Vivi Ann Flørenes

Up-regulation of ephrin-A1 during melanoma progression.

Ephrin‐A1, formerly called B61, is a new melanoma growth factor; it is angiogenic and chemoattractant for endothelial cells. EPH‐A2, or ECK (a receptor for ephrin‐A1), is ectopically expressed in most melanoma cell lines; the pathology where this expression is first manifested and the possible role of the receptor in tumor progression are unknown. To determine these, we studied the expression of this ligand and receptor in biopsies of benign and malignant melanocytic lesions. EPH‐A2 was not detected in normal melanocytes, benign compound nevi or advanced melanomas, though it was found in 2 of 9 biopsies of malignant melanoma in situ. Ephrin‐A1 was present in occasional early lesions and in advanced primary melanomas (43%) and metastatic melanomas (67%). Expression of ephrin‐A1 was induced in melanoma cells by pro‐inflammatory cytokines. Our findings are consistent with 2 possible roles for ephrin‐A1 in melanoma development: it may promote melanocytic cell growth or survival and induce vascularization in advanced melanomas. Both effects may be potentiated by inflammatory responses. Our data are consistent with earlier observations that an inflammatory infiltrate is associated with poor prognosis in thin primary melanomas. Int. J. Cancer (Pred. Oncol.) 84:494–501, 1999.

Loss of expression of receptor tyrosine kinase family genes PTK7 and SEK in metastatic melanoma

Protein tyrosine kinases (PTKs) have been implicated in the development of many common human tumours including melanoma. Previously we isolated PTK gene sequences expressed in normal melanocytes. Here we examined expression of 9 of these genes in cell lines derived from defined stages of melanoma progression, by Northern blotting and in some cases immunoblotting. We also tested cells from 2 animal models of particular stages in progression, as well as uncultured biopsies of metastatic melanoma. The expression of 2 receptor kinase family members found in melanocytes, PTK7/CCK‐4 and SEK/TYRO1, was decreased or lost in advanced melanomas. PTK7 mRNA was found in only 54% of melanoma cell lines and 20% of melanoma biopsies. Similarly, expression was lost in 2 advanced cell lines selected from an early melanoma line that did express PTK7 mRNA. SEK/TYRO1 expression was observed in 75% and 17% of cell lines from primary and metastastic melanomas, respectively. Conversely, mRNA for the non‐receptor kinase PTK6/BRK was not detected in normal melanocytes or primary melanoma lines, but was found in 9% of metastatic melanoma cell lines. Int. J. Cancer 71: 1061‐1065, 1997.

1,5-Disubstituted 1,2,3-triazoles as cis-restricted analogues of combretastatin A-4: Synthesis, molecular modeling and evaluation as cytotoxic agents and inhibitors of tubulin

A series of cis-restricted 1,5-disubstituted 1,2,3-triazole analogues of combretastatin A-4 (1) have been prepared. The triazole 12f, 2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-5-yl)aniline, displayed potent cytotoxic activity against several cancer cell lines with IC(50) values in the nanomolar range. The ability of triazoles to inhibit tubulin polymerization has been evaluated, and 12f inhibited tubulin polymerization with IC(50)=4.8microM. Molecular modeling experiments involving 12f and the colchicine binding site of alpha,beta-tubulin showed that the triazole moiety interacts with beta-tubulin via hydrogen bonding with several amino acids.

Involvement of the pRb/p16/cdk4/cyclin D1 pathway in the tumorigenesis of sporadic malignant melanomas.

Biopsies from 61 sporadic metastatic malignant melanomas and five melanoma cell lines were examined for homozygous deletions and mutations in the CDKN2 gene (p16). As the p16 protein is involved in a cell cycle regulatory pathway consisting of at least pRb, cdk4 and cyclin D1, the tumours were also screened for amplifications of the last two genes. Moreover, the transcript levels of the genes were determined and the results compared with the immunohistochemically assessed expression of pRb. Altogether, homozygous deletions of CDKN2 were found in seven tumours (11%) and two of five cell lines, whereas a mutation was detected in only one biopsy, indicating that in sporadic melanomas the former mechanism is predominant for inactivating this gene. Notably, in total 59% of the metastatic lesions lacked detectable expression of p16 mRNA, whereas all the biopsies were found to express pRb. In accordance with the postulated negative feedback loop between p16 and pRb, one melanoma cell line showed overexpression of CDKN2 mRNA together with very low levels of the Rb protein. Amplification of the other two genes may not be important in the tumorigenesis of melanomas, as only one CDK4 and no CCND1 amplification was observed. However, highly elevated CDK4 mRNA levels, compared with that seen in a panel of normal tissues, were observed in 76% of the tumours, accompanied in 71% of the cases by high expression of the CCND1 cyclin activator. Although a low frequency of CDKN2 DNA aberrations was observed, the high number of tumours that lacked CDKN2 expression but showed overexpression of CDK4 and/or CCND1, suggest that functional inactivation of pRb through this pathway may be involved in the development or progression of sporadic human melanomas.

Homozygous deletion frequency and expression levels of the CDKN2 gene in human sarcomas--relationship to amplification and mRNA levels of CDK4 and CCND1.

Homozygous deletions of the putative tumour-suppressor gene CDKN2, which encodes an inhibitor of cdk4, have been detected in a high percentage of cancer cell lines of various histological types. In the present study, 109 human sarcomas were examined for homozygous deletions and for mRNA expression levels of the CDKN2 gene. Altogether, deletions were found in only eight (7%) of the cases, but, interestingly, in two (of eight) malignant Schwannomas and in two (of five) rhabdomyosarcomas. In comparison, such deletions were seen in only one (of 21) osteosarcomas and in none of 20 MFHs and 21 liposarcomas. Notably, highly elevated CDKN2 mRNA levels were found in 33% of the sarcomas, whereas no detectable transcript was present in 12 normal tissues. Amplifications of CDK4 and CCND1 (cyclin D1) were observed in 11% and 4% of the sarcomas respectively, but never in tumours with CDKN2 deletions. The level of CDK4 mRNA expression was increased in nine tumours in addition to the 12 samples with CDK4 amplification. Increased levels of the cyclin D1 transcript was found in 37 cases, four with and 33 without amplification. The data indicate that aberrations of these functionally related genes, or in regulation of the expression of the kinase, the activator or the inhibitor, may participate in sarcoma development. Furthermore, the data suggest that homozygous CDKN2 deletions may be of dissimilar significance in different sarcoma subtypes.

E‐cadherin and α‐, β‐, and γ‐catenin protein expression is up‐regulated in ovarian carcinoma cells in serous effusions

The aims of this study were firstly, to investigate the expression of E‐cadherin complex proteins in ovarian carcinoma cells in serous effusions and in primary and metastatic lesions; and secondly to study the value of these four proteins and calretinin, a mesothelial marker, in the differential diagnosis of ovarian carcinoma cells from reactive mesothelial cells in effusions. Sixty‐seven malignant effusions and 97 corresponding primary (n=36) and metastatic (n=61) lesions were immunohistochemically stained for E‐cadherin and α‐, β‐, and γ‐catenin. Staining extent and intensity were scored. Effusion specimens were additionally analysed for calretinin immunoreactivity. Membrane immunoreactivity for E‐cadherin and α‐, β‐, and γ‐catenin was detected on carcinoma cells in the majority of the effusions, but rarely on reactive mesothelial cells (p<0.001 for all markers). Calretinin immunoreactivity was confined to mesothelial cells (p<0.001). An association was seen between E‐cadherin and α‐catenin expression, in both effusions and solid tumours, and for β‐catenin in solid tumours (range p<0.001 to p=0.014). Up‐regulation of all four cadherin complex proteins was seen in carcinoma cells in effusions, when compared with corresponding primary tumours (range p<0.001 to p=0.028). As with effusions, metastatic lesions showed up‐regulation of α‐, β‐, and γ‐catenin when compared with primary carcinomas (p=0.002–0.015). Carcinoma cells in effusions showed in addition elevated levels of E‐cadherin when compared with metastatic lesions (p<0.001). Staining results in effusions showed no association with effusion site, tumour type or histological grade. Immunoblotting on 29 malignant effusions confirmed the presence of all four proteins in the majority of samples and co‐precipitation of E‐cadherin and β‐catenin was seen in ten specimens examined. E‐cadherin complex proteins are widely expressed in ovarian carcinoma cells. Together with calretinin, they form a powerful battery of markers for the cytological diagnosis of carcinoma cells in effusions. The up‐regulation of E‐cadherin complex proteins in serous effusions and metastatic lesions may mark an early metastatic phenotype and possibly mediates survival of tumour cells at these sites through the inhibition of apoptosis. Copyright

Granulin-Epithelin Precursor Is a Novel Prognostic Marker in Epithelial Ovarian Carcinoma

The granulin‐epithelin precursor (GEP) was preferentially expressed in invasive ovarian tumor epithelium specimens compared with specimens of borderline ovarian tumors. The objective of the current study was to evaluate the anatomic site‐related and cellular expression of GEP and its association with clinicopathologic parameters and survival in patients with advanced‐stage ovarian carcinoma.

Gene Expression Signatures Differentiate Ovarian/Peritoneal Serous Carcinoma from Diffuse Malignant Peritoneal Mesothelioma

Purpose: Ovarian/primary peritoneal serous carcinoma (OC/PPC) and diffuse peritoneal malignant mesothelioma (DMPM) are highly aggressive tumors that are closely related morphologically and histogenetically. It remains unclear whether both tumors are molecularly distinct neoplasms. The current study compared global gene expression patterns in OC/PPC and DMPM. Experimental Design: Ten OC/PPC and five DMPM effusions were analyzed for gene expression profiles using the Affymetrix U133 Plus 2 arrays and the dCHIP analysis program. Differentially expressed candidate genes were validated using quantitative real-time PCR and immunohistochemistry. Results: Unsupervised hierarchical clustering using all 54,675 genes in the array classified the samples into two groups: DMPM specimens versus OC/PPC specimens. A total of 189 genes that were differentially expressed in these two groups were selected based on statistical significance. Genes overexpressed in DMPM (n = 68) included calretinin, vitronectin, claudin 15, α4 laminin, hyaluronan synthase 1, cadherin 11, RAB7, v-maf, and the epidermal growth factor–containing fibulin-like extracellular matrix protein 1. Genes overexpressed in OC/PPC (n = 121) included insulin-like growth factor II (IGF-II); IGF-II binding protein 3; cyclin E1; folate receptors 1 and 3; RAB25; MUC4; endothelin-1; CD24; kallikreins 6, 7, and 8; claudins 3, 4, and 6; Notch3; and MMP-7. Quantitative real-time PCR validated the differential expression of 13 genes, and immunohistochemistry confirmed the differences for four gene products. Conclusions: Expression profiling separates OC/PPC from DMPM and identifies a number of genes that are differentially expressed in these tumors. The molecular signatures unique to OC/PPC and DMPM should provide a molecular basis to study both tumors and new potential markers for facilitating their differential diagnosis.

Expression of Activated Akt and PTEN in Malignant Melanomas Relationship With Clinical Outcome

Our purpose was to analyze, by immunohisto-chemistry, the expression of activated serine-threonine protein kinase B (p-Akt) and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in benign nevi and primary and metastatic melanomas and to correlate the expression level with clinical variables. We observed cytoplasmic and/or nuclear expression of p-Akt in 22 (54%) of 41 benign nevi, 112 (71.3%) of 157 primary tumors, and 50 (71%) of 70 metastases. Cytoplasmic PTEN staining was observed in 0 (0%), 152 (87.7%), and 64 (90%) of 41 nevi, 162 primary tumors, and 71 metastases, respectively. A significant positive correlation was seen between PTEN and p-Akt cytoplasmic expression (P < .001) in primary melanomas. Cytoplasmic p-Akt expression showed a positive association with cyclin A in superficial spreading (P = .038) but not in nodular (P = .22) melanomas. Cytoplasmic p-Akt and PTEN expression did not have an impact on disease-free and overall survival, but complete lack of nuclear p-Akt expression was a predictor of shorter disease-free survival (P = .025) for patients with superficial spreading melanoma.

Expression of S100A4 combined with reduced E-cadherin expression predicts patient outcome in malignant melanoma

The aim of the present study was to analyze the expression of S100A4 and E-cadherin in a panel of primary and metastatic malignant melanoma, and to correlate the expression level to clinicopathological parameters. The expression of S100A4 was examined by immunohistochemistry in 99 superficial spreading and 60 nodular primary melanomas, while the expression of E-cadherin was analyzed in 92 superficial spreading and 52 nodular lesions from the same panel. The expression levels of S100A4 and E-cadherin in the biopsies were inversely correlated, with S100A4 being expressed at the highest frequency in the nodular and E-cadherin in the superficial spreading lesions, respectively. When analyzing the melanoma subgroups separately, it was revealed that expression of S100A4 had a more significant impact on patient outcome in early superficial spreading melanomas than in the nodular subtype, while E-cadherin expression did not predict patient outcome in any of the subgroups. When examining all the patients, both markers give clinical information as predictors for disease-free survival, but when combining the expression of the two markers, a stronger significant correlation between high E-cadherin expressing/S100A4 negative biopsies and increased disease-free survival (P=0.002) was revealed, demonstrating the importance of examining the expression of more than one factor involved in the metastatic cascade when predicting patient outcome. We have also evaluated the relationship between the expression of these two antigens and cell cycle and signal transduction factors.