Biomedical subjects

F Hofstaedter

Publications and source records attributed to F Hofstaedter.

At least 19 recordsLinked to original sources

PubMed2000

Clonality and genetic divergence in multifocal low-grade superficial urothelial carcinoma as determined by chromosome 9 and p53 deletion analysis.

Multifocality and recurrence are clinically important features of urothelial carcinomas of the urinary bladder. Recent molecular genetic studies have suggested that multifocal urothelial carcinomas are monoclonally derived from an identical transformed progenitor cell. However, most of these studies investigated advanced and poorly differentiated tumors. The study presented focuses on early papillary tumors, including 52 superficial well-differentiated multifocal and recurrent bladder carcinomas from 10 patients. Microdissection separating urothelium from stromal cells was considered essential to obtain pure tumor cell populations. Genetic analysis was carried out by applying two different methods. Dual color fluorescence in situ hybridization (FISH) with centromeric probes for chromosomes 9 and 17 and gene-specific probes for chromosome loci 9q22, 9p21, and 17p13 was carried out in parallel to loss of heterozygosity (LOH) analyses applying 5 microsatellite markers on these chromosomes. Overall, deletions on chromosome 9p were found in 47 tumors (90%), at chromosome 9q in 36 tumors (69%) and at chromosome 17p in 3 tumors (6%). There was a very high correlation of the results between FISH and LOH analysis. Ten early superficial papillary tumors showed deletion of chromosome 9p without deletion of 9q, suggesting 9p deletions as a very early event in the development of papillary urothelial carcinoma. Although in four patients, all investigated tumors showed identical genetic alterations and one patient showed no genetic alterations at the loci investigated, in five patients, two or more clones with different deletions were found. In four of these patients, the results are compatible with clonal divergence and selection of different cell subpopulations derived from a common progenitor cell. However, in one patient different alleles in two markers at chromosome 9 were deleted, favoring an independent evolution of two recurring tumor cell clones. In summary, we could show that there is considerable genetic heterogeneity in early multifocal and recurring urothelial carcinoma and demonstrated the occurrence of two independent clones in at least one patient as an indicator of possible initial oligoclonality of bladder cancer.

Aged↗

PubMed1999

Use of a mechanical dissociation device to improve standardization of flow cytometric cytokeratin DNA measurements of colon carcinomas.

In order to standardize dual-fluorescence DNA flow cytometry using cytokeratin (CK) antibodies, normal colonic mucosa and tumor tissue were sampled from 308 colorectal surgical specimens. Fresh colon specimens were processed directly and stored frozen until dissociation. The samples were divided into aliquots for manual dissociation with tweezers and scalpel, and parallel dissociation with an automated disaggregation device (Medimachine, DAKO Diagnostika GmbH, Hamburg, Germany). An indirect immunofluorescence method with anti-cytokeratin antibodies and propidiumiodide was applied and measured on a single-laser flow cytometer (FACScan, Becton Dickinson [BDI], Heidelberg, Germany). Evaluation with CellFit (BDI) or MultiPlus (Phoenix Flow Systems, San Diego, CA) showed that dual-parameter fluorescence propidiumiodide (DNA staining) and fluorescein-isothiocyanate (cytokeratin labeling) provides a reasonable staining method for DNA analysis of epithelial cells. No significant differences in coefficient of variation in CK-gated versus ungated cells could be observed. Normal colon mucosa served as a reliable internal, diploid DNA control. Medimachine dissociation led to a significantly higher gain of cytokeratin-positive cells compared to percentage of cytokeratin-positive cells after manual tissue disaggregation. Cytokeratin gating led to a clear-cut separation of S-phase fractions within the respective ploidy groups, irrespective of manual or automated dissociation. The S-phase fraction increased significantly from normal tissue to diploid and nondiploid tumors. In general, automated tissue preparation with the Medimachine allows simple cell-isolation for dual DNA/CK-flow cytometric measurement, improving the gain of CK-positive cells, and facilitating a standardized DNA analysis.

Carcinoma↗

PubMed1999

Ossifying lipoma.

Lipomas are very common, but osseous changes within these tumours are rare. A lipoma with osseous components is presented, with an overview of the literature and pathogenesis of this unusual lesion and considerations relating to the differential diagnosis.

Aged↗

PubMed1999

Frequent genetic alterations in simple urothelial hyperplasias of the bladder in patients with papillary urothelial carcinoma.

In order to understand the origin of bladder cancer, very early urothelial lesions must be investigated in addition to more advanced tumors. Tissue from 31 biopsies of 12 patients with urothelial hyperplasias and simultaneous or consecutive superficial papillary tumors were used to microdissect urothelium from 15- microm sections of biopsies. The biopsies were obtained with the recently developed highly sensitive diagnostic method of 5-aminolevulinic acid-induced fluorescence endoscopy (AFE). Besides flat and papillary urothelial neoplasms, the method of photodynamic diagnostics also detects simple urothelial hyperplasias as fluorescent positive lesions. In addition, 12 fluorescence-positive biopsies showing histologically normal urothelium were investigated. Fluorescence in situ hybridization was done using a dual color staining technique of biotinylated centromeric probes of chromosomes 9 and 17 and digoxigenin-labeled gene-specific P1 probes for chromosomes 9q22 (FACC), 9p21(p16/CDKI2), and 17p13(p53). Ten of 14 hyperplasias (70%) showed deletions of chromosome 9. In 7 out of 8 patients with genetic alterations in the hyperplasias the genetic change was also present in the papillary tumor. Six out of 12 samples of microdissected normal urothelium also showed genetic alterations on chromosome 9. Microdissection of urothelial lesions, obtained during AFE, has led to the first unequivocal documentation of genetic changes in urothelial lesions diagnosed as normal in histopathology. Thus, this technical approach is important to provide insight into the earliest molecular alterations in bladder carcinogenesis.

Adult↗

PubMed1998

Evaluation of flow-cytometric three-parameter analysis for EGFR quantification and DNA assessment in human bladder carcinomas.

Flow-cytometric multi-parameter staining is an excellent method for defining tumour subpopulations. This provides further understanding of tumour heterogeneity and defines the biological relevance of tumour subpopulations. A method of quantifying the epidermal growth factor receptor (EGFR) in parallel with DNA staining, which was previously established in bladder carcinoma cell lines, was applied to twenty-five biopsies of urothelium and urothelial neoplasms. Uro5, a surface glycoprotein, was used to identify urothelial cells. Objective quantification of receptor content via flow cytometry was achieved with beads of defined numbers of antigen-binding sites, and receptor numbers obtained from urothelial and nonurothelial cells were compared with staining intensity in a three-step immunoperoxidase detection of the EGFR. The data obtained matched the immunohistochemical findings and were more sensitive in the low range (ca. 5x103) of receptors. Parallel definition of the proliferative fraction and DNA-ploidy of tumour cells means that this method satisfies the requirements of objective quantification for oncological diagnosis.

DNA, Neoplasm↗

PubMed1997

Poorly differentiated colonic adenocarcinoma, medullary type: clinical, phenotypic, and molecular characteristics.

Clinicopathological evidence has accumulated that colorectal adenocarcinoma with minimal or no glandular differentiation constitutes two entities with different prognosis. In a series of 20 predominantly nonglandular, poorly differentiated adenocarcinomas, histological features, DNA content, p53 protein expression, Ki-ras mutation, and microsatellite instability were analyzed and correlated to the biology of the tumors. In addition, the presence of Epstein-Barr virus (EBV) transcripts was tested by RNA in situ hybridization and EBV DNA was demonstrated by nested polymerase chain reaction. Histologically, 13 tumors showed small uniform cells and 7 tumors showed large pleomorphic cells. Tumors with uniform cells exhibited more commonly an expansive growth pattern (69.2% versus 0%; P < 0.025) and a dense peritumor lymphoid infiltrate (84.6% versus 14.3%; P < 0.01) resembling their gastric counterpart, solid or medullary carcinoma. These tumors showed less frequent lymph node as well as hematogeneous metastases than pleomorphic carcinomas. In addition, they were usually diploid (84.6% versus 28.6%; P < 0.05) and lacked stabilization of the p53 protein (0% versus 42.9%; P < 0.05). No significant difference between the medullary and the pleomorphic tumor type was found with respect to bcl2 expression and the occurrence of Ki-ras mutations at codon 12. In contrast, microsatellite instability was almost totally restricted to poorly differentiated adenocarcinomas of the medullary type (100% versus 14.3%; P < 0.001). Finally, polymerase chain reaction revealed EBV DNA in 5 tumor specimens, which was, however, restricted to the peritumor lymphoid infiltrate as shown by in situ hybridization. Correlation with the biology of the tumors revealed that only one patient with the uniform cell type died due to metastastic disease during the follow-up period (median, 31 months), which was the case in five of the seven patients with the pleomorphic-type carcinoma (P < 0.025). Our results clearly indicate that the poorly differentiated colonic carcinoma with minimal or no glandular structures constitute two different entities, a medullary and a pleomorphic variant, which markedly differ in their phenotype, genotype, and prognosis.

Adult↗

PubMed1996

Side effects of high-energy shockwaves in the human kidney: first experience with model comparing two shockwave sources.

The side effects of high-energy shockwaves (HESW) from two different sources on kidney parenchyma obtained from 10 patients treated by radical nephrectomy for renal cell carcinoma were examined. Immediately after nephrectomy, the kidneys were perfused with cold HTK solution and kept in hypothermia (8 degrees C) for a maximum of 4 hours. In five cases, the tumor-free parenchyma was treated at the upper or lower renal pole with 2000 shocks, energy output 21 kV, in an experimental electromagnetic shockwave system (Siemens Co., Erlangen). In the other five cases, the upper or lower poles were treated with 2000 shocks, energy output 24 kV, in an electrohydraulic spark gap system (MFL 5000; Dornier Medizintechnik, Germering). The resulting tissue defects were analyzed by histologic examinations. Changes after treatment with the electromagnetic system were found mainly in the tubules and midsized blood vessels in a well-defined focal area. Treatment with the electrohydraulic system was followed by tubular and glomerular lesions combined with vessel defects in a patchy pattern. The model is able to define the side effects of HESW in the human kidney and to test the side effects of different lithotripters.

Aged↗

PubMed1995

Tumor cells in blood shed from the surgical field.

OBJECTIVES: To analyze blood shed from the surgical field during oncologic surgery for tumor cells and to assess functional characteristics of these cells. DESIGN AND PATIENTS: Series of 61 patients with cancer who underwent surgery for an abdominal, orthopedic, urological, gynecological, or head and neck malignant tumor, and blinded comparison with 15 patients with benign diseases undergoing surgery. SETTING: A 500-bed tumor center and a tertiary care hospital. MAIN OUTCOME MEASURES: Tumor cells were isolated from intraoperatively salvaged and washed blood by density gradient centrifugation. They were identified in cytospin specimens by their content of cytokeratins and nucleolar organizer regions with a sensitivity of 10 cells in 500 mL of blood. Clonogenicity was tested in a cell colony assay; invasiveness, in Boyden chambers; and tumorigenicity, in nude mice. RESULTS: In 57 of 61 patients, tumor cells were detected in the blood shed during oncologic surgery. They demonstrated proliferation capacity, invasiveness, and tumorigenicity. The total number of tumor cells identified ranged from 1 x 10(1) to 7 x 10(6), with no close correlation to the amount of blood loss. Circulating tumor cells were demonstrated in only 26% of these patients and in small numbers. CONCLUSIONS: Malignant cells identified regularly in the blood shed during tumor surgery and different from circulating tumor cells are of concern, since at the surgical site they may cause local tumor recurrence, or in the salvaged blood they may cause hematogenic metastasis after retransfusion. Therefore, the contraindication of intraoperative autotransfusion in tumor surgery is strongly supported, and a review of surgical procedures and adjuvant therapy may be indicated, as the passage of the identified cells to the shed blood is yet unknown.

Blood Loss, Surgical↗

PubMed1995

Microsatellite instability: new aspects in the carcinogenesis of colorectal carcinoma.

Very recently a new molecular mechanism in the tumorigenesis of colorectal carcinoma has been described which is closely linked to hereditary non-polyposis colonic cancer (HNPCC). Ubiquitous changes in the length of simple repetitive DNA sequences between constitutional and tumour DNA occur in about 90% of cases of HNPCC and in about 15% of cases of non-familial, sporadic colorectal carcinoma. Such microsatellite instabilities have been shown to be the phenotypical marker of mutations in the human homologues of prokaryotic mismatch repair genes (MutS, MutL, MutH). These data provide crucial new tools in the detection of patients at high risk of developing colon cancer and other HNPCC-related carcinomas. In addition, these developments provide new insights into a new, presumably primary event in oncogenesis, i.e. the occurrence of mutations in genomic stability genes leading to an increased cellular mutation rate ("mutator phenotype") and thus to cancer.

Carcinogens↗

PubMed1995

Mechanisms of shockwave action in the human kidney.

The effects on the human kidney parenchyma of high-energy shockwaves (HESW) with different energy densities were examined. Kidneys of patients treated by radical nephrectomy for renal cell carcinoma were perfused with cold HTK solution immediately after nephrectomy and kept in hypothermia (8 degrees C) for a maximum of 4 hours. The tumor-free parenchyma was treated with 2000 shocks at energy outputs of 15 kV (16 MPa, 0.15 mJ/mm2), 17 kV (32 MPa, 0.25 mJ/mm2), 19 kV (50 MPa, 0.4 mJ/mm2), and 21 kV (65 MPa, 0.6 mJ/mm2) in an experimental electromagnetic shockwave system (Siemens Co., Erlanger, Germany). Resulting tissue effects were analyzed by histologic and immunohistochemical examinations and confocal laser scanning microscopy. Different sensitivities of cell components, blood vessels, and tubules were found. Laser scanning microscopy revealed nuclear alterations in the vicinity of the focus up to a distance of approximately 10 mm. Severe histologic changes were found in a smaller zone, while immunohistochemistry studies revealed negative collagen IV staining in an area of approximately 4 x 4 mm (all distances measured within the plane perpendicular to the acoustic axis). From these results, it can be concluded that HESW directly damage the tubules and the vascular system, which might explain the clinical changes after extracorporeal shockwave lithotripsy in human patients. The extent of these effects seems to be dependent on the applied energy.

Carcinoma, Renal Cell↗

PubMed1995

Treatment of human renal cell carcinoma with high-energy shock waves--a new in vivo/in vitro model.

The effects of high-energy shock waves (HESW) on the human renal cell carcinoma were examined. The kidneys were available from 32 patients treated by radical nephrectomy due to renal cell carcinoma. Immediately after nephrectomy the kidneys were perfused with cold HTK solution and stored for a maximum of 4 h in hypothermia at 8 degrees C. The tumors were treated with 4,000 shocks (65 mPa = 0.6 mJ/mm2) in an electromagnetic lithotriptor (Siemens Co., Erlangen, Germany). Microscopic and immunohistological examinations of the tumors were performed after treatment, and cell proliferation rates of treated and untreated specimens were analyzed by cell cultures in 10 cases. HESW induce severe microscopic damage in the tumor tissue as complete rupture of the vessel walls and destruction of the tubular-formed tumor masses in the focal area. Immunohistochemistry shows intact immune reactive endothelial cells by factor 8-associated antibodies until the border to histological damage. Around this region a zone of negative antibody reaction against collagen type 4 is found. In cell cultures the proliferation rates of treated specimens were significantly lower compared to untreated. The human renal cell carcinoma seems to be susceptible for treatment with shock waves. HESW induce direct damage of tumor cells and vascular damage in the tumor which may be the primary cause of tumor necrosis.

Aged↗

PubMed1995

In vitro and ex vivo expression of nucleolar proteins B23 and p120 in benign and malignant epithelial lesions of the prostate.

The expression of two specific nucleolar antigens, p120 and B23, has been investigated in the prostatic carcinoma cell line LNCaP as well as in 40 frozen and 40 formalin-fixed tissue samples of benign and malignant prostatic lesions (15 benign hyperplasias, 5 grade 1, 15 grade 2, and 5 grade 3 carcinomas). In vitro, immunoreactivity of p120 was confined to nucleoli of proliferating cells, with virtually no negative staining during S and G2/M phases. Unlike p120, B23 was expressed in the nucleoli of all LNCaP cells independently of growth and cell cycle phases. Hence, B23 was detectable in all stromal as well as in normal and malignant epithelial prostatic cells, both in fresh and in formalin-fixed tissue sections after microwave treatment. In contrast, the immunoreactivity of p120 was almost completely restricted to the nucleoli of prostate carcinoma cells: frozen sections of benign prostatic hyperplasia (n = 15) were either totally negative for p120 (n = 13) or had a low percentage of positively stained cells (labeling index = 3.3% in 3 cases). In the carcinoma group 76% (19/25) of the specimens were p120 positive, and there was a significant rise of labeling index from 18.1% in grade 1 to 82.2% in grade 3 carcinomas (P < 0.001). In contrast to B23, p120 could not be reliably demonstrated in formalin-fixed and paraffin-embedded tissue. We therefore conclude that anti-B23 is a general marker of nucleoli, whereas expression of p120 appears to correlate with "hyperactivity" of the nucleolus and provides a new tool for flow cytometrical and immunohistochemical assessment of nucleolar activity in tumor pathology.

Adenocarcinoma↗

PubMed1994

Flow cytometric detection and quantitation of the epidermal growth factor receptor in comparison to Scatchard analysis in human bladder carcinoma cell lines.

The epidermal growth factor receptor (EGFR) is considered a tumor-related marker with potential diagnostic and prognostic value. In order to assess the sensitivity of flow cytometry to detect EGFR and to quantify receptors objectively, two human bladder carcinoma cell lines with different urothelial differentiation, RT4 and J82, were grown in vitro, and their membrane EGFR content was measured by flow cytometry. Exponential monolayers showed decrease of EGFR content after 20 min pulses with 10 ng/ml EGF in medium, as detected with the antibody EGFR1 in a double staining technique with propidium iodide for DNA evaluation. Further decrease of green fluorescence intensity was seen in cells constantly exposed to EGF. Absolute receptor numbers were determined by Scatchard analysis with radioactive EGF and resulted in relatively low receptor numbers for both cell lines (approximately 3-4 x 10(4) EGFR/cell), as well as one affinity class. These findings could be matched by absolute receptor quantification by flow cytometry, adding beads with defined antigenic sites (Quantum Simply Cellular, Microbead Corporation) to the cell suspension for staining. Our data suggest that flow cytometric EGFR detection and quantitation may be supplied to in vivo tumor samples and that measurements by multiparameter analysis may define subpopulations valuable for tumor diagnosis and judgment on tumor progression.

Biomarkers, Tumor↗

PubMed1994

The genomic structure of the human AP-2 transcription factor.

The transcription factor AP-2 is encoded by a gene located on chromosome 6 near the HLA locus. Here we describe the genomic organization of the AP-2 gene including an initial characterization of the promoter. We have mapped two mRNA initiation sites, the entire exon-intron structure and located two polyadenylation sites. The mature AP-2 mRNA is spliced from 7 exons distributed over a region of 18 kb genomic DNA. A recently cloned inhibitory AP-2 protein is generated by alternative usage of a C-terminal exon. The proline-rich transactivation motif is encoded by a single exon within the N-terminal region in contrast to the complex DNA binding and dimerization motif which involves amino acid residues located on four different exons. The sites of mRNA initiation are located 220 and 271 bases upstream from the ATG translation start site. Although the promoter contains no canonical sequence motifs for basal transcription factors, such as TATA-, CCAAT- or SP-1 boxes, it mediates cell-type-specific expression of a CAT reporter gene in PA-1 human teratocarcinoma cells and is inactive in murine F9 teratocarcinoma cells. We demonstrate that the promoter of the AP-2 gene is subject to positive autoregulation by its own gene product. A consensus AP-2 binding site is located at position -622 with respect to the ATG. This site binds specifically to bacterially expressed AP-2 as well as to multiple proteins, including AP-2, present in PA-1 and HeLa cell nuclear extracts. A partial AP-2 promoter fragment including the AP-2 consensus binding site is approximately 5-fold transactivated by cotransfection of an AP-2 expression plasmid.

Animals↗

PubMed1994

Proliferation of tumor spheroids after shock-wave treatment.

Multicellular tumor spheroids (MCTS) grown from the bladder cancer cell line RT112 and from the prostate cancer cell line PCA were exposed to 200 or 800 electromagnetically generated focused ultrasound shock waves. RT112 cells showed a distinct but transient decrease in proliferation whereas the effect of PCA cells was less pronounced. Flow-cytometric measurements of DNA content and Ki67 expression revealed no significant changes in the cell cycle distribution. The capacity of RT112 cells exposed to 800 shock waves to re-grow as MCTS was markedly decreased, indicating an alteration of intercellular adhesion.

Cell Division↗

PubMed1994

[Molecular cloning of a new AP-2 transcription factor, AP-2beta, and its function in cell differentiation].

Transcription factor AP-2 has been previously shown to play an important function in embryonal development and cell differentiation. We have investigated the possibility that AP-2 function in embryonic development is exerted by a multigene family of AP-2 related transcription factors. Here we describe the molecular cloning of such an AP-2 related gene, AP-2 beta, and prove that it encodes for a functional transcription factor. In situ hybridizations of murine embryo sections revealed a temporally restricted and tissue-specific expression pattern that indicates a function of AP-2 beta in the development of the midbrain in the differentiation of sensory neurons for taste, olfaction and palpation.

Animals↗

PubMed1993

Cloning and characterization of a fourth human somatostatin receptor.

We have isolated a gene coding for a fourth human somatostatin (somatotropin release-inhibiting factor) receptor. This additional somatostatin receptor (hSSTR4) is specifically expressed in human fetal and adult brain and lung tissue. The deduced amino acid sequence of the receptor displays both sequence and structural homology to three cloned somatostatin receptors as well as to other members of the family of GTP-binding-protein-coupled seven-helix transmembrane-spanning receptors. Pharmacological characterization of the expressed receptor reveals specific, high-affinity binding of somatostatin 14 and somatostatin 28. Surprisingly, several well-characterized synthetic somatostatin analogs fail to exhibit high-affinity binding to hSSTR4, indicating the existence of pharmacologically different receptor subtypes. Our data suggest that the diverse biological effects exerted by somatostatin are mediated by a family of receptors with discrete patterns of expression and different pharmacological properties.

Amino Acid Sequence↗

PubMed1993

Effects of high-energy shock waves on the viable human kidney.

Between September 1990 and July 1991, we treated 17 patients with renal-cell carcinoma by radical nephrectomy and two patients with urothelial carcinoma of the kidney pelvis by ureteronephrectomy. Immediately after nephrectomy, perfusion of the kidneys with cold HTK solution was performed and the organs were kept in hypothermia of 8 degrees C. The tumor-free parenchyma of the kidneys was treated 4 h later with shock waves of different energy levels in an experimental shock-wave system (Siemens Company, Erlangen). Light microscopy and examinations by scanning laser microscopy were performed after treatment. High-energy shock waves (HESW) produce significant changes in the tubulary and blood-vessel system of the viable human kidney, depending on the energy applied. Although our model is limited by hypothermia of the explanted kidneys, the effects of shock waves on the organs can be studied. Our model is suitable for testing the effects of different lithotriptors on the human kidney.

Aged↗