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Tumors Models, and
Response of Tumors
Martin Brown
April 23, 2012
Cancers comprise more than tumor cells. Do the
stromal cells affect the response to radiotherapy?
Hanahan and Weinberg, Cell 2011
Endothelial cells should be a prime target in
radiotherapy – Julie Denekamp
Capillary
Endothelial
cell
•Endothelial cells in tumors
are rapidly dividing so will
die rapidly after irradiation
•One endothelial cell
supports 2000 tumor cells!
Tumor curability by IR does not depend
on the sensitivity of the host (Budach et al, 1993)
TD50 assay for in vivo response of
tumors
(Hewitt and
Wilson, 1959)
Cell survival curve of leukemia cells irradiated
in situ and in vitro (Hewitt & Wilson,1959)
Lung colony assay (Hill and Bush)
Tumor Growth Delay Assay
Tumor growth delay following
different radiation doses.
Demonstration of Tumor Radiosensitization
using TCD50 assay (Sheldon et al, 1974)
In vivo/in vitro (excision) assay
This is the fastest, cheapest and most accurate assay of the response
of the tumor cells. However, it makes the assumption that the response
of the cells is not altered by taking them out of the tumor.
Demonstration of “hypoxic fraction” in
mouse tumors (Rockwell & Kallman, 1972)
Spheroids: an in vitro tumor model.
Mimics several aspects of
tumors
-chronic hypoxia adjacent
to necrosis
-non dividing cells
-cell to cell contact
-limited diffusion of drugs
Tumor Hypoxia results from Differences in the
Vasculature between Tumor and Normal Tissues
Chronic Hypoxia (HIF-1 staining) in Human
Head & Neck Cancers(Aebersold et al, 2001)
HIF-1 staining
Blood vessels
Necrosis
Oxygenation of human tumors is usually measured with
an oxygen electrode (“Eppendorf”)
Oxygen tensions of Normal Tissue and
Nodes of Head and Neck Cancers
20
% of Measurements
16
Normal Subcutaneous Tissue
12
8
4
0
Median pO2
0 10 20 30 40 50 60 70 80 90
100
20
16
Tumor
12
8
4
0
0
10 20 30 40 50 60 70 80 90 100
Oxygen Partial Pressure (mm Hg)
Median pO2 Values for Tumor and Normal
Tissues for 62 Head & Neck Cancer Patients (Le et al 2004)
Median pO2 values for Tumor and Normal
0.3
0.2
Proportion
of
Values
0.1
Tumor Median
= 11.8 mmHg
Normal Median
= 51.9 mmHg
0.0
0 10 20 30 40 50 60 70 80
Median pO2 (mmHg)
Effect of Tumor Hypoxia on Local Control of
Head and Neck Cancer (Brizel et al, 1999)
63 patients treated.
Similar differences
for survival and DFS.
Oxygenation of Prostate Carcinoma
by Eppendorf Electrode
Movsas et al, 2001
Parker et al, 2004
Med = 2.4mmHg
Med = 4.5 mmHg
Effect of tumor hypoxia on outcome of
prostate ca to RT (Movsas et al, 2002)
Biochemical failure by PSA
pO2 = 5.3 mmHg
pO2 = 1.0 mmHg
Median Oxygen Levels of Human Tumors
(Brown & Wilson Nat. Rev. Cancer 4: 437 2004)
It is incorrect to think that tumors are composed of
hypoxic and aerobic cells. In fact the majority are
at intermediate pO2
Hypoxic
Intermediate
Aerobic
3.0
Koch et al,1984
Whillans and
Hunt,1982
OER(p)
2.5
2.0
1.5
1.0
0.1
1
10
100
Oxygen Partial Pressure (p) (mm Hg)
When the cells at intermediate hypoxia are
considered the predicted survival to 2 Gy fractions
is dramatically altered
10 0
Surviving Fraction
10 -1
10 -2
10 -3
10 -4
10% hypoxic cells and
intermediate p02 cells with
full reoxygenation
10 -5
10 -6
10 -7
10% hypoxic cells with full
reoxygenation (binary)
10 -8
10 -9
10 -10
0
No Hypoxic Cells
10
20
30
40
50
60
Total Dose (2 Gy Fractions)
Wouters and Brown,1997
Consequences of Tumor Hypoxia for
Cancer Treatment
• Hypoxic cells are resistant to killing by IR
– Extent of hypoxia affects response to radiotherapy
• Hypoxia is associated with slowing of
proliferation:
– Leads to resistance to most anticancer drugs.
• Hypoxia promotes and selects for a more
malignant phenotype
– Hypoxic tumors are more metastatic
Hypoxia and proliferation in human H & N
tumors (Van der Kogel et al)
Green: hypoxia
Red: proliferation (IdUrd+)
Human Glioblastoma Stained with EF5
(Red) and Ki-67 (Green) (Evans and Koch,
2002)
Capillary
O2
O2
O2
Surviving Fraction
Expect gradient of cell killing from blood
vessels to necrosis
.
Radiation / Chem.
Drug
0
50
100
150
Distance from Capillary (µm)
X-ray Survival of Cells in SiHa Tumors as a
function of Distance from Blood Vessels
(Durand &
Olive, 1997)
Aerobic
Hypoxic
Expression of hypoxia induced proteins (eg CA IX) can
be used in archival tissues as a surrogate for hypoxia.
Immunohistochemical staining for CA IX
Head and Neck
Breast
Adenocarcinoma
NPC
Ovarian
Adenocarcinoma
Hui et al, 2002
Wykoff et al, 2000
Can we exploit tumor hypoxia?
• A drug specifically toxic to hypoxic cells
would kill only tumor cells - and the most
resistant ones.
Preferential Toxicity of Tirapazamine
(TPZ) to Hypoxic Cells in vitro
O
N
N
N
O
TPZ
Mouse SCCVII cells 1 hr exposure
Mouse SCCVII cells 1 hr exposure
10 0
10 -1
10 -2
10 -3
Hypoxia
10 -4
10 -5
0
Air
1000 2000 3000 4000
TPZ Conc (µM)
Surviving Fraction
10 0
10 -1
HCR=300
10 -2
10 -3
10 -4
10 -5 0
10
10 1
10 2
10 3
TPZ Conc (µM)
10 4
NH
2
Addition of a Hypoxic Cytotoxin to Standard
Treatment Could Exploit Tumor Hypoxia
O2
O2
O2
Surviving Fraction
Capillary
Hypoxic Cytotoxin
.
Radiation / Chem.
Drug
Combined
0
50
100
Distance from Capillary (µm)
150
Complementary Killing by TPZ and IR in SiHa Tumors
as a function of Distance from Blood Vessels
(Durand &
Olive, 1997)
Combined
Aerobic
Hypoxic
Primary site failure in 92 randomized
advanced H&N patients (Peters et al,2007,)
PET
hypoxia
status
Treatment
P-value
RT + cis
/no TPZ
RT +
cis/TPZ
NonHypoxic
2/27 (7%)
3/21 (14%)
NS
Hypoxic
8/18 (44%)
0/26 (0%)
0.0002
P-value
0.008
NS
Results of multicenter randomized clinical trial
of TPZ with advanced H & N cancer
All 863 patients in 89 sites in 16 countries
693 patients without major protocol violations
Need to select hypoxic tumors in future trials and have good QA for radiotherapy
Rischin et al and Peters et al, JCO, 2010
Patient Numbers Needed to Detect a
Change from 40 to 65% Response Rates
10000
90% probability
80% probability
Number
1000
of
Patients
100
0
20
40
60
80
100
% Patients with Hypoxic Tumors
SN30000 is superior to TPZ in multiple
tumor models
Single doses ( 15 or 20 Gy)
Fractionated 8 x 2 Gy SiHa tumor
Hicks et al, Clin Cancer Res 2010
Summary
• Sensitivity of tumor and normal cells can be
assayed quantitatively in situ in experimental
systems.
• Tumor hypoxia has a major negative impact
on the curability of tumors by radiotherapy,
and probably also chemotherapy
• Tumor hypoxia can be exploited using drugs
specifically toxic to hypoxic cells.