Immunohistochemistry of Melanocytic Proliferations Victor G. Prieto, MD, PhD; Christopher R. Shea, MD

Context.—Histologic analysis allows accurate clasNsification 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. ANTIBODIES COMMONLY USED IN DERMATOPATHOLOGY S100 Protein 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). There are 2 types of antibodies, polyclonal and monoclonal. Both label melanocytic lesions, with a cytoplasmic and nuclear pattern. Of the several antigens detected by anti-S100, A6 is expressed by some melanocytic lesions and is also a very helpful marker to detect neurothekeomas.1,2 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. Accepted for publication May 27, 2010. 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. Reprints: Victor G. Prieto, MD, PhD, Departments of Pathology and Dermatology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Unit 85, Houston TX 77030 (e-mail: vprieto@ mdanderson.org). Arch Pathol Lab Med—Vol 135, July 2011

Data Sources.—Personal experience and review of literature. Conclusions.—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. (Arch Pathol Lab Med. 2011;135:853–859) 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 singlecell 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 sunexposed 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 (anti–Ki-67) 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 dermalepidermal 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 Immunohistochemistry of Melanocytic Proliferations—Prieto & Shea

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Figure 1. Distinction between nevus (A) and melanoma (B). Both lesions are large, with small intraepidermal component and no pagetoid upward migration. Nevus cells mature with depth (C versus E), while melanoma cells remain approximately the same size in the top and bottom of the lesion (D versus F). HMB-45 labels only cells in the epidermis and upper dermis of the nevus (G), while it highlights scattered melanoma cells throughout the lesion (H). With anti–Ki-67, nevus cells have minimal proliferation rate (I) in contrast with melanoma cells at the base of the lesion (J) (hematoxylin-eosin, original magnifications 34 [A and B] and 340 [C through F]; HMB-45, aminoethylcarbazole with light hematoxylin, original magnifications 310 [G and H]; anti–Ki-67, diaminobenzidine with light hematoxylin, original magnifications 340 [I and J]). 854

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Figure 2. Blue nevus–type lesions. Large cellular blue nevus showing uniform distribution of melanin pigment (A). A different lesion, also large, apparently similar at low-power magnification, with a more plexiform pattern of growth (B). At higher power, the cellular blue nevus has uniform cells with interspersed melanophages (C). The other lesion has 2 morphologically different areas: uniform, small spindle cells consistent with a blue nevus (D) and larger cells with prominent nucleoli and mitotic figures, thus consistent with melanoma (E). With immunohistochemistry, the blue nevus has uniform expression of gp100 (F), similar to that of the benign melanocytes in the other lesion (G); in contrast, in the melanoma area, there is only patchy expression (H). A double immunohistochemical study with anti-MART1 and anti–Ki-67 shows very rare melanocytes proliferating in the cellular blue nevus (I) or in the benign nevus area of the other lesion (J), in contrast with the numerous melanocytes expressing both markers in the melanoma area (cytoplasmic MART1 and nuclear Ki-67) (K). A, C, F, and I, Cellular blue nevus. B, D, E, G, H, J, and K, Melanoma arising in association with a blue nevus (hematoxylin-eosin, original magnifications 34 [A and B], 320 [C and D], and 340 [E]; HMB-45, aminoethylcarbazole with light hematoxylin, original magnifications 320 [F through H]; anti-MART1, aminoethylcarbazole and anti–Ki-67, diaminobenzidine, with light hematoxylin, original magnifications 340 [I through K]). Arch Pathol Lab Med—Vol 135, July 2011

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Figure 3. Hematoxylin-eosin sections of desmoplastic melanocytic lesions (A through D). Both desmoplastic nevus and melanoma show mostly spindle cells within a markedly fibrous stroma; some areas of desmoplastic melanoma show marked pleomorphism with rare mitotic figures (D, arrow). This particular desmoplastic nevus shows diffuse expression of HMB-45 antigen (E), while melanomas have only rare cells expressing HMB45 antigen (F). Desmoplastic nevi have minimal cellular proliferation with anti–Ki-67 (G, arrow) in contrast with the high number of cells expressing Ki-67 in desmoplastic melanoma, usually more than 20/mm2 (H). A, C, E, and G, Desmoplastic nevus. B, D, F, and H, Desmoplastic melanoma (hematoxylin-eosin, original magnifications 34 [A and B] and 340 [C and D]; HMB-45, aminoethylcarbazole, original magnifications 340 [E and F]; double immunohistochemical study with HMB-45, aminoethylcarbazole and anti–Ki-67, diaminobenzidine, with light hematoxylin as counterstain, original magnifications 340 [G and H]). 856

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Tyrosinase 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, antiMART1, 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. Other antibodies/antigens that may become more popular in the future include NKI-C3,15 p16,16 galectin3,17 COX-2,18 TRP,19 survivin,20 and claudin-1.21 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 The distinction between melanoma and nevus is shown in Figures 1, A through J; 2, A through K; 3, A through H; and 4, A through C. 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 ‘‘deeppenetrating’’ nevi) and Spitz nevi, which may show Arch Pathol Lab Med—Vol 135, July 2011

Figure 4. Standard desmoplastic melanoma lesion to illustrate use of anti-MART1. Flat epidermis with hyperchromatic cells in the dermis showing irregular nuclei (inset) (A). Only intraepidermal cells express MART1 (B). In contrast, both the intraepidermal and dermal components are strongly positive for S100 protein (C) (hematoxylin-eosin, original magnifications 310 [A] and 340 [inset]; anti-MART1, aminoethylcarbazole with light hematoxylin, original magnification 340 [B]; anti-S100 protein, aminoethylcarbazole with light hematoxylin, original magnification 340 [C]).

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 Immunohistochemistry of Melanocytic Proliferations—Prieto & Shea

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Figure 5. Comparison of melanoma in situ, lentigo maligna type (A), and pigmented actinic keratosis/sun-damaged skin (B). Both specimens were taken as scouting biopsies in the same patient to determine the extent of melanoma. A pan-melanocytic cocktail (HMB-45, anti-MART1, and antityrosinase) highlights both the melanocyte body and dendrites of melanoma cells, some in clusters and with focal pagetoid upward migration (arrow). Also note the presence of a few dermal cells, likely to be macrophages10 (C). Owing to the labeling of melanocyte dendrites by the panmelanocytic cocktail, it appears that increased numbers of melanocytes (D) are present in the area of pigmented actinic keratosis. Anti-MiTF highlights the nuclei of numerous melanoma cells in the melanoma lesion, including a couple of cells with pagetoid upward migration (arrow) (E). In contrast, the area of pigmented actinic keratosis shows only rare, scattered melanocytes (F). A, C, and E, Melanoma in situ lentigo maligna type. B, D, and F, Actinically damaged skin (hematoxylin-eosin, original magnifications 340 [A and B]; HMB-45, anti-MART1, and anti-tyrosinase, original magnifications 340 [C and D]; anti-MiTF, diaminobenzidine, original magnifications 340 [E and F]; light hematoxylin as counterstain [B, C, E, and F]). Abbreviation: MiTF, microphthalmia transcription factor. 858

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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 Melanocytic desmoplastic lesions are shown in Figures 3 and 4. 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). SUMMARY 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. References 1. McNutt NS. The S100 family of multipurpose calcium-binding proteins. J Cutan Pathol. 1998;25(10):521–529.

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2. Fullen DR, Lowe L, Su LD. Antibody to S100a6 protein is a sensitive immunohistochemical marker for neurothekeoma. J Cutan Pathol. 2003;30(2): 118–122. 3. Prieto VG, McNutt NS, Lugo J, Reed JA. The intermediate filament peripherin is expressed in cutaneous melanocytic lesions. J Cutan Pathol. 1997;24(3):145–150. 4. Huttenbach Y, Prieto VG, Reed JA. Desmoplastic and spindle cell melanomas express protein markers of the neural crest but not of later committed stages of Schwann cell differentiation. J Cutan Pathol. 2002;29(9):562–568. 5. McNutt NS, Urmacher C, Hakimian J, Hoss DM, Lugo J. Nevoid malignant melanoma: morphologic patterns and immunohistochemical reactivity. J Cutan Pathol. 1995;22:502–517. 6. Busam KJ, Chen YT, Old LJ, et al. Expression of melan-A (MART1) in benign melanocytic nevi and primary cutaneous malignant melanoma. Am J Surg Pathol. 1998;22(8):976–982. 7. Jungbluth AA, Busam KJ, Gerald WL, et al. A103: an anti-melan-a monoclonal antibody for the detection of malignant melanoma in paraffinembedded tissues. Am J Surg Pathol. 1998;22(5):595–602. 8. Fetsch PA, Fetsch JF, Marincola FM, Travis W, Batts KP, Abati A. Comparison of melanoma antigen recognized by T cells (MART-1) to HMB-45: additional evidence to support a common lineage for angiomyolipoma, lymphangiomyomatosis, and clear cell sugar tumor. Mod Pathol. 1998;11(8): 699–703. 9. El Shabrawi-Caelen L, Kerl H, Cerroni L. Melan-A: not a helpful marker in distinction between melanoma in situ on sun-damaged skin and pigmented actinic keratosis. Am J Dermatopathol. 2004;26(5):364–366. 10. Trejo O, Reed JA, Prieto VG. Atypical cells in human cutaneous reexcision scars for melanoma express p75NGFR, C56/N-CAM and GAP-43: evidence of early Schwann cell differentiation. J Cutan Pathol. 2002;29(7):397– 406. 11. Rudolph P, Schubert C, Schubert B, Parwaresch R. Proliferation marker KiS5 as a diagnostic tool in melanocytic lesions. J Am Acad Dermatol. 1997;37(2, pt 1):169–178. 12. Harris GR, Shea CR, Horenstein MG, Reed JA, Burchette JL Jr, Prieto VG. Desmoplastic (sclerotic) nevus: an underrecognized entity that resembles dermatofibroma and desmoplastic melanoma. Am J Surg Pathol. 1999;23(7):786–794. 13. King R, Googe PB, Weilbaecher KN, Mihm MC Jr, Fisher DE. Microphthalmia transcription factor expression in cutaneous benign, malignant melanocytic, and nonmelanocytic tumors. Am J Surg Pathol. 2001;25(1):51–57. 14. Koch MB, Shih IM, Weiss SW, Folpe AL. Microphthalmia transcription factor and melanoma cell adhesion molecule expression distinguish desmoplastic/spindle cell melanoma from morphologic mimics. Am J Surg Pathol. 2001; 25(1):58–64. 15. Paul E, Cochran AJ, Wen DR. Immunohistochemical demonstration of S100 protein and melanoma-associated antigens in melanocytic nevi. J Cutan Pathol. 1988;15(3):161–165. 16. Reed JA, Loganzo F Jr, Shea CR, et al. Loss of expression of the p16/cyclindependent kinase inhibitor 2 tumor suppressor gene in melanocytic lesions correlates with invasive stage of tumor progression. Cancer Res. 1995;55(13): 2713–2718. 17. Prieto VG, Mourad-Zeidan AA, Melnikova V, et al. Galectin-3 expression is associated with tumor progression and pattern of sun exposure in melanoma. Clin Cancer Res. 2006;12(22):6709–6715. 18. Chwirot BW, Kuzbicki L. Cyclooxygenase-2 (COX-2): first immunohistochemical marker distinguishing early cutaneous melanomas from benign melanocytic skin tumours. Melanoma Res. 2007;17(3):139–145. 19. Hashimoto Y, Ito Y, Kato T, Motokawa T, Katagiri T, Itoh M. Expression profiles of melanogenesis-related genes and proteins in acquired melanocytic nevus. J Cutan Pathol. 2006;33(3):207–215. 20. Ding Y, Prieto VG, Zhang PS, et al. Nuclear expression of the antiapoptotic protein survivin in malignant melanoma. Cancer. 2006;105(5):1123–1129. 21. Cohn ML, Goncharuk VN, Diwan AH, Zhang PS, Shen SS, Prieto VG. Loss of claudin-1 expression in tumor-associated vessels correlates with acquisition of metastatic phenotype in melanocytic neoplasms. J Cutan Pathol. 2005;32(8): 533–536. 22. Chorny JA, Barr RJ. S100-positive spindle cells in scars: a diagnostic pitfall in the re-excision of desmoplastic melanoma. Am J Dermatopathol. 2002;24(4): 309–312. 23. Kanik AB, Yaar M, Bhawan J. p75 nerve growth factor receptor staining helps identify desmoplastic and neurotropic melanoma. J Cutan Pathol. 1996; 23(3):205–210. 24. Yan S, Brennick JB. False-positive rate of the immunoperoxidase stains for MART1/MelanA in lymph nodes. Am J Surg Pathol. 2004;28(5):596–600. 25. Wiltz KL, Qureshi H, Patterson JW, Mayes DC, Wick MR. Immunostaining for MART-1 in the interpretation of problematic intra-epidermal pigmented lesions. J Cutan Pathol. 2007;34(8):601–605.

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