3.4 The Five Rs of Radiobiology Cheat Sheet (DRAFT) by Molly

There is evidence that the blood vessels in tumours open and close in a random fashion so that different parts of the tumour become hypoxic interm­itt­ently.

Acute Hypoxia is due to transient closure of capill­aries or arterioles servicing parts of the tumour.

These vessels are usually only closed for short times but may occur during a fracti­onated dose of radiation.

The α/β ratio provides the dose in Gray where cell killing from linear and quadratic components of the linear quadratic equation are equal. At points below this value, linear cell kill is dominant; in those after quadratic cell kill takes over. This ratio may be used in many areas of radiot­herapy.

Early responding tissues (high α/β ratio) sensitive to total dose but with minimal effect with fracti­ona­tion. Late responding tissues (low α/β ratio), demons­trating increased survival at low doses and signif­icantly greater toxicity at higher doses.

Most tumours are rapidly growing and have high α/β values, making them sensitive to overall dose with less effect from fraction size. Some tumours, notably prostate cancer and melanoma, have low α/β values. This makes them more sensitive to large fraction sizes and resistant to small fraction sizes. In practice: high dose rate brachy­therapy → delivers a small number of large doses to the vicinity of the prostate, has potential for increasing cure rates of prostate cancer.

Low α/β tissues typically suffer less toxicity at low doses compared with high α/β tissues. Most tumours also possess high α/β values. In areas where late toxicity requires high dose, such as in the skin or limbs, it is possible to shorten treatment times without signif­icant late normal tissue toxicity. This becomes proble­matic in areas with organs sensitive to late effects; in these cases a more fracti­onated regime is recomm­ended to exploit the cell kill between the tumour cells and late responding cells.

Dose response curves can be generated for tumour control or normal tissue compli­cat­ions. If compared, the ratio between the two curves at a particular dose is the therap­eutic index. If the curves can be shifted (eg. by drugs, oxygen­ation etc) then the therap­eutic index is altered, possibly providing a beneficial effect.

Chemot­herapy agents which sensitise cells to radiation will usually shift the tumour control and normal tissue compli­cation curves to the left (a greater effect for a similar dose). If chemot­herapy widens the gap between tumour control and normal tissue compli­cations for a particular dose, then the therap­eutic index will be improved and chemot­herapy may be benefi­cial. This explains why, for some treatm­ents, a reduced fraction size or dose is used when concurrent treatment is used instead of radiation alone.

Another way to present these data is to plot the RBE as a function of LET.

The shape of the survival curve for mammalian cells exposed to sparsely ionising radiation strongly suggests that more than one target in the cell must be inacti­vated before the cell will lose its reprod­uctive integrity.

Low LET radiation produces ionising events that are well separated and in most cases only one event will be deposited per cell, which is insuff­icient to kill the cell. Cell kill is only possible when two separate particles pass through the cell.

This radiation is efficient at killing since no radiation is wasted.

Many cells have three or more ionising events in them, when only two are necessary to kill; this leads to a waste of radiation

In general, cells charac­terised by an x-ray survival curve with a large shoulder, indicating that they can accumulate and repair a large amount of sublethal damage, will show a large RBE for neutrons.

When consid­ering the possib­ility of using neutrons for radiation therapy, we need to know the response of both tumour cells and normal cells.

This is, the therap­eutic gain factor TGF

Relative Biological Effect­iveness (RBE) allows comparison of a test radiation with a standard radiation.

For example, if 10 Gy of 60Co gamma rays kills 50% of the mice in a group, and 1 Gy of heavy ion radiot­herapy kills the other group, the RBE would be 10/1=10

RBE depends on a number of other factors:

Fracti­onation of each dose: Fracti­onation may split the survival curves of the mice, meaning that 20 Gy of gamma rays versus 1.5 Gy of heavy ions are required, giving a RBE of 13.3.

In summary, it is found that the relative biological effect­iveness depends on: 1. radiation quality (LET) 2. radiation dose 3. number of dose fractions 4. dose rate 5. biological system or end point.

The spatial distri­bution or density of these events can be classified as sparse, interm­ediate or dense.

Neutrons give rise to recoil protons carrying unit charge but having a mass ~2000 times that of electrons. These have an interm­ediate ionising density.

Linear Energy Transfer (LET) accounts for all the energy liberated along the path of an ionising particle.

It is defined as the ratio dE/dL where dE is the average energy (keV) deposited in the medium by a charged particle of specified energy in moving a distance dL (micro­met­ers).

The rate of loss of energy by ionising particles will be affected by the velocity of and the electronic charge on such a particle.

A faster moving particle and/or one with a lesser charge will have a much smaller LET.

Energy loss events are essent­ially randomly distri­buted along the track of the photon or charged particle.

For such radiations the amount of energy deposited in a region of the track similar in dimensions to biological molecules also various widely from a few eV up to 100s of eV.

Radios­ens­itivity is the relative suscep­tib­ility of cells, tissues, organs or organisms to the harmful effect of ionising radiation.

This means that actively dividing cells or those not fully mature are most at risk from radiation.

Factors that Affect Cellular Radios­ens­itivity:

Inherent factors - biologic factors charac­ter­istic of the cell:
Mitotic rate
Degree of differ­ent­iation
Cell cycle phase

Tumours under 1 mm in size are fully oxic, but tumours over this size develop regions of hypoxia.

1. Reopening of tempor­arily occluded blood vessels occurs in the minutes following radiation exposure

3. Death of cells due to mitotic catast­rophe occurs within hours of radiot­herapy. The resorption of dead cells leads to decreased distance from capill­aries to tumour cells, improving their oxygen supply. This is the longest process, taking days.

Immedi­ately after irradi­ation, most cells in the tumour are hypoxic, but the pre-ir­rad­iation pattern tends to return because of reoxyg­ena­tion.

Fracti­onation of dose kills oxygenated cells first, allowing the hypoxic population to migrate closer to the vascular network and become oxygen­ated.

If the radiation is given in a series of fractions separated in time sufficient for reoxyg­enation to occur, the presence of hypoxic cells does not greatly influence the response of the tumour.

1. Lethal damage, which is irreve­rsible, and leads to cell death;

3. Sublethal damage, which under normal circum­stances can be repaired in hours unless additional sublethal damage is added with which it can interact to form lethal damage.

As the tumours grew larger, the necrotic centres enlarged so that the thickness of the layer of viable cells remained almost constant.

Conseq­uently, these cells could provide the focus for the subsequent regrowth of the tumour following radiot­herapy.

These chroni­cally hypoxic cells are also resistant to radiation.

Chronic hypoxia, which is a conseq­uence of the limited diffusion distance of oxygen.

When x-radi­ation is absorbed by tissue, fast charged particles are produced. These produce ion pairs. The ion pairs have a very short lifetime and produce free radicals that, because of their unpaired valance electrons, are highly reactive molecules.

If oxygen is not present many of the ionised target molecules can repair themselves and recover the ability to function normally.

Oxygen is said to 'fix' the radiation damage in the sense of making it permanent and the process is known as the 'oxygen fixation hypoth­esis'.

This is because the actions of high-LET radiations are less suscep­tible to enhanc­ement than that of low-LET radiat­ions.

High-LET radiations are, in themse­lves, very effective at producing damage and very high LET radiations are so effective that the passage through a cell by one of them is lethal.

Its main signif­icance is in radiot­herapy. It is likely that most, if not all, cancers contain cells that are hypoxic and also some which probably are severely hypoxic.

For some radiation types, there is a great difference in the biological effect seen in oxic versus anoxia condit­ions.

This is because oxygen is required to 'fix', or make permanent, the damage caused by free radicals.

The ratio of hypoxic to aerated doses needed to achieve the same biological effect (for example, the same level of cell killing) is called the oxygen enhanc­ement ratio (OER).

This is because oxygen is not required to 'fix' the damage caused by these radiat­ions, as they interact directly with the DNA molecule to cause damage.

As the dose rate is lowered and the exposure time extended, the biological effect is generally reduced.

Cells such as HeLa, which have a survival curve with a small initial shoulder, show only a modest dose-rate effect.

Three main biologic processes are involved:

2. Cell prolif­eration occurs during protracted radiation exposure if the dose rate is low enough or the cell cycle time is short enough.

The effect of this cell prolif­eration during treatment, known as repopu­lation (regen­era­tion), will be to increase the number of cells during the course of the treatment and reduce the overall response to irradi­ation.

It will be of little conseq­uence in late-r­esp­onding, slowly prolif­erating tissues (e.g., kidney), which do not suffer much early cell death and hence do not produce an early prolif­erative response to the radiation treatment.

Local control is reduced by approx­imately 0.5 percent for each day that overall treatment time is prolonged.

Repopu­lation is likely to be more important toward the end of a course of treatment, when sufficient damage has accumu­lated (and cell death occurred) to induce a regene­rative response - for tumours as well as for normal tissues.

Two effects can make the cell population more sensitive to a subsequent dose of radiation:

2. Some of the surviving cells will redist­ribute into more sensitive parts of the cell cycle.

When radiot­herapy is given to a population of cells, they may be in different parts of the cell cycle.

A small dose of radiation delivered over a short time period (external beam or high dose brachy­the­rapy) will kill a lot of the sensitive cells and less of the resistant cells.

As reasso­rtment inevitably involves cell prolif­era­tion, the survival will also be influenced by repopu­lation, which reduces the effect of reasso­rtment.

In many normal tissues (and probably in some tumours), stem cells can be in a resting phase (G0) but can be recruited into the cell cycle to repopulate the tissue.

Recrui­tment of resting cells into the prolif­erative cycle during the course of fracti­onated treatment, therefore, may tend to increase the sensit­ivity of the whole popula­tion.

By splitting radiation dose into small parts, cells are allowed to repair sublethal damage.

Malignant cells have often suppressed these pathways, often through mutation or inhibition of TP53, preventing them from undergoing efficient repair.

The shoulder on a survival curve after single radiation doses is indicative of the capacity of the cells to accumulate and repair radiation damage.

The effective slope depends on the size of the individual dose fractions, becoming shallower as the fraction size is reduced. This effect is also seen for irradi­ation of different tissues.

At this limit, essent­ially all the repairable damage is being repaired between each fraction so that the cell killing is due almost entirely to non-re­pai­rable events.

When the size of the individual dose fractions is such that the survival is repres­ented by the shoulder region of the survival curve, as for most dose fractions used clinic­ally, then repair will be maximal when equal-­sized dose fractions are given.

Cells are exposed to sufficient radiation so that any survivors will have accumu­lated the maximum amount of sublethal damage.

In experi­ments, however, the observed proportion of survivors can be greater than this. In the time between the two exposures, cells that survive the first exposure are thought to recover from damage done during that exposure.

1. Repair: First there is the prompt repair of sublethal radiation damage.

3. Repopu­lation: Third, there is an increase of surviving fraction due to cell division when the interval between the split doses exceeds the cell cycle.

The phenomenon of repair is one of the important factors in the sparing effect on normal tissues by the multi-­fra­ction dosage regimens that are commonly used in radiot­herapy, even though sublethal damage repair occurs in both tumours and normal tissues.

The existence of this form of damage is inferred from the shape of the survival curves, but so far it has not been demons­trated directly.

Sublethal damage is presumed to occur because of the steep slope in the dose range beyond the shoulder.

The sublethal injuries themselves are not detected; the only time anything is noted is when a cell is lethally injured and dies.

If we irradiate two identical cell popula­tions with single doses of low-LET radiation under identical condit­ions, the probab­ility of survival will differ according to the post- irradi­ation condit­ions.

If mitosis is delayed, DNA damage can be repaired.

In clinical radiation therapy, the importance of PLD repair is still not proved. However, it may be respon­sible for the radior­esi­stance exhibited by some tumours, that is, these tumours can repair PLD effici­ently, but this is still a matter of debate.