Immunohistochemistry in Dermatopathology

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1 Immunohistochemistry in Dermatopathology Tammie Ferringer, MD Context. Immunohistochemistry is not a diagnostic test but a highly valuable tool that requires interpretation within a context. Objective. To review the current status and limitations of immunohistochemistry in dermatopathology. Data Sources. English-language literature published between 1980 and Conclusions. Although immunohistochemistry is rarely completely specific or sensitive, it is an important adjunctive technique in dermatopathology and can be helpful in a series of diagnostic dilemmas. (Arch Pathol Lab Med. 2015;139:83 105; doi: / arpa ra) Immunohistochemistry (IHC) cannot replace routine Metastatic renal cell carcinoma and eccrine-apocrine carcinomas have shown expression. 5,6 Xanthomatous lesions, 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. 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 CUTANEOUS ADNEXAL NEOPLASMS potentially causing confusion when differentiating those Sebaceous Neoplasms neoplasms from sebaceous carcinoma; however, a granular pattern and decreased intensity of expression favor the Differentiation of sebaceous carcinoma and squamous cell former tumors. 6,7 carcinoma (SCC) can be challenging. Ansai et al 1 found that The potentially aggressive course of sebaceous carcinoma androgen receptor (AR) and adipophilin (Figure 1) expression supports the diagnosis of sebaceous carcinoma, rather neoplasms. Cabral et al 8 sought to find an immunohisto- necessitates accurate differentiation from benign sebaceous than SCC. chemical method that could aid morphologic differentiation, Sebaceous epitheliomas (sebaceomas) are mostly composed of basaloid cells with a small proportion of mature have significantly increased nuclear staining with p53 and especially in small, partial biopsies. Sebaceous carcinomas sebocytes. Thus, there is histologic overlap between the Ki-67 (MIB-1) and reduced levels of BCL2 and p21, in basaloid cells of sebaceomas and those of basal cell comparison to sebaceous adenomas and sebaceous epitheliomas. The p53 protein is a tumor suppressor. Mutations carcinoma (BCC). Although IHC is not commonly necessary in differentiation, epithelial membrane antigen (EMA) lead to an abnormally stable but inactive p53 protein that highlights most mature sebocytes in sebaceoma, whereas can be detected immunohistochemically by nuclear staining. its expression is uncommon in BCC. 2,3 Reactivity for Sebaceous carcinomas are associated with increased, adipophilin in sebaceous tumors can also distinguish them presumably defective, p53. Therefore, decreased functional from BCC. 1 The utility of Ber-EP4 in this setting is p53 and p21 tumor suppression may allow for tumor controversial; although BCCs stain positive for Ber-EP4, progression and loss of BCL2 suggests improper regulation there is varying literature on its reactivity in sebaceous of apoptosis. Ki-67 is a nuclear marker of cell proliferation. lesions. 2,4 In some cases, IHC can serve as surrogate protein markers Adipophilin is a protein on the surface of intracellular lipid for underlying genetic events. Sebaceous gland neoplasms droplets and thus shows expression in sebaceous lesions, are relatively rare, and that diagnosis should raise the particularly well-differentiated lesions. Caution is necessary possibility of Muir-Torre syndrome (MTS). The dermatopathologist may be the first to consider the possibility of this when using adipophilin to distinguish sebaceous neoplasms from other cutaneous lesions with clear cell histology. syndrome because, in some patients, only a single sebaceous tumor is the first indication. Muir-Torre syndrome is an autosomal-dominant disease associated with Accepted for publication May 14, From the Departments of Dermatology and Laboratory Medicine, multiple keratoacanthomas and visceral malignancies, Geisinger Medical Center, Danville, Pennsylvania. especially genitourinary and gastrointestinal. Less common The author has no relevant financial interest in the products or associations include tumors of the breast and lung. Muircompanies described in this article. Torre syndrome is most frequently caused by mutational Reprints: Tammie Ferringer, MD, Departments of Dermatology and Laboratory, Medicine Geisinger Medical Center, 100 N Academy inactivation of mismatch repair genes MSH2 and MLH1 Ave, MC 01-31, Danville, PA ( tferringer@geisinger. with resultant microsatellite instability. Other mismatch edu). repair proteins can be involved, such as MSH6 and PMS2. Arch Pathol Lab Med Vol 139, January 2015 Immunohistochemistry in Dermatopathology Ferringer 83

2 Figure 1. Adipophilin in sebaceous carcinoma (original magnification 3600). 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 3600 [A and B]). 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 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 84 Arch Pathol Lab Med Vol 139, January 2015 Immunohistochemistry in Dermatopathology Ferringer

3 Table 1. Differentiation of Metastatic Adenocarcinomas to Skin From Primary Cutaneous Adnexal Neoplasm (PCAN) Stain Metastatic Adenocarcinoma PCAN 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 D2-40 þ CK5/6 þ CK15 þ Sclerosing Epithelial Neoplasms p63 þ Differentiation of sclerosing epithelial neoplasms is not only of academic interest but also paramount to clinical p63 reactivity has also been supported in other studies management, and diagnosis is often requested on small, Plumb et al 24 similarly found CK5/6 expression in more than superficial biopsies. Various immunohistochemical markers 95% of PCANs, albeit most tumors studied were benign. In have been studied to help distinguish desmoplastic trichoepitheliomas (DTE), infiltrating or morpheaform BCCs, and general, metastatic adenocarcinomas expressed CK5/6 in only 33% (9 of 27) of the cases, predominantly with weak microcystic adnexal carcinomas, including CD23, CD5, intensity. However, metastatic breast carcinoma was reactive for CK5/6 in almost half of their cases. 24 pleckstrin homology-like domain, family A, member 1 CD10, CD34, CK20, CK15, stromelysin-3, BCL2, AR, protein (PHLDA1), p75 neurotrophin receptor (p75ntr), Ivan et al 25 extended the analysis of p63 to include fibroblast activation protein (FAP), Ber-EP4, p63, and metastases from adnexal carcinomas and found that 91% of others (Table 2). Much of the available data are based PCANs were strongly marked with p63 and, excluding on a few cases reported by a single group of authors and apocrine and mucinous carcinoma, their metastases labeled require additional validation. Conflicting results were found similarly. with the same marker in other studies. A few deserve further Podoplanin (D2-40) expression is also a useful adjunct in mention, but the histopathologic criteria and clinical data differentiating adenocarcinomas that have metastasized to remain the current gold standard. the skin from PCAN. Reactivity with D2-40 is seen in The panel of AR and CK20 has proven useful in primary cutaneous carcinomas and skin adnexal tumors but distinguishing DTE and morpheaform BCC. 35,39 CK20 þ 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 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 (Table 1). noted in classic or conventional trichoepitheliomas. 48 Several studies have attempted to identify immunomarkers Pagetoid Tumors to differentiate conventional trichoepithelioma or trichoblastoma from nonaggressive forms of BCC. Although Intraepidermal lesions with a pagetoid distribution can be there are supporting data for use of CD34 and BCL2 in this diagnostically difficult. CK7 positivity reportedly differentiates Paget disease of the breast and extramammary Paget setting, minimal research has been conducted evaluating those markers in the setting of sclerosing epithelial disease (EMPD) from pagetoid Bowen disease and melanoma in situ; however, the marker is not 100% sensitive, mann et al 42 found the stroma of DTEs was positive for neoplasms. As with conventional trichoepitheliomas, Kirch- and occasional cases of CK7 þ pagetoid Bowen disease have CD34, differentiating it from the negative staining stroma of been described. 27,28 Similarly CAM 5.2 expression favors morpheaform BCC and microcystic adnexal carcinoma; Paget disease or EMPD, rather than SCC in situ, but however, Costache et al 39 failed to identify stromal staining reactivity has been reported in both. 28,29 Sellheyer and in any of their 19 DTEs. BCL2 tends to show expression only Krahl 30 proposed the addition of Ber-EP4 to the panel used to evaluate pagetoid cutaneous tumors. In their study, 30 Ber- EP4 labeled all cases of EMPD but failed to label Bowen 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 disease and melanoma in situ. Other authors 31 have BCC make that distinction nebulous, as seen in the identified strong nuclear p63 in pagetoid SCC in situ, in contrast to no reactivity in EMPD. 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 Table 2. Differentiation of Morpheaform/Infiltrating weak expression, whereas DTEs are strongly and diffusely Basal Cell Carcinoma (BCC) and Desmoplastic positive. 44,45 p75 is typically expressed in the outer root Trichoepithelioma (DTE) sheath of follicles; therefore, reactivity in DTEs supports Stain BCC DTE their classification as a follicular hamartoma mimicking that p75 (tumor cells) þ portion of the follicle. Microcystic adnexal carcinomas PHLDA1 (tumor cells) þ cannot be reliably distinguished with p75 because nearly CK20 þ (Merkel cells) Absent Present one-half are strongly positive. 44 PHLDA1 is a hair-follicle AR (tumor cells) þ stem-cell marker in the bulge area of the follicle. Reactivity Abbreviations: PHLDA1, pleckstrin homology-like domain, family A, for that marker in more than 50% of tumor cells member 1 protein; AR, androgen receptor. characterizes DTEs versus the lack or minimal expression Arch Pathol Lab Med Vol 139, January 2015 Immunohistochemistry in Dermatopathology Ferringer 85

4 Table 3. Differential Diagnosis of Atypical Spindle Cell Lesions of the Dermis Stain Spindle Cell SCC AFX DM LMS AE1/AE3 þ CK903 þ p63 þ /þ /þ /þ Vimentin /þ þ þ þ S100 þ p75 /þ þ Sox10 þ NR Desmin þ Calponin /þ þ SMA /þ þ h-caldesmon NR NR þ PC1 /þ þ /þ þ/ CD10 þ/ þ /þ /þ S100A6 þ þ þ/ þ Abbreviations: AFX, atypical fibroxanthoma; DM, desmoplastic melanoma; LMS, leiomyosarcoma; NR, not reported; PC1, procollagen 1; SCC, squamous cell carcinoma; SMA, smooth muscle antigen. 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 Ber- EP4 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 papillomavirus associated 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 (34bE12) and CK5/6 to be sensitive markers in this context; however, some spindle cell SCCs do not stain with either 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 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 86 Arch Pathol Lab Med Vol 139, January 2015 Immunohistochemistry in Dermatopathology Ferringer

5 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 þ cells in a scar. D, Diffuse nuclear SOX10 expression in desmoplastic melanoma (original magnifications 3200 [A through C] and 3100 [D]). Figure 4. A, MART-1 þ pseudonests. B, S100 pseudonests (original magnification 3200 [A and B]). 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 As with all IHC markers, careful localization of the antibody staining should be established. PC1 þ 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 þ dendritic cells colonize the lesion (possibly Langerhans cells), 81,82 but the neoplastic cells are generally S100, 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 þ 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 þ spindle cells complicating differentiation, particularly of reexcision specimens, but unlike DM, the S100 þ cells are only focal and are predominantly in a horizontal pattern (Figure 3, C) Arch Pathol Lab Med Vol 139, January 2015 Immunohistochemistry in Dermatopathology Ferringer 87

6 p75, also known as nerve growth factor receptor, is a neural crest marker expressed in most desmoplastic and neurotropic melanomas, often more intensely than S ,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 þ 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 S 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 þ, 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 þ 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 ,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 KBA 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 88 Arch Pathol Lab Med Vol 139, January 2015 Immunohistochemistry in Dermatopathology Ferringer

7 melanocytes, melanophages, or pigmented keratinocytes only stain green-blue 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 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 þ, S100, 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 case 137 of SOX10 junctional nests in the setting of erythema dyschromicum perstans. MITF has shown similar issues with these pseudonests 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 MIB- 1 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 MIB- 1 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 onethird 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) 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 Arch Pathol Lab Med Vol 139, January 2015 Immunohistochemistry in Dermatopathology Ferringer 89

8 Figure 5. A, Subungual melanoma in situ. B, S100. C, MART-1 þ (hematoxylin-eosin, original magnification 3200 [A]; original magnifications 3200 [B] and 3100 [C]). Figure 6. MART-1 (red)/mib-1 (brown) in a malignant melanoma. Note the MIB-1 þ lymphocytes that lack cytoplasmic MART-1 expression, distinguishing them from the melanocytic component (original magnification 3600). 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 Table 4. Differentiation of Spitz Nevus Stain and Spitzoid Melanoma Spitz Nevus Melanoma Melanoma Prognosis Prognostic indicators of melanoma on H&E stained S100A6 þ diffusely þ patchy sections include ulceration and tumor depth, but there has HMB-45 gradient from top to bottom no gradient been increasing study into the use of IHC-assisted MIB-1,2%.10% prognostication. Identification of lymphatic invasion can p16 þ be increased with the use of D2-40 (podoplanin), a 90 Arch Pathol Lab Med Vol 139, January 2015 Immunohistochemistry in Dermatopathology Ferringer

9 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 3100 [A, C, and D] and 3200 [B and E]). lymphatic endothelial marker, or CD34, and recognition is associated with decreased disease-free and overall survival 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 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 magnification 3600 [A and B]). Figure 9. The mitotic hot spot of a melanoma highlighted with PHH3 (original magnification 3600). 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 3100 [A] and 3200 [B and C]). Arch Pathol Lab Med Vol 139, January 2015 Immunohistochemistry in Dermatopathology Ferringer 91

10 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 320 magnification, in contrast to the 340 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 PHH 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 PHH3- determined 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 ,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 þ 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 ¼ 15) in this small study 176 lacked immunoreactivity for MIB-1, but all metastases (n ¼ 40) were positive for MIB-1, usually with more than 10% of the cells being MIB-1 þ. 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 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 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. 92 Arch Pathol Lab Med Vol 139, January 2015 Immunohistochemistry in Dermatopathology Ferringer

11 Figure 11. Paranuclear reactivity of CK20 in Merkel cell carcinoma (original magnification 3600). 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 þ /CK7 pattern, CK20 /CK7 þ 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 Table 5. Blue Cell Tumors Stain Lymphoma MCC MM Mets Small Cell EWS/PNET S100 þ /þ CD45 þ a CK20 þ paranuclear TTF1 NR þ NR FLI1 b þ/ /þ þ 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. b FLI1 is positive in lymphoblastic lymphoma, but other types of lymphoma have not been closely studied. lymphoma, and CD56 as in natural killer T-cell lymphoma. 197, 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, 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 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 synaptophysin. 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 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 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 CD ,227 Arch Pathol Lab Med Vol 139, January 2015 Immunohistochemistry in Dermatopathology Ferringer 93

12 Figure 12. A, Cellular neurothekeoma (hematoxylin-eosin). B, MITF nuclear expression (original magnification 3100 [A]; original magnification 3100 [B]). 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 þ, 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 þ and factor XIIIa, and DF is CD34 and factor XIIIa þ ; however, that is not absolute 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 CD 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 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, 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- * References 74, 89, 236, 238, 239, 242, Arch Pathol Lab Med Vol 139, January 2015 Immunohistochemistry in Dermatopathology Ferringer

13 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 320 [A and B]). Figure 14. GLUT1 expression in endothelial cells of an infantile hemangioma. Note also the staining of red blood cells within the lumens (original magnification 3200). Figure 15. Sparse scattered CD30 þ cells in the inflammatory infiltrate of nodular scabies (original magnification 3200). 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 3200 [A and B]). 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, 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, 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 CD 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 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 Arch Pathol Lab Med Vol 139, January 2015 Immunohistochemistry in Dermatopathology Ferringer 95