Radiobio part 1

Question 1

6 Rs of Radiobiology (ordered according to time)

Answer 1

Repair
Redistribution
Reoxygenation
Repopulation
Radiosensitivity
Remote bystander effect

Question 2

Heirarchical or flexible - what are the 3 types of cells

Answer 2

1. Stem cells - has unlimited proliferation potential
2. Functional - fully differentiated e.g granulocytes - incapable of further division and die after finite life span.
3. Maturing partially differentiating cells - e.g. granuloblasts

Question 3

Hierarchical Tissues

Answer 3

Have all 3 populations with stem cells constantly giving rise to maturing cells which eventually differentiate to become functional cells.

Rapid turnover & high rate of cell loss

Early responding tissues - bone marrow, epidermis & intestinal epithelium

(fails when precrusor cell pool fails to generate enough differentiated cells e.g. bone marrow)

Response to radiation (cell death after irradiation occurs mostly as cells attempt to divide).
- damage becomes evident quicker.
- cells on the road to differentiation are more radioresistant - stem cells are more radiosensitive
stem cell populationis killed

Question 4

Functional subunits

Answer 4

In some tissues FSU are discrete e.g. kidneys - nephrons, liver - lobule

In others non clear anatomical demarcation ie skin, mucosa, spinal cord

survival of structurally defined fsu depends on the survival of one or more clonogenic cell within them

FSUs may be arranged in serial, parallel or combination.

An FSU is the largest tissue volume or unit of cells that can be regerated from a single surviving clonogenic cell.

Question 5

Flexible Tissues (F Type)

Answer 5

Cells rarely divide (normal conditions) but may be induced to by damage.

Cells are functional but retain ability to re-enter cell cycle if required i.e. no strict hierarchy

E.g. late responding tissues - liver, thyroid, dermis

Low turnover rate & respond slowly to irradiation damage

Response to radiation:
- radiation damage remains latent for a long period and is expressed slowly.
- particularly if dose is small because cells do not enter cell cycle immediately
- acute damage is repaired rapidly because of rapid stem cell proliferation
- late damage may be repaired to some extent but is not fully reversible.

Question 6

Serial Organs

Answer 6

FSUs structured serially (like links in chain - each one critical to organ function).
Damage of 1 fsu can lead to loss of function of whole organ
sensitive to hot spot/high point doses

E.g. spinal cord, oesophagus, rectum

Question 7

Parallel organs

Answer 7

FSUs are structured in parallel, each FSU is able to function independently of the others
Damage to one / several FSUs may not affect overall organ function
Can tolerate a higher dose in that area, more sensitive to overall volume affected e.g. lungs, liver, kidney

higher functional reserve capacity

risk of complication related to total dose rather than hot spots

Question 8

CFV (critically functioning volume)

Answer 8

HIGH CFV:
- e.g. liver & kidney
only need 30% of the organ working to maintain function
- sensitive to TOTAL VOLUME irradiation but can tolerate a much higher dose in smaller volumes
- said to have parallel like behaviour

LOW CFV
said to have serial like behaviour

Question 9

clonogenic cells

Answer 9

cells with capacity for sustained cell division (at least 7 generations)

Clonogenic tumour cell - capacity to generate new tumour

Clonogenic normal tissue cell - can regenerate functional tissue following cytotoxic insult

Radiobiologically cell is killed if it is rendered unable to divide & cause further growth

Question 10

Clonogenic survival assay

Answer 10

used to measure clonogenicity in vivo and in vitro.

= GOLD standard measure for radiotherapy sensitivity in the lab.

Question 11

Clonogenic assay in vivo

Answer 11

(living organism)
Irradiate cells ex vivo & inoculate a known number into a mouse - measuring how many colonies form in lung/spleen

Question 12

Clonogenic assay In vitro

Answer 12

(lab dish/test tube)

more relevant to clinical radiotherapy than in vivo assays of cell growth or proliferation

take a precise no. of tumour cells, plate & irradiate with set doses, measure survival and colony formation based on Radiation dose

measures reproductive intergrity of the clonogenic stem cells in tissue

Question 13

Cell survival Curves

Answer 13

Describes relationship between radiation dose & proportion of cells that survive

Mitotic death is dominant mechanism of death following irradiation

Radiobiologically, death = unable to divide and cause further growth . (if cell fully differentiated, death could be defined as loss of function)

A dose 100 Gy is necessary to destroy cell function in non proliferating systems. In contrast the mean lethal dose for loss of proliferative capacity is usually less than 2Gy.

Question 14

Process of cell survival curve

Answer 14

Specimen taken from tumour or normal tissue, prepared into single cell suspension

seeded onto culture dish, covered in growth medium, kept at 37 degrees @ aseptic conditions. If able to divide each cell develops into a colony

2 plates are produced, 1 is irradiated, 1is control & then they are compared

Under normal conditions all cells should survive - they don’t - use plating efficiency

Question 15

Plating Efficiency

Answer 15

Describes the % of cells seeded that grow into colonies

PE = colonies observed / number of cells plated
(x 100)

Question 16

Surviving fraction of irradiated plate is calculated

Answer 16

Colonies counted / (cells seeded x {plating efficiency/100})

Question 17

Shape of cell survival curve

Answer 17

Dose plotted on a linear scale and fraction on a log scale (allows measurement over huge range 0.001 to 1)

not a straight line because at low doses cells are able to regenerate

Initial linear slope - low dose region, less DNA damage, cells are able to repair

Shoulder region - higher dose, bend in curve

Exponential region as dose continues to increase ++ DNA damage overwhelms cells ability to repair

All mammalian cells, normal or malignant exhibit similar XR survival curves

Question 18

Factors affecting cell cycle curves

Answer 18

• Type of radiation, dose rate, dose delivered
• Type of tissue (acute or late responding) - a/b ratio

Rs of radiobiology

Combination with other therapies

Question 19

Multi target model for cell survival curve

Answer 19

used for many years

Described in terms of initial slope (D1), the final slope (D0) and n/Dq to represent the width of the shoulder

Width of shoulder is determined by:
- n (extrapolation number). If large (10-12)= broad shoulder, if small (1.5-2)= narrow shoulder

• Dq quasithreshold dose (almost threshold dose/sublethal dose). Defined as dose at which the straight portion of the survival cure, extrapolated backwards cuts the dose axis through a survival fraction of unity. There is no true threshold dose as there is no dose below which radiation causes no effect.

Radiosensitive cells are characterised by curves with a steep DO (or narrow shoulder). i.e. if High LET there is no shoulder and Dq is very small

Question 20

Linear Quadratic Model

Answer 20

now model of choice

assumes 2 components to cell killing by radiation. One that is proportional to the dose ( linear) and one that is proportional to the square of the dose (quadratic)

A = gradient of the shoulder (low dose). Cell death increases linearly with dose. Directly proportional

B = the gradient of the exponential (high dose) - Cell death increases in proportion to the square of the dose. (directly proportional to square of dose)

E = AD + BD^2.
E= effect, D = radiation dose
S = fraction of cells surviving dose (D)
A & B are constnats

S = e^-ad - BD^2

Question 21

Linear Quadratic Equation

Answer 21

Linear component = unrepairable cell kill

Quadratic component = repairable but cumulative cell kill

Cell kill is the result of single lethal hits, plus accumulated damage from 2 independent sublethal hits

E= aD - lethal non repairable linear term (low dose region, cells that can’t repair themselves after 1 radiation hit). Important for HIGH LET RADIATION
- Apoptotic and mitotic cell death are dominant

E = BD^2 - sublethal repairable quadratic term. Corresponds to cells that stop dividing after more than 1 radiation hit but can repair damage caused by radiation. LOW LET RADIATION. Mitotic death is dominant.

LQ model best describes data in range of 1-6 Gy and should not be used outside this range

Question 22

A/B ratio

Answer 22

dose at which death due to single lethal lesion = death due to accumulation of sublethal lesions. ie. aD =bD^2

indicates sensitivity to changes in dose fractionation

can be used to predict response to different fractionation schedules

Determines bendiness of survival curve

Larger e.g. 10Gy for early responding tissues and smaller i.e. 2Gy for late responding tissues

most tumours have a high ratio, some (e.g. melanoma, sarcoma & prostate) have a low ratio.

a= sensitivity to low doses of radiation
b = sensitivity to high doses of radiation

Question 23

Factors determining tumour growth

Answer 23

cell cycle time
growth fraction
cell loss fraction

Question 24

cell cycle time

Answer 24

quantitative assessment of the constituent parts of the cell cycle

1) Mitotic index
counts proportion of cells in mitosis
if assume all cells in population are dividing & all have the same mitotic cycle
= length of mitosis / total length of mitotic cycle/cell cycle.

2) Labelling index
cell population is first ‘flash labelled’
fraction of cells that take up titrated thymidine (ie. the fraction of cells in S) (proportion of cells that are labelled)

Both tells us the ratio of mitosis and DNA synthesis as fractions of the Tc. But not absolute duration of any part of the cycle.

Question 25

Growth Fraction

Answer 25

= fraction of cells in active cell cycle
= p/ p + q
p = proliferating cells
q = quiscient cells

Because not all tumour cells are in the growth fraction, the actual measured tumour volume doubling time is usually longer than cell cycle time.

In normal adult tissues show no net growth i.e. GF is balanced by net loss

For tumours cell cycle time roughly 1-5 days. Tumour doubling time is roughly 40-100 days.

Potential doublign time = Cell cycle time (tc) / GF
(defined as cell doubling time without any cell loss)

Question 26

Cell Loss

Answer 26

Cell loss factor = fraction of cells produced by cell division that are lost from the tumour. Tends to be large for carcinomas and small for sarcomas.

Cell loss factor = 1 - Tpot (potential doubling time) / Td (actual tumour doubling time)