Expression of heat shock protein 70 in renal cell carcinoma and its relation to tumor progression and prognosis

Heat shock proteins (HSPs) play an important role in the cellular response to environmental stress and exert a cytoprotective effect. Especially HSP70 is an effective inhibitor of apoptosis, suggesting a role of HSP70 in carcinogenesis and tumor progression. To explore the relevance of HSP70 in renal cell carcinomas (RCCs), we analyzed nuclear and cytoplasmic HSP70 protein expression in formalin-fixed tissue from 145 clear cell RCCs by immunohistochemistry as well as Western blot analysis. Nuclear HSP70 expression was found in all RCCs and 75% of the tumors also exhibited a cytoplasmic HSP70 staining. Importantly, RCCs showed significantly reduced cytoplasmic (p=0.001) and combined nuclear/cytoplasmic (p=0.0022) HSP70 expression when compared with their cells of origin. A significant (p=0.0176) decrease of nuclear HSP70 expression became evident from well to poorly differentiated clear cell RCCs. Quite similarly, a trend (p=0.0558) for reduced combined nuclear/cytoplasmic HSP70 expression was shown from early (pT1) to advanced (pT3) tumor stages. Nevertheless, no correlation between HSP70 expression and patients´ survival became evident. In conclusion, our investigation demonstrates a significant decrease of antiapoptotic HSP70 protein expression during carcinogenesis and during progression from well (G1) to poorly (G3) differentiated clear cell RCCs. Our results suggest that HSP70-mediated inhibition of apoptosis seems to be of minor importance for carcinogenesis and tumor progression in RCCs

Molecular Characterization of Commonly Used Cell Lines for Bone Tumor Research: A Trans-European EuroBoNet Effort

Usage of cancer cell lines has repeatedly generated conflicting results provoked by differences among subdones or contamination with mycoplasm or other immortal mammalian cells. To overcome these limitations, we decided within the EuroBoNeT consortium to characterize a common set of cell lines including osteosarcomas (OS), Ewing sarcomas (ES), and chondrosarcomas (CS). DNA fingerprinting was used to guarantee the identity of all of the cell lines and to distinguish subdones of osteosarcoma cell line HOS. Screening for homozygous loss of 38 tumor suppressor genes by MLPA revealed deletion of CDKN2A as the most common event (15/36), strictly associated with absence of the CDKN2A (p 16) protein. Ten cell lines showed missense mutations of the TPS3 gene while another set of nine cell lines showed mutations resulting in truncation of the TP53 protein. Cells harboring missense mutations expressed high levels of nuclear TP53, while cell lines with nonsense mutations showed weak/absent staining for TP53. TP53(wt) cell lines usually expressed the protein in 2-10% of the cells. However, seven TP53(wt) osteosarcomas were negative for both mRNA and protein expression. Our analyses shed light on the correlation between immunohistochemical and genetic data for CDKN2A and TP53, and confirm the importance of these signaling pathways. The characterization of a substantial number of cell lines represents an important step to supply research groups with proven models for further advanced studies on tumor biology and may help to make results from different laboratories more comparable. (C) 2009 Wiley-Liss, Inc

Molecular Characterization of Commonly Used Cell Lines for Bone Tumor Research: A Trans-European EuroBoNet Effort

Usage of cancer cell lines has repeatedly generated conflicting results provoked by differences among subdones or contamination with mycoplasm or other immortal mammalian cells. To overcome these limitations, we decided within the EuroBoNeT consortium to characterize a common set of cell lines including osteosarcomas (OS), Ewing sarcomas (ES), and chondrosarcomas (CS). DNA fingerprinting was used to guarantee the identity of all of the cell lines and to distinguish subdones of osteosarcoma cell line HOS. Screening for homozygous loss of 38 tumor suppressor genes by MLPA revealed deletion of CDKN2A as the most common event (15/36), strictly associated with absence of the CDKN2A (p 16) protein. Ten cell lines showed missense mutations of the TPS3 gene while another set of nine cell lines showed mutations resulting in truncation of the TP53 protein. Cells harboring missense mutations expressed high levels of nuclear TP53, while cell lines with nonsense mutations showed weak/absent staining for TP53. TP53(wt) cell lines usually expressed the protein in 2-10% of the cells. However, seven TP53(wt) osteosarcomas were negative for both mRNA and protein expression. Our analyses shed light on the correlation between immunohistochemical and genetic data for CDKN2A and TP53, and confirm the importance of these signaling pathways. The characterization of a substantial number of cell lines represents an important step to supply research groups with proven models for further advanced studies on tumor biology and may help to make results from different laboratories more comparable. (C) 2009 Wiley-Liss, Inc.Molecular tumour pathology - and tumour genetic

Microarray analysis of Ewing's sarcoma family of tumours reveals characteristic gene expression signatures associated with metastasis and resistance to chemotherapy.

In Ewing's sarcoma family of tumours (ESFT), the clinically most adverse prognostic parameters are the presence of tumour metastasis at time of diagnosis and poor response to neoadjuvant chemotherapy. To identify genes differentially regulated between metastatic and localised tumours, we analysed 27 ESFT specimens using Affymetrix microarrays. Functional annotation of differentially regulated genes revealed 29 over-represented pathways including PDGF, TP53, NOTCH, and WNT1-signalling. Regression of primary tumours (n=20) induced by polychemotherapy was found to be correlated with the expression of genes involved in angiogenesis, apoptosis, ubiquitin proteasome pathway, and PI3 kinase and p53 pathways. These findings could be confirmed by in vitro cytotoxicity assays. A set of 46 marker genes correctly classifies these 20 tumours as responding versus non-responding. We conclude that expression signatures of initial tumour biopsies can help to identify ESFT patients at high risk to develop tumour metastasis or to suffer from a therapy refractory cancer

Clear Cell Papillary Renal Cell Carcinoma and Renal Angiomyoadenomatous Tumor: Two Variants of a Morphologic, Immunohistochemical, and Genetic Distinct Entity of Renal Cell Carcinoma.

Clear cell papillary renal cell carcinoma (ccpRCC) and renal angiomyoadenomatous tumor (RAT) share morphologic similarities with clear cell (ccRCC) and papillary RCC (pRCC). It is a matter of controversy whether their morphologic, immunophenotypic, and molecular features allow the definition of a separate renal carcinoma entity. The aim of our project was to investigate specific renal immunohistochemical biomarkers involved in the hypoxia-inducible factor pathway and mutations in the VHL gene to clarify the relationship between ccpRCC and RAT. We investigated 28 ccpRCC and 9 RAT samples by immunohistochemistry using 25 markers. VHL gene mutations and allele losses were investigated by Sanger sequencing and fluorescence in situ hybridization. Clinical follow-up data were obtained for a subset of the patients. No tumor recurrence or tumor-related death was observed in any of the patients. Immunohistochemistry and molecular analyses led to the reclassification of 3 tumors as ccRCC and TFE3 translocation carcinomas. The immunohistochemical profile of ccpRCC and RAT samples was very similar but not identical, differing from both ccRCC and pRCC. Especially, the parafibromin and hKIM-1 expression exhibited differences in ccpRCC/RAT compared with ccRCC and pRCC. Genetic analysis revealed VHL mutations in 2/27 (7%) and 1/7 (14%) ccpRCC and RAT samples, respectively. Fluorescence in situ hybridization analysis disclosed a 3p loss in 2/20 (10%) ccpRCC samples. ccpRCC and RAT have a specific morphologic and immunohistochemical profile, but they share similarities with the more aggressive renal tumors. On the basis of our results, we regard ccpRCC/RAT as a distinct entity of RCCs