Immunohistochemical and molecular characterizations in urothelial carcinoma of bladder in patients less than 45 years

Bladder tumours in early-onset patients are rare and seem to exhibit unique clinicopathological features. Only few studies have investigated somatic alterations in this specific age of onset group and evidence is accumulating of a distinct molecular behaviour of early-onset bladder tumours. We collected the largest cohort of early-onset tumours of patients 45 years old or younger and aimed to test genomic alterations typically found in bladder cancer. Tumours of 118 early-onset patients were compared with a consecutive group of 113 cases. Immunohistochemistry of TP53, CK20 and Ki-67 was carried out. Molecular analysis was conducted to test for loss of heterozygosity of chromosome 9 and 17, as well as TP53 and FGFR3 mutations. Fisheŕs exact and chi-squared test were appropriately used. No differences in grade/stage characteristics were observed. Overexpressed TP53 was differentially distributed between the two groups. TP53 nuclear accumulation was significantly more frequent in early-onset papillomas, PUNLMPs and pTa low-grade tumours compared to the consecutive cohort (p=0.005). Moreover, chromosome 9 deletions (29.5% vs. 44.6%) and FGFR3 mutations (34.5% vs. 63.7%) were less often detected in early-onset patients (p=0.05 and p<0.0001). By comparing the largest cohort of early-onset bladder cancer patients with an unselected group, we demonstrated that the typical molecular features are not independent of age at diagnosis. Our study supports the hypothesis of a distinct biological behaviour in early-onset tumours

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

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

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

Effects of lithotripsy on rat kidney: evaluation with MR imaging, histology, and electron microscopy

Magnetic resonance (MR) imaging at 1.5 T was used to evaluate the effects of extracorporeal shock wave lithotripsy (ESWL) in 30 rats and the findings on T1- and T2-weighted (spin echo 600/22, 1,600-2,000/90) images were compared with histology and scanning microscopy. The observed pathologic changes increased in severity with the number of shock waves given (500-5,000 15 kV). Post-ESWL MR findings in 54 kidneys included perirenal and subcapsular fluid (n = 30), diffuse loss of corticomedullary junction definition (n = 28), intrarenal foci of increased (n = 7) or decreased (n = 6) signal intensity, focal indentation of the renal contour (n = 5), and loss of distinction between the renal, splenic, or hepatic contour (n = 7). The subcapsular and intrarenal findings corresponded pathologically to areas of hemorrhage and hematoma formation--the contour changes to foci of renal scarring or perirenal adhesions. Electron microscopy demonstrated marked alterations of the renal tubules and vasculature. The study shows the feasibility of assessing the nature and chronology of renal damage post-ESWL in a rat model by MR

NAD(P)H:Quinone Oxidoreductase 1 (NQO1) P187S Polymorphism and Prostate Cancer Risk in Caucasians

NAD(P)H:quinone oxidoreductase 1 (NQO1) catalyses the reduction of quinoid compounds to hydroquinones, preventing the generation of free radicals and reactive oxygen. A “C” to “T” transversion at position 609 of NQO1, leading to a nonsynonymous amino acid change (Pro187Ser, P187S), results in an altered enzyme activity. No NQO1 protein activity was detected in NQO1 609TT genotype, and low to intermediate activity was detected in NQO1 609CT genotype compared with 609CC genotype. Thus, this polymorphism may result in altered cancer predisposition. For prostate cancer, only sparse data are available. We therefore analyzed the distribution of the NQO1 P187S SNP (single nucleotide polymorphism) in prostate cancer patients and a healthy control group. Allelic variants were determined using RFLP analysis. Overall, 232 patients without any malignancy and 119 consecutive prostate cancer patients were investigated. The genotype distribution in our cohorts followed the Hardy–Weinberg equilibrium in cases and controls. The distribution of the NQO1 codon 187 SNP did not differ significantly between prostate cancer patients and the control group (p = 0.242). There was also no association between the allelic variants and stage or Gleason score of the tumors. The NQO1 P187S SNP was not significantly associated with an increased prostate cancer risk in our cohorts. The SNP has also no influence on histopathological characteristics of the tumors. A combined analysis of all available data from published European studies also showed no significant differences in the genotype distribution between controls and prostate cancer patients. Our data suggest a minor role of the NQO1 nucleotide 609 polymorphism in prostate carcinogenesis

NAD(P)H:Quinone Oxidoreductase 1 (&lt;em&gt;NQO1&lt;/em&gt;) P187S Polymorphism and Prostate Cancer Risk in Caucasians

NAD(P)H:quinone oxidoreductase 1 (NQO1) catalyses the reduction of quinoid compounds to hydroquinones, preventing the generation of free radicals and reactive oxygen. A “C” to “T” transversion at position 609 of &lt;em&gt;NQO1&lt;/em&gt;, leading to a nonsynonymous amino acid change (Pro187Ser, P187S), results in an altered enzyme activity. No NQO1 protein activity was detected in &lt;em&gt;NQO1 &lt;sup&gt;609&lt;/sup&gt;TT&lt;/em&gt; genotype, and low to intermediate activity was detected in &lt;em&gt;NQO1 &lt;sup&gt;609&lt;/sup&gt;CT&lt;/em&gt; genotype compared with &lt;em&gt;&lt;sup&gt;609&lt;/sup&gt;CC&lt;/em&gt; genotype. Thus, this polymorphism may result in altered cancer predisposition. For prostate cancer, only sparse data are available. We therefore analyzed the distribution of the &lt;em&gt;NQO1&lt;/em&gt; &lt;em&gt;P187S&lt;/em&gt; SNP (single nucleotide polymorphism) in prostate cancer patients and a healthy control group. Allelic variants were determined using RFLP analysis. Overall, 232 patients without any malignancy and 119 consecutive prostate cancer patients were investigated. The genotype distribution in our cohorts followed the Hardy–Weinberg equilibrium in cases and controls. The distribution of the &lt;em&gt;NQO1&lt;/em&gt; codon 187 SNP did not differ significantly between prostate cancer patients and the control group (&lt;em&gt;p&lt;/em&gt; = 0.242). There was also no association between the allelic variants and stage or Gleason score of the tumors. The &lt;em&gt;NQO1&lt;/em&gt; &lt;em&gt;P187S&lt;/em&gt; SNP was not significantly associated with an increased prostate cancer risk in our cohorts. The SNP has also no influence on histopathological characteristics of the tumors. A combined analysis of all available data from published European studies also showed no significant differences in the genotype distribution between controls and prostate cancer patients. Our data suggest a minor role of the &lt;em&gt;NQO1&lt;/em&gt; nucleotide 609 polymorphism in prostate carcinogenesis

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

Multiple intracranial metastases from a prolactin secreting pituitary tumour

A 28 year old man presented with partial hypopituitarism and signs of a pituitary tumour. A chromophobe adenoma was partially removed by right frontal craniotomy. Seven years later complete hypopituitarism and hyperprolactinaemia were documented, at which time there was no evidence of tumour recurrence of CT scan. The patient was treated with bromocriptine but the pituitary tumour redeveloped a year later. Nine years after the original operation the first metastasis was demonstrated together with very high prolactin levels. The intracranial metastasis, and the pituitary tumour were removed at a second craniotomy following which the prolactin concentration fell. Further metastases developed subsequently and the patient died 12 years after the initial diagnosis. At autopsy multiple metastases were found in the brain, tumour cells were present in the subarachnoid space and in cerebral veins. The pituitary tumour and secondaries were shown by immunocytochemistry to contain prolactin but not ACTH or growth hormone. This appears to be the third well documented case of a metastasizing, prolactin secreting pituitary tumour

Characterization of the new bladder cancer cell line HOK-1: expression of transitional, squamous and glandular differentiation patterns

The new continuous cell line HOK-1 derived from a grade-III transitional-cell bladder carcinoma with foci of squamous and glandular differentiation was shown to retain this phenotypical heterogeneity for more than 45 passages in vitro. Electron microscopy revealed transitional as well as a considerable proportion of squamous carcinoma and adenocarcinoma cells. PAS-positive mucus was detected in numerous cells. These features were principally maintained when grown as multicellular spheroids and in nude mice. More pronounced signs of differentiation (i.e., expression of cytokeratins 10 and 11, formation of glandular structures) were found in xenograft tumours. Independently, cytokeratins 13, 18 and 19 were detected in vitro and in vivo, reflecting the urothelial origin. The line forms distinct colonies in soft agar, expresses Lewis-x and Lewis-y antigens and reacts with monoclonal antibodies (MAbs) against CEA, beta-HCG and URO-5. Cytogenetic analysis revealed several related clones with a rearrangement at chromosome 1 and loss of one X chromosome as common karyotypic changes in all clones. DNA content, as quantified by image analysis, showed a DNA stemline close to 2c. The new cell line HOK-1 can be used as an in vitro model to study the mechanisms of heterogeneous differentiation patterns in bladder cancer