Biomedical subjects

Robert Stoehr

Publications and source records attributed to Robert Stoehr.

30 records · Page 2Linked to original sources

PubMed2005

High-throughput tissue microarray analysis of COX2 expression in urinary bladder cancer.

To investigate whether protein expression of cyclooxygenase 2 (COX2) is associated with tumor phenotype, immunohistochemical alterations, and clinical outcome in urinary bladder cancer (BC). Tissue microarrays (n = 776) were used to analyze COX2, P53 and the Ki-67 labeling index immunohistochemically. A monoclonal mouse antibody was used after heat-induced antigen retrieval. COX2 expression was scored semiquantitatively (0-3+). COX2 expression was detected in 60% (368/617) of urothelial BC. Positive COX2 staining was seen in 77.8% (140/182) of muscle invasive urothelial BC, compared to 35% (7/20) of muscle invasive squamous cell carcinomas (p < 0.001). COX2 protein expression was associated with advanced tumor stage (p < 0.0001), high-grade histology (p < 0.0001), solid growth pattern in invasive BC (pT1-4, p = 0.02), high Ki-67 labeling index (p < 0.0001), and positive P53 IHC (p < 0.001). COX2 expression was not associated with survival, recurrence, and progression in clinically relevant subgroups (pTa, pT1, pT2-4). Expression of COX2 is common in advanced BC with poor prognostic characteristics, supporting efforts to initiate clinical trials on the efficacy of COX2 inhibitors in the adjuvant treatment of high-risk urinary BC.

Aged↗

PubMed2004

Deletions of chromosome 8p and loss of sFRP1 expression are progression markers of papillary bladder cancer.

Many molecular alterations are known to occur in urothelial carcinoma of the bladder, but their significance for tumor progression is poorly understood. Deletions of chromosome 8p are frequently found in several tumor types and are often associated with progressive disease. In all, 99 bladder tumors were screened for deletions at 8p using loss of heterozygosity (LOH) and multicolor fluorescence in situ hybridization FISH analyses. Allelic loss on chromosome 8p in at least one marker was found in 25/99 (25%) tumors. There was a significant correlation of 8p deletions with invasive tumor growth and a highly significant association with papillary growth pattern in patients with invasive disease. cDNA array analyses revealed that secreted Frizzled-related protein 1 (sFRP1), an antagonist of Frizzled receptors and Wnt pathway activation on chromosome 8p12-11.1, is frequently downregulated in bladder cancer. To investigate sFRP1 as a candidate for a putative progression-related gene on 8p, urothelial cell lines and primary urothelial carcinomas were screened for sFRP1 expression using quantitative real-time PCR, Northern blot, immunofluorescence and immunohistochemistry (IHC). Of the investigated bladder cancers, 38% showed loss of sFRP1 expression by quantitative RT-PCR. Evaluation of the protein expression by IHC using tissue microarrays containing 776 bladder cancers revealed loss or strong reduction of sFRP1 expression in 66% of cases. SFRP1 loss was associated with higher tumor stage and grade and shorter overall survival. In addition, loss of sFRP1 was an independent indicator of poor survival in patients with papillary but not with muscle invasive bladder cancer. There were neither mutations in the coding region of sFRP1 nor homozygous deletions at 8p12-11.21. However, promoter methylation was detected using methylation-specific PCR in 29% of cases. In conclusion, we could show a close correlation of chromosome 8p deletions and progression of papillary bladder tumors. The sFRP1 gene on chromosome 8p12-11.1 could be a candidate gene for the predicted, progression-related tumor suppressor gene in bladder cancer and could contribute to urothelial carcinogenesis.

Biomarkers, Tumor↗

PubMed2004

No evidence for mutation of B-RAF in urothelial carcinomas of the bladder and upper urinary tract.

Mutational activation of the MAP kinase pathway is frequently found in various cancers. Recently, activating mutations in the B-RAF gene, an important activator of this pathway, have been described in several tumor types including melanoma, colorectal and papillary thyroid cancer. The most frequent mutation in exon 15 (V599E), as well as several other mutations within exons 11 and 15 result in constitutive activation of the oncoprotein. In addition, a significant association between mismatch-repair (MMR) deficiency and the V599E mutation in colorectal tumors has been found. The aim of our study was to investigate B-RAF mutations in 121 urothelial carcinomas of the urinary bladder (ranging from pTaG1 to pT4aG3) and 27 tumors from the upper urinary tract (UUT), including 16 tumors of the renal pelvis and 11 tumors of the ureter). Twelve of 27 UUT tumors were MMR-deficient and showed microsatellite instability. The V599E mutation was screened for by allele-specific PCR (PASA) and exons 11 and 15 of B-RAF including intron-exon-boundaries were sequenced. Overall, 116/121 bladder tumors and 23/27 tumors of the UUT were successfully investigated by PASA. None of the tumors showed the V599E mutation. Sequence analysis of exons 11 and 15 was successful in 46 urothelial tumors (bladder, n=31; UUT, n=15). No mutations within the coding region of exons 11 and 15 and the intron-exon junctions were found. The most frequent alterations in the B-RAF gene do not seem to be involved in urothelial carcinogenesis, and there is no correlation between MMR-deficiency and B-RAF mutations in urothelial tumors.

Aged↗

PubMed2004

Fluorescence in situ hybridization detects frequent chromosome 9 deletions and aneuploidy in histologically normal urothelium of bladder cancer patients.

Investigation of early urothelial lesions is essential to gain insight into the development of bladder cancer. In order to evaluate if deletions of chromosome arms 9p and 9q are preceding histomorphological changes in the urothelium, histologically normal urothelial samples of patients with previous and/or simultaneous (pre)malignant urothelial lesions were investigated. Dual colour fluorescence in situ hybridization (FISH) was performed on 96 histologically inconspicuous urothelial biopsies from 41 patients. Centromeric probes for chromosomes 9 and 17 were combined with gene locus specific probes for 9p21 (p16/CDKI2), 9q22 (FACC) and 17p (p53). Deletions of chromosome 9 (defined as presence of only one copy of centromere 9 or fewer gene-specific than centromeric signals in at least 40% of evaluated cells) were found in 21% of the samples (18/87). Deletions of chromosome 9p were present in 16% of cases (14/89), whereas deletions of chromosome 9q were encountered in 10% of specimens (9/93). A hemizygous deletion of p53 was found only once. Polysomy in more than 10% of cells was encountered in 16% of cases for both chromosome 9 and chromosome 17. Non-diploidy (defined as polysomy of both chromosomes) was found in 6% of samples (5/80). In summary, chromosome 9 deletions are frequently found in histologically normal urothelium of patients with bladder cancer, although less frequently than in hyperplasias and dysplasias. These findings support the hypothesis of multi-focal development of bladder cancer from histologically inconspicuous but already genetically altered urothelium. FISH analysis of chromosome 9 regions could provide a useful tool to detect potentially premalignant lesions in the follow-up care of patients with bladder cancer.

Aneuploidy↗

PubMed2003

WIF1, a component of the Wnt pathway, is down-regulated in prostate, breast, lung, and bladder cancer.

To detect novel Wnt-pathway genes involved in tumourigenesis, this study analysed the RNA expression levels of 40 genes of the Wnt pathway by chip hybridization of microdissected matched pairs of 54 primary prostate carcinomas. Eleven genes showed greater than two-fold differential expression in at least 10% of prostate cancers. Three of these genes encode extracellular components of the Wnt pathway (WNT2, WIF1, SFRP4); two are receptors (FZD4, FZD6); two belong to the intracellular signal cascade (DVL1, PPP2CB); one regulates transcription (TCF4); and three represent genes regulated by this pathway (CCND2, CD44, MYC). While SFRP4, FZD4, FZD6, DVL1, TCF4, and MYC are up-regulated, WIF1, WNT2, PPP2CB, CCND2, and CD44 are down-regulated in certain prostate cancer patients. Wnt inhibitory factor 1 (WIF1) and secreted frizzled related protein (SFRP4) showed the most significant aberrant expression at the RNA level. WIF1 was down-regulated in 64% of primary prostate cancers, while SFRP4 was up-regulated in 81% of the patients. Immunohistochemical analysis using a polyclonal antibody revealed strong cytoplasmic perinuclear WIF1 expression in normal epithelial cells of the prostate, breast, lung, and urinary bladder. Strong reduction of WIF1 protein expression was found in 23% of prostate carcinomas, but also in 60% of breast, 75% of non-small cell lung (NSCLC), and 26% of bladder cancers analysed. No significant association between WIF1 down-regulation and tumour stage or grade was observed for prostate, breast or non-small cell lung carcinomas, indicating that loss of WIF1 expression may be an early event in tumourigenesis in these tissues. However, down-regulation of WIF1 correlated with higher tumour stage in urinary bladder tumours (pTa versus pT1-pT4; p = 0.038).

Adaptor Proteins, Signal Transducing↗

PubMed2003

Lasermicrodissection--an important prerequisite for the molecular-genetic analysis of bladder cancer.

The past 10 years have shown that a crucial point in the molecular-genetic analysis of malignant tumors is the exact histopathological definition and separation of the investigated lesions. Laser microdissection has become an important tool allowing for the study of specific histological entities and defined preneoplastic lesions. Reliable detection of tumor-specific alterations can be compromised by the presence of normal cells. This requires microdissection of pure tumor cell populations (>80%), necessary for detecting chromosomal alterations by loss of heterozygosity analysis (LOH) and fluorescence in situ hybridization (FISH). The combination of microdissection and molecular methods could also be useful for studying multifocal lesions of the urothelial tract. This article describes in detail the use of laser microdissection, whole genome amplification by Improved Primer Extension Preamplification (I-PEP)-PCR and subsequent LOH, FISH, and sequencing analyses for the investigation of urothelial tumors and their precursors, e.g. dysplasias and hyperplasias. The combination of the described methods allows for a wide spectrum of molecular investigations in lesions with a limited number of tumor cells, and might help to understand the fundamental alterations involved in urothelial carcinogenesis.

Chromosome Aberrations↗

PubMed2003

Microsatellite instability at chromosome 8p in non-small cell lung cancer is associated with lymph node metastasis and squamous differentiation.

Genetic alterations at chromosome arm 8p are associated with advanced disease and poor patient outcome in several types of malignant tumors. We studied the frequency of microsatellite instability (MSI) and loss of heterozygosity (LOH) at chromosome 8p in early stage non-small cell lung cancer (NSCLC) of 47 patients with stage I or II disease (25 squamous cell carcinomas and 22 adenocarcinomas). Microsatellite analysis was performed after laser microdissection using 5 polymorphic tetranucleotide microsatellite markers and 4 dinucleotide markers at chromosome 8p. A pentanucleotide repeat marker at the chromosomal locus 17p13.1 (TP53.Alu) was also analyzed. Expression of the mismatch repair (MMR) proteins hMSH2, hMSH6 and hMLH1 was evaluated by immunohistochemistry. Microsatellite instability (MSI) in at least 2 markers was detected in 9 of 47 patients (19.1%) and was predominantly found at tetranucleotide repeats. Sixteen of 47 (34.0%) NSCLC demonstrated LOH at chromosome 8p. All MSI-positive tumors showed normal expression of the MMR proteins. The presence of MSI at chromosome 8p was associated with lymph node metastasis (p=0.02), squamous differentiation (8/25; 32%-p=0.03), and the presence of LOH at the p53 locus (p=0.06). None of the other investigated clinical, pathologic or molecular factors correlated with MSI. Our study showed that an elevated MSI at selected tetranucleotide sequences (EMAST) on chromosome 8p is frequent in early stage squamous cell carcinomas of the lung with lymphatic spread. The tetranucleotide marker panel used in this study was able to indicate lymph node metastasis and high risk disease in patients with resectable squamous cell lung cancer.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Frequent microsatellite instability in sporadic tumors of the upper urinary tract.

Urothelial carcinoma of the renal pelvis and ureter may develop sporadically or as a manifestation of hereditary nonpolyposis colorectal cancer. The majority of hereditary nonpolyposis colorectal cancer is caused by mutation of the human DNA mismatch repair (MMR) genes and is detected by associated microsatellite instability (MSI). Seventy-three unselected urothelial carcinomas of the ureter and/or renal pelvis were screened for MSI using the National Cancer Institute-designated reference panel (plus BAT40). Instability of at least two microsatellite markers (MSI-high) was detected in 15 samples (21%). Immunohistochemical staining of the MMR proteins (hMSH2, hMLH1, or hMSH6) was absent in 13 of 15 (87%) MSI tumors, and alteration of coding sequence microsatellites (TGFbetaRII, Bax, hMSH3, and hMSH6) was found at frequencies of 7-33% in these samples. Tumors with MSI had significantly different clinical and histopathological features including higher prevalence in female patients, low tumor stage and grade, and a papillary and frequently inverted growth pattern. Our results suggest a molecular pathway of tumorigenesis that is similar to MMR-deficient colorectal cancers and consistent with the notion that the site distributions of hereditary or sporadic MSI-high tumors may reflect tissue-specific susceptibility to lesions processed by the MMR machinery.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Clonality of multifocal urothelial carcinomas: 10 years of molecular genetic studies.

Multifocal occurrence and frequent recurrence are characteristic features of urothelial carcinomas of both the urinary bladder and the upper urinary tract. To describe the clonal nature of these tumors, 2 theories have been proposed. The monoclonality hypothesis describes the multiple tumors as descendants of a single genetically transformed cell spreading throughout the urothelium. In contrast, field cancerization caused by carcinogen exposure of the urothelium may lead to independent development of synchronous or metachronous nonrelated tumors at different sites of the urothelial tract. In the last 10 years, a multitude of molecular genetic studies have investigated the clonality of multifocal urothelial carcinomas. The majority of studies revealed a monoclonal origin of the multiple tumors. However, most of these studies investigated advanced invasive carcinomas. A small but significant proportion of multifocal urothelial carcinomas appear to arise from different clones, supporting the field-cancerization hypothesis. Oligoclonal tumors might be more common in precursor lesions and early tumor stages. The frequent monoclonality found in patients with advanced tumors could be due to outgrowth of 1 tumor cell clone with specific genetic alterations. Two important mechanisms appear to be important for the spread of malignant cells: intraluminal seeding and intraepithelial migration. Investigation of the entire urothelial lining in patients with urothelial tumors should provide further insight into the development of multifocal urothelial carcinomas.

Animals↗

PubMed2002

Histologic-genetic mapping by allele-specific PCR reveals intraurothelial spread of p53 mutant tumor clones.

Common and clinically important features of urothelial carcinomas are multifocality and a high rate of recurrence. Molecular studies demonstrated that multifocal tumors are frequently composed of one tumor clone spreading throughout the urothelial tract. A combination of histologic and genetic mapping of cystectomy specimens from bladder cancer patients is a valuable tool to study bladder carcinogenesis and tumor cell spread by correlating urothelial morphologic features and defined genetic alterations. In the present study, the primary tumors of 14 cystectomy specimens were investigated for p53 protein overexpression by immunohistochemistry and p53 gene mutation by genomic sequencing. Seven tumors showed a strong nuclear staining for the p53 protein. In six of seven tumors, a p53 gene mutation was detected. Allele-specific PCR of defined p53 mutations was established in five of six cases with a p53 mutation. Subsequent screening of the entire urothelial lining of each cystectomy specimen by allele-specific PCR revealed p53-mutant cell clones in urothelial patches with carcinoma in situ and dysplasia, but also frequently in histomorphologically normal urothelium adjacent to the tumor. The pattern of tumor cell spread indicated a continuous intraurothelial growth of the p53-mutant clone. P53 immunohistochemistry visually confirmed the presence of mutant cells in most of these samples. We conclude that allele-specific PCR is a highly sensitive and reliable method for tracking specific p53 mutant clones in the urothelium. Moreover, the detection of p53-mutant cells in histologically normal or preneoplastic urothelial areas in four patients with invasive bladder cancer indicates an extensive intraurothelial tumor cell spread. The excellent correlation of immunohistochemically positive urothelial patches with the presence of a specific mutation highlights the biologic significance of p53-positive cells in the urothelium of tumor patients.

Aged↗

PubMed2002

No evidence for involvement of beta-catenin and APC in urothelial carcinomas.

The wnt pathway plays an important role in embryonal patterning and cell fate determination, involving stabilization of nuclear and cytoplasmic beta-catenin (CTNNB1) mediated by APC, axin, and other proteins. Uncomplexed beta-catenin binds to TCF/LEF transcription factors and activates the expression of growth regulatory target genes such as c-myc or cyclin D1. In colorectal and other cancers, constitutive wnt signaling results frequently from mutations in one or more pathway components, e.g. APC and beta-catenin, resulting in nuclear and/or cytoplasmic accumulation of beta-catenin. In the present study, the most frequent alterations in the CTNNB1 and APC genes were investigated in primary urothelial bladder tumors and cell lines. Snap-frozen bladder tumors (n=99) of different stages and grades and 4 cell lines (RT4, RT112, J82, UROtsa) were investigated for APC allelic deletions by loss of heterozygosity (LOH) analysis. The most frequent mutated regions of CTNNB1 (degradation box in the third exon) and APC (mutation cluster region) were directly sequenced. Beta-catenin expression was analyzed by immunofluorescence in the cell lines. LOH at the APC gene locus on chromosome 5q21 was found in 7 of 72 (10%) of the informative cases. No mutations were found in either CTNNB1 or APC. A previously described polymorphism at codon 1493 of the APC gene was detected in 8 tumors and 3 cell lines. All cell lines showed normal membranous beta-catenin staining without evidence for nuclear or cytoplasmic accumulation. Alteration of APC and beta-catenin, which are the most frequent wnt pathway alterations in many tumor types, are rare events in urothelial carcinomas. Other wnt pathway members, such as axin, may play an important role in urothelial carcinogenesis.

Adenomatous Polyposis Coli Protein↗

PubMedDate not supplied

Low frequency of human polyomavirus BKV and JCV DNA in urothelial carcinomas of the renal pelvis and renal cell carcinomas.

Human polyomaviruses BKV and JCV establish a persistent infection in the kidney and possess oncogenic potential in experimental animal models. Their possible involvement in the pathogenesis of human urogenital tumors has been suggested by several studies but is not conclusively resolved. For the present study, highly sensitive quantitative TaqMan PCR assays were developed to detect BKV and JCV DNA sequences in matched pairs of human tumors and surrounding normal tissue. Samples from 55 patients with renal pelvic urothelial carcinomas and from 83 patients with renal cell carcinomas of all histologic subtypes were investigated. Human polyomavirus DNA was detected in renal pelvis samples with a frequency of 16% (BKV) and 15% (JCV), and in kidney samples with a frequency of 1% (BKV) and 23% (JCV), respectively. However, viral sequences were not present more often in tumors than in normal tissue. There was no integration of the potentially oncogenic viral large T-antigen in the tumors, and the viral concentration of both BKV and JCV was not higher in DNA extracted from tumors compared to normal tissue. These data provide no evidence for a causative role of the human polyomaviruses BKV and JCV in the development of tumors of the renal pelvis and the kidney.

Adult↗