Dissecting the Immunobiology of PostTransplant Skin Cancer : The unholy trio of Sun Damage, Immunosu

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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

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Outline

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

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The Former Problem in Transplantation
"The surgeon looks to the left, pivots to the
right,

transplants the organ and ... whoa! Rejected!"
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Immunosuppressive medication
Both the Blessing and Bane of Transplantation
Photo courtesy of Dr. Allan Kirk, NIH/NIDDK
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Post-transplant Complications

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

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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 whod 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.

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Immunosuppressive medication
Both the Blessing and Bane of Transplantation
Photo courtesy of Dr. Allan Kirk, NIH/NIDDK
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Warty-like lesions
Photo courtesy of Dr. Eggert Stockfleth, Charite,
Berlin
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Field Cancerization Multiple Actinic Keratoses,
Squamous Cell Carcinomas
Photo courtesy of Dr. Eggert Stockfleth, Charite,
Berlin
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What other Immunosuppressed populations exhibit
increased Skin Cancer?

• HIV/AIDS patients
• Cancer patients
• Autoimmune disease patients

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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)
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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.
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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
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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
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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
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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)
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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
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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
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What happens to UVB-induced inflammation and
carcinogenesis when therapeutic immunosuppression
is present?
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Experimental Scheme
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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.
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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
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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
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CD4-depletion increases skin production of PGE2
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30
25
20
PGE2 pg/mg protein
15
10
5
0
1 week Treatment
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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 (CD3CD4)
• 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

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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)

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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?
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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
plt0.001 compared to No UV control.
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NKT-deficient mice have exacerbated UVB-induced
inflammatory responses
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20
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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.
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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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Importance of Inflammation even after chronic UVB
exposure
Trend toward increased MPO in CD4-depleted mice
at week 11
T.M. Oberyszyn
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Mutational analysis anti-CD4 vs IgG

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

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All thats 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
621075. 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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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
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Effect of clinically relevant Immunosuppressants o
n MPO activity at 48 hours post-UVB
300
250
200
150
MPO units (x 10-4)
100
50
0
No UV
UV
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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).

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CsA vs TacWhy Different Responses?

• The simple answer We dont know
• Possibilities
• differential effects on neutrophil activity or
  trafficking.
• Differential effects on T cell function.
• Differential effects on monocyte/macrophage/dendri
  tic cell functions.

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Effect of single and dual therapies on
UVB-induced MPO activity 48 hours after a single
UVB exposure preliminary/new data
5
4
3
Fold increase in MPO
no UV vs UV
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.
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Future Plans
Basic Research

• 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)

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Future Plans
Clinical Research

• 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.

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.
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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
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Acknowledgements
VanBuskirk Laboratory Anne VanBuskirk Sagal
Ali Tyler Hoppes F Jason Duncan Kelly Johnson Nye
Kusewitt Laboratory Donna Kusewitt Allison
Parent Erin Brannick
Oberyszyn Laboratory Tatiana Oberyszyn Jennifer
Hatton Kathy Tober Brian Wulff
Stoner Laboratory Gary D. Stoner
Brutkiewicz Laboratory Randy Brutkiewicz Emily
Yin-Ling Lin
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
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