1

Eosinophilic disorders

PKI

Prof. Dr. Hans-Uwe Simon

Institutsdirektor

Institute of Pharmacology

http://www.pki.unibe.ch/

Lectures: Clinical Immunology

Bern, May 21, 2015

(Hyper)eosinophilia

Intrinsic eosinophilic disorders Extrinsic eosinophilic disorders

T cells Tumor cells

- Allergic diseases

- Autoimmune diseases

- Infectious diseases

- Graft-versus-host diseases

- Immunodeficiencies

- Clonal T cell diseases

- Drug-induced diseases

- Hodgkin‘s lymphomas

- Cutaneous T cell

lymphomas

- Acute lymphoblastic/-

cytic leukemias

- Langerhans cell

histiocytos is

- Epithelial cancers

Multipotent myeloid

stem cells

Pluripotent hematopoietic

stem cells

- Chronic eosinophilic leukemias

- Acute myeloid leukemias

Problem inside of

eosinophils

Problem outside of

eosinophils

Mutations Cytokines

- Chronic myeloid leukemias

- Chronic eosinophilic leukemias

- Acute myeloid leukemias

- Myelodysplastic syndromes

- Other myeloproliferative

diseases

Simon D & HU, J. Allergy Clin. Immunol. 2007

(Idiopathic) hypereosinophilic syndrome

(Idiopathic) hypereosinophilic syndromes (HESs)

1. Eosinophils > 1500/mm3 - with exceptions

(2. Persistant for more than 6 month)* - at 2

occasions

3. Exclusion of secondary causes of

eosinophilia

(4. Evidence for organ damage/dysfunction)* -

only when blood eosinophilia of < 1500/mm3

organ dysfunction is required

5. Heterogenous disorder

*No longer valid: Redefinition of HESs

Simon HU et al., J. Allergy Clin. Immunol. 2010

Why and how do eosinophils mediate

immunopathology?

MBP

Nucleus

Golgi

ECP

EPO

EDN

Toxicity towards

pathogens

and tissues

Eosinophils can be stained by Congo Red

CSS

Heart

Do eosinophils contain intracellular amyloid depositions?

Eosinophils are stained with amyloid-reacting dyes

Blo

od

E

oE

Thioflavin T p-FTAA

Congo Red

Bright Field

Fluorescence OC anti-MBP Overlay

CS

S

Sc

his

tos

om

a

Eo

E

CS

S

Sc

his

tos

om

a

Blo

od

Birefringence

Fluorescence

Congo Red

Thioflavin T p-FTAA OC anti-MBP Overlay

MBP is stored as an amyloid within eosinophil granules

2

MBP mediates toxic effect on bronchial epithelial cells:

Critical role for aggregation

0

20

40

60

80

0

20

40

60

80

BE

AS

-2B

ce

ll d

ea

th (

%)

a

b c

BE

AS

-2B

ce

ll d

ea

th (

%)

BE

AS

-2B

ce

ll d

ea

th (

%)

hA

EC

ce

ll d

ea

th (

%)

Me

diu

m

Co

ntr

ol

Sta

uro

sp

ori

ne

MB

P

OC

Hep

ari

n

MB

P +

OC

MB

P +

he

pa

rin

MB

P +

Q-V

D

Me

diu

m

Co

ntr

ol

Sta

uro

sp

ori

ne

MB

P

OC

He

pa

rin

MB

P +

OC

MB

P +

he

pa

rin

MB

P +

Q-V

D

Time (h) Peptide (mM) 0 1 3 5 0 8 1 2 3 4 5

p < 0.001 p < 0.001

p < 0.005

p < 0.005

p < 0.001 p < 0.001

0

20

40

60

80

100

Control

MBP

0

20

40

60

80

100

Control

MBP

In vitro

0

5

10

15

MB

P

Co

ntr

ol

MB

P +

OC

MB

P +

he

pa

rin

TU

NE

L in

ten

sit

y (

AU

)

MBP Control MBP + OC MBP + heparin

Human skin

Mouse skin

0

10

20

30

TU

NE

L in

ten

sit

y (

AU

)

p < 0.01

p < 0.01 p < 0.001

p < 0.01

p < 0.01 p < 0.001

MBP mediates toxic effect on keratinocytes:

Critical role for aggregation

In vivo and ex vivo

Take-home

message

b. Mature

granules

d. Piecemeal degranulation

e. Excessive eosinophil

infiltration & degranulation

Nucleus

c. Activated

granules

Golgi

a. Granules biogenesis

Killing of pathogens (bacteria/helminths/fungi)

Extracellular protein deposition

Soragni A et al.,

Mol Cell 57 (2015), 1011-1021

Undefined Associated Overlap Familial

Benign

Lymphocytic forms

Myeloproliferative forms

Features of myeloproliferative

disease without proof of clonality

Populations of T cells secreting

eosinophil hematopoietins

Asymptomatic with no

evidence of organ

involvement

Family history of

documented persistent

eosinophilia of unknown

cause

Organ restricted eosinophilic

disorders

Eosinophilia in association with a

defined diagnosis, such as IBD or

CSS

Clonal eosinophilia,

including FIP1L1/

PDGFRA- positive CEL

CEL

Hypereosinophilic syndromes (HESs)

Cyclical angioedema

and eosinophilia

Myeloproliferative HES

Episodic

Clonal T cells

No T cell clone

T cells often exhibit an abnormal immuno-

phenotype

Unclear

T cell activation

Idiopathic HES

No evidence for T cell-mediated

eosinophilia and myeloproliferative

disease

Classification of hypereosinophilic

syndromes (HESs)

Simon HU et al., J. Allergy Clin. Immunol. 2010

Search for

common

triggers

Extrinsic or intrinsic

disorder?

IL-5, IL-3 and

GM-CSF

analysis

(serum, T cell

supernatants,

immunohisto-

chemistry)*

- IgE-mediated allergy

- Infection

- Drug-induced

Extrinsic

eosinophilic

disorder?

- Non-IgE-mediated allergy

- Clonal T cell disease

- Immunodeficiency

- Tumor-associated

- Idiopathic

Intrinsic

eosinophilic

disorder?

- Stem cell disorder

- Idiopathic

yes

yes no

no

Work up for

underlining

disease

Hematological

work up

1

2

3 3

Simon D & HU, J. Allergy Clin. Immunol. 2007

(Hyper)eosinophilia Abnormal expression of lineage-

associated proteins in HES

NEJM 341 (1999), 1112-1120

3

Abnormal T cell clones express IL-5

A B

GM-CSF IL-5

Follow-up of patients with a T cell clone

Int Arch Allergy Immunol

124 (2001), 242-245

The lymphocytic form of HES

1. Increased levels of eosinophil differentiation

factors and TARC in blood.

2. Abnormal (or normal?) immunophenotype of T

cells. Presence or absence of a T cell clone. Often

defects in Fas signaling.

3. Chronic, stable disease. However, there is the

possibility of a transmission into a T cell

lymphoma – IFN-alpha (?) is a possible treatment

choise.

4. Eosinophils are activated and demonstrate

delayed apoptosis in vitro.

Simon HU et al.

J. Exp. Med. 1996

N. Engl. J. Med. 1999

N. Engl. J. Med. 2003

Eur. J. Immunol. 2003

Eur. J. Immunol. 2006

Anti – IL-5 antibody treatment*

0

5

10

15

20

25

30

35

40

0 1 7 14 21 28 42

Patient 1

Patient 2

Patient 3

i

i

i

0

20

40

60

80

100

120

140

0 1 7 14 21 28 42

i

Time [days]

Patient 1

Patient 2

Patient 3

Blo

od

e

os

ino

ph

ils

[%

]

Time [days]

2 x 750 mg Mepolizumab (arrows) S. Plötz et al.

Anti – IL-5 antibody treatment* reduces

clinical symptoms

* 2 x 750 mg Mepolizumab (arrow)

Patient 1 Patient 2 Patient 3

Post

Pre

S. Plötz et al.

Anti – IL-5 antibody treatment* reduces

eosinophil infiltration of the skin

* 2 x 750 mg Mepolizumab

20x

63x 63x

10x

Before After (2 weeks)

S. Plötz et al.

4

Summary: Anti –IL-5* in HES

* 2 x 750 mg Mepolizumab

1. Immediate reduction of eosinophil numbers in blood

(within 24 h).

2. Dramatic reduction of serum ECP levels, but slightly

delayed compared to eosinophil numbers.

3. Eosinophils in skin biopsies were reduced, but

significant numbers (10-50%) were still detectable.

4. Reduction of skin-infiltrating CD4+ and CD8+ T-cells.

5. Reduced numbers of Th2 effector memory T-cells in

blood.

6. Clinical improvement including markedly reduced itch

within three days.

N. Engl. J. Med. 349 (2003), 2332-2337.

Use of anti – IL-5 antibodies

in clinical studies

Eosinophilic dermatitis

Hypereosinophilic syndrome

Bronchial asthma

Eosinophilic sinus disease

Atopic dermatitis

Eosinophilic esophagitis

Ploetz SG et al., N Engl J Med 2003

Rothenberg M et al., N Engl J Med 2008

Leckie MJ et al., Lancet 2000

Flood-Page P et al., Am J Respir Crit Care Med 2007

Nair P et al., N Engl J Med 2009

Haldar P et al., N Engl J Med 2009

Gevaert P et al., J Allergy Clin Immunol 2006

Oldhoff JM et al., Allergy 2005

Stein ML et al., J Allergy Clin Immunol 2006

Straumann A et al., Gut 2010

IL-5

Targeted antibodies

C

A

Sabutoclax

Mcl-1

Apoptosis

Omacetaxine

Obatoclax

D

Restoration of protein degradation

TKI

Inhibitors of autophagy

Proteasome

Autophagosome

Autophagy

B

Constitutively active FIP1L1-PDGFRA kinase

CXCR4

Plerixafor

BM homing F

Hypoxia

PR-104H Glycolysis

PR-104A

HIF-α

G DNA-crosslinking Inhibition of DNA synthesis and repair

Pan-histone-deacetylase

inhibitors

Altered transcription

Histone-deacetylase

Indomethacin

E

SDF-1

Eosinophils as drug targets

Radonjic-Hoesli S et al.:

Annu Rev Pharmacol Toxicol 55

(2015), 633-656

Search for

common

triggers

Extrinsic or intrinsic

disorder?

IL-5, IL-3 and

GM-CSF

analysis

(serum, T cell

supernatants,

immunohisto-

chemistry)*

- IgE-mediated allergy

- Infection

- Drug-induced

Extrinsic

eosinophilic

disorder?

- Non-IgE-mediated allergy

- Clonal T cell disease

- Immunodeficiency

- Tumor-associated

- Idiopathic

Intrinsic

eosinophilic

disorder?

- Stem cell disorder

- Idiopathic

yes

yes no

no

Work up for

underlining

disease

Hematological

work up

(Hyper)eosinophilia

1

2

3 3

Simon D & HU, J. Allergy Clin. Immunol. 2007

The myeloproliferative form of HES (FIP1L1-PDGFRA positive chronic eosinophilic leukemia)

1. Normal IL-5 and B12 levels, increased tryptase

levels in blood.

2. No cytogenetic abnormalities, no increased

numbers of blasts in bone marrow.

3. FIP1-like 1 – platelet-derived growth factor

receptor a gene fusion.

4. Treatment with imatinib mesilate (tyrosine kinase

blocker).

5. Some patients develop aquired imatinib

resistance due to T674I mutation NEJM 348 (2003), 1201-1214

Blood 101 (2003), 4660-4666

Interstitial deletion

Chromosome 4q12

cen tel FIP1L1 PDGFRA

Encodes tyrosine kinase

Gene fusion PCR amplification

EO

L-1

cell l

ine

HE

S #

542

No

rmal

Neu

tro

ph

ils

H2O

DN

A m

ark

er

Detection of the

FIP1L1-PDGFRA gene fusion

Cools J et al.

N. Engl. J. Med. 348 (2003),

1201-1214

5

Case report

• 59-year-old man suffering from hypereosinophilic syndrome since 1989

• Developed end-organ damage (endocarditis fibroblastica, pulmonal hypertension, cortical ischemia, polyneuropathy, splenomegaly, renal insufficiency, restrictive pulmonary disease)

• Bone marrow: no cytogenetic abnormalities or increased numbers of blasts

• Did not respond to prednisolone, interferon-alpha, hydroxyurea, and imatinib mesilate

0

5000

10000

15000

20000

0 10 20 30 40

Time (weeks)

Eo

sin

op

hil

s (

pe

r m

m3)

100

200

Imatinib 400 mg/d

Primary imatinib resistance

Simon D. et al.: J Allergy Clin Immunol. 2008

560 bp

Imatinib-

resistant

patient

Wild-type

PDGFRA

A B

FIP1L1-PDGFRA and mutation analysis

Mutation 1 Mutation 2

TCT CCT CTA CCA

S601P L629P

Pati

en

t

Co

ntr

ol

EO

L-1

0

5000

10000

15000

20000

0 10 20 30 40 50 60 70

Time (days)

Eo

sin

op

hil

s (

pe

r m

m3)

Imatinib

400 mg/d Mepolizumab

750 mg/application

Imatinib resistance also in conjunction with

anti-IL-5 antibody treatment

Simon D et al., J Allergy Clin Immunol 2008

A

B

FIP1L1 PDGFRA

COOH NH2

Mechanism of imatinib resistance?

Simon D et al., J Allergy Clin Immunol 2008

Superposition of the active and inactive

kinase (structural modeling analysis)

Salemi S et al., Allergy 2009

6

Conclusions

• First reported FIP1L1-PDGFRA positive CEL/HES patient with primary imatinib resistance

• Concurrent neutralization of IL-5 was not effective

• Two new mutations within the PDGFRA kinase domain were detected that favor the active stage of the kinase, explaining imatinib resistance

• Whether one of the newly identified mutations alone is sufficient to cause imatinib resistance remains to be investigated

• There is a need to develop additional therapy for FIP1L1-PDGFRA positive CEL/HES patients

Salemi S et al., Allergy 2009

Simon D et al., J Allergy Clin Immunol 2008 and

IL-4

Th0 cell

IL-5

Eotaxin-1 (CCL11)

TSLP

PGD2

IL-13

IL-4

Mast cell

Eosinophil

CRTh2 antagonists (see Table 1)

Dupilumab

Pitrakinra

TSLPR

CCR3

TPI ASM8

Benralizumab

IgE

PGD2 IL-5

B cell

IL-5R

IL-4R

IL-13R

IL-4Rα

IL-4Rα

CRTh2

βγ

α

IL-5

Basophil

IL-4 IL-13

IL-13Rα1

γc

IL-4

Th2 cell

PGD2 IL-13

IL-4, IL-13

Epithelium

TSLP

Eotaxin

AMG 157

Bertilimumab

Mepolizumab Reslizumab

Tralokinumab Lebrikizumab

Omalizumab B

C

D

E

F

IL-7Rα TSLPR

A

IgE FcεRI

γ β α

GW766944

Th2 cells, Th2 cytokines and eosinophils as

drug targets in allergic diseases

Radonjic-Hoesli S et al.: Annu Rev Pharmacol Toxicol 55 (2015), 633-656

Relative Change in Forced Expiratory Volume in 1 Second (FEV1) in the Intention-to-Treat

Population.

Straumann A et al. Allergy 2013;68:375-385.

A CRTH2 antagonist mediates anti-eosinophil

activities in EoE

1 2 CD3 CRTH2 Overlay

CRTH2+CD3+

CRTH2-CD3+

Eo

sin

op

hils

T

ce

lls

Baseline Post-treatment

CRTH2 antagonism is also clinically effective in allergic rhinitis and asthma