Revealing the Immune Population Hierarchy Using Novel Interactive Visualization Tools, a Comparison of Methods: Sunburst, viSNE, and Spade on Mass Cytometry Data

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5122-5122
Author(s):  
Tiffany J Chen ◽  
Nikesh Kotecha
Keyword(s):  
New Technologies ◽  
Dimensional Space ◽  
Cell Populations ◽  
Employment Equity ◽  
Computational Tools ◽  
Bottleneck Identification ◽  
Equity Ownership ◽  
Visualization Tools ◽  
Immune Subset

Abstract The development of new technologies for high-parameter data has resulted in a critical bottleneck: identification of immune subsets is restricted to expert-based analysis, focusing on post-acquisition characterization of cell populations. Identification of cell subsets in flow cytometry has primarily focused on manual analysis, despite the fact that computational tools have proven useful for high-parameter and cross-sample comparisons. Sharing well-annotated data improves transparency and facilitates vital reproduction of results by external groups. Adoption of these new tools for immune subset discovery requires thorough collaborative investigation and validation of identified cell populations. To this end, in this study we compare the ease of discovery of immune subsets by comparing analysis through the use of three visualization tools: the sunburst hierarchy, the SPADE tree, and dimensionality reduction using viSNE. The sunburst hierarchy is a visual and interactive representation of traditional manual gating, whereas the SPADE tree is a semi-automated clustering and visualization tool for identification of cell subsets. viSNE allows interaction with high parameter data in the context of two-dimensional space where gating can be accomplished. In this study, we demonstrate the ability to automatically elucidate many immune subsets using Cytobank via an iterative analytic approach, combining computational tools (viSNE and SPADE) to recapitulate manually derived cell subsets. Disclosures Chen: Cytobank, Inc: Employment, Equity Ownership. Kotecha:Cytobank, Inc: Employment, Equity Ownership.

scholarly journals Chromatin Accessibility Mapping of Primary Erythroid Cell Populations Leads to Identification and Validation of Nuclear Factor I X (NFIX) As a Novel Fetal Hemoglobin (HbF) Repressor

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 812-812
Author(s):  
Mudit Chaand ◽  
Chris Fiore ◽  
Brian T Johnston ◽  
Diane H Moon ◽  
John P Carulli ◽  
...  
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Chromatin Accessibility ◽  
Cell Populations ◽  
Rna Seq ◽  
Globin Mrna ◽  
Employment Equity ◽  
Equity Ownership ◽  
Erythroid Maturation ◽  
Globin Gene Expression

Human beta-like globin gene expression is developmentally regulated. Erythroblasts (EBs) derived from fetal tissues, such as umbilical cord blood (CB), primarily express gamma globin mRNA (HBG) and HbF, while EBs derived from adult tissues, such as bone marrow (BM), predominantly express beta globin mRNA (HBB) and adult hemoglobin. Human genetics has validated de-repression of HBG in adult EBs as a powerful therapeutic paradigm in diseases involving defective HBB, such as sickle cell anemia. To identify novel factors involved in the switch from HBG to HBB expression, and to better understand the global regulatory networks driving the fetal and adult cell states, we performed transcriptome profiling (RNA-seq) and chromatin accessibility profiling (ATAC-seq) on sorted EB cell populations from CB or BM. This approach improves upon previous studies that used unsorted cells (Huang J, Dev Cell 2016) or that did not measure chromatin accessibility (Yan H, Am J Hematol 2018). CD34+ cells from CB and BM were differentiated using a 3-phase in vitro culture system (Giarratana M, Blood 2011). Fluorescence-activated cell sorting and the cell surface markers CD36 and GYPA were used to isolate 7 discrete populations, with each sorting gate representing increasingly mature, stage-matched EBs from CB or BM (Fig 1A, B). RNA-seq analysis revealed expected expression patterns of the beta-like globins, with total levels increasing during erythroid maturation and primarily composed of HBB or HBG transcripts in BM or CB, respectively (Fig 1C). Erythroid maturation led to progressive increases in chromatin accessibility at the HBB promoter in BM populations. In CB-derived cells, erythroid maturation led to progressive increases in chromatin accessibility at the HBG promoters through the CD36+GYPA+ stage (Pops 1-5). Chromatin accessibility shifted from the HBG promoters to the HBB promoter during the final stages of differentiation (Pops 6-7), suggesting that HBG gene activation is transient in CB EBs (Fig 1D). Hierarchical clustering and principal component analysis of ATAC-seq data revealed that cell populations cluster based on differentiation stage rather than by BM or CB lineage, suggesting most molecular changes are stage-specific, not lineage-specific (Fig 2A, B). To identify transcription factors driving cell state, and potentially beta-like globin expression preference, we searched for DNA binding motifs within regions of differential chromatin accessibility and found NFI factor motifs enriched under peaks that were larger in BM relative to CB (Fig 2C). Transcription factor footprinting analysis showed that both flanking accessibility and footprint depth at NFI motifs were also increased in BM relative to CB (Fig 2D). Increased chromatin accessibility was observed at the NFIX promoter in BM relative to CB populations, and in HUDEP-2 relative to HUDEP-1 cell lines (Fig 2E). Furthermore, accessibility at the NFIX promoter correlated with elevated NFIX mRNA in BM and HUDEP-2 relative to CB and HUDEP-1, respectively. Together these data implicated NFIX in HbF repression, a finding consistent with previous genome-wide association and DNA methylation studies that suggested a possible role for NFIX in regulating beta-like globin gene expression (Fabrice D, Nat Genet 2016; Lessard S, Genome Med 2015). To directly test the hypothesis that NFIX represses HbF, short hairpin RNAs were used to knockdown (KD) NFIX in primary erythroblasts derived from human CD34+ BM cells (Fig 3A). NFIX KD led to a time-dependent induction of HBG mRNA, HbF, and F-cells comparable to KD of the known HbF repressor BCL11A (Fig 3B-D). A similar effect on HbF was observed in HUDEP-2 cells following NFIX KD (Fig 3E). Consistent with HbF induction, NFIX KD also increased chromatin accessibility and decreased DNA methylation at the HBG promoters in primary EBs (Fig 3F, G). NFIX KD led to a delay in erythroid differentiation as measured by CD36 and GYPA expression (Fig 3H). Despite this delay, by day 14 a high proportion of fully enucleated erythroblasts was observed, suggesting NFIX KD cells are capable of terminal differentiation (Fig 3H). Collectively, these data have enabled identification and validation of NFIX as a novel repressor of HbF, a finding that enhances the understanding of beta-like globin gene regulation and has potential implications in the development of therapeutics for sickle cell disease. Disclosures Chaand: Syros Pharmaceuticals: Employment, Equity Ownership. Fiore:Syros Pharmaceuticals: Employment, Equity Ownership. Johnston:Syros Pharmaceuticals: Employment, Equity Ownership. Moon:Syros Pharmaceuticals: Employment, Equity Ownership. Carulli:Syros Pharmaceuticals: Employment, Equity Ownership. Shearstone:Syros Pharmaceuticals: Employment, Equity Ownership.

Single Cell Network Profiling (SCNP) Functionally Characterizes FLT3 Pathway Deregulation in Non-M3 Acute Myeloid Leukemia (AML) and Provides Prognostic Value Independent From Mutational Status

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2512-2512
Author(s):  
Alessandra Cesano ◽  
Santosh Putta ◽  
Kavita Mathi ◽  
David B. Rosen ◽  
Urte Gayko ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Induction Therapy ◽  
Mutational Load ◽  
Receptor Signaling ◽  
Employment Equity ◽  
Cell Network ◽  
Mutational Status ◽  
Equity Ownership

Abstract Abstract 2512 Background: FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations (FLT3 ITD+) result in constitutive activation of this receptor and have been shown to increase the risk of relapse in patients (pts) with AML; however, substantial heterogeneity in clinical outcomes still exists within both the FLT3 ITD+ and FLT3 ITD- AML subgroups, suggesting alternative mechanisms of disease relapse not accounted for by FLT3 mutational status. Single Cell Network Profiling (SCNP) is a multiparametric flow cytometry-based assay that simultaneously measures, in a quantitative fashion and at the single cell level, both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators (Kornblau et al. Clin Cancer Res 2010). Previously, we reported the use of this assay to functionally characterize FLT3 receptor signaling in healthy bone marrow and AML samples (Rosen et al. PLoS One 2010). By applying it to a separate cohort of samples collected from elderly non-M3 AML pts at diagnosis, a subclassification of AML samples beyond their “static” molecular FLT3 ITD status was generated (Rosen et al. ASH 2010 Abstr 2739). Specifically, FLT3 ITD- AML samples displayed a wide range of induced signaling, with a fraction having signaling profiles comparable to FLT3 ITD+ AML samples. Conversely, FLT3 ITD+ AML samples displayed more homogeneous induced signaling, with the exception of those with low mutational load, which had profiles more analogous to FLT3 ITD- AML samples. Due to the small numbers of pts in that exploratory study (n=44 [38 FLT3 ITD- and 6 FLT3 ITD+ pts]), an independent study was undertaken to confirm the observations, as well as to evaluate their clinical relevance (i.e., association with disease free survival (DFS) following anthracycline/cytarabine-based induction therapy). Methods: SCNP was performed as previously described on cryopreserved bone marrow or peripheral blood samples collected prior to anthracycline/cytarabine-based induction therapy from 104 elderly (>60y) non-M3 AML pts enrolled on ECOG trial 3999 or 3993 for whom ITD mutational status (including % mutational load), response and DFS data were available. Samples included 85 FLT3 ITD- and 19 FLT3 ITD+ AML, 30 and 8 of which, respectively, were collected from patients who achieved complete remission (CR). Objectives: The primary study objective was to confirm that levels of FLT3 ligand (FLT3L)-induced signaling (as measured by changes in intracellular phospho-S6 level) are more homogeneous in FLT3 ITD+ than in FLT3 ITD- myeloblasts. Four FLT3 ITD+ groups were pre-defined based on % mutation load (>0, 30%, 40%, or 50%). In addition, FLT3 ITD mutational status and signaling data from the SCNP assay (FLT3L and stem cell factor-induced phospho-S6 signaling and cytarabine/daunorubicin-induced apoptosis [cleaved PARP]) were combined to mathematically model their association with DFS among pts who achieved CR. DFS was defined as time from date of confirmed CR to date of relapse or death. Results: As shown in Figure 1a, our previous observations that variance in FLT3L-induced signaling is higher in FLT3 ITD- AML samples than in FLT3 ITD+ ones and that variance is decreased with increasing mutational load were verified in this study (Levene Test for FLT3 ITD- vs FLT3 ITD+ 50 p value=0.023). Further, when the association of DFS with FLT3 ITD mutational status and signaling data from the SCNP assay was measured using a Cox Proportional-Hazards model, the SCNP data were shown to provide independent information from FLT3 ITD mutational status (p =0.0115 for FLT3L-induced phospho-S6 signaling, Figure 1b). Conclusions: These data add to the growing body of evidence that, even within currently accepted risk stratification groups, AML is a heterogeneous disease. Functional characterization of FLT3 receptor signaling deregulation using SCNP provides prognostic information independent from FLT3 ITD mutational status and allows for more accurate pt stratification by functionally defining DFS risk sub-groups. Characterization of FLT3 signaling deregulation by SCNP could ultimately aid in the improved clinical management of AML pts and help identify candidates for FLT3 receptor inhibitor studies. Disclosures: Cesano: Nodality: Employment, Equity Ownership. Putta:Nodality Inc.: Employment, Equity Ownership. Mathi:Nodality: Employment. Rosen:Nodality Inc.: Employment, Equity Ownership. Gayko:Nodality Inc.: Employment, Equity Ownership. Hawtin:Nodality: Employment, Equity Ownership.

Characterization of MYD88 mutated Lymphoplasmacytic Lymphoma in Comparison to MYD88 mutated Chronic Lymphocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 132-132
Author(s):  
Constance Regina Baer ◽  
Frank Dicker ◽  
Wolfgang Kern ◽  
Torsten Haferlach ◽  
Claudia Haferlach
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Lymphocytic Leukemia ◽  
Bone Marrow Infiltration ◽  
Multiparameter Flow Cytometry ◽  
Employment Equity ◽  
Cytogenetic Aberration ◽  
Highly Sensitive ◽  
Equity Ownership

Abstract Introduction: MYD88 (Myeloid Differentiation Primary Response 88) mutations are the most common genetic aberration in Waldenström's macroglobulinemia/lymphoplasmacytic lymphoma (LPL). Since the initial description of MYD88 mutations in LPL, the detection has gained great importance in diagnosing the disease. However, in some patients with other B cell malignancies, including chronic lymphocytic leukemia (CLL), MYD88 mutations are detectable. Aim: We describe the molecular and cytogenetic profile of MYD88 mutated LPL in comparison to CLL, in order to identify aberration patterns potentially useful for diagnostic purposes. Patients and Methods: We analyzed bone marrow samples of 78 LPL patients for MYD88 by highly sensitive allele specific PCR (ASP) for the L265P mutation and by next-generation sequencing (NGS) for MYD88 and CXCR4 (Chemokine (C-X-C Motif) Receptor 4) mutations. For CLL, 784 blood or bone marrow samples were sequenced for MYD88 (by NGS), IGHV, TP53, NOTCH1 and SF3B1 by Sanger or NGS as well as the MYD88 mutated CLL cases for CXCR4. For all samples, cytogenetic and multiparameter flow cytometry data was available. Results: In LPL, 68/78 patients (87%) harbored a MYD88 mutation. In 13 cases with low bone marrow infiltration (median: 3%; range: 1-6%), the MYD88 mutation was detected by ASP only and not by NGS. However, one case was identified by NGS only because of a non-L265P mutation, which cannot be detected by ASP (1/68; 1%). In contrast, in CLL only 17/784 (2%) carried a MYD88 mutation. Interestingly, 5/17 (29%) were non-L265P mutations. Of the MYD88 mutated LPL, 17/68 (25%) carried a genetic lesion in the C-terminal domain of CXCR4. In contrast to MYD88, the mutation spectrum of CXCR4 was much broader including non-sense mutations at amino acid S338 (10/18) but also frame shifts resulting in loss of regulatory serine residues. One patient had two independent CXCR4 mutations (S338* and S341Pfs*25). The mean bone marrow infiltration by flow cytometry was 14% and 9% in the CXCR4 mutated and unmuted subsets, respectively (p=0.17). Besides molecular genetic aberrations, 25% (17/68) of MYD88 mutated LPL cases carried cytogenetic aberration. The most frequent cytogenetic aberration in the MYD88 positive LPL was the deletion of 6q (10/68; 15%). Other recurrent cytogenetic abnormalities were gains of 4q (n=3), 8q (n=2), and 12q (n=4), as well as loss of 11q (n=4), 13q (n=2) and 17p (n=3). In the MYD88 unmutated group, we did neither identify any CXCR4 mutation nor any del(6q), suggesting different genetic driver events in this LPL subcohort. Importantly, in the MYD88 positive CLL cohort, cytogenetic analysis did not reveal any patient with del(6q). Instead, del(13q)(q14) was the most prevalent cytogenetic aberration (12/17; 71%). Neither 11q deletions nor 17p deletions were detected. All MYD88 positive CLL had a mutated IGHV status (MYD88 unmutated CLL: 453/767; 59%; P<0.001). The TP53, NOTCH1 and SF3B1 mutational landscape did not reveal any differences between the MYD88 mutated and unmutated cohort. Finally, CXCR4 mutations were present in none of 15 analyzed MYD88 mutated CLL cases. Conclusion: Besides multiparameter flow cytometry, MYD88 mutations are the most powerful tool in the diagnosis of LPL. MYD88 mutated LPL are characterized by a high frequency of CXCR4 mutations and del(6q), while MYD88 unmutated LPLs are associated with a different pattern of genetic abnormalities. MYD88 mutated CLL is a distinct CLL subset associated with mutated IGHV status, a high frequency of 13q deletions and low frequencies of 11q and 17p deletions. MYD88 mutated CLL differs from MYD88 mutated LPL with respect to the pattern of MYD88 mutations, cytogenetic aberrations and the absence of CXCR4 mutations. Highly sensitive ASP allows the L265P mutation detection even in LPL cases with very low bone marrow infiltration; whereas highly sensitive NGS assay are best applicable for detection of more heterogenic MYD88 mutations in CLL or CXCR mutations in LPL. Thus, an integrated molecular and cytogenetic approach allows the characterization of disease specific genetic patterns and should be analyzed for its clinical impact. Disclosures Baer: MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

scholarly journals Circulating Multiple Myeloma Cells (CMMCs): A Novel Method for Detection and Molecular Characterization of Peripheral Blood Plasma Cells in Multiple Myeloma Precursor States

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2031-2031
Author(s):  
Brendan Weiss ◽  
Kate Sasser ◽  
Chandra Rao ◽  
Brad Foulk ◽  
Steven Gross ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Molecular Characterization ◽  
Research Funding ◽  
Plasma Cells ◽  
Employment Equity ◽  
Myeloma Cells ◽  
Novel Method ◽  
Equity Ownership ◽  
Precursor States

Abstract Background Circulating plasma cells (PCs) have been identified as a prognostic factor in patients with myeloma precursor states (MGUS and SMM) and active multiple myeloma (MM). Enumeration of circulating PCs by available methods is not suitable for widespread use and does not provide molecular characterization. We developed and evaluated a novel method for enumeration and molecular characterization of circulating PCs (circulating multiple myeloma cells, “CMMC”), based on the CELLSEARCH® System (Janssen Diagnostics LLC, Raritan, NJ), an automated technology for the capture, enumeration and characterization of rare cells in the peripheral blood. Methods We are performing a prospective study of patients with MGUS and SMM to evaluate CMMCs as biomarker for progression to active MM. Utilizing the CELLSEARCH® System CMMCs were captured by CD138 ferrofluid magnetic particles and identification was defined as CD38+ and CD19-, CD45-. Nonviable cells were excluded by DAPI. Isolated CMMCs were stored and FISH for t(4:14), t(14;16) and del17 was performed. Results We have enrolled 16 patients, MGUS = 3, SMM = 11, and newly diagnosed MM = 2. The Mayo Risk stratification for MGUS patients was: low risk = 2, low-intermediate = 1. All SMM patients were low risk by Mayo Model incorporating serum free light chains. The median number of bone marrow plasma cells for MGUS patients was 7 (range 7-9) and for SMM patients was 15 (range 10-40). The median CMMCs for MGUS = 6 (range 2-55), median CMMCs for SMM = 31 (5-1918). The two patients with NDMM had 5870 and 5 CMMCs, respectively. A single patient with SMM progressed with a symptomatic solitary lumbar plasmacytoma and had CMMCs of 5 and 3 at baseline and progression, respectively. Abnormalities by FISH were detected in both bone marrow and CMMCs. Accrual is ongoing and additional data will be presented at the meeting. Conclusions The CELLSEARCH® CMMC assay can detect, quantify and provide molecular characterization of circulating PCs in MGUS/SMM/MM; longer prospective follow-up is needed to test the prognostic value of CMMCs. Disclosures Weiss: Janssen: Consultancy, Research Funding. Sasser:Janssen: Employment. Rao:Janssen: Employment, Equity Ownership. Foulk:Janssen: Employment. Gross:Johnson & Johnson: Employment, Equity Ownership. Cohen:Janssen: Membership on an entity's Board of Directors or advisory committees. Vogl:Celgene Corporation: Consultancy; Amgen: Consultancy; Millennium/Takeda: Research Funding; GSK: Research Funding; Acetylon: Research Funding. Stadtmauer:Janssen: Consultancy.

Preclinical Characterization of a Potent, Selective Inhibitor of the Protein Methyltransferase DOT1L for Use in the Treatment of MLL-Rearranged Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2379-2379 ◽  
Author(s):  
Roy M Pollock ◽  
Scott R Daigle ◽  
Carly A Therkelsen ◽  
Aravind Basavapathruni ◽  
Lei Jin ◽  
...  
Keyword(s):  
Tumor Regression ◽  
Xenograft Model ◽  
Selective Inhibitor ◽  
Significant Weight Loss ◽  
Employment Equity ◽  
Nude Rat ◽  
Protein Methyltransferase ◽  
Equity Ownership ◽  
H3k79 Methylation

Abstract Abstract 2379 The enzymatic activity of the protein methyltransferase (PMT) DOT1L has been shown to be a driver of cell proliferation in MLL-rearranged leukemia. Our group has previously reported the design of potent and selective aminonucleoside inhibitors of DOT1L [Daigle et al. (2011) Cancer Cell 20: 53–65; Basavapathruni et al. (2012) Chem. Biol. Drug Design, in press]. Structure-guided design, together with robust biochemical and biological assays, was used to optimize the potency, selectivity and pharmacological features of the aminonucleosides, resulting in the compound EPZ-5676. EPZ-5676 is an S-adenosyl methionine (SAM) competitive inhibitor of DOT11L that displays a Ki value of 80 pM and a drug-target residence time of > 24 hours. The compound is highly selective for DOT1L, demonstrating > 37,000-fold selectivity against all other PMTs tested. Crystallographic studies reveal that the high affinity, durable inhibition of DOT1L by EPZ-5676 has its origin in a conformational adaptation of the protein that attends inhibitor binding, extending the compound binding pocket to include novel recognition elements beyond the SAM binding active site. Treatment of leukemia cells with EPZ-5676 results in concentration- and time-dependent reduction of H3K79 methylation without effect on the methylation status of other histone sites. The reduction of H3K79 methylation leads to inhibition of key MLL target genes and selective, apoptotic cell killing in MLL-rearranged leukemia cells, but has minimal impact on non-rearranged cells. EPZ-5676 is highly soluble in aqueous solution and can thus be formulated for intravenous administration. The effective pharmacokinetic half-life of EPZ-5676 in systemic circulation has been measured to be 0.25 and 1.5 h in rats and dogs, respectively. A nude rat subcutaneous xenograft model of MLL-rearranged leukemia has been established. Continuous intravenous infusion of EPZ-5676 for 21 days in this model leads to dose-dependent anti-tumor activity. At the highest dose, complete tumor regressions are achieved with no regrowth for up to 32 days after the cessation of treatment (Figure 1). Figure 1. EPZ-5676 causes complete and sustained tumor regression in a MV4–11 nude rat xenograft model of MLL-rearranged leukemia. No significant weight loss or obvious toxicity was observed in rats treated with EPZ-5676 during this efficacy study. EPZ-5676 is thus a potent, selective inhibitor of DOT1L that demonstrates strong efficacy in a rat xenograft model of MLL-rearranged leukemia. Details of the preclinical characterization of this compound will be presented. Figure 1. EPZ-5676 causes complete and sustained tumor regression in a MV4–11 nude rat xenograft model of MLL-rearranged leukemia. . / No significant weight loss or obvious toxicity was observed in rats treated with EPZ-5676 during this efficacy study. EPZ-5676 is thus a potent, selective inhibitor of DOT1L that demonstrates strong efficacy in a rat xenograft model of MLL-rearranged leukemia. Details of the preclinical characterization of this compound will be presented. Disclosures: Pollock: Epizyme: Employment, Equity Ownership. Daigle:Epizyme: Employment, Equity Ownership. Therkelsen:Epizyme: Employment, Equity Ownership. Basavapathruni:Epizyme: Employment, Equity Ownership. Jin:Epizyme: Employment, Equity Ownership. Allain:Epizyme: Employment, Equity Ownership. Klaus:Epizyme, Inc.: Employment, Equity Ownership. Raimondi:Epizyme: Employment, Equity Ownership. Porter Scott:Epizyme: Employment, Equity Ownership. Chesworth:Epizyme: Employment, Equity Ownership. Moyer:Epizyme: Employment, Equity Ownership. Copeland:Epizyme Inc.: Employment, Equity Ownership. Richon:Epizyme, Inc.: Employment, Equity Ownership. Olhava:Epizyme: Employment.

scholarly journals From Bivariate to Multivariate Analysis of Cytometric Data: Overview of Computational Methods and Their Application in Vaccination Studies

Vaccines ◽  
2020 ◽  
Vol 8 (1) ◽  
pp. 138 ◽  
Author(s):  
Simone Lucchesi ◽  
Simone Furini ◽  
Donata Medaglini ◽  
Annalisa Ciabattini
Keyword(s):  
Population Analysis ◽  
Single Cells ◽  
Functional Characterization ◽  
Automated Analysis ◽  
Cell Populations ◽  
Computational Tools ◽  
Multiparametric Flow Cytometry ◽  
Functional Features ◽  
Automated Tools

Flow and mass cytometry are used to quantify the expression of multiple extracellular or intracellular molecules on single cells, allowing the phenotypic and functional characterization of complex cell populations. Multiparametric flow cytometry is particularly suitable for deep analysis of immune responses after vaccination, as it allows to measure the frequency, the phenotype, and the functional features of antigen-specific cells. When many parameters are investigated simultaneously, it is not feasible to analyze all the possible bi-dimensional combinations of marker expression with classical manual analysis and the adoption of advanced automated tools to process and analyze high-dimensional data sets becomes necessary. In recent years, the development of many tools for the automated analysis of multiparametric cytometry data has been reported, with an increasing record of publications starting from 2014. However, the use of these tools has been preferentially restricted to bioinformaticians, while few of them are routinely employed by the biomedical community. Filling the gap between algorithms developers and final users is fundamental for exploiting the advantages of computational tools in the analysis of cytometry data. The potentialities of automated analyses range from the improvement of the data quality in the pre-processing steps up to the unbiased, data-driven examination of complex datasets using a variety of algorithms based on different approaches. In this review, an overview of the automated analysis pipeline is provided, spanning from the pre-processing phase to the automated population analysis. Analysis based on computational tools might overcame both the subjectivity of manual gating and the operator-biased exploration of expected populations. Examples of applications of automated tools that have successfully improved the characterization of different cell populations in vaccination studies are also presented.

scholarly journals Recombinant Human IL-12 Exerts Differential Effects on Circulating CD56bright and CD56dim NK Cells in Healthy Human Subjects

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 756-756
Author(s):  
Simmy Thomas ◽  
Chris E Lawrence ◽  
Vernon Mar ◽  
Hue Kha ◽  
Lena A Basile
Keyword(s):  
Nk Cells ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Human Subjects ◽  
Cell Populations ◽  
Employment Equity ◽  
Healthy Human ◽  
Healthy Human Subjects ◽  
Equity Ownership

Abstract Interleukin-12 (IL-12) has potent immunoregulatory and hematopoietic properties, and exerts significant biological effects on natural killer (NK) cells, inducing IFNγ production and enhancing cytotoxicity. Two distinct NK cell populations correlate with their immunoregulatory functions. Mature CD56dimCD16bright NK cells represent 90% of the NK cells resident in the blood and can exert cytotoxic effects on transformed cells. Cytokine producing immature CD56brightCD16+/- NK cells exist in the blood (10% of total circulating NK cells) but are most prominently located in secondary lymphoid tissues. In the continued clinical development of recombinant human IL-12 (HemaMax™, rHuIL-12), to be used in combination with radiotherapy or chemotherapy for the treatment of cancer patients, we have performed a clinical safety study in healthy human subjects. A single subcutaneous (sc) dose of rHuIL-12 (12μg) was administered to 17 healthy human subjects. Placebo was administered to 5 healthy subjects. Peripheral blood samples were collected before rHuIL-12 administration, and up to Day 14 post administration. Immunophenotyping of blood cell populations was conducted by FACS. rHuIL-12 caused a transient decrease in peripheral blood CD56dimCD16bright NK cells, with a nadir (60% reduction from baseline) reached on Day 2 following rHuIL-12 administration. CD56dimCD16bright NK cell levels returned almost to baseline levels on Day 5. Placebo was without effect. Conversely rHuIL-12 caused an elevation in peripheral blood CD56brightCD16+/- NK cells, particularly between Days 2 and 3 after rHuIL-12 administration, which was sustained until a peak was reached on Day 5 (265% above baseline). Levels returned to baseline by Day 11, while placebo was without effect. rHuIL-12 did not impact the less functional CD56-CD16bright NK cell subset. CD56dimCD16bright NK cells expressing the IL-12 receptor β2 subunit (IL-12Rβ2+) showed a substantial, and transient, decrease in levels on Day 2. The plasma concentration of IFNγ was elevated to a peak over 35 fold above baseline level at 10hr. after rHuIL-12 administration. Human NK cells were negatively selected from highly enriched leukapheresis-derived blood and stimulated in vitro with 10 pM rHuIL-12. After 16hr. incubation these predominantly CD56dimCD16brightNK cells showed enhanced release of IFNγ and the increased killing of K562 cells, a human erythroleukemic cell line, when compared with vehicle controls. qPCR analysis of the human NK cell lysates showed rHuIL-12-induced elevation of CD56 (302%) and IL-12Rβ2 (587%) mRNA, when compared with vehicle controls. rHuIL-12 did not influence CD16 mRNA expression, but did increase the level of CD62L (L selectin, 206%) mRNA. The rapid 60% fall in circulating mature CD56dimCD16bright NK cells after rHuIL-12 administration to healthy human subjects suggests their immediate exit from peripheral blood into the tissue compartments. This could be mediated by the observed increase in NK cell CD62L mRNA expression seen in vitro. The sustained increase in immature CD56brightCD16+/- NK cell levels between Day 3 and 6 suggests their IL-12-induced development from CD34+ hematopoietic progenitor cells. In summary rHuIL-12 administration to healthy human subjects demonstrates differential effects on the two key NK cell populations in peripheral blood, increasing CD56brightCD16+/- NK cell numbers, potentially stimulating IFNγ release from and enhancing the cytotoxicity of the CD56dimCD16bright NK cells, and preparing this population for migration into tissues. rHuIL-12 thus shows excellent potential as an immunotherapeutic and hematopoietic agent for the treatment of cancer patients, by impacting the maturation, activation, immunoregulation, and cytolytic properties of NK cells. Disclosures Thomas: Neumedicines: Employment, Equity Ownership. Lawrence:Neumedicines: Employment, Equity Ownership. Mar:Neumedicines: Employment, Equity Ownership. Kha:Neumedicines: Employment, Equity Ownership. Basile:Neumedicines: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Immune Microenvironment Analysis of Bone Marrow By Mass Cytometry and RNA Sequencing in Multiple Myeloma Patients Treated with Daratumumab and Durvalumab

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3296-3296 ◽  
Author(s):  
Frances Seymour ◽  
Mary H Young ◽  
Mark Tometsko ◽  
Jamie Cavenagh ◽  
Ethan G. Thompson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Nk Cells ◽  
Research Funding ◽  
Cell Populations ◽  
Mass Cytometry ◽  
Immune Microenvironment ◽  
Employment Equity ◽  
Equity Ownership

Abstract Introduction Relapsed and refractory multiple myeloma (RRMM) remains a challenging disease to treat due to its heterogeneity and complexity. There is an urgent need for novel combination strategies, including immunotherapy. The study of the tumour and immune microenvironment before and after treatment with combination therapy is a crucial part of understanding the underpinning of disease response. Methods Longitudinal samples of bone marrow aspirates and whole blood were collected from a phase II clinical trial, MEDI4736-MM-003 (NCT02807454) where daratumumab and durvalumab naïve patients were exposed simultaneously to both these drugs. A combination of mass cytometry (CyTOF), RNAseq and flow cytometry were performed on a subset of samples from these subjects. Specifically, paired bone marrow mononuclear cells (BMMC) samples from nine patients taken at screening and six weeks post-treatment were analysed by mass cytometry (CyTOF) using a 37-marker pan-immune panel that included both lineage and functional intracellular/extracellular markers. In addition, whole blood sample specimens were collected at screening and on treatment (8, 15, 30, and 45 days after treatment) and analysed by flow cytometry. Flow cytometry panels were designed to allow interrogation of the abundance and activation status of immune cell subsets. Finally, RNA from bone marrow aspirates at screening and C2D15 were analysed by RNA sequencing. Expression profiles from the aspirates were used to estimate cell proportions by computational deconvolution. Individual cell types in these microenvironments were estimated using the DCQ algorithm and a gene expression signature matrix based on the published LM22 leukocyte matrix (Newman et al., 2015) augmented with 5 bone marrow- and myeloma-specific cell types. Results In a heavily pre-treated population with RRMM, treatment with durvalumab and daratumumab leads to shifts in a number of key immunological populations when compared to pre-treatment. In the bone marrow, CD8 and CD4 populations rise (by CyTOF and RNAseq), while NK, DC and B cell populations fall (by CyTOF). In the bone marrow within CD8+ T lymphocyte populations, we observed a post-treatment rise in markers of degranulation (granzyme p=0.0195, perforin p=0.0078, Wilcoxon signed-rank test). This is also accompanied by a fall in PD1 expression (p=0.0078) and rise in the co-stimulatory receptor DNAM1 (p=0.0273). These changes are most marked on cells with an effector memory CD45RA+ CD8+ T cell phenotype. In the blood, similar to the bone marrow, CD8+ T cells proliferate over the course of treatment (flow cytometry). A fall in both naïve and active NK cell populations is seen following treatment in bone marrow. NK cells express high levels of CD38 and are therefore depleted by daratumumab. Those NK cells which remain have an active phenotype with increased expression of TNFa (p=0.0039) and IFNg (p=0.0195) following treatment. Across the time points sampled in peripheral blood, NK cells were also decreased and those that remained were proliferating. Dendritic cells with a tolerogenic phenotype can be identified prior to treatment and are seen to fall in abundance following treatment with durvalumab and daratumumab. Conclusions The combination of durvalumab and daratumumab leads to several immune microenvironment changes that biologically portend clinical effect. We see increases in the abundance of cell populations with functional anti-tumour activity, including granzyme B+ CD8 T cells and a reduction in PD1high T cells. Despite the treatment expectedly reducing NK cell numbers, many functionally competent NK cells remain, as evidenced by the presence of anti-tumour cytokines. This combination strategy also reduces immunosuppressive tolerogenic DCs, which suppress CD4 and CD8 T cell activity. Taken together, this suggests that this chemotherapy free, doublet treatment has the potential to up-regulate anti-tumour immunological responses, which may restore immunosurveillance mechanisms critically needed in these highly refractory patients. Disclosures Seymour: Celgene: Research Funding. Young:Celgene Corporation: Employment, Equity Ownership. Tometsko:Celgene Corporation: Employment, Equity Ownership. Cavenagh:Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Thompson:Celgene Corporation: Employment, Equity Ownership. Whalen:Celgene Corporation: Employment, Equity Ownership. Danziger:Celgene Corporation: Employment, Equity Ownership. Fitch:Celgene Corporation: Employment, Equity Ownership. Fox:Celgene Corporation: Employment, Equity Ownership. Dervan:Celgene Corporation: Employment, Equity Ownership. Foy:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment, Equity Ownership. Gribben:Acerta Pharma: Honoraria, Research Funding; Cancer Research UK: Research Funding; TG Therapeutics: Honoraria; Roche: Honoraria; NIH: Research Funding; Medical Research Council: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria; Kite: Honoraria; Pharmacyclics: Honoraria; Novartis: Honoraria; Janssen: Honoraria, Research Funding; Wellcome Trust: Research Funding; Unum: Equity Ownership.

Characterization of FLT3 Pathway Deregulation by Single Cell Network Profiling (SCNP) Stratifies AML Patients Beyond Molecular Profiling

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2739-2739
Author(s):  
David B. Rosen ◽  
Santosh Putta ◽  
Urte Gayko ◽  
Alessandra Cesano ◽  
Larry Cripe ◽  
...  
Keyword(s):  
Single Cell ◽  
Intracellular Signaling ◽  
Kinase Inhibitors ◽  
Flt3 Ligand ◽  
Employment Equity ◽  
Mutation Status ◽  
Cell Network ◽  
Equity Ownership ◽  
Pathway Deregulation

Abstract Abstract 2739 Background: Internal tandem duplication mutations of the FMS-like tyrosine kinase 3 receptor (FLT3-ITD) are among the most frequent mutations in de novo acute myeloblastic leukemia (AML) and result in constitutive activation of the receptor tyrosine kinase. Based on data showing that these mutations negatively affect outcome in AML, FLT3 receptor kinase activity is currently being targeted in the clinic. To date, only FLT3 receptor mutant AML patients have been selected for trials involving FLT3 receptor kinase inhibitors, with surprisingly variable clinical responses. Downstream targets of FLT3 receptor activation, whether by receptor mutation or FLT3 ligand binding, involve Stat5, PI3-kinase/Akt and the Ras/Raf/Erk kinase signal transduction pathways which ultimately affect cell survival and proliferation. Functional characterization of those signaling pathways in mutated versus non-mutated FLT3 receptor (FLT3 -WT) myeloblasts has significant potential to reveal heterogeneity among these genetically defined subgroups and to predict for response to kinase inhibition, independent of FLT3 receptor mutation status. Methods: Modulated single cell network profiling (SCNP) was used to evaluate the activation state of intracellular signaling molecules (i.e. nodes), including phosphorylated (p)-Akt, p-Erk, p-S6, p-Stat5 and cleaved-PARP, at baseline and after treatment with specific modulators [including cytokines (such as IL-27) growth factors (such as FLT3 ligand) and drugs (such as cytosine arabinoside)] in 7 healthy bone marrow mononuclear blasts (BMMb) and leukemic myeloblasts, characterized for FLT3 receptor mutation status, from 44 AML patients (38 FLT-WT and 6 FLT3-ITD), aged >60 years (ECOG trial E3999). A total of 64 node-metrics were analyzed. Results: Signaling profiles differed significantly in FLT3-ITD vs. FLT3-WT AML, and in FLT3-WT vs. BMMb (shown in Figure 1 for a representative node, FLT3 ligand induced p-S6). Specifically, compared to BMMb, FLT3-ITD blasts uniformly showed increased basal p-Stat5 levels, decreased FLT3 ligand-induced activation of P13K and Raf/Ras/Erk pathways, minimal IL-27 induced activation of the Jak/Stat pathway, and higher apoptotic responses to DNA-damaging agents. Two AML samples harboring a low FLT3-ITD mutant burden, however, exhibited a signaling pattern similar to FLT3-WT AML samples. By contrast, FLT3-WT samples displayed heterogeneous signaling profiles, overlapping both with those of FLT3-ITD and BMMb samples, suggesting that a fraction of FLT3-WT AML samples exhibit FLT3 receptor pathway deregulation even without FLT3-ITD. Conclusions This study showed that SCNP, which provides a detailed view of intracellular signaling networks at the single-cell level, subclassified patients with AML beyond their molecularly determined FLT3 mutation status. In particular, a fraction of FLT3-WT AML samples signaled as if containing a FLT3 receptor length mutation while FLT3-ITD samples with low mutational load signaled like FLT3-WT AML samples. The clinical relevance of this observation, both for disease prognosis and response to kinase inhibitors, will be revealed only if AML patients are accrued to kinase inhibition trials irrespective of FLT3 receptor mutation status. The wide range of signaling responses observed in FLT3-WT AML suggests that disease across FLT3-WT patients is heterogeneous, likely promoted through distinct mutations and alterations, giving rise to distinct signaling profiles in individual patients Our data also provide evidence for the co-existence of differentially signaling blast populations in individual patients. The potential impact of signaling heterogeneity on clinical response needs to be assessed and may require an individualized combination of treatment modalities. Disclosure: Rosen: Nodality Inc.: Employment, Equity Ownership. Putta:Nodality Inc.: Employment, Equity Ownership. Gayko:Nodality Inc.: Employment, Equity Ownership. Cesano:Nodality Inc.: Employment, Equity Ownership.

Automated Enumeration and Characterization of Circulating Multiple Myeloma Cells in Blood

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1825-1825 ◽  
Author(s):  
Steven Gross ◽  
Brad Foulk ◽  
Jaymala Patel ◽  
Mark Connelly ◽  
Marielena Mata
Keyword(s):  
Multiple Myeloma ◽  
Plasma Cells ◽  
Employment Equity ◽  
Myeloma Cells ◽  
Smoldering Multiple Myeloma ◽  
Healthy Donors ◽  
Two Samples ◽  
Equity Ownership ◽  
Active Disease

Abstract Abstract 1825 Detection of circulating Multiple Myeloma cells (CMMC) by flow cytometry is an indicator of active disease. In addition, circulating plasma cells can be detected in earlier stages of disease, including MGUS and Smoldering Multiple Myeloma, and appear to correlate with prognosis. The capture and characterization of these circulating plasma cells from peripheral blood may provide novel biomarkers for the management of Multiple Myeloma patients, particularly in monitoring minimal residual disease and in progression from MGUS or Smoldering Multiple Myeloma to active disease. The enumeration and characterization of circulating tumor cells (CTC) in patients with metastatic breast, prostate or colorectal cancer using the CellSearch® technology, has been shown to provide clinically relevant prognostic and predictive information. Here we describe the development of an automated assay for detecting circulating normal plasma cells (CPC) and multiple myeloma cells (CMMC) in blood using CellSearch. Assay results from Multiple Myeloma, MGUS patients, and from an aged matched control population are presented. The CellTracks® AutoPrep® System and CellTracks Analyzer II® systems were used to capture and enumerate CPC and CMMC. Magnetic particles conjugated to anti-CD138 are used to capture myeloma cells from 4.0mL of blood. Enriched cells are then stained with the nucleic acid dye DAPI and anti-CD38-Phycoerythrin (PE) antibody. Allophycocyanine (APC) conjugated anti-CD45 and anti-CD19 were used to exclude leukocytes and B-cells. In addition, FITC labeled anti-CD56 was added as a biomarker. The enriched and stained cells were transferred to a CellTracks® cartridge and MagNest® for magnetic mounting. The cartridge was scanned using the CellTracks Analyzer II®. Individual images of cells were presented to the operator for review, and scored as CMMCs, based on fluorescence and cell morphology. In a model spike-in system the assay consistently recovered ∼60% of the cells from the Multiple Myeloma cell line H929 spiked into 4.0mL of blood from healthy donors. The assay was linear over the tested range of from 0 to 2000 spiked H929 cells (r2 0.98, slope 0.50, intercept 10). The assay was validated using blood from age matched healthy donors (n=22) and patients with Multiple Myeloma (n=66) and MGUS (n=7). In 4.0mL blood from normal donors, 0 CPC were detected in 12/22 (55%) and low numbers (1–6 CPC) were detected in 10/22 (45%) samples. Interestingly, one CD56 positive CPC (CMMC?) was found in a normal donor. CMMC in Multiple Myeloma patients ranged from 0 – 17,000 /4.0mL blood. One or more CMMC were detected in 91% of the patients, > 5 in 68%, > 10 in 58% and > 100 in 35%. Expression of CD56 was highly variable in the patient population. CMMC in MGUS patients ranged from 0 – 112 /4.0mL blood. One or more CMMC were detected in 6/7 of the patients, > 5 in 4/6, > 10 in 2/6 and > 100 in 1/6. To further characterize CMMC, and differentiate CPC from CMMC, an interphase fluorescent in situ hybridization (FISH) assay was developed to be used with the capture and detection system described above. A four color FISH probe was used to simultaneously detect high risk mutations including two recurrent translocations of the IgH locus (t(4;14)(p16;q32) and t(14;16)(q32;q23)) as well as deletion of the TP53 locus (Δ17p13). The FISH assay was verified on cell lines H929, MM1s, and U266, which showed mutations at t(4;14), t(14;16) and Δ17p13, respectively. The FISH assay was tested on 9 CMMC patient samples and 8 samples yielded evaluable results. Two samples showed t(4;14)fusions, 3 patients showed aberrant FISH signal patterns indicating aneuploidy of chromosome 4 or 14 and the remaining patients showed normal FISH patterns. Well controlled prospective clinical studies are needed to establish the prognostic and predictive value of the presence, and characteristics, of CMMC in multiple myeloma or MGUS. In addition, as with CTC, this automated CMMC assay should prove useful in evaluating the effectiveness of new treatments as well as the assessment of potential treatment targets on CMMC in this difficult disease. Disclosures: Gross: Johnson and Johnson: Employment, Equity Ownership. Foulk:Johnson and Johnson: Employment, Equity Ownership. Patel:Johnson and Johnson: Employment, Equity Ownership. Connelly:Johnson and Johnson: Employment, Equity Ownership. Mata:Johnson and Johnson: Employment, Equity Ownership.

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