Comparison of Chronic Lymphocytic Leukemia patients expressing high or low level of ZAP70 mRNA: new prognostic factors, differences in microRNA expression and interaction with the

microenvironment.

B. Stamatopoulos, B. Haibe-Kains, C. Equeter, N. Meuleman, A. Sorée, C. De Bruyn, D. Hanosset, D. Bron, P. Martiat, L. Lagneaux

Experimental Hematology, Institut Jules Bordet (ULB)

23rd annual general meeting of the BHS, January 25, 2008

Introduction

CLL : 2 distinct evolutions

Prognostic factors : IgVH mutational status … and its surrogate : ZAP70, LPL,…

ZAP70 measured by flow cytometry

(Stamatopoulos B. et al, Clin. Chem, 2007)

0 50 100 150 200 2500

20

40

60

80

100ZAP70 - by qPCRZAP70 + by qPCR

P<0.0001 Median TFS: 20 months

Median TFS: 172 months

Month

Cu

mu

lati

ve

TF

S

0 100 200 3000

20

40

60

80

100

ZAP70 - by qPCRZAP70 + by qPCR

P=0.0012

Median OS: Undef.

Median OS: 152 months

Month

Cu

mu

lati

ve

OS

TFS OS

Assoc. with MS

Strength of ass. with

MS

AUC predict. of MS

Conc. with MS

Assoc. with TFS

Assoc. with OS

TFS in case of discord. with MS

Univ. Cox

predict. of TFS

Univ. Cox

predict. of OS

Multiv. Cox

predict. of TFS

1 year AUC

predictor of TFS

2 years AUC

predictor of TFS

Mut. status (MS) - - - - S S - S S NS 70% 77%

Zap-70 (qPCR) S very strong 89% 86% S S S S S S 74% 83%

Zap-70 (FC) S strong 85% 78% S S NS S S S 79% 84%

LPL (qPCR) S strong 76% 75% S NS NS S NS NS 69% 69%

CD38 (FC) S substantial 70% 67% S NS NS S NS NS 63% 66%

S: significant; NS: non significant

ZAP70 measured by quantitative real time PCR

Aims of the study

To determine gene expression profiles linked to ZAP70 expression using Affymetrix technology

To find genes associated with prognosis but also with biology

To confirm differentially expressed microRNA and link them to TFS and OS

Patient selection

0.0

200.0

400.0

600.0

800.0

1000.0

1200.0

1400.0

1600.0

1800.0

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85 88 91 94 97 100 103 106

patients

Fol

d ch

ange

Low

high

ZAP70 mRNA expression among 108 patients by qPCR

Patient characteristics

Mut. Status Treatment Death

Num Group Sex Age Stage qPCR Status % cell. Status Status Status

1 high F 69 A UM 1507.7 + 66 + yes yes

2 high M 68 C UM 1475.8 + 41 + yes no

3 high F 57 B UM 1179.2 + 57 + yes yes

4 high F 67 A M 1137.0 + 39 + yes yes

5 high M 61 C UM 1074.7 + 22 + yes yes

6 high F 74 C UM 650.3 + 42 + yes yes

7 high M 73 B UM 643.9 + 77 + yes yes

8 low F 67 A M 61.8 - 2 - no no

9 low F 68 A M 50.4 - 2.5 - no no

10 low F 59 B M 18.9 - 14 - no no

11 low M 67 B M 14.2 - 1 - no no

12 low M 72 B M 9.5 - 3 - yes no

13 low M 77 A M 7.5 - 12 - yes no

14 low M 69 A M 2.7 - 10 - yes no

PATIENTS Zap-70 by FC.Zap-70 by qPCR

Methods

Affymetrix Protocole- 50 000 oligonucleotides

- 47 000 transcripts

- 39 500 human genes

blood collection PBMC gradient isolation

Purification with anti-CD19 magnetic beads

98-100% purity

RNA extraction

ARN 56

0

1

2

3

4

5

6

7

220 240 260 280 300 320 340

Longueur d'onde

(1,5µg)

Results

937 probe sets with P<0.05

135 probe sets with P<0.001

43 probe sets with FDR<10%

Top of the list:

1 < 1e-07 < 1e-07 17.9 124.4 6.94 1555613_a_at ZAP70

2 < 1e-07 < 1e-07 31.9 168.9 5.29 214032_at ZAP70

Gene symbol

ZAP70high mean

Ratio Probe setParametric

p-valueFDR

ZAP70low mean

qPCR validation

Novel genes and clinical outcome

0 50 100 150 200 2500

20

40

60

80

100

FCRL1 -FCRL1 +

P=0.0292

Month

Cu

mu

lati

ve

TF

S

0

50

100

150

Zap-70 - Zap-70+

P=0.0004

FC

RL

1 e

xp

res

sio

n

0 100 200 3000

20

40

60

80

100

FCRL1 -FCRL1 +

P=0.0015

Month

Cu

mu

lati

ve

OS

0

50

100

150

Zap-70- Zap-70+

P<0.0001

FC

RL

2 e

xp

res

sio

n

0 50 100 150 200 2500

20

40

60

80

100

FCRL2 -

FCRL2 +

P<0.0001

Month

Cu

mu

lati

ve

TF

S

0 100 200 3000

20

40

60

80

100

FCRL2 -FCRL2 +

P=0.0010

Month

Cu

mu

lati

ve

OS

0 50 100 150 200 2500

20

40

60

80

100

FCRL3 -

FCRL3 +

P=0.0066

Month

Cu

mu

lati

ve

TF

S

0 100 200 3000

20

40

60

80

100

FCRL3 -

FCRL3 +

P=0.0038

Month

Cu

mu

lati

ve

OS

0

200

400

600

800

Zap-70- ZAP-70+

P<0.0001

FC

RL

3 e

xp

res

sio

n

0

10

20

30

40

Zap-70 - Zap-70 +

P<0.0001

FC

RL

5 e

xp

res

sio

n

0 50 100 150 200 2500

20

40

60

80

100

FCRL5 -

FCRL5 +

P=0.0006

Month

Cu

mu

lati

ve

TF

S

0 100 200 3000

20

40

60

80

100

FCRL5 -

FCRL5 +

P=0.0232

Month

Cu

mu

lati

ve

OS

0 50 100 150 200 2500

20

40

60

80

100

PDE8A -

PDE8A +

P=0.0003

Month

Cu

mu

lati

ve

TF

S

0 100 200 3000

20

40

60

80

100

PDE8A -

PDE8A +

P=0.012

Month

Cu

mu

lati

ve

OS

0

5

10

15

Zap-70 - Zap-70 +

P<0.0001

PD

E8

A e

xp

res

sio

n

0.0

0.2

0.4

0.6

0.8

1.0

Zap-70 - Zap-70 +

P=0.0102

ITG

A4

ex

pre

ss

ion

0 50 100 150 200 2500

20

40

60

80

100

ITGA4 -ITGA4 +

P=0.3558

Month

Cu

mu

lati

ve

TF

S

0 100 200 3000

20

40

60

80

100 ITGA4 -ITGA4 +

P=0.0075

Month

Cu

mu

lati

ve

OS

A.

B.

C.

D.

E.

F.

G.

H.

I.

J.

K.

L.

M.

N.

O.

P.

Q.

R.

0 50 100 150 200 2500

20

40

60

80

100

FCRL1 -FCRL1 +

P=0.0292

Month

Cu

mu

lati

ve

TF

S

0

50

100

150

Zap-70 - Zap-70+

P=0.0004

FC

RL

1 e

xp

res

sio

n

0 100 200 3000

20

40

60

80

100

FCRL1 -FCRL1 +

P=0.0015

Month

Cu

mu

lati

ve

OS

0

50

100

150

Zap-70- Zap-70+

P<0.0001

FC

RL

2 e

xp

res

sio

n

0 50 100 150 200 2500

20

40

60

80

100

FCRL2 -

FCRL2 +

P<0.0001

Month

Cu

mu

lati

ve

TF

S

0 100 200 3000

20

40

60

80

100

FCRL2 -FCRL2 +

P=0.0010

Month

Cu

mu

lati

ve

OS

0 50 100 150 200 2500

20

40

60

80

100

FCRL3 -

FCRL3 +

P=0.0066

Month

Cu

mu

lati

ve

TF

S

0 100 200 3000

20

40

60

80

100

FCRL3 -

FCRL3 +

P=0.0038

Month

Cu

mu

lati

ve

OS

0

200

400

600

800

Zap-70- ZAP-70+

P<0.0001

FC

RL

3 e

xp

res

sio

n

0

10

20

30

40

Zap-70 - Zap-70 +

P<0.0001

FC

RL

5 e

xp

res

sio

n

0 50 100 150 200 2500

20

40

60

80

100

FCRL5 -

FCRL5 +

P=0.0006

Month

Cu

mu

lati

ve

TF

S

0 100 200 3000

20

40

60

80

100

FCRL5 -

FCRL5 +

P=0.0232

Month

Cu

mu

lati

ve

OS

0 50 100 150 200 2500

20

40

60

80

100

PDE8A -

PDE8A +

P=0.0003

Month

Cu

mu

lati

ve

TF

S

0 100 200 3000

20

40

60

80

100

PDE8A -

PDE8A +

P=0.012

Month

Cu

mu

lati

ve

OS

0

5

10

15

Zap-70 - Zap-70 +

P<0.0001

PD

E8

A e

xp

res

sio

n

0.0

0.2

0.4

0.6

0.8

1.0

Zap-70 - Zap-70 +

P=0.0102

ITG

A4

ex

pre

ss

ion

0 50 100 150 200 2500

20

40

60

80

100

ITGA4 -ITGA4 +

P=0.3558

Month

Cu

mu

lati

ve

TF

S

0 100 200 3000

20

40

60

80

100 ITGA4 -ITGA4 +

P=0.0075

Month

Cu

mu

lati

ve

OS

A.

B.

C.

D.

E.

F.

G.

H.

I.

J.

K.

L.

M.

N.

O.

P.

Q.

R.

… but also TLR7, LPL, PCDH9, MYBL1, …

TFS OS

PDE8A: a novel therapeuthic target ?

R1 R2

R3 R4

100 101 102 103 104Annexin Log

100

101

102

103

104

IP L

og

4.7%

36.5%

R1 R2

R3 R4

100 101 102 103 104

Annexin Log

100

101

102

103

104

IP L

og

25.7%

56.2%

Control (ethanol) Dipyridamol 15µM

Annexine V - FITC

Propidium Ioide - PE

Gene set enrichment analysis

GO categories Description P-value

GO0015629 actin cytoskeleton 0.0021

GO0030036 actin cytoskeleton organization and biogenesis 0.0027

GO0030029 actin filament-based process 0.0014

GO0008154 actine polymerization and/or depolimerization 0.0010

GO0007155 cell adhesion 8.10 -7

GO0016387 cell-cell adhesion 0.0053

GO0007160 cell-matrix adhesion 0.0003

GO0006935 chemotaxis 0.0023

GO0005856 cytoskeleton 0.0002

GO0007010 cytoskeleton organisation and biogenesis 0.0009

GO0040011 locomotion 0.0060

GO0005874 microtubule 0.0003

GO0007018 microtubule based movement 0.0108

Kegg Pathway Description P-value

hsa04514 Cell adhesion molecules (CAMs) 0.0020hsa04530 tight junction 2.6.10

-7

hsa04520 adherens junction 2.5. 10-6

hsa04540 gap junction 3.9 . 10-5

hsa04670 transendothelial leukocyte migration <10-7

hsa04810 regulation of actin cytoskeleton <10-7

hsa04510 Focal adhesion <10-7

Broad Pathway

0.0121

0.0069

0.0011

0.0005

0.0004

1.9 . 10-5

cell_adhesion_h

h_ST_Integrin_Signaling_Pathway

cell_adhesion_receptor_activity_h

SIG_Regulation_of_the_actin_cytoskeleton_by_Rho_GTPases_h

cell_adhesion_molecule_activity_h

cell_motility_h

Biocarta pathways

h_lympathway adhesion and diapedis of lymphocytes 0.0012

h_integrinPathway Integrin Signaling Pathway 0.0015

h_lymphocytePathway Adhesion Molecules on Lymphocyte 0.0012

h_cxcr4Pathway CXCR4 Signaling Pathway 0.0006

Lagneaux L. et al, Blood, 1998

Ghia P., Semin Cancer Biol, 2002

ZAP70 and CXCR4 modulation by ME

CXCR4 on fresh samples

Adhesion and migration

MicroRNA expression

TFS OS

Conclusions

ZAP70 + and – patients :

distinc gene expression profiles

new prognostic factors : genes and microRNA

new potential therapeutic target

gene set implicated in migration and adhesion

ZAP70 + : better adhesion and migration capacities into their microenvironment

Better survival of ZAP70 + cells and the agressiveness of the disease

CLL sample collection:

Nathalie Meuleman

Dominique Bron

Philippe Martiat

Philippe Herman (St-Pierre Hospital)

Alain Kentos (Erasme Hospital –ULB)

PhD director:

Laurence Lagneaux

and ALL MEMBERS of the Laboratory of Experimental Hematology of Pr. Philippe Martiat

Thank you for your attention.

(Bordet Institute – ULB)

Acknowledgments

This work was financed by F.R.I.A. grant and the Télévie fund, both of which are affiliated with the F.R.S-F.N.R.S.