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Update on Immunohistochemical Methods Relevant to Dermatopathology

Justin Wasserman, MD; Jessica Maddox, MD; Mark Racz, MD; Vesna Petronic-Rosic, MD

Context.Dermatopathology covers a large variety of entities, some having very similar histologic appearances. Immunohistochemistry is an incredibly helpful tool that is useful in diagnosis as well as prognosis of selected skin tumors. Objective.To provide a comprehensive review of recent trends and immunohistochemical stains used by dermatopathologists. Emphasis is placed on new stains as well as novel uses of existing stains. Data Sources.All data were gathered from published journal articles available through the National Center for Biotechnology Information PubMed database. he skin houses cells of all types and origins and gives rise to an array of diseases and neoplasms, some originating primarily in the skin and some appearing secondary to metastases from other organ systems. Most can be diagnosed from standard hematoxylin-eosinstained sections with reasonable certainty. There are always, though, poorly differentiated neoplasms, or poor specimens that can make a standard diagnosis more challenging. The addition of widespread immunohistochemical stains in recent years has given the pathologist in general and the dermatopathologist specically many new weapons in their diagnostic armamentarium. Immunohistochemistry, however, is only a tool to be used appropriately in the context of clinical and histologic information; unreasonable use can be misleading and nancially cost ineffective. The goal of this review is to provide pathologists with a brief update on commonly used immunohistochemical stains in dermatopathology and an in-depth look at some of the newer stains or new applications of older stains that have appeared in the last several years. Immunohistochemistry is constantly evolving and expanding, and it is important to understand that this article is not a comprehensive guide to immunohistochemistry in dermatopathology. For example, an important area of dermatopathology not covered here is immunohistochemistry for he-

Conclusions.New immunohistochemical targets are continually being found, contributing to more accurate diagnosis and classication of skin tumors. The presence of specic markers can be used to determine the aggressiveness of malignanicies and design treatment strategies. In addition, application of existing stains can help determine intravascular spread of malignancy in primary cutaneous lesions. And use of rapid immunohistochemical staining techniques on frozen sections can assist in more complete excision of tumor margins. Immunohistochemistry is a highly versatile and growing tool of dermatopathologists. (Arch Pathol Lab Med. 2009;133:10531061) matopoetic neoplasms in the skin, which would need to be covered separately to receive adequate attention. NEURAL AND NEUROENDOCRINE NEOPLASMS Most neural tumors of the skin can be diagnosed on routine hematoxylin-eosinstained sections. Immunohistochemistry is most often needed to distinguish Merkel cell carcinoma from other small blue-celled tumors, such as lymphoma or small cell cancer of the lung. Standard immunohistochemical stains that have been used for years are listed in Table 1, and thus far have been the most effective. CK20 is the most specic stain for Merkel cell carcinoma, and when used in combination with a negative thyroid transcription factor 1 it provides strong support for the diagnosis, especially if all tumor cells stain positive. CK20 positivity has also been reported in a very small percentage (approximately 2.4%) of small cell carcinomas of the salivary gland, and rarely in other small cell carcinomas.1 Other useful adjuncts are listed in Table 1; however, they are less sensitive and less specic for Merkel cell carcinoma and are positive in other varieties of neuroendocrine carcinoma that may metastasize to the skin. CD117 examination2 has not proven to be superior to existing stains. D2-40 was recently evaluated in peripheral nerve sheath neoplasms and showed strong, diffuse, cytoplasmic staining in most schwannomas (76%) and membranous staining in epithelioid malignant peripheral nerve sheath tumors. Neurobromas and spindle cell malignant peripheral nerve sheath tumors were negative in most cases.3 SOFT TISSUE NEOPLASMS Standard stains used in the diagnosis of soft tissue neoplasms are listed in Table 2. Recent advances are discussed below.
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Accepted for publication December 11, 2008. From the Section of Dermatology, University of Chicago, Chicago, Illinois. The authors have no relevant nancial interest in the products or companies described in this article. Reprints: Justin Wasserman, MD, Section of Dermatology, University of Chicago, MC5067, 5841 S Maryland Ave, Chicago, IL 60637 (e-mail: justin.wasserman@uchospitals.edu). Arch Pathol Lab MedVol 133, July 2009

Immunohistochemical Methods in DermatopathologyWasserman et al

Table 1.
Stain

Immunostains in Neural and Neuroendocrine Neoplasms


Pattern Application

S100 Neuron-specic enolase Cytokeratin 20 Neurolament Chromogranin Synaptophysin Thyroid transcription factor 1

Nuclear/cytoplasmic staining Cytoplasmic staining Perinuclear dot staining Perinuclear dot staining Cytoplasmic staining Cytoplasmic staining Nuclear staining

Gliomas, primitive neuroectodermal tumors, schwannoma, neurobroma, and neuronal and chondroid tumors Neuroendocrine cells, Merkel cell carcinoma Most sensitive stain for Merkel cell carcinoma Neuroendocrine cells, Merkel cell carcinoma Neuroendocrine cells, Merkel cell carcinoma Neuroendocrine cells, Merkel cell carcinoma Negative in Merkel cell carcinoma

Table 2.
Stain Pattern

Immunostains in Soft Tissue Neoplasms


Application

CD34 Factor XIIIa Smooth muscle actin Desmin Vimentin Muscle-specic actin

Cytoplasmic staining Cytoplasmic staining Cytoplasmic staining Cytoplasmic staining Cytoplasmic staining Cytoplasmic staining

Dermatobrosarcoma protuberans, blood vessels, malignant brous histiocytoma, epithelioid sarcoma Dermatobroma, juvenile xanthogranuloma, multicentric reticulohistiocytosis, radiation dermatitis Glomus tumor, leiomyoma, dermatomyobroma, leiomyosarcoma Found in striated and smooth muscle Retained in mesenchymally derived cells, broblasts, endothelial cells, macrophages Found in striated and smooth muscle

Table 3.
Stain Pattern

Immunostains in Infectious Diseases


Application

Cytomegalovirus Herpes simplex virus Varicella zoster virus Epstein-Barr virus Human herpesvirus 8

Nuclear/cytoplasmic staining Nuclear/cytoplasmic staining Nuclear/cytoplasmic staining Cytoplasmic staining Nuclear/cytoplasmic staining

Cytomegalovirus infection Herpes simplex virus 1 or 2 infection Varicella zoster virus infection Posttransplantation lymphoproliferative disorders, Hodgkin lymphoma Kaposi sarcoma, primary effusion lymphoma, Castleman disease

D2-40 Most studies and the most useful applications of this immunohistochemical stain are discussed in the section on vascular diseases; here, we will talk about a potentially useful application of this stain in soft tissue neoplasms. D2-40 may represent a helpful adjunct in distinguishing between dermatobromas (DFs) and dermatobrosarcoma protuberans (DFSP), which can occasionally be difcult. Strong and diffuse staining with D2-40 was seen in all 20 DFs analyzed (100%), whereas only 4 of 24 DFSPs (16%) displayed patchy weak reactivity. Further studies will be helpful in conrming these ndings.4 CD99 Monoclonal antibodies that react against CD99, the product of the MIC2 gene, are useful, most notably in Ewing sarcoma and peripheral neuroectodermal tumor. Reactivity also is present in gastrointestinal stromal tumors, ovarian sex cord tumors, and hematopoetic malignancies, like lymphoblastic lymphoma/leukemia. CD99 has a membranous pattern of staining. Recent studies showed that CD99 labeled 17 of 17 cases (100%) and 19 of 26 cases (73%) of atypical broxanthoma, with most showing a moderate to strong staining pattern.5,6 In contrast, pleomorphic malignant brous histiocytoma displayed moderate to strong reactivity with CD99 in 35% of cases, and only 15% stained diffusely.7 None of the poorly differentiated squamous cell carcinomas stained, whereas studies have reported mixed results
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in malignant melanoma (see section on melanocytic lesions).5,6 CD99 expression also was seen in a larger series evaluating DFSP and giant cell angiobroblastoma; 23 of 29 DFSPs (79%) and 2 of 5 GCPs (40%) expressed CD99. CD99 also stains nuchal-type bromas, which have been seen in contiguity with DFSP.8 Solitary brous tumor, a neoplasm most commonly associated with the pleura, is another uncommon tumor where positive staining for anti-CD99 can be present and serve as an adjunct in diagnosis along with CD34 and BCL-2.9 INFECTIOUS DISEASE Most infectious diseases have antibodies associated with them that may be used in immunohistochemistry. In dermatopathology, only a small subset of these are useful for several reasons: (1) other laboratory methods may be more sensitive and cost efcient; (2) unless the specic cause of the infection is known or suspected it would be difcult to choose which stains to apply in given cases; and (3) chemical staining is usually sufcient for diagnosis in most cases. Table 3 lists several immunostains specic to microorganisms that have been useful in dermatopathology. Antitreponemal Antibodies In routine evaluation of suspected syphilis, spirochetes are sought using the Steiner or Warthin-Starry silver
Immunohistochemical Methods in DermatopathologyWasserman et al

Figure 1. Antitreponemal antibodies highlight spirochetes in a biopsy of a patient with syphilis (original magnication 400).

stains. Unfortunately, signicant background staining and artifact can hamper interpretation, leading to low sensitivities of these techniques, reported to range from 33% to 71%.10 Past studies, including 2 more recent ones, have shown that immunohistochemical staining can provide better sensitivity for the detection of Treponema pallidum. In one study, a polyclonal antibody to T pallidum was positive in 11 of 12 cases (91%) from patients with secondary syphilis, whereas none of the controls reacted.11 Hoang et al12 showed staining in 12 of 17 biopsies (71%) with a monoclonal antibody compared with 41% detection using a silver stain; none of the controls reacted with the monoclonal antibody. Spirochetes were demonstrated in both the epidermis and dermis, including around supercial capillaries, within follicular epithelium, and within arector pili muscles. An example of antitreponemal antibody staining in a patient diagnosed with syphilis is shown in Figure 1. MELANOCYTIC PROLIFERATIONS Immunohistochemistry is a critical step for the identication and staging of malignant melanoma. Many stains exist and help to a variable degree in establishing both the diagnosis and prognosis (Table 4). pHH3 Histone H3 is a core histone protein that complexes with other histone proteins in the nucleus and makes up most of the protein component of chromatin in eukaryotic cells. In mammalian cells, phosphorylation of the Ser-10 residue of histone H3 is minimal during interphase and
Table 4.
Stain Pattern

maximum during mitosis.13 Immunohistochemical studies performed with antiphospho-histone H3 (anti-pHH3) antibody have documented a precise temporal and spatial correlation between H3 phosphorylation and mitotic chromosome condensation, and they also have shown no phosphorylation on histone H3 during apoptosis14; therefore, pHH3 serves as a mitotic marker. Currently, pHH3 staining has been used with good results in meningioma grading systems. It is important to note that pHH3 is only specic for cells undergoing mitosis and will not distinguish between cell types. In neural tissue, this is of minimal importance because there are very few cell types present; however, in the skin it is important to keep in mind that there are many cell types present and that not all mitosis seen may be related to the cell type of interest. Double staining with cell-specic immunohistochemical markers may be a useful method to avoid this pitfall. A recent publication by Nasr and El-Zammar15 highlights the usefulness of pHH3 in melanocytic lesions. In a study of 66 melanocytic lesions, pHH3 did not stain dysplastic or compound nevi, was positive in 3 of 8 Spitz nevi but localized to the supercial dermis, and was positive in all of the malignant melanoma, with mitotic gures appearing both supercially and deep in the dermis. An example of pHH3 staining in melanoma is shown in Figure 2. CD99 CD99 has been evaluated recently in the differentiation of Spitz nevi from spitzoid melanoma. Previously, studies showed mixed results concerning CD99 expression in melanomas. Early on, it was seen only in 10%,4 whereas a second report found positive staining in 60% of melanomas,16 and most recently in 56% of spitzoid melanomas.17 Spitz nevi were negative in 55 of 58 cases (95%), with only focal staining in the 3 positive cases, whereas melanoma showed a predominately diffuse staining pattern in both studies.17 Future research is necessary to further evaluate the diagnostic utility of CD99 in melanocytic neoplasms. Microphthalmia Transcription Factor Microphthalmia transcription factor is necessary for the development of melanocytes during embryogenesis.18 It is a nuclear stain and is thus more easily interpreted, which may be useful when large amounts of cytoplasmic pigment may obscure other immunohistochemical staining patterns. Ways to avoid this issue include melanin bleaching,19 use of azure B counterstaining, or use of a red chromogen.20 Several studies have estimated sensitivity of microphthalmia transcription factor for melanoma to be between 81% and 100%.21 Although these initial investigations showed a high specicity for melanoma, more recent

Immunostains in Melanocytic Neoplasms


Application

S100 HMB-45 Melan-A/Mart-1 Tyrosinase Ki-67

Nuclear/cytoplasmic staining Cytoplasmic staining Cytoplasmic staining Fine granular cytoplasmic staining Nuclear staining

Most sensitive marker for melanoma and spindle cell/desmoplastic melanoma Higher specicity for melanoma (primary-metastatic) can help distinguish nevi from melanoma Similar sensitivity and specicity to HMB-45, but with more diffuse and intense staining Higher sensitivity than HMB-45 and specicity of 97%100%. Sensitivity decreases with increased clinical stage and in metastases Less than 5% of cells in nevi, and 13%30% of cells in malignant melanoma; higher percentages have been noted in Spitz nevi as well Immunohistochemical Methods in DermatopathologyWasserman et al 1055

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Figure 2. A, Primary cutaneous nodular melanoma (hematoxylin-eosin, original magnication 100). B, Staining with pHH3 assists in identifying mitotically active cells in this highly cellular malignant melanoma (original magnication 200).

Figure 3. Melanoma in situ on sun-damaged skin (hematoxylin-eosin, original magnication 400).

data found the specicity to be much lower in the spindle cell variant.2224 An example of microphthalmia transcription factor immunostaining is illustrated in Figures 3 and 4. MUM1 The multiple myeloma 1/interferon regulatory factor 4 (MUM1/IRF4; also known as LSIRF, Pip, and ICSAT) gene product is a member of the interferon regulatory factor family of transcription factors.2528 These factors are known to play an important role in the regulation of gene expression in response to interferon and other cytokines. Although the role of MUM1 in hematolymphoid malignancies and immune regulation has been established, expression patterns of the MUM1 protein in other tissues have only been examined recently.29,30 One study demonstrated that the MUM1 protein can be detected in neoplasms of plasmacytic differentiation as well as in a wide variety of B-cell, T-cell, and natural killer cell lymphomas, but was
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negative in all other malignant neoplasms tested except melanoma.31 MUM1 exhibits a nuclear staining pattern. In a study by Sundram et al,32 the MUM1 antibody highlighted most melanomas, with 92% of conventional primary and metastatic melanomas being positive. These data suggest it is a more sensitive marker than either HMB-45 or Melan-A in primary and metastatic melanomas. MUM1 antibody also showed strong and more diffuse staining of benign melanocytic nevi than either HMB45 or Melan-A, staining 75% of benign nevi in this study, compared with 13% and 63%, respectively. MUM1 strongly stained 80% (8 of 10) of Spitz nevi.32 Because MUM1 expression appears to be present in both nevi and in melanomas, and it does not highlight desmoplastic melanoma more effectively than well-established stains do, it is unclear whether it will have a role in the future and what that role would be. Immunostains that are currently being actively investigated for use in melanocytic neoplasms include melanocortin-1 receptor, SM5-1, PNL2, and newer antibodies to tyrosinase.33 In addition, the evolving use of immunostains in frozen sections34,35 may help decrease recurrence rates after surgery for melanoma, where ex tempore interpretation is greatly compromised by freeze artifact. VASCULAR PROLIFERATIONS Previously used stains for vascular lesions have been very effective, but still not without their drawbacks (Table 5). Recent years have seen the development of several immunohistochemical stains that have been highly useful in this category. D2-40 D2-40 is a novel monoclonal antibody against M2A antigen, an Mr 40 000 O-linked sialoglycoprotein that reacts to a xation-resistant epitope on lymphatic endothelium.36 A cytoplasmic staining pattern of endothelial cells highlights lymphatic channels. Studies have shown that this antigen is expressed in lymphatics of normal tissues, as well as Kaposi sarcoma, lymphangiomas, some angiosarcomas, and Dabska tumor.36 The utility of D2-40 extends beyond its obvious application to identify vascular tumors
Immunohistochemical Methods in DermatopathologyWasserman et al

Table 5.
Stain

Immunostains in Vascular Proliferations


Pattern Application

CD31 CD34 Factor VIIIrelated Ag Ulex Europaeus I agglutinin

Cytoplasmic membrane staining Cytoplasmic membrane staining Cytoplasmic staining, ultrastructurally correspond to Weibel-Palade bodies Cytoplasmic staining

Specic marker of endothelial cells; poor sensitivity (66%) High sensitivity for endothelial cells; poor specicity Less sensitive than CD31 for endothelial cells; infrequently used Less sensitive than CD31 for endothelial cells; infrequently used

because it is used to help determine the intralymphatic presence of primary or metastatic cancers. A recent study of melanoma by Firouzeh et al37 identied lymphatic invasion using D2-40 and found it to correlate with sentinel lymph node positivity. They hypothesized that it may be helpful in identifying candidates for sentinel lymph node biopsy.37 See Figure 5 for an example of D2-40 staining lymphatic vessels. GLUT1 GLUT1 protein is an erythrocyte glucose transporter restricted to endothelial cells in areas with blood-tissue barrier functions, such as the brain, eye, nerve, and placenta. GLUT1 stains the cytoplasmic membrane, and its primary utility is in the diagnosis of pediatric vascular lesions. Although not necessary for diagnosing pediatric vascular lesions, it has been found in juvenile hemangiomas at all stages of development38 but is not present in vascular malformations, granulation tissue, pyogenic granulomas, noninvoluting congenital hemangiomas, rapidly involuting congenital hemangiomas, or other vascular and nonvascular tumors.39 This stain is highly specic for juvenile hemangiomas; it has enabled diagnostic accuracy of these entities and yielded valuable data regarding their pathogenesis.39 Fli-1 Fli-1 protein is a member of the ETS family of DNAbinding transcription factors, characterized by a highly conserved DNA-binding domain. Originally used in the diagnosis of Ewing sarcoma/primitive neuroectodermal tumors, the only normal tissues it has been found to stain include small lymphocytes and endothelial cells.40 Fli-1 is a nuclear stain, which makes it more easily interpretable than other stains typically used as vascular markers, including CD31, CD34, and factor VIII-related antigen. Its utility for the diagnosis of vascular tumors and growths has been studied and showed very encouraging results. The sensitivity (94%) and specicity (100%) of Fli-1 reported by Folpe et al41 was equal to or exceeded those of the established vascular markers, CD31, CD34, and von Willebrand factor. Early studies used a polyclonal antibody and had wider variation in staining and interpretation, but monoclonal antibodies have been developed showing more consistency.42 Fli-1 has shown comparable sensitivity to currently used vascular markers and higher specicity than CD31, CD34, and factor VIII antigen.4042 In addition, it is more sensitive and specic than other novel vascular markers, such as VEGFR-3,43 podoplanin,44 and CD117.45 Fli-1 shows no variation in staining among vascular proliferations, and it stains benign tumors equally well as malignant tumors. Therefore, its usefulness is limited to the diagnosis of vascular tumors but cannot distinguish entities within this group.40
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EPIDERMAL AND APPENDAGEAL NEOPLASMS Both benign and malignant epidermal and appendageal neoplasms are numerous, with often-similar morphologic presentations that may also mimic other cutaneous neoplasms, such as melanoma. Table 6 lists commonly used stains. We describe below several novel stains that have shown promise in the diagnosis of these entities. CD10 CD10 antigen is a 100-kDa cell surface zinc metalloendopeptidase expressed in a variety of normal and neoplastic lymphoid and nonlymphoid tissues. It also is known as common acute lymphocytic leukemia antigen.46 Immunohistochemical staining may be membranous and/ or cytoplasmic. It serves as a marker for the common form of acute lymphocytic leukemia as well as for Burkitt lymphoma and follicular germinal center lymphoma.47 CD10 is normally present on the surface of early lymphoid cells as well as on a number of other normal cell types, such as bile canaliculi and renal and intestinal epithelial cells.4850 Within the skin, CD10 immunoreactivity was signicant in sebaceous and xanthomatous neoplasms, but not in eccrine and apocrine ones.51,52 It also is present in periadenexal mesenchymal cells of normal dermis and in tumors, such as dermatobroma, to a lesser extent in dermatobrosarcoma protuberans,53 and in melanoma.54 CD10 is found in dermal sheath cells of hair follicles.55 The dermal sheath that surrounds the outside of the hair follicle contains progenitor cells that maintain and regenerate the dermal papilla, a key component for hair growth.56 Recent ndings suggest that CD10 may be a useful marker for specialized mesenchymal cells and tumors of the skin. Specically, it has been shown to assist in differentiation between basal cell carcinoma and trichoepithelioma. One study revealed that the expression of CD10 in trichoepithelioma was in the stroma and tumor papillae and notably lacking in the epithelial component, whereas in basal cell carcinoma, CD10 was expressed peripherally in the basaloid nests but was absent in the stroma.57 p63 p63 is a nuclear stain that may be a helpful marker in skin pathology because it is expressed in the nuclei of basal and spinous cells of the epidermis. p63 is a member of the tumor suppressor protein p53 gene family and comprises at least 6 different protein isoforms with homology to p53.58 One such isoform, Np63, is the predominant isoform in mature epidermis and appears to be a master regulator of squamous stem cell differentiation.59 In addition, expression also has been established in a variety of neoplasms, including squamous cell carcinoma.60 Immunohistochemical stains for p63 have been used to differentiate squamous cell carcinoma from adenocarcinoma in
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Figure 4. Microphthalmia transcription factor highlights increased numbers of melanocytes located in the basal layer and at higher levels of the stratum spinosum (original magnication 400). Figure 5. D2-40 highlights lymphatic vessels adjacent to an unstained blood vessel (original magnication 100). 200). 200). Figure 6. Squamous cell carcinoma with individual tumor cells budding off (lower right corner; hematoxylin-eosin, original magnication Figure 7. MNF116 stains keratinocytes budding off the larger tumor as single cells and small clusters (original magnication

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Table 6.
Stain

Immunostains in Epidermal and Appendegeal Neoplasms


Pattern Application

Carcinoembryonic antigen Gross cystic disease uid protein 15 Epithelial membrane antigen Cytokeratin 7 CAM 5.2 AE1/AE3 Ber-EP4 BCL-2

Cytoplasmic staining Cytoplasmic staining Bubbly cytoplasmic staining Cytoplasmic staining Cytoplasmic staining Cytoplasmic staining Cytoplasmic staining Cytoplasmic staining

Marker of glandular differentiation; can help distinguish extramammary Paget disease Marker of apocrine differentiation Stains malignant eccrine and apocrine often, and most sebaceous glands Sensitive and specic for Paget and extramammary Paget disease Lowmolecular weight keratins present in glandular neoplasms Mixture of low and highmolecular weight cytokeratins; useful in squamous cell carcinoma Positive in basal cell carcinoma and negative in squamous cell carcinoma; stains sebaceous neoplasms Stains the basal layer of the epidermis; can be helpful in distinguishing basal cell carcinoma from trichoepithelioma

several arenas, including cervical and anal pathology.61,62 p63 is notably absent in dermal broblasts, smooth muscle, Schwann cells, and endothelial cells,63 lending itself useful for diagnosis of atypical squamous cell carcinoma.64 To differentiate spindle cell squamous cell carcinoma from other spindle cell neoplasms, such as atypical broxanthoma, spindle cell melanoma, leiomyosarcoma, and others, investigators demonstrated that spindle cell squamous cell carcinoma expressed p63 strongly and diffusely, whereas most controls were negative or displayed weaker and more focal staining.65 In addition, most primary adnexal carcinomas and their metastases express p63, whereas visceral adenocarcinomas and their cutaneous metastases do not make this stain a useful adjunct to distinguish primary cutaneous adnexal carcinomas from metastatic visceral adenocarcinomas.66 Similarly, D2-40 also was found to have a high sensitivity (94.5%) and specicity (97.2%) in identifying primary cutaneous adnexal carcinomas from metastatic adenocarcinomas to the skin.67 MNF116 MNF116 recognizes cytoplasmic polypeptides of 45, 46, and 56.5 kDa, corresponding to cytokeratins 5, 6, 8, 17, and 19. It shows a broad pattern of reactivity with human epithelial tissues from simple glandular epithelia to stratied squamous epithelia, like the epidermis, mammary gland ducts, and tracheal epithelium. In normal skin, MNF116 exhibits particularly strong staining in the basal cells of the epidermis and adnexae. MNF116 is positive in epithelial tumors and negative in all mesenchymal and melanocytic tumors.68 It has been useful in identifying unusual variants of squamous cell carcinoma, such as spindle cell squamous cell carcinoma with single-cell inltration at the periphery.64 Examples showing the usefulness of MNF116 staining in invasive squamous cell carninoma compared to hematoxylin and eosin staining are illustrated in Figures 6 through 8. 34 E12 Highmolecular weight cytokeratin antibody, also known as CK903, is a cytoplasmic marker. This antibody

recognizes keratin polypeptides of 68, 58, 56.5, and 50 kDa, corresponding to cytokeratins 1, 5, 10, and 14, which are expressed in squamous, ductal, and other complex epithelia, basal cells, and myoepithelial cells.69 It stains adenocarcinomas, breast, pancreas, bile duct, and salivary gland, as well as squamous cell and transitional cell carcinomas. This stain is positive in cutaneous adnexal and epidermal neoplasms. Its use in dermatopathology may be in diagnosing poorly differentiated carcinomas. A recent case report details the use of 34 E12 to identify an atypical cutaneous squamous cell carcinoma originally misdiagnosed as an atypical broxanthoma.70 CK 5/6 Antibodies to CK 5/6 recognize a highmolecular weight intermediate lament and are useful markers of squamous differentiation. This antibody is a cytoplasmic marker found in a wide variety of cutaneous adnexal neoplasms, both benign and malignant.71 It also has been identied in metastic adenocarcinomas, but to a much lesser extent. In a study by Plumb et al,71 it was found that 97% of cutaneous adnexal neoplasms stained positive with CK5/6, but only 33% of metastatic adenocarcinomas stained positive, and those stained weakly. This immunostain may be helpful in distinguishing primary cutaneous tumors from metastatic lesions. CONCLUSIONS Immunohistochemistry is an excellent diagnostic technique with the distinct advantage of being able to exactly locate a given protein within the tissue examined. The eld is continuously expanding, with new applications steadily increasing. Melanocytic staining carries the most potential, because many immunostains are being explored not only for diagnostic purposes but for prognostic value as well. Multipronged immunohistochemistry can help dene malignancy risk stratication and therapeutic guidelines. The evolving use of immunostains in frozen sections may help decrease recurrence rates after surgery for tumors such as melanoma, where interpretation is greatly compromised by freeze artifact. Because a variety of mo-

Figure 8. A, A spindle cell neoplasm is present in the dermis, stained with hematoxylin-eosin, and (B) demonstrates strong staining for MNF116. A prior history of invasive squamous cell carcinoma immediately adjacent to the biopsy site was present (original magnications 60 [A] and 200 [B]). Arch Pathol Lab MedVol 133, July 2009 Immunohistochemical Methods in DermatopathologyWasserman et al 1059

lecular pathways are altered in cancer, some of the alterations can be targeted in cancer therapy. Immunohistochemistry can be used to assess which tumors are likely to respond to treatment by detecting the presence or elevated levels of the molecular target. Ultimately, the possibilities of this eld are immense and the future very promising.
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