Ferdinand Hofstädter

Detection of Early Bladder Cancer by 5-Aminolevulinic Acid Induced Porphyrin Fluorescence

PURPOSE We determined whether the sensitivity of detecting dysplasia or early bladder cancer can be improved by 5-aminolevulinic acid induced porphyrin fluorescence. MATERIALS AND METHODS A 3% 5-aminolevulinic acid solution was instilled intravesically before cystoscopy in 104 patients. The 5-aminolevulinic acid induced porphyrin fluorescence was excited by violet light from a krypton ion laser (wavelength 406.7 nm.). RESULTS The sensitivity of the fluorescence cystoscopy (96.9%) was significantly (p < 0.0001) greater than that of white light cystoscopy (72.7%). There was no impact on specificity. CONCLUSIONS Due to the high sensitivity of the procedure fluorescence guided biopsies are recommended instead of random biopsies.

Multiple Mutation Analyses in Single Tumor Cells with Improved Whole Genome Amplification

Combining whole genome amplification (WGA) methods with novel laser-based microdissection techniques has made it possible to exploit recent progress in molecular knowledge of cancer development and progression. However, WGA of one or a few cells has not yet been optimized and systematically evaluated for samples routinely processed in tumor pathology. We therefore studied the value of established WGA protocols in comparison to an improved PEP (I-PEP) PCR method in defined numbers of flow-sorted and microdissected tumor cells obtained both from frozen as well as formalin-fixed and paraffin-embedded tissue sections. In addition, the feasibility of I-PEP-PCR for mutation analysis was tested using clusters of 50-100 unfixed tumor cells obtained by touch preparation of ten breast carcinomas by conventional sequencing of exon 7 and 8 of the p53 gene. Finally, immunocytochemically stained microdissected single disseminated tumor cells from bone marrow aspirates were investigated with respect to mutations in codon 12 of Ki-ras by restriction fragment length polymorphism (RFLP)-PCR after I-PEP-PCR. The modified I-PEP-PCR protocol was superior to the original PEP-PCR and DOP-PCR protocols concerning amplification of DNA from one cell (efficiency rate I-PEP-PCR 40% versus PEP-PCR 15% and DOP-PCR 30%) and five cells (efficiency rate I-PEP-PCR 100% versus PEP-PCR 33% and DOP-PCR 20%). Preamplification by I-PEP allowed 100% sequence accuracy in > 4000 sequenced base pairs and Ki-ras mutation detection in isolated single disseminated tumor cells. For reliable microsatellite analysis of I-PEP-preamplified DNA, at least 10 unfixed cells from fluorescence-activated cell sorting, 10 cells from frozen tissue, or at least 30 cells from formalin-fixed and paraffin-embedded tissue sections were required. Thus, I-PEP-PCR allowed multiple reliable microsatellite analyses suited for microsatellite instability and losses of heterozygosity and mutation analysis even at the single cell level, rendering this technique a powerful new tool for molecular analyses in diagnostic and experimental tumor pathology.

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.

Stage T1, Grade 3 Transitional Cell Carcinoma of the Bladder: An Unfavorable Tumor?

Transurethral resection only was performed in 172 patients with initial stage Ta, T1 transitional cell carcinoma of the bladder. Additional treatment during the course of disease was given to 9 patients with carcinoma in situ and to 8 patients with tumor progression. The mean followup was 106 months. The 10-year survival rates were 95 per cent for patients with stage Ta, grade 1 disease, 89 per cent for stage Ta, grade 2, 84 per cent for stage Ta, grade 3, 78 per cent for stage T1, grade 2 and 50 per cent for stage T1, grade 3. The percentage of first tumor recurrence at the same site increased with tumor grade (stage T1, grade 3 was 74 per cent). The recurrence rate in stage T1, grade 3 tumors (4.08) differed significantly from the other groups of superficial tumors. The tumor progression rate for stage T1, grade 3 tumors (32.5 per cent) was significantly higher as well. The characteristics of stage T1, grade 3 tumors with and without progression were different in regard to multiplicity, recurrence rate, mean interval to recurrence and type of tumor invasion. Of the 13 patients who died of progressive neoplastic disease 11 presented initially with stage T1, grade 3 tumors. When these results are considered it is obvious that a patient with a stage T1, grade 3 tumor deserves additional therapy, such as chemotherapy, immunotherapy or phototherapy.

INDOCYANINE GREEN: INTRACELLULAR UPTAKE AND PHOTOTHERAPEUTIC EFFECTS IN VITRO

Indocyanine green (ICG; absorption peak in human plasma 805 nm) was investigated for ICG-mediated phototherapy in vitro. The cellular uptake of ICG (1 microM-50 microM) into HaCaT keratinocytes after an incubation period of 24 h increased up to an intracellular ICG concentration of 12.1 +/- 1.3 nmol per 10(6) cells. To examine dose dependent phototoxic effects in vitro, keratinocytes were incubated with 0 microM-50 microM ICG for 24 h and irradiated by a diode laser (805 nm) with different energy densities (0, 12, 24, 48 J cm-2). All applied ICG concentrations except for 5 microM yielded a cell killing effect in combination with irradiation depending significantly on ICG concentration and light dose. Cell viability for dark control and cells incubated with 50 microM ICG and irradiated with 48 J cm-2 was 0.82 +/- 0.15 and 0.07 +/- 0.02, respectively. Sodium azide (100 mM), a quencher of reactive oxygen species, inhibited significantly the cell killing using 50 microM ICG and 24 J cm-2. Taken together, photoactivation of ICG by irradiation with a diode laser was shown to induce effectively cell killing of HaCaT keratinocytes. Moreover, this effect was inhibited by sodium azide, thus irradiation of ICG might induce a photodynamic reaction.

Global Levels of Histone Modifications Predict Prostate Cancer Recurrence

Epigenetic alterations such as DNA methylation and histone modifications play important roles in carcinogenesis. It was reported that global histone modification patterns are predictors of cancer recurrence in various tumor entities. Our study was performed to evaluate histone lysine (HxKy) and histone acetyl (HxAc) modifications in prostate tissue.

CELLULAR FLUORESCENCE OF THE ENDOGENOUS PHOTOSENSITIZER PROTOPORPHYRIN IX FOLLOWING EXPOSURE TO 5-AMINOLEVULINIC ACID

Abstract— Supplying 5‐aminolevulinic acid (ALA), a precursor in the biosynthetic pathway to heme from an external source leads to an accumulation of the endogenous fluorescent photosensitizer protoporphyrin IX (PPIX). Following instillation of ALA in the urinary bladder neoplastic tissue can be discerned by fluorescence cystoscopy or treated by illumination with light of an appropriate wavelength. In order to provide a biological rationale for the clinical findings, we have analyzed the capacity of three different cell lines to accumulate PPIX by flow cytometry. Three different urothelial cell lines, normal fibroblasts and endothelial cells were exposed to ALA under varying conditions. Urothelial cell lines J82 and RT4, derived from malignancies of the bladder displayed fluorescence intensities 9‐ and 16‐fold, respectively, above the fluorescence level of the normal urothelial cell line HCV29. Human umbilical cord endothelial cells fluoresced moderately while the fibroblast cell line Nl exhibited a fluorescence level comparable to those of the cancer cells. Fluoresence increased with increasing cell density and was also dependent on the growth of cells as monolayers or multicellular spheroids. Increasing ALA concentrations led to saturation of fluorescence after 4 h of incubation at cell type‐specific fluorescence levels obtained at different ALA concentrations. Continuous incubation in medium containing serum resulted in a linear rise of fluorescence during the first 4 h, which was followed by a saturation period (8–24 h) and a renewed rise. In the case of serum depletion, fluorescence intensities were significantly higher and increased linearly during the entire 48 h incubation period. By replacing serum with albumin, it could be shown that the emission of PPIX into the medium in the presence of serum is mainly caused by this protein. The ALA‐induced fluorescence was predominantly perinuclear after 4 h of incubation and relocated toward the cell membrane after prolonged incubation. This study demonstrated the complexity of factors influencing the ALA‐induced fluorescence and should stimulate further research in this field.

Molecular Analysis of Microdissected Tumors and Preneoplastic Intraductal Lesions in Pancreatic Carcinoma

Little or no data exist concerning the inactivation of tumor suppressor genes in intraductal lesions surrounding invasive ductal pancreatic carcinomas. Using a novel improved primer extension and preamplification polymerase chain reaction, we analyzed microdissected paraffin-embedded specimens of pancreatic carcinoma (n = 29) and their corresponding pancreatic intraductal lesions (PIL, n = 331) for loss of heterozygosity (LOH) of p16(INK4), DPC4, and p53 by microsatellite analysis and for p53 protein by immunohistochemistry. LOH at the p16(INK4) locus (9p21) was found in nine of 22 informative tumors (41%), in 15 of 25 tumors (60%) at the DPC4 locus (18q21.1), and in 22 of 27 tumors (81%) at the p53 locus (17p13). Homozygous deletions of p16(INK4) and DPC4 were found in eight of 22 (36%) and four of 25 tumors (16%), respectively. Furthermore, 24 of 29 tumors (83%) revealed considerable intratumoral genetic heterogeneity. In 165 of 277 PILs (60%) having suitable DNA for microsatellite analysis, alterations in at least one tumor suppressor gene were found. In individual PILs, up to three alterations were detected, and p53 LOH occurred even in morphologically normal-appearing ductal epithelium near the tumor. Although deletions of all three tumor suppressor genes were found in PILs without nuclear atypia, there was a tendency toward earlier LOH of p16(INK4) compared to DPC4 and p53 in these lesions. LOH in tumors accompanied positive p53 immunohistochemistry in 81% but only in 38% in PILs.

Indocyanine green (ICG) and laser irradiation induce photooxidation

Abstract The cellular uptake and subcellular localization of indocyanine green (ICG; absorption band 700– 850 nm), and cell survival and ultrastructural changes following ICG-mediated phototherapy were investigated in vitro in four different cell lines derived from human skin (SCL1 and SCL2 squamous cell carcinoma, HaCaT keratinocytes and N1 fibroblasts). The cellular uptake of ICG (1–50 μ M , incubation times 1, 4, 24 h) was saturable, highly cumulative and could be inhibited by the addition of 250 μ M bromosulphophthalein indicating the involvement of the organic anion transporting polypeptide (OATP). For HaCaT cells, the maximum cellular uptake (V max ) and the Michaelis constant (K m ) were 9.9 ± 1.1 m M and 47 ± 16 μ M , respectively, following a 24-h incubation with ICG. Fluorescence microscopy revealed a cytoplasmic distribution of ICG, probably bound to glutathione S -transferase. Following irradiation with a cw-diode laser (805 nm, 80 mW/cm 2 ) at doses of 24 or 48 J/cm 2 , the phototoxicity was determined using the MTT assay as a measure of cell viability. For all cell lines, ICG concentrations above 25 μ M produced a significant phototoxic effect. The EC 50 of ICG for HaCaT cells following irradiation at 24 J/cm 2 was 20.1 ± 3.9 μ M . Growth curves showed that even HaCaT cells treated at the EC 50 were killed within a week following treatment. Electron microscopy 1 h after ICG-mediated phototherapy revealed cytoplasmic vesiculation, dilation of the rough endoplasmic reticulum, the Golgi complex and the perinuclear cisternae and the beginning of chromatin condensation in the nucleus. These ultrastructural findings are not consistent with a photothermal action of ICG-mediated phototherapy. Taken together with those of previous studies by our group these results support photooxidation as a major cell-killing mechanism.

Ischemic-like cholangiopathy with secondary sclerosing cholangitis in critically Ill patients

OBJECTIVES:Sclerosing cholangitis in critically ill patients (SC-CIP) is a newly described entity of severe biliary disease with progression to liver cirrhosis. The mechanisms leading to this form of cholangiopathy with stricture formation and complete obliteration of bile ducts are unknown.PATIENTS AND  METHODS:In the last 2 yr, sclerosing cholangitis was diagnosed in 26 patients during or after their stay on the intensive care unit by ERCP and/or liver histology. Complete patient records were available for 17 patients. Histological evaluations of liver biopsies and of four explanted livers, parameters of cardiovascular and respiratory conditions, treatment modalities, and accompanying infections were analyzed to find further hints for the pathomechanisms leading to SC-CIP.RESULTS:With the beginning of cholestasis, the earliest endoscopic findings were intrahepatic biliary casts with impairment of the biliary flow and subsequent biliary infection, in most cases with Enterococcus faecium. Liver biopsy confirmed cholangitis and histology of explanted livers revealed ulcerated biliary epithelium with hemorrhagic exudates in the bile ducts. In the further course, progressive sclerosis with formation of multiple strictures of the bile ducts was observed. All patients suffered severe respiratory insufficiency with the need for mechanical ventilation (40.7 ± 32.9 days). The PaO2/FiO2 ratio until beginning of cholestasis was 150.5 ± 43.1. Half of the patients (9/17) were treated with high-frequency oscillatory ventilation and 12/17 patients by intermittent prone positioning. All patients required catecholamines for hemodynamic stabilization.CONCLUSIONS:SC-CIP is a severe and in most cases rapidly progressive complication of intensive care patients. Ischemic injury of the biliary tree with the formation of biliary casts and subsequent ongoing biliary infection due to multiresistant bacteria seem to be major pathogenic mechanisms in the development of this new entity of sclerosing cholangitis.