Lecture by Dr. Heidi Lyng in the context of the Course: "Tumour Hypoxia: From Biology to Therapy III". For the complete e-Course see http://www.myhaikuclass.com/MaastroClinic/metoxia

1. METOXIA Course
Tumor hypoxia
- detection and prognostic significance
Heidi Lyng
Thursday 11 October 2012
This course is funded with the support of the METOXIA project
under the FP7 Programme.

2. Learning objectives
• Why does hypoxia arise in tumors?
• How can we measure tumor hypoxia in patients?
• What is the clinical importance of tumor hypoxia?
This course is funded with the support of the METOXIA project
under the FP7 Programme.

3. Tumor development
Steps in carcinogenesis
Gene defects
Metastases
Tumor
Normal cell Cancer cell Cell population
Transformation Uncontrolled cell growth Angiogenesis
Anti-apoptosis
Hypoxia
Apoptosis: programmed cell death Spreading
Invasive growth
Angiogenesis: growth of blood vessels
Therapy
This course is funded with the support of the METOXIA project
under the FP7 Programme.

4. Pioneer work from 1955
Thomlinson and Gray describe corded structures in tumors
Vascularized
stroma
Tumor cells
Necrosis
Hypoxia
Thomlinson & Gray, 1955
Hypothesis: cells bordering necrosis were viable, but hypoxic
This course is funded with the support of the METOXIA project
under the FP7 Programme.

5. Oxygen concentrations
Air: 21% O2 (160 mmHg)
Normal tissue: 5-9% O2 (40 - 75mmHg)
Hypoxia: < 3% O2 (0 - 20 mmHg)
• Radiobiological hypoxia: < 0.5% O2 (4 mmHg)
• Activation of hypoxia inducible factor 1 (HIF1):
< 1% O2 (7-8 mmHg)
• Important clinically: < 1.5% O2 (10 mmHg)
This course is funded with the support of the METOXIA project
under the FP7 Programme.

6. Why does hypoxia arise in tumors?
Oxygen supply Oxygen demand
Normal tissue
Tumor
Tumors: imbalance between oxygen supply and demand
This course is funded with the support of the METOXIA project
under the FP7 Programme.

7. Oxygen imbalance in tumors
• Poor oxygen supply
- Inefficient vascular network
- Low oxygen saturation of hemoglobin (HbO2)
• High oxygen demand
- High metabolic activity
Oxygen
This course is funded with the support of the METOXIA project
under the FP7 Programme.

8. Tumor vascular network
Tumor grown in a window chamber
Microscopy of living tissue
Dewhirst et al. Radiother Oncol 2007
Irregular vascular network in tumors
This course is funded with the support of the METOXIA project
under the FP7 Programme.

9. Oxygen saturation of hemoglobin
Tumor grown in a window chamber
Hardee et al, Curr Mol Med 2009
Heterogeneous and often low oxygen saturation in tumors
This course is funded with the support of the METOXIA project
under the FP7 Programme.

10. Cycling (acute, transient) hypoxia
Hemoglobin oxygen saturation (%)
Time (min)
Hardee et al., Curr Mol Med 2009
Fluctuations in red cell flux and oxygen saturation
in tumor vessels
This course is funded with the support of the METOXIA project
under the FP7 Programme.

11. Why is hypoxia an adverse factor?
TUMOR HYPOXIA
Radioresistance Chemoresistance
Genomic Selection pressure
Angiogenesis Invasion
instability by apoptosis
Metastasis, treatment resistance, malignant progression
Poor clinical outcome
This course is funded with the support of the METOXIA project
under the FP7 Programme.

12. Hypoxia-induced radioresistance
Oxygen: more damage, damage fixation
Hypoxia increases the radioresistance of tumor cells
This course is funded with the support of the METOXIA project
under the FP7 Programme.

13. Detection of tumor hypoxia in patients
• Oxygen tension (pO2) electrodes
• Immunohistochemistry markers
• Gene signatures
• Imaging
This course is funded with the support of the METOXIA project
under the FP7 Programme.

14. PO2 electrodes
PO2 oxygen concentration ”Gold standard”
Invasive
This course is funded with the support of the METOXIA project
under the FP7 Programme.

15. PO2 measurements
50
Lyng et al. Radiother Oncol 1997
PO2 in human tumors:
 lower than in normal tissues
 varies within the tumor
 varies from tumor to tumor
This course is funded with the support of the METOXIA project
under the FP7 Programme.

16. PO2 measurements
Multi-center study of 397 head and neck tumors
Less hypoxic
More hypoxic
P = 0.006
Nordsmark et al. Radiother Oncol 2005
Poor outcome of patients with hypoxic head and neck tumor
This course is funded with the support of the METOXIA project
under the FP7 Programme.

17. Immunohistochemistry markers
• Pimonidazole, EF5
• Hypoxia-inducible proteins
Cell type and compartment information
Invasive
This course is funded with the support of the METOXIA project
under the FP7 Programme.

18. Pimonidazole
HYPOXIA
Mod from Varia et al. Gyn Oncol 1998
Requires administration of pimonidazole
This course is funded with the support of the METOXIA project
under the FP7 Programme.

19. Pimonidazole
Pimonidazole staining of melanoma xenograft
Necrosis
Lyng , unpublished
This course is funded with the support of the METOXIA project
under the FP7 Programme.

20. Hypoxia-inducible proteins
HIF1 regulation
Key transcription factor:
Hypoxia-inducible factor HIF1
VEGF (angiogenesis)
CA9 (pH)
GLUT1 (metabolism)
Proteins upregulated under hypoxia are potential
immunohistochemistry markers
This course is funded with the support of the METOXIA project
under the FP7 Programme.

21. HIF1α staining
HIF1α staining of cervical carcinoma
Necrosis
Lyng, unpublished
This course is funded with the support of the METOXIA project
under the FP7 Programme.

22. Multimarker staining
Staining of head and neck carcinoma
Pimo Rademakers et al. BMC Cancer 2011
HIF1
CA9
This course is funded with the support of the METOXIA project
under the FP7 Programme.

23. Gene signatures
Tumor
DNA
mRNA Whole genome screening of mRNAs
(gene expressions)
Protein
Gene signature
Can include genes from several pathways
Invasive
This course is funded with the support of the METOXIA project
under the FP7 Programme.

24. Gene signatures
Hypoxia gene signature of cervical tumors 31 hypoxia inducible genes in:
Low gene expression
Metabolism
Cell cycle regulation
Proliferation
High gene expression
Halle et al. Cancer Research 2012
Poor outcome of patients with high expression of signature genes
This course is funded with the support of the METOXIA project
under the FP7 Programme.

25. Imaging
• Dynamic contrast enhanced (DCE) MR imaging
Halle et al. Cancer Research 2012
Information on the entire tumor and its heterogeneity
Non-invasive but requires administration of contrast agent
This course is funded with the support of the METOXIA project
under the FP7 Programme.

26. DCE-MR imaging of tumor hypoxia
Hypoxic cervical tumor
HIF1
Less hypoxic cervical tumor
HIF1
This course is funded with the support of the METOXIA project Halle et al. Cancer Research 2012
under the FP7 Programme.

27. Prognostic significance
Some cancer types and methods
• Head and neck cancer, cervical cancer:
- pO2 electrodes, pimonidazole, hypoxia-inducible proteins,
hypoxia gene signature, imaging
• Prostate cancer, soft tissue sarcomas:
- pO2 electrodes
• Breast cancer, ovarian cancer:
- hypoxia-inducible proteins, hypoxia gene signature
This course is funded with the support of the METOXIA project
under the FP7 Programme.

28. Tumor hypoxia – key points
• Arises due to a higher oxygen demand than supply
• Can be chronic or cycling (acute, transient)
• Promotes metastasis, treatment resistance and malignant
progression
• Has been studied extensively in patients by complementary
methods with different strengths and weaknesses
• Is related to poor clinical outcome of many cancer diseases
This course is funded with the support of the METOXIA project
under the FP7 Programme.