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DNA aneuploidy has been demonstrated to be an independent parameter of prognostic significance in malignant melanomas. In order to improve the detection of DNA aneuploidy in malignant melanomas, and to minimize diploid non-tumor cells in the sample, we developed a two-color staining strategy for S100 protein and DNA content in paraffin embedded samples. The ability to detect aneuploidy, defined as DNA ploidy index less than or equal to 0.90 or greater than or equal to 1.10, in 37 stage I malignant melanoma samples by flow cytometry analysis was significantly improved from 10.8% of cases using traditional one-color analysis for DNA content only (propidium iodide) to 32.4% of cases using two-color analysis for simultaneous measurement of both DNA (propidium iodide) and S100 protein (fluorescein conjugated antibody) (Chi-square with Yates' correction; p less than 0.05). The largest increase in sensitivity was found in level I and II melanomas less than or equal to 0.76 mm in thickness. In addition, we report the new observation that multiple S100 protein-positive subpopulations were found significantly more frequently in malignant melanomas (23/37 cases) than in compound melanocytic nevi (5/22 cases) (Chi-square with Yates' correction; p less than 0.01). These findings suggest that there is a previously unsuspected degree of tumor heterogeneity even in thin, presumably early, malignant melanomas.

In vitro DNA amplification followed by oligonucleotide mismatch hybridization was used to study the frequency of HRAS mutations in the benign self-regressing skin tumors keratoacanthomas and in squamous cell carcinomas. We used freshly obtained keratoacanthomas as well as Formalin-fixed paraffin-embedded tissues from both types of tumors. DNA from 50 samples of each tumor type was analyzed for activating mutations involving codons 12 and 61. A relatively high percentage (30%) of HRAS mutations was found in the keratoacanthomas compared with 13% in the squamous cell carcinomas. The most frequent mutation identified is the A-T-to-T.A transversion in the second position of codon 61. The present findings demonstrate the involvement of the HRAS oncogene in human benign tumors. Moreover, they indicate that an activated HRAS oncogene is not sufficient to maintain a neoplastic phenotype and argue against a role of HRAS in the progression of skin tumorigenesis.

The involvement of the ras oncogenes in tumorigenesis was investigated in keratoacanthomas, which are benign and self-regressing skin tumors, both in humans and in a corresponding animal model system. Keratoacanthomas were induced on rabbit ears by repeated applications of 7,12-dimethylbenz(a)anthracene. About 60% of the tumor DNAs produced transformed foci after transfection into NIH 3T3 cells, and in all of them the transforming gene was identified as H-ras by Southern and Northern (RNA) hybridization. Immunoprecipitation experiments suggested that the transforming rabbit H-ras protein carried a mutation in codon 61. In addition, an activated H-ras gene was detected in a human keratoacanthoma by using a nude mouse tumorigenesis assay after transfection of tumor DNA into NIH 3T3 cells. This is the first report of ras activation in a benign human tumor. The transforming human H-ras gene showed a point mutation in codon 61 that would result in leucine instead of the glutamine present in the normal gene product. The finding of ras activation in tumors that are not only benign but also self-regressing indicates that activated ras genes are not sufficient to maintain a neoplastic phenotype, although they likely play a role in early stages of tumorigenesis.

A clinicopathologic study of eight examples of polypoid and dome-shaped cutaneous fibrous lesions with sparse cellularity but striking nuclear atypia and rare mitotic figures is presented. Positive immunohistochemical staining for vimentin and actin supported the fibroblastic nature of these lesions. All eight cases were adults whose ages ranged from 33 to 67 years (mean 52 years). Five were women and three were men. Five lesions were located on extremities, two on the trunk, and one on the face and they measured from 4 to 16 mm in greatest dimension. The lesions were clinically followed from 4 months to 5 years. They all showed benign clinical behavior, with only one local recurrence in a lesion that had been incompletely removed. The nuclear atypia seen in these fibrous lesions may be similar to that which occurs in other benign mesenchymal neoplasms, such as pleomorphic lipoma, pleomorphic leiomyoma, ancient schwannoma, and variants of dermatofibroma with atypical cells. We suggest that "pleomorphic fibroma" is an appropriate term for this lesion based on its histologic differentiation, cytologic atypia, and benign clinical course.

Forty nasopharyngeal carcinomas (NPC) were studied by immunohistochemistry using an antibody to involucrin and the following three keratin antibodies: (1) an antibody to low molecular weight keratin reactive with nonsquamous epithelium, (2) a high molecular weight keratin antibody reactive with suprabasal squamous epithelium, and (3) a keratin antibody reactive with full thickness stratified epithelium. In its pattern of reactivity, the last antibody overlaps the low and high molecular weight keratin antibodies and is used as a broad spectrum keratin antibody. By World Health Organization (WHO) classification, the cases in this article included eight keratinizing squamous cell carcinomas, eight nonkeratinizing carcinomas, 20 undifferentiated carcinomas, and four adenocarcinomas. The antibody to broad spectrum keratin had an overall sensitivity of 87.5% and was positive in all eight keratinizing squamous cell carcinomas, seven nonkeratinizing carcinomas (87.5%), 18 undifferentiated carcinomas (90%), and two adenocarcinomas (50%). Low molecular weight keratin antibody stained one additional NPC, which was negative when broad spectrum keratin antibody was used. Involucrin and high molecular weight keratin antibodies demonstrated near parallel staining in all histologic classes; there was marked localization to areas of squamous differentiation. While involucrin is a marker for foci of greater squamous differentiation, broad spectrum keratin antibody may aid in the diagnosis of all histologic subtypes of NPC.

Dull pink globules were found within the epidermis in 65% of junctional, 75% of compound, and 25% of intradermal types of Spitz's nevi (the nevi of large spindle and/or epithelioid cells). These globules were PAS-positive, diastase-resistant and also were positive with the trichrome stain. Similar-appearing eosinophilic globules were noted in the epidermis in only 2% of malignant melanomas and in but 0.9% of ordinary melanocyte nevi. The globules in malignant melanomas and in ordinary melanocytic nevi were negative with PAS and trichrome stains. Therefore, the finding of PAS- and tri-chrome-positive eosinophilic globules within the epidermis is a helpful sign for histologic differentiation of Spitz's nevus from malignant melanoma.The histologic differentiation of Spitz's nevus (the nevus of large spindle and/or epithelioid cells) from malignant melanoma is of utmost importance in dermatopathology. Most authors agree that no single histologic finding enables absolute differentiation of the benign neoplasm from the malignant one. One of us (HK), while reviewing a large series of melanocytic lesions, observed distinctive eosinophilic globules within the epidermis and the dermis of many specimens of Spitz's nevus. The microscopic appearance of these globules and their significance is the subject of this paper.