A.M. VanBuskirk Division of Surgical Oncology, OSU Department of Surgery D.F. Kusewitt

1. Dissecting the Immunobiology of Post-Transplant Skin Cancer : The unholy trio of Sun Damage, Immunosuppression and Inflammation A.M. VanBuskirk Division of Surgical Oncology, OSU Department of Surgery D.F. Kusewitt OSU Department of Veterinary Biosciences T.M. Oberyszyn OSU Department of Pathology Arthur G. James Comprehensive Cancer Center and R.J. Solove Research Institute

2. Outline • Background/scope of the problem • Data in humans (almost all epidemiological, NOT immunological) • Data in animal models • Where do we go from here?

3. The Former Problem in Transplantation "The surgeon looks to the left, pivots to the right, transplants the organ and ... whoa! Rejected!"

4. Immunosuppressive medication Both the Blessing and Bane of Transplantation Photo courtesy of Dr. Allan Kirk, NIH/NIDDK

5. Post-transplant Complications • Chronic Rejection • Infectious Diseases • Malignancies • Post-Transplant Lymphoproliferative Disorders (PTLD) • Skin Cancer (particularly Squamous Cell Carcinomas)

6. Cancer in Transplant Patients: factoids • Transplant patients are at increased risk for developing cancer (on average, a 2- to 4-fold risk of developing any cancer compared to the general population). • Non-melanoma skin cancer (NMSC) is the most common cancer after transplantation, with a 50-250-fold increase compared to the general population. • Risk factors for skin cancer in transplant recipients include older age at time of transplantation, fair skin, history of sun exposure and length of time since transplantation. • Transplant patients tend to develop multiple skin cancers that are aggressive and can be life-threatening. SCC is reported as the cause of death for 27% of Australian cardiac transplant recipients who’d survived greater than 4 years. Also recently reported to be cause of death in a significant number of Swedish transplant recipients. Data on SCC are NOT routinely collected in North America.

7. Immunosuppressive medication Both the Blessing and Bane of Transplantation Photo courtesy of Dr. Allan Kirk, NIH/NIDDK

8. Warty-like lesions Photo courtesy of Dr. Eggert Stockfleth, Charite, Berlin

9. Field Cancerization: Multiple Actinic Keratoses, Squamous Cell Carcinomas Photo courtesy of Dr. Eggert Stockfleth, Charite, Berlin

10. What other Immunosuppressed populations exhibit increased Skin Cancer? • HIV/AIDS patients • Cancer patients • Autoimmune disease patients

11. What is a commonality among transplant recipients and these other immunosuppressed populations? Exogenous/Therapeutic Immunosuppression A reduced number of circulating CD4+ cells The reduced number of CD4+ T cells is thought to impair immune surveillance. Approximately 23% of transplant patients have reduced numbers of CD4+ T cells (Hutchinson, 2003) Transplant patients with SCC have lower CD4+ T cell numbers than patients without SCC (Ducloux, 1998)

12. However, the immunobiology of skin cancer in the context of therapeutic immunosuppression or CD4 leukopenia has not been systematically investigated. Animal models are effective pre-clinical tools.

13. Experimental Models of Skin Cancer • Chemically induced (SCC, melanoma) • Ultraviolet radiation-induced (SCC) • Transplantable skin tumors • Human (SCC,melanoma) • Murine (SCC, melanoma) • Tumors arising in transplanted skin or skin cells • Human (SCC,melanoma) • Murine (SCC, melanoma) D.F. Kusewitt

14. Mouse versus Human Skin Human Mouse Epidermis Papillary dermis Reticular dermis Arrector pili Pilosebaceous unit Eccrine gland Apocrine gland Panniculus carnosus Subcutis/hypodermis D.F. Kusewitt

15. How Mouse Skin Differs from Human Skin • The skin is thinner • The skin lacks eccrine and apocrine glands • Melanocyte location is restricted • The mouse is fully haired • No known papillomaviruses infect mouse skin D.F. Kusewitt

16. Our friend, the SKH/hairless mouse Outbred Functioning immune system Develop SCC and SCC precursors upon repeated exposure to UVB Pros: Accepted model of SCC carcinogenesis, reflects outbred population, excellent for prevention studies Cons: Difficult to do immunological experiments [Inbred SKH strain has been offered to us, but must be re-derived (currently in MHV+ facility)] Also, currently breeding the hairless gene onto FVB/n (6th generation)

18. 0.6 0.5 0.4 Mean units of MPO (x 10-2) 0.3 * * 0.2 0.1 0 Wilgus et al, 2000. Celecoxib Ace UVB/ Celecoxib UVB/Ace The Importance of Inflammation to Carcinogenesis Using pre-clinical models, the link between early inflammation and the development of UV skin tumors is well established (Fischer, Pentland and Oberyszyn groups). Early inflammation under conditions of immunosuppression needs further investigation

19. 20 15 10 * 5 * * * * * * * * 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Reducing inflammation with Celecoxib results in fewer skin tumors UVB/Acetone UVB/Celecoxib Mean number of tumors per mouse Weeks UV and treatment Wilgus et al, 2003

20. What happens to UVB-induced inflammation and carcinogenesis when therapeutic immunosuppression is present?

21. Skin Parameters Skin thickness MPO Neutrophil Infiltration p53+ basal layer cells UVR 3x/week 1 week Or 1 exposure Outbred SKH/hairless Outbred SKH/hairless CD4 depletion Experimental immunosuppressant Experimental Scheme

22. p=.003 Background: CD4+ cells infiltrate the epidermis in response to UVB 3.5 3 2.5 2 average # CD4+ cells 1.5 1 0.5 0 CTRL UV UV Anti-CD4 UV Anti-CD8 **Have recently developed protocol to isolate 98% pure CD4+ CD3+ cells from the epidermis of UV-exposed mice.

23. CTRL UV UV Anti-CD4 CTRL UV UV Anti-CD4 CD4-depletion increases MPO and neutrophil infiltration 0.9 0.8 0.7 0.6 0.5 MPO MPO units ( x 10-2) 0.4 0.3 0.2 0.1 0 20 15 Neutrophil number 10 Ly6G+ cells 5 0

24. CTRL UV UV Anti-CD4 CD4-depletion increases the number of p53+ cells in the basal layer of the epidermis 6 5 4 avg # of p53+ cells/field 3 2 1 0 1 week of UVB exposures, harvest 24 hours after last UVB exposure

25. P<.006 P<.002 UV/IgG UV/anti-CD4 UV/anti-CD8 IgG CD4-depletion increases skin production of PGE2 35 30 25 20 PGE2 pg/mg protein 15 10 5 0 1 week Treatment

26. Nice, but what kind of CD4+ T cell is this and how is it modulating UVB-induced inflammation? • T-regulatory (CD3+ CD4+ CD25+), TH-3 • MHC Class 2 restricted • cell contact dependent or cytokines- IL10/TGF-b • TH-2 (CD3+CD4+) • MHC Class 2 restricted • cytokines- IL4/IL5/IL10/IL13 • CD4+ NKT (CD3+, NK+/-, TCR: Va14-Ja18+) • CD1 restricted • direct killing, cytokines -IFN-g/IL4/IL10/IL13

27. Identifying Different CD4+ cell types • Isolate epidermal infiltrating CD4+ cells in SKH mice and assess • surface phenotype, fox-p3 protein, intracellular cytokines • TCR usage by PCR • fox-p3 by PCR • Use NKT-deficient mice (Balb/c background)

28. WT Spleen CD1d-/- Spleen H2O M1 M3 Marker M4 K1 T1 Va14Ja18 268 bp B-actin 348 bp Initial data: NKT cells can be detected in hairless mice Are NKT cells present in UVB-exposed skin? Are NKT cells reduced/ depleted in anti-CD4 treated mice? Are NKT-associated cytokines reduced in CD4-depleted mice?

29. Balb/c Ja-18 -/- CD1d -/- If NKT cells modulate UVB inflammation, then NKT-deficient mice should have exacerbated inflammatory responses to UVB. 1.6 1.4 1.2 1 No UV 0.8 Skin Thickness (mm) UV 0.6 0.4 0.2 0 Mice were shaved and treated with hair remover 3 days prior to UVB exposure. Forty-eight hours after UVB exposure, animals were sacrificed and edema (skin thickness) measured. Star indicates p<0.001 compared to No UV control.

30. p<.015 p<.03 Balb/c Ja18 -/- CD1d -/- NKT-deficient mice have exacerbated UVB-induced inflammatory responses 25 20 15 Average Fold Increase in MPO 10 5 0 Dorsal skin punches were taken from wild-type and NKT deficient mice 48 hours after UVB exposure. Data are shown as the average fold increase in MPO over matched no UV controls.

31. Conclusions (1) • CD4-depletion increases neutrophil number and activity • CD4-depletion increases DNA damage, evidenced indirectly as an increase in p53+ epidermal cells • CD4-depletion results in increased PGE2 in the skin • Preliminary data indicate that CD4+ NKT cells are important regulators, as NKT deficient mice have exacerbated inflammatory responses to UVB.

32. Deplete CD4+ T-cells (inject Abs every 3 weeks) P<0.03 UVB Exposure UVB Exposure Determine tumor number Assess MPO activity 70 3x weekly 60 50 Tumor number per mouse 11 weeks 25 weeks 10 wks 40 30 SKH-1 hairless mouse UVB exposure 3 times weekly only 20 10 Continue UVB exposure 0 25 week Treatment UV/IgG UV/anti-CD4 Importance of Inflammation even after chronic UVB exposure Trend toward increased MPO in CD4-depleted mice at week 11 T.M. Oberyszyn

33. Mutational analysis: anti-CD4 vs IgG Preliminary analysis of p53, exon 8 (S. Tanner)

34. Conclusions (2) • CD4+ cells modulate inflammation after both acute and chronic UVB. • Celecoxib reduces inflammation after acute and chronic UVB. • CD4 depletion enhances tumor development after chronic UVB. • Tumors in anti-CD4 treated mice have more detectable p53 mutations.

35. All that’s nice, but what happens when clinically relevant immunosuppressants are used? In the few published studies, immunosuppressants decreased the time to tumor development and sometimes increased the number of tumors. Kelly et al. 1987. Transplantation 44(3): 429-434. Daynes et al. 1979. J. Natl. Cancer Institute 62:1075. Reeve et al. 1985. Aus. J. Exp. Biol. Med. Sci. 63: 655. However, the most commonly used immunosuppressants today were either not tested, or were tested in non-therapeutic doses. None of these studies looked at UVB-induced inflammation. None of these studies used combination therapies.

36. p=.05 P<.011 P<.007 PBS Effect of anti-CD4 and clinically relevant immunosuppressants on Con A driven proliferation 2.5 2.0 Mean Stimulation Index 1.5 1.0 0.5 0 Anti-CD4 TAC CsA CTRL IgG CsA: 20mg/kg/day, ip TAC: 2mg/kg/day, ip

37. 48 hours 140 120 100 80 MPO units (x 10-4) 60 40 No UV controls p=.01 20 0 UV IgG UV Anti-CD4 PBS CsA TAC PBS CsA TAC Effect of clinically relevant Immunosuppressants on MPO activity at 48 hours post-UVB 300 250 200 150 MPO units (x 10-4) 100 50 0 No UV UV

38. Conclusions • Systemic cyclosporine treatment reduces the splenic MLR response, but increases UVB-induced inflammation (MPO increased 4-8-fold). • Systemic tacrolimus treatment reduces the splenic MLR response, but does not increase or decrease UVB-induced inflammation (MPO activity similar to PBS controls).

39. CsA vs Tac:Why Different Responses? • The simple answer: We don’t know • Possibilities: • differential effects on neutrophil activity or trafficking. • Differential effects on T cell function. • Differential effects on monocyte/macrophage/dendritic cell functions.

40. PBS CSA MMF SIR TAC CSA + MMF TAC + MMF CSA + SIR Effect of single and dual therapies on UVB-induced MPO activity: 48 hours after a single UVB exposure **preliminary/new data** 5 4 Fold increase in MPO no UV vs UV 3 2 1 0 CSA: 20mg/kg TAC: 2mg/kg MMF: 20mg/kg SIR: 2 mg/kg Mice treated for 1 week, then exposed to UVB. MPO activity measured at 48 hours after UVB.

42. Determine patterns of cellular infiltration and whether these are altered by immunosuppression. Assess mechanisms by which immunosuppressants alter UVB-induced inflammation: effects on neutrophils, keratinocytes, endothelial cells. Assess the effects of clinically relevant immunosuppressants on skin carcinogenesis. Assess effectiveness of new topical treatments to reduce inflammation and carcinogenesis. Post-Transplant Research Group web site: funded by Research on Research Grant, TELR (1 of 10 University-wide) Future Plans Basic Research

43. What is the scope of the problem in the OSU transplant population? Assess distribution of cytokine gene polymorphisms in patients who develop skin cancer rapidly after transplantation compared to those who do not. Assess UVB-induced inflammatory responses in transplant patients. Assess new topical treatments to prevent skin cancer in transplant patients. Future Plans Clinical Research Main difficulty is a lack of dermatology infrastructure linked to the transplant program at OSU. So, currently we need outside collaborators: ITSCC and SCOPE members have offered to help with samples.

44. “When I say “I”, I mean we, when I say “we”, I mean they” -Dr. Frank Fitch Quote co-opted by Dr. Charles Orosz, and in turn, by me

45. Acknowledgements Kusewitt Laboratory Donna Kusewitt Allison Parent Erin Brannick Oberyszyn Laboratory Tatiana Oberyszyn Jennifer Hatton Kathy Tober Brian Wulff VanBuskirk Laboratory Anne VanBuskirk Sagal Ali Tyler Hoppes F Jason Duncan Kelly Johnson Nye Brutkiewicz Laboratory Randy Brutkiewicz “Emily” Yin-Ling Lin Stoner Laboratory Gary D. Stoner Tanner Laboratory Stephan Tanner OSU Comprehensive Cancer Center- RJ Solove Research Institute (MCC program and Immunology program) National Institutes of Health- NCI American Heart Association, Ohio Valley Affiliate American Cancer Society, Ohio Division

46. Got Milk? Questions?