dissecting the immunobiology of post-transplant skin cancer : the unholy trio of sun damage,...
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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
0
0.1
0.2
0.3
0.4
0.5
0.6
Mean u
nit
s of
MPO
(x 1
0-2)
* *Ace Celecoxib UVB/Ace UVB/
Celecoxib
Wilgus et al, 2000.
0
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.
0
0.5
1
1.5
2
2.5
3
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
0
5
10
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
0
1
2
3
4
5
6
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
0
5
10
15
20
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.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
CD1d -/-
Ski
n T
hic
knes
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.
0
5
10
15
20
25
Balb/c
Ave
rag
e F
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.
0
10
20
30
40
50
60
70
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.
0
0.5
1.0
1.5
2.0
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
0
50
100
150
200
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.
0
1
2
3
4
5
Fo
ld in
crea
se in
MP
On
o U
V v
s U
V
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
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