Radiation Oncology Radiobiology

1. Importance of oxygen in
radiotherapy
Dr. Namrata Das
2nd year Junior Resident
Moderator: Dr. Kannan

2. Outline
• Oxygen Effect – definition
• History
• Oxygen Enhancement Ratio
• Factors affecting OER
• Mechanism of Oxygen Effect
• Concentration of oxygen required
• Hypoxia
- Acute hypoxia
-Chronic hypoxia
• Effects of hypoxia
• Strategies to overcome hypoxia
1. Fractionated radiotherapy:
reoxygenation
2. Raising the oxygen content of
inspired gas
3. Modification based on haemoglobin
4. Hypoxic cell radiosensitizers
5. Overcoming acute hypoxia in
tumours
6. Hypoxic cell cytotoxins: bioreductive
drugs
7. Vascular targeting therapies

4. O2 : molecule of life
Oxidative phosphorylation to
generate ATP

5. RADIOTHERAPY Aim: Ionizing radiation for
killing tumour cells
To modify the biological response several chemical
and pharmacological agents have been used
Oxygen
Dramatic
response
Simple Practical

6. Definition: oxygen effect
Response of cells to ionizing radiation is strongly dependent on
oxygen. The enhancement of radiation induced damage with
increasing oxygen tension is known as the oxygen effect.
Oxygen tension Cell kill

7. History
1912, Schwartz: radium applicator placed
hard onto skin
Reduced skin reaction
1923, Petry: correlation between
radiosensitivity and oxyen
1921, Holhusen: Ascaris eggs were relatively
resistant to radiation in the absence of oxygen
All were
published in the
German language

8. History
• 1935, Mottram and his colleagues, Gray and Read: quantitative
measurement of the oxygen effect in primary root of Vicia faba (bean
plant)

9. Oxygen Enhancement Ratio
The enhancement of radiation damage by oxygen is dose-modifying
(i.e. the radiation dose that gives a particular level of cell survival is
reduced by approximately the same factor at all levels of survival).
Oxygen Enhancement
Ratio (OER) =
Radiation dose in hypoxia
Radiation dose in air

10. Oxygen Enhancement
Ratio (OER) =
Radiation dose in hypoxia
Radiation dose in air
OER = 28 Gy/10 Gy = 2.8
Mammalian Cell Survival Cure

11. Radiobiological basis: mechanism of oxygen
effect
• Biological effects of radiation occur by DNA damage
• Two types of action:
DIRECT ACTION
Critical target: DNA
• Radiation (electromagnetic or charged particles)
interacts directly with the critical target in the DNA
• Atoms of target ionized/ excited initiating chain of
events
• Dominant process with radiations of high LET:
neutrons, alpha particles
INDIRECT ACTION
• Free radical mediated damage by interaction
of radiation with water molecules
• Free radical is an atom or molecule containing
unpaired orbital electron in outer shell: high
degree of chemical reactivity
• X rays, gamma rays this predominates

12. Action of radiation INDIRECT ACTION
Absorption of
radiation in
body: production
of fast charged
particles
Charged particles
produce several
ion pairs: half life =
10-10 s
Free radical: half life
= 10-5
Highly reactive – break
chemical bonds and cause
biological damage

13. Oxygen Effect: enhances indirect action
• Absence of oxygen, or in the
presence of reducing species
(sulfhydryl group: SH): DNA
radical formed can be reduced
to normal form
• Presence of Oxygen: RO2●, an
organic peroxide formed: non
restorable -> permamnent
change in chemical composition
• Oxygen “fix”es or makes the
radiation damage permament

14. Factors influencing OER
1. Type of radiation
A) X rays and gamma rays: 2.5
B) Neutron (15 MeV): 1.6
C) Low energy alpha particles: 1

15. Linear Energy Transfer (LET) and Relative
Biological Effectiveness (RBE)
• Linear Energy Transfer (LET):
LET (L) of charged particles in medium is the quotient of dE/dl where
dE is the average energy locally imparted to the medium by a charged
particle of specified energy in traversing a distance of dl.
• Relative Biological Effectiveness (RBE):
RBE of some test radiation (r) compared with X Rays is defined as the
ratio D250/Dr where D250 and Dr are respectively the doses of x rays
and test radiation required for equal biological effect

17. Radiation Weighting Factor (WR)
• 2 Gy of photon is not biologically equivalent to 2 Gy of proton
• WR: Dimensionless multiplier used to place the biological effects
(risks) from exposure to different kinds of ionizing radition on a
common scale
• Photons, electrons: WR = 1
• Protons: WR = 2
• Equivalent dose = Absorbed dose X WR
• Aborbed dose is measured in Gray, Equivalent dose in Sievert

18. Factors influencing OER
2. Dose
Cells are more sensitive to X-Rays in the
presence of molecular oxygen than its absence
(i.e., under hypoxia). For X Rays:
(A) OER = 3.5 at high doses
(B) OER = 2.5 at lower doses

19. Factors influencing OER
3. Phase of cell cycle
• OER in S phase > OER in G1 phase
• G1: more radiosensitive -> dominate low dose region of cell survival
curve -> lower OER

20. Time at which oxygen acts
• Experiments in high pressure oxygen chambers done
• Oxygen needs to be present during irradiation but could be added
afterward, provided the delay was not long (upto 5 millisecond)

21. Concentration of oxygen required
• Oxygen tension of normal tissue is assumed to be similar to
venous blood or lymph = 20 to 40 mm Hg
• Oxygen probe measurements indicate that it varies from 1 to 100
mm Hg
• Some cells are always borderline and radiobiologically hypoxic
• Partial pressure of oxygen in air = 160 mm Hg (21% of 760 mm Hg)

22. Concentration of oxygen required
• A: Dark red, open circles: air,
160 mm Hg
• Pink, closed circle: pO2 = 1.7
mm Hg
• Blue, Open squares: pO2 = 0.25
mm Hg
• Green, closed square: pO2 =
0.075 mm Hg
• Yellow, open triangle: pO2 =
0.0075 mm Hg

23. • Radiosensitivity under anoxic
conditions arbitrarily is
assigned unity
• Relative radiosensitivity till
3mm Hg, 0.5% oxygen
• Rapid change from 0 to 30
mm Hg
• Increase to 100% oxygen has
little effect
• However the hypoxic fraction
of cells have to be borne in
mind

24. Tumour microenvironment
• Solid tumours develop their own blood supply by
angiogenesis
• Neo-vasculature usually lags behind tumour growth which
outgrows its blood supply
• This vasculature is primitive, morphologically, functionally
abnormal
• Hence microregional areas of hypoxia develop within
tumours

25. Types of hypoxia in tumours
Chronic
hypoxia
Acute hypoxia

26. • Thomlinson and Gray (1988): simplified description of microscopic regions
of necrosis in tumours
• They studied histological sections of human bronchial carcinoma
Chronic hypoxia

27. Conclusions by Thomlinson and Gray:
• < 160 μm tumour cord: no necrosis
• 100 μm: band of tumour cells separating necrosed
centre from stroma

28. Chronic hypoxia
• 70 μm: distance to which
oxygen can diffuse at
arterial end
• Intermediate region: oxygen
high enough for cells to be
clonogenic but low enough
to be relatively protected
from the effect of ionizing
radiation
Tumour cells can proliferate and grow actively only is they are
close to a supply of oxygen or nutrients
Postulated that presence of a relatively small
proportion of hypoxic cells could limit success
of radiotherapy

29. Acute hypoxia
• 1980s, Martin and Brown
• Tumour vessels open and close
randomly
• In contrast to acute hypoxia,
chronic hypoxic cells less likely to
become reoxygenated

30. First experimental demonstration of hypxic
cells in tumour
• Response to lower dose (< 9 Gy):
dominated by killing of well
oxygenated cells
• Doses (> 9 Gy): Oxygenated
compartment is depopulated
severely and response is
characteristic of response of
hypoxic cells
• Biphasic curve
99%: oxic cell,
steep slope
1%:hypoxic cell

31. Proportion of hypoxic cells
A) Fraction of hypoxic cells at any
dose level can be determined
from the ratio of survival of
completely and partially
hypoxic fractions
B) Air curve contains a mixture of
oxic and hypoxic cells. Vertical
separation between two lines
gives proportion of hypoxic
cells

32. Methods of measuring tumour hypoxia
● measurements of tumour vascularization because the oxygenation
status of tumours is strongly dependent on vascular supply
● haemoglobin–oxygen saturation, because this controls oxygen
delivery to tumours
● tumour metabolic activity, which changes under hypoxic conditions
● estimating the degree of DNA damage, since hypoxic cells are likely
to show less DNA damage than aerobic cells for a given radiation dose;

33. Methods of measuring tumour hypoxia
Oxygen probe
measurement for
measuring pO2:
gold standard

34. Oxygen probe meassurement
• Polarographic technique
• Eppendorf probe can be moved thorugh tumour in multiple tracks by
computer
• Local control of tumour correlating with probe measurements
• Predictive role in radiotherapy
• Cervical cancer, sarcoma: hypoxia predictive of tumour aggressiveness

35. Green: pimonidazole
(hypoxia)
Red: HIF-1 alpha (decreasing
oxygen tension)
Blue: Blood vessels expressing
CD31
Frozen biopsy of carcinoma
cervox patient
Hypoxic markers

36. Effects of hypoxia
1. Radioresistance, chemoresistance:
• Radioresistance: decreased fixation of biological damage
• Chemoresistance: fluctuating blood flow, drug diffusion distance,
decreased proliferation

37. Effects of hypoxia
2. Tumour progression
• German study on carcinoma cervix patients: presence of hypoxia
limited success of radiotherapy
• Hypoxia general indicator of tumour aggression rather than initial
view that hypoxia conferred radioresistance to some cells
• United States, soft tissue sarcoma: correlation between tumour
oxygenation and frequency of distant metastasis

38. Hypoxia response pathways
Many of the biological changes that occur during hypoxia result from
changes in gene expression, a process that is affected at many different
levels, including chromatin remodelling, transcription, RNA
modification, mRNA translation and protein modifications.
Two primary pathways:
1. Transcription regulation: HIF pathway
2. Protein translation regulation

39. Hypoxia inducible factor (HIF)
• HIF: Master transcriptional regulator of hypoxic response
• HIF-1, HIF-2 are transcription factors which are expressed in hypoxia
• Regulate the transcriptional induction of more than 100 different
known genes during hypoxia
• These HIF targets regulate several important processes including
erythropoiesis, metabolism, angiogenesis, invasion, proliferation and
cell survival

41. Role of HIF in Tumours
1. Tumour Angiogenesis:
VEGF-A expression
increased
2. Tumour Metabolism
3. Tumour Metastasis:
upregulation of MMPs,
lysyl oxidase, CXCR4
HIF: pleiotropic regulator of metabolism
Increase in glycolysis, decrease in oxidative phosphorylation

42. Cancer mutations that activate HIF
• Hypoxia is one only mechanism activating HIF
• HIF can be activated in normoxic conditions by mutations involving
VHL -> germline mutations predispose to highly vascular neoplasms
like hemangioblastoma of retina, CNS, renal cell carcinoma,
phaechromocytoma
• PTEN suppression results in PI3/Akt increased HIF acivity through
mTOR signalling

43. Hypoxia and protein synthesis
• HIF-mediated changes in transcription do not explain all of the
biological changes that occur during hypoxia
• On a genome-wide scale, a comparable number of genes are
influenced through changes in their rate of protein synthesis.
• In light of the often acute and transient nature of hypoxic stress, cells
utilize fast-responding and reversible mechanisms such as those
regulating protein synthesis

44. mTOR and regulation of protein translation
• Hypoxia affects protein synthesis is through inhibition of the
mammalian target of rapamycin (mTOR) kinase signaling pathway
• occurs in response to long-lasting moderate hypoxia (around 0.5
percent O2)
• hypoxic conditions, eIF4E becomes inactivated leading to reduced
rates of protein synthesis
• Because of its ability to control protein synthesis mTOR is recognized
as an important regulator of overall cellular metabolism and many
different receptor signalling pathways influence cell growth and
proliferation by this process.

46. Therapeutic approaches to tumour hypoxia
1. Fractionated radiotherapy: reoxygenation
2. Raising the oxygen content of inspired gas
3. Modification based on haemoglobin
4. Hypoxic cell radiosensitizers
5. Overcoming acute hypoxia in tumours
6. Hypoxic cell cytotoxins: bioreductive drugs
7. Vascular targeting therapies

47. 1. Fractionated radiotherapy: Reoxygenation
4 Rs of Radiotherapy:
1. Repair
2. Repopulation
3. Reassortment
4. Reoxygenation

49. • Time course changes in the
hypoxic fraction during the life
history of a tumour.
• Small lesions are well oxygenated
but as the tumour grows, the
hypoxic fraction rises perhaps in
excess of 10 %
• Large single dose of radiation kills
oxic cells and raises the hypoxic
fraction: the subsequent fall is
termed reoxygenation

51. 2. Raising the oxygen content of inspired gas
• Early clinical attempts to
eliminate hypoxia by
making patients breath high
oxygen content air
• Hyperbaric oxygen therapy
(HBO): An increase in the
barometric pressure of the
gas breathed during
radiotherapy

53. • Use of HBO resulted in improved
local control and survival.
• Benefit was greatest in patients
under the age of 55 who
presented with stage III disease.
• Slight increase in radiation
morbidity
• True improvement in the
therapeutic ratio.

54. • Similar benefit was not observed in bladder cancer and smaller
studies
• HBO therapy was discontinued with the introduction of chemical
sensitizers
• Disadvantage was patient discomfort

55. 3. Modification based on hemoglobin
• Haemoglobin concentration is an important prognostic factor for the
response to radiotherapy in certain tumour types, especially
squamous cell carcinomas
• Patients with low haemoglobin levels have a reduced local-regional
tumour control and survival probability

56. Cervix
• Controversial whether haemoglobin levels affect patient prognosis
• Usual practice: PRBC transfusion to bring Hb level > 12 g/dl
• No benefit of addition of EPO in terms of OS, RFS

58. Head and neck

59. Head and neck
Rationale behind
discontinuation of smoking
during radiotherapy

60. 4. Hypoxic cell radiosensitizers
Chemical radiosensitization was introduced by Adams and Cooke
(1969):
• Showed certain compounds can mimic oxygen and enhance oxygen
damage
• Demonstrated efficiency of sensitization directly related to electron
affinity of compound
• Postulated that these agents would diffuse out of the tumour blood
supply to reach more distant hypoxic cells since they aren’t
metabolized like oxygen

62. Res
Results of the Danish Head and Neck Trial

63. 5. Overcoming acute hypoxia in tumours
• Most of the interventions target diffusion restricted chronic hypoxia
• Experimental studies have demonstrated that nicotinamide, a vitamin
B3 analogue, can enhance radiation damage in a variety of murine
tumours using both single-dose and fractionated schedules
• Mechanism of action of nicotinamide: primarily prevents the
transient fluctuations in tumour blood flow that lead to acute hypoxia

64. ARCON
• The combination of two potentially successful strategies, Accelerated
Radiotherapy to overcome tumour cell proliferation with CarbOgen
and Nicotinamide (ARCON) has been studied in various tumour sites,
but most extensively in head and neck carcinoma (Kaanders et al.,
2002). Components:
• Accelerated, to overcome proliferation
• Hyperfractionated, to spare late-responding normal tissues
• Carbogen breathing, to overcome chronic hypoxia
• Nicotinamide, to overcome acute hypoxia

65. 6. Hypoxic cell cytotoxins: bioreductive drugs
• Bioreductive drugs selectively kill hypoxic cells
• These drugs undergo reduction under low oxygen tension to form active
cytotoxic species
Quinolones (Mitomycin C)
Nitroimidazole (RSU 1069)
N- oxides (Tirazapamine)

67. 7. Vascular targeting therapies

68. Overgaard’s metanalysis of trials of modified
tumour hypoxia
• 10,602 patients treated in 82 randomized clinical trials involving
hyperbaric oxygen, chemical sensitizers, carbogen breathing, or blood
transfusions
• sites included the bladder, uterine cervix, central nervous system,
head and neck, and lung
• local tumor control was improved by 4.6%, survival by 2.8%, and the
complication rate increased by only 0.6%
• Head and neck showed the most benefit

70. Summary