Malignant Melanoma: an update. (Special article).
When melanomas are recognized early, that is, in the radial growth phase (RGP), and the disease is still localized and restricted to the skin, proper surgical excision can result in clinical cure. (2,5,9-11,13) However, the appearance of a cell population with the capacity to develop into an expansile dermal tumor nodule, that is, in the vertical growth phase (VGP), has an important negative impact on patient survival. Melanomas in the VGP stage have metastatic capability, and once the metastatic process has started, the tumor becomes resistant to current methods of therapy. (2,5,9-14)
This review covers recent advances in the study of genetic, molecular, and immunological abnormalities associated with melanoma. We then relate these findings to the more traditional clinical and pathologic variables. We also discuss new melanoma tumor markers, which could predict clinical behavior and metastatic potential and, thus, help guide new therapeutic approaches. (1,3-7,15-21)
CLINICAL AND PATHOLOGIC CRITERIA USED IN THE DIAGNOSIS OF MELANOMA
The diagnosis of melanoma is made by the presence of proliferating atypical melanocytes along the dermal-epidermal junction that replace the basilar region and/or display a pagetoid spread within the epidermis (melanoma in situ; Figure 1). The appearance of nests or aggregates of atypical melanocytes in the papillary dermis signals an invasive pattern, which in more advanced stages form expansile nodules in the dermis with cytology different from the tumor cells in the overlying epidermis. The most important determinant of prognosis in melanoma is the type of growth, for example, RGP or VGP (Figures 2 and 3). Clinically, the RGP presents as patches or plaques. In the case of superficial spreading melanoma, the patches or plaques can measure up to 2.5 cm. Superficial spreading melanoma lesions are slightly raised and show striking variations of red, blue, white, brown, and black coloration. Acral lentiginous and lentigo maligna in the RGP are usually flat or only focally raised, with coloration usually limited to variations of brown and black, unless there are areas of regression that are blue, gray, or white. Both types of lentiginous growth, especially lentigo maligna, can extend for several centimeters. Lentigo maligna is confined to the face or sun-exposed areas of the elderly. It may evolve for several years, sometimes as long as 20 to 30 years before invasive melanoma supervenes. These melanoma cells show radial spread, usually confined to the intraepidermal compartment; if invading the papillary dermis (invasive RGP), it is only in the form of single cells or small nests of melanocytes similar in size and number (no more than 5-10 melanoma cells) (Figure 2). In RGP, melanoma mitoses are frequently seen in the epidermis but rarely in the dermis, melanoma aggregates do not become expansile, and an inflammatory lymphocytic response is present in the papillary dermis. Occasionally, the upper reticular dermis also may be infiltrated, but by isolated single cells. After complete surgical excision of the tumor, RGP melanomas are usually associated with long-term metastasis-free survival. (2,5,9-14)
[FIGURES 1-3 OMITTED]
Vertical growth phase melanomas usually present as gray-black, blue-black, or even amelanotic nodules that supervene in the preexisting RGP (Figure 3). When the VGP appears de novo without preexisting RGP, it manifests as nodular melanoma. Histologically, the VGP that supervenes on RGP appears as a new cell population with metastatic capability that determines the clinical course. (11-14) The VGP is classified into an early and a late stage. In early VGP, a small papulonodule arises in an RGP lesion and is usually darker than RGP-associated lesions, with blue-black coloration on gross examination. Histologically, small expansile cell aggregates appear in the papillary dermis of a type different than that of RGP, with larger cell numbers (often 20-25) and occasional mitotic figures. In the late or developed VGP (Figure 3), melanomas form expansile nodules in the dermis with cytology different from melanoma cells in the overlying epidermis. Mitotic figures are variably present, and tumor aggregates may extend into the reticular dermis or even subcutaneous fat. Dermal tumoral nests are larger in VGP than in RGP. Survival in VGP is directly related to the measured tumor thickness (vertical diameter) and is also influenced by the presence of regression, tumor-infiltrating lymphocyte response, dermal mitotic activity, ulceration and satellitosis, patient gender and age, and anatomic site of involvement. In general, when melanoma cells reach the VGP, they have also acquired metastatic potential.
The clinical prognosis of melanoma may also be related to the traditional subtypes. (2,5,9) For example, there is some evidence that nodular melanoma and acral lentiginous melanoma have a worse prognosis than other subtypes. (2,5,9) Conversely, lentigo maligna melanoma is limited to the skin areas most exposed to sunlight and is characterized by longer intraepidermal or radial growth phases (long RGP) (Figure 1). In superficial spreading melanoma, the intraepidermal growth phase is generally of shorter duration than in lentigo maligna melanoma. Acral lentiginous melanoma is the most common form of melanoma in dark-skinned individuals and involves predominantly the palm of the hands, soles of the feet, and nail beds (Figure 4). Similar to lentigo maligna melanoma, atypical melanocytes of acral lentiginous melanoma remain mostly at the RGP, although their behavior is more aggressive than in lentigo maligna melanoma. Nodular melanoma exhibits a pure VGP, and the disease is almost never detected at the intraepidermal growth phase or RGP. Special care should be taken with desmoplastic melanoma, a rare neoplasia of uncertain pathogenesis that is difficult to diagnose. Desmoplastic melanoma (Figure 5) is best classified as a variant of spindle cell melanoma and is characterized by a fibrous stroma with a tendency to local infiltrative growth and recurrence, as well as neurotropism. (22,23)
[FIGURES 4-5 OMITTED]
A 4-stage system for the classification of melanoma has been recommended by the American Joint Committee on Cancer (AJCC). In this classification, the localized growth phase is represented by stages I and II, regional involvement by stage III, and distant metastatic disease by stage IV. A more recent revised staging system has been submitted by the AJCC and presented for official recommendation (Table 1). (24) In this system it is proposed (1) to remove Clark's level of invasion from the T category, except for T1 melanomas measuring less than 1.0 mm (see Table 1), and add ulceration of primary tumor; (2) to include in the N category the number of metastatic lymph nodes and type of involvement (microscopic vs macroscopic); (3) to include in the M category the anatomic site of distant metastases and level of serum lactase dehydrogenase; (4) to increase the staging for patients with stages I through III when the primary tumor is ulcerated; and (5) to merge satellite metastases and in-transit metastases into a single entity under stage III disease. The only difference between pathologic (Table 1) and clinical criteria for staging relates to the upgrading in the N category of stage IIIA to N1b, stage IIIB to N2b, and stage IIIC to N2c. (24)
The clinical and histologic variables directly affecting patient survival in localized melanoma are listed in Table 2. To reiterate, disease outcome is influenced by clinical factors, such as race, age, gender, and anatomic site. Outcome is also influenced by histologic determinants, such as type of growth, tumor thickness (total vertical dimension), dermal mitoses, tumor regression, tumor-infiltrating lymphocytes, ulceration, satellitosis, and angiolymphatic spread. Ulceration of the primary tumor (its width, but not depth) has recently been recognized as an independent marker affecting survival rate. (25,26) Evaluation of most of these factors is provided in the standardized melanoma profile supplied by dermatopathology laboratories. As would be anticipated, development of regional or systemic metastases (stages III-IV) becomes a major predictive factor of decrease in patient survival (Figure 6).
[FIGURE 6 OMITTED]
The process of melanoma metastasis follows several well-defined and interrelated steps, whose temporal sequence is determined by the interaction between intrinsic behavior of melanoma cells and host response. (27) When the regional lymph nodes become involved, the 5-year survival rate decreases to approximately 30% to 40%, with worsening of prognosis as the number of involved lymph nodes increases, extranodal disease becomes apparent, and/or the primary tumor becomes thicker and ulcerated. (2,5,9-14,24-26) The prognosis for primary tumors is also worse when localized in the trunk, as opposed to its presence in the extremities. Once distant metastases are present, the disease is almost incurable and median survival is approximately 6 months. (2,8-11) A large number of metastases, involvement of visceral sites, and short duration of remission are further unfavorable prognostic factors. Gender may also influence the course of the disease, which has a worse prognosis in males than females. (5,24-26) There is no clear explanation why women have better survival. Contributing factors may include distribution of melanoma lesions localized to more favorable anatomic sites as compared to men. (5) Furthermore, men also present with a higher proportion of ulcerated lesions that are also thicker. (5,24-26)
Melanoma susceptibility is influenced by genetic factors, such as familial incidence of melanoma, race background, skin type, and gender; constitutional factors, such as age, number, size, and type of pigmented nevi; and environmental factors, such as the accumulated lifetime exposure to solar light. (1-9) Thus, in approximately 10% of cutaneous melanomas there is a history of the familial atypical multiple mole melanoma syndrome. There is also race group effect, with the highest incidence of melanoma found in white individuals and the lowest frequencies among black, dark Asian, and east Asian populations. (1-9) Melanoma is also more common in photosensitive type I skin and less frequent in the tanning-prone type IV skin. Accordingly, individuals with red or blond hair and blue eyes are more susceptible to develop melanomas. In black and Asian populations, melanomas are located predominantly on acral sites and have unfavorable prognosis.
In general, women with melanomas have better survival rates than men, and the risk of melanoma development is higher in individuals older than 15 years than in younger children. (2,5,8,9) Premalignant or malignant pigmented lesions, such as lentigo maligna or malignant melanoma of lentigo maligna type, are characteristic of older age (sixth and seventh decades). (2,5,8,9) The risk of developing a melanoma is generally enhanced with increases in the number and size of melanocytic nevi, particularly dysplastic nevi. (2,5,9) The atypical lesions most prone to melanoma transformation are acquired dysplastic melanocytic nevi, melanocytic dysplasia on acral or mucosal surface, atypical spindle cell and/or epithelioid melanocytic nevus, and dysplastic and/or congenital nevus spilus. (2,5,9) Benign lesions with relatively low risk include congenital melanocytic nevus, nevus of Ota, nevus of Ito, and cellular blue nevus. (2,5)
An important environmental consideration for the diagnosis of pigmented lesions is a history of prolonged sun exposure and frequent episodes of sunburn, since the ultraviolet light component of solar radiation promotes malignant melanoma development after a sometimes-extended latency period. (6) Experiments performed in animal models suggest that UVA and UVB may be equally responsible for malignant transformation of melanocytes. (28,29) This finding is in contrast to epithelial cancers, for which there is a strong association between carcinogenesis and UVB, but not UVA exposure. (8) These findings are of clinical and epidemiologic concern since many commercial sunscreens that provide protection from UVB are not as effective against UVA.
GENETIC ABNORMALITIES IN MELANOMA
The familial atypical multiple mole melanoma syndrome is characterized by recurrent chromosomal abnormalities detected not only in the atypical nevi, but also in fibroblasts and lymphocytes. (2-7) Several studies have demonstrated linkage of the familial atypical multiple mole melanoma syndrome to 9p21, with the p16 locus being most frequently mutated (reviewed in references 2-7 and 30). In addition, at least 1 study found a linkage to chromosome 1p36 (reviewed in references 3 and 4).
Sporadic dysplastic nevi show a high rate of loss of heterozygosity in chromosomes 1p and 9q (31); some variants show a predominant allelic deletion at chromosome 9p21 (p16 locus) that may be accompanied by deletion in 17p13 (p53 locus). (32) Cytogenetic abnormalities also have been found in Spitz nevi (33); these changes have not been detected in benign nevi. Telomerase activity, a potential tumoral marker, is higher in dysplastic than in benign common melanocytic nevi, increasing further during progression to melanoma. (34) These observations underscore a genetic predisposition for progression to melanoma in some melanocytic nevi, and further suggest that specific molecular markers may eventually predict which dysplastic or Spitz nevi have the potential for progression to malignant melanoma. The obvious therapeutic implications reside in the differential management (size of excision margin and clinical follow-up) for those dysplastic or Spitz nevi containing mutations, as opposed to the more conservative approach for nevi without genetic changes. (31-34)
In sporadic melanomas, chromosomes 1, 6, 7, 9, and 10 are most commonly affected. (2-9,30,35-40) In chromosome 1, structural rearrangements are frequent and include translocations or deletions of 1p12-22, (3) loss of heterozygosity in 1p, (3,41,42) and deletion of 1p36. (3,43) Interestingly, deletion of 1p36, usually found at higher Clark levels, is also frequent in nodular melanomas, suggesting a putative tumor suppressor gene on this locus. (3,43) In chromosome 6, the most commonly noted changes have been nonreciprocal translocations or deletions involving 6q11-6q24. (3,35,36,39,42) There is also an increased frequency of loss of heterozygosity in 6q, (44-46) and it has been proposed that this locus contains another tumor suppressor gene for melanoma. (47,48) The abnormalities in chromosome 7 consist of chromosomal loss or gain, (30,39,42,43,49) with the latter associated with increased expression of the receptor for epidermal growth factor located on 7p12-13. (50) Loss of chromosome 10 is frequently associated with melanoma progression, (3,30,49,51,52) related perhaps to loss of the nma gene, located at 10p11.2-p12.3, which is a potential inhibitor of metastatic capability. (53,54) Abnormalities in chromosomes 7 and 11 may be linked to shorter survival in melanoma patients. Recent studies show that acral melanomas have distinct molecular defects at regions 11q13, 22q11-13, and 5p15. (55) Furthermore, molecular probing has detected malignant cells (up to 3 mm) beyond the histologically detectable boundary, thereby revealing a potential mechanism for local recurrence of melanomas excised with narrow margin. (55)
Since 9p21 may encode tumor suppressor genes, cytogenetic changes in chromosome 9 are of great interest. (56-63) Thus, the tumor suppressor gene p[16.sup.INK4] maps to chromosome 9p13-22, which contains the putative locus of familial melanoma. (3-5,56-61) The p[15.sup.INK4B] tumor suppressor gene, also located on chromosome 9p21, has been linked to melanoma progression. (64)
Mutations in Tumor Suppressor Genes and Oncogenes
Sporadic and familial melanomas have been associated with mutations, loss of heterozygosity, and deletions in the p16 locus, which is important for the normal progression of the cell cycle. (2-7) Expression of p16 was further reported to correlate inversely with aggressive melanoma behavior (3,4,56-63); nevertheless, mutations at the p16 locus also have been detected in normal melanocytes and in benign compound nevi lacking signs of clinical or histologic atypia. (65) Analysis of familial melanomas in patients living in Australia suggests that mutation in other genes, but closely linked to p16 on chromosome 9, may be involved in some hereditary melanoma kindreds. (59) It has also been proposed that germline p16 mutations suffice to explain most, if not all, 9p21-linked familiar melanomas. (66) Presumed oncogenic mechanisms include the already identified mutations in the p16 binding domain of CDK4 that generate a dominant oncogene resistant to normal physiological inhibition by p16. (66) Thus, p16 is presently the best candidate for tumor suppressor gene in melanoma; studies on its role and that of other cell cycle regulatory proteins should help define their involvement in melanoma progression.
Notwithstanding the significance of p16, p15 and p19, which code for additional proteins regulating cell cycle, are also located on chromosome 9p and may be involved in melanocyte transformation. (3,37,56,60,64) Conversely, nm23, nmb, and nma, which may inhibit melanoma metastatic potential, represent potential candidates for tumor suppressors. (3,4,42,53,54) Indeed, expression of nm23 correlates inversely with tumor progression. (4,42) Mutations in p53 are the most common contributors to the etiology of neoplastic disorders, but their role in the pathogenesis of melanoma has not been established. Thus, there is no apparent correlation between p53 gene rearrangement or altered expression of p53 protein and progression of melanocytic lesion. (67) Nevertheless, some authors suggest that p53 could have a more complex role in the pathogenesis of melanoma by acting on downstream effector genes, such as MDM2, GADD45, and CIP1/WAF1. (68) It has also been suggested that the level of expression of p53 could modify the response of melanoma cells to chemotherapeutic agents. (69) Evaluation of these possibilities warrants further studies on the role of p53-activated pathways in melanoma biology. (70)
Mutated ras proto-oncogenes have been detected in approximately 5% to 6% of human melanoma specimens, (42) and mutations affecting N-ras are most frequent in the sun-exposed areas. (3,4) Mutations in Ha-, Ki-, and N-ras oncogenes have been also detected in cultured melanoma cells and in cell lines of metastatic melanomas. (3,42) The mutations of N- and Ki-ras have been associated with over-expression of epidermal growth factor receptor and dedifferentiated phenotype. (3,42) However, the Ki-ras mutation has also been found in benign melanocytic nevi, (49) and thus, while ras may play a role in malignant melanocytes proliferation, its actual contribution to melanoma development remains unclear. Other nuclear oncogenes with reported altered expression in cultured melanoma cells, but with questionable roles in melanoma development, include c-myb, c-myc, c-fos, and c-jun. (3,4,42,71) Negative results also have been reported for c-src, a non-receptor membrane-associated tyrosine kinase. (4,42) The role of the c-kit proto-oncogene that encodes the transmembrane tyrosine kinase receptor KIT/SCF-R is unclear, despite the KIT receptor involvement in melanoblast migration and differentiation toward melanocytes. (72)
GROWTH FACTORS AND THEIR RECEPTORS
Melanomas that produce growth factors and cytokines, together with the corresponding receptors, can potentially autoregulate their own tumoral behavior. For example, the expression of receptors for the proopiomelanocortin (POMC)-derived neuropeptides melanocyte-stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) are known to modify the phenotype of normal and malignant melanocytes. (1,73-75) Thus, both ACTH and MSH interact with their specific cell surface receptors to enhance melanocyte proliferation, melanogenesis, and dendrite formation. (1,73-75) The peptides also act as potent immunosuppressors, (76,77) and [alpha]-MSH down-regulates expression of the adhesion molecule NCAM-1 on normal and malignant melanocytes. (77) In melanoma cells, the expression and activity of MSH receptors (MC1-R) are selectively stimulated by UVB light, dbcAMP, MSH itself, phosphorylated isomers of L-DOPA, L-tyrosine, retinoic acid, interleukin-1, and the interferons [alpha], [beta], and [gamma]. (78-84) A possible role of MC1-R in melanoma development is suggested by the finding of mutations at the MC1-R locus (resulting in the red hair phenotype) that have been detected in melanoma. (85,86) Furthermore, it has been suggested that MC1-R status may be a determinant of risk for cutaneous melanoma development. (87,88)
Melanoma cells actually express the POMC gene and produce ACTH, MSH, and [beta]-endorphin peptides. (73,75,77,89-91) Proopiomelanocortin antigens are commonly detected in melanomas, showing more intense and diffuse immunocytochemical stain in VGP. (90,91) In contrast, POMC peptides are absent from normal skin melanocytes or benign blue nevi; a weak level of expression is seen in common and dysplastic melanocytic nevi, with a trend for higher expression levels in the latter. (91) Measurement of serum [alpha]-MSH with direct radioimmunoassay found detectable concentrations in patients with melanoma (92) that correlated positively with advances in clinical stage or level of invasion. (93) Ultraviolet light, a putative melanocyte carcinogen, can also stimulate melanocyte production of MSH and ACTH, as well as expression of the corresponding receptors. (77,81-83) Thus, it has been proposed that local expression of POMC peptides and their corresponding receptors may alter melanoma behavior through autocrine, intracrine, or paracrine mechanisms and/or through the modulation of the local immune and vascular systems. (75)
Other growth factors that regulate the phenotype in normal and malignant melanocytes include basic fibroblast growth factor (bFGF), mast cell growth factor, hepatocyte growth factor, endothelins, insulin growth factor, insulin, transforming growth factor-[alpha] (TGF[alpha]), and neuron growth factor. (1,3,94-98) It has been further proposed that bFGE an important growth regulator for normal melanocytes, can also act as a melanoma oncogene. (94,95) Thus, cultured melanoma cells produce bFGF that autostimulates their own growth; treatment with either antisense deoxynucleotide against bFGF or anti-bFGF antibodies inhibits the growth of tumor cells. In situ studies have shown that bFGF expression is absent in normal melanocytes and becomes detectable during neoplastic progression of melanocytic lesions, including desmoplastic melanoma. (99,100) Hepatocyte growth factor and mast cell growth factor stimulate melanocyte growth through respective activation of the c-met receptor and the product of c-kit proto-oncogene; both receptors are membrane bound and display intrinsic tyrosine kinase activity. (3,42,94,95) Endothelins also affect the proliferation, motility, and differentiation of melanocytes. (3,98)
A number of studies have shown that insulin and insulin growth factor stimulate melanoma cell growth, both in vivo and in vitro. (3,42) However, Kahn et al (101) found that insulin can alternatively inhibit or stimulate the proliferation of melanoma cells, depending on their genotype. Furthermore, expression of a stimulatory response to insulin was dependent on the phosphorylation of a protein of approximately 85 to 90 kd (pp90). (102) Insulin-resistant lines had defective insulin receptor because of adenosine triphosphate-dependent partial proteolysis of the [beta] sub-unit. (103)
In situ analyses showed that the pattern of expression of receptors for neuron growth factor and epidermal growth factor correlated positively with stage of development in melanocytic lesions. (104,105) Expression of TGF[beta] was also increased in advanced stages of melanoma development, as compared to its low levels of expression in benign lesions. (106-108) There are similar data for insulin growth factor, suggesting a positive correlation between melanoma progression and increased growth factor expression. (109) A higher TGF[alpha] production rate in dysplastic than benign nevi suggests a role for the TGF[alpha]/epidermal growth factor receptor system in the evolution of melanocytic lesions. (105,109)
Melanomas also produce other growth factors and cytokines, such as keratinocyte growth factor, platelet-derived growth factor-[alpha], platelet-derived growth factor-[beta], stem cell factor, melanoma growth stimulating activity, interleukin (IL)-1[alpha], IL-1[beta], IL-6, IL-7, IL-8, IL-10, IL-12, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, TNF[alpha], interferon [gamma], and interferon [beta]. (2,3,42,94-97,106-115) Through interaction with their corresponding receptors, many of those factors could act as regulators of melanoma cell proliferation, differentiation, and motility, and could stimulate angiogenesis. In addition, they could modify expression of major histocompatibility antigens, cell adhesion molecules, integrins, nonintegrin matrix adhesion receptors, and extracellular matrix proteins on melanocytes and surrounding cells, and they could affect local and systemic immune system activity. Thus, a complex signaling network is formed within the melanoma that results in the acquisition of multicytokine resistance and growth factor production autonomy in the advanced stages. (113) In this context, as compared to VGP melanoma, RGP melanomas are less effective in induction of angiogenesis, more sensitive to cytokines and growth factors, and prone to undergo apoptosis under a variety of growth conditions. (111)
MELANOGENESIS IN THE DIAGNOSIS OF MELANOMA
An important diagnostic tool that helps separate melanomas from other tumors is the capability to synthesize melanin and to express the enzymatic and structural proteins involved in the process. Melanin synthesis is initiated with the enzymatic hydroxylation of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA) and oxidation of L-DOPA to DOPAquinone. (1,116) DOPAquinone is subsequently transformed to melanin in a series of reactions accelerated by enzymes or metal cations. (1,116) The type of melanin produced depends on cellular genotype and environmental factors, resulting in the black pigment eumelanin, the reddish to yellow pigment pheomelanin, or the mixed melanin that contains both components. (1,116) Melanin can be detected with the standard histologic stains or with the specific Fontana-Masson method. Melanin concentration and type (eumelanin or pheomelanin) can be conveniently determined by electron spin resonance spectroscopy using paraffin blocks, fresh or frozen tissue. (117-121) Eumelanin is characterized by the presence of paramagnetic centers that are solely of the semiquinone type, while pheomelanin contains a hyperfine structure with an unpaired electron near the nucleus of [sup.14]N. (121) The detection and quantification of melanin provide helpful information about therapeutic options and outcome, because melanin can attenuate the effect of radiotherapy, phototherapy, or chemotherapy. (116,117,122) Moreover, intermediate products of melanogenesis can suppress local and, possibly, systemic immune responses. (122,123) Thus, the presence of melanogenesis products or of melanin itself may be responsible for unsatisfactory response to therapy. We have proposed that uncontrolled melanogenesis generates an oxidative environment and produces genotoxic and mutagenic intermediates that can destabilize tumor cells and their microenvironment and result in melanoma progression. (122,124) This effect would be further amplified by the immunosuppressive activity of melanin precursors. (122) Other authors, however, have proposed the opposite approach, the use of melanogenesis cytotoxicity as the basis for an experimental melanoma therapy that would incorporate up-regulation of melanogenesis with intratumoral delivery of potentially cytotoxic false precursors of melanin. (125,126)
The specific ultrastructural marker of melanocytes is the presence of melanosomes. (1,116) These are metabolically active organelles that affect the metabolic state and function of the host cell. (122,127,128) Melanosome biogenesis involves 4 stages. (1,116,128) In the eumelanogenic pathway, stage II melanosomes, which are devoid of melanin, develop a fibrillar matrix. Stage III melanosomes are formed after delivery of tyrosinase via vesicles from the trans-Golgi reticulum and initiation of melanin synthesis. Stage IV melanosomes are represented by organelles filled with electron-dense melanin. In pheomelanogenesis, vesiculoglobular bodies are incorporated into stage I melanosomes, while at stage II melanosome pheomelanin is deposited in the vesiculoglobular bodies. (1,116) Pheomelanin synthesis depends on availability of cysteine to conjugates with DOPAquinone and form the precursor cysteinylDOPA. (1,116) This process may become defective in melanomas, in which tyrosinase may be incorporated and activated at stage I of melanosome formation, and melanin is deposited within organelles of "granular type" that contain neither fibrillar nor vesiculoglobular matrix. (129) The structure of melanosomes is frequently abnormal in malignant lesions and may allow leakage of melanogenesisrelated cytotoxic and potentially mutagenic products into sensitive cellular compartments. (129-133) Thus, electron microscopy, which is used for the diagnosis of melanoma, could also help predict melanoma behavior on the basis of degree and type of melanosomal defects.
Among the commonly used immunocytochemical markers for melanoma, the S100 antigen has high sensitivity since it is found in almost all cases. However, S100 lacks sufficient specificity because it is also expressed in nonmelanocytic cells. Another melanogenic marker (Table 3), the HMB-45 melanosomal antigen, is less sensitive but highly specific for pigmented cells. (1) The melanosomal protein MART-1/Melan-A shows promise of becoming an important tool for the diagnosis of melanoma. (134) Tyrosinase enzyme represents a powerful marker of melanoma, since expression of the tyrosinase gene is restricted to cells of melanocytic origin. In fact, tyrosinase protein or its messenger RNA (mRNA) can be detected even in undifferentiated amelanotic melanomas, (135-137) including those lacking premelanosomes, as documented at the ultrastructural level. (129) We have also detected tyrosinase protein expression in selected cases of desmoplastic melanoma. The relative abundance of tyrosinase mRNA in some undifferentiated amelanotic melanomas (135) allows its use in in situ hybridization techniques as a valuable diagnostic option. (136) The same can be said for the determination of tyrosinaserelated protein-1 (TRP-1), recognized by the commercially available antibody MEL-5, and the tyrosinase-related protein-2 (TRP-2), which acts as dopachrome tautomerase. Expression of these genes and proteins is found predominantly in cells of melanocytic lineage. Thus, immunocytochemistry for enzymatic protein tyrosinase, TRP-1 and TRP-2, and structural melanosomal proteins, collectively called melanogenesis-related proteins (MRPs), should provide highly specific and sensitive tools for the positive identification of melanomas. The promoter region of the TRP gene family contains an M box that binds the microphthalmia gene product. (1) This is a transcription factor of the basic helix-loop-helix family that can regulate expression of the melanogenic machinery. (1,138) Recent immunocytochemical studies showing high sensitivity and specificity for the microphthalmia antigen suggest that antibodies against the M gene product could potentially become an excellent tool for the diagnosis of melanoma. (138)
One of the critical issues in the histopathologic management of melanoma is the identification of melanoma cells in the sentinel lymph node, which is negative on hematoxylin-eosin sections (Figure 7). To detect metastatic disease in those cases, HMB-45 immunocytochemistry lacks sensitivity, while highly sensitive S100 immunocytochemistry is nonspecific, for example, it also stains cells of nonmelanocytic origin, introducing a degree of subjectivity in the diagnosis. In such a setting, the use of antibodies against MART-1, tyrosinase, TRP-1, TRP-2, and M proteins could help identify true melanoma cells. These immunocytochemistry tests could be complemented with molecular analyses (see below).
[FIGURE 7 OMITTED]
Since the mRNA for MRP is frequently abundant even in amelanotic melanoma cells, the detection of mRNAs for tyrosinase, TRP-1, TRP-2, MART-1, HMB-45 using Northern blotting, reverse transcriptase-polymerase chain reaction, or in situ hybridization techniques can be helpful for the diagnosis of melanoma or for detection of metastatic disease. In fact, testing of mRNA for tyrosinase and MRPs with the reverse transcriptase-polymerase chain reaction technique in blood or bone marrow samples has confirmed that circulating metastatic cells can be detected before their clinical expression. (139-145) Some sentinel lymph nodes that are both histologically and immunocytochemically negative for melanoma cells become positive on the reverse transcriptase-polymerase chain reaction for tyrosinase mRNA. (140,144,145) However, to avoid false-positive detection by molecular techniques, analysis of regular hematoxylin-eosin-stained sections should be performed to exclude presence of benign melanocytic nevus or Schwann cells in the lymph node. Therefore, amplification of complementary DNA-encoding MRPs in conjunction with histology is probably the optimal strategy for the detection of metastatic disease in sentinel lymph node.
Biochemical Markers of Melanoma
Serum levels of tyrosinase and melanin precursors correlate positively with melanoma progression. (146-149) Thus, patients with advanced melanoma show increased plasma or even urine levels of the melanin precursors DOPA, 5-Scysteinyldopa (5-S-CD), dihydroxyindole (DHI), its carboxylic form (DHICA), and O-methyl derivatives of DHI and DHICA. (146-151) Use of these markers allows more selective melanoma staging; for example, high plasma levels of 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C) are generally correlated with tumor thickness greater than 3.0 mm, independent of the absence of metastases. The same abnormality can be occasionally seen in thinner melanomas, but only when metastases are present. (146) Therefore, humoral levels of MRPs or melanogenic products can provide information on progression, regression, or recurrence of the disease, or on the presence of occult melanoma. Serum tyrosinase can be detected with assays for measuring either enzyme activity or enzyme concentration (radioimmunoassay). (150,151) Since intermediates of melanogenesis can act as potent immunosuppressors, (122) and tyrosinase may amplify that activity (by inducing continuous oxidation of tyrosine to form lymphocytotoxic precursors of melanin), the finding of increased serum levels of intermediates of melanogenesis and tyrosinase activity may serve as an indicator of impaired host immune response to melanoma. (122,152)
IMMUNE MARKERS FOR MELANOMA THERAPY
Tyrosinase, TRP-1, TRP-2, HMB-45, and melanocyte-specific MART-1 are classified as major histocompatibility complex class I-restricted tumor antigens, (16-18,20,21,153-156) implying that their expression in relation to specific human haplotypes could be used as a guide for potential immunotherapy. The family of major histocompatibility complex class I-restricted melanoma antigens also includes the MAGE proteins, which are expressed on melanoma cells but not melanocytes. (157) The T-cell immune response to these antigens is highly heterogenous, since tyrosinase-derived peptides may be recognized by T cells in association with HLA-A2, HLA-A24, HLA-B44, HLA-A1, HLA-DR4, and HLA-DR15. (15-18,153-156) Immunization with purified tyrosinase enzyme to induce a humoral response has also been suggested as an adjuvant therapeutic strategy for malignant melanoma. (152)
Stimulation of the humoral (antibody) response against cell surface antigens, such as plasma membrane gangliosides, represents another immunotherapeutic approach for the treatment of melanoma. Plasma membrane gangliosides are good candidates as substrates for antimelanoma vaccines. (18-20,158-161) However, the pattern of ganglioside expression changes during melanoma progression. (156,162,163) Whereas in animal models of melanoma specific ganglioside patterns correlate with tumor growth rate and melanoma differentiation, (164,165) in human melanoma the expression of gangliosides in either localized or metastatic lesions is heterogenous. Thus, ganglioside pattern cannot be used as a diagnostic marker of melanoma progression. Analysis for specific ganglioside fractions, for example, GD2, GD3, 0-Ac-GD3, or GM2, is nevertheless valuable in the context of immunotherapy with antiganglioside antibodies or with ganglioside-containing antimelanoma vaccine. In a similar fashion, high-molecular-weight melanoma-associated antigens have been also proposed as targets for highly selective antimelanoma therapy. (16,18,20,21,166-168)
To summarize, information recently acquired in the fields of biochemistry and molecular biology of melanin synthesis is becoming important for the optimal management of melanoma. For example, the potential effectiveness of immunotherapy may be reduced by ongoing active melanogenesis, since intermediates of melanogenesis can suppress immune responses. (122) Moreover, uncontrolled melanogenesis that generates an oxidative environment with genotoxic and mutagenic intermediates can destabilize tumor cells and their microenvironment, triggering melanoma progression. (122) Therefore, melanoma therapy could be strengthened by the addition of differential upregulation of MRPs expression, antigen-specific immunization, and inhibition of melanogenesis. (122) Inhibition of melanogenesis could serve an adjuvant role when preparing a vaccine from nonmelanogenic antigens, for example, gangliosides, for the therapy of melanoma.
Table 1. Staging of Melanoma Melanoma TNM Classification T Classification Thickness, mm Ulceration Status T1 [less than or equal to] 1.0 a: Without ulceration and level II/III b: With ulceration or level IV/V T2 1.01-2.0 a: Without ulceration b: With ulceration T3 2.01-4.0 a: Without ulceration b: With ulceration T4 >4.0 a: Without ulceration b: With ulceration N Classification No. of Metastatic Nodes Nodal Metastatic Mass N1 1 Node a: Micrometastasis * b: Macrometastasis ([dagger]) N2 2-3 Nodes a: Micrometastasis * b: Macrometastasis ([dagger]) c: In transit metastasis(es)/ satellite(s) without metastatic nodes N3 4 or more metastatic nodes, or matted nodes, or in transit metastasis(es)/ satellite(s) with metastatic node(s) M Serum Lactase Classification Site Dehydrogenase M1a Distant skin, subcutaneous, Normal or nodal metastases M1b Lung metastases Normal M1c All other visceral Normal metastases Any distant metastasis Elevated Proposed Stage Groupings for Cutaneous Melanoma Clinical Staging Pathologic Staging ([double dagger]) ([sections]) 0 Tis N0 M0 0 Tis N0 M0 IA T1a N0 M0 IA T1a N0 M0 lB T1b N0 M0 IB T1b N0 M0 T2a N0 M0 T2a N0 M0 IIA T2b N0 M0 IIA T2b N0 M0 T3a N0 M0 T3a N0 M0 IIB T3b N0 M0 IIB T3b N0 M0 T4a N0 M0 T4a N0 M0 IIC T4b N0 M0 IIC T4b N0 M0 III Any T Any N M0 IIIA T1-4a N1a M0 T1-4a N2a M0 IIIB T1-4b N1a M0 T1-4b N2a M0 T1-4a N1b M0 T1-4a N2b M0 T1-4a N2c M0 IIIC T1-4b N1b M0 T1-4b N2b M0 T1-4b N2c M0 Any T N3 M0 IV Any T Any N Any M IV Any T Any N Any M * Micrometastases are diagnosed after sentinel or elective lymphadenectomy. ([dagger]) Macrometastases are defined as clinically detectable nodal metastases confirmed by therapeutic lymphadenectomy or when nodal metastasis exhibits gross extracapsular extension. ([double dagger]) Clinical staging includes microstaging of the primary melanoma and clinical/radiologic evaluation for metastases. By convention, it should be used after complete excision of the primary melanoma with clinical assessment for regional and distant metastases. ([section]) Pathologic staging includes microstaging of the primary melanoma and pathologic information about the regional lymph nodes after partial or complete lymphadenectomy. Pathologic stage 0 or stage IA patients are the exception; they do not require pathologic evaluation of their lymph nodes. Table 1 represents modification of the table published by Balch et al (24) and is published by permission of the American Cancer Society and of Wiley-Liss, Inc, a subsidiary of John Wiley & Sons, Inc. Table 2. Prognostic Variables in Localized Melanoma Variable Category ([dagger]) Value Tumor thickness, * mm [less than or equal Survival decreases to] 1.0 with increased 1.01-2.0 tumor thickness 2.01-4.0 >4.0 Level of invasion I (intraepidermal) Survival decreases II (into papillary with increased dermis) level of invasion III (filling papillary dermis) IV (into reticular dermis) V (into subcutaneous fat) Growth pattern RGP, VGP VGP indicates metastatic capability Histologic subtype SSM, LMM, ALM, NM, DM Unclear([double dagger]) Mitotic rate 0, 1-6, >6 Increasing numbers of (per square unit) * mitoses indicate worse prognosis Tumor-infiltrating Brisk, nonbrisk, Nonbrisk and absent lymphocytes absent indicate worse response * prognosis Regression * Presence/absence Presence increases risk for metastasis Ulceration * Presence/absence Worse prognosis with ulceration Microscopic satellites Presence/absence Presence indicates worse prognosis Vascular invasion Presence/absence Presence indicates worse prognosis Cell morphology Shape, presence or Unclear absence of melanin Gender * Female/male Better prognosis for women Anatomic location * Head and neck, trunk, Better prognosis for extremities extremities vs head and neck, palms or soles, or trunk Age Young/old Aging increases morbidity and mortality * Independent factor in some multivariate analyses. ([dagger]) RGP indicates radial growth phase; VGP, vertical growth phase; SSM, superficial spreading melanoma; LMM, lentigo maligna melanoma; ALM, acral lentiginous melanoma; NM, nodular melanoma; and DM, desmoplastic melanoma. ([double dagger]) According to some authors, NM and ALM have worse prognosis, and DM has better prognosis but higher recurrence rate. Table 3. Melanogenesis-Related Proteins * Therapeutic Protein Function Application Application TYR Enzyme Melanocyte marker (?) Immunotherapy TRP-1 Enzyme Melanocyte marker (?) Immunotherapy TRP-2 Enzyme Melanocyte marker (?) Immunotherapy Pmel-17 Enzyme Melanocyte marker (?) Immunotherapy Mart-1/ Melanosomal Melanocyte marker (?) Immunotherapy Melan-A protein MITF Transcriptional Melanocyte marker Unclear regulator P protein Regulatory/ Melanocyte marker Unclear melanosomal protein ([dagger]) LAMP (1-3) Membrane proteins Unclear Unclear Calnexin Chaperone Unclear Unclear (p90) * TYR indicates tyrosinase; TRP-1, tyrosinase-related protein-1; TRP-2, tyrosinase-related protein-2; Pmel-17, human homolog of the product of silver locus; Mart-1/Melan-A, structural melanosomal protein; MITF, microphthalmia-associated transcription factor; LAMP (1-3), lysosome-associated membrane proteins; and (?), possible. ([dagger]) Mutation of P gene causes tyrosinase positive oculocutaneous albinism.
This work was supported by grant 99-51 from the American Cancer Society, IL Division, to Dr Slominski.
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Accepted for publication June 1, 2001.
From the Department of Pathology, University of Tennessee Health Sciences Center, Memphis (Dr Slominski); the Department of Medicine, Southern Illinois University, Springfield (Dr Wortsman); the Department of Pathology and Laboratory Medicine, Albany Medical Center, Albany, NY (Dr Carlson); The Pacific Center for Dermatology and Phototherapy, Honolulu, Hawaii (Dr Matsuoka); the Departments of Surgery and Oncology, Johns Hopkins Medical Center, Baltimore, Md (Dr Balch); and the Department of Dermatopathology, Massachusetts General Hospital, Boston, Mass (Dr Mihm).
Reprints: Andrzej Slominski MD, PhD, Department of Pathology, 576 BMH, University of Tennessee Health Science Center, 899 Madison Ave, Memphis, TN 38163 (e-mail: firstname.lastname@example.org).
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