[Video-based teaching in pathology. Experience gained in the last 3 years at the RWTH Aachen University]. / Videobasierte Lehre der Pathologie. Erfahrungen der letzten drei Jahre an der RWTH Aachen.

Modern computer technology provides students with easier access to learning materials. Basic knowledge of pathological findings in organs is essential in medical education. We have produced didactic videos for teaching pathology in a clinical context in addition to regular lectures at the university. Didactic material includes macroscopic and histological findings, as well as cartoons explaining pathophysiology and clinical links. Videos can be downloaded in mv4 format as podcasts to a local hard disk or to an iPhone or iPod via iTunes University and are designed to improve classical medical literature. Analysis over 3 years of server traffic and subjective impressions by the students revealed regular use and high acceptance by users. Didactic material in clinical pathology can be successfully integrated in videos to complement lectures and practical training. Modern teaching methods in pathology make the specialty more understandable and therefore more attractive for students.

Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion.

Nuclear factor-kappa B (NF-kappaB)/Rel transcription factors play an important role in the inducible regulation of a variety of genes involved in the inflammatory and proliferative responses of cells. The present study was designed to elucidate the implication of NF-kappaB/Rel in the pathogenesis of atherosclerosis. Activation of the dimeric NF-kappaB complex is regulated at a posttranslational level and requires the release of the inhibitor protein IkappaB. The newly developed mAb alpha-p65mAb recognizes the IkappaB binding region on the p65 (RelA) DNA binding subunit and therefore selectively reacts with p65 in activated NF-kappaB. Using immunofluorescence and immunohistochemical techniques, activated NF-kappaB was detected in the fibrotic-thickened intima/media and atheromatous areas of the atherosclerotic lesion. Activation of NF-kappaB was identified in smooth muscle cells, macrophages, and endothelial cells. Little or no activated NF-kappaB was detected in vessels lacking atherosclerosis. Electrophoretic mobility shift assays and colocalization of activated NF-kappaB with NF-kappaB target gene expression suggest functional implications for this transcription factor in the atherosclerotic lesion. This study demonstrates the presence of activated NF-kappaB in human atherosclerotic tissue for the first time. Atherosclerosis, characterized by features of chronic inflammation and proliferative processes, may be a paradigm for the involvement of NF-kappaB/Rel in chronic inflammatory disease.

Can an endocytoscope system (ECS) predict histology in neoplastic lesions?

BACKGROUND AND STUDY AIMS: An endocytoscope system (ECS) has recently been developed with the possibility of super-high magnification of gastrointestinal mucosa, thus allowing in vivo imaging of living cells. The aim of the present study was to assess the potential of ECS in the prediction of histology in both normal gastrointestinal mucosa and neoplastic lesions. PATIENTS AND METHODS: In total, 76 patients (57 men, 19 women; age range 37-86 years) with neoplastic lesions in the esophagus, stomach, or colon were enrolled into the study and underwent esophagogastroduodenoscopy or colonoscopy. After staining with 1% methylene blue, the mucosa was examined with the ECS probe (x 450 and x 1100 magnification), and video sequences were recorded on video disk. Biopsies from the examined areas were taken for histology and served as the gold standard. The endocytoscope video sequences were evaluated by two blinded pathologists. Finally the results were compared with those resulting from the evaluation of an experienced endoscopist who was aware of the macroscopic endoscopic pictures and the endocytoscope image results. RESULTS: A total of 25 patients with esophageal lesions, 28 patients with colonic lesions, and 23 patients with gastric lesions were examined. The sensitivity and specificity for the evaluation of the blinded pathologists was 81% and 100%, respectively, in the esophagus, 56% and 89% in the stomach, and 79% and 90% in the colon. If an endoscopist evaluated the endocytoscopic pictures in combination with the macroscopic endoscopic images sensitivity and specificity increased significantly. CONCLUSIONS: First experiences with ECS show good sensitivity rates even by blinded assessment for esophageal and colonic lesions. Sensitivity for neoplastic lesions in the stomach is lower because of gastric mucous secretion. Combining the endoscopic and cytoscopic appearance of the lesion may further enhance the diagnostic value of the method.

[Fluorescence cystoscopy at bladder cancer: present trials]. / Die Fluoreszenzzystoskopie beim Harnblasenkarzinom : Aktueller Stellenwert.

Bladder cancer is a frequent disease and represents the second most common genitourinary neoplasm. Although many aspects of the management of not-muscle-infiltrating bladder cancer are now well established, significant challenges remain, which influence patient outcome. Early detection and treatment of recurrent disease is required to optimize bladder preservation, reduce patient morbidity and increase quality of life and survival. Fluorescence cystoscopy, often referred to as "photodynamic diagnosis" (PDD) with intravesical application of photosensitizing agents has been developed in order to enhance the early detection of bladder cancer. Since March 2005 the hexyl-ALA ester (Hexvix) has been approved for the diagnosis of bladder cancer in 27 EU/EEA countries through the European Mutual Recognition Procedure. There is growing evidence that PDD enhances the detection of bladder cancer, particularly of high-grade flat lesions. Furthermore, transurethral resection of bladder tumor under fluorescence guidance has been shown to reduce the risk of recurrent tumors. Nevertheless, a resulting relatively decreased number of recurrences have still to be verified in prospective randomized trials.

The role of salvage extended lymph node dissection in patients with rising PSA and PET/CT scan detected nodal recurrence of prostate cancer.

BACKGROUND: Non-prostatic bed recurrence of prostate cancer (PCa) is usually treated with androgen deprivation therapy (ADT). We analyzed the impact of salvage extended lymph node dissection (sLND) on cancer control in patients with rising PSA and lymph node (LN) metastases. METHODS: Between 2009 and 2016 we performed sLND in 87 patients with biochemical recurrence (BCR) and positive LNs on 18FEC and 68Ga-PSMA positron emission tomography/X-ray computer tomography (PET/CT) after primary treatment (PT) of PCa. Intra- and postoperative complications according to Clavien-Dindo were assessed and the rates of biochemical response (BR), BCR-free and clinical recurrence (CR)-free survival, as well as time to initiation of systemic treatment were evaluated. RESULTS: Mean age of patients and mean PSA at sLND was 66.7 years (46-80 years) and 2.63 ng ml-1 (1.27-3.75 ng ml-1), respectively. With 87.4% radical prostatectomy (RP) was the most common PT. In all, 57.9% of patients additionally underwent adjuvant/salvage radiation therapy (RT) and 18.4% received ADT before sLND. Complete BR (cBR) was diagnosed in 27.5% of patients and incomplete BR in 40.6%. In total, 62.2% of patients remained without ADT at follow-up. With a median follow-up of 21 months (1-75 months), the cancer-specific mortality rate was 3.7%. The 3-year BCR-free, systemic therapy-free and CR-free survival rates for patients with cBR were 69.3%, 77.0% and 75%, respectively. CONCLUSIONS: sLND can be performed without significant complications and achieves an immediate BR, thus allowing a significant postponement of systemic therapy in selected patients with BCR and nodal recurrence of PCa. Therefore, sLND following 68Ga-PSMA PET/CT should be considered as part of a multimodal diagnostic and treatment concept for selective patients.

[Urine cytology and urine markers. Significance for clinical practice]. / Urinzytologie und Urinmarker. Bedeutung für die Praxis.

Urothelial carcinoma of the bladder is a frequent disease that can be identified timely by screening patients at high risk. Due to the high rate of disease recurrence, frequent follow-up procedures are necessary. For this purpose, cystoscopy is the standard procedure, and supplementary non-invasive procedures such as cytology or tumor marker tests are used. These tests have different advantages and disadvantages in terms of their sensitivities and specificities. Thus, they provide additional information, but are not able to replace cystoscopy as the standard instrument in the diagnosis of bladder cancer.

Evidence for oligoclonality and tumor spread by intraluminal seeding in multifocal urothelial carcinomas of the upper and lower urinary tract.

Multifocality and recurrence of urothelial carcinoma may result from either the field effect of carcinogens leading to oligoclonal tumors or monoclonal tumor spread. Previous molecular studies, favoring the monoclonality hypothesis, are mostly limited to the urinary bladder. We investigated genetic alterations in a total of 94 synchronous or metachronous multifocal tumors from 19 patients with at least one tumor both in the upper and lower urinary tract. Loss of heterozygosity (LOH) was determined using eight markers on chromosome 9 and one marker on 17p13 (p53). Microsatellite instability was investigated at six loci and protein expression of MSH2 and MLH1 was evaluated by immunohistochemistry. In addition, exons 5-9 of the p53 gene were sequenced. Deletions at chromosome 9 were found in 73% of tumors and at 17p13 in 18% of tumors. There was no significant difference in the frequency of LOH in the upper and lower urinary tract. Deletions at 9p21 were significantly correlated with invasive tumor growth. The pattern of deletion revealed monoclonality of all tumors in nine patients. In five patients there were at least two tumor clones with different genetic alterations. In four of these patients the different clones occurred in the bladder and subsequently in the ureter and renal pelvis. All four patients with p53 mutations revealed identical mutations in all tumors. Thus, multifocal urothelial carcinomas are frequently monoclonal, whereas others show oligoclonality, providing molecular evidence for field cancerization. Intraluminal tumor cell seeding appears to be an important mechanism of multifocal occurrence and recurrence of urothelial carcinomas.

Development and characterization of cisplatin-resistant human testicular and bladder tumour cell lines.

Cisplatin-resistant cells were derived in vitro from a human bladder carcinoma cell line (RT112) and a testicular tumour cell line (SuSa) by continuous exposure to increasing concentrations of cisplatin for 14 and 11 months, respectively. Both resistant cell lines had a four-fold level of resistance relative to their parental cell lines, comparing the cisplatin concentration to inhibit colony forming ability by 70%. These levels of resistance were retained in the absence of cisplatin for at least 3 months. In each case, four-fold fewer micronuclei were produced in the resistant lines by the same cisplatin concentrations. Cross-resistance to carboplatin and methotrexate was observed in both resistant cell lines, but neither line was resistant to doxorubicin. Isozyme and DNA analysis with hypervariable probes confirmed the origin of each resistant cell line from its parental line. Population doubling times and intermitotic times were similar in each of the pairs of cell lines. Karyotyping showed that the resistant cell lines had gained and lost marker chromosomes, but there were no changes common to both resistant cell lines.

Detection of differentially expressed genes in human bladder cancer cells using arbitrarily primed PCR of RNA.

The aim of the present study was to detect differentially expressed genes in human bladder cancer cell lines using a non-radioactive RNA fingerprinting technique (arbitrarily primed polymerase chain reaction of RNA, RAP-PCR). The two clonal urothelial cancer cell lines, RT4 and J82, show different growth kinetics upon stimulation with EGF. By RAP-PCR we detected changes in band patterns for J82 cells treated with EGF but not for RT4 cells. Polymorphic fragments were further characterized and sequences from two of these gave a perfect match to the coding sequence of the human tropomyosin gene TM30(pl) and the human MAC30 gene, respectively. In accordance with the results of RAP-PCR downregulation in EGF-stimulated J82 cells could be demonstrated by reverse transcription PCR.

Tissue response of the biliary and digestive system of rabbits after MTBE infusion into the gallbladder.

We evaluated the tissue response of the biliary and digestive system after Methyl-tert-Butyl-Ether (MTBE) gallbladder infusion in 32 rabbits. After laparotomy, MTBE (5-11 ml) was infused into the gallbladder for eight hours. Animals were sacrificed after eight hours or after two months. Control animals received saline solution infusion into the gallbladder. Afterwards the gallbladder, the common bile duct, the liver, the pancreas and the duodenum were examined histologically. All animals receiving MTBE had different degrees of gallbladder necrosis, common bile duct necrosis and necrosis of intrahepatic bile ducts. After two months, scar formation and a hyperplastic cholecystitis were observed. Control animals did not have comparable tissue reactions; only small areas of necrosis in the gallbladder and the common bile duct occurred after eight hours. The gallbladder, common bile duct and liver remained unchanged in those animals which survived two months. Although the results of this animal study cannot be directly transferred to humans, the data suggest that MTBE should be used in gallstone therapy with caution, and that if it is used, a well-controlled follow-up of these patients is necessary.

Tumor-associated fibroblasts (part II): Functional impact on tumor tissue.

The article focuses on the functional impact of tumor-associated fibroblasts (TAF) on its surrounding cells. It intends to cover the recent knowledge on TAF, the phenotype, and expression profile of which have been described in the first part of the review series (Kunz-Schughart and Knuechel, 2002). The present review is subdivided into two main chapters: (1) functional impact of TAF on tumor cells and (2) fibroblast-host cell interactions in tumor tissue. In the first paragraph of chapter (1) about the role of fibroblasts in tumor cell growth and differentiation it is revealed, how strongly cellular interaction is dependent on fibroblast and tumor cell type as well as the spatial ratio between the cells. The variation of cellular behavior depending on quantity of molecules holds also true for the group of ECM molecules, e.g. the balance between MMPs and TIMPs, which provide an interesting therapeutic target in tumor tissue. This is one of the topics addressed in the second paragraph which focuses on tumor cell dissemination. Chapter (2) addresses the relation of TAF to other intra- or peritumoral host cells. The hypoxia-related angiogenesis induction of fibroblasts via growth factor secretion (e.g. VEGF) is considered as important as the immune modulatory properties of fibroblasts on immune cells, such as monocytes/macrophages. These cellular properties can be tested under controlled conditions in three-dimensional heterologous cultures of human cells, providing the chance for systematic modification to assess therapeutic effects in an in vivo like environment.

Tumor-associated fibroblasts (part I): Active stromal participants in tumor development and progression?

Phenotypic and functional characteristics of tumor associated fibroblasts (TAF) in contrast to normal fibroblasts are reviewed in this first synopsis (part I). Terms as tumor stroma, desmo-plasia, TAF, myofibroblast, and fetal-type fibroblast are defined, and experimental systems to study heterologous cell interactions are presented. While we only start to gather information on the genotype of TAF, a broad range of data deals with the expression profile of these cells, covering e.g. ECM and ECM-modulating molecules, growth factors and cytokines. Summarizing the recent state of knowledge indicates that TAF provide sources for tumor diagnosis and therapy, that have to be further defined in an organ-specific approach in terms of the functional impact on the tumor cell and its environment (see part II).

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.

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.

Flow cytometric DNA and phenotype analysis in pathology. A meeting report of a symposium at the annual conference of the German Society of Pathology, Kiel, Germany, 6-9 June 2000.

This meeting report summarizes the presentations of three different groups that are active in the field of flow cytometry (FCM) in relation to diagnosing and classification of proliferative disorders. The report starts with the contribution from Regensburg about the developments in DNA FCM, the progression to dual parameter determinations, and combination of immunophenotyping in combination with DNA. In the second part, the use of FCM for the detection of isolated tumor cells in the peripheral blood from patients with prostate or breast cancer is discussed in a contribution from Münster. In the third part, from Heerlen, the use of multi-parameter FCM on formalin-fixed paraffin-embedded tissues from solid tumors is discussed as a new development and application in routine surgical pathology.

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.

Fluorescence photodetection of neoplastic urothelial lesions following intravesical instillation of 5-aminolevulinic acid.

OBJECTIVES: Tiny papillary tumors and flat urothelial lesions such as dysplasia or carcinoma in situ can easily be missed during routine cystoscopy. Various methods for in vivo detection of fluorescing agents (preferentially localized in malignant tissue) have been developed. Most of them are based on systemically administered synthetic porphyrin compounds and require sensitive detection devices and image processing units for fluorescence visualization. The usefulness of intracellularly accumulated endogenous protoporphyrin IX (PPIX), induced by 5-aminolevulinic acid (ALA), for diagnosis of early bladder cancer and the correlation with cystoscopic, microscopic, and fluorescence findings was investigated. METHODS: ALA was instilled intravesically in 68 patients, followed by fluorescence cystoscopy with violet light from a krypton ion laser that produced fluorescence excitation. There were 299 biopsies obtained from fluorescing and nonfluorescing areas of the bladder. RESULTS: ALA-induced fluorescence could be easily observed with the naked eye during cystoscopy under violet light illumination. All tumor lesions were sharply marked with brightly shining red fluorescence. Correlation of fluorescence and microscopic findings gave a sensitivity of 100% and a specificity of 68.5%. There were 26 malignant or precancerous lesions that were missed during routine cystoscopy but were detected only by ALA-induced fluorescence. CONCLUSIONS: Labeling of urothelial lesions by PPIX fluorescence induced by intravesically instilled ALA seems to be a promising diagnostic procedure for malignant lesions that are difficult to visualize with standard cystoscopy.

5-aminolevulinic acid-induced fluorescence endoscopy applied at secondary transurethral resection after conventional resection of primary superficial bladder tumors.

OBJECTIVES: A prospective investigation was carried out to evaluate the use of 5-aminolevulinic acid (5-ALA)-induced fluorescence diagnosis with secondary transurethral resection (TUR). METHODS: Fifty patients underwent secondary TUR of the former resection area 6 weeks after conventional TUR for superficial bladder carcinoma. 5-ALA-induced fluorescence diagnosis was used in addition to standard white light endoscopy. All former resection areas were biopsied regardless of fluorescence findings. In addition, specific red fluorescent areas were resected, as were suspicious areas seen at white light endoscopy. RESULTS: One hundred thirty areas or tumors were resected. The sensitivity of fluorescence cystoscopy was 77.8% (95% confidence interval 52.4% to 93.6%). Residual tumors were found in the area of the former resection in 7 (14%) of 50 patients; 4 of these 7 were fluorescence negative and 3 were fluorescence positive. In an additional 7 patients (14%), exclusively fluorescing tumors not visible under white light could be detected outside the areas of former resection (n = 5, Stage pTaG1/2; n = 1, Stage pT1G1/2; n = 1, carcinoma in situ). CONCLUSIONS: Despite high sensitivity, fluorescence diagnosis at this early stage of control does not allow us to evaluate sufficiently the granulation tissue of necrotic areas after TUR without biopsy. The main advantage of the 5-ALA-induced fluorescence endoscopy is in the evaluation of untreated urothelium because of the easier detection of tumors not visible by conventional endoscopy.

New 5-aminolevulinic acid esters--efficient protoporphyrin precursors for photodetection and photodynamic therapy.

Photodetection (PD) and photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PPIX) accumulation are approaches to detect and treat dysplasia and early cancer in the gastrointestinal tract and in the urinary bladder. Because ALA-induced PPIX production is limited, we synthesized ALA ester hydrochlorides 3-22 and tested them in two different in vitro models (gastrointestinal tract: HT29-CCD18; urinary bladder: J82-UROTSA). PPIX accumulation after incubation with 0.12 mmol/L for 3 h and PPIX accumulation as a function of different incubation times were measured using flow cytometry. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were performed to check cellular dark toxicity. Phototoxicity after irradiation was tested. ALA nonafluorohexylester hydrochloride 11, ALA thiohexylester hydrochloride 13 and ALA dibenzyldiester dihydrochloride 19 induced appreciably increased PPIX levels and showed improved phototoxicity compared with the references ALA hydrochloride 1, ALA hexylester hydrochloride 3 and ALA benzylester hydrochloride 4. Thus, the new compounds 11, 13 and 19 are promising compounds for PD and PDT.

Cell-type specific protoporphyrin IX metabolism in human bladder cancer in vitro.

5-Aminolevulinic acid (ALA)-supported fluorescence endoscopy of the urinary bladder results in a detection rate of bladder cancer superior to that of white light endoscopy. The different accumulation of the metabolite protoporphyrin IX (PPIX) in tumor cells after ALA instillation is poorly understood; however, it is crucial to optimize diagnosis and potential phototherapy. For systematic analysis of cell-type specific PPIX accumulation and metabolism two human bladder carcinoma cell lines (RT4 and J82), a normal urothelial cell line (UROtsa), and a fibroblast cell line (N1) were chosen, and grown in two different growth states to model important tissue components of the urinary bladder, i.e. tumor, normal epithelium and stroma. To quantitate PPIX content, fluorescence intensities measured by flow cytometry were matched with cellular PPIX extraction values, and related to relative ferrochelatase activity, cellular iron content, number of transferrin receptors per cell and porphobilinogen deaminase (PBGD) activity. For in vitro experiments, the initial correlation of relative flow cytometric and spectrometric measurements of PPIX provides a calibration curve for consequent flow cytometric PPIX quantification. Lower fluorescence of normal cells could be explained by significant differences of ferrochelatase activity and iron content in comparison to tumor cells. However, the content of iron was not related to transferrin receptor content. PBGD activity seemed to play a minor role for the differential accumulation of PPIX in urothelial cells. In conclusion, the in vitro culture of urothelial cells and fibroblasts indicates that the most important metabolic step for PPIX accumulation in the urinary bladder is the transition from PPIX to heme. Further investigation of PPIX metabolism does support the validation of photodynamic diagnosis, and might also lead the way to a highly specific tumor related molecule.