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PATHOLOGIC PARAMETERS IN THE DIAGNOSIS AND PROGNOSIS OF PRIMARY CUTANEOUS MELANOMA
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MELANOMA
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PATHOLOGIC PARAMETERS IN THE DIAGNOSIS AND PROGNOSIS OF PRIMARY CUTANEOUS MELANOMA H. Randolph Byers, MD, PhD, and Jag Bhawan, MD
The goal of pathologic diagnosis in malignant melanoma, as any lesion, is to relate the histologic findings to potential cell behavior and clinical outcome. Pathologists recognize malignant melanoma from benign or atypical melanocytic proliferations using architectural and cellular criteria; however, criteria are not always agreed upon.2 Expert dermatopathologists demonstrate only moderate concordance even when restricting the question to simply benign versus malignant.25There is clearly a greater need for more specific formalized weighted criteria or other tools to better diagnose and predict the behavior of melanocytic proliferations. Yet enormous progress has been made in the last 30 years in the recognition of precursor lesions, tumor progre~sion,’~ and the diagnosis of melanoma. This article will focus on the histologic features of melanoma that contribute to the refinement of prognosis and treatment of individual patients. BRESLOW THICKNESS
Once the diagnosis of malignant melanoma is made, the single most important histologic criterion predicting metastatic behavior and patient survival is the Breslow tumor thickness measurement in millimeters12
From the Department of Dermatology, Boston University Medical School, Boston, Massachusetts
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(also see article on Prognostic Factors by Drs. Stadelmann and Reintgen on page 767 of this issue). In this measurement, a calibrated ocular micrometer quantifies the distance from the top of the granular cell layer of the epidermis to the base of the tumor or to the deepest infiltrating cell in the dermis or subcutaneous tissue, whichever is greater. Fortunately, studies for Breslow thickness show high c o n c ~ r d a n c eand ~ ~ numerous studies have confirmed this relationship of the melanoma thick16, %, 57, 65 The ness in millimeters to metastasis and patient su~vival.~, findings have essentially been reproduced in multiple centers in several countries. Table 1 summarizes the histopathologic variables for 5- or 8year survival in two representative studies: the Western Canada melanoma study including 650 patients with stage I, level 11, 111, IV, and V (see next section on Clark level) melan0ma,6~and the University of Pennsylvania study by Clark et all6 on 264 patients with tumorigenic vertical growth phase (VGP) melanoma (see section on VGP). Breslow thickness remains the "gold standard" and allows great Table 1. HISTOLOGIC PARAMETERS IN PRIMARY CUTANEOUS MELANOMA AND SURVIVAL IN TWO REPRESENTATIVE STUDIES Survival (%) Histologic Parameters Breslow thickness in m M <0.76 (0.76-1.49)' [0.76-1.69]t (1.50-2.99)* [1.70-3.60]t (3.00-4.99)* [>3.60]t (>5.00)* Clark level II 111 IV V Mitotic rat& (low; <0.2/HPF)* [O/mmz]t (moderate; 0.2-l/HPF)* [0.1-6.0/mmz]t (high; >l/HPF)* [>6.O/rnm2]t Tumor-infiltratinglymphocytes$ (absent) (nonbrisk) (brisk) Regression$ Absent or incomplete Present
5 year'
8 yeart
98 90 83 66 57
93 87 60 33
97 90 79 58
96 76 61 39
93 81 76
96 79 38 87 75 59 77 60
'Data (in parentheses) from Worth AJ, Gallagher RP, Elwood JM, et al: Pathologic prognostic factors for cutaneous malignant melanoma: The Western Canada Melanoma Study. Int J Cancer 43:370, 1989; included microinvasive radial growth phase (RGP) and tumorigenic vertical growth phase (VGP). tData (in brackets) from Clark W Jr, Elder DE, Guerry D IV, et al: Model predicting survival in stage I melanoma based on tumor progression. J Natl Cancer lnst 81:1893, 1989; University of Pennsylvania study, included tumorigenic VGP only. *Independent predictor in multivariable logistic regression model of Clark et aLq6 §nd = not done. A variety of other parameters of lymphocytic infiltrates were studied.
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insight into melanoma biology; it can be used to create subcategories that guide clinical treatment, that is, limited surgical margins in thinner lesions or more aggressive surgical approaches such as wider excisions or sentinel node lymphoscinitigraphy and node excision in thick lesions?, 32, 51* 60, a Nevertheless, the Breslow thickness and survival data have limitations for use in any individual patient. There are clearly individuals with thin lesions that do poorly and individuals with thick lesions that do well. Anatomic limitations of the linear measurement may under- or overestimate tumor thickness. For example, if the tumor induces an epidermal hyperplasia, the granular layer is distanced from the tumor base by proliferative keratinocytes, and an overestimation of tumor is made. Likewise, for an excoriated or ulcerated lesion or a lesion that has been subjected to previous exogenous destruction modalities such as electrocautery or freezing, an underestimation of tumor thickness may be made. In addition, other anatomic considerations such as proximity to superficial or deep vessels may influence metastasis. Furthermore, Clark17and others also investigated the role of histologic parameters other than thickness to better predict clinical outcome. Clark level and these other parameters are outlined subsequently.
CLARK LEVEL Anatomic or Clark level I is defined as confinement of melanoma cells to the intraepidermal compartment and is also known as melanoma in situ. Anatomic or Clark level I1 is defined as melanoma cells that have extended into the papillary dermis, whereas in level I11 the melanoma cells fill the papillary dermis. In level IV melanoma the tumor cells extend into the reticular dermis, and in level V there is involvement of the subcutaneous fat. Because melanoma in situ (level I) is confined to the epidermis, it is not given a Breslow thickness measurement. Nearly all level I or melanoma in situ (MIS) have 100% survival; however, for reasons that will be outlined later, rare metastases may be associated with MIS. Table 1 summarizes the relationship of Breslow thickness, Clark level, and numerous other histologic parameters to 5- and 8-year survival in the Western Canada65and University of Pennsylvania studies16; however, the Clark level, instead of being an independent predictor for survival in multivariate analysis, rather parallels that of tumor thickness. Nevertheless, it is helpful information to the pathologist and clinician to visualize from a report the extent of involvement of the different anatomic compartments by the tumor. Histologic criteria found to be independent of tumor thickness in multivariant analyses include the mitotic rate, the degree of tumor-infiltrating lymphocytic (TIL) host response, presence of regression, and ulceration. These criteria are discussed in the following sections.
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MITOTIC RATE
Numerous studies have shown that mitotic rate is an independent variable in melanoma prognosis.l6,65 A clear relationship exists, as in many other different tumor cell types, of high mitotic index and poor prognosis. Original studies on melanoma gave mitotic counts per 10 high power fields, but owing to variability in field size from microscope to microscope, number of mitoses per mm2 are now often used (after calibration of the objective field). For example, in the study by Clark et a1,16 absence of mitoses gave an 8-year survival of 95%. Counts of 0.1 up to 6.0 mitoses per mm2resulted in a drop down to 79% survival. Greater than 6.0 mitoses per mm2 resulted in a fall in survival to just 38% at 8 years. Owing to the different ranges and various percentages of survival seen in different studies, some pathologists prefer to note in the report simply whether there are no, few, or many mitoses present per 10 high power fields. TUMOR-INFILTRATING LYMPHOCYTES
The original concept of host inflammatory reaction has been sharpened by specific definition of tumor-infiltrating lymphocytes (TILs) and application of criteria that estimate their numbers.16The concept of TILs has evolved with immunobiology studies on the isolation and recognition of major histocompatability complex (MHC)-restricted T lymphocytes cytotoxic to autologous melanoma cells.36,40 The absence of TILs portends a worse prognosis (see Table l), whereas focal areas of TILs (nonbrisk infiltrate) improves prognosis by nearly 15%, and an extensive infiltrate of TILs improves 8-year survival by nearly 30%. TILs were found to be an independent predictor for survival16; however, "nonbrisk" versus "brisk infiltrates of TILs described in the original publication is subjective and needs further definition; some authors have presented diagrams to better characterize this di~tinction.~~ The concordance of evaluation of TILs has not been studied. Nevertheless, the impact on prognosis is consistent with a number of other studies that found lymphocytic host response or inflammatory reaction a favorable risk factor.65TILs, as opposed to lymphocytic host response, include lymphocytes distributed among the tumor cells and not those in focal areas in the papillary dermis adjacent or subjacent to the tumor or in necrotic foci or sclerotic portions of the tumor. REGRESSION
Regression has become defined as early (lymphocytes disrupting nests of melanoma cells), intermediate (loss of continuity of lesion with mild fibrosis), and late (extensive horizontal fibrosis) in a study with high interobserver con~ordance.~~ Regression may be identified with or
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without a marked reduction of “skip” areas of melanoma cells in the overlying epidermis and in the papillary dermis. In intermediate and late regression, scattered melanoma cells or small islands or cords of cells in the fibrotic papillary dermis may be seen. Furthermore, regression may be asymmetrically located on one end or may extend from one end of the lesion to the other.52Careful communication with the clinician is essential to determine if the fibrosis is spontaneous or secondary to a scar caused by a previous biopsy or destructive modality. The presence of regression identifies thin lesions less than 0.76 to 1.50 mm in thickness that show a greater risk of metasta~is.~, 55 Rare examples of very thin melanomas and even malignant MIS are associated with metastase~.~~ These lesions were found to exhibit striking fibrotic regression of greater than 75% of the lesion. Linking regression with greater risk of metastasis at first appears counterintuitive, because a successful cytotoxic T-lymphocytic response exists; however, these lesions may have progressed through a phase of immune recognition and destruction of the majority of melanoma cells, resulting in a subpopulation that has escaped recognition and migrated into the lymphatics. Such thin metastasizing melanomas indicate that variables other than thickness hidden from routine microscopic description influence aggressive cell behavior. Alternatively, regression may be viewed simply as an indication of underestimation of tumor thickness. Another histologic variable that may underestimate tumor thickness is ulceration.
ULCERATION
Several studies have shown that ulceration is an independent risk 65 Tumors with greater than 3.0 mm factor in melanoma prognosis.16* ulcer width likely underestimate the true potential thickness of the melanoma. First of all, the absent granular cell layer and the entire epidermis is not included in the measurement. Second, part of the upper portion of the tumor itself may be sloughed and/or necrotic; thus, the thickness is further underestimated.
MICROSCOPIC SATELLITES, VASCULAR INVASION, AND ANGIOGENESIS
Several other histologic parameters that appear related to prognosis 41 vascular invasi0n,2~ and of melanoma include microscopic ~atellites,3~, angiogenesis6,44; however, these parameters in multivariate analyses are not always independent predictors of survival.16,65 Nevertheless, these parameters are typically included in reports of level I11 to level V melanoma.
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NONTUMORIGENIC RADIAL GROWTH PHASE (RGP) AND TUMORIGENIC VERTICAL GROWTH PHASE (VG P)
In the 1980s, Clark, Elder,’6,35 and others developed the concept of melanoma tumor progression in at least three steps. First, the majority of primary melanomas begin as “in situ” or intraepidermal proliferations. Melanoma cell proliferations along the basement membrane, expanding laterally along radii, constitute in situ radial growth phase (RGP). These proliferations appear to begin as a lentiginous melanocytic hyperplasia, defined as restricted along the basement membrane. Later, in situ lesions show increasing detachment of the cells from the basement membrane and they are displaced into the upper epidermal cell layers, giving rise to the so-called pagetoid spread (Fig. 1). Second, melanoma cells gain the capacity to digest the basement membrane and migrate into the papillary dermis, hence the term invasive RGP (Fig. 2, arrows). Third, when the melanoma cells form tumors in the dermis, they are designated as being in the vertical growth phase (VGP)16(Fig. 3). The first two steps are combined into the term nonturnorigenic RGP, with the last step defined as the tumorigenic VGP. In other words, the nontumorigenic RGP melanoma includes all types (see classification section) of melanoma that are in situ (intraepidermal, level I) or microinvasive (involvement of papillary dermis, level 11). Tumorigenic VGP includes all types of melanoma (see classification section), with a predominant aggregate of cohesive cells filling the papillary dermis (level 111), or extending into reticular dermis (level IV) or subcutaneous fat (level V). This concept was developed largely in parallel with melanoma and other tumor cell biology studies that indicate acquisition of malignancy through multiple For example, mutations or specific gene transfection may lead to immortalization in vitro, but the cells retain an incapacity of tumorigenic potential in soft agar. Acquisition of additional specific mutations, chromosomal deletions, or amplifications results in tumor growth in soft agar and capacity of tumor formation in immunodeficient mice.26Furthermore, melanoma cells derived from VGP tumors exhibit a capacity for tumorigenic growth, whereas RGP cells do Finally, tumorigenic growth capacity in a number of cell types relates to capacity for experimental metastasi~.~~ Based on these experimental findings, Clark et all6 investigated survival in invasive nontumorigenic VGP melanoma. They found that of 122 invasive RGP melanoma, there were no metastases after a minimum follow-up of 8 years, with a median follow-up of 12 years. In contrast, of 264 patients with tumorigenic VGP, 8-year survival was 71%. Additional cases and studies in recent years have found that nontumorigenic RGP is not associated with metastatic risk.31Combination of this concept of tumor progression with the six independent prognostic variables including mitotic rate, TILs, tumor thickness, anatomic site of primary melanoma, gender of the patient, and histologic regression permits construction of a model that is 100% accurate in predicting survival in
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DIAGNOSIS AND PROGNOSIS OF PRIMARY CUTANEOUS MELANOMA
nontumorigenic RGP and 84% accurate for VGP tumors, with an overall accuracy of 87%.16Therefore, a statement in a pathology report to specify the presence of the tumorigenic VGP clearly conveys the risk of metastasis. CLASSIFICATION OF MELANOMA
A classification scheme must help the pathologist to recognize melanoma and guide the clinician or surgeon in treatment decision making. Table 2 lists a classification scheme by Clark, Elder, and others7,16, 22, 54 that incorporates the nontumorigenic RGP and tumorigenic VGP
Table 2. CLASSIFICATION OF MELANOMA: FREQUENCY AND GROUP SURVIVAL Frequency (%.)* Nontumorigenic (radial growth phase) In situ (level I) Superficial spreading melanoma$ Lentigo maligna melanoma* Acral lentiginous melanoma* Unclassified$ Microinvasive (level II) Superficial spreading melanoma$ Lentigo maligna melanoma$ Acral lentiginous melanoma* Unclassified* Tumorigenic (vertical growth phase; level 111, IV, and V) Superficial spreading melanoma$ Nodular melanoma* Lentigo maligna melanoma* Acral lentiginous melanoma* Variants Desmoplastic melanoma5 Neurotropic melanomas Balloon cell melanoma Minimal deviation melanoma Nevoid melanoma Other Unclassified
&Year Survival (%)t 100
100
23-28 1-3 1 1 84
37-42 22-32 3-4 2-3 <1 <1 <1 <1 <1 <1
2
'Data from Worth AJ, Gallagher RP, Elwood JM, et at: Pathologic prognostic factors for cutaneous malignant melanoma: The Western Canada Melanoma Study. Int J Cancer 43:370,1989;and Shaw HM, Balch CM, Soong SJ, et al: Prognostic histopathological factors in malignant melanoma. Pathology 17:271,1985;stage I only, in situ melanoma excluded; relative frequency will depend on population sample and pathologist's criteria. tData from Clark W Jr, Elder DE, Guerry D4, et al: Model predicting survival in stage I melanoma based on tumor progression. J Natl Cancer lnst 81:1893,1989. *Many dermatopathologists, including one of us (JB), hold that subclassification into these categories is of limited usefulness, if any, owing to poor concordance among pathologists and because group survival for each subtype is identical. §Usually, but not always, a subset of lentigo maligna melanoma.
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See legends on opposite page
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Figure 1. A, Nontumorigenic radial growth phase (RGP) melanoma in situ. There is intraepidermal melanoma cell proliferation along the basement membrane (hematoxylineosin, original magnification x 120). B, Detachment of scattered melanoma cells from the basement membrane with displacement into the upper keratinocyte layers (pagetoid spread) is easily detected at higher magnification (hematoxylin-eosin, original magnification x 240). C,lmmunohistochemical staining for tyrosinase-related protein-1 (MEL-5) improves the visualization of melanoma cells along the basement membrane and pagetoid spread (immunostaining for MEL-5, original magnification x 240). Figure 2. A, An inflammatory reaction is seen at low magnification in the papillary dermis (hematoxylin-eosin, original magnification x 60). B, Nontumorigenic RGP microinvasive melanoma. Nests of melanoma cells similar to the intraepidermal melanoma cells are observed within the papillary dermis (arrow) (hematoxylin-eosin, original magnification x 120). C, Higher magnification reveals an inflammatory infiltrate around a single invasive melanoma cell in the papillary dermis (arrow) (hematoxylin-eosin, original magnification X 240). Figure 3. A, Tumorigenic vertical growth phase (VGP) melanoma. An aggregate of melanoma cells is seen within the papillary dermis. An intraepidermal component is also present (hematoxylin-eosin, original magnification x 120). 6, The tumorigenic VGP melanoma cells in the papillary dermis are often larger and more atypical than melanoma cells of the intraepidermal component (hematoxylin-eosin, original magnification x 240).
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statistical model and includes subtypes, approximate frequency, and group survival. In situ nontumorigenic or RGP melanoma may thus be of the superficial spreading type, lentigo maligna type, acral lentiginous type, or the unclassified type.7Distinction of these types is not absolutely essential, but classification may grant the clinician greater flexibility in treatment of the lesions or help in the clinical-pathologic correlation to reach greater certainty in the diagnosis. Superficial Spreading Melanoma Superficial spreading melanoma exhibits severely atypical melanocytic hyperplasia that may reach confluency along the basal layer of the epidermis. Usually, there are also irregularly shaped nests of these severely atypical cells with abundant cytoplasm and large pleomorphic hyperchromatic vesicular nuclei. In addition, superficial spreading melanoma shows these severely atypical cells with abundant cytoplasm throughout the epidermis and into the granular cell layer in the classic "pagetoid" spreading pattern. Lentigo Maligna Melanoma In contrast to superficial spreading melanoma, lentigo maligna melanoma and its precursor lentigo-maligna is usually found on the head and neck. Lentigo maligna melanoma is associated with an atrophic epidermis and extensive solar elastosis. Early lentigo maligna lesions typically demonstrate lentiginous atypical melanocytic hyperplasia that is defined by atypical melanocytic hyperplasia restricted along the basement membrane in the intraepidermal compartment. These atypical cells show variable but frequently fusiform hyperchromatic nuclei with relatively scant cytoplasm compared with superficial spreading melanoma cells. These cells proliferate along the basement membrane and into the pilosebaceous appendages. Later lentigo maligna melanoma lesions begin to show confluence of the lentiginous melanocytic hyperplasia, irregular nesting, and eventual pagetoid spread of the melanoma cells. A subset of later lesions show evidence of invasion or tumorigenic growth. Acral Lentiginous Melanoma Acral lentiginous melanoma arises on acral skin and mucosal surfaces and exhibits a pattern of lentiginous melanocytic hyperplasia of melanoma cells similar to that seen in lentigo maligna; however, the lentiginous melanocytic proliferation extends down primarily eccrine ducts on acral skin and into salivary or mucinous ducts on mucosal surfaces as opposed to the pilosebaceous appendages. In addition, nest-
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ing and pagetoid spread is observed later in acral lentiginous melanoma as compared with superficial spreading melanoma. Micro-invasive melanoma (level 11) is seen associated with the intraepidermal RGP of all types of melanoma (see Table 2). This diagnosis is made when severely atypical melanoma cells similar to those in the intraepidermal compartment are identified extending into or "invading" the papillary dermis. These cells may form cords or nests in the papillary dermis; however, the nests are usually of similar size and number to those in the intraepithelial component. Furthermore, no single nest predominates or exhibits an expansile character, nor are any mitoses identified in the nests.22 Nodular Melanoma
Tumorigenic or VGP melanoma (including nodular melanoma) is designated when a sheet of melanoma cells fills the papillary dermis, or a nest of melanoma cells predominates over other nests and/or exhibits mitoses. These lesions may not entirely fill the papillary dermis and are thus designated level II/III, but they usually fill the papillary dermis (level 111) or extend into the reticular dermis (level IV) or subcutaneous fat (level V). Tumorigenic VGP melanoma is manifested by any of the types of melanomas (see Table 2). Infrequent or rare variants of melanoma such as desmoplastic melanoma are discussed elsewhere7and are beyond the scope of this article. In early lesions of melanoma, a distinct classification often cannot be made, and the in situ RGP is best left unclassified. Likewise, when the original biopsy does not include sufficient adjacent epidermis to evaluate lateral intraepidermal spread, classification should be withheld. Interestingly, in superficial spreading melanoma and nodular melanoma, pre-existing nevomelanocytic nevi are identified up to 30% of the time and include dysplastic nevi.7, 30, 53 On the other hand, lentigo maligna melanoma and acral lentiginous melanoma are rarely associated with a pre-existing melanocytic nevus. Genetic analysis of the familial background and acquired genetic abnormalities may eventually help segregate subtypes. All classification schemes are imperfect.34Indeed, classification is often based on histologic clues that indicate at which site on the body the lesion is located.' Furthermore, all types of melanomas show similar patient survival curves for each Breslow thickness range, and therefore subclassification is held by many dermatopathologists as irrelevant to the clinician; however, during the earliest period of melanoma growth-that is, in the in situ RGP-it is reported that there are differences in behavior of each type. Based on clinical-pathologic correlation, the duration of the in situ RGP in superficial spreading melanoma and acral lentiginous melanoma prior to microinvasion appears to be months to several years. On the other hand, in lentigo maligna, the duration of intraepidermal in situ RGP appears to range from nearly a decade to
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several decades. This difference in latency period alters the aggressiveness of follow-up and treatment. Thus, many oncologic surgeons perform simple excisions or excisions with 0.5-cm margins for RGP of superficial spreading melanoma in situ and acral lentiginous melanoma in situ, whereas in lentigo maligna they are comfortable with simple excisions with narrower margins, particularly if in critical sites on the face, in order to preserve eyebrows, eyelids, or other areas that are difficult to reconstruct. Likewise, in the elderly, radiation therapy or cryotherapy for lentigo maligna in sites requiring anatomic preservation may be performed.28,59, Thus, the classification of melanoma communicates to the clinician the potential latency period of the lesion prior to microinvasion and provides the clinician with greater flexibility of treatment options. Finally, classification is useful to the pathologist. Recognition of different types of melanoma and subtypes prepares the pathologist to search, for example, for the often subtle spindle cell melanoma or desmoplastic melanoma in lentigo maligna melanoma. Without knowledge of these subtypes and variants, for example, a diagnosis of ”scar” instead of recognition of the VGP of desmoplastic melanoma unfortunately can be made. ’
PRACTICAL TOOLS FOR THE DIAGNOSIS OF MELANOMA
Three essential tools are currently used in the differentiation of melanoma from other tumors in pathology: classical histochemical methods; electron microscopy; and immunohistochemistry. One of the earliest methods used to aid in the diagnosis of malignant melanoma is the Fontana-Masson histochemical stain. This stain reacts with melanosomes and is most often used to help identify metastatic melanoma or primary epithelioid tumors that are poorly pigmented clinically or on routine hematoxylin and eosin sections. On the other hand, highly pigmented tumors may have so many melanosomes that the nuclear characteristics are difficult to evaluate; thus, the melanin bleach technique is useful to eliminate the melanin and permit visualization of the nuclei for pleomorphism and atypical chromatin distribution. The advent of the electron microscope permitted characterization and identification of the fine structure of the stages of melanosomes. The fine structural periodicity identified in ”pie-melanosomes” permitted differentiation from other electron-dense organelles and helped differentiate so-called amelanotic or poorly pigmented melanoma tumors from other poorly differentiated epithelioid or spindle tumors. Modern immunohistochemical techniques have had a profound effect on the accuracy of distinguishing malignant melanoma from other poorly differentiated tumors such as spindle cell squamous cell carcinoma, atypical fibroxanthoma, epithelioid angiosarcoma, and several other primary or metastatic malignant tumors. The ”gold standard” is
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the use of a panel of immunohistochemical stains, rather than any single antibody, which may be subject to the vagaries of tissue preservation and the multiple variables in the steps of the technique itself. Therefore, all histochemical stains must be accompanied by positive and negative control tissue. Likewise, the pathologist must be alert to the ideal "internal" control: appropriate staining of adjacent normal tissue structure. The most helpful panel of antibodies to test for primary cutaneous melanoma are the positive markers S-100 protein,2*,27, HMB-45,29,64 vimentin, and negative markers for cytokeratins. This panel will help identify the majority of difficult cases of melanoma (Table 3). HMB-45 is considered more specific but less sensitive in detection of melanoma; however, melanoma is a derivative of the neuroectoderm and thus may not only lose expression of HMB-45 but also even gain expression of cytokeratins in metastatic lesions.46No single histochemical marker is thus infallible, and every tumor must be evaluated in the context of clinical, routine histochemical, and immunocytochemical findings. For example, simple-minded concepts such as S-100 protein specificity for neural or neural crest tissues are flawed; eccrine glands and adipose tissue show positivity as well as eccrine, breast, pancreatic carcinoma, Table 3. USEFUL DIAGNOSTIC MARKERS IN THE DIAGNOSIS OF MELANOMA Antigen (Antibody)
Result
Specificity
S-100 protein
+
Cytokeratins
-
Most melanoma, neural tumors, eccrine tumors, some breast carcinoma Rare ( + ) in primary melanoma, occasional ( + ) in recurrent and metastatic melanoma, ( + ) in epithelial tumors Specific, but not all melanomas
Premelanosomal protein (HMB-45) Vimentin Nerve growth factor receptor Tyrosinase related protein-1 (MEL-5)
+ + + +
Mesenchymal tumors including melanoma Most melanoma, neural tumors as well as HMB-45 negative desmoplastic or neurotropic spindle cell melanoma ( + ) lntraepidermal melanocytes and radial growth phase (RGP) melanoma cells; ( + ) blue nevus; ( - ) in most vertical growth phase (VGP) melanoma
Data from Duray PH, Palazzo J, Gown AM, et al: Melanoma cell heterogeneity: A study of two monoclonal antibodies compared with 5-100 protein in paraffin sections. Cancer 61:2460, 1988; Ordonez NG, Ji XL, Hickey RC: Comparison of HMB-45 monoclonal antibody and 5-100 protein in the immunohistochemical diagnosis of melanoma. Am J Clin Pathol 90:385, 1988; Gown AM, Vogel AM, Hoak D, et al: Monoclonal antibodies specific for melanocytic tumors distinguish subpopulations of melanocytes. Am J Pathol 12:195, 1986; Wick MR, Swanson PE, Rocamora A: Recognition of malignant melanoma by monoclonal antibody HMB-45, an immunohistochemical study of 200 paraffin-embedded cutaneous tumors. J Cutan Pathol 15:201, 1988; Kanik AB, Yaar M, Bhawan J: p75 nelve growth factor receptor staining helps identify desmoplastic and neurotropic melanoma. J Cutan Pathol 23:205, 1996; Bhawan J: Mel-5, a novel antibody for differential diagnosis of epidermal pigmented lesions of the skin in paraffinembedded sections. Melanoma Res 7:43, 1997
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and a subset of sarcomas may show S-100 protein positivity. These findings underscore the need for a full panel of immunohistochemical stains. A second series of antibodies should be invoked if the poorly differentiated tumor is considered endothelial in origin (CD31, Factor VIII or Ulex europaeus antigen), fibrohistiocytic (CD68, Factor XIIIa); dermatofibrosarcoma protuberans (CD34); leiomyosarcoma (desmin); or a large cell lymphoma (CD45, CD30). A variety of other markers may be included if other tumors are contemplated. In recent years we have found additional immunocytochemical stains that prove useful in a subset of malignant melanoma cases. For example, it is reported that some S-100 negative or poorly staining spindle cell or neurotrophic melanoma cases may be identified by expression of nerve growth factor receptor.38Likewise, the degree of atypical melanocytic hyperplasia may be difficult to evaluate in biopsies of pigmented actinic keratoses or actinically damaged skin. In these cases of early RGP melanoma, HMB-45 and S-100 protein may be negative in the intraepidermal component. In such cases, detection of tyrosinase related protein-1 by MEL-5 is useful to help identify the lentiginous hyperplasia and pagetoid spreads (see Fig. 1C). Table 3 also includes these recent markers that we find useful in the diagnosis of melanoma. FUTURE DIRECTIONS
A tremendous number of specific markers increase in expression as 23, 63 a function of tumor thickness, infiltration, or tumor progre~sion.'~, These markers, although providing insight into the behavior or prognosis of the melanoma lesions, are primarily experimental and have not yet been used on a routine basis. Table 4 lists a selected number of the markers with significant experimental and pathologic evidence for association with melanoma tumor progression or aggressive behavior. Further study is required to better characterize which marker may be an independent predictor of melanoma prognosis. Unfortunately, no single marker has been identified that can be used to reliably distinguish benign melanocytic lesions (such as spitz nevi and its variants) from malignant melanoma; however, recent progress has been made with cell cycle regulatory proteins or cell proliferation markers such as proliferation cell nuclear antigen (PCNA), Ki-67, or the cyclin dependent kinase inhibitors p16 and p21. These new proliferation tumor progression markers hold promise in the diagnosis and further definition of prognosis of an individual case of malignant melanoma. Despite recent data suggesting lack of significant correlation of PCNA with tumor progression,5O the pattern of distribution of PCNA may nevertheless prove useful to help distinguish spitz nevi and its variants from nevoid melanoma.45Conflicting evidence is reported on whether loss of the tumor suppressor proteins p16, p21, or Ki-67 are associated 58, Nevertheless, increase in expression with melanoma progre~sion.~~,
2
increased increased increased increased increased increased increased increased increased increased increased increased increased increased increased increased increased increased increased increased increased increased
integrin integrin integrin CD44 isoform protease
immunoglobulin super gene family immunoglobulin super gene family immunoglobulin super gene family epidermal growth factor receptor vascular endothelial growth factor TGF-p isoform small GTPase family S-100 family transmembrane receptor lysosomal proteinase G-actin binding protein actin binding protein proliferating cellular nuclear antigen
Expression
disialoganglioside integrin integrin integrin
Family/Molecule
homotypic cell adhesion homotypic cell adhesion major histocompatibility complex growth factor signaling cascade angiogenesis growth factor signal transduction Ca+ binding protein cell metabolism iron transport catabolism sequestration of G-actin cross-links actin filaments auxiliary protein for protein DNA polymerase
membrane adhesion adhesion, migration on vitronectin adhesion, migration on collagen adhesion, migration on collagen, fibronectin and laminin adhesion, migration on fibronectin adhesion, migration on fibronectin adhesion to fibrinogen adhesion and migration on hyaluronate matrix degradation
Function
Data from Weterman MA, van Muijen GN, Bloemers HP, et al: Molecular markers of melanocytic tumor progression, Lab Invest 70:593, 1994; Carrel S, Dore JF, Ruiter DJ, et al: The EORTC Melanoma Group exchange program: Evaluation of a multicenter monoclonal antibody study. Int J Cancer 48:836, 1991; Elder DE, Rodeck U, Thurin J, et al: Antigenic profile of tumor progression stages in human melanocytic nevi and melanomas. Cancer Res 49:5091, 1989; Newton BJ: Molecular pathology of melanoma. Cancer Metastasis Rev 16:141, 1997; Marcoval J, Moreno A, Graells J, et al: Angiogenesis and malignant melanoma. Angiogenesis is related to the development of vertical (tumorigenic) growth phase. J Cutan Pathol 24:212, 1997; Albino AP: Genes involved in melanoma susceptibility and progression. Curr Opin Oncol 7:162, 1995; Byers HR, Mihm MCJ: Development of cell morphologic and molecular variables in melanoma prognosis. In Hori Y, Hearing VJ and Nakayama J (eds): Melanogenesis and Malignant Melanoma: Biochemistry, Cell Biology, Molecular Biology, Pathophysiology, Diagnosis and Treatment. Amsterdam, Elsevier Science, 1996, p 205
CD44H Urokinase-type plasminogen activator CAM-1 MUC-18 Hull EGF-R VEGF TGF-P2 RAS Calcyclin Transferrin receptor Cathepsin D Thymosin p l 0 ABP PCNA
%JPB
4%
%P1
%P1
4%
%Po
GD3
Antigen
Table 4. SELECTED EXPERIMENTAL AND HISTOLOGIC MARKERS REPORTED IN LATER STAGES OF TUMOR PROGRESSION
+
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of the cell cycle protein Ki-67 (MIB-1) is reported coupled with other progression markers.lO,47,49, 58 Further studies are needed to resolve these controversies. The accumulation of genetic alterations and altered protein expression and signaling pathways in the melanocytic lineage sets in motion not only aberrant growth regulation but also abnormal migratory behavior. This migratory behavior is ultimately required for invasion and metastasis and is dependent on changes in the ~ytoske1eton.l~ For example, altered protein expression of cytoskeletal associated proteins such as actin is associated with differential motilityi9 and invasiveness of melanoma." Many other cytoskeletal proteins localized at adhesion plaques such as focal adhesion kinase3 or alpha-actininZ0are altered in melanoma and are associated with invasiveness or tumorigenic VGP. Further investigation is needed to determine whether cytoskeletal proteins may be used for prognostication of melanoma behavior. The most significant recent developments to help define the outcome of patients with melanoma is outside of histologic examination. Specifically, two important developments are the sentinel node biopsy technique following lymph~scintigraphy~l (discussed elsewhere in this issue) and the polymerase chain reaction to detect melanoma cells in the blood by amplifying tyrosinase message.39Nevertheless, histologic identification of nontumorigenic VGP melanoma helps define the subset of patients to follow not only for nodal spread but also for blood testing for metastatic disease. SUMMARY
Significant progress has been made in the last 10 years on the identification of histologic parameters that are independent predictors of melanoma prognosis, immunohistochemical markers of cells of melanocytic origin and changes in adhesion molecules, cytoskeletal proteins, growth factor receptors, cell signaling, and nuclear proliferation proteins associated with tumor progression. Histologic criteria may never be completely sufficient to predict behavior accurately, because the fundamental change that renders a cell aggressive may not be morphologically reflected and may require immunohistochemical or other molecular markers to establish behavior. To date, it is humbling that no immunohistochemical or molecular marker provides a greater predictable value for aggressive behavior than does the simple calibrated ocular micrometer to measure tumor thickness. Nevertheless, development of multiple histologic parameters with the concept of nontumorigenic RGP and tumorigenic VGP provides a reliable statistical model to predict metastases. Fortunately, nontumorigenic RGP melanomas with greater than 75% regression are rare. Thus, individual patients with melanoma without regression and without the tumorigenic VGP can be given reasonable assurance of 100% survival. Nevertheless, this assurance is based on a statistical model with a finite population studied. Additional studies are
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needed to confirm this model, as well as more definitive markers to precisely predict outcome for those individuals with tumorigenic VGP melanoma.
References 1. Ackerman AB: Malignant melanoma: A unifying concept. Hum Pathol 11:591, 1980 2. Ackerman AB Discordance among expert pathologists in diagnosis of melanocytic neoplasms. Hum Pathol 271115, 1996 3. Akasaka T, van Leeuwen RL, Yoshinaga IG, et al: Focal adhesion kinase (p125FAK) expression correlates with motility of human melanoma cell lines. J Invest Dermatol 105104,1995 4. Balch CM, Soong SJ, Murad TM, et al: A multifactorial analysis of melanoma. 11. Prognostic factors in patients with stage I (localized) melanoma. Surgery 86343,1979 5. Balch CM, Urist MM, Karakousis CP, et al: Efficacy of 2-cm surgical margins for intermediate-thickness melanomas (1 to 4 mm). Results of a multi-institutional randomized surgical trial. Ann Surg 218962, 1993 6. Bamhill RL, Fandrey K, Levy MA, et al: Angiogenesis and tumor progression of melanoma. Quantification of vascularity in melanocytic nevi and cutaneous malignant melanoma. Lab Invest 67331, 1992 7. Bamhill RL, Mihm M Jr: The histopathology of cutaneous malignant melanoma. Semin Diagnost Pathol 10:47, 1993 8. Bhawan J: Mel-5: A novel antibody for differential diagnosis of epidermal pigmented lesions of the skin in paraffin-embedded sections. Melanoma Res 743, 1997 9. Blessing K, McLaren KM, McLean A, et al: Thin malignant melanomas (less than 1.5 mm) with metastasis: A histological study and survival analysis. Histopathology 17389, 1990 10. Boni R, Doguoglu A, Burg G, et al: MIB-1 immunoreactivity correlates with metastatic dissemination in primary thick cutaneous melanoma. J Am Acad Dermatol35416,1996 11. Bouffard D, Duncan LM, Howard CA, et al: Actin-binding protein expression in benign and malignant melanocytic proliferations. Hum Pathol 25709, 1994 12. Breslow A: Thickness, cross-sectional areas and depth of invasion in the prognosis of cutaneous melanoma. Ann Surg 172:902,1970 13. Byers HR, Mihm MCJ: Development of cell morphologic and molecular variables in melanoma prognosis. In Hori Y, Hearing VJ, Nakayama J (eds): Melanogenesis and Malignant Melanoma: Biochemistry, Cell Biology, Molecular Biology, Pathophysiology, Diagnosis and Treatment. Amsterdam, Elsevier Science, 1996, p 205 14. Carrel S, Dore JF, Ruiter DJ, et al: The EORTC Melanoma Group exchange program: Evaluation of a multicenter monoclonal antibody study. Int J Cancer 48:836, 1991 15. Clark W Jr: What is inherited in neoplastic systems? Animal models of cutaneous malignant melanoma. Lab Invest 71:1, 1994 16. Clark W Jr, Elder DE, Guerry D IV, et al: Model predicting survival in stage I melanoma based on tumor progression. J Natl Cancer Inst 81:1893, 1989 17. Clark W Jr, From L, Bernardino EA, et al: The histogenesis and biologic behavior of primary human malignant melanomas of the skin. Cancer Res 29:705, 1969 18. Clemente C, Cochran AJ, Elder DE, et al: Histopathologic diagnosis of dysplastic nevi: Concordance among pathologists convened by the World Health Organization Melanoma Programme. Hum Pathol22:313,1991 19. Cunningham CC, Gorlin JB, Kwiatkowski DJ, et al: Actin-binding protein requirement for cortical stability and efficient locomotion. Science 255:325, 1992 20. Duncan LM, Bouffard D, Howard C, et a1 In situ distribution of integrin alpha 2 beta 1 and alpha-actinin in melanocytic proliferations. Mod Pathol 9:938, 1996 21. Duray PH, Palazzo J, Gown AM, et al: Melanoma cell heterogeneity. A study of two monoclonal antibodies compared with 5-100 protein in paraffin sections. Cancer 61:2460, 1988
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22. Elder D, Elenitsas R Benign pigmented lesions and malignant melanoma. In Elder D, Elenitsas R, Jaworsky C, et a1 (eds): Lever’s Histopathology of the Skin, ed 8. Philadelphia, Lippincott-Raven, 1997, p 625 23. Elder DE, Rodeck U, Thurin J, et al: Antigenic profile of tumor progression stages in human melanocytic nevi and melanomas. Cancer Res 49:5091,1989 24. Fallowfield ME, Cook MG: Vascular invasion in malignant melanomas. An independent prognostic variable? Am J Surg Pathol 13217, 1989 25. Farmer ER, Gonin R, Hanna MP: Discordance in the histopathologic diagnosis of melanoma and melanocytic nevi between expert pathologists. Hum Pathol27528,1996 26. Freedman VH, Shin SI: Cellular tumorigenicity in nude mice: Correlation with cell growth in semi-solid medium. Cell 3:355, 1974 27. Gaynor R, Irie R, Morton D, et al: SlOO protein is present in cultured human malignant melanomas. Nature 286400, 1980 28. Geara FB, Ang KK: Radiation therapy for malignant melanoma. Surg Clin North Am 76:1383, 1996 29. Gown AM, Vogel AM, Hoak D, et al: Monoclonal antibodies specific for melanocytic tumors distinguish subpopulations of melanocytes. Am J Pathol 123:195, 1986 30. Greene MH, Clark W Jr, Tucker MA, et al: High risk of malignant melanoma in melanoma-prone families with dysplastic nevi. Ann Intern Med 102458, 1985 31. Guerry D IV, Synnestvedt M, Elder DE, et a1 Lessons from tumor progression: The invasive radial growth phase of melanoma is common, incapable of metastasis, and indolent. J Invest Dermatol 100:342, 1993 32. Harris MN, Shapiro RL, Roses DF: Malignant melanoma. Primary surgical management (excision and node dissection) based on pathology and staging. Cancer 75:715, 1995 33. Harrist TJ, Rigel DS, Day C Jr, et al: “Microscopic satellites” are more highly associated with regional lymph node metastases than is primary melanoma thickness. Cancer 532183, 1984 34. Heenan PJ, Matz LR, Blackwell JB, et al: Inter-observer variation between pathologists in the classification of cutaneous malignant melanoma in western Australia. Histopathology 8:717, 1984 35. Herlyn M, Thurin J, Balaban G, et al: Characteristics of cultured human melanocytes isolated from different stages of tumor progression. Cancer Res 45:5670, 1985 36. Itoh K, Tilden AB, Balch C M Interleukin 2 activation of cytotoxic T-lymphocytes infiltrating into human metastatic melanomas. Cancer Res 46:3011, 1986 37. Kang S, Barnhill RL, Mihm M Jr, et al: Histologic regression in malignant melanoma: An interobserver concordance study. J Cutan Pathol 20126, 1993 38. Kanik AB, Yaar M, Bhawan J: p75 nerve growth factor receptor staining helps identify desmoplastic and neurotropic melanoma. J Cutan Pathol 23205, 1996 39. Kunter U, Buer J, Probst M, et al: Peripheral blood tyrosinase messenger RNA detection and survival in malignant melanoma. J Natl Cancer Inst 88:590, 1996 40. Kurnick JT, Kradin RL, Blumberg R, et al: Functional characterization of T lymphocytes propagated from human lung carcinomas. Clin Immunol Immunopathol 38:367, 1986 41. Leon P, Daly JM, Synnestvedt M, et al: The prognostic implications of microscopic satellites in patients with clinical stage I melanoma. Arch Surg 126:1461, 1991 42. Li L, Price JE, Fan D, et al: Correlation of growth capacity of human tumor cells in hard agarose with their in vivo proliferative capacity at specific metastatic sites. J Natl Cancer Inst 81:1406, 1989 43. Liotta LA, Wewer U, Rao NC, et al: Biochemical mechanisms of tumor invasion and metastases. Adv Exp Med Biol 233:161, 1988 44. Marcoval J, Moreno A, Graells J, et al: Angiogenesis and malignant melanoma. Angiogenesis is related to the development of vertical (tumorigenic) growth phase. J Cutan Pathol 24:212, 1997 45. McNutt NS, Urmacher C, Hakimian J, et al: Nevoid malignant melanoma: Morphologic patterns and immunohistochemical reactivity. J Cutan Pathol 22502, 1995 46. Miettinen M, Franssila K Immunohistochemical spectrum of malignant melanoma. The common presence of keratins. Lab Invest 61:623, 1989 47. Moretti S, Pinzi C , Berti E, et al: In situ expression of transforming growth factor beta
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55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65.
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is associated with melanoma progression and correlates with Ki67, HLA-DR and beta 3 integrin expression. Melanoma Res 7:313, 1997 Ordonez NG, Ji XL, Hickey RC: Comparison of HMB-45 monoclonal antibody and S100 protein in the immunohistochemical diagnosis of melanoma. Am J Clin Pathol 90385, 1988 Ramsay JA, From L, Iscoe NA, et al: MIB-1 proliferative activity is a significant prognostic factor in primary thick cutaneous melanomas. J Invest Dermatol 105:22, 1995 Reddy VB, Gattuso P, Aranha G, et al: Cell proliferation markers in predicting metastases in malignant melanoma. J Cutan Pathol22:248, 1995 Reintgen D, Balch CM, Kirkwood J, et a1 Recent advances in the care of the patient with malignant melanoma. Ann Surg 2251,1997 Ronan SG, Eng AM, Briele HA, et al: Thin malignant melanomas with regression and metastases. Arch Dermatol 1231326, 1987 Seykora J, Elder D Dysplastic nevi and other risk markers for melanoma. Semin Oncol 23:682, 1996 Shaw HM, Balch CM, Soong SJ, et a1 Prognostic histopathological factors in malignant melanoma. Pathology 17271, 1985 Slingluff C Jr, Seigler H F “Thin” malignant melanoma: Risk factors and clinical management. Ann Plast Surg 28:89, 1992 Slominski A, Ross J, Mihm MC: Cutaneous melanoma: Pathology, relevant prognostic indicators and progression. Br Med Bull 51:548, 1995 Soong SJ, Shaw HM, Balch CM, et al: Predicting survival and recurrence in localized melanoma: A multivariate approach. World J Surg 16:191, 1992 Talve L, Sauroja H, Collan Y, et al: Loss of expression of the pl6INK4/CDKN2 gene in cutaneous malignant melanoma correlates with tumor cell proliferation and invasive stage. Int J Cancer 74255, 1997 Tsang RW, Liu FF, Wells W, et al: Lentigo maligna of the head and neck. Results of treatment by radiotherapy. Arch Dermatol 130:1008, 1994 Veronesi U, Cascinelli N. Narrow excision (1-cm margin). A safe procedure for thin cutaneous melanoma. Arch Surg 126438,1991 Veronesi U, Cascinelli N, Adamus J, et al: Thin stage I primary cutaneous malignant melanoma. Comparison of excision with margins of 1 or 3 cm. N Engl J Med 318:1159, 1988 Wang Y, Becker D: Differential expression of the cyclin-dependent kinase inhibitors p16 and p21 in the human melanocytic system. Oncogene 121069,1996 Weterman MA, van Muijen GN, Bloemers HP, et a1 Molecular markers of melanocytic tumor progression. Lab Invest 70:593, 1994 Wick MR, Swanson PE, Rocamora A: Recognition of malignant melanoma by monoclonal antibody HMB-45. An immunohistochemical study of 200 paraffin-embedded cutaneous tumors. J Cutan Pathol 15:201, 1988 Worth AJ, Gallagher RP, Elwood JM, et al: Pathologic prognostic factors for cutaneous malignant melanoma: The Western Canada Melanoma Study. Int J Cancer 43:370,1989
Address reprint requests to H. Randolph Byers, MD, PhD Dermatopathology Section Department of Dermatology Boston University Medical School 609 Albany Street - J407 Boston, MA 02118
MELANOMA
+ .OO
PATHOLOGIC PARAMETERS IN THE DIAGNOSIS AND PROGNOSIS OF PRIMARY CUTANEOUS MELANOMA H. Randolph Byers, MD, PhD, and Jag Bhawan, MD
The goal of pathologic diagnosis in malignant melanoma, as any lesion, is to relate the histologic findings to potential cell behavior and clinical outcome. Pathologists recognize malignant melanoma from benign or atypical melanocytic proliferations using architectural and cellular criteria; however, criteria are not always agreed upon.2 Expert dermatopathologists demonstrate only moderate concordance even when restricting the question to simply benign versus malignant.25There is clearly a greater need for more specific formalized weighted criteria or other tools to better diagnose and predict the behavior of melanocytic proliferations. Yet enormous progress has been made in the last 30 years in the recognition of precursor lesions, tumor progre~sion,’~ and the diagnosis of melanoma. This article will focus on the histologic features of melanoma that contribute to the refinement of prognosis and treatment of individual patients. BRESLOW THICKNESS
Once the diagnosis of malignant melanoma is made, the single most important histologic criterion predicting metastatic behavior and patient survival is the Breslow tumor thickness measurement in millimeters12
From the Department of Dermatology, Boston University Medical School, Boston, Massachusetts
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(also see article on Prognostic Factors by Drs. Stadelmann and Reintgen on page 767 of this issue). In this measurement, a calibrated ocular micrometer quantifies the distance from the top of the granular cell layer of the epidermis to the base of the tumor or to the deepest infiltrating cell in the dermis or subcutaneous tissue, whichever is greater. Fortunately, studies for Breslow thickness show high c o n c ~ r d a n c eand ~ ~ numerous studies have confirmed this relationship of the melanoma thick16, %, 57, 65 The ness in millimeters to metastasis and patient su~vival.~, findings have essentially been reproduced in multiple centers in several countries. Table 1 summarizes the histopathologic variables for 5- or 8year survival in two representative studies: the Western Canada melanoma study including 650 patients with stage I, level 11, 111, IV, and V (see next section on Clark level) melan0ma,6~and the University of Pennsylvania study by Clark et all6 on 264 patients with tumorigenic vertical growth phase (VGP) melanoma (see section on VGP). Breslow thickness remains the "gold standard" and allows great Table 1. HISTOLOGIC PARAMETERS IN PRIMARY CUTANEOUS MELANOMA AND SURVIVAL IN TWO REPRESENTATIVE STUDIES Survival (%) Histologic Parameters Breslow thickness in m M <0.76 (0.76-1.49)' [0.76-1.69]t (1.50-2.99)* [1.70-3.60]t (3.00-4.99)* [>3.60]t (>5.00)* Clark level II 111 IV V Mitotic rat& (low; <0.2/HPF)* [O/mmz]t (moderate; 0.2-l/HPF)* [0.1-6.0/mmz]t (high; >l/HPF)* [>6.O/rnm2]t Tumor-infiltratinglymphocytes$ (absent) (nonbrisk) (brisk) Regression$ Absent or incomplete Present
5 year'
8 yeart
98 90 83 66 57
93 87 60 33
97 90 79 58
96 76 61 39
93 81 76
96 79 38 87 75 59 77 60
'Data (in parentheses) from Worth AJ, Gallagher RP, Elwood JM, et al: Pathologic prognostic factors for cutaneous malignant melanoma: The Western Canada Melanoma Study. Int J Cancer 43:370, 1989; included microinvasive radial growth phase (RGP) and tumorigenic vertical growth phase (VGP). tData (in brackets) from Clark W Jr, Elder DE, Guerry D IV, et al: Model predicting survival in stage I melanoma based on tumor progression. J Natl Cancer lnst 81:1893, 1989; University of Pennsylvania study, included tumorigenic VGP only. *Independent predictor in multivariable logistic regression model of Clark et aLq6 §nd = not done. A variety of other parameters of lymphocytic infiltrates were studied.
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insight into melanoma biology; it can be used to create subcategories that guide clinical treatment, that is, limited surgical margins in thinner lesions or more aggressive surgical approaches such as wider excisions or sentinel node lymphoscinitigraphy and node excision in thick lesions?, 32, 51* 60, a Nevertheless, the Breslow thickness and survival data have limitations for use in any individual patient. There are clearly individuals with thin lesions that do poorly and individuals with thick lesions that do well. Anatomic limitations of the linear measurement may under- or overestimate tumor thickness. For example, if the tumor induces an epidermal hyperplasia, the granular layer is distanced from the tumor base by proliferative keratinocytes, and an overestimation of tumor is made. Likewise, for an excoriated or ulcerated lesion or a lesion that has been subjected to previous exogenous destruction modalities such as electrocautery or freezing, an underestimation of tumor thickness may be made. In addition, other anatomic considerations such as proximity to superficial or deep vessels may influence metastasis. Furthermore, Clark17and others also investigated the role of histologic parameters other than thickness to better predict clinical outcome. Clark level and these other parameters are outlined subsequently.
CLARK LEVEL Anatomic or Clark level I is defined as confinement of melanoma cells to the intraepidermal compartment and is also known as melanoma in situ. Anatomic or Clark level I1 is defined as melanoma cells that have extended into the papillary dermis, whereas in level I11 the melanoma cells fill the papillary dermis. In level IV melanoma the tumor cells extend into the reticular dermis, and in level V there is involvement of the subcutaneous fat. Because melanoma in situ (level I) is confined to the epidermis, it is not given a Breslow thickness measurement. Nearly all level I or melanoma in situ (MIS) have 100% survival; however, for reasons that will be outlined later, rare metastases may be associated with MIS. Table 1 summarizes the relationship of Breslow thickness, Clark level, and numerous other histologic parameters to 5- and 8-year survival in the Western Canada65and University of Pennsylvania studies16; however, the Clark level, instead of being an independent predictor for survival in multivariate analysis, rather parallels that of tumor thickness. Nevertheless, it is helpful information to the pathologist and clinician to visualize from a report the extent of involvement of the different anatomic compartments by the tumor. Histologic criteria found to be independent of tumor thickness in multivariant analyses include the mitotic rate, the degree of tumor-infiltrating lymphocytic (TIL) host response, presence of regression, and ulceration. These criteria are discussed in the following sections.
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MITOTIC RATE
Numerous studies have shown that mitotic rate is an independent variable in melanoma prognosis.l6,65 A clear relationship exists, as in many other different tumor cell types, of high mitotic index and poor prognosis. Original studies on melanoma gave mitotic counts per 10 high power fields, but owing to variability in field size from microscope to microscope, number of mitoses per mm2 are now often used (after calibration of the objective field). For example, in the study by Clark et a1,16 absence of mitoses gave an 8-year survival of 95%. Counts of 0.1 up to 6.0 mitoses per mm2resulted in a drop down to 79% survival. Greater than 6.0 mitoses per mm2 resulted in a fall in survival to just 38% at 8 years. Owing to the different ranges and various percentages of survival seen in different studies, some pathologists prefer to note in the report simply whether there are no, few, or many mitoses present per 10 high power fields. TUMOR-INFILTRATING LYMPHOCYTES
The original concept of host inflammatory reaction has been sharpened by specific definition of tumor-infiltrating lymphocytes (TILs) and application of criteria that estimate their numbers.16The concept of TILs has evolved with immunobiology studies on the isolation and recognition of major histocompatability complex (MHC)-restricted T lymphocytes cytotoxic to autologous melanoma cells.36,40 The absence of TILs portends a worse prognosis (see Table l), whereas focal areas of TILs (nonbrisk infiltrate) improves prognosis by nearly 15%, and an extensive infiltrate of TILs improves 8-year survival by nearly 30%. TILs were found to be an independent predictor for survival16; however, "nonbrisk" versus "brisk infiltrates of TILs described in the original publication is subjective and needs further definition; some authors have presented diagrams to better characterize this di~tinction.~~ The concordance of evaluation of TILs has not been studied. Nevertheless, the impact on prognosis is consistent with a number of other studies that found lymphocytic host response or inflammatory reaction a favorable risk factor.65TILs, as opposed to lymphocytic host response, include lymphocytes distributed among the tumor cells and not those in focal areas in the papillary dermis adjacent or subjacent to the tumor or in necrotic foci or sclerotic portions of the tumor. REGRESSION
Regression has become defined as early (lymphocytes disrupting nests of melanoma cells), intermediate (loss of continuity of lesion with mild fibrosis), and late (extensive horizontal fibrosis) in a study with high interobserver con~ordance.~~ Regression may be identified with or
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without a marked reduction of “skip” areas of melanoma cells in the overlying epidermis and in the papillary dermis. In intermediate and late regression, scattered melanoma cells or small islands or cords of cells in the fibrotic papillary dermis may be seen. Furthermore, regression may be asymmetrically located on one end or may extend from one end of the lesion to the other.52Careful communication with the clinician is essential to determine if the fibrosis is spontaneous or secondary to a scar caused by a previous biopsy or destructive modality. The presence of regression identifies thin lesions less than 0.76 to 1.50 mm in thickness that show a greater risk of metasta~is.~, 55 Rare examples of very thin melanomas and even malignant MIS are associated with metastase~.~~ These lesions were found to exhibit striking fibrotic regression of greater than 75% of the lesion. Linking regression with greater risk of metastasis at first appears counterintuitive, because a successful cytotoxic T-lymphocytic response exists; however, these lesions may have progressed through a phase of immune recognition and destruction of the majority of melanoma cells, resulting in a subpopulation that has escaped recognition and migrated into the lymphatics. Such thin metastasizing melanomas indicate that variables other than thickness hidden from routine microscopic description influence aggressive cell behavior. Alternatively, regression may be viewed simply as an indication of underestimation of tumor thickness. Another histologic variable that may underestimate tumor thickness is ulceration.
ULCERATION
Several studies have shown that ulceration is an independent risk 65 Tumors with greater than 3.0 mm factor in melanoma prognosis.16* ulcer width likely underestimate the true potential thickness of the melanoma. First of all, the absent granular cell layer and the entire epidermis is not included in the measurement. Second, part of the upper portion of the tumor itself may be sloughed and/or necrotic; thus, the thickness is further underestimated.
MICROSCOPIC SATELLITES, VASCULAR INVASION, AND ANGIOGENESIS
Several other histologic parameters that appear related to prognosis 41 vascular invasi0n,2~ and of melanoma include microscopic ~atellites,3~, angiogenesis6,44; however, these parameters in multivariate analyses are not always independent predictors of survival.16,65 Nevertheless, these parameters are typically included in reports of level I11 to level V melanoma.
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NONTUMORIGENIC RADIAL GROWTH PHASE (RGP) AND TUMORIGENIC VERTICAL GROWTH PHASE (VG P)
In the 1980s, Clark, Elder,’6,35 and others developed the concept of melanoma tumor progression in at least three steps. First, the majority of primary melanomas begin as “in situ” or intraepidermal proliferations. Melanoma cell proliferations along the basement membrane, expanding laterally along radii, constitute in situ radial growth phase (RGP). These proliferations appear to begin as a lentiginous melanocytic hyperplasia, defined as restricted along the basement membrane. Later, in situ lesions show increasing detachment of the cells from the basement membrane and they are displaced into the upper epidermal cell layers, giving rise to the so-called pagetoid spread (Fig. 1). Second, melanoma cells gain the capacity to digest the basement membrane and migrate into the papillary dermis, hence the term invasive RGP (Fig. 2, arrows). Third, when the melanoma cells form tumors in the dermis, they are designated as being in the vertical growth phase (VGP)16(Fig. 3). The first two steps are combined into the term nonturnorigenic RGP, with the last step defined as the tumorigenic VGP. In other words, the nontumorigenic RGP melanoma includes all types (see classification section) of melanoma that are in situ (intraepidermal, level I) or microinvasive (involvement of papillary dermis, level 11). Tumorigenic VGP includes all types of melanoma (see classification section), with a predominant aggregate of cohesive cells filling the papillary dermis (level 111), or extending into reticular dermis (level IV) or subcutaneous fat (level V). This concept was developed largely in parallel with melanoma and other tumor cell biology studies that indicate acquisition of malignancy through multiple For example, mutations or specific gene transfection may lead to immortalization in vitro, but the cells retain an incapacity of tumorigenic potential in soft agar. Acquisition of additional specific mutations, chromosomal deletions, or amplifications results in tumor growth in soft agar and capacity of tumor formation in immunodeficient mice.26Furthermore, melanoma cells derived from VGP tumors exhibit a capacity for tumorigenic growth, whereas RGP cells do Finally, tumorigenic growth capacity in a number of cell types relates to capacity for experimental metastasi~.~~ Based on these experimental findings, Clark et all6 investigated survival in invasive nontumorigenic VGP melanoma. They found that of 122 invasive RGP melanoma, there were no metastases after a minimum follow-up of 8 years, with a median follow-up of 12 years. In contrast, of 264 patients with tumorigenic VGP, 8-year survival was 71%. Additional cases and studies in recent years have found that nontumorigenic RGP is not associated with metastatic risk.31Combination of this concept of tumor progression with the six independent prognostic variables including mitotic rate, TILs, tumor thickness, anatomic site of primary melanoma, gender of the patient, and histologic regression permits construction of a model that is 100% accurate in predicting survival in
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nontumorigenic RGP and 84% accurate for VGP tumors, with an overall accuracy of 87%.16Therefore, a statement in a pathology report to specify the presence of the tumorigenic VGP clearly conveys the risk of metastasis. CLASSIFICATION OF MELANOMA
A classification scheme must help the pathologist to recognize melanoma and guide the clinician or surgeon in treatment decision making. Table 2 lists a classification scheme by Clark, Elder, and others7,16, 22, 54 that incorporates the nontumorigenic RGP and tumorigenic VGP
Table 2. CLASSIFICATION OF MELANOMA: FREQUENCY AND GROUP SURVIVAL Frequency (%.)* Nontumorigenic (radial growth phase) In situ (level I) Superficial spreading melanoma$ Lentigo maligna melanoma* Acral lentiginous melanoma* Unclassified$ Microinvasive (level II) Superficial spreading melanoma$ Lentigo maligna melanoma$ Acral lentiginous melanoma* Unclassified* Tumorigenic (vertical growth phase; level 111, IV, and V) Superficial spreading melanoma$ Nodular melanoma* Lentigo maligna melanoma* Acral lentiginous melanoma* Variants Desmoplastic melanoma5 Neurotropic melanomas Balloon cell melanoma Minimal deviation melanoma Nevoid melanoma Other Unclassified
&Year Survival (%)t 100
100
23-28 1-3 1 1 84
37-42 22-32 3-4 2-3 <1 <1 <1 <1 <1 <1
2
'Data from Worth AJ, Gallagher RP, Elwood JM, et at: Pathologic prognostic factors for cutaneous malignant melanoma: The Western Canada Melanoma Study. Int J Cancer 43:370,1989;and Shaw HM, Balch CM, Soong SJ, et al: Prognostic histopathological factors in malignant melanoma. Pathology 17:271,1985;stage I only, in situ melanoma excluded; relative frequency will depend on population sample and pathologist's criteria. tData from Clark W Jr, Elder DE, Guerry D4, et al: Model predicting survival in stage I melanoma based on tumor progression. J Natl Cancer lnst 81:1893,1989. *Many dermatopathologists, including one of us (JB), hold that subclassification into these categories is of limited usefulness, if any, owing to poor concordance among pathologists and because group survival for each subtype is identical. §Usually, but not always, a subset of lentigo maligna melanoma.
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See legends on opposite page
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Figure 1. A, Nontumorigenic radial growth phase (RGP) melanoma in situ. There is intraepidermal melanoma cell proliferation along the basement membrane (hematoxylineosin, original magnification x 120). B, Detachment of scattered melanoma cells from the basement membrane with displacement into the upper keratinocyte layers (pagetoid spread) is easily detected at higher magnification (hematoxylin-eosin, original magnification x 240). C,lmmunohistochemical staining for tyrosinase-related protein-1 (MEL-5) improves the visualization of melanoma cells along the basement membrane and pagetoid spread (immunostaining for MEL-5, original magnification x 240). Figure 2. A, An inflammatory reaction is seen at low magnification in the papillary dermis (hematoxylin-eosin, original magnification x 60). B, Nontumorigenic RGP microinvasive melanoma. Nests of melanoma cells similar to the intraepidermal melanoma cells are observed within the papillary dermis (arrow) (hematoxylin-eosin, original magnification x 120). C, Higher magnification reveals an inflammatory infiltrate around a single invasive melanoma cell in the papillary dermis (arrow) (hematoxylin-eosin, original magnification X 240). Figure 3. A, Tumorigenic vertical growth phase (VGP) melanoma. An aggregate of melanoma cells is seen within the papillary dermis. An intraepidermal component is also present (hematoxylin-eosin, original magnification x 120). 6, The tumorigenic VGP melanoma cells in the papillary dermis are often larger and more atypical than melanoma cells of the intraepidermal component (hematoxylin-eosin, original magnification x 240).
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statistical model and includes subtypes, approximate frequency, and group survival. In situ nontumorigenic or RGP melanoma may thus be of the superficial spreading type, lentigo maligna type, acral lentiginous type, or the unclassified type.7Distinction of these types is not absolutely essential, but classification may grant the clinician greater flexibility in treatment of the lesions or help in the clinical-pathologic correlation to reach greater certainty in the diagnosis. Superficial Spreading Melanoma Superficial spreading melanoma exhibits severely atypical melanocytic hyperplasia that may reach confluency along the basal layer of the epidermis. Usually, there are also irregularly shaped nests of these severely atypical cells with abundant cytoplasm and large pleomorphic hyperchromatic vesicular nuclei. In addition, superficial spreading melanoma shows these severely atypical cells with abundant cytoplasm throughout the epidermis and into the granular cell layer in the classic "pagetoid" spreading pattern. Lentigo Maligna Melanoma In contrast to superficial spreading melanoma, lentigo maligna melanoma and its precursor lentigo-maligna is usually found on the head and neck. Lentigo maligna melanoma is associated with an atrophic epidermis and extensive solar elastosis. Early lentigo maligna lesions typically demonstrate lentiginous atypical melanocytic hyperplasia that is defined by atypical melanocytic hyperplasia restricted along the basement membrane in the intraepidermal compartment. These atypical cells show variable but frequently fusiform hyperchromatic nuclei with relatively scant cytoplasm compared with superficial spreading melanoma cells. These cells proliferate along the basement membrane and into the pilosebaceous appendages. Later lentigo maligna melanoma lesions begin to show confluence of the lentiginous melanocytic hyperplasia, irregular nesting, and eventual pagetoid spread of the melanoma cells. A subset of later lesions show evidence of invasion or tumorigenic growth. Acral Lentiginous Melanoma Acral lentiginous melanoma arises on acral skin and mucosal surfaces and exhibits a pattern of lentiginous melanocytic hyperplasia of melanoma cells similar to that seen in lentigo maligna; however, the lentiginous melanocytic proliferation extends down primarily eccrine ducts on acral skin and into salivary or mucinous ducts on mucosal surfaces as opposed to the pilosebaceous appendages. In addition, nest-
DIAGNOSIS AND PROGNOSIS OF PFUMARY CUTANEOUS MELANOMA
727
ing and pagetoid spread is observed later in acral lentiginous melanoma as compared with superficial spreading melanoma. Micro-invasive melanoma (level 11) is seen associated with the intraepidermal RGP of all types of melanoma (see Table 2). This diagnosis is made when severely atypical melanoma cells similar to those in the intraepidermal compartment are identified extending into or "invading" the papillary dermis. These cells may form cords or nests in the papillary dermis; however, the nests are usually of similar size and number to those in the intraepithelial component. Furthermore, no single nest predominates or exhibits an expansile character, nor are any mitoses identified in the nests.22 Nodular Melanoma
Tumorigenic or VGP melanoma (including nodular melanoma) is designated when a sheet of melanoma cells fills the papillary dermis, or a nest of melanoma cells predominates over other nests and/or exhibits mitoses. These lesions may not entirely fill the papillary dermis and are thus designated level II/III, but they usually fill the papillary dermis (level 111) or extend into the reticular dermis (level IV) or subcutaneous fat (level V). Tumorigenic VGP melanoma is manifested by any of the types of melanomas (see Table 2). Infrequent or rare variants of melanoma such as desmoplastic melanoma are discussed elsewhere7and are beyond the scope of this article. In early lesions of melanoma, a distinct classification often cannot be made, and the in situ RGP is best left unclassified. Likewise, when the original biopsy does not include sufficient adjacent epidermis to evaluate lateral intraepidermal spread, classification should be withheld. Interestingly, in superficial spreading melanoma and nodular melanoma, pre-existing nevomelanocytic nevi are identified up to 30% of the time and include dysplastic nevi.7, 30, 53 On the other hand, lentigo maligna melanoma and acral lentiginous melanoma are rarely associated with a pre-existing melanocytic nevus. Genetic analysis of the familial background and acquired genetic abnormalities may eventually help segregate subtypes. All classification schemes are imperfect.34Indeed, classification is often based on histologic clues that indicate at which site on the body the lesion is located.' Furthermore, all types of melanomas show similar patient survival curves for each Breslow thickness range, and therefore subclassification is held by many dermatopathologists as irrelevant to the clinician; however, during the earliest period of melanoma growth-that is, in the in situ RGP-it is reported that there are differences in behavior of each type. Based on clinical-pathologic correlation, the duration of the in situ RGP in superficial spreading melanoma and acral lentiginous melanoma prior to microinvasion appears to be months to several years. On the other hand, in lentigo maligna, the duration of intraepidermal in situ RGP appears to range from nearly a decade to
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BYERS & B H A W A N
several decades. This difference in latency period alters the aggressiveness of follow-up and treatment. Thus, many oncologic surgeons perform simple excisions or excisions with 0.5-cm margins for RGP of superficial spreading melanoma in situ and acral lentiginous melanoma in situ, whereas in lentigo maligna they are comfortable with simple excisions with narrower margins, particularly if in critical sites on the face, in order to preserve eyebrows, eyelids, or other areas that are difficult to reconstruct. Likewise, in the elderly, radiation therapy or cryotherapy for lentigo maligna in sites requiring anatomic preservation may be performed.28,59, Thus, the classification of melanoma communicates to the clinician the potential latency period of the lesion prior to microinvasion and provides the clinician with greater flexibility of treatment options. Finally, classification is useful to the pathologist. Recognition of different types of melanoma and subtypes prepares the pathologist to search, for example, for the often subtle spindle cell melanoma or desmoplastic melanoma in lentigo maligna melanoma. Without knowledge of these subtypes and variants, for example, a diagnosis of ”scar” instead of recognition of the VGP of desmoplastic melanoma unfortunately can be made. ’
PRACTICAL TOOLS FOR THE DIAGNOSIS OF MELANOMA
Three essential tools are currently used in the differentiation of melanoma from other tumors in pathology: classical histochemical methods; electron microscopy; and immunohistochemistry. One of the earliest methods used to aid in the diagnosis of malignant melanoma is the Fontana-Masson histochemical stain. This stain reacts with melanosomes and is most often used to help identify metastatic melanoma or primary epithelioid tumors that are poorly pigmented clinically or on routine hematoxylin and eosin sections. On the other hand, highly pigmented tumors may have so many melanosomes that the nuclear characteristics are difficult to evaluate; thus, the melanin bleach technique is useful to eliminate the melanin and permit visualization of the nuclei for pleomorphism and atypical chromatin distribution. The advent of the electron microscope permitted characterization and identification of the fine structure of the stages of melanosomes. The fine structural periodicity identified in ”pie-melanosomes” permitted differentiation from other electron-dense organelles and helped differentiate so-called amelanotic or poorly pigmented melanoma tumors from other poorly differentiated epithelioid or spindle tumors. Modern immunohistochemical techniques have had a profound effect on the accuracy of distinguishing malignant melanoma from other poorly differentiated tumors such as spindle cell squamous cell carcinoma, atypical fibroxanthoma, epithelioid angiosarcoma, and several other primary or metastatic malignant tumors. The ”gold standard” is
DIAGNOSIS AND PROGNOSIS OF PRIMARY CUTANEOUS MELANOMA
729
the use of a panel of immunohistochemical stains, rather than any single antibody, which may be subject to the vagaries of tissue preservation and the multiple variables in the steps of the technique itself. Therefore, all histochemical stains must be accompanied by positive and negative control tissue. Likewise, the pathologist must be alert to the ideal "internal" control: appropriate staining of adjacent normal tissue structure. The most helpful panel of antibodies to test for primary cutaneous melanoma are the positive markers S-100 protein,2*,27, HMB-45,29,64 vimentin, and negative markers for cytokeratins. This panel will help identify the majority of difficult cases of melanoma (Table 3). HMB-45 is considered more specific but less sensitive in detection of melanoma; however, melanoma is a derivative of the neuroectoderm and thus may not only lose expression of HMB-45 but also even gain expression of cytokeratins in metastatic lesions.46No single histochemical marker is thus infallible, and every tumor must be evaluated in the context of clinical, routine histochemical, and immunocytochemical findings. For example, simple-minded concepts such as S-100 protein specificity for neural or neural crest tissues are flawed; eccrine glands and adipose tissue show positivity as well as eccrine, breast, pancreatic carcinoma, Table 3. USEFUL DIAGNOSTIC MARKERS IN THE DIAGNOSIS OF MELANOMA Antigen (Antibody)
Result
Specificity
S-100 protein
+
Cytokeratins
-
Most melanoma, neural tumors, eccrine tumors, some breast carcinoma Rare ( + ) in primary melanoma, occasional ( + ) in recurrent and metastatic melanoma, ( + ) in epithelial tumors Specific, but not all melanomas
Premelanosomal protein (HMB-45) Vimentin Nerve growth factor receptor Tyrosinase related protein-1 (MEL-5)
+ + + +
Mesenchymal tumors including melanoma Most melanoma, neural tumors as well as HMB-45 negative desmoplastic or neurotropic spindle cell melanoma ( + ) lntraepidermal melanocytes and radial growth phase (RGP) melanoma cells; ( + ) blue nevus; ( - ) in most vertical growth phase (VGP) melanoma
Data from Duray PH, Palazzo J, Gown AM, et al: Melanoma cell heterogeneity: A study of two monoclonal antibodies compared with 5-100 protein in paraffin sections. Cancer 61:2460, 1988; Ordonez NG, Ji XL, Hickey RC: Comparison of HMB-45 monoclonal antibody and 5-100 protein in the immunohistochemical diagnosis of melanoma. Am J Clin Pathol 90:385, 1988; Gown AM, Vogel AM, Hoak D, et al: Monoclonal antibodies specific for melanocytic tumors distinguish subpopulations of melanocytes. Am J Pathol 12:195, 1986; Wick MR, Swanson PE, Rocamora A: Recognition of malignant melanoma by monoclonal antibody HMB-45, an immunohistochemical study of 200 paraffin-embedded cutaneous tumors. J Cutan Pathol 15:201, 1988; Kanik AB, Yaar M, Bhawan J: p75 nelve growth factor receptor staining helps identify desmoplastic and neurotropic melanoma. J Cutan Pathol 23:205, 1996; Bhawan J: Mel-5, a novel antibody for differential diagnosis of epidermal pigmented lesions of the skin in paraffinembedded sections. Melanoma Res 7:43, 1997
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and a subset of sarcomas may show S-100 protein positivity. These findings underscore the need for a full panel of immunohistochemical stains. A second series of antibodies should be invoked if the poorly differentiated tumor is considered endothelial in origin (CD31, Factor VIII or Ulex europaeus antigen), fibrohistiocytic (CD68, Factor XIIIa); dermatofibrosarcoma protuberans (CD34); leiomyosarcoma (desmin); or a large cell lymphoma (CD45, CD30). A variety of other markers may be included if other tumors are contemplated. In recent years we have found additional immunocytochemical stains that prove useful in a subset of malignant melanoma cases. For example, it is reported that some S-100 negative or poorly staining spindle cell or neurotrophic melanoma cases may be identified by expression of nerve growth factor receptor.38Likewise, the degree of atypical melanocytic hyperplasia may be difficult to evaluate in biopsies of pigmented actinic keratoses or actinically damaged skin. In these cases of early RGP melanoma, HMB-45 and S-100 protein may be negative in the intraepidermal component. In such cases, detection of tyrosinase related protein-1 by MEL-5 is useful to help identify the lentiginous hyperplasia and pagetoid spreads (see Fig. 1C). Table 3 also includes these recent markers that we find useful in the diagnosis of melanoma. FUTURE DIRECTIONS
A tremendous number of specific markers increase in expression as 23, 63 a function of tumor thickness, infiltration, or tumor progre~sion.'~, These markers, although providing insight into the behavior or prognosis of the melanoma lesions, are primarily experimental and have not yet been used on a routine basis. Table 4 lists a selected number of the markers with significant experimental and pathologic evidence for association with melanoma tumor progression or aggressive behavior. Further study is required to better characterize which marker may be an independent predictor of melanoma prognosis. Unfortunately, no single marker has been identified that can be used to reliably distinguish benign melanocytic lesions (such as spitz nevi and its variants) from malignant melanoma; however, recent progress has been made with cell cycle regulatory proteins or cell proliferation markers such as proliferation cell nuclear antigen (PCNA), Ki-67, or the cyclin dependent kinase inhibitors p16 and p21. These new proliferation tumor progression markers hold promise in the diagnosis and further definition of prognosis of an individual case of malignant melanoma. Despite recent data suggesting lack of significant correlation of PCNA with tumor progression,5O the pattern of distribution of PCNA may nevertheless prove useful to help distinguish spitz nevi and its variants from nevoid melanoma.45Conflicting evidence is reported on whether loss of the tumor suppressor proteins p16, p21, or Ki-67 are associated 58, Nevertheless, increase in expression with melanoma progre~sion.~~,
2
increased increased increased increased increased increased increased increased increased increased increased increased increased increased increased increased increased increased increased increased increased increased
integrin integrin integrin CD44 isoform protease
immunoglobulin super gene family immunoglobulin super gene family immunoglobulin super gene family epidermal growth factor receptor vascular endothelial growth factor TGF-p isoform small GTPase family S-100 family transmembrane receptor lysosomal proteinase G-actin binding protein actin binding protein proliferating cellular nuclear antigen
Expression
disialoganglioside integrin integrin integrin
Family/Molecule
homotypic cell adhesion homotypic cell adhesion major histocompatibility complex growth factor signaling cascade angiogenesis growth factor signal transduction Ca+ binding protein cell metabolism iron transport catabolism sequestration of G-actin cross-links actin filaments auxiliary protein for protein DNA polymerase
membrane adhesion adhesion, migration on vitronectin adhesion, migration on collagen adhesion, migration on collagen, fibronectin and laminin adhesion, migration on fibronectin adhesion, migration on fibronectin adhesion to fibrinogen adhesion and migration on hyaluronate matrix degradation
Function
Data from Weterman MA, van Muijen GN, Bloemers HP, et al: Molecular markers of melanocytic tumor progression, Lab Invest 70:593, 1994; Carrel S, Dore JF, Ruiter DJ, et al: The EORTC Melanoma Group exchange program: Evaluation of a multicenter monoclonal antibody study. Int J Cancer 48:836, 1991; Elder DE, Rodeck U, Thurin J, et al: Antigenic profile of tumor progression stages in human melanocytic nevi and melanomas. Cancer Res 49:5091, 1989; Newton BJ: Molecular pathology of melanoma. Cancer Metastasis Rev 16:141, 1997; Marcoval J, Moreno A, Graells J, et al: Angiogenesis and malignant melanoma. Angiogenesis is related to the development of vertical (tumorigenic) growth phase. J Cutan Pathol 24:212, 1997; Albino AP: Genes involved in melanoma susceptibility and progression. Curr Opin Oncol 7:162, 1995; Byers HR, Mihm MCJ: Development of cell morphologic and molecular variables in melanoma prognosis. In Hori Y, Hearing VJ and Nakayama J (eds): Melanogenesis and Malignant Melanoma: Biochemistry, Cell Biology, Molecular Biology, Pathophysiology, Diagnosis and Treatment. Amsterdam, Elsevier Science, 1996, p 205
CD44H Urokinase-type plasminogen activator CAM-1 MUC-18 Hull EGF-R VEGF TGF-P2 RAS Calcyclin Transferrin receptor Cathepsin D Thymosin p l 0 ABP PCNA
%JPB
4%
%P1
%P1
4%
%Po
GD3
Antigen
Table 4. SELECTED EXPERIMENTAL AND HISTOLOGIC MARKERS REPORTED IN LATER STAGES OF TUMOR PROGRESSION
+
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of the cell cycle protein Ki-67 (MIB-1) is reported coupled with other progression markers.lO,47,49, 58 Further studies are needed to resolve these controversies. The accumulation of genetic alterations and altered protein expression and signaling pathways in the melanocytic lineage sets in motion not only aberrant growth regulation but also abnormal migratory behavior. This migratory behavior is ultimately required for invasion and metastasis and is dependent on changes in the ~ytoske1eton.l~ For example, altered protein expression of cytoskeletal associated proteins such as actin is associated with differential motilityi9 and invasiveness of melanoma." Many other cytoskeletal proteins localized at adhesion plaques such as focal adhesion kinase3 or alpha-actininZ0are altered in melanoma and are associated with invasiveness or tumorigenic VGP. Further investigation is needed to determine whether cytoskeletal proteins may be used for prognostication of melanoma behavior. The most significant recent developments to help define the outcome of patients with melanoma is outside of histologic examination. Specifically, two important developments are the sentinel node biopsy technique following lymph~scintigraphy~l (discussed elsewhere in this issue) and the polymerase chain reaction to detect melanoma cells in the blood by amplifying tyrosinase message.39Nevertheless, histologic identification of nontumorigenic VGP melanoma helps define the subset of patients to follow not only for nodal spread but also for blood testing for metastatic disease. SUMMARY
Significant progress has been made in the last 10 years on the identification of histologic parameters that are independent predictors of melanoma prognosis, immunohistochemical markers of cells of melanocytic origin and changes in adhesion molecules, cytoskeletal proteins, growth factor receptors, cell signaling, and nuclear proliferation proteins associated with tumor progression. Histologic criteria may never be completely sufficient to predict behavior accurately, because the fundamental change that renders a cell aggressive may not be morphologically reflected and may require immunohistochemical or other molecular markers to establish behavior. To date, it is humbling that no immunohistochemical or molecular marker provides a greater predictable value for aggressive behavior than does the simple calibrated ocular micrometer to measure tumor thickness. Nevertheless, development of multiple histologic parameters with the concept of nontumorigenic RGP and tumorigenic VGP provides a reliable statistical model to predict metastases. Fortunately, nontumorigenic RGP melanomas with greater than 75% regression are rare. Thus, individual patients with melanoma without regression and without the tumorigenic VGP can be given reasonable assurance of 100% survival. Nevertheless, this assurance is based on a statistical model with a finite population studied. Additional studies are
DIAGNOSIS AND PROGNOSIS OF PRIMARY CUTANEOUS MELANOMA
733
needed to confirm this model, as well as more definitive markers to precisely predict outcome for those individuals with tumorigenic VGP melanoma.
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Address reprint requests to H. Randolph Byers, MD, PhD Dermatopathology Section Department of Dermatology Boston University Medical School 609 Albany Street - J407 Boston, MA 02118