RadBio Flashcards

Question

Describe how a eukaryote cell nucleus is enclosed

Answer 1

Double membrane enclosed organelle (the nuclear envelope) communication with the cytoplasm is mediated by protein lined nuclear pores

Answer 2

Introns and exons Exons are removed by splicing Each end contains a 5' and 3' untranslated region

Answer 3

Divides the nucleotide sequence into non overlapping nucleotide triplets which specify specific amino acids. An open reading frame is a continuous sequence of triplets from start codon to stop codon

Answer 4

The most common start codon is AUG. The start codon is often preceded by a 5' untranslated region (5' UTR).

Answer 5

Start and stop codons | Regulatory segments: promotors, silencers that help control expression.

Answer 6

Polynucleotides Nucleotides: - Nucleobase (adenine, guanine, cysteine, thymine) - Deoxyribose sugar group - Phosphate group The deoxyribose phosphate group forms the backbone

Answer 7

Nucleobase pairs C-G, A-T | With hydrogen bonds

Answer 8

1) Nucleosome: DNA double strand wraps around 8 histones twice (really 1.65 times) 2) Chromatosome: Nucleosomes pair with a H1 histone to form a chromatosome (DNA wrapped twice around the 9 histone configuration (about 11nm wide). Strands now 7 times shorter than fully unravelled DNA. "Beads on a string" - genes under active transcription 3) 30nm fibre: Nucleosomes coiled into compact 30nm fibre - these are less active genes During mitosis chromatin is further compacted using scaffold proteins into the classic for-arm chromosome

Answer 9

sister chromatids joined by a centromere

Answer 10

The process of creating two DNA double stands from a single double strand. Both strands of Original DNA are used to create complimentary strands. Semi-conservative because each daughter double strand contains one strand of the original DNA double strand

Answer 11

Initiator protein binds to a replication origin Starts to unwind DNA, attracts the proteins that make up the replication machine

Answer 12

Helicase (+single stranded binding protein) unwind DNA | Gyrate makes nicks to prevent supercoiling

Answer 13

Initiator protein bind replicator origin -> helicase + single-stranded binding protein and DNA gyrase recruited. 2 replicator machines built 2 replication forks formed at origin Machines move away from replication origin, as forks open in opposite directions

Answer 14

DNA polymerase, this in tern needs RNA polymerase to generate a 3' end (primer) for polymerase.

Answer 15

5' to 3' - i.e DNA polymerase adds nucleotides to the 3' end.

Answer 16

as the replication fork moves long the strand extending in the 3' to 5' direction needs to be replicated using fragments produced 5' to 3' and put together (Okazaki fragments).

Answer 17

Proofreading after each nucleotide added to ensure no mismatch. If error then 3-5' exonuclase removes

Answer 18

Ribose rather tha deoxyribose backbone Uracil instead of thymine single stranded and oft folded

Answer 19

RNA polymerase binds to promoter region (TATA box) of the gene. Once bound, the polymerase opens up the double helix. One of the DNA strands act as template for RNA synthesis which continues to the terminator site (stop site).

Answer 20

RNA is synthesized in the 5’-3’ direction. ○ Synthesized RNA is processed to increase stability: ■ Capping modifies the 5’ end ■ A poly-A tail is added at the 3’ end

Answer 21

Exons are spliced and exons stitched together

Answer 22

Ribosome searches for start codon (usually methionine) to begin translation mRNA is pulled through the ribosome in the 5’-3’ direction and is read in nucleotide triplets until stop codon reached (e.g UAG)

Answer 23

Epigenetic factors: Methylation - Increased methylation of promotor regions leads to decreased expression Acetylation - of histones makes DNA more accessible

Answer 24

○ Acetylation of histone proteins alter DNA structure, making them more accessible ○ Acetyl groups also attract proteins that promote transcription ○ Acetylation of different histones has different effects: ■ acetylation of p53 causes activation ■ acetylation of BCL-6 (transcription inhibitor) causes inactivation

Answer 25

``` DSB: MRN-ATM-CHK2-p53 SSB: RPA+ssDNA-ATR+ATRIP-TOPBP1-CHK1-p53 Direct action of CHK2: Inhibit CDC25CC which maintains CDK2 (i.e leads to DEphosphorylaed = inactivated CDK2) CHK1 also does this tion and release of E2F) ```

Answer 26

1) DNA damage. DSB =MRN-ATM-CHK2, SSB=RPA-ATRIP+ATR-CHK1 2) [Reactive Oxygen Species], mitogen stimulation: The INK4a = p16 - block Cyclin D CDK4/6 complex ARF = ARF-MDM2-p53-p21 inhibit CyclinD-CDK4/6 INK4a, ARF, INK4b are at the same location on chromosome9

Answer 27

G1: | Increased ROS -> p16 - inhibits cyclin D-CDK4/6 complex (Prevents phosphorylation of Rb to relate E2F)

Answer 28

Growth hormone | Growth factors

Answer 29

Bind Tyrosine kinase receptors: ○ promote entry into cell cycle ○ remove blocks on cell cycle progression ○ prevent apoptosis ○ enhance biosynthesis of cellular components required for cell growth and division. Can also drive cellular migration, differentiation and synthetic capacity.

Answer 30

Gain of function mutations lead to uncontrolled proliferative ability.

Answer 31

Epidermal Growth Factor and Transforming Growth Factor α

Answer 32

■ Produced by macrophages and epithelial cells ■ mitogenic for epithelial cells, hepatocytes and fibroblasts. ■ Mutations/amplifications implicated in many tumours: lung, brain, head and neck, breast.

Answer 33

■ Produced by mesenchymal cells ■ Induced by hypoxia and other factors (eg. PDGF, TGF-α) ■ stimulates lymph and angiogenesis. ■ VEGF antibodies used in treatment of renal and colon cancers.

Answer 34

Growth Hormone ● synthesized, stored and secreted by the anterior pituitary gland. ● regulated by the hypothalamus ● Activates the MAPK/ERK pathway, stimulating cell division esp of chondrocytes. ● Also stimulates insulin-like growth factor-1 which activate the PI3K-AKT-MTOR pathway, promoting proliferation and inhibiting apoptosis.

Answer 35

Receptor tyrosine kinases ○ transmembrane proteins with extracellular ligand-binding domain and intracellular tyrosine kinase domain. ○ Usually activated transiently by binding of specific growth factor, causing dimerisation ○ activated receptor autophosphorylates tyrosine residues and recruits signalling proteins. ○ Can be constitutively activated in tumours

Answer 36

EGFR: ERBB1 mutation (lung) ERBB2 mutation (breast) ALK: translocation and point mutation in lung adenoma and lymphomas. VEGF

Answer 37

``` RTK is the initial event for all 1) RAS (most important to remember, i.e. Cyclin D-CDK4 trigger, also stimulates MAPK) 2) MAPK aka the RTK-RAS-MET-ERK pathway 3) PI3K/AKT: RTK-AKT-mTOR The end-point is cell proliferation ``` All frequently involved in gain of function mutations

Answer 38

Growth factor binds RTK activated RTK tyrosine kinase phosphorylates RAS-GDP to RAS-GTP Inactivated by the GTPase neurofibromin

Answer 39

RTK-PI3K-AKT-mTOR promotion of cyclin D (ie. G1 to S) Inhibited by PTEN (i.e a tumour suppressor gene) ● AKT also inactivates BAD, a pro-apoptotic protein ○ PTEN mutations seen in endometrial cancer ● PI3K activity inhibited by PTEN (hence tumour suppressor). ○ PI3K mutation seen in breast cancers.

Answer 40

RTK binding -> RAS-RAF-MEK-ERK-transcription | BRAF mutation in hairy cell leukemias, melanomas, colon cancers

Answer 41

Tyrosine kinase Over expression of EGF is seen in pancreatic cancers, and is associated with more aggressive phenotype and poorer survival. Overexpression of EGFR, through mutation or amplification is implicated in several cancers eg. lung and breast.

Answer 42

6 Hallmarks + 2 enabling characteristics 1) Sustained proliferative signaling (e.g. autocrine, increased TKR or GFs) 2) Evading Growth Suppressors 3) Resisting Cell Death 4) Angiogenesis 5) Activating invasion and/or metastasis 6) Replicative immortality Enablers: Genome Instability Tumour-promoting inflammation Emerging hallmarks: Evading immune destruction Deregulating cellular energetics

Answer 43

Sustaining proliferative signalling: Increase receptors Mutate receptors to be more active Increase GFs or stimulate other cells to release Autocrine activation Constitutive activation of a component of signalling pathway

Answer 44

Overexpression of EGF ● Produced by macrophages and epithelial cells ● EGF binds to EGFR which dimerizes and phosphorylates its tyrosine kinase residues, activating pathways that lead to cellular proliferation ● Hence EGF is mitogenic. ● Overexpression of EGF is seen in pancreatic cancers, and is associated with more aggressive phenotype and poorer survival.

Answer 45

Signal transduction pathways are chain reactions of activated intracellular molecules leading to cellular proliferation Gain of function: ● Most relevant are Ras/Raf/MEK/ERK and PI3K/AKT/mTOR pathways. ● Loss of function of inhibitory molecules eg. GAP and PTEN also lead to oncogenesis

Answer 46

High risk subtypes HPV are 16 and 18

Answer 47

HPV mediates loss of tumour suppressor function

Answer 48

All HPV are non-enveloped double stranded DNA viruses. Encode eight major proteins, 6 located in the “early” (i.e. E1,..E6) region 2 in the “late” region

Answer 49

``` Late encode capsid Replication proteins ONCOGENES - E5,6,7 ○ L1,2 encode capsid proteins ○ E1,2,4 regulate viral replication ○ E 5,6,7 are oncogenes. ```

Answer 50

E5, E6, E7 HPC invades basal layer of epithelium which acts as a continuous reservoir of HPV DNA. Inserts into DNA (i.e it is DS-DNA virus), host expresses oncoproteins

Answer 51

(5,6,7 EGF, p53, Rb) | Activates EGF signalling pathway (i.e RAS-Raf-Mek-Erk, and PI3K-AKT-mTOR) ->Cell growth and proliferation

Answer 52

(5,6,7 EGF, p53, Rb) Binds p53 induces its degredation

Answer 53

(5,6,7 EGF, p53, Rb) Binds Rb releasing E2F: Cyclin E increases, Cylcin A-CDK2 stimulated, cell goes into S Phase. Inactivates CIP/KIP (p21, p27)

Answer 54

ORFs E5,6 &7 | Promote check point inhibition and mitosis

Answer 55

Mutated genes that encode proteins which result in continuous replicative potential of the cell

Answer 56

Genes that check and regulate cell growth and repair, preventing proliferation of mutated or damaged cells.

Answer 57

1) Point mutation = change of a single base 2) Deletions and insertions: single or pair of BASE PAIRS deleted = frameshift mutations. IFF pairs involved are in multiples of 3 then an abnormal protein results - either gaining or loosing an amino acid 3) Translocations = re-arrangement of sequences between non-homologous chromosomes 4) Amplifications: Overexpression due to reduplication and presence of multiple copies within the cell. Can be double minutes (extrachromosomal) or homog eneously staining regions (intrachromosomal).

Answer 58

Deletions usually result in loss of tumour suppressor genes eg. Rb1 and VHL genes.

Answer 59

Rearrangement of parts between nonhomologous chromosomes.

Answer 60

Proto-oncogenes are translocation mutations. Activated by loss of regulatory element or gain of promotor: ○ removal from their regulatory elements eg. Burkitt’s Lymphoma t(8,14) ○ formation of hybrid oncogenes that encode growth promoting oncoproteins. CML t(9,22)

Answer 61

Overexpression due to reduplication and presence of multiple copies within the cell. Can be double minutes (extrachromosomal) or homogeneously staining regions (intrachromosomal). Eg. ERBB2 (HER2 receptor) in breast, ovarian and stomach cancer, NMYC in neuroblastoma, Cyclin D1 gene in head and neck cancers.

Answer 62

Either: Comparative Genome Hybridisation (used to detect of chromosomal copy number changes) In-Situ Hybridisation Both methods use DNA or RNA probe(s) labelled with fluorescent dye.

Answer 63

Detection of chromosomal copy number changes ○ Test DNA and reference (normal) DNA are labelled with different fluorescent dyes. ○ The samples are hybridized to an array with DNA probes spanning all 22 autosomes and sex chromosomes. ○ The binding of the samples are compared ○ If both samples bind equally the spots will fluoresce yellow ○ If deletion or duplication is present, fluorescence will skew towards red or green

Answer 64

Technique for identifying/quantifying an RNA/DNA sequence of interest using RNA/DNA probes labelled with a fluorescent dye (FISH) that hybridise to a target sequence.

Answer 65

○ DNA denaturation by heating, separates the strands. ○ Probes are introduced, labelled with fluorescent dye (FISH). ○ Probes are hybridized to target sequence ○ Sample is washed, removing excess, unbound probes. ○ Sample is viewed under fluorescent microscope ○ Spectral karyotyping (multicolour FISH) uses different coloured fluorescent labels to visualize the entire genome.

Answer 66

Chromosomal structural aberrations are either: 1) Stable (there are for): Are not lethal to the cell and include translocations and deletions (as well as duplications and inversions) 2) Unstable: Gross changes to chromosomes that are lethal to cells - e.g Dicentrics (anaphase bridge), ring chromosomes

Answer 67

● Model of tumour suppressor gene inactivation by Knudson in familial retinoblastoma. ● Developed bilateral disease and much earlier. ● Proposed that patients inherited a germline mutation in one RB gene but required a mutation in the other (‘second hit’) to develop RB, whereas sporadic patients required mutations in both genes. ● Thus, tumour suppressor genes are recessive genes that require inactivation of both functional copies before malignancy develops, whereas loss of one functional copy leads to increased susceptibility.

Answer 68

Tumour suppressor genes Tumour suppressor genes are recessive genes that require inactivation of both functional copies before malignancy develops Infant patients inherited a germline mutation in one Rb gene but required a mutation in the other (‘second hit’) to develop RB, whereas sporadic patients required mutations in both genes. As one mutant is inherited (leaving only one functional allele) early (infant) disease has an autosomal dominant pattern.

Answer 69

Epigenetic changes. 1) Methylation - Decrease access to DNA. Hyper and hypomethylation seen in many tumours, leading to under or over production of multiple genes. 2) Acetylation - Increase access to DNA. Acetyl groups attract promotors

Answer 70

● Altered DNA methylation throughout the genome is seen in various tumours, as hyper or hypomethylation ● Hypomethylated genomes also lead to chromosomal instability. ● Local hypermethylation of the promoter regions of tumour suppressor genes lead to silencing. ● Usually hypermethylation occurs in one allele, and the other functional copy is lost through another mechanism eg. deletion or point mutation. ● One example is CDKN2A which is a locus that encodes tumour suppressors p14 and p16 that enhance p53 and pRb activity.

Answer 71

CREBBP (a histone acetyltransferase) mutation is seen in 40% of DLBCL.

Answer 72

Carcinogenesis involves Initiation and Promotion ● Initiation results from exposure to carcinogens causing permanent DNA damage. ● Promoters are non-tumourigenic but enhance the proliferation of initiated cells. Eg. unopposed oestrogen and chronic inflammation.

Answer 73

Some carcinogens require metabolic activation into | ‘ultimate carcinogens’

Answer 74

Vogelstein model of colon adenocarcinoma- the adenoma-carcinoma sequence: Hyperplasia (1→ Dysplasia (2→ Adenoma (3→ Carcinoma 1) APC - Adenomatous Polyposis Coli 2) K-Ras - GTPase mutation leading to increased proliferative signal 3) p53 - decreased apoptosis and cell cycle arrest Studies of tumour gene expression do not support this model (e.g. data sows very rare to have both K-Ras and p53 mutation)

Answer 75

1) Transformed cells are created by irradiation of embryo or fibroblast cells. 2) Malignant cells

Answer 76

● Transformed cells are created by irradiation of embryo or fibroblast cells. ○ When plated, cells replicate in disorganized fashion ● Malignant cells ○ when grown in suspension grow as spheroids = large spheric clump of cells ○ The spheroids mimic characteristics of solid tumours, with hypoxic core and cycling cells on the outside ○ They are reproducible and inexpensive ○ However they lack ECM and vasculature, hence not a fully accurate model of tumour behaviour.

Answer 77

Benign: 1) Well Differentiated 2) Few mitotic figures, appear normal 3) Normal Karyotype 4) Limited number of cell divisions 5) Exhibits contact inhibition- Stops proliferating when cells come into contact with each other Malignant: 1) Well to poorly differentiated 2) Many mitotic figures which appear bizarre 3) Abnormal karyotype: Abnormal number of chromosomes or chromosomes with abnormal structure 4) May proliferate indefinitely in culture Can produce telomerase 5) No contact inhibition- will continue to pile up into mounds of cells

Answer 78

Neovascularization : ○ Injury of local basement membrane or hypoxia ○ Pro-angiogenic factors activate endothelial cells which migrate to the site of tissue injury

Answer 79

1) Pro-angiogenic factors activate endothelial cells which migrate to the site of tissue injury 2) Endothelial cell proliferation 3) Regulated by angiogenic factors 4) After neovascularization there is a resolution phase where blood vessels mature and stabilize ■ This phase occurs less in tumour angiogenesis hence tumour blood vessels are crap (leaky, disorganised, small, prone to collapse)

Answer 80

After angiogenesis there is a resolution phase where blood vessels mature and stabilize ■ This phase occurs less in tumour angiogenesis hence tumour blood vessels are: ``` ● leaky ● disorganized ● smaller in diameter ● prone to collapse ● high permeability to large molecules ● variable blood flow ```

Answer 81

Angiogenesis is controlled by balance between pro- and antiangiogenic factors. When the balance is in favour of angiogenesis "angiogenic switch" ○ pro: bFGF (FGF-2), VEGF, TGF alpha and beta, TNF alpha ○ anti: angiostatin, endostatin, thrombospondin-1, Interferon

Answer 82

● Regulators include proteases (produced by the tumour or ECM), p53, RAS/MAPK signalling ● Hypoxia is a major trigger of angiogenesis. ● VEGF inhibitors not as effective as hoped suggesting other escape pathways.

Answer 83

1) Dissociation of cancer cells from each other Loss of E-cadherin facilitates detachment and infiltration of surrounding tissues. E.g. SNAIL and TWIST downregulate expression. 2) Degradation of basement membrane Secretion of proteases. Changes in attachment to ECM proteins. 3) This enables Locomotion 4) Movement through the basement membrane and Intravasation = Invasion of endothelium to enter the circulation 5) Formation of tumour emboli Tumour cells aggregate in clumps by adhesion with platelets and T-lymphocytes 6) Adhesion to endothelium basement membrane and extravasation Adhesion molecules involved: CD44 adhesion molecule expressed on normal T lymphocytes 7) Arrest at distant organ site, Trapping in first capillary bed, tumour organ topism 8) Survival in tumour microenvironment e.g. Angiogenesis, evasion of immune mediated destruction 9) Colony formation

Answer 84

Intravascularly, tumour cells are vulnerable to destruction ● mechanical shear stress ● apoptosis ● innate and adaptive immunity

Answer 85

1 step of Metastatic cascade. Dissociation of cancer cells from each other Loss of E-cadherin facilitates detachment and infiltration of surrounding tissues. E.g. SNAIL and TWIST downregulate expression.

Answer 86

Sigmoid (Gompertz) growth. Where the mass is typically only clinically detectable at the plateau phase. Sigmoid Characterised by: 1) Initial Exponential Growth: Small number of tumour cells with adequate nutrition undergo division. Each progeny is able to survive and divide. The growth fraction close to 100%. 2) Linear Growth: Competition for nutrients/oxygen make tumour micro environment less favourable, not all progeny survive. 3) Plateau: Tumour microenvironment increasingly unfavourable - only a reducing minority of cells are able to proliferate. Eventuallycell loss factor is equal to the growth fraction. The tumour mass is clinically detectable and causing symptoms. May also be areas of necrosis from hypoxia.

Answer 87

Y - axis: Number of cells 10^9 [0 .2, 0.4,..... 1] | X Axis: Time (Arbitrary units)

Answer 88

The ratio of proliferating cells over total number of cells Cell loss factor: The ratio of the rate of cell loss over the rate of new cell proliferation

Answer 89

Tumour doubling time is the time taken for a tumour to double its initial volume. Potential doubling time is the theoretical doubling time in the absence of cell loss. Used to calculate cell loss Fraction. Tpot = Tc/GF (Tc = cell cycle time, GF = growth fraction) i.e (time to make new cell/number of proliferating cells) = number of new cells per unit time (assuming no death) (e.g. cells/sec)

Answer 90

The ratio of the rate of cell loss over the rate of new cell proliferation ● 1- (Tpot/Td) where Tpot is the potential doubling time and Td is the actual doubling time.

Answer 91

1) Cell cycle time 2) Growth Fraction (#prolif/#cells) 3) Cell Loss Factor = 1- (potDt/Dt) 4) Accelerated re-population 5) Tumour micro environment: stroma and hypoxia

Answer 92

Hypoxia activates pathways that allow tumour cells to adapt to hypoxic stress. 3 Key adaptations: ○ anaerobic glycolysis ○ changes in blood flow ○ stimulate angiogenesis.

Answer 93

Accelerated re-population In experiments of SCC head and neck, this seems to take place approximately 28 days after starting treatment. ● The dose required to compensate for this repopulation is about 0.6Gy/day

Answer 94

1) Improved/less competitive tumour microenvironment: reduction in number of tumour cells, improvement in oxygen and nutrient supply to the remaining tumour cells resulting in a more favourable tumour microenvironment. 2) regeneration response of clonogenic cells, similar to that seen in normal epithelial tissue, potentially triggered by EGFR

Answer 95

Methods to counteract this is to reduce overall treatment time by accelerated hyperfractionated treatment, combined chemoradiotherapy, or higher total dose.

Answer 96

Tumours become detectable at about 10^9 cells

Answer 97

Phosphorylation of H2AX to gammaH2AX

Answer 98

Leads to recruitment of proteins to site of DNA damage | Form ionising radiation induced foci (IRIF) - if stained with antibodies.

Answer 99

3 related kinases can phosphorylate H2AX at sites of DNA damage: 1) ATM-MRN (the main one) 2) DNA-PKcs-KU 3) ATR-ATRIP

Answer 100

■ MRN (MRE11, RAD50, NBS1) assembles at site of DNA breaks ■ MRN recruits and activates ATM ■ ATM phosphorylates H2AX

Answer 101

Potential doubling time: Tumour doubling time when there is no cell loss Tpot = Tc/GF (Tc = cell cycle time, GF = growth fraction)

Answer 102

Cell loss factor: Ratio of rate of cell loss to rate of new cell production 1 – Tpot/VDT

Answer 103

Number of cells replicating and time for them to replicate is dependant on: 1) Number of tumour cells actively going through cell cycle and replicating (growth fraction) 2) Duration of cell cycle in these cells (cell cycle time) 3) Rate of Cell Loss

Answer 104

Number of Cells Replicating/number of cells in tumour

Answer 105

Ki67 proliferation index in a tumour is an Indication of growth fraction: Ki67 cell cycle specific protein Detected by IHC using monoclonal antibody

Answer 106

Most solid tumours have growth fractions below 50%

Answer 107

For a typical rapidly proliferating human cell with a total cycle time of 24 hours, the G1 phase might last about 11 hours, S phase about 8 hours, G2 about 4 hours, and M about 1 hour.

Answer 108

2 Examples: 1) Identification of S phase cells by incorporation of radioactive thymidine. 2) Cells at different stages of the cell cycle can also be distinguished by their DNA content. Cells in G1 are diploid so their DNA content is referred to as 2n. Cells in S have DNA contents ranging from 2n to 4n. DNA content remains at 4n for cells in G2 and M, decreasing to 2n after cytokinesis. Incubation of cells with a fluorescent dye that binds to DNA, followed by analysis of the fluorescence intensity of individual cells in a flow cytometer

Answer 109

For 1 Gy of radiation >1000 damaged bases about 500 - 1000 ss DNA breaks about 20 - 40 ds DNA breaks

Answer 110

● Damage to bases- repaired through base excision repair or nucleotide excision repair ● Single strand breaks- repaired through SSB repair ● Double stranded breaks- repaired through homologous recombination or non-homologous end-joining. ● Other lesions not directly related to radiation damage: ○ mismatch bases can occur when attempting replication- repaired through mismatch repair ○ bulky lesions or DNA adducts formed by UV light or chemotherapy- repaired through nucleotide excision repair.

Answer 111

Variant of histone H2A 10 – 15% total cellular histone H2A Distributed throughout nucleus H2AX phosphorylation occurs at site of DNA damage, within 30 mins of DNA damage Recruits proteins to sites of DNA damage and signals activation of effector pathways

Answer 112

ATM mutation: H2AX-MRN-ATM-CHK2 | Sufferers of AT or cells that have inhibition of ATM are very radiosensitive – defective DNA damage response

Answer 113

When phosphorylated to gammaH2AX recruits proteins to sites of DNA damage and signals activation of effector pathways. MRN-ATM is key activator (missing in AT patients), if missing ATR-ATRIP and DNA-PKcs-Ku will phosphorylate H2AX

Answer 114

Homologous recombination - slow, but error free Non-homologous end-joining - Fast, cell can survive DSB, but deletion and insertion errors. For Homologous recombination to occur there bust be a sister chromatid therefore DSB must occur in late S and G2.

Answer 115

Homologous Recombination uses sister DNA with the same sequence as a template for repair: 1) Single stranded filaments made around DSB 2) Filaments coated with RPA 3) RAD51 displaces RPA and SEARCHES and INVADES sister chromatid 4) Helices UNWIND and polymerases SYNTHESISE 5) Finally crossover points are CUT by RESOLVES, and ligated.

Answer 116

Detection→Recruitment/activation→Processing→Ligation: 1) Detection: DSBs detected by Ku70 and Ku80 proteins which bind to site. They protect ends of DNA strands, prevent exonucleases degrading DNA. 2) Recruit: DNA-PKcs recruited - autophosphorylates & phosphorylates other proteins. Artemis (protein complex) recruited to DNA break, forms complex with DNA-PKcs and is activated by phosphorylation. 3) Processing: Artemis endonuclease activity processes the DNA ends ready for ligation. If DNA damage or repair process causes a non-blunt end of DNA i.e. a 3’ or 5’ overhang, then the absent DNA can be generated by polymerases. 4) DNA ends ligated through ligase IV and XRRC4 and XLF proteins.

Answer 117

Artemis endonuclease activity processes the DNA ends ready for ligation. If DNA damage or repair process causes a non-blunt end of DNA i.e. a 3’ or 5’ overhang, then the absent DNA can be generated by polymerases.

Answer 118

1) Damage to Base = Base excision and repair 2) SSB = Single Stranded Brake Repair 3) DSB - Non-homologous End joining 4) DSB - Homologous Repair

Answer 119

Damaged DNA detected and removed by glycosylases: ○ Each glycosylase is specific for a particular type of base damage. ○ They cut out the damaged base without cutting the DNA backbone resulting in an abasic site. 2) AP endonuclease then cuts the DNA backbone making a nick (SSB). 3) Subsequent repairs follow one of two pathways: ■ Short patch ● involves replacing the damaged base only ● DNA synthesis carried out by DNA polymerase β ● Ligated by ligase 3 ■ Long patch ● involves cutting out and replacing up to 10 nucleotides. ● DNA polymerases δ and ε ● ligase 1

Answer 120

1) Damage detected by PARP-1. 2) Breaks are ‘dirty’ and require end-processing step by polynucleotide kinase PNK BER (ie. proceeds from the point of nick): 3)Once clean nick produced, short or long patch repair follows BER. ■ Short patch ● involves replacing the damaged base only ● DNA synthesis carried out by DNA polymerase β ● Ligated by ligase 3 ■ Long patch ● involves cutting out and replacing up to 10 nucleotides. ● DNA polymerases δ and ε ● ligase 1

Answer 121

Phosphorylated form γ-H2AX: ○ recruits proteins involved in DSB repair ○ opens tertiary structure of DNA allowing access to repair proteins ○ activates checkpoint proteins ○ involved in formation of ionizing radiation induced foci

Answer 122

ATR may also be activated by ATM-MRN pathway when processing ds DNA breaks which creates ss DNA ATR-ATRIP-CHK1 MRN-ATM-CHK2

Answer 123

1) Chromosome aberrations. Assessed for at 1st metaphase after exposure. 2) Assays for DNA damage i. γ-H2AX - Radiation induced foci immunofluresence ii. Comet Assay - i.e the more fragmented the DNA the longer the tail behind the head (nucleus) iii. Pulse field Gel electrophoresis - post electrification (current applied in diff directions) fragmentation corresponds to DNA damage iv. Micronucleus assay v. Plasmid assay

Answer 124

● Single cell electrophoresis ● Irradiated cells embedded in agarose gel on microscope slide ● Cells lysed using high salt and detergent solution ● DNA electrophoresed ● Fluorescent staining of gel performed with ethidium bromide or propidium iodide which is specific for DNA ● DNA fragments move away from nucleus, forming a ‘comet’ with head formed by the nucleus and tail composed of smaller DNA fragments. ● Comet tail length correlates with DNA damage.

Answer 125

Pulse field Gel electrophoresis - post electrification (current applied in diff directions) fragmentation: ● Used to assess DNA DSB and large fragments up to 10 Mbp ● Comet assays not effective in base pairs >50kb ● irradiated cells suspended in agarose gel and poured into moulds ● cells lysed and gel electrophoresis performed ● Current applied switching from different directions: straight across and 60 degrees either side ● Larger pieces will react slower to change in direction ● Over the course of time bands will begin to separate ● Gel is read after staining with fluorescent ethidium bromide ● Fragmentation of DNA correlates with amount of DNA damage.

Answer 126

``` Mitotic catastrophe Necrosis Apoptosis (Type 1 Death) Autophagy (Type 2 Death) “Senescence” = Reproductive Death ```

Answer 127

Occurs after mitosis because cells have entered mitosis with DNA damage still present. Cells can no longer replicate due to the chromosomal damage = Reproductive death. This form of cell death can trigger other cell death pathways such as apoptosis. Morphological changes: multinucleated giant cells, chromosome aberrations, dicentric nuclei

Answer 128

Occurs due to unfavourable growth conditions, e.g change in pH in cellular environment, but also due to insults which damage DNA such as radiation. Morphological changes: cellular swelling and rupture, In nucleus – uncondensed chromosomes, micronuclei, clumping of DNA In Cytoplasm - organelle degeneration, mitochondrial swelling, release of lysosomal enzymes, vacuolation of cytoplasm

Answer 129

Regulated form of cell death. Essential component of development and in maintaining tissue homeostasis Triggered from within cell or by external cell signals Changes in apoptosis control can contribute to cancer development

Answer 130

1) Chromatin condensation and nuclear fragmentation 2) DNA laddering 3) Cell membrane blebbing 4) Formation of apoptotic bodies (vesicle containing parts of a dying cell) - Prevents leakage of potentially damaging cellular proteins

Answer 131

NECROSIS Cell body: Cellular Swelling Chromatin: Does not condense Cell integrity: Blebbing -> cell becomes leaky Resolution: Cellular and nuclear lysis causes inflammation ``` APOPTOSIS Cell body: Shrinks Chromatin: Condenses Cell integrity: Budding Resolution: Buds become apoptotic bodies which are phagocytised without inflammation ```

Answer 132

Sensor molecules = sensor caspases Involved in initiating apoptosis Activation of sensor caspase (8 or 9) Effector molecules = Effector caspases Subsequent activation of downstream effector caspases (including caspase 3) Sequential activation of caspases Usually in inactive form (procaspase) Inhibitors of apoptosis proteins (IAP)

Answer 133

Extrinsic pathway: ■ sensor caspase 8, activated by binding of extracellular ligand which activates death receptor. Not usually activated by radiation damage. ■ Extracellular ligands include – TNF, TRAIL, FAS. Intrinsic pathway: ■ sensor caspase 9, activated in cell by presence of cell damage (e.g p53 signal). ■ Balance of Pro and anti-apoptotic factors in cell (around or in mitochondria) will determine if sensor caspase 9 activated.

Answer 134

Extrinsic pathway: 1) sensor caspase 8, activated by binding of extracellular ligand which activates death receptor. Eff ■ Extracellular ligands include – TNF, TRAIL, FAS. 2) Effector caspases activated (Caspase 3, also 6 and 7) 3) Apotosis

Answer 135

Intrinsic pathway: 1) Presence of cell damage. Signalled by p53, PI3K-AKT ■ Pro and anti-apoptotic factors in cell (around or in mitochondria) will determine if sensor caspase 9 activated. ■ Normally anti-apoptotic factors predominate and caspase 9 is inactive, but if cell damage occurs pro-apoptotic factors activated (e.g. by p53) and balance changes in favour of caspase 9 activation. ■ Pro-apoptotic proteins include BAX, BAK, PUMA. BAD from PI3K - AKT activates BAX and BAK ■ Anti-apoptotic: BCL-2 IAP family inhibit Caspase 9 and 3. 2) If balance altered: cytochrome C released from mitochondria into cytoplasm - apoptosome formed Caspase 9 activated 3) Effector caspases especially 3 (others 6 and 7) mediate apotosis.

Answer 136

Part of cytoplasm of cell digested to release molecules and energy Can be activated in response to loss of nutrients or growth factors Regulated by mTOR kinase (i.e. PI3K-AKT-mTOR) Can prolong cell survival in adverse conditions Can also lead to a form of programmed cell death (Type II) that is not caspase dependent Autophagy acting as a tumour suppressor function – if genes associated with initiation of autophagy lost then increased cancer development in mice studies

Answer 137

Autophagy acting as a tumour suppressor function – if genes associated with initiation of autophagy lost then increased cancer development in mice studies

Answer 138

Autophagy can lead to a form of programmed cell death (Type II) that is not caspase dependent: Looks like Apotosis BUT no: DNA laddering, vesicles no apototic bodies (but still vesicles present)

Answer 139

Metabolically active but Reproductive Death - Cell-cycle stopped - "G0" Usually associated with telomere shortening in aging DNA damage Premature senescence can be induced by cellular stress and IR DNA damage (not related to telomere length, activated by other pathways).

Answer 140

Different cell types have different propensity to undergo senescence – common in fibroblast cells after radiation

Answer 141

Heterochromatic nuclei Increased granularity of cytoplasm Flattening of cytoplasm

Answer 142

DNA Damage Response (DDR) DDR activation of apoptosis: upregulation of pro-apoptotic proteins and activation of p53 Genes regulating apoptosis can influence radiosensitivity of cell in this situation

Answer 143

After cells have attempted to divide or after 2 or 3 replications DDR activates cell cycle checkpoints and DNA repair DDR does not lead to apoptosis even if p53 activated. Signal for cell death is the inability to undergo mitosis properly

Answer 144

Death of cells owing to irradiation of neighbouring cells, not as a direct action of irradiation ● Not fully understood but thought to be: ○ secretion of factors by irradiated cells that are damaging to unirradiated cells ○ Sharing of reactive oxygen species between cells ○ This phenomenon is seen more clearly when cells are connected by gap junctions.

Answer 145

Phenomenon where local irradiation results in cell kill outside the radiation field, even in a distant site. ○ Mechanisms ■ Release of cytokines which mediate a systemic inflammatory reaction to radiation ■ Enhances innate immune recognition of tumour cells

Answer 146

Large tumours require higher doses to control compared to small tumours ● This is true even if intrinsic radiosensitivity, hypoxia and repopulation rates are equal

Answer 147

Sensors consists of a group of proteins that actively survey the genome for presence of damage. These proteins then signal this damage to three main effector pathways: ○ programmed cell death ○ DNA repair ○ Cell cycle arrest

Answer 148

Main source of phosphorylation of H2AX at ds DNA breaks: MRN-ATM pathway. MRN complex: MRE11 RAD50 NBS1 (directly binds to ATM)

Answer 149

Recruits ATM (inactive in cell until recruited to ds DNA break by MRN) Activated ATM phosphorylates H2AX -Spreads over large area chromatin in both directions Other proteins also phosphorylated in this pathway including p53 and MDM2

Answer 150

1) DNA-PKcs-Ku. Alternative mechanism to ATM-MRN but delayed kinetics 2) ATR-ATRIP. Does not play a substantial role in the initial recognition of DSBs (is SSB dominant pathway). Can be activated by the SSBs produced during processing of DSBs.

Answer 151

Structurally similar to ATM

Answer 152

Ku70-Ku80 complex binds to ends of DSBs and recruits DNA-PKcs which phosphorylate H2AX

Answer 153

In G1 cell cycle arrest is mediated by p53 which upregulates p21, a CDK inhibitor

Answer 154

In S and G2, CDK activity is regulated by CDC25. The inactivation of CDC25 by CHK1/2 results in S and G2 phase arrest. MRN-ATM-CHK2 ATR-ATRIP-CHK1

Answer 155

Colongenic cells are cell able to form colonies of >50

Answer 156

Histone acetyltransferases acetylate histones ● This modification alters chromatin structure, allowing greater accessibility to the underlying DNA ● Acetyl groups also attract transcription factors.

Answer 157

γH2AX recruits other repair proteins to form the ionizing radiation induced foci (IRIF). Proteins include: RAD51, BRCA1 and 2, MDC1, 53BP1

Answer 158

ATM is involved in phosphorylation of 1) Repair Proteins: H2AX, BRCA1, SMC1, p53 2) Cell cycle proteins: p53, CHK 2

Answer 159

Autosomal recessive

Answer 160

Nijmegan Breakage Syndrome (NBS1 is the part of MRN that attracts ATM) Similar therefore to Ataxia telangetasia Syndrome

Answer 161

Patients have similar radiosensitivity to AT. ``` Patients have phenotype of: short stature microcephaly, distinctive facial features immunodeficiency increased risk of cancer ```

Answer 162

- progressive cerebellar ataxia - oculocutaneous telangiectasia - immunodeficiency - increased risk of cancer. Hypersensitive to ionising radiation and agents that result in DNA breaks. Not hypersensitive to UV light.

Answer 163

Fanconi Anemia ● Autosomal recessive disorder ● Spontaneous chromosomal instability, sensitivity to DNA crosslinking, and IR. ● FANCD2 group of genes (including BRCA2) that play an important role in DNA repair and homologous recombination.

Answer 164

Artemis-deficient Severe Combined Immunodeficiency Syndrome (SCID) ● Artemis complexes with DNA-PK, plays an important role in NHEJ and VDJ recombination (for B- and T- cell genes). ● Artemis-deficient cells are radiosensitive and exhibit increased chromosomal instability.

Answer 165

1. Cells from actively growing culture prepared into suspension using trypsin to help them detach from vessel 2. Number of cells/volume counted (electronically) 3. Cells seeded onto plate and incubated 1-2 weeks. Number of cells seeded recorded 4. Colonies counted. 5. Calculate plating efficiency (PE = #colonies/#seeded) 6. Multiple plates seeded (one for each dose level being studied), if surviving fraction likely to be very low use more cells for that plate. # of cells seeded recorded. 7. Exposure. Incubation 1-2 weeks. Staining and counting of colonies. 8. Surviving fraction (SF) for each dose: SF = #colonies/(PE*number cells seeded) 9. Plot serving fraction (y-axis) versus Dose (Gy)

Answer 166

Basically count number of something (jejunal crypts, renal tubules ect)/volume and plot for dose. 1) Jejunal crypt cells - animals exposed to range of doses, sacrificed day 3, number of jejunal crypts/volume vs dose 2) Testes stem cells - similar to above but 6 weeks 3) Kidney tubules - For late responding tissue. Count regenerating tubules after 60 weeks. 4) Spleen colony assay - For lymphomas 5) Lung colony assay - transplant mouse tumours that are known to met to lung

Answer 167

Biological effect of a given dose is reduced with lowering dose-rate and increasing exposure time. ● This results from the repair of sublethal damage that occurs during long radiation exposure.

Answer 168

● For acute exposures at high dose rates, the SF has a significant initial shoulder (more "beta-like"). ● As dose rate decreases and treatment time increases, more SLD can be repaired during radiation exposure. ● Hence, SF curve becomes more shallow and the shoulder tends to disappear (could draw up arrows w/"SLD repair" labelled on)

Answer 169

For some cell lines, if the dose rate becomes slow enough (1.5cGy/min) damage isn't significant enough to trigger cell-cycle arrests in less sensitive /resistant phases of the cell cycle (e.g G1, S) and so cycle continues until sensitive phase G2 reached where the cell becomes arrested. This accumulated minor damage leading to increased cell kill at a low rate. This can be exploited to give low dose rate treatments (implants) to susceptible tumours

Answer 170

S = e^-1(alphaD + betaD^2) hence, - ln(S) = alphaD + betaD^2 Where Dose is in Gy, alphaD/betaD^2 = alpha/(beta Gy) Therefore in units Gy^-1

Answer 171

(Its just the simplest 2nd -order equation describing a monotonically decreasing function - this over interpretation is dumb) The LQ model assumes two components to radiation cell kill: ○ one that is proportional to dose and is the result of a single electron track (α) ○ one that is proportional to the square of the dose, resulting from chromosome breaks from two separate electron tracks (β) ● The components of cell killing are equal at a dose that is equal to the ratio of α and β ○ D=α/β (ie. where single and double tracks equally as likely)

Answer 172

The demarcation value between low and high LET is at about 10 keV/μm.

Answer 173

The α and β components of mammalian cell killing are equal at approximately α/β = 10 Gy and α/β = 3 Gy for early and late responding tissue, respectively.

Answer 174

Oxygenated carcinomas of head and neck, and lung have α/β values similar to acutely responding tissues

Answer 175

● Based on in vitro data – For example does not reflect fundamental in vivo processes like radiation induced T-cell response and revascularisation ect ● At very low doses per fraction <1 Gy, the LQ model could underestimate the biological effect of a given dose, due to the low-dose hyper-radiosensitivity phenomenon. ● At very high doses per fraction >6 Gy, the LQ model underestimates the biological effect due to factors such as vascular and stromal damage not being taken into account. ● Difficult to relate to underlying radiobiological mechanisms ● The LQ formula does not include a time factor and assumes sufficient time between fractions for repair of sublethal damage ● It does not take into account tissue/tumour repopulation over time ● It has not been validated with concurrent chemotherapy.

Answer 176

Y axis: Surviving fraction [10^-4, 10^-3 ...... 10^0] X axis: Dose (Gy): 0, 2, 4, 6, 8, 10, 12 (allows α/β <3, and α/β > 10 Gy) 1) High LET (e.g. α particle): Pretty much a straight line terminating at 4Gy intersection w/10^-3. Low LET (e.g. XR) draw a normal curve: a bit of the shoulder, then hits 10^-3 around 10Gy 2) Draw horizontal line x=10^0. Draw 2 limit slopes 1st. The straighter high α/β should go under the high α/β at until 6Gy then terminate at 10^-3 at >12Gy. Draw limit slope in way that α/β = >10Gy. Terminate Low α/β line around 10Gy, α/β <3Gy

Answer 177

D0 (Dzero) = On exponential part of curve the change in dose that results in a scaling of survival by e^-1 (ie. SF x 0.37). This has historical roots in Target Theory D1 = Is like D0, but is the dose required for the initial 37% reduction in SF n = where a straight line from D0 would intersect y-axis (once thought related to number of target in cells) Dq = where above line to n intersects the horizontal line y=1.

Answer 178

The Effective Survival curve = Where sufficient time is allowed for SLD repair, then effective curve is a composite of initial shoulder curve repeated for the number fractional doses. If the initial shoulder is repeated then early responding tissues (straight line/high α/β) should be more suited to fractionation. I.e late responding tissues need big enough fractions to overcome the shoulder and reach the exponential portion of the curve.

Answer 179

1) Repair 2) Repopulation 3) Redistribution 4) Re-oxygenation 5) Radiosensitivity (intrinsic)

Answer 180

● Late responding tissues have low α/β ratio and are more sensitive to changes in dose per fraction ● Early responding tissues have high α/β ratio and are less sensitive to changes in dose per fraction

Answer 181

1) Decrease in isoeffective dose 2) Decreased tissue tolerance Determined by the repair halftime of the tissues ○ time required between fractions in low dose rate treatments, for half the maximum repair to take place. ○ 4-5 hours for some organs, possibly longer for spinal cord and brain.

Answer 182

The repair halftime of the tissues ○ time required between fractions in low dose rate treatments, for half the maximum repair to take place. ○ 4-5 hours for some organs, possibly longer for spinal cord and brain.

Answer 183

Relates a tissue response/Captures the relationship between the dose required to produce a particular effect (BED)The total dose, delivered in infinitely small fractions, that would be required to produce a particular effect as indicated by the LQ-equation. ● BED=total dose (nd) x relative dose effectiveness (rde) rde = 1+(dose/(α/β ) BED for different α/β ratios are not directly comparable. i.e. different tissues, also differnt endpoints

Answer 184

Equivalent dose in 2Gy #s: EQD2 = D(d+α/β )/(2+ (α/β )) where D = total dose given with fraction doses d. It is critically dependent on the end-point being considered.

Answer 185

The reduction in biological effectiveness (BED) can be determined by considering the potential doubling time of tumour BED- gamma(t/Tpot) where gamma is ln(2)/Tpot - which relates to the doubling time of the tumour within the between fractions time window t.

Answer 186

A hot spot within a treatment field receives BOTH: 1) a higher dose, and 2) higher dose per fraction Markedly increases the biological effect (i.e the Biologically effective dose increases). ● It is important to note where hotspots are in the treatment plan and if they could lead to more normal tissue toxicity than expected.

Answer 187

LDR equivalent to multiple infinitely small fractions without interfraction intervals ○ damage induction and repair takes place simultaneously.

Answer 188

The RBE is: The ratio of biological effectiveness of one type of ionizing radiation (E.g LET) relative to another (Dome standard), given the same amount of absorbed energy. The RBE is an empirical value that varies depending on: 1) type of ionizing radiation, 2) energies involved, 3) biological effects being considered such as cell death, 4) oxygen tension of the tissues (oxygen effect).

Answer 189

Difference bettwen high LET (e.g. α particle) and conventional photons. Make a Cell Survival curve. Graph a straightish line terminating at 4-5Gy for high LET. A "mid" responding tissue line for XR. Pick an outcome (e.g 10^-2 SF) draw a horizontal line between the points on each graph at this outcome - thats the RBE.

Answer 190

BED gives the total dose required (give n in small fractional doses) to produce a particular effect. Which is dependedent on total dose, dose per fraction and α/β d=dose BED = (d x num #s)(1 + d/αβratio) T is time between doses ln(2) = 0.693 Proflif during Tx = BED - (0.693xT)/(αTpot) i.e the equivalent dose is less due to interval time and proliferation scaled by sensitivity

Answer 191

Biologically effective dose EQD2 = Equivalent dose BED=total dose (D) x relative effectiveness (RE) RE = 1+(d/αβratio) EQD2 = D(d+α/β )/(2+ (α/β )) Hence, EQD2 = BED/1+(2/αβratio) So while one if more concerned with finding a biologically effective dose, and the other an equivalent dose, they use the same terms, and hence can be related to each other.

Answer 192

Easier to under stand More clinically practical More familiar to clinicians More easily related to clinical experience

Answer 193

Dose rate Time interval of dose rate Repair rate constant α/β

Answer 194

1) The volume effect (less volume of normal tissue treated) and 2) Low dose rate effect on normal tissues. Hence, ‘double benefit’

Answer 195

Normal cells have intact repair pathways to repair sub-lethal DNA damage before the next fraction is given but cell cycle checkpoints and repair mechanisms are usually lacking in tumour cells (from lack of tumour suppressor function eg p53), leading to preferential cell kill.

Answer 196

The regeneration response of early-responding tissues in between fractions, leading to increased tolerance for normal tissues and increased radioresistance for tumours, with increasing overall treatment time. I.e the longer the treatment the more regeneration with radioresistant tumour cells. BUT The more regeneration of tolerant normal tissue.

Answer 197

Repopulation is important in: * tumours whose stem cells are capable of rapid proliferation * acutely responding normal tissues, e.g. skin, GI tract, oral mucosa has little consequence in late-responding, slowly proliferating tissues, e.g. kidney

Answer 198

Repopulation, occurring at 2-4 weeks after start of RT, is due to increase in proliferation of clonogenic cells and results in:  Decreased local control of cancer cells  Decreased acute toxicity Whithers 1988 - review of H&N SCC - little diff in dose for 50% tumour control for shedules <4weeks, but control dose incre 0.4 - 0.8 Gy/# after 4 weeks

Answer 199

Radiosensitivity of cells varies through cell cycle (M > G2 > G1 > S early > S late) The population that survive irradiation become more synchronous (e.g. more cells in S phase have maintained their reproductive integrity) At the next fraction a greater proportion of surving cells may therefore be in a more sensitive phase. Also some damage accumulated in less sensitive phases may cause arrest in G2 where cells may still be when next fraction delivered (and therefore be more sensitive).

Answer 200

Redistribution makes the cell population more sensitive to fractionated treatment as compared with a single dose. For high dose/low or single fraction treatments cells in less sensitive phase of cycle may evade death.

Answer 201

1) When oxic cells are killed by radiation, the hypoxic fraction moves closer to the blood supply and become relatively oxic and hence more radiosensitive with the next fraction. 2) In acute hypoxia tumour cells that are transiently hypoxic could be oxic when the next fraction is given.

Answer 202

Often described in terms of surving fraction after 2Gy (SF2) - Alternative is area under survival curve.

Answer 203

1) Radiosensitive tumours: Neuroblastoma, lymphoma, myeloma 0.19 SF2 of about 0.19 2) Intermediate: Breast, bladder, cervix ca. Lung SCC SF2 of about 0.46, 3) Resistant: Melanoma, osteosarcoma, glioblastoma, renal ca. SF2 of about 0.52

Answer 204

α/β Ratio for tumours of: 1) Prostate = 1.1 2) Breast = 4.6 3) Melanoma = 0.6 4) Skin = 8.5 5) General H&N = 10.5

Answer 205

Hypo fractionation causes more hypoxia = less oxic enhanced cell killing (it has been argued enhanced immune response offsets this). Hypo fractionation may benefit slow growing tumours (low α/β e.g. melanoma/prostate) where IR sensitivity is low (i.e. larger dose per/# needed) Shorter courses limit accelerated repopulation Hypo fractionation may not benefit greatly from redistribution BUT more damage per fraction may also be more difficult to repair.

Answer 206

Hypo fractionation may improve cell kill for late responding tumours, But the effects of hypofractionation on normal tissue will depend very much on whether the normal tissue is a serial or parallel arranged organ. (i.e serial much more sensitive)

Answer 207

For external beam RT of tumors... Must fractionate treatment: 1) To overcome hypoxia 2) So fractionation spares late-responding tissues compared to most tumours Must prolong treatment: To limit early sequelae Would like to shorten treatment: To prevent accelerated repopulation

Answer 208

Normal tissue repair is typically around 6hrs. But in some cases (e.g. spinal cord) may be as long as 24hrs. The influence of incomplete repair is described by the repair half time (T-half)

Answer 209

The implication of incomplete time for repair between fractions is accumulation of unprepared damage, and an interaction with damage from previous fraction(s) and the next. Therefore much less dose needed to produce the same effect.

Answer 210

Modify EQD2 by incorporating incomplete repair with variable Hm which depends on the number m of fractions, the time interval between them, and the Thalf of tissue. EQD2 = D( d(1+Hm)+α/β / 2+ α/β )

Answer 211

Hyper fractionation = | Treatment schedule where dose per fraction is reduced below the conventional 1.8-2Gy fractions.

Answer 212

Aims to increase total dose to the tumour while sparing late normal tissue side effects. Due to more time for normal tissue repair. But conversely increased possibility of tumour repopulation. Therefore usually given twice daily

Answer 213

Treatment schedule where dose per fraction more than 1.8-2 Gy is used

Answer 214

1) Decrease treatment time 2) Increased late responding tumour cell kill 3) Stereotactic radiotherapy- ablative doses while sparing surrounding normal tissues 4) Palliative radiotherapy- less concern for late side effects, more convenient for the patient.

Answer 215

Due to increased effects on normal tissue (especially serial arranged tissue), total dose is usually decreased.

Answer 216

Accelerated fractionation = Increased weekly dose >10Gy/ week

Answer 217

○ Shortens overall treatment time to counter tissue repopulation ○ normal early effects expected to increase ○ often hyperfractionated to reduce late side effects. ○ Care must be taken to ensure sufficient repair time between fractions

Answer 218

Early: Total dose, Treatment time (increasing T causes more acute effects), dose per fraction less important. Late: Total dose, dose per fraction, overall treatment time has little effect.

Answer 219

Increase acute effects, but not change severity of late effects. Increase late side effects, not change degree of acute side effects very much.

Answer 220

Can be broken into 3 considerations: 1) Aim: Symptom control vs reproductive death of all tumour cells. 2) Dose required for desired effect: e.g symptom relief balanced by toxicity vs eradicate all clonogens, tolerate more toxicity 3) Acute and late toxicities have different emphasis in these 2 groups.

Answer 221

Brachy = infinite number infinite small #s. Repair - modifies radio sensitivity at higher dose rates (favours late responding tissue relative to tumours) Repopulation - modifies sensitivity at very low dose rates (favours tumours) Redistribution/reoxygenation are intermediate. Radiosensitivity determine how effective treatment is with increasing distance from source.

Answer 222

● As dose rate decreases below 1 Gy/min, radiosensitivity decreases, curves become straighter. ● As dose decreases below <1 cGy/min repopulation further improves cell survival. ● Inverse dose rate effect ○ low-dose hyper-radiosensitivity ○ slight increase in cell kill with further lowering of the dose rate ○ Thought to be due to accumulation of cells in G2, increasing radiosensitivity and radiation damage.

Answer 223

Hydroxyl free radicals May then go on to damage DNA.

Answer 224

The Oxygen Fixation Hypothesis: 1) Sparsely ionising (i.e. low LET) radiation causes damage via free radical production 2) Under hypoxic conditions free radicals are repaired by sulfhydral groups 3) In normoxic conditions damage is not repaired rather it is 'fixed' (in the "stuck with"/permanent sense of the word) permanently and irreversibly

Answer 225

● Concentration of oxygen needed to produce an effect is low. ○ 3 mmHg or 0.5% oxygen concentration results in radiosensitivity halfway between hypoxia and fully oxygenated conditions ○ Radiosensitivity at 30 mmHg or 5% O2 is almost at fully oxygenated levels

Answer 226

1) Chronic hypoxia Death of oxygenated cells leads to diffusion of oxygen to hypoxic cells, rendering them more sensitive to radiation at the next fraction. 2) Acute hypoxia Caused by temporary blockage or closure of a blood vessel. causing intermittent hypoxia ■ hypoxic cells could become oxygenated during the next fraction

Answer 227

Due to outgrowing of blood supply. Distance of oxygen diffusion from capillaries is about 70 μm with variations from arterial to venous ends. Creates three layers of cells: 1. proliferating well 2. region where cells can survive and remain clonogenic but radioresistant 3. dead or dying cells.

Answer 228

The ratio of dose under hypoxic conditions to the dose under aerated conditions to produce the same biological effect.

Answer 229

Low LET radiation: Usually 2.5-3 High LET: Closer to 1 for high LET (ie. no oxygen effect) Neutrons ~ 1.6

Answer 230

1) Animal studies: Mouse sarcomas air breathing vs Nitrogen asphyxiated 2) Clonogenic assays (normal vs clamped conditions) 3) DNA damage assays 4) Clinical: i) Observation H&N cancers Eppendorph probes ii) Pharma Interventional: E.g. DAHANCA 2 and 5, ACRON

Answer 231

1) Fractionation 2) High LET XRT 3) Hypoxic cell sensitisers (the nitronazoles: misonidazole, nimonidazole) 4) Blood transfusion - Nil sigbif benefit 5) Nicotinamide and carbogen gas (95%02, 5%C02) - ACRON trial - improved local control in H&N Ca 6) Hyperthermia - ?evidence 7) Hyperbaric O2 - Small benefit, but difficult to deliver 8) Hypoxic cell cytotoxins: Inactive in aerobic cells. Highly toxic but big effect.

Answer 232

Nitroimidazoles. E.g Misonidazole (old more toxic), Nimonidazole (new, less toxic - DAHANCA 5) MOA: ○ Mimics sensitizing effect of oxygen ○ Diffuses out of tumour vasculature to reach hypoxic cells. Dose limited by neurotoxicity

Answer 233

``` H&N Ca studies: DAHANCA 2 (Misonidazole - effective dose limited by toxicity). Less effect than hoped. More effective for worse prognosis Ca. DAHANCA 5 (Nimonidazole - higher dose could be given as less toxic): both local control and PFS increased. ```

Answer 234

Overcome BOTH acute and chronic hypoxia. Vit B3 - prevents transient fluctuations in tumour blood flow - prevents Acute Hypoxia. Carbogen (5%CO2, 95% O2): Overcome chronic hypoxia. ACRON trial - improved local control in H&N Ca

Answer 235

Hypoxic tumours are more sensitive to effects of hyperthermia ■ reduced blood flow ■ reduced pH

Answer 236

Hypoxia and reoxygenation causes aberrant DNA synthesis, leading to over-replication and gene amplification Hypoxia also reduces expression and function of DNA repair genes. Cells have increased mutation frequency and genetic instability when grown in hypoxic conditions in vitro.

Answer 237

1) Causes aberrant DNA synthesis, leading to over-replication and gene amplification 2) Reduces expression and function of DNA repair genes.

Answer 238

1) Directly Ionising: ○ Interacts directly with ionizing electron and ionizes atom. ○ Charged particles ■ alpha particle ■ beta particle ■ heavy charged particles eg protons and ions 2) Indirectly Ionising: ○ requires two step process- liberation of a charged particle that goes on to ionize other atoms ○ Uncharged particles ■ photons: capable of ionizing atoms, majority of energy is deposited via a two-step process hence considered indirectly ionizing ■ neutron: interacts with nucleus, liberates protons

Answer 239

The energy deposited by a particle per unit length while traversing through matter. Units: keV/um

Answer 240

Divide track of radiation: 1) Into intervals of equal length and take average energy in each = TRACK AVERAGE or, 2) Divide track into intervals each with equal energy, take average interval length = ENRGY AVERAGE

Answer 241

LET = 0.2 keV/um Often the reference radiation for RBE calculations (which is the ratio of doses required to produce a given biological effect) The alternative reference for Low LET is 250 keV XRs = 2 keV/um

Answer 242

For a given charged particle, the higher the energy the lower the LET, as secondary electrons travel away from the area of interest. E.g: 150MeV protons have LET of 0.5keV/um whereas 10MeV protons have LET 4.7 keV/um

Answer 243

250 keV XRs = 2 keV/um Cobalt-60 gamma rays = 0.2 keV/um Alpha particles = 166 keV/um

Answer 244

● High LET ○ Tends to cause direct DNA damage ○ Less reliant on free radical production ■ hence less effect of free radical scavengers ○ Less reliant on oxygen effect (due to less indirect action) ○ Less reliant on position of cell cycle ○ Steeper cell survival curve with less shoulder

Answer 245

● Low LET ○ tends to cause indirect damage ■ production of hydroxyl radicals by ionization of water, which cause DNA damage ● hence affected by free radical scavengers ■ Also affected by presence or absence of Oxygen. ■ Affected by position of cell cycle

Answer 246

RBE := The ratio dose required to cause biological effect using reference radiation (Dref) - typically low LET cobalt-60 or 250keV Divided by dose required to cause same biological effect using test radiation (Dtest) RBE = Dref/Dtest

Answer 247

As LET increases the RBE increase, reaching a peak (optimum) around 100keV/um, after this more damage is done than is needed to kill cells - so high LET cannot get anymore effective

Answer 248

y-axis (left) RBE [0 to 10] y axis (right) OER [1, 2, 3] x-axis LET (units keV/um): log10[0.1, 1, 10, 100, 1000] RBE exponential curve peaking at a little over 100 (point of inflexion around 80), with steep drop off OER slowly decreasing with til about ~50, then steep drop off

Answer 249

At 100 keV/μm the separation of ionizing events is roughly equal to the diameter of DNA double helix (2nm) ● Hence 100 keV/μm is the LET that deposits energy causing just enough DNA DSB to cause cell death.

Answer 250

The 2 dose response curves: 1) Tumour Control Probability 2) Normal Tissue Control Probability Can be modelled by Poissonian statistics: E.g if dose sufficient to on average cause one lethal hit per cell then, number of cells receiving lethal hits is 67%

Answer 251

Gamma, which is low at start and end, highest at point of inflexion. Gamma at 50% response is Gamma50 and is often included in tables.

Answer 252

1) Intrinsic radiosensitivity 2) Tumour heterogeneity 3) Chemical factors: oxygen, radiosensiters and protectors 4) Organisation of tissue 5) Host factors: age, syndromes/co-morbities 6) Radiation quality

Answer 253

``` risk factors eg. ■ smoking ■ previous treatment to the area ■ age ■ concurrent chemotherapy ■ co-morbidities eg. COPD for risk of radiation fibrosis ■ use of radioprotectors ■ hyperbaric oxygen ```

Answer 254

Dose to achieve a desired level of tumour control compared with the normal tissue response at that dose. When TR is high good tumour control is achieved low normal tissue damage.

Answer 255

1) Sensitisers: I.e increase tumour cell kill relative to normal tissue. 2) Fractionation: I.e Hyperfractionation spares late responding tissue 3) Radioprotectors - protect normal tissue from radiation.

Answer 256

Hyperfractionation: spares late-responding tissues relative to tumours

Answer 257

The possibility of eradicating the tumour at its primary site with radiotherapy

Answer 258

The overall level of clinical response to radiotherapy

Answer 259

1) Physical: Dose/rate, fractionation, beam quality, temp 2) Chemical: Sensitisers, protectors, Oxygen 3) Biological: Intrinsic radiosensitivity, tissue organisation, tumour size/clonogen number, host factors 4) Technical factors = inaccuracies - hotspots, geographic miss.

Answer 260

1) Oxygen: nimodazole 2) Sensitisers: Chemotherapy: 5-FU, cisplatin, taxanes 3) Radioprotectors: Amifostine

Answer 261

Amifostine detoxifies reactive metabolites of platinum and alkylating agents, as well as scavenges free radicals. Used to prevent or lessen the damage to the kidneys caused by cisplatin (a chemotherapy) or damage to the salivary glands, caused by radiation therapy,

Answer 262

``` Physical factors ● Total dose ● Dose rate ● Dose/fraction ● Radiation quality ● Hyperthermia ○ T>45 C results in direct cellular killing ○ T 40-43 C increases radiosensitivity by stimulating vascular activity, increasing oxygenation. ```

Answer 263

``` Biological factors ● radiosensitivity ○ position in cell cycle ○ cell type ● organization of tissue ○ serial vs parallel ● clonogen number/size of tumour ● host factors ○ age ○ radiosensitivity syndromes ○ comorbidities eg diabetes, scleroderma ○ Volume of normal tissue irradiated !!!!!!! ```

Answer 264

``` host factors ○ age ○ radiosensitivity syndromes ○ comorbidities eg diabetes, scleroderma ○ Volume of normal tissue irradiated !!!!!!! ```

Answer 265

Inaccuracies in delivery ○ hot spots- double trouble ○ geographic miss- leads to insufficient treatment of tumour and higher volume of normal tissue being irradiated

Answer 266

Cells that are less differentiated, have greater proliferative capacity and divide more rapidly are more radiosensitive

Answer 267

In normal tissue most effects of radiation are due to the depletion of a cell population by cell-killing

Answer 268

○ Inherent cellular radiosensitivity ○ Kinetics of the tissue = Law of Bergonie and Tribondeau ○ Organization of the tissue

Answer 269

Within 90 days Usually tissues with rapid turnover rate. Therefore results in progressive cell depletion However rapid growth me imply repaired rapidly and injury may be completely reversible

Answer 270

Persistent severe acute normal tissue radiation effects could result in chronic late effect, the consequential late effects

Answer 271

The time-course of the early radiation effects is determined by: 1) The overall cell turnover time 2) Dose: I.e fewer stem cells survive - slower repair.

Answer 272

1) Proinflammatory phase: Proteins COX-2, IL-1, TNF-A 2) Cellular depletion: Due to stem cell loss - leads to inability to sufficiently re-populate functional compartment. 3) Recovery - determined by dose (surviving stem cells) and cell growth

Answer 273

Onset after 90 days Most often occur in late responding tissue Much more sensitive to changes in fractionation

Answer 274

1) Vascular injury - loss capillaries, loss of arteriolar smooth muscle (telangiectasia) = impaired diffusion 2) Fibroblast proliferation - Increased collagen synthesis. 3) Loss of parenchymal cells

Answer 275

The time period between radiation and clinical signs of cell injury. Latency also described as time period during which cells are undergoing mitotic death but there is no evidence of injury clinically.

Answer 276

1) In rapidly dividing cells latency is short, and independent of dose (above some critical amount) 2) In slowly dividing tissues latency is long, and is dose dependent

Answer 277

Normal tissue tolerance depends on number of surviving cells able to maintain organ function

Answer 278

Generally the total dose that can be tolerated by a normal tissue is dependent on the volume that is irradiated (with larger volumes tolerating less dose). Of critical importance to a tissue's sensitivity to volume irradiated is the arrangement of functional subunits (FSUs) within the tissue.

Answer 279

Organs with a serial arrangement of FSUs exhibit a binary (all or nothing) response to radiation - loosing function when a critical dose/volume threshold is crossed. Organ

Answer 280

1) Arrangement: Serial vs parallel | 2) Structure: Defined vs undefined

Answer 281

Anatomically delineated structures with clear corresponding tissue function eg. nephrons, liver lobules. Each FSU is small and autonomous. Implication for repair: ■ Surviving clonogens cannot migrate from one FSU to another. Dose response pattern: ■ Depleted by small doses.

Answer 282

Michaelowski Classification of Tissues: described tissues as either hierarchical or flexible, based on their cell populations comprised of a combination of 3 types of cell: Stem cell, Mature functional cell, in between = maturing. ● Hierarchical type: Populations of all three types, with stem cells constantly giving rise to maturing cells which eventually fully differentiate and become functional cells. Hierarchical tissues include most epithelial layers and the bone marrow. ● Flexible cells: Cells rarely divide but may be induced to by damage. Their cells are functional but retain the ability to re-enter the cell cycle if required. F-type tissues include the liver, thyroid and the dermis of the skin. ○ no compartments and strict hierarchy.

Answer 283

In general, H-type tissues 1) Respond rapidly to radiation damage as the stem cell population is killed. 2) This prevents the production of more maturing cells and leads to symptoms related to this – eg: thinning of epithelium or decrease in blood cell production.

Answer 284

F-type tissues may not display damage for some time, particularly if the dose is small, because not all cells enter the cell cycle immediately. ○ can be triggered to divide by damage to the tissue ○ no compartments and strict hierarchy.

Answer 285

Release of cytokines, hormones and immune (e.g Tcell mediated) sensitising. A symptom due to the abscopal effect is fatigue.

Answer 286

○ thought to be due to release of cell-damaging enzymes during death ○ seen in cells connected with gap junctions.

Answer 287

In relation to normal tissue injury, in broad terms stem cell depletion is a response to dose. Therefore stem cell asymmetry loss is a dose response as may be accelerated repopulation. Whereas tissue hypoplasia reflects both dose and volume. Therefore accelerated proliferation and abortive proliferation are more responses to volume.

Answer 288

○ Within minutes to hours there is repair of sublethal damage ○ In days to weeks there is radiation induced tissue regeneration ○ In months to years some tissues exhibit long term restoration

Answer 289

1) Acceleration of stem cell proliferation 2) Stem cell asymmetry 3) Abortive proliferation

Answer 290

■ New stem cells are produced to replace those that are killed by irradiation ■ Normally, stem cells divide into one stem cell and one differentiating cell asymmetric division) ■ Under duress, stem cells can divide into two stem cells to repopulate the pool (asymmetry loss)

Answer 291

■ Shortening of cell cycle time to compensate for cell loss | ■ Dose dependent: higher dose = faster acceleration

Answer 292

Abortive proliferation plays a role in post radiation generation of normal tissue. ■ Irradiated cells can undergo limited number of divisions before death, to maintain cell numbers.

Answer 293

Asymmetry loss is controlled by stem cell depletion to compensate for dose, whereas tissue hypoplasia induces acceleration of proliferation and abortive proliferation to compensate for dose and cell loss.

Answer 294

The maximum dose that can be given without causing an unacceptably high probability of treatment complications.

Answer 295

Radiation quality, volume, concurrent treatments, patient/organ status

Answer 296

In FSUs arranged in parallel there is a marked volume effect which determines tolerance dose

Answer 297

It is important to remember clinical tolerance, as opposed to just objective measures of some kind of injury. E.g. Large skin ulcer leads to pain and slow healing whereas small ulcers heal quickly

Answer 298

1) Parenchymal cells: Decreases in sensitivity according to Casarett from type I to IV 2) Connective tissue 3) Vascular system - commonly implicated in late effects. Includes vessels and heart. 4) Immune system

Answer 299

○ 4 classes according to Casarett ○ Decreases in sensitivity from I to IV Type: I) Rapidly dividing undifferentiated = most sensitive. E.g germinal epidermis. II) Dividing cell becoming differentiated. E.g myleocytes III) Functional cells that can re-enter mitosis: E.g hepatocytes, renal cells IV) Fully differentiated post mitotic cells - Cardiac myocytes, neurons, lymphocytes (lymphocytes are super radiosensitive however - go into apoptosis easily)

Answer 300

1) Fibroblasts: Radiation drives differentiation into fibrocytes (via TGF-B), similar to hypoxia. This results in collegen production and deposition and can clinically appear as radiation fibrosis.

Answer 301

Radiation drives differentiation of fibroblasts into fibrocytes (via TGF-B), similar to hypoxia. This results in collegen production and deposition and can clinically appear as radiation fibrosis.

Answer 302

1) Cartilage: Very sensitive to radiation. Results in death of condroblasts. Dose >20Gy results in irreversible deficit. Adult cartilage is more resistant. 2) Bone: OsteoRADIOnecrosis - due to vascular effects.

Answer 303

Thrombosis and capillary necrosis Smooth muscle cells diminish over several years resulting in telangestsia (larger capillaries and veins), and stenosis where college has replaced muscle. In larger vessels, sub intimal deposition of lipids may result in atherosclerosis.

Answer 304

Arteries - more myocytes/active tissue >50Gy is threshold for arteries >40Gy is threshold for capillaries

Answer 305

■ Acute pericarditis occurs after 12 months ■ Threshold dose is 20 Gy ■ 11% incidence at 45-50 Gy

Answer 306

■ Radiation induced cardiomyopathy evolves over a period of years. ■ Results from dense and diffuse fibrosis ■ Threshold dose ~30 Gy if most of the heart treated. I.e volume effect in parallel tissue.

Answer 307

Lymphocytes most sensitive | Macrophages the least

Answer 308

Lymphocytes are very sensitive to IR, with sufficient dose they quickly go into apoptosis ■ Total lymphoid irradiation of 30-40 Gy leads to prolonged T-cell lymphopoenia

Answer 309

1) Estimate the physical circumstances: The field, position in the field and duration spent there. 2) Biological dosimetry: - Clinical symptoms. In particular onset of vommiting. Relate to acute radiation syndrome (e.g. provided by CDC) - Lab investigations i) Blood count - especially lymphocyte count over time (typically 6hrly/48hrs) ii) Chromosome aberrations in peripheral lymphocytes iii) Assays - especially Comet assay iv) gammaH2AX

Answer 310

● Data from survivors of atom bombs and nuclear accidents | ● Occurs with short exposure of dose >0.7 Gy to the whole body.

Answer 311

○ If onset of vomiting greater than 2 hours after exposure suggests absorbed dose <2 Gy ○ If onset less than 30 mins suggests dose >6-8 Gy

Answer 312

After radiation exposure lymphocyte count is typically monitored every 6 hours for 48hrs Chromosome aberrations in peripheral lymphocytes

Answer 313

Rapidly reducing numbers of lymphocytes following exposure may make studying them difficult. As peripheral lymphocytes are typically in G0, PHA is needed to make them re-enter cell cycle. Colchicine is then used to arrest cells in metaphase. Chromosomes are then assessed by some form of assay (e.g. dicentric assay, translocation assay ect)

Answer 314

1) Prodromal syndrome 2) Latent period 3) Manifest illness 4) Recovery or death

Answer 315

Prodromal syndrome of ARS: | Time of onset, severity, and duration depends on dose.

Answer 316

Dose>10Gy (But as low as 6Gy) Onset within a few hours, lasts about 2 days. The LD100 is about 10Gy

Answer 317

● Depopulation of the gastrointestinal epithelium ● Latent stage of a few days ● Death within 2 weeks from infection, dehydration and electrolyte imbalance

Answer 318

1) Hematopoietic Syndrome: Dose>0.7Gy. 2) Gastrointestinal Syndrome: Dose >=10Gy (can be as low as 6) 3) Cerebrovascular Syndrome: Dose <50Gy

Answer 319

``` Haematopoietic Syndrome ● >0.7 Gy ● Bone marrow failure. ● Latent phase 1-6 weeks ● Death in a few months from infection and haemorrhage. ```

Answer 320

``` Cerebrovascular Syndrome ● >50 Gy ● Onset within minutes ● Uncontrolled intracranial hypertension and circulatory collapse, likely due to damage of microvasculature ● death within 3 days ```

Answer 321

The LD50/60 is the dose necessary to kill 50% of the exposed population in 60 days. ‡ The LD100 is the dose necessary to kill 100% of the exposed population

Answer 322

>2Gy, usually begins around day 15

Answer 323

Diarrhoea is associated with total body doses greater than 6Gy

Answer 324

Treatment of ARS ● Predominantly supportive. ● Protective isolation and antibiotics can double LD50 to ~7 Gy ● Narrow window of bone marrow transplant ○ <7 Gy probably no benefit ○ >10 Gy patient will die from gastrointestinal syndrome.

Answer 325

Cutaneous Radiation Injury ● Radiation burns Differ from thermal and chemical burns 1) latent period between exposure and effect 2) undergo recurrent breakdown even after scar formation ● Threshold for epilation is 3 Sv, erythema 6 Sv, desquamation and ulceration >10 Sv. ● Can persist to develop chronic ulceration and infection.

Answer 326

1) Preimplantation 0-9 days 2) Organogenesis 10 days to 6 weeks 3) Foetal period 6 weeks to 9 months The threshold dose for all is 0.1Gy

Answer 327

Preimplantation 0-9 days. | All or nothing embryonic death response with threshold around 0.1Gy

Answer 328

Organogenesis 10 days to 6 weeks ○ Radiation causes major structural congenital anomalies ○ Severe intrauterine growth reduction from cellular depletion ○ Microcephaly Threshold dose 0.1 Gy

Answer 329

Foetal period 6 weeks to 9 months 1) Mental retardation - 25 IQ points per Gy, threshold 0.1Gy, due to failure of neurons to migrate peripherally. 2) Microcephaly 3) Eye, skeletal, genital abnormalities 4) Intrauterine growth restriction

Answer 330

10mSv (i.e. Wr for XRs = 1, so 0.01Gy) The excess absolute risk coefficient at this level of exposure is approximately 6% per gray

Answer 331

The dose required to double the spontaneous anomalies within a population. Atomic bomb data suggests this is about 2 Sv.

Answer 332

Epigenetic changes - leading to transgenerational genomic instability.

Answer 333

Current recommendations to wait 6 months before attempting pregnancy following exposure of radiation to the gonads.

Answer 334

○ 0.15 Gy- oligospermia ○ 0.5 Gy- temporary sterility ○ Cumulative dose >2 Gy- permanent sterility

Answer 335

○ Does not produce changes in hormone balance, libido or physical capability ■ controlled by testosterone which is secreted by Leydig cells ■ >20 Gy

Answer 336

○ Division of spermatogonia to mature sperm takes about 64 days ○ Mature and maturing cells more resistant to radiation ○ Hence latent period after exposure to radiation until clinical oligo/azoospermia is seen

Answer 337

``` ○ Oocytes present at birth ○ No cell division until puberty ○ Radiation above a threshold dose produces permanent sterility ■ 12 Gy prepuberty ■ 2 Gy premenopausal ``` ○ Radiation sterility produces hormonal changes

Answer 338

1) simple way of describing severity of toxicity 2) specific for each organ, 3) specific endpoints seen following radiotherapy for that particular organ 4) standardized internationally, enables comparisons between investigators, institutions, studies and treatment modalities

Answer 339

○ Grade 0: no effect ○ Grade 1: mild, reversible and spontaneously heal ○ Grade 2: moderate, patient still able to self care. Requires outpatient treatment without cessation of therapy ○ Grade 3: severe, medically significant, interfere with activities of daily living. Requiring inpatient treatment and supportive care. Radiotherapy may require altering or temporary cessation ○ Grade 4: Life-threatening, requiring intensive treatment and supportive care, and immediate cessation of radiotherapy ○ Grade 5: Death due to radiotherapy toxicity

Answer 340

○ RTOG/EORTC ○ CTCAE v3 ○ WHO ○ LENT-SOMA (for late effects)

Answer 341

Late Effects Normal Tissues (LENT)-Subjective, Objective, Management, Analytic (SOMA) scales by the joint efforts of the EORTC and RTOG in 1995 was an attempt to produce a universal system for measuring and recording the late effects of radiotherapy. (NB the Analytic part refers to imagining/laboratory tests that may be done)

Answer 342

● Published in 2010 | ● Summarizes dose/volume/outcome data for many organs, extracted from publications.

Answer 343

○ Based on DVHs ■ not ideal representation of 3D dose distribution ■ do not consider fraction size variations ■ do not account for anatomic variations during therapy ○ Most data relate to conventionally fractionated regimens ○ Does not factor concurrent chemotherapy ○ Does not include host factors ○ Not available for all endpoints

Answer 344

○ Based on DVHs ■ not ideal representation of 3D dose distribution ■ do not consider fraction size variations ■ do not account for anatomic variations during therapy

Answer 345

Not available for all endpoints

Answer 346

Brain: 54Gy (TD50 70Gy) Spinal cord: 50Gy (TD50 60Gy) Heart: 55Gy (TD50 65Gy) Lung: 20Gy (TD50 30Gy)

Answer 347

Testes (sterility): 1Gy (TD50 2Gy) Ovaries (sterility): 6Gy (TD50 12Gy) Lymphocytes: 2Gy (TD50 10Gy)

Answer 348

Leukemia has the shortest latency post radiation. Appears invert few years and peaks at 6-7years.

Answer 349

For irradiated mice the incidence of radiation induced leukaemia is a bell curve with fat right tail (Peak of 40% at 2.5-3Gy). Where x-axis is Dose (Gy) and y axis on left is incidence. On right linear surviving fraction - peak incidence occurring at 0.4Gy. In contrast For solid tumours ERR/Gy is relatively linear ■ Breast cancer in Hodgkins patients, and A-bomb data

Answer 350

The incidence of radiation induced leukaemia is a bell curve with fat right tail (Peak of 40% at 2.5-3Gy). Where x-axis is Dose (Gy) and y axis on left is incidence. On right linear surviving fraction - peak incidence occurring at 0.4Gy. Gray suggested this was due to increased induction of transformed cells with dose causing the increase in incidence making left tail of the curve, the right-sided decline in incidence is due to increased cell-kill with dose.

Answer 351

Currently radiation protection committee adopts the Linear No-Threshold model. ● LNT states that there is no threshold dose below which exposure to radiation is safe. ● Assumes that radiation carcinogenesis is a stochastic effect

Answer 352

● LNT states that there is no threshold dose below which exposure to radiation is safe. ● Assumes that radiation carcinogenesis is a stochastic effect ○ No threshold ○ Risk increases with increasing dose ○ Severity of cancer is not dose related

Answer 353

1) Justification 2) Optimisation 3) Dose limitation

Answer 354

Planned exposure to radiation should be justified (eg. diagnostic imaging) and optimized (shielding), and dose to public, patient and staff should not exceed individual dose limits, social and economic factors taken into account.

Answer 355

Severity of cancer is not dose related

Answer 356

Occupational dose limits do not include radiation doses received by the occupationally exposed individual while 1) undergoing a medical examination, 2) any radiation dose from natural radiation sources, E.g cosmic rays and naturally occurring radioactivity

Answer 357

To obtain an effective dose, the calculated absorbed organ dose DT is first corrected for the radiation type using factor WR to give a weighted average of the equivalent dose quantity HT received in irradiated body tissues, and the result is further corrected for the tissues or organs being irradiated using factor WT, to produce the effective dose quantity E.

Answer 358

Used only if there has been non-uniform irradiation of a body. If the body has been subject to uniform irradiation, the effective dose = whole body equivalent dose, and only the radiation weighting factor WR is used.

Answer 359

One sievert carries with it a 5.5% chance of eventually developing cancer. Based on the linear no-threshold model.

Answer 360

Effective Dose: 20 mSv/yr over 5 years 50 mSv/yr Equivalent Dose: 150 mSv/yr to the lens 500 mSv/yr to Skin

Answer 361

The effective dose (sometimes referred to as the whole body dose) limit is concerned with carcinogenic risk. The equivalent dose limits are designed to ensure that individual doses are kept below the dose thresholds for deterministic effects.

Answer 362

Effective Dose: 1 mSv/yr (20 times less than worker) Equivalent Dose (10 times less than worker): 15 mSv/yr to the lens 50 mSv/yr to Skin

Answer 363

Integral Dose = Volume x density x average Dose to that volume. Whole body exposure events (e.g. A-Bomb survivors) often provide the data for predicting risk associated with radiation exposure, however relating ID to second malignancy data may be more relevant for radiotherapy exposures.

Answer 364

Breast Ca: | ○ lung ca risk of <0.6% after 10 years

Answer 365

0.3% risk of any malignancy

Answer 366

Hodgkins Lymphoma ○ breast cancer in patients <30- 11.7% at 25 years ■ combination of chemotherapy ■ older techniques

Answer 367

Consequential late effects develop through interactions between early and late effects in the same organ. Predominantly found in organ systems where the acute response (of the epithelial lining) degrades barrier against mechanical or chemical stress, which may cause additional trauma to the underlying tissues. Therefore, CLE are mainly found in the urinary and intestinal system, in mucosa and, to some extent, in skin. Amelioration of the acute response to irradiation may be a useful approach to minimize late side effects of effective radiation therapy

Answer 368

1) Tumour localisation: May be easier to delineate tumour site pre-resection 2) Treatment volume. Larger post op. 3) Clonogen number. Reduced by surg, increased probability therefore of complete killing all colonogens 4) Blood supply - More hypoxia post, (intra op tumours may be normoxic) 5) Time of delivery (repopulation) 6) Normal tissue effects - neoadj may complicate wound healing, post causes higher rates of late effects 7) Diagnostic tissue - obvious

Answer 369

With Chemo 1) Spatial co-operation: i - micrometastatic disease outside field ii - Treat 'Sanctuary sites' (e.g brain) that cannot be reached by CTx 2) Cytotoxic enhancement: i - independent action - given sequentially ii - Additive effect - concurrent, w/reduced dose iii - Synergistic interaction- concurrent, w/reduced dose Radio sensitisers: 3) Biological co-operation: i - Hypoxic sensitizers- Nitroimidazoles, ii - Hypoxic cytotoxins - Tirapazamine 4) Temporal modulators. Preclinical evidence tumours increase EGFR expression post XRT. Cetuxumab binds EGFR.

Answer 370

Cytotoxic enhancement: i - independent action - given sequentially ii - Additive effect - concurrent, w/possible reduced dose iii - Synergistic interaction- concurrent, w/possible reduced dose

Answer 371

Dose Modifying Factor is the ratio of isoeffective doses in the absence and presence of radiosensitizer.

Answer 372

1) Spatial co-operation: i - micrometastatic disease outside field ii - Treat 'Sanctuary sites' (e.g brain) that cannot be reached by CTx 2) Cytotoxic enhancement: i - independent action - given sequentially ii - Additive effect - concurrent, w/reduced dose iii - Synergistic interaction- concurrent, w/reduced dose

Answer 373

1) Biological co-operation - Hypoxic sensitisers - Hypoxic cytotoxins 2) Temporal Modulators - Hormonal therapy - EGFR inhibitor - Cituxumab

Answer 374

Hypoxic cell radiosensitizers ■ nitroimidazoles: eg misonidazole, nimorazole ■ able to diffuse into hypoxic cells and has the same effect as oxygen on DNA damage ■ Requires concurrent delivery with radiotherapy

Answer 375

Preclinical evidence that EGFR is activated following RT in EGFR mutated cells: ○ Tumour cells that overexpress EGFR repopulate more rapidly following RT than non-EGFR mutated cells ○ EGFR inhibitors if given concurrently might therefore reduce their proliferation Cetuximab has been delivered concurrently with RT for SCC head and neck, but clinical results not as promising as predicted.

Answer 376

Sulfhydryl compounds: ○ eg. amifostine ○ act as free radical scavengers ○ and removes active metabolites from platinum and alkylating agents ○ prodrug, requires activation by alkaline phosphatase which is found in higher concentrations in normal cells

Answer 377

Selective activity in normal cells but not 100%, possible protection of tumour cells as well ○ Requires administration <30 mins before RT ○ associated with side effects: hypotension, anaphylaxis, Steven-Johnson syndrome, nausea and vomiting

Answer 378

Effect of these drugs called the Dose Reduction Factor | ○ Ratio of dose with drug to dose without drug

Answer 379

● Early toxicity is based on damage to stem cells which are unable to repopulate the cell pool ● Chemotherapy is also active in proliferating cells ● Concurrent treatments therefore increase the likelihood of early side effects When treatments are given sequentially, normal tissue has time to repair and toxicity is not as high as concurrent treatments

Answer 380

● Late effects are seen after a latent period of months to years ● Tend to be irreversible ● Can also be due to consequential late effects ● Chemotherapies that interfere with DNA repair can increase the risk of late side effects. ● Risk is also increased in combined modality treatments when drugs have a specific tissue toxicity ○ eg bleomycin for lung, doxorubicin for heart

Answer 381

This could result in doses over the tolerance doses for that organ or surrounding normal tissue Use EQD2 to add both previous and proposed treatments (if doses were/are not given to standard fractionations). ■ α/β ratio used should be that of normal tissues at risk. ■ Retreatment should only be considered if total doses given previously have not exceeded tolerance doses (but consider 'forgotten dose over time')

Answer 382

○ Initial dose and proposed re-irradiation dose ○ Dose per fraction -Large doses per fraction will more likely lead to early and late effects ○ Irradiated volume ○ Irradiated tissues. I.e unique repair kinetics ○ Time interval - Most tissues especially early-reacting tissues exhibit ‘forgotten dose’ if enough time for recovery is given ○ Radiation technique ○ Modifiers of radiation dose ○ Other treatment options ○ Patient fitness and life expectancy

Answer 383

Most tissues especially early-reacting tissues exhibit ‘forgotten dose’ if enough time for recovery is given = a dose higher than the difference between tolerance dose and initial dose may be given safely ○ eg total of 160% of tolerance dose could be given with a time interval of 3 years. BUT: Tolerance of some organs worsen over time ● eg kidney, due to progressive fibrosis of glomeruli

Answer 384

1) Skin rodent studies Early skin reactions recover and tolerate almost full repeated doses. Late skin reaction (use limb deformation) - very poor retreatment tolerance. 2) Primate spinal cord: 167% of EQD2 tolerated after 3 years.

Answer 385

!!!!!Caution interpreting data: small n, changing techniques and fractionations, no control groups.

Answer 386

○ Studies of reirradiation of recurrent glioma ○ High risk of radionecrosis if combined total dose >100 Gy ○ No correlation with time interval!!!!!!! ○ Higher retreatment doses possible with more conformal techniques

Answer 387

Substantial recovery provided initial treatment did not exceed 90% of tolerance dose and there was a time interval of at least year between treatments ○ Data suggest cumulative dose of 130-135% of the acceptable tolerance dose ■ hence 30-35% recovery in a year ○ Consider conformal therapies and dose per fraction <2Gy

Answer 388

○ Retreatment doses of 50-60 Gy required to be effective!!!!! ○ Associated with very high rates of complications including treatment related death ○ Increased risk of complications with shorter time interval and higher combined total dose

Answer 389

○ Retreatment using electron beams or interstitial brachytherapy can be used with low incidence of side effects ○ High doses can be applied to the chest wall with sparing of heart and lungs

Answer 390

Pro-drug actived by Alk-phos which is higher in normal tissue. Activated drug acts as free radical scavenger, decreasing the effect of indirectly ionising radiation.