Specificity of tyrosinase and HMB45 PCR in the detection of melanoma metastases in sentinel lymph node biopsies.

AIMS: Sentinel lymph node biopsy is an increasingly established procedure in the primary staging of high-risk melanoma patients. However, the laboratory evaluation of sentinel lymph node biopsies is a matter of controversy. The aim of this study was to determine the specificity of polymerase chain reaction (PCR) techniques for the evaluation of lymph nodes with regard to melanoma metastases in comparison with histology and immunohistology. METHODS AND RESULTS: Sentinel lymph nodes (n = 41) from 29 melanoma patients and 29 lymph nodes from 27 patients without melanoma were analysed by histology (H&E) and immunohistology (Melan A, HMB45). cDNA of these lymph nodes was subjected to LightCycler PCR amplification using primers specific for tyrosinase and HMB45. Two melanoma sentinel lymph nodes contained naevus cells by histology and immunohistology and were therefore excluded from further evaluation. Eight (20.5%) of the remaining 39 melanoma sentinel lymph nodes were positive by histology and immunohistology and tyrosinase PCR, 15.4% (6/39) were positive only by tyrosinase PCR, 2.6% (1/39) were positive only by histology and immunohistology. HMB45 PCR revealed positive results in 7.7% (3/39) sentinel lymph nodes, which were also positive by tyrosinase PCR and histology and immunohistology. Of non-melanoma lymph nodes 13.8% (4/29) and 14.8% (4/27) of non-melanoma patients were positive by tyrosinase PCR but negative by histology and immunohistology and HMB45 PCR. Thus, tyrosinase PCR had a specificity of only 85.2%. CONCLUSIONS: The specificity of tyrosinase PCR and the sensitivity of HMB45 PCR are too low to recommend these PCR examinations for the guidance of therapy, in particular complete regional lymph node dissection.

Successful treatment of anogenital Bowen's disease with the immunomodulator imiquimod, and monitoring of therapy by DNA image cytometry.

Imiquimod (Aldara, 3M) is an immune response modifier used for the treatment of anogenital warts. We report a 55-year-old non-immunocompromised woman with extensive, human papillomavirus (HPV) 16-positive anogenital Bowen's disease. After 5 months of local treatment with imiquimod, the lesions completely regressed clinically and histologically, and HPV 16 DNA was no longer detectable. Moreover, DNA image cytometry revealed DNA aneuploidy (an indicator of prospective malignancy) in pretreatment samples but not in post-treatment samples. Therefore, imiquimod might be a treatment option for Bowen's disease, particularly in patients where other treatment modalities such as surgery are contraindicated.

[Pyoderma gangraenosum. Case report and comparison with necrotizing fasciitis]. / Pyoderma gangraenosum. Fallbeispiel und Vergleich mit nekrotisierender Fasziitis.

The differential diagnosis of patients with ulcerative trachelophyma repeatedly causes difficulties. Particularly in view of the possibly fatal outcome, early differentiation between two clinical pictures is very important. Necrotizing fasciitis is often misdiagnosed or the diagnosis is delayed with a mortality rate of approximately 25-40%. It is characterized by local disintegration of the subcutaneous fascia and extensive gangrene of the skin. Sometimes it is not even possible to control the disease using combined antibiotics. Early surgical exploration is mandatory to stop progression of the disease. Pyoderma gangrenosum develops following an initial lesion with pustules and bullae in an ulceration with slight bleeding. Therapy consists of i.v. glucocorticoids (60-200 mg prednisolone/day) administered as early as possible. In complete contrast to the therapy for necrotizing fasciitis, surgical intervention should be strictly avoided in cases of pyoderma gangrenosum.

Differentiation between malignant and benign follicular adnexal tumours of the skin by DNA image cytometry.

BACKGROUND: We have demonstrated in previous studies that DNA image cytometry (DNA ICM) can be helpful in detecting malignancy in sebaceous tumours of the Muir-Torre syndrome and sweat gland tumours. However, little is known about DNA ICM in cutaneous adnexal tumours with follicular differentiation. OBJECTIVES: To study a larger series of benign and malignant follicular adnexal tumours with DNA ICM. METHODS: We studied 13 malignant follicular tumours (seven trichilemmal carcinomas, five malignant proliferating pilar tumours, one pilomatrix carcinoma) and 55 benign follicular tumours (four tumours of the follicular infundibulum, seven Winer's pores, eight trichilemmomas, two trichofolliculomas, 16 trichoepitheliomas, 13 pilomatrixomas, five trichoblastomas) by DNA ICM. All cases were clear-cut as malignant or benign, respectively, on histopathological criteria. The stemline interpretation according to Böcking et al. (DNA distribution in gastric cancer and dysplasia. In: Precancerous Conditions and Lesions of the Stomach, Zhang YC, Kawai K, eds. Berlin: Springer-Verlag, 1993: 103-20) was performed in all cases. In addition, 5[c]-exceeding events (5cEE) and the 2[c] deviation index (2cDI) were calculated, except in one histopathologically benign tumour, which revealed euploid polyploidization, as the analysis of 5cEE and 2cDI is not valid in that case. RESULTS: A 2cDI threshold of 0.24 proved to be the most reliable marker for the distinction between malignant and benign follicular tumours. On the basis of this feature, all malignant and benign tumours were correctly classified. A specificity of 100% was achieved by all three interpretation methods, but the sensitivity of 2cDI for the detection of malignant tumours was superior to the analysis of 5cEE (sensitivity 77%) and to the stemline interpretation (sensitivity 23%). CONCLUSIONS: DNA ICM may be helpful in distinguishing between malignant and benign follicular tumours.

Tissue eosinophilia in acute and chronic atopic dermatitis: a morphometric approach using quantitative image analysis of immunostaining.

BACKGROUND: The frequency and amount of tissue eosinophilia in spontaneous lesions of acute and chronic atopic dermatitis (AD) are still a matter of controversy, and little is known about the distribution of eosinophilia in skin. OBJECTIVES: To give a quantitative description of tissue eosinophilia in spontaneous lesions of acute and chronic AD based on morphometric data. METHODS: Thirty-one lesional skin biopsies of AD were evaluated using our recently described method for the quantitative assessment of eosinophilic granule protein (EGP) deposition by image analysis of immunostaining using the antibodies EG1, EG2, MBP, EPO and neutrophil elastase (NE). The frequency, amount and distribution of protein deposition including extracellular EGP deposition as an indicator of complete activation and degranulation of eosinophils were determined. Eosinophil count was performed in addition. Histopathological parameters of acute dermatitis (spongiosis) and chronic dermatitis (epidermal hyperplasia) were scored to look for a correlation with tissue eosinophilia. RESULTS: Tissue eosinophilia was found in nearly all biopsies (30 of 31). The most protein was detected by EG2, followed by EG1, MBP and EPO, with very small amounts of NE. A superficial tissue distribution of eosinophilia was found, with < 10% of total EGP deposition below a depth of 1.39 mm from the epidermis. Eosinophils were involved in acute, spongiotic dermatitis, but more tissue eosinophilia including EGP deposition was detected in lesions with pronounced epidermal hyperplasia than in biopsies without. CONCLUSIONS: These data provide further evidence for the involvement of activated eosinophils in acute and chronic AD by a new quantitative in situ approach. Pronounced tissue eosinophilia, especially EGP deposition as the result of complete activation of eosinophils, is found in chronic AD and may be involved in the development or maintenance of chronicity.

[Chemotherapy-induced erythema nodosum leprosum: successful treatment with thalidomide]. / Erythema nodosum leprosum unter Chemotherapie. Erfolgreiche Behandlung mit Thalidomid.

The severity and outcome of a chronic granulomatous infection caused by M. leprae depend on the cell-mediated immunity towards the pathogen. The disease classification is based on the host's response to M. leprae ranging from high to low resistance (polar tuberculoid leprosy to polar lepromatous leprosy). The host's position in the spectrum is not stable; leprosy reactions reflecting changed immune status may occur spontaneously or during chemotherapy. The type II reaction or erythema nodosum leprosum can most often be seen in patients with lepromatous leprosy, a multiorgan disease characterized by an unrestricted bacillary replication. Clinically, this reaction is characterized by crops of painful bright pink, dermal and subcutaneous nodules arising in clinically normal skin, in association with fever, malaise, glomerulonephritis and arthralgias. Therefore, prompt institution of immunosuppressive therapy with corticosteroids or thalidomide is recommended. This case report describes the development of erythema nodosum leprosum during chemotherapy treated successfully with thalidomide. Furthermore, immunologic effects and potential side effects of this drug are discussed.

Detection of clonal T cell receptor gamma gene rearrangements in cutaneous T cell lymphoma by LightCycler-polymerase chain reaction.

Cutaneous T cell lymphoma is thought to be characterized by a monoclonal T cell infiltrate in the skin that can be detected by polymerase chain reaction-based amplification of T cell receptor gamma gene rearrangements. We sought to establish a new, simple, and fast LightCycler-based real-time polymerase chain reaction assay for the detection of monoclonality in cutaneous T cell lymphoma, which was suitable for routine laboratory application. Monoclonal T cell receptor gamma gene rearrangements were detected by polymerase chain reaction with consensus primers using: (i) a thermocycler followed by polyacrylamide gel electrophoresis; (ii) a Light Cycler followed by melting curve analysis; and (iii) a LightCycler and subsequent polyacrylamide gel electrophoresis. The detection limit of monoclonal Jurkat T cells diluted in polyclonal peripheral blood mononuclear cells was: (i) 1--3% by thermocycler--polymerase chain reaction and polyacrylamide gel electrophoresis; (ii) 10% by LightCycler--polymerase chain reaction and melting curve analysis; and (iii) 1% by LightCycler--polymerase chain reaction and polyacrylamide gel electrophoresis. In skin biopsies of 22 cutaneous T cell lymphoma patients, a monoclonal or biclonal T cell infiltrate was detected in: (i) 15 of 22 (68%) by thermocycler--polymerase chain reaction and polyacrylamide gel electrophoresis; (ii) 13 of 22 (59%) by LightCycler--polymerase chain reaction and melting curve analysis; and (iii) 16 of 22 (72%) by LightCycler--polymerase chain reaction and polyacrylamide gel electrophoresis. All three techniques revealed negative results in skin biopsies from 26 patients with benign dermatitis. In conclusion, LightCycler--polymerase chain reaction and melting curve analysis is a fast, simple and specific method to detect monoclonal T cell infiltrates in cutaneous T cell lymphoma. Sensitivity of LightCycler--polymerase chain reaction and polyacrylamide gel electrophoresis is slightly higher compared with sensitivity of thermocycler--polymerase chain reaction and polyacrylamide gel electrophoresis. Melting curve analysis, however, is less sensitive compared with polyacrylamide gel electrophoresis, and in case of negative results of the melting curve analysis, it is recommended to resolve LightCycler--polymerase chain reaction samples by gel electrophoresis.

Characterization of the CC chemokine receptor 3 on human keratinocytes.

CC chemokine receptors are expressed on hematopoietic cells, and these may impart selective homing of monocyte, leukocyte, and lymphocyte subsets to sites of inflammation. CC chemokine receptor 3 is the major receptor on eosinophils and is also expressed on other inflammatory cells suggesting its important role for allergic diseases such as atopic dermatitis and bronchial asthma. Eotaxin, eotaxin-2 and eotaxin-3 have been identified as ligands that only activate CC chemokine receptor 3. CC chemokine receptor 3 is also activated by other promiscuous ligands, however, such as RANTES and monocyte chemotactic protein 4. To date, CC chemokine receptor 3 has not been reported to be expressed on nonhematopoietic cells. In this study, we investigated whether keratinocytes possess autocrine and paracrine mechanisms for CC chemokine secretion and receptor expression as reported for the expression of interleukin 8 and its receptors. Reverse transcriptase polymerase chain reaction analysis demonstrated that CC chemokine receptor 3 mRNA is expressed constitutively in cultured keratinocytes. The signal quantities of the CC chemokine receptor 3 amplicons showed lower intensities for keratinocytes than for eosinophils. In situ hybridization techniques exhibited that basal cell layers of the epidermis were stained homogeneously for CC chemokine receptor 3 mRNA with a decreasing signal to the upper epidermis showing that differentiating and proliferating keratinocytes did express mRNA specific for CC chemokine receptor 3. Immunohistochemical studies confirmed low expression of CC chemokine receptor 3 protein on epidermal keratinocytes compared to the high level observed on infiltrating eosinophils. Furthermore, stimulation of cultured keratinocytes with eotaxin resulted in an increased [3H]thymidine incorporation indicating a role of CC chemokine receptor 3 in epidermal proliferation and differentiation. These data demonstrate that CC chemokine receptor 3 is expressed not only on hematopoietic cells but also on keratinocytes as nonhematopoietic cells with ectodermal origin. Therefore, the identification of CC chemokine receptor 3 on epidermal keratinocytes may indicate a role for CC chemokine receptor 3 and its ligands in skin physiology and pathophysiology.

[Pyoderma gangrenosum: successful topical therapy with tacrolimus (FK506)]. / Pyoderma gangraenosum: erfolgreiche topische Therapie mit Tacrolimus (FK506).

Pyoderma gangrenosum is a distinct clinical entity characterized by chronic, recurring, destructive ulcerations. Although the pathogenesis of pyoderma gangrenosum is unknown, immunologic aberrations of neutrophil granulocytes seem to be important. Systemic steroids and macrolide lactones such as cyclosporin A and tacrolimus have been reported to be useful in the clinical management of disease. Pyoderma gangrenosum has been found to be associated with several systemic diseases. The association with chronic ulcerative colitis is well known, but the diagnosis may be complicated by early administration of systemic steroids. Therefore, local immunosuppression with topically applied agents could be an efficient therapeutic alternative especially for mild or early cutaneous lesions.We describe the successful topical treatment of a patient with multiple lesions of pyoderma gangrenosum with 0,1% tacrolimus (FK506) ointment which is known to have better dermal penetration and higher immunosuppressive potency than topical cyclosporin A. In addition, other indications for topical tacrolimus are discussed.

[Post-zoster granuloma with detection of varicella zoster virus DNA in the granulomas]. / Postzosterische Granulome mit Nachweis von Varizella-zoster-Virus-DNA in den Granulomen.

Granulomatous skin changes following herpes zoster are uncommon and their pathogenesis is unclear. We demonstrated varicella-zoster virus in the granuloma tissue of an immunocompromised patient with postherpetic granulomas and use this finding as basis for discussing the pathogenesis of these lesions.

Allelic loss at the neurofibromatosis type 1 (NF1) gene locus is frequent in desmoplastic neurotropic melanoma.

Mutations of the tumour-suppressor gene NF1 (neurofibromatosis 1) have been observed in neurofibromas and neurofibrosarcomas of patients with von Recklinghausen's disease and in sporadic nerve sheath tumours. In contrast, melanoma, another tumour type of neuroectodermal origin, rarely shows NF1 alterations. Desmoplastic neurotropic melanoma (DNM) is an uncommon melanoma subtype that shares morphological characteristics with nerve sheath tumours. Therefore, we analysed 15 DNM and 20 melanomas without morphological features of desmoplasia or neuroid differentiation (common melanomas) for loss of heterozygosity (LOH) at the NF1 locus and flanking regions. Allelic loss was detected in 10/15 (67%) DNM but only in 1/20 (5%) common melanomas. LOH was most frequently observed at marker IVS38, located in intron 38 of NF1. These data suggest a role for NF1 in the pathogenesis of DNM and support an earlier hypothesis that exon 37 might encode a functional domain. DNM may represent an interesting tumour model tor the further elucidation of the cellular functions and tumour-suppressive potential of neurofibromin.

Aluminium-induced granulomas after inaccurate intradermal hyposensitization injections of aluminium-adsorbed depot preparations.

BACKGROUND: The development of persistent subcutaneous nodules at the injection sites of aluminium-adsorbed hyposensitization solutions is rare. These nodules have been interpreted as a delayed, granulomatous hypersensitivity reaction to aluminium. We report for the first time a case of persistent intradermal granulomas that developed at the sites of inaccurate intradermal, instead of subcutaneous, hyposensitization injections. METHODS: An intradermal nodule was excised and processed for histopathology, scanning electron microscopy, and X-ray microanalysis. Intradermal and patch tests with aluminium hydroxide were performed. RESULTS: Histologically, the nodule presented a pattern of granulomatous inflammatory reaction surrounding foci of necrotic tissue. Scanning electron microscopy and X-ray microanalysis revealed deposits of aluminium within the granulomas. Patch tests with aluminium hydroxide were negative, and intradermal tests caused persistent intradermal granulomas. Subsequent hyposensitization therapy in our department with the usual subcutaneous injections of aluminium-adsorbed allergen extracts was well tolerated by the patient. CONCLUSIONS: Local toxic effects of aluminium may be crucial in the development of persistent intradermal injection-site granulomas. Such intradermal nodules may develop even if the subcutaneous route is well tolerated. We conclude that inaccurate intradermal injections of aluminium-containing solutions have to be strictly avoided.

11q23 allelic loss is associated with regional lymph node metastasis in melanoma.

Genetic alterations of the long arm of chromosome 11 have been implicated in melanoma pathogenesis, and we recently identified two distinct regions of common allelic loss in chromosomal band 11q23. To establish the point in time of melanoma tumorigenesis at which these two putative tumor suppressor loci become relevant, we investigated allelic loss [loss of heterozygosity (LOH)] in both chromosomal regions in tumors of progressing patients. We analyzed 102 tumor samples from 23 patients for whom at least two (10 patients) or three (13 patients) tumor samples from different clinical progression steps (such as primary tumor and/or in-transit metastasis and/or regional lymph node metastasis and/or distant metastasis) were available. We detected no 11q23 LOH at any stage in 3 of 23 patients and detected LOH at all stages tested in 8 of 23 patients. In 8 of the remaining 12 (67%) patients with 11q23 LOH at some stage during tumor progression, we found this to occur first at regional lymph node metastasis. Two of these patients retained constitutional heterozygosity in several in-transit metastases that developed up to 7 months after lymph node metastases that already had loss. We therefore conclude that 11q23 LOH is associated with regional lymph node metastasis in melanoma. Finally, we detected an allele shift restricted to a histomorphologically distinct part of a primary melanoma and found that the same parental chromosome was affected by allelic loss in a subsequently occurring lymph node metastasis. These findings support our conclusion and give additional evidence for the hypothesis of molecular heterogeneity of early tumor cell populations in melanoma.

[Trigeminotrophic ulceration of the ala nasi in Wallenberg syndrome]. / Trigeminotrophe Ulzeration des Nasenflügels bei Wallenberg-Syndrom.

A patient developed a trophic ulceration of the nose after an acute bulbar ischemia with infarction of the right trigeminal nuclei. Neurologic examination showed symptoms of a Wallenberg syndrome including ipsilateral hyp- and paresthesia of the second trigeminal branch and disturbed sensibility and temperature sense on the contralateral half of the body. The right ala nasi showed the characteristic sickle-shaped defect (ulcération en arc). The differential diagnosis and therapeutic approaches are discussed.

DNA image cytometry in malignant and benign sweat gland tumours.

The histopathological differentiation between well-differentiated carcinomas and atypical adenomas of sweat gland origin may be difficult, even if immunohistochemical methods are used. Therefore, additional techniques may be helpful. We previously demonstrated that DNA image cytometry (ICM-DNA) can be useful in distinguishing between malignant and benign clear cell hidradenoma. In the present study, a larger series of sweat gland tumours, with a clear-cut diagnosis as malignant or benign on histopathological criteria, was examined by ICM-DNA. Enzymatic cell separation specimens were prepared from paraffin-embedded tissues of 18 sweat gland carcinomas (14 porocarcinomas, one classic eccrine adenocarcinoma, two microcystic adnexal carcinomas and one mostly ductal apocrine carcinoma) and 47 benign sweat gland tumours (three syringocystadenomas, five spiradenomas, 14 cylindromas, three syringomas, seven nodular hidradenomas, 10 cutaneous mixed tumours, four poromas and one apocrine hidrocystoma). Specimens were examined by ICM-DNA according to the current recommendations of the European Society for Analytical Cellular Pathology with the AutoCyte QUIC-DNA workstation using mesenchymal cells as an internal reference. DNA aneuploidy was detected by the stemline interpretation according to Böcking and/or at least three 5[c]-exceeding events. DNA aneuploidy was detected in 16 of 18 (89%) of the sweat gland carcinomas, but in none of the 47 adenomas. These results suggest that the detection of DNA aneuploidy in sweat gland tumours using ICM-DNA is a clear and specific indicator of prospective malignancy.

Retiform hemangioendothelioma: another tumor associated with human herpesvirus type 8?

Retiform hemangioendothelioma is a rare low-grade angiosarcoma of the skin. It shares some clinical characteristics with Kaposi's sarcoma, a tumor with known human herpesvirus 8 (HHV-8) association. We report a case of retiform hemangioendothelioma in which we detected HHV-8 DNA sequences.

Accuracy of frozen section measurements for the determination of Breslow tumour thickness in primary malignant melanoma.

AIMS: Microstaging of primary malignant melanoma (MM) and the width of surgical margins depend mainly on Breslow tumour thickness (BTT). The use of frozen section (FS) measurements of BTT has been doubted, and previous reports have shown conflicting results regarding the comparability to paraffin sections (PS). To look for significant differences of BTT due to freezing or paraffin embedding, we evaluated a larger series of melanocytic lesions as far as possible excluding other technical influences. METHODS AND RESULTS: Paired 'mirror sections' of 112 melanocytic lesions (33 MM and 79 melanocytic naevi) were measured according to Breslow on single corresponding PS and FS of the same tumour specimen. Comparing measurements on FS and PS, we found very small differences of BTT on average and an almost equal distribution of BTT in the two sets of values with no statistically significant difference by applying the Wilcoxon signed rank test. Concerning the clinically most important 1 mm-threshold of BTT, 110 (98.2%) of the lesions gave equal measurements in FS and PS. CONCLUSIONS: Frozen sections can be used for accurate measurements of Breslow tumour thickness. Consequently, intraoperative frozen section diagnosis of thick melanoma immediately followed by excision with wide surgical margins is possible in experienced centres.

Quantification of eosinophilic granule protein deposition in biopsies of inflammatory skin diseases by automated image analysis of highly sensitive immunostaining.

Eosinophilic granulocytes are major effector cells in inflammation. Extracellular deposition of toxic eosinophilic granule proteins (EGPs), but not the presence of intact eosinophils, is crucial for their functional effect in situ. As even recent morphometric approaches to quantify the involvement of eosinophils in inflammation have been only based on cell counting, we developed a new method for the cell-independent quantification of EGPs by image analysis of immunostaining. Highly sensitive, automated immunohistochemistry was done on paraffin sections of inflammatory skin diseases with 4 different primary antibodies against EGPs. Image analysis of immunostaining was performed by colour translation, linear combination and automated thresholding. Using strictly standardized protocols, the assay was proven to be specific and accurate concerning segmentation in 8916 fields of 520 sections, well reproducible in repeated measurements and reliable over 16 weeks observation time. The method may be valuable for the cell-independent segmentation of immunostaining in other applications as well.

[Amicrobial intertriginous pustulosis in autoimmune diseases--a new entity?]. / Amikrobielle intertriginöse Pustulose bei Autoimmunerkrankungen--Eine neue Entität?

During the last decade an unusual amicrobial intertriginous pustulosis has been described in association with autoimmune disease in sixteen female patients. The clinical hallmark is a sterile pustular dermatosis preferentially located in intertriginous regions that responds to local or systemic corticosteroids. Histologic features are subcorneal sometimes spongiform neutrophilic pustules. We report an additional patient suffering from this unusual dermatosis. An overview of the patients described to date and a review of the literature are given in an attempt to delineate this amicrobial intertriginous pustulosis from the known pustular dermatoses.