intratumoral mrna expression of genes involved in angiogenesis and hif-1 pathway to predict outcome...

Intratumoral mRNA expression of genes involved in angiogenesis and HIF-1 pathway to predict outcome to

VEGFR tyrosine kinase inhibitor (TKI) in patients enrolled in CONFIRM1 and CONFIRM2

Peter M. Wilson, Ph.D.D. Yang, M. M. Shi, W. Zhang, C. Jacques, J. C. Barrett, K. Danenberg,

T. Trarbach, G. Folprecht, G. Meinhardt, H. J. Lenz Department of Pathology and Division of Medical Oncology

GI Oncology Program

The Sharon E Carpenter Laboratory

USC Norris Comprehensive Cancer Center

USC Keck School of Medicine

Los Angeles, CA.

Disclosures

Response Genetics

Novartis Pharmaceutical Corp

Bayer Schering Pharma

Background

Colorectal cancer is 3rd most common cause of cancer in the US.

In 2008 there will be an estimated†: 149,000 cases 50,000 deaths

Prognosis depends on: Stage Patient performance status Tumor differentiation Molecular Markers (MSI, 18q status)

† - American Cancer Society – Estimated New Cancer Cases and Deaths, US, 2008

CONFIRM Trials

PTK/ZK – Oral anti-angiogenic agent1

Competitive inhibitor at the ATP-binding site of VEGF receptors 1-3, platelet-derived growth factor and c-kit.

1. Wood JM, Bold G, Buchdunger E, et al. Cancer Res, 2000; 60:2178-89.

Randomized, double-blind, placebo-controlled, phase III trials in patients with metastatic adenocarcinoma of the colon or rectum.

VEGFR-1

/ Flt-1

VEGFR-2

/ KDR

VEGFR-3

/ Flt-4

• Angiogenesis• Lymphangiogenesis

• Metastasis

X X X X X XPTK/ZK PTK/ZK PTK/ZK

VEGF-AVEGF-B

PIGF

VEGF-AVEGF-CVEGF-D

VEGF-CVEGF-D

PTK/ZK Inhibits All Known VEGF Receptors

Extracellular

Intracellular

Inhibition of angiogenic signals1

Inhibition of metastasis

Sensitization to chemo-therapeutics2

2. Sini P, Samarzija I, Baffert F, et al. Cancer Res 2008;68:1581-1592.

Bevacizumab

1. Wood JM, Bold G, Buchdunger E, et al. Cancer Res, 2000; 60:2178-89.

CONFIRM Trials

Randomized

FOLFOX4/PTK 585 Patients

FOLFOX4/Placebo 583 PatientsCONFIRM 1

• 1ST Line• 1168 Patients

Randomized

FOLFOX4/PTK 429 Patients

FOLFOX4/Placebo 426 PatientsCONFIRM 2

• 2ND Line• 855 Patients

Progressed from irinotecan-based

therapy

Stratification Factors

PS 0, 1-2

LDH ≤, > 1.5 x ULN

ULN – Upper limit of normal; PS – Performance status; LDH – Lactate dehydrogenase

Response to PTK/ZK

Overall Response

Complete Response

Partial Response

Stable Disease

Progressive Disease

PTK/ZK Placebo

46 47 19 18

4 5 2 1

42 43 17 17

32 32 42 43

FOLFOX4 +

17 17 34 37

CONFIRM1 CONFIRM2

PTK/ZK Placebo

PFS and OS in CONFIRM1

Overall Survival High LDHProgression Free Survival High LDH

Hecht et al. European Cancer Organization,2007

No statistically significant increase in PFS or OS in total population with PTK/ZK treatment

PFS and OS in CONFIRM2

Overall Survival High LDH

Progression Free Survival Progression Free Survival High LDH

Kohne et al. Journal of Clinical Oncology, 2007 ASCO Annual Meeting Proceedings Part I. Vol 25, No. 18S, 2007: 4033

Overall Survival

M

Clinical Significance of LDH

Patients with high serum LDH appear to benefit most from PTK/ZK

Facilitates anaerobic glycolysis Marker for increased tumor burden and

aggressive disease Associated with poor prognosis

LDH may be a surrogate marker for HIF-1 signaling and proangiogenic propensity

M

M

M

LDHA Gene

M

LDH5

Enzyme

Koukourakis et al. Journal of Clinical Oncology, 2007 ASCO Annual Meeting Proceedings Part I. Vol 25, No. 18S (June 20 Supplement), 2007: 4107

Koukourakis et al. Journal of Clinical Oncology, 2006 Sep 10;24(26):4301-8.

Koukourakis et al. Clin Exp Metastasis. 2005;22(1):25-30.

LDH Correlates with HIF1 Signaling

Serum LDH level was associated with activation of HIF-related

intratumoral gene expression in 92 specimens from 36 mCRC patients

Azuma et al. Pharmacogenomics. 2007 Dec;8(12):1705-13.

VEGFR1

VEGFA

Serum LDHp=0.006

p=0.004

mCRC – metastatic colorectal cancer

HIF2α

VEGF

LDHA

p=0.001

HIF1α

Glut1

p=0.013p=0.044

p=0.007

Intratumoral

Objectives

Identify a panel of molecular markers predicting outcome to PTK/ZK in patients enrolled in CONFIRM1 and CONFIRM2

To measure intratumoral mRNA expression of genes involved in: Hypoxia Glycolysis Angiogenesis

HIF-1 Signaling

Gene Expression

Oncogene Signaling

PI3KPKCRASSRC

HIF-1α

HIF-1α

Hypoxia

Nucleus

VEGF (angiogenesis)

GLUT1 (glucose

transport)

LDH-A

(glycolysis)

EPO

(erythropoiesis)

VHL

NOS

ARNT

HRE

Normoxia

VHL

Methods I

191 formalin-fixed paraffin-embedded tumor samples were obtained from CONFIRM 1 and 2.

CONFIRM 1• 85 Patients

FOLFOX4/PTK 52 Patients

FOLFOX4/Placebo 54 Patients

CONFIRM 2• 106 Patients

FOLFOX4/PTK 43 Patients

FOLFOX4/Placebo 42 Patients

Specimens obtained at time of diagnosis.

Patient Characteristics

CONFIRM1 CONFIRM2

PTK/ZK Placebo PTK/ZK Placebo

Characteristics (n=43) (n=42) (n=52) (n=54)

Median age, y 63 62 64 62

Male/Female 31/12 25/17 34/18 40/14

(72%/28%) (60%/40%) (65%/35%) (74%/26%)

Low / High LDH 35/8 31/11 36/16 43/11

(81%/19%) (74%/26%) (69%/31%) (80%/20%)

WHO PS, 0/1 or 2 23/20 26/16 25/27 29/25

(53%/47%) (62%/38%) (48%/52%) (54%/46%)

Patient characteristics for CONFIRM 1 and CONFIRM 2 are well balanced between treatment groups, all p-values >0.05

Methods II

RNA Extracted

Reverse Transcription

PCR with TaqMan®

Data Analysis

RNA

cDNA

Laser Capture Micro-dissection

Candidate Genes

PTK/ZK

HIF-1α

Nucleus

ANGIOGENESIS• VEGF

• VEGFR 1/2

GLYCOLYSIS• Glut1

HYPOXIA• HIF1α• LDHA

Ability to predict outcome to PTK/ZK

Gene Expression Medians and Range

CONFIRM1 CONFIRM2

FOLFOX4+PTK/ZK FOLFOX4+Placebo FOLFOX4+PTK/ZK FOLFOX4+Placebo

Gene* N Median (range) N Median (range) N Median (range) N Median (range)

LDHA 42 1.11 (0.07-2.54) 41 1.09 (0.14-3.63) 52 0.91 (0.23-3.46) 50 1.04 (0.20-2.66)

Glut1 42 2.11 (0.26-23.86) 41 2.49 (0.36-38.59) 51 2.39 (0.45-13.37) 50 3.45 (0.50-35.48)

HIF1a 42 1.62 (0.59-3.68) 41 1.62 (0.31-4.98) 52 1.43 (0.60-6.45) 50 1.43 (0.55-3.65)

VEGF 42 6.21 (3.05-21.90) 41 5.79 (1.29-15.90) 52 5.71 (2.32-35.33) 50 7.57 (2.54-65.06)

VEGFR1 41 6.28 (0.49-23.25) 36 5.59 (0.56-28.06) 48 5.33 (1.53-20.62) 40 6.15 (2.54-17.83)

VEGFR2 42 1.91 (0.02-8.16) 41 1.64 (0.62-11.63) 50 2.11 (0.76-10.46) 50 1.95 (0.49-14.48)

No significant differences between trials or treatment groups; all p-values >0.05

Response to PTK/ZK

>LDHA 0.033

>Glut1 0.045

>VEGFR1 0.012

<HIF-1α 0.021

CONFIRM 1

Gene p-value Gene p-value

CONFIRM 2

Response to PTK/ZK

0%

10%

20%

30%

40%

50%

60%

70%

80%

<0.36 >0.36 <1.50 >1.50 <3.78 >3.78

Response Rate

N

Glut 1 VEGFR1 LDHA

6 35 11 30 10 30

P = 0.033 P = 0.045 P = 0.012

CONFIRM 1

Response to PTK/ZK

CONFIRM2

0%

10%

20%

30%

40%

50%

60%

<1.18 > 1.18

Response Rate

NHif1a

19 32

P = 0.021

Response to PTK/ZK

VEGFR1(n=42)

<3.85

Group 1

(10%)

≥ 3.85

10

1Group 3

(61%)32

20

Confirm 1

Hif1a(n=51)

≥1.21 <1.21

Group 2

(53%)19

10Group 1

(13%)32

4

Confirm 2

Response(n=93)

Multivariate Analysis:

- Serum LDH

- Age

- Gender

- Performance Status

PFS with PTK/ZK

>HIF-1α 9.4 v 3.5

>LDHA 11.3 v 7.6

>VEGFR2 8 v 4.1

CONFIRM 1

Gene Months Gene Months

CONFIRM 2

<HIF-1α 7.6 v 2.7

<LDHA 7.6 v 1.7

0.075

0.021 0.021

0.031

0.001

P Value

P Value

P Value

P Value

P Value

VEGFR2 CONFIRM1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 5 10 15 20 25 30 35 40

Months since randomization

Est

ima

ted

P

rob

ab

ility

o

f P

rog

ress

ion

-Fre

e S

urv

iva

lP for interaction between treatment and

VEGFR2 expression = 0.001

VEGFR2 <2.98 (n=34)

with PTK/ZK

VEGFR2 > 2.98 (n=8)with PTK/ZK

VEGFR2 <2.98 (n=34)w/o PTK/ZK

CONFIRM 1

VEGFR2 > 2.98 (n=7) w/o PTK/ZK

Confirm 2

Hif1a(n=52)

<0.85

Glut1(n=42)

≥3.25

≥0.85

Group 3HR=1.25(n=10)

Group 5HR=7.96(n=16)

<3.25

Group 4HR=3.02(n=26)

PFS with PTK/ZK

PFS(n=95)

LDHA(n=43)

≥ 0.92

Group 1HR=1(n=28)

< 0.92

Group 2HR=1.94(n=49)

Confirm 1

Multivariate Analysis:

- Serum LDH

- Age

- Gender

- Performance Status

Overall Survival with PTK/ZK

<VEGFR2 0.012

CONFIRM 1

Gene p-value Gene p-value

CONFIRM 2

<Glut1 0.021

Confirm 2

Glut1(n=52)

< 3.28

Glut1(n=36)

≥ 2.12 < 2.12

≥ 3.28

Group 5HR=12.9(n=16)

Group 3HR=2.00(n=17)

Group 4HR=4.32(n=19)

OS in CONFIRM 1 and 2

Overall Survival(n=95)

VEGFR2(n=43)

Confirm 1

< 1.78

Group 1HR=1(n=17)

≥ 1.78

Group 2HR=2.49(n=26)

Multivariate Analysis:

- Serum LDH

- Age

- Gender

- Performance Status

VEGFR2 Predicts OS in CONFIRM1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 6 12 18 24 30 36 42 48

Months since randomization

Est

imat

ed

Pro

bab

ilit

y o

f S

urv

ival

Adjusted P value = 0.012

VEGFR2 <1.76 (n=38)

VEGFR2 >1.76 (n=45)

CONFIRM 1

VEGFR2 >1.76

35.8 mo v 20 mo

Conclusions

Genes involved in HIF-1α and angiogenesis pathway are

associated with response and PFS with VEGFR TKI

treatment.

These molecular markers were independent of serum

LDH.

Genes in this pathway (VEGFR2) may have prognostic

value for overall survival.

Provides an initial panel of markers which warrant further

testing in clinical trials with future VEGFR TKIs.

Acknowledgements

The patients and their families and Investigators who participated in CONFIRM 1 and

2.

Response Genetics: Kathleen D. Danenberg.

USC Statistics: Dongyun Yang.

Novartis / Bayer Schering AG CONFIRM team:

Michael M. Shi, Christian Jacques, J. Carl Barrett, Bee Chen and Gerold Meinhardt.

Funding: Dhont Foundation and Carpenter Lab