Jevon Cutler

Standardization of flow cytometry in myelodysplastic syndromes: a report from an international consortium and the European LeukemiaNet Working Group

Flow cytometry (FC) is increasingly recognized as an important tool in the diagnosis and prognosis of myelodysplastic syndromes (MDS). However, validation of current assays and agreement upon the techniques are prerequisites for its widespread acceptance and application in clinical practice. Therefore, a working group was initiated (Amsterdam, 2008) to discuss and propose standards for FC in MDS. In 2009 and 2010, representatives from 23, mainly European, institutes participated in the second and third European LeukemiaNet (ELN) MDS workshops. In the present report, minimal requirements to analyze dysplasia are refined. The proposed core markers should enable a categorization of FC results in cytopenic patients as ‘normal’, ‘suggestive of’, or ‘diagnostic of’ MDS. An FC report should include a description of validated FC abnormalities such as aberrant marker expression on myeloid progenitors and, furthermore, dysgranulopoiesis and/or dysmonocytopoiesis, if at least two abnormalities are evidenced. The working group is dedicated to initiate further studies to establish robust diagnostic and prognostic FC panels in MDS. An ultimate goal is to refine and improve diagnosis and prognostic scoring systems. Finally, the working group stresses that FC should be part of an integrated diagnosis rather than a separate technique.

Rationale for the clinical application of flow cytometry in patients with myelodysplastic syndromes: position paper of an International Consortium and the European LeukemiaNet Working Group

Abstract An international working group within the European LeukemiaNet gathered, aiming to determine the role of flow cytometry (FC) in myelodysplastic syndromes (MDS). It was agreed that FC has a substantial application in disease characterization, diagnosis and prognosis. FC may also be useful in predicting treatment responses and monitoring novel and standard therapeutic regimens. In this article the rationale is discussed that flow cytometry should be integrated as a part of diagnostic and prognostic scoring systems in MDS.

Rationale for the clinical application of flow cytometry in patients with myelodysplastic syndromes: position paper of an International Consortium and the European LeukemiaNet Working Group.

Abstract An international working group within the European LeukemiaNet gathered, aiming to determine the role of flow cytometry (FC) in myelodysplastic syndromes (MDS). It was agreed that FC has a substantial application in disease characterization, diagnosis and prognosis. FC may also be useful in predicting treatment responses and monitoring novel and standard therapeutic regimens. In this article the rationale is discussed that flow cytometry should be integrated as a part of diagnostic and prognostic scoring systems in MDS.

Phenotypic Abnormalities Strongly Reflect Genotype in Patients with Unexplained Cytopenias

In patients with unexplained cytopenias, abnormal karyotyping studies can be found with inconclusive light microscopic findings. Multidimensional flow cytometry (FCM) can identify myelomonocytic cells with aberrant phenotypes often not seen by standard morphology.

A multi-omic analysis of human naïve CD4+ T cells

BackgroundCellular function and diversity are orchestrated by complex interactions of fundamental biomolecules including DNA, RNA and proteins. Technological advances in genomics, epigenomics, transcriptomics and proteomics have enabled massively parallel and unbiased measurements. Such high-throughput technologies have been extensively used to carry out broad, unbiased studies, particularly in the context of human diseases. Nevertheless, a unified analysis of the genome, epigenome, transcriptome and proteome of a single human cell type to obtain a coherent view of the complex interplay between various biomolecules has not yet been undertaken. Here, we report the first multi-omic analysis of human primary naïve CD4+ T cells isolated from a single individual.ResultsIntegrating multi-omics datasets allowed us to investigate genome-wide methylation and its effect on mRNA/protein expression patterns, extent of RNA editing under normal physiological conditions and allele specific expression in naïve CD4+ T cells. In addition, we carried out a multi-omic comparative analysis of naïve with primary resting memory CD4+ T cells to identify molecular changes underlying T cell differentiation. This analysis provided mechanistic insights into how several molecules involved in T cell receptor signaling are regulated at the DNA, RNA and protein levels. Phosphoproteomics revealed downstream signaling events that regulate these two cellular states. Availability of multi-omics data from an identical genetic background also allowed us to employ novel proteogenomics approaches to identify individual-specific variants and putative novel protein coding regions in the human genome.ConclusionsWe utilized multiple high-throughput technologies to derive a comprehensive profile of two primary human cell types, naïve CD4+ T cells and memory CD4+ T cells, from a single donor. Through vertical as well as horizontal integration of whole genome sequencing, methylation arrays, RNA-Seq, miRNA-Seq, proteomics, and phosphoproteomics, we derived an integrated and comparative map of these two closely related immune cells and identified potential molecular effectors of immune cell differentiation following antigen encounter.

Differential signaling through p190 and p210 BCR-ABL fusion proteins revealed by interactome and phosphoproteome analysis

Two major types of leukemogenic BCR-ABL fusion proteins are p190BCR-ABLand p210BCR-ABL. Although the two fusion proteins are closely related, they can lead to different clinical outcomes. A thorough understanding of the signaling programs employed by these two fusion proteins is necessary to explain these clinical differences. We took an integrated approach by coupling protein–protein interaction analysis using biotinylation identification with global phosphorylation analysis to investigate the differences in signaling between these two fusion proteins. Our findings suggest that p190BCR-ABL and p210BCR-ABL differentially activate important signaling pathways, such as JAK-STAT, and engage with molecules that indicate interaction with different subcellular compartments. In the case of p210BCR-ABL, we observed an increased engagement of molecules active proximal to the membrane and in the case of p190BCR-ABL, an engagement of molecules of the cytoskeleton. These differences in signaling could underlie the distinct leukemogenic process induced by these two protein variants.

Prediction of Gene Activity in Early B Cell Development Based on an Integrative Multi-Omics Analysis

An increasingly common method for predicting gene activity is genome-wide chromatin immuno-precipitation of ‘active’ chromatin modifications followed by massively parallel sequencing (ChIP-seq). In order to understand better the relationship between developmentally regulated chromatin landscapes and regulation of early B cell development, we determined how differentially active promoter regions were able to predict relative RNA and protein levels at the pre-pro-B and pro-B stages. Herein, we describe a novel ChIP-seq quantification method (cRPKM) to identify active promoters and a multi-omics approach that compares promoter chromatin status with ongoing active transcription (GRO-seq), steady state mRNA (RNA-seq), inferred mRNA stability, and relative proteome abundance measurements (iTRAQ). We demonstrate that active chromatin modifications at promoters are good indicators of transcription and steady state mRNA levels. Moreover, we found that promoters with active chromatin modifications exclusively in one of these cell states frequently predicted the differential abundance of proteins. However, we found that many genes whose promoters have non-differential but active chromatin modifications also displayed changes in abundance of their cognate proteins. As expected, this large class of developmentally and differentially regulated proteins that was uncoupled from chromatin status used mostly post-transcriptional mechanisms. Strikingly, the most differentially abundant protein in our B-cell development system, 2410004B18Rik, was regulated by a post-transcriptional mechanism, which further analyses indicated was mediated by a micro-RNA. These data highlight how this integrated multi-omics data set can be a useful resource in uncovering regulatory mechanisms. This data can be accessed at: https://usegalaxy.org/u/thereddylab/p/prediction-of-gene-activity-based-on-an-integrative-multi-omics-analysis

A minority of concurrent monoclonal lymphocytes and plasmacytic cells sharing light chains are genetically related in putative lymphoplasmacytic lymphoma

Flow cytometric cell sorting combined with molecular gene rearrangement analysis was used to detect and to further characterize simultaneously occurring phenotypically distinct B cell monoclonal lymphoid and monoclonal plasma cell populations from 38 individual specimens. By sorting and subsequent gene rearrangement analysis, separate or identical monoclonality genotypes could be revealed and confirmed. In only 13 of 38 specimens, the B lymphoid cells and plasma cell populations showed an identical genotypic profile, while 25 had non-identical profiles (including 4 process control specimens). The majority of the genotypically identical group had a phenotype consistent with Waldenströms/lymphoplasmacytic lymphoma (WM/LPL), while WM/LPL phenotype was present in 16/25 of the non-identical cases. Proof of an identical monoclonal genotype for plasmacytic and B-lymphoid cell populations must be used to define WM/LPL as a distinct entity in the clinical setting of monoclonal lymphoid and plasma cells expressing the same light chains. Conversely, the confirmation of genotypically distinct populations can significantly improve confidence in diagnostic and prognostic decisions in specimens with B lymphoid lymphomas and a concurrent, possibly smoldering myeloma or multiple myeloma. These techniques are requisite in future clinical studies for diagnosis and prognosis in these diseases.

BioSITe: A Method for Direct Detection and Quantitation of Site-Specific Biotinylation

Biotin-based labeling strategies are widely employed to study protein-protein interactions, subcellular proteomes and post-translational modifications, as well as, used in drug discovery. While the high affinity of streptavidin for biotin greatly facilitates the capture of biotinylated proteins, it still presents a challenge, as currently employed, for the recovery of biotinylated peptides. Here we describe a strategy designated Biotinylation Site Identification Technology (BioSITe) for the capture of biotinylated peptides for LC-MS/MS analyses. We demonstrate the utility of BioSITe when applied to proximity-dependent labeling methods, APEX and BioID, as well as biotin-based click chemistry strategies for identifying O-GlcNAc-modified sites. We demonstrate the use of isotopically labeled biotin for quantitative BioSITe experiments that simplify differential interactome analysis and obviate the need for metabolic labeling strategies such as SILAC. Our data also highlight the potential value of site-specific biotinylation in providing spatial and topological information about proteins and protein complexes. Overall, we anticipate that BioSITe will replace the conventional methods in studies where detection of biotinylation sites is important.

The TRUElncRNA workflow: a Galaxy based workflow for identification of novel long non-coding RNAs.

Results