Determinants of Tumor Cure

www.rwww.radbioladbiol.ucla.edu.ucla.edu

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Tumor Responses to RTTumor Responses to RT

Bill McBrideBill McBrideDept. Radiation OncologyDept. Radiation Oncology

David Geffen School MedicineDavid Geffen School MedicineUCLA, Los Angeles, Ca.UCLA, Los Angeles, Ca.

[email protected]@mednet.ucla.edu


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Determinants of Tumor CureDeterminants of Tumor Cure

• Size of the clonogenic pool (stem cells)Size of the clonogenic pool (stem cells)• Intrinsic radiosensitivityIntrinsic radiosensitivity

– S.F. 2Gy (pro-apoptotic tendency?)S.F. 2Gy (pro-apoptotic tendency?)• RepairRepair

– T1/2 (HR, NHEJ, SLDR, PLDR, fast and slow repair?) T1/2 (HR, NHEJ, SLDR, PLDR, fast and slow repair?) • Rate of repopulation/regeneration during therapyRate of repopulation/regeneration during therapy

– Tpot (L/I., Ki67?)Tpot (L/I., Ki67?)• Reoxygenation (extent of hypoxia)Reoxygenation (extent of hypoxia)

– POPO22 (dependence on tissue type, vascularity?) (dependence on tissue type, vascularity?)• RedistributionRedistribution

– Growth fraction (dependence on cell type, growth factors?)Growth fraction (dependence on cell type, growth factors?)


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Determinants of Tumor Cure (continued)Determinants of Tumor Cure (continued)

Heterogeneity:Heterogeneity:• BiologicalBiological

– Number of clonogenic “stem cells”Number of clonogenic “stem cells”• Intrinsic radiosensitivityIntrinsic radiosensitivity• Proliferative potentialProliferative potential

– Tumor microenvironmentTumor microenvironment• HypoxiaHypoxia• MetabolismMetabolism• Host cell infiltratesHost cell infiltrates• Interstitial pressureInterstitial pressure

– GeneticGenetic• OncogenesOncogenes• Tumor suppressor genesTumor suppressor genes• Single Nucleotide Polymorphisms (SNPs)?Single Nucleotide Polymorphisms (SNPs)?

• PhysicalPhysical– Dose heterogeneityDose heterogeneity– Geographic missGeographic miss


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TD50 AssayTD50 Assay1. Inject varying numbers of tumor cells into mice1. Inject varying numbers of tumor cells into mice

2. Determine the number of cells that are needed to form tumors in 50% of mice.2. Determine the number of cells that are needed to form tumors in 50% of mice.

100100

5050

00

Percent ofPercent ofmice with mice with

tumorstumors

10 1010 1022 10 1033 10 1044 10 1055 10 1066 10 1077

Size of tumor inoculumSize of tumor inoculum

To grow, tumors must have To grow, tumors must have arisen in that specific strain of arisen in that specific strain of mice, or the mice must be mice, or the mice must be immune deficient. Even then, immune deficient. Even then, not all tumors will grow, and not all tumors will grow, and most need an inoculum size of most need an inoculum size of at least 10at least 1044 cells cells

Concept: Only cancer Concept: Only cancer “stem” cells will grow“stem” cells will grow


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Renewing stem cell

Non-stem cell

Tumor regenerationfrom stem cell pool

The cancer stem cell hypothesis suggests that there are a small number of clonogenic stem cells in a tumor and that, if they are therapy-resistant, they are responsible for recurrences, and accelerated tumor repopulation during therapy.

stem cell

Tumor cure


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MCF-7 Breast Cancer Stem Cells are MCF-7 Breast Cancer Stem Cells are Radioresistant and are enriched Following Radioresistant and are enriched Following

IrradiationIrradiation

“Stem” cells

At least some human tumors have a clonogenic subpopulation with stem-like characteristics that can be grown in cytokines as spheres and that are radioresistant and are selected for by fractionated irradiation. Phillips et al J Natl Cancer Inst 98:1777, 2006


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TCD50 AssayTCD50 Assay1. Inject mice with enough cells to form a tumor1. Inject mice with enough cells to form a tumor

3. Determine the dose of radiation that is needed to cure 50% of mice.3. Determine the dose of radiation that is needed to cure 50% of mice.

100100

5050

00

Percent ofPercent ofmice with mice with

tumorstumors

2. Irradiate when 6mm diam2. Irradiate when 6mm diam

0 10 20 30 40 50 60 70 800 10 20 30 40 50 60 70 80

GyGy

Threshold-sigmoid curve Threshold-sigmoid curve that goes from 10% to that goes from 10% to 90% cure over about 90% cure over about 10Gy in a clinical 10Gy in a clinical fractionation scheme fractionation scheme (which is hard to do in (which is hard to do in mice).mice).


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Tumor Control ProbabilityTumor Control Probability

• In order to cure a tumor, the last surviving clonogen must be killed, In order to cure a tumor, the last surviving clonogen must be killed, and even then it is a probability function of dose.and even then it is a probability function of dose.

• TCP = eTCP = e-x-x = e = e-(m. SF) -(m. SF)

or eor e-m.e-(-m.e-(d+d+D2)D2) or e or e -(m. e -(D/D0))-(m. e -(D/D0))

– Where x is the number of surviving clonogenic stem cells, Where x is the number of surviving clonogenic stem cells, – m is the initial number of clonogensm is the initial number of clonogens

• If there is an average of 1 cell surviving TCP=37%If there is an average of 1 cell surviving TCP=37%


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Tumor Control ProbabilityTumor Control Probability

• The slope represents extent of heterogeneity The slope represents extent of heterogeneity in tumor responsein tumor response

• The normalized dose response gradient (The normalized dose response gradient () ) measures the change in TCP in % points for measures the change in TCP in % points for a 1% increase in dosea 1% increase in dose

• Often 1-3%Often 1-3%


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DOSE (Gy)DOSE (Gy)

00

2020

4040

6060

8080

100100

00 1010 2020 3030 4040 5050 6060 7070 8080 9090 100100 110110 120120 130130

N=10N=109 9

SFSF22=0.3=0.3

SFSF22=0.4=0.4

SFSF22=0.5=0.5

SFSF22=0.6=0.6

SFSF22=0.7=0.7TCP (%)TCP (%)

Heterogeneity in RadiosensitivityHeterogeneity in Radiosensitivity

Rafi Suwinski


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DOSE (Gy)DOSE (Gy)

00

2020

4040

6060

8080

100100

00 1010 2020 3030 4040 5050 6060 7070 8080 9090

N=10N=10992Gy2Gy

= 0.5= 0.5

N=10N=101010

N=10N=101111

SFSF

TCP (%)TCP (%)

Heterogeneity in Clonogen NumberHeterogeneity in Clonogen Number

AverageAverage

Rafi Suwinski


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DOSE (Gy)DOSE (Gy)

PE

RC

EN

T R

ED

UC

TIO

N I

NP

ER

CE

NT

RE

DU

CT

ION

IN

ME

TA

ST

AS

ES

RIS

KM

ET

AS

TA

SE

S R

ISK

00

2020

4040

6060

8080

100100

00 1010 2020 3030 4040 5050 6060 7070

n=10n=1055

N =10-10

N =10-108 8

SFSF2Gy2Gy =0.5=0.5

N=10N=10 88N=10N=103 3 N=10N=10

Heterogeneity in tumor volumeHeterogeneity in tumor volume

Micrometastatic DiseaseMicrometastatic Disease


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Tumor Growth and Tumor Growth and RegressionRegression

The The kinetics kinetics of tumor growth and regression depend uponof tumor growth and regression depend upon

• Cell cycleCell cycle• Growth fraction (G.F.)Growth fraction (G.F.)

• G.F. is the proportion of proliferating cellsG.F. is the proportion of proliferating cells• G.F. = P / (P + Q) where P = proliferating cells and Q = non-G.F. = P / (P + Q) where P = proliferating cells and Q = non-

proliferating cells (quiescent/senescent/differentiated cells)proliferating cells (quiescent/senescent/differentiated cells)

• Cell loss factorCell loss factor• Cell Loss Factor Cell Loss Factor measures loss of cells from a tissuemeasures loss of cells from a tissue• If If = 0, Td = Tpot= 0, Td = Tpot where Td is the actual volume doubling time where Td is the actual volume doubling time

and Tpot is potential volume doubling time and Tpot is potential volume doubling time • = 1 - Tpot / Td= 1 - Tpot / Td• if G.F. = 1 then Tpot = Tc if G.F. = 1 then Tpot = Tc • Under steady state conditions, constant cell number is maintained Under steady state conditions, constant cell number is maintained

by the balance between cell proliferation and cell loss i.e. by the balance between cell proliferation and cell loss i.e. = 1.0. = 1.0. In tumors (and embryos) In tumors (and embryos) < 1.0 < 1.0


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Tumor KineticsTumor Kinetics

Tc Cell cycle timeTc Cell cycle time

G.F. Growth fraction G.F. Growth fraction

Tpot Pot. doubling timeTpot Pot. doubling time

TdTd Actual doubling time Actual doubling time

Cell loss factorCell loss factor

Human SCCHuman SCC

36 hrs36 hrs

0.250.25

6 days6 days

60 days60 days

0.90.9

Rate of tumor growth and rate of tumor regression after therapy are determined Rate of tumor growth and rate of tumor regression after therapy are determined largely by the cell loss factor, that varies greatly from tumor to tumorlargely by the cell loss factor, that varies greatly from tumor to tumor

(36hr x 4)(36hr x 4)

(1-6/60)(1-6/60)


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Tumor Growth and RegressionTumor Growth and Regression

• Slow growing tumors may regress rapidlySlow growing tumors may regress rapidly• Slow regression is Slow regression is notnot an indication of treatment failure an indication of treatment failure• Rapidly growing tumors would be expected to regress and regrow Rapidly growing tumors would be expected to regress and regrow

rapidlyrapidly

• In general, the rate of tumor regression after Tx is not prognosticIn general, the rate of tumor regression after Tx is not prognostic


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Tumor RegenerationTumor Regeneration

Rat rhabdomyosarcomaRat rhabdomyosarcomaHermans and Barendsen, 1969Hermans and Barendsen, 1969

Tumors can Tumors can regenerate at the regenerate at the

same time as same time as they regress!they regress!

ControlControlIrradiatedIrradiated

Surviving clonogensSurviving clonogensmeasured in vitromeasured in vitro

Growth delay Growth delay

TimeTime

Relative tumor Relative tumor volumevolume

20Gy X-rays20Gy X-rays


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The regrowth rate of The regrowth rate of surviving clonogens varies surviving clonogens varies with the surviving fraction with the surviving fraction - Lewis Lung Carcinoma- Lewis Lung Carcinoma(Stephens and Steel)(Stephens and Steel)

ControlControl15 Gy15 Gy25 Gy25 Gy35 Gy35 Gy


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EVIDENCE FOR ACCELERATED EVIDENCE FOR ACCELERATED REPOPULATION IN TUMORSREPOPULATION IN TUMORS

• After RT, tumors recur faster than than would be After RT, tumors recur faster than than would be expected from the original growth rate expected from the original growth rate

• Split-course RT often gives poor resultsSplit-course RT often gives poor results• Protraction of treatment time often gives poor Protraction of treatment time often gives poor

resultsresults• Accelerated treatment is sometimes of benefit.Accelerated treatment is sometimes of benefit.


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Accelerated Tumor RepopulationAccelerated Tumor Repopulation

• T2 and T3 SCC head and neckT2 and T3 SCC head and neck (excluding nasopharynx and vocal cord). TCD (excluding nasopharynx and vocal cord). TCD5050 values values

are consistent with are consistent with onset of repopulation at 4 weeksonset of repopulation at 4 weeks followed by accelerated followed by accelerated repopulation with a 3-4 day doubling time, implying a loss in dose of about 0.6 Gy/dyrepopulation with a 3-4 day doubling time, implying a loss in dose of about 0.6 Gy/dy

• If the red line is correct, onset may be about day 21 and repopulation may not be If the red line is correct, onset may be about day 21 and repopulation may not be constant. It may increase from 0.6 Gy/dy around week 3-4 to even 1.6 – 1.8 Gy/day constant. It may increase from 0.6 Gy/dy around week 3-4 to even 1.6 – 1.8 Gy/day around week 6-7.around week 6-7.

T2 T3T2 T3local controllocal control

no local controlno local control

Withers et al, 1988Withers et al, 1988Maciejewski et al., 1989Maciejewski et al., 1989

Total DoseTotal Dose(2 Gy equiv.)(2 Gy equiv.)

Treatment DurationTreatment Duration

local controllocal control

no local controlno local control

7070

5555

4040

T2 T3T2 T3


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476 patients

Tpot in a Large Multicenter Tpot in a Large Multicenter HNSC TrialHNSC Trial

(Begg (Begg et alet al 1999) 1999)

• It was thought that shortening treatment time by accelerated It was thought that shortening treatment time by accelerated hyperfractionation and that this might be predicted by Thyperfractionation and that this might be predicted by Tpot pot , but a , but a large multicenter trial was unable to confirm thislarge multicenter trial was unable to confirm this

• But note that TBut note that Tpotpot in HNSCC was 3-5dys for most patients, confirming in HNSCC was 3-5dys for most patients, confirming the potential for very rapid growththe potential for very rapid growth


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Sources of HeterogeneitySources of Heterogeneity

• Biological DoseBiological Dose– Number of clonogenic “stem cells”Number of clonogenic “stem cells”

• Intrinsic radiosensitivityIntrinsic radiosensitivity• Proliferative potentialProliferative potential

– Tumor microenvironmentTumor microenvironment• HypoxiaHypoxia• MetabolismMetabolism

• Physical DosePhysical Dose– Need to know the importance of dose-volume Need to know the importance of dose-volume

constraintsconstraints


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• HistoryHistory– 19091909

• Schwarz - radium dose on human skinSchwarz - radium dose on human skin

– 1930-19501930-1950• Gray, Mottram, Flanders - oxygen effects in biologyGray, Mottram, Flanders - oxygen effects in biology

– 19551955• Thomlinson & Gray - tumor cordsThomlinson & Gray - tumor cords

– 1960-19651960-1965• Powers & Tolmach - survival curves Powers & Tolmach - survival curves in vivoin vivo• Churchill Davidson - HBO in patientsChurchill Davidson - HBO in patients


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• Chronic hypoxia is a result largely of Chronic hypoxia is a result largely of – Limited OLimited O22 diffusion due to diffusion due to

• oxygen consumption (”diffusion limited hypoxia”)oxygen consumption (”diffusion limited hypoxia”)• irregular vascular geometryirregular vascular geometry

• Acute/transient/intermittent hypoxia is a result largely of Acute/transient/intermittent hypoxia is a result largely of – Chaotic vasculature and interstitial pressureChaotic vasculature and interstitial pressure

• vascular stasisvascular stasis• flow instabilitiesflow instabilities

Hypoxia in TumorsHypoxia in Tumors


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• Within areas of need, oxygen is released from red blood cells and enters Within areas of need, oxygen is released from red blood cells and enters tumor tissue by diffusion. It is metabolized by respiring cells. As a result, tumor tissue by diffusion. It is metabolized by respiring cells. As a result, at distances greater than about 100 µm from the nearest blood vessel at distances greater than about 100 µm from the nearest blood vessel insufficient oxygen remains to maintain cell viability. insufficient oxygen remains to maintain cell viability.

Chronic HypoxiaChronic Hypoxia

VVVV

VV

VVVV

VVVV

VVVV

VV VVVVVV

VVVVVVVV

VV

VV

BLOODBLOODVESSELVESSEL

NecrosisNecrosis

HypoxiaHypoxia

ProliferationProliferation

100100HIGH.................LOWHIGH.................LOW

Proliferation, O2, Proliferation, O2, pH, cell viabilitypH, cell viability

• Adjacent to areas of necrosis, one may Adjacent to areas of necrosis, one may find a region 1-2 cell layers thick where find a region 1-2 cell layers thick where oxygen tensions are hypoxic. Within a oxygen tensions are hypoxic. Within a solid tumor mass, mitotic index and solid tumor mass, mitotic index and viability decrease with distance from the viability decrease with distance from the nearest blood vessel (Tomlinson and nearest blood vessel (Tomlinson and Gray; Tannock, Cancer Res 30: 2470, Gray; Tannock, Cancer Res 30: 2470, 1970)1970)

• Hypoxia does NOT correlate with tumor Hypoxia does NOT correlate with tumor volumevolume


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Brown & Giaccia, 1994

Normal Tissue

Neoplastic tissue

Konerding et al., 1998

• The vascular network The vascular network that develops in tumors that develops in tumors is structurally abnormalis structurally abnormal

• Vessels are dilated, Vessels are dilated, tortuous, elongated, tortuous, elongated, with A-V shunts and with A-V shunts and blind endsblind ends

• Pericytes are frequently Pericytes are frequently absentabsent

• The basement The basement membrane is thinmembrane is thin

• Vessels are more Vessels are more permeable giving permeable giving increased interstitial increased interstitial pressure pressure

• The abnormal The abnormal vasculature results in vasculature results in spatial and temporal spatial and temporal heterogeneity in blood heterogeneity in blood flow that in turn produce flow that in turn produce regions of regions of temporary or temporary or acuteacute hypoxia, acidity hypoxia, acidity and nutrient depletionand nutrient depletion

Acute HypoxiaAcute Hypoxia


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THE OXYGEN EFFECTTHE OXYGEN EFFECT• Oxygen is a powerful oxidizing agent and therefore acts as a radiosensitizer Oxygen is a powerful oxidizing agent and therefore acts as a radiosensitizer

if it is present at the time of irradiation (within if it is present at the time of irradiation (within secs)secs)• The magnitude of the OER is critically dependent upon oxygen tension. The The magnitude of the OER is critically dependent upon oxygen tension. The

greatest increase occurs between 0-20 mm Hg with further modest greatest increase occurs between 0-20 mm Hg with further modest increases to air (155 mm Hg) and above (760 mm Hg=100% oxygen). increases to air (155 mm Hg) and above (760 mm Hg=100% oxygen).

• Its effects are measured as the oxygen enhancement ratio (O.E.R.)Its effects are measured as the oxygen enhancement ratio (O.E.R.)– O.E.R. = the ratio of doses needed to obtain a given level of biological effect under anoxic O.E.R. = the ratio of doses needed to obtain a given level of biological effect under anoxic

and oxic conditions = D(anox)/D(ox)and oxic conditions = D(anox)/D(ox)– For low LET radiation the O.E.R. is 2.5-3.0 and in the higher range at higher dosesFor low LET radiation the O.E.R. is 2.5-3.0 and in the higher range at higher doses– For neutrons, O.E.R is about 1.6For neutrons, O.E.R is about 1.6

Dose (Gy)Dose (Gy)

S.F.S.F.

O.E.R.= 2.67O.E.R.= 2.67

0 2 4 6 8 10 0 2 4 6 8 10

1.01.0

0.10.1

0.010.01

oxicoxic

hypoxichypoxic

1.0

1.5

2.0

2.5

3.0

O.E.R.

76020050403020100Partial Pressure of Oxygen (mm Hg) at 37o C

air 100% oxygen


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RBE and OER as a function of LETRBE and OER as a function of LET

Linear Energy Transfer (LET in keV/Linear Energy Transfer (LET in keV/m)m)

RBERBE(for cell kill)(for cell kill)

1000100010010010101100

22

44

66

88

RBERBEDiagnosticDiagnostic

X-raysX-rays

Fast Fast NeutronsNeutrons

Alpha Alpha ParticlesParticles

0.10.1

Co-60Co-60gamma raysgamma rays

00

11

22

33

44

OEROER

OEROER

OER is the inverse of RBE because it depends on the indirect action of ionizing radiation


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• Differential radiation sensitivityDifferential radiation sensitivity• Eppendorf polarographic electrodeEppendorf polarographic electrode • ImmunohistochemistryImmunohistochemistry

– MisonidazoleMisonidazole– Hypoxyprobe™ immunohistochemistry with pimonidazoleHypoxyprobe™ immunohistochemistry with pimonidazole– HIF-1 and productsHIF-1 and products

• PET imaging PET imaging – 18F-fluoromisonidazole (FMISO-PET)18F-fluoromisonidazole (FMISO-PET)– EF5 - EF5 - etanidazoleetanidazole – Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone (Cu-ATSM)Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone (Cu-ATSM)

Demonstrating hypoxic Demonstrating hypoxic regions/cells within tumorsregions/cells within tumors


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14 Days

Plate cells

Colony assay

IRRADIATEtumor

After 24hrs make cell suspension

Tumor Cell Survival : In vivo-in vitro assayTumor Cell Survival : In vivo-in vitro assay

0 2 4 6 8 10 12 14 16 18 20 22 0 2 4 6 8 10 12 14 16 18 20 22

11

1010

1010

1010

1010

1010

-1-1

-2-2

-3-3

-4-4

-5-5

1010-6-6

DOSE (Gy)DOSE (Gy)

HYPOXICHYPOXIC

AIRAIR

S.F.S.F.

OXICOXIC

Hypoxic Hypoxic FractionFraction

• If solid tumors in mice are irradiated with single doses of radiation under hypoxic conditions or in air and an in vitro clonogenic assay performed, normally a dog-leg curve is obtained in air indicating a radioresistant population whose magnitude can be estimated by extrapolation onto the Y axis. After Rockwell and Kalman, 1973


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Tumor HypoxiaTumor Hypoxia

• If murine tumors are If murine tumors are irradiated with varying sized irradiated with varying sized single doses of radiation single doses of radiation under clamped (hypoxic) and under clamped (hypoxic) and normal conditions and the % normal conditions and the % of tumors controlled plotted, of tumors controlled plotted, the TCP curve is shifted to the TCP curve is shifted to the right by hypoxia and the the right by hypoxia and the O.E.R. can be calculated.O.E.R. can be calculated.

Moulder and Rockwell, 1984Moulder and Rockwell, 1984


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Probe Probe CasingCasing300 300 mm

Gold WireGold Wire12 12 m m

Insulating Insulating glassglass

MembraneMembrane

Eppendorf Polarographic Fine Eppendorf Polarographic Fine Needle pONeedle pO22 Probe Probe

• A 700 mV polarizing voltage is applied against the Ag/AgCl anode. The A 700 mV polarizing voltage is applied against the Ag/AgCl anode. The measured current is proportional to the local oxygen tensionmeasured current is proportional to the local oxygen tension

• No longer sold, but other versions are possibleNo longer sold, but other versions are possible


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0%

10%

20%

30%

40%

50%

<6 <12 <18 <24 <30 <36 <42 <48

NFSANFSA IL7

0%

10%

20%

30%

40%

<6 <12 <18 <24 <30 <36 <42 <48

NFSANFSA TNF

Proportion of measures

Eppendorf Polarographic ProbeEppendorf Polarographic Probe

mmHg


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Pimonidazole staining of human CRC tumorPimonidazole staining of human CRC tumor

• Misonidazole forms adducts in hypoxic cells in vitro and in vivo with thiol groups in proteins, peptides and amino acids. Hypoxia (pO2 < 10 mmHg) is required for binding.

• FMISO-PET is one of 2 commonly used PET tracers (the other being Cu-ATSM), but it accumulates slowly. Other imidazoles are under study.

• EF5 is a fluorinated derivative of etanidazole

• Pimonidazole is generally injected in vivo and the adducts stained using antibodies.

• Intracellular Cu-ATSM is a non-nitroimidazole that has been shown to be bioreduced and trapped in hypoxic cells and is used for PET.

Bioreductive DrugsBioreductive Drugs


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Hypoxia and proliferation in a solid tumorHypoxia and proliferation in a solid tumor

blood vessels

proliferating cells(IdUrd +)

Hypoxia(pimonidazole +)

Biopsy of head/neck squamous cell carcinoma

necrosis

From: Albert Van der Kogel


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Chronic Acute

Pimonidazole (green) and vascular staining (red)in human head and neck tumor


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Hypoxia-induced gene Hypoxia-induced gene expressionexpression• Transcription factorsTranscription factors

– AP-1, NF-kB, SP-1 activation AP-1, NF-kB, SP-1 activation • which can mediate radioresistancywhich can mediate radioresistancy

– p53 inductionp53 induction• which can cause apoptosis with hypoxia-driven p53 mutant which can cause apoptosis with hypoxia-driven p53 mutant

selection and increasing genetic instabilityselection and increasing genetic instability

– HIF-1HIF-1 and products and products eg VEGF, CA IX, OPN etceg VEGF, CA IX, OPN etc • HIF-1alpha is a target for prolyl hydroxylation by HIF prolyl-HIF-1alpha is a target for prolyl hydroxylation by HIF prolyl-

hydroxylase, targeting it for rapid degradation in normoxic hydroxylase, targeting it for rapid degradation in normoxic conditions. Under hypoxia, HIF prolyl-hydroxylase is inhibited, conditions. Under hypoxia, HIF prolyl-hydroxylase is inhibited, since it utilizes oxygen as a cosubstrate, stabilizing HIF-1α. This since it utilizes oxygen as a cosubstrate, stabilizing HIF-1α. This upregulates several genes to promote survival in low-oxygen upregulates several genes to promote survival in low-oxygen conditions, including glycolytic enzymes and VEGF, which conditions, including glycolytic enzymes and VEGF, which promotes angiogenesis. promotes angiogenesis.

• In general these surrogate markers do not correlate well with In general these surrogate markers do not correlate well with hypoxia, probably because more than hypoxia stabilizes HIF-1hypoxia, probably because more than hypoxia stabilizes HIF-1


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• Enhances resistance to radiation and chemotherapy because of Enhances resistance to radiation and chemotherapy because of classic oxygen effectclassic oxygen effect

• Induces expression of genes that Induces expression of genes that – confer resistance to radiation and other pro-apoptotic insultsconfer resistance to radiation and other pro-apoptotic insults– triggers genetic instabilitytriggers genetic instability– cause angiogenesis and potentiate metastasiscause angiogenesis and potentiate metastasis

Contribution of hypoxia to tumor progressionContribution of hypoxia to tumor progression

From Giaccia, 1999


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Regulation of hypoxia-induced gene Regulation of hypoxia-induced gene expressionexpression

HIF-1HIF-1 synthesissynthesisPI3KPI3K

HER2HER2

FRAPFRAP

PTENPTEN

RapamycinRapamycin

LY294002LY294002

AKTAKT

IGFRIGFR EGFREGFR

SrcSrc

HIF-1HIF-1 mRNAmRNAHIF-1HIF-1proteinprotein

Target geneTarget gene

expressionexpression

VEGFVEGF

IGF-2IGF-2

GlucoseGlucoseProlyl Prolyl hydroxylationhydroxylation

UbiquitinationUbiquitinationVHLVHL

p53p53

HYPOXIAHYPOXIA

HIF-1HIF-1FIH-1FIH-1

AngiogenesisAngiogenesis

MetabolismMetabolism

HIF-1HIF-1 degradationdegradation

transporterstransporters



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angiogenesis

energymetabolism

HIF-1

apoptosis/necrosis

proliferation

Glucose transporters Glut1,3Glycolytic enzymes

ALDA, PGK1, PKM, PFKL, LDHAALDA, PGK1, PKM, PFKL, LDHA

BNip3(BCl2 family)

VEGFVEGFR

EPO EGFEGFR

PDGF-BPDGF-BIGF-1IGF-2

carbonic Anhydrases

CA9

pH regulation

Redoxregulation

InflammatoryCytokines

IL-1IL-1, IL-8, IL-8Heme oxygenase 1,Heme oxygenase 1,metallothionein, metallothionein, diaphorase, diaphorase, GSH,GSH,


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Clinical Relevance of Tumor Clinical Relevance of Tumor HypoxiaHypoxia

• Evidence for hypoxia in human tumorsEvidence for hypoxia in human tumors– Hyperbaric chambers anecdotally show benefitHyperbaric chambers anecdotally show benefit

• normobaric oxygen/carbogen has alsonormobaric oxygen/carbogen has also been applied and, at times, combined been applied and, at times, combined with nicotinamide, a Bwith nicotinamide, a B66

vitamin analog thought to counteract the acute hypoxia vitamin analog thought to counteract the acute hypoxia (ARCON)(ARCON)

– Anemia correction has benefit especially in cervix caAnemia correction has benefit especially in cervix ca• Note that erythropoietin has a deleterious effect in HNSCC due to stimulating Note that erythropoietin has a deleterious effect in HNSCC due to stimulating

tumor growthtumor growth– Nitroimidazoles - immunohistochemistry and PET Nitroimidazoles - immunohistochemistry and PET – Microelectrode measurements - several studies have correlated hypoxia with poor Microelectrode measurements - several studies have correlated hypoxia with poor

local response and survivallocal response and survival• Nordsmark et al. Radiother Oncol 41, 31, 1996 showed local tumor control correlates Nordsmark et al. Radiother Oncol 41, 31, 1996 showed local tumor control correlates

with pre-treatment oxygen levels in head and neck ca. with pre-treatment oxygen levels in head and neck ca. • Brizel et al IJROBP 38:285, 1997 showed DFS correlates with hypoxia in T3 and T4 and Brizel et al IJROBP 38:285, 1997 showed DFS correlates with hypoxia in T3 and T4 and

large node mets from head and necklarge node mets from head and neck• Hockel et al Cancer Res 56:4509, 1996 showed hypoxia correlated with local invasion Hockel et al Cancer Res 56:4509, 1996 showed hypoxia correlated with local invasion

and survival in cases treated with RT or and survival in cases treated with RT or only with surgeryonly with surgery


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Tumor type Median pO2 fraction <10 mm Hg

Breast C. 23-28 26-32

Cervical C. 2-21 21-46

Rectal C. 19-25 -

Lung Ca 14 36

Soft tissue sarcomas 18-27 44

Glioblastomas 7 61

Head & Neck C. 19-26 33

H&N lymph nodes 9-25

14-54

Melanoma 10 49

From Vaupel et al., 1998

Clinical HypoxiaClinical Hypoxia


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Hypoxia and Local Tumor Hypoxia and Local Tumor ControlControl

Small Hypoxic FractionSmall Hypoxic Fraction

Large Hypoxic FractionLarge Hypoxic Fraction

• Local tumor control correlates Local tumor control correlates with pre-treatment oxygen levels with pre-treatment oxygen levels in head and neck ca., as in head and neck ca., as measured with an Eppendorf measured with an Eppendorf electrode. Tumors were electrode. Tumors were stratified by whether the fraction stratified by whether the fraction of pO2 values less than 2.5 mm of pO2 values less than 2.5 mm Hg was above or below the Hg was above or below the median (15%).median (15%).66-68 Gy was given in 33-34 Fx.66-68 Gy was given in 33-34 Fx.

• Nordsmark et al Radiother Nordsmark et al Radiother Oncol 41, 31, 1996Oncol 41, 31, 1996


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Tumor Hypoxia and DFSTumor Hypoxia and DFS

• DFS in cervix ca depends on DFS in cervix ca depends on pOpO22, irrespective of type of , irrespective of type of

treatment, surgery/RT. treatment, surgery/RT. Hockel et al, Sem. Radiat. Hockel et al, Sem. Radiat. Oncol. 6:30, 1996.Oncol. 6:30, 1996.

• This suggests that hypoxia is This suggests that hypoxia is linked to tumor aggression linked to tumor aggression


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RadiosensitizersRadiosensitizers

From Zeman, 2000


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RadiosensitizersRadiosensitizers

• Radiosensitizers such as nitroimidazoles can Radiosensitizers such as nitroimidazoles can “mimic” oxygen and fix damage“mimic” oxygen and fix damage– Associated with some toxicity and there were only rarely efforts Associated with some toxicity and there were only rarely efforts

to determine if the tumors were hypoxic in advance of treatmentto determine if the tumors were hypoxic in advance of treatment

– However there have been positive trialsHowever there have been positive trials

• DAHANCA 5 trial using nimorazole in treatment of advanced squamous cell carcinoma of the head and neck


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And a meta-analysis by Jens Overgaard has shown significantly improved survival and loco-regional control

Journal of Clinical Oncology, 25: pp. 4066-4074, 2007


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Hypoxic CytotoxinsHypoxic Cytotoxins• QuinonesQuinones

– Mitomycin CMitomycin C• NitroaromaticsNitroaromatics• Benzotriazine di-N-oxidesBenzotriazine di-N-oxides

– TirapazamineTirapazamine• Phase III clinical trials with Phase III clinical trials with

cisplatincisplatin• Phase II with RTPhase II with RT• Currently off the market!Currently off the market!


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Tumor ReoxygenationTumor Reoxygenation

• Since well oxygenated cells are more sensitive than hypoxic Since well oxygenated cells are more sensitive than hypoxic cells to ionizing radiation, one might reasonably expect that the cells to ionizing radiation, one might reasonably expect that the hypoxic fraction (hypoxic fraction (i.e.i.e. the proportion of hypoxic cells) to increase the proportion of hypoxic cells) to increase during the course of radiation therapy during the course of radiation therapy

• In fact, Putten & Kallman and others demonstrated that the In fact, Putten & Kallman and others demonstrated that the proportion of hypoxic cells present within a tumor varies a lot, proportion of hypoxic cells present within a tumor varies a lot, but does not but does not increaseincrease during a course of fractionated radiation during a course of fractionated radiation therapy showing therapy showing REOXYGENATIONREOXYGENATION exists. Multiple exists. Multiple mechanisms exist and the variation seems considerable from mechanisms exist and the variation seems considerable from tumor to tumor.tumor to tumor.


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Days post RT

Hypoxic Fraction

N.B. All single dose!


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• Development of new blood vessels from pre-existing capillaries.Development of new blood vessels from pre-existing capillaries.• Although tumors smaller than approximately 1 mm can receive Although tumors smaller than approximately 1 mm can receive

sufficient oxygen and nutrients by diffusion, continued growth sufficient oxygen and nutrients by diffusion, continued growth depends upon the development of an adequate blood supply. In depends upon the development of an adequate blood supply. In the absence of angiogenesis, tumors do not increase in size the absence of angiogenesis, tumors do not increase in size and remain localizedand remain localized

• Angiogenesis also occurs Angiogenesis also occurs duringduring – wound repairwound repair– pregnancypregnancy– certain times in the menstrual cycle certain times in the menstrual cycle

AngiogenesisAngiogenesis


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• In response to various tissue-derived pro-angiogenic signals, In response to various tissue-derived pro-angiogenic signals, endothelial cells in nearby blood vesselsendothelial cells in nearby blood vessels

Major Steps in the Angiogenic ProcessMajor Steps in the Angiogenic Process

Degradation of basement membraneDegradation of basement membrane

MigrationMigration


DifferentiationDifferentiation

Lumen formationLumen formation

Angiogenic factors:Angiogenic factors:

VEGF, FGF, PDGF,VEGF, FGF, PDGF,

EGF, HGF, TGF EGF, HGF, TGF

HypoxiaHypoxia

COCO22

COX-2COX-2NONO

Tumor suppressor genesTumor suppressor genes

OncogenesOncogenes

Ischemia and reperfusionIschemia and reperfusion

– Degrade their basement Degrade their basement membrane and invade the membrane and invade the adjacent extravascular spaceadjacent extravascular space

– Endothelial cells behind the Endothelial cells behind the leading edge proliferate to replace leading edge proliferate to replace the migrating cellsthe migrating cells

– Newly generated endothelial cells Newly generated endothelial cells migrate through connective tissue migrate through connective tissue toward the source of pro-toward the source of pro-angiogenic signalsangiogenic signals

– Endothelial cells assemble into a Endothelial cells assemble into a new vessel, form a lumen, lay new vessel, form a lumen, lay down a basement membrane and down a basement membrane and join other vessels to allow flow.join other vessels to allow flow.


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• There is a relationship between There is a relationship between microvascular density and the probability of microvascular density and the probability of metastasis, relapse free survival and/or metastasis, relapse free survival and/or prognosis. prognosis.

• Weidner Weidner et alet al (N Engl J Med 324: 1, 1991) (N Engl J Med 324: 1, 1991) demonstrated that the likelihood of demonstrated that the likelihood of developing metastasis increased directly as developing metastasis increased directly as the density of tumor associated blood the density of tumor associated blood vessels increasedvessels increased

Tumor Vasculature and MetastasisTumor Vasculature and Metastasis

# microvessels/unit area%

with

met

asta

sis

1/7

9/20

5/7

15/15


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Inhibition of tumor growthInhibition of tumor growth• chronic exposurechronic exposure

Induction of tumor necrosisInduction of tumor necrosis• acute exposureacute exposure

Blood vesselBlood vesselBlood vesselBlood vessel

Vascular TargetingVascular Targeting Induction of selective and Induction of selective and

irreversible damage to irreversible damage to establishedestablished tumor-associated tumor-associated

blood vesselsblood vessels

Anti-AngiogenesisAnti-AngiogenesisPreventing the growth of Preventing the growth of

newnew tumor-associated tumor-associated blood vesselsblood vessels


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• Since many thousands of tumor cells depend upon each blood Since many thousands of tumor cells depend upon each blood vessel for the delivery of oxygen and nutrients, theoretically even vessel for the delivery of oxygen and nutrients, theoretically even limited damage to tumor vasculature may occlude a vessel and limited damage to tumor vasculature may occlude a vessel and cause “an avalanche of tumour cell death”.cause “an avalanche of tumour cell death”.

• Since cells being targeted are in contact with the blood stream, Since cells being targeted are in contact with the blood stream, delivery problems that limit the efficacy of therapies directed delivery problems that limit the efficacy of therapies directed toward tumor cells are not an issuetoward tumor cells are not an issue

• Since endothelial cells are genetically stable and non-Since endothelial cells are genetically stable and non-transformed, treatment-related resistance is less likely to emergetransformed, treatment-related resistance is less likely to emerge

Advantages of vascular targetingAdvantages of vascular targeting


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Questions:Questions: Tumor Responses to RadiotherapyTumor Responses to Radiotherapy


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The probability of tumor cure (TCP) in a The probability of tumor cure (TCP) in a series of tumor when there is on average 1 series of tumor when there is on average 1 cell surviving iscell surviving is

1.1. 00

2.2. 0.370.37

3.3. 0.50.5

4.4. 1.01.0


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If a tumor contains 10If a tumor contains 1099 clonogenic cells and clonogenic cells and RT reduces survival by 10RT reduces survival by 10-9-9, what is the , what is the probability of tumor cureprobability of tumor cure

1.1. minimalminimal

2.2. 37%37%

3.3. 50%50%

4.4. 90%90%


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If a tumor contains 10If a tumor contains 1099 clonogenic cells and clonogenic cells and RT reduces survival by 10RT reduces survival by 10-10-10, what is the , what is the probability of tumor cureprobability of tumor cure

1.1. 10%10%

2.2. 37%37%

3.3. 50%50%

4.4. 90%90%


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Tumorigenicity is a stem cell property of Tumorigenicity is a stem cell property of tumors that is best assessed bytumors that is best assessed by

1.1. TD50 assayTD50 assay

2.2. TCD50 assayTCD50 assay

3.3. In vivo - in vitro assayIn vivo - in vitro assay

4.4. Tumor regrowth assayTumor regrowth assay


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The rate of tumor regression after RT is The rate of tumor regression after RT is determined primarily bydetermined primarily by

1.1. Tumor cell cycle timeTumor cell cycle time

2.2. Tumor growth fractionTumor growth fraction

3.3. TpotTpot

4.4. Labeling indexLabeling index

5.5. Cell loss factor Cell loss factor


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If the cell cycle time of a tumor is 48hrs and If the cell cycle time of a tumor is 48hrs and the growth fraction 10%, what is the the growth fraction 10%, what is the potential volume doubling time?potential volume doubling time?

1.1. 2 days2 days

2.2. 10 days10 days

3.3. 20 days20 days

4.4. 3 months 3 months


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If the Tpot for a tumor is 3 days and the If the Tpot for a tumor is 3 days and the actual volume doubling time is estimated as actual volume doubling time is estimated as 30 days, what is the cell loss factor30 days, what is the cell loss factor

1.1. 0.10.1

2.2. 0.30.3

3.3. 0.50.5

4.4. 0.90.9

5.5. 1.01.0


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The increase in tumor control probability The increase in tumor control probability with dose for clinically detectable tumors is with dose for clinically detectable tumors is theoretically best described by which of the theoretically best described by which of the followingfollowing1.1. A log-linear curveA log-linear curve2.2. A sigmoid curve with a dose thresholdA sigmoid curve with a dose threshold3.3. A sigmoid curve with no thresholdA sigmoid curve with no threshold4.4. A curve that is close to linear with no A curve that is close to linear with no

threshold threshold


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The probability of eliminating metastatic The probability of eliminating metastatic disease theoretically increases with dose disease theoretically increases with dose and is best described by which of the and is best described by which of the followingfollowing1.1. A log-linear curveA log-linear curve2.2. A sigmoid curve with a dose thresholdA sigmoid curve with a dose threshold3.3. A sigmoid curve with no thresholdA sigmoid curve with no threshold4.4. A flat curve that is close to linear with no A flat curve that is close to linear with no

thresholdthreshold


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Which of the following is NOT correct about Which of the following is NOT correct about accelerated repopulationaccelerated repopulation

1.1. It involves a decrease in the cell loss It involves a decrease in the cell loss factorfactor

2.2. It can be promoted by treatment breaksIt can be promoted by treatment breaks

3.3. It explains why tumor recur faster than It explains why tumor recur faster than expected after RTexpected after RT

4.4. It occurs only in tumors It occurs only in tumors


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In T2T3 HNSCC, what percent of a 2Gy In T2T3 HNSCC, what percent of a 2Gy dose is estimated may be lost to accelerated dose is estimated may be lost to accelerated repopulation later in the courserepopulation later in the course

1.1. 5%5%

2.2. 10%10%

3.3. 30%30%

4.4. 59%59%


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In T2T3 HNSCC, when is accelerated In T2T3 HNSCC, when is accelerated repopulation thought to be initiatedrepopulation thought to be initiated

1.1. 1 week1 week

2.2. 4 weeks4 weeks

3.3. 8 weeks8 weeks

4.4. 12 weeks12 weeks


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In T2T3 HNSCC, tumor doubling time may In T2T3 HNSCC, tumor doubling time may become close to Tpot, which on average is become close to Tpot, which on average is

1.1. 2-7 days2-7 days

2.2. 1-4 weeks1-4 weeks

3.3. 1-2 months1-2 months

4.4. 2-6 months2-6 months


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What occurs in tumors with distance from a What occurs in tumors with distance from a blood vesselblood vessel

1.1. Increased cell proliferationIncreased cell proliferation

2.2. Poor oxygen diffusionPoor oxygen diffusion

3.3. Decreased oxygen levels due to high Decreased oxygen levels due to high consumption near the vesselconsumption near the vessel

4.4. Increased pHIncreased pH


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At what distance from a blood vessel does At what distance from a blood vessel does radiobiologically relevant hypoxia occurradiobiologically relevant hypoxia occur

1.1. 50 micrometers50 micrometers





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Hypoxic areas in tumors are best assessed Hypoxic areas in tumors are best assessed byby

1.1. Pimonidazole uptakePimonidazole uptake

2.2. Carbonic anhydrases, like CAIXCarbonic anhydrases, like CAIX

3.3. Expression of hypoxia inducible factor Expression of hypoxia inducible factor (HIF-1)(HIF-1)

4.4. VEGF expressionVEGF expression


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Which of the following is true of tumor vasculatureWhich of the following is true of tumor vasculature

1.1. Its abnormal vasculature results in acute transient areas of Its abnormal vasculature results in acute transient areas of hypoxiahypoxia

2.2. It has a thin basement membrane but no increase in It has a thin basement membrane but no increase in permeabilitypermeability

3.3. It is responsible for low interstitial tumor pressureIt is responsible for low interstitial tumor pressure

4.4. Poor angiogenesis limits it, causing hypoxiaPoor angiogenesis limits it, causing hypoxia


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The concentration of oxygen that gives half-The concentration of oxygen that gives half-maximal radiosensitization ismaximal radiosensitization is

1.1. 1mm Hg1mm Hg

2.2. 3mm Hg3mm Hg

3.3. 10mm Hg10mm Hg

4.4. 100 mm Hg100 mm Hg


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For low LET radiation, the OER for an For low LET radiation, the OER for an isoeffect isisoeffect is

1.1. 1.0-1.61.0-1.6

2.2. 1.7-2.31.7-2.3

3.3. 2.3-3.02.3-3.0

4.4. 3.0-3.7 3.0-3.7


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For fast neutrons, the OER is approximatelyFor fast neutrons, the OER is approximately

1.1. 1.61.6

2.2. 2.32.3

3.3. 3.03.0

4.4. 3.7 3.7


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For alpha particle radiation, the OER is For alpha particle radiation, the OER is closest toclosest to

1.1. 1.01.0

2.2. 1.71.7

3.3. 2.32.3

4.4. 3.0 3.0


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The OER is lowest at an LET closest toThe OER is lowest at an LET closest to

1.1. 1 keV/1 keV/mm

2.2. 10 keV/10 keV/mm

3.3. 100 100 keV/keV/mm

4.4. 1000 1000 keV/keV/mm


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Which tracer is NOT used to detect hypoxic Which tracer is NOT used to detect hypoxic areas in tumors by PETareas in tumors by PET

1.1. FMISOFMISO

2.2. HypoxyprobeHypoxyprobe

3.3. EF5 - etanidazoleEF5 - etanidazole

4.4. Cu-ATSMCu-ATSM


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Which of the following is NOT induced by Which of the following is NOT induced by hypoxiahypoxia

1.1. Carbonic anhydrasesCarbonic anhydrases

2.2. VEGFVEGF

3.3. ErythropoietinErythropoietin

4.4. NF-NF-BB

5.5. TGF-betaTGF-beta


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The generally accepted gold standard in The generally accepted gold standard in measuring hypoxia in a human tumor ismeasuring hypoxia in a human tumor is

1.1. Polarographic needle probesPolarographic needle probes

2.2. HydroxyprobeHydroxyprobe

3.3. Carbonic anhydrase levelsCarbonic anhydrase levels

4.4. Osteopontin levelsOsteopontin levels

5.5. HIF-1 expressionHIF-1 expression


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Clinically, pretreatment hypoxia has NOT Clinically, pretreatment hypoxia has NOT been correlated withbeen correlated with1.1. Distant failure following surgeryDistant failure following surgery2.2. Loco-regional failure in HNSCC following Loco-regional failure in HNSCC following

RTRT3.3. Decreased tumor recurrence in clinical Decreased tumor recurrence in clinical

trials of erythropoietin with RT in HNSCCtrials of erythropoietin with RT in HNSCC4.4. Microvessel densityMicrovessel density


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Which of the following is true for nitroimidazolesWhich of the following is true for nitroimidazoles1.1. They mimic oxygen and radiosensitize tumorsThey mimic oxygen and radiosensitize tumors2.2. They radioprotect normal tissue by scavenging reactive They radioprotect normal tissue by scavenging reactive

oxygen speciesoxygen species3.3. They are unable to sensitize acute hypoxic areasThey are unable to sensitize acute hypoxic areas4.4. A metanalysis by Overgaard has shown that they improve A metanalysis by Overgaard has shown that they improve

locoregional control to RT, but not survivallocoregional control to RT, but not survival5.5. They act as hypoxic cell cytotoxinsThey act as hypoxic cell cytotoxins


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Which of the following is correct for Which of the following is correct for TirapazamineTirapazamine

1.1. It is a radiosensitizerIt is a radiosensitizer

2.2. It has been shown to effectively It has been shown to effectively radiosensitize tumors in phase III clinical radiosensitize tumors in phase III clinical trails in HNSCCtrails in HNSCC

3.3. it is a hypoxic cell cytotoxinit is a hypoxic cell cytotoxin

4.4. It radioprotects normal tissuesIt radioprotects normal tissues


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Vascular targeting refers toVascular targeting refers to1.1. The effects of agents like Avastin on The effects of agents like Avastin on

tumor angiogenesistumor angiogenesis2.2. The interaction of anti-angiogenesis The interaction of anti-angiogenesis

factors with RTfactors with RT3.3. The selective effects of agents on The selective effects of agents on

established vasculatureestablished vasculature4.4. The effects of agents like erythropoietin The effects of agents like erythropoietin

on oxygen delivery into tumors on oxygen delivery into tumors


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AnswersAnswers1. NA2. 13. 44. 35. 26. 27. 18. 39. 210. 211. 212. 313. 114. 4

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Determinants of Tumor Cure

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