Update on immunohistochemical methods relevant to dermatopathology.
Abstract: * 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.

Conclusions.--New immunohistochemical targets are continually being found, contributing to more accurate diagnosis and classification of skin tumors. The presence of specific 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.
Article Type: Report
Subject: Immunohistochemistry (Methods)
Immunohistochemistry (Usage)
Skin cancer (Diagnosis)
Skin cancer (Genetic aspects)
Skin cancer (Identification and classification)
Skin cancer (Care and treatment)
Pathology (Practice)
Authors: Wasserman, Justin
Maddox, Jessica
Racz, Mark
Petronic-Rosic, Vesna
Pub Date: 07/01/2009
Publication: Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2009 College of American Pathologists ISSN: 1543-2165
Issue: Date: July, 2009 Source Volume: 133 Source Issue: 7
Topic: Event Code: 200 Management dynamics
Accession Number: 230152037
Full Text: The 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-eosin-stained 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 specifically 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 financially 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 hematopoetic 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-eosin--stained 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 specific 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 specific for Merkel cell carcinoma and are positive in other varieties of neuroendocrine carcinoma that may metastasize to the skin. CD117 examination (2) 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. Neurofibromas 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.

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 dermatofibromas (DFs) and dermatofibrosarcoma protuberans (DFSP), which can occasionally be difficult. 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 confirming these findings. (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 fibroxanthoma, with most showing a moderate to strong staining pattern. (5,6) In contrast, pleomorphic malignant fibrous 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 in malignant melanoma (see section on melanocytic lesions). (5,6)

CD99 expression also was seen in a larger series evaluating DFSP and giant cell angiofibroblastoma; 23 of 29 DFSPs (79%) and 2 of 5 GCPs (40%) expressed CD99. CD99 also stains nuchal-type fibromas, which have been seen in contiguity with DFSP. (8)

Solitary fibrous 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 efficient; (2) unless the specific cause of the infection is known or suspected it would be difficult to choose which stains to apply in given cases; and (3) chemical staining is usually sufficient for diagnosis in most cases. Table 3 lists several immunostains specific 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 stains. Unfortunately, significant 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 superficial 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.

[FIGURE 1 OMITTED]

MELANOCYTIC PROLIFERATIONS

Immunohistochemistry is a critical step for the identification 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 maximum during mitosis. (13) Immunohistochemical studies performed with anti--phospho-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 apoptosis (14); 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 specific 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-specific immunohistochemical markers may be a useful method to avoid this pitfall.

A recent publication by Nasr and El-Zammar (15) 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 superficial dermis, and was positive in all of the malignant melanoma, with mitotic figures appearing both superficially 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 specificity for melanoma, more recent data found the specificity to be much lower in the spindle cell variant. (22-24) An example of microphthalmia transcription factor immunostaining is illustrated in Figures 3 and 4.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

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. (25-28) 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 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 sections (34,35) 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 fixation-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 because it is used to help determine the intralymphatic presence of primary or metastatic cancers. A recent study of melanoma by Firouzeh et al (37) identified 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 development (38) 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 specific 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 DNA-binding 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 specificity (100%) of Fli-1 reported by Folpe et al (41) 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 specificity than CD31, CD34, and factor VIII antigen. (40-42) In addition, it is more sensitive and specific 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)

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. (48-50) Within the skin, CD10 immunoreactivity was significant 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 dermatofibroma, to a lesser extent in dermatofibrosarcoma 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 findings suggest that CD10 may be a useful marker for specialized mesenchymal cells and tumors of the skin. Specifically, 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, [DELTA]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 several arenas, including cervical and anal pathology. (61,62) p63 is notably absent in dermal fibroblasts, 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 fibroxanthoma, 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 specificity (97.2%) in identifying primary cutaneous adnexal carcinomas from metastatic adenocarcinomas to the skin. (67)

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

[FIGURE 7 OMITTED]

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 stratified 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 infiltration 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.

340E12

High--molecular 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[beta]E12 to identify an atypical cutaneous squamous cell carcinoma originally misdiagnosed as an atypical fibroxanthoma. (70)

[FIGURE 8 OMITTED]

CK 5/6

Antibodies to CK 5/6 recognize a high--molecular weight intermediate filament 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 identified 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 field 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 define malignancy risk stratification 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 molecular 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 field are immense and the future very promising.

References

(1.) Chan J, Suster S, Wenig B, et al. Cytokeratin 20 immunoreactivity distinguishes merkel cell (primary cutaneous neuroendocrine) carcinomas and salivary gland small cell carcinomas from small cell carcinomas of various sites. Am J Surg Pathol. 1997;21(2):226-234.

(2.) Bobos M, Hytiroglou P, Kostopoulos I, Karkavelas G, Papadimitriou CS. Immunohistochemical distinction between merkel cell carcinoma and small cell carcinoma of the lung. Am J Dermatopathol. 2006;28(2):99-104.

(3.) Jokinen CH, Dadras SS, Goldblum JR, de Rijn M, West RB, Rubin BP. Diagnostic implications of podoplanin expression in peripheral nerve sheath neoplasms. Am J Clin Pathol. 2008;129(6):886-893.

(4.) Bandarchi B, Hafezi S, SiadatF, RastyG. D2-40anovel immunohistochemical stain as a complementory or possible replacement to factorXIIIa and CD34 immunoperoxidase stains in differentiating dermatofibroma and dermatofibrosarcoma protuberance. Mod Pathol. Jan 2008;21(suppl 1):90A.

(5.) Monteagudo C, Calduch L, Navarro S, Joan-Figueroa A, Llombart-Bosch A. CD99 immunoreactivity in atypical fibroxanthoma: a common feature of diagnostic value. Am J Clin Pathol. 2002;117(1):126-131.

(6.) Mirza B, Weedon D. Atypical fibroxanthoma: a clinicopathological study of 89 cases. Australas J Dermatol. 2005;46(4):235-238.

(7.) Hartel PH, Jackson J, Ducatman BS, Zhang P. CD99 immunoreactivity in atypical fibroxanthoma and pleomorphic malignant fibrous histiocytoma: a useful diagnostic marker. J Cutan Pathol. 2006;33(suppl 2):24-28.

(8.) Diwan AH, Skelton HG 3rd, Horenstein MG, et al. Dermatofibrosarcoma protuberans and giant cell fibroblastoma exhibit CD99 positivity. J Cutan Pathol. 2008;35(7):647-650.

(9.) Erdag G, Qureshi HS, Patterson JW, Wick MR. Solitary fibrous tumors of the skin: a clinicopathologic study of 10 cases and review of the literature. J Cutan Pathol. 2007;34(11):844-850.

(10.) Engelkens HJ, ten Kate FJ, Judanarso J, etal. Thelocalisation of treponemes and characterisation of the inflammatory infiltrate in skin biopsies from patients with primary or secondary syphilis, or early infectious yaws. Genitourin Med. 1993;69(2):102-107.

(11.) Buffet M, Grange PA, Gerhardt P, et al. Diagnosing treponema pallidum in secondary syphilis by PCR and immunohistochemistry. J Invest Dermatol. 2007;127(10):2345-2350.

(12.) Hoang MP, High WA, Molberg KH. Secondary syphilis: a histologic and immunohistochemical evaluation. J Cutan Pathol. 2004;31(9):595-599.

(13.) Juan G, Traganos F, James WM, et al. Histone H3 phosphorylation and expression of cyclins A and B1 measured in individual cells during their progression through G2 and mitosis. Cytometry. 1998;32(2):71-77.

(14.) Hendzel MJ, Nishioka WK, Raymond Y, Allis CD, Bazett-Jones DP, Th'ng JP. Chromatin condensation is not associated with apoptosis.

J Biol Chem. 1998; 273(38):24470-24478.

(15.) Nasr MR, El-Zammar O. Comparison of pHH3, ki-67, and survivin immunoreactivity in benign and malignant melanocytic lesions. Am J Dermatopathol. 2008;30(2):117-122.

(16.) Wilkerson AE, Glasgow MA, Hiatt KM. Immunoreactivity of CD99 in invasive malignant melanoma. J Cutan Pathol. 2006;33(10):663-666.

(17.) King MS, Porchia SJ, Hiatt KM. Differentiating spitzoid melanomas from spitz nevi through CD99 expression. J Cutan Pathol. 2007;34(7):576-580.

(18.) Dabbs DJ, ed. Diagnostic Immunohistochemistry. 2nd ed. Philadelphia, PA: Churchill Livingstone; 2006.

(19.) Kligora CJ, Fair KP, Clem MS, Patterson JW. A comparison of melanin bleaching and azure blue counterstaining in the immunohistochemical diagnosis of malignant melanoma. Mod Pathol. 1999;12(12):1143-1147.

(20.) Kamino H, Tam ST. Immunoperoxidase technique modified by counterstain with azure B as a diagnostic aid in evaluating heavily pigmented melanocytic neoplasms. J Cutan Pathol. 1991;18(6):436-439.

(21.) Miettinen M, Fernandez M, Franssila K, Gatalica Z, Lasota J, Sarlomo-Rikala M. Microphthalmia transcription factor in the immunohistochemical diagnosis of metastatic melanoma: comparison with four other melanoma markers. Am J Surg Pathol. 2001;25(2):205-211.

(22.) Granter SR, Weilbaecher KN, Quigley C, Fisher DE. Role for microphthalmia transcription factor in the diagnosis of metastatic malignant melanoma. Appl Immunohistochem Mol Morphol. 2002;10(1):47-51.

(23.) Busam KJ, Iversen K, Coplan KC, Jungbluth AA. Analysis of microphthalmia transcription factor expression in normal tissues and tumors, and comparison of its expression with S-100 protein, gp100, and tyrosinase in desmoplastic malignant melanoma. Am J Surg Pathol. 2001;25(2):197-204.

(24.) 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.

(25.) Grossman A, Mittrucker HW, Nicholl J, et al. Cloning of human lymphocyte-specific interferon regulatory factor (hLSIRF/hIRF4) and mapping of the gene to 6p23-p25. Genomics. 1996;37(2):229-233.

(26.) Yamagata T, Nishida J, Tanaka S, et al. A novel interferon regulatory factor family transcription factor, ICSAT/Pip/LSIRF, that negatively regulates the activity of interferon-regulated genes. Mol Cell Biol. 1996;16(4):1283-1294.

(27.) Matsuyama T, Grossman A, Mittrucker HW, et al. Molecular cloning of LSIRF, a lymphoid-specific member of the interferon regulatory factor family that binds the interferon-stimulated response element(ISRE). Nucleic Acids Res. 1995; 23(12):2127-2136.

(28.) Eisenbeis CF, Singh H, Storb U. Pip, a novel IRF family member, is a lymphoid-specific, PU.1-dependent transcriptional activator. Genes Dev. 1995; 9(11):1377-1387.

(29.) Falini B, Fizzotti M, Pucciarini A, et al. A monoclonal antibody(MUM1p) detects expression of the MUM1/IRF4 protein in a subset of germinal center B cells, plasma cells, and activated T cells. Blood. 2000;95(6):2084-2092.

(30.) Tsuboi K, Iida S, Inagaki H, et al. MUM1/IRF4 expression as a frequent event in mature lymphoid malignancies. Leukemia. 2000;14(3):449-456.

(31.) Natkunam Y, Warnke RA, Montgomery K, Falini B, van De Rijn M. Analysis of MUM1/IRF4 protein expression using tissue microarrays and immunohisto chemistry. Mod Pathol. 2001;14(7):686-694.

(32.) Sundram U, Harvell JD, Rouse RV, Natkunam Y. Expression of the B-cell proliferation marker MUM1 by melanocytic lesions and comparison with S100, gp100 (HMB45), and MelanA. Mod Pathol. 2003;16(8):802-810.

(33.) Ohsie SJ, Sarantopoulos GP, Cochran AJ, Binder SW. Immunohistochemical characteristics of melanoma. J Cutan Pathol. 2008;35(5):433-444.

(34.) Albertini JG, Elston DM, Libow LF, Smith SB, Farley MF. Mohs micrographic surgery for melanoma: a case series, a comparative study of immunostains, an informative case report, and a unique mapping technique. Dermatol Surg. 2002; 28(8):656-665.

(35.) Zalla MJ, Lim KK, Dicaudo DJ, Gagnot MM. Mohs micrographic excision of melanoma using immunostains. Dermatol Surg. 2000;26(8):771-784.

(36.) Fukunaga M. Expression of D2-40 in lymphatic endothelium of normal tissues and in vascular tumours. Histopathology. 2005;46(4):396-402.

(37.) Niakosari F, Kahn HJ, McCready D, etal. Lymphatic invasion identified by monoclonal antibody D2-40, younger age, and ulceration: predictors of sentinel lymph node involvement in primary cutaneous melanoma. Arch Dermatol. 2008; 144(4):462-467.

(38.) North PE, Waner M, Mizeracki A, Mihm MC Jr. GLUT1: a newly discovered immunohistochemical marker for juvenile hemangiomas. Hum Pathol. 2000; 31(1):11-22.

(39.) North PE, Waner M, Mizeracki A, et al. A unique microvascular phenotype shared by juvenile hemangiomas and human placenta. Arch Dermatol. 2001; 137(5):559-570.

(40.) Folpe AL, Hill CE, Parham DM, O'Shea PA, Weiss SW. Immunohistochemical detection of FLI-1 protein expression: a study of 132 round cell tumors with emphasis on CD99-positive mimics of ewing's sarcoma/primitive neuroectodermal tumor. Am J Surg Pathol. 2000;24(12):1657-1662.

(41.) Folpe AL, Chand EM, Goldblum JR, Weiss SW. Expression of fli-1, a nuclear transcription factor, distinguishes vascular neoplasms from potential mimics. Am J Surg Pathol. 2001;25(8):1061-1066.

(42.) Rossi S, Orvieto E, Furlanetto A, Laurino L, NinfoV, Dei Tos AP. Utility of the immunohistochemical detection of FLI-1 expression in round cell and vascular neoplasm using a monoclonal antibody. ModPathol. 2004;17(5):547-552.

(43.) Folpe AL, Veikkola T, Valtola R, Weiss SW. Vascular endothelial growth factor receptor-3 (VEGFR-3): a marker of vascular tumors with presumed lymphatic differentiation, including kaposi's sarcoma, kaposiform and dabska-type hemangioendotheliomas, and a subset of angiosarcomas. Mod Pathol. 2000; 13(2):180-185.

(44.) Breiteneder-Geleff S, Soleiman A, Kowalski H, et al. Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium. Am J Pathol. 1999;154(2): 385-394.

(45.) Miettinen M, Sarlomo-Rikala M, Lasota J. KIT expression in angiosarcomas and fetal endothelial cells: Lack of mutations of exon 11 and exon 17 of C-kit. Mod Pathol. 2000;13(5):536-541.

(46.) Pesando JM, Ritz J, Lazarus H, Costello SB, Sallan S, Schlossman SF. Leukemia-associated antigens in ALL. Blood. 1979;54(6):1240-1248.

(47.) Chu PG, Chang KL, Arber DA, Weiss LM. Immunophenotyping of hematopoietic neoplasms. Semin Diagn Pathol. 2000;17(3):236-256.

(48.) Fais S, Pallone F. Ability of human colonic epithelium to express the 4F2 antigen, the common acute lymphoblastic leukemia antigen, and the transferrin receptor. studies in inflammatory bowel disease and after in vitro exposure to different stimuli. Gastroenterology. 1989;97(6):1435-1441.

(49.) Loke SL, Leung CY, Chiu KY, Yau WL, Cheung KN, Ma L. Localisation of CD10 to biliary canaliculi by immunoelectron microscopical examination. J Clin Pathol. 1990;43(8):654-656.

(50.) Schulz WW, Hagler HK, Buja LM, Erdos EG. Ultrastructural localization of angiotensin I-converting enzyme (EC 3.4.15.1) and neutral metalloendopeptidase (EC 3.4.24.11) in theproximal tubuleofthehuman kidney. Lab Invest. 1988; 59(6):789-797.

(51.) Bahrami S, Malone JC, Lear S, Martin AW. CD10 expression in cutaneous adnexal neoplasms and a potential role for differentiating cutaneous metastatic renal cell carcinoma. Arch Pathol Lab Med. 2006;130(9):1315-1319.

(52.) Perna AG, Smith MJ, Krishnan B, Reed JA. CD10 is expressed in cutaneous clear cell lesions of different histogenesis. J Cutan Pathol. 2005;32(5):348-351.

(53.) Kanitakis J, Bourchany D, Claudy A. Expression of the CD10 antigen (neutral endopeptidase) by mesenchymal tumors of the skin. Anticancer Res. 2000; 20(5B):3539-3544.

(54.) Carrel S, Zografos L, Schreyer M, Rimoldi D. Expression of CALLA/CD10 on human melanoma cells. Melanoma Res. 1993;3(5):319-323.

(55.) Lee KJ, Choi YL, Kim WS, et al. CD10 is expressed in dermal sheath cells of the hair follicles in human scalp. Br J Dermatol. 2006;155(4):858-860.

(56.) Jahoda CA, Reynolds AJ. Hair follicle dermal sheath cells: unsung participants in wound healing. Lancet. 2001;358(9291):1445-1448.

(57.) Pham TT, Selim MA, Burchette JL Jr, Madden J, Turner J, Herman C. CD10 expression in trichoepithelioma and basal cell carcinoma. J Cutan Pathol. 2006; 33(2):123-128.

(58.) Yang A, Kaghad M, Caput D, McKeon F. On the shoulders of giants: p63, p73 and the rise of p53. Trends Genet. 2002;18(2):90-95.

(59.) Di Como CJ, Urist MJ, Babayan I, et al. P63 expression profiles in human normal and tumor tissues. Clin Cancer Res. 2002;8(2):494-501.

(60.) Sniezek JC, Matheny KE, Westfall MD, Pietenpol JA. Dominant negative p63 isoform expression in head and neck squamous cell carcinoma. Laryngoscope. 2004;114(12):2063-2072.

(61.) Wang TY, Chen BF, Yang YC, et al. Histologic and immunophenotypic classification of cervical carcinomas by expression of the p53 homologue p63: a study of 250 cases. Hum Pathol. 2001;32(5):479-486.

(62.) Owens SR, Greenson JK. Immunohistochemical staining for p63 is useful in the diagnosis of anal squamous cell carcinomas. Am J Surg Pathol. 2007;31(2): 285-290.

(63.) Takeuchi Y, Tamura A, Kamiya M, Suzuki K, Ishikawa O. Immunohistochemical analyses ofp63 expression in normal human skin. Br J Dermatol. 2004; 151(1):232-235.

(64.) Ko CJ, McNiff JM, Glusac EJ. Squamous cell carcinomas with single cell infiltration: a potential diagnostic pitfall and the utility of MNF116 and p63. J Cutan Pathol. 2008;35(4):353-357.

(65.) Dotto JE, Glusac EJ. P63 is a useful marker for cutaneous spindle cell squamous cell carcinoma. J Cutan Pathol. 2006;33(6):413-417.

(66.) Ivan D, Nash J, Prieto V, et al. Use of p63 expression in distinguishing primary and metastatic cutaneous adnexal neoplasms from adenocarcinoma to the skin. J Cutan Pathol. 2007;34(6):474-480.

(67.) Liang H, Wu H, Giorgadze T, et al. Podoplanin is a highly sensitive and specific marker to distinguish primary skin adnexal carcinoma from adenocarcinomas metastatic to skin. Am J Surg Pathol. 2007;31(2):304-310.

(68.) Prieto VG, Lugo J, McNutt NS. Intermediate- and low-molecular-weight keratin detection with the monoclonal antibody MNF116: an immunohistochemical study on 232 paraffin-embedded cutaneous lesions. J Cutan Pathol. 1996; 23(3):234-241.

(69.) Chu PG, Weiss LM. Keratin expression in human tissues and neoplasms. Histopathology. 2002;40(5):403-439.

(70.) Gray Y, Robidoux HJ, Farrell DS, Robinson-Bostom L. Squamous cell carcinoma detected by high-molecular-weight cytokeratin immunostaining mimicking-atypical fibroxanthoma. Arch Pathol Lab Med. 2001;125(6):799-802.

(71.) Plumb S, Argenyi Z, Stone M, DeYoung B. Cytokeratin 5/6 immunostaining in cutaneous adnexal neoplasms and metastatic adenocarcinoma. Am J Dermatopathol. 2004;26(6):447-451.

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

Accepted for publication December 11, 2008.

From the Section of Dermatology, University of Chicago, Chicago, Illinois.

The authors have no relevant financial 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).
Table 1. Immunostains in Neural and Neuroendocrine Neoplasms

Stain             Pattern                Application

S100              Nuclear/cytoplasmic    Gliomas, primitive
                  staining               neuroectodermal
                                         tumors, schwannoma,
                                         neurofibroma, and
                                         neuronal and
                                         chondroid tumors

Neuron-specific   Cytoplasmic staining   Neuroendocrine
enolase                                  cells, Merkel cell
                                         carcinoma

Cytokeratin 20    Perinuclear dot        Most sensitive stain
                  staining               for Merkel cell
                                         carcinoma

Neurofilament     Perinuclear dot        Neuroendocrine
                  staining               cells, Merkel cell
                                         carcinoma

Chromogranin      Cytoplasmic staining   Neuroendocrine
                                         cells, Merkel cell
                                         carcinoma

Synaptophysin     Cytoplasmic staining   Neuroendocrine
                                         cells, Merkel cell
                                         carcinoma

Thyroid           Nuclear staining       Negative in Merkel
transcription                            cell carcinoma
factor (1)

Table 2. Immunostains in Soft Tissue Neoplasms

Stain       Pattern                Application

CD34        Cytoplasmic staining   Dermatofibrosarcoma
                                   protuberans, blood
                                   vessels, malignant
                                   fibrous histio-cytoma,
                                   epithelioid sarcoma

Factor      Cytoplasmic staining   Dermatofibroma,
XIII (a)                           juvenile
                                   xanthogranuloma,
                                   multicentric
                                   reticulohistiocy-
                                   tosis, radiation
                                   dermatitis

Smooth      Cytoplasmic staining   Glomus tumor,
muscle                             leiomyoma,
actin                              dermatomyofibroma,
                                   leiomyosarcoma

Desmin      Cytoplasmic staining   Found in striated
                                   and smooth muscle

Vimentin    Cytoplasmic staining   Retained in
                                   mesenchymally
                                   derived cells,
                                   fibroblasts,
                                   endothelial cells,
                                   macrophages

Muscle-     Cytoplasmic staining   Found in striated
specific                           and smooth muscle
actin

Table 3.   Immunostains in Infectious Diseases

Stain                  Pattern                Application

Cytomegalovirus        Nuclear/cytoplasmic    Cytomegalovirus
                       staining               infection

Herpes simplex virus   Nuclear/cytoplasmic    Herpes simplex
                       staining               virus 1 or 2
                                              infection

Varicella zoster       Nuclear/cytoplasmic    Varicella
virus                  staining               zoster virus
                                              infection

Epstein-Barr virus     Cytoplasmic staining   Posttransplantation
                                              lymphoproliferative
                                              disorders,
                                              Hodgkin
                                              lymphoma

Human herpesvirus 8    Nuclear/cytoplasmic    Kaposi sarcoma,
                       staining               primary
                                              effusion
                                              lymphoma,
                                              Castleman
                                              disease

Table 4. Immunostains in Melanocytic Neoplasms

Stain        Pattern                Application

S100         Nuclear/cytoplasmic    Most sensitive
             staining               marker for melanoma
                                    and spindle
                                    cell/desmoplastic
                                    melanoma

HMB-45       Cytoplasmic staining   Higher specificity
                                    for melanoma
                                    (primary-metastatic)
                                    can help distinguish
                                    nevi from melanoma

Melan-A/     Cytoplasmic staining   Similar sensitivity
Mart-1                              and specificity to
                                    HMB-45, but with
                                    more diffuse and in-
                                    tense staining

Tyrosinase   Fine granular          Higher sensitivity
             cytoplasmic staining   than HMB-45 and
                                    specificity of
                                    97%-100%.
                                    Sensitivity
                                    decreases with
                                    increased clinical
                                    stage and in
                                    metastases

Ki-67        Nuclear staining       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

Table 5. Immunostains in Vascular Proliferations

Stain          Pattern                Application

CD31           Cytoplasmic membrane   Specific marker of
               staining               endothelial cells;
                                      poor sensitivity
                                      (66%)

CD34           Cytoplasmic membrane   High sensitivity for
               staining               endothelial cells;
                                      poor specificity

Factor         Cytoplasmic            Less sensitive than
VIII-related   staining,              CD31 for endothelial
Ag             ultrastructurally      cells; infrequently
               Weibel-Palade bodies   used

Ulex           Cytoplasmic staining   Less sensitive than
Europaeus I    correspond to          CD31 for endothelial
agglutinin                            cells; infrequently
                                      used

Table 6. Immunostains in Epidermal and
Appendegeal Neoplasms

Stain              Pattern        Application

Carcinoembryonic   Cytoplasmic    Marker of glandular
antigen            staining       differentiation; can
                                  help distinguish
                                  extra-mammary Paget
                                  disease

Gross cystic       Cytoplasmic    Marker of apocrine
disease fluid      staining       differentiation
protein 15

Epithelial         "Bubbly"       Stains malignant
membrane           cytoplasmic    eccrine and apocrine
antigen            staining       often, and most
                                  sebaceous glands

Cytokeratin 7      Cytoplasmic    Sensitive and
                   staining       specific for Paget
                                  and extramammary
                                  Paget dis-

CAM 5.2            Cytoplasmic    Low-molecular weight
                   staining       keratins present in
                                  glandular
                                  neo-plasms

AE1/AE3            Cytoplasmic    Mixture of low-and
                   staining       high-molecular
                                  weight cytokeratins;
                                  useful in squamous
                                  cell carcinoma

Ber-EP4            Cytoplasmic    Positive in basal
                   staining       cell carcinoma and
                                  negative in squamous
                                  cell carcinoma;
                                  stains sebaceous
                                  neoplasms

BCL-2              Cytoplasmic    Stains the basal
                   staining       layer of the
                                  epidermis; can be
                                  helpful in
                                  distinguishing basal
                                  cell carcinoma from
                                  trichoepithelioma
Gale Copyright: Copyright 2009 Gale, Cengage Learning. All rights reserved.