Anton Platz

Cytoplasmic and nuclear accumulation of β-catenin is rarely caused by CTNNB1 exon 3 mutations in cutaneous malignant melanoma

β‐catenin plays an important role in the Wnt signaling pathway by activating T‐cell factor (Tcf)/lymphoid enhancer factor (Lef)‐regulated gene transcription. The level of β‐catenin is regulated through GSK‐3β phosphorylation of specific serine and threonine residues, all of which are encoded for in exon 3 of the β‐catenin gene (CTNNB1). Mutations altering the GSK‐3β phosphorylation sites lead to cellular accumulation of β‐catenin and constitutive transcription of Tcf/Lef target genes. Such mutations have previously been found in melanoma cell lines. In our study, primary melanomas and their corresponding metastases were screened for CTNNB1 exon 3 mutations using single‐strand conformation polymorphism and nucleotide sequence analysis. One of 31 primary tumors and 1 of 37 metastases, both originating from the same patient, had a TCT to TTT mutation at codon 45, changing serine to phenylalanine. Immunohistochemical analysis revealed membranous localization of β‐catenin in a majority of the samples. The mutated primary tumor and metastasis, however, displayed widespread cytoplasmic and nuclear expression of β‐catenin. An additional 30% of the primary tumors showed focal cytoplasmic and nuclear staining. Thus, β‐catenin exon 3 mutations are rare in primary as well as metastatic melanomas and do not explain the abnormal cytoplasmic and nuclear localization of β‐catenin found in a relatively large fraction of primary melanomas.

Biallelic deletions in INK4 in cutaneous melanoma are common and associated with decreased survival.

Purpose: Both the retinoblastoma and p53 pathways are often genetically altered in human cancers and their complex regulation is in part mediated by the three gene products p16, p14ARF, and p15 of the INK4 locus on chromosome 9p21. Partial or complete biallelic deletions of the INK4 locus have been recognized in a variety of malignant tumors, including malignant melanoma. We have in the present study measured the frequency of INK4 deletions in a large number of melanoma metastases and determined their association with clinicopathologic variables and survival data. Experimental Design: Quantitative real-time PCR, as well as fluorescence-based fragment analysis, has been used to perform measurements of the relative allelic concentrations of the INK4 genes in 112 human melanoma tumor samples from 86 patients. Results: Thirty-eight of 86 melanoma patients (44%) had metastases with biallelic losses in INK4. Ten of 20 patients with multiple metastases showed similar deletion patterns in all analyzed tumors. There was no significant association between any of the clinicopathologic variables and loss of INK4. However, loss of INK4 had an adverse effect on median survival from time of diagnosis. Patients with tumors with diploid INK4 had a median survival of 142 months, whereas those with monoallelic or biallelic loss in INK4 had a median survival of only 47 months (P = 0.006). Conclusions: Our results point to homozygous deletions in the INK4 region as being one of the most common genetic alterations in malignant cutaneous melanoma. INK4 deletions are associated with an adverse prognosis.

Mutations in the tp53 gene in human malignant melanomas derived from sun-exposed skin and unexposed mucosal membranes

Mutations in the p53 tumour suppressor gene (TP53) have been linked to several types of cancer. We therefore investigated whether such mutations occur in malignant melanomas and, if so, whether they are linked to ultraviolet (sun) light exposure. For the first time, TP53 mutations in mucosal membranes and adjacent tissues shielded from sunlight were compared with those in cutaneous melanomas from sun-exposed skin. Archival tissues were obtained from 35 patients with a primary melanoma taken from unexposed mucosal areas and from 34 patients with a primary melanoma located in chronically sun-exposed head and neck skin. TP53 was characterized by means of polymerase chain reaction amplification and single-strand conformation polymorphism assay followed by nucleotide sequencing. The results showed that 17.6% of the primary cutaneous and 28.6% of the primary mucosal melanomas had point mutations in TP53. Among the cutaneous melanomas, one showed three mutations in exon 7, and one had two mutations in exon 5; the mutation was in the same allele in both cases. One mucosal melanoma had two mutations in exon 7, both in the same allele, and another had two mutations, one in exon 7 and one in intron 6, both in the same allele. C←T mutations at dipyrimidine sites, considered fingerprints for ultraviolet light-induced mutations, were about equally distributed among patients with melanomas from chronically sun-exposed areas (six out of nine; 67%) and those with melanomas from unexposed mucosal areas and adjacent skin (eight out of 14; 57%). Our data, demonstrating the presence of such mutations even in melanomas from mucosal membranes, clearly suggest that factors other than, or additional to, ultraviolet radiation are operational in the induction of TP53 mutations in melanomas.

Chromosomal localization of human glutathione transferase genes of classes alpha, mu and pi

SummaryThe numerous human glutathione transferases may be divided into three classes, mu, alpha and pi. Using a panel of human-rodent somatic cell hybrids and DNA probes specific for each of the three classes, we have mapped a class mu gene to chromosome 3, a class alpha gene to chromosome 6 and a class pi gene to chromosome 11. The two latter assignments confirm earlier reports, whereas the assignment of the class mu gene represents a new addition to the human gene map.

Immunohistochemical analysis of the N-ras p21 and the p53 proteins in naevi, primary tumours and metastases of human cutaneous malignant melanoma: increased immunopositivity in hereditary melanoma

Immunohistochemical analysis of the N-ras p21 and the p53 proteins was carried out on formalin-fixed sections of naevi, primary melanomas and metastases from patients with sporadic melanoma (SCMM) and with hereditary melanoma (HCMM)/dysplastic naevus syndrome (DNS). Seven out of 11 (64%) common naevi and three out of nine (33%) dysplastic naevi showed increased cytoplasmic N-ras expression. No p53 immunopositivity could be recognized in any of the naevus samples. However, strong N-ras expression as well as immunopositivity for p53 was recognized among primary melanomas and metastases with significantly higher frequency among samples from patients with HCMM compared with samples from SCMM cases (for N-ras, 40% vs 10%, P < 0.01; and for p53 43% vs 17%, P < 0.05). We have earlier registered N-ras codon 61 mutations among metastases from 59% of patients with HCMM and from 24% of subjects with SCMM. A comparison of the genetic data with the immunohistochemical results showed occurrence of increased N-ras p21 expression in the presence and absence of detectable N-ras mutant alleles. Increased expression of wildtype N-ras p21 may contribute to tumorigenicity in the absence of mutational activation, at least in a subset of melanomas. Altogether, N-ras p21 alterations are registered at earlier stages than p53 alterations in melanoma development and may be of aetiological importance, whereas p53 alterations may be associated with tumour progression in the late stages. The increased frequency of phenotypic alterations registered among samples from HCMM/DNS patients may be a result of genetic instability and hypermutability and may be caused by various genetic changes such as altered gene dosage or regulatory as well as structural mutations.

p53 protein expression and TP53 mutations in malignant melanomas of sun-sheltered mucosal membranes versus chronically sun-exposed skin.

In this paper, we compare the expression of the TP53 gene product, p53 protein (p53p), in primary malignant melanomas from sun-shielded mucous membranes and from chronically sun-exposed skin. Archival tissues from 29 patients with mucosal melanomas and from 27 with cutaneous melanomas in facial skin were subjected to immunohistochemical procedures using the monoclonal antibody DO-1. p53p expression did not differ significantly between the two groups of melanomas. A comparison with previously obtained data on TP53 mutations from the same tumours showed closer concordance amongst mucosal than amongst skin tumours. Primary mucosal melanomas and their satellites showed identical patterns, focal or diffuse, of p53p expression. Thus, expression of altered p53p could well participate in the clonal expansion of these mucosal melanomas and in tumour progression. The p53p characteristics recognized in our investigations are amongst the first hallmarks in the emerging molecular pathological profiling of mucosal melanomas, and may therefore be useful in exploring the aetiology of UV-independent melanomas.

Chemotherapy Resistance Mechanisms

Resistance to chemotherapy is a major problem in oncology. The drug resistance in breast cancer shares most traits with other solid tumours. As a result of studies of cell lines and animal tumours a number of drug resistance factors have been identified but only limited data from human tumours are available.

Glutathione Transferase P1–1 Expression in Human Melanoma Metastases: Correlation to N-RAS mutations and expression

Expression of the detoxication enzyme glutathione transferase P1-1 (GST P1-1) at elevated levels has been noted in many types of human tumors, including melanomas. The products of the human H-RAS, K-RAS and N-RAS genes play a key role in intracellular signal transduction leading to transcriptional activation of AP-1 (Fos/Jun) responsive genes. The oncogenic mutated forms of the ras proteins are constitutively active and interfere with normal signal transduction. Mutated RAS genes as well as increased expression of wild-type ras proteins are common features in human tumors including melanoma. We have characterized 30 melanoma metastases from 23 melanoma patients with reference to N-RAS expression and mutation as well as to GST P1 expression (immunohistochemistry and genetic analysis). Twenty-three of 30 samples (70%) had high N-Ras p21 and/or N-RAS codon 61 mutations and 18 of these 23 samples also had high GST P1-1 immunoreactivity. Seven of 30 (23%) samples had low N-Ras p21 immunoreactivity and no detectable N-RAS codon 61 mutations. Six of these 7 samples (86%) also had low GST P1-1 immunoreactivity. The results indicate a statistically significant correlation (Spearman correlation coefficient, r = 0.56, p = 0.001, 2-tailed test) and provide, for the first time, indirect evidence for a possible coregulation of N-RAS and GST P1 in human malignant melanoma which should be further evaluated.