Objectives: Know the characteristics of ionizing radiation that make it useful for RT

1. Interaction of Radiation with Biological Matter: (what is biological dose?)Bill McBrideDivision of Cellular and Molecular OncologyDept. Radiation OncologyDavid Geffen School MedicineUCLA, Los Angeles, Ca.wmcbride@mednet.ucla.eduRoom B3-109, x47051

2. Objectives: • Know the characteristics of ionizing radiation that make it useful for RT • Define LET and RBE and what is meant by quality of radiation • Know the difference between direct and indirect action of radiation and the role of free radicals • Recognize the impact of oxygen on initial radiation damage and of hypoxia in tumor RT • Understand how biological radiation dose and physical radiation dose differ

3. Radiation Therapy • Approximately 50% of cancer patients receive RT with curative intent • Approximately half of these are cured Radiation Therapy has a long history!

4. Roentgen with his wife’s hand, 1895 X-rays were rapidly adapted for use as a clinical treatment, initially for non-cancerous conditions, but soon for cancer, as well.

5. FIRST CURE OF CANCER BY X-RAYS 1899 - BASAL CELL CARCINOMA X-rays were used to cure cancer very soon after their discovery

6. And rapidly became a standard treatment Hammersmith Hospital, London, 1905

7. Although side-effects were encountered! This is a picture of a 70 year old person who was irradiated by Freund at the of age 5 in Austria 1896 for nevus pigmentosus piliferus. L. Freund, Ein mit Rontgenstrahlen behandelter fall von nevus pigmentosus piliferus. Wein. Med. Wochschr. 47, 428-434 (1987).

8. Epilepsy Initially more non-cancerous diseases were treated that cancer (still popular in Europe) Lupus

9. The Nobel Prize in Physiology or Medicine 1946 "for the discovery of the production of mutations by means of X-ray irradiation However, its use for benign conditions has been limited in most countries for fear of radiation-induced cancer. The carcinogenic effects of X-rays was discovered using fruit flies by Muller in 1946. Hermann J. Muller

10. Maltese cross Natural radioactivity was discovered by Becquerel, who was awarded the Nobel Prize in Physics in 1903 along with Marie and Pierre Curie "in recognition of the extraordinary services they have rendered by their joint researches on the radiation phenomena" Marie Curie Henri Becquerel “One wraps a Lumiere photographic plate with a bromide emulsion in two sheets of very thick black paper, such that the plate does not become clouded upon being exposed to the sun for a day. One places on the sheet of paper, on the outside, a slab of the phosphorescent substance, and one exposes the whole to the sun for several hours. When one then develops the photographic plate, one recognizes that the silhouette of the phosphorescent substance appears in black on the negative. If one places between the phosphorescent substance and the paper a piece of money or a metal screen pierced with a cut-out design, one sees the image of these objects appear on the negative. One must conclude from these experiments that the phosphorescent substance in question emits rays which pass through the opaque paper and reduces silver salts.” Paris 1896

11. Natural Radioactivity •  particles • Positively charged, helium nucleus •  particles • Negatively charged, electrons • -rays • No charge, EMR

12. Radioisotopes also were soon being used to treat and cure cancer. First cure of cancer by radium plaque - 1922 Radioactive plaques and implants are still in common use, for example in prostate implant seeds. Radium applicators were used for many other conditions!

13. Therapeutic Benefit and R.T. There is always a need to derive a therapeutic benefit from RT. There are 2 main ways by which this is achieved: 1. Physical means • distributing dose by treatment planning 2. Biological means • dose fractionation

14. 1-25 MeV Megavoltage 500 KeV Orthovoltage 150 KeV Superficial Therapy 50 KeV Contact Therapy 20 KeV Grenz Rays Major improvements in RT during the mid-1900s came from improved penumbra and decreased skin dose associated with higher energy x-rays, cobalt, and high energy photons. More recently conformal RT, IMRT, IGRT, Gammaknife, Cyberknife, tomotherapy, SRS, SRT, protons, heavy ions, etc. have added considerable variety to the choices for physical radiation delivery and present radiobiological challenges.

15. Freund - treated hairy nevus with fractionated doses Stenbeck - cured skin cancer with single doses Bergonie and Trubandeau introduced the “Law” that radiosensitivity is related to cell proliferation (NOT TRUE!) to explain why fractionated doses sterilized rams without skin reactions Regaud - treated uterine cancer with fractionated doses Schwartz - Fractionation is superior because of cell cycle redistribution 1919 Coutard cures deep-seated H&N tumors 1932 Coutard shows fractionation superior to single dose Strandquist - empirical laws for changing dose per fraction Ellis - Nominal Standard Dose (NSD) formula 1980s Linear Quadratic formula gains favor History of Fractionation

16. The First Radiation Dosimeter! Early x-ray machines took a long time to deliver effective dose and gave skin reactions that could be circumvented by dose fractionation.

17. From Amaldi and Kraft, “Radiotherapy with beams of carbon ions, Reports on Progress in Physics, 68, (2005)

18. History has repeatedly shown that dose fractionation results in a therapeutic advantage “In order to save machine time, a 3-day-a-week schedule was initiated in 1962. This schedule was quickly abandoned in pre-operative irradiation because of increased wound healing problems. Although acute reactions in the 3-day-a-week schedule for protracted radical irradiation were not excessive, late radiation sequelae are probably more pronounced as observed 2 or more years later.” Fletcher, 1966. 3 x 3.3 Gy 5 x 2 Gy

19. Clinical RT is Changing, which PresentsChallenges and Opportunities for Radiobiology Conventional treatment: Tumors are irradiated to a specified dose with 2Gy fractions delivered, more or less homogeneously, in a 6 week time period • Varying this schedule impacts outcome • Radiobiological modeling attempts to provide guidelines for customization of RT using • Radiobiological principles derived from preclinical data • Radiobiological parameters derived from clinical altered fractionation protocols Modern treatment: IMRT etc allows optimized non-homogeneous dose distributions, concomitant boosts, dose painting - dose heterogeneity SRS, SRT, HDR, Protons, Heavy Ions - high dose/fx issues Molecular and chemical targeting - dose adjustment Molecular prognosis and diagnosis promise individualized treatment plans and biological treatment planning

20. Radiobiology has derived means of understanding why dose fractionation gives a therapeutic benefit. • New physical delivery methods need to incorporate and/or modify these concepts. • In order to understand either conventional or newer treatment effects, one needs to know the differences between physical and biological radiation dose

21. What is Radiation? • Radiation is classified into two main categories: • Non-ionizing radiation • Ionizing radiation

22.  (cms) 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 1 10 102 103 104 E (eV) 1.24x107 1.24x102 1.24x10-13 v i s i b l e Radar T.V. Radio Microwaves Short Waves rays Infra Red Radio Waves X-rays U.V. IONIZING RADIATION NON-IONIZING RADIATION ELECTROMAGNETIC RADIATIONS Photon E = h(energy = Planck’s const x frequency) = hc/ (c = speed of light,  = wave length)

23. excitation and ionization particle excitation ionization -ray ’-ray • Non-ionizing radiation • Is a particle or wave that has enough kinetic energy to raise the thermal energy of an outer shell electron and cause excitation with emission of low energy EMR (infrared) • Ionizing radiation • Ionizing radiation has enough kinetic energy to detach at least one electron from an atom or molecule, creating ions • Charged particles such as electrons, protons, heavy ions, alpha and beta particles are directly ionizing because they can interact directly with atomic electrons through coulombic forces and transfer a major part of their kinetic energy directly • In contrast, photons (x rays,  rays) and neutrons are chargeless and therefore more penetrating. They areindirectlyionizing. They have sufficient kinetic energy to free an orbital electron producing a ‘fast’ recoil or Compton electron that is, in turn, directly ionizing • Energy is deposited in “packets”, which is why, when it is deposited in DNA, ionizing radiation is an efficient cytotoxic agent • Ionizing radiation has an energy in excess of 124 eV, which corresponds to a l < about 10-6 cm.

24. Ionization produces ions, ion radicals, and free radicals concentrated along tracks and especially at Bragg peak of primary and secondary electrons. They are highly reactive and cause damage to biological matter SECS Absorption of energy Physical effects Chemical lesions Chemical repair Enzyme repair/lesion Cellular effects Tissue effects Systemic effects Ion formation – H2O+ and e- Excitation and H and OH radical formation ION RADICAL LIFETIME FREE RADICAL LIFETIME BREAKAGE OF BONDS CHEMICAL REPAIR / MISREPAIR ENZYMIC REPAIR / MISREPAIR EARLY BIOLOGICAL EFFECTS LATE BIOLOGICAL EFFECTS 10-18 10-16 10-14 10-12 10-6 100 Mins-Hrs Hrs-Days Days-Years 106 • Ion - atom or molecule that has lost an electron and is charged. • Free radical - atom or group of atoms that contains an unpaired electron and is highly reactive • Aqueous electron - has lost kinetic energy and has been captured by water - a powerful reducing agent.

25. The Gray is the Physical Unit of Radiation • 1 GRAY, the unit of absorbed dose (1 joule / Kg), • Causes 1-2 x 105 ionization events / cell • 1% in DNA • A single cobalt 60 ray will deposit about 1mGy in a cell • Rad (Radiation Absorbed Dose) is the old unit = cGy

26. Direct and Indirect Action of Radiation • Indirectly ionizing radiation can act directly or indirectly on biological targets • If the ion pairs and free radicals are produced in a biologic target (DNA) then this is direct action • If water or other atoms or molecules are ionized, diffusible free radicals can act as intermediaries to cause damage - this is indirect action

27. H2O OH. e- R. Direct and Indirect Action of Ionizing Radiation on DNA 4 nm photon e- p+ INDIRECT ACTION photon p+ 2 nm DIRECT ACTION

28. Reactive Oxygen Species (ROS) • Since H2O is the major component in cells, the most common ionization event is radiolysis of water, producing reactive oxygen species (ROS) • The most relevant water is within 2nm of the DNA and tightly bound • ROS produced include: H. - reducing; OH. - oxidizing; HO2.- oxidizing (O2 + H.); H2O2 - oxidizing • The net effect is oxidation of cellular constituents • About 60% of DNA damage caused by x-rays is due to ROS • About 75% of the indirect action of radiation is due to hydroxyl radicals (OH.)

29. Free OH. radicals generateorganic radicals by: • Addition R + OH..ROH • Hydrogen abstraction RH + OH. R. + H2O • Electron transfer R + OH. R. + OH - Where R is the organic moiety

30. Free Radicals and their Scavengers Matter • Biological effects of ionizing radiation are determined in large part by free radicals • Free radicals are involved in many biological processes, including cellular respiration • We have defenses against free radicals • Endogenous free radical scavengers - most relevant within 2nm of the DNA • Anti-oxidants • eg superoxide dismutase, especially in mitochondria, and catalase • Free radical scavengers can protect normal tissue from radiation • eg Amifostine • Depleting free radical scavengers will radiosensitize • What interacts with free radicals, in particular radicals in biological materials will be important in determining outcome at this level • Oxygen interacts with free radicals

31. Oxygen Matters • Binds H radicals forming hydrogen peroxide H. + O2 HO2. (+HO2. ) H2O2 (+O2) • Binds electrons to give superoxide e- + O2 O2- + (H2O) HO2. + OH- • Binds organic radicals to form peroxides R. + O2 RO2. (radical peroxide) RO2. + R’ H ROOH + R’ (hydroperoxide) RO2. + R’. ROOR’ (peroxide) • Oxygen “fixes” the radical lesions in DNA in a form that can not be easily chemically repaired and therefore is a very powerful radiosensitizer.

32. . . . . . . . . 1.0 0.1 0.01 S.F. hypoxic oxic 0 2 4 6 8 10 Dose (Gy) Oxygen Enhancement Ratio (OER) = Dose required to produce a specific biological effect in the absence of oxygen Dose required for the same effect in its presence OER varies with level of effect but can be 2.5 - 3 fold 1) Culture Cells 4) irradiate under oxic or hypoxic conditions 5) Plate cells and grow for about 12 days 0 Gy 2Gy 4Gy 6Gy 2) Suspend Cells trysinization) 6) Count colonies ( 3) Count cells in hemocytometer Physical Dose = Biological Dose

33. Hypoxic areas occur almost solely in tumors and are more radioresistant than oxic areas. Hypoxia contributes to treatment failure Reoxygenation occurs between radiation dose fractions giving a rationale for dose fractionation The oxygen effect is greater for low LET than high LET radiation Clinical Relevance of Hypoxia The effects of hypoxia were first discovered in 1909 by Schwarz who showed that strapping a radium source on the arm gave less of a skin reaction than just placing it there. This was used to give higher doses to deep seated tumors. Giacca and Brown Pimonizadole (oxygen mimetic) staining colorectal carcinoma

34. RADIATION QUALITY AND BIOLOGICAL EFFECTIVENESS

35. LINEAR ENERGY TRANSFER LOW LET Radiation Separation of ion clusters in relation to size of biological target gamma rays deep therapy X-rays soft X-rays alpha-particle LET is average energy (dE) imparted by excitation and Ionization events caused by a charged particle traveling a set distance (dl) - LET = dE/dl (keV/ m) HIGH LET Radiation

36. excitation and ionization particle excitation ionization -ray ’-ray • A dose of 1 Gy will give 2x103 ionization events in 10-10 g (the size of a cell nucleus). This can be achieved by: • 1MeV electrons • 700 electrons which give 6 ionization events per m. • 30 keV electrons • 140 electrons which give 30 ionization events per m. • 4 MeV protons • 14 protons which give 300 ionization events per m. • The biological effectiveness of these different radiations vary!

37. 1.0 0.1 0.01 0.001 S.F. Low LET, HDR High LET DOSE Gy Physical Dose = Biological Dose Relative Biological Effectiveness (RBE) of the Radiation Matters Dose of 250 kVp x-rays required to produce an effect Dose of test radiation required for the same effect =

38. 8 4 6 3 4 2 2 1 0 0 1 10 100 1000 RBE and OER as a function of LET Fast Neutrons Alpha Particles RBE (for cell kill) OER overkill RBE Co-60 gamma rays Diagnostic X-rays OER 0.1 Linear Energy Transfer (LET keV/m) OER is the inverse of RBE because OER depends considerably on the indirect action of ionizing radiation RBE is maximal when the average distance between ionization events = distance between DNA strands = 2nm

39. DNA is the Primary, but not the only, Cellular Target for Radiation • Microbeam irradiation of cell cytoplasm does not generally cause cell death, but irradiation of the nucleus does • Tritiated thymidine incorporated into cells can kill them • Radiation-induced chromosomal abnormalities correlate with cell death and carcinogenesis • However, irradiation of the cytoplasm is not without biological consequences

40. OH . eaqu OH . eaqu OH . eaqu OH . eaquv OH . eaqu OH . eaqu OH . eaqu OH . eaqu OH . eaqu OH . eaqu OH . eaqu OH . eaqu OH . eaqu The lesions in DNA that are associated with cell death and carcinogenesis after radiation exposure are large Spur 4 nm diam 3 ion pairs 100 eV energy 95% of energy deposition events Lesion size about 15-20 nucleotides Blob 7 nm diam. 12 ion pairs The high cytotoxic efficiency of ionizing radiation can be ascribed to the deposition of low levels of energy in small packets within the DNA that cause lesions large enough to be fatal

41. DOUBLE STRAND BREAK 30/ CELL / GRAY SINGLE STRAND BREAK 1000 / CELL / GRAY BASE CHANGE (eg C - U) BASE LOSS 1000 / CELL / GRAY BASE MODIFICATION (eg thymine/cytosine glycol) SUGAR DAMAGE (abstraction of hydrogen atom) INTRASTRAND CROSSLINK 0.5 / CELL / GRAY INTERSTRAND CROSSLINK * DNA-PROTEIN CROSSLINK 1 / CELL / GRAY

42. Not all ionization events are lethal!! • As a rough guide the fraction of cells surviving 2Gy (SF2Gy) is about 0.5 • If the S.F. 2Gy is 0.5, what is the S.F. after 60Gy? = 0.530 = 0.9x10-9 • If the S.F. 2Gy is 0.7, what is the S.F. after 60Gy? = 0.730 = 2.2x10-5

43. What is the Lethal Lesion?

44. X- or -radiation is sparsely ionizing; most damage can be repaired 4 nm Repairable Sublethal Damage 2 nm

45. It is hypothesized that the lethal lesions are large double strand breaks with Multiply Damaged Sites (MDS) that can not be repaired. They are more likely to occur at the end of a track 4 nm Unrepairable Multiply Damaged Site Single lethal hit Also known as  - type killing 2 nm

46. At high dose, intertrack repairable Sublethal Damage may Accumulate forming unrepairable, lethal MDS Also known as  - type killing

47. Physical Dose = Biological Dose Dose Rate Matters 1.0 0.1 0.01 0.001 Low Dose Rate allows continuous SLDR S.F. Low LET, HDR DOSE Gy

48. 840nm Chromatin Structure Matters • Each cell contains about 2m of DNA • The basic structure is the nucleosome, which is 146 base pairs of DNA wrapped around 2 copies of histones H2A, H2B, H3, and H4 • Nucleosomes are in turn wrapped around other proteins to form compacted chromatin • Chromatin is maximally compacted during mitosis • Transcription requires decompaction to facilitate initiation (binding of transcription factors and RNAP II) and elongation miniband - 30nm

49. 1 .1 S.F. LATE S .01 EARLY S Physical Dose = Biological Dose G1 PHASE G2/M PHASE 0 0 4 8 12 16 20 Dose (Gy) Chromatin Structure and Radiation Responses • Compact chromatin is more radiosensitive than non-compacted • Mitotic cells • are 2.8 times more sensitive to DNA breaks than interphase cells • have a lower OER (eg 2.0 compared with 2.8) • do not have much of a “shoulder” on their survival curve • Actively transcribing genes are less sensitive to damage • Decompaction and compaction require acetylation and deacetylation of histones by acetyltransferases (HAT) and deacetylases (HDAC) • HDAC inhibitors are entering the clinic as anti-cancer agents and can radiosensitize • Radiation Damage to DNA is not randomly distributed. • It varies with cell cycle phase and level of gene expression

50. 700R 1500R Repopulation Redistribution 12.5Gy 14.0Gy Repair 15.5Gy 17.0Gy Withers, H. R. and Elkind, M. M. Radiology 91:998, 1968 Used the macrocolony assay in mouse jejunum to assessed the effects of 2 radiation doses given varying times apart to measure the time to and extent of repair, redistribution, and repopulation (regeneration) between dose fractions. Colony derived from a single surviving clonogen