**3. Other molecules and molecular approaches**

#### **3.1. Melanoma Associated Antigens (MAA)**

Malignant transformation of melanocytes is associated to changes in gene definition. This leads to the expression of melanoma associated antigens molecules called (MAA), which are more or less specifically associated with the malignant phenotype (Table 1). In fact sometimes these MAA can also be expressed in normal melanocytes. MAA play an important role in triggering the immune response agains melanoma cells. These antigens were mainly identified by immunological approaches, including in vitro and in vivo reactions and serological tests. These antigens can be defined by their ability to interact with T cells or B and peptides derived from

**Figure 5.** MIA is a 12kDa soluble protein, whose role has been characterized as an inhibitor of cell autocrine growth. It can be expressed by melanoma cells and chondrocytes.

these antigens have been used to induce or maintain a specific immune response. Mage-1 was the first identified MAA and now belongs to a large family of at least 12 antigens differentially expressed by benign and malignant melanocytic cells. Immune responses to these genes can be used as markers of progression and / or immunological response.

Tyrosinase RT-PCR detection in patients with melanoma is correlated with a higher risk of relapse (55% of these patients have a clinical relapse), but the specificity of this technique has yet to be optimized [15, 16]. When combined with a dosage of S-100, Domingo-Domenech showed that tyrosinase RT-PCR adds valuable prognostic information in patients with S-100 <0.15μg / l, although the team showed that S-100 had a higher predictive value. Curry et al. suggested that RT-PCR detection of tyrosinase can be useful to determine a subgroup of patients with an increased risk of metastases [17].

Profiling of autoantibodies associated with certain MAA was different by Sabel et al as potentially useful to select patients with melanoma who should benefit from the research of a sentinel node.

These results have yet to be validated [18].

melanoma cells and inflammatory cells. Gal-3 plays an important role in cell proliferation, cell differentiation, cell adhesion, cell migration, angiogenesis and metastasis. Thus, Gal-3 deserves special attention. Clarification of the role of extracellular Gal-3 should help us to understand the significance of elevated serum levels of this molecule in patients with advanced

**Figure 4.** S100B is a 21-kD dimeric protein, consists of two β subunits. This protein is a member of a family of 19 pro‐ tein and was first isolated from bovine brain in the mid sixties. S100B is expressed by glial cells and melanocytes and is

produced by brain tumors and melanomas. The roles of S100B are probably multiple and underestimated.

Malignant transformation of melanocytes is associated to changes in gene definition. This leads to the expression of melanoma associated antigens molecules called (MAA), which are more or less specifically associated with the malignant phenotype (Table 1). In fact sometimes these MAA can also be expressed in normal melanocytes. MAA play an important role in triggering the immune response agains melanoma cells. These antigens were mainly identified by immunological approaches, including in vitro and in vivo reactions and serological tests. These antigens can be defined by their ability to interact with T cells or B and peptides derived from

**3. Other molecules and molecular approaches**

**3.1. Melanoma Associated Antigens (MAA)**

melanoma [13].

32 Highlights in Skin Cancer

**Antigen HLA restriction**

MAGE-A2 A\*0201, B\*3701

MAGE-4 A\*0201 MAGE-A6 A\*3402, B\*3701 MAGE-A10 A\*0201 MAGE-A12 A\*0201 MAGE-B1 A\*0201 MAGE-B2 A\*0201 BAGE Cw\*1601 GAGE-1 Cw\*6 LAGE-1 A\*0201 PRAME A\*24 NY-ESO-1 A\*02, A\*31 DAM-6 A\*02

MAGE-A3 A\*01, A\*02, A\*2402, B\*3701, B\*44, DR\*11

Tyrosinase A\*01, A\*0201, A\*2402, B\*44, DRβ1\*0401

TRP-2 A\*31, A\*33, A\*0201, C\*0802, A\*68011

Gp100 A\*0201, A\*03, A\*0301, A\*1101, A\*2402, C\*0802, DRβ1\*0401

MART-1/Melan-A A\*0201, A\*02, B\*4501

TRP-1 A\*31

MC1R A\*0201

MUM-1 B\*44 CDK4 A\*02 B-catenin A\*24 P15 A\*24 GnT-V A\*02 TPI DRβ1\*0101 Annexin II DRβ1\*0401 CDC27 DRβ1\*0401

HER2/Neu A\*0201

**Table 1.** Melanoma associated antigens (adapté de Visser et al. [14]).

MAGE-A1 A\*01, A\*03, A\*24, A\*28, B\*3701, B\*53, Cw\*0201, Cw\*0301, Cw\*1601

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*Oncospermatogonal antigens*

*Melanocytic differentiation antigens*

*Mutated antigens*

*Oncogene-derived antigens*

**Figure 6.** Galectin-3 is a member of the family of lectins that can bind to β-galactoside residues. Many members of the galectin family are differentially expressed in cancer. Gal-3 is a molecule that contains an NH2-terminal domain, a COOH-terminal domain.

#### **3.2. Melanine metabolites**

5-S-cysteinyl dopa (5SCD) is a precursor of phaeomelanin and is produced by melanocytes and melanoma cells, as a product of the binding of a molecule named dopaquinone and highly reactive with cysteine. 5SCD is detectable in the urine and serum of melanoma patients and correlates with disease progression. In patients with progressive disease, the level of 5SCD can rise before the onset of clinical signs. A comparative report stated that with the LDH and S100B, 5SCD has an interesting value even if the authors of this report concluded that S100B could be considered as the most sensitive of the three markers. Due to the effect of UV exposure on melanin, the use of this 5SCD as a biomarker may be limited in Caucasians, whereas its use in other populations, particularly in Japan, would be more reliable. In addition, patients with metastatic but non-pigmented melanoma lesions do not usually have elevated serum 5SCD. 3,4-dihydroxyphenylalanine (L-DOPA) is the first metabolite involved in melanogenesis and its plasma levels were also correlated with melanoma progression and tumor burden, as well as the ratio of plasma L-dopa / L tyrosine-which represents an index of the activity of tyrosinase and tyrosine hydroxylase. Stoitchkov et al. showed that the latter ratio has predictive value, especially in patients with stage III, and advocated the use of multiple biomarkers [19].


**Table 1.** Melanoma associated antigens (adapté de Visser et al. [14]).

**3.2. Melanine metabolites**

COOH-terminal domain.

34 Highlights in Skin Cancer

5-S-cysteinyl dopa (5SCD) is a precursor of phaeomelanin and is produced by melanocytes and melanoma cells, as a product of the binding of a molecule named dopaquinone and highly reactive with cysteine. 5SCD is detectable in the urine and serum of melanoma patients and correlates with disease progression. In patients with progressive disease, the level of 5SCD can rise before the onset of clinical signs. A comparative report stated that with the LDH and S100B, 5SCD has an interesting value even if the authors of this report concluded that S100B could be considered as the most sensitive of the three markers. Due to the effect of UV exposure on melanin, the use of this 5SCD as a biomarker may be limited in Caucasians, whereas its use in other populations, particularly in Japan, would be more reliable. In addition, patients with metastatic but non-pigmented melanoma lesions do not usually have elevated serum 5SCD. 3,4-dihydroxyphenylalanine (L-DOPA) is the first metabolite involved in melanogenesis and its plasma levels were also correlated with melanoma progression and tumor burden, as well as the ratio of plasma L-dopa / L tyrosine-which represents an index of the activity of tyrosinase and tyrosine hydroxylase. Stoitchkov et al. showed that the latter ratio has predictive value, especially in patients with stage III, and advocated the use of multiple biomarkers [19].

**Figure 6.** Galectin-3 is a member of the family of lectins that can bind to β-galactoside residues. Many members of the galectin family are differentially expressed in cancer. Gal-3 is a molecule that contains an NH2-terminal domain, a

### **3.3. Matrix Metalloproteinases (MMP)**

MMPs are a family of 24 structurally related endopeptidases. These zinc-dependent enzymes are defined by their own substrates and can lyse components of the extracellular matrix (ECM) (eg, collagen type IV, which is a major component of the basement membrane by gelatinases such as MMP-2 and MMP-9 ) and play a role in angiogenesis and the renewal of the ECM. MMPs can also cleave different molecules such as other proteinases, proteinase inhibitors, growth factors, adhesion molecules and thereby modulate the inflammatory response, the process of tumor growth, tumor invasion and metastasis.

described as a potent mitogen for endothelial cells and its expression, which can be increased in hypoxic conditions, was also correlated with tumor progression and poor prognosis.

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Different VEGF have been described, but none has been reported as an independent prognostic factor. At most a few studies have correlated the presence / absence of certain molecules in very specific situations, eg serum VEGF-C is decreased in patients with metastatic cutaneous

An imbalance in the ratio of serum angiopoietin 1 and angiopoietin 2 than could, according et

Integrins are cell components that ensure adhesion to other cells, ECM, or other proteins. Other important roles are played by integrins such as the transmission of information between the

Integrins are heterodimeric receptors consisting of two subunits α and β,. On the basis of their common subunit, heterodimers can be classified αv, β1 β2 integrins. The main integrins involved in the progression of melanoma include avß3 (vitronectin receptor and fibronectin),

Some reports have shown that increased serum concentrations of β integrins were associated

CD44 is a transmembrane glycoprotein surface, originally described as a lymphocyte receptor. CD44 is a cell surface receptor for hyaluronic acid, While some studies have highlighted its role in tumor invasion and metastasis, it is clear that no study has identified a prognostic value

ICAM-1 is a new intercellular adhesion molecule, located in the cell membranes of leukocytes and endothelial cells. ICAM-1 is a ligand of LFA-1 (lymphocyte function-associated antigen-1) in T cells, B cells, macrophages, and neutrophils. Leukocyte migration is facilitated by the binding ICAM-1/LFA-1. One study showed that serum ICAM-1 is increased in patients with

Ever, new publications emerge with new molecular perspectives (protein profiling, micro‐

metastatic but has no independent prognostic value in multivariate analysis [27].

with shorter survival. The clinical impact of this has not yet been defined.

metastases [25].

*3.6.1. Integrins*

*3.6.2. CD44*

for serum CD44.

*3.6.3. ICAM-1*

**3.7. Others**

RNAs dosage,...).

al Gardizi sign a metastatic progression. [26]

extra-and intracellular space, and angiogenesis.

α2β1 (collagen), α4β1 (fibronectin) and α6β1 (laminin).

**3.6. Cell surface and adhesion molecules**

A balance between MMPs and tissue inhibitors of metalloproteinases (TIMP) can be broken by upregulation of MMP and TIMP downregulation, this is shown in malignant phenotype.

Another important role, angiogenesis, has been attributed to MMP, which could allow a therapeutic target possible. Batimastat (BB-94, a synthetic broad-spectrum inhibitor of metalloproteinases), for example, has shown efficacy for inhibiting angiogenesis of liver metastases in a mouse model.

MMP overexpression has been reported in melanoma progression, and elevated serum MMP, ie MMP-1 and MMP-3, have been correlated with poor survival [20].

#### **3.4. Cytokins, chimiokins and their réceptors [21]**

Chemokines are small polypeptides signaling can bind to and activate G protein-coupled receptors, a family of seven transmembrane molecules. Multiple roles have been attributed to these chemokines, and are involved in tumor transformation and metastatic process. The differential expression of these chemokines and their receptors may explain the organ specificity of metastases.

Melanoma cells expressing the chemokine CXCL8, also known as interleukin-8 (IL-8) have been described and a report showed that serum levels of IL-8 are associated with tumor burden and a poor prognosis. This track is interesting and could be exploited for therapeutic purposes. In vivo studies have indeed shown that anti-IL8 humanized antibodies are able to reduce tumor growth and angiogenesis.

A recent study of 29 serum cytokines assayed simultaneously in 179 melanoma patients (versus 378 control individuals, healthy) showed a profile of specific serum cytokines in patients compared to controls: higher serum concentrations of interleukin (IL )-1alpha, IL-1beta, IL-6, IL-8, IL-12p40, IL-13, G-CSF, MCP-1, MIP-1alpha, MIP-1 beta, IFN-alpha, TNF-alpha, EGF, VEGF and TNF receptor II. [22, 23, 24].

#### **3.5. Growth and angiogenic factors**

Angiogenesis is an important step in tumor growth as it ensures the supply of oxygen and substrates to tumor cells. This process is in fact the result of complex interactions between proangiogenic factors and anti-angiogenic released by tumor cells, endothelial, epithelial, mesothelial cells and leukocytes. Vascular Endothelial Growth Factor (VEGF) has been described as a potent mitogen for endothelial cells and its expression, which can be increased in hypoxic conditions, was also correlated with tumor progression and poor prognosis.

Different VEGF have been described, but none has been reported as an independent prognostic factor. At most a few studies have correlated the presence / absence of certain molecules in very specific situations, eg serum VEGF-C is decreased in patients with metastatic cutaneous metastases [25].

An imbalance in the ratio of serum angiopoietin 1 and angiopoietin 2 than could, according et al Gardizi sign a metastatic progression. [26]

#### **3.6. Cell surface and adhesion molecules**

#### *3.6.1. Integrins*

**3.3. Matrix Metalloproteinases (MMP)**

36 Highlights in Skin Cancer

metastases in a mouse model.

specificity of metastases.

tumor growth and angiogenesis.

VEGF and TNF receptor II. [22, 23, 24].

**3.5. Growth and angiogenic factors**

process of tumor growth, tumor invasion and metastasis.

ie MMP-1 and MMP-3, have been correlated with poor survival [20].

**3.4. Cytokins, chimiokins and their réceptors [21]**

MMPs are a family of 24 structurally related endopeptidases. These zinc-dependent enzymes are defined by their own substrates and can lyse components of the extracellular matrix (ECM) (eg, collagen type IV, which is a major component of the basement membrane by gelatinases such as MMP-2 and MMP-9 ) and play a role in angiogenesis and the renewal of the ECM. MMPs can also cleave different molecules such as other proteinases, proteinase inhibitors, growth factors, adhesion molecules and thereby modulate the inflammatory response, the

A balance between MMPs and tissue inhibitors of metalloproteinases (TIMP) can be broken by upregulation of MMP and TIMP downregulation, this is shown in malignant phenotype.

Another important role, angiogenesis, has been attributed to MMP, which could allow a therapeutic target possible. Batimastat (BB-94, a synthetic broad-spectrum inhibitor of metalloproteinases), for example, has shown efficacy for inhibiting angiogenesis of liver

MMP overexpression has been reported in melanoma progression, and elevated serum MMP,

Chemokines are small polypeptides signaling can bind to and activate G protein-coupled receptors, a family of seven transmembrane molecules. Multiple roles have been attributed to these chemokines, and are involved in tumor transformation and metastatic process. The differential expression of these chemokines and their receptors may explain the organ

Melanoma cells expressing the chemokine CXCL8, also known as interleukin-8 (IL-8) have been described and a report showed that serum levels of IL-8 are associated with tumor burden and a poor prognosis. This track is interesting and could be exploited for therapeutic purposes. In vivo studies have indeed shown that anti-IL8 humanized antibodies are able to reduce

A recent study of 29 serum cytokines assayed simultaneously in 179 melanoma patients (versus 378 control individuals, healthy) showed a profile of specific serum cytokines in patients compared to controls: higher serum concentrations of interleukin (IL )-1alpha, IL-1beta, IL-6, IL-8, IL-12p40, IL-13, G-CSF, MCP-1, MIP-1alpha, MIP-1 beta, IFN-alpha, TNF-alpha, EGF,

Angiogenesis is an important step in tumor growth as it ensures the supply of oxygen and substrates to tumor cells. This process is in fact the result of complex interactions between proangiogenic factors and anti-angiogenic released by tumor cells, endothelial, epithelial, mesothelial cells and leukocytes. Vascular Endothelial Growth Factor (VEGF) has been Integrins are cell components that ensure adhesion to other cells, ECM, or other proteins. Other important roles are played by integrins such as the transmission of information between the extra-and intracellular space, and angiogenesis.

Integrins are heterodimeric receptors consisting of two subunits α and β,. On the basis of their common subunit, heterodimers can be classified αv, β1 β2 integrins. The main integrins involved in the progression of melanoma include avß3 (vitronectin receptor and fibronectin), α2β1 (collagen), α4β1 (fibronectin) and α6β1 (laminin).

Some reports have shown that increased serum concentrations of β integrins were associated with shorter survival. The clinical impact of this has not yet been defined.

### *3.6.2. CD44*

CD44 is a transmembrane glycoprotein surface, originally described as a lymphocyte receptor. CD44 is a cell surface receptor for hyaluronic acid, While some studies have highlighted its role in tumor invasion and metastasis, it is clear that no study has identified a prognostic value for serum CD44.

#### *3.6.3. ICAM-1*

ICAM-1 is a new intercellular adhesion molecule, located in the cell membranes of leukocytes and endothelial cells. ICAM-1 is a ligand of LFA-1 (lymphocyte function-associated antigen-1) in T cells, B cells, macrophages, and neutrophils. Leukocyte migration is facilitated by the binding ICAM-1/LFA-1. One study showed that serum ICAM-1 is increased in patients with metastatic but has no independent prognostic value in multivariate analysis [27].

#### **3.7. Others**

Ever, new publications emerge with new molecular perspectives (protein profiling, micro‐ RNAs dosage,...).

Luo et al showed in well constructed paper that isoenzymes of aldehyde dehydrogenase 1A (ALDH) appear as stem cell markers of melanoma, but their presence in serum and their prognostic significance has yet to be defined [28].

Research into new biomarkers in melanoma is important issue because i twill lead to better understand the biology of this tumor, and thus it improve patient monitoring, early detection and treatment of secondary lesions and open new perspectives for targeted therapies. Multiple

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CLIDERM (Clinics in Dermatology), European Insititute for Dermatology Practice and Re‐

[1] Garbe, C, Peris, K, Hauschild, A, Saiag, P, Middleton, M, Spatz, A, Grob, J. J, Malhe‐ vy, J, Newton-bishop, J, Stratigos, A, Pehamberger, H, & Eggermont, A. M. Diagnosis and treatment of melanoma. European consensus-based interdisciplinary guideline-

[2] Balch, C. M, Gershenwald, J. E, Soong, S. J, Thompson, J. F, Atkins, M. B, Byrd, D. R, Buzaid, A. C, Cochran, A. J, Coit, D. G, Ding, S, Eggermont, A. M, Flaherty, K. T, Gi‐ motty, P. A, Kirkwood, J. M, Mcmasters, K. M, Mihm, M. C, Morton, D. L, Ross, M. I, Sober, A. J, & Sondak, V. K. Final version of (2009). AJCC melanoma staging and c

[3] Palmer, S. R, Erickson, L. A, Ichetovkin, I, Knauer, D. J, & Markovic, S. N. Circulating serologic and molecular biomarkers in malignant melanoma. Mayo Clin Proc (2011). ,

[4] Hamberg, A. P, & Korse, C. M. Bonfrer JMG, De Gast GC. Serum S100B is suitable for prediction and monitoring of response to chemoimmunotherapy in metastatic malig‐

[5] Hauschild, A, Michaelsen, J, Brenner, W, et al. Prognostic significance of serum S100B detection compared with routine blood parameters in advanced metastatic

[6] Weide, B, Elsässer, M, Büttner, P, Pflugfelder, A, Leiter, U, Eigentler, T. K, Bauer, J, Witte, M, Meier, F, & Garbe, C. Serum Markers lactate dehydrogenase and S100B predict independently disease outcome in melanoma patients with distant metasta‐

nant melanoma. *Melanoma Research*. (2003). PubMed], 13(1), 45-49.

melanoma patients. *Melanoma Research*. (1999). PubMed], 9(2), 155-161.

molecular changes of melanoma progression are currently intensively studied.

search (EIDPR), CHIREC et CHIREC CANCER INSTITUTE, Brussels, Belgium

Address all correspondence to: dr.vereecken@dermatologist.be

Update 2012. Eur J Cancer (2012). , 48(15), 2375-90.

lassification. J Clin Onclo 2009 ; , 27(36), 6199-206.

sis. Br J Cancer (2012). , 107(3), 422-8.

**Author details**

Pierre Vereecken\*

**References**

86(10), 981-90.

Adhesion molecule type 1 related to carcinoembryonic antigen (CEACAM 1) has also recently been shown as a promising biomarker by an Israeli team: measuring the serum in a retrospec‐ tive study is correlated with metastatic progression and survival patients with melanoma [29].

The utility of serum DNA of mutated BRAF gene has also recently been presented as a prognostic factor and predictor of response to biochetherapies. This would probably be very useful for patients treated with vemurafenib. These results need to be confirmed [30].
