The Immune SystemYA Hidden Treasure For Biomarker Discovery 2012 PDF

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From: Monica Neagu, The Immune System—A Hidden Treasure for Biomarker
Discovery in Cutaneous Melanoma. In Gregory S. Makowski, editor:
Advances in Clinical Chemistry, Vol. 58,
Burlington: Academic Press, 2012, pp. 89-140.
ISBN: 978-0-12-394383-5
© Copyright 2012 Elsevier Inc.
Academic Press
Author's personal copy
ADVANCES IN CLINICAL CHEMISTRY, VOL. 58
THE IMMUNE SYSTEM—A HIDDEN TREASURE FOR

BIOMARKER DISCOVERY IN CUTANEOUS MELANOMA
Monica Neagu1
Immunobiology Laboratory, ‘‘Victor Babes’’ National

Institute of Pathology, Bucharest, Romania
1. Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
2. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
3. Skin Immune System—Where the Story Begins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
3.1. Immune Surveillance at the Skin Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
4. Cutaneous Melanoma Develops in a Skin Immune System Controlled
Microenvironment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
5. Immune Markers—The Road Ahead . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
5.1. Infiltrating Immune Cells—Markers for Local Immune Response . . . . . . . . . . 108
5.2. Peripheral Immune Markers—Monitoring the Disease . . . . . . . . . . . . . . . . . . . . . 114
6. Monitoring Immune Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
7. Instead of Conclusions—Few Answered Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
7.1. Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Abbreviations
Ag antigen
AGT angiotensinogen
APCs antigen-presenting cells
ATPase adenosine triphosphatase
bFGF basic fibroblast growth factor
BMI-1 B-cell-specific moloney murine leukemia virus
integration site-1
CC (beta) beta-chemokine
CCL chemokine-beta receptor ligand
1
Corresponding author: Monica Neagu, e-mail: neagu.monica@gmail.com
89
0065-2423/12 $35.00 Copyright 2012, Elsevier Inc.

DOI: 10.1016/B978-0-12-394383-5.00011-4 All rights reserved.
90 MONICA NEAGU
CCR chemokine-beta receptor

CD44v3 CD44 splice variant 3
Cdc42 cell division control protein 42
CLA cutaneous lymphocyte antigen
CRP C-reactive protein
CSC cancer stem cells
CT cancer/testis
CTACK cutaneous T-cell-attracting chemokine
CTL cytotoxic T lymphocytes
CXC (alpha) alpha-chemokine
CXCR chemokine-alpha receptor
DAF decay-accelerating factor
DBP vitamin D-binding protein
DCs dendritic cells
DJ1 Parkinson disease (autosomal recessive, early onset) 7
DTH delayed-type hypersensitivity
FGF fibroblast growth factors
FOXP3 forkhead box protein P3
G-CSF granulocyte colony-stimulating factor
GM-CSF granulocyte-macrophage colony-stimulating factor
gp100 glycoprotein 100
GRO growth-regulated oncogene
HE4 human epididymis protein 4
HECA high endothelial cell antigen
HSP90AA2 heat-shock protein 90 kDa alpha
ICAM-1 intercellular adhesion molecule-1
IDEC inflammatory dendritic epidermal cells
IL interleukin
iNOS inducible NO synthase
IP-10 interferon-inducible protein-10
ISG interferon-stimulated genes
ITAC alpha interferon-induced T-activating chemokine alpha
KIR2DL1 killer cell immunoglobulin-like receptor 2DL1
LCs Langerhans cells
LDH lactate dehydrogenase
LFA-1 leukocyte function-associated antigen-1
LYVE-1 lymphatic vessel endothelial receptor-1
MAGE melanoma antigen-encoding
MART-1/ melanocyte antigen-1
Melan-A
M-CSF monocyte-macrophage colony-stimulating factor
BIOMARKERS DISCOVERY IN THE IMMUNE SYSTEM 91
MDC macrophage-derived chemokine

MHC major histocompatibility complex
MIC macrophage inhibitory cytokine-1
MIF immune-related macrophage migration inhibitory
factor
MIG monokine induced by IFN-gamma
MMP matrix metalloproteinase
MUC-18/ melanoma-associated cell adhesion molecule
MCAM1
MxA myxovirus resistance protein A
NKR NK-associated receptor
NKT natural killer T cells
NO nitric oxide
PAS periodic acid Schiff’s
PBMC peripheral blood mononuclear cell
pDC plasmacytoid DC
PLS3 plastin 3
PPIB peptidylprolyl isomerase B
PRDX peroxiredoxin family
ROS reactive oxygen species
SALT skin-associated lymphoid tissue
SC stratum corneum
SIS skin immune system
SLN sentinel lymph node
STAT-1 signal transducer and activator of transcription-1
TAMs tumor-associated macrophages
TARC thymus and activation-regulated chemokine
TCR T-cell receptor
TGF transforming growth factor
Th T-helper
TIA-1 T-cell intercellular antigen-1
TIL tumor-infiltrating lymphocytes
TJ tight junctions
TLR toll-like receptor
TNF tumor necrosis factor
Treg regulatory T cell
TTR transthyretin
TUBB1 tubulin beta-1 chain
VCAM-1 vascular cell adhesion molecule-1
VEGFR vascular endothelial growth factor receptor
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1. Abstract
This chapter describes how skin immune system (SIS) is specifically

involved in the development of cutaneous melanoma. Local immune surveil-
lance is presented as a complex process that comprises markers to be moni-
tored in disease’s evolution and in therapy. The ranking of tissue or soluble
immune markers in a future panel of diagnostic/prognostic panel are eval-
uated. Taking into account the difficulties of cutaneous melanoma patients’
management, this chapter shows the immune surveillance at the skin level,
the conditions that favor the tumor escape from the immunological arm, the
immune pattern of skin melanoma with diagnostic/prognostic relevance, the
circulatory immune markers, and, last but not least, how immune markers
are used in immune-therapy monitoring. The chapter cannot be exhaustive
but will give the reader a glimpse of the complex immune network that lies
within tumor escape and where to search for immune-therapeutical targets in
skin melanoma.
2. Introduction
Immune-related markers have proven their diagnostic and prognostic value

and were detected at both local tumor site and circulatory level. Thus, the
ranking of tissue or soluble immune markers in a future panel of diagnostic/
prognostic panel are evaluated.
Taking into account the difficulties of cutaneous melanoma patients
management, this chapter shows the immune surveillance at the skin level,
the conditions that favor the tumor escape from the immunological arm, the
immune pattern of skin melanoma with diagnostic/prognostic relevance, the
circulatory immune markers, and, last but not least, how immune markers
are used in immune-therapy monitoring. The chapter cannot be exhaustive
but will give the reader a glimpse of the complex immune network that lies
within tumor escape and where to search for immune-therapeutical targets
in skin melanoma.
Among the hundreds of papers published in the past 5 years regarding
immune markers in cutaneous melanoma, there are actually only tens that
focus on circulatory immune markers that prove a diagnostic/prognostic/
therapy monitoring power.
Taking into account our day-to-day experience in cutaneous melanoma, we
felt that the patient’s quantifiable immune parameters, namely the immune
status, are important tools that could be exploited in the continuous effort to
improve the patient’s clinical management. We, always, try to match our
experimental results with cell lines and clinical cases in the larger framework of
‘‘reviewing the literature.’’ This is a commonly shared ‘‘symptom’’ for rapid
‘‘validation’’ of one’s own results with other findings, matching that could
confirm/infirm the ‘‘first-hand’’ acquired knowledge. This ‘‘symptom’’ shows
a more complicated ‘‘disease,’’ namely, the fact that although research has
flourished in disease biomarkers, discovery and major investments have been
made, no new major cancer biomarkers have been approved for clinical use in
the past two decades. In the United States, the generously founded Early
Detection Research Network did not provide a single FDA-approved marker.
Human epididymis protein 4 for ovarian cancer was approved by FDA in 2009
and, as it was only approved for monitoring recurrence, was considered a
‘‘minor’’ biomarker [1]. Work is currently ongoing, as extended research net-
works and renowned institutes collaborate on melanocytic tumor for establish-
ing a tissue microarray for evaluation and validation of candidate biomarkers
[2]. Through this sustained work, the obtained tumor progression tissue micro-
array proved high biomarker expression heterogeneity in cutaneous melanoma.
Up to our knowledge, immune-related markers were not incriminated for
distinguishing benign skin disease from cutaneous melanoma. They were
used for their prognostic value, whether at tumoral site, or characterizing
the overall immune status of the treated/untreated patient, but we feel that
this domain is still insufficiently used.
3. Skin Immune System—Where the Story Begins
Exposed, during evolution, to complex agents like physical (sun rays) and
biological assault (microbes and or allergens), the skin has developed a specific
immune system [3]. The skin represents actually a peripheral immune organ
being a complex network of cell and molecules that interrelate. The local
immune network stands at the basic concept of immunodermatology [4].
In the perfect case scenario, the innate and adaptive cellular effectors cooper-
ate toward a concentrated, robust, and effective antitumoral response [5].
The immune response triggered against tumor antigens (Ags) and tumor
immune surveillance is not a new concept; this last term was suggested almost
40 years ago by Burnet [6,7]. It was postulated that the immune system
removes aberrant, somatically mutated cells, preventing these cells to develop
into tumors. The initial postulation of the concept was further sustained by
clear clinical evidences. Thus, spontaneous regression of tumors, including
melanoma, was observed, while suppressed immune system patients (AIDS
patients, medically immunosuppressed renal transplant recipients, and the
aged) have an increased incidence of tumors [8,9].
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3.1. IMMUNE SURVEILLANCE AT THE SKIN LEVEL

The largest organ with immune function, the skin, with its average of 2 m2
surface [10] has a dual role, provides the principal physical barrier to the
environment [11], and holds within a complex network of humoral and
cellular immune effectors, named SIS [3,4,12]. The cellular component
includes keratinocytes, dendritic antigen-presenting cells (APCs), mono-
cytes/macrophages, granulocytes, mast cells, lymphatic/vascular endothelial
cells, and T lymphocytes [6]. Subsequently, molecules secreted by the cellular
component of SIS, cytokines, neuropeptides, eicosanoids, prostaglandins,
free radicals, antimicrobial peptides, complement components, immunoglo-
bulins, and fibrinolysins, comprise the large array of humoral components [4]
(Table 1).
The cellular immune component of SIS is skin-associated lymphoid tissue
(SALT) that consists of circulating cells with an established route skin–
lymph nodes. These cells are mainly Langerhans cells (LCs) and dermal
APCs. The nonimmune cells, keratinocytes, and endothelial cells produce a
wide range of immune and growth regulatory cytokines. The highly specific
arm of SALT is represented by lymphocytes that extravasate from circulation
into the skin [17,28,29]. All these elements interplay to accomplish the skin
TABLE 1
IMMUNE CELLS ASSOCIATED TO SKIN AND MOLECULES THAT SUSTAIN THE CELLULAR CROSS TALK
Cells associated to the

Secreted immune-related molecules immune response References
Pro- and anti-inflammatory cytokines, chemokines Keratinocytes [13–16]

(IL-1, IL-8, CCL27, CCL5, CCL17, CXCL10, MIG,
IP9, CCL20), colony-stimulating factors (M-CSF/
GM-CSF), complement components (C3, factor
B and H, C9), neuropeptides, thymopoietin
Cytokines: IFN, IL-17, IL-4 T lymphocytes [17,18]
Perforins NK cell [19]
Complement and complement regulatory proteins, Granulocytes [4]
cathelicidins, defensins, chemokines, ROS
Leukotrienes, IL-1beta, IL-4, IL-5, IL-6, IL-13, TNF, Mast cells [20,21]
GM-CSF, prostaglandins, eicosanoids
TGF-beta1, 2, and 3 Tissue macrophage [22]
IL-1beta Langerhans cell [17,23–25]
IL-12 Mature tissue dendritic [26,27]
cell
IL-8 Vascular/lymphatic [6]
endothelial cell
immune surveillance [5,30] with a crucial role in maintaining homeostasis.

Innate and adaptive immune systems corroborate to maintain an effective
cutaneous immune surveillance [5,10]. The high importance of SALT was
proven by the increased cutaneous malignancies and infections when skin
immune functions are limited [5,8].
3.1.1. Cellular Components of SIS

Besides the already known cellular components of the adaptive immune
system (B and T cells), APCs represented by mononuclear phagocytes and
dendritic cells (DCs) have an important contribution. The epidermis con-
tributes to the local immune response through LC, keratinocytes, and endo-
thelial cells.
It is clear that two cellular components conjoin their functions: innate and
adaptive immune response [5]. The innate arm is more rapid, but nonspecific,
while the adaptive one refines the immune response and, although slower, has
a greater specificity and elicits long-term memory.
3.1.1.1. Adaptive Immune Surveillance
At the skin level, this process was somewhat didactically ranked as prima-
ry, secondary, and tertiary immune surveillance. Primary immune surveil-
lance takes place in skin-draining lymph nodes where professional APCs
present Ags to naive T; secondary immune surveillance produces Ag-specific
effector memory T cells expressing homing receptors that direct their migra-
tion to the tissue where the Ag was encountered. Tertiary immune surveil-
lance is the long-term acquired immune response, including the production
of central memory and effector cells potentially directed to tissues other than
the site of primary exposure [5].
3.1.1.1.1. Primary immune surveillance. Activated DCs (derived from
epidermal LC or dermal DCs) [31,32] are professional APCs that present
efficiently Ags and induce the maturation of naive T cells to cells having a
memory/effector phenotype [33]. Dermal DCs are the central mediators be-
tween innate and adaptive immune responses [34]. Activated APCs process the
whole Ags particles and undergo maturation as they emigrate through the
afferent lymphatics to the local skin-draining lymph nodes [33]; their matura-
tion enhances Ag processing and upregulates the expression of MHC mole-
cules and costimulatory molecules [35]. The function of the local draining
lymph nodes, major immune stations in skin melanoma, is to promote fre-
quent and supervised contact with Ags. These Ags are derived from skin
and are carried by DCs (migrated through afferent lymphatics) to the
T cells entering the lymph node through high endothelial venules [5].
Naive T cells that had encountered Ags and were activated along with mature
DC will undergo proliferation and clonal expansion. T cells will produce
96 MONICA NEAGU
autocrine growth factors and will differentiate into memory/effector T cells in

the secondary immune surveillance stage.
3.1.1.1.2. Secondary immune surveillance. The activation of T cells in the
local draining lymph nodes results in the production of Ag-specific effector
cells that express homing receptors for that site [5]. T cells recruited at the sites
of inflammation will be subjected to the action of a range of inflammatory
mediators triggered by innate immune mechanisms. Activated dermal DCs
and inflammatory dendritic epidermal cells (IDECs) can present Ag and
provide costimulatory signals to T cells that express appropriate counter-
receptors [36]. When recruiting T cells into the skin, the earliest step in this
process is the tethering and rolling of T cells on E-selectin and/or P-selectin
expressed by dermal postcapillary venules. Skin-homing T cells can be identi-
fied by expression of the normal cell surface carbohydrate epitope cutaneous
lymphocyte antigen (CLA), which binds E-selectin. CLA is expressed by
30% of circulating memory T cells and is virtually absent on naive T cells
[5]. Recent results have shown that in the majority of metastatic melanoma
samples, there is no expression of the vascular adhesion receptors E-selectin
(CD62E), P-selectin (CD62P), and ICAM-1 (CD54) on vessels within the
tumor boundaries. Strong adhesion receptor expression was noted on vessels
within adjacent tissue. Tumor-associated T lymphocytes accumulate prefer-
entially in these adjacent areas and are not enriched for skin- or lymph node-
homing receptor phenotype. On the vasculature of metastatic melanoma, the
expression of leukocyte homing receptors is deregulated, blocking therefore
the recruitment of activated tumor-specific cytotoxic T lymphocytes (CTL) to
melanoma metastases [37]. There are still matters to be clarified regarding
homing function of T cells [19]. The majority of alpha–beta T cells in normal
skin express CCR8 and produce proinflammatory cytokines. There are reports
showing that human dermal cell suspensions contain populations of Vdelta1þ
gammadelta T cells and CD56þCD16 NK cells but lack the subsets of
Vdelta2þ gammadelta T cells and CD56þCD16þ NK cells, which predominate
in peripheral blood. The skin-homing receptors CCR8 and CLA were
expressed by a large fraction of both cell types, whereas chemokine receptors
associated with lymphocyte migration to inflamed skin were absent. Stimula-
tion of cutaneous Vdelta1þ gammadelta T cell lines induced secretion of large
amounts of tumor necrosis factor-alpha (TNF-alpha), interferon-gamma
(IFN-gamma), and the CCR8 ligand CCL1. In contrast to cutaneous alphabeta
T cells, gammadelta T cells, and NK cells had the capacity to produce intracel-
lular perforin and displayed strong cytotoxic activity against melanoma cells. It
was proposed that gammadelta T cells and NK cells are regular constituents of
normal human skin having the potency to clear tumor cells [19]. CLA was
found on most T cells present in cutaneous lymphocytic infiltrates of almost
all skin diseases [38–41]. The skin-draining lymph nodes microenvironment
promotes the expression of CLA by newly activated effector T cells [42].

Studying in vitro CLA induction, it was demonstrated that its expression is
enhanced by CD3 activation in the presence of IL-12 and is not restricted to
T-cell subsets [26,27]. Several chemokines and their receptors are associated
with skin-homing T cells [43–45] including CC-chemokine receptor 4 (CCR4)
and its ligands CCL17 (thymus and activation-regulated chemokine, TARC)
and CCL22 (macrophage-derived chemokine, MDC). CCR4–CCL17 interac-
tions can lead to the arrest of rolling T cells if they are provided an integrin
ligand. A chemokine, preferentially produced by epidermal keratinocytes
CCL27 (cutaneous T-cell-attracting chemokine, CTACK), has also been impli-
cated in skin homing. It was demonstrated in vitro that when bound to CCR10,
CTACK becomes chemotactic for T cells [13–15]. It seems that skin-homing
memory cells that express CLA, CCR4, and leukocyte function-associated
antigen-1 (LFA-1) accumulate in the skin, whereas E-selectin, CCL17, and
ICAM-1 are constitutively and inducibly expressed on postcapillary venules
[5]. In noninflamed skin, T cells express high levels of CLA and CCR4 as well
as other chemokine receptors [46,47]. These cells are observed to tether and
roll constitutively on low levels of selectin expressed in dermal postcapillary
venules [48,49]. It was hypothesized that T cells continuously scan the endo-
thelial cell surfaces of their target tissue and detect the activation signals
ready to elicit an immediate immune response [5]. Constitutive expression
of E-selectin, CCL17, and ICAM-1 on cutaneous microvessels has been
described [50–52], and due to these constitutive interactions, a fraction
of T cells continuously enter the skin and traffic through it, seeking
Ag-dependent activation [5].
3.1.1.1.3. Tertiary immune surveillance
After an initial Ag encounter, Ag-specific memory cells, expressing CD62
ligand (CD62L) and CCR7, circulate through lymph nodes [53]. These cells
can then emigrate from the lymph node in which they were originally pro-
duced to other lymph nodes throughout the body (including those draining
noncutaneous epithelial-cell interfaces). In other sites, these cells may en-
counter DCs expressing the same Ag. In this way, the immune system is ready
to elicit a rapid and effective response even if the next Ag encounter occurs at
a different interface.
Recently, it has been demonstrated in transgenic animal models that the
bone marrow and tumors contain CD8þ T cells specific for the melanoma
Ag tyrosinase-related protein 2, cells with an effector memory phenotype.
Moreover, these data indicate that functionally active bone marrow-derived
melanoma-specific memory T cells are detectable at the phase of microscopic
tumor load, suggesting that they could control disseminated melanoma cells
[54]. Memory T cells can enter tissues because the local endothelium
expresses appropriate counter-receptors and chemoattractants. Only after
98 MONICA NEAGU
they have exited the blood, they can respond to their specific Ag that is
productively presented [5].
3.1.1.1.3.1. ROLE OF T LYMPHOCYTES IN SKIN IMMUNE SURVEILLANCE.
Healthy human skin does not contain B lymphocytes, and human gamma/
delta TCR subtypes do not appear to preferentially migrate to the skin.
Human skin contains alpha/beta T cells out numbering gamma/delta T cells
by a ratio of 10:1, which is identical to the ratio of alpha/beta to gamma/delta
T cells in the blood [55]. In comparison to mouse skin, it seems that in humans,
a nonpolymorphic, cutaneous specific Ag stimulates these T cells. Thus, there
is a population of T lymphocytes evolutionarily selected via receptor specificity
to conserved Ags epitopes of commensal skin organisms. These conserved
epitopes can be presented by LC in either Class I or Class II MHC-restricted
manner or in the context of CD1 if there is to be presented a glycolipid,
lipoprotein, or a phospholipid [56]. Only about 2% of skin-associated lympho-
cytes are located intraepidermally, while the rest reside in the dermis. The
majority of T cells are CD8þ cells and CD45RO memory cells [10] and about
50% of epidermal T cells express CLA. This Ag is a sialyl Lewis-X glycoprotein
that is highly studied and discussed in the context of cutaneous melanoma.
In the skin, the CD4þ to CD8þ ratio is 1:1, different from the peripheral
2:1 ratio; therefore, it is possible that T cells from the epidermis are a special
population of memory cells, which have selectively extravasated into the
epidermis [11]. Twenty years ago, it was known that dermal T lymphocytes
represent around 98% of all skin T cells, most of them being memory cells
grouped in perivascular cuffs around the small venules and arterioles [57].
The difference between dermal and epidermal populations of CD4þ and
CD8þ T cells may indicate that CD8 T cells in the epidermis are continually
being renewed by low-level exposure to cutaneous Ags presented by Class
I MHC but not Class II MHC. Additionally, T cells possess a homing ligand,
HECA-452, which is present on about 16% of circulating T cells including
CD4þ and CD8þ memory cells. About 85% of T cells in inflammatory skin
lesions express HECA-452 as opposed to only about 5% in other tissues; this
finding supports the theory that lymphocytes may preferentially circulate
between the skin and the lymph nodes [58]. Tumor Ag-specific CD4þ T cell is
elicited by melanoma Ags in MHC Class II-restricted manner, inducing long-
lasting CD8þ antitumor memory [59]. In experimental exposure to UVB—
one of the incriminating factors in triggering melanoma—reported data
show that in vivo T-cell responses are prone to UVB-mediated immune
regulation. UVB affects both the activated T-cell pool size and the develop-
ment of memory T cells in peripheral compartments [60].
Cytokines produced by recruited T cells can influence the content of
the ongoing infiltrate by modifying the balance of produced chemokines [5].
For example, IFN-gamma can induce keratinocytes to produce a range of
products, including CXCL10 (IFN-inducible protein 10, IP-10), CXCL9

(monokine induced by IFN-gamma, MIG), and CXCL11 (IFN-inducible
T-cell a-chemoattractant, ITAC), which act to recruit T cells that express
the chemokine receptor CXCR3 [16]. UVB was proven to induce in normal
skin preferentially epidermal infiltration of CD4þ T cells. LFA-1/ICAM-1
pathway and psoriasin are both involved in the accumulation of CD4þ T cells
into UVB-irradiated skin, possibly via a recruitment mechanism. In response
to UVB exposure, the expression of psoriasin mRNA, but not of IL-16
mRNA, was upregulated [61]. T cells, infiltrating the epidermis after UV
exposure, were almost exclusively CD4þCD45ROþ T cells, expressing an
alpha/beta type T cell receptor, but lacking the activation markers HLA-
DR, VLA-1, and IL-2R. Therefore, in humans, the changes of the cutaneous
T cell population may also contribute to skin UVB-induced immune suppres-
sion [62]. Regulatory T cells, a T-cell subset, might traffic to the skin
using similar pathways to those used by effector cells [43] having crucial
immunosuppressive functions as elaborated in the following sections.
3.1.1.2. Innate Immune Surveillance
The innate immune surveillance resides in epidermis on keratinocytes
and LC and in derm resides on mast cells, DCs, and macrophages. All
these cells are cross talking through chemotactic proteins and cytokines
[63–65]. The APCs that are related to the skin comprise macrophages, LC,
and dermal DC [10]. The majority of papillary dermal leukocytes are macro-
phages; these cells extravasate across the dermal venules walls and are end-
differentiated. In the dermal microenvironment, macrophages clear away
Ags and participate in immune effector mechanisms [66].
3.1.1.2.1. Langerhans cells. LCs have a close and permanent interaction
with keratinocytes and with recirculating T lymphocytes in the integrated
system of SALT that ascertains cutaneous immunosurveillance [29]. LCs
(phenotype HLA-DRþ CD207þ) are CD1þ, and during fetal development,
they migrate to the skin [67]. The immature DCs are located in the suprabasal
portion of the epidermis [11]. These cells have the property to cover almost
25% of the surface area due to their long dendritic elongations and their
horizontal orientation, although they constitute less than 5% of the population
of epidermal cells [17,58]. LCs develops an integrated network for entrapping
most of the Ags that penetrate the skin. To loose their adherence, LC needs to
be stimulated by proinflammatory cytokines and migrates to draining lymph
nodes. During the migration, LC matures into lymphoid DC. During this
migration, LCs upregulate cell surface marker HLA-DR and costimulatory
molecules, while the LC-specific C-type lectin Langerin is downregulated [68].
Maturing into DC, they can efficiently present the Ag to naive T cells. There is
a permanent cooperation between immune-related cells and cutaneous nerve
fibers that release various neuropeptides affecting local immune activity [69].
100 MONICA NEAGU
LCs are located especially near the external surface of the basement membrane,
adjacent to the cutaneous free nerve endings in position to communicate with
other LCs and T cells [70]. Dendritic extensions can be found stretching betw-
een keratinocytes within the stratum granulosum, making physical contact
with keratinocytes in the lower epidermis [10].
The APC function of LC is sustained by the expression of Class I and Class
II MHC molecules along with CD1 molecules [11]. LCs also exhibit mem-
brane adenosine triphosphatase activity, contain vimentin-type intermediate
filaments, express surface S-100 protein and CD34, and in normal epidermis
(exception acrosyringeal keratinocytes) are the only cells that express Class II
MHC Ags [11].
How acquisition of external Ags by LC occurs in spite of the stratum
corneum (SC) and tight junctions (TJs) barriers is still a matter of thorough
study. Quite recently, it was demonstrated that upon activation, LCs elon-
gate their dendrites to penetrate keratinocyte’s TJs and survey the ‘‘extra-TJ
environment,’’ beneath the SC. Penetrated dendrites uptake Ags from the tip
where Ags colocalize with Langerin/Birbeck granules. Thus, stealthy under
barriers, LC and keratinocyte cooperate for LC to gain access to external
Ags that have penetrated the SC barrier [71].
It was recently demonstrated in mixed leukocyte reactions that both LC and
dermal DC significantly induce in peripheral blood T cells IL-22-producing
CD4þ and CD8þ subsets. The induced IL-22 production correlated mostly
with IFN-gamma than IL-17. Therefore, LC and dermal DCs preferentially
induced T-helper (Th) cells to produce only IL-22, and cutaneous DC, espe-
cially LCs, may control the generation of distinct IL-22 producing Th22 cells
infiltrating the skin [67].
UVB exposure of LC revealed that the cells migrating from UVB-exposed
epidermal sheets have a decrease in HLA-DRþLC percentage, as well as a
reduced capacity to induce proliferation of allogeneic T cells, when compared
with cells migrating from nonexposed sheets. It was stated that a reduced
number of CD1aþ LC migrated from the UVB-exposed full-thickness skin
and that there was a reduction in CD1aþ LC in the epidermis. This implies
that UVB induces the death of LC as well as the loss of cell surface molecules
rather than altering their migration, whereas the LCs that were still able
to migrate fully retained the capacity to activate allogeneic T cells [72]. The
human dermis possesses specialized APCs, dermal DC (phenotype HLA-
DRhiCD11cþBDCA-1þ) expressing high levels of Class II MHC and CD1
molecules, with potent Ag presentation function similar to that of LC [67].
Other cells, like eosinophils and mast cells, can guide the local immune
response. Therefore, mast cells store and secrete TNF-alpha. When these
cells degranulate, they trigger cytokine cascades, most important in the local
inflammatory response. Both eosinophils and mast cells are known to play a
role in the pathology of various skin diseases [17] but still to be established if
in cutaneous melanoma. Mast cells releasing different patterns of cytokines
and bioactive compounds, including leukotrienes, IL-1beta, IL-4, IL-5, IL-6,
IL-13, TNF, and granulocyte-macrophage colony-stimulating factor (GM-
CSF), in response to various TLR ligands are another crucial component of
the cutaneous immune response apparatus [20,21]. These and other mast-cell
products have an important role in both the initiation and modulation of
innate immune responses and the generation of adaptive immune responses.
3.1.1.2.2. Nonimmune cells involved in the development of the immune
response
3.1.1.2.2.1. KERATINOCYTES. In the epidermis, 90% of the cells are kerati-
nocytes, and besides their role in maintaining the keratin barrier, they support
other cellular components of the epithelial microenvironment [11]. Their im-
munological role is not minor, and besides cooperation with classical immune
cells, they secrete immune response-eliciting cytokines [10]. For example, they
produce large quantities of interleukin-1alpha (IL-1alpha), tumor necrosis
factor (TNF) cytokines and neuropeptides in response to various stimuli,
including kinetic and thermal trauma, UVB radiation [73–76]. IL-1alpha
(and IL-1beta from epidermal LC) is a potent stimulator of local immune
function and is one of the key immunoregulatory cytokines [17,23–25]. All
these molecules affect skin resident innate immune cells, mast cells, DCs and
macrophages, resulting in the upregulation of other inducible mediators and
recruitment of additional immune cells from the blood [77]. For example,
secreting IL-7, growth and survival factor, keratinocytes support the epider-
mal T-cell population. The interrelation with T cell is complex and sustained
by an array of up- and downregulatory secreted molecules that influence T-cell
activity (Fig. 1). Keratinocytes secrete also monocyte-macrophage colony-
stimulating factor (M-CSF)/GM-CSF that sustain and activate LC. Keratino-
cyte synthesizes IL-1alpha but does no secrete it. When IL-1alpha ‘‘leaks’’ out,
it contributes to the inflammatory cascade initiation. The consequence is
that the neighboring keratinocytes produce increased IL-alpha, along with
IL-1beta, TNF-alpha, and IL-6; all these cytokines amplify the immune
response [78]. Keratinocytes produce CXC chemokines including IL-8 and
members of the growth-regulated oncogene (GRO) family. The expression of
CXC chemokines leads to initial recruitment of granulocytes over a mononu-
clear influx. Finally, keratinocyte secretion of M-GSF, GM-CSF, IL-7, and
IL-15 serves as growth factors for leukocytes [65]. Keratinocytes respond to
the secreted immune-related molecules, and their function being regulated by
the cytokines released by the skin-infiltrating T lymphocytes. IFN-gamma,
produced by T cells, increases keratinocyte cell adhesion through increased
expression of ICAM-1 and initiates expression of Class II MHC molecules
[17]. Activated CD4þ T cells produce IL-17 that stimulates epithelial cells.
102 MONICA NEAGU
Downregulatory cytokines
IL-1Ra, IL-10, alpha-MSH,

CXCL10, Contra IL-1, PGE2
Epidermis
Keratinocyte Chemokines
T-cell
T-cell cytokines
(IFN-alpha, IL-17, IL-4)
IL-1, GM-CSF, TNF-alpha,

IL-6, 7, 12, 15, 18
Upregulatory cytokines
FIG. 1. Interrelation mediated by humoral factors between keratinocytes and T cell. Kerati-
nocytes produce cytokines that upregulates T-cell functions, proinflammatory cytokines such as:
IL-1, GM-CSF, TNF-alpha, IL-6, 7, 12, 15, 18. Keratinocytes produce as well T-cell down-
regulatory cytokines: IL-1Ra, IL-10, alpha-MSH, CXCL10, Contra IL-1, PGE2. T cell produces
IFN-alpha, IL-17, IL-4 that influences keratinocyte’s functions. Chemoattractant cytokines
produced by keratinocytes influence T-cell trafficking: IL-1, IL-8, CCL27, CCL5, CCL17,
CXCL10, MIG, IP9, CCL20.
IL-17 is a proinflammatory cytokine, which could amplify the development of

cutaneous inflammation through stimulation of keratinocytes augmenting
their secretion of proinflammatory cytokines [18]. Comparing the effects
of IL-17 and IFN-gamma, upon keratinocytes, it was proven that IL-17
enhanced the mRNA and protein production of the proinflammatory cyto-
kines IL-6 and IL-8, inducing a weak expression of ICAM-1 and HLA-DR.
IFN-gamma augmented the production of IL-6, IL-8, and IL-15 and strongly
induced cell surface molecules ICAM-1 and HLA-DR expression. IL-17 and
IFN-gamma showed marked synergism in the stimulation of IL-6 and IL-8
protein secretion and, to a lesser extent, in the induction of ICAM-1 and HLA-
DR expression. Keratinocytes also synthesize thymopoietin, influencing skin’s
T-cell maturation. Similar to endothelial cells, keratinocytes are induced to
express Class II MHC by IFN-gamma and are unable to express sufficient
costimulatory molecules to drive T-cell activation [11].
In the skin, NF-kB signaling pathway regulates the expression of numer-

ous genes that are involved in the initiation of the inflammatory response [5],
including adhesion molecules, chemokines and cytokines (such as IL-1 and
TNF), matrix metalloproteases, nitric oxide (NO) synthase, and enzymes that
control prostanoid synthesis [79]. All these molecules recruit additional
leukocytes to the site of activation. In humans, genes regulated by NF-kB
include the endothelial adhesion molecules E-selectin and P-selectin, ICAM-1,
vascular cell adhesion molecule-1 (VCAM-1), and various chemokines and
cytokines [80] for the initiation of the leukocyte adhesion–extravasation cas-
cade that recruits circulating leukocytes from the periphery [81]. From the
periphery, Ag-nonspecific leukocytes (neutrophils and NK cells) and effector
T cells are recruited (Fig. 2).
There is also a cross talk between keratinocytes and macrophages. In vitro
experiments showed that cultured human keratinocytes express monocyte
chemoattractant protein (MCP), decay-accelerating factor (DAF), and
CD59 and that the supernatant of activated mononuclear cells and the
recombinant forms of transforming growth factor (TGF-beta) beta1, 2,
and 3 upregulate MCP and CD59 but not DAF. TGF-beta released by
macrophages can be responsible for upregulating the expression of MCP
and CD59 on keratinocytes [22]. Interestingly, flow cytometric analysis
showed that MCP and DAF, but not CD59, were expressed on cultured
human melanocytes. DAF protects melanocytes against complement attack
[82], this protective role is important as the complement system is highly
involved in host defense and inflammation. In skin, the keratinocytes are
known to produce two soluble complement components, C3 and factor
B. Eleven years ago, it was first reported that cultured keratinocytes constitu-
tively produce C3 and factor B and that their synthesis is regulated by some
cytokines, known to be produced by inflammatory cells [83]. Later, it was
demonstrated that keratinocytes are capable of synthesizing factor H and that
this synthesis IFN-gamma-regulated [84]. Afterward, it was demonstrated that
human keratinocytes constitutively express factor I mRNA, produce factor
I protein and that IFN-gamma regulates its synthesis. This factor produced by
keratinocytes cleaves C3b [85]. Recently, the possibility of human keratino-
cytes to synthesize the final complement components, C5–C9 was studied [86].
Keratinocytes are capable of synthesizing some of the terminal complement
components, and C9 synthesis is regulated by TNF-alpha [86].
As for all other skin cells, keratinocytes respond to factors that induce
various skin pathologies. Therefore, UVB exposure results in an increased
production of several cytokines and inflammatory cells infiltration enhance-
ment. UVB has been reported to upregulate membrane cofactor protein,
DAF, and CD59 on keratinocytes without affecting the constitutive release
of C3 and factor B. Thus, UVB can increase the resistance of keratinocytes
104 MONICA NEAGU
Keratinocyte
Langerhans cell
Epidermis
Memory T cell Dermal dentritic cell

CLA+
Mast cell
Dermis
CCL17
E-selectin NK cell
ICAM-1
Monocyte/
macrophage
Granulocyte
Naive
T cell
Dermal postcapillary venule
FIG. 2. Immune-response elements in noninflamed skin (adapted after Ref. [5]). Immune cells
resident in the epidermis include specialized dendritic cells (DCs) known as Langerhans cells and
intraepithelial lymphocytes. The dermis is mainly composed of connective tissue having as
resident immune cells, dermal DCs, mast cells, and a small number of cutaneous lymphocyte
antigen CLAþ memory T cells. Dermal postcapillary venules constitutively express low levels of
against their own complement components known to be produced excessive-

ly in response to inflammatory cytokines following UVB exposure [87].
The process is complex and since neutrophils invade UVB-exposed skin
and, like macrophages, express CD11b and HLA-DR, their contribution in
releasing IL-10 was studied [88]. Upon UVB exposure, IL-10 staining could
be detected in CD11bþ HLA-DRþ CD36þ macrophages in epidermis and
dermis. The abundant IL-10 expression was found in CD11bþ HLA-DRþ
elastaseþ CD66bþ neutrophils. Thus, neutrophils in UVB-exposed skin ex-
press IL-10 and are active contributors in the UVB-induced immunosuppres-
sive microenvironment [88].
3.1.1.2.2.2. ENDOTHELIAL CELLS. Other cells involved in immune surveil-
lance, the microvascular endothelial cells, are among the main immune
regulators in the skin [10]. Although these cells line the dermal venules and
arterioles having a more or less ‘‘passive’’ role in the mentioned context, they
are transversed by leukocytes during their extravasation. Endothelial cells
mediate the transduction of chemotactic and stimulatory signals linking the
dermal to the intravascular compartment. IFN-gamma secreted by cells from
the inflammatory situs induces endothelial cells to express Class II MHC,
activate cells that can phagocytose Ags, and present them to the T cells. The
interaction of ICAM-1 bound to LFA-1 and CD2 on T cells trigger costi-
mulatory signals; therefore, endothelial cells take the function of APCs to
intravascular T cells.
Human skin is composed of three distinct compartments involved in the
immune response. The epidermis composed of keratinized epithelial cells
functions as a physical barrier and as an early warning system. In the epider-
mis specialized DCs as LCs and intraepithelial lymphocytes are the resident
immune cells. The dermis is mainly composed of connective tissue produced
by dermal fibroblasts. In noninflamed dermis, dermal DCs, mast cells, and a
small number of CLAþ memory T cells are the resident immune cells. The
dermal postcapillary venules constitutively express low levels of E-selectin,
CC-chemokine ligand 17, and ICAM-1; these molecules support the margin-
ation and baseline emigration of CLAþ memory T cells into noninflamed skin.
CLA-T cells, including both naive cells and memory/effector cells that are
targeted to other tissues, as well as granulocytes and other immune cells, lack
E-selectin, CC-chemokine ligand 17 (CCL17), and intercellular adhesion molecule-1 (ICAM-1).

These support the emigration of CLAþ memory T cells into noninflamed skin. CLA-T cells,
including both naive cells and memory/effector cells that are targeted to other tissues, granulo-
cytes, and other immune cells, like NK cells lack the appropriate receptors to attach to dermal
vessels and migrate to the skin layers.
106 MONICA NEAGU
the appropriate receptors to attach dermal vessels and emigrate into nonin-
flamed skin.
The cutaneous immune reactions are discussed in the framework of SIS
where keratinocytes and dermis provide a structural context in which such
immune responses are initiated and where pathological lesions may devel-
op. SIS and neoplastic cells are interconnected processes and structures
from the outset of tumor genesis including initiation, promotion/progres-
sion, toward tumor invasion and metastases. Central to this interaction are
mechanisms which explain how cutaneous tumor cells bypass immune
attack [6]. Data had gathered regarding both UVB and chemical carcino-
gens as factors that can disrupt the local immune response by altering LC
structure and function, resulting in both cases in the generation of tolerance
to growing aberrant cells [89].
The immune surveillance is a complex and interrelated mechanism.
It consists of three stages where the primary response initiates the engage-
ment of the adaptive immune response in which Ags encountered in the skin
are carried by activated DCs and presented to naı̈ve and central memory
T cells circulating through the node. T cells that encounter the Ag prolifer-
ate and differentiate into effector cells expressing homing receptors. The
secondary immune surveillance ensures rapid and effective local adaptive
immune responses to previously encountered Ags, upregulating the expres-
sion of adhesion molecules and presentation of specific chemokines on the
local endothelium. Effector memory T cells are recruited in an Ag nonspe-
cific manner. The tertiary immune surveillance enhances adaptive immune
responses to Ags encountered in tissues distinct from those in which they
were previously encountered. Central memory T cells produced in skin-
draining lymph nodes recirculate through lymph nodes throughout the
body, providing enhanced responses to Ag encountered through a different
environmental interface [30].
All these levels, cellular or humoral, can comprise markers that indicate
the triggering of an immune response.
4. Cutaneous Melanoma Develops in a Skin Immune

System Controlled Microenvironment
SIS and neoplastic cells interconnect from the outset of tumorigenesis

including initiation, promotion/progression, toward tumor invasion and
metastases. The immune-specific arm has as main cells, the lymphocytes,
that extravasate from circulation into the skin. Central to this interaction
are the mechanisms which explain how cutaneous tumor cells bypass im-
mune attack incorporating tumor-induced impairment of Ag presentation,
activation of negative costimulatory signals, and immunosuppressive factors

[7]. The cellular components of SIS, mainly regulatory T cells, natural killer
T cells (NKT), and distinct subsets of immature and DCs are the main cells
that comprise the immunosuppressive network [90]. Recent studies have
suggested that tumors can overcome immunosurveillance following ‘‘immu-
nosculpting’’ by the immune system [91]. Therefore, the tumor-induced
immune tolerance and suppression, the loss of immunogenicity observed in
advanced stage tumors could be the result of immune-distortion, and these
immune-induced alterations could contribute to cancer pathogenesis [91].
The local immune suppression comprises the complex mechanism for
melanoma development. Melanocytic proliferation is restrained by the
immune system, but the mechanisms that underlie immune suppression,
and which therefore permit the development of cutaneous melanoma, are
not yet fully understood [92]. Important questions have arisen, given the
fact that clinical outcome of patients subjected to immune therapy has not
been satisfactory, in spite of the fact that antitumor T cells were detected
[93]. The tumor microenvironment nurtures several protumor mechanisms:
reduced chemokine-mediated trafficking of effector cells, negative regulato-
ry pathways that inhibit T-cell function, the already known immune escape
of cancer cells, and their adaptation to a vigorous immune pressure [93].
The failure of antitumor immune responses resides in the local immuno-
suppressive cells and factors. In this context, immature DC, neutrophils,
T-regulatory cells, myeloid-derived suppressor cells, and tumor-associated
macrophages (TAMs) have important roles.
In this complex cellular microenvironment, melanocytes interactions with
all these generated factors can lead to the promotion of malignant transfor-
mation and in the end to acquiring the invasive potential.
5. Immune Markers—The Road Ahead
Although in the past 50 years, the histopathology of cutaneous melanoma

was the focus point of the world wide clinical/research studies and intensive
efforts were made by the Melanoma Committee of AJCC regarding the
correlation of histological features and patient survival, the need for good
‘‘prognosticators’’ remained more than ever important [94]. We would em-
phasize that in spite of the numerous tissue markers, whether proteins and/or
genes, only few have real prognostic value [95] and have just started the
validation road [96]. The molecular basis of the 2010 AJCC melanoma
staging system [97], recently reviewed, could be improved by the development
of computerized platforms that could calculate the biomarker’s diagnostic,
prognostic, and therapy response prediction.
108 MONICA NEAGU
In this complex domain, the need for immune markers that can complete the
pathological ‘‘picture’’ of cutaneous melanoma development is still unmet.
5.1. INFILTRATING IMMUNE CELLS—MARKERS FOR LOCAL IMMUNE RESPONSE

As seen in the previous section, normal skin holds within an array of
lymphocytes, gammadelta T lymphocytes, and NK cells being the main cells
involved in the antitumoral local response in cutaneous melanoma [98]. The
role of the immune cells that infiltrate the tumor is crucial in developing the
local response. Therefore, the number, phenotype, and functional character-
istics of infiltrating immune cells are important biomarker candidates.
5.1.1. Local Response

5.1.1.1. Tumor-Infiltrating Immune Cells—Lymphocytes, TAMs, DCs,
Sustain the Local Immune Battle? Evaluating the biomarker value of infil-
trating immune cells, few years ago, researchers have studied the distribution,
the density of T lymphocyte subsets, macrophages, and DCs [99]. Out of
these cells, only the number of CD8þ-infiltrating T lymphocytes and the
number of human leukocyte Ag-D-related cells were related to a favorable
prognostic of the disease. The presence of CD68þ macrophages did not prove
any prognostic value. Seeking to establish the role of tumor-infiltrating
lymphocytes (TILs) and their involvement in the development of melanoma,
authors pointed out that in the transition from normal skin toward a neo-
plastic transformed one, there is a gradual increase in the number of TILs
[100] with a higher proportion of T versus B cells. Moreover, the expression
of surface Ags for the leukocyte common Ag (LCAþ), CD20þ, CD3þ, and
resting cytotoxic T cells (TIA-1þ) increased with the transition toward
malignization. Interestingly, the authors reported [100] that, in the melano-
ma metastatic samples, there was a decrease of TILs in comparison to
primary tumors. We assume that there is a fine tuning of the cellular immune
response in primary tumors, and when metastasis occurs, the immune factors
of the humoral immune response are more involved.
As previously presented, DCs can have the important role of presenting
tumoral Ags to the innate immune cells and thus trigger the antitumoral
response. Few years ago studying the role of infiltrating DCs as potential
immune biomarkers, it was reported [101] that the DCs density correlated
with the activated CD25þ T lymphocytes. Authors show that a robust local
immune response depicted by an increased density of mature DCs and
activated T lymphocytes can be good prognostic markers for cutaneous
melanoma. Another group, in the same year [102], showed that the density
of CD83þ DCs and T lymphocytes decreased with the thickening of the
tumor, and that this feature is an indicative of progression toward advanced
stages. Although they are crucially involved in the local immune suppression,
only several years ago, Tregs were studied at the tumoral site [103]. Investi-
gating all types of benign nevi and melanomas, primary or metastasis, the
authors reported the presence of Tregs in all samples. Their conclusion was
interesting as they asserted that Tregs’ presence is the mark of local immune
suppression that can drive the junctional and compound atypical nevi toward
melanoma development. Later, other authors showed that Tregs have a
higher density in vertical than in radial growth phase of the melanomas
[104]. The presence of infiltrating Tregs is increased in more advanced stages
in comparison with earlier ones. It is still to be established whether this is a
clear immune marker of immune suppression and an indicator of melanoma
progression [105]. Recently, it was reported that Tregs infiltrate did not
correlate with the patient’s survival, the increased patient’s survival being
correlated with a lower density of T lymphocytes and NK CD16þ cells [105].
Not only can the actual density have biomarker significance, but more so
their functional pattern. Therefore, tumors displaying a higher amount of
CD69þ lymphocytes (activation marker) correlated with an increased surviv-
al rate of the patient. From the immunohistological point of view, authors
indicate that a decreased survival can be indicated by the abundance of TILs
in tumors with periodic acid Schiff’s (PAS) positive loops (PAS reagent) that
can be candidate markers for aggressiveness [105]. TILs, as a cellular ensem-
ble, are still a questionable marker as its actual link to tumor progression is
still to be demonstrated [106]. But, as TILs are composed of many cell types,
the role of local Treg was evaluated [106]. Tregs percentage was significantly
higher in patients with recurrence of the disease in comparison with the other
ones. Therefore, authors point out that infiltrating Tregs have started the
validation road for prognostic marker.
Our experience [107] confirmed that peri- and intratumoral inflammatory
infiltrate consists mainly of T lymphocytes, and that the inflammatory reac-
tion is well represented when the tumor is in the first three stages of Clark
invasion. Only in ulcerated melanomas, the inflammatory infiltrate contained
identified plasmocytes.
Recently, the presence of TILs, macrophages, DCs, and CD34þ microves-
sels was studied in order to asses the prognostic values [108]. Intratumoral
microvessel density quantified did not correlate with the above-mentioned
immune cells, except in the thick melanomas where CD68þ macrophage
density was strongly associated with microvessel density and to a lesser extent
with B lymphocytes and DCs.
Part of the innate immune arm of defense, tissue macrophages, and tumor-
associated macrophages (TAM) have a long research history regarding their
role, function, and actual involvement in the tumoral progression. In cutane-
ous melanoma, their role in tumor development is still a subject of research
110 MONICA NEAGU
[109]. Authors report that TAMs can produce ornithine through arginase
activity, compound with pro-proliferative action. In contrast, TAMs can
produce nitric oxide via inducible NO synthase (iNOS), NO being a highly
cytotoxic molecule. Therefore, these dual role, anti/pro-tumoral compounds,
synthesized by TAMs, can offer good grounds for biomarker discovery. The
report shows [109] that in early stages the percentage of iNOSþ TAMs is
increased and overriding TAMs that have arginase activity, the overall action
being anti-proliferative. Moreover, it was reported that genetic variants of
neuronal NOS can be candidate biomarkers for risk of development cutane-
ous melanoma [110].
5.1.1.2. Chemokines Receptors and Ligands. A huge amount of scientific
research was published regarding the chemokine and the chemokine receptor
expression at tumor site. This recorded fact can be explained by both the
importance of these molecules in the metastatic processes and their main role
in immune cell intercommunication.
Few years ago, chemokine and their specific ligand were investigated aiming
to demonstrate their biomarker potency. Authors reported for the first time, 6
years ago [111], that CXC chemokine receptor type 4 (CXCR4) expression,
presence of ulceration, and sentinel lymph node (SLN) grade were indepen-
dent prognostic factors. Continuing their work, seeking to establish functional
patterns, and using its ligand, CXCL12, the same authors showed that
CXCR4 is active in metastases, contributing to the less favorable prognostic
of the disease [112].
Another chemokine receptor, CXCR3, expressed primarily on activated
T lymphocytes and NK cells [113], was the subject of study in order to
evaluate its biomarker capacity in cutaneous melanoma [114]. The authors
reported the association of CXCR3 positivity with tumor thickness, lack of
lymphocyte infiltration, and the appearance of metastasis.
Thus, CXCR3, having as ligands chemoattractants like MIG, IFN-inducible
T-cell alpha-chemoattractant (IP-10) is probably involved in tumor progres-
sion and can join the list of potential immune markers in cutaneous melanoma.
CC-chemokine receptor type 10 (CCR10), expressed among other cells, on
T cells and in skin-derived LCs [115] was studied. When CCR10 and its
ligand were concomitantly studied in tumor, they were proven to have an
inverse correlation with the intratumoral density of T lymphocytes CD3þ
and CD8þ [116]. Authors state that the CCR10/CCL27 couple can be a
marker for invasion, dissemination, and metastasis, having therefore prognos-
tic value. Recently, in the continuous effort to improve their prognostic
significance, the patterns of chemokine receptors/specific ligands were reported
[117]. Therefore, 18 chemokine receptors were studied both at mRNA and at
protein levels. CXCR6 was found in primary melanomas and metastases,
while CCR1 expression was reported as increased in correlation to melanoma
progression. CCR10, CCR7, and CCR5 had a specific pattern of expression in

primary and/or metastatic tumors. Authors show that this ‘‘chemokine print-
ing’’ should be corroborated with solid functional studies, but their presence is
definitely detecting progression stages in the tumors.
5.1.1.3. Adhesion Molecules. Immune cells that are subjected to traffick-
ing processes have a key family of guiding molecules. In several research
papers, adhesion molecules, mainly ICAM-1, are subject for biomarker
discovery in cutaneous melanoma. It is known that ICAM-1 is associated
with patient survival in early stages [118], and its polymorphism could be
related to tumor growth [118]. Moreover, its single nucleotide polymorph-
isms, R241 allele, were associated with the risk of developing cutaneous
melanoma, while K469E polymorphism did not [119]. Its soluble form was
shown to be as well related to the susceptibility of developing cutaneous
melanoma [119].
Another molecule, transmembrane glycoprotein CD44, splice variant 3
(CD44v3), has been reported as involved in tumor progression. It is involved
in controlling innate immunity cells’ migrations and PMNs’ recruitment
during inflammation. CD44v3 specifically binds to CD11b/CD18, and
recently, it was demonstrated that this binding is mediated by heparan sulfate
moieties [120].
CD44v3 expression can be correlated with a favorable prognostic without
having an independent predictive power [121]. Recently, it has been shown
that another variant of CD44, CD44v6, can be involved in the migration of
transformed melanocytes and having a possible role in metastasis [122].
5.1.1.4. Tumor Stem Cells Are Related to Immune-Related Processes.
The main relation of cancer stem cells (CSCs) and the immune response is
reported in the framework of (immune)-therapy. Few research papers have
focused on this subject; CSC domain raises more questions than any other
subject related to cutaneous melanoma. The known therapeutical resistance
of melanoma [123] can be accounted to the recently discovered CSCs. The
tumor cells characterized by the phenotype CD133þ can be the so-called
CSCs that can express cancer/testis (CT) antigens [123]. In an experimental
cell line model, authors showed that these types of CSCs could be specifically
targeted by cytotoxic T lymphocytes, therefore opening new possibilities for
immune-intervention in melanoma’s therapy.
A new class of molecules, semaphorins and their receptors, plexins and
neuropilins, were involved in various immune function related to tumor
progression and to melanocyte stem cells neoplastic transformation [124].
Recently analyzing these molecules, it was shown that in metastasis there is
significant loss of Plexin C1 compared to the primary tumors [124], results
that suggest both its tumor suppressor function and its biomarker capacity.
112 MONICA NEAGU
Lately, abundant data piled up regarding CSC markers, namely, CD133þ,

ABCB5þ, CD166, nestin, BMI-1 in relation to melanoma progression. Thus,
nestin was found increased in advanced stages and could be a biomarker for
aggressiveness and poor prognostic [125]. Another group combined nestin
with BMI-1 expression in various melanoma stages and melanoma cell lines
[126]. The results showed that BMI-1 expression is related to the metastatic
potential of the tumor and a tissue pattern with high BMI-1/low p16 (ink4a)
expression can have predictive power. CSCs through their surface markers
are related to an aggressive biological behavior. Last year, stem cell markers
were studied and shown that the simultaneous overexpression of CD133þ
and nestin can indicate a dedifferentiation pattern. Moreover, the existence
of distinct melanoma stem cell population can open new possibilities in
cancer therapy [127].
5.1.1.5. Immune-Related Factors’ Involvement in the Metastatic Process.
Immune cell can contribute to the escape of tumor cell from the original
tissue favored by the deregulation of chemokine receptors. The capacity of
cutaneous melanoma to progress toward metastasis is one of the key pro-
cesses in biomarker discovery, and the immune markers should give an
important ‘‘helping hand’’ in this matter. The need to clearly discriminate
between patients that will develop sentinel node metastasis, and the ones that
will further develop metastasis to other sites is obvious [128]. Immune
markers should indicate when and why this first immune defense station,
the SLN, will allow the melanoma to disseminate. We can affirm that
published literature in this domain focuses on adhesion molecules that favors
the motility of cells and induces spreading outside the primary tumor; angio/
lymphangiogenesis factors, chemokine, and their specific receptors are also
taken into account in this processes.
It is known that the lymphatic network sustains the cutaneous melanoma
spreading; therefore, all the molecules related to tumorigenesis, angiogenesis,
and lymphangiogenesis can be future markers predicting lymph node metas-
tasis [129]. It was observed an inverse correlation between SLN metastasis
grade and VEGFR-3/panvascular marker CD31 expression [129]. Angiogen-
ic markers like VEGF and bFGF combined with microvessel density could
enhance the information regarding tumor progression [130]. Last year, the
classical angiogenic markers, VEGF and fibroblast growth factors (FGFs),
were studied in correlation with S100A13 [131]. Analyzing all types of
cutaneous melanoma and their metastases, authors reported that VEGF-A
and S100A13 cooperate in angiogenesis, and this finding may enlarge the
future panel of prognostic markers in melanoma.
Adhesion molecules and chemokine families are another intensively stud-
ied topic in relation to metastasis in cutaneous melanoma. Investigating the
complex array of processes governating tumor cell migration [132], authors
found out that Breslow index (tumor thickness) was correlated with a higher
mobility of tumor cells thus an enhanced invasion capacity. Authors proved
that the low E-cadherin expression and overexpression of Cdc42 and
CXCR4 can have prognostic power and can indicate a poor outcome of
the disease [132].
The metastatic capacity of cutaneous melanoma to develop brain tumoral
foci can be marked by the presence of CCR4, which can be a new biomarker
candidate for predicting brain metastasis of cutaneous melanoma [133].
Other molecules involved in cellular motility like CD9 [134] or involved in
the modulation of cell adhesion and migration were studied in relation to
cutaneous melanoma metastases [135]. Neutral endopeptidase (CD10), a
marker present, besides other cells, on the surface of early lymphoid cells,
was found inversely correlated with CD9, their pattern indicating an invasive
cutaneous melanoma and a possible future prognostic set [135].
Chemokines and their receptor are mainly involved in distant sites metas-
tasis; recently, it was proven that CCR9þ melanoma cells are migrating
toward small bowel expressing CCL25, thus favoring tumor metastasis
[136]. CCR9 expression was found in over 80% of the small intestinal metas-
tases and no expression in other metastasis; thus, authors clearly showed
that this marker can indicate the progression of cutaneous melanoma and
that the couple CCR9–CCL25 is the molecular pair that favors this specific
site metastasis.
Using proteomic platforms from metastatic tissues were identified 120 pro-
teins with significantly changed expression compared to primary tumors [137].
Among the identified proteins, there were immune response, inflammation-
related, and adhesion molecules. Molecules found upregulated in metastasis
were tubulin beta-1 chain, plastin 3, immune-related macrophage migration
inhibitory factor, peptidylprolyl isomerase B, Parkinson disease (autosomal
recessive, early onset) 7 (DJ1), peroxiredoxin family (PRDX5, PRDX2,
PRDX6), heat-shock protein 90 kDa alpha (HSP90AA2). All these proteins
can be valuable candidates for tumor progression.
Other adhesion related molecule, MUC-18, was found related to poor
clinical outcome [138]. MUC-18, a member of the immunoglobulin super-
family, is related to the cellular immune arm through B lymphocytes. There is
a MUC18-dependent cross talk between a certain B-cell subpopulation and
melanoma cells that, in the end, has a prometastatic action [139] and that can
be considered a progression marker [140].
5.1.1.6. Sentinel Lymph Node—The Immune Station Related to
Metastasis. In the past years, the golden standard for evaluating the initia-
tion of the metastatic processes is the evaluation of SLN [141]. Studying
the lymphangiogenesis of the tumor, it was reported that the extent of
this parameter was the most sensitive prognostic marker for metastasis.
114 MONICA NEAGU
Moreover, lymphangiogenesis proved 83% sensitivity and 89% specificity for

predicting metastasis to SLNs [142]. Different results were published when
LYVE-1 (lymphatic vessel endothelial receptor-1) staining indicated that
in spite of a thorough lymphatic vessel network, cutaneous melanoma
metastatic potential cannot be prognosticated using lymphatic counts [143].
Another marker related to microvessel physiology, inducible NOS, was
found correlated with the density of lymphatic vessels; therefore, a molecular
marker involved in lymphatic vascular neoformation [144]. The authors did
not find any correlation of patients’ clinical outcome with microvessel or
lymphatic vessel density.
At the SLN level, immune cells that can sustain the immune suppression
were one of the first cells searched in relation to metastasis. Analyzing TCR
zeta and other immune cells, authors reported that the loss of TCR zeta is
inversely correlated with the tumor burden [145]. The zeta chain of TCR has
a key role in T-cell functionality, and its downregulation was associated with
various diseases, including cancer [146]. Finding this inverse correlation
suggests that prior to metastasis, there is a lowering of the specific immune
response in the SLN [145]. The immune milieu of SLN is sustained by T-cell
activation. Investigating the SLNs, authors reported that OX40þ CD4þ
T lymphocytes were lower as percentage in ulcerated tumors and in positive
sentinel nodes, a possible immune parameter for the metastatic capacity of
the tumor [147].
Tissue immune markers are involved in clinical diagnosis, prognostic
classification, patients’ subgrouping, but immunohistochemical biomarker
molecules’ identification is only a part of the puzzle that is the immune
suppression stated in this disease, as cutaneous melanoma is a ‘‘paradox
among all solid tumors’’ [97]. Thus, it has one of the best prognostic markers
for solid tumors, but their biological significance is still under investigation.
5.2. PERIPHERAL IMMUNE MARKERS—MONITORING THE DISEASE

5.2.1. Immune Molecules
There are several immune-related molecules that were detected in patient’s
circulation and proved to be noticeable among the candidate biomarkers
panel.
5.2.1.1. Immune Communication Intercellular Messengers. Cytokines
control immune recognition, proliferation, and the immune cells’ effector
functions. From the beginning of the research in the biomarkers discovery
field, cytokines and their receptors were searched in association to diagnostic,
prognostic, and/or therapy, and some of them even became lately therapeu-
tical agents.
IL-6 that acts as both proinflammatory and anti-inflammatory cytokine is

secreted by T cells, and macrophages was associated to cutaneous melanoma.
IL-6 is involved in advanced stage disease and in tumor recurrence. Overall,
survival of patients was shown to be related to IL-6 serum level. Patients with
low IL-6 serum concentration had higher overall survival when compared to
the high IL-6 group [148]. Pretreatment serum levels of IL-6 and TNF-alpha
were reported increased in patients with weight loss and who were nonre-
sponders to chemotherapy. In contrast to serum TNF-alpha, serum level of
IL-6 was found to be a prognostic factor as valuable as serum lactate
dehydrogenase (LDH) in metastatic melanoma patients [149]. Further,
IL-6, LDH, and tumor burden were reported as significant prognostic factors
for overall survival, while the pretreatment serum IL-6 level is a predictive
factor [148].
Not only singular cytokines but also cytokines’ patterns produced by
lymphocytes may help to explain the impairment of T-cell immune response.
Th1 cytokines (IL-2, IFN-gamma, TNF-alpha) and Th2 cytokines (IL-4,
IL-10) are meaningful to be monitored in patient’s serum and can represent
valuable markers of immune response. Therefore, IL-10, a human cytokine
synthesis inhibitory factor, an anti-inflammatory cytokine, was found signif-
icantly increased in melanoma patients with respect to normal donors [150].
In contrast, other group showed that serum IL-4 and IL-10 were not detect-
able, while serum TGF-beta level was significantly higher in stage I and stage
IV melanoma patients compared to normal controls [151]. Overall, indepen-
dent of stage, the percentage of IFN-gamma producer cells was significantly
lower [150].
Using multiplex technology, in the sera of stages IIB–III melanoma patients,
29 cytokines, chemokines, angiogenic/growth factors, and soluble receptors
were quantified. The proinflammatory cytokines IL-1beta, IL-1alpha, IL-6,
TNF-alpha, and chemokines like macrophage inflammatory protein (MIP-
1alpha and MIP-1beta) were found significantly elevated in the serum of
patients with longer relapse-free survival and were noted as prognostic markers
of clinical outcome [152].
Last year, the increased level of plasmatic TGF-beta in primary melanoma
patients was reported [153] and was found at higher levels in patients diag-
nosed with metastatic melanoma. An interesting correlation was found with
the plasma levels of MMP-2 and the fact that TGF-beta favors the MMP-2
release. The tissue and plasma levels of these molecules are increased,
indicating the tumor progression [153].
Downregulation of several immune modulation genes, with emphasis on
IFN pathways, was reported [154]. The first study to report defects in IFN
signaling in immune cells of patients with melanoma showed that the reduced
expression of STAT-1 and interferon-stimulated genes (ISGs) in T and
116 MONICA NEAGU
B cells in patients indicate a perturbation in IFN signaling. These signaling

markers may constitute immune signatures associated to the evolution of the
disease. The defects in type I IFN signaling in T and B cells, but not in NK
cells, in patients with metastatic melanoma, induce a reduced activation of
cells after in vitro stimulation. The impairment can be partially overcome
by prolonged high-dose IFN-alpha treatment, suggesting a potential mecha-
nism for the efficacy of IFN-alpha used in the therapy of melanoma. More-
over, these signaling markers can improve immunotherapeutic strategies for
cancer [155]. Molecules related to intracellular signaling involved in the
immune response can be important markers for cellular apoptosis, prolifera-
tion, functions highly involved in tumor progression, and invasion.
The research domain of immune-related circulatory molecules remains
extremely interesting as these possible markers can be noninvasively detected
in body fluids.
5.2.2. Immune Cells

Although there are concerns regarding the meaning of peripheral blood
lymphocytes investigations in relation to their actual value as biomarkers
[106], we believe that local immune status can be mirrored by the circulatory
immune parameters. As presented in Fig. 3, the immune cells circulate and
thus can indicate an on-site immune response, and moreover, therapy moni-
toring postsurgery can reside in these types of immune markers giving a good
reflection on immune status of the patient.
5.2.2.1. T Lymphocytes as Biomarkers for Melanoma. Data are now
gathering in particularly for peripheral Th (CD4þ) and T cytotoxic lympho-
cytes (CD8þ) as possible immune markers in cutaneous melanoma.
Reports studying cellular dysfunction associated with melanoma showed
that patients with advanced melanoma had severe CD4þ and CD8þ T-cell
lymphopenia associated with reduced T-cell proliferation [156]. Our results
confirmed that, in advanced stages of melanoma, the lymphopenia is asso-
ciated with a clear disproportion of CD4þ and CD8þ, and the low CD4þ/
CD8þ ratio is correlated with the diagnosed stage [157], an indicative of the
suppressive status of cutaneous melanoma patients [158] (Fig. 4). Early
activation markers detected on immune cells are related to patient’s survival.
Melanoma patients that displayed peripheral low proportions of
CD3þCD4þCD69þ cells and CD3þCD56þ cells were reported to have an
improved disease-free survival compared to those with high proportions, and
that these cellular parameters were reported as independent prognostic fac-
tors for overall survival [159]. The specific pattern of circulating CD8þ
T cells, namely, a distinct phenotype characterized by downregulation of
costimulatory molecules and higher expression of NK-associated receptors
(NKRs) can be a marker for the immune response against melanoma [160].
Peripheral blood
immune cells and molecules
Lymph
node
Skin
Sentinel Skin immune system
lymph node
Immune cells
Lymph
node Infiltrating immune cells
Immune-related molecules
Tumor
FIG. 3. Interrelation of local tumoral immune molecules/cells with circulatory pool. Immune
cells circulate through the lymphatic/blood system in and out of the tumor. At the tumor site, the
infiltrating immune cells and all other immune-related molecules are mirrored in circulation.
Peripheral immune parameters can indicate an on-site immune response. Therapy efficacy
monitoring through circulatory immune parameters can be a proper reflection of both on-site
local immune response and the overall immune status of the patient.
An abnormal phenotypic marker CD8þ DRþ, found in other diseases [161],

was reported in 85% of cutaneous melanoma patients [162]. A few years ago,
it was published a peculiar pattern of circulating CD8þ T lymphocytes
bearing the CD161þ marker [162] related to the advanced stages of cutane-
ous melanoma.
The cytotoxic subpopulation of T lymphocytes was reported as elevated in
the blood circulation, namely, the cells bearing the phenotype CD3þ TCR
gammadeltaþ [163]. In stages I–III, the number of these cells were increased
compared to healthy, probably a good indicator of the ongoing antitumoral
cytotoxic immune response.
Tregs are a population of CD4þ that naturally develops functions that
sustain immune self-tolerance and inhibitory control mechanism of the host
immune response [164]. These cells can act on both innate and adaptive
118 MONICA NEAGU
60 2.5
50
2
% Subpopulation
40
CD4:CD8 ratio
1.5
30
1
20
0.5
10
0 0
I II III IV
Cutaneous melanoma stage
CD4 CD8 ratio
FIG. 4. Peripheral CD4þ and CD8þ T cells in correlation with the diagnosed cutaneous
melanoma staging. Investigated patients present a marked lymphopenia associated with a clear
disproportion of peripheral percentage of CD4þ and CD8þ. No matter the stage, the percentage
of peripheral CD4þ subpopulation statistically remains in the same range. The peripheral CD8þ
subpopulation increases clearly with the patient’s stage, thus doubling its value in stage IV in
comparison to stage I. Thus, the low CD4þ:CD8þ ratio can be correlated with the stage in which
the patient is diagnosed. This ratio can thus be considered an indicative of the suppressive status
of cutaneous melanoma patients and moreover a good marker for therapy efficacy.
immune arms. Up to now, it is known that Treg suppression is based on

soluble factors that can induce distant suppression and/or they can induce
suppressive mechanisms upon cell–cell contact [165].
Several cellular experimental models revealed some aspects regarding
Tregs action: they can be induced in normal peripheral blood mononuclear
cells (PBMCs) by melanoma-related factors, they enhance protumor
cytokines secretion like TGF-beta [166], and they proliferate in an Ag-
independent manner in inflammatory mimicking conditions like a tumor
would induce [167].
In cutaneous melanoma, Tregs suppress the immune responses, by inhibit-
ing the development of tumor-specific T cells and suppressing their effector
functions [168]. Peripheral Tregs can be detected in cutaneous melanoma
patients, and moreover in stage IV, they are double in absolute counts
compared to both normal subjects and stage I melanoma patients [151].
Looking for detailed molecular markers that can better characterize
Tregs associated to melanoma, recently, it has been demonstrated that the
transcriptional profile of activated Treg clones includes a TGF-beta signa-

ture. Data were published regarding the production of the latent form of
TGF-beta by both activated Treg and Th clones. The bioactive form of TGF-
beta is produced only by activated Tregs. TGF-beta has an autocrine action
on Tregs and can have paracrine actions on other T cells. Authors underline
that the transcriptional profile, namely, the reported epigenetic marker, was
better correlated with the suppressive activity of the Tregs, even in compari-
son with FOXP3 mRNA or protein expression [169].
CD4þCD25þ Tregs expressing FOXP3 are cells normally involved in im-
mune regulatory processes but can be powerful immune markers in tumor
pathologies like cutaneous melanoma, especially when a thorough characteri-
zation of patient’s immune status is needed along with a better monitoring of
therapy efficacy.
5.2.2.2. NK Cells—Marker for Antitumor Immune Response. Another
well-known cell with high antitumor activity, NK cell, was studied in relation
to cutaneous melanoma. It was proven, at least in animal models, that Tregs
can influence the antitumoral activity of NK cell [170]. Few years ago, the first
report on peripheral NK cells’ activation in melanoma patients was published
[162]. Killer cell immunoglobulin-like receptor 2DL1 (KIR2DL1) can interact
with HLA-C [171]. Authors found that patients with HLA-C (Lys80) had an
increased number of NK with the phenotype CD56þCD158aþ cells and that
these cells can be a prognosis marker.
Recently, melanoma patients’ were reported as having several significant NK
alterations: decreased NK activity, reduced NK-cell IFN-gamma production,
redistribution of NK-cell subsets with an increase in CD16dim, and a reduction
in CD16bright NK subsets. Confirming the previously described work, the
research group states that NKG2D and CD158b expression in patients can
be good candidates for suppressed NK function associated to skin melanoma
and another molecular marker related to disease progression [172].
The studies regarding peripheral NK and their activity in relation to
melanoma are not abundant, but there is a strong possibility that immune-
phenotyped NK cell along with their actual in vitro tested antitumoral
cytotoxicity should be taken into account when patients are immune moni-
tored during therapy and/or disease progression.
5.2.2.3. Dendritic Cells. As an important part of the innate immune
surveillance, the skin-related APCs comprise macrophages, LC, and dermal
DC [10]. In periphery, there are few studies that report correlations with
cutaneous melanoma. Stages I and IV melanoma patients have significantly
higher number of peripheral DCs in comparison with healthy controls [151].
Stage I melanoma patients had a significantly higher number of peripheral
DCs with CD123þ phenotype as compared to normal subjects. No matter the
stage, melanoma patients have a higher peripheral concentration of CD11cþ
120 MONICA NEAGU
myeloid DCs than controls. Especially for advanced stages, authors have
shown that in peripheral blood increased numbers of circulating DC and
Tregs can be detected, finding that suggests the immunosuppressive status of
the patient. They reported that stage I is characterized by a significantly
higher number of plasmacytoid DCs (pDCs) than normal subjects [151]. The
same group showed that in stage IV, there is an inverse correlation between
the peripheral DCs and Tregs, while only the peripheral Tregs appeared to be
associated with a shorter survival [173]. Last year, it was reported that no
difference between circulatory pDCs and mDCs in melanoma patients com-
pared with normal subjects was reported and that their specific molecular
pattern would drive them from the blood to the tumor [174].
Tumor microenvironment and cellular elements of the blood/lymph circu-
lation interrelate. Cellular communications go further than the tumor itself
and comprise signals coming in both senses from and to the circulation. The
deregulated mechanisms having as central immunosuppressive cell Tregs join
toward tumor initiation, development, and later toward metastasis (Fig. 5).
5.2.2.4. Circulating Cancer Cells. One of the newest research domains in
cutaneous melanoma is the study of CSCs [175]. Using several techniques,
among which quantitative reverse transcription polymerase chain reaction,
in peripheral blood of patients with melanoma, nestin was found in higher
quantities in stage IV patients, positively correlated with the expression of
tyrosinase and Melan-A [176]. The same group showed that in adjuvant
treated or untreated patients, this expression indicated an increased risk for
metastasis [177].
In the domain of circulating melanoma cells detection, the key words are
multimarker assay [178] and proteomic fingerprint that can detect in minute
quantities these circulating cancer cells [179]. Thus, several proteins were
identified as increased, transthyretin (TTR) and angiotensinogen (AGT),
while others were decreased, vitamin D-binding protein (DBP). After thera-
py, these values entered in the normal ranges. Authors proposed the DBP
decrease as a marker related to immune processes and an overall prognos-
ticator of disease [179].
We believe that, as for many other type of tumors, circulatory CSCs’
detection incubates one of the most important biomarker in a future prog-
nostic markers panel.
6. Monitoring Immune Therapy
Immune markers related to therapy monitoring are an important field for

at least two reasons: the lack of good clinical responses in immune therapy
and the need for accumulating knowledge regarding the molecular processes
Treg
Activation
CD4+ Effector T cells
TAM or DC IDO
FOXP3+
TGF-beta
CD8+ CD8+
CCR4
CCL22 FOXP3+
Control of
tumor
Recruitment
FOXP3+
Proliferation
FOXP3+ FOXP3+
TGF-beta
VGEF
CD4+ PGE2
T cell TGF-beta
IL-10
TGF-beta
Conversion
FOXP3+
DC
Tumor cell
FIG. 5. Immune suppression developed at tumoral site (reproduced from Neagu et al. [140]).
Macrophages secrete indoleamine 2,3-dioxygenase (IDO) that induce an inhibition of T-cell
proliferation due to tryptophan depletion (activation). Moreover, IDO recruits regulatory T cells
(FOXP3þ) at the tumoral site. Recruiting more TGF-beta-secreting Tregs, the suppression
induced on the effector couple CD4–CD8 increases and therefore the control of tumor develop-
ment decreases. Tumoral cells by themselves secrete TGF-beta, IL-10, VEGF, PGE2 that induce
DCs to secrete more TGF-beta contributing to the conversion of CD4þ T cells to Tregs
phenotype enhancing the cellular immune suppression (conversion). Skin-homing T cells CC-
chemokine receptor 4 (CCR4) bind to the CCL22 (macrophage-derived chemokine) of the
tumor-associated macrophages (TAM) and are recruited to the tumoral site (recruitment).
On the whole, a favorable microenvironment is created by the concerted action that has as a
result the proliferation of Tregs that hinder the cooperation CD4þ–CD8þ and therefore abolishes
the effector activity of antitumoral cytotoxic cells.
that governate tumor progression [180]. The intent of this section is not to
describe the immune-therapy approaches but to show the importance of
immune markers in monitoring therapy efficacy.
Peripheral immune cells were quantified both in relation to the monitor-
ing of the patients’ immune status, as already described, but in therapy
efficacy as well. Our experience showed that peripheral CD3þ population is
an extremely stable immune parameter and that the CD4/CD8 ratio can
monitor a good therapeutic response. Monitoring the dynamics of CD4/
CD8 ratio shows that the low registered value prior to therapy raises
122 MONICA NEAGU
toward normal values when therapy is established [140]. In the first reported
study of high-risk melanoma patients immunized with gp100 and tyrosinase
peptides [181], no difference in nevi tissue was found regarding CD3, CD4,
CD8, MHC-I, MHC-II, CD1a, HMB-45, MART-1, tyrosinase, but an
increase in p53 and bcl-2 staining, in the nevi posttreatment. Authors
explain that activating melanoma-specific T cells for preventing melanoma
recurrence a response mediated by p53 and bcl-2 is triggered in benign
melanocytes [181]. Another group showed that following transcutaneous
delivery, gp100 vaccination activates LC and antibody production, markers
of definitely immune activation [182].
In a phase I/II trial for melanoma vaccine comprising six melanoma-
associated peptides (MAGE proteins, MART-1/Melan-A, gp100, and tyros-
inase), patients’ follow-up was performed using the in vitro proliferation of
CD4þ lymphocytes. After vaccination, the monitoring of a good response
was marked by an increased proliferation of T cells to relevant peptides in
over 80% of patients correlated with good clinical response as well [183].
Another study enrolling stage III/IV melanoma patients showed the data
regarding patients vaccinated with Melan-A/Mart-1 peptide and Klebsiella
outer membrane protein p40 as an adjuvant. In this trial, the therapy was
monitored by ex vivo analysis of Melan-A/Mart-1-specific CD8 T cells.
Increased percentages of T cells, memory/effector T-cell differentiation, pos-
itive IFN-gamma, and antibody responses to p40 were observed in all
patients and positive clinical response in half of the treated patients [184].
As known, CD28 and CTLA-4 are major surface T molecules involved in
the regulation of immune activation and tolerance; while CD28 provides
positive modulatory signals in the early stages of an immune response,
CTLA-4 signaling inhibits T-cell activation, particularly during strong T-cell
responses. Both clinical and preclinical data indicate that CTLA-4 blockade
using anti-CTL-4 antibodies results in direct activation of CD4þ and CD8þ
effector cells in melanoma [185]. After anti-CTLA-4 treatment, patients were
monitored for Treg functions in PBLs. While prior to therapy, patients with
advanced melanoma had a severe CD4þ and CD8þ T-cell lymphopenia
assigned to Treg, after therapy, the effector and memory CD4þ and CD8þ
T-cell pool and TCR-dependent T-cell proliferation was restored. In this case,
progression-free survival and overall survival were directly correlated with
the resistance of peripheral lymphocytes to Treg-inhibitory effects [156].
The authors state that the biological activity marker of memory T-cell resis-
tance to Treg resulting from anti-CTLA-4 treatment is a good efficacy marker
[156,186]. In the biopsies of patients treated with anti-CTL4 diffuse intratu-
moral infiltrates of CD8þ T cells were found in good clinical outcome patients.
Patients with regressing tumors had an increased frequency of CD8þ cells
with/without a concomitant increase in CD4þ cells [186]. Last year, the results
of a phase II trial cutaneous melanoma patients receiving CTLA-4 antibody

for 1 year were published [187]. These results showed that the Ag-specific T-cell
immune responses had a degree of variability while no decrease in Tregs was
found. Better immune markers were Th-17 inducible frequency that pin-
pointed a surrogate marker. A classic plasma immune marker, C-reactive
protein (CRP), was found increased before treatment, and it was associated
with a good clinical outcome.
In a phase II, multicenter, open-label study using systemic administration
of TLR7 agonist (852A), several immune-related markers were taken as
therapy efficacy markers [188]. Immune effects of 852A were monitored by
measuring serum type I IFN and IP-10 together with the assessment of
immune cell markers in peripheral blood. Authors reported an increase in
monocyte’s CD86 expression and an increased serum IFN-alpha [188]. Stud-
ies regarding immune cells infiltrating the tumor after therapy with a novel
synthetic compound acting through the TLR7 revealed somewhat similar
results. Immune phenotyping revealed that during treatment, an increased
population of T lymphocytes positive for CD3, CD4, and CD8 as well as a
considerable number of cytotoxic cells (TIA-1þ, granzyme Bþ) and pDC CD
123þ was found [189]. The same compound acting on TLR7 was reported
to increase lymphocytic inflammation, associated with several markers to be
accounted for an active immune response: increased expression of chemokine
receptor CXCR3, a strong expression of myxovirus resistance protein
A (MxA), and type I IFN-inducible protein [190].
Cell’s functionality, like NK cytotoxicity, can be considered an efficacy
marker. Therefore, in another treatment approach, an open-label, multicen-
ter, single-arm, phase II pilot trial using a TLR9-stimulating oligodeoxynu-
cleotide patient was evaluated for immunological parameters as well. The
immunological parameters involved peripheral activated phenotype of pDC,
elevation of serum levels of 20 ,50 -oligoadenylate, a surrogate marker of IFN
I production, and a significant stimulation of NK cytotoxicity [191].
Phase I clinical studies with DC therapy [192] used as efficacy markers
T and NK cells [193,194]. When using isolated DCs in vitro activated with
CD40L, loaded with specific melanoma antigenic peptides autologously
injected in patients the monitoring was performed using skin reactions to
peptides alone or peptide-pulsed DCs along with circulating T cells [195].
The immune cells’ infiltration in delayed-type hypersensitivity (DTH) skin
biopsies is considered as efficacy markers. Punch biopsies taken from positive
DTH sites proved clusters of CD2þ- and CD3þ-infiltrating cells, out of which
over 50% were CD4þ, the rest being CD8þ T cells [196]. In another pilot trial,
infusions of DC pulsed with autologous tumor lysate were inoculated intra-
venously along with IL-2, IFN-alpha, and GM-CSF. The patients had
biopsies taken from the DTH site, and these biopsies showed an induction
124 MONICA NEAGU
of CD8þ T cells as well as both Th1- and Th2-type cells. Authors monitored
these parameters and considered them markers for effective Ag presentation
and further antitumor immune responses [197].
Melanoma is resistant to standard chemotherapy, having a response rate
for any single agent or combination of agents of 15–25%. Using combina-
tions of chemotherapy, IFN and IL-2, the response rate improved, with no
clear effect on overall survival. All the promising new therapeutic agents have
to be related to identification of predictors of response leading toward
personalized therapy [198]. Various lymphokines involved in the generation
of immune responses (particularly IL-2) were used in cutaneous melanoma
immune-therapy in the past 20 years. A couple of years ago, despite
promising phase II data, phase III studies have failed to show meaningful
clinical benefit for the combination of cytokines with cytotoxic chemothera-
py [199], thus identifying good predictors for therapeutic response is a
prerequisite. Following high-dose IL-2 administration, the number and fre-
quency of Tregs were monitored in patients with progressive disease and
the values returned to normal when patients had an objective good clinical
response [200]. In therapy comprising IL-2 and Melan-A-specific CTL, an
efficient antitumor response was monitored by an elevated frequency of
circulating Melan-A þ T cells, an increase in eosinophils and a selective loss
of Melan-A expression in lymph node metastases [201].
Investigating patients with intralesional IL-2 treatment, authors pointed out
an increase in the CD4:CD8 ratio and a rise in the percentage of CD25þ cells in
the CD4þ population, the majority of this population being activated T cells.
The local IL-2 is able to induce a systemic beneficial immunological effect [202].
IFN-alpha is one of the most used immune-therapy agent and significantly
prolonged relapse-free survival of patients diagnosed in stages IIB–III mela-
noma. When these patients were subjected to IFN-alpha2b therapy, a signifi-
cant decrease in serum levels of immunosuppressive/tumor angiogenic/
growth stimulatory factors was noted, decrease that correlated with a good
clinical outcome [152]. As therapeutic monitoring tools, gene microarray
analysis of the transcriptional profile of peripheral T cells, NK, and mono-
cytes was demonstrated. Authors pointed out that the transcriptional profiles
of PBMCs from IFN-alpha-treated patients may be a useful predictor of the
in vivo response of immune cells to IFN-alpha immunotherapy [203].
Viewing immunotherapy from a different perspective, namely the immune
response elicited toward the therapeutic agents, authors found other candi-
dates for immune-related biomarkers in melanoma. In stages II–III melano-
ma patients treated with IFN, various autoantibodies were tested. Obtained
data clearly indicated that the appearance of an autoimmune reaction is
associated with significant improvement in relapse-free survival and overall
survival [183].
In vitro studies demonstrated that in the absence of IL-2, Ag stimulation

resulted in T-cell activation and acquisition of effector function without
induction of FOXP3, indicating that the acquisition of effector function is
FOXP3 independent in CD8þ T cells [204]. IL-15, but not IL-7 or IL-21, led
to de novo induction of FOXP3 in Ag-specific CD8þ T cells, suggesting that
signaling by IL-2/IL-15Rbeta chain is crucial for the induction of FOXP3 in
human CD8þ T cells. These findings indicate that in vitro-induced expression
of FOXP3 cannot be simply interpreted as an indicator of Treg activity or
activation marker [204].
IL-12 as therapy agent has both immunoregulatory function and antitumor
activity mediated by the stimulation of T and NK effector cells. Authors
propose IL-12 as a therapeutical agent using a protocol to prescreen melanoma
patients for IL-12Rbeta2 expression to stratify the potential responders, admin-
istrate nontoxic doses, and target IL-12Rþ tumor cells, by local administration
or injection of IL-12 fused to an antibody specific to tumor cells [205].
Immune-therapy monitoring is one of the key points in quantifying effica-
cy, and in this framework, immune markers are the hallmark of patient’s
monitoring.
Up to date, immunotherapy approaches that are in the stage of clinical
development include cytokines (IL-2, IFN, TNF, IL-7, IL-12, IL-21), cytokine-
antibody fusion proteins or immunocytokines, whole tumor cell vaccines,
genetically modified tumor cells, heat-shock protein vaccines, peptide vaccines,
DCs pulsed with tumor Ags, tumor Ag-naked DNA vectors, recombinant
viral vectors, adoptive transfer of cloned tumor Ag-specific T cells, TLR
ligands, antagonistic antibodies CTLA4 (CD152), and activating antibodies
to CD40 and CD137 [206]. All these approaches seek to induce cytotoxic T-cell
responses to tumors ranging from monospecific immunotherapy, targeting
only one specific tumor Ag, to polyvalent immunotherapy, which attempts
to induce immune responses to multiple antigenic components, to gene-based
therapy, which manipulates the immunogenicity of the tumor. The goal of
each of these therapies is to induce proliferation and differentiation of anti-
tumoral Ag-specific memory T cells.
Each of the immunosuppressive mechanisms already presented can be
targets for clinical manipulation, and it is obvious that immune-related
parameters found their utility in immunotherapy monitoring.
7. Instead of Conclusions—Few Answered Questions
Cutaneous melanoma is the leader in skin tumor-induced death worldwide

[207], and from the immunological point of view, there are ‘‘black holes’’
regarding tumor initiation in the skin, unfavorable progression toward
126 MONICA NEAGU
dissemination and metastasis. Molecules that arise in the processes of neo-

plastic transformation, invasion, and metastasis are interrelated with the
components of the immune responses. Disseminating through lymphatic
network is a complex, multistep process, and therefore, several different
biomarkers in which immune markers have to be combined in order to
increase the prognostic power [129]. It is commonly agreed upon the fact
that markers like immune cells and cytokines are related to early stages and
can discriminate between responders and nonresponders patients subjected
to immunotherapy. Melanoma Ags recognized by the immune cells can be
found in body fluids and correlated more to the prognostic of the disease.
The failure of immune-therapies probably resides in the immunological
profile of the patient; thus, the need of finding predictive markers is manda-
tory in order to detect treatment outcome in melanoma.
Markers that were qualified in preclinical stages have failed in clinical trials
probably due to the immune status particularities of different patient popu-
lations [97] and other still undiscovered intimate interactions of established
parameters with immune-related factors.
A complete, individualized immunological profile of the patient can reorient/
personalize immune therapy and monitor the efficacy of the therapeutic
approach. The molecular diagnostic of skin melanoma will have important
immune components in the near future. As the pathophysiology of melanoma
is complex, the means for monitoring will use state-of-the-art proteomics/geno-
mics technology. It is more convincing that only a combination of markers from
genomics, transcriptomics, metabolomics, and proteomics domain can cover
the array of processes comprising the development of cutaneous melanoma.
7.1. CONSIDERATIONS
The immunosuppressive mechanisms that overpower the antitumoral
immune response are sustained by immature DC, neutrophils, Tregs,
myeloid-derived suppressor cells, and tumor-associated macrophages.
Tumor progression is associated with a subset of B-infiltrating lympho-
cytes and mature DCs combined with activated T lymphocytes—good
prognosis predictors [101].
The expression of tumoral MHC-II [208] is associated to the metastatic
potential of the tumor, while chemokine receptors’ expression in SLNs
favors tumor dissemination [209].
In the peripheral blood of the patients, a low ratio of CD4þ:CD8þ is
stage correlated [140], while circulatory Tregs indicate the immune sup-
pression status of the patient [210].
Low proportion of activated T (CD3þCD4þCD69þ) and NKT cells
(CD3þCD56þ) can be independent prognostic factors for overall
survival [159] and when combined with serum concentrations of cyto-

kines like IL-6 and IL-8, their predictive power increases [148].
For proper monitoring of immune-therapy efficacy markers to be
considered comprise Tregs [156,200], CD3þ, CD4þ, CD8þ, pDC
CD123þ [189].
Circulatory biomarkers whether immune cells and/or molecules that
sustain the cellular cross talk can be promising biomarker candidates in
cutaneous melanoma.
We acknowledge that this chapter is far from being exhaustive, but we
gathered the updated results of mainly preclinical and clinical trials and
discussed the main points of interest from the wealth of modern information
in order to prove the position of immune markers in the management of
cutaneous melanoma patient.
ACKNOWLEDGMENTS
Authors would thank for technical assistance and graphics to students Simina Neagu and
Irina Radu. This work was partially supported by the National Grants PN 09.33-01.01/2008,
NATO SfP 982838/2007.
REFERENCES
[1] E.P. Diamandis, Cancer biomarkers: can we turn recent failures into success? Commen-
tary. J. Natl. Cancer Inst. 102 (2010) 1462–1467, doi:10.1093/jnci/djq306.
[2] R.M. Nazarian, V.G. Prieto, D.E. Elder, L.M. Duncan, Melanoma biomarker expression
in melanocytic tumor progression: a tissue microarray study, J. Cutan. Pathol. 37 (1)
(2010) 41–47.
[3] J.D. Bos, The skin as an organ of immunity, Clin. Exp. Immunol. 107 (1) (1997) 3–5.
[4] J.D. Bos, Skin Immune System: Cutaneous Immunology and Clinical Immunodermatol-
ogy, third ed., CRC Press, Boca Raton/New York, 2005 pp. 3–13.
[5] T.S. Kupper, R.C. Fuhlbrigge, Immune surveillance in the skin: mechanisms and clinical
consequences, Nat. Rev. Immunol. 4 (3) (2004) 211–222.
[6] G.M. Woods, R.C. Malley, H.K. Muller, The skin immune system and the challenge of
tumour immunosurveillance, Eur. J. Dermatol. 15 (2) (2005) 63–69.
[7] F.M. Burnet, The concept of immunological surveillance, Prog. Exp. Tumor Res. 13
(1970) 1–27.
[8] S. Uthayakumar, R. Nandwani, T. Drinkwater, A.T. Nayagam, C.R. Darley, The preva-
lence of skin disease in HIV infection and its relationship to the degree of immunosuppres-
sion, Br. J. Dermatol. 137 (1997) 595–598.
[9] G. Lugo-Janer, J.L. Sánchez, E. Santiago-Delpin, Prevalence and clinical spectrum of skin
diseases in kidney transplant recipients, J. Am. Acad. Dermatol. 24 (1991) 410–414.
[10] C. Debenedictis, S. Joubeh, G. Zhang, M. Barria, R.F. Ghohestani, Immune functions of
the skin, Clin. Dermatol. 19 (2001) 573–585.
[11] B. Spellberg, The cutaneous citadel, a holistic view of skin and immunity, Life Sci. 67
(2000) 477–502.
128 MONICA NEAGU
[12] J.D. Bos, M.L. Kapsenberg, The skin immune system. Its cellular constituents and their
interactions, Immunol. Today 7 (1986) 235–240.
[13] Y. Reiss, A.E. Proudfoot, C.A. Power, J.J. Campbell, E.C. Butcher, CC chemokine
receptor (CCR)4 and the CCR10 ligand cutaneous T cell-attracting chemokine
(CTACK) in lymphocyte trafficking to inflamed skin, J. Exp. Med. 194 (2001) 1541–1547.
[14] J. Morales, B. Homey, A.P. Vicari, S. Hudak, E. Oldham, J. Hedrick, R. Orozco,
N.G. Copeland, N.A. Jenkins, L.M. McEvoy, A. Zlotnik, CTACK, a skin-associated
chemokine that preferentially attracts skin-homing memory T cells, Proc. Natl. Acad.
Sci. U.S.A. 96 (1999) 14470–14475.
[15] B. Homey, H. Alenius, A. Müller, H. Soto, E.P. Bowman, W. Yuan, L. McEvoy,
A.I. Lauerma, T. Assmann, E. Bünemann, M. Lehto, H. Wolff, D. Yen, H. Marxhausen,
W. To, J. Sedgwick, T. Ruzicka, P. Lehmann, A. Zlotnik, CCL27–CCR10 interactions
regulate T cell mediated skin inflammation, Nat. Med. 8 (2002) 157–165.
[16] S. Klunker, A. Trautmann, M. Akdis, J. Verhagen, P. Schmid-Grendelmeier, K. Blaser,
C.A. Akdis, A second step of chemotaxis after transendothelial migration: keratinocytes
undergoing apoptosis release IFN-gamma-inducible protein 10, monokine induced by
IFN-gamma, and IFN-gamma-inducible alpha-chemoattractant for T cell chemotaxis
toward epidermis in atopic dermatitis, J. Immunol. 171 (2003) 1078–1084.
[17] M.V. Dahl, Clinical Immunodermatology, third ed., Mosby, New York, 1996, pp. 121–133.
[18] M.B. Teunissen, C.W. Koomen, R. de Waal Malefyt, E.A. Wierenga, J.D. Bos, Interleu-
kin-17 and interferon-gamma synergize in the enhancement of proinflammatory cytokine
production by human keratinocytes, J. Invest. Dermatol. 111 (4) (1998) 645–649.
[19] L.M. Ebert, S. Meuter, B. Moser, Homing and function of human skin gammadelta T cells
and NK cells: relevance for tumor surveillance, J. Immunol. 176 (7) (2006) 4331–4336.
[20] V. Supajatura, H. Ushio, A. Nakao, S. Akira, K. Okumura, C. Ra, H. Ogawa, Differential
responses of mast cell Toll-like receptors 2 and 4 in allergy and innate immunity, J. Clin.
Invest. 109 (2002) 1351–1359.
[21] J.D. McCurdy, T.J. Olynych, L.H. Maher, J.S. Marshall, Cutting edge: distinct Toll-like
receptor 2 activators selectively induce different classes of mediator production from
human mast cells, J. Immunol. 170 (2003) 1625–1629.
[22] M.C. Pasch, J.D. Bos, M.R. Daha, S.S. Asghar, Transforming growth factor-beta iso-
forms regulate the surface expression of membrane cofactor protein (CD46) and CD59 on
human keratinocytes, Eur. J. Immunol. 29 (1) (1999) 100–108.
[23] R.C. Fuhlbrigge, T.S. Kupper, D.Y.M. Leung, M.W. Greaves (Eds.), Molecular Mechan-
isms of Allergic Skin Responses In: Allergic Skin Disease, New York, Marcel Dekker,
2000, pp. 29–51.
[24] A. Takashima, P.R. Bergstresser, Cytokine-mediated communication by keratinocytes
and Langerhans cells with dendritic epidermal T cells, Semin. Immunol. 8 (1996) 333–339.
[25] S. Stoll, G. Müller, M. Kurimoto, J. Saloga, T. Tanimoto, H. Yamauchi, H. Okamura,
J. Knop, A.H. Enk, Production of IL-18 (IFN-gamma-inducing factor) messenger RNA
and functional protein by murine keratinocytes, J. Immunol. 159 (1997) 298–302.
[26] D. Armerding, T.S. Kupper, Functional cutaneous lymphocyte antigen can be induced
in essentially all peripheral blood T lymphocytes, Int. Arch. Allergy Immunol. 119 (1999)
212–222.
[27] M. Akdis, S. Klunker, M. Schliz, K. Blaser, C.A. Akdis, Expression of cutaneous lympho-
cyte-associated antigen on human CD4þ and CD8þ TH2 cells, Eur. J. Immunol. 30
(2000) 3533–3541.
[28] J.W. Streilein, L.W. Lonsberry, P.R. Bergstresser, Depletion of epidermal Langerhans cells
and Ia immunogenicity from tape-stripped mouse skin, J. Exp. Med. 155 (1982) 863–871.
[29] G.B. Toews, P.R. Bergstresser, J.W. Streilein, Langerhans cells: sentinels of skin associated
lymphoid tissue, J. Invest. Dermatol. 75 (1980) 78–82.
[30] C. Robert, T.S. Kupper, Inflammatory skin diseases, T cells, and immune surveillance,
N. Engl. J. Med. 341 (1999) 1817–1828.
[31] K. Inaba, M. Inaba, N. Romani, H. Aya, M. Deguchi, S. Ikehara, S. Muramatsu,
Generation of large numbers of dendritic cells from mouse bone marrow cultures supple-
mented with granulocyte/macrophage colony-stimulating factor, J. Exp. Med. 176 (1992)
1693–1702.
[32] A. Lenz, M. Heine, G. Schuler, N. Romani, Human and murine dermis contain dendritic
cells, J. Clin. Invest. 92 (1993) 2587–2596.
[33] J. Banchereau, R.M. Steinman, Dendritic cells and the control of immunity, Nature 392
(1998) 245–252.
[34] D.N.J. Hart, Dendritic cells: unique leukocyte populations which control the primary
immune response, Blood 90 (1997) 3245–3287.
[35] P. Pierre, S.J. Turley, E. Gatti, M. Hull, J. Meltzer, A. Mirza, K. Inaba, R.M. Steinman,
I. Mellman, Developmental regulation of MHC class II transport in mouse dendritic cells,
Nature 388 (1997) 787–792.
[36] E. Schuller, B. Teichmann, J. Haberstok, M. Moderer, T. Bieber, A. Wollenberg, In situ
expression of the co-stimulatory molecules CD80 and CD86 on Langerhans cells and
inflammatory dendritic epidermal cells (IDEC) in atopic dermatitis, Arch. Dermatol.
Res. 293 (2001) 448–454.
[37] C. Weishaupt, K.N. Munoz, E. Buzney, T.S. Kupper, R.C. Fuhlbrigge, T-cell distribution
and adhesion receptor expression in metastatic melanoma, Clin. Cancer Res. 13 (9) (2007)
2549–2556.
[38] L.J. Picker, S.A. Michie, L.S. Rott, E.C. Butcher, A unique phenotype of skin-associated
lymphocytes in human. Preferential expression of the HECA-452 epitope by benign and
malignant T cells at cutaneous sites, Am. J. Pathol. 136 (1990) 1053–1068.
[39] C. Pitzalis, A. Cauli, N. Pipitone, C. Smith, J. Barker, A. Marchesoni, G. Yanni,
G.S. Panayi, Cutaneous lymphocyte antigen-positive T lymphocytes preferentially migrate
to the skin but not to the joint in psoriatic arthritis, Arthritis Rheum. 39 (1996) 137–145.
[40] L.A. Noorduyn, R.C. Beljaards, S.T. Pals, P. van Heerde, T. Radaszkiewicz, R. Willemze,
C.J. Meijer, Differential expression of the HECA-452 antigen (cutaneous lymphocyte
associated antigen, CLA) in cutaneous and non-cutaneous T-cell lymphomas, Histopa-
thology 21 (1992) 59–64.
[41] Y. Tanaka, A. Wake, K.J. Horgan, S. Murakami, M. Aso, K. Saito, S. Oda, I. Morimoto,
H. Uno, H. Kikuchi, Y. Izumi, S. Eto, Distinct phenotype of leukemic T cells with various
tissue tropisms, J. Immunol. 158 (1997) 3822–3829.
[42] J.C. Dudda, J.C. Simon, S. Martin, Dendritic cell immunization route determines CD8 þ
T cell trafficking to inflamed skin: role for tissue microenvironment and dendritic cells in
establishment of T cell-homing subsets, J. Immunol. 172 (2004) 857–863.
[43] L. Colantonio, A. Iellem, F. Sinigaglia, D. D’Ambrosio, Skin-homing CLA þ T cells and
regulatory CD25þ T cells represent major subsets of human peripheral blood memory
T cells migrating in response to CCL1/I-309, Eur. J. Immunol. 32 (2002) 3506–3514.
[44] T. Biedermann, C. Schwärzler, G. Lametschwandtner, G. Thoma, N. Carballido-Perrig,
J. Kund, J.E. de Vries, A. Rot, J.M. Carballido, Targeting CLA/E-selectin interactions
prevents CCR4-mediated recruitment of human TH2 memory cells to human skin in vivo,
Eur. J. Immunol. 32 (2002) 3171–3180.
[45] S. Hudak, M. Hagen, Y. Liu, D. Catron, E. Oldham, L.M. McEvoy, E.P. Bowman,
Immune surveillance and effector functions of CCR10þ skin homing T cells, J. Immunol.
169 (2002) 1189–1196.
130 MONICA NEAGU
[46] K. Ferenczi, R.C. Fuhlbrigge, J. Pinkus, G.S. Pinkus, T.S. Kupper, Increased CCR4
expression in cutaneous T cell lymphoma, J. Invest. Dermatol. 119 (2002) 1405–1410.
[47] E.J. Kunkel, J. Boisvert, K. Murphy, M.A. Vierra, M.C. Genovese, A.J. Wardlaw,
H.B. Greenberg, M.R. Hodge, L. Wu, E.C. Butcher, J.J. Campbell, Expression of the
chemokine receptors CCR4, CCR5, and CXCR3 by human tissue-infiltrating lympho-
cytes, Am. J. Pathol. 160 (2002) 347–355.
[48] G.H. Janssen, G.J. Tangelder, M.G. Oude Egbrink, R.S. Reneman, Spontaneous leuko-
cyte rolling in venules in untraumatized skin of conscious and anesthetized animals, Am.
J. Physiol. 267 (1994) H1199–H1204.
[49] C. Robert, R.C. Fuhlbrigge, J.D. Kieffer, S. Ayehunie, R.O. Hynes, G. Cheng, S. Grabbe,
U.H. von Andrian, T.S. Kupper, Interaction of dendritic cells with skin endothelium: a
new perspective on immunosurveillance, J. Exp. Med. 189 (1999) 627–636.
[50] B.F. Chong, J.-E. Murphy, T.S. Kupper, R.C. Fuhlbrigge, E-selectin, thymus- and activa-
tion-regulated chemokine/CCL17, and intercellular adhesion molecule-1 are constitutively
coexpressed in dermal microvessels: a foundation for a cutaneous immunosurveillance
system, J. Immunol. 172 (2004) 1575–1581.
[51] W. Weninger, L.H. Ulfman, G. Cheng, N. Souchkova, E.J. Quackenbush, J.B. Lowe,
U.H. von Andrian, Specialized contributions by alpha(1,3)-fucosyltransferase-IV and
FucT-VII during leukocyte rolling in dermal microvessels, Immunity 12 (2000)
665–676.
[52] R.W. Groves, E. Ross, J.N. Barker, J.S. Ross, R.D. Camp, D.M. MacDonald, Effect of
in vivo interleukin-1 on adhesion molecule expression in normal human skin, J. Invest.
Dermatol. 98 (1992) 384–387.
[53] F. Sallusto, D. Lenig, R. Forster, M. Lipp, A. Lanzavecchia, Two subsets of memory
T lymphocytes with distinct homing potentials and effector functions, Nature 401 (1999)
708–712.
[54] V. Umansky, O. Abschuetz, W. Osen, M. Ramacher, F. Zhao, M. Kato, D. Schadendorf,
Melanoma-specific memory T cells are functionally active in Ret transgenic mice without
macroscopic tumors, Cancer Res. 68 (22) (2008) 9451–9458.
[55] C.A. Foster, H. Yokozeki, K. Rappersberger, F. Koning, B. Volc-Platzer, A. Rieger,
J.E. Coligan, K. Wolff, G. Stingl, Human epidermal T-cells predominantly belong to the
lineage expressing alpha/beta T-cell receptor, J. Exp. Med. 171 (1990) 997–1013.
[56] K. Uyemura, R.J. Deans, H. Band, J. Ohmen, G. Panchamoorthy, C.T. Morita, T.H. Rea,
R.L. Modlin, Evidence for clonal selection of gamma/delta T cells in response to a human
pathogen, J. Exp. Med. 174 (1991) 83–92.
[57] J.D. Bos, I. Zonneveld, P.K. Das, S.R. Krieg, C.M. van der Loos, M.L. Kapsenberg, The
skin immune system (SIS): distribution and immunophenotype of lymphocyte subpopula-
tions in normal human skin, J. Invest. Dermatol. 88 (1987) 569–573.
[58] A.K. Abbas, A.H. Litchman, J.S. Pober, Cellular and molecular immunology, fourth ed.,
WB Saunders, Philadelphia, 2000, pp. 222–237.
[59] D.L. Norton, A. Haque, Insights into the role of GILT in HLA class II antigen processing
and presentation by melanoma, J. Oncol. 2009 (2009) 142959.
[60] S. Rana, S.N. Byrne, L.J. MacDonald, C.Y. Chan, G.M. Halliday, Ultraviolet
B suppresses immunity by inhibiting effector and memory T cells, Am. J. Pathol. 172 (4)
(2008) 993–1004.
[61] S. Di Nuzzo, R.M. Sylva-Steenland, C.W. Koomen, M.A. de Rie, P.K. Das, J.D. Bos,
M.B. Teunissen, Exposure to UVB induces accumulation of LFA-1þ T cells and enhanced
expression of the chemokine psoriasin in normal human skin, Photochem. Photobiol. 72
(3) (2000) 374–382.
[62] S. Nuzzo, R.M. Sylva-Steenland, M.A. de Rie, P.K. Das, J.D. Bos, M.B. Teunissen, UVB
radiation preferentially induces recruitment of memory CD4þ T cells in normal human
skin: long-term effect after a single exposure, J. Invest. Dermatol. 110 (6) (1998) 978–981.
[63] T.S. Kupper, R.W. Groves, The interleukin-1 axis and cutaneous inflammation, J. Invest.
Dermatol. 105 (1995) 62S–66S.
[64] J.E. Murphy, C. Robert, T.S. Kupper, Interleukin-1 and cutaneous inflammation: a
crucial link between innate and acquired immunity, J. Invest. Dermatol. 114 (2000)
602–608.
[65] C. Heufler, G. Topar, A. Grasseger, U. Stanzl, F. Koch, N. Romani, A.E. Namen,
G. Schuler, Interleukin 7 is produced by murine and human keratinocytes, J. Exp. Med.
178 (1993) 1109–1114.
[66] M. Allgower, G.A. Schoenenberger, B.G. Sparkes, Burning the largest immune organ,
Burns 21 (Suppl 1) (1995) S7–S47.
[67] H. Fujita, K.E. Nograles, T. Kikuchi, J. Gonzalez, J.A. Carucci, J.G. Krueger, Human
Langerhans cells induce distinct IL-22-producing CD4 þ T cells lacking IL-17 production,
Proc. Natl. Acad. Sci. U.S.A. 106 (51) (2009) 21795–21800.
[68] M.A. de Jong, L. de Witte, T.B. Geijtenbeek, Isolation of immature primary Langerhans
cells from human epidermal skin, Methods Mol. Biol. 595 (2010) 55–65.
[69] J. Hosol, G.F. Murphy, C.L. Egan, E.A. Lerner, S. Grabbe, A. Asahina, R.D. Granstein,
Regulation of Langerhans cell function by nerves containing calcitonin gene-related
peptide, Nature 363 (1993) 159–163.
[70] A.D. Hogan, A.W. Burks, Epidermal Langerhans’ cells and their function in the skin
immune system, Ann. Allergy Asthma Immunol. 75 (1995) 5–10.
[71] A. Kubo, K. Nagao, M. Yokouchi, H. Sasaki, M. Amagai, External antigen uptake by
Langerhans cells with reorganization of epidermal tight junction barriers, J. Exp. Med. 206
(13) (2009) 2937–2946.
[72] I.B. Kremer, R.M. Sylva-Steenland, J.D. Bos, M.B. Teunissen, Despite the presence of
UVB-induced DNA damage, HLA-DR þ cells from ex vivo UVB-exposed human skin
are able to migrate and show no impaired allostimulatory capacity, J. Invest. Dermatol.
109 (5) (1997) 626–631.
[73] T.S. Kupper, A.O. Chua, P. Flood, J. McGuire, U. Gubler, Interleukin 1 gene expression
in cultured human keratinocytes is augmented by ultraviolet irradiation, J. Clin. Invest. 80
(1987) 430–436.
[74] L.C. Wood, P.M. Elias, C. Calhoun, Barrier disruption stimulates interleukin-1alpha
expression and release from a preformed pool in murine epidermis, J. Invest. Dermatol.
106 (1996) 397–403.
[75] T.A. Luger, T. Scholzen, S. Grabbe, The role of alpha-melanocyte-stimulating hormone in
cutaneous biology, J. Investig. Dermatol. Symp. Proc. 2 (1997) 87–93.
[76] C.A. Dinarello, Interleukin-1, interleukin-1 receptors and interleukin-1 receptor antago-
nist, Int. Rev. Immunol. 16 (1998) 457–499.
[77] M. Steinhoff, T. Brzoska, T.A. Luger, Keratinocytes in epidermal immune responses,
Curr. Opin. Allergy Clin. Immunol. 1 (2001) 469–476.
[78] T.S. Kupper, Biology of the interleukin-1 receptor, J. Invest. Dermatol. 94 (1990)
146S–150S.
[79] R. Medzhitov, C.A. Janeway, Innate immunity: the virtues of a nonclonal system of
recognition, Cell 91 (1997) 295–298.
[80] P.J. Barnes, Nuclear factor-kB, Int. J. Biochem. Cell Biol. 29 (1997) 867–870.
[81] U.H. Von Andrian, C.R. Mackay, T-cell function and migration. Two sides of the same
coin, N. Engl. J. Med. 343 (2000) 1020–1034.
132 MONICA NEAGU
[82] G.T. Venneker, R.M. Vodegel, N. Okada, W. Westerhof, J.D. Bos, S.S. Asghar, Relative
contributions of decay accelerating factor (DAF), membrane cofactor protein (MCP) and
CD59 in the protection of melanocytes from homologous complement, Immunobiology
198 (4) (1998) 476–484.
[83] M.C. Pasch, N.H. Van Den Bosch, M.R. Daha, J.D. Bos, S.S. Asghar, Synthesis
of complement components C3 and factor B in human keratinocytes is differentially
regulated by cytokines, J. Invest. Dermatol. 114 (1) (2000) 78–82.
[84] K.K. Timár, M.C. Pasch, N.H. van den Bosch, H. Jarva, S. Junnikkala, S. Meri, J.D. Bos,
S.S. Asghar, Human keratinocytes produce the complement inhibitor factor H: synthesis is
regulated by interferon-gamma, Mol. Immunol. 43 (4) (2006) 317–325.
[85] K.K. Timár, S. Junnikkala, A. Dallos, H. Jarva, Z.A. Bhuiyan, S. Meri, J.D. Bos,
S.S. Asghar, Human keratinocytes produce the complement inhibitor factor I: synthesis
is regulated by interferon-gamma, Mol. Immunol. 44 (11) (2007) 2943–2949.
[86] K.K. Timár, A. Dallos, M. Kiss, S. Husz, J.D. Bos, S.S. Asghar, Expression of terminal
complement components by human keratinocytes, Mol. Immunol. 44 (10) (2007)
2578–2586.
[87] M.C. Pasch, N. Okada, J.D. Bos, S.S. Asghar, Effects of UVB on the synthesis of
complement proteins by keratinocytes, J. Invest. Dermatol. 111 (4) (1998) 683–688.
[88] G. Piskin, J.D. Bos, M.B. Teunissen, Neutrophils infiltrating ultraviolet B-irradiated
normal human skin display high IL-10 expression, Arch. Dermatol. Res. 296 (7) (2005)
339–342.
[89] H.K. Muller, C.D. Bucana, M.L. Kripke, Antigen presentation in the skin: modulation by
u.v. radiation and chemical carcinogens, Semin. Immunol. 4 (1992) 205–215.
[90] G.A. Rabinovich, D. Gabrilovich, E.M. Sotomayor, Immunosuppressive strategies that
are mediated by tumor cells, Annu. Rev. Immunol. 25 (2007) 267–296.
[91] J.M. Reiman, M. Kmieciak, M.H. Manjili, K.L. Knutson, Tumor immunoediting and
immunosculpting pathways to cancer progression, Semin. Cancer Biol. 17 (4) (2007)
275–287.
[92] E. Zattra, A.B. Fortina, M. Bordignon, S. Piaserico, M. Alaibac, Immunosuppression and
melanocyte proliferation, Melanoma Res. 19 (2) (2009) 63–68.
[93] T.F. Gajewski, Failure at the effector phase: immune barriers at the level of the melanoma
tumor microenvironment, Clin. Cancer Res. 13 (18 Pt. 1) (2007) 5256–5261.
[94] A.N. Crowson, C.M. Magro, M.C. Mihm, Prognosticators of melanoma, the melanoma
report, and the sentinel lymph node, Mod. Pathol. 19 (Suppl. 2) (2006) S71–S87.
[95] A.K. Bosserhoff, Novel biomarkers in malignant melanoma, Clin. Chim. Acta 367 (1–2)
(2006) 28–35.
[96] J. Utikal, D. Schadendorf, S. Ugurel, Serologic and immunohistochemical prognostic
biomarkers of cutaneous malignancies, Arch. Dermatol. Res. 298 (10) (2007) 469–477.
[97] A. Spatz, G. Batist, A.M. Eggermont, The biology behind prognostic factors of cutaneous
melanoma, Curr. Opin. Oncol. 22 (3) (2010) 163–168.
[98] L.M. Ebert, S. Meuter, B. Moser, Homing and function of human skin gammadelta T cells
and NK cells: relevance for tumor surveillance, J. Immunol. 176 (7) (2006) 4331–4336.
[99] F. Piras, R. Colombari, L. Minerba, D. Murtas, C. Floris, C. Maxia, A. Corbu,
M.T. Perra, P. Sirigu, The predictive value of CD8, CD4, CD68, and human leukocyte
antigen-D-related cells in the prognosis of cutaneous malignant melanoma with vertical
growth phase, Cancer 104 (6) (2005) 1246–1254.
[100] M.R. Hussein, D.A. Elsers, S.A. Fadel, A.E. Omar, Immunohistological characterisation
of tumour infiltrating lymphocytes in melanocytic skin lesions, J. Clin. Pathol. 59 (3)
(2006) 316–324.
[101] A. Ladányi, J. Kiss, B. Somlai, K. Gilde, Z. Fejos, A. Mohos, I. Gaudi, J. Tı́már, Density
of DC-LAMP(þ) mature dendritic cells in combination with activated T lymphocytes
infiltrating primary cutaneous melanoma is a strong independent prognostic factor,
Cancer Immunol. Immunother. 56 (9) (2007) 1459–1469.
[102] O. Simonetti, G. Goteri, G. Lucarini, C. Rubini, D. Stramazzotti, L. Lo Muzio,
G. Biagini, A. Offidani, In melanoma changes of immature and mature dendritic cell
expression correlate with tumor thickness: an immunohistochemical study, Int.
J. Immunopathol. Pharmacol. 20 (2) (2007) 325–333.
[103] V. Mourmouras, M. Fimiani, P. Rubegni, M.C. Epistolato, V. Malagnino, C. Cardone,
E. Cosci, M.C. Nisi, C. Miracco, Evaluation of tumour-infiltrating CD4þCD25þFOXP3
þ regulatory T cells in human cutaneous benign and atypical naevi, melanomas and
melanoma metastases, Br. J. Dermatol. 157 (3) (2007) 531–539.
[104] G. De Panfilis, N. Campanini, M. Santini, G. Mori, E. Tognetti, R. Maestri, M. Lombardi,
E. Froio, D. Ferrari, R. Ricci, Phase- and stage-related proportions of T cells bearing the
transcription factor FOXP3 infiltrate primary melanoma, J. Invest. Dermatol. 128 (3) (2008)
676–684.
[105] F. Hillen, C.I. Baeten, A. van de Winkel, D. Creytens, D.W. van der Schaft,
V. Winnepenninckx, A.W. Griffioen, Leukocyte infiltration and tumor cell plasticity are
parameters of aggressiveness in primary cutaneous melanoma, Cancer Immunol. Immun-
other. 57 (1) (2008) 97–106.
[106] C. Miracco, V. Mourmouras, M. Biagioli, P. Rubegni, S. Mannucci, I. Monciatti,
E. Cosci, P. Tosi, P. Luzi, Utility of tumour-infiltrating CD25þFOXP3þ regulatory
T cell evaluation in predicting local recurrence in vertical growth phase cutaneous mela-
noma, Oncol. Rep. 18 (5) (2007) 1115–1122.
[107] M. Costache, M. Neagu, A. Petrescu, C. Constantin, G. Manda, C.D. Vrabie, M. Waller,
C.S. Petrescu, Simionescu O Statistical correlations between peripheral blood lymphocyte
subpopulations and tumor inflammatory infiltrate in stage I of skin melanoma, Rom.
J. Morphol. Embryol. 51 (4) (2010) 693–699.
[108] J. Kiss, Examination of different factors influencing the vascularization of human cutane-
ous melanoma, Magy. Onkol. 52 (4) (2008) 385–389.
[109] D. Massi, C. Marconi, A. Franchi, F. Bianchini, M. Paglierani, S. Ketabchi, C. Miracco,
M. Santucci, L. Calorini, Arginine metabolism in tumor-associated macrophages in cuta-
neous malignant melanoma: evidence from human and experimental tumors, Hum.
Pathol. 38 (10) (2007) 1516–1525.
[110] C. Li, Z. Hu, Z. Liu, L.E. Wang, J.E. Gershenwald, J.E. Lee, V.G. Prieto, M. Duvic,
E.A. Grimm, Q. Wei, Polymorphisms of the neuronal and inducible nitric oxide synthase
genes and the risk of cutaneous melanoma: a case-control study, Cancer 109 (8) (2007)
1570–1578.
[111] S. Scala, A. Ottaiano, P.A. Ascierto, M. Cavalli, E. Simeone, P. Giuliano, M. Napolitano,
R. Franco, G. Botti, G. Castello, Expression of CXCR4 predicts poor prognosis in
patients with malignant melanoma, Clin. Cancer Res. 11 (5) (2005) 1835–1841.
[112] S. Scala, P. Giuliano, P.A. Ascierto, C. Ieranò, R. Franco, M. Napolitano, A. Ottaiano,
M.L. Lombardi, M. Luongo, E. Simeone, D. Castiglia, F. Mauro, I. De Michele,
R. Calemma, G. Botti, C. Caracò, G. Nicoletti, R.A. Satriano, G. Castello, Human
melanoma metastases express functional CXCR4, Clin. Cancer Res. 12 (8) (2006)
2427–2433.
[113] S. Qin, J.B. Rottman, P. Myers, N. Kassam, M. Weinblatt, M. Loetscher, A.E. Koch,
B. Moser, C.R. Mackay, The chemokine receptors CXCR3 and CCR5 mark subsets
of T cells associated with certain inflammatory reactions, J. Clin. Invest. 101 (4) (1998)
746–754.
134 MONICA NEAGU
[114] C. Monteagudo, J.M. Martin, E. Jorda, A. Llombart-Bosch, CXCR3 chemokine receptor

immunoreactivity in primary cutaneous malignant melanoma: correlation with clinico-
pathological prognostic factors, J. Clin. Pathol. 60 (6) (2007) 596–599.
[115] W. Wang, H. Soto, E.R. Oldham, M.E. Buchanan, B. Homey, D. Catron, N. Jenkins,
N.G. Copeland, D.J. Gilbert, N. Nguyen, J. Abrams, D. Kershenovich, K. Smith,
T. McClanahan, A.P. Vicari, A. Zlotnik, Identification of a novel chemokine (CCL28),
which binds CCR10 (GPR2), J. Biol. Chem. 275 (2000) 22313–22323.
[116] O. Simonetti, G. Goteri, G. Lucarini, A. Filosa, T. Pieramici, C. Rubini, G. Biagini,
A. Offidani, Potential role of CCL27 and CCR10 expression in melanoma progression
and immune escape, Eur. J. Cancer 42 (8) (2006) 1181–1187.
[117] H. Seidl, E. Richtig, H. Tilz, M. Stefan, U. Schmidbauer, M. Asslaber, K. Zatloukal,
M. Herlyn, H. Schaider, Profiles of chemokine receptors in melanocytic lesions: de novo
expression of CXCR6 in melanoma, Hum. Pathol. 38 (5) (2007) 768–780.
[118] W.M. Howell, M.J. Rose-Zerilli, J.M. Theaker, A.C. Bateman, ICAM-1 polymorphisms
and development of cutaneous malignant melanoma, Int. J. Immunogenet. 32 (6) (2005)
367–373.
[119] M. Vinceti, G. Pellacani, B. Casali, C. Malagoli, D. Nicoli, E. Farnetti, S. Bassissi,
M. Bergomi, S. Seidenari, High risk of cutaneous melanoma amongst carriers of the
intercellular adhesion molecule-1 R241 allele, Melanoma Res. 16 (1) (2006) 93–96.
[120] K. Zen, D.-Q. Liu, L.-M. Li, C.X.-J. Chen, Y.-L. Guo, B. Ha, X. Chen, C.-Y. Zhang,
Y. Liu, The heparan sulfate proteoglycan form of epithelial CD44v3 serves as a CD11b/
CD18 counter-receptor during polymorphonuclear leukocyte transepithelial migration,
JBC 284 (2009) 3768–3776.
[121] M.D. Pacifico, R. Grover, P.I. Richman, F.M. Daley, F. Buffa, G.D. Wilson, CD44v3
levels in primary cutaneous melanoma are predictive of prognosis: assessment by the use of
tissue microarray, Int. J. Cancer 118 (6) (2006) 1460–1464.
[122] S. Damm, P. Koefinger, M. Stefan, C. Wels, G. Mehes, E. Richtig, H. Kerl, M. Otte,
H. Schaider, HGF-promoted motility in primary human melanocytes depends on CD44v6
regulated via NF-kappa B, Egr-1, and C/EBP-beta, J. Invest. Dermatol. 130 (7) (2010)
1893–1903.
[123] C. Gedye, J. Quirk, J. Browning, S. Svobodová, T. John, P. Sluka, P.R. Dunbar, D. Corbeil,
J. Cebon, I.D. Davis, Cancer/testis antigens can be immunological targets in clonogenic
CD133þ melanoma cells, Cancer Immunol. Immunother. 58 (10) (2009) 1635–1646.
[124] R. Lazova R, B.E. Gould Rothberg, D. Rimm, G. Scott, The semaphorin 7A receptor Plexin
C1 is lost during melanoma metastasis, Am. J. Dermatopathol. 31 (2) (2009) 177–181.
[125] S. Brychtova, M. Fiuraskova, A. Hlobilková, T. Brychta, J. Hirnak, Nestin expression
in cutaneous melanomas and melanocytic nevi, J. Cutan. Pathol. 34 (5) (2007) 370–375.
[126] D. Mihic-Probst, A. Kuster, S. Kilgus, B. Bode-Lesniewska, B. Ingold-Heppner,
C. Leung, M. Storz, B. Seifert, S. Marino, P. Schraml, R. Dummer, H. Moch, Consistent
expression of the stem cell renewal factor BMI-1 in primary and metastatic melanoma, Int.
J. Cancer 121 (8) (2007) 1764–1770.
[127] B.K. Sharma, V. Manglik, E.G. Elias, Immuno-expression of human melanoma stem cell
markers in tissues at different stages of the disease, J. Surg. Res. 163 (1) (2010) e11–e15.
[128] V.K. Sondak, J.L. Messina, Current status of biomarkers for melanoma metastasis,
IDrugs 9 (9) (2006) 627–631.
[129] M. Wobser, C. Siedel, D. Schrama, E.B. Bröcker, J.C. Becker, C.S. Vetter-Kauczok,
Expression pattern of the lymphatic and vascular markers VEGFR-3 and CD31 does
not predict regional lymph node metastasis in cutaneous melanoma, Arch. Dermatol. Res.
297 (8) (2006) 352–357.
[130] C. Demirkesen, N. Büyükpinarbaşili, R. Ramazanoğlu, O. Oğuz, N.M. Mandel,

G. Kaner, The correlation of angiogenesis with metastasis in primary cutaneous melano-
ma: a comparative analysis of microvessel density, expression of vascular endothelial
growth factor and basic fibroblastic growth factor, Pathology 38 (2) (2006) 132–137.
[131] D. Massi, M. Landriscina, A. Piscazzi, E. Cosci, A. Kirov, M. Paglierani, C. Di Serio,
V. Mourmouras, S. Fumagalli, M. Biagioli, I. Prudovsky, C. Miracco, M. Santucci,
N. Marchionni, F. Tarantini, S100A13 is a new angiogenic marker in human melanoma,
Mod. Pathol. 23 (6) (2010) 804–813.
[132] M.G. Tucci, G. Lucarini, D. Brancorsini, A. Zizzi, A. Pugnaloni, A. Giacchetti, G. Ricotti,
G. Biagini, Involvement of E-cadherin, beta-catenin, Cdc42 and CXCR4 in the progres-
sion and prognosis of cutaneous melanoma, Br. J. Dermatol. 157 (6) (2007) 1212–1216.
[133] S. Izraely, A. Klein, O. Sagi-Assif, T. Meshel, G. Tsarfaty, D.S. Hoon, I.P. Witz, Chemo-
kine-chemokine receptor axes in melanoma brain metastasis, Immunol. Lett. 130 (1–2)
(2010) 107–114.
[134] K.J. Radford, R.F. Thorne, P. Hersey, CD63 associates with transmembrane 4 superfami-
ly members, CD9 and CD81, and with beta 1 integrins in human melanoma, Biochem.
Biophys. Res. Commun. 222 (1) (1996) 13–18.
[135] Y. Wu, X.B. Zhang, H.J. Liu, L.D. Zheng, J.W. Li, Y. Lin, Expression of neutral
endopeptidase and motility-related protein-1 in cutaneous malignant melanoma,
Zhonghua Bing Li Xue Za Zhi 36 (7) (2007) 466–469.
[136] F.F. Amersi, A.M. Terando, Y. Goto, R.A. Scolyer, J.F. Thompson, A.N. Tran,
M.B. Faries, D.L. Morton, D.S. Hoon, Activation of CCR9/CCL25 in cutaneous melano-
ma mediates preferential metastasis to the small intestine, Clin. Cancer Res. 14 (3) (2008)
638–645.
[137] S.K. Huang, M.M. Darfler, M.B. Nicholl, J. You, K.G. Bemis, T.J. Tegeler, M. Wang,
J.P. Wery, K.K. Chong, L. Nguyen, R.A. Scolyer, D.S. Hoon, LC/MS-based quantitative
proteomic analysis of paraffin-embedded archival melanomas reveals potential proteomic
biomarkers associated with metastasis, PLoS One 4 (2) (2009) e4430.
[138] B.E. Gould Rothberg, M.B. Bracken, D.L. Rimm, Tissue biomarkers for prognosis in
cutaneous melanoma: a systematic review and meta-analysis, J. Natl. Cancer Inst. 101 (7)
(2009) 452–474.
[139] F.I. Staquicini, A. Tandle, S.K. Libutti, J. Sun, M. Zigler, M. Bar-Eli, F. Aliperti,
E.C. Pérez, J.E. Gershenwald, M. Mariano, R. Pasqualini, W. Arap, J.D. Lopes, A subset
of host B-lymphocytes control melanoma metastasis through a MCAM/MUC18-dependent
interaction: evidence from mice and humans, Cancer Res. 68 (20) (2008) 8419–8428.
[140] M. Neagu, C. Constantin, C. Tanase, Immune-related biomarkers for diagnosis/prognosis
and therapy monitoring of cutaneous melanoma, Expert Rev. Mol. Diagn. 10 (7) (2010)
897–919.
[141] V.B. Shidham, C.C. Chang, R. Komorowski, MCW melanoma cocktail for the evaluation
of micrometastases in sentinel lymph nodes of cutaneous melanoma, Expert Rev. Mol.
Diagn. 5 (3) (2005) 281–290.
[142] S.S. Dadras, B. Lange-Asschenfeldt, P. Velasco, L. Nguyen, A. Vora, A. Muzikansky,
K. Jahnke, A. Hauschild, S. Hirakawa, M.C. Mihm, M. Detmar, Tumor lymphangiogen-
esis predicts melanoma metastasis to sentinel lymph nodes, Mod. Pathol. 18 (9) (2005)
1232–1242.
[143] D. Sahni, A. Robson, G. Orchard, R. Szydlo, A.V. Evans, R. Russell-Jones, The use of
LYVE-1 antibody for detecting lymphatic involvement in patients with malignant mela-
noma of known sentinel node status, J. Clin. Pathol. 58 (7) (2005) 715–721.
136 MONICA NEAGU
[144] D. Massi, M.C. De Nisi, A. Franchi, V. Mourmouras, G. Baroni, J. Panelos, M. Santucci,

C. Miracco, Inducible nitric oxide synthase expression in melanoma: implications in
lymphangiogenesis, Mod. Pathol. 22 (1) (2009) 21–30.
[145] B. Negin, D. Panka, W. Wang, M. Siddiqui, N. Tawa, J. Mullen, S. Tahan, L. Mandato,
A. Polivy, J. Mier, M. Atkins, Effect of melanoma on immune function in the regional
lymph node basin, Clin. Cancer Res. 14 (3) (2008) 654–659.
[146] M. Baniyash, TCR z-chain downregulation: curtailing an excessive inflammatory immune
response, Nat. Rev. Immunol. 4 (2004) 675–687.
[147] M. Sarff, D. Edwards, B. Dhungel, K.W. Wegmann, C. Corless, A.D. Weinberg,
J.T. Vetto, OX40 (CD134) expression in sentinel lymph nodes correlates with prognostic
features of primary melanomas, Am. J. Surg. 195 (5) (2008) 621–625.
[148] C. Soubrane, O. Rixe, J.B. Meric, D. Khayat, R. Mouawad, Pretreatment serum interleu-
kin-6 concentration as a prognostic factor of overall survival in metastatic malignant
melanoma patients treated with biochemotherapy: a retrospective study, Melanoma Res.
15 (3) (2005) 199–204.
[149] F. Tas, H. Oguz, A. Argon, D. Duranyildiz, H. Camlica, V. Yasasever, E. Topuz, The
value of serum levels of IL-6, TNF-alpha, and erythropoietin in metastatic malignant
melanoma: serum IL-6 level is a valuable prognostic factor at least as serum LDH in
advanced melanoma, Med. Oncol. 22 (3) (2005) 241–246.
[150] R. Botella-Estrada, M. Escudero, J.E. O’Connor, E. Nagore, B. Fenollosa, O. Sanmartı́n,
C. Requena, C. Guillén, Cytokine production by peripheral lymphocytes in melanoma,
Eur. Cytokine Netw. 16 (1) (2005) 47–55.
[151] M.D. McCarter, J. Baumgartner, G.A. Escobar, D. Richter, K. Lewis, W. Robinson,
C. Wilson, B.E. Palmer, R. Gonzalez, Immunosuppressive dendritic and regulatory T cells
are upregulated in melanoma patients, Ann. Surg. Oncol. 14 (10) (2007) 2854–2860.
[152] Z.R. Yurkovetsky, J.M. Kirkwood, H.D. Edington, A.M. Marrangoni, L. Velikokhatnaya,
M.T. Winans, E. Gorelik, A.E. Lokshin, Multiplex analysis of serum cytokines in melanoma
patients treated with interferon-alpha2b, Clin. Cancer Res. 13 (8) (2007) 2422–2428.
[153] G. Malaponte, A. Zacchia, Y. Bevelacqua, A. Marconi, R. Perrotta, M.C. Mazzarino,
V. Cardile, F. Stivala, Co-regulated expression of matrix metalloproteinase-2 and trans-
forming growth factor-beta in melanoma development and progression, Oncol. Rep. 24 (1)
(2010) 81–87.
[154] K. Hoek, D.L. Rimm, K.R. Williams, H. Zhao, S. Ariyan, A. Lin, H.M. Kluger, A.J. Berger,
E. Cheng, E.S. Trombetta, T. Wu, M. Niinobe, K. Yoshikawa, G.E. Hannigan, R. Halaban,
Expression profiling reveals novel pathways in the transformation of melanocytes to
melanomas, Cancer Res. 64 (15) (2004) 5270–5282.
[155] R.J. Critchley-Thorne, N. Yan, S. Nacu, J. Weber, S.P. Holmes, P.P. Lee, Down-regula-
tion of the interferon signaling pathway in T lymphocytes from patients with metastatic
melanoma, PLoS Med. 4 (5) (2007) e176 897–911.
[156] C. Ménard, F. Ghiringhelli, S. Roux, N. Chaput, C. Mateus, U. Grohmann, S. Caillat-
Zucman, L. Zitvogel, C. Robert, CTLA-4 blockade confers lymphocyte resistance to
regulatory T-cells in advanced melanoma: surrogate marker of efficacy of tremelimumab?
Clin. Cancer Res. 14 (16) (2008) 5242–5249.
[157] M. Neagu, C. Constantin, G. Manda, I. Margaritescu, Biomarkers of metastatic melano-
ma, Biomark. Med. 3 (1) (2009) 71–89.
[158] D. Ilkovitch, D.M. Lopez, Immune modulation by melanoma-derived factors, Exp. Der-
matol. 17 (12) (2008) 977–985.
[159] M. Hernberg, P.S. Mattila, M. Rissanen, J. Hansson, S. Aamdal, L. Bastholt, H. von der
Maase, H. Schmidt, U. Stierner, J. Tarkkanen, The prognostic role of blood lymphocyte
subset distribution in patients with resected high-risk primary or regionally metastatic
melanoma, J. Immunother. 30 (7) (2007) 773–779.
[160] J.G. Casado, R. Soto, O. DelaRosa, E. Peralbo, M. del Carmen Muñoz-Villanueva,
L. Rioja, J. Peña, R. Solana, R. Tarazona, CD8 T cells expressing NK associated receptors
are increased in melanoma patients and display an effector phenotype, Cancer Immunol.
Immunother. 54 (12) (2005) 1162–1171.
[161] J.-F. Viallard, P. Blanco, M. André, G. Etienne, F. Liferman, D. Neau, E. Vidal,
J.-F. Moreau, J.-L. Pellegrin, CD8þHLA-DRþ T lymphocytes are increased in common
variable immunodeficiency patients with impaired memory B-cell differentiation, Clin.
Immunol. 119 (1) (2006) 51–58.
[162] J.A. Campillo, J.A. Martı́nez-Escribano, M.R. Moya-Quiles, L.A. Marı́n, M. Muro,
N. Guerra, A. Parrado, M. Campos, J.F. Frı́as, A. Minguela, A.M. Garcı́a-Alonso,
M.R. Alvarez-López, Natural killer receptors on CD8 T cells and natural killer cells
from different HLA-C phenotypes in melanoma patients, Clin. Cancer Res. 12 (16)
(2006) 4822–4831.
[163] J.A. Campillo, J.A. Martı́nez-Escribano, A. Minguela, R. López-Alvarez, L.A. Marı́n,
A.M. Garcı́a-Alonso, A. Bensussan, M.R. Alvarez-López, Increased number of cytotoxic
CD3þ CD28 gammadelta T cells in peripheral blood of patients with cutaneous malig-
nant melanoma, Dermatology 214 (4) (2007) 283–288.
[164] F. Avogadri, J. Yuan, A. Yang, D. Schaer, J.D. Wolchok, Modulation of CTLA-4 and
GITR for Cancer Immunotherapy, Curr. Top. Microbiol. Immunol. 344 (2010) 211–244.
[165] Z. Fehérvari, S. Sakaguchi, CD4þ Tregs and immune control, J. Clin. Invest. 114 (9)
(2004) 1209–1217.
[166] J. Baumgartner, C. Wilson, B. Palmer, D. Richter, A. Banerjee, M. McCarter, Melanoma
induces immunosuppression by up-regulating FOXP3(þ) regulatory T cells, J. Surg. Res.
141 (1) (2007) 72–77.
[167] R.A. Clark, T.S. Kupper, IL-15 and dermal fibroblasts induce proliferation of natural
regulatory T cells isolated from human skin, Blood 109 (1) (2007) 194–202.
[168] L. Vence, A.K. Palucka, J.W. Fay, T. Ito, Y.J. Liu, J. Banchereau, H. Ueno, Circulating
tumor antigen-specific regulatory T cells in patients with metastatic melanoma, PNAS 18
(2) (2007) 59–70.
[169] J. Stockis, W. Fink, V. François, T. Connerotte, C. de Smet, L. Knoops, P. van der
Bruggen, T. Boon, P.G. Coulie, S. Lucas, Comparison of stable human Treg and Th
clones by transcriptional profiling, Eur. J. Immunol. 39 (3) (2009) 869–882.
[170] F. Ghiringhelli, C. Ménard, M. Terme, C. Flament, J. Taieb, N. Chaput, P.E. Puig,
S. Novault, B. Escudier, E. Vivier, A. Lecesne, C. Robert, J.Y. Blay, J. Bernard, S. Caillat-
Zucman, A. Freitas, T. Tursz, O. Wagner-Ballon, C. Capron, W. Vainchencker, F. Martin,
L. Zitvogel, CD4þCD25þ regulatory T cells inhibit natural killer cell functions in a trans-
forming growth factor-beta-dependent manner, J. Exp. Med. 202 (8) (2005) 1075–1085.
[171] E. Baba, R. Erskine, J.E. Boyson, G.B. Cohen, D.M. Davis, P. Malik, O. Mandelboim,
H.T. Reyburn, J.L. Strominger, N-linked carbohydrate on human leukocyte antigen-C
and recognition by natural killer cell inhibitory receptors, Hum. Immunol. 61 (12) (2000)
1202–1218.
[172] G. Konjević, K. Mirjacić Martinović, V. Jurisić, N. Babović, I. Spuzić, Biomarkers of
suppressed natural killer (NK) cell function in metastatic melanoma: decreased NKG2D
and increased CD158a receptors on CD3-CD16þ NK cells, Biomarkers 14 (4) (2009)
258–270.
138 MONICA NEAGU
[173] J.M. Baumgartner, R. Gonzalez, K.D. Lewis, W.A. Robinson, D.A. Richter, B.E. Palmer,
C.C. Wilson, M.D. McCarter, Increased survival from stage IV melanoma associated with
fewer regulatory T Cells, J. Surg. Res. 154 (1) (2009) 13–20.
[174] J. Charles, J. Di Domizio, D. Salameire, N. Bendriss-Vermare, C. Aspord, R. Muhammad,
C. Lefebvre, J. Plumas, M.T. Leccia, L. Chaperot, Characterization of circulating dendrit-
ic cells in melanoma: role of CCR6 in plasmacytoid dendritic cell recruitment to the tumor,
J. Invest. Dermatol. 130 (6) (2010) 1646–1656.
[175] M. Sabatino, D.F. Stroncek, H. Klein, F.M. Marincola, E. Wang, Stem cells in melanoma
development, Cancer Lett. 279 (2) (2009) 119–125.
[176] A. Fusi, S. Ochsenreither, A. Busse, A. Rietz, U. Keilholz, Expression of the stem cell
marker nestin in peripheral blood of patients with melanoma, Br. J. Dermatol. 163 (2010)
107–114.
[177] A. Fusi, S. Collette, A. Busse, S. Suciu, A. Rietz, M. Santinami, W.H. Kruit, A. Testori,
C.J. Punt, A.G. Dalgleish, A. Spatz, A.M. Eggermont, U. Keilholz, Circulating melanoma
cells and distant metastasis-free survival in stage III melanoma patients with or without
adjuvant interferon treatment (EORTC 18991 side study), Eur. J. Cancer 45 (18) (2009)
3189–3197.
[178] S. Medic, R.L. Pearce, P.J. Heenan, M. Ziman, Molecular markers of circulating melano-
ma cells, Pigment Cell Res. 20 (2) (2007) 80–91.
[179] M. Greco, M.D. Mitri, F. Chiriacò, G. Leo, E. Brienza, M. Maffia, Serum proteomic
profile of cutaneous malignant melanoma and relation to cancer progression: association
to tumor derived alpha-N-acetylgalactosaminidase activity, Cancer Lett. 283 (2) (2009)
222–229.
[180] A.I. Riker, R. Jove, A.I. Daud, Immunotherapy as part of a multidisciplinary approach to
melanoma treatment, Front. Biosci. 11 (2006) 1–14.
[181] D.S. Cassarino, W.J. Miller, A. Auerbach, A. Yang, R. Sherry, P.H. Duray, The effects of
gp100 and tyrosinase peptide vaccinations on nevi in melanoma patients, J. Cutan. Pathol.
33 (5) (2006) 335–342.
[182] S. Frankenburg, I. Grinberg, Z. Bazak, L. Fingerut, J. Pitcovski, R. Gorodetsky, T. Peretz,
R.M. Spira, Y. Skornik, R.S. Goldstein, Immunological activation following transcutane-
ous delivery of HR-gp100 protein, Vaccine 25 (23) (2007) 4564–4570.
[183] C.L. Slingluff Jr., G.R. Petroni, W. Olson, A. Czarkowski, W.W. Grosh, M. Smolkin,
K.A. Chianese-Bullock, P.Y. Neese, D.H. Deacon, C. Nail, P. Merrill, R. Fink,
J.W. Patterson, P.K. Rehm, Helper T-cell responses and clinical activity of a melanoma
vaccine with multiple peptides from MAGE and melanocytic differentiation antigens,
J. Clin. Oncol. 26 (30) (2008) 4973–4980.
[184] D. Lienard, M.F. Avril, F.A. Le Gal, P. Baumgaertner, W. Vermeulen, A. Blom,
C. Geldhof, D. Rimoldi, S. Pagliusi, P. Romero, P.Y. Dietrich, N. Corvaia,
D.E. Speiser, Vaccination of melanoma patients with Melan-A/Mart-1 peptide and Kleb-
siella outer membrane protein p40 as an adjuvant, J. Immunother. 32 (8) (2009) 875–878.
[185] D. Schrama, A. Hauschild, J.C. Becker, Immunmodulatory antibodies in the treatment of
skin cancer, Hautarzt 59 (10) (2008) 806–813.
[186] L.F. Langer, T.M. Clay, M.A. Morse, Update on anti-CTLA-4 antibodies in clinical trials,
Expert Opin. Biol. Ther. 7 (8) (2007) 1245–1256.
[187] A.A. Sarnaik, B. Yu, D. Yu, D.R. Morelli, M.S. Hall, D. Bogle, L. Yan, S.R. Targan,
J. Snively, G. Nichol, M. Yellin, J.S. Weber, Extended dose ipilimumab with a peptide
vaccine: immune correlates associated with clinical benefit in patients with resected high-
risk stage IIIc/IV melanoma, Clin. Cancer Res. 17 (4) (2011) 896–906.
[188] R. Dummer, A. Hauschild, J.C. Becker, J.J. Grob, D. Schadendorf, V. Tebbs, et al.,
An exploratory study of systemic administration of the toll-like receptor-7 agonist 852A
in patients with refractory metastatic melanoma, Clin. Cancer Res. 14 (3) (2008)
856–864.
[189] I.H. Wolf, K. Kodama, L. Cerroni, H. Kerl, Nature of inflammatory infiltrate in superfi-
cial cutaneous malignancies during topical imiquimod treatment, Am. J. Dermatopathol.
29 (3) (2007) 237–241.
[190] J. Wenzel, M. Uerlich, O. Haller, T. Bieber, T. Tueting, Enhanced type I interferon
signaling and recruitment of chemokine receptor CXCR3-expressing lymphocytes into
the skin following treatment with the TLR7-agonist imiquimod, J. Cutan. Pathol. 32 (4)
(2005) 257–262.
[191] M. Pashenkov, G. Goëss, C. Wagner, M. Hörmann, T. Jandl, A. Moser, C.M. Britten,
J. Smolle, S. Koller, C. Mauch, I. Tantcheva-Poor, S. Grabbe, C. Loquai, S. Esser,
T. Franckson, A. Schneeberger, C. Haarmann, A.M. Krieg, G. Stingl, S.N. Wagner,
Phase II trial of a toll-like receptor 9-activating oligonucleotide in patients with metastatic
melanoma, J. Clin. Oncol. 24 (36) (2006) 5716–5724.
[192] A. Delcayre, H. Shu, J.B. Le Pecq, Dendritic cell-derived exosomes in cancer immuno-
therapy: exploiting nature’s antigen delivery pathway, Expert Rev. Anticancer Ther. 5 (3)
(2005) 537–547.
[193] S. Hao, O. Bai, J. Yuan, M. Qureshi, J. Xiang, Dendritic cell-derived exosomes stimulate
stronger CD8 þ CTL responses and antitumor immunity than tumor cell-derived exo-
somes, Cell. Mol. Immunol. 3 (3) (2006) 205–211.
[194] B. Escudier, T. Dorval, N. Chaput, F. André, M.P. Caby, S. Novault, C. Flament,
C. Leboulaire, C. Borg, S. Amigorena, C. Boccaccio, C. Bonnerot, O. Dhellin,
M. Movassagh, S. Piperno, C. Robert, V. Serra, N. Valente, J.B. Le Pecq, A. Spatz,
O. Lantz, T. Tursz, E. Angevin, L. Zitvogel, Vaccination of metastatic melanoma patients
with autologous dendritic cell (DC) derived-exosomes: results of the first phase I clinical
trial, J. Transl. Med. 3 (1) (2005) 10–23.
[195] I.D. Davis, Q. Chen, L. Morris, J. Quirk, M. Stanley, M.L. Tavarnesi, P. Parente,
T. Cavicchiolo, W. Hopkins, H. Jackson, N. Dimopoulos, T.Y. Tai, D. MacGregor,
J. Browning, S. Svobodova, D. Caron, E. Maraskovsky, L.J. Old, W. Chen, J. Cebon,
Blood dendritic cells generated with Flt3 ligand and CD40 ligand prime CD8 þ T cells
efficiently in cancer patients, J. Immunother. 29 (5) (2006) 499–511.
[196] E.H. Aarntzen, C.G. Figdor, G.J. Adema, C.J. Punt, I.J. de Vries, Dendritic cell vaccina-
tion and immune monitoring, Cancer Immunol. Immunother. 57 (10) (2008) 1559–1568.
[197] N. Nakai, N. Katoh, W.T. Germeraad, T. Kishida, E. Ueda, H. Takenaka, O. Mazda,
S. Kishimoto, Immunohistological analysis of peptide-induced delayed-type hypersensi-
tivity in advanced melanoma patients treated with melanoma antigen-pulsed mature
monocyte-derived dendritic cell vaccination, J. Dermatol. Sci. 53 (1) (2009) 40–47.
[198] P.B. Chapman, Melanoma vaccines, Semin. Oncol. 34 (6) (2007) 516–523.
[199] M.H. Andersen, J. Gehl, S. Reker, L.Ø. Pedersen, J.C. Becker, P. Geertsen, P. Thor
Straten, Dynamic changes of specific T cell responses to melanoma correlate with IL-2
administration, Semin. Cancer Biol. 13 (6) (2003) 449–459.
[200] G.C. Cesana, G. DeRaffele, S. Cohen, D. Moroziewicz, J. Mitcham, J. Stoutenburg,
K. Cheung, C. Hesdorffer, S. Kim-Schulze, H.L. Kaufman, Characterization of CD4
þCD25þ regulatory T cells in patients treated with high-dose interleukin-2 for metastatic
melanoma or renal cell carcinoma, J. Clin. Oncol. 24 (7) (2006) 1169–1177.
[201] A. Mackensen, N. Meidenbauer, S. Vogl, M. Laumer, J. Berger, R. Andreesen, Phase
I study of adoptive T-cell therapy using antigen-specific CD8þ T cells for the treatment of
patients with metastatic melanoma, J. Clin. Oncol. 24 (31) (2006) 5060–5069.
140 MONICA NEAGU
[202] D.S. Green, A.G. Dalgleish, N. Belonwu, M.D. Fischer, M.D. Bodman-Smith, Topical
imiquimod and intralesional interleukin-2 increase activated lymphocytes and restore the
Th1/Th2 balance in patients with metastatic melanoma, Br. J. Dermatol. 159 (3) (2008)
606–614.
[203] J.M. Zimmerer, G.B. Lesinski, A.S. Ruppert, M.D. Radmacher, C. Noble, K. Kendra,
M.J. Walker, W.E. Carson 3rd, Gene expression profiling reveals similarities between the
in vitro and in vivo responses of immune effector cells to IFN-alpha, Clin. Cancer Res. 14
(18) (2008) 5900–5906.
[204] M. Ahmadzadeh, P.A. Antony, S.A. Rosenberg, IL-2 and IL-15 each mediate de novo
induction of FOXP3 expression in human tumor antigen-specific CD8 T cells, J. Immun-
other. 30 (3) (2007) 294–302.
[205] C. Cocco, V. Pistoia, I. Airoldi, New perspectives for melanoma immunotherapy: role of
IL-12, Curr. Mol. Med. 9 (4) (2009) 459–469.
[206] A. Ribas, Update on immunotherapy for melanoma, J. Natl. Compr. Canc. Netw. 4 (7)
(2006) 687–694.
[207] S. Dalle, T. Martin-Denavit, L. Thomas, Genotypic hypervariability of melanoma: a
therapeutic challenge, Med. Sci. (Paris) 22 (2) (2006) 178–182.
[208] I. Martins, K. Sylla, F. Deshayes, J. Lauriol, S. Ghislin, M.C. Dieu-Nosjean, M. Viguier,
O. Verola, D. Charron, C. Alcaide-Loridan, R. Al-Daccak, Coexpression of major
histocompatibility complex class II with chemokines and nuclear NFkappaB p50 in
melanoma: a rational for their association with poor prognosis, Melanoma Res. 19 (4)
(2009) 226–237.
[209] R. Franco, M. Cantile, S. Scala, E. Catalano, M. Cerrone, G. Scognamiglio, A. Pinto,
M.G. Chiofalo, C. Caracò, A.M. Anniciello, A. Abbruzzese, M. Caraglia, G. Botti,
Histomorphologic parameters and CXCR4 mRNA and protein expression in sentinel
node melanoma metastasis are correlated to clinical outcome, Cancer Biol. Ther. 9 (6)
(2010) 423–429.
[210] N. Nakai, N. Katoh, T. Kitagawa, E. Ueda, H. Takenaka, S. Kishimoto, Immunoregula-
tory T cells in the peripheral blood of melanoma patients treated with melanoma antigen-
pulsed mature monocyte-derived dendritic cell vaccination, J. Dermatol. Sci. 54 (1) (2009)
31–37.

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ADVANCES IN CLINICAL CHEMISTRY, VOL. 58

THE IMMUNE SYSTEM—A HIDDEN TREASURE FOR

Immunobiology Laboratory, ‘‘Victor Babes’’ National

0065-2423/12 $35.00 Copyright 2012, Elsevier Inc.

CCR chemokine-beta receptor

BIOMARKERS DISCOVERY IN THE IMMUNE SYSTEM 91

MDC macrophage-derived chemokine

This chapter describes how skin immune system (SIS) is specifically

Immune-related markers have proven their diagnostic and prognostic value

BIOMARKERS DISCOVERY IN THE IMMUNE SYSTEM 93

3. Skin Immune System—Where the Story Begins

3.1. IMMUNE SURVEILLANCE AT THE SKIN LEVEL

Cells associated to the

Pro- and anti-inflammatory cytokines, chemokines Keratinocytes [13–16]

BIOMARKERS DISCOVERY IN THE IMMUNE SYSTEM 95

immune surveillance [5,30] with a crucial role in maintaining homeostasis.

3.1.1. Cellular Components of SIS

autocrine growth factors and will differentiate into memory/effector T cells in

BIOMARKERS DISCOVERY IN THE IMMUNE SYSTEM 97

promotes the expression of CLA by newly activated effector T cells [42].

BIOMARKERS DISCOVERY IN THE IMMUNE SYSTEM 99

products, including CXCL10 (IFN-inducible protein 10, IP-10), CXCL9

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BIOMARKERS DISCOVERY IN THE IMMUNE SYSTEM 101

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IL-1Ra, IL-10, alpha-MSH,

IL-1, GM-CSF, TNF-alpha,

IL-17 is a proinflammatory cytokine, which could amplify the development of

BIOMARKERS DISCOVERY IN THE IMMUNE SYSTEM 103

In the skin, NF-kB signaling pathway regulates the expression of numer-

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Memory T cell Dermal dentritic cell

BIOMARKERS DISCOVERY IN THE IMMUNE SYSTEM 105

against their own complement components known to be produced excessive-

E-selectin, CC-chemokine ligand 17 (CCL17), and intercellular adhesion molecule-1 (ICAM-1).

106 MONICA NEAGU

4. Cutaneous Melanoma Develops in a Skin Immune

SIS and neoplastic cells interconnect from the outset of tumorigenesis

BIOMARKERS DISCOVERY IN THE IMMUNE SYSTEM 107

activation of negative costimulatory signals, and immunosuppressive factors

5. Immune Markers—The Road Ahead

Although in the past 50 years, the histopathology of cutaneous melanoma

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5.1. INFILTRATING IMMUNE CELLS—MARKERS FOR LOCAL IMMUNE RESPONSE

5.1.1. Local Response

BIOMARKERS DISCOVERY IN THE IMMUNE SYSTEM 109

110 MONICA NEAGU

BIOMARKERS DISCOVERY IN THE IMMUNE SYSTEM 111

progression. CCR10, CCR7, and CCR5 had a specific pattern of expression in

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Lately, abundant data piled up regarding CSC markers, namely, CD133þ,

BIOMARKERS DISCOVERY IN THE IMMUNE SYSTEM 113

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Moreover, lymphangiogenesis proved 83% sensitivity and 89% specificity for

5.2. PERIPHERAL IMMUNE MARKERS—MONITORING THE DISEASE

BIOMARKERS DISCOVERY IN THE IMMUNE SYSTEM 115

IL-6 that acts as both proinflammatory and anti-inflammatory cytokine is

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B cells in patients indicate a perturbation in IFN signaling. These signaling

5.2.2. Immune Cells

BIOMARKERS DISCOVERY IN THE IMMUNE SYSTEM 117