Clear cell melanoma is a rare clear cell malignancy. Accurate diagnosis of clear cell melanoma requires integration of immunohistochemical and morphologic findings, with molecular studies to rule out clear cell sarcoma. The differential diagnosis includes melanoma, carcinoma, perivascular epithelioid cell tumor, and epidermotropic clear cell sarcoma. We use a case of a lesion on the helix of an 86-year-old man as an example. Histologic examination revealed an ulcerated clear cell malignant tumor. Tumor cell cytoplasm contained periodic acid-Schiff–positive, diastase-sensitive glycogen. Tumor cells showed positive labeling for S100, HMB-45, and Melan-A, and negative labeling for cytokeratins, p63, and smooth muscle actin. Molecular studies demonstrated BRAF V600E mutation, copy gains at the 6p25 (RREB1) and 11q13 (CCND1) loci, and absence of EWSR1-ATF1 fusion. These findings supported a diagnosis of clear cell melanoma. The rare pure clear cell morphology occurs due to accumulation of intracytoplasmic glycogen. We review the differential diagnosis of clear cell melanoma and describe the utility of immunohistochemical and molecular studies in confirming this diagnosis.
Clear cell melanoma is a rare clear cell malignancy. Accurate diagnosis of clear cell melanoma requires integration of immunohistochemical and morphologic findings, with molecular studies to rule out clear cell sarcoma. We use a clinical example to introduce this topic.
An 86-year-old man with a history of multiple cutaneous squamous cell carcinomas presented with an ulcerated tumor of unknown duration on the left helix. Clinically, the lesion resembled a squamous cell carcinoma. A shave biopsy was performed.
PATHOLOGIC AND MOLECULAR FINDINGS
At low magnification (Figure 1, A) the tumor extended from the ulcerated epidermis into the subcutis and consisted of sheets and poorly formed nests of atypical clear cells. At high magnification the tumor displayed marked cytologic atypia, numerous mitotic figures, foci of tumor necrosis, and poorly formed nests (Figure 1, B). There was pagetoid spread of tumor cells as nests and single cells (Figure 1, C). Multinucleated giant cells were not identified.
On immunohistochemical staining, the neoplastic cells were diffusely positive for S100 (Figure 1, D) and Melan-A. HMB-45 expression was strong and diffuse, without evidence of diminished expression with depth (Figure 1, E). Expression of p16 was retained throughout the tumor. Cyclin D1 displayed strong, diffuse expression. The neoplastic cells were negative for cytokeratin (CK MNF 116; Figure 1, F), smooth muscle actin (Figure 1, G), and p63. The tumor cells showed cytoplasmic staining with periodic acid–Schiff (PAS) that was sensitive to diastase treatment (Figure 1, H and I).
Given these findings, the differential diagnosis rested between clear cell melanoma (CCM) and clear cell sarcoma. Clear cell sarcoma is classically characterized by a t(12;22) translocation resulting in EWSR1-ATF1 fusion.1–3 Reverse transcription–polymerase chain reaction (RT-PCR) was negative for EWSR1-ATF1 fusion transcript variants 1 to 3. Consistent with PCR findings, no EWSR1 rearrangement was detected by dual-color, break-apart fluorescence in situ hybridization (FISH; Figure 2, A). Polymerase chain reaction amplification and sequencing of BRAF exon 15 from tumor DNA demonstrated heterozygous V600E mutation (Figure 2, B). Single-nucleotide polymorphism array interrogation identified copy number changes, including gains at 6p25 (RREB1) and 11q13 (CCND1; Figure 2, C, and Supplemental Table S1; see supplemental material file at www.archivesofpathology.org in the October 2014 table of contents), both of which are frequent sites of copy gain in melanoma.4,5 Integrating these morphologic, immunophenotypic, and molecular findings, including the lack of EWSR1 rearrangement, the diagnosis of CCM was made.
Clinicopathologic Features of CCM
Clear cell melanoma is an extremely rare variant of melanoma that has been reported at cutaneous and extracutaneous sites.6–10 Using immunohistochemistry, classic melanoma markers, such as S100 and Melan-A, are expressed. Cytoplasmic glycogen is demonstrated by diastase-sensitive PAS staining. Molecular studies of CCM have been limited to date. Cytogenetic analysis of one case of uveal CCM revealed gains at 6p and 8q, with losses at 6q and 16p.6 A FISH analysis of a case of acral CCM demonstrated a chromosomal break at 12q13 near ATF1.11 Given the limited data available, it is unknown whether CCM has recurrent genetic changes distinct from other melanoma subtypes. However, the BRAF V600E mutation and chromosomal gains at 6p and 11q identified in our case are common findings in conventional melanoma.12
Pathologic Differential Diagnosis
The differential diagnostic considerations of a clear cell lesion in the skin are broad and include melanocytic lesions (balloon cell nevus or balloon cell melanoma), epithelial neoplasms (benign adnexal neoplasm and carcinoma with clear cells), xanthomas, and various mesenchymal neoplasms (eg, perivascular epithelioid cell tumor or clear cell sarcoma; Table 1).13,14 Although diagnosis is often straightforward if clear cell change is focal in an otherwise conventional lesion, lesions predominantly composed of clear cells present a diagnostic challenge. Morphologic analysis of architecture, epidermal involvement, and degree of cytologic atypia may help to narrow the differential diagnosis (Table 2). Immunohistochemical stains are essential for accurate diagnosis in many cases. A panel of immunohistochemical stains including cytokeratins, smooth muscle actin, S100, and additional melanocytic markers (eg, Melan-A) is helpful in assigning epithelial, melanocytic, or mesenchymal differentiation (Table 3).
Carcinomas with Clear Cells
A number of carcinomas may display predominant clear cells in the skin, including squamous cell carcinoma (Figure 3, A) and adnexal carcinomas, such as tricholemmal carcinoma, sebaceous carcinoma, and hidradenocarcinoma. Like melanoma, squamous and adnexal carcinomas may have an in situ component with pagetoid scatter.13 Sebaceous carcinoma can often be diagnosed without the aid of immunohistochemistry, by recognition that the clear cell component displays the distinctive features of sebaceous differentiation characterized by microvesicular cytoplasm and scalloped nuclei (Figure 3, B).
Metastatic carcinoma may also be a consideration, especially metastatic renal clear cell carcinoma (RCC; Figure 3, C). Metastatic RCC presents as one or multiple nodules of large polygonal clear cells in a nested arrangement, which may appear deceptively bland and be mistaken for clear cell hidradenoma. Metastatic RCC may have associated prominent chicken wire vasculature with hemorrhage, which would not be expected in clear cell hidradenoma. If metastatic RCC is a consideration, a useful panel of immunohistochemical stains may include renal cell carcinoma marker (specific for RCC, although not completely sensitive), PAX8 (positive in RCC and negative in cutaneous adnexal carcinomas), and p63 (negative in RCC and positive in most primary cutaneous carcinomas).15–19
Melanocytic Lesions with Clear Cell Appearance
Benign and malignant melanocytic lesions may display focal or diffuse clear cell change. Balloon cell nevi (Figure 4, A) and cellular blue nevi (Figure 4, B) may be distinguished from CCM using the same criteria that distinguish nevi from conventional melanoma, such as the absence of both nuclear atypia and dermal mitoses. Clear cell melanoma (Figure 4, C) displays morphologic overlap with balloon cell melanoma (Figure 4, D), which has abundant finely vacuolated cytoplasm attributed to a melanosomal defect. Special staining for PAS aids in distinction between CCM and balloon cell melanoma; the former displays PAS-positive, diastase-sensitive cytoplasmic staining, whereas the latter displays PAS-positive, diastase-resistant cytoplasmic granules.20,21 Some investigators consider CCM to be a subtype of balloon cell melanoma given the overlap between these entities,22 but further studies are needed to examine clinical and genetic distinctions that may exist between these morphologic variants.
For melanocytic lesions predominantly or entirely composed of clear cells, immunohistochemical stains may be necessary for distinction from nonmelanocytic lesions (Table 3). S100 is useful because the vast majority of melanomas are S100 positive.23,24 However, S100 has limited specificity in this context. Although some morphologically similar lesions lack S100 expression (such as cellular neurothekeoma),25 S100 is expressed in a subset of sarcomas and may be expressed in carcinomas, especially those from the breast and salivary gland.23,26 Melan-A (MART1) and HMB-45 are expressed in most melanomas, although expression is often lost in spindled/desmoplastic melanoma.26 Positive staining for Melan-A or HMB-45 reasonably excludes carcinomas and most sarcomas.23 MiTF is less specific, with expression in nonmelanocytic lesions, including histiocytic lesions and cellular neurothekeoma.23,25,26 Immunohistochemical stains cannot reliably distinguish CCM from perivascular epithelioid cell tumor (PEComa) and CCS, both of which express melanocytic markers.1,27,28
Clear Cell Sarcoma
Clear cell sarcomas are typically deep-seated sarcomas of proposed neural crest origin that tend to present in the extremities of young adults.1,3 Microscopically, CCS presents as a multinodular mass composed of cells with clear or eosinophilic cytoplasm and scattered multinucleate wreathlike giant cells, arranged in nests or fascicles (Figure 5, A and B). Eosinophilic cells predominate in some cases, which may lead to confusion with conventional melanoma or poorly differentiated carcinoma (Figure 5, C). Melanin and glycogen are common findings.3 The presence of pagetoid scatter and true junctional nests argues strongly for melanoma; however, a case of CCS with an intraepidermal component mimicking a compound nevus has been reported.29 Unlike melanoma, CCS typically lacks significant nuclear pleomorphism. Using immunohistochemistry, CCS expresses melanocytic markers, including HMB-45 (90%), MiTF (71%), S100 (64%), and Melan-A (43%).3 Because CCS expresses melanocytic markers, immunohistochemistry is not useful for distinction from melanoma, and molecular studies may be required for accurate diagnosis. BRAF and KIT mutations are rare in CCS.1,30 Unlike melanoma, CCS classically displays the t(12;22)(q13;q12); EWSR1-ATF1 translocation in most cases.1,2 This translocation may be detected by RT-PCR for fusion transcript or dual-probe FISH for EWSR1 rearrangement. Of these, RT-PCR is specific for the fusion transcript associated with CCS but will not identify tumors with rare fusion variants unless primers specific for these variants are included. FISH for EWSR1 rearrangement is predicted to be more sensitive in this respect but does not distinguish CCS from other neoplasms with EWSR1 rearrangement.
Perivascular epithelioid cell tumors are mesenchymal neoplasms of uncertain histogenesis and are in the family of angiomyolipomas and clear cell (sugar) tumors of the lung. There is a strong female predominance (6:1).31 The most common sites of involvement are the uterus or skin and soft tissue. Microscopically, PEComas are composed of usually bland clear cells with epithelioid features, clear or granular cytoplasm, round to oval central nuclei, and prominent nucleoli. Scattered multinucleate giant cells and areas of radial arrangement around vessels are present (Figure 6, A). Intraepidermal involvement has not been described for PEComas. Melanotic PEComas have been described at extracutaneous sites.32,33 Most PEComas are cytologically bland. However, some display large tumor size, high-grade atypia (Figure 6, B), dense cellularity, infiltrative growth, necrosis, significant mitotic activity (>1 per 50 high-power fields), or angiolymphatic invasion; cases with these concerning features warrant classification as PEComa of uncertain malignant potential if one feature is present, or malignant PEComa if multiple features are present.28,31
Using immunohistochemistry, PEComas are generally described as having melanocytic and smooth muscle differentiation. Almost all cases express HMB-45, although staining may be focal.28 Most cases also express Melan-A and MiTF. Most cutaneous PEComas are S100 negative.27,28,31 Despite their classification as myomelanocytic tumors, cutaneous PEComas may lack detectable muscle marker expression in up to 50% of cases.31 Smooth muscle actin is likely to be expressed in extracutaneous PEComas (82%) but is negative in most cutaneous PEComas.27,31 Desmin may be more sensitive than smooth muscle actin in the context of cutaneous PEComas.31 Caldesmon and calponin may also be expressed.31 MUM-1 may be a useful additional marker, given that it is weakly expressed (25%) or negative (75%) in PEComa but strongly expressed in most melanomas.34 Taken together, muscle marker expression or melanocytic marker expression in the absence of S100 argues strongly against melanoma and should raise consideration for PEComa. However, the overlap in morphologic and immunohistochemical features can present a diagnostic challenge when distinguishing PEComa from melanoma. This distinction is critical because most PEComas are clinically benign.
Unlike CCS, no single genetic driver has been described for PEComas. Allelic loss at the TSC2 locus on chromosome 16p13 occurs in most extracutaneous cases; however, deletion at this locus has also been described in uveal CCM.6,31 Rearrangement of the TFE3 gene has been reported in a minority of PEComas, although this has not been identified in cutaneous PEComas.35 Therefore, molecular studies are of limited use for confirming or excluding the diagnosis of PEComa.
MATERIALS AND METHODS
All studies were conducted in accordance with protocols approved by the University of Michigan Institutional Review Board (No. HUM00045834). Hematoxylin-eosin–stained sections were prepared according to standard protocols at the Department of Pathology, University of Michigan.
Immunohistochemistry was performed on the Ventana Benchmark (Ventana, Tucson, Arizona) as per standard protocols in the Immunohistochemistry Laboratory of the Department of Pathology, University of Michigan. Antibodies and dilutions are listed in Table S2. Photomicrographs were captured using a SPOT Flex camera mounted on an Olympus BX41 microscope (Olympus America Inc, Center Valley, Pennsylvania), with SPOT Basic software (SPOT Imaging Solutions, Sterling Heights, Michigan).
Single-Nucleotide Polymorphism Array Analysis
A section with more than 80% tumor purity was selected for DNA extraction and single-nucleotide polymorphism array analysis. Formalin-fixed, paraffin-embedded tissue was deparaffinized, and tumor DNA was extracted using the QIAgen DNeasy Blood & Tissue DNA extraction kit (QIAgen, Germantown, Maryland). A total of 900 ng of DNA was used for analysis by Cytoscan HD single-nucleotide polymorphism microarray (Affymetrix, Santa Clara, California) on the GeneChip Fluidics Station 450Dx (Affymetrix). Scanning was performed by a GeneChip Scanner 3000 (Affymetrix). Results were analyzed by the Chromosome Analysis Suite (Affymetrix) and further confirmed by Nexus Copy Number software (BioDiscovery, Hawthorne, California). Copy number variations were considered significant if estimated copy number was greater than 2.5 or less than 1.5, and size was greater than 0.5 MB for gains and losses, respectively. Cancer-relevant genes within area of copy change were identified using the cancer gene census of the Catalog of Somatic Mutations in Cancer (http://cancer.sanger.ac.uk/cancergenome/projects/census/; accessed February 25, 2014).
EWSR1 Gene Rearrangement Studies
Reverse transcription–polymerase chain reaction for ESWR1-ATF1 transcripts was performed as per standard protocol in the Molecular Diagnostics Laboratory of the Department of Pathology, University of Michigan. Briefly, four 10-micronometer paraffin scrolls of the tissue block were deparaffinized and digested in cell lysis buffer (Gentra Puregene, Qiagen) with 125 μg of proteinase K overnight. RNA was extracted using Trizol LS reagent (Life Technologies/Invitrogen, Carlsbad, California). One-step RT-PCR was performed using the GeneAmp Gold RNA PCR Core Kit (Life Technologies/Applied Biosystems) on an Applied Biosystems 9700 thermal cycler with primer sets specific for 3 EWSR1-ATF1 fusion transcripts: type 1 (EWSR1 exon 8/ATF1 exon 4), type 2 (EWSR1 exon 7/ATF1 exon 5), and type 3 (EWSR1 exon 10/ATF1 exon 5). Each reaction included control primers to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Amplification products were analyzed by capillary electrophoresis on a 3130xl Genetic Analyzer (Applied Biosystems).
Fluorescence in situ hybridization for EWSR1 rearrangement was performed using a previously reported protocol36 with bacterial artificial chromosome clones RP11-367E7 5′ probe (green detection) and RP11-91J21 3′ probe (red detection) used as a break-apart FISH probe.
BRAF Mutation Detection
BRAF mutation status was determined by PCR amplification of BRAF exon 15 from 50 ng of input tumor DNA, using the following primers previously reported37 : (forward) 5′-CCTAAACTCTTCATAATGCTTGCT-3′ and (reverse) 5′-AGTAACTCAGCAGCATCTCAGG-3′. Polymerase chain reaction products were purified by QIAquick spin column (QIAgen) and submitted for Sanger sequencing at the University of Michigan's DNA Sequencing Core. Sequencing chromatograms were visualized with Sequence Scanner 2 software (Applied Biosystems, Carlsbad).
We would like to acknowledge Tina Fields, BS, for her technical assistance with immunohistochemistry. This study was supported by the Dermatopathology Research Career Development Award of the Dermatology Foundation (Dr Harms).
The authors have no relevant financial interest in the products or companies described in this article.
Supplemental digital content is available for this article at www.archivesofpathology.org in the October 2014 table of contents.
Presented in part at the New Frontiers in Pathology: An Update for Practicing Pathologists meeting; University of Michigan; September 26–28, 2013; Ann Arbor, Michigan.