Circulating Biomarkers in Malignant Melanoma

Estibaliz Alegre*; Miguel Sammamed † , ‡; Sara Fernández-Landázuri*; Leyre Zubiri ‡; Álvaro González* , 1     * Laboratory of Biochemistry, University Clinic of Navarra, Pamplona, Spain
† Centro de Investigación Médica Aplicada (CIMA), University of Navarra, Pamplona, Spain
‡ Department of Oncology, University Clinic of Navarra, Pamplona, Spain
1 Corresponding author: email address: agonzaleh@unav.es

1 Some Clinical and Epidemiological Aspects of Melanoma

Melanoma is a neoplastic disorder caused by the malignant transformation of normal melanocytes, the cells responsible for production of the melanin pigment. In the first trimester of fetal life, melanocyte precursors migrate from neural crest to skin, meninges, mucus membranes, and eyes. Melanocytes can become potentially malignant in any of these locations, but the most common site is the skin, specifically the dermal–epidermal junction. Malignant melanoma is the sixth most common cancer in the United States and its incidence is increasing worldwide [1]. According to data from the National Cancer Database (NCD), 91% of melanomas are cutaneous, 5.3% are localized in the eye, 1.3% are mucosal melanomas, and 2.2% are melanomas of an unknown primary origin [2].

There are four classical patterns of histological growth: superficial spreading melanoma, nodular melanoma, acral lentiginous melanoma, and lentigo malignant melanoma [3]. Melanoma staging is based mainly on the thickness of the tumor (described using the Breslow scale) localized (stages I and II) or has spread to lymph nodes (stage III) or other parts of the body (stage IV) [4] (Fig. 1).

Classical treatment for the metastatic disease included chemotherapy (dacarbazine) and immunotherapy (high doses of interleukin-2 (IL-2)), but its efficacy was very limited with low objective response rate (< 10%) and no demonstrated improvement in overall survival. However, in the last 10 years, new treatments have emerged with improvement in both response rate and survival in advanced melanoma patients. Treatment with specific BRAF inhibitors (iBRAF) has shown to be effective in patients harboring BRAF V600 mutation [5]. Melanoma patients with BRAF V600 mutation present with hyperactivation of mitogen-activated protein kinases (MAPK) pathway. Other drugs like MEK inhibitors target MAPK downstream to BRAF with the intention to blocking this oncogenic pathway. MEK inhibitors have demonstrated an improvement in overall survival when used as monotherapy [6] and in combination with iBRAF have demonstrated an improvement in progression-free survival in comparison with iBRAF alone [7,8]. Immunotherapeutic molecules recently approved by the FDA include Ipilimumab (March 2011) and Pembrolizumab (September 2014). Ipilimumab is a blocking antibody against cytotoxic T-lymphocyte-associated protein 4 (CTLA4) that specifically increases T-lymphocytes activation and, in general, increases immune response against tumor cells [9]. Pembrolizumab is a humanized commercial antibody that blocks PD-1 [10], an inhibitory signaling receptor expressed on the surface of activated T-cells. This antibody blocks PD-1 binding by its ligand PD-L1 which is overexpressed on certain cancer cells resulting in activation of T-cell-mediated immune responses against tumor cells. Treatments blocking PD-L1/PD-1 pathway have demonstrated very promising clinical outcomes in terms of response rate and survival [11,12]. Recently, the results in an ongoing clinical trial combining anti-CTLA-4 and anti-PD-1 monoclonal antibodies have been published with an unprecedented antitumor response in melanoma [13].

1.1 The need of circulating biomarkers in melanoma

As in other types of cancer, the search for tumor markers in melanoma is being intensively investigated to provide better tools for less invasive disease management. Historically, the lack of effective therapies against advanced melanoma has limited the utility of these markers. Fortunately, encouraging results obtained with new therapeutic strategies, i.e., BRAF inhibitors or blocking PD-1/PD-L1, has stimulated renewed interest in this field.

Melanoma cells release a myriad of compounds into circulation either by active secretion or as result of cell death. Others are endogenously produced in response to the disease process. As can be expected, the concentration of these substances, i.e., biomarkers, can change during the course of the illness in response to progression or therapeutic intervention. These markers include nucleic acid, protein, metabolites, and microvesicles (Fig. 2). In addition, during disease progression, some cells can detach from the primary tumor and be incorporated into the circulatory compartment and as such serve as biomarkers themselves.

Among the required characteristics, an ideal tumor marker should have high sensitivity and specificity, ideally 100%, and should be easily quantified in accessible samples. Blood is a very accessible specimen that can be obtained repeatedly providing a more dynamic picture of the disease process versus a tissue biopsy that encompasses a single point in time. Circulating biomarkers offer information related to the diagnosis, staging, prognosis, and monitoring of the disease process. Herein, we summarize the most important tumor markers in melanoma.

2 Tumor Biology

Recent discoveries in cell signaling have provided greater understanding of the underlying biology of melanoma [3,14]. Elucidation of these biochemical mechanisms has enabled improvements in diagnosis and staging, and has led to the design of better therapeutic agents. A number of oncogenic pathways predominate in different melanoma subtypes supporting the concept that different melanomas are distinct genetically [15]. The crucial cell-signaling pathways governing disease etiopathogenesis is discussed below.

2.1 MAP kinase pathway

The MAPK pathway includes the proteins RAS, RAF, MEK, and ERK that participate in a signaling cascade to transfer extracellular signals from the cell membrane to the nucleus. The successful activation of the pathway leads to activation of genes that promote cell proliferation. In melanoma, MAPK pathway can be activated by the mutational activation of growth-factor receptors such as c-KIT. c-KIT mutations have been predominantly described in acral and mucosal melanomas, and in melanomas of chronically sun-damaged skin [15,16]. A more common mechanism is through function-gain mutations in N-RAS, one of the three Ras genes in humans (together with HRAS and KRAS). N-RAS is mutated in 15–30% of melanomas and the most common mutation is a glutamine to leucine substitution at position 61 (Q61L) [17]. In addition to MAPK pathway, RAS simultaneously activates the phosphatidylinositol 3-kinase (PI3K) pathway (Fig. 3). However, the most commonly mutated component in this pathway is BRAF, one of the three RAF isoforms in humans (together with A-RAF and C-RAF). BRAF mutations have been described predominantly in melanomas from not chronically sun-damaged, but intermittently exposed skin, being mutated in 50% of all melanomas [18]. In more than 90% of cases, the mutation consists in a substitution of valine at codon 600 by glutamic acid (V600E) [19]. BRAFV600E stimulates constitutive ERK signaling, inducing proliferation, survival, and neoangiogenesis. Several genes downstream of BRAFV600E were identified and subsequently implicated in various aspects of melanoma induction and progression...