Robert Bristow MD PhD FRCPC

1

Translational Research in Prostate Cancer
Treatment and Progression

• Robert Bristow MD PhD FRCPC
• Departments of Radiation Oncology and Medical
  Biophysics,
• University of Toronto and
• The Princess Margaret Hospital-
• University Health Network

MBP1008-March 2009
PMH-Terry Fox Hypoxia Program
2
Prostate Cancer Topics

• Lecture I
• Introduction to Prostate Cancer
• Concepts relating to DNA-DSB repair
• Clinical radiotherapy
• DNA repair and radiotherapy
• Biomarkers of DNA repair
• Synthetic lethality-PARP inhibition

3
Prostate Cancer Topics

• Lecture II
• Genetic instability and cancer risk
• Models of prostate cancer progression
• Hypoxia, genetics, environment, nomograms
• Surgery
• male lumpectomy?
• Chemotherapy
• Taxanes, Mitoxantrone, others
• Hormone therapy
• Androgen axis, autocrine androgens
• Novel molecular targeting agents

4
Prostate Cancer Some Basics
Risk factors age, family history, high-fat diet,
African ancestry Currently, the extent and
prognosis of prostate depends on (1) a digital
rectal exam (DRE) and spread of disease (TNM) (2)
the prostate specific antigen (PSA) blood test
(3) the pathologic grade (Gleason score)
5
Prostate Cancer Some Basics
6
Canadian Prostate Cancer Statistics

• In 2007 22,300 men diagnosed with prostate
  cancer and 4,300 will die from it
• On average, 439 men are diagnosed each week
• One in 8 men will be diagnosed with prostate
  cancer, mostly after the age of 60
• One in 27 will die from it
• Test at age 40-50 depending on risk factors

7
New Cases of Cancer in Men 2007
8
Treatment Options Side Effects
Indolent Disease
Aggressive Disease
Active Disease
Surgery Radical Prostatectomy
Hormone Therapy (injections/tablets)
WATCH THE PSA CAREFULLY
Robots or laprascopic
Active Surveillance
Radiotherapy or brachytherapy (seeds)
Chemotherapy
Combinations
Increasing Stage and Aggression
9
Risk Groupings and Treatment(Active
Surveillance, Surgery, Radiotherapy, Hormone
Therapy and Chemotherapy)

• PROGNOSTIC FACTORS
• Traditional T-stage, PSA, Gleason Score
• Newer Percent Positive Biopsies, Ki-67, PSA DT lt
  10 months
• Promising p53, BAX-BCL2, EGFR,MDM2, SURVIVIN,
  p16INK4a, Hypoxia, Repair
• Future New targets and stem cells

• RISK GROUPS
• LOW T1/T2 PSA lt10 GS 4-6 (Brachy, HIFU, Cryo,
  EBRT Surgery, AS)
• INTERMEDIATE T1/T2 GS 7 PSA 10-20 (Brachy/EBRT
  /- Hormones Surgery, Other)
• HIGH PSA gt 20 GS 8-10 T3-T4 (EBRT
  Hormones/- Chemo New Agents, Surgery)

10
The New Era of Prostate Cancer Research

• The 20th century approach to cancer Seek and
  destroy
• The 21st century approach target and control
• Personalized genetic medicine
• To treat patients with fewer side effects.
• To prevent deaths in patients who are currently
  incurable.

11
Individual Oncology Predict-Change Treatment
Biomarker Data
Bristow-2004 Adapted after press2.nci.nih.gov/sc
iencebehind/snps_cancer
12
Precision-Guided Radiotherapy to Kill Cancer
Cells and Protect Normal Cells
High Dose To Cancer
Low Dose To Normal Cells
13
Radiotherapy for Cancer

• High-energy (6-25 MV) photons that deposit their
  energy deep within tissue and spare skin
• 1) Curative Given as a series of daily
  treatments or fractions of 1.8-2.5 Gy over 6.5
  to 8.5 weeks fractionated radiotherapy
• 2) Palliative Single 8 Gy or 20 Gy in 5 fraction
  doses for pain control or other symptomatic
  end-points
• Other interstitial radioactive implants
  brachytherapy (ie. some prostate, cervix, head
  and neck cancers) or stereotactic radiotherapy-a
  few high doses (20-30Gy) to a small area (brain
  metastases, small lung tumors)

14
Radiotherapy for Cancer

• Understand anatomy of local and distant spread
• Image the limits of the tumour as best as
  possible
• Contour the tumour and normal tissues to deliver
  high dose to tumour (e.g. increase local control)
  and low doses to critical structures (e.g. less
  side-effects)

Lecchi, 2007
15
Use Special Scans and Knowledge of Organ
Movement To Plan Radiotherapy
-the patient will have a CT scan of the region of
interest -the radiation oncologist will contour
the tumour (gross tumour volume GTV) and
normal structures to void -the dosimetrist will
execute beam arrangements to deliver a specific
dose to the tumour and sparing normal tissues -a
margin will be added to the contours (planning
target volume) that will take into account organ
movement and microscopic spread
Tumour Perfusion
Overlay Information
MRI Anatomy
Prostate
Sharpe and Dawson. 2005
16
A Final 3-D Plan Bladder Cancer
M Milosevic, 2006
17
Dose-Volume Histograms (DVH)

• a way to calculate the radiotherapy dose to
  normal and tumour tissues
• OPTIMIZE TREATMENT high dose to tumour low
  dose to normal tissues

18
Radiotherapy for Cancer

• Make sure the patient does not move make sure if
  the organs and tumour moves (prostate, lung, etc)
  that you can track the movement
• Create a safe and effective radiotherapy plan and
  verify always on target every treatment to ensure
  no geographical miss
• Decide on whether combined chemo-molecular
  targeted drug with radiotherapy and take this
  into account
• Follow patient every week for side-effects and
  then afterwards for result

19
(No Transcript)
20
Radiotherapy for Cancer

• Local tumour control is dictated by the
  eradication of all TUMOUR CLONOGENS
• Fractionated radiotherapy is used to maximize the
  THERAPEUTIC RATIO in order to kill more tumour
  cells than normal cells
• Fractionated radiotherapy sensitizes cells to a
  greater extent than single doses due to multiple
  factors 5 Rs of Radiotherapy (radiosensitiivity,
  repair, redistribution, reoxygenation,
  repopulation)

21
Erythema Moist Desquamation
Telangiectasia
NORMAL TISSUE EFFECTS
22
REPAIR NORMAL TISSUE RADIOBIOLOGY

• The time to expression of normal tissue injury
  depends on its turnover.
• ACUTE RESPONDING TISSUES
• Are rich in stem cells and proliferative
  progenitor cells that differentiate into
  functional cells with a high turnover and a high
  rate of cell loss
• Eg. Gut, Skin, Bone Marrow, Mucosa
• LATE RESPONDING TISSUES
• Have a slow turnover rate and stem cells play a
  smaller role in regeneration, which generally is
  from the functional cellular pools after a longer
  lag time.
• Eg. Brain, Spinal Cord, Kidney, Lung, Bladder

23
Favourable Treatment
Probability of normal tissue damage ()
Probability of tumour control ()
70
Complication-free cure 35
40
35
Complication-free cure 35
5
70 Gy
75 Gy
Radiation dose
Tumour dose-response curve
Normal tissue dose-response curve
Harrington and Nutting, Curr Opin Investig Drugs
2002
24
Radiosensitisation and Radioprotection
25
DNA Breaks A Way To Kill Cancer Cells With
Therapy
Pisansky, 2006
26
DNA Repair Radiation Oncology

• Responses to DNA damage
• checkpoints
• Repair of DNA damage
• BER, SSB, DSB
• Measuring DSBs
• Intranuclear foci
• Importance of DSB repair and clinical relevance ?

27
DNA Breaks A Way To Kill Cancer Cells With
Therapy
28
DNA Repair and Cancer
Cancer
Treatment
29
DNA Repair and Clinical Syndromes Increased
Sensitivity Chromosomal Instability and
Increased Cancer Risk
30
ATR
ATM
MRE11 NBS1 RAD50
MDC1
?-H2AX
53BP1
RFN8
CHK2
CHK1
Repair
Checkpoints
HR (S, G2)
NHEJ (G1, G2)
S Arrest CDC25A BRCA1/SMC1 NBS1
BER
G1 Arrest CDC25A p53/p21WAF RB
G2 Arrest CDC25B CDC25C Wee1
KU70 KU80 DNA-PKcs XRCC4 LIGASE IV XLF ARTEMIS
RAD51 RPA BRCA2 RAD51B/C/D XRCC3 MUS81 p53
PARP
CyclinA/E CDK2
CyclinA/E CDK2
CyclinB CDK1
Liu, Olive and Bristow, Can Met Rev, 2008
31
IR Induces 4 Distinct Checkpoints
Cyclin B ATM ATR CHK1 CHK2
Cyclin D/E ATM p53 CHK2
ATM Cyclin E/A cdc25A
32
(No Transcript)
33
(No Transcript)
34
ATM-p53 Signaling In Patient Prostate Biopsies
Pre-Operative IMRT 25Gy/5 Bristow, Warde, Klotz,
Fleshner, Sweet
p16INK4a
p21WAF
ATMSer1981
N15 all WTp53 by direct DNA sequencing
PRE-EBRT (Fiducial Markers) pO2
Measurements Biopsies MRSi
POST-EBRT MRSi Biopsy
INTRA-EBRT MRSi Biopsy
35
Cells Must Repair DNA Damage To Prevent Cancer
Development and Progression
36
Helleday, 2008
37
Summary of repair pathways after X-rays
Ionizing radiation
Double strand breaks
Base damage
Single strand breaks
38
Base Excision Repair Single Strand Break Repair
39
SSBR
BER
glycosylase
X
glycosylase removes damaged base
APE1
APE1 makes nick (SSB)
P
OH
C T A T T G G T
POLd/e
FEN1
G A T A C A C C A
POLB
XRCC1
PCNA
common intermediate
T G G
pol beta inserts single correct base
replicative pols replace 2-10 bases
SHORT PATCH
LONG PATCH
LIG1
LIG3
XRCC1
ligation by ligase3
ligation by ligase1
40
BER and SSBR affect radiosensitivity
DNA polymerase beta
XRCC1
WT
XRCC1 KO
mouse embryo fibroblasts
BER base excision repair SSBR single strand
break repair WT wildtype, KO knock-out
41
DNA-dsb Repair Pathways

• HR
• Homologous template
• Error free
• S G2 predominates
• Defects common in
• tumours
• Germline cells

BRCA2
RAD51
p53
Paralogs
RPA
MUS81
BLM
BRCA1

• NHEJ
• No homology
• Error prone
• G1 predominates
• Defects uncommon
• Somatic cells

DNA-PKcs
Ku80
DNA Ligase IV
Ku70
ARTEMIS
ATM
XRCC4
42
DSB
43
DSB
44
Homologous recombination affects radiosensitivity
45
Non-homologous endjoining affects radiosensitivity
wildtype
KU80 mutants
Erami A et al NAR 1998
46
Non-homologous endjoining affects radiosensitivity
Kurimasa A et al PNAS 1999
47
ATM is an important determinant of
radiosensitivity
100
Fibroblasts from Ataxia Telangiectasia and normal
patients
10-1
normal
Surviving fraction
10-2
ATM-/-
10-3
0
8
12
4
Radiation dose (Gy)
ATM mutated in Ataxia Telangiectasia protein
kinase
48
DSB repair in mutant cells
LIG4 -/-
DNA-PK -/-
ATM -/-
gH2AX
Wildtypes
Riballo E et al, Mol Cell 16715, 2004
a measure of DSB
49
Summary

• DSBs are the most important damage produced by IR
• DSBs are sensed primarily by ATM and MRN
• Apoptosis (rarely)
• Checkpoint activation
• DNA repair
• Repair requires large repair factories containing
  many proteins
• NHEJ (DNAPKcs, Ku70/80, Artemis, XRCC4, Ligase)
• HR (BRCA1/2, Rad51/52, FANCD2)
• Impaired DNA repair machinery causes extreme
  radiosensitivity

50
Summary

• Base damage and single strand breaks are repaired
  by related processes (BER, SSBR)
• Double strand breaks are the most lethal type of
  damage produced by radiation
• DSBs are repaired by NHEJ and HR
• Both are important
• HR works only if DNA is replicated (proliferating
  cells)
• MRN complex activates ATM, which is crucial in
  signaling
• Activates DNA repair (both NHEJ and HR)
• Activates checkpoints and apoptosis

51
DNA Repair Biomarkers-Some Issues

• Normal
• Dose-dependent
• Time-dependent
• Accessible ? (surrogates for solid tissues ?)
• Acute vs Late reacting
• Prove that the target is indeed being inhibited
  or not (is the drug working?)
• Or show that target not inhibited in normal
  tissues but it is in the tumour

• Tumour
• Same as for normal but have to account for tumour
  environment hypoxia and other goodies
• Non-invasive imaging could help
• Need better data to decide when to use agents-up
  front or at end of a treatment regimen

52
Potential DNA-Dsb Repair Biomarkers
Enzyme activity
kinase
substrate
substrate
WB
Protein
Post-translational modification
phosphorylation
IHC
kinase
kinase

• Surrogate tissue
• skin biopsy
• PBLs
• fluids (urine, serum)
• buccal mucosa
• hair follicle

IF
expression level
Protein A
Protein B
Protein C
Protein expression (Proteomics)
or
DSBs DNA repair foci
Tumor
DNA
Comet assay

-
electrophoresis
Chromatin

-
CFGE / PFGE
Liu, Olive and Bristow, Can Res Met, 2008
electrophoresis
53
DNA-dsbs Dose- and Time-Dependent
Rejoining Assays
DNA-dsb Remaining
54
DNA Repair Foci Sensitive to Planning/Diagnostic
CT
Bristow and Hill, 2008
55
Nuclear
Nuclear 3D
DNA Breaks
56
Microscopic Human DNA Repair Foci In Vitro
gH2AX-53BP1
gH2AX-PML
57
UV Microbeam Recruitment to DNA-dsbs
laser path
DNA Breaks
58
In situ Cell-Cycle Markers
In asynchronous populations, have to control for
cell cycle given phosphoforms can be activated by
cell cycle
59
(No Transcript)
60
Counting DNA Breaks Murine Skin
Initial Breaks
2Gy-30
Radiation Dose (Gy)
61
DNA Repair and Foci in Human Skin During
Radiotherapy
Simonsson et al., RadOncol, 2008
62
BRCA1/2 Primer for Carcinogenesis
Autosomal Dominant
-patients with either mutation have a lifetime
risk of 80 of breast cancer -median age at Dx
40(BRCA1) 45(BRCA2) -tumours higher grade and
proliferation, medullary, ER/PR neg. -increased
risk of contralateral cancers (60-70 lifetime) -h
owever, survival may be similar to sporadic -both
are tumour suppressor proteins and involved in
some forms of DNA-dsb break repair -in tumours
one allele is mutant and the other becomes
inactivated by mutation or other means
63
Are BRCA1/2 Patients More Sensitive ?

• Fibroblasts from BRCA1/2 heterozygote patients
  had SF2 in low-normal range (Brock 2001
  Peacock-McMillan, 2004)
• Mice lacking BRCA1/2 function and cancer cell
  lines lacking BRCA1/2 function had decreased
  survival and chromosomal re-arrangements
  following IR
• Suggested increased DNA damage and/or faulty
  repair in BRCA1/2-negative cells
• Worries about BRCA1/2 heterozygote patients
  receiving adjuvant radiotherapy
• Increased acute/late effects ?
• Increased XRT-induced tumours within ipsilateral
  breast or contralateral breast (scatter) ?

CONTRALATERAL TUMOURS IS IT INHERENT TISSUE
RISK OR XRT-INDUCED RISK ?
64
BRCA1/2-Summary

• To date, although pre-clinical data suggested
  increased radiosensitivity clinical data fail
  to support increased acute or late reactions in
  patients receiving adjuvant radiotherapy
• BCT can be delivered safely as long as patient
  counseled about risk of new primary tumours
  bilaterally
• Risk-reduction strategies oophorectomy and
  tamoxifen
• Careful follow-up MRI-based imaging
• BRCA1/2-associated tumours may be sensitive to
  HR-targeted agents (MMC or Gemcitabine) or PARP
  inhibitors
• Normal tissues in heterozygotes may have enough
  HR function to protect and preserve therapeutic
  ratio
• Secondary malignancies in clinical cohorts
  following XRT and/or chemotherapy ?

65
Synthetic Lethality Therapeutic Ratio
Defect in Gene X or Gene Y Alone Not
lethal Defect in Both Genes Lethal (e.g.
BRCA1/2-/- PARPi)
(Bryant et al. Nature, 2005Farmer el al, 2005)
66
PARP Inhibitors and Clinical Trials
-AZD2281 trials (RMH, NKI)-BRCA1/2 carriers with
hereditary ovarian cancer (cDDP resistant)
responses seen up to 15 months -biomarker gH2AX
foci in hair and tissue PARP activity New trials
PARP TMZ RT
67
Selective Treatment Based on Repair Defect
DNA Repair As A Target ? Altered
checkpoints/repair Differential HR/S-G2
fractions Repair defects in hypoxia
Individual Treatment
DNA Repair Pathway
Example Genes in Pathway
Unique Sensitivity If Genes Abnormal
Rad51, BRCA1, BRCA2, DSS1, Bard, Rad54, Xrcc2/3,
Rad52, etc.
1) HR DNA-dsb Repair
PARP inhibitors, HU MMC, cisplatinum, IR
Ku70, Ku80, DNA-PKcs, XRCC4, DNA Lig IV, XLF,
Artemis, etc.
2) NHEJ DNA-dsb Repair
Topoisomerase inhibitors, IR, Bleomycin,
MSH2, MSH3, MSH6, MLH1, PSM1, PSM2, HMGB1 etc.
3) Mismatch Repair (MMR)
Temozolomide, 5-FU, MNNG
OGG1, MYH, PCNA, REF1, FEN-1, Neil1, etc.
4) Base Excision Repair ( BER)
5-FU, IR
5) Nucleotide Excision Repair (NER)
XP-A/B/C/D/E/F/G, TFIIH, Ercc1, etc.
Cisplatinum, MMC, nitrogen mustards
68
DNA Breaks A Way To Kill Cancer Cells With
Therapy
- hypoxia leads to 2.5-3.0 less DSBs
69
Hypoxia HR Functional Data
Bindra, Glazer, Bristow MCB-2004 Meng,Bristow
RadOncol-2005
Breast and Prostate cell lines independent of
cell cycle and HIF1a
FUNCTIONAL OUTCOME ?
70
Defective HR And Cancer Cell Therapy
71
Acute ? Prolonged (Chronic) Hypoxia
Princess Margaret Hospital University Health
Network
72
In Vivo Quantitative Tracking of Repair
Cellular
Quantitation in vivo
Prostate TMA Post-XRT
Oxic
2Gy-24h
Hypoxic
73
ATR
ATM
MRE11 NBS1 RAD50
MDC1
?-H2AX
53BP1
RFN8
CHK2
CHK1
Repair
Checkpoints
HR (S, G2)
NHEJ (G1, G2)
S Arrest CDC25A BRCA1/SMC1 NBS1
BER
G1 Arrest CDC25A p53/p21WAF RB
G2 Arrest CDC25B CDC25C Wee1
KU70 KU80 DNA-PKcs XRCC4 LIGASE IV XLF ARTEMIS
RAD51 RPA BRCA2 RAD51B/C/D XRCC3 MUS81 p53
PARP
CyclinA/E CDK2
CyclinA/E CDK2
CyclinB CDK1
Liu, Olive and Bristow, Can Met Rev, 2008
74
Resistant clones
Sensitive Clones
Start
F/U
DRUG
PREDICTOR
Start
F/U
DRUG
PREDICTOR
75
Biomarker Schema in DNA-Dsb Repair Inhibition
Biopsy Inhibition
Biopsy Residual Dsbs
Biopsy Initial Dsbs
XRT
24h
0
1h
Drug
NeoAdjuvant
Concurrent
Adjuvant
PK/PD Enzyme Inhibition In Oxic and
Hypoxic Cells ?
Initial Breaks and Inhibition of DNA
Damage Signaling Pathways in Oxic and
Hypoxic Cells
Excess Residual Breaks and Cell Death in Oxic
and Hypoxic Cells
76
Signal Transduction DNA Repair

• EGFR inhibitors (Cetuximab) lead to decreased
  levels of DNA-PKcs and RAD51 and lead to elevated
  residual DNA-dsbs
• Radioresistant cells that over-express the RAS
  oncogene have elevated levels of Ku80 and DNA-dsb
  repair
• Effects of Farnesyl Transferase Inhibitors on
  this finding ?
• HDAC inhibitors (TSA, SAHA, MS-275) decrease
  RAD51 expression and lead to increased residual
  DNA-dsbs
• IGF1-R inhibitors decrease to altered HR and NHEJ
• Gemcitabine/cDDP XRT decreased repair of
  DNA-dsbs
• ATM, DNA-PK, PARP inhibitors lead to increased
  DNA-dsbs

77
Conclusions and Questions

• Biomarkers and Repair More Complex in Tumours
• Glycolysis and pH needed
• Need biomarkers to predict acute/chronic hypoxia
• Need to develop non-phosphorylation-based
  biomarkers (given clinical use of kinase
  inhibitors)
• For fractionated radiotherapy complex pattern of
  re-oxygenation, cell death, S/G2M phase in 4-D
• Focus on first XRT fraction for residual breaks
  at 24-48 hrs for inhibitor trials with
  pre-clinical work-up
• Can we image repair ?
• Could be prognostic/predictive for therapy and
  lead to individualized treatment options
• How do we calculate molecular therapeutic ratio ?