The oncogenic B-raf V599E mutation occurs more frequently in melanomas at sun-protected body sites.

Downstream of Ras, the serine/threonine kinase, B-raf, has recently been reported to be mutated, among other carcinomas, in a substantial subset of primary melanomas with a preponderance of the oncogenic V599E transition. As the risk of melanoma is enhanced by intermittent ultraviolet (UV) exposure but is less with chronic UV exposure, we here studied B-raf kinase domain (exon 15) mutations in primary cutaneous melanomas with respect to anatomical locations reflecting chronic versus intermittent UV exposure. Investigating a representative number of 101 primary melanoma resection specimens for the presence of mutations within the activation segment (exon 15) of the B-raf kinase domain by polymerase chain reaction and single-strand conformation polymorphism gel electrophoresis, followed by DNA cloning and sequencing, we found 32 cases (32%) which harbour somatic B-raf exon 15 mutations. As to the B-raf protein sequence, the V599E mutation was predicted in 66% of these positive melanomas, followed in frequency by the V599K transition (16%). Only two Cright curved arrow T transitions, considered to be induced by UV irradiation, occurred in two melanomas located on the head. Among 23 melanomas located at body sites with chronic UV exposure, only a single tumour harboured the B-raf V599E mutation (4%), which was a significantly lower frequency in comparison to melanomas from sun-protected body sites (26%; Fisher's exact test, p=0.038; odds ratio, 7.59). Our observation parallels the epidemiological data of intermittent sunlight exposure on unacclimatised skin increasing the risk of melanoma development.

Decline in angiogenic factors, such as interleukin-8, indicates response to chemotherapy of metastatic melanoma.

Serum concentrations of angiogenic factors have been reported to correlate with tumour burden and prognosis in metastatic melanoma. The present study was performed to assess the value of angiogenic factors in serum in indicating response or failure to chemotherapy and immunochemotherapy in stage IV melanoma. Thirty-five patients suffering from stage IV melanoma according to the American Joint Committee on Cancer (AJCC) criteria were included in this prospective study. Before and following chemotherapy or immunochemotherapy, serum levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-AB), vascular cell adhesion molecule-1 (VCAM-1) and interleukin-8 (IL-8) were measured. Staging examinations following chemotherapy revealed 15 patients with response to therapy (complete response, partial response, stable disease), 14 patients with progressive disease and six patients with mixed response. Patients who responded to therapy showed a significant decrease in the serum level of IL-8 at the time of staging examinations, whereas patients with progressive disease did not. Following chemotherapy, serum concentrations of PDGF-AB had significantly decreased in both patients with response and patients with progressive disease. Comparing the VEGF and bFGF levels of responders and non-responders after a single administration of cytostatics showed significantly lower concentrations in patients with response to therapy. In all patients, a high intra- and inter-individual variability of serum values was observed during application of therapy. It can be concluded that low IL-8 serum levels after chemotherapy indicate response to chemotherapy in stage IV melanoma patients. The persistence of elevated serum levels of VEGF and bFGF following the initial cytostatic administration may help to identify patients resistant to chemotherapy. The distinct variability of serum levels indicates that processes other than tumour angiogenesis also influence the serum concentration of the examined angiogenic factors.

Preponderance of the oncogenic V599E and V599K mutations in the B-raf kinase domain is enhanced in melanoma lymph node metastases.

Downstream of Ras, the serine/threonine kinase B-raf has been reported to be mutated, amongst other carcinomas, in a substantial subset of primary melanomas, with a preponderance of mutations within the kinase domain, including the activating V599E and V599K transitions. We investigated a representative series of 54 resection specimens of melanoma lymph node metastases for the presence of mutations within the activation segment (exon 15) of the B-raf kinase domain by polymerase chain reaction (PCR) and single-strand conformational polymorphism (SSCP) gel electrophoresis. Sequencing of cloned PCR-SSCP amplicons resulted in 24 (44%) samples harbouring somatic mutations, which is not significantly different from the mutation frequency found in recently investigated primary cutaneous melanomas (Deichmann M, Thome M, Benner A, Näher H. B-raf exon 15 mutations are common in primary melanoma resection specimens but not associated with clinical outcome. Oncology 2004; 66:411-419). The activating mutation T1796A was present in 20 (83%) of these resection specimens, followed in frequency by the oncogenic g1795A mutation in five (21%) cases. With regard to the B-raf protein sequence, the acidic amino acid transitions V599E and V599K were predicted in 15 (62%) and five (21%) of the 24 positive metastases, respectively. The detection of mutations at this hot spot codon was significantly associated with subsequent visceral metastasis (P=0.03; Fisher's exact test). During the transition from primary melanomas (see reference above) to lymph node metastases, the spectrum of B-raf mutations narrows significantly (P=0.00047). The oncogenic B-raf mutations V599E and V599K, as early events in melanocyte transformation, persist throughout metastasis with important prognostic implications.

The chemoresistance gene ABCG2 (MXR/BCRP1/ABCP1) is not expressed in melanomas but in single neuroendocrine carcinomas of the skin.

BACKGROUND: As the molecular mechanisms in melanoma chemoresistance remain unknown to date, we hypothesized these tumors to express the ATP-binding cassette (ABC) transporter G2 (ABCG2/MXR/BCRP1/ABCP1), a recently detected membrane transporter and putative stem-cell marker. Besides melanoma, we addressed the neuroendocrine carcinoma of the skin (Merkel cell carcinoma), another cutaneous cancer supposed to originate from neuroectoderm and usually chemoresistant. METHODS AND RESULTS: Upon semiquantitative reverse transcription polymerase chain reaction, ABCG2 mRNA expression was not upregulated in 18 melanoma resection specimens when compared with 19 acquired melanocytic nevi from which melanomas are known to often arise (Mantel-Haenszel test, p=0.3). At protein level, immunohistochemistry was negative in all 66 investigated melanoma resection specimens (50 primary melanomas and 16 cutaneous/subcutaneous metastases) and in 19 acquired melanocytic nevi. Among 29 neuroendocrine carcinomas of the skin, ABCG2 protein was detected in single clusters of cells in three tumors. As a positive control, three dermatofibrosarcomas were also stained and showed ABCG2 protein expression of the endothelial cells of the blood vessels. CONCLUSION: Altogether, chemoresistance of melanomas and neuroendocrine carcinomas of the skin cannot be explained by expression of the ABCG2-chemoresistance gene. Most of these tumors do not exhibit this potential stem-cell feature.

Preponderance of the oncogenic V599E and V599K mutations in B-raf kinase domain is enhanced in melanoma cutaneous/subcutaneous metastases.

BACKGROUND: Downstream of Ras, the serine/threonine kinase B-raf has been reported to be mutated, among other carcinomas, in a substantial subset of primary melanomas with a preponderance of mutations within the kinase domain including the activating V599E and V599K transitions. METHODS: We here investigated a representative series of 60 resection specimens of cutaneous and subcutaneous melanoma metastases for the presence of mutations within the activation segment (exon 15) of the B-raf kinase domain by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) gel electrophoresis. RESULTS: Sequencing of cloned PCR-SSCP amplicons resulted in 24 (40%) samples harbouring somatic mutations which is not exceeding the mutation frequency in recently investigated primary melanomas. The activating mutation T1796A was present in 24/60 (40%) resection specimens, followed in frequency by the oncogenic g1795A mutation in 8/60 (13%) cases. As to the B-raf protein sequence, the acidic amino acid transitions V599E and V599K were predicted in 19/60 (32%) and 6/60 (10%) cases, respectively, but were not associated with enhanced risk for subsequent metastasis in patients' follow up. In comparison to the primary melanomas that we recently investigated, the spectrum of predicted B-raf protein mutations narrowed significantly in the cutaneous/subcutaneous metastases. Unexpectedly, V599 and V599E mutations were absent in cutaneous/subcutaneous metastases derived from acrolentiginous melanomas as preceding primary tumours. CONCLUSION: During transition from primary melanomas towards cutaneous/subcutaneous metastases, the spectrum of predicted B-raf mutations narrows significantly. Focusing on the V599E and V599K, these oncogenic mutations are likely to affect melanocyte-specific pathways controlling proliferation and differentiation.

Detection of reverse transcriptase activity in human melanoma cell lines and identification of a murine leukemia virus contaminant.

BACKGROUND: Stimulated by earlier reports on the presence of retroviruses in mouse and hamster melanoma cell lines, we addressed the question as to whether human melanoma cell lines might also harbour a retrovirus. METHODS AND RESULTS: The melanoma cell lines SK-MEL-25, SK-MEL-28, MEL-JUSO, MML-I, MeWo, A-375, Colo-38, BS-780 were confirmed to be human by human leucocyte antigen (HLA) typing, and supernatants were tested by the product-enhanced reverse transcriptase (PERT) assay for reverse transcriptase (RT) activity. Cell lines SK-MEL-25, SK-MEL-28, MEL-JUSO and MML-I were positive, whereas cell lines MeWo, A-375, Colo-38 and BS-780 were negative. The RT activity peaked at a buoyant density in sucrose typical for retroviruses. From this peak fraction an R-U5 sequence indistinguishable from murine leukemia virus (MLV) was identified by particle-associated retrovirus RNA amplification (PARRA). Semiquantitative MLV-specific RNA-PCR demonstrated colocalization of the MLV-like RNA and RT activity on the sucrose gradient of SK-Mel-25. MLV RNA and DNA were also detectable in culture supernatants of SK-MEL-28, MEL-JUSO and MML-I, but not of MeWo, A-375, Colo-38 and BS-780 by semiquantitative polymerase chain reaction (PCR). Sequence comparison revealed highest homology with the RET sequence previously identified in mouse myeloma SP2/0-AG14 cells. Taken together, our data strongly suggest that certain human melanoma cell lines are productively infected by a MLV which was probably introduced during tumour passage in mice or by laboratory contamination many years ago and subsequently spread to other lines. CONCLUSION: We recommend mandatory testing of melanoma and other human cell lines for contamination with infectious MLV or other animal retroviruses, similar to mycoplasma screening, in order to avoid artificial experimental data.

BRAF mutations distinguish anorectal from cutaneous melanoma at the molecular level.

BACKGROUND & AIMS: Anorectal melanoma (AM) is a rare but highly malignant tumor, displaying histologic and immunohistochemical features very similar to cutaneous melanoma (CM). Because BRAF mutations were recently identified in the majority of CM and nevi, we investigated AM for BRAF mutations and mutations of NRAS , an additional component of the MAPK-signalling pathway. METHODS: DNA from formalin-fixed and paraffin-embedded AM was PCR amplified and sequenced. RESULTS: We detected BRAF mutations in 2 of 19 cases and NRAS mutations in none of the cases. Mutations in exon 15 of BRAF were present in only 1 tumor (1 of 19 cases). The A1800T base exchange represented a novel mutation and resulted in a K600N transition in an AM from a 96-year-old white man who presented with rectal bleeding and painful sitting of a few weeks' duration. The second positive AM case, a 69-year-old white man who presented with painless rectal bleeding and clinical symptoms of an intestinal constipation showed a novel missense mutation (C1327T leading to R443W conversion) in BRAF exon 11. None of the AM cases displayed the oncogenic V599E mutation preponderating in CM. CONCLUSIONS: With regard to the frequency of V599E BRAF mutations, AM significantly differs from CM (P < or = .0001), which suggests that BRAF mutations distinguish anorectal from cutaneous melanoma at the molecular level.

Phenotypical and molecular profiling of the extraneuronal cholinergic system of the skin.

We present molecular and protein profiling of all acetylcholine receptors (ACh-R) in human scalp skin using PCR, in situ hybridization and double-labeling immunofluorescence. Within the epidermis, the nicotinic (n)ACh-R subunits, alpha3, alpha5, beta2, and beta4 were expressed in the basal cell layer (BCL) and in a single cell layer in the stratum granulosum; alpha9 was expressed in the basal and lower spinous layers. alpha7, alpha10, and beta1 were preferentially detected in the upper spinous and granular layers. Of the muscarinic (m)ACh-R, m1 and m4 were found in the suprabasal layers, whereas m2, m3, and m5 remained restricted to the lower layers. In the outer root sheath of the hair follicle, all ACh-R except alpha9, beta1, and m4 were found in the BCL whereas the alpha9, m4, and m5 ACh-R were restricted to the central cell layer. The alpha5, beta1, beta2, m1-m4 chains were strongly expressed in the inner root sheath. Undifferentiated sebocytes expressed the alpha3, alpha9, beta4, m3-m5 ACh-R whereas alpha7, beta2, beta4, m2, and m4 were found in mature sebocytes. In sweat glands, the alpha3*, alpha7, and m2-m5 ACh-R were most prominent in the myoepithelial cells whereas alpha9, beta2, m1, m3, and m4 ACh-R were present in the acinar cells. Taken together, our data result in a complete molecular map of the extraneuronal cholinergic system of the skin that may be translated into distinct functional reaction patterns.

B-raf exon 15 mutations are common in primary melanoma resection specimens but not associated with clinical outcome.

Downstream of Ras, the serine/threonine kinase B-raf has recently been reported to be mutated, among other carcinomas, in a majority of melanoma cell lines with a preponderance of mutations within the kinase domain including the activating V599E transition. We therefore investigated a representative number of 50 primary melanoma resection specimens for the presence of mutations within the activation segment (exon 15) of the B-raf kinase domain. Applying polymerase chain reaction and single-strand conformation polymorphism gel electrophoresis, followed by DNA cloning and sequencing, we found 19 cases (38%) to harbor somatic B-raf exon 15 mutations. With respect to the B-raf protein sequence, the V599E mutation was predicted in 63% of these positive melanomas, followed in frequency by the V599K transition (31%). Detection of B-raf exon 15 mutations or prediction of the activating mutation V599E were not statistically associated with the risk for subsequent metastasis in the follow-up of patients. Altogether, the B-raf oncogene is affected in a substantial subset of melanoma resection specimens. As B-raf alterations possibly affect melanocyte-specific pathways controlling proliferation and differentiation, activation of this oncogene may contribute to the development of melanoma.

Melanoma metastasis is associated with enhanced expression of the syntenin gene.

Primary cutaneous melanomas and melanoma metastases were examined for differential gene expression using subtractive suppression hybridization in a search for any genes associated with metastasis. Generating a subtracted library of candidate genes up-regulated in melanoma metastases, this library contained 8 different cDNAs, among them a cDNA fragment of the syntenin gene which was overexpressed in further independent melanoma resection specimens on cDNA Southern blots when compared to acquired melanocytic nevi from which melanomas are known to arise. Upon immunohistochemistry, the syntenin protein expression was detected in the cytoplasm of primary cutaneous melanomas and melanoma metastases. Melanoma metastases exhibited higher proportions of tumour cells positive for syntenin immunostaining in comparison to acquired melanocytic nevi or non-metastasizing primary melanomas which was statistically significant. In addition to melanoma, gastric cancer tissues exhibited a higher syntenin mRNA expression on a matched tumour/normal cDNA array than their normal counterparts which was statistically significant. Altogether, we here first describe the detection of the syntenin protein in resection specimens of melanocytic lesions. We conclude that melanoma metastasis is associated with increased expression of the syntenin gene which may participate in signal transduction and cell adhesion via the multifunctional protein-binding properties of its tandem PDZ domains.

Expression of the heat shock cognate protein HSP73 correlates with tumour thickness of primary melanomas and is enhanced in melanoma metastases.

Seeking to identify melanoma-associated genes by comparing gene expression in uncultured primary melanoma specimens with those in nevi, from which melanomas often are known to arise, we applied subtractive suppression hybridization. Generating a subtracted library of candidate genes up-regulated in primary melanomas, this library contained cDNA fragments of the genes encoding heat shock cognate protein (HSP73) and major histocompatibility complex (HLA-DR) which were overexpressed in further 19 independent melanoma resection specimens on cDNA Southern blots when compared to 19 acquired melanocytic nevi. Upon immunohistochemistry, HSP73 protein expression was detected in the cytoplasm of melanoma cells in primary tumours and metastases. In primary melanomas, the proportion of HSP73 protein expressing cells correlated with tumour thickness according to Breslow which was statistically significant. HSP73 immunostaining was stronger in melanoma metastases when compared with acquired melanocytic nevi which was statistically significant. In addition to melanoma, gastric and uterus cancer tissues exhibited higher HSP73 mRNA expression on a matched tumour/normal cDNA array than their normal counterparts which was statistically significant. Participating in the regulation of folding, assembly and degradation of proteins and protecting cellular proteins from the damage caused by cellular stress like hypoxia or changes in cellular pH, elevated HSP73 expression possibly confers proliferative advantage on melanoma cells.

Somatic mitochondrial mutations in melanoma resection specimens.

As epidemiological data suggest ultraviolet (UV) radiation to be the major environmental factor for the development of melanoma, we screened this malignancy for UV-specific C right curved arrow T and CC right curved arrow TT DNA mutations as described in squamous and basal cell carcinomas of the skin. Mitochondrial (mt) DNA was addressed which is known to be highly more susceptible for mutations than nuclear DNA. In the mt genome we chose part of the non-coding displacement-loop (D-loop) containing two hypermutable (C)n tracts especially prone for C right curved arrow T and CC right curved arrow TT changes. A total of 69 melanoma resection specimens was investigated by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) electrophoresis, followed by DNA cloning and sequencing. We detected alterations of the mt D-loop fragment in 4/34 (12%) melanoma primary tumours and in 7/35 (20%) cutaneous and subcutaneous metastases which was no statistically higher proportion upon Fisher's exact test (p=0.17). Among these 11 positive cases, 9 exhibited alterations in the (C)n microsatellite sequences indicating microsatellite instability (MSI) of mtDNA (C)n tracts. Besides 7 insertions and 2 deletions of nucleotides, 11 mutations occurred including only 4 UV-specific C right curved arrow T mutations in a nodular melanoma of the skin and in two subcutaneous metastases. CC right curved arrow TT mutations were not detected in any tissue sample. As (C)n tract alterations occurred in 2/9 primary melanomas with subsequent metastasis, whereas all 20 non-metastasizing cutaneous melanomas were not affected, these somatic changes may reflect genomic imbalance during tumour progression and may be useful for the assessment of patients' prognosis.

Adhesion molecules CD171 (L1CAM) and CD24 are expressed by primary neuroendocrine carcinomas of the skin (Merkel cell carcinomas).

BACKGROUND: The neuroendocrine carcinoma of the skin is a rare malignant neuroendocrine tumor, which frequently metastasizes in regional lymph nodes or visceral organs. As adhesive interactions with endothelia, leukocytes, or thrombocytes enable malignant cells to penetrate the endothelium and to circulate in blood or lymphatic vessels, we here addressed the adhesion molecules CD171 (L1CAM) and CD24, which are known to be expressed by neurons, neuroblastomas, and other malignant tumors. METHODS: Thirty-one neuroendocrine carcinomas of the skin (22 primary tumors, four recurrent tumors, and five metastases) were included in the study. Immunohistochemical staining of CD171 and CD24 was performed by the streptavidin-biotin-peroxidase-complex technique and a nickel-enhanced diaminobenzidine (DAB) reaction using the monoclonal antibodies UJ 127.11 and ML-5, respectively. RESULTS: CD171 expression was detected in most neuroendocrine carcinomas of the skin, and staining was less frequent in metastases and recurrences in comparison with primary tumors which was statistically significant. The majority of neuroendocrine carcinomas of the skin was also positive for the mucin-like adhesion protein CD24. In contrast to tumor cells, cytokeratin 20-positive Merkel cells in 12 trichoblastomas and one fibroepithelioma of Pinkus were all negative for CD171 and CD24 staining. CONCLUSIONS: Expression of CD171 and CD24 is found in most neuroendocrine carcinomas of the skin, which may be used diagnostically. Further studies will assess whether this feature may contribute to metastasis of neuroendocrine carcinomas of the skin by facilitating transendothelial migration or tumor cell dissemination as has been suggested for other malignancies.

Expression of MUC 1 and Ep-CAM in Merkel cell carcinomas: implications for immunotherapy.

The Merkel cell carcinoma (MCC) is a highly malignant carcinoma of the skin that is characterized by granules containing neuroendocrine peptides and by the expression of simple epithelial type cytokeratins. The glycoprotein Ep-CAM is a homophilic cell-cell adhesion molecule, present in most simple, pseudostratified and transitional epithelia and the tumors derived therefrom. MUC 1 is a well-established marker for squamous cell carcinomas and is generally secreted by glandular epithelial cells. We compared the expression of Ep-CAM and MUC 1 in Merkel cells and 33 cases of MCC and 12 MCC metastases using immunohistochemistry on paraffin-embedded sections. In addition, we examined the glycosylation status of MUC 1 with specific monoclonal antibodies. MUC 1 and Ep-CAM were expressed in Merkel cells and in about 82% and 70% of all MCC irrespective of clinical outcome. Both antigens were expressed in 66% of metastases. Similar to breast cancer, the presence of MUC 1 was not correlated with clinical outcome, but the staining intensity of monoclonal antibodies against glycosylation-independent and hypoglycosylated epitopes was. In MCC we found an altered glycosylation pattern in the immunodominant APDTR region of MUC 1 as compared to normal Merkel cells. Hyperglycosylated MUC 1 epitopes were not present in either MCC or normal Merkel cells. There was no correlation between glycosylation pattern and clinical outcome. Ep-CAM expression seemed to be stronger in primary MCC that metastasized than in those that did not. In conclusion, Merkel cells and the majority of MCC express Ep-CAM and MUC 1. This opens the door for treatments based on monoclonal antibodies or vaccination strategies against these antigens, already established for other tumor entities.

Frequent downregulation of DMBT1 and galectin-3 in epithelial skin cancer.

DMBT1 and galectin-3 are potential interacting proteins with presumably complex roles in tumorigenesis. While at present a variety of mechanisms are discussed for DMBT1 and its participation in cancer, galectin-3 is commonly known to exert tumor-promoting effects. However, in vitro studies in a rodent system have suggested that DMBT1/galectin-3 interaction in the ECM triggers epithelial differentiation, which would point to tumor-suppressive properties. To improve the understanding of DMBT1/galectin-3 action in cancer, we carried out studies in skin cancer of different origins. Mutational analyses of DMBT1 identified a missense mutation in 1 of 13 melanoma cell lines. It led to an exchange of an evolutionary conserved proline residue for serine and located within the second CUB domain of DMBT1. Immunohistochemical analyses demonstrated absence of DMBT1/galectin-3 expression from melanocytes but induction of DMBT1 expression in 1 of 8 nevi and 1 of 11 melanomas and of galectin-3 expression in 3 of 8 nevi and 4 of 8 melanomas. These data suggest that DMBT1 and galectin-3 are unlikely to act as classical tumor suppressors in melanomas. DMBT1 and galectin-3 appear to be secreted to the ECM by epithelial cells within the epidermis and the hair follicle. Compared to the flanking normal epidermis, skin tumors of epithelial origin frequently displayed downregulation of DMBT1 (18 of 19 cases) and galectin-3 (12 of 12 cases). Thus, loss of DMBT1/galectin-3 expression may play a role in the genesis of epithelial skin cancer. This would support the view that galectin-3 can exert tumor-suppressive effects in certain scenarios, and DMBT1/galectin-3-mediated differentiation represents a candidate mechanism for this effect.