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Immunohistochemistry in Dermatopathology.

Immunohistochemistry (IHC) cannot replace routine histology and is not required in most dermatopathology cases. However, an understanding of the ever growing number of commercially available antibodies and their limitations can maximize the pathologist's ability to make a definitive diagnosis.

CUTANEOUS ADNEXAL NEOPLASMS

Sebaceous Neoplasms

Differentiation of sebaceous carcinoma and squamous cell carcinoma (SCC) can be challenging. Ansai et al (1) found that androgen receptor (AR) and adipophilin (Figure 1) expression supports the diagnosis of sebaceous carcinoma, rather than SCC.

Sebaceous epitheliomas (sebaceomas) are mostly composed of basaloid cells with a small proportion of mature sebocytes. Thus, there is histologic overlap between the basaloid cells of sebaceomas and those of basal cell carcinoma (BCC). Although IHC is not commonly necessary in differentiation, epithelial membrane antigen (EMA) highlights most mature sebocytes in sebaceoma, whereas its expression is uncommon in BCC. (2,3) Reactivity for adipophilin in sebaceous tumors can also distinguish them from BCC. (1) The utility of Ber-EP4 in this setting is controversial; although BCCs stain positive for Ber-EP4, there is varying literature on its reactivity in sebaceous lesions. (2,4)

Adipophilin is a protein on the surface of intracellular lipid droplets and thus shows expression in sebaceous lesions, particularly well-differentiated lesions. Caution is necessary when using adipophilin to distinguish sebaceous neoplasms from other cutaneous lesions with clear cell histology.

Metastatic renal cell carcinoma and eccrine-apocrine carcinomas have shown expression. (5,6) Xanthomatous lesions, including xanthelasma and xanthogranuloma, reportedly show adipophilin reactivity, but in contrast to the membranous vesicular pattern in sebaceous neoplasms, xanthomatous lesions show a more granular pattern. (5) This granular pattern has also been reported in clear cell BCCs and SCCs potentially causing confusion when differentiating those neoplasms from sebaceous carcinoma; however, a granular pattern and decreased intensity of expression favor the former tumors. (6,7)

The potentially aggressive course of sebaceous carcinoma necessitates accurate differentiation from benign sebaceous neoplasms. Cabral et al (8) sought to find an immunohistochemical method that could aid morphologic differentiation, especially in small, partial biopsies. Sebaceous carcinomas have significantly increased nuclear staining with p53 and Ki-67 (MIB-1) and reduced levels of BCL2 and p21, in comparison to sebaceous adenomas and sebaceous epitheliomas. The p53 protein is a tumor suppressor. Mutations lead to an abnormally stable but inactive p53 protein that can be detected immunohistochemically by nuclear staining. Sebaceous carcinomas are associated with increased, presumably defective, p53. Therefore, decreased functional p53 and p21 tumor suppression may allow for tumor progression and loss of BCL2 suggests improper regulation of apoptosis. Ki-67 is a nuclear marker of cell proliferation.

In some cases, IHC can serve as surrogate protein markers for underlying genetic events. Sebaceous gland neoplasms are relatively rare, and that diagnosis should raise the possibility of Muir-Torre syndrome (MTS). The dermatopathologist may be the first to consider the possibility of this syndrome because, in some patients, only a single sebaceous tumor is the first indication. Muir-Torre syndrome is an autosomal-dominant disease associated with multiple keratoacanthomas and visceral malignancies, especially genitourinary and gastrointestinal. Less common associations include tumors of the breast and lung. Muir-Torre syndrome is most frequently caused by mutational inactivation of mismatch repair genes MSH2 and MLH1 with resultant microsatellite instability. Other mismatch repair proteins can be involved, such as MSH6 and PMS2.

Molecular techniques to identify microsatellite instability are time consuming and expensive. Immunohistochemistry has become a rapid and cost-effective screening tool for select patients with a high probability of MTS. The absence of nuclear staining with a mismatch repair protein in a sebaceous tumor suggests MTS (Figures 2, A and B). Mathiak et al (9) identified a positive correlation between IHC and molecular analysis in 93% of sebaceous tumors.

Almost three-quarters of those tumors without MSH6 also demonstrated a loss of MSH2 but a loss of MSH6 alone was identified in a small percentage. (10) This is not unexpected because MSH2 and MSH6 form a heterodimer; thus, a mutation in one could lead to the absence of the other. Loss of both MSH6 and MSH2 has a positive predictive value of 55% for MTS, similar to the positive predictive value for the loss of MSH2 alone (55%-66%). Loss of both MLH1 and MSH6 is 100% predictive. (11)

It is reasonable to check this panel (MSH2, MLH1, MSH6, with or without PMS2) in sebaceous neoplasms (other than sebaceous hyperplasia), especially in patients younger than 50 years, when multiple lesions are present, neoplasms involve nonfacial sites, or when cystic or keratoacanthomalike architecture is present. (11,12) Although some authors are conservative and recommend screening only in patients with a personal or family history of colorectal cancer, (13) most others (14-15) recommend screening regardless of age or other clinical characteristics. Routine screening with PMS2 may not be necessary because of low prevalence in MTS. Loss of staining in a tumor is not necessarily secondary to a germline mutation and when loss of expression is identified, further testing is warranted, including evaluation of the personal and family history of malignancy. (16) If genetic testing is desirable, IHC can indicate the specific gene most likely to be involved, allowing targeted mutational screening. When expression is intact, MTS is less likely, but it does not completely exclude the syndrome. If there is still clinical suspicion, polymerase chain reaction (PCR) can detect the loss of MSH2, MLH1, and other proteins. In older patients, the importance of IHC evaluation may lie in screening relatives at increased risk of malignancy and because visceral tumor development can have a late onset.

Trichilemmomas

Solitary trichilemmomas are typically sporadic but can be associated with Cowden syndrome, which is an autosomal dominant disorder characterized by multisystem hamartomatous growths and carcinomas. This syndrome is linked to a germline mutation in the tumor suppressor PTEN (phosphatase and tensin homolog), located on band 10q23.3. Complete loss of immunoreactivity with PTEN was reported in 5 of 6 patients (83%) with Cowden syndrome-associated trichilemmomas, but in only 1 of 33 patients (3%) with sporadic lesions. (17) Shon et al (18) reported similar results with 13 Cowden syndrome-associated lesions and 19 sporadic trichilemmomas. This suggests a potential role for immunohistochemical screening similar to its use in MTS.

Partial biopsies of desmoplastic tricholemmoma may require distinction from basal cell carcinoma. CD34 immunodetection in the epithelial cells of the tricholemmoma can help in the differentiation. (19)

Primary Cutaneous Adnexal Neoplasms From Metastatic Adenocarcinoma

Differentiation of metastatic adenocarcinomas from primary cutaneous adnexal neoplasms (PCAN), especially malignant ones, can be difficult. Qureshi et al (20) found that most PCANs with sweat gland differentiation were positive for p63 and high-molecular-weight CK5/6, whereas expression was rare in metastatic adenocarcinomas. Differential p63 reactivity has also been supported in other studies. (21-23) Plumb et al (24) similarly found CK5/6 expression in more than 95% of PCANs, albeit most tumors studied were benign. In general, metastatic adenocarcinomas expressed CK5/6 in only 33% (9 of 27) of the cases, predominantly with weak intensity. However, metastatic breast carcinoma was reactive for CK5/6 in almost half of their cases. (24)

Ivan et al (25) extended the analysis of p63 to include metastases from adnexal carcinomas and found that 91% of PCANs were strongly marked with p63 and, excluding apocrine and mucinous carcinoma, their metastases labeled similarly.

Podoplanin (D2-40) expression is also a useful adjunct in differentiating adenocarcinomas that have metastasized to the skin from PCAN. Reactivity with D2-40 is seen in primary cutaneous carcinomas and skin adnexal tumors but is absent in metastatic adenocarcinomas to the skin. (21,26)

A panel of immunohistochemical stains, as suggested by Mahalingam et al, (22) may provide the best sensitivity and specificity in distinction of metastatic adenocarcinomas and PCANs. Their recommended panel included p63, D2-40, and CK15. Positive staining with all 3 favors a PCAN diagnosis, rather than that of metastatic adenocarcinoma (Table 1).

Pagetoid Tumors

Intraepidermal lesions with a pagetoid distribution can be diagnostically difficult. CK7 positivity reportedly differentiates Paget disease of the breast and extramammary Paget disease (EMPD) from pagetoid Bowen disease and melanoma in situ; however, the marker is not 100% sensitive, and occasional cases of [CK7.sup.+] pagetoid Bowen disease have been described. (27,28) Similarly CAM 5.2 expression favors Paget disease or EMPD, rather than SCC in situ, but reactivity has been reported in both. (28,29) Sellheyer and Krahl (30) proposed the addition of Ber-EP4 to the panel used to evaluate pagetoid cutaneous tumors. In their study, (30) BerEP4 labeled all cases of EMPD but failed to label Bowen disease and melanoma in situ. Other authors (31) have identified strong nuclear p63 in pagetoid SCC in situ, in contrast to no reactivity in EMPD.

Several studies have attempted to use IHC to differentiate primary from secondary EMPD, but the immunoprofiles tend to overlap, including occasional CK20 and prostatespecific antigen expression in primary EMPD. (32,33) Thus far, CDX2 reactivity has only been reported in EMPD secondary to anorectal adenocarcinoma. (33)

Sclerosing Epithelial Neoplasms

Differentiation of sclerosing epithelial neoplasms is not only of academic interest but also paramount to clinical management, and diagnosis is often requested on small, superficial biopsies. Various immunohistochemical markers have been studied to help distinguish desmoplastic trichoepitheliomas (DTE), infiltrating or morpheaform BCCs, and microcystic adnexal carcinomas, including CD23, CD5, CD10, CD34, CK20, CK15, stromelysin-3, BCL2, AR, pleckstrin homology-like domain, family A, member 1 protein (PHLDA1), p75 neurotrophin receptor (p75NTR), fibroblast activation protein (FAP), Ber-EP4, p63, and others (34-48) (Table 2). Much of the available data are based on a few cases reported by a single group of authors and require additional validation. Conflicting results were found with the same marker in other studies. A few deserve further mention, but the histopathologic criteria and clinical data remain the current gold standard.

The panel of AR and CK20 has proven useful in distinguishing DTE and morpheaform BCC. (35,39) [CK20.sup.+] Merkel cells are identified as colonizing DTEs but not BCCs and microcystic adnexal carcinomas. (49) However, the Merkel cells identified in DTE may be few, requiring serial sections, and care is needed to avoid misinterpretation of Merkel cells in preexisting vellus follicles. Nuclear AR expression is focally present in most morpheaform BCCs and is absent in most DTEs. (35,39) Androgen receptor reactivity has also been noted in classic or conventional trichoepitheliomas. (48)

Several studies have attempted to identify immunomarkers to differentiate conventional trichoepithelioma or trichoblastoma from nonaggressive forms of BCC. Although there are supporting data for use of CD34 and BCL2 in this setting, minimal research has been conducted evaluating those markers in the setting of sclerosing epithelial neoplasms. As with conventional trichoepitheliomas, Kirchmann et al (42) found the stroma of DTEs was positive for CD34, differentiating it from the negative staining stroma of morpheaform BCC and microcystic adnexal carcinoma; however, Costache et al (39) failed to identify stromal staining in any of their 19 DTEs. BCL2 tends to show expression only at the periphery of the tumor islands in conventional trichoepitheliomas but is present throughout the tumor island in BCC, yet the small islands of DTE and morpheaform BCC make that distinction nebulous, as seen in the study by Costache et al. (39)

Recent studies have evaluated the utility of p75NTR (CD271) and PHLDA1. Morpheaform BCCs tend to lack expression of p75 in the tumor cells or only show focal or weak expression, whereas DTEs are strongly and diffusely positive. (44,45) p75 is typically expressed in the outer root sheath of follicles; therefore, reactivity in DTEs supports their classification as a follicular hamartoma mimicking that portion of the follicle. Microcystic adnexal carcinomas cannot be reliably distinguished with p75 because nearly one-half are strongly positive. (44) PHLDA1 is a hair-follicle stem-cell marker in the bulge area of the follicle. Reactivity for that marker in more than 50% of tumor cells characterizes DTEs versus the lack or minimal expression seen in infiltrating or morpheaform BCCs. (37,38,40) Colonizing melanocytes and tumor cells in proximity to an ulcer can be positive requiring caution in interpretation. (37)

As noted above with CD34, some studies have concentrated on tumoral stroma when attempting to differentiate these tumors. Fibroblast activation protein is a glycoprotein expressed in the granulation tissue of healing wounds and has recently (47) been identified as a marker of peritumoral stromal fibroblasts in multiple epithelial cancers, including morpheaform/infiltrative BCC, in contrast to the absence of FAP in DTE.

MALIGNANT EPITHELIAL NEOPLASMS

The morphologic overlap of basaloid SCC and BCC with squamous metaplasia may complicate diagnosis, especially in view of the ever-shrinking size of biopsies. (50) Beer et al (51) found that BCCs in their study stained positively with BerEP4 and negatively with EMA. In contrast, SCCs showed no staining with Ber-EP4, but most SCC tumors expressed EMA. (51) However, Merkel cell carcinoma (MCC) and benign follicular and sweat gland tumors, such as trichoepitheliomas and porocarcinomas, also stain positive for Ber-EP4. (2,52) Labeling may be absent from squamoid areas of a BCC, potentially complicating interpretation of small superficial biopsies. (53,54)

CDKN2A (p16) is a tumor suppressor that induces arrest in the G1 phase of the cell cycle. Immunohistochemistry for p16 is a well-established tool in the diagnosis of squamous dysplasia of the cervix. Overexpression of p16 is present in most cervical carcinomas, dysplasias, and human papillomavirus-associated vulvar SCCs. Bandlike staining with p16, especially in the upper portions of the epithelium, correlates with high-grade anal intraepithelial neoplasia. (55) p16 staining is better at differentiating human papillomavirus-related vulvar SCC and nonhuman papillomavirusassociated SCC than morphologic criteria. (56) Mutations of the p16 gene (CDKN2A) have been found in actinic keratoses and SCCs, with increased expression during progression of disease. Bowen disease is generally positive for p16 with full-thickness expression. Harvey et al (57) pointed out that the pattern of reactivity typically spares the palisaded basal cells. Actinic keratoses, on the other hand, have shown variable results with many being negative or, when positive, showing weak to moderate staining, or staining limited to the lower portions of the epidermis. (57,58) Although only a few cases have been studied, irritated seborrheic keratoses tend to have p16 expression similar to actinic keratoses, rather than the full-thickness pattern seen in Bowen disease, possibly aiding distinction of this difficult differential diagnosis. (57) Condyloma reactivity is similarly negative, sporadic (<5% isolated cells), or focal (<25%, small clusters), in comparison to the diffuse, full-thickness positivity seen in bowenoid papulosis. (59)

D2-40, an antibody to podoplanin, is a lymphatic endothelial marker that can enhance detection of lymphatic tumor invasion. However, expression of D2-40 is not limited to lymphatic endothelial cells, and reactivity has been noted within the SCC tumor itself. Interestingly, SCCs showing tumoral D2-40 expression, most often at the periphery of the tumor islands, are associated with locoregional recurrence, lymphatic metastases, and decreased overall survival. (60,61) Podoplanin is involved in actin remodeling and may be associated with tumor invasion by increasing cell motility. (60)

CUTANEOUS SPINDLE CELL TUMORS

The differential diagnosis of atypical spindle cell neoplasms on sun-damaged skin includes atypical fibroxanthoma (AFX), spindle cell or desmoplastic melanoma (see melanocytic neoplasms below), spindle cell SCC, and leiomyosarcoma. Because of potential overlapping reactivity and rare, anomalous expression, a panel of IHC markers should be selected that will support or refute each of those diagnoses.

Spindle Cell SCC

Squamous cell carcinomas, in general, stain with cytokeratin and not with vimentin. However, spindle cell SCCs that may lack an obvious origin in the epidermis or evidence of keratinization are often positive for vimentin and negative or only focally positive with routine cytokeratin stains, including AE1/AE3. Folpe and Cooper (52) found high-molecular-weight cytokeratins, such as CK903 (34PE12) and CK5/6 to be sensitive markers in this context; however, some spindle cell SCCs do not stain with either. (62-65)

p63, a member of the p53 gene family, is a transcription factor involved in the proliferative capacity of epidermal stem cells. It is normally expressed in keratinocytes of the basal and lower spinous layers. Many authors (66) have reported nuclear p63 expression in spindle cell SCC. However, p63 is not entirely specific and shows focal labeling in rare cases of AFX and leiomyosarcoma. (67-69) As part of an IHC panel, p63 can be helpful in the differential diagnosis of spindle cell neoplasms (Table 3). (67)

Atypical Fibroxanthoma

Atypical fibroxanthoma is a pleomorphic, superficial spindle cell tumor of low-grade malignancy that must be distinguished histologically from other atypical spindle cell tumors of the dermis. Although there are immunohistochemical markers for many of those other tumors, the diagnosis of AFX is generally one of exclusion. A variety of markers have been identified with reactivity in AFX, but those markers alone are not specific (Table 3). Procollagen 1 (PC1), CD10, and S100A6 are typically reactive in AFX, but those markers often stain a variety of other neoplasms.

Procollagen is secreted by fibroblasts and is cleaved to form collagen in the extracellular matrix. Expression of PC1 in most AFXs supports classification of this tumor as fibrohistiocytic. However, PC1 staining should seldom be used in isolation. Reactivity with keratin, S100, or desmin should outweigh PC1 positivity because approximately three-fourths of leiomyosarcomas and one-third of desmoplastic melanomas (DMs) and spindle cell SCCs are also PC1 positive. (70-72) As with all IHC markers, careful localization of the antibody staining should be established. [PC1.sup.+] tumor cells must be distinguished from positive background fibroblasts.

S100A6 (calcyclin) is a calcium-binding protein of the S100 family, which has been isolated from melanocytes, Schwann cells, Langerhans cells, and dermal dendrocytes. Aside from staining nevi and some melanomas, numerous fibrohistiocytic lesions stain positive with S100A6. A small study (73) revealed S100A6 in 80% (4 of 5) of the AFX cases. However, other neoplasms in the spindle cell differential have also revealed reactivity with S100A6, including a significant proportion of DMs, leiomyosarcomas, and spindle cell SCCs. (74)

Similarly, CD10 is sensitive but not specific for AFX. In one small study, (75) 94% (15 of 16) of the AFXs showed strong expression of CD10, but on the other hand, reactivity was also identified in one-third (3 of 9) of the DMs and one-half (5 of 10) of the spindle cell SCCs. Reactivity in AFX has ranged from 100% to 78%. (68,69,76-79)

Focal expression of myogenic markers (calponin, SMA, HHF35), indicative of myofibroblastic differentiation, can be seen in up to one-third of AFXs. (62,80) Therefore, use of SMA or calponin alone may be insufficient to diagnosis leiomyosarcoma, and a panel should include another muscle marker, such as desmin or h-caldesmon.

Caution is required in interpreting S100 in AFXs because [S100.sup.+] dendritic cells colonize the lesion (possibly Langerhans cells), (81,82) but the neoplastic cells are generally [S100.sup.-], essentially excluding DM. MART-1, HMB-45, and Melan-A expression in the multinucleate giant cells of AFX can be another pitfall to avoid. (83,84)

Historically other nonspecific markers with lower reactivity used in AFX have included, CD68, (82) antichymotrypsin, antitrypsin, (85) and CD99.69, (86) Studies have shown that CD163 is more specific for histiocytes than is CD68, which is an organelle-specific marker that stains lysosomes. Because CD68 reactivity is seen in some AFXs, it is not surprising that CD163 expression was reported (87); however, further studies were unable to replicate those results. (88,89) Although CD117 expression has been reported in AFXs, the percentage of reactive cells was very low and likely correlates with colonizing mast cells. (81,90)

Caution is required in distinguishing AFX with pseudoangiomatous or hemorrhagic features from angiosarcoma. That can be further complicated by occasional expression of D2-40, FLI1, and CD31 in AFX. (68,77,91,92) CD34 and ERG may be useful to differentiate those entities. (91)

Desmoplastic Melanoma

Desmoplastic melanoma often lacks melanin and, sometimes, junctional involvement. When considering its differential diagnosis, it must be remembered that cytokeratin (2%), (93) SMA, (94) and desmin (62) can be anomalously expressed in DMs. (95) [CD68.sup.+] melanomas may also be mistaken for AFX. (62) HMB-45 is usually negative, and Kucher et al (96) found that Melan-A was positive in only 7% of DMs (Figure 3, A). Microphthalmia transcription factor (MITF) similarly has low sensitivity in DM. (97) S100 is a sensitive, albeit not specific, marker for spindle/desmoplastic melanoma (Figure 3, B). However, many scars contain [S100.sup.+] spindle cells complicating differentiation, particularly of reexcision specimens, but unlike DM, the [S100.sup.+] cells are only focal and are predominantly in a horizontal pattern (Figure 3, C). (98-100) p75, also known as nerve growth factor receptor, is a neural crest marker expressed in most desmoplastic and neurotropic melanomas, often more intensely than S100. (101,102) Leiomyosarcomas, AFX, and 81% of spindle cell SCCs are negative for p75, and those spindle cell SCCs that do react show only focal nests of positivity. (103) However, many other malignant spindle cell tumors are also positive for p75, including peripheral nerve sheath tumors, dermatofibrosarcoma protuberans (DFSP), rhabdomyosarcoma, synovial sarcoma, and neurotized nevi. Spindle cell melanoma without desmoplasia or neurotropism, many typical epithelioid melanomas, and nevi fail to stain with p75 or do so only focally. (102) Scars may reveal p75+ cells, similar to (S100) (possibly myofibroblasts, nerve twigs, or Schwann cells) requiring caution in distinguishing scars from DMs. (104) SOX10 has proven to be as sensitive a marker for DM as S100 with improved specificity (Figure 3, D). Atypical fibroxanthoma and spindle cell SCCs fail to show SOX10 expression, but malignant peripheral nerve sheath tumors show scattered positivity similar to S100.105 SOX10 has become an important alternative in identification of DM, especially in reexcision specimens. Ramos-Herberth et al (100) reported only rare, weak SOX10 expression in spindled fibroblasts of scars, in comparison to greater MITF and S100 expression in histiocytes and fibroblasts of scars.

Use of a panel of immunohistochemical markers in the setting of atypical dermal spindle cell tumors is required to prevent misdiagnosis; some combination of S100 and SOX10 for DM, high-molecular-weight keratin and p63 for spindle cell SCC, desmin and h-caldesmon for leiomyosarcoma (Table 3), and if hemorrhagic, ERG or CD34 for angiosarcoma. Atypical fibroxanthoma is the diagnosis of exclusion.

MELANOCYTIC NEOPLASMS

The melanocytic origin of a tumor may not always be apparent. In addition to morphologic clues, IHC can assist in distinguishing melanocytic from nonmelanocytic lesions. Staining with S100 was one of the first and most enduring markers for melanocytic lesions. Although most melanomas are [S100.sup.+], that marker lacks specificity and stains neural tissue, Langerhans cells, other tumors, and Rosai-Dorfman disease. Thus, other antibodies may be necessary to confirm the melanocytic nature of [S100.sup.+] neoplasms. Both the MART-1 (melanoma antigen recognized by T cells) and Melan-A antibodies recognize the same gene product and are expressed by normal melanocytes, nevi, and melanoma, but less frequently by DM. (106) HMB-45 (anti-gp100), a premelanosome marker, has been shown to mark the intraepidermal and superficial dermal components of melanocytic nevi, with the exception of diffuse dermal staining in blue nevi. (107) When compared with MART-1, HMB-45 has been found to have weaker and more-focal staining in both primary and metastatic melanomas. Therefore, HMB-45 is relatively specific, staining only rare other tumors (eg, perivascular epithelioid cells tumors [PEComas]) but is not very sensitive for melanoma. (106,108)

MITF is responsible for the normal embryonic development of melanocytes, mast cells, cells of the retinal pigment epithelium, and osteoclasts. (109) Melanocytes express nuclear MITF. Most melanomas retain MITF reactivity; however, a large proportion of desmoplastic and spindle cell melanomas fail to stain. (109,110) Positivity with MITF has been reported in 88% (235 of 266) of the conventional metastatic melanomas. (110) However, MITF is not specific, and staining has also been reported in neurofibromas, dermatofibromas, AFX, leiomyosarcomas, schwannomas, malignant peripheral nerve sheath tumors, solitary fibrous tumor, giant cell tumors of the tendon sheath, dermal scars, and granular cell tumors. (106,111)

SOX10 (sex-determining region Y-box 10), a nuclear transcription factor expressed in neural crest cells, is crucial for differentiation of Schwann cells and melanocytes. Nuclear staining is noted in normal melanocytes, Schwann cells, secretory cells of the eccrine coil, myoepithelial cells, and predominantly cytoplasmic staining in mast cells. (112,113) Expression has been shown in all types of nevi (blue, neurotized, dysplastic, Spitz, capsular) and melanoma (epithelioid, spindled, desmoplastic, metastatic). (114,115) The specificity is also high, with expression in only a few other tumors, including granular cell tumor, schwannoma, neurofibroma, myoepithelioma, clear cell sarcoma, and some ductal breast carcinomas. It is notably absent in cellular neurothekeoma, Langerhans cell histiocytosis, perineurioma, MCC, spindle cell SCC, AFX, and leiomyosarcoma. (112-114,116,117) SOX10 is particularly useful in the setting of junctional melanocytic proliferations on sun-damaged skin, DM, and in evaluation of sentinel lymph node biopsies.

Several other melanocytic markers have been studied, including NKI-C3, TRP-1, and TRP-2 (antibodies to tyrosinase), SM5-1, PNL2, and KBA62. (118-123) MUM1 (multiple myeloma 1), a known hematolymphoid marker, shows positive immunostaining in nevi; in more than 90% of primary melanomas, with the exception of DM; and in 80% of metastatic melanomas. (124,125) To date, none of these have shown any significant benefit over those previously discussed.

Some melanomas exhibit aberrant immunohistochemical expression and may express desmin, SMA, CD68, carcinoembryonic antigen (CEA), and EMA. (126) Korabiowska et al (127) found cytokeratin expression in 6% of malignant melanomas, with staining tending to be focal, and only 3% or more of the cells staining. Use of a panel of immunostains, including the previously mentioned melanocytic markers, should prevent misdiagnosis.

PEComas have immunophenotypic features of smooth muscle and melanocytes. Although many are retroperitoneal or visceral, a subset arises in the skin. Those lesions can mimic melanocytic neoplasms on routine sections and immunohistochemically. HMB-45 is the most-sensitive marker, but Melan-A and MITF are also expressed in many cases. PEComas differ from melanocytic lesions in absence of S100 expression. (128)

Heavily pigmented melanocytic neoplasms are difficult to assess on routine hematoxylin-eosin (H&E)-stained slides because pigmented melanocytes can be confused with pigmented keratinocytes and melanophages. Immunostaining using diaminobenzidine, which forms a brown product, as the chromogen is challenging to distinguish from melanin. Alternatives include use of a red chromogen or use of melanin bleach; however, the bleaching may result in loss of antigenicity, incomplete melanin removal, or loss of cytologic detail. Kamino et al (129) were the first to report replacement of hematoxylin by azure B, which stains melanin green-blue and can be easily contrasted with diaminobenzidine. Thus, immunohistochemically stained melanocytes appear brown, whereas melanin granules in melanocytes, melanophages, or pigmented keratinocytes only stain green-blue. (129-131)

Junctional Melanocytic Proliferations

It can be difficult to reliably identify intraepidermal melanocytes in hematoxylin-eosin-stained sections, especially on sun-damaged skin. Even close inspection may not unequivocally discriminate pigmented keratinocytes from melanocytes or clearly delineate the melanocyte density. Thus, many pathologists employ IHC to differentiate melanoma in situ from its mimics. S100 also highlights intraepidermal Langerhans cells making it less favored. MART-1/Melan-A has historically been valued in this setting, but studies (132-134) have shown that those markers may artificially increase the perceived number of melanocytes because of the labeling of cytoplasmic dendrites encircling neighboring keratinocytes. Available nuclear melanocytic markers are sensitive, specific, and circumvent the problem of cytoplasmic dendrites. (133) SOX10 is such a nuclear marker, with the same or better sensitivity as S100, and it does not stain additional distracting cells, such as Langerhans cells of the epidermis. (133) Nuclear expression of MITF has similar benefits to SOX10 but is much less sensitive in identifying subtle underlying DM. Caution is required with initial use of nuclear melanocytic markers and should be performed in parallel with cytoplasmic markers until one is comfortable with the altered staining pattern. Confluence, as seen with cytoplasmic markers, may not be as obvious at the outset because nuclear markers lack reactivity in the juxtaposed melanocytic cytoplasm.

MART-1/Melan-A also binds to melanosomes in keratinocytes falsely labeling nonmelanocytic cells in the epidermis that are damaged by inflammation, particularly in the setting of interface processes. These "pseudomelanocytic nests" have been described as MART-1/Melan-[A.sup.+], [S100.sup.-], nonmelanocytic cellular aggregates at the dermal epidermal junction (Figure 4, A and B). They can lead to a false impression of melanoma in situ. The origin of those aggregates has not been proven, but they appear to be a compilation of degenerated keratinocytes, macrophages, lymphocytes, and possibly a few melanocytes. (135,136) SOX10 does not appear to be immune to the "pseudonest" phenomenon, with one reported case1 (37) of SOX10 junctional nests in the setting of erythema dyschromicum perstans. MITF has shown similar issues with these pseudonests. (137-139) In sum, the H&E morphology must be taken in context with the clinical scenario and the results of more than one melanocytic marker, especially in the setting of interface damage.

An additional caveat to keep in mind is the weak or negative expression of S100 in benign and malignant junctional melanocytes of the nail matrix. If performed alone, S100 may be misleading. HMB-45 and MART-1 are more sensitive than S100 for intraepithelial melanocytic proliferations of the nail (Figure 5, A through C). However, S100 is essential for differentiating invasive melanoma, particularly DM, where HMB-45 and Melan-A may fail. (140)

Benign Versus Malignant

Of most interest, but of least availability, are antibodies that contrast benign and malignant melanocytic lesions. No single marker or even panel proves the diagnosis of melanoma unequivocally. HMB-45 marks the intraepidermal and superficial dermal components of melanocytic nevi with the exception of diffuse dermal staining in blue nevi.

This gradient of HMB-45 in benign nevi contrasts with weaker and more-focal staining in both primary and metastatic melanomas. Caution may be required in interpretation of HMB-45 in the setting of traumatized nevi that may show strong labeling of cells in and below the scar. (141)

MIB-1, an antibody that detects Ki-67, is a marker of cell proliferation. There is nuclear expression in cells in the G1, M, G2, and S phases of the cell cycle but not in G0, the resting phase. Increasing expression and thus increasing proliferation is present in malignant melanocytic lesions and is highest in metastatic melanomas. (106) The pattern of staining can also be of assistance. Melanomas reveal MIB1 staining throughout the lesion, whereas nevi reveal staining superficially, if at all. (142) MIB-1 is not cell type-specific and thus labels any proliferative cell. Lymphocytes can be numerous in melanocytic lesions and often express MIB-1. It can be difficult to distinguish these cells from MIB1 positive melanocytes. Dual labeling with a cytoplasmic melanocytic marker, such as MART-1, using a conflicting chromogen (AEC versus diaminobenzidine) improves differentiation of cells that express both markers thus representing proliferating melanocytes (Figure 6). (143,144)

Elastic fibers, as identified by elastin IHC, are preserved between nests in nevi, whereas they are markedly decreased in melanoma and are compressed at its base. Similarly, in areas of regression, there is a thin layer of compressed elastic fibers present at the base. In contrast, the base of scars lack that compressed elastic layer. (145,146)

Differentiation of Spitz nevus from spitzoid melanoma can be problematic, as exemplified by the number of cases sent for consultation. In addition, lesions are often only partially sampled, further complicating diagnosis. Several immunohistochemical markers have been investigated to aid in characterization. However, the search for a conclusive way to reliably distinguish those entities, with vastly different implications, continues.

HMB-45 and MIB-1 have been used similarly in spitzoid lesions as in other melanocytic tumors (Figure 7, A and B). Vollmer (147) found that a proliferation index, as determined by MIB-1, greater than 10% favors melanoma, whereas a proliferation index less than 2% favors Spitz nevus.

The S100 antibody reacts with many proteins, mainly staining cells containing the S100B polypeptide chains. S100A6 (calcyclin) is a S100 subtype that stains Spitz nevi in a strong and diffuse pattern (Figure 7, C), whereas only one-third of melanomas express S100A6 and only in a weak and patchy pattern, with minimal to no reactivity at the junction. (148) It is important to recognize that nevi, other than Spitz, react with S100A6 in a pattern similar to melanoma, and S100A6 also stains fibrohistiocytic lesions.

Loss of 9p21, which encodes the cell cycle inhibitor p16 (cyclin-dependent kinase inhibitor 2A), has been identified in malignant spitzoid tumors by comparative genomic hybridization and fluorescent in situ hybridization (FISH) and correlates with loss of nuclear, with or without loss of cytoplasmic p16, immunohistochemically (Figure 7, D and E). (149-152) However, heterogeneity within the same tumor has been reported, (153) and Mason et al (154) were not able to confirm those findings. The conflicting studies may partially be due to considerable variability in study design: inclusion of different types of melanocytic lesions, use of different p16 antibody clones, and definition of positive expression. George et al (152) found that the cellular region evaluated affects the validity, with loss of both nuclear and cytoplasmic expression being more specific for melanoma than is nuclear loss alone.

Several other markers, including CD99, have been studied in spitzoid lesions but have not been particularly useful in our laboratory. (155,156) No isolated marker is diagnostic, but a panel of HMB-45, S100A6, MIB-1, and p16 may be helpful in this differential diagnosis (Table 4).

The cells of DM can appear remarkably bland, and mitoses are often sparse. Therefore, DM can be confused with desmoplastic nevi, as well as the other spindle cell tumors mentioned above. HMB-45 is usually negative, and MART-1/Melan-A is positive in only 7% of DM, whereas desmoplastic nevi are uniformly positive with MART-1/ Melan-A. Therefore, diffuse, strong staining with one of these markers strongly supports the diagnosis of desmoplastic nevus. Unlike desmoplastic nevi, MIB-1 expression in more than 10% of cells favors DM; however, lesser expression does not rule out DM. (96) Absence or only focal reactivity with p16 favors DM rather than desmoplastic nevus. (157)

Melanoma Prognosis

Prognostic indicators of melanoma on H&E-stained sections include ulceration and tumor depth, but there has been increasing study into the use of IHC-assisted prognostication. Identification of lymphatic invasion can be increased with the use of D2-40 (podoplanin), a lymphatic endothelial marker, or CD34, and recognition is associated with decreased disease-free and overall survival. (158-160) Identification is even further enhanced by use of dual markers, D2-40/MART-1 (Figure 8, A and B). (161)

The compressed elastic fibers at the base of melanomas, as seen with elastin IHC, can help clarify the Breslow depth, especially in melanomas that evolve in a nevus, where the elastic fibers are preserved between nests and often around individual melanocytes. (145)

The presence of dermal mitoses in melanoma is a critical variable in management and prognostic considerations. Identification of mitoses has become increasingly important in staging melanoma in the 2010 American Joint Committee on Cancer (AJCC) guidelines, as identification of one mitotic figure in a melanoma less than 1 mm thick will alter the stage. (162) However, it can be time consuming to search for mitoses, and distinguishing apoptotic, pyknotic, and hyperchromatic nuclei from mitotic figures can be difficult and subjective. Histone H3 is phosphorylated during mitotic chromatin condensation in the late G2 and M phases of the cell cycle. Immunohistochemical staining of PHH3 (phosphor-histone H3) facilitates mitotic counting, even at X20 magnification, in contrast to the X40 magnification used when counting on H&E. PHH3 is specific to the M phase of the cell cycle, unlike MIB-1, and is not expressed in apoptotic cells. PHH3 aids identification of the mitotic "hot spot" (Figure 9), shortens the time required to determine the mitotic count, and improves reproducibility beyond counts on standard hematoxylin-eosin-stained sections. (163) It is not surprising that PHH3-determined counts are higher than those from H&E-stained sections because the prophase that comprises a large fraction of the entire mitotic phase is essentially undetectable on standard sections but is well highlighted with PHH3. (164-167) The PHH3 count may also be falsely elevated by misinterpretation of nonmelanocytic mitotic figures. Dual staining with a cytoplasmic melanocytic marker (PHH3-Melan-A) enables the pathologist to make a clear distinction between mitotically active melanocytes and lymphocytes, histiocytes, or endothelial cells and shows more concordant results when compared with standard section counts. (167-169) PHH3determined mitotic counts have proven to be a stronger prognostic indicator than mitotic count determined from H&E-stained sections. (170) Although use of PHH3 improves accurate identification of mitoses and is known to show an association with survival, (171) the current melanoma staging criteria are based on a mitotic rate determined from H&E stained slides. PHH3-based cutoff levels for pT1b will need to be determined.

Diagnosis of metastatic melanoma in sentinel lymph nodes can be difficult, especially when attempting to identify small foci. Although adequate sampling and sectioning is required, IHC can further highlight small clusters of cells. Protocols for processing sentinel lymph nodes for metastatic melanoma vary from institution to institution and varied immunohistochemical markers are used, but commonly HMB-45, MART-1, and/or S100 are employed. (172,173) S100 is highly sensitive; however, it is expressed in multiple cell types, including dendritic cells and certain sinus macrophages and, thus, can make interpretation of lymph nodes for melanoma troublesome. Zubovits et al (108) reported S100 staining in 98% (123 of 126) of metastases in lymph nodes but only one-half showed diffuse staining of more than 50% of the tumor. In contrast, MART-1 stained only 82% (103 of 126) of metastases but with 83% (85 of 103) of those stains being diffuse. In addition, MART-1 does not exhibit the same background dendritic cell staining that S100 does, but on occasion, macrophages, particularly pigmented ones, can be weakly labeled with MART-1. (108,174) In one study, (175) MITF tended to result in overdiagnosis of metastases in sentinel lymph nodes, probably because of expression in histiocytes.

Identification of [MART-1.sup.+] cells within a lymph node does not necessarily mean the patient has metastatic melanoma. Nodal nevi have been detected in approximately 1% of axillary nodes in patients with breast cancer and in 3.9% of lymph nodes in patients with melanoma. (176) Nodal nevi are typically confined to the fibrous capsule or trabeculae of lymph nodes but have been reported in the node parenchyma, whereas metastatic melanoma is typically subcapsular (Figure 10, A). (177) These nodal nevi can be a potential diagnostic pitfall in interpretation of immunohistochemically stained nodes. Identification of a positive signal requires close examination of the corresponding H&E-stained sections for cytologic atypia and comparison with the primary tumor. According to Lohmann et al, (176) MIB-1 may be helpful in making the distinction between nodal nevi and metastatic melanoma. All nodal nevi (n = 15) in this small study (176) lacked immunoreactivity for MIB-1, but all metastases (n = 40) were positive for MIB-1, usually with more than 10% of the cells being MIB-1+. Combined MART-1-MIB-1 can improve the distinction between proliferating melanocytes in metastases and proliferating lymphocytes or histiocytes. (144) Just as HMB-45 does not label intradermal nevi, Biddle et al (178) found that an intranodal nevus is negative for HMB-45, unless it is a nodal blue nevus. Therefore, detection of HMB-45 in the melanocytic aggregate supports a diagnosis of metastatic melanoma; however, as mentioned previously, HMB-45 does not detect all metastatic melanomas, and its absence does not exclude melanoma. (178) HMB-45 also occasionally labels pigmented macrophages. (179) Other small studies have shown the absence of nuclear p16, positive expression of IMP3 (insulin-like growth factor-II messenger RNA binding protein-3), and reticulin staining around groups of melanocytes, rather than between individual cells, in metastatic melanoma. (180-182)

SOX10 is a highly sensitive marker for metastatic melanoma in sentinel lymph nodes but, unlike S100, is not expressed in follicular dendritic cells or sinus histiocytes, which improves the identification of small micrometastases and isolated cells (Figure 10, B). (112,115,183) Unlike HMB-45, SOX10 does not differentiate nodal nevi (Figure 10, C) from metastases, and distinction requires attention to the nodal location, cytology, and comparison to the primary. (115,184)

Promising treatments for some patients with metastatic melanoma target BRAF. Use of those drugs requires the patient's BRAF mutational status be determined before initiation of treatment. Previously, DNA-based techniques were employed, such as allele-specific PCR and direct DNA sequencing, but both are expensive, time consuming, and can be confounded by suboptimal DNA extraction. Recently an antibody directed against V600E (anti-B-raf [VE1]) has become available, and cytoplasmic expression has thus far shown high sensitivity, specificity, and interobserver reliability for detection of this mutation. (185-188) It provides a rapid, inexpensive, and accessible, first-line screening tool, even for small tumor foci, to assess candidates for BRAF inhibitors. (188,189) Immunohistochemistry provides the additional benefit of allowing the pathologist to visualize the cytology of positive cells because nevi can harbor this mutation, complicating interpretation of melanoma developing in a nevus. (190) This antibody does not show positive staining for the other less-common BRAF genotypes, such as V600K. Because the V600E genotype has been reported in other tumors, including lung, gastrointestinal, and thyroid carcinoma, individual protocols based on the tumor type are required to optimize results. (191) Although most studies have reported homogeneous staining throughout a tumor and the same patient's metastases, (186,192) a small subset of cases have been reported that show heterogeneous staining, further supported by microdissected DNA analysis. (193,194) Further study to determine the correlation with treatment response is warranted.

MALIGNANT SMALL BLUE CELL TUMORS

The small blue cell appearance of MCC elicits a differential diagnosis. including Ewing sarcoma/primitive neuroectodermal tumor (EWS/PNET), metastatic small cell carcinoma of the lung, neuroblastoma, lymphoma, and melanoma (Table 5). Small cell melanoma can be distinguished immunohistochemically because MCC does not typically express S100 but does express neuroendocrine markers, such as chromogranin, neuron-specific enolase, and synaptophysin, and displays paranuclear dot staining with neurofilament protein and CK20 (Figure 11). (195)

Although metastatic small cell carcinoma of the lung is typically negative for CK20 and neurofilament protein, it is classically positive for CK7 and TTF1 (thyroid transcription factor), a finding not usually seen in MCC. (196,197) CK20 is present in most MCCs (87%), whereas CK7 is rarely identified. (197) Despite the classic [CK20.sup.+]/[CK7.sup.-] pattern, CK20-/CK7+ MCCs have been described. (197,198) It is important to be aware of the staining pattern of the primary tumor when evaluating sentinel lymph nodes if IHC is needed.

The absence of CD45/LCA in MCC is typically sufficient to exclude lymphoma, but some markers used in evaluation of lymphoma can be detected in MCC, leading to erroneous diagnoses, including ALK1 (depending on the clone used) as in anaplastic large cell lymphoma (ALCL), BCL2 as in B-cell lymphomas, TdT and PAX5 as in B-lymphoblastic lymphoma, and CD56 as in natural killer T-cell lymphoma. (197,199-202)

As in MCC, EWS/PNET also has the potential to express neuron-specific enolase, chromogranin, and synaptophysin. CD99 is a marker used for EWS/PNET, but it is not specific and has been seen in lymphoblastic lymphoma, selected rhabdomyosarcomas, small cell carcinomas, carcinoid tumors, melanomas, and even a few MCCs. (155,156,203,204) The FLI1 antibody is also a nuclear marker for EWS/PNET, as well as for vascular tumors. However, it is also expressed in subsets of lymphomas, MCCs, and melanomas. (203,205-208)

Pankeratin reactivity is usually absent in EWS/PNET, and CK20 expression has not been reported, unlike MCCs. The surest way to distinguish EWS/PNET from MCC is cytogenetic analysis revealing t(11;22), usually involving EWS and FLI1 genes, in EWS/PNET. (204,209)

The c-kit proto-oncogene codes for a transmembrane receptor tyrosine kinase (KIT receptor/CD117). CD117 is expressed in a variety of tumors, including acute myeloid leukemia, mast cell disease, melanoma, small cell lung cancer, and gastrointestinal stromal tumors. CD117 is expressed by most MCCs. (210,211) In small cell lung cancer and gastrointestinal stromal tumors, abnormalities in KIT receptor signal transduction seem to have a role in tumor pathogenesis, and those tumors, therefore, show response to imatinib mesylate by tyrosine kinase inhibition. (210) Although KIT expression is present in MCC, response to imatinib mesylate has been poor and anecdotal. (211-213)

Rarely, MCC can mimic BCC, and both can and often do express Ber-EP4, BCL2, and NCAM/CD56. In addition, BCC can on occasion express chromogranin and synaptophy sin. (2,199,214,215)

Clonal integration of the newly discovered Merkel cell polyomavirus has been shown in most MCCs, most commonly by PCR-based techniques, and MCC cases previously reported as negative may be due to limited primers used for detection. (216) Merkel cell polyomavirus large T antigen can be detected immunohistochemically with the antibody CM2B4. Although PCR can target other structural proteins in addition to large T, results have generally been concordant. (217) The specificity of Merkel cell polyomavirus for MCC has been questioned because of the presence of Merkel cell polyomavirus DNA in non-MCC skin cancers; however, when present, the DNA load is considerably less than that seen in MCCs and is usually not detected by IHC. (218,219)

In univariate and multivariate studies, expression of p63 has been associated with shortened survival in patients with MCC, especially in its early stages. (220-223) Use of p63 in MCC staging may be a future consideration.

FIBROHISTIOCYTIC LESIONS

Cellular Neurothekeoma

Nerve sheath myxomas, believed to be of peripheral nerve sheath origin, were redesignated as neurothekeoma by Gallager and Helwig (224) in 1980. In 1986, the more-cellular form was designated cellular neurothekeoma (CNT) by Rosati et al. (225) Currently, there are 3 recognized subtypes of neurothekeoma: myxoid, cellular, and mixed. Controversy exists about the lineage of CNT, with arguments for myofibroblastic, nerve sheath, melanocytic, and leiomyomatous differentiation. Immunoreactive markers span a spectrum of cell types, including focal staining with SMA, CD68, and CD10. (226,227)

PGP9.5 (protein gene product 9.5, also known as ubiquitin carboxyl-terminal hydroxylase-1) reactivity can be helpful in the diagnosis of neurothekeoma but also suffers from low specificity. (228) PGP9.5 is positive in nerve sheath tumors, including granular cell tumors; fibroblastic tumors, including dermatofibromas; vascular tumors; and other tumors, such as leiomyomas. (229,230)

D2-40, as discussed previously, is expressed in lymphatics and PCANs, but reactivity has also been reported in the 15 CNTs thus far studied. (231) Cellular neurothekeomas and fibrous histiocytomas can share morphologic features, and both have shown expression of D2-40, further complicating differentiation.

Although myxoid neurothekeomas are [S100.sup.+], CNTs are not, arguing strongly against a peripheral nerve sheath or melanocytic histogenesis. However, CNTs are positive for S100A6 protein (calcyclin). (228,232) Reactivity with that protein is not equivalent to a diagnosis of CNT because it is also expressed in melanocytes and dermal dendrocytes. (228) Cellular neurothekeomas also can be confused with melanocytic lesions, histologically. When considering this differential diagnosis, S100A6 reactivity, with negative S100 staining, essentially eliminates a melanocytic lesion. S100 expression of colonizing antigen-presenting cells can be seen and should not be confused with lesional cell reactivity, causing an erroneous diagnosis.

Although the typical melanocytic markers, S100, HMB-45, and Melan-A are absent in CNT, other less-specific melanocytic markers, such as S100A6 have been identified. (227,228) Many CNTs express NKI-C3 (CD63), but that marker lacks specificity and has been identified in nevi, melanomas, granular cell tumors, and some fibrohistiocytic lesions. (228,233) Expression of KBA.62, an antimelanoma monoclonal antibody, has recently been reported (234) in 18 of 18 CNTs (100%). MITF is also a melanocytic marker that highlights most CNTs (Figure 12, A and B). (226)

By virtue of morphologic and phenotypic similarities, some experts believe CNTs are related to plexiform fibrohistiocytic tumors. There is significant overlap in the immunohistochemical staining pattern with both expressing S100A6, PGP9.5, CD68, SMA, NKI/C3, and CD10 to varying degrees. However, 2 small studies (231,235) have identified immunohistochemical differences, with MITF being positive in 9 CNTs but failing to react in 5 plexiform fibrohistiocytic tumors. (235) Kaddu and Leinweber (231) noted D2-40 expression in the 15 CNTs they evaluated but not in either of the 2 plexiform fibrohistiocytic tumors studied.

Dermatofibroma and DFSP

Most dermatofibromas (DFs) can be easily distinguished from DFSPs; however, morphologic features alone do not always allow reliable distinction between deep or cellular DF and DFSP. Commonly, CD34 and factor XIIIa have been used in differentiation. Typically, DFSP is [CD34.sup.+] and factor [XIIIa.sup.-], and DF is [CD34.sup.-] and factor [XIIIa.sup.+]; however, that is not absolute. (236-238) Some DFs exhibit focal staining with CD34, especially at the periphery of cellular and deep DFs (Figure 13, A), and occasional DFSPs are negative for CD34. (239) Caution is required in interpretation of DFSP margins with CD34 because CD34 disappears from scars but proliferates in pericicatricial tissue. (240) CD34 positivity is not exclusive to DFSP. It is also an endothelial marker and stains solitary fibrous tumor, spindle cell lipoma, superficial acral fibromyxoma, sclerotic fibroma, Kaposi sarcoma, neurofibroma, tricholemmoma, scleromyxedema, and nephrogenic systemic fibrosis, among others. (241-243) Factor XIIIa is typically weak and only identified at the periphery of DF or more diffusely in spindle cell-dominant cellular DFs. (237)

Many alternative markers have been explored but mostly in isolated studies with few cases. Those markers include HMGA1 and 2, CD163, ApoD, tenascin, S100A6, MMP2 and 11, IGFBP7, cathepsin K, and D2-40. *

Nestin, a neuroectodermal and mesenchymal stem cell marker, was evaluated in a larger cohort of nearly 200 cases of DFSP and DF in 4 different studies, (247-250) which showed similar results. Strong nestin expression was noted in DFSP, and no or only rare focal expression was seen in DFs. Unlike CD34, which can be absent or decreased in fibrosarcoma tous areas of DFSP (Figure 13, B), nestin remains unaltered. (250)

Immunohistochemistry for stromelysin 3, a member of the matrix metalloproteinase family, MMP-11, has also been studied in at least 6 separate studies (46,238,251-254) with similar outcomes. Stromelysin 3 has a role in tissue remodeling during wound healing and tumor invasion, and there is diffuse cytoplasmic staining of the spindle cells in most DFs, whereas it has rarely been noted in DFSPs. (46,238,251-254)

Most DFSPs harbor the COL1A1-PDGFB translocation, and FISH can be used in cases showing confusing histopathologic and/or immunohistochemical features. (255)

Positivity with CD117, an immunohistochemical marker that binds the KIT receptor, has shown an association with response to imatinib in gastrointestinal stromal tumors. Sporadic case reports of a partial response of DFSP to this tyrosine kinase inhibitor, led to evaluation of CD117 staining in these tumors, but the absence of staining suggests that adjuvant treatment with this drug should not be based on immunohistochemical assessment with CD117. (256)

VASCULAR NEOPLASMS

Several endothelial markers are available including: factor VIII-related antigen, CD34, CD31 (platelet-endothelial cell adhesion molecule type 1), FLI1, and ERG. Factor VIII-related antigen (von Willebrand factor) is specific for endothelial cells but has a low sensitivity, and because it circulates in the serum, it can be seen in zones of necrosis and hemorrhage. CD34 is sensitive but is also expressed in several nonvascular tumors, including DFSP and epithelioid sarcoma. CD34 has been considered a sensitive marker for Kaposi sarcoma, but Kaposi sarcoma-associated herpesvirus 8 latent nuclear antigen has supplanted use of CD34 in that context. (62,257) CD31 has been considered the most-sensitive marker for vascular endothelium and vascular tumors. It is expressed in endothelial cells but is not specific and has been reported in a subset of carcinomas, lymphomas, AFXs, epithelioid sarcoma, EWS/PNET, macrophages, platelets, and plasma cells. (91,258) More recently, FLI1 and ERG were reported as nuclear markers of endothelial differentiation. Folpe et al (95) found FLI1 expression in 94% of benign and malignant vascular tumors, but FLI1 is also a marker for EWS/PNET, with expression in approximately 90% of cases, and it shows expression in some melanomas, leiomyosarcomas, SCCs, and AFXs. (91) Thus far, ERG has proven a sensitive and specific marker for vascular lesions, showing expression only in a subset of prostate carcinomas, EWS/ PNET, epithelioid sarcomas, and myeloid sarcoma. (259-261) Rao et al (258) evaluated 34 angiosarcomas in a tissue microarray and found ERG and FLI1 to be the most sensitive immunomarkers. Angiosarcomas often show loss of expression of one or more endothelial markers, especially epithelioid angiosarcomas, which can also show keratin expression, suggesting that a panel should be used in diagnosis.

Markers that distinguish blood vessel endothelial cells from lymphatic endothelial cells have also been investigated. Prox1, a transcription factor important in the regulation and maintenance of the lymphatic endothelial phenotype, is expressed in lymphangiomas, Kaposi sarcoma, tufted angiomas, and Kaposiform hemangioendothelioma. (262) The monoclonal antibody D2-40 binds to podoplanin, a glycoprotein highly expressed in lymphatic endothelium. D2-40 has been identified as a sensitive marker for lymphatic endothelium in normal tissue and in a subset of vascular lesions, including Kaposi sarcoma, Dabska tumor, lymphangioma, hobnail hemangioma (targetoid hemosid erotic hemangioma) and in a subset of angiosarcomas, especially those with epithelioid endothelial cells. In general, classic hemangiomas do not express D2-40. (263,264)

D2-40 is variably expressed in a wide variety of lesions, including sebaceous neoplasm, epithelioid sarcoma, AFX, SCCs, CNTs, DFs, and PCANs. ([dagger]) Expression of lymphatic markers, including Prox1 and D2-40, in angiosarcoma suggests that it may show a mixed endothelial phenotype. (92,258,262)

Infantile hemangiomas become apparent postnatally, enlarge rapidly during the first year of life, and spontaneously involute in the first decade. The characteristic histopathologic features diminish during that evolution, and differentiating them from vascular malformations can be difficult. Endothelial immunoreactivity for the erythrocyte-type glucose transporter protein (GLUT1), normally restricted to endothelia with blood-tissue barrier function, as in the brain and the placenta, is identified in all phases of infantile hemangiomas, whereas reactivity is absent in vascular malformations, pyogenic granulomas, and granulation tissue. (267-269) Normal reactivity in erythrocytes must be discounted when interpreting the expression (Figure 14). Expression of GLUT1 has also been demonstrated in normal perineural cells and perineuriomas. (270) These [S100.sup.-], benign peripheral nerve sheath tumors are also positive with EMA but often in a focal and weak pattern because of their very thin and widely separated cytoplasmic processes. Higher antisera concentrations or longer incubation times may be required for EMA in perineuriomas, in comparison to that used for epithelial tumors. Claudin-1, a component of tight junctions, and GLUT1 are stronger and stain more diffusely in perineurioma than does EMA. (271)

Infantile hemangiomas and vascular malformations have distinct clinical courses, and the distinction is important for management considerations. Wilms tumor 1 (WT1) cytoplasmic endothelial expression has been reported in vascular tumors, including infantile hemangiomas, noninvoluting congenital hemangiomas, rapidly involuting congenital hemangiomas, tufted angiomas, pyogenic granulomas, microvenular hemangiomas, and cherry hemangiomas, but is lacking in lymphatic and venous vascular malformations. (272-274) WT1 positivity was, however, reported in the 5 stage 2 arteriovenous malformations studied, (273) possibly because of active proliferation and clinical enlargement during that stage. Current data are limited, but WT1 results are conflicting for verrucous hemangioma and hobnail hemangioma, further drawing into question the appropriate classification of those lesions as neoplasms or malformations. (274-276)

Another important distinction includes distinguishing rapidly involuting congenital hemangioma and noninvoluting congenital hemangioma from infantile hemangioma. Those lesions behave differently, as the names suggest, but have histologic features that overlap with infantile heman gioma. However, neither rapidly involuting congenital hemangioma nor noninvoluting congenital hemangioma expresses GLUT1. (269,277)

Vascular lesions of the breast can be diagnostically problematic, especially in small-sample biopsies, yet pathologists are often asked to differentiate angiosarcoma from atypical vascular lesions in postradiation and lymphedematous patients. MYC amplification has been identified in postradiation and lymphedema-associated angiosarcomas by FISH but is not present in the atypical vascular lesions. (278-281) Genetic or epigenetic alterations may lead to increased c-MYC protein expression noted by IHC without concurrent gene amplifications identified by FISH, (282) and likewise, low-level gene amplification may not be accompanied by detectable amounts of protein. However, studies have shown good concordance between MYC amplification and MYC protein expression by IHC in mammary vascular lesions. (279,281) The MYC immunohistochemical marker shows promise in determining margins in these ill-defined lesions, (279) and MYC may serve as a target for future therapy. Interestingly, most primary angiosarcomas from other sites typically do not show the same amplification, and thus, MYC IHC does not appear useful in identification of other primary cutaneous angiosarcomas unrelated to prior radiation. (279,282)

HEMATOPOIETIC AND HISTIOCYTIC TUMORS

Cutaneous B-Cell Lymphomas

CD20 is a B-cell-specific marker expressed in 98% of B-cell lymphomas, but it may be lost in the lymphomas treated with the anti-CD20 antibody (rituximab). CD79a is expressed in the precursor B-cell stage and disappears later than CD20 in B-lymphocyte differentiation, explaining why plasma cells are positive for CD79a but not for CD20. CD79a, therefore, stains most B-cell lesions, even rituximab-treated B-cell lymphomas. PAX5 is a nuclear marker expressed in early B-cell development and is, therefore, negative in plasma cells, but it can stain recurrent B-cell lymphoma following rituximab therapy. (283,284) CD38 and CD138 (syndecan-1) are expressed in plasma cells.

Aside from lymphocytoma cutis, a significant B-cell infiltrate in the skin is rarely reactive and should suggest possible B-cell lymphoma. Architecturally, the infiltrate may be nodular or diffuse. The diffuse pattern includes follicle center cell lymphoma, diffuse large B-cell lymphoma in both the leg type and the otherwise-not-specified type. The nodular pattern is seen in lymphocytoma cutis, marginal zone lymphoma, and a small proportion of follicle center cell lymphomas (Table 6).

Normal germinal centers are [BCL6.sup.+], [BCL2.sup.-], and [CD10.sup.+] with a [CD21.sup.+] and/or [CD23.sup.+] follicular dendritic cell network and high proliferative index. Benign lymphoid follicles can be seen in lymphocytoma cutis and marginal zone lymphoma. The interfollicular component of marginal zone lymphoma is neoplastic, and immunohistochemically is [BCL2.sup.+], whereas BCL6 and CD10 are negative. When there is plasmacytoid differentiation, light-chain restriction can be evaluated immunohistochemically; 2:1 is the normal [kappa]:[lambda] ratio, but above 5:1 or a 3:1 [lambda]:[kappa] ratio supports clonality. However, reproducibility for this ratio is low. (285)

Cutaneous follicle center cell lymphoma is of germinalcenter cell origin and is thus [BCL6.sup.+] and [BCL2.sup.-]. If BCL2 expression is present, it suggests spread of the disease to the skin from the lymph nodes. (283) T cells are [BCL2.sup.+] and comprise a proportion of the lymphoid cells in a follicle center cell lymphoma. BCL2 expression must be interpreted in the neoplastic cells alone and must not be confused with those colonizing T cells. The leg-type diffuse large B-cell lymphoma is also [CD20.sup.+] and [BCL6.sup.+], but unlike follicle center cell lymphoma, it expresses BCL2, MUM1, and FOXP1. (286) MUM1 is also expressed in melanoma and ALCL. (287)

Many lymphomas that develop in the setting of immune impairment, including senile Epstein-Barr virus-associated B-cell lymphoproliferative disorder, and posttransplant, AIDS, or methotrexate-related lymphomas, are attributable to Epstein-Barr virus. Immunohistochemistry for LMP1 should be considered in any type of lymphoma in the setting of immune suppression, but EBER in situ hybridization has higher sensitivity. (285)

A dense monomorphous chronic lymphocytic leukemia infiltrate can be associated with epithelial neoplasms, especially SCC. The B-cell leukemic infiltrate is [CD19.sup.+] and [CD20.sup.+] but also aberrantly expresses the T-cell markers CD43 and CD5. (288)

Myeloid Cells

Myeloperoxidase and lysozyme are helpful for identifying myeloid cells. Myeloperoxidase is positive in most granulocytic cells but less so in monocytic cells. Lysozyme is a marker for granulocytes, monocytes, and macrophages. Myelogenous leukemia cutis is typically [lysozyme.sup.+], [myeloperoxidase.sup.+], and [CD43.sup.+]. (289) CD68 is a lysosome marker with expression in myeloid, monocytic, and histiocytic cells. CD68/KP1 produces cytoplasmic, granular expression in acute myelogenous leukemia, but CD68/PG-M1 is more often restricted to monocytic cells and histiocytes. (283,290) CD163 is specific for monocytes/macrophages but is not a good marker for myeloid sarcoma or acute myelogenous leukemia of monocytic origin. (283) Pileri et al (291) found that the most commonly expressed markers in myeloid sarcoma were CD68/KP1, myeloperoxidase, and CD117.

Cutaneous T-Cell Lymphomas

Cutaneous lymphoproliferative disorders can be one of the most vexing problems in dermatopathology. Mycosis fungoides-type cutaneous T-cell lymphoma (MF) must be differentiated from reactive T-cell infiltrates. Polymerase chain reaction analysis of T-cell receptors for clonality has become helpful in this context; however, many cases of early MF are polyclonal by PCR, and monoclonality has been identified in several cases of benign dermatoses. (292) Like benign inflammatory infiltrates, most cases of MF have a [CD4.sup.+] T helper cell phenotype but, in contrast, may show loss of expression of other T-cell markers in the epidermal component. The most frequent down-regulated antigen is CD7, closely followed by CD5. (285) However, that downregulation is not absolute, and some benign inflammatory infiltrates, especially acute ones, show loss of CD7. (293) Therefore, interpretation of CD7 should be made in the context of the clinical and histologic features. The relative absence of a pan-T-cell marker CD3, CD5, or CD43, or the concordant expression or loss of both CD4 and CD8, are more likely to be seen in MF than in benign conditions.

Some studies have suggested that a CD4:CD8 epidermal ratio greater than 2 supports MF, rather than an inflammatory process. (294) However, there is significant overlap, with inflammatory conditions showing a ratio up to 6. (295) Ortonne and colleagues (296) attempted to improve diagnosis of early MF and found an epidermal CD8:CD3 ratio of less than 25% to be suggestive, but not specific, for MF. Use of CD3, rather than CD4, obviates concern about misinterpretation of [CD4.sup.+] Langerhans cells. In practice, application of these ratios is not reproducibly measured for precise diagnosis. In contrast, there is a subset of [CD8.sup.+] MF, which is most common in children and hypopigmented cases. (285) The International Society for Cutaneous Lymphoma proposed an algorithm for diagnosis of early MF, based on a scoring system involving clinical, histopathologic, biomolecular, and immunopathologic criteria. The immunohistologic features included in the score are CD2, CD3, or CD5 positivity in less than 50% of T cells, CD7 positivity in less than 10% of T cells, and epidermal/dermal discordance in expression of CD2, CD3, CD5, or CD7 expression. (297)

Subcutaneous panniculitis-like T-cell lymphoma is a [CD8.sup.+] cytotoxic T-cell neoplasm, by definition, of the [alpha][beta] receptor class and is, therefore, [beta]-[F1.sup.+], distinguishing it from cutaneous [gamma][delta] lymphoma. Subcutaneous panniculitis-like T-cell lymphoma is typically positive for TIA1, granzyme B, and perforin. The adipocyte rimming by T cells, particularly when they are [Ki-67.sup.+], is a useful feature in diagnosis. (285)

[CD30.sup.+] Lymphoproliferative Disorders

One subset of [CD4.sup.+] cutaneous T-cell lymphomas is characterized by expression of CD30 (BerH2, Ki-1). In addition to the original description of expression in Reed-Sternberg cells in Hodgkin disease, [CD30.sup.+] cells are a feature of lymphomatoid papulosis, especially types A, C, D, and E; large cell transformation of mycosis fungoides and ALCL. (298,299) CD30 positivity alone does not imply a diagnosis of lymphomatoid papulosis or ALCL. [CD304.sup.+] lymphocytes may be observed in benign processes, including bug bites, drug eruptions, nodular scabies (Figure 15), molluscum, syphilis, and orf virus and herpetic infections. (283,300) However, clusters or sheets of CD304 cells are typically only seen in lymphomatoid papulosis and ALCL. (301)

Cutaneous ALCL must be separated into those of cutaneous origin and those of extracutaneous origin. Primary cutaneous ALCL is usually not associated with a t2:5 translocation and is thus negative for the ALK1 protein that is typically expressed by nodal cases of ALCL or systemic ALCL involving the skin (Figure 16, A and B). (302) Similarly, unlike the systemic form, primary cutaneous ALCL is also negative for EMA. (303,304)

Blastic Plasmacytoid Dendritic Cell Neoplasm

Patients with blastic plasmacytoid dendritic cell neoplasms, previously known as blastic NK-cell tumors or [CD4.sup.+] [CD56.sup.+] hematodermic neoplasms, typically present with cutaneous lesions on initial exam, with rapid spreading to blood, bone marrow, and lymph nodes, and death ensues within a few years. (305) Histopathologically, blastic plasmacytoid dendritic cell neoplasm is characterized by a dense monomorphous infiltrate of blastoid cells that are positive for CD4, CD56, CD123, and T-cell leukemia/lymphoma-1 (TCL1), in the absence of T-cell (CD2, CD3), B-cell (CD20), and myelomonocytic cell lineage-specific markers (myeloperoxidase, lysozyme). (306,307) The finding of CD123 expression led to the consensus that the plasmacytoid dendritic cell is the cell of origin and thus the current name. Plasmacytoid dendritic cells are the primary source of interferon a, and has a role in antigen presentation, linking innate and adaptive immunity. Plasmacytoid dendritic cells are normally rare in skin but accumulate in certain inflammatory skin disorders, including lupus erythematosus and psoriasis. (308) Negativity of 1 of the 4 characteristic markers (CD4, CD56, CD123, TCL1), most often CD4, can be seen in one-third of cases of blastic plasmacytoid dendritic cell neoplasm, and interestingly, there can be phenotypic variability in the same patient. (307) In one small study, presence of [CXCL12.sup.+] cells correlated with leukemic change and poor prognosis. (305) Staining with CD123 and TCL1 is useful in differentiation from other [CD56.sup.+] cutaneous tumors, such as the nasal type of extranodal NK-cell lymphoma and myelomonocytic leukemia. Varying numbers of blastic plasmacytoid dendritic cell neoplasms express CD68, TdT, bcl-6, MUM1, and FOXP1, potentially leading to diagnostic errors.

Mast Cell Disorders

Mast cell disease can be difficult to diagnose because of the variety of clinical presentations and unusual morphology of mast cells in these disorders. Mast cells have cytoplasmic granules that can be identified with metachromatic stains, such as Giemsa and toluidine blue. However, metachromatic stains fail to label degranulated mast cells. Mast cells are also rich in chloroacetate esterase, but histologic identification of that enzyme is also seen in neutrophilic myelocytes. Therefore, immunophenotypic studies may be more informative.

KIT interacts with its ligand, stem cell factor, regulating mast cell proliferation and maturation. Thus, CD117 (c-kit) is expressed on normal and abnormal mast cells but is also reactive with myeloid precursor cells. Mast cell tryptase is a more-lineage specific marker but may have high background staining. (309,310) Some mast cell disorders can mimic myeloid leukemia cutis. Sundram and Natkunam (311) found that mast cell tryptase and MITF were sensitive and specific markers for distinguishing mast cell disease from myeloid leukemia. Immunohistochemical expression of tryptase was identified in some cases of acute myeloid leukemia cutis, but staining was very faint and present in less than 5% of lesional cells. MITF is a transcription factor that is important in mast cell development but is also expressed in melanocytes. (311) Although not expressed in normal mast cells, CD25 can be expressed in neoplastic mast cells, and one small study showed expression in cutaneous mastocytosis associated with systemic involvement. (312)

Langerhans Cell Disorders

Langerhans cells are dendritic, antigen-presenting cells found predominantly in the epidermis. They are [S100.sup.+] but are distinguished by expression of CD1a and the presence of Birbeck granules, ultrastructurally. The World Health 0rganization's Committee on Histiocytic/Reticulum Cell Proliferations requires identification of Birbeck granules by electron microscopy or CD1a expression by IHC for a diagnosis of Langerhans cell histiocytosis. CD1a however, can be negative in Langerhans cell histiocytosis, and expression has been rarely observed in juvenile xanthogranuloma, Rosai-Dorfman disease, or histiocytic sarcoma. (313) Langerin (CD207) plays a role in the formation of Birbeck granules, and immunohistochemical expression has become a surrogate for the presence of those granules, without the need for electron microscopy. (314) Langerin has shown diagnostic sensitivity similar to CD1a but with improved specificity. (313,315) Indeterminate cells are also [S100.sup.+] and [CD1a.sup.+] but lack the Birbeck granules typical of Langerhans cells; therefore, indeterminate cell tumors are negative for langerin. (316) B-lymphoproliferative and Tlymphoproliferative disorders, as well as benign inflammatory skin conditions, including scabetic nodules, can demonstrate numerous Langerhans cells, requiring correlation with the clinicopathologic setting. (317,318)

CUTANEOUS METASTASES

Adenocarcinomas can metastasize to the skin, and rarely, that can be the first manifestation of the primary diagnosis. Identification of the site of origin has profound prognostic and therapeutic effect. Immunohistochemistry has been useful in a number of cases. Differential cytokeratin staining (CK7 and CK20) may be helpful in this setting but is not specific. 0f the most-common metastases to skin, breast and lung adenocarcinomas are [CK7.sup.+]/[CK20.sup.-], and colorectal carcinoma is [CK7.sup.-]/[CK20.sup.+]. (3,19) 0ther potentially useful immunohistochemical stains include CDX2 and TTF1. CDX2, a nuclear marker, is expressed in almost all colorectal carcinomas but is also expressed in a few other adenocarcinomas, including gastric, biliopancreatic, and mucinous ovarian adenocarcinomas. (320,321) TTF1 is a relatively specific marker for tumors of lung or thyroid origin. Around two-thirds of lung adenocarcinomas express TTF1. However, expression is typically absent in squamous cell carcinoma of the lung and lung mucinous adenocarcinomas/bronchioloalveolar carcinomas. (320,322)

CDX2 and TTF1 are reasonable additions to the antibody panel that aims to identify the origin of a metastatic adenocarcinoma of unknown primary (Table 7). Park et al (323) proposed and evaluated a panel of antibodies to improve prediction of the primary site. In addition to CK7, CK20, TTF1, and CDX2, the panel included CEA, epithelial mucin genes MUC2 and MUC5AC, ER, and GCDFP15 (gross cystic disease fluid protein 15). GCDFP15 is a relatively specific marker for breast carcinoma with reactivity ranging from 43% to 77%. However, it has limited sensitivity. Mucin expression varies between carcinomas arising from different organs. MUC2 and MUC5AC are expressed predominantly in colon adenocarcinoma and mucinous ovarian adenocarcinoma, respectively; however, there are overlapping patterns. The combined phenotypes correctly predicted the primary site in 75% of cases. (323)

Cutaneous metastases occur in up to 11% of patients with renal cell carcinoma (RCC) and may be the presenting sign of disease. (324) CD10 is a metallomembrane endopeptidase first identified in acute lymphoblastic leukemia and also expressed in follicular lymphoma, Burkitt lymphoma, hepatocellular carcinoma, urothelial carcinoma, and prostatic carcinoma. (325) CD10 may be used to confirm the diagnosis of renal carcinomas, especially clear cell carcinomas and less so papillary or chromophobe RCCs. (326) The histologic differential diagnosis of metastatic RCCs includes primary cutaneous adnexal tumors. CD10 was evaluated in adnexal tumors by Bahrami et al324 to determine its utility in the distinction. Six percent of eccrine and apocrine neoplasms and 40% of sebaceous neoplasms demonstrated CD10 reactivity. Renal cell carcinoma metastatic to the skin can also simulate other clear cell lesions. Perna et al (327) evaluated CD10 in clear cell mimickers, including xanthomas, xanthelasma, and xanthogranulomas. CD10 stained most of those lesions; however, that was predominantly in a membranous pattern compared with the cytoplasmic pattern of RCC. CD10 was also expressed in 25% and 33% of balloon cell nevi and clear cell hidradenomas, respectively, but expression was limited to less than 10% of the clear cells. That overlap of reactivity between metastatic RCC and other cutaneous clear cell lesions is not present with the renal cell carcinoma marker (RCC-Ma). Perna et al328 found RCC-Ma expression in 62.5% of cutaneous RCC metastases and no reactivity in clear cell mimics, including xanthomas, xanthogranulomas, balloon cell nevi, clear cell hidradenoma, and sebaceous neoplasms. PAX8 is also a renal tumor marker with greater sensitivity than RCC-Ma. PAX8 also stains thyroid, gynecologic, and neuroendocrine tumors, including MCC. (329,330) Currently there are limited data on PAX8 reactivity in cutaneous tumors, other than a few porocarcinomas that may mimic metastatic RCC. (331)

INFECTIOUS DISEASE

Traditionally, diagnosis of infectious disease has been based on culture or microscopy, all with limitations. Molecular techniques, such as PCR, in situ hybridization, and IHC, have emerged as adjuvant tools to diagnose cutaneous infection. Immunohistochemistry may be particularly useful when organisms are difficult to detect on special stains, are present in low numbers, or are not culturable. (332) Established and commercially available antibodies of use in dermatopathology include CMV, Epstein-Barr virus, HSV1 and HSV2, VZV, HHV8, Bartonella henselae, Aspergillus spp, Candida albicans, Leishmaniasis, and Treponema pallidum. (332,333) The latter has shown better sensitivity than silver stains because of the significant background staining and reactivity of melanin. (332)

CONCLUSION

Numerous commercially available antibodies applicable to formalin-fixed, paraffin-embedded tissues have greatly expanded the armamentarium in dermatopathology. Immunohistochemistry will continue to be a rapidly evolving field with ever-increasing new antibodies. Growth in this area parallels the increasing understanding of pathogenesis and molecular genetics and paves the way for new therapeutic targets and improved prognostication.

Please Note: Illustration(s) are not available due to copyright restrictions.

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Tammie Ferringer, MD

Accepted for publication May 14, 2014.

From the Departments of Dermatology and Laboratory Medicine, Geisinger Medical Center, Danville, Pennsylvania.

The author has no relevant financial interest in the products or companies described in this article.

Reprints: Tammie Ferringer, MD, Departments of Dermatology and Laboratory, Medicine Geisinger Medical Center, 100 N Academy Ave, MC 01-31, Danville, PA 17822 (e-mail: tferringer@geisinger. edu).

* References 74, 89, 236, 238, 239, 242, 244-246.

([dagger]) References 21, 26, 61, 92, 231, 246, 260, 265, 266.

Caption: Figure 1. Adipophilin in sebaceous carcinoma (original magnification x600).

Caption: Figure 2. Sebaceous carcinoma. A, Normal nuclear staining with MLH1. B, Loss of nuclear staining of MSH2 and MSH6 (latter shown here), consistent with microsatellite instability and an association with Muir-Torre syndrome (original magnification x600 [A and B]).

Caption: Figure 3. A, Absent MART-1 reactivity in the dermal component of desmoplastic melanoma. B, Diffuse S100 expression in desmoplastic melanoma. C, Focal, horizontal, [S100.sup.+] cells in a scar. D, Diffuse nuclear SOX10 expression in desmoplastic melanoma (original magnifications x200 [A through C] and x100 [D]).

Caption: Figure 4. A, [MART-1.sup.+] pseudonests. B, [S100.sup.-] pseudonests (original magnification x200 [A and B]).

Caption: Figure 5. A, Subungual melanoma in situ. B, [S100.sup.-]. C, [MART-1.sup.+] (hematoxylin-eosin, original magnification x200 [A]; original magnifications x200 [B] and x100 [C]).

Caption: Figure 6. MART-1 (red)/MIB-1 (brown) in a malignant melanoma. Note the [MIB-1.sup.+] lymphocytes that lack cytoplasmic MART-1 expression, distinguishing them from the melanocytic component (original magnification x600).

Caption: Figure 7. A, HMB-45 gradient in a Spitz nevus. B, Minimal cells with both MART-1 (red) and MIB-1 (brown) staining in the dermal component of a Spitz nevus. C, Diffuse S100A6 reactivity in a Spitz nevus. D, Strong p16 expression in a Spitz nevus. E, Absence of p16 in malignant melanoma (original magnifications x100 [A, C, and D] and x200 [B and E]).

Caption: Figure 8. A, Dual staining with MART-1 (red) and D2-40 (brown) reveals lymphovascular invasion in a melanoma. B, Dual staining with MART-1 (brown) and D2-40 (red) confirms that the retraction space around a focus of melanoma is not endothelial lined (original magnification3600 [A and B]).

Caption: Figure 9. The mitotic "hot spot" of a melanoma highlighted with PHH3 (original magnification x600).

Caption: Figure 10. A, Lymph node with capsular nevus (left) and subcapsular metastatic melanoma (right) with S100. B, Subcapsular metastatic melanoma in a lymph node with SOX10. C, Capsular nevus with SOX10 (original magnifications x100 [A] and x200 [B and C]).

Caption: Figure 11. Paranuclear reactivity of CK20 in Merkel cell carcinoma (original magnification3600).

Caption: Figure 12. A, Cellular neurothekeoma hematoxylineosin). B, MITF nuclear expression (original magnification x100 [A]; original magnification x100 [B]).

Caption: Figure 13. A, Peritumoral CD34 accentuation in a dermatofibroma. B, CD34 expression in dermatofibrosarcoma protuberans with loss in the adjacent fibrosarcomatous component (left) (original magnification x20 [A and B]).

Caption: Figure 14. GLUT1 expression in endothelial cells of an infantile hemangioma. Note also the staining of red blood cells within the lumens (original magnification x200).

Caption: Figure 15. Sparse scattered [CD30.sup.+] cells in the inflammatory infiltrate of nodular scabies (original magnification x200).

Caption: Figure 16. A, CD30 expression in systemic anaplastic large cell lymphoma (ALCL) involving the skin. B, ALK-1 expression in systemic ALCL involving the skin (original magnification x200 [A and B]).
Table 1. Differentiation of Metastatic
Adenocarcinomas to Skin From Primary Cutaneous
Adnexal Neoplasm (PCAN)

Stain    Metastatic Adenocarcinoma     PCAN

D2-40                -                   +
CK5/6                -                   +
CK15                 -                   +
p63                  -                   +

Table 2. Differentiation of Morpheaform/Infiltrating
Basal Cell Carcinoma (BCC) and Desmoplastic
Trichoepithelioma (DTE)

Stain                          BCC       DTE

p75 (tumor cells)               -         +
PHLDA1 (tumor cells)            -         +
[CK20.sup.+] (Merkel cells)   Absent   Present
AR (tumor cells)                +         -

Abbreviations: PHLDA1, pleckstrin homology-like domain,
family A, member 1 protein; AR, androgen receptor.

Table 3. Differential Diagnosis of Atypical Spindle
Cell Lesions of the Dermis

Stain         Spindle Cell SCC   AFX   DM    LMS

AE1/AE3              +            -     -     -
CK903                +            -     -     -
p63                  +           -/+   -/+   -/+
Vimentin            -/+           +     +     +
S100                 -            -     +     -
p75                 -/+           -     +     -
Sox10                -            -     +    NR
Desmin               -            -     -     +
Calponin             -           -/+    -     +
SMA                  -           -/+    -     +
h-caldesmon          NR           -    NR     +
PC1                 -/+           +    -/+   +/-
CD10                +/-           +    -/+   -/+
S100A6               +            +    +/-    +

Abbreviations: AFX, atypical fibroxanthoma; DM, desmoplastic
melanoma; LMS, leiomyosarcoma; NR, not reported; PC1, procollagen
1; SCC, squamous cell carcinoma; SMA, smooth muscle antigen.

Table 4. Differentiation of Spitz Nevus
and Spitzoid Melanoma

Stain    Spitz Nevus                   Melanoma

S100A6   + diffusely                   + patchy
HMB-45   gradient from top to bottom   no gradient
MIB-1    <2%                           >10%
p16      +                             -

Table 5. "Blue" Cell Tumors

Stain   Lymphoma        MCC        MM    Mets    EWS/PNET
                                         Small
                                         Cell

S100       -             -          +      -       -/+
CD45       +a            -          -      -        -
CK20       -       + paranuclear    -      -        -
TTF1       NR            -          -      +        NR
FLI1       b            +/-        -/+     -        +

Abbreviations: EWS/PNET, Ewing sarcoma/primitive neuroectodermal
tumor; MCC, Merkel cell carcinoma;mets small cell, metastatic small
cell carcinoma of the lung; MM, malignant melanoma; NR, not
reported; TTF1, thyroid transcription factor 1.

(a) Unlike most lymphomas, lymphoblastic lymphoma is
often [CD45.sup.-].

(b) FLI1 is positive in lymphoblastic lymphoma, but other types
of lymphoma have not been closely studied.

Table 6. Cutaneous B-Cell Lymphomas

Stain                     BCL6   BCL2   CD10   MUM1

MZL                        -      +      -      - (a)
FCCL, primary cutaneous    +      b     -/+     -
DLBCL, leg type            +      +      -      +

Abbreviations: DLBCL, diffuse large B-cell lymphoma; FCCL, follicle
center cell lymphoma; MZL, cutaneous marginal zone lymphoma.

(a) Plasma cells within the MZL may be [MUM1.sup.+].

(b) T cells are BCL2+ and comprise a proportion of the lymphoid
cells in FCCL.

Table 7. Most Common Metastatic
Adenocarcinomas to Skin

Stain      Colon   Breast   Lung

TTF1         -       -       + (a)
CDX2         +       -       -
CK7          -       +       +
CK20         +       -       -
GCDFP-15     -      +/-      -
ER           -      +/-      -

Abbreviations: ER, estrogen receptor; GCDFP-15, gross cystic
disease fluid protein-15; TTF1, thyroid transcription factor-1.

(a) Mucinous adenocarcinomas/bronchioloalveolar carcinomas are
rarely positive with TTF1.
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Author:Ferringer, Tammie
Publication:Archives of Pathology & Laboratory Medicine
Date:Jan 1, 2015
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