Abstract. Background: Immunosuppression favors the
development of skin cancer. Experimental data suggest that
sirolimus (SRL) has antitumoral and antiangiogenic
properties. An investigation was undertaken into the effects of
SRL on squamous cell carcinoma (SCC) developing in organ
transplant recipients (OTR) receiving immunosuppressive
treatments, with special emphasis on vascularization.
Materials and Methods: SCC that developed in eight OTR
before and after conversion from calcineurin inhibitors (CNI)
to SRL were compared for thickness, differentiation,
ulceration, perineural invasion, density of peritumoral
infiltrate, peritumoral vascularization, density of T-regulatory
cells and of intratumoral Langerhans cells and growth
fraction. Results: SCC developing under SRL showed lower
peritumoral vascularization and thickness, and higher growth
fraction and density of peritumoral T-regulatory cells.
Conclusion: Conversion from CNI to SRL at clinically
relevant doses is associated in vivo with a reduced
vascularization and thickness of post-transplant human
cutaneous SCC. This effect could account for the beneficial
effect of SRL on immunosuppression-induced skin
carcinogenesis in humans.
Organ transplant recipients (OTR) are at increased risk for
developing cancer because of the chronic immunosuppression
necessary to prevent allograft rejection (1). Immuno-
suppressive drugs indirectly favor the development of tumors
through decreased immune surveillance and antitumor
defence, and occasionally also via direct oncogenic effects.
Cutaneous squamous cell carcinoma (SCC) accounts for the
majority of de novo malignancies developing in OTR. They
are usually multiple, may have an aggressive course and cause
significant morbidity and mortality. Their management relies
on the usual treatment modalities of skin carcinomas and on
revision of the immunosuppression (1, 2). In this setting,
sirolimus (SRL), a mammalian target of rapamycin (mTOR)
inhibitor, is a particularly interesting molecule since it
combines immunosuppressive and antitumoral properties.
Indeed, recent studies have shown that SRL-based
immunosuppressive treatments administered to OTR are
associated with a lower risk for developing post-transplant
cancer, including cutaneous ones (3). SRL seems to exert
antitumoral properties even in combination with cyclosporine
A (CSA), although the risk for skin cancer is lower when SRL
is given with steroids rather than with CSA (4, 5). Preliminary
data also suggest that conversion from calcineurin inhibitors
(CNI) to SRL decreases the incidence of skin cancer in renal
graft recipients (6). The mTOR signalling pathway, which
involves several tumor suppressor genes and proto-oncogenes
(including PTEN, PIK3, Akt and elFAE), is deregulated in
several malignancies, and its inhibition by SRL could account
for the antitumoral properties of this drug (7-9). At therapeutic
doses, SRL inhibits the growth of epidermoid cancer cell lines
with an activated Akt signalling pathway in vitro and in
xenograft models (10). Along with preventing cardiac allograft
rejection, SRL also inhibited the growth of CT-26
adenocarcinoma cells and B16 melanoma cells in a mouse
model (11). The in vivo antitumoral properties of SRL seem to
be due to an antiangiogenic effect, mediated via vascular
endothelial growth factor inhibition (12). On the other hand,
SRL has been shown to up-regulate T-regulatory cells in vitro
and to increase the density of epidermal Langerhans cells in
an animal model (3, 14).
In this work we studied several immunopathological
features, with particular emphasis on peritumoral
vascularization, of SCC developing in OTR prior and after
conversion from CNI to SRL.
Correspondence to: Jean Kanitakis, Department of Dermatology,
Ed. Herriot Hospital, 69437 Lyon Cedex 03, France. Tel: +33
472110301, Fax: +33 472110323, e-mail: jean.kanitakis@univ-
Key Words: Organ transplantation, sirolimus, rapamycin, squamous
cell carcinoma, vascularization.
ANTICANCER RESEARCH 29: 1927-1932 (2009)
Conversion from Calcineurin Inhibitors to Sirolimus
Reduces Vascularization and Thickness of Post-transplant
Cutaneous Squamous Cell Carcinomas
ANNE-LAURE RIVAL-TRINGALI1, SYLVIE EUVRARD1, EVELYNE DECULLIER2,
ALAIN CLAUDY1, MICHEL FAURE1and JEAN KANITAKIS1
1Hospices Civils de Lyon, Hôpital Edouard Herriot, Clinique Dermatologique (Pav. R), Lyon;
2Hospices Civils de Lyon, Pôle IMER, Université Lyon 1, Lyon, France
Materials and Methods
Patients and tumors. Patients considered for this study included
OTR that had developed cutaneous SCC before and after conversion
from CNI to SRL (because of multiple skin carcinomas). They
belonged to a cohort of over 3,000 OTR followed in our specialized
Outpatient Dermatology Clinic, and were examined by the same
physician (SE) every three months following the development of a
first skin carcinoma. Eight OTR of Caucasian origin were included
in the study. Their main demographic data are shown in Table I.
SRL introduction was associated with discontinuation of CNI
(except for one patient in whom CSA was maintained at low doses
of 50 mg/d). In another patient, azathioprine was discontinued.
Our study was performed on a total of 26 primary SCC excised
surgically under local anesthesia from these OTR. Fifteen tumors had
developed before (SCC1) and 11 after (SCC2) conversion from CNI
(mostly CSA) to SRL. Relevant data on these lesions are shown in
Table I. Recurring tumors and SCC that had developed less than 6
months after conversion to SRL were not studied in order to exclude
a confounding effect of the previous treatment with CNI.
Pathologic study. The specimens of SCC studied were formalin-
fixed and paraffin-embedded. Representative routinely-stained
sections of each tumor were re-examined by the same
dermatopathologist (JK) blindly as to the group (SCC1 or SCC2),
and the following pathological features were assessed: presence of
ulceration and perineural invasion, density of the peritumoral cell
infiltrate (scored semi-quantitatively as 1: weak, 2: moderate, or
3: dense), micrometric thickness measured (in mm) with an ocular
grid, and degree of differentiation (scored as 1: good, 2: moderate,
or 3: poor) (Figure 1).
Immunohistochemical study. This was performed on paraffin-
embedded tissue sections according to an avidin-biotin amplification
immunoperoxidase technique after antigen retrieval. Peritumoral
vascularization, Tregcells, Langerhans cells and tumor growth
fraction were immunohistochemically assessed with the following
monoclonal antibodies respectively: a) clone JC/70A to the
endothelial antigen CD31 (Dako, Copenhagen, Denmark); b) clone
236A/E7 to the nuclear transcription factor FoxP3; c) clone 808E10
to CD207/langerin (Dendritics, Dardilly, France); and d) clone
MIB-1 to the Ki-67 antigen (Dako) (Figures 2-3).
Evaluation of labeled structures was performed by an
automated image analyzer using the Histolab software
(Microvision, Haverhill, UK). The areas of the sections to be
counted were selected randomly in a blinded fashion as to the
group (SCC1 or SCC2) by the same observer (ALRT).
Peritumoral vascularization was expressed both as surface of
endothelial cells plus vessel lumen) per surface of dermis,
evaluated on 5 fields adjacent to the tumors. Results on CD207+
Langerhans cells and growth fraction (Ki-67+tumor cells) were
expressed both as number and surface of cells or nuclei,
respectively, per tumor surface. The number of infiltrating
FoxP3+Tregcells was counted visually (to exclude occasional
cells showing non-specific cytoplasmic staining that could have
been counted by the image analyzer) on 10 dermal fields
adjacent to the tumor, and the results expressed both per surface
of dermis and per total number of infiltrating cells counted by
the image analyzer.
endothelial cells and total vascular surface (i.e.
Statistical analysis. Statistical analysis was performed with SAS®
software (version 9.1) (SAS Institute Inc., Cary, NC, USA).
Quantitative data were compared with the non-parametric Mann-
Whitney test. Qualitative data were compared with Fisher’s exact
test. A p-value of 0.05 or less was considered significant.
The immunopathological features of the two SCC groups
studied are shown in Table II. Compared with SCC1,
SCC2 showed a statistically significantly lower thickness
vascularization, expressed both as endothelial (5.35×10–2
vs. 8.2×10–2, p=0.02) and total vascular surface
(6.30×10–2vs. 9.72×10–2, p=0.03) per dermal surface.
SCC2 contained significantly higher numbers of
peritumoral Tregcells, expressed both per total infiltrating
cells (1.85×10–2vs. 1.24×10–2, p=0.02) and per dermal
surface (1.41×10–4vs. 9.2×10–5/m2p=0.02). The growth
fraction (expressed both as number and surface of Ki-67+
nuclei per tumor surface) was higher in SCC2 vs. SCC1
(respectively 13.81×10–2vs. 4.75×10–2, p=0.01, and
18.35×10–4vs. 8.02×10–4, p=0.004). The remaining
features studied (density of intratumor Langerhans cells,
ulceration, degree of differentiation, perineural invasion,
density of the peritumoral infiltrate scored semi-
quantitatively) did not show statistically significant
differences between SCC1 and SCC2.
p=0.003) and peritumoral
ANTICANCER RESEARCH 29: 1927-1932 (2009)
Table I. Demographic characteristics of the patients and tumors studied.
6 Men/2 women
6 Kidney/2 heart
CNI + steroids: 2
CNI + steroids + MMF: 4
Steroids + Aza: 1
CNI + steroids + Aza: 1
n: 15 (5 head/neck,
6 upper limbs, 4 legs)
n: 11 (3 head/neck,
4 upper limbs, 4 legs)
Mean age at first
SCC occurrence (range)
Mean duration of immunosuppression
at SRL introduction (range)
SCC developed before
SRL introduction (SCC1)
SCC developed after SRL
CNI, Calcineurin inhibitors (6 cyclosporine A/1 tacrolimus); MMF:
mycophenolate mofetil, Aza: Azathioprine.
Several studies have shown that SRL, used at therapeutic doses
in organ transplantation, exerts an antitumoral effect both in
vitro and in vivo. In vivo studies include namely a murine
model of hepatic metastasis from CT-26 colon adenocarcinoma
cell lines (12) and a rat model of hepatocarcinoma (15). In
these models, SRL was shown to decrease tumoral
vascularization and size. In OTR, the use of SRL-based
immunosuppression is associated with a decreased incidence
of tumors, including cutaneous ones (4, 5, 16). Accordingly,
conversion of CSA to SRL, associated or not with withdrawal
of other immunosuppressants, usually induces regression of
Kaposi’s sarcoma, a tumor of endothelial origin (17-19).
The antitumoral effects of SRL seem to be due to
inhibition of tumor neovascularization, exerted through a
decrease of VEGF synthesis and secretion and inhibition of
stimulatory signals induced by the binding of VEGF on
endothelial cells (12). VEGF, fibroblast growth factor 2 and
angiopoietins secreted by inflammatory cells and tumor cells
under hypoxia induce growth of newly-formed vessels from
Rival-Tringali et al: Sirolimus and Vascularization of Squamous Cell Carcinomas
Figure 1. Squamous cell carcinoma of moderate differentiation (grade
2) surrounded by a weak (grade 1) peritumoral infiltrate (hematoxylin-
eosin stain, original magnification ×100).
Figure 2. Squamous cell carcinoma. Left panel: peritumoral vessels
revealed with immunolabeling for the CD31 endothelial antigen. Right
panel: tumor infiltrating FoxP3+T regulatory cells (immunoperoxidase,
original magnification ×200).
Figure 3. Squamous cell carcinoma. Left panel: Expression of Ki-67 by
tumor cells. Right panel: intratumor CD207/langerin+Langerhans cells
(immunoperoxidase, original magnification ×200).
Table II. Immunopathological features of the SCC studied.
SRL (n:15) SRL (n:11)
Degree of differentiation
Density of peritumor cell infiltrate
Endothelial surface/dermal surface
Total vascular surface/dermal surface
Treg cells/total infiltrating cells
Treg cells/dermal surface (μm2)
Langerhans cells /tumor surface (μm2) 1.32×10–4
Langerhans cells surface/tumor surface 2.00×10–3
Ki67+ surface/tumor surface
Ki67+ cells/tumor surface (μm2)
4 (26.7%)6 (54.5%) 0.23
0 (0%)2 (18%)0.17
pre-existing ones; this increase of peritumor vascularization
enhances further tumor growth. SRL inhibits the PI3K-Akt-
p70S6 signalling pathway, which is necessary for the
stimulation of endothelial cells by VEGF (20, 21) and is
activated in Kaposi’s sarcoma cells (22). The antiangiogenic
effects of SRL via reduced VEGF production have also been
shown in non-tumor cells, including a model of corneal
neoangiogenesis in rabbits (23) and in endometriosis (24).
Additional mechanisms that likely contribute to the
antiangiogenic effects of SRL include inhibition of
proliferation and differentiation of endothelial cell progenitors
(25) and increasing sensitivity of endothelial cells to
apoptotic signals (26). SRL also inhibits cytokine-driven
smooth muscle cell proliferation and migration (27-29), an
effect likely contributing to its antiangiogenic properties.
On the basis of the aforementioned data, we postulated that
SRL-based immunosuppression would also reduce peritumoral
angiogenesis in cutaneous SCC developing in OTR receiving
this drug in comparison with SCC developing under non SRL-
based immunosuppressive treatments. In an animal model, it
was recently shown that SRL reduces vascularity of tumors
developing in mice previously irradiated with UV (30). The
results of our study are in keeping with this finding and show
for the first time in humans that peritumoral vascularization is
reduced in cutaneous SCC developing under immuno-
suppressive doses of SRL. The anti-CD31 antibody, which
was used in our study and those of the literature, reveals both
vascular and lymphatic vessels, therefore the antiangiogenic
effect could be directed against both vascular and lymphatic
vessels. This is in keeping with recent results showing that
SRL exerts an antilymphangiogenic effect both in vitro and in
vivo via a decrease of synthesis of VEGF-C, the isoform
involved in (tumor) lymphangiogenesis and lymphatic
metastatic spread (31, 32).
Since CNI have angiogenic properties, it could be
speculated that their discontinuation may have contributed to
the reduction of vascularization observed in our study in
SCC2. However, in one of our patients CSA was withdrawn
precociously: SCC1 appeared after CSA discontinuation and
azathioprine was replaced by SRL. This finding is in favor
of the direct antiangiogenic effect of SRL. On the other
hand, the different localizations of tumors could introduce a
bias regarding peritumor vascularization, since the dermis in
some body areas (such as the face) contains a denser
vascular network. In this respect, the two groups of tumors
we studied were comparable since the proportion of tumors
located on the face or the limbs was very similar between
SCC1 and SCC2.
Regarding the histological features of aggressiveness,
SCC2 were significantly thinner as compared with SCC1.
This finding is not due to bias in patient follow-up, since all
OTR included in this study were followed on a regular three-
month interval basis after the occurrence of the first SCC,
i.e. before conversion to SRL, and were therefore followed
regularly before and after conversion to SRL. The
antiangiogenic effect of SRL could account for the reduced
tumor thickness after SRL introduction, since tumor growth
largely depends on blood supply. This result is in accordance
with an experimental study performed on mice, showing that
tumors developing under SRL had a smaller size than those
developing under CSA (30).
The remaining pathological features studied (degree of
differentiation, density of the peritumoral inflammatory cell
infiltrate, ulceration and perineural invasion) did not show
statistically significant differences between SCC1 and SCC2.
Concerning the density of peritumoral inflammation, our
findings are in keeping with those of the murine study,
reporting no significant effect of SRL on peritumoral
inflammation (30). Our finding of increased tumor growth
fraction in SCC2 vs. SCC1 is somewhat unexpected and
suggests that at therapeutic doses SRL does not inhibit the
proliferation of SCC keratinocytes. This is at variance with
results reported for other cell lines, but in those studies SRL
was used at doses unsuitable for therapeutic use (12). The
increased growth fraction in SCC2 vs. SCC1 may seem
contradictory to their lower thickness and vascularization, but
this discrepancy could be due to an increased apoptotic rate of
SCC2 vs. SCC1. This is also consistent with the fact that the
expression of Ki-67 does not seem to be correlated with the
clinical aggressiveness of SCC developing in OTR (33).
Although in our study we did not find obvious differences
in the overall density of the peritumoral cell infiltrate, we found
an increased density of infiltrating FoxP3+Tregcells in SCC2
vs. SCC1. FoxP3+
regulatory/suppressive function on immune reactions; they play
a key role in the maintenance of peripheral tolerance toward
self-antigens and in the control of inflammatory immune
responses to maintain homeostasis. In the setting of organ
transplantation, an increase of Tregcells could favor allograft
tolerance, (34, 35) although it was recently reported that
FoxP3 expression in kidney transplant biopsies is associated
with rejection and not favorable outcomes (36). Compared
with CNI, SRL has been shown to up-regulate circulating Treg
cells in renal graft patients (37, 38) and in mice (14), and to
expand human Tregcells in vitro (39). FoxP3 cells could
function in some cancer cell lines as a transcriptional repressor
of cancer via S-phase kinase-associated proteins 2 (SKP2) and
27 (40). Conversely, however, tissue-infiltrating Tregcells could
inhibit natural killer cells (41), thereby facilitating escape of
the tumor from immune surveillance. Therefore the
significance of increased numbers of Tregcells in SCC under
SRL remains to be further studied.
Langerhans cells are dendritic epidermal immunocompetent
cells that are able to elicit immune responses by presenting
antigens to naive T-cells, and to induce peripheral immune
tolerance. In the setting of organ transplantation, their role
are natural Treg cells exerting a
ANTICANCER RESEARCH 29: 1927-1932 (2009)
seems equivocal since these cells are able to induce both
allograft tolerance and rejection. The effect of SRL on dendritic
cells is debated. One study (42) reported a stimulatory effect
of SRL on dendritic cells, via an increase of CCR7 expression
and migration to lymph nodes. However, other studies claimed
that, both in vitro and in vivo, SRL inhibits the maturation (43),
the secretion of IL10 and IL12, endocytosis and the capacity
for T-cell stimulation of dendritic cells (44). Recently in a
mouse model of contact dermatitis, SRL was reported to
increase the density of epidermal Langerhans cells by
inhibiting their migration to lymph nodes (13). In our study,
we did not find statistically significant differences in the
numbers of intratumoral Langerhans cells between SCC1 and
SCC2. Further studies are needed in order to better assess the
effects that SRL exerts in vivo on these cells.
In conclusion, our findings show that conversion from
CNI to SRL at clinically relevant (immunosuppressive)
doses is associated in vivo with an antiangiogenic effect on
human SCC developing in OTR that could account, at least
partly, for reduced tumor thickness. These results confirm
relevant animal data from the literature and provide a
pathological basis for understanding the favorable effect of
SRL on skin carcinogenesis in OTR.
The skilful technical assistance of Evelyne Walch and Nicolas
Gadot (Anipath, Université de Lyon, Faculté Laënnec, 69372 Lyon
Cedex 08) is gratefully acknowledged.
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Received December 20, 2008
Revised March 3, 2009
Accepted April 2, 2009
ANTICANCER RESEARCH 29: 1927-1932 (2009)