jean tang: lessons learned

Lessons Learned in T1 Research: mouse to human

Jean Y. Tang MD PhD

Assistant Professor

Department of Dermatology

Mechanism of disease

In vitro experiments

Animal studies

Human clinical trials

Epidemiological studies

New drugs

http://www.nida.nih.gov/about/roadmap/index.html

Lessons learned: challenges

• Jack of all trades, Master of none– Journals– Conferences– Students and trainees

• Not that many role models – find a true believer and the experts

Slower time to publication

Grants: enthusiasm from NIH

KL2/K23 Mentors: – Ervin Epstein, Children’s Hospital Oakland

Research Institute– Mary-Margaret Chren, Dept of

Dermatology, UCSF– Charles McCulloch, Steve Cummings, Dept

of Epidemiology and Biostatistics, UCSF

http://www.nida.nih.gov/about/roadmap/index.html

Clinical trialsin PTCH1 +/-

BCNS patients

Observational studies at Kaiser/UCSF

Screen for drugs in cell lines andPtch1+/- mice

Translational Research in BCC

In vitro MiceObservational

studies Clinical

Trials

Celecoxib (oral) ↓ ↓ mixed ↓ in subset

Statin No No No N/A

Vitamin D3 ↓↓ ↓↓ ↓ Biomarker study

Hh Antagonist (Genentech)

↓↓↓ ↓↓↓↓ none Enrolling RCT

Itraconazole (antifungal)

↓↓ ↓↓ none Biomarker study

Summary of chemopreventive agents against BCCs

Challenge Lesson Learned

Celecoxib (oral) Combining mouse and RCT Statistics, finding the experts

StatinsDifficulty in combining mouse and epidemiologic results (Kaiser)

OK to disagree

Vitamin D3Difficulty of doing it all yourself: lab, mouse, pilot clinical trial

-1°Mentor and his lab -SFCC, EpiBio mentors

-Public databases

Hh Antagonist Collaborations with Genentech-Good to have lab skills

-RCT design

Itraconazole Collaborations with basic lab

FDA approved drug

-Nice to collaborate

-Time to start the pilot trial

Epidemiology of BCC

• 1 million BCC cases per yr in US• Estimated annual incidence of 0.1% to 0.5% • Rare risk of metastasis: < 0.5%

• 5th most costly cancer for Medicare

• The age-adjusted incidence per 100,000 white individuals: 475 cases in men, 250 cases in women

• The estimated lifetime risk of BCC in the white population is 33-39% in men and 23-28% in women.

• Risk of second BCC: 44% in 3 yr

BCC basic science:

• Almost all BCCs have mutations in PTCH1 tumor suppressor gene

• All BCCs have increased Hedgehog signaling

Ptch

Smo

Gli off

HH

Ptch

Smo

Gli on

Mutant Ptch

Smo

Gli on

CPN

Smo

Gli off

Ptch

Smo

Gli off

Ptch

Smo

Gli off

HH

Ptch

Smo

Gli on

HH

Ptch

Smo

Gli on

Mutant Ptch

Smo

Gli on

Mutant Ptch

Smo

Gli on

Smo

Gli

Hedgehog signaling pathway regulates cell proliferation and growth

Basal cell nevus syndrome

Basal cell nevus syndrome are PTCH1+/-

Ptch1+/- mice mimic BCNS phenotype: develop BCC tumors after IR or UV treatment

Goodrich and Scott, Science 1997Aszterbaum, Oro, Scott, Epstein Nature Medicine 1999

(A) Photo of multiple circled BCCs on the back of a patient with Basal Cell Nevus Syndrome. (B) Photo of Ptch1+/- K14-Cre-ER2 p53 fl/fl mice with multiple BCCs



Animal models


Hedgehog pathway

BCC cell lines

Ptch1 +/- mice

PTCH1+/- Basal cell nevus syndrome patients

Roadmap for finding new therapeutics

Epidemiological studiesPatients with sporadic

BCCs


studies Clinical

Trials


Statin No No No N/A







Genetic deletion of Cox1 or Cox2 decreases microscopic BCCs in Ptch1+/- mice

IR-treated Ptch1+/- mice wild type (n=24), deleted for Cox1 (n=12) or for Cox2 (n=6). Mean and SEM. p<0.05

Cox1 and Cox 2 KO: Smithies Cell 1995*

*

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Cox WT Cox1 KO Cox2 KO

BC

C B

urde

n (m

m2)

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

Control Celecoxib240

Celecoxib480

Celecoxib1600

BC

C b

urd

en

(m

m2

)Celecoxib decreases microscropic BCC burden in

Ptch1+/- mice (p<0.05)

• Study design: Phase II randomized, double-blinded, placebo controlled trial

• Subjects: 60 patients with Basal Cell Nevus Syndrome (BCNS)

• Treatment: oral Celecoxib at 200mg BID versus placebo for 24 months followed by 12 months of observation

• Primary endpoint: change in BCC numbers during study periods

Baseline characteristics of study participants are similar in two groups (mean and SD)

Placebo(N=27)

Celecoxib(N=33)

Age 42 ± 12 47 ± 12

Number of BCC tumors greater than 3mm 24 ± 27 45 ± 76

Body Mass Index 31 ± 1.3 30 ± 1.1

% Male 48% 58%

% Caucasian 96% 100%

% With >15 BCCs at baseline 41% 39%

% Seen at California site 59% 55%

BCNS subject with low number of BCCs at baseline (<15 tumors)

BCNS subject with high number of BCCs at baseline (>15 tumors)

How to analyze BCC development

• Regession technique: Linear mixed models

• Calcuates a slope or rate (number of BCCs/yr) for each patient

• Compare percent change in rate of BCCs in placebo and celecoxib groups

• Accounts for drop-outs

• Adjust for age, gender, BCC at baseline

050

010

0015

000

500

1000

1500

050

010

0015

000

500

1000

1500

180 200 220 240 180 200 220 240 180 200 220 240

180 200 220 240 180 200 220 240

3254 3282 3292 3309 3310

3313 3314 3321 3323 3329

3330 3347 3350 3352 3353

3354 3362

tum

or

daysGraphs by mouse

Celecoxib reduces BCC development in subjects with less severe disease (< 15 BCCs)

Lessons learned: importance of finding a reliable mouse model

• Ptch1+/- mice are a reliable model for testing new anti-BCC agents in humans– Moderate effect of celecoxib on BCCs in mice

and in BCNS patients– Greater effect on tumor size rather than

number in both mice and BCNS patients

Lessons learned: statistics• Statistics – linear mixed models for

determining slope of BCCs in RCT• Regression models for tumors in mice

– Go to class

• Building a database (and managing)– Go to class

Lessons learned:• Long time to publication• Journals and co-authors disagree on

whether to present data from mice and clinical trial study together

• Cancer Prevention Research


studies Clinical

Trials


Statin No No No N/A







Cyclopamine

Smo

Gli

New and relatively safe agents that decrease Hedgehog signaling

Corcoran and Scott, Proceedings of the Natl Acad Sci 2006Bijlsma and Peppelenbosch, PLOS Biology 2007

Vitamin D3, Statins



Animal models


Statins blocks Hedgehog pathway

BCC cell lines

Ptch1 +/- mice

Epidemiological studiesStatin therapy and risk of

subsequent BCCs in Kaiser cohort

7dehydrocholesterol →→ Vitamin D3

Skin

Liver

Vit D3

25(OH)D

Kidney

1,25(OH)2D

Vitamin D Receptor (VDR)

24OHase

Excretion

Diet

Figure 4B2.3. Synthesis and metabolism of vitamin D3


Skin

Liver

Vit D3

25(OH)D

Kidney

1,25(OH)2D


24OHase

Excretion

Diet


Skin

Liver

Vit D3

25(OH)D

Kidney

1,25(OH)2D


24OHase

Excretion

Diet

Figure 4B2.3. Synthesis and metabolism of vitamin D3

Lessons learned

• Have a good biomarker or target gene (Gli1 mRNA)

• Have a good way to measure bioavailability (24OHase)

• Have a reliable cell line

Vitamin D3 decreases Gli mRNA in BCC cells

Vitamin D3 and 1,25(OH)2 D activate the Vitamin D receptor

(A) Ptch1+/- K14-Cre-ER2 p53 fl/fl mice treated with vehicle control (Left) versus topical vitamin D3 (right) at 7 months of age.

(B) Example of large BCC tumor on the dorsal skin these transgenic mice(C) Histological confirmation of BCC.

Topical Vitamin D3 decrease BCC development by 50% (p<0.05)

Unadjusted p value Adjusted* p value

Vitamin D3 -62% .008 -53% .042

1,25(OH)2D -36% .39 -33% .43

Statin** -58% .10 -40% .30

Topical vitamin D3 decreases BCC development in mice

*Adjusted for gender and coat color of mouse

Lessons learned

• Have a good biomarker or target gene (Gli1 mRNA)

• Have a good way to measure bioavailability (24OHase)

• Pilot trial of topical and oral vitamin D on human BCC



Animal models


Vit D3 blocks Hedgehog pathway

BCC cell lines

Ptch1 +/- mice

PTCH1+/- Basal cell nevus syndrome patients

Epidemiological studies Vit D3 levels in BCC pts

Lesson Learned

• Translating from mouse studies to epidemiologic studies (skip the human clinical trial/pilot)

• Mouse to Epi (Ralph Gonzalez)

The association of serum vitamin D with skin cancer risk in elderly men

Jean Y. Tang1,3, Neeta Parimi2, Angela Wu1,3, John Boscardin1, Meg Chren1,Steven R. Cummings1,2,

Ervin Epstein3, and Douglas C. Bauer1,2 1 University of California San Francisco, 2 San Francisco Coordinating

Center, California Pacific Medical Center Research Institute, 3

Children’s Hospital Oakland Research Institute

Table 3 Association of increasing serum 25(OH)D levels with non- melanoma skin cancer

* Adjusted for age (continuous variable), BMI (continuous variable), season of blood draw, and clinic site † Adjusted for age, BMI, season of blood draw, clinic site, outdoor walking activity (continuous variable), and cigarette smoking (yes/no)


studies Clinical

Trials


Statin No No No N/A







Cyclopamine

Smo

Gli

Williams and Wang , Proceedings of the Natl Acad Sci 2003

Topical Cur-414 creamOral Hh antag

Placebo cream does not reduce BCC tumors

Day 0 Day 20 Day 35

Untreated BCC

Topical Hh Antagonist BCCs decreases BCC tumors

Day 0

Day 35Day 20

0.00

200.00

400.00

600.00

800.00

1000.00

1200.00

1400.00

32

82

-S-t

rea

ted

32

82

-Tb

32

82

-Td

32

82

-Un

tre

ate

d

33

10

-S

33

10

-T

33

21

-S

33

29

-Ta

33

29

-Tc

33

50

S

33

50

Tu

-a

33

50

Tu

-b

33

50

Tu

-d

38

24

-S

38

24

-Ta

38

24

-Tb

Re

lati

ve

mR

NA

lev

els

(20

->

0;

45

d;

by

21

d)

(9 -

> 1

0;

13

d)

(17

->

16

; 1

3d

)

(8 -

> 0

; 4

4d

; b

y 3

8d

)

(10

->

11

; 4

4d

)

(6 -

> 5

; 4

4d

)

(9 -

> 1

1;

45

d)

S Tu ReRe ReTu Tu S S Tu S Tu S Tu Tu

Hh Antagonist cream Placebo

Tu

Hh Antagonist cream decreases Gli1 mRNA in BCC tumors

Lesson: benefits of collaborator

Lesson: Benefits of UCSF Cancer Center core labs

Topical Cur-414 treated BCC

Lessons learned: mechanism of disease

1.Topical and oral HH antagonists significantly reduce murine BCCs by decreasing tumor proliferation and/or inducing follicular differentiation.

2.We know how to collect tumors, do these assays, get to mechanism of disease in future trials

Investigator sponsored trial in BCC prevention

• Genentech Hh antagonist GDC 0449: 150mg daily

• Phase II, placebo controlled RCT in 41 Basal Cell Nevus Syndrome subjects

• Primary endpoint: change in BCCs at 12 mo and 18 mo

Cyclopamine

Smo

Gli

New and relatively safe agents that decrease Hedgehog signaling

J Kim and P Beachy, Stanford

Itraconazole



Animal models


Small molecule library screen for inhibitors of

Hedgehog pathway

Cell based assays

Ptch1 +/- mice

Patients with sporadic BCCs

Day 0, BCC A: 10mm

Day 4 – irritation and necrosis, 11 mm

Day 11 – residual BCC, 3mm

Itraconazole#178

Cyclodextrin#5460

Day 0 – BCC B (11 mm), BCC D (10 mm), BCC G (9mm)

Day 7 – BCC B (11 mm), BCC D (10 mm), BCC G (9mm)

Lessons learned:

• Collaboration with another basic lab – Post-doc fellows who are experts at specific

assays (5 years) vs training someone new

• Focus on getting the first pilot clinical trial of itraconazole on human BCCs– Measure Gli1mRNA in BCCs– Measure Ki67– Paired t-test (at biopsy and at excision)

• New opportunities/markets

Lessons learned:

• Easier to translate FDA approved drug or a drug manufactured and tested by Pharma - already have paid the $$$

• New agent (vitamin D) – investigator pays– Efficacy– Stability– GMP grade for human – IND

Challenge Lesson Learned

Celecoxib (oral) Combining mouse and RCT Statistics, finding the experts

StatinsDifficulty in combining mouse and epidemiologic results (Kaiser)

OK to disagree

Vitamin D3Difficulty of doing it all yourself: lab, mouse, pilot clinical trial

-1°Mentor and his lab -SFCC, EpiBio mentors

-Public databases

Hh Antagonist Collaborations with Genentech-Good to have lab skills

-RCT design

Itraconazole Collaborations with basic lab

FDA approved drug

-Nice to collaborate

-Time to start the pilot trial

Children’s Hospital OaklandErvin EpsteinPo Lin SoTony Zheng XiaoElana ShpallAngela WuKris ChangYefim Khaimsky

UCSF EpiBiostatCharles McCulloch Ralph Gonzalez Steve HulleySteve CummingsDoug BauerNeeta ParimiJohn BoscardinMichael Kohn

Kaiser Division of ResearchMaryam Asgari

GenentechFred de SauvageTracy TangChris Callahan

UCSF DermMeg ChrenDan BikleLoretta Chan

Funding SourcesNRCC – CTSA KL2NIAMS – K23Prevent Cancer FoundationAmerican Skin Association

jean tang: lessons learned

Documents

bcc cases

hh ptch smo gli

treated bcc

epidemiology of bcc

mice ptch1

residual bcc

smo gli hedgehog

mutant ptch smo gli