LaboratoryPublications
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An Integrated Multi-omic Single-Cell Atlas of Human B Cell Identity.
Immunity
Glass, D. R., Tsai, A. G., Oliveria, J. P., Hartmann, F. J., Kimmey, S. C., Calderon, A. A., Borges, L., Glass, M. C., Wagar, L. E., Davis, M. M., Bendall, S. C.
2020; 53 (1): 217–32.e5
B cells are capable of a wide range of effector functions including antibody secretion, antigen presentation, cytokine production, and generation of immunological memory. A consistent strategy for classifying human B cells by using surface molecules is essential to harness this functional diversity for clinical translation. We developed a highly multiplexed screen to quantify the co-expression of 351 surface molecules on millions of human B cells. We identified differentially expressed molecules and aligned their variance with isotype usage, VDJ sequence, metabolic profile, biosynthesis activity, and signaling response. Based on these analyses, we propose a classification scheme to segregate B cells from four lymphoid tissues into twelve unique subsets, including a CD45RB+CD27- early memory population, a class-switched CD39+ tonsil-resident population, and a CD19hiCD11c+ memory population that potently responds to immune activation. This classification framework and underlying datasets provide a resource for further investigations of human B cell identity and function.
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Multiplexed single-cell morphometry for hematopathology diagnostics.
Nature medicine
Tsai, A. G., Glass, D. R., Juntilla, M., Hartmann, F. J., Oak, J. S., Fernandez-Pol, S., Ohgami, R. S., Bendall, S. C.
2020; 26 (3): 408–17
The diagnosis of lymphomas and leukemias requires hematopathologists to integrate microscopically visible cellular morphology with antibody-identified cell surface molecule expression. To merge these into one high-throughput, highly multiplexed, single-cell assay, we quantify cell morphological features by their underlying, antibody-measurable molecular components, which empowers mass cytometers to 'see' like pathologists. When applied to 71 diverse clinical samples, single-cell morphometric profiling reveals robust and distinct patterns of 'morphometric' markers for each major cell type. Individually, lamin B1 highlights acute leukemias, lamin A/C helps distinguish normal from neoplastic mature T cells, and VAMP-7 recapitulates light-cytometric side scatter. Combined with machine learning, morphometric markers form intuitive visualizations of normal and neoplastic cellular distribution and differentiation. When recalibrated for myelomonocytic blast enumeration, this approach is superior to flow cytometry and comparable to expert microscopy, bypassing years of specialized training. The contextualization of traditional surface markers on independent morphometric frameworks permits more sensitive and automated diagnosis of complex hematopoietic diseases.
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Human Chromosomal Translocations at CpG Sites and a Theoretical Basis for Their Lineage and Stage Specificity
CELL
Tsai, A. G., Lu, H., Raghavan, S. C., Muschen, M., Hsieh, C., Lieber, M. R.
2008; 135 (6): 1130-1142
We have assembled, annotated, and analyzed a database of over 1700 breakpoints from the most common chromosomal rearrangements in human leukemias and lymphomas. Using this database, we show that although the CpG dinucleotide constitutes only 1% of the human genome, it accounts for 40%-70% of breakpoints at pro-B/pre-B stage translocation regions-specifically, those near the bcl-2, bcl-1, and E2A genes. We do not observe CpG hotspots in rearrangements involving lymphoid-myeloid progenitors, mature B cells, or T cells. The stage specificity, lineage specificity, CpG targeting, and unique breakpoint distributions at these cluster regions may be explained by a lesion-specific double-strand breakage mechanism involving the RAG complex acting at AID-deaminated methyl-CpGs.
DOI 10.1016/j.cell.2008.10.035
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Terminal deoxynucleotidyl transferase and CD84 identify human multi-potent lymphoid progenitors.
Nature communications
Kim, Y., Calderon, A. A., Favaro, P., Glass, D. R., Tsai, A. G., Ho, D., Borges, L., Greenleaf, W. J., Bendall, S. C.
2024; 15 (1): 5910
Lymphoid specification in human hematopoietic progenitors is not fully understood. To better associate lymphoid identity with protein-level cell features, we conduct a highly multiplexed single-cell proteomic screen on human bone marrow progenitors. This screen identifies terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase intrinsic to VDJ recombination, broadly expressed within CD34+ progenitors prior to B/T cell emergence. While these TdT+ cells coincide with granulocyte-monocyte progenitor (GMP) immunophenotype, their accessible chromatin regions show enrichment for lymphoid-associated transcription factor (TF) motifs. TdT expression on GMPs is inversely related to the SLAM family member CD84. Prospective isolation of CD84lo GMPs demonstrates robust lymphoid potentials ex vivo, while still retaining significant myeloid differentiation capacity, akin to LMPPs. This multi-omic study identifies human bone marrow lymphoid-primed progenitors, further defining the lympho-myeloid axis in human hematopoiesis.
- Author Correction: Advances and prospects for the Human BioMolecular Atlas Program (HuBMAP). Nature cell biology Jain, S., Pei, L., Spraggins, J. M., Angelo, M., Carson, J. P., Gehlenborg, N., Ginty, F., Goncalves, J. P., Hagood, J. S., Hickey, J. W., Kelleher, N. L., Laurent, L. C., Lin, S., Lin, Y., Liu, H., Naba, A., Nakayasu, E. S., Qian, W., Radtke, A., Robson, P., Stockwell, B. R., Van de Plas, R., Vlachos, I. S., Zhou, M., HuBMAP Consortium, Borner, K., Snyder, M. P., Ahn, K. J., Allen, J., Anderson, D. M., Anderton, C. R., Curcio, C., Angelin, A., Arvanitis, C., Atta, L., Awosika-Olumo, D., Bahmani, A., Bai, H., Balderrama, K., Balzano, L., Bandyopadhyay, G., Bandyopadhyay, S., Bar-Joseph, Z., Barnhart, K., Barwinska, D., Becich, M., Becker, L., Becker, W., Bedi, K., Bendall, S., Benninger, K., Betancur, D., Bettinger, K., Billings, S., Blood, P., Bolin, D., Border, S., Bosse, M., Bramer, L., Brewer, M., Brusko, M., Bueckle, A., Burke, K., Burnum-Johnson, K., Butcher, E., Butterworth, E., Cai, L., Calandrelli, R., Caldwell, M., Campbell-Thompson, M., Cao, D., Cao-Berg, I., Caprioli, R., Caraccio, C., Caron, A., Carroll, M., Chadwick, C., Chen, A., Chen, D., Chen, F., Chen, H., Chen, J., Chen, L., Chen, L., Chiacchia, K., Cho, S., Chou, P., Choy, L., Cisar, C., Clair, G., Clarke, L., Clouthier, K. A., Colley, M. E., Conlon, K., Conroy, J., Contrepois, K., Corbett, A., Corwin, A., Cotter, D., Courtois, E., Cruz, A., Csonka, C., Czupil, K., Daiya, V., Dale, K., Davanagere, S. A., Dayao, M., de Caestecker, M. P., Decker, A., Deems, S., Degnan, D., Desai, T., Deshpande, V., Deutsch, G., Devlin, M., Diep, D., Dodd, C., Donahue, S., Dong, W., Dos Santos Peixoto, R., Duffy, M., Dufresne, M., Duong, T. E., Dutra, J., Eadon, M. T., El-Achkar, T. M., Enninful, A., Eraslan, G., Eshelman, D., Espin-Perez, A., Esplin, E. D., Esselman, A., Falo, L. D., Falo, L., Fan, J., Fan, R., Farrow, M. A., Farzad, N., Favaro, P., Fermin, J., Filiz, F., Filus, S., Fisch, K., Fisher, E., Fisher, S., Flowers, K., Flynn, W. F., Fogo, A. B., Fu, D. A., Fulcher, J., Fung, A., Furst, D., Gallant, M., Gao, F., Gao, Y., Gaulton, K., Gaut, J. P., Gee, J., Ghag, R. R., Ghazanfar, S., Ghose, S., Gisch, D., Gold, I., Gondalia, A., Gorman, B., Greenleaf, W., Greenwald, N., Gregory, B., Guo, R., Gupta, R., Hakimian, H., Haltom, J., Halushka, M., Han, K. S., Hanson, C., Harbury, P., Hardi, J., Harlan, L., Harris, R. C., Hartman, A., Heidari, E., Helfer, J., Helminiak, D., Hemberg, M., Henning, N., Herr, B. W., Ho, J., Holden-Wiltse, J., Hong, S., Hong, Y., Honick, B., Hood, G., Hu, P., Hu, Q., Huang, M., Huyck, H., Imtiaz, T., Isberg, O. G., Itkin, M., Jackson, D., Jacobs, M., Jain, Y., Jewell, D., Jiang, L., Jiang, Z. G., Johnston, S., Joshi, P., Ju, Y., Judd, A., Kagel, A., Kahn, A., Kalavros, N., Kalhor, K., Karagkouni, D., Karathanos, T., Karunamurthy, A., Katari, S., Kates, H., Kaushal, M., Keener, N., Keller, M., Kenney, M., Kern, C., Kharchenko, P., Kim, J., Kingsford, C., Kirwan, J., Kiselev, V., Kishi, J., Kitata, R. B., Knoten, A., Kollar, C., Krishnamoorthy, P., Kruse, A. R., Da, K., Kundaje, A., Kutschera, E., Kwon, Y., Lake, B. B., Lancaster, S., Langlieb, J., Lardenoije, R., Laronda, M., Laskin, J., Lau, K., Lee, H., Lee, M., Lee, M., Strekalova, Y. L., Li, D., Li, J., Li, J., Li, X., Li, Z., Liao, Y., Liaw, T., Lin, P., Lin, Y., Lindsay, S., Liu, C., Liu, Y., Liu, Y., Lott, M., Lotz, M., Lowery, L., Lu, P., Lu, X., Lucarelli, N., Lun, X., Luo, Z., Ma, J., Macosko, E., Mahajan, M., Maier, L., Makowski, D., Malek, M., Manthey, D., Manz, T., Margulies, K., Marioni, J., Martindale, M., Mason, C., Mathews, C., Maye, P., McCallum, C., McDonough, E., McDonough, L., Mcdowell, H., Meads, M., Medina-Serpas, M., Ferreira, R. M., Messinger, J., Metis, K., Migas, L. G., Miller, B., Mimar, S., Minor, B., Misra, R., Missarova, A., Mistretta, C., Moens, R., Moerth, E., Moffitt, J., Molla, G., Monroe, M., Monte, E., Morgan, M., Muraro, D., Murphy, B. R., Murray, E., Musen, M. A., Naglah, A., Nasamran, C., Neelakantan, T., Nevins, S., Nguyen, H., Nguyen, N., Nguyen, T., Nguyen, T., Nigra, D., Nofal, M., Nolan, G., Nwanne, G., O'Connor, M., Okuda, K., Olmer, M., O'Neill, K., Otaluka, N., Pang, M., Parast, M., Pasa-Tolic, L., Paten, B., Patterson, N. H., Peng, T., Phillips, G., Pichavant, M., Piehowski, P., Pilner, H., Pingry, E., Pita-Juarez, Y., Plevritis, S., Ploumakis, A., Pouch, A., Pryhuber, G., Puerto, J., Qaurooni, D., Qin, L., Quardokus, E. M., Rajbhandari, P., Rakow-Penner, R., Ramasamy, R., Read, D., Record, E. G., Reeves, D., Ricarte, A., Rodriguez-Soto, A., Ropelewski, A., Rosario, J., Roselkis, M., Rowe, D., Roy, T. K., Ruffalo, M., Ruschman, N., Sabo, A., Sachdev, N., Saka, S., Salamon, D., Sarder, P., Sasaki, H., Satija, R., Saunders, D., Sawka, R., Schey, K., Schlehlein, H., Scholten, D., Schultz, S., Schwartz, L., Schwenk, M., Scibek, R., Segre, A., Serrata, M., Shands, W., Shen, X., Shendure, J., Shephard, H., Shi, L., Shi, T., Shin, D., Shirey, B., Sibilla, M., Silber, M., Silverstein, J., Simmel, D., Simmons, A., Singhal, D., Sivajothi, S., Smits, T., Soncin, F., Song, Q., Stanley, V., Stuart, T., Su, H., Su, P., Sun, X., Surrette, C., Swahn, H., Tan, K., Teichmann, S., Tejomay, A., Tellides, G., Thomas, K., Thomas, T., Thompson, M., Tian, H., Tideman, L., Trapnell, C., Tsai, A. G., Tsai, C., Tsai, L., Tsui, E., Tsui, T., Tung, J., Turner, M., Uranic, J., Vaishnav, E. D., Varra, S. R., Vaskivskyi, V., Velickovic, D., Velickovic, M., Verheyden, J., Waldrip, J., Wallace, D., Wan, X., Wang, A., Wang, F., Wang, M., Wang, S., Wang, X., Wasserfall, C., Wayne, L., Webber, J., Weber, G. M., Wei, B., Wei, J., Weimer, A., Welling, J., Wen, X., Wen, Z., Williams, M., Winfree, S., Winograd, N., Woodard, A., Wright, D., Wu, F., Wu, P., Wu, Q., Wu, X., Xing, Y., Xu, T., Yang, M., Yang, M., Yap, J., Ye, D. H., Yin, P., Yuan, Z., Yun, C. J., Zahraei, A., Zemaitis, K., Zhang, B., Zhang, C., Zhang, C., Zhang, C., Zhang, K., Zhang, S., Zhang, T., Zhang, Y., Zhao, B., Zhao, W., Zheng, J. W., Zhong, S., Zhu, B., Zhu, C., Zhu, D., Zhu, Q., Zhu, Y. 2024
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Rapid Setup of Tissue Microarray and Tiled Area Imaging on the Multiplexed Ion Beam Imaging Microscope using the Tile/SED/Array Interface.
Journal of visualized experiments : JoVE
Piyadasa, H., Oberlton, B., Kong, A., Camacho Fullaway, C., Reddy Varra, S., Sowers, C., Tsai, A. G.
2023
Multiplexed ion beam imaging (MIBI) is a next-generation mass spectrometry-based microscopy technique that generates 40+ plex images of protein expression in histologic tissues, enabling detailed dissection of cellular phenotypes and histoarchitectural organization. A key bottleneck in operation occurs when users select the physical locations on the tissue for imaging. As the scale and complexity of MIBI experiments have increased, the manufacturer-provided interface and third-party tools have become increasingly unwieldy for imaging large tissue microarrays and tiled tissue areas. Thus, a web-based, interactive, what-you-see-is-what-you-get (WYSIWYG) graphical interface layer - the tile/SED/array Interface (TSAI) - was developed for users to set imaging locations using familiar and intuitive mouse gestures such as drag-and-drop, click-and-drag, and polygon drawing. Written according to web standards already built into modern web browsers, it requires no installation of external programs, extensions, or compilers. Of interest to the hundreds of current MIBI users, this interface dramatically simplifies and accelerates the setup of large, complex MIBI runs.
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Advances and prospects for the Human BioMolecular Atlas Program (HuBMAP).
Nature cell biology
Jain, S., Pei, L., Spraggins, J. M., Angelo, M., Carson, J. P., Gehlenborg, N., Ginty, F., Gonçalves, J. P., Hagood, J. S., Hickey, J. W., Kelleher, N. L., Laurent, L. C., Lin, S., Lin, Y., Liu, H., Naba, A., Nakayasu, E. S., Qian, W. J., Radtke, A., Robson, P., Stockwell, B. R., Van de Plas, R., Vlachos, I. S., Zhou, M., Börner, K., Snyder, M. P.
2023
The Human BioMolecular Atlas Program (HuBMAP) aims to create a multi-scale spatial atlas of the healthy human body at single-cell resolution by applying advanced technologies and disseminating resources to the community. As the HuBMAP moves past its first phase, creating ontologies, protocols and pipelines, this Perspective introduces the production phase: the generation of reference spatial maps of functional tissue units across many organs from diverse populations and the creation of mapping tools and infrastructure to advance biomedical research.
- UNRAVELLING HUMAN HEMATOPOIETIC PROGENITOR CELL DIVERSITY THROUGH ASSOCIATION WITH INTRINSIC REGULATORY FACTORS Favaro, P., Glass, D., Borges, L., Baskar, R., Reynolds, W., Ho, D., Bruce, T., Tebaykin, D., Tsai, A., Shestopalov, I., Bendall, S. ELSEVIER SCIENCE INC. 2023: S79
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Supervised dimensionality reduction for exploration of single-cell data by HSS-LDA.
Patterns (New York, N.Y.)
Amouzgar, M., Glass, D. R., Baskar, R., Averbukh, I., Kimmey, S. C., Tsai, A. G., Hartmann, F. J., Bendall, S. C.
2022; 3 (8): 100536
Single-cell technologies generate large, high-dimensional datasets encompassing a diversity of omics. Dimensionality reduction captures the structure and heterogeneity of the original dataset, creating low-dimensional visualizations that contribute to the human understanding of data. Existing algorithms are typically unsupervised, using measured features to generate manifolds, disregarding known biological labels such as cell type or experimental time point. We repurpose the classification algorithm, linear discriminant analysis (LDA), for supervised dimensionality reduction of single-cell data. LDA identifies linear combinations of predictors that optimally separate a priori classes, enabling the study of specific aspects of cellular heterogeneity. We implement feature selection by hybrid subset selection (HSS) and demonstrate that this computationally efficient approach generates non-stochastic, interpretable axesamenable to diverse biological processes such as differentiation over time and cell cycle. We benchmark HSS-LDA against several popular dimensionality-reduction algorithms and illustrate its utility and versatility for the exploration of single-cell mass cytometry, transcriptomics, and chromatin accessibility data.
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An optimized protocol for phenotyping human granulocytes by mass cytometry.
STAR protocols
Vivanco Gonzalez, N., Oliveria, J., Tebaykin, D., Ivison, G. T., Mukai, K., Tsai, M. M., Borges, L., Nadeau, K. C., Galli, S. J., Tsai, A. G., Bendall, S. C.
2022; 3 (2): 101280
Granulocytes encompass diverse roles, from fighting off pathogens to regulating inflammatory processes in allergies. These roles are represented by distinct cellular phenotypes that we captured with mass cytometry (CyTOF). Our protocol enables simultaneous evaluation of human basophils, eosinophils, and neutrophils under homeostasis and upon immune activation by anti-Immunoglobulin E (anti-IgE) or interleukin-3 (IL-3). Granulocyte integrity and detection of protein markers were optimized so that rare granulocyte populations could be deeply characterized by single cell mass cytometry. For complete details on the use and execution of this protocol, please refer to Vivanco Gonzalez etal. (2020).
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Longitudinal study of 2 patients with cyclic thrombocytopenia, STAT3, and MPL mutations.
Blood advances
Zhang, H., Chien, M., Hou, Y., Shomali, W., Brar, R., Ho, C., Han, P., Xu, D., Zhang, B. M., Guo, X., Tolentino, L., Wu, N. C., Tsai, A. G., Jin, J., Witteles, W. H., Chen, Z., Abidi, P., Jangam, D., Krieger, M. S., Craig, M., Bussel, J. B., Gotlib, J. R., Zehnder, J. L.
2022
Cyclic thrombocytopenia (CTP) is a rare disease of periodic platelet count oscillations. The pathogenesis of CTP remains elusive. To study the underlying pathophysiology and genetic and cellular associations with CTP, we applied systems biology approaches to two patients with stable platelet cycling and reciprocal thrombopoietin (TPO) cycling at multiple time points through 2 cycles. Blood transcriptome analysis revealed cycling of platelet-specific genes, which are in parallel with and precede platelet count oscillation, indicating that cyclical platelet production leads platelet count cycling in both patients. Additionally, neutrophil and erythrocyte-specific genes also showed fluctuations correlating with platelet count changes, consistent with TPO effects on hematopoietic progenitors. Moreover, we found novel genetic associations with CTP. One patient had a novel germline heterozygous loss-of-function (LOF) thrombopoietin receptor (MPL) c.1210G>A mutation, and both had pathogenic somatic gain-of-function (GOF) variants in signal transducer and activator of transcription 3 (STAT3). In addition, both patients had clonal T-cell populations that remained stable throughout platelet count cycles. These mutations and clonal T cells may potentially involve in the pathogenic baseline in these patients rendering exaggerated persistent thrombopoiesis oscillations of their intrinsic rhythm upon homeostatic perturbations. This work provides new insights into the pathophysiology of CTP and possible therapies.
- Chromatin Content Capture Reveals Acute Leukaemia Oncogenic Vulnerability Point in Human B Cell Development Baskar, R., Favaro, P., Reynolds, W. D., Domizi, P., Tsai, A. G., Davis, K. L., Bendall, S. AMER SOC HEMATOLOGY. 2021
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Mass Cytometry Phenotyping of Human Granulocytes Reveals Novel Basophil Functional Heterogeneity.
iScience
Vivanco Gonzalez, N., Oliveria, J., Tebaykin, D., Ivison, G. T., Mukai, K., Tsai, M. M., Borges, L., Nadeau, K. C., Galli, S. J., Tsai, A. G., Bendall, S. C.
2020; 23 (11): 101724
Basophils, the rarest granulocyte, play critical roles in parasite- and allergen-induced inflammation. We applied mass cytometry (CyTOF) to simultaneously asses 44 proteins to phenotype and functionally characterize neutrophils, eosinophils, and basophils from 19 healthy donors. There was minimal heterogeneity seen in eosinophils and neutrophils, but data-driven analyses revealed four unique subpopulations within phenotypically basophilic granulocytes (PBG; CD45+HLA-DR-CD123+). Through CyTOF and fluorescence-activated cell sorting (FACS), we classified these four PBG subpopulations as (I) CD16lowFcepsilonRIhighCD244high (88.5± 1.2%), (II) CD16highFcepsilonRIhighCD244high (9.1± 0.4%), (III) CD16lowFcepsilonRIlowCD244low (2.3± 1.3), and (IV) CD16highFcepsilonRIlowCD244low (0.4± 0.1%). Prospective isolation confirmed basophilic-morphology of PBG I-III, but neutrophilic-morphology of PBG IV. Functional interrogation via IgE-crosslinking or IL-3 stimulation demonstrated that PBG I-II had significant increases in CD203c expression, whereas PBG III-IV remained unchanged compared with media-alone conditions. Thus, PBG III-IV could serve roles in non-IgE-mediated immunity. Our findings offer new perspectives in human basophil heterogeneity and the varying functional potential of these new subsets in health and disease.
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Impact of somatic and germline mutations on the outcome of systemic mastocytosis.
Blood advances
Munoz-Gonzalez, J. I., Jara-Acevedo, M., Alvarez-Twose, I., Merker, J. D., Teodosio, C., Hou, Y., Henriques, A., Roskin, K. M., Sanchez-Munoz, L., Tsai, A. G., Caldas, C., Matito, A., Sanchez-Gallego, J. I., Mayado, A., Dasilva-Freire, N., Gotlib, J. R., Escribano, L., Orfao, A., Garcia-Montero, A. C.
2018; 2 (21): 2814–28
Systemic mastocytosis (SM) is a highly heterogeneous disease with indolent and aggressive forms, with the mechanisms leading to malignant transformation still remaining to be elucidated. Here, we investigated the presence and frequency of genetic variants in 34 SM patients with multilineal KIT D816V mutations. Initial screening was performed by targeted sequencing of 410 genes in DNA extracted from purified bone marrow cells and hair from 12 patients with nonadvanced SM and 8 patients with advanced SM, followed by whole-genome sequencing (WGS) in 4 cases. Somatic mutations were further investigated in another 14 patients with advanced SM. Despite the fact that no common mutation other than KIT D816V was found in WGS analyses, targeted next-generation sequencing identified 67 nonsynonymous genetic variants involving 39 genes. Half of the mutations were somatic (mostly multilineal), whereas the other half were germline variants. The presence of ≥1 multilineal somatic mutation involving genes other than KIT D816V, ≥3 germline variants, and ≥1 multilineal mutation in the SRSF2, ASXL1, RUNX1, and/or EZH2 genes (S/A/R/E genes), in addition to skin lesions, splenomegaly, thrombocytopenia, low hemoglobin levels, and increased alkaline phosphatase and beta2-microglobulin serum levels, were associated with a poorer patient outcome. However, the presence of ≥1 multilineal mutation, particularly involving S/A/R/E genes, was the only independent predictor for progression-free survival and overall survival in our cohort.
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Bone marrow morphology is a strong discriminator between chronic eosinophilic leukemia, not otherwise specified from reactive idiopathic hypereosinophilic syndrome.
Haematologica
Wang, S. A., Hasserjian, R. P., Tam, W., Tsai, A. G., Geyer, J. T., George, T. I., Foucar, K., Rogers, H. J., Hsi, E. D., Rea, B. A., Bagg, A., Bueso-Ramos C, C., Arber, D. A., Verstovsek, S., Orazi, A.
2017
Chronic eosinophilic leukemia, not otherwise specified can be difficult to distinguish from idiopathic hypereosinophilic syndrome according to the current World Health Organization guideline. To examine whether the morphological features of bone marrow might aid in the differential diagnosis of these two entities, we studied a total of 139 patients with a diagnosis of chronic eosinophilic leukemia, not otherwise specified (n=17) or idiopathic hypereosinophilic syndrome (n=122). As a group, abnormal bone marrow morphological features, resembling myelodysplastic syndromes, myeloproliferative neoplasm or myelodysplastic/myeloproliferative neoplasm, were identified in 40/139 (27%) patients: 16 (94%) of those with chronic eosinophilic leukemia and 24 (20%) of those with hypereosinophilic syndrome. Abnormal bone marrow correlated with older age (P<0.001), constitutional symptoms (P<0.001), anemia (P=0.041), abnormal platelet count (P=0.002), organomegaly (P=0.008), elevated lactate dehydrogenase concentration (P=0.005), abnormal karyotype (P<0.001), as well as the presence of myeloid neoplasm-related mutations (P<0.001). Patients with abnormal bone marrow had shorter survival (48.1 months versus not reached, P<0.001), a finding which was independent of other confounding factors (P<0.001). The association between abnormal bone marrow and shorter survival was also observed in hypereosinophilic syndrome patients alone. In summary, most patients with chronic eosinophilic leukemia, not otherwise specified and a proportion of those with idiopathic hypereosinophilic syndrome show abnormal bone marrow features similar to the ones encountered in patients with myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasm or BCR-ABL1-negative myeloproliferative neoplasm. Among patients who are currently considered to have idiopathic hypereosinophilic syndrome, abnormal bone marrow is a strong indicator of clonal hematopoiesis. Similar to other myeloid neoplasms, bone marrow morphology should be one of the major criteria to distinguish patients with chronic eosinophilic leukemia, not otherwise specified or clonal hypereosinophilic syndrome from those with truly reactive idiopathic hypereosinophilic syndrome.
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Targeted next-generation sequencing identifies a subset of idiopathic hypereosinophilic syndrome with features similar to chronic eosinophilic leukemia, not otherwise specified.
Modern pathology
Wang, S. A., Tam, W., Tsai, A. G., Arber, D. A., Hasserjian, R. P., Geyer, J. T., George, T. I., Czuchlewski, D. R., Foucar, K., Rogers, H. J., Hsi, E. D., Bryan Rea, B., Bagg, A., Dal Cin, P., Zhao, C., Kelley, T. W., Verstovsek, S., Bueso-Ramos, C., Orazi, A.
2016; 29 (8): 854-864
The distinction between chronic eosinophilic leukemia, not otherwise specified and idiopathic hypereosinophilic syndrome largely relies on clonality assessment. Prior to the advent of next-generation sequencing, clonality was usually determined by cytogenetic analysis. We applied targeted next-generation sequencing panels designed for myeloid neoplasms to bone marrow specimens from a cohort of idiopathic hypereosinophilic syndrome patients (n=51), and assessed the significance of mutations in conjunction with clinicopathological features. The findings were further compared with those of 17 chronic eosinophilic leukemia, not otherwise specified patients defined by their abnormal cytogenetics and/or increased blasts. Mutations were detected in 14/51 idiopathic hypereosinophilic syndrome patients (idiopathic hypereosinophilic syndrome/next-generation sequencing-positive) (28%), involving single gene in 7 and ≥2 in 7 patients. The more frequently mutated genes included ASXL1 (43%), TET2 (36%), EZH2 (29%), SETBP1 (22%), CBL (14%), and NOTCH1 (14%). Idiopathic hypereosinophilic syndrome/next-generation sequencing-positive patients showed a number of clinical features and bone marrow findings resembling chronic eosinophilic leukemia, not otherwise specified. Chronic eosinophilic leukemia, not otherwise specified patients showed a disease-specific survival of 14.4 months, markedly inferior to idiopathic hypereosinophilic syndrome/next-generation sequencing-negative (P<0.001), but not significantly different from idiopathic hypereosinophilic syndrome/next-generation sequencing-positive (P=0.117). These data suggest that targeted next-generation sequencing helps to establish clonality in a subset of patients with hypereosinophilia that would otherwise be classified as idiopathic hypereosinophilic syndrome. In conjunction with other diagnostic features, mutation data can be used to establish a diagnosis of chronic eosinophilic leukemia, not otherwise specified in patients presenting with hypereosinophilia.Modern Pathology advance online publication, 13 May 2016; doi:10.1038/modpathol.2016.75.
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Human lymphoid translocation fragile zones are hypomethylated and have accessible chromatin.
Molecular and cellular biology
Lu, Z., Lieber, M. R., Tsai, A. G., Pardo, C. E., Müschen, M., Kladde, M. P., Hsieh, C.
2015; 35 (7): 1209-1222
Chromosomal translocations are a hallmark of hematopoietic malignancies. CG motifs within translocation fragile zones (typically 20 to 600 bp in size) are prone to chromosomal translocation in lymphomas. Here we demonstrate that the CG motifs in human translocation fragile zones are hypomethylated relative to the adjacent DNA. Using a methyltransferase footprinting assay on isolated nuclei (in vitro), we find that the chromatin at these fragile zones is accessible. We also examined in vivo accessibility using cellular expression of a prokaryotic methylase. Based on this assay, which measures accessibility over a much longer time interval than is possible with in vitro methods, these fragile zones were found to be more accessible than the adjacent DNA. Because DNA within the fragile zones can be methylated by both cellular and exogenous methyltransferases, the fragile zones are predominantly in a duplex DNA conformation. These observations permit more-refined models for why these zones are 100- to 1,000-fold more prone to undergo chromosomal translocation than the adjacent regions.
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BCL6 breaks occur at different AID sequence motifs in Ig-BCL6 and non-Ig-BCL6 rearrangements
BLOOD
Lu, Z., Tsai, A. G., Akasaka, T., Ohno, H., Jiang, Y., Melnick, A. M., Greisman, H. A., Lieber, M. R.
2013; 121 (22): 4551-4554
BCL6 translocations are common in B-cell lymphomas and frequently have chromosomal breaks in immunoglobulin heavy chain (IgH) switch regions, suggesting that they occur during class-switch recombination. We analyze 120 BCL6 translocation breakpoints clustered in a 2156-bp segment of BCL6 intron 1, including 62 breakpoints (52%) joined to IgH, 12 (10%) joined to Ig light chains, and 46 (38%) joined to non-Ig partners. The BCL6 breaks in Ig-BCL6 translocations prefer known activation-induced cytosine deaminase (AID) hotspots such as WGCW and WRC (W = A/T, R = A/G), whereas BCL6 breaks in non-Ig rearrangements occur at CpG/CGC sites in addition to WGCW. Unlike previously identified CpG breaks in pro-B/pre-B-cell translocations, the BCL6 breaks do not show evidence of recombination activating gene or terminal deoxynucleotidyl transferase activity. Both WGCW/WRC and CpG/CGC breaks at BCL6 are most likely initiated by AID in germinal center B-cells, and their differential use suggests subtle mechanistic differences between Ig-BCL6 and non-Ig-BCL6 rearrangements.
DOI 10.1182/blood-2012-10-464958
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Both CpG Methylation and Activation-Induced Deaminase Are Required for the Fragility of the Human bcl-2 Major Breakpoint Region: Implications for the Timing of the Breaks in the t(14;18) Translocation
MOLECULAR AND CELLULAR BIOLOGY
Cui, X., Lu, Z., Kurosawa, A., Klemm, L., Bagshaw, A. T., Tsai, A. G., Gemmell, N., Mueschen, M., Adachi, N., Hsieh, C., Lieber, M. R.
2013; 33 (5): 947-957
The t(14;18) chromosomal translocation typically involves breakage at the bcl-2 major breakpoint region (MBR) to cause human follicular lymphoma. A theory to explain the striking propensity of the MBR breaks at three CpG clusters within the 175-bp MBR region invoked activation-induced deaminase (AID). In a test of that theory, we used here minichromosomal substrates in human pre-B cell lines. Consistent with the essential elements of the theory, we found that the MBR breakage process is indeed highly dependent on DNA methylation at the CpG sites and highly dependent on the AID enzyme to create lesions at peak locations within the MBR. Interestingly, breakage of the phosphodiester bonds at the AID-initiated MBR lesions is RAG dependent, but, unexpectedly, most are also dependent on Artemis. We found that Artemis is capable of nicking small heteroduplex structures and is even able to nick single-base mismatches. This raises the possibility that activated Artemis, derived from the unjoined D to J(H) DNA ends at the IgH locus on chromosome 14, nicks AID-generated TG mismatches at methyl CpG sites, and this would explain why the breaks at the chromosome 18 MBR occur within the same time window as those on chromosome 14.
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IgH partner breakpoint sequences provide evidence that AID initiates t(11;14) and t(8;14) chromosomal breaks in mantle cell and Burkitt lymphomas
BLOOD
Greisman, H. A., Lu, Z., Tsai, A. G., Greiner, T. C., Yi, H. S., Lieber, M. R.
2012; 120 (14): 2864-2867
Previous studies have implicated activation-induced cytidine deaminase (AID) in B-cell translocations but have failed to identify any association between their chromosomal breakpoints and known AID target sequences. Analysis of 56 unclustered IgH-CCND1 translocations in mantle cell lymphoma across the ~ 344-kb bcl-1 breakpoint locus demonstrates that half of the CCND1 breaks are near CpG dinucleotides. Most of these CpG breaks are at CGC motifs, and half of the remaining breaks are near WGCW, both known AID targets. These findings provide the strongest evidence to date that AID initiates chromosomal breaks in translocations that occur in human bone marrow B-cell progenitors. We also identify WGCW breaks at the MYC locus in Burkitt lymphoma translocations and murine IgH-MYC translocations, both of which arise in mature germinal center B cells. Finally, we propose a developmental model to explain the transition from CpG breaks in early human B-cell progenitors to WGCW breaks in later stage B cells.
DOI 10.1182/blood-2012-02-412791
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Heterogeneity and Randomness of DNA Methylation Patterns in Human Embryonic Stem Cells
DNA AND CELL BIOLOGY
Tsai, A. G., Chen, D. M., Lin, M., Hsieh, J. C., Okitsu, C. Y., Taghva, A., Shibata, D., Hsieh, C.
2012; 31 (6): 893-907
DNA methylation has been proposed to be important in many biological processes and is the subject of intense study. Traditional bisulfite genomic sequencing allows detailed high-resolution methylation pattern analysis of each molecule with haplotype information across a few hundred bases at each locus, but lacks the capacity to gather voluminous data. Although recent technological developments are aimed at assessing DNA methylation patterns in a high-throughput manner across the genome, the haplotype information cannot be accurately assembled when the sequencing reads are short or when each hybridization target only includes one or two cytosine-phosphate-guanine (CpG) sites. Whether a distinct and nonrandom DNA methylation pattern is present at a given locus is difficult to discern without the haplotype information, and the DNA methylation patterns are much less apparent because the data are often obtained only as methylation frequencies at each CpG site with some of these methods. It would facilitate the interpretation of data obtained from high-throughput bisulfite sequencing if the loci with nonrandom DNA methylation patterns could be distinguished from those that are randomly methylated. In this study, we carried out traditional genomic bisulfite sequencing using the normal diploid human embryonic stem (hES) cell lines, and utilized Hamming distance analysis to evaluate the existence of a distinct and nonrandom DNA methylation pattern at each locus studied. Our findings suggest that Hamming distance is a simple, quick, and useful tool to identify loci with nonrandom DNA methylation patterns and may be utilized to discern links between biological changes and DNA methylation patterns in the high-throughput bisulfite sequencing data sets.
- t(X;14)(p22;q32)/t(Y;14)(p11;q32) CRLF2-IGH translocations from human B-lineage ALLs involve CpG-type breaks at CRLF2, but CRLF2/P2RY8 intrachromosomal deletions do not BLOOD Tsai, A. G., Yoda, A., Weinstock, D. M., Lieber, M. R. 2010; 116 (11): 1993-1994
- The t(14;18)(q32;q21)/IGH-MALT1 translocation in MALT lymphomas is a CpG-type translocation, but the t(11;18)(q21;q21)/API2-MALT1 translocation in MALT lymphomas is not BLOOD Tsai, A. G., Lu, Z., Lieber, M. R. 2010; 115 (17): 3640-3641
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Nonhomologous DNA end joining (NHEJ) and chromosomal translocations in humans.
Sub-cellular biochemistry
Lieber, M. R., Gu, J., Lu, H., Shimazaki, N., Tsai, A. G.
2010; 50: 279-296
Double-strand breaks (DSBs) arise in dividing cells about ten times per cell per day. Causes include replication across a nick, free radicals of oxidative metabolism, ionizing radiation, and inadvertent action by enzymes of DNA metabolism (such as failures of type II topoisomerases or cleavage by recombinases at off-target sites). There are two major double-strand break repair pathways. Homologous recombination (HR) can repair double-strand breaks, but only during S phase and typically only if there are hundreds of base pairs of homology. The more commonly used pathway is nonhomologous DNA end joining, abbreviated NHEJ. NHEJ can repair a DSB at any time during the cell cycle and does not require any homology, although a few nucleotides of terminal microhomology are often utilized by the NHEJ enzymes, if present. The proteins and enzymes of NHEJ include Ku, DNA-PKcs, Artemis, DNA polymerase mu (Pol micro), DNA polymerase lambda (Pol lambda), XLF (also called Cernunnos), XRCC4, and DNA ligase IV. These enzymes constitute what some call the classical NHEJ pathway, and in wild type cells, the vast majority of joining events appear to proceed using these components. NHEJ is present in many prokaryotes, as well as all eukaryotes, and very similar mechanistic flexibility evolved both convergently and divergently. When two double-strand breaks occur on different chromosomes, then the rejoining is almost always done by NHEJ. The causes of DSBs in lymphomas most often involve the RAG or AID enzymes that function in the specialized processes of antigen receptor gene rearrangement.
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H3K4me3 Stimulates the V(D)J RAG Complex for Both Nicking and Hairpinning in trans in Addition to Tethering in cis: Implications for Translocations
MOLECULAR CELL
Shimazaki, N., Tsai, A. G., Lieber, M. R.
2009; 34 (5): 535-544
The PHD finger of the RAG2 polypeptide of the RAG1/RAG2 complex binds to the histone H3 modification, trimethylated lysine 4 (H3K4me3), and in some manner increases V(D)J recombination. In the absence of biochemical studies of H3K4me3 on purified RAG enzyme activity, the precise role of H3K4me3 remains unclear. Here, we find that H3K4me3 stimulates purified RAG enzymatic activity at both the nicking (2- to 5-fold) and hairpinning (3- to 11-fold) steps of V(D)J recombination. Remarkably, this stimulation can be achieved with free H3K4me3 peptide (in trans), indicating that H3K4me3 functions via two distinct mechanisms. It not only tethers the RAG enzyme complex to a region of DNA, but it also induces a substantial increase in the catalytic turnover number (k(cat)) of the RAG complex. The H3K4me3 catalytic stimulation applies to suboptimal cryptic RSS sites located at H3K4me3 peaks that are critical in the inception of human T cell acute lymphoblastic lymphomas.
DOI 10.1016/j.molcel.2009.05.011
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Conformational Variants of Duplex DNA Correlated with Cytosine-rich Chromosomal Fragile Sites
JOURNAL OF BIOLOGICAL CHEMISTRY
Tsai, A. G., Engelhart, A. E., Hatmal, M. M., Houston, S. I., Hud, N. V., Haworth, I. S., Lieber, M. R.
2009; 284 (11): 7157-7164
We found that several major chromosomal fragile sites in human lymphomas, including the bcl-2 major breakpoint region, bcl-1 major translocation cluster, and c-Myc exon 1-intron 1 boundary, contain distinctive sequences of consecutive cytosines exhibiting a high degree of reactivity with the structure-specific chemical probe bisulfite. To assess the inherent structural variability of duplex DNA in these regions and to determine the range of structures reactive to bisulfite, we have performed bisulfite probing on genomic DNA in vitro and in situ; on duplex DNA in supercoiled and linearized plasmids; and on oligonucleotide DNA/DNA and DNA/2'-O-methyl RNA duplexes. Bisulfite is significantly more reactive at the frayed ends of DNA duplexes, which is expected given that bisulfite is an established probe of single-stranded DNA. We observed that bisulfite also distinguishes between more subtle sequence/structural differences in duplex DNA. Supercoiled plasmids are more reactive than linear DNA; and sequences containing consecutive cytosines, namely GGGCCC, are more reactive than those with alternating guanine and cytosine, namely GCGCGC. Circular dichroism and x-ray crystallography show that the GGGCCC sequence forms an intermediate B/A structure. Molecular dynamics simulations also predict an intermediate B/A structure for this sequence, and probe calculations suggest greater bisulfite accessibility of cytosine bases in the intermediate B/A structure over canonical B- or A-form DNA. Electrostatic calculations reveal that consecutive cytosine bases create electropositive patches in the major groove, predicting enhanced localization of the bisulfite anion at homo-C tracts over alternating G/C sequences. These characteristics of homo-C tracts in duplex DNA may be associated with DNA-protein interactions in vivo that predispose certain genomic regions to chromosomal fragility.
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Unexpected complexity at breakpoint junctions in phenotypically normal individuals and mechanisms involved in generating balanced translocations t(1;22)(p36;q13)
GENOME RESEARCH
Gajecka, M., Gentles, A. J., Tsai, A., Chitayat, D., Mackay, K. L., Glotzbach, C. D., Lieber, M. R., Shaffer, L. G.
2008; 18 (11): 1733-1742
Approximately one in 500 individuals carries a reciprocal translocation. Balanced translocations are usually associated with a normal phenotype unless the translocation breakpoints disrupt a gene(s) or cause a position effect. We investigated breakpoint junctions at the sequence level in phenotypically normal balanced translocation carriers. Eight breakpoint junctions derived from four nonrelated subjects with apparently balanced translocation t(1;22)(p36;q13) were examined. Additions of nucleotides, deletions, duplications, and a triplication identified at the breakpoints demonstrate high complexity at the breakpoint junctions and indicate involvement of multiple mechanisms in the DNA breakage and repair process during translocation formation. Possible detailed nonhomologous end-joining scenarios for t(1;22) cases are presented. We propose that cryptic imbalances in phenotypically normal, balanced translocation carriers may be more common than currently appreciated.
- RAGs found "not guilty": cleared by DNA evidence BLOOD Tsai, A. G., Lleber, M. R. 2008; 111 (4): 1750-1750
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Single-stranded DNA ligation and XLF-stimulated incompatible DNA end ligation by the XRCC4-DNA ligase IV complex: influence of terminal DNA sequence
NUCLEIC ACIDS RESEARCH
Gu, J., Lu, H., Tsai, A. G., Schwarz, K., Lieber, M. R.
2007; 35 (17): 5755-5762
The double-strand DNA break repair pathway, non-homologous DNA end joining (NHEJ), is distinctive for the flexibility of its nuclease, polymerase and ligase activities. Here we find that the joining of ends by XRCC4-ligase IV is markedly influenced by the terminal sequence, and a steric hindrance model can account for this. XLF (Cernunnos) stimulates the joining of both incompatible DNA ends and compatible DNA ends at physiologic concentrations of Mg2+, but only of incompatible DNA ends at higher concentrations of Mg2+, suggesting charge neutralization between the two DNA ends within the ligase complex. XRCC4-DNA ligase IV has the distinctive ability to ligate poly-dT single-stranded DNA and long dT overhangs in a Ku- and XLF-independent manner, but not other homopolymeric DNA. The dT preference of the ligase is interesting given the sequence bias of the NHEJ polymerase. These distinctive properties of the XRCC4-DNA ligase IV complex explain important aspects of its in vivo roles.
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HotPatch: A statistical a pproach to finding biologically relevant features on protein surfaces
JOURNAL OF MOLECULAR BIOLOGY
Pettit, F. K., Bare, E., Tsai, A., Bowie, J. U.
2007; 369 (3): 863-879
We describe a fully automated algorithm for finding functional sites on protein structures. Our method finds surface patches of unusual physicochemical properties on protein structures, and estimates the patches' probability of overlapping functional sites. Other methods for predicting the locations of specific types of functional sites exist, but in previous analyses, it has been difficult to compare methods when they are applied to different types of sites. Thus, we introduce a new statistical framework that enables rigorous comparisons of the usefulness of different physicochemical properties for predicting virtually any kind of functional site. The program's statistical models were trained for 11 individual properties (electrostatics, concavity, hydrophobicity, etc.) and for 15 neural network combination properties, all optimized and tested on 15 diverse protein functions. To simulate what to expect if the program were run on proteins of unknown function, as might arise from structural genomics, we tested it on 618 proteins of diverse mixed functions. In the higher-scoring top half of all predictions, a functional residue could typically be found within the first 1.7 residues chosen at random. The program may or may not use partial information about the protein's function type as an input, depending on which statistical model the user chooses to employ. If function type is used as an additional constraint, prediction accuracy usually increases, and is particularly good for enzymes, DNA-interacting sites, and oligomeric interfaces. The program can be accessed online (at http://hotpatch.mbi.ucla.edu).
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Analysis of non-B DNA structure at chromosomal sites in the mammalian genome
DNA REPAIR, PT B
Raghavan, S. C., Tsai, A., Hsieh, C., Lieber, M. R.
2006; 409: 301-316
Changes at sites of genetic instability ultimately involve DNA repair pathways. Some sites of genetic instability in the mammalian genome appear to be unstable because they adopt a non-B DNA conformation. We describe two structural approaches for determination of whether a genomic region is configured in a non-B DNA conformation. Our studies indicate that at least some chromosomal fragile sites can be explained by such altered DNA conformations. One of the methods that we describe is called the bisulfite modification assay. This is a powerful assay because it provides information on individual DNA molecules. The second approach uses preexisting DNA structural reagents, but describes our specific application of them to analysis of DNA in vivo.
DOI 10.1016/S0076-6879(05)09017-8