new test allows to perform a valid historical comparison of patient survival times when only a historical survival curve rather than the full historic data is available. It thus applies in settings where the two-sample log-rank test is not applicable as method of choice due to non-availability of historic individual patient survival time data. We develop sample size calculation formulas, give an example application and study the performance of the new test in a simulation study.

Statistical analysis plan for the biomarker-guided intervention to prevent acute kidney injury after major surgery (BigpAK-2) study: An international randomised controlled multicentre trial.

Objective: This article describes the statistical analysis plan for the Biomarker-guided intervention to prevent AKI after major surgery (BigpAK-2) trial. Design: Adaptive trial design with an interim analysis after enrolment of 618 evaluable patients. Setting: The BigpAK.-2 trial is an international, prospective, randomised controlled multicentre study. Participants: The BigpAK-2 study enrols patients after major surgery who are admitted to the intensive care or high dependency unit and are at high-risk for postoperative AKI as identified by urinary biomarkers (tissue inhibitor of metalloproteinases-2 and insulin-like growth factor binding protein 7 ([TIMP-2]∗[IGFBP7]) will be enrolled. Intervention: Patients are randomly and evenly allocated to standard of care (control) group or the implementation of a nephroprotective care bundle (intervention group), as recommended by the Kidney Disease: Improving Global Outcomes (KDIGO) guidelines. The KDIGO care bundle recommends discontinuation of nephrotoxic agents if possible, ensuring adequate volume status and perfusion pressure, considering functional haemodynamic monitoring, regular monitoring of serum creatinine and urine output, avoiding hyperglycemia, and considering alternatives to radiocontrast procedures when possible. Results: The BigpAK-2 study investigates whether the biomarker-gudied implementation of the KDIGO care bundle reduces the incidence of moderate or severe AKI (stage 2 or 3), according to the KDIGO 2012 criteria, within 72 h after surgery. Conclusion: AKI is a common and often severe complication after major surgery. As no specific treatments exist, prevention of AKI is of high importance. The BigpAK-2 study investigates a promising approach to prevent AKI after major surgery. Trial registration: The trial was registered prior to start at clinicaltrials.gov; NCT04647396.

Characterizing Foveal Hypoplasia Using Optical Coherence Tomography Angiography: Evaluation of Microvascular Abnormalities and Clinical Significance.

This study aimed to evaluate foveal avascular zone (FAZ) features and macular flow density (FD) in various retinal layers in a cohort of patients with foveal hypoplasia (FH) using optical coherence tomography angiography (OCTA), in order to characterize microvascular abnormalities and explore their potential clinical significance. FAZ parameters and FD, as well as retinal thickness and volume values were analyzed and compared between patients with FH and an age- and gender-matched control cohort. Correlations between disease severity and visual acuity (VA), as well as between disease severity and FAZ features were evaluated. A total of 19 eyes with FH and 19 control eyes were included. The study group showed significantly higher FD values in the foveal sectors of the superficial and deep capillary plexus compared to controls. FAZ area, perimeter, and acircularity index (ACI) were noticeably altered in eyes with FH; however, they did not correlate with disease severity. Visual acuity was negatively correlated with disease severity. The results of this study provide evidence of altered microvasculature architecture specifically in the foveal sectors of patients with FH. The higher FD values in the foveal sectors of FH patients suggest a potential compensatory response of the retinal microvasculature. FAZ parameters and FD values of the foveal sectors could be used as part of an OCTA-based grading system in FH patients.

Evaluation of Retinal Nerve Fiber Layer and Macular Ganglion Cell Layer Thickness in Relation to Optic Disc Size.

To investigate whether optic nerve ganglion cell amount is dependent on optic disc size, this trial analyzes the correlation between Bruch's membrane opening area (BMOA) and retinal nerve fiber layer (RNFL) thickness as well as macular ganglion cell layer thickness (mGCLT). Additionally, differences in RNFL and mGCLT regarding various optic disc cohorts are evaluated. This retrospective, monocentric study included 501 healthy eyes of 287 patients from the University Hospital Münster, Germany, who received macular and optic disc optical coherence tomography (OCT) scans. Rank correlation coefficients for clustered data were calculated to investigate the relationship between BMOA and thickness values of respective retinal layers. Furthermore, these values were compared between different optic disc groups based on BMOA. Statistical analysis did not reveal a significant correlation between BMOA and RNFL thickness, nor between BMOA and mGCLT. However, groupwise analysis showed global RNFL to be significantly decreased in small and large discs in comparison to medium discs. This was not observed for global mGCLT. This study extends existing normative data for mGCLT taking optic disc size into account. While the ganglion cell amount represented by the RNFL and mGCLT seemed independent of BMOA, mGCLT was superior to global RNFL in displaying optic nerve integrity in very small and very large optic discs.

Influence of Cilioretinal Arteries on Flow Density in Glaucoma Patients Measured Using Optical Coherence Tomography Angiography.

It has long been speculated whether the presence of a cilioretinal artery (CRA) can influence the development of glaucomatous damage in patients with open-angle glaucoma. Studies involving healthy patients have shown a change in flow density (FD) depending on the presence of a CRA. Similarly, studies that compared the optical coherence tomography angiography (OCTA) results of healthy controls and glaucoma cohorts identified a reduction in FD in certain retinal layers for glaucoma patients. These observations raise the question of whether FD is altered in glaucoma patients depending on the presence of CRA, with possible implications for the progression of glaucomatous damage. In this prospective study, 201 eyes of 134 primary and secondary open-angle glaucoma patients who visited the Department of Ophthalmology at the University of Muenster Medical Center, Germany were included. The patients were allocated to different groups according to the presence of CRAs and the level of glaucoma severity. The FD results obtained using OCTA for the CRA and non-CRA groups were compared. While FD differed noticeably between the CRA and non-CRA cohorts in the deep macular plexus, no differences in FD were observed between the two groups when adjusted for glaucoma severity. In both the CRA and non-CRA eyes, increasing glaucoma severity correlated most strongly with a reduction in peripapillary FD. Our results suggest that the presence of CRAs does not significantly affect retinal perfusion in glaucoma patients.

Nasolacrimal intubation in transcanalicular endoscopic dacryoplasty: a long-term follow-up study.

Nowadays, transcanalicular endoscopic dacryoplasty represents the majority of lacrimal duct surgery procedures performed in adults in specialised centers. However, there are still hardly any data available regarding the intra- and postoperative care, particularly regarding the duration of silicone tube intubation (STI). Our aim was to evaluate the relation between tube duration and recurrence of symptoms in patients who underwent transcanalicular microdrill dacryoplasty (MDP) in a long-term setting. Medical records of 576 adult patients after MDP were retrospectively reviewed. A total of 256 eyes of 191 patients could be included. The median follow-up time was 7.83 [7.08; 9.25] years. In 57.0% of the cases there was still full resolution of symptoms at the time of the survey. The median duration of the STI was 6 [3.00; 6:00] months. When distinguishing between a tube duration < 3 months and ≥ 3 months there was a significant difference in the long-term success rate (< 3 months: 38%; ≥ 3 months: 61%; p = 0.011). In conclusion, an early removal of the STI (< 3 months) after transcanalicular MDP seems to be associated with a higher incidence of recurrence of symptoms. This should be considered in the intra- and postoperative care of patients following this minimally invasive first-step procedure.

Ultrahypofractionated Low-Dose Total Skin Electron Beam in Advanced-Stage Mycosis Fungoides and Sézary Syndrome.

PURPOSE: The aim of this study was to assess the safety and efficacy of an ultrahypofractionated low-dose total skin electron beam therapy (TSEBT) regimen in patients with advanced mycosis fungoides (MF) or Sézary syndrome (SS). METHODS AND MATERIALS: In this multicenter observational study from 5 German centers, 18 total patients with MF or SS underwent TSEBT with a total dose of 8 Gy in 2 fractions. The primary endpoint was the overall response rate. RESULTS: Fifteen of 18 patients with stage IIB-IV MF or SS were heavily pretreated with a median of 4 prior systemic therapies. The overall response rate was 88.9% (95% confidence interval [CI], 65.3-98.6), with 3 complete responses (16.9%; 95% CI, 3.6-41.4). At a median follow-up period of 13 months, the median time to next treatment (TTNT) was 12 months (95% CI, 8.2-15.8), and the median progression-free survival was 8 months (95% CI, 2-14). A significant reduction in the modified severity-weighted assessment tool, total Skindex-29 score (Bonferroni-corrected P < .005), and all subdomains (Bonferroni-corrected P < .05) was observed after TSEBT. Half of the irradiated patients (n = 9) developed grade 2 acute and subacute toxicities. One patient had confirmed grade 3 acute toxicity. Chronic grade 1 toxicity has been observed in 33% of patients. Patients with erythroderma/SS or prior radiation therapy appear at higher risk of skin toxicities. CONCLUSIONS: TSEBT with 8 Gy in 2 fractions achieves good disease control and symptom palliation with acceptable toxicity, greater convenience, and fewer hospital visits.

Reference curve sampling variability in one-sample log-rank tests.

The one-sample log-rank test is the method of choice for single-arm Phase II trials with time-to-event endpoint. It allows to compare the survival of patients to a reference survival curve that typically represents the expected survival under standard of care. The one-sample log-rank test, however, assumes that the reference survival curve is known. This ignores that the reference curve is commonly estimated from historic data and thus prone to sampling error. Ignoring sampling variability of the reference curve results in type I error rate inflation. We study this inflation in type I error rate analytically and by simulation. Moreover we derive the actual distribution of the one-sample log-rank test statistic, when the sampling variability of the reference curve is taken into account. In particular, we provide a consistent estimate of the factor by which the true variance of the one-sample log-rank statistic is underestimated when reference curve sampling variability is ignored. Our results are further substantiated by a case study using a real world data example in which we demonstrate how to estimate the error rate inflation in the planning stage of a trial.

Development of two age estimation models for buccal swab samples based on 3 CpG sites analyzed with pyrosequencing and minisequencing.

The analysis of DNA methylation levels of specific CpG sites is one of the most promising molecular techniques to estimate an individual's age. Numerous studies were published recently presenting age estimation models based on DNA methylation patterns from blood samples, with only a few using saliva or buccal swabs. The aim of this study was to identify age-dependent methylation of 88 CpG sites in eight different marker regions (PDE4C, ELOVL2, ITGA2B, ASPA, EDARADD, SST, KLF14 and SLC12A5) in buccal swab samples. A total of 141 buccal swabs from individuals with age ranging from 21 to 69 years were split into a training set (n = 95) and a validation set (n = 46). Samples of the training set were analyzed by pyrosequencing and markers with best age correlation were identified. Stepwise linear regression analysis was performed resulting in an age estimation model including three of the examined CpG sites and showing a mean absolute deviation of estimated from chronological age of 5.11 years. To allow easy implementation into forensic laboratories without the need for pyrosequencing equipment, a multiplex minisequencing reaction was developed, including the same CpG sites previously identified by pyrosequencing. An adjusted age estimation model was evaluated with a mean absolute deviation of estimated from chronological age of 5.16 years. The independent validation set of 46 buccal swab samples was used to test model performances. Mean absolute deviation of estimated from chronological age was 5.33 years and 6.44 years for the pyrosequencing model and the minisequencing model, respectively. Comparison of the two methods showed a high concordance of results, both, qualitatively and quantitatively. In conclusion, buccal swabs offer a suitable alternative to blood samples for molecular age estimation with the additional advantage of being collected non-invasively. Furthermore we showed that minisequencing offers a cost-effective and easy-to-integrate alternative to pyrosequencing for the analysis of methylation status of individual CpG sites.