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Basic radiobiology: fractionation,

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5 Rs, α/β ratio, QUANTEC

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Gill Barnett

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Addenbrookes Hospital
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Radiotherapy (RT) Fractionation

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n
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eg
• RT is an important component of multi-modality cancer treatment

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• used in approximately 60% of patients treated with curative intent

fo
cs
• In the 1930’s, RT pioneers discovered that splitting radiation dose into a number of small

si
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fractions yielded better outcomes than a single exposure

y
og
• RT schedule depends on cancer type & surrounding normal tissue tolerance

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nc
total radiation dose overall treatment time

O
in
dose per fraction volume irradiated
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number of fractions
la
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Therefore RT schedules are designed to

as
M

maximize tumour kill

Therapeutic
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minimize normal tissue damage ratio

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Aims of RT: the therapeutic ratio

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in
eg
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fo
cs
100%

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y
Probability

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Tumour

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50% control

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O
Normal tissue

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damage
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Radiation dose (Gy)

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as

Best chance of cure, with least normal tissue side

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O

effects
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Barnett GC. et al, Nat Rev Cancer 2009

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Radiotherapy adverse effects

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eg
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fo
Acute effects Late effects

cs
si
Ba
y
og
• Occur during or shortly after RT • Occur months to years after RT

ol
• Affect rapidly-proliferating tissues due • Can be permanent

nc
to cell death • Wide individual variation in amount and

O
•E.g. skin, rectum severity of late effects after RT

in
• Tend to cause inflammation ss • 5–10% patients have marked toxicity
la
• Generally manageable • Serious late side effects impact negatively
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• Usually reversible due to proliferation on quality-of-life of cancer survivors

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& repopulation by surviving stem cells  debilitating (e.g. bowel incontinence)

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 life-threatening (e.g. bowel obstruction)

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The 5 Rs of Radiobiology

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• Initially described to provide a means of understanding the success or

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failure of radiotherapy 1,2

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cs
• Relevant to both tumour and normal cells

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•

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Repair
• Repopulation

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• Redistribution

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• Reoxygenation

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• Radiosensitivity

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• Provide framework to examine new therapeutic strategies from point
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of view of both tumour and normal cells
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• Enabled development of safe and effective dose-fractionation regimens

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1 Withers HR Adv Radiat Biol 1975

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2 Steel GG IJROBP 1989

 Repair

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• Principal damaging effects of ionizing radiation is due to its ability to

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eject (ionize) electrons from molecules within cells

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• Most biological damage is done by the ejected electrons:

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• which go on to cause further ionizations in molecules with which they collide

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• progressively slowing down as they go

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og
• At the end of e- tracks, interactions with other molecules become

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more frequent, giving rise to clusters of ionization

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O
in
 These clusters are a unique characteristic of IR
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 Many ionizations can occur within a few base
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pairs of DNA
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 Difficult for cell to deal with damage clusters

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How much damage needs to be repaired?

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• 1 Gy of RT causes approximately

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• 105 ionizations

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• >1000 damages to DNA bases

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• 1000 single strand breaks

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• 20-40 double strand breaks (DSBs)

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in
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• Kills approximately 30% of human cells, as a consequence of
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efficient DNA repair
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Repair and DNA Damage Response (DDR)

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• Complex series of pathways (DDR) for ensuring that

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DNA remains intact in the face of internal and

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external attack

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• Effectors of DDR: determine the outcome for the cell

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• Programmed cell death pathways: apoptosis

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• DNA repair pathways

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• Damage checkpoints that cause temporary or

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permanent blocks in progress of cells through

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the cell cycle

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• E.g mitotic delay allows repair of DNA damage in
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cells prior to undergoing DNA replication or mitosis
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• Preventing acquisition of genetic instability in
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future cell generations

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• In clinical courses of fractionated RT, fractions are

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given daily allowing sufficient time for repair to occur

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Hein et al Int J of Oncology

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DNA Damage Repair Pathways

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Pathway Target Notes

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Base excision repair (BER) Damaged bases

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Single Strand Break Repair Single strand breaks Similar to BER

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(SSBR)

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Homologous Recombination Double strand breaks

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(HR)

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Non-homologous end-joining Double strand breaks

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(NHEJ)

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Mismatch Repair (MMR) ss
Correcting mismatches of bases in DNA Less relevant to RT
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Nucleotide Excision Repair Repairing bulky lesions or DNA adducts Less relevant to RT
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formed e.g. by UV light or cisplatin

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Reviewed in Shibata and Jeggo Clin Onc 2014

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Repopulation

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• Each fraction of RT results in a decrease in the number of surviving

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clonogenic tumour cells

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• i.e. cells that have the ability to repopulate

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• Clonogenic tumour cells that survive irradiation can repopulate the

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tumour by increasing their rate of proliferation and/or reduced cell loss

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Thereby reduce efficacy of RT

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Rapid repopulation of clonogenic tumour cells is important in treatment resistance

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• Time factor of radiotherapy ss
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If tumour can repopulate, increasing overall treatment time will result in

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more clonogenic cells that need to be killed

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And therefore a higher dose to achieve local control

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Repopulation

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• Repopulation is important in

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• tumours whose stem cells are capable of rapid proliferation

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• acutely responding normal tissues, e.g. skin, GI tract, oral mucosa

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y
• In humans accelerated repopulation tends to occur 2-4 weeks after

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the start of RT

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• Repopulation has little consequence in late-responding, slowly

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proliferating tissues, e.g. kidney
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do not suffer much early cell death and do not produce an early
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proliferative response to RT
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Evidence for repopulation during fractionated RT for

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Head and Neck (H&N) cancer: Withers 1988

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fo
• Reviewed published studies using various

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fractionated schedules

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• Total dose required to give 50% control of

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H&N tumours plotted against time of RT

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• For overall time less than about 3-4 weeks

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there is little change in dose required for

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50% tumour control ss
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• At longer times: substantial increase in total
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dose required as treatment time increases

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Withers et al 1988
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Repopulation in Squamous cell cancer of Head and Neck (H&N)

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• Treatment times longer than 3-4 weeks

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• Effect of proliferation equivalent to loss of dose of about

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0.4-0.8 Gy a day

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• Loss of local control with treatment gaps

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• gaps compensated by giving 2 fractions per day

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• >6 hours apart to allow repair of normal tissues

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• evidence for the benefit of accelerated RT schedules

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in
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Repopulation, occurring at 2-4 weeks after start of RT, is due to increase
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in proliferation of clonogenic cells and results in decreased

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 local control of cancer cells

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 acute toxicity
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Redistribution

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• Radiosensitivity of cells varies considerably as

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they pass through cell cycle

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• Cells in S phase (especially late S phase) are most

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resistant

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• Cells in very late G2 and M are most sensitive

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• Variations probably due to ability of cells to Sinclair and Morton 1965

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repair damage by homologous recombination
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• Sensitivity:
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M > G2 > G1 > S early > S late

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Redistribution

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• Effect of on a group of cells which are at various points in the cell cycle is

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to make the population that survive irradiation more synchronous

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• e.g. more cells in S phase have maintained their reproductive integrity

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• During a course of fractionated RT, proliferating cells will move from 1

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phase to another between doses

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• 2 effects can make the cell population more sensitive to subsequent

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radiation

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• 1) Some of cells will be blocked in G2 phase of cycle (mitotic delay) which is a

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sensitive phase of the cycle
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• 2) Some of surviving cells will redistribute into more sensitive parts of the cycle
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Redistribution makes the cell population more sensitive to

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fractionated treatment as compared with a single dose

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 Reoxygenation

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• Process by which hypoxic clonogenic cells become better oxygenated

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during period after irradiation of a tumour

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• Tumours <1mm fully oxygenated

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• Above this size they usually become partially hypoxic

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• Supply of oxygen affects the potency of RT

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• thought to be caused by the generation of ROS (reactive oxygen species)

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• Oxygen assists in making radiation-induced damage permanent

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• More double-strand breaks occur in cells irradiated in the presence of than
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in the absence of oxygen

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Reoxygenation

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• If tumours are irradiated with a large single dose of RT: most of

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radiosensitive aerobic cells will be killed

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• Cells that survive will predominantly be hypoxic

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radiobiological hypoxic fraction immediately after RT will be close to 100%

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• Subsequently hypoxic fraction falls due to reoxygenation and

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approaches its initial starting value

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Reoxygenation can result in a substantial increase in sensitivity of tumours
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during fractionated RT ss
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• Probably major reason why fractionating treatment leads to an improvement in
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therapeutic ratio as compared to single large doses

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Cell survival curve illustrating Rexoygenation

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• Wouters and Brown 1997

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Radiosensitivity

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• Cellular radiosensitivity

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• Sensitivity of cells to ionizing radiation in vitro

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• Tissue radiosensitivity

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• Tissue vary in radiosensitivity due to functional organisation,

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level of proliferation and ability to undergo apoptosis

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• Individual radiosensitivity

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• Intrinsic radiosensitivity is that which is genetically
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determinedss
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• Actual radiosensitivity is influenced by cell/tissue type and

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assay (cellular/tissue) and lifestyle factors (individual)

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Measuring radiosensitivity

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• Cellular radiosensitivity usually measured using a

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radiation survival curve using parameters such as

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• surviving fraction at low doses (e.g. SF2)

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• Dbar (area under a radiation survival curve)

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Surviving fraction

0.1
• Gold standard for measuring radiosensitivity is a

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clonogenic assay

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• Single cells plated and colonies counted after 1-4 weeks

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0.01
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• A variety of assays have been/are studied to measure
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tumour radiosensitivity
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• Many assays not sufficiently reliable to be used in clinical studies

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0 2 4 6 8 10
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Dose (Gy)
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Differences in radiosensitivity reflect response to RT

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• Variable susceptibility to cell death

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Cell lines derived from SF2 • contributes to radiosensitivity

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Neuroblastoma, lymphoma, myeloma 0.19

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• may partly explain clinical heterogeneity

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Medullablastoma, small-cell lung ca. 0.22 within and between cancer types

y
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Breast, bladder, cervix ca. 0.46
• Interest in investigating new

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Pancreas, colorectal, squamous lung ca. 0.43
technologies for measuring multiple

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Melanoma, osteosarcoma, 0.52

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glioblastoma, renal ca. genes and deriving molecular

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la profiles/signature
Deacon et al. 1984
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Summary: 5 Rs of radiobiology

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Total dose required for a Tumours Early-responding Late-responding

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given level of damage normal tissues normal tissues

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Repair √ √ √

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Repopulation √ √

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Redistribution √ √

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Reoxygenation √
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Radiosensitivity √ √ √
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Further reading: Potential relationships between the 5Rs
Fig. 2

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of radiobiology and the hallmarks of cancer

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K. Harrington, et al. Clinical Oncology Volume 19, p561, 2007

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J.S. Good, K. Harrington, Clinical Oncology Volume 25, p569, 2013

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Cell survival and the alpha-beta ratio

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The linear quadratic model

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• Cell survival curve can be described by equation:

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-Ln (Survival) = αD + βD2

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y
• α/β describes bendiness of the cell-survival curve

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• Often used to quantify fractionation sensitivity of tissues

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O
• Initially established that α/β was

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High: for most tumours and acutely responding tissues (>8 Gy)
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Low: for late-responding normal tissues (3-4 Gy)

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The alpha-beta ratio

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• Late responding tissues have lower

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α/β ratio and so curve is more bendy

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si
• Late tissues are more sensitive to

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change in fraction size

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og
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in
• LQ model used to calculate effectiveness of various RT dose schedules
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• E.g. 20 Gy in 5#
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• E.g. calculate equivalent schedule in 2 Gy fractions which would give same

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biological effect (EQD2)

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• Use different vales of α/β to study acute and late effects

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The alpha-beta ratio: New data

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eg
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• New evidence suggests that α/β for some tumours is lower than that

fo
of surrounding tissues

cs
• Clinical and pre-clinical data suggest that prostate cancer has a low

si
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α/β ratio of 1.4-1.9 Gy

y
og
 tumour cells should be more sensitive to increased dose / fraction

ol
• 5 year efficacy and toxicity outcomes from 4 phase III trials

nc
(n>6000) have been published since 2016

O
in
• Moderate hypofractionation (3.0-3.4 Gy / fraction) non-inferior to
ss
conventional fractionation (1.8-2.0 Gy per fraction)
la
• Consistent with α/β of 1.3 -1.8 Gy
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Reviewed in The Role of Hypofractionated Radiotherapy in Prostate Cancer,

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Benjamin, Tree, Dearnaley; Curr Oncol Rep (2017)

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But it is a bit more complicated……

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eg
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fo
• Also depends on the ‘volume effect’….whether an organ at risk

cs
exhibits serial or parallel behaviour

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Ba
• Serial: disabling a single functional subunit disables the whole organ,

y
og
e.g. spinal cord

ol
• Parallel: functional subunits arranged in parallel so organ can still

nc
O
function even if several functional subunits disabled, e.g. lung

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O

The Radiobiology of Hypofractionation, A. Nahim, Clin Onc 27 (2016)

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 RT Fractionation

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Tumour control Normal tissue damage

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Relevant Rs

fo
cs
Repair
Acute effects Late effects

si
Repopulation

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Redistribution

y
og
Reoxygenation

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Radiosensitivity • Increased with shorter treatment time • Less dependent on overall

nc
due to lower repopulation of early treatment time

O
responding tissues • α/β range 0.5-6 Gy

in
• α/β range 7-20 Gy •More sensitive to changes in
ss •Less sensitive to changes in dose/fraction dose/fraction
la
Relevant Rs
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Repair Relevant Rs
α/β = Ratio of the parameters α
as

Repopulation Repair
and β in the linear-quadratic model
M

Redistribution Radiosensitivity
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Radiosensitivity
 s
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QUANTEC

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eg
• Quantitative Analysis of Normal Tissue Effects in the Clinic

rB
fo
• Int J Radiat Oncol Biol Phys 76 (3 Supplement) 2010 (open access)

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si
• QUANTEC reviews in 2010 provide focused summaries of the

Ba
dose/volume/outcome information for 16 organs at risk

y
og
• E.g. brain, spinal cord, parotid gland, lung, kidney, bowel etc.

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ES
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QUANTEC

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QUANTEC

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eg
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Limitations Aims

fo
To assist in determining acceptable dose constraints

cs
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Difficulty synthesizing results

Ba
from different publications

y
og
Incomplete reporting results Provide recommendations on reporting and gathering data on

ol
in studies dose-volume dependencies of treatment outcome

nc
O
Use of incompatible or Provide recommendations for appropriate toxicity endpoints

in
ambiguous endpoints to use
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To suggest areas for future research
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Adoption of these recommendations will facilitate future

as

meta-analysis and therefore increase use of individual studies

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Further Reading

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Thank you for your attention

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