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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
Lessons learned: challenges
• Jack of all trades, Master of none
– Journals
– Conferences
– Students and trainees
• Not th...
KL2/K23 Mentors:
– Ervin Epstein, Children’s Hospital Oakland
Research Institute
– Mary-Margaret Chren, Dept of
Dermatolog...
Clinical trials
in PTCH1 +/-
BCNS patients
Observational studies
at Kaiser/UCSF
Screen for drugs in
cell lines and
Ptch1+/...
In vitro Mice
Observational
studies
Clinical
Trials
Celecoxib (oral) ↓ ↓ mixed ↓ in subset
Statin No No No N/A
Vitamin D3 ...
Challenge Lesson Learned
Celecoxib (oral) Combining mouse and RCT
Statistics, finding the
experts
Statins
Difficulty in co...
Epidemiology of BCC
• 1 million BCC cases per yr in US
• Estimated annual incidence of 0.1% to 0.5%
• Rare risk of metasta...
BCC basic science:
• Almost all BCCs
have mutations in
PTCH1 tumor
suppressor gene
• All BCCs have
increased
Hedgehog sign...
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
G...
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 1997
Aszterbau...
Mechanism of disease
In vitro experiments
Animal models
Human clinical trials
Hedgehog pathway
BCC cell lines
Ptch1 +/- mi...
In vitro Mice
Observational
studies
Clinical
Trials
Celecoxib (oral) ↓ ↓ mixed ↓ in subset
Statin No No No N/A
Vitamin D3 ...
Genetic deletion of Cox1 or Cox2 decreases
microscopic BCCs in Ptch1+/- mice
IR-treated Ptch1+/- mice wild type (n=24),
de...
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
Control Celecoxib
240
Celecoxib
480
Celecoxib
1600
BCCburden(mm2)
Celecoxib dec...
• Study design: Phase II randomized, double-
blinded, placebo controlled trial
• Subjects: 60 patients with Basal Cell Nev...
Baseline characteristics of study participants
are similar in two groups (mean and SD)
Placebo
(N=27)
Celecoxib
(N=33)
Age...
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) ...
050010001500050010001500050010001500050010001500
180 200 220 240 180 200 220 240 180 200 220 240
180 200 220 240 180 200 2...
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-BC...
Lessons learned: statistics
• Statistics – linear mixed models for
determining slope of BCCs in RCT
• Regression models fo...
Lessons learned:
• Long time to publication
• Journals and co-authors disagree on
whether to present data from mice and
cl...
In vitro Mice
Observational
studies
Clinical
Trials
Celecoxib (oral) ↓ ↓ mixed ↓ in subset
Statin No No No N/A
Vitamin D3 ...
Cyclopamine
Smo
Gli
New and relatively safe agents that decrease
Hedgehog signaling
Corcoran and Scott, Proceedings of the...
Mechanism of disease
In vitro experiments
Animal models
Human clinical trials
Statins blocks Hedgehog
pathway
BCC cell lin...
7dehydrocholesterol →→ Vitamin D3
Skin
Liver
Vit D3
25(OH)D
Kidney
1,25(OH)2D
Vitamin D
Receptor (VDR)
24OHase
Excretion
D...
Lessons learned
• Have a good biomarker or target gene
(Gli1 mRNA)
• Have a good way to measure
bioavailability (24OHase)
...
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 ...
Unadjusted p value Adjusted* p value
Vitamin D3 -62% .008 -53% .042
1,25(OH)2D -36% .39 -33% .43
Statin** -58% .10 -40% .3...
Lessons learned
• Have a good biomarker or target gene
(Gli1 mRNA)
• Have a good way to measure
bioavailability (24OHase)
...
Mechanism of disease
In vitro experiments
Animal models
Human clinical trials
Vit D3 blocks Hedgehog
pathway
BCC cell line...
Lesson Learned
• Translating from mouse studies to
epidemiologic studies (skip the human
clinical trial/pilot)
• Mouse to ...
The association of serum vitamin D with skin
cancer risk in elderly men
Jean Y. Tang1,3
, Neeta Parimi2
, Angela Wu1,3
, J...
Table 3 Association of increasing serum 25(OH)D levels with non-
melanoma skin cancer
* Adjusted for age (continuous varia...
In vitro Mice
Observational
studies
Clinical
Trials
Celecoxib (oral) ↓ ↓ mixed ↓ in subset
Statin No No No N/A
Vitamin D3 ...
Cyclopamine
Smo
Gli
Williams and Wang , Proceedings of the Natl Acad Sci 2003
Topical Cur-414 cream
Oral 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
3282-S-treated
3282-Tb
3282-Td
3282-Untreated
3310-S
3310-T
3321-...
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 tum...
Investigator sponsored trial in BCC
prevention
• Genentech Hh antagonist GDC 0449:
150mg daily
• Phase II, placebo control...
Cyclopamine
Smo
Gli
New and relatively safe agents that decrease
Hedgehog signaling
J Kim and P Beachy, Stanford
Itraconaz...
Mechanism of disease
In vitro experiments
Animal models
Human clinical trials
Small molecule library
screen for inhibitors...
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...
Lessons learned:
• Easier to translate FDA approved drug or
a drug manufactured and tested by
Pharma - already have paid t...
Challenge Lesson Learned
Celecoxib (oral) Combining mouse and RCT
Statistics, finding the
experts
Statins
Difficulty in co...
Children’s Hospital Oakland
Ervin Epstein
Po Lin So
Tony Zheng Xiao
Elana Shpall
Angela Wu
Kris Chang
Yefim Khaimsky
UCSF ...
Jean Tang: Lessons Learned
Jean Tang: Lessons Learned
Jean Tang: Lessons Learned
Jean Tang: Lessons Learned
Jean Tang: Lessons Learned
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Jean Tang: Lessons Learned

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Jean Tang: Lessons Learned

  1. 1. Lessons Learned in T1 Research: mouse to human Jean Y. Tang MD PhD Assistant Professor Department of Dermatology
  2. 2. Mechanism of disease In vitro experiments Animal studies Human clinical trials Epidemiological studies New drugs
  3. 3. 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
  4. 4. 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
  5. 5. Clinical trials in PTCH1 +/- BCNS patients Observational studies at Kaiser/UCSF Screen for drugs in cell lines and Ptch1+/- mice Translational Research in BCC
  6. 6. In vitro Mice Observational 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
  7. 7. Challenge Lesson Learned Celecoxib (oral) Combining mouse and RCT Statistics, finding the experts Statins Difficulty in combining mouse and epidemiologic results (Kaiser) OK to disagree Vitamin D3 Difficulty 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
  8. 8. 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
  9. 9. BCC basic science: • Almost all BCCs have mutations in PTCH1 tumor suppressor gene • All BCCs have increased Hedgehog signaling
  10. 10. 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
  11. 11. Basal cell nevus syndrome Basal cell nevus syndrome are PTCH1+/-
  12. 12. Ptch1+/- mice mimic BCNS phenotype: develop BCC tumors after IR or UV treatment Goodrich and Scott, Science 1997 Aszterbaum, 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
  13. 13. Mechanism of disease In vitro experiments Animal models Human clinical trials Hedgehog pathway BCC cell lines Ptch1 +/- mice PTCH1+/- Basal cell nevus syndrome patients Roadmap for finding new therapeutics Epidemiological studies Patients with sporadic BCCs
  14. 14. In vitro Mice Observational 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
  15. 15. 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 BCCBurden(mm2)
  16. 16. 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 Control Celecoxib 240 Celecoxib 480 Celecoxib 1600 BCCburden(mm2) Celecoxib decreases microscropic BCC burden in Ptch1+/- mice (p<0.05)
  17. 17. • 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
  18. 18. 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%
  19. 19. BCNS subject with low number of BCCs at baseline (<15 tumors)
  20. 20. BCNS subject with high number of BCCs at baseline (>15 tumors)
  21. 21. 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
  22. 22. 050010001500050010001500050010001500050010001500 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 tumor days Graphs by mouse
  23. 23. Celecoxib reduces BCC development in subjects with less severe disease (< 15 BCCs)
  24. 24. 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
  25. 25. 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
  26. 26. 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
  27. 27. In vitro Mice Observational 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
  28. 28. Cyclopamine Smo Gli New and relatively safe agents that decrease Hedgehog signaling Corcoran and Scott, Proceedings of the Natl Acad Sci 2006 Bijlsma and Peppelenbosch, PLOS Biology 2007 Vitamin D3, Statins
  29. 29. Mechanism of disease In vitro experiments Animal models Human clinical trials Statins blocks Hedgehog pathway BCC cell lines Ptch1 +/- mice Epidemiological studies Statin therapy and risk of subsequent BCCs in Kaiser cohort
  30. 30. 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 7dehydrocholesterol →→ Vitamin D3 Skin Liver Vit D3 25(OH)D Kidney 1,25(OH)2D Vitamin D Receptor (VDR) 24OHase Excretion Diet 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
  31. 31. Lessons learned • Have a good biomarker or target gene (Gli1 mRNA) • Have a good way to measure bioavailability (24OHase) • Have a reliable cell line
  32. 32. Vitamin D3 decreases Gli mRNA in BCC cells
  33. 33. Vitamin D3 and 1,25(OH)2 D activate the Vitamin D receptor
  34. 34. (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)
  35. 35. 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
  36. 36. 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
  37. 37. Mechanism of disease In vitro experiments Animal models Human clinical trials Vit D3 blocks Hedgehog pathway BCC cell lines Ptch1 +/- mice PTCH1+/- Basal cell nevus syndrome patients Epidemiological studies Vit D3 levels in BCC pts
  38. 38. Lesson Learned • Translating from mouse studies to epidemiologic studies (skip the human clinical trial/pilot) • Mouse to Epi (Ralph Gonzalez)
  39. 39. 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
  40. 40. 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)
  41. 41. In vitro Mice Observational 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
  42. 42. Cyclopamine Smo Gli Williams and Wang , Proceedings of the Natl Acad Sci 2003 Topical Cur-414 cream Oral Hh antag
  43. 43. Placebo cream does not reduce BCC tumors Day 0 Day 20 Day 35
  44. 44. Untreated BCC
  45. 45. Topical Hh Antagonist BCCs decreases BCC tumors Day 0 Day 35Day 20
  46. 46. 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00 1400.00 3282-S-treated 3282-Tb 3282-Td 3282-Untreated 3310-S 3310-T 3321-S 3329-Ta 3329-Tc 3350S 3350Tu-a 3350Tu-b 3350Tu-d 3824-S 3824-Ta 3824-Tb RelativemRNAlevels (20->0;45d;by21d) (9->10;13d) (17->16;13d) (8->0;44d;by38d) (10->11;44d) (6->5;44d) (9->11;45d)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
  47. 47. Lesson: Benefits of UCSF Cancer Center core labs
  48. 48. Topical Cur-414 treated BCC
  49. 49. 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
  50. 50. 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
  51. 51. Cyclopamine Smo Gli New and relatively safe agents that decrease Hedgehog signaling J Kim and P Beachy, Stanford Itraconazole
  52. 52. Mechanism of disease In vitro experiments Animal models Human clinical trials Small molecule library screen for inhibitors of Hedgehog pathway Cell based assays Ptch1 +/- mice Patients with sporadic BCCs
  53. 53. Day 0, BCC A: 10mm Day 4 – irritation and necrosis, 11 mm Day 11 – residual BCC, 3mm Itraconazole #178
  54. 54. 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)
  55. 55. 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
  56. 56. 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
  57. 57. Challenge Lesson Learned Celecoxib (oral) Combining mouse and RCT Statistics, finding the experts Statins Difficulty in combining mouse and epidemiologic results (Kaiser) OK to disagree Vitamin D3 Difficulty 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
  58. 58. Children’s Hospital Oakland Ervin Epstein Po Lin So Tony Zheng Xiao Elana Shpall Angela Wu Kris Chang Yefim Khaimsky UCSF EpiBiostat Charles McCulloch Ralph Gonzalez Steve Hulley Steve Cummings Doug Bauer Neeta Parimi John Boscardin Michael Kohn Kaiser Division of Research Maryam Asgari Genentech Fred de Sauvage Tracy Tang Chris Callahan UCSF Derm Meg Chren Dan Bikle Loretta Chan Funding Sources NRCC – CTSA KL2 NIAMS – K23 Prevent Cancer Foundation American Skin Association

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