PTEN/MMAC1 expression in melanoma resection specimens.

PTEN/MMAC1, a tumour suppressor gene located on chromosome 10q23.3, has been found to be deleted in several types of human malignancies. As the chromosomal region 10q22-qter commonly is affected by losses in melanomas, we addressed this gene as tumour suppressor candidate in melanomas. Investigating PTEN/MMAC1 expression at mRNA level by semi-quantitative reverse transcription-polymerase chain reaction, we did not find a statistically significant down-regulation in melanoma resection specimens in comparison to acquired melanocytic nevi from which melanomas quite often are known to arise. Upon immunohistochemistry, PTEN/MMAC1 protein expression in melanomas was not lost. Sequencing the PTEN/MMAC1 cDNAs in 26 melanoma resection specimens (21 primary melanomas, five metastases), we detected three point mutations and two nucleotide deletions which did not represent genetic polymorphisms. With respect to the predicted protein sequences, all three point mutations were silent whereas the two frame shifts at the extreme C-terminus resulted in a loss of the putative PDZ-targeting consensus sequence. As loss of this motif possibly impairs localization and function of PTEN/MMAC1 in the two corresponding primary tumours, alterations of this tumour suppressor protein may participate in some melanomas.

Analysis of losses of heterozygosity of the candidate tumour suppressor gene DMBT1 in melanoma resection specimens.

Deleted in malignant brain tumours 1 (DMBT1), a candidate tumour suppressor gene located on chromosome 10q25.3-q26.1, has recently been identified and found to be deleted in several different types of human tumours. In melanomas, the chromosomal region 10q22-qter is commonly affected by losses, hence we screened primary melanoma samples for losses of heterozygosity (LOH), and acquired melanocytic naevi and melanomas for transcription of DMBT1 and protein expression. Of 38 informative melanomas, 1 nodular melanoma and 2 subcutaneous metastases showed LOH of both microsatellites flanking the gene, suggesting loss of 1 DMBT1 allele. Three further melanomas showed LOH at 1 informative locus but were heterozygous for the second marker. Applying reverse-transcription polymerase chain reaction (RT-PCR), DMBT1 transcription was not found in melanomas. However, DMBT1 transcription was also absent from the majority of naevi from which melanomas frequently arise, making down-regulation of gene transcription during transformation from naevus to melanoma unlikely. Immunohistochemistry showed nerves, sweat glands and the stratum spinosum of the epidermis to be DMBT1 protein positive, whereas the naevi and melanoma cells themselves were negative. All considered, the candidate tumour suppressor gene DMBT1 does not appear to be a major inactivation target in the development of melanomas.

The protein phosphatase 2A subunit Bgamma gene is identified to be differentially expressed in malignant melanomas by subtractive suppression hybridization.

Several genes implicated in the development of various malignancies appear to be of minor relevance in melanoma. We therefore aimed to find a tumour suppressor candidate involved in this malignancy by comparing gene expression in uncultured primary melanoma specimens with those in acquired melanocytic naevi, from which quite often melanomas are known to arise. Applying the subtractive suppression hybridization technique, we generated a subtracted library of candidate genes downregulated in melanoma. Among the cDNA fragments identical to known genes, this library included a cDNA fragment 630 bp in length that is identical to the gene for the human protein phosphatase 2A (PP2A) regulatory subunit B (B56) gamma isoform (PP2A-Bgamma, PPP2R5C). On further evaluation of 15 primary melanoma and 16 acquired melanocytic naevus tissue specimens from independent patients using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis, expression of this gene was found to be suppressed in melanomas compared with naevi; the difference was statistically significant. As PP2A is known to be a major cellular serine-threonine phosphatase, and has been implicated not only in the regulation of cell growth and division but also in the control of gene transcription and growth factor signal transduction, alterations in the pattern of the regulatory subunits may affect substrate specificity and subcellular localization of the PP2A holoenzyme in melanoma cells.

[Extracorporeal photopheresis--treatment option in scleromyxedema?]. / Extrakorporale Photopherese--Therapieoption beim Skleromyxödem?

Scleromyxedema is an uncommon disease of unclear etiology. Therapy is difficult. Two patients with scleromyxedema were treated with extracorporeal photopheresis (ECP). The first patient has been treated unsuccessfully for 3 months with PUVA-bath-therapy and for one year with cyclophosphamide and prednisolone. Thus supplementary treatment with ECP was initiated, as the cyclophosphamide and prednisolone were gradually reduced. After 29 cycles of ECP, the skin lesions had almost disappeared and the associated myopathy also resolved. In the second patient initial monotherapy with ECP was started after PUVA-bath-therapy for 3 months did not show any effect. After temporary improvement with ECP every four weeks, the skin lesions relapsed, so oral cyclophosphamide was added. These two cases confirm the effect of ECP in scleromyxedema, but probably combination therapy is at least initially more successful.

Are responses to therapy of metastasized malignant melanoma reflected by decreasing serum values of S100beta or melanoma inhibitory activity (MIA)?

In metastatic melanoma S100beta as well as melanoma inhibitory activity (MIA) are elevated in the serum in the majority of patients. Elevation has been found to correlate with shorter survival, and changes in these parameters in the serum during therapy were recently reported to predict therapeutic outcome in advanced disease. However, the value of these markers with respect to other possible markers by multivariate analysis has not yet been proven for individual patients. In this prospective study, S100beta and MIA were measured in the serum of 67 consecutive patients before and following treatment. Analysing both the sensitivity and the specificity of the serum parameters by the areas under the receiver operating characteristics (ROC) curves, decreases in S100beta and MIA during therapy were associated with response to therapy, while increases indicated progressive disease. Unexpectedly, the individual diagnostic value of changes in tumour markers during therapy was not superior to one-point measurements at restaging. Moreover, S100beta and MIA were not superior to the conventional parameters lactate dehydrogenase and C-reactive protein (CRP) on multiple logistic regression analysis. Applying classification and regression trees (CARTs), one-point measurements of CRP was shown to be the most relevant overall parameter.

Combined mechanical and antibiotic periodontal therapy in a case of Papillon-Lefèvre syndrome.

BACKGROUND: Papillon Lefèvre syndrome (PLS) is a rare entity and, as such, it is almost impossible to evaluate an effective therapy in a randomized controlled study. The amount of success reported after therapy for prepubertal periodontitis (PP) in PLS is highly variable from case to case. The goal of this case report is to evaluate the effects of a combined mechanical and antibiotic periodontal therapy regimen in the management of PLS. METHODS: A male patient was diagnosed as suffering from PP associated with PLS at the age of 7 years. He showed hyperkeratosis of the palms and soles, as well as advanced periodontal disease already affecting permanent teeth with maximal probing depth and vertical attachment loss of 12 mm and 11 mm, respectively. Subgingival debridement was performed with simultaneous administration of oral 250 mg amoxicillin 3 times daily and 250 mg metronidazole twice daily for one week. Clinical parameters were assessed and subgingival plaque was collected from all teeth prior to therapy and 7 and 26 months after treatment. Selective cultures for A. actinomycetemcomitans were incubated for each individual tooth and DNA probe analysis was performed for various periodontal pathogens. RESULTS: Prior to combined mechanical and antibiotic treatment, all teeth but one harbored Actinobacillus actinomycetemcomitans subgingivally. However, at 7 and 26 months after therapy A. actinomycetemcomitans could be detected neither by culture nor by DNA probes. Clinical parameters improved markedly and teeth erupting after therapy did not exhibit attachment loss of more than 1.5 mm during the observation period. CONCLUSIONS: Eradication (suppression beneath detection levels) of A. actinomycetemcomitans seems to play a significant role in the successful treatment of localized prepubertal periodontitis in PLS.

[Therapy of metastatic malignant uveal melanoma]. / Therapie des metastasierten malignen Uveamelanoms.

The uvea is the most common site for extra-cutaneous melanoma and uveal melanoma is the most frequent primary intraocular tumour in adults. Because its different location, biology, histology, genetic features and prognosis in comparison to cutaneous melanoma, this tumour is considered as a distinct entity in the group of malignant melanoma. While primary uveal melanoma is usually treated by ophthalmologic oncologists, metastatic diseases is often managed by dermatologic oncologists. Hematogenous spread predominantly involves the liver and is often restricted to this organ for a long period. Metastatic uveal melanoma is usually resistant to chemotherapeutic regimens established for the therapy of cutaneous melanoma. Newer therapeutic modalities, such as local intra-arterial chemotherapy into the hepatic artery, perhaps combined with embolisation of feeder blood vessels of liver metastases, improves the prognosis of metastatic uveal melanoma. Currently the nitrosourea derivate fotemustine is the drug of choice in the local hepatic and systemic treatment and seems to be superior to other chemotherapeutic agents. Following the characterisation of primary uveal melanoma, we summarize the results of different treatment protocols for metastatic disease and give an overview of new strategies.

Prognosis of metastatic melanoma: no correlation of tyrosinase mRNA in bone marrow and survival time.

Recent publications suggest that tyrosinase mRNA in blood as well as in bone marrow is detectable only in a subgroup of patients with metastatic melanoma. This would imply that tyrosinase mRNA is of limited value as a tumor marker. We addressed the question of whether patients with metastatic melanoma and RT-PCR-detectable tyrosinase mRNA in blood or bone marrow have a different prognosis than tyrosinase mRNA-negative patients. Twenty melanoma patients with widespread clinical metastases were enrolled; the survival time after first diagnosis of visceral metastases was correlated to tyrosinase mRNA presence in blood and bone marrow samples. The time of survival of eight patients with metastatic melanoma and detectable tyrosinase mRNA in either blood or bone marrow was not different from the prognosis of 12 patients without detectable tyrosinase mRNA in either blood or bone marrow. Detection of tyrosinase mRNA in blood or bone marrow samples of melanoma patients with advanced disease seems to have no substantial relevance for survival time and outcome of disease. In this constellation, detection of tyrosinase mRNA by RT-PCR is not a valid tumor marker. Nevertheless, tyrosinase positivity in bone marrow in earlier tumor stages might indicate increased risk for the development of distant metastases. This should be addressed in further studies.

Transient complete remission of metastasized Merkel cell carcinoma by high-dose polychemotherapy and autologous peripheral blood stem cell transplantation.

Merkel cell carcinoma (MCC) is a rare cutaneous tumour with neuroendocrine differentiation. Metastasis occurs preferentially to regional lymph nodes but distant and multiple visceral metastases may occur. Chemotherapy has been performed with a variety of protocols based largely on agents active in small-cell lung cancer. Owing to the rarity of MCC, there is no standard protocol for the treatment of metastatic disease. We report a 59-year-old patient with systemic metastatic MCC. After diagnosis of distant metastases, first-line polychemotherapy (cisplatin 80 mg m(-2), doxorubicin 50 mg m(-2), etoposide 300 mg m(-2) and bleomycin 30 mg) was administered four times at 3-weekly intervals and resulted in partial remission of metastases. Subsequently, high-dose chemotherapy according to the PEI regimen (ifosfamide 12 g m(-2), carboplatin 900 mg m(-2) and etoposide 1500 mg m(-2)) was applied, followed by autologous blood stem cell transplantation (ABSCT). This protocol resulted in a complete remission that lasted for 6 months. This is the first report on a complete remission of metastatic MCC after high-dose polychemotherapy and ABSCT. High-dose chemotherapy might be a therapeutic option in chemosensitive metastatic MCC, and further evaluation is warranted.

No correlation of tyrosinase mRNA in bone marrow with prognosis of metastatic melanoma.

BACKGROUND: Recent publications suggest that tyrosinase mRNA in blood as well as in bone marrow is detectable only in a subgroup of patients with metastatic melanoma. OBJECTIVE: We addressed the question, whether patients with metastatic melanoma and with RT-PCR-detectable tyrosinase mRNA in blood or bone marrow have a different prognosis compared to tyrosinase mRNA-negative patients. METHODS: 20 melanoma patients with widespread clinical metastases were enrolled and the survival time after first diagnosis of visceral metastases was correlated to tyrosinase mRNA presence in blood and bone marrow samples. RESULTS: The time of survival of 8 patients with metastatic melanoma and detectable tyrosinase mRNA in either blood or bone marrow was not different from the prognosis of 12 patients without detectable tyrosinase mRNA in either blood or bone marrow. CONCLUSION: Although based on a limited number of patients our results suggest that detection of tyrosinase mRNA in blood or bone marrow samples of melanoma patients with advanced disease seems to have no substantial relevance for survival time and outcome of disease. For this purpose, detection of tyrosinase mRNA by RT-PCR is not a valid tumor marker. Nevertheless, tyrosinase positivity in bone marrow in earlier tumor stages might indicate increased risk for the development of distant metastases. This should be addressed in further studies.

Seropositivity for MIA and S100 in patients with gastrointestinal carcinomas.

Serum levels of melanoma inhibiting activity (MIA) and S100, both markers in malignant melanoma, are increased only in few patients with non-melanocytic tumors. We examined a series of serum samples from patients with colorectal (CRC) (N=56), gastric (GC) (N=43), pancreatic (PC) (N=29), hepatocellular (HCC) (N=30), cholangiocellular and gallbladder carcinoma (CCC) (N=18). MIA and S100 were measured by commercially available assays. Positive serum levels for MIA and S100 were found in 16.1% and 5.4% of the patients with CRC, 11.6% and 9.3% with GC, 34.5% and 13.8% with PC, 0% and 30% with HCC and 16.7% with CCC, respectively. All patients with sera positive for either MIA or S100 suffered from advanced tumors and received palliative treatment. Elevated serum levels of MIA and S100 are frequent in patients with gastrointestinal cancer. Further investigation is warranted to define the role of MIA or S100 seropositivity in gastrointestinal cancer with regard to follow-up.

Interleukin-6 and its surrogate C-reactive protein are useful serum markers for monitoring metastasized malignant melanoma.

Malignant melanoma cells are known to secrete interleukin-6, and elevated interleukin-6 serum levels were reported to correlate with shorter median survival rates. We, therefore, investigated serum values of interleukin-6 and its surrogate C-reactive protein for the ability to discriminate progressive from non-progressive metastatic melanoma disease. Just prior to re-staging examinations, interleukin-6, C-reactive protein and the conventional parameter lactate dehydrogenase were determined in 74 patients with stage IV malignant melanoma according to the criteria of the American Joint Committee on Cancer. We found all tested serum parameters to be significantly elevated in progressive disease. Calculating sensitivities and specificities by logistic regression analysis, the highest sensitivities, according to the established thresholds, were found for interleukin-6 and C-reactive protein with 86% and 76%, respectively. Lactate dehydrogenase had the highest specificity with 94%. Calculating Somers' D rank correlation and the area under the "Receiver Operating Characteristic" curve, all three parameters showed high ability to driscriminate progressive from non-progressive disease. By multiple logistic regression, lactate dehydrogenase was identified to be the most statistically significant marker for progressive disease. We conclude that, comparable to lactate dehydrogenase, interleukin-6 and its surrogate C-reactive protein are useful serum markers for monitoring metastatic malignant melanoma.

S100-Beta, melanoma-inhibiting activity, and lactate dehydrogenase discriminate progressive from nonprogressive American Joint Committee on Cancer stage IV melanoma.

PURPOSE: Monitoring advanced malignant melanoma, serum levels of S100-beta (S100beta) and melanoma-inhibiting activity (MIA) were assessed for the ability to discriminate progressive from nonprogressive disease. S100beta and MIA were supposed to be superior to conventional variables, such as lactate dehydrogenase (LDH) level. PATIENTS AND METHODS: Seventy-one patients with stage IV malignant melanoma according to the criteria of the American Joint Committee on Cancer (AJCC) were included in the study. Results of restaging examinations were used as an independent reference standard for diagnosing progressive disease, and S100beta, MIA, LDH level, and erythrocyte sedimentation rate (ESR) were determined in venous blood just before restaging. Sensitivities and specificities of the parameters were calculated by logistic regression analysis. Discrimination ability was assessed by Somers' D(xy) rank correlation and the area under the receiver-operating characteristic curve (ROC-AUC). RESULTS: All tested serum parameters were significantly elevated in patients with progressive disease. The highest sensitivities according to the established thresholds were found for S100beta and MIA (91% and 88%, respectively). LDH had the highest specificity (92%). ESR was dropped from the analysis because of low specificity. In calculating Somers' D(xy) and ROC-AUC values, S100beta, MIA, and LDH showed high discrimination ability. By multiple logistic regression, LDH was identified to be the only statistically significant marker for progressive disease. S100beta and MIA did not provide additional significant information because of their high correlation with LDH with respect to clinical outcome. CONCLUSION: Elevated serum levels of S100beta, MIA, and LDH indicate current disease progression in AJCC stage IV melanoma. LDH was the most relevant overall parameter.

[Pathogenesis of malignant melanoma. Molecular biology aspect]. / Pathogenese des malignen Melanoms. Molekularbiologische Aspekte.

The incidence of melanoma, the most aggressive tumor of the skin, is increasing worldwide. The genetic mechanisms responsible for the initiation and progression of melanoma are poorly understood. Mutations of p16 (CDKN2), p53, ras, neurofibromatosis type I gene (NF-1), bcl2 and the retinoblastoma gene have been described, but none are common. Suggesting heterogeneous mechanisms of carcinogenesis. Both familial inheritance of potential tumor suppressor genes, e.g. p16, and differences in DNA-repair capacity contribute to the individual risk for melanoma. The most important carcinogen for melanoma seems to be u.v. exposition whose mutagenic effects can be demonstrated by molecular analysis of detected point mutations in relevant genes. The u.v.-induced DNA damage generates mutations which are capable of activating proto-oncogenes or inactivating tumor suppressor genes, demonstrating the molecular link between u.v. exposition, DNA damage, mutations and tumor initiation and/or progression. A stage-dependent model of melanoma carcinogenesis analogous to colorectal cancer remains to be established, despite the existence of morphologically and histopathologically well defined melanoma precursor lesions in the skin.

The human herpesvirus-type 8 is not involved in malignant melanoma.

Malignant melanomas were supposed to harbour the human herpesvirus-type 8 (HHV-8) genome, as melanoma cells were reported to express interleukin-6 and a homologue of interleukin-6 was found in the HHV-8 genome. We therefore investigated 33 primary malignant melanomas by polymerase chain reaction, but could not find this tumorigenic gamma-herpesvirus in any tumour.

[S-100 beta protein in serum, a tumor marker in malignant melanoma-- current state of knowledge and clinical experience]. / S-100 beta-Protein im Serum als Tumormarker beim malignen Melanom. Aktueller Kenntnisstand und Klinische Erfahrungen.

S100 is an acidic-calcium-binding protein, composed as a heterodimer of two isomeric subunits alpha and beta and was first described in cells of neuroendocrine origin. It plays an important role in various cellular processes such as cell differentiation and proliferation and interacts with the tumour suppressor gene p53.S100 is also present in melanoma cells and its immunhistochemical detection is widely used in the histopathological diagnosis of malignant melanoma. S100 has been detected in the serum of patients with malignant melanoma and many clinical studies have been performed to establish this protein as a tumor marker in different stages of the disease. The data suggest that S-100 beta-protein in serum of patients with malignant melanoma could be an independent prognostic marker and an additional clinical parameter for progression of metastatic disease and serological monitoring during systemic therapy. However there are patients in stage of lymph node- or systemic metastasis with negative S-100 beta-serum levels and no correlation to the course of disease. Our results confirm the findings for patients in stage III/IV. However, the percentage of S-100 beta-positive patients in stage III/IV is lower than reported in the literature, if repeatedly positive samples are excluded from statistical analysis. For monitoring in stage I and II it seems to be not helpful.

The detection of tyrosinase-specific mRNA in bone marrow is not more sensitive than in blood for the demonstration of micrometastatic melanoma.

Recent reports suggest that as a marker of progression of malignant melanoma, the detection of tyrosinase mRNA in blood is of limited value. In the present study, we investigated whether the detection of tyrosinase mRNA by reverse transcription-polymerase chain reaction (RT-PCR) in bone marrow samples might be a more useful method for the detection of micrometastatic melanoma. The presence of tyrosinase mRNA was analysed in blood and in bone marrow samples from 20 melanoma patients with widespread clinical metastases. Of these 20 patients, 12 were negative for tyrosinase mRNA in both blood and bone marrow. The remaining eight patients had tyrosinase mRNA in either blood or bone marrow: six in bone marrow and blood, one in bone marrow but not blood, and one in blood but not bone marrow. The sensitivity of tyrosinase mRNA detection by RT-PCR in bone marrow samples apparently does not exceed that in blood samples from metastatic melanoma patients. This seems to be independent of prior chemotherapy or immunotherapy. In contrast to different solid tumours, in melanoma, bone marrow seems not to be a significant reservoir for micrometastatic tumour cells.

Massive lethal cerebral bleeding in a patient with melanoma without intracranial metastasis.

The case history is reported of a patient with melanoma and advanced metastases, who died from massive cerebral bleeding. The lethal event was not caused by intracerebral metastasis but by thrombocytopenia. Depression of the bone marrow resulted from tumour infiltration of the skeleton, chemotherapy and vertebral irradiation. An increase of intracranial pressure triggered the cerebral bleeding, caused by haematemesis from a gastric metastasis directly preceding sudden somnolence.

Non-human immunodeficiency virus Kaposi's sarcoma can be effectively treated with low-dose interferon-alpha despite the persistence of herpesvirus-8.

Three patients, negative for human immunodeficiency virus (HIV), with histologically and polymerase chain reaction-proven non-HIV Kaposi's sarcoma who received low-dose interferon (IFN) as first-line treatment because of disseminated symptomatic disease are reported. Applying 3-18 million IU of IFN-alpha 2a per day, 3 days a week, subcutaneously for 8-20 months, major responses were achieved in all three cases. Tumour regression was observed within 4 months and has continued for 57 and 18 months to date (cases 1 and 2, respectively). Influenza-like symptoms, including fever, headaches and fatigue, were mild side-effects. However, in the third patient interferon injections had to be stopped because of hepatic enzyme elevation. Including this case report, 27 non-HIV Kaposi's sarcoma patients subcutaneously treated with IFN-alpha have been reported in literature. Most therapy regimens included 3-18 million IU IFN-alpha per day for 3 days a week. Twenty of 27 patients, or 74%, responded to therapy, whereas seven patients or 26% had stable or progressive disease. Relapse after IFN withdrawal can occur but is frequently delayed and limited, as in case 1. Following the response to IFN treatment, human herpesvirus-8 DNA was detected in the blood mononuclear cells of all three patients, possibly contributing to future relapses.