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Genetic Analysis of Malignant Melanoma: Do We Still Need Histology?


Victor G. Prieto
University of Texas MD Anderson Cancer Center
Houston, TX


The incidence of reported melanoma cases has risen strikingly over the past several decades. Melanoma is the skin cancer with highest mortality. Since there is no cure for advanced disease, it is crucial to detect melanoma in early stages, either in-situ (confined to the epidermis) or when it involves the upper dermis (around 95% of such lesions are cured by complete surgical removal). Exposure to ultraviolet radiation (UVR) is a major melanoma risk factor, but also fair skin, history of sunburn, blue or green eyes, red hair, number of typical acquired, congenital, or atypical (dysplastic) nevi, ephelides, and lentigines. Immunosuppression and personal and family history of melanoma are also associated with increased risk of melanoma.

Despite multiple studies analyzing the pathogenesis of melanoma, it is still mostly unknown for several reasons. There appears to be different types of melanoma; as mentioned before, a number of melanomas is associated with sun / ultraviolet light exposure (mostly lentigo maligna) but melanomas do occur in sun-protected acral and genital locations. Also, some patients have relatives with melanoma while most cases do not appear to be familial so it is more difficult to detect any possible genetic alterations. Finally, skin lesions are easily observed by patient, family, and doctor. Therefore, lesions that are suspicious for melanoma are likely to undergo biopsy before they reach large size. And since the determination of prognosis is essentially based upon the histologic examination of the lesion, very little material can be spared for the non-routine histologic studies currently necessary for analyzing genetic anomalies.

Although some rare authors disagree, there seems to be some sort of progression between benign and malignant melanocytic lesions. As such, it has been proposed that mature melanocytes develop in nevi and that some nevi (common or atypical) develop in melanomas in situ, then invasive, and finally metastatic. Therefore, before analyzing the possible genetic and molecular changes associated with this purported progression we will briefly analyze these possible steps.

The first step in melanoma progression may be benign nevus. Number of nevi is an independent risk factor for melanoma. However, since nevi are so prevalent while melanomas are much less common, it is obvious that the vast majority of nevi are not premalignant. Precursor nevi are identified in contiguity with melanoma in 10-40% of all cases, in particular large congenital nevi. Conversely a majority of melanomas seem to originate from epidermal melanocytes so the nevus is probably not an obligatory stage in the pathogenesis of melanoma.

The terms atypical/ dysplastic/ Clark nevus are synonymous. After much controversy started against the original term ("dysplastic nevus") proposed by Clark et el, the NIH Consensus Conference recommended using "nevus with architectural disorder and cytologic atypia", but this longer name has not completely caught up. Although much controversy has attended this concept, in essence it is a nevus that resembles melanoma, clinically and histologically: large size (generally >5 mm), poor circumscription, border irregularity, a play of colors, and asymmetry. Atypical nevi occur at a prevalence of 2-9% in unselected white populations in most series. These nevi represent a strong, independent risk factor for development of melanoma, whether they occur sporadically or in the context of a melanoma-prone familial predisposition (dysplastic nevus syndrome). Moreover, this association shows a dose-response relationship, with melanoma risk rising proportionately with number of atypical nevi present. Histologically, these lesions are described as having cytologic and architectural atypia. Both the degree of cytologic atypia and the extent of architectural disorder tend to be positively correlated with each other, so it seems that histologic analysis is successful in detecting these markers for melanoma (dysplastic nevi).

The next step in melanoma progression is melanoma. Early melanoma has been arbitrarily defined as those lesions with Breslow thickness <1 mm (measured from the top of the granular layer to the deepest tumor cell). Some such lesions also are in radial growth phase (RGP), early phase in which melanomas are considered to have little if any potential for metastasis. By definition, all MIS (Clark level I) and some superficially invasive (Clark level II, lesions involving but not filling or expansion of papillary dermis, and lacking large nests in the dermis or dermal mitotic figures) are in RGP. Beyond this point, some authors consider that melanoma enters a tumorigenic phase (vertical growth phase, VGP). In this early melanomas less than 1 mm in Breslow thickness, the overall survival after complete excision is greater than 90% at 5 years.

Several genetic changes are associated with the proposed melanoma progression. There are anomalies in chromosome 1 (1p36) in patients with the dysplastic nevus syndrome/familial melanoma. Such families also exhibit abnormalities in a second gene, in chromosome 9, that encodes an inhibitor of a kinase involved in the cell cycle, named p16INK4a or cyclin-dependent kinase inhibitor-2 (CDKN2A). Loss of heterozygosity for p16 is an early event in melanomas, having been reported also in atypical nevi. The addition of activating mutations in N-ras to melanoma cells already containing mutations in p16 may induce the VGP phenotype. Such N-ras mutations are common in melanomas from sun-exposed skin.

In addition to p16 mutations, atypical nevi may occur due to abnormalities in DNA repair in response to UV light. These patients with dysplastic nevus syndrome have chromosomal instability, as documented in normal skin, atypical nevi, and blood lymphocytes. The lesions contain increased amounts of pheomelanin compared with common nevi, a fact that might increase their susceptibility to UVR damage. Shifts among these melanin types may reflect changes in pigment regulatory genes such as that for the melanocortin-1 receptor.

A number of genes show relatively consistent mutations in thick melanomas, possibly correlating with a decreased survival: c-myc (nuclear protein required for transition from G1 to S phase), p53 (gatekeeper protein preventing cell proliferation after DNA damage), bcl-2 (anti-apoptotic factor), transforming growth factor-b (TGF- b , protein involved in angiogenesis and wound healing), CD40 (receptor that induces escape from apoptosis), and the cyclin-dependent kinase inhibitors p27 and p21 (KIP1 and Waf-1/SDI-1, respectively). Enzymes that degrade collagen have been proposed to facilitate permeation of the dermis and access to blood vessels; in particular, collagenases-1 and -3 are expressed in invasive and metastatic but not in in situ melanomas. The Ets-1 transcription factor is overexpressed in melanoma and may play a role in tumor vascularization and invasion by regulating expression of matrix-degrading proteases in endothelial cells and fibroblasts in the tumor stroma. Melastatin (TRPM1) is expressed in nevi and melanomas, but it is usually lost in metastatic lesions.

Similarly, some integrins (molecules involved in cell-to-cell recognition), such as the beta3 subunit of the vitronectin receptor, are first detected in VGP melanomas, as is interleukin-8 (IL-8). Since tumors must recruit new vessels in order to grow, it seems obvious that there should be secretion of angiogenic factors at some point. There is a relationship between numbers of mast cells (which contain high quantities of angiogenic factors) and invasiveness in melanomas. Also some studies have shown increased vascularity in VGP versus radial growth phase melanomas. Two candidate proteins are VEGF (vascular endothelial growth factor) and b-FGF, which have mitogenic effects on endothelial cells. Supporting this view, by immunohistochemistry or in situ hybridization, b-FGF is expressed in invasive but not in situ melanomas.

An exciting discovery has been the recent description of activating mutations in 60% to 80% of cutaneous melanomas and benign melanocytic nevi in the BRAF gene, a component of the RAS/RAF/MAPK signaling cassette. In particular, several groups are currently studying the possible use of anti-BRAF compounds as treatment for melanoma. Furthermore, some authors have suggested that there is an inverse relationship between activation of BRAF and loss of PTEN. Interestingly, Spitz nevi lack the T1796A BRAF mutation that is commonly detected in other nevi and melanoma.

Due to the need for sufficient tissue for specialized studies, most genetic changes have been described in advanced lesions, either deeply invasive primary lesions or metastatic lesions, or in cell lines. Generally, only small quantities of tissue are available for analysis in common nevi, atypical nevi, and early MM. Breakthrough techniques such as laser microdissection now allow removal and analysis of minute quantities of tissue, potentially allowing molecular analysis of the earliest stages of incipient melanoma. This approach has been employed to detect chromosomal differences between RGP and VGP melanoma: losses of chromosomes 9 and 10 early, and gains of chromosome 7 later. Using similar techniques we found an alteration in Chr 20q13, in an area where is located a gene involved in chromosome function (aurora2, STK15, or STK6). This modification is present in superficial melanomas and in dysplastic nevi, but not in common nevi and may therefore be important in early malignancy transformation. Similar studies have shown that superficial spreading melanomas are genetically different from acral lentiginous melanomas, which may explain the different phenotype that these lesions present. Furthermore, the same genetic alterations seen in acral lentiginous melanoma are also present in individual, morphologically benign melanocytes located away from the main lesion ("field effect"). Also using microdissection, gene array profiling may be able to detect genetic differences between benign and malignant melanocytic lesions.

So what is the current status in analysis of melanocytic lesions? Are we at a point in which a clinician may place a piece of skin into a machine and then retrieve a diagnosis of melanoma vs. nevus and a list of prognostic factors? Despite the existence of a spectrum of melanocytic atypia, ranging from banal nevi to outright melanoma, we believe that the experienced pathologist can confidently render a diagnosis of nevus versus melanoma in the majority of cases encountered in routine practice. We make this assertion despite some studies wherein experts reviewed cases of melanoma or of nevus sharing features with melanoma, and were asked to classify them as malignant, indeterminate, or benign, then obtaining complete agreement in only 30% of cases. In part, the discordance may be the result of case-selection at the high end of the spectrum (where concordance regarding malignancy versus benignity is most problematical). In addition, when morphologic features may be ambiguous, immunohistochemical studies may be helpful. The use of antibodies against antigens typically expressed in melanocytic cells, such as S100 protein, gp100 (detected with HMB45), or MART-1 (melanoma antigen recognized by T-cells) allows confirmation of melanocytic lineage in a given neoplasm and distinction in small biopsies of pigmented basal cell carcinoma versus small cell melanoma. Also, when examining sentinel node specimens, immunohistochemistry may help detect small clusters of or even single melanoma cells within the lymph node. An exciting but controversial field is the use of immunohistochemistry for the differential diagnosis of melanoma and nevus. Although some authors reject this approach we strongly feel that, in selected cases and along with clinical and histologic criteria, immunohistochemical data are helpful in the diagnosis of melanocytic lesions. An example is the assessment of maturation in the dermal components. Superficial, round, type A nevus cells share many immunohistochemical markers with neurons while the deep, spindle, type C nevus cells resemble Schwann cells. This morphology is reflected in the decreased expression of gp100 by those melanocytes located at the base of the nevus and not in melanomas. Also, the almost universal lack of mitotic figures in the deep regions of nevi correlates with the very sparse expression of proliferation markers such as Ki-67 by the more deeply located melanocytes in nevi. In contrast, melanomas do not show this orderly pattern, but instead had a random pattern of immunoreactivity, especially concentrated at the deep tumor borders (see table below).


Pattern of expression: Nevus Melanoma
gp100 (with HMB45) Superficial region Throughout
Tyrosinase Superficial region Throughout
Peripherin Superficial region Throughout
Ki67 (nuclear, proliferation marker) Rare at base Throughout

In summary, the take home message of this talk is that despite the very exciting findings described in the last 10 years, the gold standard (and by far cheapest) method to study melanocytic lesions remains histologic analysis.

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