Multiparameter flow cytometry reveals myelodysplasia-related aberrant antigen expression in myelodysplastic/myeloproliferative neoplasms

Abstract Abstract 595 Diagnosis and classification of myelodysplastic syndromes (MDS) is based on cytomorpholgy (CM) and cytogenetics (CG). By the identification of MDS-related aberrant antigen expression multiparameter flow cytometry (MFC) may add important diagnostic information. Examples include an aberrant expression pattern of CD13 and CD16 in granulocytes, an aberrante expression pattern of HLA-DR and CD11b in monocytes and the expression of lymphoid markers on myeloid blasts (Haematologica 2009:94:1124). To evaluate the potential role of MFC in the diagnostic setting of MDS we analyzed 1013 cases with suspected MDS by CM, CG, and MFC in parallel. Cases were classified by CM as refractory anemia (RA, n=31, 3.1%), refractory anemia with ring sideroblasts (RARS, n=27, 2.7%), refractory cytopenia with multilineage dysplasia (RCMD, n=64, 6.3%), refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS, n=49, 4.8%), refractory anemia with excess of blasts 1 (RAEB-1, n=133, 13.1%), RAEB-2 (n=81, 8.0%), 5q- syndrome (n=24, 2.4%), chronic myelomonocytic leukemia (CMML, n=65, 6.4%), myelodysplastic syndrome unspecified (MDS-u, n=15, 1.5%), MDS borderline to acute myeloid leukemia (MDS/AML, n=6, 0.6%), MDS/myeloproliferative neplasia overlap (MDS/MPN, n=16, 1.6%), suspected MDS (n=225, 22.2%), reactive condition (n=266, 26.3%), and normal findings (n=11, 1.1%). Cytogenetic findings were normal karyotype (n=768, 75.8%), isolated deletion of long arm of chromosome 5 (del(5q), n=43, 4.2%), isolated aberrations of chromosome 7 (n=14, 1.4%), isolated trisomy 8 (n=30, 3.0%), isolated deletion of long arm of chromosome 20 (del(20q), n=21, 2.1%), complex karyotype (n=23, 2.3%), loss of Y-chromosome (n=43, 4.2%), other aberrations (n=71, 7.0%). Concordance between CM and MFC was 82.0% for diagnostic results in 788 cases with unequivocal CM. 277 of these 788 cases were classified by CM as not having MDS, 13 (4.7%) of which showed MDS-typical features by MFC. Additional 225 cases showed only minor dysplastic features by CM, 51 (22.7%) of which showed clear evidence of MDS by MFC. To further analyze the significance of MDS-related findings by MFC we then focused on cytogenetically aberrant cases without unequivocal MDS by CM. In 6/12 (50.0%) cases with no indication of MDS by CM and MDS-typical cytogenetic aberrations MFC revealed MDS characteristics. In another 11/23 (47.8%) cases with minor dysplastic features by CM and MDS-typical cytogenetic aberrations MFC revealed MDS characteristics. Furthermore, we compared blast counts as determined by CM and MFC and found a strong correlation (p<0.001) although the mean±SD percentage was higher as determined by CM as compared to MFC (4.67±4.18 vs. 3.78±2.97). Frequencies of aberrantly expressed antigens significantly differed between cases rated by CM as MDS (median number of aberrantly expressed antigens: 3), suspected MDS (1), and no MDS (0, p<0.001). In various cases MFC identified MDS-typical aberrant antigen expression in cell compartments not rated dysplastic by CM. Spearman rank correlation confirmed a highly significant relation between the number of aberrantly expressed antigens and IPSS (r=0.409, p<0.001). In 257 cases with data on overall survival (OS) the presence of MDS-related findings (≥3 aberrantly expressed antigens or a blast count >5% in MFC or a reduced side-scatter signal) resulted in significantly inferior 6-year-OS (68% vs. 100% p=0.008). The present analysis clearly demonstrates a diagnostic yield of MFC in addition to cytomorphology and cytogenetics in cases with suspected MDS. Disclosures: Kern: MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

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In Myelodysplastic/Myeloproliferative Neoplasms Aberrant Antigen Expression As Assessed by Multiparameter Flow Cytometry Is Related to Molecular Genetic Mutations

Blood ◽

10.1182/blood.v118.21.5152.5152 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5152-5152

Author(s):

Wolfgang Kern ◽

Susanne Schnittger ◽

Tamara Alpermann ◽

Claudia Haferlach ◽

Torsten Haferlach

Keyword(s):

Myeloproliferative Neoplasms ◽

Molecular Genetic ◽

Antigen Expression ◽

Multiparameter Flow Cytometry ◽

Employment Equity ◽

Aberrant Expression ◽

Equity Ownership ◽

Diagnostic Work ◽

Work Up ◽

Diagnostic Work Up

Abstract Abstract 5152 Background: Immunophenotyping by multiparameter flow cytometry (MFC) is increasingly used in the diagnostic work-up of patients with cytopenias and suspected myelodysplastic syndromes (MDS). Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) comprise a group of diseases with some features of MDS and is separately classified in the current WHO system. While the immunophenotype of chronic myelomonocytic leukemia has been described in detail, data is scarce on the use of MFC in myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPNu) as well as on refractory anemia with ring sideroblasts and thrombocytosis (RARS-T), which is a provisional entity in the current WHO classification. Aim: To assess patients with MDS/MPNu and RARS-T for MDS-related aberrant immunophenotypes in the context of a comprehensive diagnostic work-up including cytomorphology, cytogenetics, and molecular genetics. Patients and Methods: A total of 91 patients were analyzed in parallel by cytomorphology, cytogenetics, and MFC applying an antibody panel designed to diagnose MDS. MFC was used to detect expression of mature antigens in myeloid progenitors; abnormal CD13-CD16- and CD11b-CD16-expression patterns, aberrant expression of myeloid markers and reduced side scatter signal in granulocytes; reduced expression of myelomonocytic markers in monocytes; aberrant expression of CD71 in erythroid cells; as well as expression of lymphoid markers in all myeloid cell lines. In 77/91 patients molecular genetic markers were investigated. The median age of the patients was 75.1 years (range, 35.3–87.4). The male/female ratio was 60/31. Six patients had RARS-T and 85 had MDS/MPNu. Results: In 54/91 (59.3%) patients MFC identified an MDS-immunophenotype. This was true in 4/6 (66.7%) RARS-T and in 50/85 (58.8%) MDS/MPNu (n.s.). Cases with MDS-immunophenotype displayed aberrancies significantly more frequently than those without as follows: in myeloid progenitor cells (number of aberrantly expressed antigens, mean±SD: 0.5±0.6 vs. 0.2±0.4, p=0.002), granulocytes (2.7±1.3 vs. 1.2±1.1, p<0.001), and monocytes (1.7±1.2 vs. 0.5±0.7, p<0.001). Accordingly, there was a significant difference in the total number of aberrantly expressed antigens (4.9±2.4 vs. 2.0±1.4, p<0.001). The presence of an aberrant karyotype was not related to an MDS-immunophenotype which was observed in 11/18 (61.1%) cases with aberrant karyotype and in 43/73 (58.9%) with normal karyotype (n.s.). Mutations in RUNX1 and TET2 as well as FLT3-ITD were predominantly present in cases with an MDS-immunophenotype (10/33, 30.3%) and occurred less frequently in cases without (1/7, 9.1%, n.s.). In detail, RUNX1 mutations were present in 4/26 (10.3%) vs. 0/2, TET2 mutations were present in 4/6 (66.7%) vs. 1/2 (50%), and FLT3-ITD was present in 3/29 (10.3%) vs. 0/5. Accordingly, in cases with RUNX1 or TET2 mutations or with FLT3-ITD a significantly higher number of aberrantly expressed antigens was observed as compared to cases with none of these mutations (mean±SD, 6.4±2.0 vs. 4.4±2.5, p=0.024). In contrast, JAK2V617F mutations occurred at identical frequencies in patients with and without MDS-immunophenotype (11/38, 28.9% vs. 9/31, 29.0%). Regarding prognosis, the presence of an MDS-immunophenotype had no impact on overall survival. Conclusions: These data demonstrates that MDS-related aberrant antigen expression is present in the majority of patients with RARS-T and MDS/MPNu. While there is no association between the presence of an MDS-immunophenotype and the detection of JAK2 mutations cases with an MDS-immunophenotype tended to more frequently carry mutations in RUNX1 and TET2 as well as FLT3-ITDs. These data therefore suggests that MDS/MPNu may be subdivided based on molecular genetics and on the immunophenotype into cases with MDS-related features and those without. Further analyses are needed to validate these findings and their potential significance in RARS-T. Disclosures: Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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The potential of proliferative and apoptotic parameters in clinical flow cytometry of myeloid malignancies

Blood Advances ◽

10.1182/bloodadvances.2020004094 ◽

2021 ◽

Vol 5 (7) ◽

pp. 2040-2052

Author(s):

Stefan G. C. Mestrum ◽

Anton H. N. Hopman ◽

Frans C. S. Ramaekers ◽

Math P. G. Leers

Keyword(s):

Flow Cytometry ◽

Myeloproliferative Neoplasms ◽

Diagnostic Process ◽

Multiparameter Flow Cytometry ◽

Major Step ◽

Differentiation Markers ◽

Myeloid Malignancies ◽

Cell Lineages ◽

Apoptotic Markers ◽

Multiparameter Analysis

Abstract Standardization of the detection and quantification of leukocyte differentiation markers by the EuroFlow Consortium has led to a major step forward in the integration of flow cytometry into classification of leukemia and lymphoma. In our opinion, this now enables introduction of markers for more dynamic parameters, such as proliferative and (anti)apoptotic markers, which have proven their value in the field of histopathology in the diagnostic process of solid tumors and lymphoma. Although use of proliferative and (anti)apoptotic markers as objective parameters in the diagnostic process of myeloid malignancies was studied in the past decades, this did not result in the incorporation of these biomarkers into clinical diagnosis. This review addresses the potential of these markers for implementation in the current, state-of-the-art multiparameter analysis of myeloid malignancies. The reviewed studies clearly recognize the importance of proliferation and apoptotic mechanisms in the pathogenesis of bone marrow (BM) malignancies. The literature is, however, contradictory on the role of these processes in myelodysplastic syndrome (MDS), MDS/myeloproliferative neoplasms, and acute myeloid leukemia. Furthermore, several studies underline the need for the analysis of the proliferative and apoptotic rates in subsets of hematopoietic BM cell lineages and argue that these results can have diagnostic and prognostic value in patients with myeloid malignancies. Recent developments in multiparameter flow cytometry now allow quantification of proliferative and (anti)apoptotic indicators in myeloid cells during their different maturation stages of separate hematopoietic cell lineages. This will lead to a better understanding of the biology and pathogenesis of these malignancies.

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Detection of Aberrant Antigen Expression in Acute Myeloid Leukemia by Multiparameter Flow Cytometry

Recent Advances in Cell Biology of Acute Leukemia - Recent Results in Cancer Research ◽

10.1007/978-3-642-84895-7_16 ◽

1993 ◽

pp. 185-196 ◽

Cited By ~ 11

Author(s):

B. Wörmann ◽

M. Safford ◽

S. Könemann ◽

T. Büchner ◽

W. Hiddemann ◽

...

Keyword(s):

Flow Cytometry ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Antigen Expression ◽

Multiparameter Flow Cytometry ◽

Acute Myeloid

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Flow cytometric assessment of human T-cell differentiation in thymus and bone marrow

Blood ◽

10.1182/blood.v79.3.666.666 ◽

1992 ◽

Vol 79 (3) ◽

pp. 666-677 ◽

Cited By ~ 152

Author(s):

LW Terstappen ◽

S Huang ◽

LJ Picker

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Cell Differentiation ◽

T Cell ◽

Cell Surface ◽

Expression Patterns ◽

Antigen Expression ◽

T Cell Differentiation ◽

Multiparameter Flow Cytometry ◽

Human T Cell

Abstract Using multidimensional flow cytometry we have defined and quantified the human T-cell differentiation pathway, focusing on those events occurring among the most immature thymocytes and putative bone marrow (BM) T-precursors. Early thymocytes were found to express the CD34 antigen and consisted of a mean 1.2% of cells within human pediatric (n = 9) and 2.0% in fetal thymi (n = 4). All CD34+ thymocytes were atypical blast by morphology, expressed intracytoplasmatic, but not cell surface, CD3, and were cell surface CD2+, CD5+, CD7+, CD38+, CD45+, CD45RA+, CD49d+, and LECAM-1(Leu8)high. CD34high thymocytes lacked surface expression of CD4 and CD8, but as CD34 expression diminished there was a coordinate increase in CD4 levels, followed by the appearance of CD8. The expression of CD1 and CD10 also increased concomitant with the loss of CD34, whereas expression of LECAM-1 diminished with CD34 downregulation. The differential expression of these antigens on early thymocytes (as well as the number of thymocytes displaying these patterns) was highly reproducible among the nine pediatric and four fetal specimens examined, suggesting a precise, stereotyped regulation of early differentiation events. Cell populations with antigen expression patterns suggestive of pluripotent stem cell (CD34high, CD38-), or non-T-lineage committed stem cells (CD34+, CD33+ or CD34+, CD19+) were not identified in either fetal or pediatric thymi (sensitivity = 1/10(4)). The presence of cells with the antigenic profile of the earliest CD34+ thymocytes was explored in human BM. Putative BM T-cell precursors with the appropriate phenotype (CD34+, CD7+, CD5+, CD2+, LECAM-1high) were readily identified in fetal specimens (constituting +/- 2% of the CD34+ population), but could not be reliably detected in adults. In contrast with thymi, only 13% of these cells expressed cytoplasmatic CD3, suggesting the presence of the immediate precursor of the putative prothymocyte population. This was further supported by the detection of CD34bright, CD7+, CD2-, CD5-, LECAM-1moderate cells in fetal specimens. Our results document the flow of cell surface differentiation during T-lymphopoiesis and suggest that T-lineage features are first acquired in the BM. The ability to reproducibly identify and isolate T-cell precursor populations of precisely defined maturational stage in marrow and thymus by multiparameter flow cytometry will facilitate characterization of the molecular events controlling T-lineage differentiation.

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Flow cytometric assessment of human T-cell differentiation in thymus and bone marrow

Blood ◽

10.1182/blood.v79.3.666.bloodjournal793666 ◽

1992 ◽

Vol 79 (3) ◽

pp. 666-677 ◽

Cited By ~ 2

Author(s):

LW Terstappen ◽

S Huang ◽

LJ Picker

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Cell Differentiation ◽

T Cell ◽

Cell Surface ◽

Expression Patterns ◽

Antigen Expression ◽

T Cell Differentiation ◽

Multiparameter Flow Cytometry ◽

Human T Cell

Using multidimensional flow cytometry we have defined and quantified the human T-cell differentiation pathway, focusing on those events occurring among the most immature thymocytes and putative bone marrow (BM) T-precursors. Early thymocytes were found to express the CD34 antigen and consisted of a mean 1.2% of cells within human pediatric (n = 9) and 2.0% in fetal thymi (n = 4). All CD34+ thymocytes were atypical blast by morphology, expressed intracytoplasmatic, but not cell surface, CD3, and were cell surface CD2+, CD5+, CD7+, CD38+, CD45+, CD45RA+, CD49d+, and LECAM-1(Leu8)high. CD34high thymocytes lacked surface expression of CD4 and CD8, but as CD34 expression diminished there was a coordinate increase in CD4 levels, followed by the appearance of CD8. The expression of CD1 and CD10 also increased concomitant with the loss of CD34, whereas expression of LECAM-1 diminished with CD34 downregulation. The differential expression of these antigens on early thymocytes (as well as the number of thymocytes displaying these patterns) was highly reproducible among the nine pediatric and four fetal specimens examined, suggesting a precise, stereotyped regulation of early differentiation events. Cell populations with antigen expression patterns suggestive of pluripotent stem cell (CD34high, CD38-), or non-T-lineage committed stem cells (CD34+, CD33+ or CD34+, CD19+) were not identified in either fetal or pediatric thymi (sensitivity = 1/10(4)). The presence of cells with the antigenic profile of the earliest CD34+ thymocytes was explored in human BM. Putative BM T-cell precursors with the appropriate phenotype (CD34+, CD7+, CD5+, CD2+, LECAM-1high) were readily identified in fetal specimens (constituting +/- 2% of the CD34+ population), but could not be reliably detected in adults. In contrast with thymi, only 13% of these cells expressed cytoplasmatic CD3, suggesting the presence of the immediate precursor of the putative prothymocyte population. This was further supported by the detection of CD34bright, CD7+, CD2-, CD5-, LECAM-1moderate cells in fetal specimens. Our results document the flow of cell surface differentiation during T-lymphopoiesis and suggest that T-lineage features are first acquired in the BM. The ability to reproducibly identify and isolate T-cell precursor populations of precisely defined maturational stage in marrow and thymus by multiparameter flow cytometry will facilitate characterization of the molecular events controlling T-lineage differentiation.

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Aberrant Expression of CD Markers in Acute Myeloid Leukaemia

Haematology Journal of Bangladesh ◽

10.37545/haematoljbd201812 ◽

2018 ◽

Vol 2 (01) ◽

pp. 14-16

Author(s):

Abul Kalam Azad ◽

Md. Rafiquzzaman Khan ◽

ABM Hasan Habib ◽

Md. Abdul Wadud Miah ◽

Masuda Begum

Keyword(s):

Flow Cytometry ◽

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Acute Leukaemia ◽

Antigen Expression ◽

Aberrant Expression ◽

Female Ratio ◽

Cd Antigen ◽

Cd Markers ◽

Acute Myeloid

Background: Aberrant expression of cluster differentiation (CD) antigen marker is associated with poor outcome of acute leukaemia. Objective: Aim of this study is to determine the frequency and pattern of aberrant expression of CD markers in acute myeloid leukaemia patients in Bangladesh. Methods: This retrospective data analysis was conducted in the Department of Haematology, Bangabandhu Sheikh Mujib Medical University (BSMMU) to assess the frequency of aberrant CD antigen expression in acute myeloid leukaemia from October 2016 to September 2017. During this period, we did one hundred flow cytometry of acute leukaemia patients and among them we found 48 acute myeloid Leukaemia (AML) who were included in this study. Result: Mean age of patients was 35 years (SD +14 years; Rang 3 to 50 years) with male: female ratio of 0.92. Four colour flow cytometry was done on fresh bone marrow aspirates and peripheral blood. Among 48 AML patients, aberrant CD expression was observed in 58% cases. CD5 and cCD79a lymphoid markers were seen to be expressed in 32% cases of AML. Aberrant cCD3 and CD7 were expressed in 29% and 25% cases respectively and aberrant CD10, CD19, cCD22 were expressed in 11%, 3%, 3% cases acute myeloid leukaemia patients respectively. Conclusion: Aberrant CD antigen expression is not uncommon in AML patients of Bangladeshi population that may adversely affect the treatment outcome of the disease.

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