Dissecting the Immunobiology of Post-Transplant Skin Cancer :

The unholy trio of Sun Damage, Immunosuppression and

Inflammation

A.M. VanBuskirkDivision of Surgical Oncology, OSU Department of Surgery

D.F. KusewittOSU Department of Veterinary Biosciences

T.M. OberyszynOSU Department of Pathology

Arthur G. James Comprehensive Cancer Center and R.J. Solove Research Institute

Outline

Background/scope of the problem Data in humans (almost all

epidemiological, NOT immunological) Data in animal models Where do we go from here?

"The surgeon looks to the left, pivots to the right, transplants the organ and ... whoa! Rejected!"

The Former Problem in Transplantation

Photo courtesy of Dr. Allan Kirk, NIH/NIDDK

Immunosuppressive medicationBoth the Blessing and Bane of Transplantation

Post-transplant Complications

Chronic Rejection Infectious Diseases Malignancies

– Post-Transplant Lymphoproliferative Disorders (PTLD)

– Skin Cancer (particularly Squamous Cell Carcinomas)

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.

Photo courtesy of Dr. Allan Kirk, NIH/NIDDK

Immunosuppressive medicationBoth the Blessing and Bane of Transplantation

Warty-like lesions

Photo courtesy of Dr. Eggert Stockfleth, Charite, Berlin

Field Cancerization: Multiple Actinic Keratoses, Squamous Cell Carcinomas

Photo courtesy of Dr. Eggert Stockfleth, Charite, Berlin

What other Immunosuppressed populations exhibit increased Skin Cancer?

HIV/AIDS patients Cancer patients Autoimmune disease patients

The reduced number of CD4+ T cells is thought to impair immune surveillance.

What is a commonality among transplant recipients and these other

immunosuppressed populations?

Exogenous/Therapeutic ImmunosuppressionA reduced number of circulating CD4+ cells

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)

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.

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

Mouse versus Human Skin

Epidermis

Papillary dermis

Reticular dermis

Arrector pili

Pilosebaceous unit

Eccrine gland

Apocrine gland

Panniculus carnosus

Subcutis/hypodermis

Mouse Human

D.F. Kusewitt

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

Our friend, the SKH/hairless mouseOutbredFunctioning immune systemDevelop 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)

The Importance of Inflammation to CarcinogenesisUsing 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

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Mean u

nit

s of

MPO

(x 1

0-2)

* *Ace Celecoxib UVB/Ace UVB/

Celecoxib

Wilgus et al, 2000.

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5

10

15

20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

UVB/Acetone

UVB/Celecoxib

Mean

nu

mb

er

of

tum

ors

per

mou

se

* ** * * *

* **

Reducing inflammation with Celecoxibresults in fewer skin tumors

Weeks UV and treatment

Wilgus et al, 2003

What happens to UVB-induced inflammation and carcinogenesis

when therapeutic immunosuppression is present?

OutbredSKH/hairless

OutbredSKH/hairless

Skin Parameters Skin thicknessMPONeutrophil Infiltrationp53+ basal layer cells

Experimental Scheme

UVR 3x/week 1 week

Or 1 exposure

CD4 depletion

Experimental immunosuppressant

Background: CD4+ cells infiltrate the epidermis in response to UVB

**Have recently developed protocol to isolate 98% pure CD4+ CD3+ cells from the epidermis of UV-exposed mice.

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1

1.5

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2.5

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3.5avera

ge #

CD

4+

cells

CTRL UV UVAnti-CD4

UVAnti-CD8

p=.003

00.10.20.30.40.50.60.70.80.9

MPO

unit

s (

x 1

0-2)

MPO

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15

20

Ly6

G+

cells

CTRL

CTRL

UV

UV

UVAnti-CD4

UVAnti-CD4

Neutrophilnumber

CD4-depletion increases MPO and neutrophil infiltration

avg

# of

p53

+ c

ells

/fie

ld

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CTRL UV UVAnti-CD4

CD4-depletion increases the number of p53+ cells in the basal layer of the

epidermis

1 week of UVB exposures, harvest 24 hours after last UVB exposure

CD4-depletion increases skin production of PGE2

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25

30

35

UV/anti-CD8

1 week Treatment

PG

E2 p

g/

g p

rote

in

IgG UV/IgG

P<.002

UV/anti-CD4

P<.006

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-

TH-2 (CD3+CD4+)– MHC Class 2 restricted– cytokines- IL4/IL5/IL10/IL13

CD4+ NKT (CD3+, NK+/-, TCR: V14-J18+)– CD1 restricted– direct killing, cytokines -IFN-/IL4/IL10/IL13

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)

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?

Marker M1 M3 M4 K1 T1WT

Spleen CD1d-/-Spleen H2O

V14J18268 bp

-actin348 bp

Initial data: NKT cells can be detected in hairless mice

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.

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CD1d -/-

Ski

n T

hic

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s (m

m)

No UV

UV

J-18 -/-Balb/c

If NKT cells modulate UVB inflammation, then NKT-deficient mice should have exacerbated inflammatory responses to UVB.

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Balb/c

Ave

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old

Incr

ease

in M

PO

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.

CD1d -/-

p<.015

J18 -/-

p<.03

NKT-deficient mice have exacerbated UVB-induced inflammatory responses

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.

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25 week Treatment

Tum

or

num

ber

per

mouse

UV/IgG UV/anti-CD4

P<0.03

Importance of Inflammation even after chronic UVB exposure

T.M. Oberyszyn

SKH-1 hairless mouse

UVB ExposureDeplete CD4+ T-

cells(inject Abs every

3 weeks)

UVB Exposure

3x weekly

10 wks

UVB exposure 3 times weekly

only

Assess MPOactivity

25 weeks

Continue UVB

exposure

11 weeks

Determine tumor number

Trend toward increased MPO in CD4-depleted mice at week 11

Group Number of Tumors

Number with mutations

Type of Mutation

Codon

UVB/ IgG 21 3 C>TC>T C>T

R270C, P275SP275SR270C, P275S

UVB/ anti-CD4

24 6 C>T C>TC>TC>TC>TC>T

R270C, R270CP275SR270CR270CR270CR270C

Mutational analysis: anti-CD4 vs IgG

Preliminary analysis of p53, exon 8 (S. Tanner)

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.

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.

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2.5

CsAAnti-CD4 TAC

Mean S

tim

ula

tion Ind

ex

Effect of anti-CD4 and clinically relevant immunosuppressants on Con A driven proliferation

P<.007

PBSCTRLIgG

p=.05 P<.011

CsA: 20mg/kg/day, ipTAC: 2mg/kg/day, ip

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250

300

PBS PBSCsA CsATAC TAC

No UV UV

MPO

unit

s (x

10

-4)

020406080

100120140

MP

O u

nit

s (

x 1

0-4) 48 hours

No UVcontrols

UVIgG

UVAnti-CD4

Effect of clinically relevant Immunosuppressantson MPO activity at 48 hours post-UVB

p=.01

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).

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.

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SIRMMF CSA+

MMF

PBS CSA TAC 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**

CSA: 20mg/kgTAC: 2mg/kgMMF: 20mg/kgSIR: 2 mg/kg

Mice treated for 1 week, then exposed to UVB. MPO activity measured at 48 hours after UVB.

Future Plans

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)

Basic Research

Future Plans

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.

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.

“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

Acknowledgements

VanBuskirk LaboratoryAnne VanBuskirk

Sagal AliTyler Hoppes

F Jason DuncanKelly Johnson Nye

OSU Comprehensive Cancer Center- RJ Solove Research Institute(MCC program and Immunology program)

National Institutes of Health- NCIAmerican Heart Association, Ohio Valley Affiliate

American Cancer Society, Ohio Division

Kusewitt LaboratoryDonna KusewittAllison ParentErin Brannick

Stoner LaboratoryGary D. Stoner

Oberyszyn LaboratoryTatiana Oberyszyn

Jennifer HattonKathy ToberBrian Wulff

Brutkiewicz LaboratoryRandy Brutkiewicz

“Emily” Yin-Ling Lin

Tanner LaboratoryStephan Tanner

Got

Milk?

Questions?