Biomarkers from the Tumor Microenvironment: Are they Useful?

George Coukos, M.D, Ph.D.Director, Gynecologic Malignancy Research Programs

University of Pennsylvania

Biomarkers from the tumor microenvironment

• Tumor microenvironment events affect tumor outcome

• Tumor microenvironment events are identifiable through cellular or molecular markers

• Markers are mechanistically relevant• Modulation of tumor microenvironment affects

tumor outcome• Modulation of tumor microenvironment can be

traced through these markers

Tumor Microenvironment

Cancer CellsCancer Cells

Immune Cells

Stroma Cells

Endothelial Cells

The Reductionist View A Heterotypic Cell Biology

Do Leukocytes Play a Role in Ovarian Cancer ?

CD45 CD11c CD3

Cellular BiomarkersIntratumoral T cellsT regulatory cellsVascular leukocytes

CD3+

Intratumoral T cells

54.8%

38.7%

6.5%N=174

Zhang et al., NEJM 2003

Impact of TILs on Outcome in Ovarian CancerStage III/IV – All patients (n=174)

PFS Survival

+ TILs- TILs

N=174

Zhang et al., NEJM 2003

TILs correlate with tumor-reactive PBLs

Patient No. ELISPOT TILs

OV22 + +

OV23 -/+ +

OV24 -- --

OV26 -- --

OV27 + +

OV28 -- --

OV29 + +

OV31 + --

OV32 + +

OV34 -- --

P < 0.045

Intratumoral T cells exhibit oligoclonal expansion

A

B D

C

Vβ8

TILs Recognize TAAs in Ovarian Cancer

CD

8S

SC

CD

14-C

D4

Her2-369-APCEBV-PE TERT-PE Her2-689-PETax-PE

CD8FSC

.

Tumor Biopsyor Ascites

Disassociation

PE

APC-CY7

PE-Cy5

APC-Cy7

+Flow Cytometric

Analysis

Antigen Specific TILs

Cy5

PE

FreshlyIsolated

.

Bob Vonderheide

Proposed Biomarkers for Tumor Immune Surveillance

• Intratumoral CD8+ T cells

• IFN-γ, MIG, IP-10

• pLCK, pZap-70

• NKG2D ligands

CD4+CD25+ Treg

R3 live cells/CD3+

Ascites panel.019

1 10 100 1000 10000

FL2-H: CD25 PE

1

10

100

1000

10000

FL4-H: CD4 APC

R3 live cells/CD3+ Ascites panel.018

1 10 100 1000 10000 FL2-H: CD25 PE

1

10

100

1000

10000

FL4-H: CD8 APC

26.3

CD25

CD8 CD4

Numbe r of CD4+ cells added

0 1000 5000 10000 200000

20

40

60

80

100

120

CD4+C D25+

CD4+C D25-

B

% o

f M

axim

um

Res

pons

e

Numbe r of CD4+ cells added

0 1000 5000 10000 200000

20

40

60

80

100

120

CD4+C D25+

CD4+C D25-

B

% o

f M

axim

um

Res

pons

e

Numbe r of CD4+ cells added

0 1000 5000 10000 200000

20

40

60

80

100

120

CD4+C D25+

CD4+C D25-

CD4+C D25+

CD4+C D25-

B

% o

f M

axim

um

Res

pons

e

CD4+CD25+

CD4+CD25-

CTLA-4

CTLA-4

CD4

CD

25

CD4+CD25+

CD4+CD25-

CTLA-4

CTLA-4

CD4

CD

25

Woo et al., Cancer Res 2002; J Immunol 2003

Regulatory CD4+CD25+ T Cells in Ovarian Cancer

0

10

20

30

40

50

0 5 15 25 35 45 55 65 75 85 95

mRNA FOXP3 (M idpoint)

Pe

rce

nt

Curiel, Coukos et al., Nature Med 2004

Proposed Biomarkers for Tolerance

• CD4+CD25+ T cells

• foxP3

• GITR, CTLA-4

• Neuropilin-1

• IL-10, TGF-β• PD-1

CD11c+ cells with DC / endothelial phenotype and

vasculogenic competence

Vascular Leukocytes

Conejo-Garcia et al., Nature Med 2004

Ascites

Endothelial cells and vascular leukocytes coexist in human ovarian cancer

Conejo-Garcia et al., Blood 2004

Proposed Biomarkers for Angiogenesis

• CD45+CD11c+VE-cadherin+ cells

• VEGF-A

Activity of Biological Therapy in Ovarian Cancer

Hodi et al., PNAS 2003

0 1 2 3 4Years

0.2

0.4

0.6

0.8

1.0

PF

S (

%)

CP (n=68)

CP+IFN-γ (n=65)

P=0.031

Conejo-Garcia et al. Nat Med. 2004Windbichler GH, et al. Br J Cancer. 2000

Acknowledgements

Coukos Lab•Lin Zhang•Jose R. Conejo-Garcia•Fabian Benencia•Ron Buchanovitz•Alisha Mohamed•Cecilia Coureges•Heidi Gray•Ann Jenkins•David Holtz•Eugene Kang•Cristina Vezzani•Giorgia Regnani•Shun Liang•Karen Nishida

Dept. Biostatistics•Phyllis Gimotty

Abramson•Carl June

U. of Torino•Dionyssis Katsaros•Marco Massobrio