IMPROVINGTHERAPEUTIC RATIO RADIOBIOLOGICAL BACKGROUND DR ARNAB BOSE Dept. of Radiotherapy NRS Medical College, Kolkata
Introduction Radiobiology is a branch of science concerned with the action of ionizing radiation on biological tissues and living organisms Objective of this presentation - To understand why ionising radiation can be used to treat malignant cells To know the type of radiation that does this best To identify factors of significance to the success of this process
Introduction The interaction of radiation with a cell is a matter of chance [probability]. If an interaction occurs, the damage may not be expressed, in fact damage is more frequently repaired The initial deposition of energy occurs very quickly The radiation is deposited in the cell randomly Expression of damage occurs after a latent period, ranging from hours to years or even generations The DNA is the sensitive target in the cell
Cell Cycle The cell proliferation cycle is defined by two well defined time periods: Mitosis (M), where division takes place & the period of DNA synthesis (S). The S and M portions of the cell cycle are separated by two periods (gaps) G1 and G2 when, respectively, DNA has not yet been synthesized or has been synthesized but other metabolic processes are taking place. The time between successive divisions (mitoses) is called the cell cycle time.
Cell Cycle In general, the G2/M phases are the most radiosensitive and late S phase is most radioresistant. Transition through the cell cycle is governed by cyclins and cyclin-dependent kinases (cdk). List of important checkpoints: G1 →S governed by p53, Rb, Cyclin D1/Cdk4/6, and Cyclin E/Cdk2 S governed by Cyclin A/Cdk2 G2 →M governed by Cyclin B/A/Cdk1 For a typical mammalian cell, a single fraction of radiation (1–2 Gy) results in >1,000 base damage, 1,000 SSB, and 40 DSBs. DSBs are the most relevant in terms of cell-killing
Cell Death Cell death of non-proliferating (static) cells is defined as the loss of specific function, while for stem cells and other cells capable of many divisions it is defined as the loss of reproductive integrity (reproductive death). A surviving cell that maintains its reproductive integrity and proliferates almost indefinitely is said to be clonogenic. When cells are exposed to ionizing radiation the standard physical effects between radiation and the atoms or molecules of the cells occur first and the possible biological damage to cell functions follows later.
Classification of Radiations inRadiobiology For use in radiobiology and radiation protection the physical quantity that is useful for defining the quality of an ionizing radiation beam is the linear energy transfer (LET). The ICRU defines the LET as follows: “LET of charged particles in a medium is the quotient 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.” Unit usually used for the LET is keV/µm.
Typical LET values for commonly used radiations are: 250 kVp X rays: 2 keV/µm. Cobalt-60 gamma rays: 0.3 keV/µm. 3 MeV X rays: 0.3 keV/µm. 1 MeV electrons: 0.25 keV/µm. LET values for other, less commonly used radiations are: 14 MeV neutrons: 12 keV/µm. Heavy charged particles: 100–200 keV/µm. 1 keV electrons: 12.3 keV/µm. 10 keV electrons: 2.3 keV/µm. X rays and gamma rays are considered low LET (sparsely ionizing) radiations, while energetic neutrons, protons and heavy charged particles are high LET (densely ionizing) radiations. The demarcation value between low and high LET is at about 10 keV/µm.
Cell Damage by Radiation The biological effects of radiation result mainly from damage to the DNA, which is the most critical target within the cell When directly ionizing radiation is absorbed in biological material, the damage to the cell may occur in one of two ways:4. Direct5. Indirect.
Direct Action In direct action the radiation interacts directly with the critical target in the cell. The atoms of the target itself may be ionized or excited through Coulomb interactions, leading to the chain of physical and chemical events that eventually produce the biological damage. Direct action is the dominant process in the interaction of high LET particles with biological material.
Indirect Action In indirect action the radiation interacts with other molecules and atoms (mainly water, since about 80% of a cell is composed of water) within the cell to produce free radicals, which can, through diffusion in the cell, damage the critical target within the cell. In interactions of radiation with water, short lived yet extremely reactive free radicals such as H2O+ (water ion) and OH• (hydroxyl radical) are produced. The free radicals in turn can cause damage to the target within the cell.
A free radical is a molecule or atom, which is not combined to anything (free) and carries an unpaired electron in its outer shell. It is in a state associated with a high degree of chemical reactivity. If the water molecule is ionised H2O = H2O+ + e- (H2O is the water molecule ; H2O+ is an ion radical ) Ion meaning it is electrically charged, because it has lost an electron and a radical because it has an unpaired electron in the outer shell, making it very reactive. Ion radicals have a short life, usually no more than 10-10 s, before they decay to form free radicals
Free radicals are not charged, but do have an unpaired electron in the outer shell. The water ion radical can, for example, do the following: H2O+ + H2O = H3O+ + OH*(H2O+, H3O+ are the ion radicals H2O is a water molecule) OH* is a highly reactive hydroxyl radical, with 9 electrons, therefore one is unpaired. Hydroxyl radicals (OH*), are highly reactive and can go on to react with DNA. It is estimated that 2/3 of the x-ray damage to mammalian DNA is by hydroxyl radicals
Types of DNA Damage DNA damage to the cell can come in several forms: 1. Base damage/single-strand breaks (SSBs) – repaired via base excision repair, not a major contributor to radiosensitivity. 2. Double-strand breaks (DSBs) – repaired via homologous recombination repair (in late S/G2, a DNA template is available) which is accurate, or non homologous end-joining which is error-prone. DSBs are a major contributor to radiosensitivity; ~40 DSBs are required to kill cell. 3.Chromosome aberrations – result from unrepaired or misrepaired DSBs. Symmetric chromosome damage (e.g., translocations) tends to be nonlethal, whereas asymmetric damage (e.g., rings) tends to be lethal due to the loss of large amounts of DNA.
Cell Survival Curve A cell survival curve describes the relationship between the surviving fraction of cells (i.e. the fraction of irradiated cells that maintain their reproductive integrity (clonogenic cells)) and the absorbed dose. Cell survival as a function of radiation dose is graphically represented by plotting the surviving fraction on a logarithmic scale on the ordinate against dose on a linear scale on the abscissa.
The type of radiation influences the shape of the cell survival curve. Densely ionizing radiations exhibit a cell survival curve that is almost an exponential function of dose, shown by an almost straight line on the log–linear plot. For sparsely ionizing radiation, however, the curves show an initial slope followed by a shoulder region and then become nearly straight at higher doses.
Linear Quadratic Model During the 1980s the linear-quadratic model has gained wide acceptance as a mathematical description of biological response to irradiation. The dose range where the LQ model is well supported by data is roughly 1–5Gy per fraction. Extrapolations made outside this range should be done with extreme caution It is mainly used for the calculation of treatment parameters of schedules supposed to be isoeffective. The simplest adequate mathematical description of these data is provided by a linear-quadratic function:
There is a hypothesis considering two types of radiation damage The first type of damage, responsible for the linear component, is assumed to result from a single event. This damage is lethal for the cell if it is not or insufficiently repaired. The probability to produce such a damage is proportional to dose, while its probability to be repaired insufficiently is assumed to be dose independent within the range of clinically relevant doses.
The second type of damage, responsible for the quadratic component, is by itself not lethal for the cell. It is a so-called sublethal damage. Only the combination of two such lesions can yield a lethal event for the cell. The probability to produce a single sublethal damage is again proportional to dose. The probability to produce two of such lesions is proportional to the square of dose, i.e. Again the probability of insufficient repair is assumed to be dose independent within the range of clinically relevant doses.
Typically, survival curves are continuously bending, with a slope that steepens as the dose increases. The ratio α/ β gives the relative importance of the linear dose term and the quadratic dose term for those cells, and controls the shape of the survival curve. When α/β is large, the linear term predominates, so a plot of log (SF) against d is relatively straight, while if α/β is small, the quadratic term is more important, giving a plot with greater curvature. For cells whose survival curves have a lower α/β ratio, doubling the dose leads to more than doubling of the effect on log (SF). Such cells will be particularly sensitive to changes in fraction size when radiation is given as fractionated schedule.
The earlier multitarget single hit model described the slope of the survival curve by D0 (the dose to reduce survival to 37% of its value at any point on the final near exponential portion of the curve) and the extrapolation number n (the point of intersection of the slope on the log survival axis). Dq was the quasi-threshold dose. However, this model does not have any current biological basis.
The linear quadratic model assuming that there are two components to cell kill by radiation where S(D) is the fraction of cells surviving a dose D; alpha is a constant describing the initial slope of the cell survival curve; beta is a smaller constant describing the quadratic component of cell killing. The ratio gives the dose at which the linear and quadratic components of cell killing are equal (8 Gy in the example shown)
High α/β [straighter curve], characteristic of cell with little repair capability e.g. tumour cells [from 5 - 20 Gy] Low α/β [more curved], characteristic of high repair potential e.g. late responding normal tissue [1-4 Gy] This difference in cell survival curves provides rationale for fractionated radiation therapy treatment and explains therapy treatment and explains radiobiological advantage The biological equivalent dose (BED) refers to the effective total absorbed dose (in Gy) for a given fractionation scheme if it were given by standard fractionation (1.8–2.0 Gy/day). BED = nd[1+d/(a(alpha)b(beta))], where n = number of fractions and d = the dose per fraction.
In the past few decades great efforts have been made to apply radiobiological concepts to design safer and more effective therapeutic strategies Withers (1975) suggested four basic mechanisms that contribute to the diverse reactions of different tissues to irradiation: Re distribution of cells in the cell cycle Re oxygenation of hypoxic cells in the tumor Repair of cellular radiation damage Re population of surviving cells during radiotherapy treatment
Re distribution The radio sensitivity of cells varies considerably when they transit through the cell cycle Radiation-induced partial synchrony is a consequence from selective killing of cells in a sensitive phase of the cell cycle as well as by progression delay in late G2- phase Cells surviving irradiation are preferentially those which were in relatively resistant phases during fractionated radiotherapy
Re distribution Redistribution of surviving cells within the mitotic cycle results in self-sensitization of proliferating cell populations This process, however, only affects cells that divide frequently during the 4 to 8 weeks commonly taken to administer a course of curative radiotherapy, but there is little or no such an effect in slowly or non-proliferating tissues Assuming a proliferating tumor surrounded by non- proliferating normal tissue, small doses per fraction and time intervals sufficient for redistribution, should result in an improved therapeutic differential
Re oxygenation Hypoxic cells are about 2.5 to 3.0 times more resistant to X-irradiation than euoxic cells In tumors, hypoxic cells arise because of imbalances between the rate of production of new cells and the vascularization of the tumor Cells are well oxygenated to a distance of about 100 mm from a capillary. At greater distances partial oxygen pressure is so low that cells die and later become necrotic. At intermediate distances, the oxygen concentration is high enough to keep cells viable but at the same time low enough to increase their resistance to X-rays These chronically hypoxic cells might limit radio curability of the tumor
Oxygen “fixes” the free radical damage to DNA caused by X-rays. For this effect to be observed, oxygen must be present in the target at the time of irradiation or microseconds afterwards. Generally, at least 2% oxygen concentration results in maximum radiosensitization. In addition to rendering cells more radioresistant, both chronic and acute hypoxia also contribute to malignant and metastatic progression.
Re oxygenation Irradiation preferentially sterilizes cells that are adequately oxygenated. If a mixed population is irradiated, a biphasic dose response curve results which is steep at low doses but shallower at higher doses due to preferential survival of the more resistant hypoxic cells Between fractions hypoxic cells may be re oxygenated which increases radio curability of the tumor There had been many attempts to overcome hypoxia by specific radio sensitizers, by improving oxygenation pharmacologically or by irradiation under hyperbaric oxygen pressure or by breathing carbogen
Oxygen Enhancement Ratio The ratio of doses without and with oxygen (hypoxic versus well oxygenated cells) to produce the same biological effect is called the oxygen enhancement ratio (OER). OER = Dose to produce a given effect without oxygen Dose to produce the same effect with oxygen The OER for X rays and electrons is about three at high doses and falls to about two for doses of 1–2 Gy. The OER decreases as the LET increases and approaches OER = 1 at about LET = 150 keV/mm,
Relative Biological Effectiveness The relative biological effectiveness (RBE) compares the dose of test radiation to the dose of standard radiation to produce the same biological effect. The standard radiation has been taken as 250 kVp X rays for historical reasons, but is now recommended to be 60Co g rays. RBE = Dose from standard radiation to produce a given biological effect Dose from test radiation to produce the same biological effect The RBE varies not only with the type of radiation but also with the type of cell or tissue, biologic effect under investigation, dose, dose rate and fractionation. In general, the RBE increases with the LET to reach a maximum RBE of 3–8 (depending on the level of cell kill) at LET ª 200 keV/m and then decreases because of energy overkill
Repair The influence of repair of molecular injury on cell survival and the response of tissue to irradiation can be inferred from in vitro survival curves and from changes in the total dose required to produce a certain level of injury as a function of changes in dose per fraction, i.e. from isoeffect curves Fractionation responses can be modeled in terms of two types of radiation-induced cellular injury, one resulting in a logarithmic decline in target cell survival that is linear with dose , and another in which the decline increases proportionally to the square of the dose
Repair The linear component is assumed to reflect cell kill from a single molecular event, while the quadratic component might be due to two independent so-called sub lethal events that have to interact to become lethal for the cell Sub lethal events may be repaired with half-times in the order of 20 minutes to some hours If a dose is split into two fractions with a time interval of several hours then a substantial portion of sub lethal damage induced by the first fraction is already repaired when the second fraction is given
Repair Thus the likelihood for interaction of two sub lethal damages is diminished, resulting in less cell kill due to the quadratic component , as compared to the same dose given in a single session Thus not only total dose but also the number of fractions or the dose per fraction, respectively, determine the magnitude of the radiation effect.
If radiation dose is delivered in a series of equal fractions (F), separated by a time interval that allows complete SLD repair, the effective dose survival curve becomes an exponential function of dose Shoulder of the survival curve is repeated many times; the effective survival curve is a straight line from the origin through point on the single-dose survival curve corresponding to the daily dose (F) D0 (the reciprocal of the slope), has a value close to 3 Gy for human cells
In mammalian cells 3 types of radiation damage described : Lethal damage Sub lethal damage Potentially lethal damage Lethal Damage - Irreversible and irreparable Leads to cell death Potentially Lethal Damage - Component of radiation damage that can be modified by post irradiation environmental conditions
Sub lethal Damage - Under normal circumstances can be repaired in hours usually considered to be complete within 24 h If additional sub lethal damage added within this time then can interact to form lethal damage Sub lethal damage repair observed as an increase in survival if a dose of radiation is split into 2 equal fractions separated by a time interval fractions
If dose is split into 2 fractions separated by a time interval more cells survive than for the same total dose given in a single fraction, because the shoulder of the curve must be repeated each time.
As time interval between 2 F increases see rapid increase in SF, usually complete within 2 h in culture but longer in vivo, particularly for some late responding tissues As time interval increases may see dip in SF due to movement of surviving cells through the cell cycle; only observed in cycling cells If time interval exceeds the cell cycle, see increase in SF due to proliferation
Conventional fractionation is explained as follows: division of dose into multiple fractions spares normal tissues through repair of sublethal damage between dose fractions and repopulation of cells. The former is greater for late reacting tissues and the latter for early reacting tissues. Concurrently, fractionation increases tumour damage through reoxygenation and redistribution of tumour cells. A balance is achieved between the response of tumour and early and late reacting normal tissues, so that small doses per fraction spare late reactions preferentially, and a reasonable schedule duration allows regeneration of early reacting tissues and tumour reoxygenation to likely occur.
The current standard fractionation is based on five daily treatments per week and a total treatment time of several weeks. This regimen reflects the practical aspects of dose delivery to a patient, successful outcome of patient treatments and convenience to the staff delivering the treatment. Conventional fractionation consists of daily fractions of 1.8 to 2.0 Gy, 5 days per week; the total dose is determined by the tumor being treated and the tolerance of critical normal tissues in the target volume (usually 60 to 75 Gy).
Hyperfractionation uses an increased total dose, with the size of dose per fraction significantly reduced and the number of fractions increased; overall time is relatively unchanged In accelerated fractionation, overall time is significantly reduced; the number of fractions, total dose, and size of dose per fraction are unchanged or somewhat reduced, depending on the overall time reduction Accelerated hyperfractionation has features of both hyperfractionation and accelerated fractionation. Hypofractionation uses decreased number of fractions with increased fraction size
Concomitant boost is an additional dose delivered 1 or more times per week to selected target volumes (i.e., gross tumor volume) through smaller field(s), along with the conventional dose to larger irradiated volumes. To achieve an increase in tolerance of late-responding tissues through dose fractionation, the time interval between the dose fractions must be long enough (6 hours) to allow cellular repair to approach completion.
Dose-rate effect refers to repair of SLD that occurs during long radiation exposure. Smaller doses per fraction lead to a repeat of the shoulder on the survival curve. Continuous low-date irradiation (such as I-125 seeds) would be considered an infinite number of infinitely small fractions leading to a survival curve with no shoulder and far shallower compared to acute exposures. The inverse-dose effect occurs when decreasing dose rate actually increases cell killing. This is because higher dose rates (HDRs) would cause arrest in radioresistant phases of the cell cycle
Together with the total dose and fractionation schedule, target volume is a major variable in radiotherapy. For a given fractionation regimen, higher doses can usually be given when volumes at the same site are small rather than large Volume is also an important determinant of normal tissue response to a given dose, first because larger volumes provide less opportunity for tissues to draw on their ‘functional reserve’ and second because larger irradiated volumes make it more likely that a critical volume element will exceed some upper dose limit
Re population Early reacting tissues like skin and mucosa counteract cell depletion by repopulation, usually after a delay that depends on the degree of denudation. Cells in late reacting tissues proliferate very slowly if at all. Prolongation of treatment time might spare acute normal tissue damage but not late reactions Proliferation of surviving tumor cells during treatment is one of the main factors that determine the outcome of fractionated radiotherapy An increase in the number of viable tumor cells between fractions or during treatment interruptions is assumed to result in a failure to control the tumor. Irradiation treatment should be performed in as short a time as possible
Radiation kills cells randomly, which means that each tumour cell has the same probability of surviving irradiation, that probability depending on the given dose. SF2 is the probability of any cell surviving a single dose of 2 Gy, the most commonly used fraction size. Generally, after F fractions, the final survival probability will be (SF2)F.