Ágnes Bégány

EGFR gene copy number alterations in primary cutaneous malignant melanomas are associated with poor prognosis

Copy number alterations of the epidermal growth factor receptor (EGFR) gene have been extensively analyzed in different cancers, but no data are available for primary malignant melanoma. The aim of the present study was to simultaneously investigate the EGFR gene and chromosome 7 copy number alterations in 81 cutaneous malignant melanomas by interphase FISH and correlate the data with clinicopathological parameters of patients. EGFR mRNA levels were detected by Affymetrix GeneChip Human Genome U133 Plus 2.0 expression arrays for 16 lesions. Both increased gene dosage and chromosome 7 alterations were found in 70% of tumors. Extra EGFR copies were detected in an additional 10% of samples. Polysomy 7 was associated with EGFR gene amplification. Significant correlation was found between EGFR alterations and histological subtypes, tumor thickness, ulceration and metastases formation. Amplification was significantly higher in lesions that developed metastases within 2 years after surgical excision of the primary tumor. Gene copy alterations were associated with elevated mRNA expression in 77% of lesions when compared to tumors with disomic EGFR status, the correlation was not directly proportional to gene copy number. Associations between protein expression and mRNA levels were even less prominent. In conclusion, our study indicates that amplification of the EGFR gene and polysomy 7 are frequent alterations in primary melanomas and are associated with bad prognosis. Further studies are required to clarify whether melanoma patients with EGFR alterations can benefit from anti‐EGFR therapy.

Characterization of candidate gene copy number alterations in the 11q13 region along with BRAF and NRAS mutations in human melanoma

Amplification of the 11q13 chromosomal region is a common event in primary melanomas. Several candidate genes are localized at this sequence; however, their role in melanoma has not been clearly defined. The aim of this study was to develop an accurate method for determining the amplification pattern of six candidate genes that map to this amplicon core and to elucidate the possible relationship between BRAF, NRAS mutations and CCND1 copy number alterations, all of which are key components of the MAP kinase pathway. Characterization of gene copy numbers was performed by quantitative PCR and, as an alternative method, fluorescence in situ hybridization was used to define the CCND1 amplification pattern at the single cell level. Samples with amplified CCND1 (32%) were further analyzed for copy number alterations for the TAOS1, FGF3, FGF19, FGF4 and EMS1 genes. Coamplification of the CCND1 and TAOS1 was present in 15% of tumors and was more frequent in ulcerated lesions (P=0.017). Furthermore, 56% of primary melanomas had either BRAF or NRAS mutations, but these two mutations were not present in any of the lesions analyzed. Of these cases, 34% also had CCND1 amplification. There was a significant relationship between NRAS activating mutations and UV exposure (P=0.005). We did not find correlations between CCND1 gene amplification status and any of the patients’ clinicopathological parameters. However, CCND1 amplification simultaneously with either BRAF or NRAS activation mutations was observed mainly in primary tumors with ulcerated surfaces (P=0.028). We assume that coamplification of these candidate genes in the 11q13 region or CCND1 gene alterations along with either BRAF or NRAS mutations might be more important for prognosis than the presence of these alterations alone.

Extra copies of c-myc are more pronounced in nodular melanomas than in superficial spreading melanomas as revealed by fluorescence in situ hybridisation

Amplification of c‐myc is a common genetic alteration and associated with a poor prognosis in a variety of cancers. Extra copies of the gene have been found in large numbers of melanoma metastases, but only few primary tumours have been studied. We investigated the c‐myc copy number alterations in two different subtypes of primary melanomas with different biological behaviours.

Characterization of 9p21 copy number alterations in human melanoma by fluorescence in situ hybridization

Alteration of the CDKN2A (alias p16) tumor suppressor gene, located on 9p21, occurs frequently in familial and sporadic melanomas. Beside CDKN2A, other genes (e.g., CDKN2B, and ARF/p14(ARF), long considered distinct from CDKN2A) on this locus are often deleted or mutated in a large number of tumors including glioma, bladder cancer, and lung cancer. The aim of this study was to evaluate the deletion pattern of the 9p21 locus on a cell-by-cell basis in a large number of melanoma samples using fluorescence in situ hybridization (FISH). In an analysis of 81 primary lesions targeting the 9p21 region and chromosome 9 centromere, high frequency of 9p21 loss (84%) was found. Deletion of 9p21 was present in both early- and late-stage melanomas with similar frequencies. Extra 9p21 copies were rarely seen; they were always associated with polysomy 9 and were observed only in advanced stage melanomas (6 tumors). This FISH study strengthens the hypothesis that the loss of 9p21 occurs frequently in primary melanoma, that the deletion is present in early and late stages of the disease with similar frequency, and that it affects a large extent of the locus.

Marked genetic differences between braf and nras mutated primary melanomas as revealed by array comparative genomic hybridization

Somatic mutations of BRAF and NRAS oncogenes are thought to be among the first steps in melanoma initiation, but these mutations alone are insufficient to cause tumor progression. Our group studied the distinct genomic imbalances of primary melanomas harboring different BRAF or NRAS genotypes. We also aimed to highlight regions of change commonly seen together in different melanoma subgroups. Array comparative genomic hybridization was performed to assess copy number changes in 47 primary melanomas. BRAF and NRAS were screened for mutations by melting curve analysis. Reverse transcription PCR and fluorescence in-situ hybridization were performed to confirm the array comparative genomic hybridization results. Pairwise comparisons revealed distinct genomic profiles between melanomas harboring different mutations. Primary melanomas with the BRAF mutation exhibited more frequent losses on 10q23–q26 and gains on chromosome 7 and 1q23–q25 compared with melanomas with the NRAS mutation. Loss on the 11q23–q25 sequence was found mainly in conjunction with the NRAS mutation. Primary melanomas without the BRAF or the NRAS mutation showed frequent alterations in chromosomes 17 and 4. Correlation analysis revealed chromosomal alterations that coexist more often in these tumor subgroups. To find classifiers for BRAF mutation, random forest analysis was used. Fifteen candidates emerged with 87% prediction accuracy. Signaling interactions between the EGF/MAPK–JAK pathways were observed to be extensively altered in melanomas with the BRAF mutation. We found marked differences in the genetic pattern of the BRAF and NRAS mutated melanoma subgroups that might suggest that these mutations contribute to malignant melanoma in conjunction with distinct cooperating oncogenic events.

Involvement of Chromosome Losses in the Progression and Metastasis Formation of a Human Malignant Melanoma

To characterize the possible cytogenetic link between a primary tumor and its metastasis, interphase cytogenetic analysis was performed on tumor cells and cutaneous metastasis from a male patient with malignant melanoma by using fluorescence in situ hybridization. The numbers of distinct hybridization domains specific for ten different pericentromeric sequences were used as indicators of copy numbers of these chromosomes. In the primary tumor, the majority of cells had two copies of these chromosomes, but significant numbers of nuclei also were present with one and three copies. In addition, in almost all cells, both sex chromosomes were abnormal; nullisomy of the Y chromosome was associated with X disomy. The corresponding metastatic tumor cells were predominantly monosomic; only the distribution of chromosomes 11 and 7 was similar to the primary tumor. In the metastatic tumor, the sex chromosomes had a normal copy number; that is, one Y and one X were detected. These data indicate that both the initiation and the progression of this melanoma are associated with chromosome losses.

Immunohistochemical Examination of P-cadherin in Bullous and Acantholytic Skin Diseases

Autoimmune blistering diseases (pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, dermatitis herpetiformis) and certain genodermatoses with acantholysis (Darier-disease, Hailey-Hailey disease) have different aetiological factors, but all result in bulla formation and/or in acantholysis. Cadherins are Ca++-dependent cell-cell adhesion molecules which play an important role in the cellular connection between normal cells. P-cadherin is involved in the selective adhesion of epidermal cells, and is expressed only on the surfaces of the two basal layers. We examined the expression of P-cadherin in some autoimmune bullous skin diseases and Dariers disease using immunohistochemistry and found P-cadherin to be strongly upregulated. We believe the upregulation is compensatory to the primary pathophysiological events in the various bullous dermatoses.

Cutaneous cryptococcosis mimicking basal cell carcinoma in a patient with Sézary syndrome

Cryptococcosis is an opportunistic yeast infection that is the most common systemic fiangal infection in immunocompromised patients. Skin involvement is a feature in 10-20% of cases of disseminated cryptococcal infection (1). We report here a case of a 63-year-old woman with Sézary syndrome (T4, N3, MO, Bl) with an ulcerated preauricular tumour that developed during photopheresis with a combination of methotrexate and steroid treatment. We highlight the importance of differential diagnosis of cryptococcosis in the case of any atypical or non-healing lesions observed in an immunosuppressed patient.

HHV-8 ELISA based on a one-step affinity capture of biotinylated K8.1 antigen.

The immunogenic envelope antigen gp35-37 of human herpesvirus-8 (HHV-8) is encoded by orfK8.1. An ELISA is described using streptavidin capture of bacterially expressed and biotinylated recombinant K8.1 antigen. The antigen capture strategy provides a simple and reliable method, which does not require high yield production and purification of the recombinant antigen before the serological assay. The specificity and sensitivity of the K8.1 ELISA were validated by gp35-37 envelope antigen Western blot and anti-lytic membrane immunofluorescence assay using lytically induced HHV-8 infected BCBL-1 cells. Under the established ELISA conditions, eight of the 10 Kaposis sarcoma patients and five of the 180 healthy blood donors had IgG antibodies to K8.1 envelope antigen.

A Study on Cell-Mediated Immunity in Polymorphic Light Eruption

Polymorphic light eruption (PLE) can be defined as a delayed abnormal response to sunlight. In this paper some features of the presumable immune response were studied in 55 patients with PLE and the results were compared with the findings available in 58 cases with porphyria cutanea tarda (PCT). The mean intracutaneous reactivity index measured by skin tests of delayed type was normal in both diseases. The number of the active and total E-rosette-forming peripheral lymphocytes was significantly lower in the active stage of PLE whereas in remission normal values were found. In active PCT only the number of the total E-rosette-forming cells was decreased. The percentage of lymphocytes with so-called dot-like alpha-naphthyl acetate esterase enzyme reaction was reduced significantly in the active stage of PLE whereas in remission their number was normal. Changes observed in PLE seem to be functional and suggest the involvement of T lymphocytes in some phases of the hypersensitive cutaneous reaction induced by sunlight.