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

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

CD34/CD117 CD34 CD117

CD34/CD36 CD36 CD34

CD34/HLA-DR HLA-DR CD34

CD34/CD13 CD34 CD13

CD34/CD33 CD34 CD33

CD38/CD117 CD117 CD38

CD38/CD36 CD36 CD38

CD33/CD14 CD14 CD33

CD14/CD34 CD34 CD14

CD34/CD11b CD11b CD34

CD11b/HLA-DR HLA-DR CD11b

CD34/CD15 CD15 CD34

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

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

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

ELN website: www.leukemia-net.org

Identification of cell compartments by CD45-SSC

CD11b/CD16 expression pattern in granulocytes

CD13/CD16 expression pattern in granulocytes

CD11b/CD13 expression pattern in granulocytes

CD56 expression in granulocytes

SSC signal in granulocytes

CD2 expression in monocytes

CD4/CD14 expression in monocytes

CD56 expression in monocytes

CD13/CD11b expression in monocytes

HLA-DR/CD11b expression in monocytes

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

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)

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

Diagnosis in AML Definition of AML M7: positive for CD41

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

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

Typical findings in AML with t(8;21): Subclassification in AML Typical findings in AML with t(8;21): Coexpression of CD19 Coexpression of CD56

Typical findings in AML with inv(16): Subclassification in AML Typical findings in AML with inv(16): Coexpression of CD65 and CD34 Coexpression of CD2

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

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

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

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

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

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

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

Antibody panel FITC PE PC5 FITC PE PC5 CD34 CD2 CD33 CD90 CD117 CD34 CD11b CD117 CD34 CD64 CD4 CD45 CD34 CD13 CD19 CD65 CD87 CD34 CD15 CD34 CD33 HLA-DR CD33 CD34 CD4 CD13 CD14 CD34 CD135 CD117 CD34 CD116 CD33 FITC PE PC5 CD90 CD117 CD34 CD34 7.1 CD33 CD38 CD133 CD34 CD61 CD14 CD45 CD36 CD235a CD45 CD15 CD13 CD33 TdT CD33 CD45 MPO LF CD15 TdT CD22 CD3 TdT CD79a CD3

LAIP+ cells in normal bone marrow Kern et al. Haematologica 2003;88:646-653 n Normal BM, analyzed samples total 26 per LAIP (median, range) 24, 11-26 Analyses, total 2863 Median frequency of LAIP+ cells in normal BM median (range) all LAIP (n=140) 0.07% (0.00%-1.20%) only 1 LAIP per patient (n=68) 0.05% (0.00%-0.43%)

LAIP+ cells in normal and leukemic bone marrow Kern et al. Haematologica 2003;88:646-653 Frequency of LAIP+ cells in AML-BM median (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 (0.99-4.23) only 1 LAIP per patient (n=68) 2.82 (1.58-4.23)

CD34+CD56+CD33+ cells Serial dilution of AML cells in normal BM 100 10 Kern et al. Haematologica 2003;88:646-653 0.0001 0.001 0.01 0.1 1 10 100 1E-05 % calculated % measured

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

Day 16 MRD—Detection of cytoreduction % bone marrow blasts 0.01 0.1 1 10 100 Cytomorphology A % LAIP+ cells in bone marrow 0.01 0.1 1 10 100 Multiparameter flow cytometry B day 1 day 16 Kern et al., Haematologica 2004;89(5):528-540

Day 16 MRD—Detection of cytoreduction % bone marrow blasts 0.01 0.1 1 10 100 Cytomorphology A % LAIP+ cells in bone marrow 0.01 0.1 1 10 100 Multiparameter flow cytometry B day 1 day 16 Kern et al., Haematologica 2004;89(5):528-540

Day 16 MRD—Multivariate analysis Parameter CR EFS RFS OS LD day 16 p 0.062 0.004 0.031 n.s. RR 1.490 0.678 0.555 Favorable karyotype p n.s. 0.044 n.s. n.s. RR 0.289 Unfavorable karyotype p 0.032 0.007 n.s. 0.021 RR 0.330 2.293 Kern et al., Haematologica 2004;89(5):528-540

Day 16 MRD—Relapse-free survival 1095 730 365 1.00 0.75 0.50 0.25 0.00 days Kern et al., Haematologica 2004;89(5):528-540

Separation according to 25-percentile Log-difference (=1.70) Prognostic impact of MRD after induction Separation according to 25-percentile Log-difference (=1.70) RFS 1.00 LD >25%ile: median EFS 12.0 mos. LD <25%ile: median EFS 3.8 mos. p=0.0004 0.75 0.50 0.25 0.00 365 730 1095 days Kern et al., Blood 2004;104(10):3078-3085

Separation according to 75-percentile Log-difference (=2.94) Prognostic impact of MRD after consolidation Separation according to 75-percentile Log-difference (=2.94) RFS 1.00 0.75 0.50 0.25 LD >75%ile: 2-year-EFS 83.3% LD <75%ile: 2-year-EFS 25.7% p=0.0034 0.00 365 730 1095 days Kern et al., Blood 2004;104(10):3078-3085

MRD after induction and after consolidation (multivariate) MRD MRD (ind.) (cons.) Parameter RFS OS RFS OS LD p 0.006 n.s. 0.006 0.005 RR 0.348. 0.397 0.408 Unfavorable karyotype p 0.0001 n.s. 0.006 n.s. RR 7.178 4.370 Kern et al., Blood 2004;104(10):3078-3085

MRD assessment, extended cohort Patients Patients (n) 286 3y-OS 54% MRD assessments (n) 550 EFS, median 14.5 M. Standard therapy LAIP+ BM cells at Dx 16.04% (2.54%-76.14%) LAIP+ cells normal BM 0.02% (0.00%-1.01%) Follow-up assessments n Log-difference (median) Up to day 28 85 2.02 Day 29 to day 60 122 2.29 Day 61 to day 120 158 2.39 Day 121 to day 365 137 2.53 After day 365 48 2.81 Kern et al., ASH 2005

Prognostic impact of MRD EFS 3y-OS Median p Months p (Months) Up to day 28 21.1 vs. 9.1 0.001 71% vs. 56% 0.035 Day 29 to day 60 21.5 vs. 9.3 <0.001 83% vs. 42% <0.001 Day 61 to day 120 39.3 vs. 13.5 <0.001 82% vs. 63% 0.011 Day 121 to day 365 57.1 vs. 13.7 <0.001 95% vs. 65% <0.001 After day 365 n.r. vs. 29.0 0.001 n.s. Median Log-difference diagnosisMRD-assessment as separator Kern et al., ASH 2005

Prognostic impact of MRD levels day 121 to day 365 RFS OS median 57.1 vs. 13.7 95% vs. 65% at 3 years p<0.001 p<0.001 Kern et al., ASH 2005

Prognostic impact of MRD (multivariable) EFS 3y-OS RR p RR p Up to day 28 0.831 0.085 n.s. Day 29 to day 60 0.542 <0.001 0.538 0.001 Day 61 to day 120 0.754 0.035 n.s. Day 121 to day 365 0.510 <0.001 0.422 <0.001 After day 365 0.413 <0.001 n.s. Kern et al., ASH 2005

Impact of MRD levels on RFS in cytogenetic subgroups favorable intermediate unfavorable CG = 1 CG = 2 CG = 3 1.00 1.00 LD <2.53: 25% at 2 years 1.00 LD >2.53: 75% at 2 years p=0.0221 0.75 0.75 0.75 p p p 0.50 0.50 0.50 LD <2.53: 37% at 2 years LD <2.53: 0% at 2 y. 0.25 0.25 0.25 LD >2.53: 80% at 2 years LD >2.53: 88% at 2 y. p=0.0029 p=0.0014 0.00 0.00 0.00 365 730 1095 1460 365 730 1095 1460 365 730 1095 1460 days days days Kern et al., ASH 2005

Improvement of MRD assessment by CD45-SSC-gating Kern et al., Crit Rev Oncol Hematol 2005;56:283-309

Improvement of MRD assessment by CD45-SSC-gating AML without CD45 gating 69.714% Normal BM without CD45 gating 0.511% AML with CD45 gating 66.675% Normal BM with CD45 gating 0.002% Kern et al., Hematol J 2004;5:410-418

Impact of CD45-gating on sensitivity/specificity without CD45 gating with CD45 gating LAIP+ LAIP+ LD LAIP+ LAIP+ LD AML normal BM AML normal BM Median 20.86% 0.15% 2.26 20.16% 0.02% 3.07 Min 2.33% 0.02% 1.09 2.24% 0.01% 1.22 Max 82.52% 0.58% 3.34 81.94% 0.42% 4.01 Kern et al., Hematol J 2004;5:410-418

Improvement of MRD assessment by 5-color-staining FITC PE ECD PC5 PC7 CD64 CD87 CD4 CD56 CD45 CD65 CD2 CD34 CD13 CD9 HLA-DR CD33 CD11b CD116 CD117 CD19 CD15 CD7 CD36 CD61 CD14 CD235a 7.1 CD38 CD135 CD90 CD133 MPO LF TdT CD22 CD3 CD79a Voskova et al., Leuk Lymphoma 2007;48(1):80-88

Improvement of MRD assessment by 5-color-staining 51.70% 0.004% SSC CD33-PC5 SSC CD33-PC5 CD45-PC7 SSC CD45-PC7 SSC CD7-PE CD7-PE CD7-PE CD7-PE CD15-FITC CD34-ECD CD15-FITC CD34-ECD FSC FSC SSC SSC Voskova et al., Leuk Lymphoma 2007;48(1):80-88

Impact of 5-color analysis 4-color 5-color n=139 n=139 LAIP+ LAIP+ LD LAIP+ LAIP+ LD AML normal BM AML normal BM Median 19.09% 0.030% 2.86 13.65% 0.003% 3.66 Min 1.90% 0.001% 0.77 1.90% 0.001% 1.98 Max 84.83% 3.600% 4.91 77.57% 0.040% 4.89 Voskova et al., Leuk Lymphoma 2007;48(1):80-88

Course of MRD using 5-color analysis

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

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 Improvements of method by CD45-gating and 5-color-staining Further assessment and standardization needed

AML with limited differentiation (AML-LD) Kern et al., Leukemia 2009;23:1361-1364

AML with limited differentiation (AML-LD) Kern et al., Leukemia 2009;23:1361-1364

AML with limited differentiation (AML-LD) Kern et al., Leukemia 2009;23:1361-1364

Patient cohort of present GEP study AML-LD 27 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

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

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

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

Diagnosis of MPAL

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

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: erythroid cells (also ringed sideroblasts >15% considered diagnostic) neutrophils and their precursors megakaryocytes

Prognosis in MDS Karyotype favorable: normal, -Y, del(5q), del(20q) Points 0.5 1 1.5 2 % bone marrow blasts 5 5-10 11-20 21-30 Karyotype favorable intermediate unfavorable Cytopenias 0/1 2/3 Karyotype favorable: normal, -Y, del(5q), del(20q) Karyotype unfavorable: complex aberrant (≥3 aberrations), aberrations of chromosome 7 Points 0.5 to 1.0 1.5 to 2.0 ≥2.5 Risk group Low Int-1 Int-2 High

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;31:727-736

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;31:727-736

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;31:727-736

ELN working conference Amsterdam, March 27/28 2008 Munich, October 29/30 2009 London, November 5/6 2010 Pavia, November 4/5 2011 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

Evaluation of MFC in MDS Wells et al. Blood 2003 115 pts. with MDS, 104 pts. with various disorders, 25 healthy donors Van de Loosdrecht et al. Blood 2008 50 pts. with MDS, 15 healthy volunteers, 3 pts. undergoing surgery Kern et al. Cancer 2010 1013 pts. with suspected MDS

Parameters scored as aberrant in immature compartment van de Loosdrecht et al., Haematologica 2009;94:1124-1134

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

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

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

Parameters in maturing myeloid and monocytic compartment van de Loosdrecht et al., Haematologica 2009;94:1124-1134

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

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

Cytomorphologic findings Aberrant antigen expression in granulocytes MFC findings Cytomorphologic findings p-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%) CD56+ 10 (3.6%) 90 (17.6%) 23 (10.2%) CD33- 18 (6.5%) 53 (10.4%) 19 (8.4%) n.s. CD64- 14 (2.7%) 8 (3.6%) 0.011 # of aberrant antigens 0.0±0.21,2 0.2±0.61 0.1±0.62 1<0.001 20.003 Reduced SSC signal 14 (5.1%) 286 (56.0%) 42 (18.7%) SSC-ratio G:L (mean±SD) 7.47±1.091,2 6.55±2.321 7.38±1.172 2n.s. Kern et al., Cancer 2010

Aberrant antigen expression in granulocytes 406 cases without dysgranulopoiesis by cytomorphology aberrant CD13/CD16 expression pattern 104 (25.6%) aberrant CD11b/CD16 expression pattern 62 (15.3%) CD56 expression 38 (9.4%) lack of CD33 expression 44 (10.8%) lack of CD64 expression 2 (0.5%) Aberrant expression ≥2 antigens RA 16/31 (51.6%) RARS 15/27 (55.6%) Kern et al., Cancer 2010

Parameters scored as aberrant in erythroid compartment van de Loosdrecht et al., Haematologica 2009;94:1124-1134

Proposed marker combinations van de Loosdrecht et al., Haematologica 2009;94:1124-1134

Example of a screening panel for 4-color floy cytometry van de Loosdrecht et al., Haematologica 2009;94:1124-1134

Lymphatic/Granulocyte MDS 10 color panel Blast/Granulocyte Tube Monocyte/Erythroid Lymphatic/Granulocyte FITC CD14 CD71 CD7 PE CD13 CD2 CD10 ECD CD38 CD64 CD8 PC5.5 CD123 CD56 CD5 PC7 CD117 CD4 APC CD11b CD36 CD3 APC-Alexa Fluor 700 CD34 APC-Alexa Fluor 750 CD33 CD19 Pacific Blue CD16 HLA-DR CD15 Krome Orange CD45

List of pathological controls to determine the specificity van de Loosdrecht et al., Haematologica 2009;94:1124-1134

Recommended minimal requirements to assess MDS by MFC BONE MARROW SUBSET RECOMMENDED 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 relation of HLA-DR and CD11b relation of CD36 and CD14 expression of CD56*** Progenitor B cells enumeration as fraction of total CD34+ based on CD45/CD34/SSC in combination with CD10 or CD19 Westers et al., Leukemia 2012

Diagnostic results in MFC and cytomorphology 1,013 patients with cytopenias and suspected MDS analyzed Non-MDS malignancies excluded MFC Cytomorphology MDS no MDS suspected MDS MDS 382 (74.8%) 13 (4.7%) 51 (22.7%) no MDS 129 (25.2%) 264 (95.3%) 174 (77.3%) Total 511 (100%) 277 (100%) 225 (100%) Overall concordance 646/788 (82.0%) Kern et al., Cancer 2010

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

Diagnostic results in MFC and cytogenetics MFC Cytogenetics aberrant karyotype normal karyotype MDS 189 (77.1%) 257 (33.5%) no MDS 56 (22.9%) 511 (66.5%) Total 245 (100%) 768 (100%) Kern et al., Cancer 2010

Results in MFC, Cytomorphology, Cytogenetics 25 cases with aberrant karyotype and without clear-cut MDS MFC Cytomorphology no MDS suspected MDS MDS 6 (50.0%) 11 (47.8%) no MDS 6 (50.0%) 12 (52.2%) Total 12 (100%) 23 (100%) Kern et al., Cancer 2010

Correlation Immunophenotyping and Cytomorphology Wells et al., Blood 2003;102:394-403

Correlation Immunophenotyping and Cytomorphology van de Loosdrecht et al., Blood 2008;111:1067-1077

Correlation of MFC with cytogenetics and IPSS van de Loosdrecht et al., Blood 2008;111:1067-1077

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

Correlation of MFC with outcome following allogeneic Tx Wells et al., Blood 2003;102:394-403

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

OS according to IPSS 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 Kern et al., 11th Int. Symposium on MDS, Edinburgh, UK, 2011

OS according to MFC OS according to number of aberrantly expressed antigens OS according to flow score 0-1 (n=492) 2-4 (n=395) 0-1 vs. 2-4: p=0.004 0-1 vs. >4: p<0.001 >4 (n=94) Flow score=0 (n=463) Flow score=1 (n=520) Flow score 0 vs. 1: p=0.001 Kern et al., 11th Int. Symposium on MDS, Edinburgh, UK, 2011

OS according to diagnostic result Cytomorphology MFC 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 Kern et al., 11th Int. Symposium on MDS, Edinburgh, UK, 2011

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

OS in cases with MDS by cytomorphology OS according to number of aberrantly expressed antigens OS according to flow score 0-1 (n=117) 2-4 (n=293) 0-1 vs. >4: p=0.009 >4 (n=82) Flow score=0 (n=115) Flow score=1 (n=378) Flow score 0 vs. 1: p=0.008 Kern et al., 11th Int. Symposium on MDS, Edinburgh, UK, 2011

OS according to diagnostic result by MFC 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 Kern et al., 11th Int. Symposium on MDS, Edinburgh, UK, 2011

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

OS in cytogenetic subgroups OS according to number of aberrantly expressed antigens in IPSS CG=0.5 in IPSS CG=0.0 in IPSS CG=1.0 0-1 (n=474) 2-4 (n=306) 0-1 vs. >4: p=0.000 2-4 vs. >4: p=0.016 >4 (n=72) >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=0.006 Kern et al., 11th Int. Symposium on MDS, Edinburgh, UK, 2011

OS according to flow score in IPSS CG=0.0 OS in cytogenetic subgroups 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 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. Flow score=0 (n=4) Flow score 0 vs. 1: n.s. ge25ssc63 =1 (n=30) Kern et al., 11th Int. Symposium on MDS, Edinburgh, UK, 2011

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