Context.—Histologic analysis allows accurate classification of most melanocytic lesions as benign or malignant. Only in a minority of lesions is it necessary to use other techniques as an aid in the diagnosis. Among them, most authors recommend immunohistochemistry.
Objective.—To describe how to apply immunohistochemistry to particular differential diagnoses and the potential pitfalls.
Data Sources.—Personal experience and review of literature.
Conclusions.—here is no single marker, or combination thereof, that establishes an unequivocal diagnosis of melanoma or nevus. Thus it is necessary to carefully analyze the pattern of expression (patchy versus diffuse) and localization (maturation) in the context of morphologic standard features.
ANTIBODIES COMMONLY USED IN DERMATOPATHOLOGY
More than 95% of primary cutaneous melanomas express S100. However, several conditions may affect its expression, such as too much or too little fixation time, previously frozen tissue, and enzymatic pretreatment with trypsin (see also troubleshooting).
gp100 (As Detected With the Antibody HMB-45)
gp100 is fairly specific for melanocytic differentiation; rarely do other lesions express this marker (angiomyolipoma, sugar cell tumor of the lung, and so-called pecoma). HMB-45 is particularly helpful in detecting the pattern of “maturation” of nevi. Thus, superficial, type A melanocytes (epithelioid cells, intraepidermal or close to the epithelium, and mostly pigmented) express neuronal markers and gp100, while the deeply located type C melanocytes (spindle cells) express schwannian markers.3,4 The only exceptions to this rule include blue nevi and related lesions (eg, deep-penetrating nevi) and some Spitz nevi, in which the entire lesion is labeled with HMB-45.
In contrast to nevi, primary cutaneous melanomas usually express gp100 in a patchy pattern, with isolated or clustered cells throughout the dermis; such pattern is also seen in nevoid melanoma.5 By also labeling the intraepidermal component, HMB-45 highlights a single-cell pattern of growth or pagetoid upward migration.
Melanoma Antigen Recognized by T Cells–1
It is one of the most important melanocytic markers is melanoma antigen recognized by T cells–1 (MART1).6 Detected by 2 different antibodies (Melan-A and A-103), this antigen is expressed by most melanocytic lesions, benign and malignant. Therefore, it is very helpful in detecting melanocytic differentiation.7 Conversely, strong and diffuse expression of this marker in a spindle cell melanocytic lesion makes a diagnosis of desmoplastic melanoma unlikely.
Other cells may also express this marker. In particular, steroid-producing tumors may react with A-103. Similarly to gp100, angiomyolipoma, sugar cell tumor of lung, lymphangioleiomyomatosis, and pecoma also express MART1.8 Since the antibody is so sensitive, in sun-exposed skin the labeling of the cell processes may give the appearance that more melanocytes are present than normal, thus raising the consideration of melanoma in situ9 (see below). On occasion, macrophages (particularly pigmented ones) are labeled with anti-MART1.10
MIB1 is a proliferation marker expressed in proliferating cells. Its pattern of expression, similar to that of gp100, highlights the presence or absence of “maturation.” Common nevi and dysplastic nevi exhibit reactivity in fewer than 1% of cells, generally disposed at the dermal-epidermal junction or in the more superficial dermal compartment. In contrast, melanomas have a random pattern of immunoreactivity, with a mean proliferative fraction of more than 10%, particularly at the deep edge of the lesion.11 Similarly, desmoplastic melanomas have a much higher proliferation rate, as detected with MIB1, than do desmoplastic nevi.12
Tyrosinase is an enzyme that participates in melanogenesis and is therefore fairly specific for melanocytic differentiation. In our hands, tyrosinase expression is very similar to HMB-45 labeling.
Microphthalmia Transcription Factor
Microphthalmia transcription factor (MiTF) is a nuclear protein involved in the development of melanocytes and the regulation of melanin synthesis in melanocytic lesions13,14; it may be expressed by macrophages, lymphocytes, fibroblasts, Schwann cells, and smooth muscle cells. Owing to its nuclear pattern of expression, we find that anti-MiTF is very helpful when quantifying the number of intraepidermal melanocytes in areas of pigmented epidermis.
To increase the sensitivity of immunohistochemistry, several cocktails have been developed to include more than 1 antibody. A popular combination is designated “pan-melanocytic cocktail” and consists of HMB-45, anti-MART1, and anti-tyrosinase. Analogously, at our institution, we have developed a cocktail including anti-MART1 and anti–Ki-67. Since MART1 is a cytoplasmic marker and Ki-67 is a nuclear marker, by using 2 different systems (eg, ABC and alkaline phosphatase) with 2 different chromogens (eg, diaminobenzidine [brown] and aminoethylcarbazole [red]), it is relatively easy to identify which cells express both markers, thus representing the fraction of melanocytes that are proliferating.
USE OF IMMUNOHISTOCHEMISTRY IN SPECIFIC DIFFERENTIAL DIAGNOSES
For the analysis of sentinel lymph nodes, please see the corresponding section.
1. Distinction Between Melanoma and Nevus
Most nevi show a pattern of maturation, that is, a change in expression of several immunohistochemical markers from the top to the bottom of the lesion (with the exception of Spitz nevi and blue nevi and related nevi). In our experience, the 2 most helpful markers to detect the pattern of maturation appear to be HBM-45 antigen (gp100) and Ki-67. Both markers are predominantly expressed in those melanocytes located in the epidermis/adnexa and the periepithelial dermis (papillary and adventitial). Therefore, a pattern in which HMB-45 antigen and Ki-67 are expressed in the intraepithelial and periepithelial components but are almost completely absent from the deep areas of the lesion is more consistent with a nevus than with a melanoma (Figure 1, G through J). In our practice, rather than actually counting the number of cells that are positive for Ki-67, we prefer to compare the top and the bottom of the lesion, since all nevi should have many fewer cells at the base of the lesion than the top areas (intraepithelial and periepithelial), regardless of the amount of positive cells.
The 2 types of nevi that diverge from this pattern are blue nevi (including cellular blue, plexiform, and “deep-penetrating” nevi) and Spitz nevi, which may show diffuse labeling with HMB-45 throughout the lesion (Figure 2, F through H). However, as with common nevi, these lesions show a very low proliferation rate with anti–Ki-67 (Figure 2, I through K).
The use of these 2 immunohistochemical markers may help also in the distinction between the invasive component of melanoma and an associated nevus. In contrast with the associated dermal nevus, the invasive component of melanoma is usually positive with HMB-45 and has a higher rate of Ki-67–positive cells. Furthermore, by examining the pattern of expression of gp100 in a blue nevus–type lesion, it may be possible to distinguish a primary blue nevus–type melanoma from a metastatic melanoma. This type of melanoma resembling blue nevus (so-called malignant blue nevus) sometimes arises in association with a blue nevus. In such lesions, gp100 is strongly and diffusely expressed in the benign, preexistent blue nevus, while its expression becomes patchy in the malignant areas (Figure 2, F through H).
2. Analysis of Melanocytic Desmoplastic Lesions
Desmoplastic melanomas usually display high numbers of Ki-67–positive cells and may show rare cells labeled with HMB-4512 (Figure 3, E through H). Another marker that may be helpful in distinguishing desmoplastic nevus from desmoplastic melanoma is MART1. As mentioned above, most melanocytic lesions express this marker, with the notable exception of spindle cell melanoma. Therefore, a spindle cell melanocytic lesion that does not express MART1 is more likely to be a melanoma than a nevus.
Immunohistochemistry is also helpful in the diagnosis of desmoplastic melanoma, since the tumor cells will express S100 protein. Furthermore, anti-S100 may help in delineating the extent of the lesion to determine the depth of invasion. In the differential diagnosis, even though scar tissue shows the presence of scattered S100-positive cells,10,22 when compared with scars, desmoplastic melanomas will have many more positive cells for this marker (Figure 4, B and C). Another marker typically expressed in desmoplastic melanoma and not in scars is p75.23
3. Diagnosis of Lentigo Maligna
Lentigo maligna is shown in Figure 5, A through F.
Anti-MART1 or HMB-45 will label the atypical melanocytes of lentigo maligna (melanoma in situ) in the epidermis, highlighting a confluent pattern, in contrast to the scattered, atypical melanocytes that can be seen in actinically damaged skin/pigmented actinic keratosis. Since anti-MART1 extensively labels the melanocyte dendrites, thus mimicking a confluent pattern of growth (Figure 5, C and D), some authors discourage the use of anti-MART1.24 However, addition of other antibodies, such as HMB-4525 or anti-MiTF, may be helpful since these markers label the cytoplasm of the melanocyte body or only the nuclei, respectively (Figure 5, E and F).
It is our opinion that immunohistochemistry has an important role in the diagnosis of melanocytic lesions. However, there is no single marker, or combination thereof, that establishes an unequivocal diagnosis of melanoma or nevus. Thus it is necessary to carefully analyze the pattern of expression (patchy versus diffuse) and localization (maturation) in the context of morphologic standard features.
From the Departments of Pathology and Dermatology, University of Texas M. D. Anderson Cancer Center, Houston (Dr Prieto); and Section of Dermatology, University of Chicago, Chicago, Illinois (Dr Shea).
The authors have no relevant financial interest in the products or companies described in this article.