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Normal and abnormal maturation patterns in myeloid cells, myeloid neoplasms Wolfgang Kern MLL Munich Leukemia Laboratory www.mll.com.

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Presentation on theme: "Normal and abnormal maturation patterns in myeloid cells, myeloid neoplasms Wolfgang Kern MLL Munich Leukemia Laboratory www.mll.com."— Presentation transcript:

1 Normal and abnormal maturation patterns in myeloid cells, myeloid neoplasms Wolfgang Kern MLL Munich Leukemia Laboratory

2 Antigen expression in myelopoiesis Neutrophil Maturation Antigen Density CD16 CD11b CD33 CD15 CD45 CD34 CD117 CD13 HLA-DR

3 CD34 CD117 CD34/CD117

4 CD36 CD34 CD34/CD36

5 HLA-DR CD34 CD34/HLA-DR

6 CD34 CD13 CD34/CD13

7 CD34 CD33 CD34/CD33

8 CD117 CD38 CD38/CD117

9 CD36 CD38 CD38/CD36

10 CD14 CD33 CD33/CD14

11 CD34 CD14 CD14/CD34

12 CD11b CD34 CD34/CD11b

13 HLA-DR CD11b CD11b/HLA-DR

14 CD15 CD34 CD34/CD15

15 Multiparametric flow cytometry of normal human bone marrow: analysis and display strategies GEIL-GTLLF 2008 Part one : Leukocyte subsets A colour code is applied trhoughout this atlas: Granulocytes in red Monocytes in green Lymphocytes in purple All other cells, in a region of maturation defined by the exclusion of mature cell types and thus dubbed « bermudes » in cyan I.1

16 CD11b/CD16 I.6 CD45APC FSC-Height CD11bFITC SSC-Height CD16PC7 FLx FLy FLx FLy CD45 SSC FSC SSC FLx FLy FLx FLy

17 CD11b/CD117 I.9 CD45APC FSC-Height CD45APC CD11bFITC SSC-Height CD117PE FLx FLy FLx FLy CD45 SSC FSC SSC FLx FLy FLx FLy

18 ELN website:

19 Identification of cell compartments by CD45-SSC

20 CD11b/CD16 expression pattern in granulocytes

21 CD13/CD16 expression pattern in granulocytes

22 CD11b/CD13 expression pattern in granulocytes

23 CD56 expression in granulocytes

24 SSC signal in granulocytes

25 CD2 expression in monocytes

26 CD4/CD14 expression in monocytes

27 CD56 expression in monocytes

28 CD13/CD11b expression in monocytes

29 HLA-DR/CD11b expression in monocytes

30 Indications for immunophenotyping Consensus: Davis et al. Cytometry Part B 2007;72B:S5-S13 Indicationen: Clinical signs Cytopenias Leukocytosis Atypical cells / blasts, evaluation of body fluids Plasmacytosis / monoclonal gammopathy Organomegaly / tissue masses Monitoring No indication: Neutrophilia Polyclonal hypergammaglobulinemia Polycythemia Thrombocytosis Basophilia

31 Diagnosis in AML Diagnosis and subclassification of AML is based on: Cytomorphology and cytochemistry Cytogenetics/FISH Molecular genetics Immunophenotyping Immunophenotyping for diagnosis of AML: AML M7 AML M0 BAL Immunophenotyping for subclassification of AML: Hint to genetic abnormalities t(15;17), t(8;21), inv(16)

32 Diagnosis in AML Definition of AML M0: positive for myeloid Antigens negative for lymphatic Antigens

33 Diagnosis in AML Definition of AML M7: positive for CD41 positive for CD61

34 Diagnosis in AML CD7+CD33+MPO+LF-TdT+cyCD3+ Biphenotypic acute leukemia Mixed phenotype acute leukemia

35 Typical findings in APL: characteristic SSC/FSC-pattern high auto-fluorescence CD33+/HLA-DR- AML M3 Normal BMAML M2 Subclassification in AML

36 Typical findings in AML with t(8;21): Coexpression of CD19Coexpression of CD56

37 Subclassification in AML Typical findings in AML with inv(16): Coexpression of CD65 and CD34Coexpression of CD2

38 Differential using CD45-SSC-Gate Immunophenotyping 11% blasts Cytomorphology 8% blasts

39 Differential using CD45-SSC-Gate Immunophenotyping 27% blasts Cytomorphology 27% blasts

40 Differential using CD45-SSC-Gate Cytomorphology 82% blasts Immunophenotyping 88% blasts

41 Differential using CD45-SSC-Gate Immunophenotyping 6% blasts Cytomorphology 18% blasts

42 Differential using CD45-SSC-Gate Cytomorphology 18% blasts Immunophenotyping 14% monocytic cells Immunophenotyping 6% blasts

43 Background Prognostic factors in AML Pre-therapeutic parameters: Karyotype, molecular genetics, age, sAML Heterogeneous prognosis within defined groups Prognosis dependent on therapy Therapy-dependent prognostic parameters

44 Monitoring of minimal residual disease (MRD) CD34 CD33 CD56 Diagnosis Day 0 After 1 st induction Day 18 After 2 nd induction Day 68 After alloTx Day 100

45 Antibody panel FITCPEPC5 CD34CD2CD33 CD7CD33CD34 CD34CD56CD33 CD11bCD117CD34 CD64CD4CD45 CD34CD13CD19 CD65CD87CD34 CD15CD34CD33 HLA-DRCD33CD34 CD4CD13CD14 CD34CD135CD117 CD34CD116CD33 FITCPEPC5 CD90CD117CD34 CD347.1CD33 CD38CD133CD34 CD61CD14CD45 CD36CD235aCD45 CD15CD13CD33 TdTCD33CD45 MPOLFCD15 TdTCD22CD3 TdTCD79aCD3

46 LAIP+ cells in normal bone marrow Kern et al. Haematologica 2003;88: n Normal BM, analyzed samples total26 per LAIP (median, range)24, Analyses, total2863 Median frequency of LAIP+ cells in normal BMmedian (range) all LAIP (n=140)0.07% (0.00%-1.20%) only 1 LAIP per patient (n=68)0.05% (0.00%-0.43%)

47 LAIP+ cells in normal and leukemic bone marrow Kern et al. Haematologica 2003;88: Frequency of LAIP+ cells in AML-BMmedian (range) all LAIP (n=140)25.10% (10.13%-76.14%) only 1 LAIP per patient (n=68)25.81% (10.13%-76.14%) log-difference of LAIP+ cells (normal BM / AML) median (range) all LAIP (n=140)2.47 ( ) only 1 LAIP per patient (n=68)2.82 ( )

48 Serial dilution of AML cells in normal BM Kern et al. Haematologica 2003;88: E % calculated % measured CD34+CD56+CD33+ cells

49 Day 16 blasts by cytomorphology Kern et al. Blood 2003;101:64-70

50 Day 16 MRD—Detection of cytoreduction % bone marrow blasts % LAIP+ cells in bone marrow day 1day 16 A B Cytomorphology Multiparameter flow cytometry Kern et al., Haematologica 2004;89(5):

51 Day 16 MRD—Detection of cytoreduction % bone marrow blasts % LAIP+ cells in bone marrow day 1day 16 A B Cytomorphology Multiparameter flow cytometry Kern et al., Haematologica 2004;89(5):

52 Day 16 MRD—Multivariate analysis Kern et al., Haematologica 2004;89(5): ParameterCREFSRFSOS LD day 16p n.s. RR Favorable karyotypepn.s.0.044n.s.n.s. RR0.289 Unfavorable karyotypep n.s RR

53 Day 16 MRD—Relapse-free survival Kern et al., Haematologica 2004;89(5): days

54 Prognostic impact of MRD after induction Separation according to 25-percentile Log-difference (=1.70) LD >25%ile: median EFS 12.0 mos. LD <25%ile: median EFS 3.8 mos. p= days RFS Kern et al., Blood 2004;104(10):

55 Prognostic impact of MRD after consolidation Separation according to 75-percentile Log-difference (=2.94) RFS days LD >75%ile: 2-year-EFS 83.3% LD <75%ile: 2-year-EFS 25.7% p= Kern et al., Blood 2004;104(10):

56 MRD after induction and after consolidation (multivariate) Kern et al., Blood 2004;104(10): MRD (ind.)(cons.) ParameterRFSOSRFSOS LDp0.006n.s RR Unfavorable karyotypep0.0001n.s.0.006n.s. RR

57 MRD assessment, extended cohort Patients Patients (n)2863y-OS54% MRD assessments (n)550EFS, median14.5 M. Standard therapy LAIP+ BM cells at Dx16.04% (2.54%-76.14%) LAIP+ cells normal BM0.02% (0.00%-1.01%) Follow-up assessments n Log-difference (median) Up to day Day 29 to day Day 61 to day Day 121 to day After day Kern et al., ASH 2005

58 Prognostic impact of MRD EFS3y-OS MedianpMonthsp (Months) Up to day vs % vs. 56%0.035 Day 29 to day vs. 9.3< % vs. 42%<0.001 Day 61 to day vs. 13.5< % vs. 63%0.011 Day 121 to day vs. 13.7< % vs. 65%<0.001 After day 365n.r. vs n.s. Median Log-difference diagnosis  MRD-assessment as separator Kern et al., ASH 2005

59 Prognostic impact of MRD levels day 121 to day 365 Kern et al., ASH 2005 RFS OS median 57.1 vs % vs. 65% at 3 yearsp<0.001

60 Prognostic impact of MRD (multivariable) Kern et al., ASH 2005 EFS3y-OS RRpRRp Up to day n.s. Day 29 to day < Day 61 to day n.s. Day 121 to day < <0.001 After day <0.001n.s.

61 Impact of MRD levels on RFS in cytogenetic subgroups Kern et al., ASH days p LD <2.53: 37% at 2 years LD >2.53: 80% at 2 years p= CG = days p LD <2.53: 25% at 2 years LD >2.53: 75% at 2 years p= CG = days p LD <2.53: 0% at 2 y. LD >2.53: 88% at 2 y. p= CG = 3 favorableintermediateunfavorable

62 A B Improvement of MRD assessment by CD45-SSC-gating Kern et al., Crit Rev Oncol Hematol 2005;56:

63 Improvement of MRD assessment by CD45-SSC-gating Kern et al., Hematol J 2004;5: AML without CD45 gating AML with CD45 gating Normal BM without CD45 gating Normal BM with CD45 gating 0.002% 0.511% % %

64 Impact of CD45-gating on sensitivity/specificity Kern et al., Hematol J 2004;5: without CD45 gatingwith CD45 gating LAIP+LAIP+LDLAIP+LAIP+LD AMLnormal BMAMLnormal BM Median20.86%0.15% %0.02%3.07 Min2.33%0.02% %0.01%1.22 Max82.52%0.58% %0.42%4.01

65 Improvement of MRD assessment by 5-color-staining Voskova et al., Leuk Lymphoma 2007;48(1):80-88 FITCPEECDPC5PC7 CD64CD87CD4CD56CD45 CD65CD2CD34CD13CD45 CD9HLA-DRCD34CD33CD45 CD11bCD116CD34CD117CD45 CD34CD56CD19CD33CD45 CD15CD7CD34CD33CD45 CD36CD61CD14CD235aCD45 CD47.1CD14CD13CD45 CD38CD135CD34CD90CD45 CD15CD133CD34CD117CD45 MPOLFCD34CD33CD45 TdTCD22CD3CD79aCD45

66 Improvement of MRD assessment by 5-color-staining % 0.004% CD45-PC7SSC CD15-FITCCD34-ECD SSC FSC CD7-PE SSC CD33-PC5 CD45-PC7SSC CD15-FITCCD34-ECD SSC FSC CD7-PE SSC CD33-PC5 Voskova et al., Leuk Lymphoma 2007;48(1):80-88

67 Impact of 5-color analysis Voskova et al., Leuk Lymphoma 2007;48(1): color5-colorn=139 LAIP+LAIP+LDLAIP+LAIP+LD AMLnormal BMAMLnormal BM Median19.09%0.030% %0.003%3.66 Min1.90%0.001% %0.001%1.98 Max84.83%3.600% %0.040%4.89

68 Course of MRD using 5-color analysis

69 Stability of LAIP between diagnosis and relapse A DC B Voskova et al., Clin Cytometry 2004;62B:25-38

70 MRD assessment by multiparameter flow cytometry in AML 1.Applicable to the vast majority of patients 2.Prognostic information in addition to cytogenetics 3.MRD useful as stratification parameter in clinical trials 4.Improvements of method by CD45-gating and 5-color-staining 5.Further assessment and standardization needed

71 AML with limited differentiation (AML-LD) Kern et al., Leukemia 2009;23:

72 AML with limited differentiation (AML-LD) Kern et al., Leukemia 2009;23:

73 AML with limited differentiation (AML-LD) Kern et al., Leukemia 2009;23:

74 Patient cohort of present GEP study n AML-LD27 AML with NPM1 type A mutation and without cytogenetic abnormalities, no AML-LD immunophenotype (NPM1-A)24 AML with NPM1 mutation other than type A and without cytogenetic abnormalities, no AML-LD immunophenotype (NPM1-other)12 AML without NPM1 mutation and with normal karyotype, no AML-LD immunophenotype (AML-NK)30 Acute promyelocytic leukemia (APL)15

75 Cluster analysis, four groups (excluding APL).. AML-LD

76 Cluster analysis, five groups (including APL) AML-LD APL

77 Diagnosis of BAL BAL: Score >2 for myeloid and B- or T-lymphatic

78 Diagnosis of MPAL

79 Immunophenotyping in acute leukemias Determination of cell size and heterogeneity Analysis of expression of multiple antigens on one cell Characterization of cell populations by antigen expression pattern Quantification of cell populations

80 Definition of MDS Group of myeloid neoplasms Bone marrow failure with peripheral cytopenia Morphologic dysplasia in one or more of the following hematopoietic cell lineages: (i)erythroid cells (also ringed sideroblasts >15% considered diagnostic) (ii)neutrophils and their precursors (iii)megakaryocytes

81 Prognosis in MDS Points % bone marrow blasts Karyotypefavorableintermediateunfavorable Cytopenias0/12/3 Karyotype favorable: normal, -Y, del(5q), del(20q) Karyotype unfavorable: complex aberrant (≥3 aberrations), aberrations of chromosome 7 Points00.5 to to 2.0≥2.5 Risk groupLowInt-1Int-2High

82 Minimal diagnostic criteria in MDS (A) Prerequisite criteria Constant cytopenia in one or more of the following cell lineages: erythroid (hemoglobin <11 g/dl) or neutrophilic (ANC < 1,500/µl) or megakaryocytic (platelets <100,000/µl) Exclusion of all other hematopoietic or non-hematopoietic disorders as primary reason for cytopenia/dysplasia (B) MDS-related (decisive) criteria Dysplasia in ≥10% of all cells in one of the following lineages in bone marrow smear: erythroid or neutrophilic or megakaryocytic or >15% ringed sideroblasts (iron stain) 5–19% Blast cells in bone marrow smears Typical chromosomal abnormality (by conventional karyotyping or FISH) MDS: both (A) criteria and one (B) criterion Valent et al., Leuk Res 2007;31:

83 Minimal diagnostic criteria in MDS (A) Prerequisite criteria Constant cytopenia in one or more of the following cell lineages: erythroid (hemoglobin <11 g/dl) or neutrophilic (ANC <1,500/µl) or megakaryocytic (platelets <100,000/µl) Exclusion of all other hematopoietic or non-hematopoietic disorders as primary reason for cytopenia/dysplasia (C) Co-criteria Abnormal phenotype of bone marrow cells clearly indicative of a monoclonal population of erythroid or/and myeloid cells, determined by flow cytometry Clear molecular signs of a monoclonal cell population in HUMARA assay, gene chip profiling, or point mutation analysis (e.g. RAS mutations) Markedly and persistently reduced colony-formation (±cluster formation) of bone marrow or/and circulating progenitor cells (CFU-assay) Highly suspective of MDS: both (A) criteria and one (C) criterion Valent et al., Leuk Res 2007;31:

84 Idiopathic cytopenia of uncertain significance (ICUS) (A) Definition Cytopenia in one or more of the following cell lineages (for ≥6months): erythroid (Hb <11 g/dl) neutrophilic (<1,500/µl) platelet (<100,000/µl) MDS excluded (see ‘B’ and ‘C’) All other causes of cytopenia also excluded (see ‘B’ and ‘C’) (B) Initial investigations required to establish the diagnosis of ICUS Detailed case history (toxins, drugs, mutagenic events, etc.) Thorough clinical investigations including X-ray and sonography of spleen Differential blood count (microscopic) and complete serum chemistry Bone marrow histology and immunohistochemistry Bone marrow smear including an iron stain Flow cytometry of bone marrow and peripheral blood cells Chromosome analysis including FISH Molecular analysis where appropriate (e.g. T cell receptor rearrangement— neutropenia) Exclusion of viral infections (HCV, HIV, CMV, EBV, others) (C) Recommended investigations in the follow-up Blood count and differential count as well as serum chemistry (1–6 months) Suspicion for MDS becomes evident: bone marrow examination Valent et al., Leuk Res 2007;31:

85 ELN working conference Arjan A van de Loosdrecht, Canan Alhan, Marie Christine Béné, Matteo G Della Porta, Angelika M Dräger, Jean Feuillard, Patricia Font, Ulrich Germing, Detlef Haase, Christa H Homburg, Robin Ireland, Joop H Jansen, Wolfgang Kern, Luca Malcovati, Jeroen G te Marvelde, Gulham J Mufti, Kiyoyuki Ogata, Alberto Orfao, Gert J Ossenkoppele, Anna Porwit, Frank W Preijers, Steve Richards, Gerrit Jan Schuurhuis, Dolores Subirá, Peter Valent, Vincent HJ van den Velden, August H Westra, Theo M de Witte, Denise A Wells, Michael Loken, Theresia M Westers Amsterdam, March 27/ Munich, October 29/ London, November 5/ Pavia, November 4/5 2011

86 Evaluation of MFC in MDS Wells et al. Blood pts. with MDS, 104 pts. with various disorders, 25 healthy donors Van de Loosdrecht et al. Blood pts. with MDS, 15 healthy volunteers, 3 pts. undergoing surgery Kern et al. Cancer pts. with suspected MDS

87 Parameters scored as aberrant in immature compartment van de Loosdrecht et al., Haematologica 2009;94:

88 Quantification of myeloblasts Cytomorphology vs. MFC Mean 4.67±4.18 vs. 3.78±2.97, r=0.362, p<0.001 Kern et al., Cancer 2010

89 Differential using CD45-SSC-Gate Immunophenotyping 6% blasts Cytomorphology 18% blasts Kern et al., Cancer 2010

90 Differential using CD45-SSC-Gate Cytomorphology 18% blasts Immunophenotyping 14% monocytic cells Immunophenotyping 6% blasts Kern et al., Cancer 2010

91 Parameters in maturing myeloid and monocytic compartment van de Loosdrecht et al., Haematologica 2009;94:

92 CD13/CD16 expression pattern in granulocytes Normal BM MDS Kern et al., Cancer 2010

93 CD11b/CD16 expression pattern in granulocytes Normal BM MDS Kern et al., Cancer 2010

94 Aberrant antigen expression in granulocytes MFC findingsCytomorphologic findingsp-value No MDS (n=277) MDS (n=511) Suspected MDS (n=225) Abnormal CD13/CD16 25 (9.0%)219 (42.9%)54 (24.0%)<0.001 Abnormal CD11b/CD16 9 (3.2%)143 (28.0%)25 (11.1%)<0.001 CD56+10 (3.6%)90 (17.6%)23 (10.2%)<0.001 CD33-18 (6.5%)53 (10.4%)19 (8.4%)n.s. CD (2.7%)8 (3.6%)0.011 # of aberrant antigens 0.0±0.2 1,2 0.2± ± < Reduced SSC signal14 (5.1%)286 (56.0%)42 (18.7%)<0.001 SSC-ratio G:L (mean±SD) 7.47±1.09 1,2 6.55± ± < n.s. Kern et al., Cancer 2010

95 Aberrant antigen expression in granulocytes 406 cases without dysgranulopoiesis by cytomorphology aberrant CD13/CD16 expression pattern104 (25.6%) aberrant CD11b/CD16 expression pattern62 (15.3%) CD56 expression38 (9.4%) lack of CD33 expression44 (10.8%) lack of CD64 expression2 (0.5%) Aberrant expression ≥2 antigens RA16/31 (51.6%) RARS15/27 (55.6%) Kern et al., Cancer 2010

96 Parameters scored as aberrant in erythroid compartment van de Loosdrecht et al., Haematologica 2009;94:

97 Proposed marker combinations van de Loosdrecht et al., Haematologica 2009;94:

98 Example of a screening panel for 4-color floy cytometry van de Loosdrecht et al., Haematologica 2009;94:

99 MDS 10 color panel Blast/Granulocyte Tube Monocyte/Erythroid Tube Lymphatic/Granulocyte Tube FITCCD14CD71CD7 PECD13CD2CD10 ECDCD38CD64CD8 PC5.5CD123CD56CD5 PC7CD117 CD4 APCCD11bCD36CD3 APC-Alexa Fluor 700CD34 APC-Alexa Fluor 750CD33 CD19 Pacific BlueCD16HLA-DRCD15 Krome OrangeCD45

100 List of pathological controls to determine the specificity van de Loosdrecht et al., Haematologica 2009;94:

101 Recommended minimal requirements to assess MDS by MFC Westers et al., Leukemia 2012 BONE MARROW SUBSETRECOMMENDED ANALYSES Erythroid compartment*% of nucleated erythroid cells relation CD71 and CD235a expression of CD71 expression of CD36 expression of CD117 Immature myeloid and monocytic progenitors% of cells in nucleated cell fraction**; expression of CD45; expression of CD34; expression of CD117; expression of HLA-DR; expression of CD13 and CD33; asynchronous expression of CD11b, CD15; expression of CD5, CD7, CD19, CD56***; Maturing neutrophils% of cells as ratio to lymphocytes SSC as ratio vs. SSC of lymphocytes relation of CD13 and CD11b relation of CD13 and CD16 relation CD15 and CD10 Monocytes% of cells as ratio to lymphocytes relation of HLA-DR and CD11b relation of CD36 and CD14 expression of CD13 and CD33; expression of CD56*** Progenitor B cellsenumeration as fraction of total CD34+ based on CD45/CD34/SSC in combination with CD10 or CD19

102 Diagnostic results in MFC and cytomorphology 1,013 patients with cytopenias and suspected MDS analyzed Non-MDS malignancies excluded MFCCytomorphology MDSno MDSsuspected MDS MDS382 (74.8%)13 (4.7%)51 (22.7%) no MDS129 (25.2%)264 (95.3%)174 (77.3%) Total511 (100%)277 (100%)225 (100%) Overall concordance646/788 (82.0%) Kern et al., Cancer 2010

103 Numbers of aberrantly expressed antigens Kern et al., Cancer 2010 Cytomorphology: no MDS Cytomorphology: suspected MDS Cytomorphology: MDS

104 Diagnostic results in MFC and cytogenetics MFCCytogenetics aberrant karyotypenormal karyotype MDS189 (77.1%)257 (33.5%) no MDS56 (22.9%)511 (66.5%) Total245 (100%)768 (100%) Kern et al., Cancer 2010

105 Results in MFC, Cytomorphology, Cytogenetics 25 cases with aberrant karyotype and without clear-cut MDS MFCCytomorphology no MDSsuspected MDS MDS6 (50.0%)11 (47.8%) no MDS6 (50.0%)12 (52.2%) Total12 (100%)23 (100%) Kern et al., Cancer 2010

106 Correlation Immunophenotyping and Cytomorphology Wells et al., Blood 2003;102:

107 Correlation Immunophenotyping and Cytomorphology van de Loosdrecht et al., Blood 2008;111:

108 Correlation of MFC with cytogenetics and IPSS van de Loosdrecht et al., Blood 2008;111:

109 Correlation of MFC with IPSS IPSS Aberrantly expressed antigens (mean) Kern et al., Cancer 2010

110 Correlation of MFC with outcome following allogeneic Tx Wells et al., Blood 2003;102:

111 Correlation of MFC with outcome Survival after diagnosis 6-year-OS 68% vs. 100% p=0.008 Kern et al., Cancer 2010

112 IPSS low (n=309) IPSS lnt-1 (n=435) IPSS lnt-2 (n=112) IPSS high (n=23) IPSS low vs. IPSS Int-2: p=0.001 IPSS low vs. IPSS high: p=0.000 IPSS Int-1 vs. IPSS Int-2: p=0.001 IPSS Int-1 vs. IPSS high: p=0.000 OS according to IPSS Kern et al., 11th Int. Symposium on MDS, Edinburgh, UK, 2011

113 OS according to number of aberrantly expressed antigens OS according to flow score Flow score=0 (n=463) Flow score=1 (n=520) Flow score 0 vs. 1: p= (n=492) 2-4 (n=395) 0-1 vs. 2-4: p= vs. >4: p<0.001 >4 (n=94) OS according to MFC Kern et al., 11th Int. Symposium on MDS, Edinburgh, UK, 2011

114 CytomorphologyMFC MDS suspected (n=217) MDS excluded (n=274) MDS (n=493) MDS vs. MDS suspected: p=0.095 MDS vs. MDS excluded: p=0.070 No MDS (n=554) MDS (n=430) MDS vs. No MDS: p<0.001 OS according to diagnostic result Kern et al., 11th Int. Symposium on MDS, Edinburgh, UK, 2011

115 Cytomorphology: no MDS Cytomorphology: MDS No MDS (n=124) MDS (n=369) MDS vs. No MDS: p=0.013 No MDS (n=261) MDS (n=13) MDS vs. No MDS: p=0.012 OS according to diagnostic result by MFC Kern et al., 11th Int. Symposium on MDS, Edinburgh, UK, 2011

116 Flow score=0 (n=115) Flow score=1 (n=378) Flow score 0 vs. 1: p= (n=117) 2-4 (n=293) 0-1 vs. >4: p=0.009 >4 (n=82) OS in cases with MDS by cytomorphology OS according to number of aberrantly expressed antigens OS according to flow score Kern et al., 11th Int. Symposium on MDS, Edinburgh, UK, 2011

117 OS according to IPSS CG OS in IPSS CG=0.0 IPSS CG 0,0 (n=855) IPSS CG 0,5 (n=95) IPSS CG 1,0 (n=10) IPSS CG 1,0 vs. IPSS CG 0,0: p=0.000 IPSS CG 1,0 vs. IPSS CG 0,5: p=0.000 No MDS (n=533) MDS (n=322) MDS vs. No MDS: p=0.003 OS according to diagnostic result by MFC Kern et al., 11th Int. Symposium on MDS, Edinburgh, UK, 2011

118 OS in IPSS CG=0.5 OS in IPSS CG=1.0 MDS (n=77) No MDS (n=18) MDS vs. No MDS: n.s. No MDS (n=3) MDS (n=31) MDS vs. No MDS: n.s. OS according to diagnostic result by MFC Kern et al., 11th Int. Symposium on MDS, Edinburgh, UK, 2011

119 in IPSS CG=0.0 OS according to number of aberrantly expressed antigens in IPSS CG=0.5 in IPSS CG=1.0 >4 (n=19) 2-4 (n=62) n.s. 0-1 (n=4) 2-4 (n=27) >4 (n=3) 2-4 vs. >4: p= (n=474) 2-4 (n=306) 0-1 vs. >4: p= vs. >4: p=0.016 >4 (n=72) OS in cytogenetic subgroups Kern et al., 11th Int. Symposium on MDS, Edinburgh, UK, 2011

120 Flow score=0 (n=444) Flow score=1 (n=410) Flow score 0 vs. 1: p=0.004 Flow scor =0 (n=15) Flow score=1 (n=80) Flow score 0 vs. 1: n.s. ge25ssc63 =1 (n=30) Flow score=0 (n=4) Flow score 0 vs. 1: n.s. OS according to flow score in IPSS CG=0.0 OS according to flow score in IPSS CG=0.5 OS according to flow score in IPSS CG=1.0 OS in cytogenetic subgroups Kern et al., 11th Int. Symposium on MDS, Edinburgh, UK, 2011

121 Conclusions MFC may significantly add to the present standard diagnostic work-up of suspected MDS by CM and CG The diagnostic result by MFC and the degree of aberrancies detected by MFC may be used to estimate prognosis and to stratify patients Additional studies should be performed applying CM, CG and MFC in parallel to further validate these findings


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