Christoph Schubert

Telomerase Activity in Melanocytic Lesions : A Potential Marker of Tumor Biology

Telomerase activation, being a cardinal requirement for immortalization, is a crucial step in the development of malignancy. With a view toward diagnostic and biological aspects in melanocytic neoplasia, we investigated the relative levels of telomerase activity in 72 nevi and 16 malignant melanomas by means of a modified telomeric repeat amplification protocol (TRAP) assay, including an internal amplification standard. We further compared telomerase activity with the expression of two different proliferation-specific proteins, Ki-67 and repp86, a protein expressed exclusively in the cell cycle phases S, G2, and M. Telomerase activity was associated with the overall growth fraction (Ki-67) but showed a closer correlation with the expression of repp86. Both telomerase activity and proliferation indices discriminated clearly between malignant melanomas and nevi, but not between common and dysplastic nevi. Nonetheless, a portion of nevi exhibited markedly elevated telomerase activity levels without proportionally increased proliferation. This was independent of discernible morphological changes. Clinicopathological correlations showed an association between high telomerase activity and early metastatic spread in melanomas, linking telomerase to tumor biology. Our results provide arguments in favor of an occasional progression from nevi to melanomas and imply that proliferation measurements in combination with telomerase assays may help to elicit early malignant transformation that is undetectable by conventional morphology.

Proliferation marker Ki-S5 as a diagnostic tool in melanocytic lesions

BACKGROUND A discrimination between benign and malignant melanocytic lesions is not always possible by conventional histology. OBJECTIVE Our purpose was to determine the proliferative activity in various types of melanocytic neoplasms and to appraise its value as a diagnostic adjunct. METHODS The growth fraction was assessed immunohistochemically in 398 melanocytic lesions by means of the monoclonal antibody Ki-S5 directed to the Ki-67 antigen. In this way, a cut-off level was defined and applied to 112 lesions of equivocal histology. The revised diagnoses were correlated to the clinical courses. RESULTS Common nevi, Spitz nevi, and melanomas exhibited significantly different proliferation indices and distinctive distribution patterns of cycling cells. In agreement, malignancy diagnosed on the basis of a growth fraction greater than 5% was confirmed by disease progression in 68% of our cases with uncertain diagnosis. CONCLUSION Determination of the proliferative activity in melanocytic lesions may usefully complement conventional histology to improve diagnostic accuracy.

Giant cell collagenoma: A benign dermal tumor with distinctive multinucleate cells

We present five cases of a hitherto unreported cutaneous neoplasm. The tumors appeared as solitary slow-growing flesh-colored nodules arising in young and middle-aged adults. They were located on the trunk, the upper extremities, and the face, and did not recur after complete excision. Clinically, they were diagnosed as dermal nevus, Spitzs nevus, fibroma, or neurofibroma. Histology revealed polypoid flat-dome-shaped lesions with a sharply demarcated matrix consisting of coarse hyalinized collagen bundles arranged in a prominent storiform pattern and separated by mucin-containing clefts. Despite a low overall cellularity, the tumors contained numerous, occasionally bizarre-shaped, multinucleate giant cells with crowded vesicular nuclei and a pale staining foamy cytoplasm, as well as plump fibroblastlike cells with analogous nuclear morphology. Atypical nuclei or mitotic figures were not observed. The cells were strongly positive for vimentin but negative for cytokeratin, smooth muscle actin, desmin, S-100 protein, CD34, factor XIIIa, and the macrophage markers KP1, Mac 387, and Ki-M1p, suggesting a fibroblastic origin. Based on the overall architecture, we conclude that these tumors probably represent a distinctive variant of solitary circumscribed storiform collagenoma (sclerotic fibroma) and propose the designation of giant cell collagenoma.

Immunophenotyping of dermal spindle cell tumors: diagnostic value of monocyte marker Ki-M1p and histogenetic considerations.

Various studies have reported the utility of anti-CD34 staining in the differential diagnosis of dermal spindle-cell tumors. To investigate whether monoclonal antibody Ki-M1p might add practical diagnostic information, we examined a total of 120 cutaneous spindle cell neoplasms using a panel of markers. Anti-CD34 antibody QBEnd/10 consistently stained dermatofibrosarcomas, Kaposis sarcomas, neurofibromas, and, to a lesser extent, hemangiopericytomas. A positive reaction was also found in > 18% of the dermatofibromas. Ki-M1p staining showed an intense immunoreaction in all dermatofibromas, whereas no reactivity was observed in dermatofibrosarcomas. In addition, a subset of cells was labeled in atypical fibroxanthomas and Kaposis sarcomas. Neurofibromas, spindle-cell hemangioendotheliomas, and hemangiopericytomas were negative. Dermatofibrosarcomas and atypical fibroxanthomas also moderately expressed smooth muscle-specific actin. Immunohistochemically, a discrimination between dermatofibrosarcomas and neurofibromas was possible only by means of an antibody against the nerve growth factor receptor. We conclude that the combination of several antibodies, in particular anti-CD34 and Ki-M1p, may improve the accuracy of diagnostic immunohistochemistry in the field of cutaneous spindle cell tumors. We speculate that dermatofibroma is primarily a macrophage-rich inflammatory lesion in which cytokine secretion induces a secondary proliferation of fibroblasts, whereas dermatofibrosarcoma is likely to issue from primitive dermal cells of uncertain origin.

Differential expression of CD34 and Ki-M1p in pleomorphic fibroma and dermatofibroma with monster cells.

Pleomorphic fibroma (PF) and dermatofibroma with monster cells (DFMC) are characterized by the presence of numerous cells with large atypical nuclei. Despite cytologic similarities, the two entities are likely to be unrelated, but their histogenesis is poorly understood. In this study, we examined six cases of PF and eleven cases of DFMC by immunohistochemistry using antibodies against vimentin, alpha-smooth muscle actin, S-100 protein, CD34, factor XIIIa, and the pan-monocytic marker Ki-M1p. Strong vimentin expression was seen in all tumors, whereas none of them expressed S-100 protein. PF consistently exhibited CD34 staining but appeared to be depleted of Ki-M1p positive cells compared with the surrounding normal skin. Conversely, all cases of DFMC contained numerous Ki-M1p positive cells including atypical multinucleate cells, but virtually no CD34 reactivity was observed. A weak staining for alpha-smooth muscle actin was occasionally seen in a subset of the cells of both entities. Our results indicate that PF and DFMC are histogenetically distinct entities that may arise from two different types of dermal dendritic cells defined by their reactivity for CD34 and Ki-M1p, respectively. Immunohistochemistry using these two antibodies permits an easy and reliable discrimination between PF and DFMC.

Immunolocalization of defensins and cathelicidin in human glands of Moll.

The human gland of Moll located at the margin of the eyelids is a specialized apocrine gland, the function of which is not exactly known. The presence of antimicrobial proteins was identified in this gland recently, suggesting a function in the external ocular defense barrier against pathogens. In this study, we have demonstrated beta-defensin-1, beta-defensin-2 and cathelicidin (LL-37) in the secretory endpieces of the glands of Moll using immunohistochemical methods. beta-Defensin-1, beta-defensin-2 and cathelicidin (LL-37) showed a weak to moderately intensive staining pattern. The strongest immunolocalization of beta-defensin-1 was observed in the apical protrusions of the gland, which could also be observed but to a lesser extent in the case of beta-defensin-2 and cathelicidin. In active glandular cells, a granular staining pattern could be observed. beta-Defensin-1 and beta-defensin-2 varied in staining intensities, and even within one section strongly and weakly stained cells can coexist side by side. Also cells that, according to morphological criteria, appeared to be inactive still had an apical beta-defensin-1 immunolabeling. We assume that beta-defensin-1, beta-defensin-2 and cathelicidin (LL-37) work together with other antimicrobial peptides and proteins to create a defensive barrier against microbial invasion at the ocular surface.

Early mycosis fungoides: molecular analysis for its diagnosis and the absence of p53 gene mutations in cases with progression.

The histological diagnosis of initial mycosis fungoides (MF) and the molecular mechanisms that are responsible for its progression and transformation to the more highly malignant variants of MF remain largely unknown. Because of the rare occurrence of these tumours, the need for snap frozen skin biopsy specimens and the difficulty to obtain suitable material for karyotypic and genotypic analysis, specific cytogenetic and molecular lesions have not yet been identified. In particular the role of known oncogenes and tumour suppressor genes, including the p53 gene, in the pathogenesis and clinical progression of MF has not been extensively investigated. The present study was carried out using the polymerase chain reaction (PCR) technique combined with temperature gradient gel electrophoresis (TGGE) to detect mutations of the p53 gene in 58 patients with MF. TGGE analysis was also used in combination with clonality analysis by means of T-cell receptor gamma (TCRG) gene rearrangement studies to distinguish parapsoriasis en plaque and initial MF from patch/plaque stage MF. More than 83% of the diagnoses of initial MF could be confirmed using PCR-TGGE analysis. However, although the sensitive TGGE analysis was used for all exons, p53 gene polymorphisms were found in 4 and p53 gene mutation in only 1 of 58 biopsy specimens. It appears unlikely that p53 gene mutations play a role in either the pathogenesis of parapsoriasis and initial MF or their progression to advanced stages of MF. However, TCRG gene rearrangement studies by means of TCR-TGGE analysis may be useful for distinguishing histologically discordant cases of initial MF.

White sponge nevus of the vulva

The white sponge nevus is a hereditary leukokerotosis localized preferably in the oral mucosa, but may simultaneously manifest itself in other regions, e.g. perianally.

Histological and histochemical observations on the neurosecretory cells in the diencephalon of Chthonerpeton indistinctum and Ichthyophis paucisulcus (Gymnophiona, Amphibia).

SummaryIn the diencephalon of two species of Gymnophiona (Amphibia) two neurosecretory nuclei were examined with histological (Alcian Blue, Aldehyde Fuchsin, Brookes Trichrome stain) and enzyme histochemical techniques (acid phosphatase, α-naphthyl acetate esterase, acetylcholinesterase (AChE)). In the preoptic nucleus two categories of secretory neurons were distinguished: large and medium sized neurons. The perikarya of both cell types contain very little neurosecretory material. The Alcian Blue method stained the medium sized neurons faintly but selectively. The tractus praeopticohypophyseus is marked by the presence of Herring bodies, which, however, are relatively scarce. The neurohypophysis, in contrast, contains large amounts of neurosecretory material. Both cell types of the preoptic nucleus are characterized by their very strong AChE and α-naphthylacetate esterase activity. The AChE also marks the tractus praeoptico-hypophyseus. In the large neurons acid phosphatase is present around the nucleus; in the medium sized neurons this enzyme is concentrated close to the origin of the axon. In the dorso-caudal hypothalamus a small group of neurons is stained with Alcian-Blue. These neurons, which also contain AChE, are located immediately under the ependyma which seems to be specialized in this region.

Investigations on the thyroid gland of embryonic larval and adult Ichthyophis glutinosus and Ichthyophis kohtaoensis (Gymnophiona, amphibia)

SummaryDifferent developmental stages of two species of the genus Ichthyophis have been investigated. In the late embryo the follicular cells of the thyroid gland exhibit various degrees of cytodifferentiation. Well differentiated cells show a polar organization and contain numerous granular inclusions, but a colloid-containing lumen is rare. Most cells at this stage contain large lipid inclusions. In young and older larvae the cells contain well-developed rough ER and Golgi systems, numerous mitochondria, and abundant granular and vesicular inclusions. Tentative identifications were made of primary lysosomes, secondary lysosomes, residual bodies, and two types of small apical vesicles—containing resorbed colloid or transporting material into the follicular lumen. In the larvae the number of apical microvilli is relatively high. The thyroid cells of the older larvae seem to contain more granular and vesicular inclusions than those of the younger larvae. In the adult the size of the follicles greatly increases, the height of the epithelium decreases, microvilli become rare, residual bodies are more frequent, and the small primary lysosomes are replaced by larger ones. Colloid droplets have been found only rarely in the cytoplasm of the thyroid cells of adult animals. In the immediate neighbourhood of the follicular epithelium, profiles of nerve fibres were found in all animals. Radioiodide investigations—measurements of conversion ratio and thyroid uptake factor—show, if compared with the results of corresponding studies in other amphibians, only relatively small differences between the larvae on the one hand and larvae and adults on the other. The absolute counts of the thyroid region are lowest in the adult and highest in the older larvae, shortly before metamorphosis. Furthermore our results indicate, on the basis of four animals tested, that in Ichthyophis the activity of the thyroid gland is temperature dependent. The results in Ichthyophis show that the classical stages of metamorphosis, in other amphibians characterized among other things by different levels of thyroid activity, are very indistinct in this animal.