Winfried Hindermann

Genetic subtyping of renal cell carcinoma by comparative genomic hybridization.

The prognosis of renal cell carcinoma (RCC) varies dependent on histologic tumor subtypes. However, differentiation between RCC types may sometimes be difficult on histologic grounds alone. Furthermore, the prognostic value of histologic parameters for the individual prognosis is limited. Additional information on the molecular level seems necessary to obtain more certainty in diagnostic and prognostic evaluation. By investigating genetic alterations in different RCC subtypes, we sought to obtain a genotype-phenotype correlation. Eighty-two clear-cell, 53 papillary, 23 chromophobe RCCs and 26 renal oncocytomas were investigated. Comparative genomic hybridization (CGH) was performed on DNA from paraffin-embedded tissue samples. DNA was isolated from tumor areas by microdissection and amplified by degenerated oligonucleotide primed polymerase chain reaction (DOP-PCR). CGH was performed according to standard protocols. We were able to detect specific alterations in each RCC subtype: clear cell RCC showed -3p, +5/5q, -8p, -9, -14, -18; papillary (chromophilic) RCC gains of chromosomes 7, 17, 16, 3, 12; chromophobe RCC loss of chromosomes 1, 2, 6, 10, 13, 17, 21; renal oncocytomas loss of chromosomes 1/1p and 14. Furthermore, for clear cell RCC, it was possible to define alterations which are associated with metastatic disease: loss of 9, 10, 14. Our results demonstrate that each RCC subtype is characterized by distinct genetic alterations. The definition of genetic alterations seems helpful for a tumor typing especially when morphology is equivocal. Therefore, genetic analyses represent a powerful diagnostic and prognostic tool for RCC.

Synthesis and protein distribution of the unspliced large tenascin‐C isoform in oral squamous cell carcinoma

The inclusion or omission of the alternatively spliced region in the tenascin‐C (Tn‐C) mRNA gives rise to the large (Tn‐CL) or small (Tn‐CS) variant, respectively. Tn‐CL is thought to be a typical component of provisional extracellular matrices (ECMs) and is expressed during tumour stroma remodelling. Tn‐CL synthesis has been studied using RNA/RNA in situ hybridization, and Tn‐CL protein distribution, using immunohistochemistry (clone BC‐2), in 18 oral squamous cell carcinomas (OSCCs) of different grades of malignancy. While the Tn‐CL protein was demonstrated within the whole stromal compartment regardless of grade of malignancy, the majority of the Tn‐CL mRNA signal‐bearing cells were carcinoma cells. Only a few stromal myofibroblasts were able to synthesize Tn‐CL, as revealed by α‐smooth muscle actin double staining. In well‐differentiated carcinomas (G1), the Tn‐CL synthesizing carcinoma cells were localized as a single positive cell layer in the tumour stroma interface, particularly in invasive areas. A higher grade of malignancy (G2/G3) is associated with a significantly increased number of Tn‐CL synthesizing carcinoma cells randomly distributed within the invading tumour areas. Double‐staining experiments (Tn‐CL mRNA ISH/BC‐2 immunohistochemistry) indicate that these cells are capable of organizing and depositing a three‐dimensional Tn‐CL matrix. Even though an instructive and/or inductive role of the carcinoma cells in tumour stroma formation cannot be excluded, these results demonstrate that carcinoma cells can directly produce the ECM components of tumour stroma. Copyright

Frequent DNA hypomethylation of human juxtacentromeric BAGE loci in cancer

The BAGE (B melanoma antigens) sequence family contains 15 nearly identical sequences that are in the juxtacentromeric regions of chromosomes 9, 13, 18, and 21. BAGE loci are expressed in male germ tissue and in a high percentage of cancers and cancer cell lines. We analyzed the DNA methylation state of the sequences in or near the promoters of the BAGE loci by a quantitative bisulfite and PCR‐based assay (multiplex COBRA) using MboI and HphI in 18 somatic tissue samples, 4 testis and 4 sperm samples, and 48 tumors and tumor cell lines. In 94% of the control somatic tissue samples, DNA was highly methylated in the analyzed regions. In contrast, 98% of tumor DNA samples displayed hypomethylation. Also, DNA from testes and sperm was hypomethylated in at least one of the BAGE loci. BAGE transcripts were observed in only 47% of the analyzed tumor samples. Consequently, we propose BAGE hypomethylation as a new, highly informative epigenetic biomarker for the diagnosis of cancer, whose hypomethylation in cancer may be causally related to that of juxtacentromeric satellite DNA.

An improved version of the DNA methylation database (MethDB)

Cytosine methylation is a characteristic property of prokaryotic and eukaryotic genomes. In the latter, it is indispensable for a healthy development of the organism and uncontrolled changes in the distribution of 5-methylcytosine (5mC) have been linked to severe disorders, in particular cancer. The growing scientific interest in DNA methylation has led to a considerable amount of data about this epigenetic phenomenon. In order to make these data readily available, we have established a dedicated database. The DNA Methylation database (MethDB) is currently the only public database for DNA methylation (http://www.methdb.net). This constantly growing database has become a key resource in the field of DNA methylation research. The database contains currently methylation patterns, profiles and total methylation content data for 46 species, 160 tissues and 72 phenotypes coming from a total of 6667 experiments (as of September 4, 2002). About 14% of the data have not been published elsewhere. These data can be conveniently searched and represented in different ways. Recently, we have included an on-line submission tool that permits the scientific public to directly enter new data into MethDB.

Clonal Origin of Multifocal Renal Cell Carcinoma as Determined by Microsatellite Analysis

PURPOSE The reported incidence of satellite tumor lesions in renal cell carcinoma (7% to 25%) suggests that there is a risk of local recurrence after nephron sparing surgery. It remains largely unknown whether small satellite tumors show malignant features and whether they are metastases from the primary tumor. Therefore, we determined the clonality of multifocal tumors by molecular genetic analysis. MATERIALS AND METHODS A total of 19 multifocal clear cell renal cell carcinomas were investigated by microsatellite analysis using 6 markers for chromosome 3p, namely D3S1560, D3S1289, D3S1766, D3S1300, D3S1566 and D3S1663. Polymerase chain reaction was performed according to standard protocols, followed by gel electrophoresis and automated analysis using an automated DNA sequencer (Li-Cor, Lincoln, Nebraska). RESULTS All primary clear cell tumors were characterized by loss of heterozygosity on 3p. Multifocal tumors showed identical microsatellite alterations with at least 1 marker in 17 of the 19 cases. In 2 cases different microsatellite patterns were detected in tumors from the same kidney. CONCLUSIONS Identical loss of heterozygosity and shift patterns detected in different tumors in the same kidney strongly suggest that multifocal clear cell renal cell carcinomas have a common clonal origin in most cases. These findings indicate that satellite tumors are the result of intrarenal metastasis from the primary tumor. The clinical implications of these results must be correlated with the clinical disease course in patients with multifocal renal cell carcinoma.

mRNA expression and protein distribution of the unspliced tenascin-C isoform in prostatic adenocarcinoma.

The inclusion or omission of the alternatively spliced region in the tenascin‐C (Tn‐C) mRNA gives rise to the large (Tn‐CL) or small (Tn‐CS) variant, respectively. Tn‐CL is thought to be a typical component of provisional extracellular matrices (ECMs) and is expressed during tumour stroma remodelling in cancer. Tn‐CL mRNA expression and protein distribution have been studied in 44 prostatic adenocarcinomas using RNA/RNA in situ hybridization supplemented by reverse transcriptase‐polymerase chain reaction (RT‐PCR), and immunohistochemistry (clone BC‐2). While the Tn‐CL protein was demonstrated within tumour stroma, Tn‐CL mRNA expression was mainly observed in carcinoma cells, regardless of the histological grade of the tumour. Carcinoma cells containing Tn‐CL mRNA were particularly localized at the tumour invasion front. Tn‐CL mRNA was also identified in benign prostatic hyperplasia, where it was present exclusively in the basal cell layer, and in prostatic intraepithelial neoplasia in which there was partial loss of positive basal cells and increasing positivity of luminal cells. Furthermore, newly formed tumour blood vessels and inflammatory and stromal cells take part in the expression of Tn‐CL and are involved in the formation of a provisional tumour matrix. It is concluded that deposits of Tn‐CL indicate rebuilding processes in non‐neoplastic as well as in neoplastic prostatic tissues. In respect of the Tn‐CL synthesis in budding prostatic carcinoma cells, the results demonstrate that tumour cells can directly produce the ECM components of carcinoma stroma, creating conditions that facilitate the process of invasion. Copyright

Quantitative Evaluation of Apoptosis and Proliferation in Renal Cell Carcinoma. Correlation to Tumor Subtype, Cytological Grade According to Thoenes-Classification and the Occurrence of Metastasis

To analyse growth characteristics of human renal cell tumors, 66 renal cell carcinomas and one oncocytoma were investigated concerning the proliferative activity by immunohistochemical demonstration of the Ki-67 antigen (clone MIB1) and the apoptotic rate using the terminal deoxynucleotidyl-transferase mediated dUTP-fluorescin nick end labelling (TUNEL) method. The TUNEL method indicates DNA double strand breaks considered as a hallmark of programmed cell death (apoptosis). Apoptotic cells were observed in 57 of 67 cases. The apoptotic rate (percentage of stained tumor cells) varied from 0% to 54.1%. GI carcinomas possessed a statistically significant higher apoptotic rate than GII/GIII carcinomas. The proliferation index (percentage of Ki-67 labelled cells) ranged from 0.09% to 22.3%. The well differentiated carcinomas (GI) showed statistically lower proliferative activity than moderate and poorly differentiated carcinomas (GII/GII). The clear cell variant of renal cell carcinoma expressed a higher apoptotic rate than the chromophilic variant. A statistical correlation between apoptosis/proliferation and occurrence of metastasis could not be established. In progression from well to less differentiated renal cell carcinoma the decrease of apoptotic rate, as well as the increase of the proliferative activity, contributes to a rapid tumor growth.

Immunohistochemical demonstration of the γ2 chain of laminin-5 in urinary bladder urothelial carcinoma: Impact for diagnosis and prognosis

Abstract The heterotrimeric molecule laminin-5 (Ln-5) represents a main protein of the epithelial adhesions complex. It links the basement membrane (BM) with the hemidesmosomes of the basal urothelial cells. The study was aimed to evaluate invasion associated changes of the epithelial adhesion complex in urothelial carcinoma (UC) monitored by immunohistochemical demonstration of the Ln-5 γ2 chain. For correlation to UC phenotype and patients outcome, a semiquantitative immunohistochemical analysis of 100 routinely processed paraffin embedded samples (non-invasive and invasive UC) using the antibody D4B5 specific for the Ln-5 γ2 chain was performed. An increased risk of death is associated with an increased Ln-5 loss from BM ( P =0.001), an increase of stroma deposition ( P =0.001), as well as an increase of cellular retention of Ln-5 protein ( P =0.001) (Kruskal–Wallis test). As shown in multivariate analysis, in addition to tumor stage the cellular retention of Ln-5 is the most important prognostic parameter. In consequence, the modulation of Ln-5 is recommended as a diagnostic marker of invasive UC phenotype.

BAGE Hypomethylation, A New Epigenetic Biomarker for Colon Cancer Detection

Early detection of colorectal cancer is a decisive step in the successful and complete cure of the disease. Epigenetic markers, in particular, those based on aberrant DNA methylation, can be used to diagnose cancer. B melanoma antigens (BAGE) are a family of genes and truncated genes located in the heterochromatic regions of several human chromosomes. Our previous work showed that BAGE loci (i.e., genes and truncated genes) were hypermethylated in normal tissues and hypomethylated in 98% of human cancers. In the present study, we analyzed DNA methylation of the BAGE loci in 54 colon cancers and in neighboring histopathologic normal tissue samples. Using a combined bisulfite restriction assay, we showed that BAGE loci were hypomethylated in 81% of carcinoma samples. Colon cancer could be diagnosed with 94% specificity, 83% sensitivity, and 89% accuracy. No correlation was found between DNA methylation of BAGE loci and age, gender of patients, nor with the tumor stage or site. Based on the hypothesis that during neoplastic transformation, hypomethylation occurs in juxtacentromeric CpG islands, we suggest that other genes located in the heterochromatic compartment should be tested. These new markers enrich the list of currently studied epigenetic alterations in colon cancer and could be associated with hypermethylation markers to develop reliable diagnostic tests. (Cancer Epidemiol Biomarkers Prev 2008;17(6):1374–9)

Analysis of activated EGFR signalling pathways and their relation to laminin-5 γ2 chain expression in oral squamous cell carcinoma (OSCC)

Overexpression of epidermal growth factor receptor (EGFR) was shown for the majority of squamous cell carcinomas. The EGFR expression correlates to tumour size, stage and cytoplasmic accumulation of the laminin-5 γ2 chain (Ln-5/γ2), which is known as a marker of invading tumour cells. There is only limited knowledge if and how EGFR signalling pathways are important for invasion-associated processes and for the regulation of Ln-5/γ2. Therefore the distribution of phosphorylated Erk1/2, p38 MAPK and Akt was immunohistochemically defined in oral squamous cell carcinoma (OSCC) of different histological grade and compared to histological criteria of invasion and cytoplasmic Ln-5/γ2 deposition. With raising histological grade, there is a slight increase in nuclear pErk1/2-stained tumour cells (P=0.398) and a loss of nuclear (P=0.593) and increased cytoplasmic staining (P=0.144) of pAkt mainly in invading OSCC cells. Nuclear pp38 MAPK could only be sporadically detected in few cases. In case of pErk1/2 and pAkt, only a partial co-localisation could be revealed in cases with abundant kinases and Ln-5/γ2. Among the investigated kinases, only pAkt shows a relation to histological grade and invasion in OSCC. pErk1/2, pp38 MAPK and pAkt do not represent a direct link between EGFR and Ln-5 synthesis. Therefore, enhanced Ln-5/γ2 may be a secondary phenomenon of EGFR-induced tumour cell proliferation and dissemination.