Simultaneous detection of HER2/neu gene amplification and protein overexpression in paraffin-embedded breast cancer.

The determination of HER2/neu status in breast carcinomas has become essential for the selection of breast cancer patients for Herceptin therapy. Herceptin treatment is used in patients with metastatic breast carcinoma with HER2/neu protein overexpression detected by immunohistochemistry (IHC) or gene amplification analysed by fluorescence in situ hybridization (FISH). A multiparametric fluorescent approach based on the simultaneous detection of HER2/neu gene amplification and protein expression was established to increase the accuracy, and to improve the reproducibility, of HER2/neu diagnostics. Based on four paraffin-embedded breast cancer cell lines, a combined fluorescent immunostaining (FIHC) and FISH method was developed by using the PathVysion HER2 DNA Probe Kit (VYSIS) and the polyclonal antibody from the HercepTest (DAKO). Diagnostic applicability was documented on 215 formalin-fixed primary breast carcinomas. Criteria for immunofluorescence quantification were chosen by analogy with the FDA-approved HercepTest scoring, ranging from 0 to 3+. There was 97.7% concordance between conventional IHC and fluorescence IHC. The FISH data resulting from the multiparametric approach did not differ from conventional FISH. Breast carcinomas with HER2/neu protein overexpression and simultaneous gene amplification were detected with 100% sensitivity. In addition, five of the 215 cases (2.3%) had HER2/neu gene amplification without protein overexpression. The main advantage of this novel approach is that polysomy, aneuploidy, gene amplification, and protein content can be analysed simultaneously in the same cell.

Distribution and subcellular localization of a water-soluble hematoporphyrin-platinum(II) complex in human bladder cancer cells.

The water-soluble porphyrin-platinum complex diammine[7,12-bis[1-(polyethyleneglycol-750-monomethylether-1-yl)ethyl]-3,8,13,17-tetramethylporphyrin-2,18-dipropionato]platinum(II) (PEG-HPPt) was studied with respect to cellular accumulation, subcellular localization, behavior in 3D-cell aggregates and degree of DNA platination on the low-differentiated J82 cells, a model of invasive bladder cancer, and UROtsa, a normal urothelial cell line. Accumulation studies with 2D and spheroid cell cultures revealed that the concentration of PEG-HPPt was 1.7-times higher in J82 cancer cells than in UROtsa cells. Despite its high molecular weight, penetration of PEG-HPPt was not restricted to the peripheral cells of the spheroids. Fluorescence microscopic analysis showed that PEG-HPPt was localized in essential cellular targets of photodynamic therapy. DNA platination in J82 and UROtsa cells was higher by PEG-HPPt than by cisplatin, whereas there was no significant difference between the two cell lines.

Hexaminolevulinate-induced fluorescence endoscopy in patients with rectal adenoma and cancer: a pilot study.

BACKGROUND: Fluorescence endoscopy is a promising new method for detection and treatment of premalignant and malignant lesions. The aim of this pilot study was to investigate the feasibility of hexaminolevulinate-based photodetection of rectal adenoma and cancer, including safety, dose finding, and efficacy. METHODS: Ten patients with known rectal adenoma or cancer were sensitized by instillation of 3.2 mM of hexaminolevulinate as an enema. Fluorescence endoscopy was performed after retention of the enema for 30 to 60 minutes, followed by a rest time of up to 30 minutes before endoscopy. Biopsy specimens were taken from fluorescent and non-fluorescent areas and fluorescence microscopy studies were performed to assess the distribution of protoporphyrin IX fluorescence in different tissue layers. Adverse events were reported by direct questioning of all patients; skin photosensitivity, changes in biochemical tests of liver function, blood pressure and heart rate, and the occurrence of GI symptoms (nausea, vomiting) were recorded for 5 patients. OBSERVATIONS: Hexaminolevulinate-induced fluorescence endoscopy produced selective fluorescence of all rectal adenomas with intraepithelial neoplasia. For rectal cancer, there was only weak fluorescence or none at all. No hexaminolevulinate-induced side effect was observed. In two patients, fluorescence differentiated adenomas and hyperplastic polyps. CONCLUSIONS: Hexaminolevulinate-based fluorescence endoscopy (3.2 mM administered as an enema) in patients with rectal cancer and adenoma was well tolerated and produced no significant skin sensitivity or other side effects. The optimal duration of application is 30 to 45 minutes, with a rest time of 30 minutes. Selective fluorescence of adenoma with intraepithelial neoplasia suggests that hexaminolevulinate-based fluorescence endoscopy may be useful for detection of premalignant lesions.

Combined chemotherapeutic and photodynamic treatment on human bladder cells by hematoporphyrin-platinum(II) conjugates.

Four porphyrin-platinum complexes, conceived as a new approach in cancer therapy by combining the cytostatic activity of cisplatin or oxaliplatin and the photodynamic effect of hematoporphyrin in the same molecule, were studied in detail with respect to solubility and stability in cell culture medium as well as in terms of cytotoxicity and phototoxicity against J82 bladder cancer cells and UROtsa, normal urothelial cells. This study demonstrated that the most active and promising compound among the porphyrin-platinum conjugates investigated was the water-soluble porphyrin-platinum complex 4 (diammine[7,12-bis[1-(polyethyleneglycol-750-monomethylether-1-yl)ethyl]-3,8,13,17-tetramethylporphyrin-2,18-dipropionato]platinum(II)) which exhibited a synergistic antiproliferative effect compared to cisplatin and hematoporphyrin alone or a combination of the drugs.

Hematoporphyrin-derived soluble porphyrin-platinum conjugates with combined cytotoxic and phototoxic antitumor activity.

To combine the cytotoxic activity of cisplatin and the phototoxicicity of hematoporphyrin derivatives in the same molecule, hematoporphyrin was derivatized at the two secondary alcohol positions by etherification with oligo- and poly(ethylene glycol) units. The two carboxylic acid groups of the propionate side chains were used to bind platinum fragments. The antiproliferative activity of 35 platinum complexes (0.5, 1, and 5 microM) differing in solubility and type of the platinum fragment and the corresponding porphyrin ligands were studied in tests with TCC-SUP and J82 transitional bladder cancer cells in the dark and after irradiation (lambda = 600-730 nm, 24 J/cm(2)). The most active compounds were found among the porphyrin-platinum conjugates bearing the diammine and (RR/SS)-trans-1,2-diaminocyclohexane ligand. These porphyrin-platinum conjugates, especially the water-soluble species, such as diammine(7,12-bis[1-(poly(ethylene glycol)-750-monomethyl ether-1-yl)ethyl]-3,8,13,17-tetramethylporphyrin-2,18-dipropionato)platinum(II), are promising candidates for the development of a novel type of photosensitizers with intrinsic cytotoxicity, which due to the porphyrin constituent may selectively enrich in tumor tissues.

Soluble tetraarylporphyrin-platinum conjugates as cytotoxic and phototoxic antitumor agents.

A series of asymmetric tetraarylporphyrins was synthesized from pyrrole, para-substituted oligo- or poly(ethylene glycol) monomethyl ether benzaldehyde and from 4-hydroxybenzaldehyde etherified with diethyl bromomalonate according to the Lindsey method. After hydrolysis of the tetraarylporphyrin esters, the resulting carboxylic acid groups were used to bind platinum fragments. In comparison to analogous hematoporphyrin-platinum conjugates, the title compounds are characterized by a 30 nm bathochromic shift of their absorption bands. The antiproliferative activity of 18 platinum complexes (1, 5, and 10 microM) differing in solubility, type of the platinum fragment, and the corresponding tetraarylporphyrin ligands were studied on TCC-SUP transitional bladder cancer cells in the dark and after irradiation (lambda = 600-730 nm; 24 J/cm(2)). The most active compounds were among the tetraarylporphyrin-platinum conjugates bearing the diammine and (RR/SS)-trans-1,2-diaminocyclohexane ligands.