Computational flow cytometry as a diagnostic tool in suspected-myelodysplastic syndromes

The diagnostic work-up of patients suspected for myelodysplastic syndromes is challenging and mainly relies on bone marrow morphology and cytogenetics. In this study, we developed and prospectively validated a fully computational tool for flow cytometry diagnostics in suspected-MDS. The computational diagnostic workflow consists of methods for pre-processing flow cytometry data, followed by a cell population detection method (FlowSOM) and a machine learning classifier (Random Forest). Based on a six tubes FC panel, the workflow obtained a 90% sensitivity and 93% specificity in an independent validation cohort. For practical advantages (e.g., reduced processing time and costs), a second computational diagnostic workflow was trained, solely based on the best performing single tube of the training cohort. This workflow obtained 97% sensitivity and 95% specificity in the prospective validation cohort. Both workflows outperformed the conventional, expert analyzed flow cytometry scores for diagnosis with respect to accuracy, objectivity and time investment (less than 2 min per patient)

CoNVaDING:Single Exon Variation Detection in Targeted NGS Data

We have developed a tool for detecting single exon copy number variations (CNVs) in targeted next-generation sequencing data: CoNVaDING (Copy Number Variation Detection In Next-generation sequencing Gene panels). CoNVaDING includes a stringent quality control metric, that excludes or flags low quality exons. Since this quality control shows exactly which exons can be reliably analysed and which exons are in need of an alternative analysis method, CoNVaDING is not only useful for CNV detection in a research setting, but also in clinical diagnostics. During the validation phase, CoNVaDING detected all known CNVs in high quality targets in 320 samples analysed, giving 100% sensitivity and 99.998% specificity for 308,574 exons. CoNVaDING outperforms existing tools by exhibiting a higher sensitivity and specificity and by precisely identifying low-quality samples and regions. This article is protected by copyright. All rights reserved.</p

Multidetector CT imaging of mechanical prosthetic heart valves: quantification of artifacts with a pulsatile in-vitro model

Item does not contain fulltextOBJECTIVES: Multidetector computed tomography (MDCT) can detect the cause of prosthetic heart valve (PHV) dysfunction but is hampered by valve-induced artifacts. We quantified artifacts of four PHV using a pulsatile in-vitro model and assessed the relation to leaflet motion and valve design. METHODS: A Medtronic Hall tilting disc (MH), and Carbomedics (CM), St Jude (SJM), and ON-X bileaflet valves underwent CT in an in-vitro model using retrospective gating with a 64 detector CT system in stationary and pulsatile conditions. Artifacts and radiopaque component volumes were quantified with thresholds based on surrounding structures and valvular components. RESULTS: Hypodense artifacts volumes (mm(3)) were 1,029 +/- 147, 535 +/- 53, 371 +/- 16, and 366 +/- 18 for the SJM, MH, CM and ON-X valves (p < 0.001 except for the latter two valves p = 0.43). Hyperdense artifact volumes were 3,546 +/- 141, 2,387 +/- 103, 2,003 +/- 102, and 3,033 +/- 31 for the SJM, MH, CM and ON-X valve, respectively (all differences p < 0.001). Leaflet motion affected hypodense (F = 41.5, p < 0.001) and hyperdense artifacts (F = 53.7, p < 0.001). Closed and moving leaflets were associated with the least and the most artifacts respectively (p < 0.001, both artifact types). CONCLUSION: Both valve design and leaflet motion affect PHV-induced artifacts. Best imaging results may be expected for the CM valve during phases in which the leaflets are closed

Feasibility of Follow-Up Studies and Reclassification in Spinocerebellar Ataxia Gene Variants of Unknown Significance

Spinocerebellar ataxia (SCA) is a heterogeneous group of neurodegenerative disorders with autosomal dominant inheritance. Genetic testing for SCA leads to diagnosis, prognosis and risk assessment for patients and their family members. While advances in sequencing and computing technologies have provided researchers with a rapid expansion in the genetic test content that can be used to unravel the genetic causes that underlie diseases, the large number of variants with unknown significance (VUSes) detected represent challenges. To minimize the proportion of VUSes, follow-up studies are needed to aid in their reclassification as either (likely) pathogenic or (likely) benign variants. In this study, we addressed the challenge of prioritizing VUSes for follow-up using (a combination of) variant segregation studies, 3D protein modeling, in vitro splicing assays and functional assays. Of the 39 VUSes prioritized for further analysis, 13 were eligible for follow up. We were able to reclassify 4 of these VUSes to LP, increasing the molecular diagnostic yield by 1.1%. Reclassification of VUSes remains difficult due to limited possibilities for performing variant segregation studies in the classification process and the limited availability of routine functional tests

Prospective ECG triggering reduces prosthetic heart valve-induced artefacts compared with retrospective ECG gating on 256-slice CT

Item does not contain fulltextOBJECTIVES: Multidetector computed tomography (MDCT) has diagnostic value for the evaluation of prosthetic heart valve (PHV) dysfunction but it is hampered by artefacts. We hypothesised that image acquisition using prospective triggering instead of retrospective gating would reduce artefacts related to pulsating PHV. METHODS: In a pulsatile in vitro model, a mono- and bileaflet PHV were imaged using 256 MDCT at 60, 75 and 90 beats per minute (BPM) with either retrospective gating (120 kV, 600 mAs, pitch 0.2, CTDI(vol) 39.8 mGy) or prospective triggering (120 kV, 200 mAs, CTDI(vol) 13.3 mGy). Two thresholds (>175 and <-45HU), derived from the density of surrounding structures, were used for quantification of hyper- and hypodense artefacts. Image noise and artefacts were compared between protocols. RESULTS: Prospective triggering reduced hyperdense artefacts for both valves at every BPM (P = 0.001 all comparisons). Hypodense artefacts were reduced for the monoleaflet valve at 60 (P = 0.009), 75 (P = 0.016) and 90 BPM (P = 0.001), and for the bileaflet valves at 60 (P = 0.001), 90 (P = 0.001) but not at 75 BPM (P = 0.6). Prospective triggering reduced image noise at 60 (P = 0.001) and 75 (P < 0.03) but not at 90 BPM. CONCLUSIONS: Compared with retrospective gating, prospective triggering reduced most artefacts related to pulsating PHV in vitro. KEY POINTS: * Computed tomographic images are often degraded by prosthetic heart valve-induced artefacts * Prospective triggering reduces prosthetic heart valve-induced artefacts in vitro * Artefact reduction at 90 beats per minute occurs without image noise reduction * Prospective triggering may improve CT image quality of moving hyperdense structures.1 juni 201

Targeting Toll-like receptor 7/8 enhances uptake of apoptotic leukemic cells by monocyte-derived dendritic cells but interferes with subsequent cytokine-induced maturation

Therapeutic vaccination with dendritic cells (DC) is an emerging investigational therapy for eradication of minimal residual disease in acute myeloid leukemia. Various strategies are being explored in manufacturing DC vaccines ex vivo, e.g., monocyte-derived DC (MoDC) loaded with leukemia-associated antigens (LAA). However, the optimal source of LAA and the choice of DC-activating stimuli are still not well defined. Here, loading with leukemic cell preparations (harboring both unknown and known LAA) was explored in combination with a DC maturation-inducing cytokine cocktail (CC; IL-1β, IL-6, TNF-α, and PGE2) and Toll-like receptor ligands (TLR-L) to optimize uptake. Since heat shock induced apoptotic blasts were more efficiently taken up than lysates, we focused on uptake of apoptotic leukemic cells. Uptake of apoptotic blast was further enhanced by the TLR7/8-L R848 (20–30%); in contrast, CC-induced maturation inhibited uptake. CC, and to a lesser extent R848, enhanced the ability of MoDC to migrate and stimulate T cells. Furthermore, class II-associated invariant chain peptide expression was down-modulated after R848- or CC-induced maturation, indicating enhanced processing and presentation of antigenic peptides. To improve both uptake and maturation, leukemic cells and MoDC were co-incubated with R848 for 24 h followed by addition of CC. However, this approach interfered with CC-mediated MoDC maturation as indicated by diminished migratory and T cell stimulatory capacity, and the absence of IL-12 production. Taken together, our data demonstrate that even though R848 improved uptake of apoptotic leukemic cells, the sequential use of R848 and CC is counter-indicated due to its adverse effects on MoDC maturation

A genome-wide association study of Hodgkin Lymphoma identifies new susceptibility loci at 2p16.1 (REL), 8q24.21, and 10p14 (GATA3)

To identify predisposition loci for classical Hodgkin Lymphoma (cHL) we conducted a genome-wide association study of 589 cHL cases and 5,199 controls with validation in 4 independent samples totaling 2,057 cases and 3,416 controls. We identified three new susceptibility loci at 2p16.1 (rs1432295, REL; odds ratio [OR]=1.22, Pcombined=1.91×10−8), 8q24.21 (rs2019960, PVT1; OR=1.33, Pcombined=1.26×10−13) and 10p14 (rs501764, GATA3; OR=1.25, Pcombined=7.05×10−8). Furthermore, we confirmed the role of the MHC in disease etiology by revealing a strong HLA association (rs6903608; OR=1.70, Pcombined=2.84×10−50). These data provide new insight into the pathogenesis of cHL

Cytomorphology review of 100 newly diagnosed lower-risk MDS patients in the European LeukemiaNet MDS (EUMDS) registry reveals a high inter-observer concordance

Objectives To examine contemporary survival patterns in the general population of patients diagnosed with chronic myeloid leukaemia (CML), and to identify patient groups with less than optimal outcomes. Design Prospective population-based cohort. Setting The UK's Haematological Malignancy Research Network (catchment population 3.6 million, with >2000 new haematological malignancies diagnosed annually). Participants All patients newly diagnosed with CML, from September 2004 to August 2011 and followed up to 31 March 2013. Main outcome measure Incidence and survival. Results With a median diagnostic age of 59 years, the CML age standardised (European) incidence was 0.9/100 000 (95% CIs 0.8 to 0.9), 5-year overall survival was 78.9% (72.3 to 84.0) and 5-year relative survival 88.6% (81.0 to 93.3). The efficacy of treatment across all ages was clearly demonstrated; the relative survival curves for those under 60 and over 60 years being closely aligned. Survival findings were similar for men and women, but varied with deprivation; the age and sex adjusted HR being 3.43 (1.89 to 6.22) for deprivation categories 4–5 (less affluent) versus 1–3 (more affluent). None of these differences were attributable to the biological features of the disease. Conclusions When therapy is freely provided, population-based survival for CML is similar to that reported in clinical trials, and age loses its prognostic significance. However, although most of the patients with CML now experience close to normal lifespans, those living in more deprived areas tend to have poorer outcomes, despite receiving the same clinical care. A significant improvement in overall population outcomes could be achieved if these socioeconomic differences, which may reflect the treatment compliance, could be eliminated

Recurrent Coding Sequence Variation Explains only A Small Fraction of the Genetic Architecture of Colorectal Cancer

Whilst common genetic variation in many non-coding genomic regulatory regions are known to impart risk of colorectal cancer (CRC), much of the heritability of CRC remains unexplained. To examine the role of recurrent coding sequence variation in CRC aetiology, we genotyped 12,638 CRCs cases and 29,045 controls from six European populations. Single-variant analysis identified a coding variant (rs3184504) in SH2B3 (12q24) associated with CRC risk (OR = 1.08, P = 3.9 × 10-7), and novel damaging coding variants in 3 genes previously tagged by GWAS efforts; rs16888728 (8q24) in UTP23 (OR = 1.15, P = 1.4 × 10-7); rs6580742 and rs12303082 (12q13) in FAM186A (OR = 1.11, P = 1.2 × 10-