GastroIntestinal Stromal tumors:

Immunomodulation for Cure ?

Sylvie RusakiewiczGustave Roussy Institute

VillejuifFrance

Understanding the mechanism of action of chemotherapy/ Targeted-therapy…

Investigating Imatinib mesylate off targets : GIST

GISTGIST: : Gastrointestinal stromal TumorGastrointestinal stromal Tumor

Cajal cells origineCajal cells origine

cKIT cKIT ++ CD34 CD34++ (DOG-1 (DOG-1++))

15 cases/106/year; >50 years c-KIT/PDGFRA mutation screening

Clinical Classification Miettinen: (localisation, size, mitotic index, surgery marges)1st Line Treatment: Imatinib Mesylate

60%

<1%

1-2%

4-5%

4%

30%

Imatinib Mesylate (IM; Gleevec) revolutionized Imatinib Mesylate (IM; Gleevec) revolutionized the control of the diseasethe control of the disease

Courtesy Maria Debiec-Rychter - EGAM-EORTC 2005

(years)0 1 2 3 4

0102030405060708090

100

Wild type

Kit-ex9

Kit-ex11

3-year PFS: 32%3-year OS: 58%

63.7%

14.9%

16%

% P

FS

Imatinib mesylate revolutionized the control of GIST

Anti-cancer effect of IM via immunomodulation (1)

+

GIST

Teff/Treg ratio

HLA class I negative

Balachandran et al. Nat Med. Sept. 2011; Rusakiewicz et al., Cancer Res 2011

CD3

EMR8-5

CD8

H&E

MHC class I

N=39N=10

** P=0.002

Negative Heterogenous Positive

KITSCF

Homeostatic negative control IFN

NK activation

TNFα

Anti-cancer effect of IM via immunomodulation (2)

B7H6

NKp30

Ménard et al., Cancer res. 2009, Delahaye et al., Nat Med. 2011

Alternatively spliced NKp30 isoforms affect affect the prognosis of GISTthe prognosis of GIST

Ove

rall

surv

ival

Time from Gleevec* treatment (months)

Log-Rank P =0.001

GIST Profile AB (n=44)GIST Profile C (n=36)

Delahaye et al., Nat Med. 2011

M1 M2

IL-10C/EBP

Cavnar et al., J Exp Med. 2013

What is the relevance at the tumor What is the relevance at the tumor site ?site ?

GIST are enriched with Tumor infiltrating GIST are enriched with Tumor infiltrating Lymphocytes and with NK cells Lymphocytes and with NK cells

Via

bil

ity

mar

ker

CD

3

CD56CD45

C-K

IT

CD34

NK cells

GIST

GIST

BloodTumor

STS

*** NS

% o

f C

D3

- C

D5

6+/C

D4

5+

Via

bil

ity

mar

ker

CD45 CD45

CD

3

STS

Does immune infiltrates impact disease Does immune infiltrates impact disease outcome ?outcome ?

NKp46 CD3 FoxP3

PF

S (

%)

Time from diagnosis (years)

**P=0.001 NS

Time from diagnosis (years)Time from diagnosis (years)

N=53 N=53 N=53

**P=0.007

PF

S (

%)

PF

S (

%)

NKp46 and CD3 infiltrates are prognostic factors for progression-free survival in

localized GIST

Rusakiewicz et al., Can Res. 2013

Time from diagnosis (years)

**P=0.0062P

FS

(%

)

NKp46 and CD3 infiltrates add information to the Miettinen classification of GIST

Miettinen Score

***P=0.0001

Time from diagnosis (years)

PF

S (

%)

***P=0.0001

Time from diagnosis (years)

NK and Miettinen Score CD3 and Miettinen Score

Validation on a 2nd cohort of more advanced GIST in process

No IM IM

Peri-tumor

Tumor nest

Peri-tumor Tumor nest

At Diagnosis

NKp46+ cells accumulates in the peri-tumoral area. IM promotes NK relocalization in tumor nests

N=25 N=14

ns *** p<0.0001

N=34 N=19

Tumor NestsPeri-tumor

*** p=0.0001*p=0.05

N=11 N=19 N=7 N=9

NeoAdjuvantAdjuvantNeoAdjuvant

p=0.05

N=8 N=13 N=6 N=5

Adjuvant

No IMIM

Tumor NestsPeri-tumor

NK cells

N=34 N=19

* p=0.04

NeoAdjuvantN=11 N=18 N=7 N=9

Adjuvant

* p=0.036nsFoxP3 cells

KIT-11 Oncogene-desaddiction modulate immunity

Peri-tumor Tumor

HGF

MIF

s-VCAM-1

IL-16

F=4

F=2

F=1.2

F=3

No IM IM

F=10

F=1.5

F=1

F=1

Cxcl9

Cxcl10

Cxcl8

CcL2

F=1.5

F=2.5

F=1

F=1.5

F=1.5

F=2-10

F=1

F=1.5

=

=

No IM IM

N=5Collaboration with Dr N. Halama

Chemokines/cytokines analysis by micro-dissection of GIST frozen samples:running test

HGF

MIF

s-VCAM-1

IL-16

F=4

F=2

F=1.2

F=3

No IM IM

F=10

F=1.5

F=1

F=1

Cxcl9

Cxcl10

Cxcl8

CcL2

F=1.5

F=2.5

F=1

F=1.5

F=1.5

F=2-10

F=1

F=1.5

=

=

No IM IM

N=5

Chemokines/cytokines analysis by micro-dissection of GIST frozen samples:running test

N=7N=5N=3

NK Blood

NK TILs

CD56

CD

16

CD56

CX

3C

R1

CD56C

D9

4

CD56

NK

G2

A

CD56

NK

G2

C

CD15

8a/h

CD15

8e/k

CD15

8b

CD56CD56CD56

CD56

NK

p3

0

NK TILs have a particular phenotype: CD56bright CD16-KIR- and CD94+ but NKG2A-

NKG2C-

TNFα

Blood Tumor

IFN

γ

Blood

**

Tumor

CD

69

NK-TIL are activatedNK-TIL phenotype

Rusakiewicz et al., Can Res. 2013

Hypothesis: Are NK cells differentiated « in situ » ?

Progenitors in GIST

GIST is mesenchymal tumor: niche for NK cell differentiation ?

CD34

C-K

IT

CD45

Via

bil

ity

Mar

ker

Hypothesis: Are NK cells differentiated « in situ » ?

Progenitors in GIST

Stromal cells: niche

for NK cell differentiation ?

CD34

C-K

IT

CD45

Via

bil

ity

Mar

ker

NK-TIL microarray analysis from GIST specimens

What Immunomodulation is required to control What Immunomodulation is required to control GIST ?GIST ?

Incubation with Immuno-modulators in culture plate

Cytokine release Proliferation

GIST

Cut into pieces

Gentle MACs Dissociation

Enzymatic dissociation in 2 hours

Cell suspension:GIST and TILs

TILs Phenotyping

In-vitro assay to test TIL reactivity: Immunobiogram

+/- IFNa2b

ICS: Cytokine releaseat 24 hours

Proliferationat 3-6 days

CD107a degranulation test?

Apoptosis of tumor cells ?

Milieu seul  

mIgG1 11711

anti PD1 B7H1m.3

anti PDL1 5H1

anti CTLA-4 Ipilimumab 

PolyIC Ampligen

IFN-a (Roferon) Roferon

Ngoat IgG polyclonal

anti Trail neutra. TRAIL R3

mouse IgG2b 133303

anti FAS-L 100419

Rat IgG2b JES3-19F1

anti IL-10 R35-95

IL-2 Proleukine

Antibodies/recombinant molecules Read out

CytoPlex: Cytokine/Chemokines

releaseat 24 hours

In-vitro assay to test TIL reactivity: Immunobiogram

Patients Extend of disease Miettinen Classification

Targeted Therapy Type of targeted Therapy

VINJE Localized 1 None NA

CLEGI Localized 3 Adjuvant IM

DUCAN Localized 2 Adjuvant IM

TORFR Localized 1 Neo-Adj IM

BOUFE Metastatic 3 Adjuvant Masatinib

GADJU Localized ? Neo-Adj IM

JATMA Localized ? Neo-Adj IM

Feasibility test: GIST patients’ characteristics

Neo-Adjuvant

Cytokine secretion at baseline is not correlated with tumor invasion or CD45+ infiltrates frequency (but

neoAdj IM)

IFNg

Tumor invasion* **

* * * * * *

*

Immunobiogram: overall response

NK IFNg production

CD8+ KI67 CD4+ KI67

CD4

CD8

NK

% o

f Ki6

7 po

sitiv

e ce

lls%

of K

i67

posi

tive

cells

% o

f Ki6

7 po

sitiv

e ce

lls

Patient 3: DUC AN no IM, KIT Ex11

Medium

Iso aIL10

aIL10

Iso aFasL

aFasL

CD4 CD8 NKProliferation Assay Ki67 J6 Cytokines/Chemokines at 24hIL-10 blockade on GIST derived TILs

induce T cells proliferation at day 5

CD4

CD8

NK

% o

f pos

itive

cel

ls%

of p

ositi

ve c

ells

% o

f pos

itive

cel

ls

Patient 4: TOR FR IM, KIT Ex17

IFNγ (% of positive cells)

Not done

IL-10 blockade promote NK cells and CD8+ T cells IFNg in IM Neoadjuvant GIST specimens

Cytokines/Chemokines at 24h

IFNa2b responses in GIST patients

28

Patients Treatment Time Read out

VINJE None 24h ICS (NK cells :IFNγ et TNFα)ELISA: IFNγ

CLEGI None 24h ICS (NK cells :IFNγ et TNFα)

DUCAN None 24h ICS (NK cells :IFNγ et TNFα)

TORFR IM 24h ELISA: CXCL-10, IL-6, IL-1B

GADJU IM 24h ELISA: CXCL-10

JATMA None 24h ICS (NK cells :(IFNγ)ELISA: TNFα, IL-6, IL-1B, IL-10

6/7 GIST patients responded to IFN2b

Blocking IL-10 responses in GIST

29

Patients Treatment Time Read out

VINJE None 24h & J6 ELISA: IFNγ, TNFα, IL-5, IL13KI67:CD4, CD8 and NK proliferation

CLEGI None 24h & J6 ICS (NK cells :IFNγ )ELISA: TNFα, IL-5, IL-6, IL-13, CXCL-10KI67: CD4 and CD8 proliferation

DUCAN None 24h & J6 ELISA: IFNγ, TNFα, IL-2, IL-5, IL-9, IL-13KI67: CD4 and CD8 proliferation

TORFR IM 24h ICS (NK, CD4 and CD8 :IFNγ )ELISA: IFNγ, IL-1B, IL-4, IL-13, CXCL-10

BOUFE AB1010 24h & J6 ICS (NK cells :IFNγ and TNFα )ELISA: IFNγ, IL-5, IL-9, IL-13KI67:CD4 and CD8 proliferation

JATMA None 24h ELISA: TNFα, IL-1B, IL-6

6/7 GIST patients responded to anti-IL-10

Patients Treatment Time Read out

CLEGI None 24h ELISA: IL-13

DUCAN None J6 KI67: CD4 and CD8 Proliferation

Patients Treatment Time Read out

CLEGI None 24h ICS (NK cells :IFNγ et TNFα)

Blocking FasL responses: 2/7 GIST tested

Blocking TRAIL responses: 1/7 GIST tested

NK IFNg production

CD8+ KI67 CD4+ KI67

No response to ICP inhibitors !

AB

AB

C

C

C

C

AB

CD34

Tumeur

C-k

it

B7H1HLA-DR B7H6 MICA/B HLA class I B7H3

ND

ND

Isotype ctlMarker

NKp30

GIST don’t express B7H1 Ligand

X40 X40

GIST don’t express B7H1 Ligand:Validation by IHC

And T cells don’t express PD-1

1/20 postive, Pediatric GIST

GIST

PD-1

Tim-3 Tim-3

PD-1

Melanoma

IM/Glivec

GISTGIST

IFNa2bIL15« sushi »IL15IL-2

NKp30ab profil

Anti-GD2 and anti-NKp30 bispécifiques Ab

(or with anti-KIT in GIST?)

Neutralizing anti-sB7-H6 Ab

Towards novel therapeutic strategies

IM/Glivec

GIST

Methyltransferase inhibitors

Anti-IL-10 or anti-IL-10R Ab

NKp30c profil

siRNA NKp30c specificCollab. Dr. Mirco Ponzoni

GIST

IFNa2bIL15« sushi »IL15IL-2

NKp30ab profil

Anti-GD2 and anti-NKp30 bispécifiques Ab

(or with anti-KIT in GIST?)

Neutralizing anti-sB7-H6 Ab

Towards novel therapeutic strategies

Anti-GD2/Anti-B7-H6 T CARs

IM/Glivec

GIST

Methyltransferase inhibitors

Anti-IL-10 or anti-IL-10R Ab

NKp30c profil

siRNA NKp30c specificCollab. Dr. Mirco Ponzoni

GIST

IFNa2bIL15« sushi »IL15IL-2

NKp30ab profil

Anti-GD2 and anti-NKp30 bispécifiques Ab

(or with anti-KIT in GIST?)

Neutralizing anti-sB7-H6 AbAnti-GD2/Anti-B7-H6 T CARs

anti-PD-1 Ab, anti-PDL-1 ? Anti-CTLA-4, anti-Tim-3, anti-LAG3…

T cells

Towards novel therapeutic strategies

anti-KIT Ab anti-CSFR1

Gustave Roussy U1015/ CIC BT 1428Nicolas Delahaye

Michaela SemeraroCedric Menard

Nathalie ChaputLaurence Zitvogel

Alexander EggermontDepartment of Pathology,

Sarcoma committeeAxel LecesneOlivier Mir

Philippe TerrierJulien Adam

Paule OpolonDepartment of surgery

Sylvie BonvalotDepartment of Biostatistique

David Enot

Centre Leon Berard, Lyon

Jean-Yves Blay

CHU Jean Minhoz, Besançon

Loic Chaigneau

Centre Georges-Francois Leclerc, Dijon

Nicolas Isambert

Hopital Ambroise Pare, Boulogne

Jean-Francois Emile

Institut Bergonie, Bordeaux

Jean-Michel Coindre

Hopital Europeen Georges Pompidou, Paris

Bruno Landi, Anne Berger

Institut Mutualiste Montsouris, Paris

Thierry Perniceni, Pierre Validire

University of Heidelberg

Niels Halama

Acknowledgements All the patients….