Multiparameter Flow Cytometry Provides Independent Prognostic Information in Patients with Suspected Myelodysplastic Syndromes: A Study on 804 Patients

Abstract Abstract 1722 Backgroundn: Multiparameter flow cytometry (MFC) has been demonstrated capable of identifying aberrant antigen expression related to myelodysplastic syndromes (MDS). The exact role and place of MFC in the diagnostic work-up of patients with suspected MDS, however, remains to be defined. Aim: Evaluation of the diagnostic impact of MFC in relation to cytomorphology (CM) and cytogenetic (CG) by determining the association of MFC results to overall survival (OS). Patients and Methods: In 1,013 patients with suspected MDS bone marrow samples had been analyzed in parallel by MFC, CM, and CG. CM confirmed MDS in 511 patients, excluded MDS in 277 patients, and showed dysplastic features but not sufficient to unequivocally diagnose MDS by CM in 225. The MFC diagnostic result was in agreement with MDS (“MDS by MFC”) in 446 patients including 382/511 patients with MDS proven by CM. CG revealed an aberrant karyotype in 245/1,013 patients. The median follow-up time amounted to 14.8 months, a total of 156 deaths was recorded. Results: The first set of analyses was performed on the cohort of 511 patients with MDS confirmed by CM. The median total number of aberrantly expressed antigens amounted to 3 (range, 0–11) and included 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 (SSC) 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. A higher total number of aberrantly expressed antigens as a continuous variable correlated with a shorter OS (Cox analysis, p=0.008). Next, patients were categorized based on the three parameters i) at least 3 aberrantly expressed antigens, ii) significantly reduced SSC in granulocytes, and iii) >5% myeloid progenitor cells in MFC. Patients with at least one of these criteria had a significantly shorter OS than those without (median 48.5 months vs. not reached (n.r.), p<0.001). Overall, the global diagnostic rating of “MDS by MFC” was the strongest MFC parameter: Patients with “MDS by MFC” had a shorter OS as compared to patients without (median 56.8 months vs. n.r., p=0.001). Non-MFC parameters related to OS in univariable Cox analysis included WBC count, thrombocyte count, CG (grouped according to IPSS), % blasts by CM (p<0.001 each), Hb level (p=0.001), and age (p=0.002). In order to determine the clinical relevance of “MDS by MFC” a multivariable analysis for OS was performed on this parameter together with non-MFC parameters (blood counts excluded due to incomplete data sets). It revealed an independent relation between “MDS by MFC” and OS (p=0.045). This was also true for relation of OS to the other parameters (CG, p<0.001; age, p=0.001, % blasts by CM p=0.014). Given this strong prognostic value of “MDS by MFC” in cases with MDS proven by CM a second set of analyses on the relation between MFC findings and OS were performed for the complete cohort of 1,013 patients, i.e. additionally including all cases with a diagnostic result by CM of “no MDS” or “dysplastic features not sufficient to diagnose MDS”. Again, significant relations to OS was found for the total number of aberrantly expressed antigens as a continuous variable (Cox analysis, p<0.001), for at least one of the above mentioned criteria i), ii) or iii) (median 75.6 months vs. n.r., p<0.001), as well as for “MDS by MFC” (median 60.5 months vs. n.r., p<0.001). Again, “MDS by MFC” proved to be the most relevant MFC parameter. Multivariable Cox analysis for OS including “MDS by MFC” and non-MFC parameters revealed a trend only for “MDS by MFC” (p=0.135) and significance for the other parameters (age, p<0.001; CG, p<0.001; blasts by CM, p=0.045). Conclusions: 1) The present data indicates the diagnostic use of MFC for MDS results in independent prognostic information for cases with MDS as proven by CM. 2) Furthermore, the diagnosis of MDS by MFC has a strong prognostic impact even without prove of MDS by CM which strengthens the diagnostic value of MFC even more. 3) This analysis therefore argues in favour of diagnosing MDS not only based on a combination of CM and CG but of adding also MFC for better classification and even prognostication in the future. Disclosures: Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Multiparameter flow cytometry provides independent prognostic information in patients with suspected myelodysplastic syndromes: A study on 804 patients

Cytometry Part B Clinical Cytometry ◽

10.1002/cyto.b.21224 ◽

2015 ◽

Vol 88 (3) ◽

pp. 154-164 ◽

Cited By ~ 15

Author(s):

Wolfgang Kern ◽

Ulrike Bacher ◽

Claudia Haferlach ◽

Tamara Alpermann ◽

Susanne Schnittger ◽

...

Keyword(s):

Flow Cytometry ◽

Myelodysplastic Syndromes ◽

Multiparameter Flow Cytometry ◽

Prognostic Information

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Multiparameter Flow Cytometry Identifies Dysplasia in Granulocytic, Monocytic, and Erythrocytic Lineages and Blasts in Patients with Myelodysplastic Syndromes: Correlation with Cytomorphology and Cytogenetics.

Blood ◽

10.1182/blood.v104.11.2376.2376 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2376-2376

Author(s):

Wolfgang Kern ◽

Daniela Voskova ◽

Claudia Schoch ◽

Wolfgang Hiddemann ◽

Susanne Schnittger ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Myelodysplastic Syndromes ◽

Median Number ◽

Multiparameter Flow Cytometry ◽

Strong Correlations ◽

Trisomy 8 ◽

Prognostic Information ◽

Accurate Identification ◽

Hla Dr

Abstract Myelodysplastic syndromes (MDS) are diagnosed and classified based on cytomorphology (CM) and cytogenetics. The accurate identification of dysmyelopoiesis and quantification of bone marrow blasts requires a high degree experience in CM. Multiparameter flow cytometry (MFC) has been shown capable of identifying dysplastic features in all lineages and to reveal prognostic information in MDS. We analyzed 23 bone marrow samples from patients with untreated MDS by MFC, CM, and cytogenetics in parallel. In addition, four normal bone marrow samples and one bone marrow sample with hereditary sideroachrestic anemia (HSA) were analyzed by MFC for comparison. Diagnoses included RA (n=4), RARS (n=1), RAEB-1 (n=7), RAEB-2 (n=6), 5q- syndrome (n=2), and CMML (n=2). CM revealed dysgranulopoiesis and dyserythropoiesis in 16 and 14 cases, respectively, and blasts counts ranging from 3% to 20% (median, 9%). Karyotype aberrations included 5q- (n=3); trisomy 8 (n=2); del(11q) (n=2); t(3;11) (n=1); loss of chromosome X (n=1); and trisomy X, trisomy 8, and 5q- (n=1); while n=13 cases had normal karyotypes. Blast counts as determined by MFC ranged from 1.9% to 14.8% (median, 4.7%; correlation with CM: r=0.466, p=0.029). The median number of aberrant features detected by MFC were 4 for blasts (range, 2 to 9), 3 for granulocytes (1 to 8), 3 for monocytes (0 to 7), and 0 for erythrocytes (0 to 2). The presence of dysgranulopoiesis (CM) correlated with hypogranulation (MFC; 50% vs. 17%, p>0.05) and expression of HLA-DR (38% vs. 17%, p>0.05) and CD7 (13% vs. 0%, p>0.05) in granulocytes as well as with the lack of CD16 expression (63% vs. 0%, p=0.035), the expression of CD2 (25% vs. 0%, p>0.05), and the dim expression of CD66 (63% vs. 40%, p>0.05) on monocytes. Combining these parameters led to strong correlations between dysgranulopoiesis (CM) and at least one dysplastic feature by MFC for granulocytes (75% vs. 17%, p=0.023) and monocytes (81% vs. 33%, p=0.054). Combining the granulocytic and the monocytic MFC scores revealed dysplastic features in 100% cases with dysgranulopoiesis (CM) and in 50% without (p=0.013). No clear-cut correlations were found between dyserythropoiesis (CM) and dysplastic features in erythroid cells as detected by MFC. The most striking MFC features of cases with 5q- syndrome included CD2-positivity (100% vs. 6%, p=0.003) and CD34-positivity (100% vs 28%, p=0.042) in monocytes as well as hypogranulation (100% vs. 32%, p=0.055) and HLA-DR-positivity (100% vs 21%, p=0.023) in granulocytes. Dysplastic features that were detected by MFC were related to each other. Thus, in blasts the expression of CD56 was strongly related to the expression of CD64 (p=0.0003) and the dim expression of CD45 (p=0.0003). In addition, the expression of CD11b was associated with the expression of CD15 (p=0.001). In granulocytes, the lack of CD33 was associated with a lack of CD64 expression (p=0.0004). In monocytes, the dim expression of CD45 was associated with a lack of CD14 expression (p=0.0005). The case with HSA displayed an aberrant pattern of CD71 expression. These data suggest that MFC is capable of reproducibly detecting dysplastic features in different compartments of the bone marrow. Strong correlations between CM and cytogenetic finding and MFC are detectable. Future studies will define the role of MFC in optimizing the diagnosis of MDS and the prognostication in patients with MDS.

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Application of Multiparameter Flow Cytometry for the Identification of Dysplasia in Granulocytic, Monocytic, and Erythrocytic Lineages and Blasts in Patients with Myelodysplastic Syndromes.

Blood ◽

10.1182/blood.v108.11.2615.2615 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2615-2615

Author(s):

Wolfgang Kern ◽

Claudia Schoch ◽

Susanne Schnittger ◽

Torsten Haferlach

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Myelodysplastic Syndromes ◽

Erythroid Cell ◽

Multiparameter Flow Cytometry ◽

Cell Populations ◽

Aberrant Expression ◽

Hla Dr ◽

Cd16 Expression

The diagnosis and classification of myelodysplastic syndromes (MDS) are based on cytomorphology (CM) and cytogenetics. A high degree of experience in CM is required to allow the accurate identification of dysmyelopoiesis and quantification of bone marrow blasts. The identification of dysplastic features in all lineages by multiparameter flow cytometry (MFC) has been shown feasible. To further analyze the potential role of MFC in the diagnostic work-up of MDS we analyzed 224 bone marrow samples from patients with suspected of proven MDS by MFC, CM, and cytogenetics in parallel. Blast counts as determined by CM and MFC, respectively, ranged from 0% to 21% (median, 5%) and from 0% to 33% (median, 4%; correlation: r=0.192, p=0.018). The median number of aberrant features detected by MFC were 0 for blasts (range, 0 to 4), 2 for granulocytes (0 to 7), 1 for monocytes (0 to 5), and 0 for erythrocytes (0 to 2). The most frequent dysplastic features observed in the blast populations included aberrant coexpression of CD11b (20.5%), CD15 (14.3%) and CD64 (14.3%). The most frequent dysplastic features observed in the granulocytic cell populations included reduced side-scatter signal corresponding to hypogranulation (71.4%), aberrant coexpression of CD56 (29.0%), aberrant pattern of CD13/CD16 expression (26.3%), aberrant pattern of CD11b/CD16 expression (25.9%), reduced expression of CD64 (17.0%), and aberrant expression of HLA-DR (14.7%). The most frequent dysplastic features observed in the monocytic cell populations included aberrant coexpression of CD56 (31.3%), aberrant coexpression of CD16 (26.3%), an aberrant pattern of CD11b/HLA-DR expression (6.7%), and aberrant coexpression of CD2 (5.8%). The most frequent dysplastic features observed in the erythroid cell populations included an aberrantly strong expression of CD71 and CD235a (23.7%), a lack of CD71 expression (10.7%), and an aberratly homogeneous expression of CD71 (7.1%). The presence of dysplastic features by CM as well as the presence of cytogenetic aberrations tended to be associated with a higher number of dysplastic features by MFC. These data suggest that the identification of dysplastic features by MFC is feasible although there is a large heterogeneity in aberrantly expressed antigens. Thus, a comprehensive panel of antibodies must be applied to allow the detection of dysplasia. Future studies will define the role of MFC in optimizing the diagnosis of MDS in cooperation with CM and cytogenetics.

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(D8) Multiparameter flow cytometry in patients with myelodysplastic syndromes

Leukemia Research ◽

10.1016/0145-2126(91)90358-z ◽

1991 ◽

Vol 15 ◽

pp. 9

Author(s):

Inger Marie Jensen ◽

Jergen Ellegaard ◽

Marianne Hokland ◽

Peter Hokland

Keyword(s):

Flow Cytometry ◽

Myelodysplastic Syndromes ◽

Multiparameter Flow Cytometry

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Detection of Dysplastic Features by Multiparameter Flow Cytometry in Myelodysplastic Syndromes in Relation to Cytomorphology and Cytogenetics.

Blood ◽

10.1182/blood.v110.11.2461.2461 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2461-2461

Author(s):

Wolfgang Kern ◽

Claudia Haferlach ◽

Susanne Schnittger ◽

Torsten Haferlach

Keyword(s):

Flow Cytometry ◽

Myelodysplastic Syndromes ◽

Erythroid Cell ◽

Multiparameter Flow Cytometry ◽

Cell Populations ◽

Erythroid Cells ◽

Monocytic Cell ◽

Aberrant Expression ◽

Cell Lineages ◽

Cd16 Expression

Abstract Dysplastic features can be detected in different cell lineages in myelodysplastic syndromes (MDS) by multiparameter flow cytometry (MFC). The aim of the present study has been the assessment of the flow cytometric detection of dysplastic features previously published to occur in MDS in relation to findings in cytomorphology (CM) and cytogenetics (CG). We analyzed 307 bone marrow samples from patients with suspected (n=130) or proven (n=177) MDS by MFC, CM, and CG in parallel. Blast counts as determined by CM and MFC, respectively, ranged from 0% to 21% (median, 3.5%) and from 0% to 23% (median, 3%; r=0.271, p<0.0001). The median number of aberrant features detected by MFC were 0 for blasts (range, 0 to 4), 2 for granulocytes (0 to 5), 1 for monocytes (0 to 5), and 0 for erythroid cells (0 to 2); median total number=3, range 0–11. The most frequent dysplastic features observed in the blast populations included aberrant coexpression of CD11b (13.7%), CD15 (10.4%) and CD64 (10.4%). The most frequent dysplastic features observed in the granulocytic cell populations included reduced side-scatter signal corresponding to hypogranulation (67.1%), aberrant coexpression of CD56 (32.9%), aberrant pattern of CD13/CD16 expression (31.6%), aberrant pattern of CD11b/CD16 expression (24.1%), and reduced expression of CD33 (13.4%). The most frequent dysplastic features observed in the monocytic cell populations included aberrant coexpression of CD56 (42.7%) and of CD16 (18.9%). The most frequent dysplastic features observed in the erythroid cell populations included a lack of CD71 expression (15.0%) and an aberrantly homogeneous expression of CD71 (9.1%). As compared to cases with no indication of MDS by CM (=non-MDS) cases with MDS according to CM were significantly associated with a reduced side-scatter signal in granulocytes (ratio granulocytes:lymphocytes 6.53±1.27 vs. 7.44±1.17, p<0.0001) as well as a higher number of dysplastic features in granulocytes (1.98±1.09 vs. 1.00±1.31, p<0.0001), monocytes (0.81±0.84 vs. 0.35±0.63, p<0.0001), and erythroid cells (0.33±0.47 vs. 0.20±0.40, p=0.061). Particularly, an aberrant expression of CD56 in monocytes occurred more frequently in 33 cases with CMML as compared to non-MDS cases (84.8% vs. 15.7%, p<0.0001). In cases with possible MDS according to CM the differences to non-MDS cases were less pronounced (reduced side-scatter signal in granulocytes 7.77±1.47 vs. 7.44±1.17, n.s., dysplastic features in granulocytes 1.90±1.21 vs. 1.00±1.31, p=0.003, monocytes 0.50±0.63 vs. 0.35±0.63, n.s., and erythroid cells 0.30±0.47 vs. 0.20±0.40, n.s.). In cases with aberrant cytogenetics (n=80, excluding those with –Y as sole aberration) dysplastic features by MFC occurred more frequently as compared to cases with normal karyotypes (reduced side-scatter signal in granulocytes 6.35±1.18 vs. 7.09±1.38, p<0.0001, dysplastic features in granulocytes 2.05±0.95 vs. 1.68±1.31, p=0.021, monocytes 0.80±0.89 vs. 0.71±0.81, n.s., and erythroid cells 0.38±0.49 vs. 0.31±0.48, n.s.). A total of more than two dysplastic features in blasts, granulocytes, and monocytes and/or a blast count >5% occurred in 68.4%, 63.3%, and 35.3% of cases with MDS, possible MDS, and non-MDS according to CM (p=0.0005). Interestingly, in some cases aberrant antigen expression has been observed in cell lineages not rated dysplastic by CM. This evaluation suggests that MFC may be used to identify dysplastic features in patients with suspected MDS. Sensitivity and specificity varies between MDS subtypes and should be clearly defined in future studies.

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A quantitative evaluation of erythropoiesis in myelodysplastic syndromes using multiparameter flow cytometry

Leukemia Research ◽

10.1016/0145-2126(93)90149-f ◽

1993 ◽

Vol 17 (10) ◽

pp. 839-846 ◽

Cited By ~ 9

Author(s):

Inger Marie Jensen ◽

Marianne Hokland ◽

Peter Hokland

Keyword(s):

Flow Cytometry ◽

Quantitative Evaluation ◽

Myelodysplastic Syndromes ◽

Multiparameter Flow Cytometry

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Multiparameter flow cytometry for assessment of minimal residual disease in patients with myelodysplastic syndromes treated with allogeneic stem cell transplantation

Acta Haematologica Polonica ◽

10.2478/ahp-2020-0017 ◽

2020 ◽

Vol 51 (2) ◽

pp. 88-94

Author(s):

Katarzyna Wiśniewska-Piąty ◽

Joanna Dziaczkowska-Suszek ◽

Agata Wieczorkiewicz-Kabut ◽

Karolina Chromik ◽

Anna Koclęga ◽

...

Keyword(s):

Flow Cytometry ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Minimal Residual Disease ◽

Cell Transplantation ◽

Allogeneic Stem Cell Transplantation ◽

Myelodysplastic Syndromes ◽

Residual Disease ◽

Multiparameter Flow Cytometry ◽

Minimal Residual

AbstractBackgroundMyelodysplastic syndromes (MDSs) are a heterogeneous group of clonal myeloid neoplasms. Allogeneic stem cell transplantation (allo-SCT) remains the curative method for MDS treatment. Little is known about the monitoring of minimal residual disease (MRD) in patients with MDS after allo-SCT.AimWe aimed to evaluate the significance of leukemia-associated immunophenotypes (LAIPs) identified in acute myeloid leukemia (AML) for MRD monitoring in patients with MDS after allo-SCT.Material and methodsSeven males and 4 females with a median age of 55 years were included. The diagnosis of MDS was established according to 2016 World Health Organization (WHO) criteria. The significance of eight LAIPs in bone marrow samples using multiparameter flow cytometry (MFC) was evaluated for MRD.ResultsEight patients were positive for several LAIPs before allo-SCT. The identified LAIPs included the presence of aberrant lymphoid antigens on myeloblasts and lack of CD33 expression on myeloblasts. All studied MDS patients were negative for LAIPs at Day +30 after the procedure. This was followed by full-donor chimerism in all cases. The Ogata score after allo-SCT decreased in all patients in whom it was indicative for MDS before allo-SCT.ConclusionsMFC could be useful in monitoring MRD in MDS patients after allo-SCT. Further studies in this field are needed.

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Determination of relapse risk based on assessment of minimal residual disease during complete remission by multiparameter flow cytometry in unselected patients with acute myeloid leukemia

Blood ◽

10.1182/blood-2004-03-1036 ◽

2004 ◽

Vol 104 (10) ◽

pp. 3078-3085 ◽

Cited By ~ 176

Author(s):

Wolfgang Kern ◽

Daniela Voskova ◽

Claudia Schoch ◽

Wolfgang Hiddemann ◽

Susanne Schnittger ◽

...

Keyword(s):

Flow Cytometry ◽

Acute Myeloid Leukemia ◽

Complete Remission ◽

Myeloid Leukemia ◽

Residual Disease ◽

Multiparameter Flow Cytometry ◽

Consolidation Therapy ◽

Prognostic Information ◽

Minimal Residual ◽

Acute Myeloid

Abstract Quantification of minimal residual disease (MRD) reveals significant prognostic information in patients treated for acute myeloid leukemia (AML). The application of multiparameter flow cytometry (MFC) for MRD assessment has resulted in significant prognostic information in selected cases in previous analyses. We analyzed MRD in unselected patients with AML in complete remission (CR) after induction (n = 58) and consolidation (n = 62) therapies. By using a comprehensive panel of monoclonal antibodies we identified at least one leukemia-associated aberrant immunophenotype (LAIP) in each patient. The degree of reduction between diagnosis and CR in LAIP-positive cells (log difference [LD]) as a continuous variable was significantly related to relapse-free survival (RFS) both after induction (P = .0001) and consolidation (P = .000 08) therapies, respectively. The LD determined after consolidation therapy was the only parameter related to overall survival (OS) (P = .005). Separation of patients based on the 75th percentile of LD after consolidation therapy resulted in groups with highly different RFS (83.3% versus 25.7%, P = .0034) and OS (87.5% versus 51.4%, P = .0507) at 2 years. Multivariate analysis identified LD as an independent prognostic factor for RFS at both checkpoints. MFC-based quantification of MRD reveals important prognostic information in unselected patients with AML in addition to cytogenetics and should be further evaluated and used in clinical trials.

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