Concordance of copy number abnormality detection using SNP arrays and Multiplex Ligation-dependent Probe Amplification (MLPA) in acute lymphoblastic leukaemia

In acute lymphoblastic leukaemia, MLPA has been used in research studies to identify clinically relevant copy number abnormality (CNA) profiles. However, in diagnostic settings other techniques are often employed. We assess whether equivalent CNA profiles are called using SNP arrays, ensuring platform independence. We demonstrate concordance between SNP6.0 and MLPA CNA calling on 143 leukaemia samples from two UK trials; comparing 1,287 calls within eight genes and a region. The techniques are 99% concordant using manually augmented calling, and 98% concordant using an automated pipeline. We classify these discordant calls and examine reasons for discordance. In nine cases the circular binary segmentation (CBS) algorithm failed to detect focal abnormalities or those flanking gaps in IKZF1 probe coverage. Eight cases were discordant due to probe design differences, with focal abnormalities detectable using one technique not observable by the other. Risk classification using manually augmented array calling resulted in four out of 143 patients being assigned to a different CNA risk group and eight patients using the automated pipeline. We conclude that MLPA defined CNA profiles can be accurately mirrored by SNP6.0 or similar array platforms. Automated calling using the CBS algorithm proved successful, except for IKZF1 which should be manually inspected

Accurate sample assignment in a multiplexed, ultrasensitive, high-throughput sequencing assay for minimal residual disease

High-throughput sequencing (HTS) (next-generation sequencing) of the rearranged Ig and T-cell receptor genes promises to be less expensive and more sensitive than current methods of monitoring minimal residual disease (MRD) in patients with acute lymphoblastic leukemia. However, the adoption of new approaches by clinical laboratories requires careful evaluation of all potential sources of error and the development of strategies to ensure the highest accuracy. Timely and efficient clinical use of HTS platforms will depend on combining multiple samples (multiplexing) in each sequencing run. Here we examine the Ig heavy-chain gene HTS on the Illumina MiSeq platform for MRD. We identify errors associated with multiplexing that could potentially impact the accuracy of MRD analysis. We optimize a strategy that combines high-purity, sequence-optimized oligonucleotides, dual indexing, and an error-aware demultiplexing approach to minimize errors and maximize sensitivity. We present a probability-based, demultiplexing pipeline Error-Aware Demultiplexer that is suitable for all MiSeq strategies and accurately assigns samples to the correct identifier without excessive loss of data. Finally, using controls quantified by digital PCR, we show that HTS-MRD can accurately detect as few as 1 in 10(6) copies of specific leukemic MRD

Guidance in an uncertain world

In the face of the coronavirus pandemic, clinicians are looking to multiple sources for guidance. Good guidance provides a helpful tool to support decision making by experienced and highly-trained healthcare professionals. However, in the context of a readily changing landscape there are risks of guidance that hinders rather than helps, duplicates effort and fails to consider the front-line implications. Conversely, an overly conservative approach may result in good guidance never seeing the light of day, or being published too late. We suggest that there are key principles that may help guideline producers to improve the process. These include: addressing directly and transparently the competing risks and benefits to individual patients, staff and the wider community; making greater efforts to find reliable data to inform recommendations; ensuring duplication of effort and conflict with extant guidance is minimised; involving front-line staff in development and consideration of real-world implications of delivery; and ensuring that feedback and revisions are integral to the process. The MORAL Balance framework has previously been advocated for making complex individual patient level decision in critical care. We believe the same process can be appliedas a framework for guideline development groups

Single nucleotide polymorphism (SNP) array-based signature of low hypodiploidy in acute lymphoblastic leukemia.

Low hypodiploidy (30-39 chromosomes) is one of the most prevalent genetic subtypes among adults with ALL and is associated with a very poor outcome. Low hypodiploid clones can often undergo a chromosomal doubling generating a near-triploid clone (60-78 chromosomes). When cytogenetic techniques detect a near triploid clone, a diagnostic challenge may ensue in differentiating presumed duplicated low hypodiploidy from good risk high hyperdiploid ALL (51-67 chromosomes). We used single-nucleotide polymorphism (SNP) arrays to analyze low hypodiploid/near triploid (HoTr) (n=48) and high hyperdiploid (HeH) (n=40) cases. In addition to standard analysis, we derived log2 ratios for entire chromosomes enabling us to analyze the cohort using machine-learning techniques. Low hypodiploid and near triploid cases clustered together and separately from high hyperdiploid samples. Using these approaches, we also identified three cases with 50-60 chromosomes, originally called as HeH, which were, in fact, HoTr and two cases incorrectly called as HoTr. TP53 mutation analysis supported the new classification of all cases tested. Next, we constructed a classification and regression tree model for predicting ploidy status with chromosomes 1, 7 and 14 being the key discriminators. The classifier correctly identified 47/50 (94%) HoTr cases. We validated the classifier using an independent cohort of 44 cases where it correctly called 7/7 (100%) low hypodiploid cases. The results of this study suggest that HoTr is more frequent among older adults with ALL than previously estimated and that SNP array analysis should accompany cytogenetics where possible. The classifier can assist where SNP array patterns are challenging to interpret. This article is protected by copyright. All rights reserved

Acute and chronic kidney disease in elderly patients with hip fracture: prevalence, risk factors and outcome with development and validation of a risk prediction model for acute kidney injury

Background Hip fracture is a common injury in older people with a high rate of postoperative morbidity and mortality. This patient group is also at high risk of acute kidney injury (AKI) and chronic kidney disease (CKD), but little is known of the impact of kidney disease on outcome following hip fracture. Methods An observational cohort of consecutive patients with hip fracture in a large UK secondary care hospital. Predictive modelling of outcomes using development and validation datasets. Inclusion: all patients admitted with hip fracture with sufficient serum creatinine measurements to define acute kidney injury. Main outcome measures – development of acute kidney injury during admission; mortality (in hospital, 30-365 day and to follow-up); length of hospital stay. Results Data were available for 2848 / 2959 consecutive admissions from 2007-2011; 776 (27.2%) male. Acute kidney injury occurs in 24%; development of acute kidney injury is independently associated with male sex (OR 1.48 (1.21 to 1.80), premorbid chronic kidney disease stage 3B or worse (OR 1.52 (1.19 to 1.93)), age (OR 3.4 (2.29 to 5.2) for >85 years) and greater than one major co-morbidities (OR 1.61 (1.34 to 1.93)). Acute kidney injury of any stage is associated with an increased hazard of death, and increased length of stay (Acute kidney injury: 19.1 (IQR 13 to 31) days; no acute kidney injury 15 (11 to 23) days). A simplified predictive model containing Age, CKD stage (3B-5), two or more comorbidities, and male sex had an area under the ROC curve of 0.63 (0.60 to 0.67). Conclusions Acute kidney injury following hip fracture is common and associated with worse outcome and greater hospital length of stay. With the number of people experiencing hip fracture predicted to rise, recognition of risk factors and optimal perioperative management of acute kidney injury will become even more important

Neuroprotective properties of levosimendan in an in vitro model of traumatic brain injury

<p>Abstract</p> <p>Background</p> <p>We investigated the neuroprotective properties of levosimendan, a novel inodilator, in an in vitro model of traumatic brain injury.</p> <p>Methods</p> <p>Organotypic hippocampal brain slices from mouse pups were subjected to a focal mechanical trauma. Slices were treated after the injury with three different concentrations of levosimendan (0.001, 0.01 and 0.1 μM) and compared to vehicle-treated slices. After 72 hrs, the trauma was quantified using propidium iodide to mark the injured cells.</p> <p>Results</p> <p>A significant dose-dependent reduction of both total and secondary tissue injury was observed in cells treated with either 0.01 or 0.1 μM levosimendan compared to vehicle-treated slices.</p> <p><b>Conclusion</b></p> <p>Levosimendan represents a promising new pharmacological tool for neuroprotection after brain injury and warrants further investigation in an in vivo model.</p