Clear cells in acral melanoma

ARTICLE

Malignant melanomas as well as melanocytic nevi are known to be composed of cells that may present morphologically with one to several of ten different phenotypes [1], namely small round, large round, oval, epithelioid, spindle-shaped, dendritic, wavy, pagetoid, ballooned and giant cell. Some of these phenotypes show a predilection for certain body areas such as dendritic shape in acral and mucosal melanoma [2, 3], epithelioid in ocular melanoma [4] and spindle-shaped in acral and so called "lentigo maligna melanoma" of the head and neck region [1]. In the present study we assessed one additional cell phenotype of melanocytic lesions, clear cells. Recognition is important since differential diagnosis includes a variety of other clear cell tumors.

Materials and methods

We defined clear cells as melanoma cells with clear or empty cytoplasm distinct from balloon and pagetoid melanocytes. The former show a vacuolated to xanthomatized cytoplasm with a central nucleus, the latter a finely to coarsely granular cytoplasmic pigmentation.

Over the last 5 years we have become aware of several cases of clear cell melanoma, interestingly all from acral location. In a work-up of all acral malignant melanomas that had been diagnosed in the Dermatohistopathological Laboratory, University of Innsbruck, Austria, during a time period of 15 years between 1982 and 1997, we analysed 49 acral malignant melanomas for features of clear cell changes.

Basic clinical details were obtained from histopathological request forms and patients' files. Serial sections were cut and mounted from formalin fixed, paraffin embedded material of the biopsies. Staining with hematoxylin and eosin (H & E) was obtained in all cases and was supplemented by special stains including periodic acid Schiff (PAS) and Gomori methenamine silver in some instances. Immunohistochemistry was carried out on paraffin sections with a panel of antibodies using a three step avidin-biotinylated peroxidase complex (ABC) method with diaminobenzidine as the final visualisation product. A polyclonal antibody was used for S100 (Dako, Glostrup, Denmark, 1:100 dilution) whereas all other antibodies were monoclonal including HMB45 (Immunotech, Marseille, France, 1:1), A103 (MelanA) (Dako, 1:50), and NK1C3 (CD57) (Monosan, Uden, Netherlands, 1:5).

Specimens were analysed for criteria of malignant melanoma comprising asymmetry of the silhouette, distribution of melanocytes, single or in nests, form and confluence of nests, cytological features of melanocytes, type and distribution of inflammatory infiltrate, distribution of dermal pigment and melanophages as well as fibroplasia of the papillary dermis. The level of invasion was determined according to Clark's classification and Breslow's tumor thickness in mm. After reevaluating the diagnosis of malignant melanoma, assessment of clear cell changes and the immunohistochemical staining profile was performed independently by two investigators (MS, BZ). The extent of clear cell features was defined according to its abundance within the lesion as prominent (> 50% of tumor cells), focal (10-50%), or sparse to none (< 10%).

Resu lts

Clinical information

The clinical information is summarized in Table I. There was a total of 49 cases of acral melanoma. The age range of the patients was 15 to 94 years (median 64.5 years). The female to male ratio was 26:23. Five tumors developed on the hands (10%), 44 tumors were located on the feet (90%, Fig. 1). Metastases were present in four patients at the time of diagnosis.

In the group of patients with prominent clear cell features three patients were female and four male. The age ranged from 49 to 73 years (median age 58.5). Tumor thickness ranged between melanoma in situ and 14 mm. In four of seven tumors with prominent clear cell features irregular dermal pigment deposition was detectable. In five of these seven specimens tumors were thicker than 0.7 mm. With the exception of one case (hand) all specimens originated from the lower extremities in this group of patients. In one case, infiltration of an inguinal lymph node with extensive diffuse infiltration by tumor cells with prominent clear cell features (> 95% of cells) was observed.

Histology

At scanning magnification the lesions showed an asymmetric silhouette, irregular inflammatory infiltrates and an eccentric accumulaton of dermal pigment and melanophages. There was a varying degree of fibroplasia. Clear cell features were prominent (> 50% of tumor cells) in seven cases, focal (10-50%) in 21 cases and sparse in two cases, respectively. Nineteen acral melanomas did not show any features of clear cell changes. Pagetoid epidermal spread was seen in 42 cases. The areas of the tumors with clear cell features appeared pale as opposed to adjacent areas of "conventional" melanocytes or nevus cells. Clear cell changes were seen in dermal as well as epidermal parts of melanomas.

Viewed at high power the tumor parts with clear cell changes were composed of irregularly shaped and variably sized cells with clear cytoplasm and irregularly placed, hyperchromatic nuclei containing one or more nucleoli (Fig. 2). Intranuclear (pseudo-) inclusions were seen in some cases. Nuclear pleomorphism and mitoses were seen in all cases, but exhibited a varying degree in individual lesions. The cytoplasm appeared to be reduced in content or was empty, resembling lipid storing cells that have been extracted by fixation procedures. PAS staining was negative in these cells. In some areas disruption of the thin cytoplasmic membranes with coalescence of tumor cells was seen (Fig. 2).

Immunohistochemistry

Immunohistochemical studies demonstrated moderate to weak staining for HMB45 (10-80% of tumor cells) and S100 (10-100%). Staining intensity for these markers was reduced in areas with prominent clear cell changes, however in all of our specimens staining was detectable at least in part of the tumors. In contrast, staining was clearly present for MelanA (> 90%) and NK1C3 (CD57) (> 70%) in all specimens.

Discussion

In this series we describe clear cells in acral melanoma in addition to well established other cell types (small round, large round, oval, epithelioid, spindle-shaped, dendritic, wavy, pagetoid, ballooned and giant cell) [1]. Clear cell changes in malignant melanoma are probably more common than previously thought. These cells were arranged in nests or sheets of variably sized cells with clear cytoplasm. Distinctive criteria from balloon cell and pagetoid differentiation are the absence of cytoplasmic xanthomatisation and granulation as well as the location of nuclei (center - balloon, eccentric - clear). Clear cells can be observed in tumors of different origin and cases with wide spread clear cell changes may pose diagnostic problems [5-9]. Differential diagnosis includes balloon cell nevus, clear cell papulosis, clear cell fibrous papule of the nose, hibernoma, xanthogranuloma, clear cell syringoma, atypical fibroxanthoma, sebaceous neoplasms, clear cell sarcoma, renal cell carcinoma, liposarcoma, hidradenocarcinoma ("clear cell acrospiroma") and clear cell dermatofibroma [10-12] (for details in differential diagnosis see Table 2). As in our series coexisting diagnostic features such as "lentigo-type", non-equidistantly distributed, atypical melanocytes in the basal area of the adjacent epidermis (melanoma in situ) or areas with "conventional" invasive melanoma cells are clues for correct diagnosis.

Immunohistochemically, clear cell rich areas show moderate to weak or no reactivity for S100 and HMB45. In contrast, most cases retain staining for NK1C3 (CD57) and A103 (Melan A). In difficult cases molecular cytogenetics may be helpful for distinction from various soft tissue tumors. Some tumors diagnosed as clear cell sarcoma may indeed be unrecognized clear cell melanoma with absent or not adequately interpreted epidermal and dermal melanocytic features [13]. As opposed to malignant melanoma the translocation t(12;22)(q13;q12) is characteristically detected in clear cell sarcoma [14, 15].

The reason for the cytoplasm of tumor cells giving an "empty" clear cell appearance has not been elucidated. The plasma membrane of melanocytes may be susceptible to shrinkage phenomenon due to dendritic morphology. Accordingly, clear cell features on routine H & E stained sections may not represent an actual phenotype, but a shrinkage phenomenon from tissue preparation. Moreover, electron microscopy of clear cells has limited validity due to fixation artefacts. However, in pigmented lesions with balloon cell differentiation representing lesions with a similar faint appearance, electron microscopy studies are available and suggest a melanocytic origin of the cells by demonstrating remnants of premelanosomes and melanosomes as well as dendritic processes [16, 17]. A defect in melanosome formation with failure of premelanosomes to develop an internal structure and subsequent accumulation of membrane-bound vesicles has been suspected [16, 18]. In some cases the cytoplasm of balloon cells has been shown to contain abundant lipids [19] as suggested by their morphological appearance [9]. Other authors assume a self-destructive process of melanocytes involving cytoplasm and organelles [7].

Yet, it is tempting to speculate that the clear cell phenotype in our series may be of biological significance and may correspond to the distinct gene amplification patterns that have been described for acral melanoma [20]. Clear cell changes have been previously reported in choroid melanoma and melanoma of other species (e.g. cats) [21, 22]. Irrespective of the underlying pathophysiology, it is important to recognise clear cell features to assure proper diagnosis of malignant melanomas, which in the case of acral localization can be delayed due to atypical presentation. While clear cell changes have diagnostic value, our follow up data do not indicate a prognostic significance (Table I)..

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