8/15/2011

1

Preclinical and Clinical

Hypoxia Imaging:

Current and Emerging

Methods and Technology

Joint Imaging-Therapy Symposium

Moderator: Ted Graves, Stanford University

TU–E–214

Tumor Hypoxia

R.H. Thomlinson and L.H. Gray, Brit. J. Cancer, 1955

Capillary

Necrosis

Tumor

100 mm

Tumor Hypoxia

Necrosis Necrosis

Tumor Tumor

StromaStroma

Hypoxic

100-180mm100-180mm

Tumor Hypoxia

J.M. Brown and W.R. Wilson, Nat. Rev. Cancer, 2004

8/15/2011

2

Hypoxia and Radiation

L.H. Gray et al., Brit. J. Cancer, 1953

• Resistance to radiotherapy

• Oxygen dependence of DNA

damage

• Resistance to chemotherapy

• Poor drug delivery

• Decreased cell proliferation

• Aggressive phenotype

• Genomic instability

• Reduced apoptosis

• Increased metastasis

Hypoxic cancers exhibit:

Hypoxia and Outcome

B. Movsas et al., Urology, 2002D.M. Brizel et al., Radiother. Oncol., 1999

Prostate cancerHead and neck cancer

Immunohistochemistry

Imaging Oxygen

0.0

0.1

0.2

0.3

10 20 30 40 50 60 700 80

pO2 (mm Hg)

0.0

0.1

0.2

0.3

10 20 30 40 50 60 700 80

pO2 (mm Hg)

Eppendorff electrode

Near-infrared spectroscopy

19F MRI/MRS

BOLD MRI

EPR/Overhauser Imaging

Probe

Probe*

Cell Permeable

Cell Impermeable

Probe

O2

Probe

Probe*

Cell Permeable

Cell Impermeable

Probe

O2

106

105

104

103

102

101

100

0

10

20

30

40

50

60

70

80

90

100

F-Miso

Cu-ATSM

Oxygen Concentration (ppm)

Up

tak

e (

%)

PET

Symposium

Ted Graves “Preclinical Hypoxia PET for Treatment Prognosis and

Stanford University Response Assessment”

Dimitre Hristov “Overhauser Oxygenation Imaging: Physics,

Stanford University Instrumentation and Pre-Clinical Applications”

Robert Jeraj “Preclinical and Clinical Hypoxia Imaging: PET”

University of Wisconsin

Ralph Mason “NMR Assessment of Tumor Hypoxia and Oxygen

University of Texas SW Dynamics”

8/15/2011

3

2-Nitroimidazoles

N N

NO2

R N N

NH2

R

Reductases

O2

2-nitroimidazole

2-nitroimidazole uptake is dependent on:

• Vascular delivery

• Cell number

• Reductase expression

• Oxygen

Koch et al., 2010

EF5

18FNO2

N

N

O

NH

F

FF

F

EF5 is a 2-nitroimidazole-based hypoxia

probe that was developed by Cameron

Koch and Sydney Evans at UPenn as an

immunohistochemical marker in the 90’s.

Because of the 5 fluorines in EF5, a

radiolabeling procedure with 18F was

developed to produce a PET radiotracer

by Dolbier et al. in 2001.

EF5 is significantly more hydrophobic

than other nitroimidazole probes

(pimonidazole, FAZA, FMISO).

NO2

N

N

O

NH

F

F

F

+ 18F-19F

EF5 MicroPET

EF5

• Intravenous injection of ~200

mCi EF5

• Allow 2.5 hours for the probe to

distribute

• Acquire coincidence data for 10

minutes using an R4 MicroPET

scanner (Siemens, Knoxville TN)

• Reconstruct using OSEM

algorithm

• Calibrate reconstructed

intensities to units of %ID/g

• Calculate mean tumor:muscle

uptake ratio

EF5 MicroPET

0

0.5

1

1.5

2

2.5

3

3.5

HT29 (n=9)

A549 (n=9)

RKO (n=7) 22B (n=10)

FaDu (n=10)

SAS (n=6) SAS (n=7)

T/M

Variation in Mean EF5 T/M in Different Cell Line Derived Subcutaneous Tumors

Anti-EF5 stain H&E StainAnti-EF5 stain H&E Stain

FAZA

RKO Tumors (3hrs post-IP injection)

8/15/2011

4

0

0.5

1

1.5

2

2.5

3

HT29 2R

HT29 1L

HT29 3R

HT29 4R

HT29 2L

HT29 4L

HT29 3L

HT29 dyn

HT29 1R

HT29 dyn

A549 2R

A549 4L

A549 3R

A549 2L

A549 4R

A549 3L

A549 dyn

A549 1R

A549 1L

RKO dyn

RKO 4L

RKO 4R

RKO 3R

RKO 3L

RKO 2L

RKO 1L

T/M

Rat

ios

; c

m3

Tumors by Cell Line

T/M

Volumes

Hypoxia Imaging for Prognosis?

Bad?

Good?

R.S. Ali et al., in preparation

Hypoxia Imaging for Prognosis?

Treat subcutaneous A549

tumors after EF5 imaging

Associate response with pre-

treatment EF5 uptake

Good response?

Bad response?

Study Protocol

0

2

4

6

8

10

12

14

16

11/4/06 11/9/06 11/14/06 11/19/06 11/24/06 11/29/06 12/4/06 12/9/06 12/14/06 12/19/06

Ma

xim

um

Dia

me

ter

(mm

)

Days after tumor inoculation

Tumor

inoculation

EF5 PET EF5 PET EF5 PET EF5 PET

RT

0 5 10 15 20 25 30 35 40 45

Measure tumor size daily with calipers

Tumor Volume Response

0.90

0.95

1.00

1.05

1.10

1.15

1.20

1.25

-5.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00

No

rmal

ize

d T

um

or

Vo

lum

e

Days Post RT

0.90

0.95

1.00

1.05

1.10

1.15

1.20

1.25

-5.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00

No

rmal

ize

d T

um

or

Vo

lum

e

Days Post RT

0.90

0.95

1.00

1.05

1.10

1.15

1.20

1.25

-5.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00

No

rmal

ize

d T

um

or

Vo

lum

e

Days Post RT

0.90

0.95

1.00

1.05

1.10

1.15

1.20

1.25

-5.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00

No

rmal

ize

d T

um

or

Vo

lum

e

Days Post RT

8/15/2011

5

EF5 and RT Response

0.80

0.90

1.00

1.10

1.20

1.30

1.40

-5.00 5.00 15.00 25.00

0.80

0.90

1.00

1.10

1.20

1.30

1.40

-5.00 5.00 15.00 25.00

0.80

0.90

1.00

1.10

1.20

1.30

1.40

-5.00 5.00 15.00 25.00

EF5 T/M > 1.5 EF5 T/M < 1.5

Days post-RT

No

rma

lized

tu

mo

r vo

lum

e

1x10 Gy 1x20 Gy 1x40 Gy

EF5 and RT Response

0.80

0.90

1.00

1.10

1.20

1.30

1.40

-5.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00

0.80

0.90

1.00

1.10

1.20

1.30

1.40

-5.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00

0.80

0.90

1.00

1.10

1.20

1.30

1.40

-5.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00

0.80

0.90

1.00

1.10

1.20

1.30

1.40

-5.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00

2x5 Gy 2x10 Gy

4x5 Gy 4x10 Gy

EF5 T/M > 1.5 EF5 T/M < 1.5

Conclusions: Prognosis

• Pre-treatment EF5 uptake measured by PET predicts

response to large single fraction radiotherapy

• Pre-treatment EF5 uptake is less predictive of response

to fractionated radiotherapy• Agrees with conventional radiobiology

• EF5 may be useful in the selection of patients for

emerging hypofractionated radiotherapy regimens

• What to do for patients with EF5-avid tumors?• Fractionated radiotherapy

• Hypoxic cell radiosensitizers

• Hypoxic cytotoxins

EF5 Post-RT

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Pre-RT Post-RT

Me

an

%ID

/g

1x20 Gy Control

8/15/2011

6

OxyluciferinLuciferin

O2ATP

Firefly Luciferase

Luciferyl

Adenylate

hn

Bioluminescence

Luciferin

Luciferase-expressing

tumor

Bioluminescence Post-RT

0.00E+00

5.00E+08

1.00E+09

1.50E+09

2.00E+09

2.50E+09

3.00E+09

3.50E+09

4.00E+09

-10 0 10 20 30

To

tal R

ad

ian

ce

1x20 Gy

0.80

0.90

1.00

1.10

1.20

1.30

1.40

-5.00 5.00 15.00 25.00

No

rmal

ize

d T

um

or

Vo

lum

e

EF5 T/M > 1.5 EF5 T/M < 1.5

BLI and Oxygen

IK Cecic et al., Mol. Im., 2007

Bio

lum

ine

sc

en

ce 2.25 x 108

0

Photons/sec·cm2·sr

t = 0 minutes

Luciferin added

t = 5 minutes t = 10 minutes

Luciferin added

t = 15 minutes

Conclusions: Response

• Reoxygenation in this subcutaneous A549 model

appears to happen on a timescale of 5-10 days• Reductions in EF5 uptake relative to untreated controls

• Spike in BLI in tumors that were EF5-avid prior to radiation

• Imaging methods may shed light on the optimal dosing

regimen for hypofractionated radiotherapy

8/15/2011

7

Conclusions and Future Work

• Hypoxia PET can provide prognostic information for

tumors undergoing radiotherapy

• Importantly, radioresistance identified by pre-treatment

hypoxia PET may be overcome by dose fractionation• Concurrent radiosensitization/chemotherapy studies in progress

• Reoxygenation following radiotherapy is also evident

based on multimodal imaging data

• Many challenges remain for the physicist and the

biologist• Improving the quantitative accuracy of hypoxia PET

• Establishing the dynamic range of hypoxia PET and identifying

clinically-relevant stratification thresholds

• Proving the utility of hypoxia PET in prospective trials

Acknowledgements

Funding: NIH R01 CA131199, NIH 1P50CA114747-01, Varian Biosynergy

Imaging

Radiobiology

Laboratory

graveslab.stanford.edu

Stanford

Radiation

Oncology

Molecular Imaging

Program At Stanford