Guntram Bezold

Coexpression patterns of EGFR, HER2, HER3 and HER4 in non-melanoma skin cancer

The receptor tyrosine kinases (RTKs) epidermal growth factor receptor (EGFR), HER2, HER3 and HER4 are involved in the pathogenesis of multiple human malignant neoplasias. However, their role in the carcinogenesis of basal cell carcinomas (BCC) and squamous cell carcinomas (SCC) remains to be elucidated. In order to further define the role of these RTKs, 56 human skin tissue samples of normal skin, BCC and SCC were studied by conventional and differential and quantitative reverse transcriptase-polymerase chain reaction (rtPCR). EGFR and HER3 were predominantly expressed in the BCCs and SCCs, while HER2 was ubiquitously expressed. HER4 was not expressed in any sample. Since in vitro studies have provided compelling evidence that heterodimer formation of these receptors are associated with different signal transduction processes, coexpression patterns might be decisive for the induction and maintenance of a malignant phenotype. These results confirm this concept: isolated HER2 expression and EGFR/HER2 were predominantly found in normal skin, while HER2/HER3 and the triple expression of EGFR/HER2/HER3 were seen more frequently in the BCCs and SCCs compared with normal skin (50% and 40% compared with 26%, respectively). The activation of HER3, in addition to EGFR and HER2, might therefore be associated with the malignant phenotype. However, due to the small numbers in this study, further confirmation of the patterns is needed.

Extra c-myc oncogene copies in high risk cutaneous malignant melanoma and melanoma metastases

Amplification and overexpression of the c-myc gene have been associated with neoplastic transformation in a plethora of malignant tumours. We applied interphase fluorescence in situ hybridization (FISH) with a locus-specific probe for the c-myc gene (8q24) in combination with a corresponding chromosome 8 α-satellite probe to evaluate genetic alterations in 8 primary melanomas and 33 advanced melanomas and compared it to 12 melanocytic nevi, 7 safety margins and 2 cases of normal skin. Additionally, in metaphase spreads of 7 melanoma cell lines a whole chromosome 8 paint probe was used. We investigated the functionality of the c-myc gene by detecting c-myc RNA expression with RT-PCR and c-myc protein by immunohistochemistry. 4/8 primary melanomas and 11/33 melanoma metastases showed additional c-myc signals relative to the centromere of chromosome 8 copy number. None of the nevi, safety margins or normal skin samples demonstrated this gain. In 2/7 melanoma cell lines (C32 and WM 266–4) isochromosome 8q formation with a relative gain of c-myc copies and a loss of 8p was observed. The highest c-myc gene expression compared to GAPDH was found in melanoma metastases (17.5%). Nevi (6.6%) and primary melanomas (5.0%) expressed the c-myc gene on a lower level. 72.7% of the patients with c-myc extra copies had visceral melanoma metastases (UICC IV), patients without c-myc gain in 35.0% only. The collective with additional c-myc copies also expressed the gene on a significantly higher level. These results indicate that a c-myc gain in relation to the centromere 8 copy number might be associated with advanced cutaneous melanoma.

Direct detection of five common dermatophyte species in clinical samples using a rapid and sensitive 24-h PCR-ELISA technique open to protocol transfer.

Identification of dermatophytes is usually based on morphological characteristics determined by time‐consuming microscopic and cultural examinations. An effective PCR–ELISA method has been developed for rapid detection of dermatophyte species directly from clinical specimens within 24 h. Isolated genomic DNA of skin scrapings and nail samples from patients with suspected dermatophyte infections is amplified with species‐specific digoxigenin‐labelled primers targeting the topoisomerase II gene. The subsequent ELISA procedure with biotin‐labelled probes allows a sensitive and specific identification of the five common dermatophytes –Trichophyton rubrum, T. interdigitale, T. violaceum, Microsporum canis and Epidermophyton floccosum. PCR–ELISA, based on the new polyphasic species concept, was assessed using 204 microscopy‐positive samples in two university mycological laboratories in Munich and Tübingen, and 316 consecutive specimens – regardless of mycological findings – in a dermatological practice laboratory in Neu‐Ulm. One of the five dermatophytes was confirmed by PCR–ELISA in 163 of 204 (79.9%) of the clinical samples from the university hospitals found positive using microscopy. Culture was positive for dermatophytes in 59.8% of the same cases. A significant difference between these two methods could be demonstrated using the McNemar test (P < 0.005). Analysis of specimens from Neu‐Ulm confirmed the results in a dermatological practice laboratory as 25.0% of the specimens had positive PCR results, whereas only 7.3% were positive according to culture. Direct DNA isolation from clinical specimens and the PCR–ELISA method employed in this study provide a rapid, reproducible and sensitive tool for detection and discrimination of five major dermatophytes at species level, independent of morphological and biochemical characteristics.

Langerhans cell histiocytosis in a child presenting as a pustular eruption

Sir, Langerhans cell histiocytosis (LCH) includes a spectrum of disorders with overlapping clinical features, i.e. Letterer±Siwe disease, Hand±SchuÈ ller±Christian syndrome, eosinophilic granuloma and congenital self-healing reticulohistiocytosis. The typical cutaneous findings comprise seborrhoeic dermatitis-like lesions, reddish-brown purpuric papules and nodules, oozing erosions, and ulcerations. A child with LCH is described, who presented with an uncommon pattern of pustular lesions on the forehead, hands, feet and genital region, following an atypical clinical course. One week after birth, a 14-month-old girl developed multiple, sharply demarcated itchy pustules on the forehead, together with red papules, and pustules partly covered with haemorrhagic crusts, which were predominantly on the soles of the feet (Fig. 1a). Some pustules occurred on the hands and in the genital and gluteal region. No lymphadenopathy was detectable. During topical treatment with lotio zinci (lotion with zinc oxide, talcum, glycerol and distilled water) the cutaneous lesions regressed slowly, but new lesions developed during the following months. After 5 months, an ulcer developed in the maxillary region. There was no specific family history of skin diseases. A skin biopsy of a pustule from the gluteal region revealed a dense upper dermal infiltrate of large histiocytes with a reniform nucleus, and intraepidermal collections of histiocytic cells (Fig. 1b). Immunohistochemical stains were positive for CD1 and for S-100 antigen. Electron microscopy revealed intracytoplasmic Birbeck granules within the histiocytic cells, thus confirming the diagnosis of LCH. Bacteriological and mycological examinations from skin lesions were negative. Full blood count, liver and kidney function were normal. Scintiscanning and radiography showed distinct involvement of the maxilla. Abdominal sonography was normal. Based on the occurrence of new cutaneous lesions and the subsequent involvement of the osseous part of the maxilla, chemotherapy was initiated with oral prednisolone 40 mg m daily for 4 weeks; then reduction of dose, vinblastine 0 ́2 mg kg once weekly for 6 weeks, then once monthly; and 6-mercaptopurine 50 mg m daily. Cutaneous lesions on the scalp, hands and feet, and the osseous lesion of the maxilla healed during the chemotherapy, which was performed for 1 years. In 1955, Lichtenstein summarized the three entities Letterer±Siwe disease, Hand±SchuÈ ller±Christian disease and eosinophilic granuloma under the term histiocytosis X. The cause of histiocytosis X (LCH) is unknown, but it is believed to be a proliferative process of Langerhans cells. Lesional histiocytes in the child described here were positive for S-100 antigen and CD1, as well as for intracytoplasmic Birbeck granules. The skin lesions did not clear completely

Detection of cutaneous herpes simplex virus infections by immunofluorescence vs. PCR

Background Detection of cutaneous infections with herpes simplex virus (HSV) has proven difficult, as serum antibody tests sometimes are not sensitive and specific enough for that purpose.

Varicella zoster viraemia during herpes zoster is not associated with neoplasia.

Background Shingles are caused by an endogenous or exogenous reinfection with varicella zoster virus (VZV). Up to 50% of individuals with Hodgkins disease develop herpes zoster; however, no association could be shown between the occurrence of herpes zoster and underlying subclinical malignancies.

Quantitation of Herpes simplex DNA in Blood during Aciclovir Therapy with Competitive PCR ELISA

Background: Monitoring viral load in blood has already been introduced into clinical routine for human immunodeficiency virus and hepatitis C virus. Objective: This study was conducted to monitor the decline of herpes simplex (HSV) viral load in the blood of a patient with gingivostomatitis herpetica prior and during aciclovir therapy. Methods: Analysis was done by quantitative PCR ELISA using an internal quantitation standard. Results: Copy numbers were 66/µl blood prior to therapy, 60 during oral medication with valaciclovir, 97 and 72 copies/µl blood during the first 2 days of intravenous aciclovir therapy, followed by a sharp decline to 8 and 9 copies on days 3 and 4. During the following days, HSV was no longer detectable. Conclusion: As this quantitative approach can be easily adjusted to any other PCR, it provides a reliable, easy-to-apply method for monitoring therapy, also during new antiviral clinical trials.

Sneddon's syndrome in a child.

Sir, We read with interest the recent report by Cox et al. on the association of atopic dermatitis with the beta subunit of the high-affinity IgE receptor (Fc1RI-b). The gene was originally identified as a candidate for atopy on human chromosome 11q. Fc1RI-b has been shown to be involved in the amplification of Fc1RI-mediated signalling that might be related to the pathogenesis of atopic disease. Because of the predominantly maternal transmission of atopy, it has been strongly suggested that the gene for Fc1RI-b may show a parent-of-origin effect such as genomic imprinting. One previous report consistent with this notion showed maternal transmission of the I181L allele in a British population, while another showed no parent-of-origin effect of the transmission of the E237G allele in an Australian population. The report by Cox et al. again raises this issue by demonstrating exclusive maternal transmission of Fc1RI-b alleles to offspring with atopic dermatitis. None the less, as far as we know, there has been no direct test of possible allele-specific expression of this gene. To study the possible involvement of a genomic imprinting mechanism, we have used the widely applicable mouse model system. We find, as detailed below, that the Fc1RI-b gene shows no parent-of-origin-specific expression, using F1 hybrids between a laboratory mouse strain (C57BL/6J) and wild mouse strains [either Mus musculus molossinus (MOLF/Ei) or M. spretus (SPRET/Ei)]. The parental mouse strains (C57BL/6J, MOLF/Ei, SPRET/ Ei) were purchased from Jackson Laboratory (Bar Harbor, ME, U.S.A.). Cloning, sequencing, total RNA extraction and reverse transcription±polymerase chain reaction (PCR) analysis of Fc1RI-b were done as described previously. Based on a human genomic DNA sequence (GenBank M89796) and a mouse cDNA sequence (GenBank J05019), we designed a PCR primer pair spanning exon 6, intron 6 and exon 7 of the Fc1RI-b gene, so that an intron could be included in the PCR target. This allowed us to measure the gene expression directly, without interference by possible contamination of genomic DNAs in RNA samples. Sequence polymorphisms among C57BL/6J, MOLF/Ei and SPRET/Ei were identified by sequencing PCR products from each strain (deposited in GenBank; accession numbers U90217±U90219). A 395-base pair (bp) fragment of the gene for Fc1RI-b was amplified from cDNAs with primers up6: 5 0-ATCCTGGCCTTTTGCAGTGC-3 0 and dn10: 5 0-TGTATGTGAAATTGTGACAC-3 0. To obtain enough DNA for analyses, a shorter fragment (361 bp) was reamplified from the PCR products with primers up6 and dn4: 5 0-GGAGTGAATGATATCCGCAA-3 0. Allele-specific expression in reciprocal crosses between C57BL/6J and MOLF/Ei was examined by using a Sau3AI restriction fragment length polymorphism (RFLP) in a 361-bp PCR product (Fig. 1A). For RFLP analysis, 3 mL of unpurified PCR products were digested with 5 U of Sau3AI in a 15-mL reaction mixture at 37 8C for 3 h, then electrophoresed in a 12% polyacrylamide gel. The gel was stained by ethidium bromide and photographed under ultraviolet radiation. Both paternally and maternally derived alleles were expressed in all examined tissues, i.e. embryo, placenta and yolk sac at 14 ́5 days postconception, whole body and skin at the newborn stage (Fig. 1B), and spleen, lung and colon in adults (not shown). The data clearly demonstrate the biallelic expression of the gene for Fc1RI-b. To confirm these data, interspecific hybrids between a laboratory mouse strain and M. spretus were used. In these crosses, the C57BL/6J-specific allele (296 bp) can be distinguished from the SPRET/Ei-specific allele (279 bp) by length

Receptor Tyrosine Kinase and p16/CDKN2 Expression in a Case of Tripe Palms Associated with Non-Small-Cell Lung Cancer

Background: Tripe palms is a descriptive term for a cutaneous paraneoplastic keratoderma. Tripe palms are frequently associated with gastric and pulmonary carcinoma. The pathogenetic mechanism remains unknown. Objective: To determine the influence of receptor tyrosine kinases, which are both expressed in pulmonary carcinomas and in human skin, we performed expression studies on epidermal growth factor receptor (EGFR), HER2, HER3 in a skin sample of tripe palms obtained from a patient with non-small-cell lung cancer with lymph node involvement. Two months after diagnosis, the patient had developed palmoplantar ‘tripe palms’. Additionally, the expression of SRC, c-myc and p16/ CDKN2 were studied. Method: Conventional reverse-transcription polymerase chain reaction was performed on a tissue sample of tripe palms. Results: Weak expression of HER2 and of p16/CDKN2 was found. EGFR, HER3, c-myc and SRC were not expressed. Conclusion: Receptor tyrosine kinases of subclass I, the tyrosine kinase SRC and the oncogene c-myc play no major role in the pathogenesis of this case of tripe palms.