Sebaceous Carcinoma of the Eyelid Carlos Prieto-Granada, MD, and Paul Rodriguez-Waitkus, MD, Phd

Helpful immunostains for the

differential diagnosis of PSC include EMA,

Ber-Ep4, AR, and adipophilin.

Photo courtesy of Lynn E. Harman, MD. Half Dome From Glacier Point.

Sebaceous Carcinoma of the Eyelid Carlos Prieto-Granada, MD, and Paul Rodriguez-Waitkus, MD, PhD

Background: Periocular sebaceous carcinoma (PSC) is a rare but aggressive neoplasm that tends to clinically and histopathologically mimic other conditions. PSC can be challenging to diagnose using histomorphology alone given its overlap with 2 more common tumors that occur in this area (basal cell carcinoma [BCC] and squamous cell carcinoma [SCC]). Use of immunohistochemistry can help resolve this differential diagnosis. Methods: A review of the literature was performed, focusing on the epidemiology, morphology, and immunohistochemical features of PSC. Results: The most useful immunostains in the differential diagnosis of PSC are epithelial membrane antigen, Ber-Ep4, androgen receptor (AR), and adipophilin. To discern PSC from BCC, one should use EMA, Ber-Ep4, AR, and adipophilin, whereas discerning PSC from SCC can be achieved by evaluating AR and adipophilin. In addition, p53 and ERBB2 (formally known as HER2/neu) are other potentially useful immunohistochemical markers for the differential diagnosis of PSC. Conclusions: Use of new immunohistochemical techniques, as well as the elucidation of molecular alterations, such as the presence of ERBB2 amplification, will advance our understanding of PSC.

Introduction 5% of all malignant neoplasms arising in the periocu- Cutaneous sebaceous carcinoma is a rare disease that lar region, followed basal cell carcinoma (BCC), which makes up an estimated 0.05% of all cutaneous malig- makes up 90% of eyelid malignancies.4-6 nancies.1,2 However, because the eyelid and periocular In general, periocular sebaceous carcinoma (PSC) regions harbor a high density of sebaceous glands ei- presents in the elderly (average of 70 years of age).1-3 ther associated with eyelashes (Zeis) or the tarsal plate Predisposing factors, such as immunosuppression, pri- (meibomian), it is not surprising that, when consider- or history of radiotherapy to the head and neck, and ing all cutaneous sebaceous carcinomas, up to 40% of Muir–Torre syndrome, occur in a minority of patients.7 these tumors arise in this particular anatomical site.3 PSC can clinically present as a diffuse thickening of the In the United States, sebaceous carcinoma represents eyelid, often exhibiting inflammatory features. More advanced cases are characterized as a discrete, palpe- bral mass. The clinical presentation of early-stage PSC From the Departments of Dermatology & Cutaneous Surgery (CP- has the potential of mimicking non-neoplastic lesions, G, PR-W) and Pathology & Cell Biology (PR-W), Morsani College of Medicine, University of South Florida, Tampa, Florida. notably chalazion, keratoconjunctivitis, and other in- 5,8,9 Address correspondence to Carlos Prieto-Granada, MD, Universi- flammatory, non-neoplastic processes. Thus, those ty of South Florida, 13330 USF Laurel Drive, Fourth floor, Tampa, with PSC are at an increased risk for delayed diagnosis. FL 33612. E-mail: [email protected] Historically, a diagnosis of PSC was associ- Submitted August 15, 2015; accepted December 17, 2015. ated with a poor prognosis and a mortality rate that No significant relationships exist between the authors and the 10,11 companies/organizations whose products or services may be ref- reached 50%. However, with increased disease erenced in this article. awareness and the introduction of better diagnostic

126 Cancer Control April 2016, Vol. 23, No. 2 techniques, rates of survival have improved (mortal- In a similar fashion to cutaneous sebaceous carci- ity rates of 2%–11%).4,11,12 Early identification is crucial noma, cytologically PSC tumors are composed of 3 ba- and relies on a high level of clinical suspicion, together sic cell types: basaloid, sebaceous/vacuolated cells, and with an accurate histopathological diagnosis that often intermediate forms. These cell types are usually inter- depends on small or limited specimens on biopsy. mixed at varying ratios, which will determine the level of differentiation of a given tumor, among other factors Histomorphology (see Figs 2D, 2E, and 2G). Broadly speaking, the per- When examining the invasive component of PSC at centage of basaloid cells will be inversely correlated low magnification, 4 basic morphological patterns with the level of differentiation, which ranges from well- can be recognized: trabecular, lobular, papillary, and differentiated tumors to poorly differentiated and ana- BCC-like (Figs 1 and 2).7 Similar to BCC, a mixture of plastic/de-differentiated tumors. 2 or more of these patterns can be seen in a single le- Another factor to consider when grading a lesion is sion. However, all of the classic features of BCC are the architecture of the tumor. Well-differentiated PSC tu- not found, such as peripheral palisading, tumor–stro- mors are often nodular, whereas poorly differentiated mal cleft artifacts, and mucinous/myxoid spindle cell tumors are more infiltrative. Taking into account all of stroma. The trabecular pattern of PSC has a tendency these features, including grading, architecture, presence to reveal areas of central comedo-like necrosis. An in or absence of an in situ component/pagetoid spread, as situ component of PSC may be encountered, either as a well as presence of spread to distant sites, the following standalone lesion as well as adjacent or overlying an in- 4-tiered classification has been proposed13: PSC in situ, vasive lesion (see Figs 1C–F). The intraepithelial lesions low-grade, high-grade infiltrating PSC with or without are often bowenoid in appearance and can exhibit exu- pagetoid spread, and PSC with extraocular/extracutane- berant pagetoid spread (see Figs 1D and 1E). They can ous spread, including distant metastases. also radially spread by undermining the normal con- In addition to its ability to clinically mimic other le- junctival epithelium (see Fig 1F). Keratinization is not sions, PSC can also overlap the histopathology of several usually a feature of PSC. The presence of changing ke- benign and malignant entities encountered in the peri- ratinization should prompt the health care professional ocular region. Among the benign processes, the health to consider squamous cell carcinoma (SCC) with seba- care professional should consider sebaceous adenoma,14 ceous differentiation rather than PSC.7 follicular adnexal tumors (eg, desmoplastic trichoepithe-

A B C

D E F

Fig 1A–F. Composite demonstrating different morphological patterns of PSC. (A) Basal cell carcinoma–like type of configuration. H & E, ×40. (B) Complete with superficial ulceration with the presence of vacuolated cells. H & E, ×200. (C, D) Usual presentation of PSC with extensive intraepidermal involvement exhibiting pagetoid spread. A subjacent main tumor mass is present with tumor cells expanding into the epidermis through the follicular structures. C: H & E, ×20. D: H & E, ×100 (E) High-power imaging shows the nuclear features of PSC: vesicular chromatin with prominent yet small nucleoli. H & E, ×200. (F) The radial extension of PSC cells through normal epithelium reveals conjunctival mucosa undermined by tumor cells. H & E, ×40. H & E = hematoxylin and eosin, PSC = periocular sebaceous carcinoma.

April 2016, Vol. 23, No. 2 Cancer Control 127 A B C

D E F

G H I

Fig 2A–I. — Panel exhibiting immunohistochemical staining patterns of 3 markers useful for the differential diagnosis of PSC. (A–C) Epithelial membrane antigen often exhibits patchy but intense membranous and cytoplasmic positivity in PSC. An internal control is present and represented by normal sebaceous glands (C, arrow). A: H & E, ×20. B: H & E, ×20. C: H & E, ×200. (A, D, E) Although this case exhibits a basal cell carcinoma–like pattern (D and E: H & E, ×200), (F) Ber-Ep4 is completely negative. F: H & E, ×200. An internal control is present in the germinative portion of an adjacent follicular structure (arrow). (G) The presence of intermediate cells with lipid vacuoles (arrows) is highlighted with adipophilin. G: H & E, ×400. (H, I) A “vacuolar-type” staining pattern is seen. H: H & E, ×200. I: H & E, ×400. H & E = hematoxylin and eosin, PSC = periocular sebaceous carcinoma. lioma),15 trichoadenoma,16 and squamous papilloma.17-19 Merkel cell carcinoma, which have washed-out, neu- When considering malignancies, the 2 main possibilities roendocrine-type chromatin. By contrast, melanoma are BCC — particularly with sebaceous differentiation cells often display large nuclei with vesicular chroma- — and SCC with sebaceous differentiation (rare). The tin and macronucleoli. In a proportion of poorly dif- latter also includes a basaloid variant. A rare but plau- ferentiated types of PSC (approximately 23%–77%), sible differential diagnostic possibility — particularly morphological assessment will be hampered by the for poorly differentiated types of PSC — is Merkel cell presence of overlapping features with other entities carcinoma, which is a malignant neoplasm with a predi- included the differential diagnosis.4,22,23 In such situa- lection to arise in the head and neck area.20,21 When con- tions, immunohistochemical stains may be helpful. fronted with in situ PSC with abundant pagetoid spread, other pagetoid malignancies such as pagetoid SCC in Immunohistochemistry situ, melanoma in situ, and intraepidermal Merkel cell Among the malignant tumors that arise in the perioc- carcinoma should be considered. ular region, the 3 most common are BCC, squamous Morphologically, the nuclear features in PSC tu- cell carcinoma (SCC), and PSC.5,6 Several immunohis- mor cells have a distinctive shape — namely, they tochemical markers are useful in the differential diag- are round, oval, or indented — and contain fine, ink- nosis, including epithelial antigens, as are hormonal drop, or smudgy chromatin with 1 or 2 small nucleoli markers and other markers highlighting fat vacuole (see Figs 1E and 2G).7 These features are not found in production. Immunohistochemical stains will also re-

128 Cancer Control April 2016, Vol. 23, No. 2 flect subjacent molecular alterations. The different expres- Table. — Three Commonly Used Immunohistochemical Markers for the Differential Diagnosis of Periocular Sebaceous Carcinoma sion profiles using the most useful markers for PSC, BCC, Type of Carcinoma Epithelial Ber-Ep4 Androgen Adipophilin p53 ERBB2 and SCC are summarized in Membrane Receptor Antigen the Table. Periocular sebaceous + −/+ + + + + Epithelial Markers Basal cell − + +/− − −/+ − Epithelial antigens are tools Squamous cell + − − − +/− − that can be used to help in the differential diagnosis of PSC. The 2 epithelial markers deemed to be most useful in in epithelial malignancies, the ability of cytokeratins the differential diagnosis of PSC, BCC, and SCC are epi- to discriminate among the common tumors around the thelial membrane antigen (EMA) and Ber-Ep4.7,24 eye is limited. Epithelial Membrane Antigen: Also known as Thomsen–Friedenreich (T) and its Precursor mucin 1, cluster designation 227, and cancer antigen (Tn) Antigen: These markers are composed of O- 15-3 (particularly when detected in serum), EMA is a glycosylated, mucin-type complex carbohydrates and 75-kDa transmembrane glycoprotein coded by the are — in addition to the glycophorin protein system 1q21 locus. It is commonly used as an epithelial marker — part of the MNS blood group system. They are ex- in conjunction with cytokeratins. In normal skin and pressed in carcinomas from a visceral origin while be- conjunctiva, EMA is expressed in glandular structures, ing hidden from the immune system via the addition of including sebocytes (see Fig 2C, arrow), as well as in covalently linked carbohydrates and sialic acid in the squamous epithelium, but not in follicular structures.25 majority of normal tissues, with the exception of ma- EMA exhibits a membranous and cytoplasmic signal ture sebocytes, the luminal aspect of the sweat glands, (see Figs 2B and 2C) and is prominently expressed in and in the intercellular junctions of the spinous layer of the majority of cases of PSC and SCC, while being pre- the epidermis.31,32 A small study specifically testing the dominantly negative in BCC.7,24 immunohistochemical expression of the Thomsen– Ber-Ep4: Ber-Ep4 is a monoclonal antibody di- Friedenreich (T) antigen found a strong expression of rected toward the epithelial cell adhesion molecule, a this marker in the majority of sebaceous carcinoma transmembrane glycoprotein coded by the 2p21 locus cases (n = 8) tested as well as in the sebaceous ade- and normally expressed at the basolateral membrane of noma (n = 15) and sebaceoma (n = 9) samples, while cells by the majority of epithelial tissues, except in adult being negative in the all but 1 of the tested BCC cases squamous epithelium and by some specific epitheli- (n = 13); only 1 case of BCC was positive for sebaceous um cell types (eg, hepatocytes).26,27 This marker is ex- differentiation.13,32 Although no follow-up studies were pressed in the secretory portion of eccrine glands and found in the literature regarding this marker, one could follicular germinative cells in normal skin and eyelids consider incorporating T-antigen immunohistochemis- (see Fig 2F, arrow).28 This antibody exhibits a predomi- try to resolve this differential diagnosis. nant membranous pattern of staining; it is most useful in distinguishing BCC from PSC and SCC because it is Lipid-Processing Markers frequently positive for Ber-Ep4 (70%–100%).24,28 By con- Sebaceous carcinomas are related to the produc- trast, PSC and SCC are negative for Ber-Ep4 in 74% to tion of lipids (mainly triglycerides). Historically, the 94% and 100% of cases, respectively (see Fig 2F).24,28 health care professional was only able to rely on the Cytokeratin: Intermediate filaments known as cy- often cumbersome histochemical demonstration of tokeratins are expressed in carcinomas (and rarely in a lipids utilizing Oil red O or Sudan black IV, inconve- few mesenchymal tumors). Because cytokeratin is also nient stains that required a frozen tissue sample and expressed in other entities included in the differential were associated with both poor rates of sensitivity diagnosis of PSC, expression of CAM 5.2 (a cytokeratin (40%) and stain fading over time.33 However, another cocktail) is not particularly useful to resolve the differ- group of immunohistochemical markers demonstrat- ential diagnosis for PSC.29 Cytokeratin 7 is expressed in ing lipid production are now available. This group of a higher percentage in PSC cases (88%) compared with markers is composed of 3 lipid droplet–associated pro- BCC cases (28%); this is especially true in SCC cases teins comprised of the so-called perilipin, adipophilin, (9%), so it could be used as an adjunctive tool.24 In addi- and TIP47 group (also known as perilipins 1, 2, and 3, tion, the expression of cytokeratin 19 may be helpful in respectively). These molecules are associated with the distinguishing sebaceous carcinomas (17% focally posi- phospholipid monolayer membrane surrounding the tive) from BCC (64% strongly positive and 14% focally triglyceride core of the intracytoplasmic lipid droplets positive).30 However, given the ubiquity of cytokeratins and are related in the formation, maintenance, modifi-

April 2016, Vol. 23, No. 2 Cancer Control 129 cation, and involution of such structures.34 All of these glands.25,41,42 Because it is a transcription factor and markers appear specific for lipid droplets in normal a hormone receptor from the steroid class, its immu- tissues as well as in a variety of malignant neoplasms nohistochemical signal is predominantly nuclear (see with lipogenic features.28,35-38 Although adipophilin ap- Fig 3). This marker can be of particularly help when pears to be the most sensitive marker of the group, per- differentiating between PSC (predominantly positive, ilipin is considered to have superior rates of specificity, 33%–83%), SCC (predominantly negative, 90%–100%), and both immunostains have proven to be of particu- and BCC (occasionally negative, 50%–86%).27,28 lar utility in the differential diagnosis of PSC.7,23,25,33,39,40 Adipophilin: Also known as adipocyte differentia- Underlying Mutations/Genetic Aberrations tion–related protein and perilipin 2, adipophilin is a lip- p53 Protein: The p53 protein is a transcription fac- id vesicle–associated protein coded by a gene in 9p22.1. tor coded by TP53 (17p13.1), which induces apoptosis This marker is expressed in normal sebocytes. However, when DNA damage occurs. Approximately 67% of PSC when using this stain, the health care professional must tumors will harbor missense or nonsense TP53 muta- be aware of its different staining patterns, including its tions that are not of the ultraviolet (UV) signature type diffuse cytoplasmic signal pattern, which is often en- (unrelated to UV damage).43 Patients with germline countered in well-differentiated areas of PSC tumors; its TP53 mutations (Li–Fraumeni syndrome) frequently vacuolar, vesicular-rimming pattern (see Figs 2G and 2I); develop PSC, along with other malignancies that char- or the presence of both of these, thus highlighting the acterize the condition.44 Because the gene mutation intermediate cells of a PSC tumor; and a fine particu- rate is quite high in the setting of PSC, many of these late, granular, or dusty pattern (considered nonspecific tumors (40%–72%) will show an overexpression (ac- and could be found in other tumors [eg, SCC, BCC]). The cumulation) of p53 protein on immunohistochemistry health care professional must remember the poorly dif- translated by a strong nuclear signal.7,45 By contrast, ferentiated areas of PSC tumors will often be lipid-poor this pattern of p53 staining occurs in fewer than 20% and basaloid cell-rich; thus, they may be predominantly of BCC cases and 50% to 60% in SCC cases.7,46,47 Thus, negative or weakly positive for adipophilin in an overall some have advocated for use of p53 immunohisto- patchy pattern.7,23,25,33,39 Adipophilin can be useful when chemistry as a diagnostic aid in the differential diagno- differentiating PSC that is predominantly immunoreac- sis of PSC.7 Both the presence of TP53 mutations and tive, showing either vacuolar or diffuse patterns, or is predomi- A nantly negative or weakly positive with a finely granular pattern in the setting of BCC and SCC. Perilipin: Perilipin is a trans- membranous protein related to lipid vesicles coded by the 15q26 locus that is expressed in sebocytes. Perilipin appears to be more specific but less sensitive than adipophilin.33,39 The different patterns of staining are similar to those of adipophilin, and the health care professional could use B C this marker in cases for which adipophilin was unsatisfactory.

Androgen Receptor Androgen receptor (AR) is coded by Xq12 and forms part of a fam- ily of nuclear-located, steroid hormone transcription factors/ receptors that bind testoster- one and dihydrotestosterone. Fig 3A–C. — Pattern of immunohistochemical staining with androgen receptor. (A) In this case, In normal skin and conjunctiva, periocular sebaceous carcinoma exhibited a striking bowenoid appearance predominantly composed of basaloid cells. H & E, ×40. (B, C) Androgen-receptor immunohistochemical stain dem- AR is predominantly expressed onstrated diffuse and strong nuclear positivity in the tumor cells with a weak cytoplasmic blush. in follicular and adnexal struc- B: H & E, ×100. C: H & E, ×200. tures, especially in the sebaceous H & E = hematoxylin and eosin.

130 Cancer Control April 2016, Vol. 23, No. 2 the consequent p53 overexpression are more frequent- ting of PSC have been correlated with poor rates of sur- ly encountered in PSC than extraocular sebaceous car- vival.55,57 Loss, diminution, and aberrant E-cadherin ex- cinoma and are inversely correlated with the presence pression as well as CDH1 promoter methylation58 can of alterations of the mismatched repair (MMR) proteins also be found in both BCC59-62 and SCC,61,63 so these and Muir–Torre syndrome.45 findings preclude use of ZEB2 as a diagnostic marker Mismatched Repair Proteins: Assessing the ex- in this setting. pression of MMR proteins with immunohistochemistry for mutL homolog 1, mutS homolog 2, mutS ho- Conclusions molog 6, and PMS1 homolog 2 (MLH1, MSH2, MSH6 Several markers are differentially expressed in perioc- and PMS2, MMR system components) in cutaneous ular sebaceous carcinoma (PSC) compared with lesions neoplasms — particularly in sebaceous adenoma — in the differential diagnosis. A panel of immunohis- is becoming an easy and convenient way to screen for tochemical stains is recommended because different Muir–Torre syndrome, a variant of the Lynch syndrome combinations of immunoprofiles characterize these spectrum.48-50 However, by contrast to the extraocular entities, of which epithelial membrane antigen (EMA), forms of sebaceous neoplasms, most PSC tumors ap- Ber-Ep4, androgen receptor (AR), and adipophilin pear to be unrelated to loss of MMR expression. There- appear to be the most useful. An optimal stain panel fore, PSC should be considered as a poor “index lesion” to discriminate PSC from basal cell carcinoma (BCC) to screen for Muir–Torre syndrome.45,51,52 Nevertheless, is presented by EMA, Ber-Ep4, AR, and adipophilin, periocular sebaceous adenoma appears to follow the while distinguishing PSC from squamous cell carcino- same trend as extraocular sebaceous neoplasms in ma (SCC) can be achieved by exploring the expressions terms of its relationship with Muir–Torre syndrome.53 of AR and adipophilin. ERBB2 (formally known as HER2/neu): The 3 most typical immunophenotypes are the PSC has been shown to exhibit 2+ and 3+ patterns of following: (1) PSC is positive for EMA, AR, and adipo- ERBB2 (formally known as HER2/neu) immunohisto- philin but negative for Ber-Ep4, (2) SCC shows a pre- chemical positivity in up to 82% of cases, with 75% of dominant immunoprofile of EMA positivity but is neg- these cases showing ERBB2 amplifications by fluores- ative for AR, Ber-Ep42, and adipophilin, and (3) BCC is cence in situ hybridization.50 Based on the results of a predominantly negative for EMA and adipophilin but study reporting on a series of 14 cases, these findings positive for Ber-Ep4. Two other potential immunohis- have diagnostic value because both ERBB2 immuno- tochemical markers that may be useful in the differ- reactivity of more than 1+ and gene amplification are ential diagnosis are p53 and HER2/neu, both of which infrequently found in mimickers and they provide a will be predominantly expressed in PSC. Mismatched possible therapeutic avenue for PSC.50,54 repair protein immunohistochemical expression aber- Adhesion Protein Expression and Epithelial rations appear to be absent in the majority of cases of Mesenchymal Transition–Related Markers: Simi- PSC, because they appear to harbor TP53 mutations. lar to other types of aggressive carcinomas, such as PSC as an index case to screen of Muir–Torre syndrome lobular carcinoma of the breast, PSC (in particular, is currently not recommended. poorly differentiated PSC) has been shown to disrupt The introduction of new immunohistochemical the intercellular and cell stroma adhesion machin- stains for PSC and the expansion of our knowledge ery with loss or diminished membranous E-cadherin regarding the underlying molecular aberrations of and beta-catenin protein expression in 53% to 83% PSC, such as the presence of ERBB2 amplifications, and 56% to 61% of cases, respectively.55,56 Loss or di- represent an exciting new era in understanding of minished expression of these markers was correlated this entity. These advancements could facilitate the with the presence of the epigenetic promoter meth- early detection of PSC and possibly result in improved ylation of CDH1 (16q22.1).55 patient outcomes. Similarly, PSC also appears to overexpress ZEB2 in up to 68% of cases.57 ZEB2 is a transcription factor gene References closely related to the epithelial to mesenchymal transi- 1. Anderson HL, Joseph AK. A pilot feasibility study of a rare skin tion phenomenon that also interacts with CDH1-pro- tumor database. Dermatol Surg. 2007;33(6):693-696. 2. Kyllo RL, Brady KL, Hurst EA. Sebaceous carcinoma: review of the ducing gene repression and the subsequent decrease literature. Dermatol Surg. 2015;41(1):1-15. of E-cadherin expression. The epithelial to mesenchy- 3. Dasgupta T, Wilson LD, Yu JB. A retrospective review of 1349 cases of sebaceous carcinoma. Cancer. 2009;115(1):158-165. mal transition is an embryonic process to which poorly 4. Shields JA, Demirci H, Marr BP, et al. 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