Improving the role of Flow Cytometry

for the characterization of

Myelodysplastic Syndromes

Arjan A. van de Loosdrecht, MD, PhD

Department of Hematology

VU University Medical Center

Cancer Center Amsterdam (CCA)

Amsterdam, The Netherlands

December 6th, 2013

New Orleans

MDS- Foundation

ASH 2013 Symposium

Major Therapeutic and Molecular Advances in MDS

ASH 2013 Symposium

Major Therapeutic and Molecular Advances in MDS

Arjan A. van de Loosdrecht, MD, PhD

Department of Hematology

VU University Medical Center

Cancer Center Amsterdam (CCA)

Amsterdam, The Netherlands

December 6th, 2013

New Orleans

DISCLOSURE

I have no relevant financial

relationships to disclose.

Role of Flow Cytometry in diagnosis of AML:

impact on clinical decision making

• if > 20% -blasts- by morphology:

• Flow cytometry: diagnosis/monitoring

– Immature vs mature leukemia

– Lymphoid vs myeloid AL

– B vs T-lymphoid AL

– Lymphoid differentiation: pro-B-ALL mature B-ALL

– Specific immunophenotypes: APL (t(15;17); AML t(8;21); AML/ALL with t(9;21)

– Mixed Phenotype Acute Leukemia

– Minimal Residual Disease

– Leukemic Stem Cells

Points B T Myeloid

2 cD79a

cIgM

cCD22

CD3 (c or s)

TCR αβ

TCR γ

cMPO

1 CD19

CD10

CD20

CD2

CD5

CD8

CD10

CD13

CD33

CD65

CD117

0.5 cTdT

CD24

cTdT

CD7

CD1a

CD14

CD15

CD64

Biphenotypic and Bilinear Acute Leukemia: The European

Group for the Immunological Classification of Leukemias (EGIL)

Bene MC, et al., Leukemia 1995;10:1783-6; Borowitz MJ, et al., In: WHO classification of

tumours of haematopoietic and lymphoid tissues 2008; pp150-155

B cell lineage T cell lineage Myeloid lineage

Strong CD19

with :

cCD22, CD10 or

cCD79a

CD3 (c or s)

cMPO

CD19 with at least

two of:

cCD22, CD10 or

cCD79a

2 or more of: CD11c,

CD14, CD36, CD64

WHO-2008: Mixed Phenotype Acute

Leukemia (MPAL)

Borowitz MJ, et al., In: WHO classification of tumours of haematopoietic

and lymphoid tissues 2008; pp150-155

B/My EGIL + EGIL-

WHO

MPAL+

4 1

WHO

MPAL -

21 482

T/My EGIL + EGIL-

WHO

MPAL+

1 1

WHO

MPAL -

5 482

UPN EGIL WHO

UPN 1

BAL

B/T/My

T/My

UPN 2

BAL

B/T/My

T-ALL

• 5 of 517 patient did not express any

lineage defining markers and are

defined as MPAL-

Van den Ancker, Van de Loosdrecht AA, et al., Leukemia 2010:24;1392-96

Comparison of EGIL and WHO-2008 criteria

MRD-MRD+Logrank

N109

55

F4433

P =0.008

MRD-MRD+

At risk:10955

8128

6222

4515

11

8

MRD-

MRD+

0

25

50

75

100

months0 12 24 36 48

Cu

mu

lative

pe

rce

nta

ge

After Cycle I

MRD-MRD+Logrank

N141

42

F6231

P <.001

MRD-MRD+

At risk:14142

9613

7812

57

6

18

2

MRD-

MRD+

0

25

50

75

100

months0 12 24 36 48

Cu

mu

lative

pe

rce

nta

ge

After Cycle II

MRD-MRD+Logrank

N9724

F3417

P <.001

MRD-MRD+

At risk:9724

69

8

53

5

24

3

51

MRD-

MRD+

0

25

50

75

100

months0 12 24 36 48

Cu

mu

lative

pe

rce

nta

ge

After Consolidation

MRD-MRD+Logrank

N3814

F10

7P =0.07

MRD-MRD+

At risk:3814

31

7

24

7

18

5

62

MRD-

MRD+

0

25

50

75

100

months0 12 24 36 48

Cu

mu

lative

pe

rce

nta

ge

After Cycle II - Good risk

MRD-MRD+Logrank

N8819

F4315

P <.001

MRD-MRD+

At risk:8819

58

6

48

5

36

1

11

0

MRD-

MRD+

0

25

50

75

100

months0 12 24 36 48

Cu

mu

lative

pe

rce

nta

ge

After Cycle II - Intermediate risk

F99

P =0.006

N15

9

MRD-MRD+Logrank

MRD-MRD+

At risk:159

70

60

30

10

MRD-

MRD+0

25

50

75

100

months0 12 24 36 48

Cu

mu

lative

pe

rce

nta

ge

After Cycle II - Poor risk

A.

B.

Relapse free survival after different therapy cycles (A)

and after cycle II in different risk groups (B)

Terwijn M, Ossenkoppele GJ, Schuurhuis GJ. et al., JCO 2013;31:3889-97

Roughly 90% of patients have Leukemia Associated ImmunoPhenotypes (LAIP)

Relative risk (95% CI) P-value

8.5 (2.6-27.5) 0.0004 0.09 2.6 (0.9-7.8)

P-value Relative risk (95% CI)

Leukemic Stem Cell Assessment in Remission Bone Marrow of

Acute Myeloid Leukemia Patients Is a New Prognostic Parameter

Terwijn M, et al., Int Lab Hematol 2012;34:432-41;

Schuurhuis GJ, et al. PlosOne 2013;8:e78897

Role of FCM in Myelodysplastic Syndromes

• if cytopenic and/or < 5% (-20) -blasts- by morphology +/-

dysplastic features and no specific cytogenetics abn

• Flow cytometry: diagnosis

• Flow cytometry: prognosis

• Flow cytometry: prediction of response on drugs

• Flow cytometry: disease monitoring

– Minimal Residual Disease

– Leukemic Stem Cells

Minimal diagnostic criteria in MDS

consensus (Vienna 2006)

• A. Prerequisite Criteria – constant cytopenia in one or more cell lineages

– exclusion of all other hematopoietic or non-hematopoietic disorders

• B. MDS-related (Decisive) Criteria – dysplasia in > 10% of all cells in one of the lineages

or > 15% ring sideroblasts (iron stain)

– 5–19% blast cells in bone marrow smears

– typical chromosomal abnormality (karyotyping or FISH)

Valent P, et al., Leuk Res 2007;31:727-31

Platzbecker U, et al., Leuk Res 2012;36:264-70

FISH = fluorescence in situ hybridization.

Dysplasia: multinucleated megakaryocytes, bi-nucleated

erythroblasts, hypogranularity (by courtesy of R. Ireland, King’s London)

Cazzola M, Haematologica 2011;96:349

WHO-2008 and Overall Survival in MDS Classification based on morphology!

• C. Co-criteria

– abnormal phenotype of bone marrow cells by flow cytometry

– molecular signs of a monoclonal cell population

o HUMARA assay, mutation analysis

o markedly and persistently reduced colony-formation (CFU-assay)

Valent P et al., Leuk Res 2007;31:727-31

Loosdrecht AA van de et al., Leuk Res 2008;32:205-7

Loken MR, et al., Leuk Res 2008;32:5-17

Loosdrecht AA van de et al., Blood 2008;111;1067-77

Loosdrecht AA van de et al., Haematologica 2009;94:1124-1134

Platzbecker U et al., Leuk Res 2012;36:264-70

Westers TM et al., Leukemia 2012;;36:422-30

Della Porta M et al., Haematologica 2012;97:1209-1217

Van de Loosdrecht AA et al., Leuk and Lymph 2013;54:472-475

Minimal diagnostic criteria in MDS (Vienna 2006)

Antigen expression during neutrophil

differentiation: the concept

103

102

101

Adapted from: A Orfao, ELNet Flow MDS 2008, Amsterdam

BAND/

NEUTROPHIL METAMYELOCYTE MYELOCYTE PROMYELOCYTE MYELOBLAST

CD13

CD11b

CD13

CD16

Antigen expression during neutrophil

differentiation: the concept

103

102

101

BAND/

NEUTROPHIL METAMYELOCYTE MYELOCYTE PROMYELOCYTE MYELOBLAST

CD34

HLA-DR CD117

CD13

CD33

CD11b

CD64

CD65

CD54

CD10

CD35

CD13

MPO

CD15

CD16

Adapted from: A Orfao, ELNet Flow MDS 2008, Amsterdam

Flow cytometry of myeloid progenitor cells:

normal BM

Granulocytic differentiation by flow cytometry:

normal BM

CD16-CD117-CD45-CD11b-CD34-CD13

Antigen expression during monocytic

differentiation: the concept

Adapted from: A Orfao, ELNet Flow MDS 2008, Amsterdam

Monocytic differentiation by flow cytometry:

normal BM

CD36-CD33-CD45-CD11b-CD34-CD14 / CD15-HLA-DR-CD45-CD11b-CD34-CD13

Antigen expression during erythroid

differentiation: the concept

Adapted from: A Orfao, ELNet Flow MDS 2008, Amsterdam

Diagnostic tool Diagnostic value Priority

Peripheral blood

smear

• Evaluation of dysplasia in one or more cell lines

• Enumeration of blasts Mandatory

Bone marrow

aspirate

• Evaluation of dysplasia in one or more

myeloid cell lines

• Enumeration of blasts

• Enumeration of ring sideroblasts

Mandatory

Bone marrow biopsy • Assessment of cellularity, CD34+ cells, and fibrosis Mandatory

Cytogenetic analysis

• Detection of acquired clonal chromosomal

abnormalities that can allow a conclusive diagnosis

and also prognostic assessment

Mandatory

FISH

• Detection of targeted chromosomal abnormalities

in interphase nuclei following failure of standard G-

banding

Recommended

Flow cytometry

immunophenotype

• Detection of abnormalities in erythroid,

immature myeloid, maturing granulocytes,

monocytes, immature and mature lymphoid

compartments

Recommended*

If according to

ELN guidelines

SNP-array

• Detection of chromosomal defects at a high

resolution in combination with metaphase

cytogenetics

Suggested (likely to

become a

diagnostic tool in

the near future)

Mutation analysis of

candidate genes*

• Detection of somatic mutations that can allow a

conclusive diagnosis and also reliable prognostic

evaluation

Suggested (likely to

become a

diagnostic tool in

the near future)

Diagnostic approach to MDS 2013

Malcovati L, et al., ELN guidelines. Blood 2013;122:2943-64; Greenberg P et al., J Nat Compr

Netw Canc 2013;11:838-74; *Westers TM, et al., Leukemia 2012;26:1730-41

Standardization of flow cytometry in MDS: ELNet 2013 recommendations

Westers TM et al., Leukemia 2012;36:422-30

Van de Loosdrecht AA et al., J Natl Comp Canc Netw 2013;11:892-902

A C E

B D F

Normal and dysplastic flow cytometric profiling A-C-E: normal bone marrow; B-D-F: myelodysplastic bone marrow

Adapted from Van de Loosdrecht AA and Westers TM. MDS Foundation News Letter 2013;19:2-4

• CD34+ myeloid progenitor cells (%)

among all nucleated cells

• CD34+ B cell precursors (%) among all

CD34+ cells

FCM in diagnostics: Cardinal Parameters

Ogata et al., Blood;2006; Ogata et al., Haematologica 2009;

Della Porta et al., [ELNet] Haematologica 2012;97:1209-17

Granulocytes

Lymph Lymph

SSC peak channel ratio between CD10- granulocytes and lymphocytes

Mbl

CD45 expression (MFI) ratio between myeloblasts and lymphocytes

FCM in diagnostics: Cardinal Parameters

Ogata et al., Blood;2006; Ogata et al., Haematologica 2009;

Della Porta et al., [ELNet] Haematologica 2012;97:1209-17

MDS: example with internal reference population

lymphocytes as internal

reference population

Diagnostic power of FCM in MDS:

the learning (validation) cohort

Ogata et al., Blood;2006; Ogata et al., Haematologica 2009;

Della Porta et al., [ELNet] Haematologica 2012;97:1209-17

FCM of dyserythropoiesis: a sensitive and powerful diagnostic

tool for myelodysplastic syndromes: The RED score

Mathis S, et al., Leukemia 2013;27:1981-198; Westers TM et al., [iMDSflow ELN WG]

Munich 2013 [in preparation]

Red score treshold points

CD71: CV <80; ≥80 0 vs 3

CD36: CV <65; ≥65 0 vs 2

Hb level >10.5f or

>11.5m;

≤10.5f or

≤11.5m

0 vs 2

Red score ≥ 3: 80% correctly scored

MDS/non-MDS;

with Ogata score: sensitivity of 70%

88%

Kern et al. Haematologica 2013;98:201-207

Role of FCM: serial assesment of suspected

MDS by FCM [no initial diagnosis of MDS by CM/CG]

myeloid blasts granulocytes

(maturing myeloid cells)

monocytes

increased percentage decreased myeloid/lymphoid ratio

(<1)

decreased/increased number as

compared to lymphocytes

abnormal granularity* abnormal granularity* abnormal granularity*

abnormal expression** of CD45 abnormal expression of CD45 abnormal expression of CD45

abnormal expression of CD34 abnormal CD11b/CD13 pattern abnormal expression of CD14

abnormal expression of CD117 abnormal CD16/CD13 pattern abnormal CD11b/HLA-DR pattern

abnormal expression of CD13 abnormal expression of CD15 abnormal expression of CD13

abnormal expression of CD33 abnormal expression of CD33 abnormal expression of CD33

abnormal expression of HLA-DR expression of HLA-DR abnormal expression of CD36

expression of CD11b expression of CD34 abnormal expression of HLA-DR

expression of CD15 asynchronous shift to the left expression of CD34

expression of lineage infidelity

markers CD5, CD7, CD19 or

CD56

expression of lineage infidelity

markers CD5, CD7, CD19

expression of lineage infidelity

markers CD5, CD7, CD19

over expression of CD56 over expression of CD56

Flow Cytometric myeloid dysplasia

Loosdrecht AA van de, et al., Blood 2008;111:1067-1077; Westers TM et al.,

Leukemia 2012;36:422-30; Wells D, et al., Blood 2003:102

Flow Cytometric Scoring System (FCSS)

Wells D, et al., Blood 2003;102 Loosdrecht AA van de, Westers TM, et al., Blood 2008;111

total flow score of:

0-1: normal-mild

2-3: moderate

4-9: severe

score definition 0 no FC aberrancies in either subpopulation analyzed 1 - a single aberrancy in either granulocytes or monocytes 2 - a single aberrancy in both granulocytes and monocytes or …

- two or three aberrancies in either granulocytes and monocytes or … - expression of CD34 or lineage infidelity markers on either granulocytes or monocytes

3 - four or more aberrancies in either granulocytes and monocytes 4 - two or three aberrancies in both granulocytes and monocytes

+ 1 - decreased myeloid/lymphoid ratio (<1) - normal percentage of myeloid blasts (<5%) with flow cytometric aberrancies

+ 2 - increased percentage of abnormal myeloid blasts (5-10%) + 3 - increased percentage of abnormal myeloid blasts (11-20%) + 4 - increased percentage of abnormal myeloid blasts (>20%)

Loosdrecht AA van de, Westers TM, et al., Blood 2008;111:1067-1077

Wells D, et al., Blood 2003:102; Cutler J, et al., Cytometry 2011

Flow cytometric scoring system

and WHO2008 (n=261; controls: n=55)

01-04-2012

n=59 n=29 n=82 n=34 n=57 n=41 n=14

Van de Loosdrecht and Westers et al., Blood, 2008

Westers et al., unpublished data (May; 2012)

Flow Cytometric Scoring System in MDS-RCMD is

associated with worse overall survival

P = .019

non-MDS RA/MDS-U RCMD±RS RAEB-1 RAEB-2

** *

Chu SC et al., Leuk Res 2011:35:868-73; Ogata K. Leuk Res 2011:35:848-9

Van de Loosdrecht AA et al., Leuk Res 2011:35:850-2

Van de Loosdrecht AA et al., J Natl Comp Canc Netw 2013;11:892-902

Aberrant immunophenotype as biomarker for prediction of

response to treatment

Westers TM, van de Loosdrecht AA, et al. Blood 2010:115:1779

– Myeloid: granulocytic cells [immature and maturing cells]

– Myeloid: monocytic cells

-Percentages myeloid progenitor cells

-Aberrant subpopulations

-Abnormal maturation patterns

Due to lack of (prospective) validation studies:

do not report on:

– (dys) Erythropoiesis

– (dys) Megakaryopoiesis/trombocytes

How to report to clinicians? Guidelines of the

IMDSflow WG on FCM and MDS 2013

Loosdrecht AA van de, Westers TM. J Nat Compr Cancer Netw 2013;11:892-902

*Westers TM, et al., Leukemia 2012;26:1730-41; Porwit A, et al., [2013 IMDSflow WG report

submitted]

• A:

FCM analysis: no not show features MDS-related features

• B:

FCM analysis: show changes often seen in MDS (dys

[myelo/mono]-poiesis)

• C:

Results of FCM analysis are consistent with MDS

How to report to clinicians? Guidelines of the

IMDSflow WG on FCM in MDS 2013

Loosdrecht AA van de, Westers TM. J Nat Compr Cancer Netw 2013;11:892-902

*Westers TM, et al., Leukemia 2012;26:1730-41; Porwit A, et al., [2013 IMDSflow WG report

submitted]

Additional comments in final integrated report

• Flow cytometry is 1 diagnostic approach; the report should include:

– PB –penia; differential

– Cytology

– Immunohistochemistry/biopsy

– Cytogenetics/FISH

• Note: dyserythropoiesis and dysmegakaryopoiesis is not included in the flow cytometric analysis yet!

• Note: repeat analysis after 6 month in inconclusive cases and/or if disconcordance between diagnostic tools is evident

• Note: no prognostic and prediction of response information yet!

Loosdrecht AA van de, et al., J Nat Compr Cancer Netw 2013;11:892-902

*Westers TM, et al., Leukemia 2012;26:1730-41; Porwit A, et al., [2013

IMDSflow WG report submitted]

Conclusions:

Clinical significance of FCM in MDS

• FCM can be performed in MDS based on well-defined standard

criteria

• FCM is instrumental in the diagnosis or exclusion of MDS

• FCM is part of the recommended diagnostic work-up [ELN and

NCCN]

• Aberrancies on CD34+ cells and myelo-monocytic cells by FC

are correlated to clinical prognostic parameters

• FCSS identifies patients with a short OS within the IPSS-revised

low risk and good cytogenetic subgroups

• Aberrant CD34+ phenotype predicts response to Epo/G-CSF in

low risk MDS

• The time is ready to report FCM results in an integrated

diagnostic report in MDS

Perspectives of FCM in MDS: 2014

• Full Implementation in an integrated diagnostic report including

prognostic value of FCM results

• FCM should be part of the next WHO-classification

• Identification of prognostic subgroups in MDS specific disease

entities [i.e. (del)5q]

• confirmation of FCM results in (dys) erythropoiesis in 2014

• explorative studies on (dys) megakaryopoisis by FCM are ongoing

within IMDSflow WG

Acknowledgements

VU University Medical Center, Cancer Center Amsterdam

Department of Hematology, Amsterdam, The Netherlands

Marisa Westers, Claudia Cali,

Canan Alhan, Eline Cremers

Gert Ossenkoppele

Dutch Society of Cytometry/WP MDS

International/ELNet MDS [IMDSflow]: Spain, Austria,

Australia, Greece, Germany, French, Italy, Sweden,

UK, Bulgaria, Japan, Taiwan, USA, Canada,

Netherlands

MLL Munich; King’s NHS London; University of Pavia

Wolfgang Kern, Robin Ireland, Matteo Della Porta

Grants/support

- MDS Foundation Inc. USA

- VUmc-Cancer Center Amsterdam

- Dutch Society for Cytometry

- ELN WP8/WP10 on MDS

- HOVON The Netherlands

- Dutch Cancer Foundation (KWF)

- European Science Foundation (ESF)