Reflectance confocal microscopy for noninvasive examination of nonmelanocytic tumors and virus-associated skin lesions in organ transplant recipients.

BACKGROUND: Drug-induced immunosuppression is necessary to prevent rejection of the foreign organ in transplanted patients, but neoplastic and virus-associated skin diseases are frequent complications. Reflectance confocal microscopy (RCM) recently emerged as a promising tool for the early diagnosis of skin lesions. MATERIALS AND METHODS: A total of 61 skin lesions, among them 20 basal cell carcinomas, six Bowen's diseases, 23 actinic keratoses, and 12 verrucae, were analyzed. All lesions were clinically evaluated followed by RCM evaluation by two independent dermatologists and histological examination. RESULTS: For the diagnosis of basal cell carcinoma, a sensitivity of 100% by both investigators (INV I + II) and a specificity of 100% by INV I and 80% by INV II were achieved. The sensitivity average rate for RCM features reached by both investigators ranged between 60% and 100%, and the specificity between 55% and 90%. For the diagnosis of actinic keratosis, a concordant sensitivity of 94.4% and a specificity of 80% (INV I) and 60% (INV II) were detected. The sensitivity average rate of specific RCM criteria ranged between 72.3% and 97.2%, whereas specificity ranged between 20% and 90%. Regarding verrucae, RCM confirmed the histological diagnosis with a sensitivity of 85.7% (INV I) and 100% (INV II), while specificity was 100% and 80%, respectively. CONCLUSION: Reflectance confocal microscopy resulted to be a reliable tool for the noninvasive diagnosis of neoplastic and virus-associated skin changes in organ transplant recipients. Nevertheless, given the frequency and diagnostic complexity of the hyperkeratotic lesions occurring post-transplantation, larger cohorts of patients are required to confirm and consolidate these findings.

Bowen's disease with features resembling myrmecia wart.

We report the clinical and pathological findings of two cases of Bowen's disease (BD) with features resembling myrmecia wart, and tried to find evidence of human papillomavirus (HPV) infection in such lesions by immunohistological staining, genotyping systems, polymerase chain reaction (PCR) and electron microscopy. Both cases manifested unique barnacle-like hyperkeratotic nodules or plaques clinically, and microscopically proliferation of atypical keratinocytes involving the entire thickness of the epidermis, hypergranulosis with eosinophilic and/or basophilic inclusion bodies, features that mimicked myrmecia wart. Electron microscopy revealed myrmecia inclusion-like large intranuclear and cytoplasmic electron-dense bodies. Immunohistological staining with anti-HPV antibody, genotyping systems for HPV infection and specific PCR designed to detect HPV-1 L1 sequences failed to detect evidence of HPV infection. P16(INK4a) was overexpressed in the atypical keratinocytes of both cases. This finding suggests that the pathogenesis of these two BD may involve certain unknown or undetectable HPV, or reflect disturbances of the Rb signaling pathway unrelated to HPV infection. The unique "myrmecioid" clinicopathological features in our cases suggest that this type of lesion may be a new variant of BD.

Ultrastructural Examination of a Case of Pagetoid Bowen Disease Exhibiting Immunohistochemical Features in Common With Extramammary Paget Disease.

A panel of immunohistochemical markers may be used to differentiate between pagetoid Bowen disease (PBD) and primary extramammary Paget disease (EMPD) in selected cases. Although diffuse staining with cytokeratin 7 (CK7), CAM5.2, carcinoembryonic antigen, epithelial membrane antigen (EMA), and gross cystic disease fluid protein 15 generally supports diagnosis of EMPD, cases have been reported where PBD also expressed CK7, EMA, and CAM5.2. Based on these findings, some authors suggested that the 2 entities may arise from the same multipotent stem cell, capable of further differentiation toward squamous and secretory lines. To the best of our knowledge, this issue has never been investigated by comparing PBD and EMPD at the ultrastructural level. We performed the first ultrastructural study of a case of PBD exhibiting common immunohistochemical features with EMPD. The lesion displayed some ultrastructural features often observed in Bowen disease and some that are typically found in EMPD. These findings indicate the presence of a bidirectional differentiation--secretory and squamous--within the same lesion, thus supporting the hypothesis that PBD and primary EMPD may arise from a common progenitor cell.

Electron microscopic in situ DNA nick end-labeling in combination with immunoelectron microscopy.

We describe an in situ DNA nick end-labeling method that can be performed at the electron microscopic level and can also be combined with immunoelectron microscopy. As the materials, we used skin tissues from normal skin and from Bowen's disease that had been cryofixed, freeze-substituted, and embedded in Lowicryl K11M resin. Ultrathin sections were cut and incubated with a reaction buffer containing digoxigenin-dUTP and terminal deoxynucleotidyl transferase. Digoxigenin nucleotides were labeled with anti-digoxigenin antibodies conjugated with colloidal gold. Specific signals were detected in the condensed chromatin of differentiated epidermal cells and hair follicles in normal skin and of dyskeratotic cells in Bowen's disease. The labeling density over chromosomal areas of apoptotic cells was significantly higher than that over chromosomal areas of mitotic cells or cytoplasmic areas. Ultrastructure was well preserved and double staining with an anti-keratin antibody was also successfully performed. This simple method has a wide range of applications to identify the nature of apoptotic cells and explore the mechanisms of apoptosis.

Ultrastructural study of extramammary Paget's disease--histologically showing transition from bowenoid pattern to Paget's disease pattern.

Histological, immunohistochemical, and ultrastructural studies were performed on two cases of histologically unusual extramammary Paget's disease. Histologically, the central area of the lesions showed a bowenoid pattern, and the peripheral area showed typical extramammary Paget's disease. The transition zone showed an intermediate pattern. All these areas were positive for CEA and EMA, and negative for S-100 protein. Ultrastructurally, in the intermediate pattern, the tumour cells had abundant cytoplasmic glycogen, and the widened intercellular spaces contained numerous glycogen particles, which were probably secreted by the tumour cells. It is well known that eccrine glands, but not apocrine glands, secrete glycogen particles. Therefore, the present findings suggest that some cases of extramammary Paget's disease are a proliferation of germinative cells with eccrine gland differentiation.

Tenascin expression in hyperproliferative skin diseases.

The expression of tenascin, a recently discovered extracellular matrix glycoprotein, was studied by immunohistochemistry in normal human skin and in a number of skin diseases with epidermal hyperproliferation such as psoriasis, basal cell carcinoma, Bowen's disease and solar keratosis. Tenascin expression in the upper dermis of normal skin was found to vary from almost absent to patchy along the basal membrane. Staining was continuous and intense around blood vessels, hair follicles and eccrine sweat ducts. In basal cell carcinoma a marked expression of tenascin was found in the tumour stroma, especially adjacent to the basal membrane surrounding the tumour cell nests. In Bowen's disease and solar keratosis, tenascin expression was found in the dermis next to the keratinocytes. In psoriasis the dermal papillae of clinically involved skin were intensely stained and a continuous band of tenascin was present in the upper dermis along the basal membrane. The distribution of tenascin differed from other known extracellular matrix components.

Merkel cell carcinoma of the head and neck associated with Bowen's disease.

The Merkel cell carcinoma occurs primarily in the skin of the head and neck, and develops in the dermis with a trabecular growth pattern. Immunohistochemistry reveals positive staining for neuron-specific enolase, neurofilaments, cytokeratin and chromogranin A. Electron microscopically, the tumor cells contain dense-core granules, spinous cytoplasmic processes, desmosomes, zonulae adherentes and paranuclear filament aggregates besides frequent mitoses, focal necroses and lymphocyte and plasma cell infiltrates. The Merkel cell carcinoma is often co-existent with other malignancies such as squamous cell carcinoma or, as in the present study, with Bowen's disease. The definite diagnosis of the Merkel cell carcinoma can be effected only by electron microscopic examination of the tumor.

[A rare complication of ulcerative colitis: Bowen's disease in perianal condyloma acuminatum]. / Colitis ulcerosa ritka szövödménye: Bowen-kór, perianalis condyloma acuminatumban.

Discharging, itching perianal pointed condyloma acuminatum in a 35 years old male patient who had been suffering from ulcerative colitis for 20 years. Two years following its development the condyloma was excised. Histological examination revealed in the perianal condyloma in situ cancer, characteristic of Bowen's disease. With electronmicroscopic examination the tumor cells were found to be keratinocytes of medium differentiation. Virus could not be detected in the tumor cells. The author was the first to describe in the literature the development of perianal Bowen's disease on the ground of ulcerative colitis. On the basis of literary data the author emphasizes the importance of the radical removal and regular control.

Apoptosis in Bowen's disease. An ultrastructural study.

We performed ultrastructural studies of apoptosis (previously referred to as "malignant dyskeratosis") in a case of genital Bowen's carcinoma in which human papillomavirus (HPV) type 33 genome was identified and in two cases of cutaneous Bowen's disease with no detectable viral DNA; herein we present the sequential stages in the development of apoptotic bodies. The apoptotic process in the HPV-containing genital Bowen's disease was similar to that in the cutaneous lesions with no detectable HPV. The presence of a large number of apoptotic bodies in Bowen's disease may be responsible for the slow progression and noninvasive growth of this carcinoma in situ.

Genetic convergence during serial in vitro passage of a polyclonal squamous cell carcinoma.

A cell line was established from an in situ squamous cell carcinoma of the skin (Bowen's disease), and its in vitro karyotypic evolution was cytogenetically analyzed. Initially, considerable genetic heterogeneity was evident. Nine cytogenetically abnormal clones, eight of which were apparently unrelated, were found among the 83 metaphases analyzed from the primary culture and the first passage. With increasing time in culture this complexity was reduced, so that a single clone dominated passages 7-11. The clone that emerged from this genetic convergence had a t(12;17)(p13;q21) as the sole abnormality. Our findings indicate that the cytogenetic multiclonality that has been repeatedly detected in short-term cultures of squamous cell carcinomas is not caused by the in vitro conditions. Instead, the principles of Darwinian selection apply: the altered, but stable, selection pressure facing a newly established and initially multiclonal cell line will lead to a reduction of genetic heterogeneity until the one clone that now has the proliferative advantage outgrows the other subpopulations.

Hidroacanthoma simplex showing variation in the appearance of tumor cells in different nests.

We report a case of hidroacanthoma simplex on the inframammary area of an 86-year-old woman. The tumor cells showed considerable variation in appearance in different nests from the same lesion. By light and electron microscopy 5 types of tumor cells were identified. This tumor was thought to originate from the outer cells of the intraepidermal eccrine duct.

Large cell acanthoma. A cytologic variant of Bowen's disease?

Although it was first described by Pinkus in 1970, the entity known as large cell acanthoma (LCA) has been largely ignored. In this paper, we review the clinical and histopathologic features of 11 solitary and three multiple cases of LCA. We also compare this series with two previous reports, one by Rahbari and Pinkus and one by Rabinowitz. From the analysis of these 70 cases, we conclude that although LCA is not uncommon (it occurs in 1-2.55 per 1,000 cutaneous biopsies), as an asymptomatic, slightly keratotic lesion, usually less than 10 mm in size, it has been largely underestimated. It is more common in women than in men and mainly affects middle-aged and elderly patients. LCA is found mainly on the face and upper limbs yet in the multiple cases, the lesions seem to occur on the limbs and the back. Histologically, it can be distinguished by the large size of the malpighian cells, both nucleus and cytoplasm. The lesion is sharply limited from normal epidermis and usually shows acanthosis, hypergranulosis, and hyperorthokeratosis. Finally, we discuss the nature of LCA. Based on the frequent disordered arrangement of the malpighian cells, its nuclear variability and the occasional finding of dyskeratoses and suprabasal mitoses, as well as the involvement of skin appendages, we conclude that LCA is probably a cytologic variant of Bowen's disease.

Distribution of actin filaments in human malignant keratinocytes.

Distribution of actin filaments in human malignant keratinocytes was examined by immunofluorescence staining. The primary cultures were obtained from a squamous cell carcinoma, a basal cell carcinoma, and Bowen's disease. Rhodamine-phalloidin staining revealed that actin filaments were occasionally organized to form stress fibers, many short bundles with a ripple appearance, and regular arrays of actin patches. Some of these structures appeared in untransformed keratinocytes as a result of a brief exposure to a tumor promotor, TPA. These findings suggest that regulation of actin functions is involved in neoplastic processes from the very early stages and that alteration is persistent in neoplastic cells.

Bowenoid papulosis. A clinicopathologic study with ultrastructural observations.

One hundred eight patients were studied who had anogenital lesions showing microscopic features as seen in bowenoid papulosis (BP), a recently described condition occurring most commonly in young adults. Patients typically show multiple papules, small nodules, or plaques that clinically mimic verrucae or nevocellular nevi. Although the lesions show microscopic cytologic atypia, a distinction from Bowen's disease, erythroplasia of Queyrat, and other forms of carcinoma in situ can usually be made on the basis of histologic and clinical criteria. The disorder responds to conservative treatment, although recurrences are not uncommon. Evolution of the lesions to invasive carcinoma was not observed. Mounting evidence links the development of BP to infection with human papilloma virus, but other viruses, as well as hormonal and immunologic factors, may also play a role.

Ricinus communis agglutinin I binding to the cell membrane in benign, premalignant and malignant epidermal lesions.

Membrane changes in keratinocytes were studied in a selected series of skin biopsies from 58 patients comprising cases of healing wounds, keratoachanthomas, actinic keratoses, Bowen's disease and squamous and basal cell carcinomas. The changes were demonstrated by means of a fluorescein-conjugated lectin ricinus communis agglutinin I, which specifically binds to beta-D-galactopyranosyl residues normally present on the keratinocyte surface. The RCA I binding equalled the binding of the normal epidermis in hyperplastic epidermis adjacent to healing wounds and in keratoachanthomas, but was slightly decreased in actinic keratoses and cases of Bowen's disease. In epidermal outgrowths from the edges of healing wounds and in squamous and basal cell carcinomas a heavy loss of RCA I binding was seen. The results are supported by previous in vivo and in vitro studies of normal and transformed cells, and it is suggested that the presented histochemical RCA I binding technique could be a valuable diagnostic tool.

[Light and electron microscopic and cytophotometric investigations in multiple Bowenoid papules (author's transl)]. / Histologie, Ultrastruktur und Zytophotometrie bei "Bowenoider Papulose der Anogenitalregion".

In to find a satisfactory nosological position of so-called Bowenoid Papules, biopsies of three patients were examined by light- and electron microscopy order as well as by cytophotometry and the findings were compared to the results of two cases of Bowen's disease. The dysplasia of the epidermis, hardly definable from Bowen's disease by light microscopy, revealed numerous ultrastructural alterations which also can be observed in the intraepidermal carcinoma, in addition single alterations indicate a viral genesis (which couldn't be proved until now). Cytophotometric investigations showed an only slight increase of DNA in the nuclei. It is interesting, however, that biopsies of a recurrent Bowenoid Papulosis exhibited a distinct increase of the DNA content. The not aggressive postoperative behaviour of two cases faces a third case with advanced dysplasia (increased DNA-content) and recurrence. The origin and/or the releasing moments for this diverging behaviour are not quite clear yet.