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Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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Page 1: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Cancer Chemoprevention & Surrogate End Point Markers

JianYu Rao, M.D.

Associate Prof. Of Pathology

UCLA

Page 2: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

CANCER PREVENTION

• PRIMARY• STOP THE EXPOSURE

• SECONDARY• INTERVENTION OR CHEMOPREVENTION

• TERTIARY• TREATMENT

Page 3: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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

Page 4: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Cancer

Precancerous Intraepithelial Lesions, (PIN, CIN, PaIN..)

Birth

Exposure to Carcinogen Additional Molecular Event

CHEMOPREVENTION

Page 5: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Multiyear progression from initiation and early precancerous lesions to invasive disease in major

cancer target organs

Kelloff et al. 2000 (Fig. 1)

Page 6: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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

Page 7: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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.

Page 8: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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.

Page 9: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Chemoprevention:Some Terminologies

• INDIVIDUAL RISK AND STRATIFICATION

• INTERMEDIATE END POINT MARKER (SURROGATE END POINT MARKER)

• FIELD CANCERIZATION

• MULTI-PATH OF CARCINOGENESIS

Page 10: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

RISK STRATIFICATION

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

LEGEND:Not at risk to develop disease At risk of developing disease, biology A, responsive to agent XAt risk of developing disease, biology B, NOT responsive to agent X

Page 11: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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 individual’s risk for developing cancer and to monitor the effectiveness of intervention methods.

Page 12: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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.

Page 13: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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.

Page 14: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

TARGET POPULATION

INDIVIDUALS AT RISK =

LATENCY (20 YEARS) x # EXPECTED TO DIE IN ONE YEAR

(1.1 MILLION)

= 22 MILLION

Page 15: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

CHEMOPREVENTION IN DIFFERENT RISK CATEGORIES

ParameterRisk category

General Population High Risk

Agent toxicity Trivial to none Slight

Selection method Public Health Clinical

Other consideration Use dietary supplements Need biomarkersmay be applicable

From lee W. Wattenberg, P.S.E.B.M., 1997 216:133-141.

Page 16: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Phase I Trial

Objectives:• To determine the intervention’s short-term (<1

yr.) dose-toxicity relationship

• To determine the intervention’s human pharmacokinetics

Design:• Single arm, nonrandomized

• Multiple dose levels

• Less than 1 yr. duration

• Accrual 25-100

Page 17: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Phase II Trial

Objectives:• To determine the intervention’s 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

Page 18: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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

Page 19: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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

Page 20: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

CATEGORIES OF CHEMOPREVENTIVE AGENTS

• BLOCKING CARCINOGEN METABOLISM AND EXPOSURE

• INCREASE TISSUE RESISTANCE/DIFFERENTIAITON

• TARGETING ONCOGENIC PATHWAYS

Page 21: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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

Page 22: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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 functionCastration - reduce risk of prostate ca

• Decrease cell proliferationLow fat diet decrease epithelial proliferation rate in intestinal tract - reduce colon cancer risk

Page 23: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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)

Page 24: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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

• PCP=18.4% vs 24.8% in treated vs ctrl group

• Ongoing Trial: Selenium/Vit E trial

Page 25: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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

Page 26: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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.

Page 27: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

PROBLEMS OF CHEMOPREVENTION

• TOO LONG

• TOO LARGE COHORT

• TOO MUCH COST

ANSWER:

NEED TO DEVELOP RELIABLE SEMS

Page 28: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

BIOMARKERS OF CANCER

• CLINICAL SETTINGS (TUMOR MARKERS)

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

Page 29: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

CURRENT CLINICALLY USED TUMOR MARKERS

• PSA - Prostate Adenocarcinoma

• Alpha FP - Hepatoma & some Ovarian Ca

• HCG - Choriocarcinoma

• CEA - Ovarian CA

Page 30: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

BIOMARKERS

• Genetic susceptibility markers

• Markers of exposure

• Markers of biological effects

-Detect early lesions

-Prognostic indicators

Page 31: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

GENETIC SUSCEPTIBILITY MARKERS

• Glutathione S-transferase (GST) M1 and T1

• N-acetyl transferase (NAT)

• Cytochrome P-450

• DNA repair gene defect (Lynch syndrome)

Page 32: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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

Page 33: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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”

Page 34: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

HOW TUMOR MARKERS ARE USED CLINICALLY

• Early detection

• Predict the biological potential of cancer (metastasize and recurrence)

• Monitor the effectiveness of therapy

Page 35: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Cancer

Precancerous Intraepithelial Lesions, (PIN, CIN, PaIN..)

Birth

Genetic Suscep. Marker

Markers for Exposure

Markers ofEffect

Tumor Markers

Exposure to Carcinogen Additional Molecular Event

Surrogate End Point Markers

CHEMOPREVENTION

Page 36: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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

Page 37: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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

Page 38: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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

Page 39: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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

Page 40: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

CATEGORIES OF SEM

• HISTOLOGICAL AND MORPHOMETRIC MARKERS

• PROLIFERATION, DIFFERENTIATION AND INVASION MARKERS

• SPECIFIC ONCOGENES/GROWTH REGULATORS

• MARKERS OF GENETIC AND EPIGENETIC INSTABILITY

Page 41: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

POTENTIAL SEMS FOR BREAST, COLON AND PROSTATE

Breast ColonProstate

Histological DCIS, LCIS, ADHAdenomatous polyps

Aberrant polyps PIN

Proliferation S-phase fraction S-phase fraction PCNAKi-67 Brdu Uptake, PCNA Ki-67

Differentiation Myoepithelial (s-100 BGA, Mucin core ag HM CytokVimentin), 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

Page 42: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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.

Page 43: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Genome Wide Genotypic SEM Analysis

• Identify high risk population

• Identify individuals with genetic susceptibility for treatment (pharmacogenomics)

• Monitoring/analyzing individual’s treatment response

Page 44: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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.

Page 45: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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

Page 46: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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

Page 47: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

BIOMARKER STUDY DSEIGN

a. Untargeted Design:

Register Randomize

Treatment

Control

b. Untargeted Design:

Register Randomize

Treatment

Control

Test Biomarker

Biomarker +

Page 48: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

BIOMARKER STUDY DSEIGN

Biomarker by Treatment Interaction Design:

Register

Randomize

Treatment

Control

Test Biomarker

Biomarker +

Stratify

Biomarker -

Randomize

Treatment

Control

Page 49: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

BIOMARKER STUDY DSEIGN

Biomarker Based Strategy Design:

Register Randomize

Treatment A

No BiomarkerEvaluation

Test Biomarker

Biomarker +

Biomarker -

Treatment B

Treatment B

Page 50: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

BIOMARKER STUDY DSEIGN

Modified Biomarker Based Strategy Design:

Register Randomize

Treatment A

No BiomarkerEvaluation

Test Biomarker

Biomarker +

Biomarker -

Treatment B

Treatment B

Randomize

Treatment A

Page 51: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Actin Remodeling As a Target for Biomarker

Development

Page 52: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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

Page 53: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

WHAT TO DO WITH THIS?

Page 54: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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

Page 55: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

VASP

TenuinF-Actin

Zyxin

Actinin

p-Tyr?

Vinculin

- Ras Sup. Family (Rac/Rho/CDC42)- pp60sro

- pp125FAK-Abl

PM

Substrate

ECM Integrin b a b a

Talin R/E/M

Paxillin

Tensin

Model in Focal Adhesions

Page 56: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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

Page 57: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Increased cellular F-actin is a marker of cellular differentiation

Using leukemic cell lines:HL-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)

Page 58: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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)

Page 59: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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)

Page 60: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

QFIABiomarker Profile

G-actin: Texas-Red conjugated DNase I

M344: FITC (or Rhodamin) 3-Step

Immunofluorescence DNA: Hoechst or DAPI

Page 61: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA
Page 62: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Test Our Biomarker Profile

to Detect Bladder Cancer

in Workers Exposed to

Cancer Causing Chemicals

Test Our Biomarker Profile

to Detect Bladder Cancer

in Workers Exposed to

Cancer Causing Chemicals

Page 63: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Study Design in Worker Risk Assessment Study

373controls

1788 Workers

Screen Workers Markers Exposure Physical Exam Questionnaire Smoking Asses.

Monitor in 1 yr

Monitor in 3 yrs

Positive TREATVery High Risk

High Risk

Moderate Risk

Low Risk

Classify Risk

Action/Intervention

Negative

Cystoscopy

Page 64: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Procedures in Screening Program

Notification of exposed workers.Selection of matching controls.Administration of questionnaire.

Occupational history.Medical history.Genitourinary tract history.Smoking assessment.

Physical examination.UrinalysisPapanicolaou cytologyDNA, M344, G-actin biomarkers

Notification of exposed workers.Selection of matching controls.Administration of questionnaire.

Occupational history.Medical history.Genitourinary tract history.Smoking assessment.

Physical examination.UrinalysisPapanicolaou cytologyDNA, M344, G-actin biomarkers

Page 65: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Pathology Summary

30 Cancers detected29 Transitional Cell Carcinoma 1 Squamous Cell Carcinoma

4 Cases of Muscle Invasion (>T2)

20 Cases Grades 1-2; 8 Cases Grade 3.

Page 66: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Incidence Rate (per 100,000 person-year) of Bladder

Cancer in the Cohort Exposed to Benzidine ( 1991-1997)

CohortNo. of

Subjects Followed

Age ( Mean ± SD )Cancer Cases

Incidence

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

Page 67: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

TEST POSITIVE PRIOR TO OR AT THE TIME OF DIAGNOSIS

TEST POSITIVE PRIOR TO OR AT THE TIME OF DIAGNOSIS

BIOMARKERS NO. OF

POSITIVE/NO. OF CASES

RATE POSITIVE %

QFIA HIGH OR MODERATE

RISK28/29 96.5

PAP CYTOLOGY

15/28 53.6

HEMATURIA 4/28 14.3

Page 68: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Biomarker Results of Cohort StudyBiomarker Results of Cohort Study Detection Detection

Page 69: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Biomarker Results of Cohort StudyBiomarker Results of Cohort StudyRisk AssessmentRisk Assessment

Sensitivity Specificity OR PPV

DNA 88 87 46 2.7

M344 50 98 46 9.1

High Risk 63 91 17.3 2.9

HM or P 88 70 16.6 1.3

PAP 14 99 24.7 6.7

Page 70: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Cox Proportional Regression Model with Cox Proportional Regression Model with Time Dependent CovariatesTime Dependent Covariates

Page 71: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Lead Time to Tumor Detection During the Follow-up (1991-1997)

Total Cases=25*

N % CasesMean

Months

Moderate Risk 10** 40 33

High Risk 23 92 15

Pap 16 64 8

Hematuria 4 16 3

** 8 out of 10 cases progressed to high risk prior to tumor detection.

* 4 cases were excluded due to their detection at the initial screen. 1 case was excluded due to the error of sample collection.

** 8 out of 10 cases progressed to high risk prior to tumor detection.

* 4 cases were excluded due to their detection at the initial screen. 1 case was excluded due to the error of sample collection.

Page 72: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Abnormal G-actin in the Field Predicts Tumor Recurrence

Page 73: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA
Page 74: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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)

Page 75: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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

Page 76: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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

Page 77: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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

• ….

Page 78: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Fig. 1. Schematic of an AFM tip (a) approaching, (b) indenting and (c) retracting from a cell

(a)

(b) (c)Force

Displacement

(a)

(b)

(c)

(a)

(b) (c)Force

Displacement

(a)

(b)

(c)Force

Displacement

(a)

(b)

(c)

Page 79: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

A v e r a g e Y o u n g ’ s M o d u lu s ( E ) va lu e s fo r A 5 4 9 h u m a n lu n g a d e n o c a r c in o m a c e l ls t r e a te d w ith o r w ith o u t ( c tr l ) 4 0 u g / m L g r e e n te a e x tr a c t ( G T E ) fo r 6 a n d 1 2 h o u r s , r e s p e c t iv e ly .

E f f e c t s o f G T E o n t h e m i g r a t i o n o f A 5 4 9 c e l l s . C o n fl u e n tm o n o l a y e r s o f c e l l s w e r e m a i n t a in e d in a s e r u m f r e e m e d i a a n d a l a n e w a s s c r a p e d t h r o u g h t h e m o n o l a y e r s o f t h e c e l l s w i t h a p l a s t i c m ic r o p i p e t t e t i p . T h e c e l l s w e r e a l l o w e d t o m i g r a t e a c r o s s t h e l a n e a t 3 7 o C f o r 6 o r 2 4 h i n t h e p r e s e n c e ( 4 0 µ g /m l ) o r a b s e n c e o f G T E . T h e d i s t a n c e th a t c e l l s m i g r a t e d i n t o th e a r e a o f t h e w o u n d a t d i f f e r e n t p o i n t s w a s p h o t o g r a p h e d u s i n g a c o m p u t e r i m a g i n g s y s t e m . T o p p a n e l s : G T E u n t r e a t e d ; l o w e r p a n e l s : G T E t r e a t e d ( 4 0 μ g /m l ) .

0 h r 6 h r 2 4 h r

C t r l

G T E

Page 80: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

0 50 100 150 200 250 300 350 4000

1

2

3

4

5

6

7

8

Co

un

ts

Young's Modulus (E)

Adhesion Force Measured between Mesothelial Cells and Cancer Cells

050

100150200250300

E AF

AFM MeasurementsM

easu

rem

ent

Mesothelial cells Cancer cells

A B

C DPhase-contrast

Mesothelial cells

Tumor

Page 81: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

c

db

(i)(ii)

a

Page 82: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA
Page 83: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

“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

Page 84: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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

Page 85: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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 (<12 months) with

prior superficial bladder ca

StageTa T1CIS

Placebo TreatmentPE Erlotinib

Page 86: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Specimen Types

• Blood

• Urine cytological specimens

– Voided urine– Catheterized urine– Bladder wash

• Tissue

– Biopsy– Cystectomy specimen

Page 87: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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

Page 88: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

Biomarker Core

Urinary Cytology(VU/CU/BW, 100 cc each)

Tissue(ca, random)

Blood(20 cc)

10 cc 2-bFGFBrook’s labThin Prep (3)

1 slide 2 slide

2-QFIARao’s Lab

2- Cytology

Extract Genomic DNA

2- M.S.I.(Core facility)

3- Genetic Polymorphism(Zhang’s lab)

Fresh Frozen

Store(Rao’s lab)

ParaffinEmb.

1- Histology

3- Tissue ArrayEGFR, Ki67, Gelsolin, p53, etc(Seligson’s lab)

Leukocyte

3- Genetic Polym.(Zhang’s lab)

4- Polyphenol(Heber’s lab)

Plasma

Store

5 cc

1- Primary end point2- Secondary end point3-Tertiary end point4- Compliance marker

Page 89: Cancer Chemoprevention & Surrogate End Point Markers JianYu Rao, M.D. Associate Prof. Of Pathology UCLA

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