# Radiation Oncology/Radiobiology/Hypoxia

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Hypoxia

## Overview

* Oxygen "fixes" (makes permanent) damage caused by free radicals (ion pair -> free radical -> DNA damage -> O2 fixation by making permanent DNA-peroxide bond)
** Must be present during or immediately (~5 msec) after RT
* Oxygen enhancement ratio (OER) ~2.5x
** Decreases with increasing LET: photons 2.5, neutrons 1.5, alpha 1.0
** Similarly, decreases with increasing RBE
** For photons, increases with dose and dose rate. If dose/fx <2 Gy OER ~1.5x, if dose/fx >2 Gy OER ~3x
* Measurements of oxygenation
** Evaluated directly by polarographic Eppendorf oxygen probes
** Exogenous: Nitroimidazoles (reduced and irreversibly bound under low O2 tension), EF5, carbon black
** Endogenous compounds: carbonic anhydrase (CA9), HIF, GLUT1 (need biopsy)
** Noninvasive imaging: PET F-18-miso, PET Cu-64-Cu-ATSM, SPECT I-123-azomycin arabinoside
* Hypoxia markers:
* O2 concentration
** Air 155 mmHg, 100% oxygen 760 mmHg
** O2 tension in tissues varies between 1 - 100 mmHg, venous blood ~30 mmHg. Many tissues normally borderline hypoxic
** Cell survival very sensitive to low level O2. At 0.2% O2 (1 mm Hg), survival curve noticeably different
** At 0.5% O2 (3 mm Hg), survival halfway to aerated
** Most rapid change between 0 and 30 mmHg
** Virtually no change in survival curve from venous to arterial to 100% oxygen
* O2 diffusion distance in metabolic active tissue 100-200 µm (Tomlinson-Gray hypothesis, PMID: 13106296)
* Hypoxia varies
** Spatially: within tumor
** Temporary: chronic (diffusion-mediated due to distance from blood vessels) vs acute (perfusion-mediated due to transient fluctuations in blood flow due to malformed vascular supply)
** From patient to patient
* Hypoxic fraction
** Can estimate by extrapolating back from shallow portion of the biphasic survival curve (steep portion is for oxygenated and shallow portion is for hypoxic cells)
** Ranges from 0-50%, on average ~15%
* Reoxygenation
** RT preferentially kills oxygenated cells. Hypoxic cells survive, but typically become re-oxygenated within 24 hours, just in time for the next fraction
** Therefore, if re-oxygenation is achieved, hypoxic cells do not have a significant effect on outcome of fractionated RT
** Lack of reoxygenation is potentially a concern for single fraction SRS/SBRT treatment approaches
** The extent and rapidity of re-oxygenation varies dramatically from tumor to tumor, and depend on proportion of chronic vs acute hypoxia present
* Hypoxic conditions may play a role in malignant progression, by decreasing apoptosis, increasing genomic instability and gene amplification, and by promoting angiogenesis
* Radiosensitization of hypoxic cells
** Improved oxygen delivery: Hyperbaric oxygen, perfluorocarbons, carbagen
** Tobacco cessation
** Drugs: nitroimidazoles (misonidazole showed limited effect, nimorazole significant improvement in a Danish H&N trial) for chronic hypoxia, nicotinamide for acute hypoxia
** Concurrent chemo: Mitomycin C, Tirapazamine
* Hypoxia imaging (PMID: 28540739): the principal noninvasive approaches to imaging tumor hypoxia currently include magnetic resonance and radionuclides (PET and single-photon emission computed tomography), but other techniques, such as optical imaging or electron spin resonance, are under investigation
* Angiogenesis (see more below):
** Pro-angiogenesis: HIF-1α, VEGF, PDGF, FGF
** Anti-angiogenesis:
*** Natural: VHL, TSP-1, Angiostatin, Endostatin, Heparin
*** Drugs: bevacizumab, sunitinib, sorafenib, thalidomide

## Hypoxia Inducible Factor (HIF)

* HIF proteins are a constitutively expressed family, including HIF-1α, HIF-1β, and HIF-2α
* EGLN2 enzyme acts as a cellular oxygen sensor. It is a prolyl hydroxylase (PHD)
** Under normoxic conditions, it hydroxylates HIF-1α using available oxygen
** Hydroxylated HIF-1α is recognized by Von Hippel-Lindau (VHL) protein, and marked for degradation by ubiquination
** Under hypoxic conditions EGLN2 does not have access to oxygen, and thus does not hydroxylate HIF-1α
* HIF-1α subsequently binds to HIF-1β, and the complex acts as a transcription factor on DNA hypoxia-responsive elements (HREs). Targets include
** VEGF: promotes angiogenesis
** GLUT-1: promotes glycolysis and oxygen-independent ATP production
** Stimulation of erythropoiesis
** Increase in apoptosis by interaction with Bcl-2 protein family and p53
* However, HIF-1α levels are also influenced by Ras and PI3K pathways, so that HIF-1α activity may not directly correlate with hypoxia
Review
* Duke 2005 PMID 16098463 -- "Pleiotropic effects of HIF-1 blockade on tumor radiosensitivity." (Moeller BJ, Cancer Cell. 2005 Aug;8(2):99-110.)
** Radiation increases HIF-1 activity in tumors, both sensitizing and protective:
*** Radiosensitization: promotes ATP metabolism, proliferation, p53 activation
*** Radioresistance: stimulates endothelial cell survival
** Net effect of HIF-1 blockade highly dependent on treatment sequencing, with "radiation first" being more effective due to preventing development of radioresistance effect
** Comment from MSKCC PMID 16098459

## Vascular Endothelial Growth Factor (VEGF)

* Family of proteins resulting from alternate splicing of mRNA from a single VEGF gene
* Bind to tyrosine kinase receptors (VEGF-R) on cell surface, causing them to dimerize
* VEGF-R2 appears to modulate most known cellular responses
** Angiogenesis (endothelial cell migration, mitosis, creation of blood vessel lumen, fenestrations, etc)
** Chemotaxis for macrophages and granulocytes
** Vasodilation through NO release
* VEGF-R3 appears to mediate lymphangiogenesis
* Anti-VEGF therapies
** Monoclonal antibodies: bevacizumab for cancer indications, ranibizumab for macular degeneration
** Small molecule TKIs: sunitinib, sorafenib

## Lymphangiogenesis

* 2007 PMID 17878481 -- "Role of lymphangiogenesis in cancer." (Sundar SS, J Clin Oncol. 2007 Sep 20;25(27):4298-307.)
** Review