Double CEBPA mutations, but not single CEBPA mutations, define a subgroup of acute myeloid leukemia with a distinctive gene expression profile that is uniquely associated with a favorable outcome

Mutations in CCAAT/enhancer binding protein α (CEBPA) are seen in 5% to 14% of acute myeloid leukemia (AML) and have been associated with a favorable clinical outcome. Most AMLs with CEBPA mutations simultaneously carry 2 mutations (CEBPAdouble-mut), usually biallelic, whereas single heterozygous mutations (CEBPAsingle-mut) are less frequently seen. Using denaturing high-performance liquid chromatography and nucleotide sequencing, we identified among a cohort of 598 newly diagnosed AMLs a subset of 41 CEBPA mutant cases (28 CEBPAdouble-mut and 13 CEBPA single-mut cases) CEBPAdouble-mut associated with a unique gene expression profile as well as favorable overall and event-free survival, retained in multi-variable analysis that included cytoge-netic risk, FZT3-ITD and NPM1 mutation, white blood cell count, and age. In contrast, CEBPA single-mut AMLs did not express a discriminating signature and could not be distinguished from wild-type cases as regards clinical outcome. These results demonstrate significant underlying heterogeneity within CEBPA mutation-positive AML with prognostic relevance

Integrated Bioluminescent Immunoassays for High-Throughput Sampling and Continuous Monitoring of Cytokines

Immunoassays show great potential for the detection of low levels of cytokines, due to their high sensitivity and excellent specificity. There is a particular demand for biosensors that enable both high-throughput screening and continuous monitoring of clinically relevant cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα). To this end, we here introduce a novel bioluminescent immunoassay based on the ratiometric plug-and-play immunodiagnostics (RAPPID) platform, with an improved intrinsic signal-to-background and an &gt;80-fold increase in the luminescent signal. The new dRAPPID assay, comprising a dimeric protein G adapter connected via a semiflexible linker, was applied to detect the secretion of IL-6 by breast carcinoma cells upon TNFα stimulation and the production of low concentrations of IL-6 (∼18 pM) in an endotoxin-stimulated human 3D muscle tissue model. Moreover, we integrated the dRAPPID assay in a newly developed microfluidic device for the simultaneous and continuous monitoring of changes in IL-6 and TNFα in the low-nanomolar range. The luminescence-based read-out and the homogeneous nature of the dRAPPID platform allowed for detection with a simple measurement setup, consisting of a digital camera and a light-sealed box. This permits the usage of the continuous dRAPPID monitoring chip at the point of need, without the requirement for complex or expensive detection techniques.</p

Assessing quality of hepato-pancreato-biliary surgery: nationwide benchmarking

Background: Clinical auditing is a powerful tool to evaluate and improve healthcare. Deviations from the expected quality of care are identified by benchmarking the results of individual hospitals using national averages. This study aimed to evaluate the use of quality indicators for benchmarking hepato-pancreato-biliary (HPB) surgery and when outlier hospitals could be identified. Methods: A population-based study used data from two nationwide Dutch HPB audits (DHBA and DPCA) from 2014 to 2021. Sample size calculations determined the threshold (in percentage points) to identify centres as statistical outliers, based on current volume requirements (annual minimum of 20 resections) on a two-year period (2020–2021), covering mortality rate, failure to rescue (FTR), major morbidity rate and textbook/ideal outcome (TO) for minor liver resection (LR), major LR, pancreaticoduodenectomy (PD) and distal pancreatectomy (DP). Results: In total, 10 963 and 7365 patients who underwent liver and pancreatic resection respectively were included. Benchmark and corresponding range of mortality rates were 0.6% (0 −3.2%) and 3.3% (0–16.7%) for minor and major LR, and 2.7% (0–7.0%) and 0.6% (0–4.2%) for PD and DP respectively. FTR rates were 5.4% (0–33.3%), 14.2% (0–100%), 7.5% (1.6%–28.5%) and 3.1% (0–14.9%). For major morbidity rate, corresponding rates were 9.8% (0–20.5%), 28.1% (0–47.1%), 36% (15.8%–58.3%) and 22.3% (5.2%–46.1%). For TO, corresponding rates were 73.6% (61.3%–94.4%), 54.1% (35.3–100), 46.8% (25.3%–59.4%) and 63.3% (30.7%–84.6%). Mortality rate thresholds indicating a significant outlier were 8.6% and 15.4% for minor and major LR and 14.2% and 8.6% for PD and DP. For FTR, these thresholds were 17.9%, 31.6%, 22.9% and 15.0%. For major morbidity rate, these thresholds were 26.1%, 49.7%, 57.9% and 52.9% respectively. For TO, lower thresholds were 52.5%, 32.5%, 25.8% and 41.4% respectively. Higher hospital volumes decrease thresholds to detect outliers. Conclusion: Current event rates and minimum volume requirements per hospital are too low to detect any meaningful between hospital differences in mortality rate and FTR. Major morbidity rate and TO are better candidates to use for benchmarking

Assessing quality of hepato-pancreato-biliary surgery: nationwide benchmarking

Background: Clinical auditing is a powerful tool to evaluate and improve healthcare. Deviations from the expected quality of care are identified by benchmarking the results of individual hospitals using national averages. This study aimed to evaluate the use of quality indicators for benchmarking hepato-pancreato-biliary (HPB) surgery and when outlier hospitals could be identified. Methods: A population-based study used data from two nationwide Dutch HPB audits (DHBA and DPCA) from 2014 to 2021. Sample size calculations determined the threshold (in percentage points) to identify centres as statistical outliers, based on current volume requirements (annual minimum of 20 resections) on a two-year period (2020–2021), covering mortality rate, failure to rescue (FTR), major morbidity rate and textbook/ideal outcome (TO) for minor liver resection (LR), major LR, pancreaticoduodenectomy (PD) and distal pancreatectomy (DP). Results: In total, 10 963 and 7365 patients who underwent liver and pancreatic resection respectively were included. Benchmark and corresponding range of mortality rates were 0.6% (0 −3.2%) and 3.3% (0–16.7%) for minor and major LR, and 2.7% (0–7.0%) and 0.6% (0–4.2%) for PD and DP respectively. FTR rates were 5.4% (0–33.3%), 14.2% (0–100%), 7.5% (1.6%–28.5%) and 3.1% (0–14.9%). For major morbidity rate, corresponding rates were 9.8% (0–20.5%), 28.1% (0–47.1%), 36% (15.8%–58.3%) and 22.3% (5.2%–46.1%). For TO, corresponding rates were 73.6% (61.3%–94.4%), 54.1% (35.3–100), 46.8% (25.3%–59.4%) and 63.3% (30.7%–84.6%). Mortality rate thresholds indicating a significant outlier were 8.6% and 15.4% for minor and major LR and 14.2% and 8.6% for PD and DP. For FTR, these thresholds were 17.9%, 31.6%, 22.9% and 15.0%. For major morbidity rate, these thresholds were 26.1%, 49.7%, 57.9% and 52.9% respectively. For TO, lower thresholds were 52.5%, 32.5%, 25.8% and 41.4% respectively. Higher hospital volumes decrease thresholds to detect outliers. Conclusion: Current event rates and minimum volume requirements per hospital are too low to detect any meaningful between hospital differences in mortality rate and FTR. Major morbidity rate and TO are better candidates to use for benchmarking

Epigenetic alterations affecting hematopoietic regulatory networks as drivers of mixed myeloid/lymphoid leukemia

Leukemias with ambiguous lineage comprise several loosely defined entities, often without a clear mechanistic basis. Here, we extensively profile the epigenome and transcriptome of a subgroup of such leukemias with CpG Island Methylator Phenotype. These leukemias exhibit comparable hybrid myeloid/lymphoid epigenetic landscapes, yet heterogeneous genetic alterations, suggesting they are defined by their shared epigenetic profile rather than common genetic lesions. Gene expression enrichment reveals similarity with early T-cell precursor acute lymphoblastic leukemia and a lymphoid progenitor cell of origin. In line with this, integration of differential DNA methylation and gene expression shows widespread silencing of myeloid transcription factors. Moreover, binding sites for hematopoietic transcription factors, including CEBPA, SPI1 and LEF1, are uniquely inaccessible in these leukemias. Hypermethylation also results in loss of CTCF binding, accompanied by changes in chromatin interactions involving key transcription factors. In conclusion, epigenetic dysregulation, and not genetic lesions, explains the mixed phenotype of this group of leukemias with ambiguous lineage. The data collected here constitute a useful and comprehensive epigenomic reference for subsequent studies of acute myeloid leukemias, T-cell acute lymphoblastic leukemias and mixed-phenotype leukemias.</p

Perforin, granzyme B, and FasL expression by peripheral blood T lymphocytes in emphysema

<p>Abstract</p> <p>Background</p> <p>It is generally accepted that emphysematous lungs are characterized by an increase in the numbers of neutrophils, macrophages, and CD8⁺T lymphocytes, the lasts having increased cytotoxic activity. Because systemic inflammation is also a component of emphysema, we hypothesize that peripheral CD8⁺T lymphocytes of emphysematous smokers who show evidence of systemic inflammation will have higher expression of cytotoxic molecules.</p> <p>Methods</p> <p>We assessed parameters of systemic inflammation in normal individuals (smokers or non-smokers) and in emphysematous subjects with an active smoking history by measuring serum interleukine-6, C-reactive protein, and tumor necrosis factor. Expression of perforin, granzyme B, and FasL protein by CD8⁺T lymphocytes, CD4⁺T lymphocytes, and natural killer cells were assessed by flow cytometry while perforin, granzyme B, and FasL mRNA expression were measured on purified systemic CD8⁺T lymphocytes by real-time PCR.</p> <p>Results</p> <p>Emphysematous smokers had higher levels of serum interleukine-6 than normal subjects. Even with the presence of systemic inflammation in emphysematous smokers, the percentage of peripheral CD8⁺T lymphocytes, CD4⁺T lymphocytes, and NK cells expressing perforin and granzyme B protein was not different between the three groups.</p> <p>Conclusion</p> <p>Despite evidence of systemic inflammation, peripheral T lymphocytes of emphysematous smokers did not show higher levels of cytotoxic markers, suggesting that increase of activated T lymphocytes in the emphysematous lung may be due to either activation in the lung or specific peripheral recruitment.</p

SARS-CoV-2 outbreaks in secondary school settings in the Netherlands during fall 2020: silent circulation

BACKGROUND: In fall 2020 when schools in the Netherlands operated under a limited set of COVID-19 measures, we conducted outbreaks studies in four secondary schools to gain insight in the level of school transmission and the role of SARS-CoV-2 transmission via air and surfaces. METHODS: Outbreak studies were performed between 11 November and 15 December 2020 when the wild-type variant of SARS-CoV-2 was dominant. Clusters of SARS-CoV-2 infections within schools were identified through a prospective school surveillance study. All school contacts of cluster cases, irrespective of symptoms, were invited for PCR testing twice within 48 h and 4-7 days later. Combined NTS and saliva samples were collected at each time point along with data on recent exposure and symptoms. Surface and active air samples were collected in the school environment. All samples were PCR-tested and sequenced when possible. RESULTS: Out of 263 sampled school contacts, 24 tested SARS-CoV-2 positive (secondary attack rate 9.1%), of which 62% remained asymptomatic and 42% had a weakly positive test result. Phylogenetic analysis on 12 subjects from 2 schools indicated a cluster of 8 and 2 secondary cases, respectively, but also other distinct strains within outbreaks. Of 51 collected air and 53 surface samples, none were SARS-CoV-2 positive. CONCLUSION: Our study confirmed within school SARS-CoV-2 transmission and substantial silent circulation, but also multiple introductions in some cases. Absence of air or surface contamination suggests environmental contamination is not widespread during school outbreaks

Text-derived concept profiles support assessment of DNA microarray data for acute myeloid leukemia and for androgen receptor stimulation

BACKGROUND: High-throughput experiments, such as with DNA microarrays, typically result in hundreds of genes potentially relevant to the process under study, rendering the interpretation of these experiments problematic. Here, we propose and evaluate an approach to find functional associations between large numbers of genes and other biomedical concepts from free-text literature. For each gene, a profile of related concepts is constructed that summarizes the context in which the gene is mentioned in literature. We assign a weight to each concept in the profile based on a likelihood ratio measure. Gene concept profiles can then be clustered to find related genes and other concepts. RESULTS: The experimental validation was done in two steps. We first applied our method on a controlled test set. After this proved to be successful the datasets from two DNA microarray experiments were analyzed in the same way and the results were evaluated by domain experts. The first dataset was a gene-expression profile that characterizes the cancer cells of a group of acute myeloid leukemia patients. For this group of patients the biological background of the cancer cells is largely unknown. Using our methodology we found an association of these cells to monocytes, which agreed with other experimental evidence. The second data set consisted of differentially expressed genes following androgen receptor stimulation in a prostate cancer cell line. Based on the analysis we put forward a hypothesis about the biological processes induced in these studied cells: secretory lysosomes are involved in the production of prostatic fluid and their development and/or secretion are androgen-regulated processes. CONCLUSION: Our method can be used to analyze DNA microarray datasets based on information explicitly and implicitly available in the literature. We provide a publicly available tool, dubbed Anni, for this purpose

External validation of six COVID-19 prognostic models for predicting mortality risk in older populations in a hospital, primary care, and nursing home setting

Objectives To systematically evaluate the performance of COVID-19 prognostic models and scores for mortality risk in older populations across three health-care settings: hospitals, primary care, and nursing homes. Study Design and Setting This retrospective external validation study included 14,092 older individuals of ≥70 years of age with a clinical or polymerase chain reaction-confirmed COVID-19 diagnosis from March 2020 to December 2020. The six validation cohorts include three hospital-based (CliniCo, COVID-OLD, COVID-PREDICT), two primary care-based (Julius General Practitioners Network/Academisch network huisartsgeneeskunde/Network of Academic general Practitioners, PHARMO), and one nursing home cohort (YSIS) in the Netherlands. Based on a living systematic review of COVID-19 prediction models using Prediction model Risk Of Bias ASsessment Tool for quality and risk of bias assessment and considering predictor availability in validation cohorts, we selected six prognostic models predicting mortality risk in adults with COVID-19 infection (GAL-COVID-19 mortality, 4C Mortality Score, National Early Warning Score 2-extended model, Xie model, Wang clinical model, and CURB65 score). All six prognostic models were validated in the hospital cohorts and the GAL-COVID-19 mortality model was validated in all three healthcare settings. The primary outcome was in-hospital mortality for hospitals and 28-day mortality for primary care and nursing home settings. Model performance was evaluated in each validation cohort separately in terms of discrimination, calibration, and decision curves. An intercept update was performed in models indicating miscalibration followed by predictive performance re-evaluation. Main Outcome Measure In-hospital mortality for hospitals and 28-day mortality for primary care and nursing home setting. Results All six prognostic models performed poorly and showed miscalibration in the older population cohorts. In the hospital settings, model performance ranged from calibration-in-the-large −1.45 to 7.46, calibration slopes 0.24–0.81, and C-statistic 0.55–0.71 with 4C Mortality Score performing as the most discriminative and well-calibrated model. Performance across health-care settings was similar for the GAL-COVID-19 model, with a calibration-in-the-large in the range of −2.35 to −0.15 indicating overestimation, calibration slopes of 0.24–0.81 indicating signs of overfitting, and C-statistic of 0.55–0.71. Conclusion Our results show that most prognostic models for predicting mortality risk performed poorly in the older population with COVID-19, in each health-care setting: hospital, primary care, and nursing home settings. Insights into factors influencing predictive model performance in the older population are needed for pandemic preparedness and reliable prognostication of health-related outcomes in this demographic