Long-term (6 and 12 months) follow-up of two prospective, randomized, controlled phase III trials of photodynamic therapy with BF-200 ALA and methyl aminolaevulinate for the treatment of actinic keratosis.

BACKGROUND: Two phase III trials of photodynamic therapy (PDT) with BF-200 ALA, a recently approved nanoemulsion formulation of 5-aminolaevulinic acid (ALA) demonstrated high clearance rates in mild-to-moderate actinic keratosis (AK). The comparison to a registered methyl aminolaevulinate (MAL) cream demonstrated significantly superior total patient clearance rates. OBJECTIVES: To evaluate long-term efficacy and safety of PDT for AK 6 and 12 months after the last PDT with BF-200 ALA, MAL or placebo. METHODS: The follow-up phase (FUP) was performed with patients of two phase III studies. Both studies compared BF-200 ALA with placebo, one of the studies additionally with MAL. Overall recurrence rates and various subgroups (light source, lesion severity, lesion location, complete responders after first PDT) were assessed 6 and 12 months after the last PDT. RESULTS: Recurrence rates were similar for BF-200 ALA and MAL, with a tendency to lower recurrence rates for BF-200 ALA. The proportion of patients who were fully cleared during PDT and remained completely clear for at least 12 months after PDT were 47% for BF-200 ALA (both studies) and 36% for MAL treatment. The subgroup that was illuminated with narrow wavelength LED lamps reached 69% and 53% for BF-200 ALA (both studies, respectively) and 41% for MAL. No safety concerns were reported. CONCLUSIONS: The FUP data confirmed the high efficacy and safety of PDT with BF-200 ALA. The slightly lower recurrence rates after BF-200 ALA treatment compared with MAL treatment enhanced the better treatment outcome due to the significantly superior efficacy.

Photodynamic therapy with BF-200 ALA for the treatment of actinic keratosis: results of a multicentre, randomized, observer-blind phase III study in comparison with a registered methyl-5-aminolaevulinate cream and placebo.

BACKGROUND: Photodynamic therapy (PDT) with 5-aminolaevulinic acid (ALA) or its methylester [methyl-5-aminolaevulinate (MAL) or 5-amino-4-oxopentanoate] was recently ranked as first-line therapy for the treatment of actinic keratosis (AK) and is an accepted therapeutic option for the treatment of neoplastic skin diseases. BF-200 ALA (Biofrontera Bioscience GmbH, Leverkusen, Germany) is a gel formulation of ALA with nanoemulsion for the treatment of AK which overcomes previous problems of ALA instability and improves skin penetration. OBJECTIVES: To evaluate the efficacy and safety of PDT of AKs with BF-200 ALA in comparison with a registered MAL cream and with placebo. METHODS: The study was performed as a randomized, multicentre, observer-blind, placebo-controlled, interindividual trial with BF-200 ALA, a registered MAL cream and placebo in a ratio of 3:3:1. Six hundred patients, each with four to eight mild to moderate AK lesions on the face and/or the bald scalp, were enrolled in 26 study centres in Germany, Austria and Switzerland. Patients received one PDT. If residual lesions remained at 3months after treatment, PDT was repeated. RESULTS: PDT with BF-200 ALA was superior to placebo PDT with respect to patient complete clearance rate (78·2% vs. 17·1%; P<0·0001) and lesion complete clearance rate (90·4% vs. 37·1%) at 3months after the last PDT. Moreover, superiority was demonstrated over the MAL cream regarding the primary endpoint patient complete clearance (78·2% vs. 64·2%; P<0·05). Significant differences in the patient and lesion complete clearance rates and severity of treatment-related adverse events were observed for the narrow- and broad-spectrum light sources. CONCLUSIONS: BF-200 ALA is a very effective, well-tolerated new formulation for AK treatment with PDT and is superior to a registered MAL medication. Efficacies and adverse events vary greatly with the different light sources used.

Diagnosing melanoma patients entering American Joint Committee on Cancer stage IV, C-reactive protein in serum is superior to lactate dehydrogenase.

Lactate dehydrogenase (LDH) in serum has recently been introduced into the American Joint Committee on Cancer (AJCC) staging system for cutaneous melanoma because of its prognostic value. We hypothesised LDH to be of value in discriminating melanoma patients entering AJCC stage IV from patients staying in AJCC stages I, II or III. Lactate dehydrogenase was compared to the acute phase protein C-reactive protein (CRP), which we observed to reflect the course of melanoma metastasis in a previous report. In this prospective study, we measured LDH and CRP in the serum of 91 consecutive melanoma patients progressing into AJCC stage IV in comparison to 125 patients staying in AJCC stages I, II or III. Comparing distributions of the parameters by median values and quartiles by Mann-Whitney test, LDH was not significantly elevated in patients entering AJCC stage IV melanoma (P=0.785), whereas CRP was (P<0.001). Analysing the sensitivity and the specificity jointly by the areas under the receiver operating characteristics curves (ROC-AUC), LDH did not discriminate between the defined groups of patients (AUC=0.491; 95% confidence interval, 0.410, 0.581), whereas CRP did (AUC=0.933; 95% confidence interval, 0.900, 0.966; P<0.001). Upon logistic regression analysis to calculate the ROC-AUC values upon the predictive probabilities, LDH provided no additional information to CRP. Choosing a cutoff point of 3.0 mg l(-1), CRP yielded a sensitivity of 0.769 together with a specificity of 0.904 in diagnosing AJCC stage IV entry. Altogether, for first diagnosing AJCC stage IV melanoma, CRP is the superior serum marker when compared to the conventional LDH.

Pyrolysis of a tubular aromatic compound.

[reaction: see text] In the attempt to close the walls of a small tubular system that is a substructure of a [4,4] armchair nanotube a very unusual rearrangement reaction was observed.

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.

Absence of mutations in the pleckstrin homology (PH) domain of protein kinase B (PKB/Akt) in malignant melanoma.

During recent years it has become evident that protein kinase B (PKB)/Akt plays an important role in oncogenic transformation. The gene for PKB/Akt has been found to be overexpressed in certain human tumours and a viral fusion protein gains transforming capacity. Recruitment to the plasma membrane is mandatory for the physiological activation of PKB/Akt; this shift from cytoplasm to the membrane is achieved by the N-terminal pleckstrin homology (PH) domain. We attempted to find out whether mutations of this domain were present in human malignant melanoma. RNA from 18 primary melanoma lesions of different sizes and histological subtypes and two melanoma metastases from 20 Caucasian patients were used for reverse transcription and subsequent polymerase chain reaction (PCR) amplification of the PH domain of PKB/Akt alpha. Cycle sequencing of the purified PCR products showed that mutations of the PH domain of PKB/Akt were absent in all 20 melanoma specimens. In virtual Northern hybridizations PKB/Akt showed a low expression in both melanomas and acquired melanocytic naevi; however, no overexpression of PKB/Akt was detected. Thus in human melanoma PH domain mutations of PKB/Akt do not play a major role in melanoma carcinogenesis.

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.

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.

[Disseminated melanoma cells in blood and bone marrow. Significance and detection by potential tumor markers]. / Disseminierte Melanomzellen in Blut und Knochenmark. Bedeutung und Nachweis durch potenzielle Tumormarker.

As the majority of primary malignant melanomas can be cured by surgical excision, the prognosis of melanomas is dependent on whether tumor cells have disseminated orare capable of doing so at the time of surgery. A prospective and valid detection of this minimal residual disease is not currently possible. The most important known so-called markers of melanoma disease, tyrosinase, S100 and MIA, all are more likely to be present in patients with more advanced disease. A valid prognostic effect has only been shown for S100 in patients with already identified metastatic disease. Further prospective studies are required to determine the potential gain of information by routine determination of these markers in melanoma patients.

Anorectal melanomas do not harbour the Kaposi sarcoma-associated human herpesvirus type 8 DNA.

Anorectal melanomas are similar to cutaneous melanomas with regard to the mode of spread and to the immunophenotype. When compared with patients with cutaneous melanoma, those suffering from anorectal melanoma have a much worse outcome. The etiology of anorectal melanomas is as yet completely unknown. For anatomical reasons, ultra-violet (UV-B) radiation can not cause anorectal melanomas as in cutaneous tumours, that are associated with exposure of the skin to UV-B radiation. As the cytokine interleukin-6 (IL-6) is known to stimulate melanoma tumour cell proliferation and a functional homologue of human IL-6 has been identified recently in the HHV-8 genome, this tumorigenic virus might be involved in the pathogenesis of anorectal melanomas. Twelve formalin fixed and paraffin embedded primary anorectal melanomas from seven female and five male patients with a mean age at diagnosis of 71 years (range 38-88 years) were investigated for the presence of HHV-8 DNA. Using a specific and highly sensitive polymerase chain reaction protocol, this tumorigenic gamma-herpesvirus was not detectable in any tumour. This data indicates that HHV-8 is not involved in the development of anorectal melanomas.

[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.

[Determining the severity of venous insufficiency with duplex sonography]. / Bestimmung des Schweregrades einer venösen Insuffizienz mittels Duplexsonographie.

OBJECTIVE: The volume flow in the common femoral vein (SV) as a representative parameter of the venous hemodynamics of the leg can be measured using duplex-sonography. A direct correlation between the SV-data and the clinical grade of the venous disease was postulated. PATIENTS/METHODS: SV was measured in 36 patients (62 limbs) with complete varicosis of the long saphenous vein, 18 patients (24 limbs) with an incomplete form of varicosis and 40 healthy persons (78 limbs). The assessments were done under standardized conditions. We found a significant difference of the SV-data between the three groups. RESULTS: The values of SV were significantly elevated in varicosis compared with the healthy limbs. The data determined in legs with complete varicosis were significantly raised compared to incomplete varicosis. The means were 0.38 l/min in complete varicosis, 0.26 l/min in incomplete varicosis and 0.13 l/min in healthy limbs. During a time course of 30 minutes the values were stable. CONCLUSIONS: The results demonstrate a significant correlation between the measured SV data and the grade of venous disease.

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.