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

Normal and abnormal maturation patterns in myeloid cells, myeloid neoplasms

Wolfgang Kern

MLL Munich Leukemia Laboratorywww.mll.com

Antigen expression in myelopoiesis

Neutrophil Maturation

An

tig

en D

ensi

ty

CD16

CD11b

CD33

CD15

CD45

CD34

CD117

CD13

HLA-DR

CD34C

D11

7

CD34/CD117

CD36C

D34

CD34/CD36

HLA-DRC

D34

CD34/HLA-DR

CD34C

D13

CD34/CD13

CD34C

D33

CD34/CD33

CD117C

D38

CD38/CD117

CD36C

D38

CD38/CD36

CD14C

D33

CD33/CD14

CD34C

D14

CD14/CD34

CD11bC

D34

CD34/CD11b

HLA-DRC

D11

b

CD11b/HLA-DR

CD15C

D34

CD34/CD15

Multiparametric flow cytometry ofnormal human bone marrow:

analysis and display strategies

GEIL-GTLLF

2008

Part one : Leukocyte subsetsA colour code is applied trhoughout this atlas:Granulocytes in redMonocytes in greenLymphocytes in purpleAll 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

CD45APCFSC-Height CD11bFITC

CD11bFITCCD11bFITC CD11bFITC

SS

C-H

eig

ht

SS

C-H

eig

ht

CD

16

PC

7

CD

16

PC

7

CD

16

PC

7

CD

16

PC

7

FLx

FL

y

FLx

FL

y

CD45

SS

C

FSC

SS

C

FLx

FL

y

FLx

FL

y

CD11b/CD117

I.9

CD45APCFSC-Height CD45APC

CD11bFITCCD11bFITC CD11bFITC

SS

C-H

eig

ht

SS

C-H

eig

ht

SS

C-H

eig

ht

CD

117

PE

CD

117

PE

CD

117

PE

FLx

FL

y

FLx

FL

y

CD45

SS

C

FSC

SS

C

FLx

FL

y

FLx

FL

y

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


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 cytochemistryCytogenetics/FISHMolecular geneticsImmunophenotyping

Immunophenotyping for diagnosis of AML:

AML M7AML M0BAL

Immunophenotyping for subclassification of AML:

Hint to genetic abnormalitiest(15;17), t(8;21), inv(16)

Diagnosis in AML

Definition of AML M0:

positive for myeloid Antigensnegative for lymphatic Antigens

Diagnosis in AML

Definition of AML M7:

positive for CD41positive for CD61

Diagnosis in AML

CD7+CD33+ MPO+LF- TdT+cyCD3+

Biphenotypic acute leukemiaMixed phenotype acute leukemia

Typical findings in APL:

characteristic SSC/FSC-pattern

high auto-fluorescence

CD33+/HLA-DR-

AML M3

Normal BM AML M2

Subclassification in AML


Typical findings in AML with t(8;21):

Coexpression of CD19 Coexpression of CD56


Typical findings in AML with inv(16):

Coexpression of CD65 and CD34 Coexpression of CD2

Differential using CD45-SSC-Gate

Immunophenotyping11% blasts

Cytomorphology8% blasts












Immunophenotyping14% monocytic cells


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)

CD34CD33

CD

56

DiagnosisDay 0

After 1st inductionDay 18

After 2nd inductionDay 68

After alloTxDay 100

Antibody panel

FITC PE PC5

CD34 CD2 CD33

CD7 CD33 CD34

CD34 CD56 CD33

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. Haematologica2003;88:646-653

nNormal BM, analyzed samples

total 26per 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. Haematologica2003;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)

Serial dilution of AML cells in normal BM

Kern et al. Haematologica2003;88:646-653

0.0001

0.001

0.01

0.1

1

10

100

1E-050.00010.0010.010.1110100

% calculated

% m

easu

red

CD34+CD56+CD33+ cells

Day 16 blasts by cytomorphology

Kern et al.Blood2003;101:64-70

Day 16 MRD—Detection of cytoreduction

0.01

0.1

1

10

100

0.01

0.1

1

10

100

% bone marrow blasts

% LAIP+ cells in bone marrow

day 1 day 16

A

B

Cytomorphology

Multiparameterflow cytometry

Kern et al., Haematologica 2004;89(5):528-540

Day 16 MRD—Detection of cytoreduction

0.01

0.1

1

10

100

0.01

0.1

1

10

100

% bone marrow blasts

% LAIP+ cells in bone marrow

day 1 day 16

A

B

Cytomorphology

Multiparameterflow cytometry


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

Day 16 MRD—Relapse-free survival


10957303650

1.00

0.75

0.50

0.25

0.00

days

Prognostic impact of MRD after induction

10957303650

1.00

0.75

0.50

0.25

0.00

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=0.0004

days

RFS

Kern et al., Blood 2004;104(10):3078-3085

Prognostic impact of MRD after consolidation

Separation according to 75-percentile Log-difference (=2.94)

RFS

10957303650

1.00

0.75

0.50

0.25

0.00

days

LD >75%ile: 2-year-EFS 83.3%LD <75%ile: 2-year-EFS 25.7%p=0.0034

Kern et al., Blood 2004;104(10):3078-3085

MRD after induction and after consolidation (multivariate)

Kern et al., Blood 2004;104(10):3078-3085

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

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 diagnosisMRD-assessment as separator


Prognostic impact of MRD levels day 121 to day 365


543210

Years

1,00

0,75

0,50

0,25

0,00

pCP4 EFS

543210

Years

1,00

0,75

0,50

0,25

0,00

p

CP4 OSRFS OS

median 57.1 vs. 13.7 95% vs. 65% at 3 yearsp<0.001 p<0.001

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.

Impact of MRD levels on RFS in cytogenetic subgroups


0 365 730 1095 1460

days

0.00

0.25

0.50

0.75

1.00

p

LD <2.53: 37% at 2 years

LD >2.53: 80% at 2 years

p=0.0029

CG = 1

0 365 730 1095 1460

days

0.00

0.25

0.50

0.75

1.00

p

LD <2.53: 25% at 2 years

LD >2.53: 75% at 2 years

p=0.0221

CG = 2

0 365 730 1095 1460

days

0.00

0.25

0.50

0.75

1.00

p

LD <2.53: 0% at 2 y.

LD >2.53: 88% at 2 y.

p=0.0014

CG = 3favorable intermediate unfavorable

A

B

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

Kern et al., Hematol J 2004;5:410-418

AMLwithout CD45 gating

AMLwith CD45 gating

Normal BMwithout CD45 gating

Normal BMwith CD45 gating

0.002%

0.511%

69.714%

66.675%

Impact of CD45-gating on sensitivity/specificity

Kern et al., Hematol J 2004;5:410-418

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

Improvement of MRD assessment by 5-color-staining

Voskova et al., Leuk Lymphoma 2007;48(1):80-88

FITC PE ECD PC5 PC7

CD64 CD87 CD4 CD56 CD45


CD9 HLA-DR CD34 CD33 CD45

CD11b CD116 CD34 CD117 CD45



CD36 CD61 CD14 CD235a CD45

CD4 7.1 CD14 CD13 CD45


CD15 CD133 CD34 CD117 CD45

MPO LF CD34 CD33 CD45

TdT CD22 CD3 CD79a CD45

Improvement of MRD assessment by 5-color-staining

51.70%

0.004%

CD45-PC7 SSC

CD15-FITC CD34-ECD

SSC

FS

C

CD

7-P

E

CD

7-P

ES

SC

CD

33

-PC

5

CD45-PC7 SSC

CD15-FITC CD34-ECD

SSC

FS

C

CD

7-P

E

CD

7-P

ES

SC

CD

33

-PC

5


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

Course of MRD using 5-color analysis

LAIP+ in %, CD15-CD7+CD34+CD33+CD45(+)

0,0001

0,001

0,01

0,1

1

10

100

Okt 06 Apr 07 Okt 07 Apr 08 Okt 08

Stability of LAIP between diagnosis and relapse

A

DC

B

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

4. Improvements of method by CD45-gating and 5-color-staining

5. Further assessment and standardization needed

AML with limited differentiation (AML-LD)

Kern et al., Leukemia 2009;23:1361-1364





Patient cohort of present GEP study

n

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

Points B-cell lineage T-cell lineage myeloid lineage

2 CD79a cy/sCD3 Anti-MPO

2 cyIgM Anti-TCRαβ

2 cyCD22 Anti-TCRγδ

1 CD19 CD2 CD13

1 CD10 CD5 CD33

1 CD20 CD8 CD65

1 CD10 CD117

0,5 TdT TdT CD14

0,5 CD24 CD7 CD15

0,5 CD1a CD64

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 hematopoieticcell lineages:(i) erythroid cells (also ringed sideroblasts >15% considered diagnostic)(ii) neutrophils and their precursors(iii) megakaryocytes

Prognosis in MDS

Points

0 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 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 criteriaConstant cytopenia in one or more of the following cell lineages:

erythroid (hemoglobin <11 g/dl) orneutrophilic (ANC < 1,500/µl) ormegakaryocytic (platelets <100,000/µl)

Exclusion of all other hematopoietic or non-hematopoietic disorders asprimary reason for cytopenia/dysplasia

(B) MDS-related (decisive) criteriaDysplasia in ≥10% of all cells in one of the following lineages in bone marrow smear:

erythroid orneutrophilic ormegakaryocytic 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 criteriaConstant cytopenia in one or more of the following cell lineages:

erythroid (hemoglobin <11 g/dl) orneutrophilic (ANC <1,500/µl) ormegakaryocytic (platelets <100,000/µl)

Exclusion of all other hematopoietic or non-hematopoietic disorders asprimary reason for cytopenia/dysplasia

(C) Co-criteriaAbnormal 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


Idiopathic cytopenia of uncertain significance (ICUS)

(A) DefinitionCytopenia 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 ICUSDetailed case history (toxins, drugs, mutagenic events, etc.)Thorough clinical investigations including X-ray and sonography of spleenDifferential blood count (microscopic) and complete serum chemistryBone marrow histology and immunohistochemistryBone marrow smear including an iron stainFlow cytometry of bone marrow and peripheral blood cellsChromosome analysis including FISHMolecular 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-upBlood count and differential count as well as serum chemistry (1–6 months)Suspicion for MDS becomes evident: bone marrow examination


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 27/28 2008Munich, October 29/30 2009London, November 5/6 2010Pavia, November 4/5 2011

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







Immunophenotyping14% monocytic cells



Parameters in maturing myeloid and monocytic compartment


CD13/CD16 expression pattern in granulocytes

Normal BM MDS



Normal BM MDS


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%) <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.

CD64- 0 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<0.00120.003

Reduced SSC signal 14 (5.1%) 286 (56.0%) 42 (18.7%) <0.001

SSC-ratio G:L (mean±SD)

7.47±1.091,2 6.55±2.321 7.38±1.172 1<0.0012n.s.


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%)


Parameters scored as aberrant in erythroid compartment


Proposed marker combinations


Example of a screening panel for 4-color floy cytometry


MDS 10 color panelBlast/Granulocyte

TubeMonocyte/Erythroid

TubeLymphatic/Granulocyte

Tube

FITC CD14 CD71 CD7

PE CD13 CD2 CD10

ECD CD38 CD64 CD8

PC5.5 CD123 CD56 CD5

PC7 CD117 CD117 CD4

APC CD11b CD36 CD3

APC-Alexa Fluor 700 CD34 CD34 CD34

APC-Alexa Fluor 750 CD33 CD33 CD19

Pacific Blue CD16 HLA-DR CD15

Krome Orange CD45 CD45 CD45

List of pathological controls to determine the specificity


Recommended minimal requirements to assess MDS by MFC

Westers et al., Leukemia 2012

BONE MARROW SUBSET RECOMMENDED ANALYSES

Erythroid compartment* % of nucleated erythroid cellsrelation CD71 and CD235aexpression of CD71expression of CD36expression 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 lymphocytesSSC as ratio vs. SSC of lymphocytesrelation of CD13 and CD11b relation of CD13 and CD16 relation CD15 and CD10

Monocytes % of cells as ratio to lymphocytesrelation of HLA-DR and CD11b relation of CD36 and CD14expression of CD13 and CD33;expression of CD56***

Progenitor B cells enumeration as fraction of total CD34+based on CD45/CD34/SSC in combination with CD10 or CD19

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%)


Numbers of aberrantly expressed antigens


Cytomorphology: no MDS

Cytomorphology: suspected MDS

Cytomorphology: MDS

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%)


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%)


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

2

2,5

3

3,5

4

4,5

0 0,5 1 1,5 2 2,5 3

IPSS

Ab

erra

ntl

y ex

pre

ssed

an

tig

ens

(mea

n)


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


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

OS according to number of aberrantly expressed antigens

OS according to flow score

Flow score=0 (n=463)


Flow score 0 vs. 1: p=0.001

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)

OS according to MFC


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

OS according to diagnostic result


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)


OS according to diagnostic result by MFC





0-1 (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 flow score


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)




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.



in IPSS CG=0.0


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=0.006

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)

OS in cytogenetic subgroups





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 accordingto flow score inIPSS CG=1.0

OS in cytogenetic subgroups


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