Lecture by Prof. Lambin in the course: "Tumour Hypoxia: From Biology to Therapy III". For the complete e-Course see http://www.myhaikuclass.com/MaastroClinic/metoxia

1. Imaging of hypoxia
Philippe Lambin
Octobre 12th 2012
This course is funded with the support of the METOXIA project
under the FP7 Programme.

2. Learning objectives
1. Know the advantages and disadvantages of hypoxia
imaging
2. Be able to name the various hypoxia imaging
modalities with some basic features

3. Advantage of imaging: 3D & non invasive
Advantageous compared to IHC, gene-miRNA signatures
because tumours are heterogeneous in space
Courtesy of B. van der Kogel

4. Disadvantage: The oxygen distribution
inside a voxel
Measured voxel pO2 is average
pO2 of the cells inside it.
Each voxel contains a mixture
of cells at different oxygen
concentrations.
Ignoring this will affect the
correct choice of dose.
4

5. Why should we do it?
For prognosis (Give prognosis of disease outcome
regardless of therapy)
For prediction (Predict outcome with a specific therapy &
allow to adapt treatment (drugs, boost BTV…)
As endpoint (secondary or primary: e.g. reduction of
hypoxia with nitroglycerine patch, pattern of
relapse…)
For research purposes (no immediate clinical benefit)

6. How to measure tumor oxygenation ?
Non Imaging methods Imaging methods
• Polarographic measurements • Hypoxia markers
• Eppendorf® • Nitroimidazoles PET/SPECT
• CA9 ligands PET/SPECT
• Optical measurements • MR
• Phosphorescence • 19F relaxation
• Fluorescence (OxyLite ®) • BOLD
• DCE-MRI
• Hypoxia markers • 1H relaxation
• Nitroimidazoles
• Immunohistochemistry • EPR-related methods
• Gene signatures
• Blood biomarkers
B. Gallez, NMR Biomed. 2004, 17, 240

7. How to measure tumor oxygenation ?
Oxygen-sensitive
Real oxygen
measurements
measurements
• NMR
• Polarographic measurements • DCE-MRI
• Eppendorf® • BOLD
• Optical measurements • 1H relaxation
• Fluorescence (OxyLite ®)
• EPR-related methods • Hypoxia markers
• Nitroimidazoles
• MRI • Immunohistochemistry
19F relaxation
• • PET/SPECT
• CA9 ligands PET/SPECT
• Gene signatures
• Blood biomarkers

8. How to measure tumor oxygenation ?
Invasive Imaging
Or not immediately Clinically applicable
clinically applicable
• NMR
• Polarographic measurements • DCE-MRI
• Eppendorf® • BOLD
• 1H relaxation
• Optical measurements
• Fluorescence (OxyLite ®)
• Hypoxia markers
• Nitroimidazoles PET/SPECT
• EPR-related methods • CA9 ligands PET/SPECT
• MRI
• 19F relaxation

9. Ideal clinical oxygen imaging modality
• Able to distinguish normoxia/hypoxia/anoxia/necrosis
• Able to distinguish between perfusion-related and diffusion-related
hypoxia (sensitive to changes of hypoxia)
• Able to reflect cellular oxygenation in preference to vascular oxygenation
• Be applicable to any tumor site
• Simple to perform, non toxic and allowing repeated measurements
• Sensitive at pO2 relevant to tumor therapies A. Padhani, Eur. Radiol. 2007, 17, 861.
• Results independant of the timing of imaging

10. EPR (Electron Paramagnetic Resonance) Oximetry
B. Gallez, NMR Biomed. 2004,17, 240
O2 dependent broadening of the EPR
linewidth (LW) of a paramagnetic O2 nitrogen
sensor implanted in the tumor air
A particular material can be calibrated
in terms of the effect of oxygen on the
LW
3168 3318 3468
Magnetic Field (G)
When introduced in vivo, the 40
measurement of LW can be 30
interpreted in terms of oxygenation in
LW (G)
20
the vicinity of the probe 10
0
0 7 14 21
% O2

11. Non-invasive imaging of chronic and
cycling tumour hypoxia in xenografts with
EPR
Yasui et al., 2010, Can Res, 70:6427-6436.

12. EPR (Electron Paramagnetic Resonance) Oximetry
 
• Absolute measurement of pO2
• 1GHz: penetration depth of 1 cm
• High sensitivity at low pO2: variations
lower than 1 mm Hg can be • Not applicable immediately into the
measured clinic: pionneer clinical studies
• Minimally invasive: few ongoing in Dartmouth Medical
microparticles should be introduced School on superficial tumors
in the tissue (invasive only the first
time)
• Measurements can be repeated from
the same site over long periods of
time (hours, days, months)
N. Khan, Antiox. Redox. Signal. 2007, 9, 1169

13. 19F-relaxometry
• Intratumoral injection of PFC (i.e. hexafluorobenzene, single 19F line)
• Measurement of the R1 relaxation of 19F relaxation that is strongly dependent on pO2
• Maps of pO2
Mason RP et al, IJROBP 1998, 42, 747; and following works of Mason’s group

14. 19F-relaxometry
Rapid estimation of R1 using SNAP-IR sequence
Simultaneous monitoring using 19F-MRI and fiber optic probes
B. Jordan
MRM 2009, 61, 634-638

15. 19F-relaxometry
 
 Rather sensitive method  Problem to inject the PFC in the
 Quantitative method: pO2 entire tumor
values  The technique is likely to slightly
 No 19F NMR background overestimate pO2
signal in tissues  Not applicable for chronic
 Good temporal & spatial purposes (only acute studies)
resolutions for O2 mapping  Toxicity concerns with some PFCs
 Lack of translation into the clinic
(invasiveness, 19F coils)

16. DCE-MRI
Attempt to correlate DCE-MRI parameters with tumor hypoxia
Ktrans significantly higher
for radiation sensitive tumors
without hypoxia
than for radiation resistant
tumors with hypoxic regions
(Gribbestad., Dynamic Contrast-Enhanced
Magnetic Resonance Imaging in Oncology, 2006)
K Gullisksrud, Radiother. Oncol. 2011, 98, 360

17. Dynamic MRI of cervix carcinoma
A. T2-weighted spin echo image
a B. [Gd] max image
C. [Gd] time-to-peak image
2
4
3
A B 1
[Gd] 1
2
3
a
4
t
C C D D
Modified from:J. Barentsz

18. Tumor Oxygenation
Perfusion Oxygen
consumption

19. High O2 consumption rate by tumor cells
 Cancer cells consume oxygen at
high rate, even if they generally
present a highly glycolytic
metabolism
 This high consumption rate
significantly contributes to the
tumor hypoxia resulting from the
imbalance between oxygen
delivery and oxygen consumption

20. Strategies to decrease the oxygen consumption
by tumor cells and potentiate radiotherapy
Theoretical simulations:
To alleviate tumor hypoxia,
decreasing the O2 consumption rate of
tumor is more effective than increasing
oxygen delivery
Secomb, Acta Oncol. 1995 34, 313
Pre-clinical studies
• Meta-iodobenzylguanidine JE Biaglow, IJROBP 1998, 42, 871
• Nitric oxide donors B. Jordan, Int. J. Cancer 2004, 109, 768
• Insulin B. Jordan, Cancer Res. 2002, 62, 3555
• NSAIDs N. Crokart, Cancer Res. 2005, 65, 7911
• Glucocorticoids N. Crokart, Clin. Cancer Res. 2007, 13, 630
• SU5416, ZD6474 R. Ansiaux, Cancer Res. 2006, 66, 9698; Radiat. Res. 2009, 172, 584
• Propylthiouracil B. Jordan, Radiat. Res. 2007, 168, 428
• Arsenic trioxide C. Diepart, submitted

21. DCE-MRI
 
 Trend of perfusion  No absolute values of pO2
parameters to differentiate
 It is unlikely that a treshold value
between hypoxic vs normoxic
of a DCE-MRI parameter will
tumors
predict the radiosensitivity
 Clinically usable
 Tumor perfusion is not the main
or sole factor that is responsible
for the tumor oxygenation
(oxygen consumption)

22. BOLD-MRI – R2*
From fMRI …… to tumor oxygenation
Carbogen
breathing
Level of blood oxygenation 
Oxy-Hemoglobin: diamagnetic DeoxyHb  /OxyHb  blood content 
Deoxy-Hemoglobin: Paramagnetic  SI
S. Ogawa et al, PNAS 1990, 87, 9868
GS Karczmar, NMR Biomed 1994, 12, 881
SP Robinson, IJROBP 1995, 33, 855
F Howe, MRI 1999, 17, 1307

23. BOLD-MRI
T2* is sensitive to the relative Hb/HbO2 ratio in
vessels:
Blood oxygen saturation
Hematocrit
Blood volume

24. Basal R2* and tumor oxygenation
Correlation between pimonidazole uptake Inverse correlation between pimoninazole
and high R2* in prostate cancer uptake and R2* values in mammary tumors
PJ Hoskin, IJROBP 2007, 68, 1065 McPhail, Radiology 2010, 254, 110
R2*: phenotype specific ?
Requires simultaneous measurements of vasculature function ?
AR Padhani, Radiology 2010, 254, 1

25. Change in R2* and change in tumor oxygenation
Comparison with OxyLite: simultaneous measurement of R2* and pO2
BOLD signal response correctly reflected the evolution of tumor
oxygenation in carbogen challenge
pO2
C. Baudelet and B. Gallez
Magn.Reson. Med.
2002;48:980.
Local
BOLD signal
Whole tumor
T2*w SI T2*

26. Tumor Oxygenation
Perfusion Oxygen
consumption

27. Change in R2* and change in tumor oxygenation
Increase in pO2 through changes in O2 consumption
Insulin infusion Control NS-398
Changes in BOLD signal and R2* in tumors
do not depend uniquely on changes in oxygenation status
B. Jordan, MRM 2006, 56, 637

28. BOLD-MRI and R2*
 
 Variations in tumor oxygenation  No absolute values of pO2
can be qualitatively measured
 The value of basal R2* is
during carbogen breathing
debatable
 Rapid dynamic measurement
 Variation in R2* cannot predict
 Clinically usable changes in oxygenation induced
by treatments modulating the
oxygen consumption

29. T1-based measurements
O2
H20 H20
H20 H20 H20
O2 O2 O2 O2 O2
H20 H20 Carbogen H20 H20
H20
H20 breathing O2 H20
O2
Dissolved oxygen acts
as a T1-shortening paramagnetic contrast agent
Oxygen produces changes in relaxation rate R1 of water
KI Matsumoto, MRM 2006, 56, 240

30. T1-based measurements
MOBILE
Mapping of Oxygen By Imaging Lipids Relaxation Enhancement
R1 (Lipids @ ~ 3.5 ppm)
R1 (H2O) 50
0.2
40
pO2 (mmHg)
relative change (%)
30
0.0
20
air carbogen postmortem post
10
mortem
-0.2 0 air carbogen
0 50 100
time (min)
-0.4
20
R1 (Lipids @ ~ 3.5 ppm)
relative change (%)
15 R1 (H2O)
10
Pooled results 5
N=5
0
en
r
ai
og
-5
rb
ca

31. Nitroimidazole-based PET: How does it work?
Nitro-imidazoles mechanism of uptake
vascular space cellular compartment
e- reduction e- reduction
RNO2 RNO2 RNO2- RNH2
necrosis
hypoxia
normoxia
retention
metabolites
• Initial distribution is flow dependent
• Local oxygen tension = main determinant for long-term retention
• NO2 reduction in radical anion;
• if O2 is present, back to the original structure
• in absence of O2, second reduction with product binding to macromolecules
• depends on the nitroreductase activity
• generally assumed to have retention under 10 mm Hg

32. Rat rhabdomyosarcoma: optimizing imaging conditions with HX4
NS NS
**
8
max Tissue to Blood ratio
*
6
*
4
2
0
0 1 2 3 4 5 6
4h p.i. Time (hours) after injection
Dubois et al. PNAS 2011

33. Is there a significant correlation between pimonidazole
staining (“the gold standard”) and HX4 uptake?

34. Validation of 18F-HX4 uptake using IHC (setup)
HEAD
b
a d
c
a b c d
DORSAL VENTRAL
TAIL
region selection
%HX4 per region
based on CT
Dubois et al. PNAS 2011

35. Validation with pimonidazole IHC: first results
Courtesy of B. van der Kogel
Dubois et al. PNAS 2011

36. Validation of 18F-HX4 uptake using IHC (results)
%PIMO vs %HX4 summary
Spearman R p-value
total 0.6000 0.2848
regions 0.7222 < 0.0001
III-1 0.7334 0.0028
III-2 0.6588 0.0055
I-2 0.9046 < 0.0001
II-3 0.8202 0.0002
III-3 0.6627 0.0733
n = 76
Dubois et al. PNAS 2011

37. Is there a causal relationship between hypoxia and HX4
uptake?

38. [18F]HX4 accumulation is oxygen dependent
Basal scan Carbogen/Nicotinamide
Basal scan 7% oxygen breathing
Dubois et al. PNAS 2011

39. Which hypoxic biomarker is the best?

40. Hypoxia PET tracers
Nitro-imidazoles:
18F-FMISO
Hypoxia 18F-FAZA 18F-HX4
sensitive
Rest part
group
Positron-
emitting
radionuclide
• Clearance: Liver –intestine – Kidney – intestine – Kidney – bladder
kidney liver
• Hydrophilicity:

41. Tumor to blood
S. Peeters et al. In preparation

42. Imaging Hypoxia
Prognostic value of outcome
F-MISO PET in 73 patients
with H&N cancer
FMISO Tumor/Blood
is a prognostic measure
of the outcome
JG Rajendran, Clin. Cancer Res. 2006, 12, 5435
Correlation also found in:
FMISO PET in 40 patients with H&N cancer
SM Eschmann, J. Nucl. Med. 2005, 46, 253
No correlation found in:
FMISO PET in 20 patients with H&N cancer
NL Lee, IJROBP 2009, 75, 201

43. (A) Baseline [18F]-fluorodeoxyglucose (FDG) positron emission tomography (PET) of patient with
T2N2b squamous cell carcinoma of the pyriform fossa with left nodal mass
(A) Baseline [18F]-fluorodeoxyglucose
(FDG) positron emission
tomography (PET) of patient with
T2N2b squamous cell carcinoma of
the pyriform fossa with left nodal
mass.
(B) (B) [18F]-fluoromisonidazole
(FMISO) -PET at baseline,
nonhypoxic primary tumor, and
hypoxic node.
(C) C) FDG-PET 12 weeks after
chemoboost, complete response in
nonhypoxic primary tumor, and
poor response in hypoxic node.
Residual tumor in nodal mass was
confirmed pathologically after neck
dissection.
Rischin, D. et al. J Clin Oncol; 24:2098-2104 2006
Copyright © American Society of Clinical Oncology

44. Phase 1 HX4 imaging: clinically feasible?
Van Loon et al, EJNM 2010
1,6
1,2
T/M
CT delineated tumor 0,8
[18F]HX4 accumulation
0,4
0
30 60 120
Tim e (m in) after injection
Van Loon J. et al. EJNM 2010

45. NSCLC Stage IIIB
18HX4-PET
CT
Van Loon et al. Eur J Nucl Med Mol Imaging. 2010

46. Time to local failure (Kaplan-Meier method) by treatment arm and hypoxia in the primary
tumor (censored times are indicated as tick marks on the curves)
Rischin, D. et al. J Clin Oncol; 24:2098-2104 2006
Copyright © American Society of Clinical Oncology

48. Zips et al. Radiother Oncol 2012

49. Zips et al. Radiother Oncol 2012

50. Biomarker: Hypoxia (F-MISO PET)
0 Gy 10 Gy 20 Gy 40 Gy
[18F]Misonidazol
Zips et al
[18F]Misnidazole
[18F]FDG
Zips , Kotzerke, Baumann et al.
Department of Radiation Oncology M. Baumann |Regaud Lecture 2012

51. Radiolabelled nitroimidazoles
 
 Hypoxia-sensitive method  No absolute values of pO2
 Relevant to estimate  Poor correlation with pO2 values
radioresistant relevant for some tracers
hypoxia (< 10 mm Hg)
 Accumulation dependent on the
 Clinically usable level of nitroreductases
 Prognostic value (especially if
repeated)

52. Imaging of tumor acute hypoxia
Spontaneous fluctuations in tumor oxygenation/perfusion
Technique Spatial Temporal Characteristics Reference
resolution resolution
NITRO-PET 4.2 mm Day 1,2,5 Hypoxia Wang,
Med. Phys.
(More or less 2009, 36, 4400
than 10 mm Hg)
DCE-MRI 0.5x0.2 mm3 15 min Flow variation Brurberg,
MRM
2007, 58, 473
T2*w-GE MRI 470x470 µm2 12.8s Oxygen/flow Baudelet,
Phys. Med. Biol.
variation 2004, 49, 3389
19F-MRI 1.88 mm 1.5 min Quantitative Magat,
Med. Phys.
pO2 2010, 37, 5434
EPRI 1.8 mm 3 min Quantitative Yasui,
Cancer Res.
pO2 2010, 70, 6427

53. In vivo CAIX specific sulfonamide accumulation is reversible upon
reoxygenation
Dubois et al, Radiother & Oncol 2009
Dubois et al, Radiother & Oncol 2009
*P < 0.05; **P < 0.01

54. Imaging of CAIX with antibody
MAb-N-succinyldesferal-89Zr (MAb-N-sucDf-89Zr) (*)
PET images of mice with tumor located subcutaneously on
right hind leg at 4 (A) and 24 h (B) after injection. p.i. = after
injection; SUV = standardized uptake value.
This course is funded with the support of
Hoeben, Kaanders et al. 2010 Jul;51(7):1076-83.
the METOXIA project under the FP7
Programme.

55. How to include hypoxia imaging in
the clinic?

56. Exploiting intra patient heterogeneity for dose painting of radiation

57. Intensity modulated radiation therapy (IMRT)
Galvin, J. Clin. Oncol. 2007, 25, 924
Possibility to sculpt the doses as a function of possible needs

58. Hypoxia Guided IMRT:
dose escalation in hypoxic areas
Dose Painting by Contours
Proof-of-concept using Cu-ASTM
KSC Chao, IJROBP 2001, 49, 1171
Dose Painting by Numbers
Theoretical feasibility
0.4
using 18F-FMISO
0.6
1.9 Dose prescription
1.1
based on tumor hypoxia
2.7
D. Thorwarth, IJROBP 2007, 68, 291
2.9 Z. Lin, IJROBP 2008, 70, 1219
NY Lee, IJROBP 2008, 70, 2
I. Toma-Dasu, Acta Oncol. 2009, 48, 1181
W. Choi, Radiother. Oncol. 2010, 97, 176

59. Hypoxia Guided IMRT:
dose escalation in hypoxic areas
Dose Painting by Contours
Proof-of-concept using Cu-ASTM
KSC Chao, IJROBP 2001, 49, 1171
Dose Painting by Numbers
Theoretical feasibility
0.4
using 18F-FMISO
0.6
1.9 Dose prescription
1.1
based on tumor hypoxia
2.7
D. Thorwarth, IJROBP 2007, 68, 291
2.9 Z. Lin, IJROBP 2008, 70, 1219
NY Lee, IJROBP 2008, 70, 2
I. Toma-Dasu, Acta Oncol. 2009, 48, 1181
W. Choi, Radiother. Oncol. 2010, 97, 176

60. Conclusions
• To bridge the gap between hypoxia-induced
radioresistance and optimized radiotherapeutic
treatment with drugs
– Oxygenation imaging is mandatory
– Qualification of oxygenation biomarkers is still mandatory
at the pre-clinical and the clinical level
– There is a crucial need to validate the value of hypoxia-
gimaging inprospective trials with interventions
ISMRM 2011: Clinical Needs and Research Promises

61. Acknowledgements
University of Maastricht
– Ludwig Dubois * University of Nijmegen (The Netherlands)
– Judith van Loon – Albert van der Kogel
– Jan Bussink
– Sarah Peeters – Hans Kaanders
– Karen Zeghers
University of Amsterdam (VUmc)
– Guus van Dongen
– Bert Windhorts
– Jonas Eriksson
University of Florence (Italy)
– Andrea Scozzafava
– Claudiu Supuran
Euroxy-Metoxia 6th & 7th
Framework University of Brussels (UCL)
– Bernard Gallez*
– Vincent Grégoire
Our patients (No immediate benefit for
them)
NIH (USA)
Siemens MI