ihc relevant to dermatopathology

Arch Pathol Lab Med—Vol 133, July 2009 Immunohistochemical Methods in Dermatopathology—Wasserman et al 1053

Update on Immunohistochemical MethodsRelevant to Dermatopathology

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

● Context.—Dermatopathology covers a large variety ofentities, some having very similar histologic appearances.Immunohistochemistry is an incredibly helpful tool that isuseful in diagnosis as well as prognosis of selected skintumors.

Objective.—To provide a comprehensive review of re-cent trends and immunohistochemical stains used by der-matopathologists. Emphasis is placed on new stains as wellas novel uses of existing stains.

Data Sources.—All data were gathered from publishedjournal articles available through the National Center forBiotechnology Information PubMed database.

Conclusions.—New immunohistochemical targets arecontinually being found, contributing to more accurate di-agnosis and classification of skin tumors. The presence ofspecific markers can be used to determine the aggressive-ness of malignanicies and design treatment strategies. Inaddition, application of existing stains can help determineintravascular spread of malignancy in primary cutaneouslesions. And use of rapid immunohistochemical stainingtechniques on frozen sections can assist in more completeexcision of tumor margins. Immunohistochemistry is ahighly versatile and growing tool of dermatopathologists.

(Arch Pathol Lab Med. 2009;133:1053–1061)

The skin houses cells of all types and origins and givesrise to an array of diseases and neoplasms, some orig-

inating primarily in the skin and some appearing second-ary to metastases from other organ systems. Most can bediagnosed from standard hematoxylin-eosin–stained sec-tions with reasonable certainty. There are always, though,poorly differentiated neoplasms, or poor specimens thatcan make a standard diagnosis more challenging.

The addition of widespread immunohistochemicalstains in recent years has given the pathologist in generaland the dermatopathologist specifically many new weap-ons in their diagnostic armamentarium. Immunohisto-chemistry, however, is only a tool to be used appropriatelyin the context of clinical and histologic information; un-reasonable use can be misleading and financially cost in-effective.

The goal of this review is to provide pathologists witha brief update on commonly used immunohistochemicalstains in dermatopathology and an in-depth look at someof the newer stains or new applications of older stains thathave appeared in the last several years. Immunohisto-chemistry is constantly evolving and expanding, and it isimportant to understand that this article is not a compre-hensive guide to immunohistochemistry in dermatopa-thology. For example, an important area of dermatopa-thology not covered here is immunohistochemistry for he-

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: [email protected]).

matopoetic neoplasms in the skin, which would need tobe covered separately to receive adequate attention.

NEURAL AND NEUROENDOCRINE NEOPLASMS

Most neural tumors of the skin can be diagnosed onroutine hematoxylin-eosin–stained sections. Immunohis-tochemistry is most often needed to distinguish Merkelcell carcinoma from other small blue-celled tumors, suchas lymphoma or small cell cancer of the lung. Standardimmunohistochemical stains that have been used for yearsare listed in Table 1, and thus far have been the mosteffective. CK20 is the most specific stain for Merkel cellcarcinoma, and when used in combination with a negativethyroid transcription factor 1 it provides strong supportfor the diagnosis, especially if all tumor cells stain posi-tive. CK20 positivity has also been reported in a verysmall percentage (approximately 2.4%) of small cell car-cinomas of the salivary gland, and rarely in other smallcell carcinomas.1 Other useful adjuncts are listed in Table1; however, they are less sensitive and less specific forMerkel cell carcinoma and are positive in other varietiesof neuroendocrine carcinoma that may metastasize to theskin. CD117 examination2 has not proven to be superiorto existing stains. D2-40 was recently evaluated in periph-eral nerve sheath neoplasms and showed strong, diffuse,cytoplasmic staining in most schwannomas (76%) andmembranous staining in epithelioid malignant peripheralnerve sheath tumors. Neurofibromas and spindle cell ma-lignant peripheral nerve sheath tumors were negative inmost cases.3

SOFT TISSUE NEOPLASMS

Standard stains used in the diagnosis of soft tissue neo-plasms are listed in Table 2. Recent advances are dis-cussed below.

1054 Arch Pathol Lab Med—Vol 133, July 2009 Immunohistochemical Methods in Dermatopathology—Wasserman et al

Table 1. Immunostains in Neural and Neuroendocrine Neoplasms

Stain Pattern Application

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

Neuron-specific enolase Cytoplasmic staining Neuroendocrine cells, Merkel cell carcinomaCytokeratin 20 Perinuclear dot staining Most sensitive stain for Merkel cell carcinomaNeurofilament Perinuclear dot staining Neuroendocrine cells, Merkel cell carcinomaChromogranin Cytoplasmic staining Neuroendocrine cells, Merkel cell carcinomaSynaptophysin Cytoplasmic staining Neuroendocrine cells, Merkel cell carcinomaThyroid transcription factor 1 Nuclear staining Negative in Merkel cell carcinoma

Table 2. Immunostains in Soft Tissue Neoplasms


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

Factor XIIIa Cytoplasmic staining Dermatofibroma, juvenile xanthogranuloma, multicentric reticulohistiocy-tosis, radiation dermatitis

Smooth muscle actin Cytoplasmic staining Glomus tumor, leiomyoma, dermatomyofibroma, leiomyosarcomaDesmin Cytoplasmic staining Found in striated and smooth muscleVimentin Cytoplasmic staining Retained in mesenchymally derived cells, fibroblasts, endothelial cells,

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

Table 3. Immunostains in Infectious Diseases


Cytomegalovirus Nuclear/cytoplasmic staining Cytomegalovirus infectionHerpes simplex virus Nuclear/cytoplasmic staining Herpes simplex virus 1 or 2 infectionVaricella zoster virus Nuclear/cytoplasmic staining Varicella zoster virus infectionEpstein-Barr virus Cytoplasmic staining Posttransplantation lymphoproliferative disorders, Hodgkin lymphomaHuman herpesvirus 8 Nuclear/cytoplasmic staining Kaposi sarcoma, primary effusion lymphoma, Castleman disease

D2-40

Most studies and the most useful applications of thisimmunohistochemical stain are discussed in the section onvascular diseases; here, we will talk about a potentiallyuseful application of this stain in soft tissue neoplasms.D2-40 may represent a helpful adjunct in distinguishingbetween dermatofibromas (DFs) and dermatofibrosarcomaprotuberans (DFSP), which can occasionally be difficult.Strong and diffuse staining with D2-40 was seen in all 20DFs analyzed (100%), whereas only 4 of 24 DFSPs (16%)displayed patchy weak reactivity. Further studies will behelpful in confirming these findings.4

CD99

Monoclonal antibodies that react against CD99, theproduct of the MIC2 gene, are useful, most notably in Ew-ing sarcoma and peripheral neuroectodermal tumor. Re-activity also is present in gastrointestinal stromal tumors,ovarian sex cord tumors, and hematopoetic malignancies,like lymphoblastic lymphoma/leukemia. CD99 has amembranous 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 pat-tern.5,6 In contrast, pleomorphic malignant fibrous histio-cytoma displayed moderate to strong reactivity withCD99 in 35% of cases, and only 15% stained diffusely.7None of the poorly differentiated squamous cell carcino-mas stained, whereas studies have reported mixed results

in malignant melanoma (see section on melanocytic le-sions).5,6

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

Solitary fibrous tumor, a neoplasm most commonly as-sociated with the pleura, is another uncommon tumorwhere positive staining for anti-CD99 can be present andserve as an adjunct in diagnosis along with CD34 andBCL-2.9

INFECTIOUS DISEASE

Most infectious diseases have antibodies associated withthem that may be used in immunohistochemistry. In der-matopathology, only a small subset of these are useful forseveral reasons: (1) other laboratory methods may be moresensitive and cost efficient; (2) unless the specific cause ofthe infection is known or suspected it would be difficultto choose which stains to apply in given cases; and (3)chemical staining is usually sufficient for diagnosis inmost cases. Table 3 lists several immunostains specific tomicroorganisms that have been useful in dermatopathol-ogy.

Antitreponemal Antibodies

In routine evaluation of suspected syphilis, spirochetesare sought using the Steiner or Warthin-Starry silver


Figure 1. Antitreponemal antibodies highlight spirochetes in a biopsyof a patient with syphilis (original magnification �400).

Table 4. Immunostains in Melanocytic Neoplasms


S100 Nuclear/cytoplasmic staining Most sensitive marker for melanoma and spindle cell/desmoplastic melanomaHMB-45 Cytoplasmic staining Higher specificity for melanoma (primary-metastatic) can help distinguish nevi

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

tense stainingTyrosinase Fine granular cytoplasmic staining Higher sensitivity than HMB-45 and specificity of 97%–100%. Sensitivity de-

creases with increased clinical stage and in metastasesKi-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

stains. Unfortunately, significant background staining andartifact can hamper interpretation, leading to low sensitiv-ities of these techniques, reported to range from 33% to71%.10 Past studies, including 2 more recent ones, haveshown that immunohistochemical staining can providebetter sensitivity for the detection of Treponema pallidum.In one study, a polyclonal antibody to T pallidum was pos-itive in 11 of 12 cases (91%) from patients with secondarysyphilis, whereas none of the controls reacted.11 Hoang etal12 showed staining in 12 of 17 biopsies (71%) with amonoclonal antibody compared with 41% detection usinga silver stain; none of the controls reacted with the mono-clonal antibody. Spirochetes were demonstrated in boththe epidermis and dermis, including around superficialcapillaries, within follicular epithelium, and within arec-tor pili muscles. An example of antitreponemal antibodystaining in a patient diagnosed with syphilis is shown inFigure 1.

MELANOCYTIC PROLIFERATIONSImmunohistochemistry is a critical step for the identi-

fication and staging of malignant melanoma. Many stainsexist and help to a variable degree in establishing both thediagnosis and prognosis (Table 4).

pHH3Histone H3 is a core histone protein that complexes

with other histone proteins in the nucleus and makes upmost of the protein component of chromatin in eukaryoticcells. In mammalian cells, phosphorylation of the Ser-10residue of histone H3 is minimal during interphase and

maximum during mitosis.13 Immunohistochemical studiesperformed with anti–phospho-histone H3 (anti-pHH3)antibody have documented a precise temporal and spatialcorrelation between H3 phosphorylation and mitotic chro-mosome condensation, and they also have shown no phos-phorylation on histone H3 during apoptosis14; therefore,pHH3 serves as a mitotic marker. Currently, pHH3 stain-ing has been used with good results in meningioma grad-ing systems. It is important to note that pHH3 is onlyspecific for cells undergoing mitosis and will not distin-guish between cell types. In neural tissue, this is of min-imal importance because there are very few cell typespresent; however, in the skin it is important to keep inmind that there are many cell types present and that notall mitosis seen may be related to the cell type of interest.Double staining with cell-specific immunohistochemicalmarkers may be a useful method to avoid this pitfall.

A recent publication by Nasr and El-Zammar15 high-lights the usefulness of pHH3 in melanocytic lesions. In astudy of 66 melanocytic lesions, pHH3 did not stain dys-plastic or compound nevi, was positive in 3 of 8 Spitz nevibut localized to the superficial dermis, and was positivein all of the malignant melanoma, with mitotic figuresappearing both superficially and deep in the dermis. Anexample of pHH3 staining in melanoma is shown in Fig-ure 2.

CD99

CD99 has been evaluated recently in the differentiationof Spitz nevi from spitzoid melanoma. Previously, studiesshowed mixed results concerning CD99 expression in mel-anomas. Early on, it was seen only in 10%,4 whereas asecond report found positive staining in 60% of melano-mas,16 and most recently in 56% of spitzoid melanomas.17

Spitz nevi were negative in 55 of 58 cases (95%), with onlyfocal staining in the 3 positive cases, whereas melanomashowed a predominately diffuse staining pattern in bothstudies.17 Future research is necessary to further evaluatethe diagnostic utility of CD99 in melanocytic neoplasms.

Microphthalmia Transcription Factor

Microphthalmia transcription factor is necessary for thedevelopment of melanocytes during embryogenesis.18 It isa nuclear stain and is thus more easily interpreted, whichmay be useful when large amounts of cytoplasmic pig-ment may obscure other immunohistochemical stainingpatterns. Ways to avoid this issue include melanin bleach-ing,19 use of azure B counterstaining, or use of a red chro-mogen.20 Several studies have estimated sensitivity of mi-crophthalmia transcription factor for melanoma to be be-tween 81% and 100%.21 Although these initial investiga-tions showed a high specificity for melanoma, more recent


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

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

data found the specificity to be much lower in the spindlecell variant.22–24 An example of microphthalmia transcrip-tion factor immunostaining is illustrated in Figures 3and 4.

MUM1The multiple myeloma 1/interferon regulatory factor 4

(MUM1/IRF4; also known as LSIRF, Pip, and ICSAT) geneproduct is a member of the interferon regulatory factorfamily of transcription factors.25–28 These factors are knownto play an important role in the regulation of gene ex-pression in response to interferon and other cytokines. Al-though the role of MUM1 in hematolymphoid malignan-cies and immune regulation has been established, expres-sion patterns of the MUM1 protein in other tissues haveonly been examined recently.29,30 One study demonstratedthat the MUM1 protein can be detected in neoplasms ofplasmacytic differentiation as well as in a wide variety ofB-cell, T-cell, and natural killer cell lymphomas, but was

negative in all other malignant neoplasms tested exceptmelanoma.31 MUM1 exhibits a nuclear staining pattern.

In a study by Sundram et al,32 the MUM1 antibodyhighlighted most melanomas, with 92% of conventionalprimary and metastatic melanomas being positive. Thesedata suggest it is a more sensitive marker than eitherHMB-45 or Melan-A in primary and metastatic melano-mas. MUM1 antibody also showed strong and more dif-fuse staining of benign melanocytic nevi than either HMB-45 or Melan-A, staining 75% of benign nevi in this study,compared with 13% and 63%, respectively. MUM1 strong-ly stained 80% (8 of 10) of Spitz nevi.32 Because MUM1expression appears to be present in both nevi and in mel-anomas, and it does not highlight desmoplastic melanomamore effectively than well-established stains do, it is un-clear whether it will have a role in the future and whatthat role would be.

Immunostains that are currently being actively investi-gated for use in melanocytic neoplasms include melano-cortin-1 receptor, SM5-1, PNL2, and newer antibodies totyrosinase.33 In addition, the evolving use of immunostainsin frozen sections34,35 may help decrease recurrence ratesafter surgery for melanoma, where ex tempore interpre-tation is greatly compromised by freeze artifact.

VASCULAR PROLIFERATIONSPreviously used stains for vascular lesions have been

very effective, but still not without their drawbacks (Table5). Recent years have seen the development of several im-munohistochemical stains that have been highly useful inthis category.

D2-40D2-40 is a novel monoclonal antibody against M2A an-

tigen, an Mr 40 000 O-linked sialoglycoprotein that reactsto a fixation-resistant epitope on lymphatic endothelium.36

A cytoplasmic staining pattern of endothelial cells high-lights lymphatic channels. Studies have shown that thisantigen is expressed in lymphatics of normal tissues, aswell as Kaposi sarcoma, lymphangiomas, some angiosar-comas, and Dabska tumor.36 The utility of D2-40 extendsbeyond its obvious application to identify vascular tumors


Table 5. Immunostains in Vascular Proliferations


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

CD34 Cytoplasmic membrane staining High sensitivity for endothelial cells; poor specificityFactor VIII–related Ag Cytoplasmic staining, ultrastructurally correspond

to Weibel-Palade bodiesLess sensitive than CD31 for endothelial cells; infre-

quently usedUlex Europaeus I agglutinin Cytoplasmic staining Less sensitive than CD31 for endothelial cells; infre-

quently used

because it is used to help determine the intralymphaticpresence of primary or metastatic cancers. A recent studyof melanoma by Firouzeh et al37 identified lymphatic in-vasion using D2-40 and found it to correlate with sentinellymph node positivity. They hypothesized that it may behelpful in identifying candidates for sentinel lymph nodebiopsy.37 See Figure 5 for an example of D2-40 staininglymphatic vessels.

GLUT1GLUT1 protein is an erythrocyte glucose transporter re-

stricted to endothelial cells in areas with blood-tissue bar-rier functions, such as the brain, eye, nerve, and placenta.GLUT1 stains the cytoplasmic membrane, and its primaryutility is in the diagnosis of pediatric vascular lesions. Al-though not necessary for diagnosing pediatric vascular le-sions, it has been found in juvenile hemangiomas at allstages of development38 but is not present in vascular mal-formations, granulation tissue, pyogenic granulomas, non-involuting congenital hemangiomas, rapidly involutingcongenital hemangiomas, or other vascular and nonvas-cular tumors.39 This stain is highly specific for juvenilehemangiomas; it has enabled diagnostic accuracy of theseentities and yielded valuable data regarding their patho-genesis.39

Fli-1Fli-1 protein is a member of the ETS family of DNA-

binding transcription factors, characterized by a highlyconserved DNA-binding domain. Originally used in thediagnosis of Ewing sarcoma/primitive neuroectodermaltumors, the only normal tissues it has been found to staininclude small lymphocytes and endothelial cells.40 Fli-1 isa nuclear stain, which makes it more easily interpretablethan other stains typically used as vascular markers, in-cluding CD31, CD34, and factor VIII-related antigen. Itsutility for the diagnosis of vascular tumors and growthshas been studied and showed very encouraging results.The sensitivity (94%) and specificity (100%) of Fli-1 re-ported by Folpe et al41 was equal to or exceeded those ofthe established vascular markers, CD31, CD34, and vonWillebrand factor. Early studies used a polyclonal anti-body and had wider variation in staining and interpreta-tion, but monoclonal antibodies have been developedshowing more consistency.42 Fli-1 has shown comparablesensitivity to currently used vascular markers and higherspecificity than CD31, CD34, and factor VIII antigen.40–42

In addition, it is more sensitive and specific than othernovel vascular markers, such as VEGFR-3,43 podoplanin,44

and CD117.45 Fli-1 shows no variation in staining amongvascular proliferations, and it stains benign tumors equal-ly well as malignant tumors. Therefore, its usefulness islimited to the diagnosis of vascular tumors but cannotdistinguish entities within this group.40

EPIDERMAL AND APPENDAGEAL NEOPLASMS

Both benign and malignant epidermal and appendagealneoplasms are numerous, with often-similar morphologicpresentations that may also mimic other cutaneous neo-plasms, such as melanoma. Table 6 lists commonly usedstains. We describe below several novel stains that haveshown promise in the diagnosis of these entities.

CD10

CD10 antigen is a 100-kDa cell surface zinc metalloen-dopeptidase expressed in a variety of normal and neo-plastic lymphoid and nonlymphoid tissues. It also isknown as common acute lymphocytic leukemia antigen.46

Immunohistochemical staining may be membranous and/or cytoplasmic. It serves as a marker for the common formof acute lymphocytic leukemia as well as for Burkitt lym-phoma and follicular germinal center lymphoma.47 CD10is normally present on the surface of early lymphoid cellsas well as on a number of other normal cell types, suchas bile canaliculi and renal and intestinal epithelialcells.48–50 Within the skin, CD10 immunoreactivity was sig-nificant in sebaceous and xanthomatous neoplasms, butnot in eccrine and apocrine ones.51,52 It also is present inperiadenexal mesenchymal cells of normal dermis and intumors, such as dermatofibroma, to a lesser extent in der-matofibrosarcoma protuberans,53 and in melanoma.54

CD10 is found in dermal sheath cells of hair follicles.55 Thedermal sheath that surrounds the outside of the hair fol-licle contains progenitor cells that maintain and regeneratethe dermal papilla, a key component for hair growth.56

Recent findings suggest that CD10 may be a useful markerfor specialized mesenchymal cells and tumors of the skin.Specifically, it has been shown to assist in differentiationbetween basal cell carcinoma and trichoepithelioma. Onestudy revealed that the expression of CD10 in trichoepi-thelioma was in the stroma and tumor papillae and no-tably lacking in the epithelial component, whereas in basalcell carcinoma, CD10 was expressed peripherally in thebasaloid nests but was absent in the stroma.57

p63

p63 is a nuclear stain that may be a helpful marker inskin pathology because it is expressed in the nuclei of bas-al and spinous cells of the epidermis. p63 is a member ofthe tumor suppressor protein p53 gene family and com-prises at least 6 different protein isoforms with homologyto p53.58 One such isoform, �Np63, is the predominantisoform in mature epidermis and appears to be a masterregulator of squamous stem cell differentiation.59 In ad-dition, expression also has been established in a variety ofneoplasms, including squamous cell carcinoma.60 Immu-nohistochemical stains for p63 have been used to differ-entiate squamous cell carcinoma from adenocarcinoma in


Figure 4. Microphthalmia transcription factor highlights increased numbers of melanocytes located in the basal layer and at higher levels of thestratum spinosum (original magnification �400).

Figure 5. D2-40 highlights lymphatic vessels adjacent to an unstained blood vessel (original magnification �100).

Figure 6. Squamous cell carcinoma with individual tumor cells budding off (lower right corner; hematoxylin-eosin, original magnification �200).

Figure 7. MNF116 stains keratinocytes budding off the larger tumor as single cells and small clusters (original magnification �200).


Table 6. Immunostains in Epidermal and Appendegeal Neoplasms


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

Gross cystic disease fluid protein 15 Cytoplasmic staining Marker of apocrine differentiationEpithelial membrane antigen ‘‘Bubbly’’ cytoplasmic staining Stains malignant eccrine and apocrine often, and most seba-

ceous glandsCytokeratin 7 Cytoplasmic staining Sensitive and specific for Paget and extramammary Paget dis-

easeCAM 5.2 Cytoplasmic staining Low–molecular weight keratins present in glandular neo-

plasmsAE1/AE3 Cytoplasmic staining Mixture of low– and high–molecular weight cytokeratins; use-

ful in squamous cell carcinomaBer-EP4 Cytoplasmic staining Positive in basal cell carcinoma and negative in squamous

cell carcinoma; stains sebaceous neoplasmsBCL-2 Cytoplasmic staining Stains the basal layer of the epidermis; can be helpful in dis-

tinguishing basal cell carcinoma from trichoepithelioma

←

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 magnifications �60 [A] and�200 [B]).

several arenas, including cervical and anal pathology.61,62

p63 is notably absent in dermal fibroblasts, smooth mus-cle, Schwann cells, and endothelial cells,63 lending itselfuseful for diagnosis of atypical squamous cell carcinoma.64

To differentiate spindle cell squamous cell carcinoma fromother spindle cell neoplasms, such as atypical fibroxantho-ma, spindle cell melanoma, leiomyosarcoma, and others,investigators demonstrated that spindle cell squamous cellcarcinoma expressed p63 strongly and diffusely, whereasmost controls were negative or displayed weaker andmore focal staining.65 In addition, most primary adnexalcarcinomas and their metastases express p63, whereas vis-ceral adenocarcinomas and their cutaneous metastases donot make this stain a useful adjunct to distinguish pri-mary cutaneous adnexal carcinomas from metastatic vis-ceral adenocarcinomas.66 Similarly, D2-40 also was foundto have a high sensitivity (94.5%) and specificity (97.2%)in identifying primary cutaneous adnexal carcinomasfrom metastatic adenocarcinomas to the skin.67

MNF116MNF116 recognizes cytoplasmic polypeptides of 45, 46,

and 56.5 kDa, corresponding to cytokeratins 5, 6, 8, 17, and19. It shows a broad pattern of reactivity with human ep-ithelial tissues from simple glandular epithelia to stratifiedsquamous epithelia, like the epidermis, mammary glandducts, and tracheal epithelium. In normal skin, MNF116exhibits particularly strong staining in the basal cells of theepidermis and adnexae. MNF116 is positive in epithelialtumors and negative in all mesenchymal and melanocytictumors.68 It has been useful in identifying unusual variantsof squamous cell carcinoma, such as spindle cell squamouscell carcinoma with single-cell infiltration at the periphery.64

Examples showing the usefulness of MNF116 staining ininvasive squamous cell carninoma compared to hematox-ylin and eosin staining are illustrated in Figures 6through 8.

34�E12High–molecular weight cytokeratin antibody, also

known as CK903, is a cytoplasmic marker. This antibody

recognizes keratin polypeptides of 68, 58, 56.5, and 50kDa, corresponding to cytokeratins 1, 5, 10, and 14, whichare expressed in squamous, ductal, and other complex ep-ithelia, basal cells, and myoepithelial cells.69 It stains ad-enocarcinomas, breast, pancreas, bile duct, and salivarygland, as well as squamous cell and transitional cell car-cinomas. This stain is positive in cutaneous adnexal andepidermal neoplasms. Its use in dermatopathology may bein diagnosing poorly differentiated carcinomas. A recentcase report details the use of 34�E12 to identify an atyp-ical cutaneous squamous cell carcinoma originally misdi-agnosed as an atypical fibroxanthoma.70

CK 5/6Antibodies to CK 5/6 recognize a high–molecular

weight intermediate filament and are useful markers ofsquamous differentiation. This antibody is a cytoplasmicmarker found in a wide variety of cutaneous adnexal neo-plasms, both benign and malignant.71 It also has beenidentified in metastic adenocarcinomas, but to a muchlesser extent. In a study by Plumb et al,71 it was found that97% of cutaneous adnexal neoplasms stained positivewith CK5/6, but only 33% of metastatic adenocarcinomasstained positive, and those stained weakly. This immu-nostain may be helpful in distinguishing primary cuta-neous tumors from metastatic lesions.

CONCLUSIONSImmunohistochemistry is an excellent diagnostic tech-

nique with the distinct advantage of being able to exactlylocate a given protein within the tissue examined. Thefield is continuously expanding, with new applicationssteadily increasing. Melanocytic staining carries the mostpotential, because many immunostains are being explorednot only for diagnostic purposes but for prognostic valueas well. Multipronged immunohistochemistry can help de-fine malignancy risk stratification and therapeutic guide-lines. The evolving use of immunostains in frozen sectionsmay help decrease recurrence rates after surgery for tu-mors such as melanoma, where interpretation is greatlycompromised by freeze artifact. Because a variety of mo-


lecular pathways are altered in cancer, some of the alter-ations can be targeted in cancer therapy. Immunohisto-chemistry can be used to assess which tumors are likelyto respond to treatment by detecting the presence or ele-vated levels of the molecular target. Ultimately, the pos-sibilities of this field are immense and the future verypromising.

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