Imaging Mass Cytometry Reveals the Spatial Architecture of Myelodysplastic Syndromes and Secondary Acute Myeloid Leukemias

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 44-45
Author(s):  
Karolyn A. Oetjen ◽  
Diane E. Bender ◽  
Marianna B. Ruzinova ◽  
Daniel A.C. Fisher ◽  
Stephen T. Oh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndromes ◽  
Marrow Cell ◽  
Cell Populations ◽  
Normal Bone Marrow ◽  
Mass Cytometry ◽  
Normal Bone ◽  
Lineage Markers ◽  
Spatial Architecture ◽  
Acute Myeloid

Histologic review of bone marrow trephine biopsies is a central component of the diagnostic and treatment response evaluation of hematologic malignancies. Well-validated antibody reagents are routinely used for immunohistochemistry of these samples to provide additional insight into abnormal antigen expression. However, current immunohistochemistry staining protocols are typically limited to only one or two markers simultaneously. Dysplastic changes in cellular morphology and dyssynchronous expression of lineage markers are common features of myelodysplastic syndromes, myeloproliferative neoplasms and secondary acute leukemias. We have integrated the use of multiple diagnostic validated antibody clones with additional antibodies for hematologic lineages and structural proteins to create a 30-marker panel for imaging mass cytometry (IMC). Antibodies included in this panel identify myeloid, lymphoid, erythroid, macrophage, vascular, megakaryocyte and stromal markers as well as markers of cellular proliferation and apoptosis. Through conjugation to elemental metal tags, the entire panel is stained simultaneously on the tissue sample, then acquired by time-of-flight mass spectrometry on a Hyperion instrument (Fluidigm). Antibody staining concentrations and antigen retrieval conditions were optimized for formalin-fixed paraffin-embedded (FFPE) bone marrow to obtain consistent staining for all markers on the panel. Redundant markers for cell populations were selected to provide further internal validation of the observed staining patterns. After data acquisition, cell segmentation algorithms using CellProfiler and ilastik were applied to quantify marker expression in single cells and Phenograph in HistoCAT was used for cell population clustering. Cluster identities for all cells are associated with the original image location in order to plot the spatial arrangement of populations. Using this highly multiplexed panel, we have imaged sets of bone marrow specimens from patients with normal bone marrow morphology and those with myeloid malignancies. We initially confirmed the staining patterns expected for each antibody patterns of co-expression of lineage markers in normal bone marrow samples. We then extended this panel to examine biopsies from patients with myelodysplastic syndrome, myelofibrosis, and secondary acute myeloid leukemia. We found a clear population of CD71+ CD235a+ erythroid cells with strong expression of the proliferative marker Ki67 located within erythroid islands in normal bone marrow samples and MDS. Cell markers of apoptosis and DNA damage are scattered at low frequency throughout the bone marrow in samples with normal bone marrow morphology, but increased clusters of the DNA damage marker phospho-H2AX are observed in selected cases of myelodysplastic syndromes. Overall, this IMC imaging approach is able to extend the current clinical immunostaining for myeloid malignancies by identifying all major bone marrow cell populations. Through highly multiplexed analysis of bone marrow cell populations, the spatial architecture of cell populations and stromal structures can be elucidated, including erythroid islands, lymphoid aggregates and changes in vascular structures with increasing severity of myelofibrosis. In ongoing studies, the development of these imaging techniques for analysis of archived FFPE bone marrow samples is being applied to translational research on hematologic diseases. Disclosures Oh: Kartos Therapeutics: Consultancy; Disc Medicine: Consultancy; PharmaEssentia: Consultancy; Constellation: Consultancy; CTI Biopharma: Consultancy; Celgene/Bristol Myers Squibb: Consultancy; Blueprint Medicines: Consultancy; Novartis: Consultancy; Gilead Sciences: Consultancy; Incyte Corporation: Consultancy.

CD34+ Cells from Acute Myeloid Leukemia, Myelodysplastic Syndromes, and Normal Bone Marrow Display Different Apoptosis and Drug Resistance–Associated Phenotypes

2004 ◽  
Vol 10 (22) ◽  
pp. 7599-7606 ◽  
Author(s):  
Lilia Suárez ◽  
María-Belén Vidriales ◽  
José García-Laraña ◽  
Guillermo Sanz ◽  
María-José Moreno ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Acute Myeloid Leukemia ◽  
Myelodysplastic Syndromes ◽  
Myeloid Leukemia ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Cd34 Cells ◽  
Acute Myeloid

scholarly journals Análisis inmunofenotípico de muestras normales de médula ósea: aplicaciones en el control de calidad en los laboratorios de citometría

2010 ◽  
Vol 15 (3) ◽  
pp. 206 ◽  
Author(s):  
Diana Carolina Roa-Higuera ◽  
Susana Fiorentino-Gómez ◽  
Viviana Marcela Rodríguez-Pardo ◽  
Alba Myriam Campos-Arenas ◽  
Elvira Antonia Infante-Acosta ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Quality Control ◽  
Reference Values ◽  
Cell Populations ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Cell Subpopulations ◽  
Hematologic Neoplasia ◽  
Routine Quality Control ◽  
Normal Leukocyte

<p><strong></strong><strong>Objective.</strong> To describe a standardized flow cytometry protocol for the relative and absolute quantification of hematopoietic cell subpopulations from normal bone marrow, and to evaluate the expression of different lineage-specific cell markers with a reactivity associated to cell differentiation to be used as part of the routine quality control in cytometry laboratories. <strong>Materials and methods. </strong>The immunophenotypical analysis of different cell subpopulations was done with samples from normal bone marrow using a panel of monoclonal and polyclonal antibodies useful in the characterization of acute leukemias with four different fluorescences, by means of a protocol that combines cell labeling of membrane and cytoplasm antigens. Expression analysis was done in terms of mean fluorescence intensity (MFI). Fluorescent beads at a known concentration were added for calculating the absolute count of cells.  <strong>Results.</strong> The antibody panel used allowed the identification and quantification of different normal leukocyte subpopulations of lymphatic and myeloid origin, including CD34+ stem cells and more differentiated cell populations in the granulocytic, monocytic, and erythroid cell lines. We established reference values for cell populations and cell marker expression ranges as part of routine quality control of cytometry laboratories. <strong>Conclusion.</strong> Immunophenotypic patterns identified as well as absolute and relative reference values for the different normal leukocyte populations from bone marrow can be used by cytometry laboratories as a basis for establishing reference parameters in phenotypic analyses of hematologic neoplasia.<strong> </strong></p> <p><strong>Key words: </strong>multiparametric flow cytometry, immunophenotype, hematologic neoplasia, normal bone marrow, reference values, quality control.</p>

A CD123-Targeting Antibody-Drug Conjugate (ADC), IMGN632, Designed to Eradicate Acute Myeloid Leukemia (AML) Cells While Sparing Normal Bone Marrow Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 768-768 ◽  
Author(s):  
Yelena Kovtun ◽  
Gregory Jones ◽  
Charlene Audette ◽  
Lauren Harvey ◽  
Baudouin Gerard ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Cell Populations ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Healthy Donors ◽  
Marrow Cells ◽  
Myeloid Progenitors

Abstract Current AML therapies are effective in a subset of patients, but often lead to prolonged myelosuppression. CD123 is an attractive AML target due to its elevated expression on AML compared to normal bone marrow cells. Still, severe myelosuppression and myeloablation have been reported in preclinical studies for some CD123-targeted therapies. Here, we present a novel ADC which selectively kills CD123-positive AML cells over normal bone marrow cells. A novel humanized anti-CD123 antibody with two engineered cysteines for payload conjugation was generated. Indolinobenzodiazepine dimers, termed IGNs, were chosen as payload molecules for the antibody due to their high potency against AML cells. The IGN dimers containing mono-imines alkylate DNA, whereas the di-imine containing IGNs can both alkylate and crosslink DNA. To select an optimal IGN payload, we compared the cytotoxicity of an ADC with a mono-imine IGN (A-ADC) to one with a di-imine IGN (C-ADC) on AML cells, as well as normal bone marrow cells in vitro. Potency of the ADCs was evaluated using AML cell lines that have CD123 levels similar to patient cells and carry markers of poor prognosis (FLT3-ITD , MDR1, EVI1, DNMT3A and TP53), as well as on samples from 11 AML patients. AML cells exposed to either ADC displayed markers of DNA damage, cell cycle arrest and apoptotic cell death by flow cytometry. Both ADCs were highly cytotoxic, generating IC50 values between 0.4 to 60 pM on the cell lines in WST-8 assays and killing 90 percent of progenitors from AML patients between 2 to 46 pM in CFU assays. The C-ADC was, on average, two-fold more active than the A-ADC. The cytotoxicity of both ADCs was CD123 dependent, since masking CD123 with a competing anti-CD123 antibody reduced the potency by more than 100-fold. Toxicity of the ADCs was assessed using bone marrow cells from a healthy human donor. The cells were exposed to the ADCs at 100 pM (a concentration highly potent against all AML samples) for 72 hours, and then markers of apoptosis were detected in different cell populations by flow cytometry. Neither ADC affected the viability of monocytes, lymphocytes and multipotential progenitors, consistent with low CD123 levels in these cell populations. In contrast, an apoptotic signal was detected in myeloid progenitors, the population with the highest CD123 level, following exposure to the C-ADC, but not to the A-ADC. The toxicity of the ADCs was also tested in CFU assays on bone marrow cells from 7 healthy donors, as the assays have been reported to predict clinical myelosuppression. Surprisingly, the C-ADC was, on average, 50-fold more cytotoxic to normal myeloid progenitors than the A-ADC (40 pM vs 2,000 pM IC90 values, respectively) (Figure 1). Finally, we compared CD123 independent toxicity of the ADCs in CD-1 mice. The C-ADC showed significantly reduced tolerability, and unlike the A-ADC, was associated with delayed toxicity manifested by weight loss 30 days after administration. Based on its potent yet highly selective toxicity to AML cells and more favorable tolerability profile, the A-ADC was selected for further study, and renamed as IMGN632. To compare IMGN632 to an ADC previously approved for the treatment of AML, the potency of IMGN632 and gemtuzumab ozogamicin (GO) was tested on bone marrow cells from 11 healthy donors and 17 AML patients, including 4 relapsed/refractory and 8 with strong multidrug resistance (Figure 1). Only 6 of 17 AML samples were sensitive to GO at concentrations that did not impact normal progenitors. In contrast, AML progenitors from all 17 patients were highly sensitive to IMGN632. Importantly, normal progenitors were only affected by IMGN632 at 150-fold higher concentrations. The pronounced difference between AML and normal progenitors in their sensitivity to IMGN632 likely reflects both higher CD123 levels on AML progenitors and the lower sensitivity of normal progenitors to the mono-imine IGN payload we observed in CFU assays. In conclusion, through use of a mono-imine IGN payload, IMGN632 demonstrated potent activity in all tested AML samples at concentrations far below levels that impact normal bone marrow cells, suggesting the potential for efficacy in AML patients in the absence of or with limited myelosuppression. These findings together with strong efficacy in multiple AML xenograft models (Kovtun et al., 21st EHA congress, 2016; Adams et al., 58th ASH annual meeting, 2016) support advancing IMGN632 into clinical trials. Disclosures Kovtun: ImmunoGen, Inc.: Employment. Jones:ImmunoGen, Inc.: Employment. Audette:ImmunoGen, Inc.: Employment. Harvey:ImmunoGen, Inc.: Employment. Gerard:ImmunoGen, Inc.: Employment. Wilhelm:ImmunoGen, Inc.: Employment. Bai:ImmunoGen, Inc.: Employment. Adams:ImmunoGen, Inc.: Employment. Goldmacher:ImmunoGen, Inc.: Employment. Chari:ImmunoGen: Employment. Chittenden:ImmunoGen, Inc.: Employment.

scholarly journals CELLS INVOLVED IN THE IMMUNE RESPONSE

1969 ◽  
Vol 130 (1) ◽  
pp. 141-163 ◽  
Author(s):  
Nabih I. Abdou ◽  
Maxwell Richter
Keyword(s):  
Bone Marrow ◽  
Immune Response ◽  
Glass Bead ◽  
Mononuclear Cells ◽  
Marrow Cell ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Immunological Specificity ◽  
Rabbit Bone ◽  
Free Antigen

The antigen-reactive cells in normal rabbit bone marrow could be isolated from a suspension of marrow cells by passage of the cells through an antigen-sensitized glass bead column. The cells which passed through the column were deficient in antigen-reactive cells directed to the antigen used to sensitize the glass beads, whereas the cells eluted from the column could transfer antibody-forming capacity to irradiated recipients only with respect to the specific sensitizing antigen. Separation of the bone marrow antigen-reactive cells could not be achieved by passage of the cells through nonsensitized glass bead columns or in the presence of excess free antigen in the column. Cells which were retained by, and later eluted from, the antigen-sensitized glass bead columns were mostly small mononuclear cells, whereas cells which passed through the columns were morphologically similar to the original unfractionated bone marrow cell suspension. The data indicate the presence of an antibody or antibody-like structure, with defined immunological specificity, on the surface of the normal bone marrow antigen-reactive cell.

Immunoreactivity of MIC2 (CD99) and Terminal Deoxynucleotidyl Transferase in Bone Marrow Clot and Core Specimens of Acute Myeloid Leukemias and Myelodysplastic Syndromes

2006 ◽  
Vol 130 (2) ◽  
pp. 153-157 ◽  
Author(s):  
Loveleen C. Kang ◽  
Cherie H. Dunphy
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Myelodysplastic Syndromes ◽  
Lymphoblastic Leukemia ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Normal Bone Marrow ◽  
Cell Precursor ◽  
Normal Bone ◽  
Myeloid Leukemias

Abstract Context.—MIC2 (“thymus leukemia”) antigen has been shown to be expressed by T cells and monocytes, as well as B cells and granulocyte-lineage cells. It is most intensely expressed by the most immature thymus T-lineage cells and is more intensely expressed by CD34-positive/CD33-positive myeloid cells (compared to more mature myeloid cells) and the earliest CD34-positive/CD10-positive B-cell precursor cells (compared to cells of later B-cell precursor stages). CD99 (MIC2) is characteristically expressed in precursor B- and T-cell lymphoblastic lymphomas/leukemias, as well as in Ewing sarcoma/primitive neuroectodermal tumors (ES/PNET). It has also been shown to be expressed in a few terminal deoxynucleotidyl transferase (TdT)–positive myeloid processes, but has been uniformly negative in TdT-negative myeloid processes. A more recent study showed that 43% of acute myeloid leukemias (AMLs) and 55% of chloromas express CD99, concluding that CD99 is commonly expressed in AML and rarely seen in myeloproliferative disorders, myelodysplastic syndromes, or normal bone marrow. Although this study speculated that MIC2 expression was probably not limited to TdT-positive AML, there was no comparison with TdT reactivity in this study. Objective.—Since AML and high-grade myelodysplastic syndrome may occasionally be difficult to distinguish morphologically from acute lymphoblastic leukemia and ES/ PNET, we undertook a study to analyze MIC2 expression in conjunction with TdT reactivity in distinguishing AML or high-grade myelodysplastic syndrome from acute lymphoblastic leukemia and ES/PNET. Design.—We studied bone marrow core and clot paraffin specimens from AML (classified according to criteria of the World Health Organization; n = 49), myelodysplastic syndromes (n = 4), precursor B-cell acute lymphoblastic leukemia (n = 4), ES/PNET (n = 1), and normal bone marrow (n = 3) with MIC2 (CD99) and TdT immunohistochemistry. Results.—Overall, CD99 was expressed in 24 (49%) of 49 AML cases, including all (11/11) TdT-positive cases. CD99 was expressed in all subtypes of AML except M5. Myelodysplastic syndromes and normal bone marrow specimens were uniformly CD99 negative. Expression of TdT was limited to a subset of AML-M0, -M1, -M2, and -M4, and AML with multilineage dysplasia. Conclusions.—In contrast to a previous study, CD99 expression was not restricted to TdT-positive hematologic proliferations. In particular, the CD99-positive M3 and M7 AMLs were TdT negative. An M5 AML may likely be excluded based on a uniform TdT-negative/CD99-negative immunophenotype. In addition, in our experience, CD99 should be routinely evaluated on bone marrow clots, owing to decreased reactivity or loss of reactivity in rapid decalcifying (RDO) solution–decalcified specimens.

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