Cancer Chemoprevention

1
Cancer Chemoprevention Surrogate End Point
Markers

• JianYu Rao, M.D.
• Associate Prof. Of Pathology
• UCLA

2
CANCER PREVENTION

• PRIMARY
• STOP THE EXPOSURE
• SECONDARY
• INTERVENTION OR CHEMOPREVENTION
• TERTIARY
• TREATMENT

3
CHEMOPREVENTION

• Administrating specific amounts of a particular
  natural or synthetic chemical in an attempt to
  identify agents that will prevent, halt or
  reverse the process of carcinogenesis
• The basic assumption is that treating early
  stages of malignant process will halt the
  progression of malignancy
• The key is to define early lesions, and treat the
  malignant field

4
Additional Molecular Event
Exposure to Carcinogen
Precancerous Intraepithelial Lesions, (PIN,
CIN, PaIN..)
Cancer
Birth
CHEMOPREVENTION
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Multiyear progression from initiation and early
precancerous lesions to invasive disease in major
cancer target organs
Kelloff et al. 2000 (Fig. 1)
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THEORIES SUPPORT FOR CHEMOPREVENTION

• EPIDEMIOLOGICAL EVIDENCE
• OVER 50 CANCERS HAVE NO KNOWN RISK FACTORS
• NUMEROUS EVIDENCE TO DEMONSTRATE THE INVERSE
  RELATIONSHIPS OF SOME NUTRIENT FACTORS WITH
  CANCER RISKS

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THEORIES SUPPORT FOR CHEMOPREVENTION (Cont.)

• EXPERIMENTAL EVIDENCE
• ALTHOUGH CARCINOGENESIS IS REGARDED AS
  NONREVERSIBLE PROCESS, STUDIES SHOWED THIS IS
  ONLY TRUE AT LATE STAGE. IN FACT, A LARGE
  PORTION OF THE LONG LATENCY PERIOD OF
  CARCINOGENIC PROCESS IS REVERSIBLE.
• IN VITRO CULTURE AND IN VIVO ANIMAL STUDIES
  IDENTIFIED NUMEROUS AGENTS THAT CAN REVERSE, OR
  HALT THE CARCINOGENESIS PROCESS, PARTICULARLY AT
  THE EARLY STAGE.

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THEORIES SUPPORT FOR CHEMOPREVENTION (Cont.)

• CLINICALLY
• ADVANCES IN CERTAIN TYPES OF CANCER TREATMENT
  HAVE LIMITED SUCCESS IN REDUCING THE OVERALL
  INCIDENCE, OR EVEN MORTALITY OF CANCER.

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ChemopreventionSome Terminologies

• INDIVIDUAL RISK AND STRATIFICATION
• INTERMEDIATE END POINT MARKER (SURROGATE END
  POINT MARKER)
• FIELD CANCERIZATION
• MULTI-PATH OF CARCINOGENESIS

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RISK STRATIFICATION

• Identification of AT-RISK subjects who are also
  SUSCEPTIBLE to treatment

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INTERMEDIATE END POINT MARKER (SURROGATE END
POINT MARKER)

• These are prevention biomarkers which are
  specifically related to early stages of
  carcinogenesis.
• These markers are used to identify individuals
  risk for developing cancer and to monitor the
  effectiveness of intervention methods.

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FIELD CANCERIZATION

• The whole field of tissue of a particular organ
  is exposed to the carcinogenic insult and is at
  increased risk for developing cancer.
• Although only a few foci eventually develop
  malignancy, the other areas are not necessary
  entirely normal.
• Most common epithelia cancers are developed
  through this mechanism. Examples of such cancers
  are Head and neck ca, bladder ca, breast ca,
  lung ca, GI ca, etc.

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MULTI-PATH OF CARCINOGENESIS

• The current model of carcinogenesis is that
  cancer develops through multiple events which are
  not necessary through linear steps, but rather
  through overlapping networks.

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TARGET POPULATION

• INDIVIDUALS AT RISK
• LATENCY (20 YEARS) x EXPECTED TO DIE IN ONE
  YEAR (1.1 MILLION)
• 22 MILLION

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CHEMOPREVENTION IN DIFFERENT RISK CATEGORIES
Risk category
Parameter
General Population High Risk
Agent toxicity Trivial to none Slight Selectio
n method Public Health Clinical Other
consideration Use dietary supplements Need
biomarkers may be applicable
From lee W. Wattenberg, P.S.E.B.M., 1997
216133-141.
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Phase I Trial

• Objectives
• To determine the interventions short-term (lt1
  yr.) dose-toxicity relationship
• To determine the interventions human
  pharmacokinetics
• Design
• Single arm, nonrandomized
• Multiple dose levels
• Less than 1 yr. duration
• Accrual 25-100

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Phase II Trial

• Objectives
• To determine the interventions side effects
• To determine optimal recruitment methods of the
  target population
• To determine retention of study participants to
  the study intervention and procedures
• To determine optimal methods for the conducting
  of a phase III trial
• To determine the effect of the intervention on
  biomarkers of carcinogenesis (phase II b)
• Design
• Randomized, double-blind, placebo-controlled
• Multiple dose levels or agents
• One to five years in duration
• Accrual 100s-1000s

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Phase III Trial

• Objectives
• To determine the effect of the intervention on
  the cancer incidence (total and specific cancer
  type)
• To determine the effect of the intervention on
  death rate and disease incidence
• To determine the long-term side effects of the
  intervention
• To determine the nature history of specific
  biomarkers of carcinogenesis (placebo group) and
  the effect of the intervention agent (treatment
  group) on these markers.
• Design
• Randomized, double-blind, placebo-controlled
• Multiple dose levels or agents, alone or in
  combination
• Five to ten years in duration
• Accrual 1000s-10,000s

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UNIQUE FEATURES OF CHEMOPREVENTION

• Participants are usually healthy or at least
  cancer free
• The degree and incidence of side effects that are
  acceptable are low
• The end point is disease prevention, not disease
  response
• The incidence of the study end point is low

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CATEGORIES OF CHEMOPREVENTIVE AGENTS

• BLOCKING CARCINOGEN METABOLISM AND EXPOSURE
• INCREASE TISSUE RESISTANCE/DIFFERENTIAITON
• TARGETING ONCOGENIC PATHWAYS

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CATEGORIES OF CHEMOPREVENTIVE AGENTS

• BLOCKING AGENTS
• Prevent metabolic activation of carcinogens or
  tumor promoters
• Enhance detoxification
• Glutathione-S-transferase,Oltipraz
• Trap reactive carcinogenic species
• Glutathione, N-Acetylcysteine
• Vaccines HBV, HPV

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CATEGORIES OF CHEMOPREVENTIVE AGENTS (Cont.)

• INCREASING TISSUE RESISTANCE
• Induce tissue maturation/differentiaiton
• Pregnancy or hormonal induced maturation of
  terminal ducts of breast - decrease breast cancer
• Retinoids, DMFO, etc
• Decrease target tissue function
• Castration - reduce risk of prostate ca
• Decrease cell proliferation
• Low fat diet decrease epithelial proliferation
  rate in intestinal tract - reduce colon cancer
  risk

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CATEGORIES OF CHEMOPREVENTIVE AGENTS (Cont.)

• PATHWAY SPECIFIC AGENTS
• Cox-2 inhibitors
• Anti-angiogenesis
• Anti-EGFR
• Hormone antagonists
• Augmenting tumor suppressor functions
• Inhibiting oncogenic activities (e.g., Ras)

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CHEMOPREVENTION TO HUMANS - UPDATE

• BREAST CANCER
• Two agents showed promising results Tamoxifen
  and retinoids
• Animal model well established
• PROSTATE CANCER
• SCID model established
• Hormonal modulation may have potential
• PCPT Trial Finasteride (5-a-reductase, 5mg/day)
• 2-arm trial, 18,882 subjects, 7 yrs
• PCP18.4 vs 24.8 in treated vs ctrl group
• Ongoing Trial Selenium/Vit E trial

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CHEMOPREVENTION TO HUMANS - UPDATE (CONT.)

• GASTRIC AND ESOPHAGEAL CANCER
• A combination of beta carotene, vitamin E, and
  selenium may be effective in early stage lesions,
  but not late severe dysplastic lesions.
• LUNG CANCER
• Beta-carotene or alpha-tocopherol showed reverse
  effect in lung cancer risk in heavy smokers in
  Finland
• Ongoing trials with COX-2 inhibitor in former
  smokers here at UCLA

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CHEMOPREVENTION TO HUMANS - UPDATE (CONT.)

• COLON CANCER
• Sulindac, a nonsteroidal anti-inflammatory
  compound hold great promise. Others, such as
  Oltiparz, selenium, and antioxidants vit. E/A,
  etc, may also be effective.
• HEAD AND NECK CANCER
• Retinoids showed promising results in both animal
  models and human studies.

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PROBLEMS OF CHEMOPREVENTION

• TOO LONG
• TOO LARGE COHORT
• TOO MUCH COST
• ANSWER
• NEED TO DEVELOP RELIABLE SEMS

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BIOMARKERS OF CANCER

• CLINICAL SETTINGS (TUMOR MARKERS)
• EPIDEMIOLOGICAL AND PREVENTIVE SETTINGS
  (INTERMEDIATE END POINT OR SURROGATE END POINT
  MARKERS).

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CURRENT CLINICALLY USED TUMOR MARKERS

• PSA - Prostate Adenocarcinoma
• Alpha FP - Hepatoma some Ovarian Ca
• HCG - Choriocarcinoma
• CEA - Ovarian CA

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BIOMARKERS

• Genetic susceptibility markers
• Markers of exposure
• Markers of biological effects
• -Detect early lesions
• -Prognostic indicators

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GENETIC SUSCEPTIBILITY MARKERS

• Glutathione S-transferase (GST) M1 and T1
• N-acetyl transferase (NAT)
• Cytochrome P-450
• DNA repair gene defect (Lynch syndrome)

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MARKERS OF EXPOSURE

• Metabolic product of carcinogen in urine
• DNA, RNA and hemoglobin adducts
• -Reflects only current exposure
• -Only a small fraction of DNA adducts will
  result in mutation
• DNA repair targets

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BIOMARKERS OF EFFECT

• Reflect the interactions of genetics and
  exposures and so the first choice for SEM
• If they persist, may also be the markers of
  disease
• Histopathologic evaluation is the gold standard

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HOW TUMOR MARKERS ARE USED CLINICALLY

• Early detection
• Predict the biological potential of cancer
  (metastasize and recurrence)
• Monitor the effectiveness of therapy

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Additional Molecular Event
Exposure to Carcinogen
Precancerous Intraepithelial Lesions, (PIN,
CIN, PaIN..)
Cancer
Birth
Surrogate End Point Markers
Markers for Exposure
Markers of Effect
Tumor Markers
Genetic Suscep. Marker
CHEMOPREVENTION
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CRITERIA FOR SELECTING SEM

• FITS EXPECTED BIOLOGICAL MECHANISM
• BIOMARKER AND ASSAY PROVIDE ACCEPTABLE
  SENSITIVITY, SPECIFICITY, AND ACCURACY
• BIOMARKER IS EASILY MEASURED
• BIOMARKER MODULATION CORRELATES TO DECREASED
  CANCER INCIDENCE

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FITS EXPECTED BIOLOGICAL MECHANISM

• DIFFERENTIALLY EXPRESSED IN NORMAL AND HIGH RISK
  TISSUE
• CLOSELY LINKED, EITHER DIRECTLY OR INDIRECTLY, TO
  CAUSAL PATHWAY FOR CANCER
• MODULATED BY CHEMOPREVENTIVE AGENTS
• LATENCY IS SHORT COMPARED WITH CANCER

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ASSAY VALIDITY

• ASSAY SHOULD BE STANDARDIZED AND VALIDATED
• DOSE-RELATED RESPONSE TO THE CHEMOPREVENTIVE
  AGENT IS OBSERVED
• STATISTICALLY SIGNIFICANT DIFFERENCE BETWEEN
  LEVELS IN TREATMENT GROUPS AND CONTROLS

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OTHER ASSAY ISSUES

• BIOMARKER CAN BE OBTAINED BY NON-INVASIVE
  TECHNIQUES
• ASSAY IS NOT TECHNICALLY DIFFICULT
• MULTIPLE MARKERS CAN BE EVALUATED SIMULTANEOUSLY
  IN LIMITED SAMPLE VOLUMES
• COST
• FALSE POSITIVE OR FALSE NEGATIVE RESULTS ARE LESS
  IMPORTANT, IN COMPARING WITH CLINICAL TUMOR
  MARKERS

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CATEGORIES OF SEM

• HISTOLOGICAL AND MORPHOMETRIC MARKERS
• PROLIFERATION, DIFFERENTIATION AND INVASION
  MARKERS
• SPECIFIC ONCOGENES/GROWTH REGULATORS
• MARKERS OF GENETIC AND EPIGENETIC INSTABILITY

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POTENTIAL SEMS FOR BREAST, COLON AND PROSTATE

• Breast Colon Prostate

Adenomatous polyps
Histological
DCIS, LCIS, ADH
Aberrant polyps
PIN
Proliferation S-phase fraction S-phase
fraction PCNA
Ki-67 Brdu Uptake, PCNA Ki-67
Differentiation Myoepithelial (s-100 BGA, Mucin
core ag HM Cytok
Vimentin), etc Cytokeratins BGA, actin
Genetic Onc (erb-2, myc Onc (ras, myc, src) Onc
(erb-2) fos, ras) Suppressor (p53, Suppressor
(p53) DCC)
Biochemical Estradiol Ornithine Decarboxylase

Polyamine TGF-beta, PSA
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SEM Modulation in Chemoprevention

• Complete Phenotypic Response -idea
• Less Than Complete Phenotypic Response -Genotypic
  markers to distinguish chemoprevention from
  selecting regressing of existing disease
• true effect is seen if post-treated lesion has
  less genotypic change than baseline or control)
• No Response.

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Genome Wide Genotypic SEM Analysis

• Identify high risk population
• Identify individuals with genetic susceptibility
  for treatment (pharmacogenomics)
• Monitoring/analyzing individuals treatment
  response

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Issues in Using SEM

• The observed SEM change may not correlate with
  end point (cancer incidence).
• Can not measure the quality of life.
• Adverse effect may not be observed in short term
  SEM studies.

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Lessons learned from SELDI-TOF

• Initial study on patient serum from cancer
  patients (ovarian, prostate, etc) versus cancer
  showed very promising results (nearly 100
  sensitivity/specificity to separate cancer from
  normal)
• Used case-control design
• Only 2 group-comparison (cancer vs. normal)
• No validation
• However, recent validation studies were rather
  disappointing

46
Biomarker-Directed Targeted Design

• Increase the efficiency of the trial, but
  depends on
• The performance of the biomarker test
  (sensitivity/specificity)
• Size of the treatment effect for target-negative
  patients

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BIOMARKER STUDY DSEIGN
a. Untargeted Design
Treatment
Register
Randomize
Control
b. Untargeted Design
Treatment
Biomarker
Test Biomarker
Randomize
Register
Control
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BIOMARKER STUDY DSEIGN
Biomarker by Treatment Interaction Design
Treatment
Biomarker
Randomize
Control
Test Biomarker
Stratify
Register
Treatment
Biomarker -
Randomize
Control
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BIOMARKER STUDY DSEIGN
Biomarker Based Strategy Design
Biomarker
Treatment A
Test Biomarker
Biomarker -
Treatment B
Register
Randomize
No Biomarker Evaluation
Treatment B
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BIOMARKER STUDY DSEIGN
Modified Biomarker Based Strategy Design
Biomarker
Treatment A
Test Biomarker
Biomarker -
Treatment B
Register
Randomize
Treatment A
No Biomarker Evaluation
Randomize
Treatment B
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Actin Remodeling As a Target for Biomarker
Development
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NORMAL
CA

• Morphological hallmarks of cancer cells
• Altered N/C-ratio
• Altered membrane (cytoplasmic and nuclear)
• Loss of cell adhesion
• Increased motility/invasion/met.
• etc..
• ALMOST All ARE RELATED TO ACTIN REMODELING

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WHAT TO DO WITH THIS?
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HYPOTHESIS/RATIONALE

• Altered cytoskeletal proteins, e.g., actin
  remodeling, is the foundation for malignant
  morphological phenotype
• Thus, signaling pathways associated actin
  remodeling may provide a potential target for
  anti-cancer drug development as well as
  biomarkers for a more objective assessment of
  malignant transformation and progression
• These targets can be identified through
  genomic/proteomic approach

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Model in Focal Adhesions
F-Actin
Tenuin
VASP
Zyxin
- Ras Sup. Family (Rac/Rho/CDC42) -
pp60sro - pp125FAK -Abl
Actinin
Vinculin
Paxillin
p-Tyr?
Talin
R/E/M
Tensin
Integrin
ECM
a
a
b
b
PM
Substrate
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ACTIN ASSCOIATED MOLECULARS IMPLICATED IN
MALIGNANT TRANSFORMATION

• Oncogene signal transduction pathways
• Ras family ( GTPase)
• Rho (stress fibers)
• Rac (lamellipodia)
• Cdc42 (filopodia)
• Src family (tyrosine kinase)
• FAK
• Relate to intergin signaling

• Tumor Suppresor
• Gelsolin
• Tropomyosin/merlin
• Alpha-actinin
• E-cadhelin
• Beta-Catanin
• Vinculin
• Fodrin
• Implicated in apoptosis

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Increased cellular F-actin is a marker of
cellular differentiation

• Using leukemic cell linesHL-60-
  Transformed/Differentiable
• Daudi- Transformed/Undifferentiable
• RPMI - Nontransfomed
• We demonstrated that increased F-actin content is
  associated with cellular differentiation
• (J. Rao, Cancer Res., 1990)

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In contrast, loss of F-actin is a marker for
cellular transformation and bladder cancer risk

• Bladder wash samples from a spectrum of cases
  with various risk for TCC show a strong
  correlation of loss of cellular F-actin contents
  with increased bladder cancer risk.
• (J. Rao, Cancer Res., 1991)

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Furthermore, actin alteration is a field disease
marker for bladder cancer

• A careful mapping analysis on touch prep slides
  obtained from distant, adjacent and tumor tissues
  showed that increased G-actin is seen in over 50
  of the distant field epithelial cells of cancer
  bearing bladder.
• (J. Rao, P.N.A.S., 1993)

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QFIABiomarker Profile
G-actin Texas-Red conjugated DNase I M344
FITC (or Rhodamin) 3- Step Immunofluorescence
DNA Hoechst or DAPI
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Test Our Biomarker Profile
Test Our Biomarker Profile
)
to Detect Bladder Cancer
to Detect Bladder Cancer
in Workers Exposed to
in Workers Exposed to
Cancer Causing Chemicals
Cancer Causing Chemicals
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Study Design in Worker Risk Assessment Study
TREAT
Very High
Positive
Risk
1788
High Risk
Cystoscopy
Workers
Negative
Monitor in
Moderate
1 yr
Risk
373
Monitor in 3 yrs
controls
Low Risk
Action/Intervention
Screen Workers
Classify
Markers
Risk
Exposure
Physical Exam
Questionnaire
Smoking Asses.
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Procedures in Screening Program
Notification of exposed workers.
Notification of exposed workers.
!
!
Selection of matching controls.
!
Selection of matching controls.
!
Administration of questionnaire.
Administration of questionnaire.
!
!
Occupational history.
Occupational history.
6
6
Medical history.
Medical history.
6
6
Genitourinary tract history.
Genitourinary tract history.
6
6
Smoking assessment.
Smoking assessment.
6
6
Physical examination.
Physical examination.
!
!
Urinalysis
Urinalysis
!
!
Papanicolaou cytology
!
Papanicolaou cytology
!
DNA, M344, G-actin biomarkers
DNA, M344, G-actin biomarkers
!
!
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Pathology Summary
30 Cancers detected
6
29 Transitional Cell Carcinoma
)
1 Squamous Cell Carcinoma
)
4 Cases of Muscle Invasion
(gtT2)
6
20 Cases Grades 1-2 8 Cases Grade 3.
6
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Incidence Rate (per 100,000 person-year) of
Bladder
Cancer in the Cohort Exposed to Benzidine (
1991-1997)
No. of
Cancer
Cohort
Subjects
Age ( Mean SD )
Incidence
Cases
Followed
Unexposed
373
57.7 10.8
2
87.23
Exposed
1788
55.4 10.5
28
263.35
Total
2161
55.8 10.5
30
232.11
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TEST POSITIVE PRIOR TO OR AT
TEST POSITIVE PRIOR TO OR AT
THE TIME OF DIAGNOSIS

THE TIME OF DIAGNOSIS
NO. OF
RATE
BIOMARKERS
POSITIVE/NO.
POSITIVE
OF CASES
QFIA HIGH OR
MODERATE
28/29
96.5
RISK
PAP
15/28
53.6
CYTOLOGY
HEMATURIA
4/28
14.3
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Biomarker Results of Cohort Study Detection
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Biomarker Results of Cohort StudyRisk Assessment
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Cox Proportional Regression Model with Time
Dependent Covariates
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Abnormal G-actin in the Field Predicts Tumor
Recurrence
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Cellular actin levels can be used to monitor the
effectiveness of chemoprevention

• Cellular F/G-actin levels in the non-tumor field
  epithelial cells after tumor was removed by TUR
  predicted the recurrence potential of the tumor.
• In addition, cellular F/G-actin levels fluctuate
  from abnormal to normal as results of
  chemopreventive effect of differentiation agent
  DMSO.
• (G.P. Hemstreet, J. Rao, Cancer Det. And Prev.,
  1999)

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SUMMARY Actin Remodeling in Cancer

• Actin remodeling as a generalized marker for
• Cancer field changes
• Precancerous lesions
• and thus, a candidate for chemopreventive SEM
• However
• Measuring actin remodeling is technically
  challenging
• New method/tools are needed

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Nanomechanical analysis of cancer cell
softness/elasticity

• Atomic Force Microscope
• A new tool for cancer research
• Ideal for analyzing the functional role of actin
  remodeling in various cellular events in single
  living cells
• Combine functional analysis with morphology at
  nanometer level

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NEWS HEADLINES

• Nanotechnology shows cancer cells are 'softer'
  than normal cells
• Microscopic 'tools' can identify cancer cells by
  'feel
• Nano breakthrough in cancer detection study
• .

78

• Fig. 1. Schematic of an AFM tip
• approaching,
• indenting and
• retracting from a cell

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A
B
Mesothelial cells
Tumor
Phase-contrast
D
C
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Chemoprevention of Superficial Bladder Cancer in
Former Smokers Parallel, Randomized,
Double-blind, Placebo-Controlled, Phase II
Adjuvent Studies of Erlotinib and Polyphenol E to
Prevent the Recurrence and Progression of
Tobacco-Related, High-grade Superficial Bladder
CancerU01-CA-96116
84
Study Objectives

• Primary
• To evaluate the effects of a daily dose of PE,
  Erlotinib, and placebo on tumor recurrence for
  pts with superficial bladder ca (former smokers)
• Secondary
• To assess toxicities of PE and Erlotinib
• To correlate the modulation of biomarkers with
  tumor recurrence/progression
• To assess the effects of PE and Erlotinib on
  tumor progression

85
Study Design

• Phase II, randomized, double-blinded,
  placebo-controlled, 3-arm trials
• A random permuted study design with one
  stratification factor (Ta vs T1 vs CIS)
• Two agents PE- 800mg/daily, Erlotinib (up to
  100mg/daily
• 330 former smokers (lt12 months) with prior
  superficial bladder ca

Placebo
Treatment PE Erlotinib
Stage Ta T1 CIS
86
Specimen Types

• Blood
• Urine cytological specimens
• Voided urine
• Catheterized urine
• Bladder wash
• Tissue
• Biopsy
• Cystectomy specimen

87
Key Secondary Biomarkers

• Cytology
• QFIA Profile
• DNA G-actin by LSC
• M344/19A211/LDQ10 by Immunocyt kit
• Microsatellite Instability Markers (M.S.I.)
• bFGF
• Survivin

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Biomarker Core
5 cc
Urinary Cytology (VU/CU/BW, 100 cc each)
Tissue (ca, random)
Blood (20 cc)
Store
10 cc
2-bFGF Brooks lab
Leukocyte
Plasma
3- Genetic Polym. (Zhangs lab)
Thin Prep (3)
Fresh Frozen
Paraffin Emb.
2 slide
1 slide
Store (Raos lab)
2-QFIA Raos Lab
2- Cytology
1- Histology
4- Polyphenol (Hebers lab)
3- Tissue Array EGFR, Ki67, Gelsolin, p53,
etc (Seligsons lab)
Extract Genomic DNA
1- Primary end point 2- Secondary end
point 3-Tertiary end point 4- Compliance marker
2- M.S.I. (Core facility)
3- Genetic Polymorphism (Zhangs lab)
89
Summary

• Biomarker is needed in Chemoprevention Trial to
• Detect early preventable lesions
• Monitoring the efficacy
• Actin remodeling and associated cellular
  nanomechanical changes provide a wealth of
  targets for chemopreventive biomarker selection
• Actin change occurs in premalignant field lesion
• Chemopreventive agents (e.g., green tea)
  modulates actin remodeling
• Actin change can be detected either by
  traditional biochemical assays or AFM
  measurements of cellular nanomechanics