CombiFlow:Flow cytometry-based identification and characterization of genetically and functionally distinct AML subclones

Summary: Many cancers, including leukemias, are dynamic oligoclonal diseases. Tools to identify and prospectively isolate genetically distinct clones for functional studies are needed. We describe our CombiFlow protocol, which is a combinatorial flow cytometry-based approach to identify and isolate such distinct clones. CombiFlow enables the visualization of clonal evolution during disease progression and the identification of potential relapse-inducing cells at minimal residual disease (MRD) time points. The protocol can be adapted to various research questions and allows functional studies on live sorted cell populations.For complete details on the use and execution of this protocol, please refer to de Boer et al. (2018)

Quantification of proteins in whole blood, plasma and DBS, with element-labelled antibody detection by ICP-MS

Over recent years, quantification of multiple proteins in body fluids has become increasingly prominent, which is beneficial to a number of scientific fields, not least biomedical. Several techniques have been developed based on conventional ELISA; one of these techniques is analysis of proteins labelled with element-tagged antibodies by ICP-MS in serum, allowing quantification of multiple targets within a single sample. This research aimed to quantify albumin and immunoglobulin G (IgG) levels in plasma, whole blood and dried blood spots using NANOGOLD and Europium labelled antibodies analysed by ICP-MS. Before the proteins were quantified simultaneously, albumin and IgG concentrations were measured separately and compared to protein levels obtained by ELISA. It was found that protein concentrations for both albumin and IgG obtained with element-labelled antibody detection correspond to those determined by ELISA. Furthermore, albumin and IgG levels measured simultaneously by ICP-MS correspond to concentrations found when the proteins were analysed separately by ICP-MS. Finally, development of this method has provided a positive indication that it can be extended to quantification of additional proteins, which could be related to a disease or as a minimum provide additional information for a protein profile of an individual. [Abstract copyright: Copyright © 2019. Published by Elsevier Inc.

CombiFlow:combinatorial AML-specific plasma membrane expression profiles allow longitudinal tracking of clones

Acute myeloid leukemia (AML) often presents as an oligoclonal disease whereby multiple genetically distinct subclones can coexist within patients. Differences in signaling and drug sensitivity of such subclones complicate treatment and warrant tools to identify them and track disease progression. We previously identified >50 AML-specific plasma membrane (PM) proteins, and 7 of these (CD82, CD97, FLT3, IL1RAP, TIM3, CD25, and CD123) were implemented in routine diagnostics in patients with AML (n = 256) and myelodysplastic syndrome (n = 33). We developed a pipeline termed CombiFlow in which expression data of multiple PM markers is merged, allowing a principal component–based analysis to identify distinctive marker expression profiles and to generate single-cell t-distributed stochastic neighbor embedding landscapes to longitudinally track clonal evolution. Positivity for one or more of the markers after 2 courses of intensive chemotherapy predicted a shorter relapse-free survival, supporting a role for these markers in measurable residual disease (MRD) detection. CombiFlow also allowed the tracking of clonal evolution in paired diagnosis and relapse samples. Extending the panel to 36 AML-specific markers further refined the CombiFlow pipeline. In conclusion, CombiFlow provides a valuable tool in the diagnosis, MRD detection, clonal tracking, and understanding of clonal heterogeneity in AML

CREB signaling activity correlates with differentiation and survival in medulloblastoma

While there has been significant progress in the molecular characterization of the childhood brain cancer medulloblastoma, the tumor proteome remains less explored. However, it is important to obtain a complete understanding of medulloblastoma protein biology, since interactions between proteins represent potential new drug targets. Using previously generated phosphoprotein signaling-profiles of a large cohort of primary medulloblastoma, we discovered that phosphorylation of transcription factor CREB strongly correlates with medulloblastoma survival and associates with a differentiation phenotype. We further found that during normal cerebellar development, phosphorylated CREB was selectively expressed in differentiating cerebellar granule neuron progenitor (CGNP) cells. In line, we observed increased differentiation in CGNPs treated with Forskolin, Bmp6 and Bmp12 (Gdf7), which induce CREB phosphorylation. Lastly, we demonstrated that inducing CREB activation via PKA-mediated CREB signaling, but not Bmp/MEK/ERK mediated signalling, enhances medulloblastoma cell sensitivity to chemotherapy

The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia

In an attempt to unravel functionality of the non-canonical PRC1.1 Polycomb complex in human leukemogenesis, we show that USP7 and TRIM27 are integral components of PRC1.1. USP7 interactome analyses show that PRC1.1 is the predominant Polycomb complex co-precipitating with USP7. USP7 inhibition results in PRC1.1 disassembly and loss of chromatin binding, coinciding with reduced H2AK119ub and H3K27ac levels and diminished gene transcription of active PRC1.1-controlled loci, whereas H2AK119ub marks are also lost at PRC1 loci. TRIM27 and USP7 are reciprocally required for incorporation into PRC1.1, and TRIM27 knockdown partially rescues USP7 inhibitor sensitivity. USP7 inhibitors effectively impair proliferation in AML cells in vitro, also independent of the USP7-MDM2-TP53 axis, and MLL-AF9-induced leukemia is delayed in vivo in human leukemia xenografts. We propose a model where USP7 counteracts TRIM27 E3 ligase activity, thereby maintaining PRC1.1 integrity and function. Moreover, USP7 inhibition may be a promising new strategy to treat AML patients

A comparison of psoriasis severity in pediatric patients treated with methotrexate vs biologic agents

This cohort study compares the use of methotrexate vs biologic agents in children with moderate to severe psoriasis. Question What is the association between use of methotrexate vs biologics and psoriasis severity and drug survival (rate and duration of adherence to a specific drug regimen) in pediatric patients with moderate to severe psoriasis? Findings In this cohort study including 234 pediatric patients with moderate to severe psoriasis, those receiving biologics were more likely than those treated with methotrexate to achieve a Physician Global Assessment status of clear/almost clear and 75% or more improvement of the Psoriasis Area and Severity Index rating at 6 months. In addition, biologics were associated with better drug survival rates at 1, 3, and 5 years, with comparable discontinuation rates owing to lack of response. Meaning In pediatric patients with psoriasis, treatment with biologics may be associated with a significantly greater reduction in psoriasis severity than methotrexate; nevertheless, with 35.6% of the patients achieving clear/almost clear and 40.0% reaching 75% or more improvement on the Psoriasis Area and Severity Index, methotrexate remains an effective treatment for pediatric psoriasis. Importance Few studies have compared the use of methotrexate and biologics, the most commonly used systemic medications for treatment of moderate to severe psoriasis in children. Objective To assess the real-world, 6-month reduction in psoriasis severity and long-term drug survival (rate and duration of adherence to a specific drug) of methotrexate vs biologics in plaque psoriasis in children. Design, Setting, and Participants A retrospective medical records review was conducted at 20 European and North American centers. Treatment response was based on site-reported Psoriasis Area and Severity Index (PASI) and/or Physician Global Assessment (PGA) scores at baseline and within the first 6 months of treatment. Participants included all 234 consecutively seen children with moderate to severe psoriasis who received at least 3 months of methotrexate or biologics from December 1, 1990, to September 16, 2014, with sufficient data for analysis. Data analysis was performed from December 14, 2015, to September 1, 2016. Main Outcomes and Measures PASI, with a range from 0 to 72 (highest score indicating severe psoriasis), and/or PGA, with a scale of 0 (clear), 1 (minimal), 2 (mild), 3 (moderate), 4 (severe), and 5 (very severe). Results Of 234 pediatric patients (103 boys [44.0%]; 131 girls [56.0%]) treated with methotrexate and/or biologics, 163 patients (69.7%) exclusively received methotrexate, 47 patients (20.1%) exclusively received biologics, and 24 children (10.2%) received methotrexate and biologics sequentially. Of the latter cohort, 23 children were treated initially with methotrexate. Mean (SD) age at initiation was 11.6 (3.7) years for methotrexate and 13.3 (2.9) years for biologics (73.2% for etanercept) (P = .002). Among patients evaluated by a scoring method at 6-month follow-up, 75% or greater improvement in PASI (PASI75) was achieved in 12 of 30 patients (40.0%) receiving methotrexate and 20 of 28 patients (71.4%) receiving biologics, and PGA was clear/almost clear (PGA 0/1) in 41 of 115 patients (35.6%) receiving methotrexate and 18 of 37 patients (48.6%) receiving biologics. Achieving PASI75 and/or PGA 0/1 between baseline and 6 months was more likely with biologics than methotrexate (PASI75: odds ratio [OR], 4.56; 95% CI, 2.02-10.27; P < .001; and PGA 0/1: OR, 2.00; 95% CI, 0.98-4.00; P = .06). Decreased mean PASI and PGA scores were associated with biologics more than with methotrexate (PASI effect, -3.13; 95% CI, -4.33 to -1.94; P < .001; and PGA effect, -0.31; 95% CI, -0.56 to -0.06; P = .02). After 1, 3, and 5 years of use, overall drug survival rates for methotrexate were 77.5%, 50.3%, and 35.9%, and for biologics, the rates were 83.4%, 64.3%, and 57.1%, respectively. Biologics were associated with a better confounder-corrected drug survival than methotrexate (hazard ratio [HR], 2.23; 95% CI, 1.21-4.10; P = .01). Discontinuation owing to lack of response was comparable (HR, 1.64; 95% CI, 0.80-3.36; P = .18). Conclusions and Relevance Methotrexate and biologics appear to be associated with improvement in pediatric psoriasis, although biologics seem to be associated with greater reduction in psoriasis severity scores and higher drug survival rates than methotrexate in the real-world setting. Additional studies directly comparing these medications should be performed for confirmation

COVID-19 and Airborne Transmission: Science Rejected, Lives Lost. Can Society Do Better?

This is an account that should be heard of an important struggle: the struggle of a large group of experts who came together at the beginning of the COVID-19 pandemic to warn the world about the risk of airborne transmission and the consequences of ignoring it. We alerted the World Health Organization about the potential significance of the airborne transmission of SARS-CoV-2 and the urgent need to control it, but our concerns were dismissed. Here we describe how this happened and the consequences. We hope that by reporting this story we can raise awareness of the importance of interdisciplinary collaboration and the need to be open to new evidence, and to prevent it from happening again. Acknowledgement of an issue, and the emergence of new evidence related to it, is the first necessary step towards finding effective mitigation solutions