Proteomics as a tool for detection of nuclear matrix proteins and new biomarkers for screening of early tumors stage.

Nuclear matrix proteins are involved in control and co-ordination of gene expression. They have been isolated by extraction procedures, followed by gel electrophoresis and matrix- or surface-enhanced laser desorption, and ionisation, with direct detection of retained proteins by time-of-flight mass spectrometry. Some nuclear matrix proteins from tumor tissues demonstrated tumor-specific expression which led to the development of highly tumor-specific nuclear matrix protein assays. In bladder cancer, NMP22 is twice as sensitive as cytology in detecting early stage cancers, and up to 90% sensitive and 99% specific. NMP179 in squamous intraepithelial cervical lesions detects high-grade lesions with 96% sensitivity. Recently new technological approaches using ProteinChips, tracer-free biomolecular interaction, mass spectroscopy and nanotechnology have helped to successfully identify, and apply specific biomarkers for cancer of the prostate, breast and colon and to develop new approaches for simultaneous screening of early cancer with several new biomarkers.

PET clínico en oncología / Clinical PET in Oncology

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[Inhibition of 18F-FDG uptake in glioblastoma cells by FDG and glucose]. / Hemmung der 18F-FDG-Aufnahme in Glioblastoma-Zellen durch FDG und Glucose.

It was the aim of the study to compare the inhibition of 18F-2-Fluor-D-deoxy-glucose uptake (18F-FDG) in tumor cells by various concentrations of FDG carrier or D-glucose in an experimental model using tissue culture and positron emission tomography (PET). Glioblastoma cells in culture were incubated with 18F-FDG with and without added carrier or in presence of glucose concentrations in the range from 0-5 mmol/L. Cellular uptake of 18F-FDG was measured after 20 min. of incubation in PBS-buffer containing different sugar concentrations. The uptake was determined with a PET camera. The similarity of the kinetics of the FDG and glucose uptake are backing the hypothesis that both substrates use the same carrier system. The more intense inhibition of the 18F-uptake by FDG can be explained by the different intracellular metabolism of both substrates. The results explain the clinical experience that there is an optimal 18F-FDG uptake in the patient's tumor when the blood glucose level is as low as possible and the specific activity of 18F-FDG is very high.

[Toxicity and radiation dosage of 2-(18F)-2-desoxy-D-glucose in positron emission tomography]. / Toxizität und Strahlendosis von 2-[18F]-2-Desoxy-D-Glukose in der Positronenemissionstomographie.

2-[18F]-FDG, a non-physiological glucose analogue, is the most important positron-emission- tomography (PET) radiopharmaceutical. As an example we refer to the production of 2-[18F]-FDG at the research center in Karlsruhe. 2-[18F]-FDG is synthesized in a "no carrier added" process. It is delivered at a maximal filling volume of 10 ml from a 14.5 ml batch with a batch-to-batch yield fluctuation from 5075 to 50,750 MBq and a specific activity from 1 to 10 GBq/mumol. The residual remaining synthesis reagents like solvents or catalysts have no toxicological relevance. The applicated dose per patient is in a range from 185 to 370 MBq and 1000 times lower than the correlating concentrations of stable FDG which can be regarded harmless in animals. 2-[18F]-FDG does not interfere with normal glucose metabolism. It is taken up by cells and phosphorylated to 2-[18F]-FDG-6-phosphate. The following dephosphorylation step is slow and the labeled compound is retained over several hours within the cells. Non-metabolized 2-[18F]-FDG is excreted rapidly in the urine to an extent of about 16% after 60 min, and 50% after 135 min, respectively. Fluorine-18F decays by emission of 511 KeV gamma photons. The whole body effective dose is reported to be 21 to 27 microSv/MBq. In case of an intravenous injection of 370 MBq this leads to a total dose of 7.8 to 10 mSv. The critical organ is the bladder wall (radiation dose 120 to 170 microSv/MBq or 80 to 100 mrem/mCi). The risk of a radiation induced late malignoma at 10 mSv can be estimated to be 1:2000. The genetical risk as a consequence of FDG-PET diagnostics would be 1:100,000 to 2:100,000 for dominant, and 5 times higher for recessive mutations.

Fluorodeoxyglucose whole-body positron emission tomography in colorectal cancer patients studied in routine daily practice.

PURPOSE: To evaluate the routine clinical value of attenuation-corrected whole-body fluorodeoxyglucose positron emission tomography in colorectal cancer, a total of 59 patients who were referred for evaluation of suspected or proven colorectal cancers were studied. METHODS: Positron emission tomography scans were recorded using a Siemens ECAT Exact 921/47. RESULTS: Median follow-up after the positron emission tomography study was 11 (mean, 12.3; range, 1-21) months. According to computed tomography, coloscopy, and ultrasound, we recorded eight apparently false-positive results. During later follow-up, however, three of those cases, which were negative with computed tomography, magnetic resonance imaging, sonography, or laparoscopy, turned out to be true-positive instead. In 3 patients, a primary colorectal cancer was suspected; in 26 patients, a recurrence of colorectal cancer was suspected. Eight patients were studied for follow-up after the history of colorectal cancer with no suspicion of recurrence. In 12 patients, the rise of serum tumor marker concentrations was the reason for the positron emission tomography study; 12 patients with known metastatic disease were also included ("restaging"). With regard to the entire patient population, we found an overall sensitivity of 100 percent, a specificity of 67 percent, and positive and negative predictive values of 92 and 100 percent, respectively. Being merely confirmative with respect to tumor recurrence or distant metastases in the majority of patients, positron emission tomography revealed a primary tumor in one patient and confirmed metastatic foci in several patients that had not been delineated by other imaging modalities. CONCLUSION: A whole-body positron emission tomography scan provides optimum conditions to locate metastatic lesions that might not be seen otherwise. There is a trend showing that positron emission tomography diagnostics as a consequence of early increased tumor markers is a highly sensitive combination, because computed tomography and magnetic resonance imaging were not as sensitive in early recurrences. Positron emission tomography, as performed in daily clinical practice, proved to be a powerful diagnostic tool in our subset of colorectal cancer patients.

Computer modeling of cytokeratin release in clinical oncology.

The levels of cytokeratins (CK) in serum of cancer patients have been widely used for monitoring progression of cancer growth and the effectiveness of cancer treatment. Previous studies have shown that the release of CK by tumors in patients is a complex process which depends on the rate of cell damage caused by an increasing tumor mass, or by the tumor treatment, but is not in any simple manner correlated to the number of proliferating cells or to the total tumor mass (1). The complexity of the CK-releasing process has been analyzed by a computer model which mimics the progress of tumor growth, allows the introduction of different types of treatment (i.e. irradiation, chemotherapy and surgery), and computes the amount of CK released by the tumor, and the level of CK in blood and blood clearance. The computer model can be used to obtain a better understanding of the interactions of various factors, for scheduling of treatment and CK sampling, and for analyzing the effects of treatment.

[PET scan in general practice for diagnosis of breast carcinoma]. / PET in der Praxis bei der Diagnostik des Mammakarzinoms.

The value of whole body positron emission tomography using 18F-fluoro-deoxy-glucose (18FDG) in primary work-up and follow-up was evaluated retrospectively in 104 patients with primary or metastatic breast cancer. Compared to other imaging methods, FDG-PET sensitivity was superior to sonography, CT or MRT. Another advantage is the possibility of whole body imaging and the earlier detection of lymph node metastasis due to the recognition of functional metabolic changes compared to structural changes found with conventional imaging methods.

Heterologous double-determinant immunoradiometric assay CA 125 II: reliable second-generation immunoassay for determining CA 125 in serum.

The new CA 125 II (Centocor) serum assay utilizing the M11 mouse monoclonal antibody as capture antibody and OC125 as tracer antibody, was investigated for its technical and clinical performance against the original CA 125 assay. The CA 125 II test revealed a quadruple increase in signal-to-noise ratio, good intra- and interassay precision (with CVs < 5% and 7%, respectively), improved dilution linearity, and a minimal detectable dose of 0.38 units/mL. Sera were obtained from healthy females (n = 192), women with benign conditions (n = 208), and patients with various cancers (n = 379). Both assays measured highly similar CA 125 distributions with equal reference ranges and nearly identical positivity (> 35 units/mL) rates, resulting in similar receiver-operating characteristic curves and monitoring graphs. Linear regression analysis of results by the two assays (CA 125 = x, CA 125 II = y) in ovarian cancer patients showed, for CA 125 assay values between 0 and 1000 units/mL, a slope of 1.00 and a y-intercept of 12.6 (n = 254, r = 0.8617, P < 0.0001). The heterologous CA 125 II assay appeared to be more accurate in patients who had human anti-mouse antibodies after immunoscintigraphy. The CA 125 II immunoradiometric assay is sensitive and reliable for measuring serum CA 125, and fully retains the cutoff values of 35 and 65 units/mL that were defined with the original CA 125 immunoradiometric assay.

The application of immunotargeting into cancer chemotherapy with carboplatin: in vitro and in vivo studies.

Anti-tumor effects of the following 2 cis-diammin (1, 1-cyclobutandicarboxylato) platin II (carboplatin, Bristol-Myers-Squibb) conjugates were evaluated through both in vitro and in vivo experiments: (1) carboplatin coupled with anti-cytokeratin monoclonal antibody (MAb), TS1 via carboxymethyl dextran (carboplatin-carboxymethyl dextran-TS1), and (2) carboplatin-carboxymethyl dextran-avidin targeted to biotinylated TS1. Using 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide, carboplatin was conjugated to carboxymethyl dextran, TS1, or avidin, at high molar ratios. The staining positivity of carboplatin-carboxymethyl dextran-TS1 in indirect immunofluorescence was almost identical to that of the original MAb. The average dose of carboplatin given in each treatment was about 60% of a clinical dose. Regarding cytotoxicity, the free drug showed the strongest effect and the best dose-dependency in cell lines: HeLa and ZR-75-1. An in vivo study giving carboplatin-MAb conjugates or free drug to HeLa tumor bearing nude rats proved that the efficacy of carboplatin-carboxymethyl dextran-TS1 in HeLa tumor was not greater than that of the free carboplatin.

Serum TPS versus TPA in Egyptian bladder cancer patients.

This study included 328 cases (106 with bladder cancer, 152 with non-malignant urinary tract diseases and 70 healthy controls). Serum TPA was determined using the Prolifigen TPA IRMA kit supplied by AB Sangtec Medical, Bromma, Sweden and serum TPS was determined using the TPS IRMA kit supplied by Beki Diagnostics AB, Bromma, Sweden. The results of this study revealed that serum TPA had better sensitivity than serum TPS while no marked difference was found in the false-positivity rates in the non-malignant urinary tract diseases. A correlation coefficient of 0.83 was found between serum TPA and TPS. No relation was found between either TPA or TPS and histopathological stage, grade or association of the tumor with bilharziasis. As regards the histopathological type of the tumor, serum TPS was slightly higher in squamous cell than transitional cell carcinoma but TPA showed no difference. In the follow-up of bladder cancer patients after surgery both TPA and TPS showed an excellent concordance with the clinical state of the patients. In conclusion, TPS does not seem to be an optimal test in Egyptian patients with bladder cancer but serial determinations of one of the two markers can be used in the follow-up of these patients after surgery.

Idiotypic cascades after injection of the monoclonal antibody OC125: a study in a mouse model. Induction of antibodies against OC125 and CA125 after immunization with an anti-CA 125 (MAb OC125) monoclonal antibody by activation of the idiotypic network.

By immunization of mice with the anti-CA 125 monoclonal antibody OC125, we tried to induce antibodies directed against the tumour-associated antigen CA125, via activation of the idiotypic network. Mice immunized by repeated administrations of F(ab')2-fragments of the OC125 antibody produced anti-idiotypic antibodies, imitating the original target antigen of the OC125. After induction of these anti-idiotypic antibodies (Ab2 beta) a murine IgG-anti-CA125 (Ab3) response was detected. The induction of idiotypic cascades offers the possibility of immunization against tumour-associated antigens, without using the original antigen and breaking antitumour tolerance.

Simultaneous determination of urinary CEA, ferritin and TPA in Egyptian bladder cancer patients.

Urinary carcinoembryonic antigen (CEA), ferritin (Fer) and tissue polypeptide antigen (TPA) were determined in 328 cases (106 with bladder cancer, 152 with non-malignant urinary tract disease and 70 healthy controls). CEA was determined by the kit supplied by Roche Diagnostica (CEA EIA Doumab 60), ferritin by the Tandem-E Fer kit supplied by Hybritech and TPA by the Prolifigen TPA-IRMA kit supplied by Sangtec Medical. The results of this work revealed that combined determination of urine CEA and Fer, CEA and TPA or Fer and TPA showed higher sensitivity than determination of the individual markers. There was no significant difference between combined and individual marker determination with respect to false positivity in non-malignant urinary tract diseases. At 97% specificity, the sensitivities of urine CEA, Fer and TPA were 82.1%, 71.7% and 90.6%, respectively, while combined urine CEA & Fer, CEA & TPA and Fer & TPA showed sensitivities of 92.5%, 99.1% and 98.1%, respectively. When the specificity was related to the entire non-cancer group (patients with benign urinary tract diseases and normal controls), some reduction in the sensitivities of the combined markers was noted compared to the normal group only. In conclusion, combined determination of urine markers is superior to determination of individual markers in the diagnosis of bladder cancer.

The role of nuclear medicine in oncology.

Nuclear Medicine offers screening methods for oncology such as bone and bone marrow scintigraphy. During the last two decades, special procedures have gained widespread application. This paper is centered around the "tumor-specific" radiopharmaceuticals. In patients with thyroid cancer, I-131 still plays a significant role. Ga-67 still has its indications in lymphoma, while in other diseases Tl-201 chloride is now the agent of choice. Especially in thyroid cancer, Tl-201 has proved to be a reliable tumor imaging radiopharmaceutical. More recently, Tc-99m MIBI was introduced for tumor imaging. Tc-99m HMPAO may also be used for tumor scintigraphy, especially in brain lesions. In addition, I-123 IMP has successfully been used for imaging malignant melanoma. Another promising field of tumor diagnosis is receptor imaging. In neuroblastoma and malignant pheochromocytoma, I-131/123 mIBG is the radiopharmaceutical of choice and may be considered as a receptor imaging agent also. First clinical results with In-111 octreotide show potentials as somatostatin-receptor radiopharmaceutical in insulinoma, islet cell carcinoma, medullary and lung cancer, while I-123 estradiol needs some improvement until it may be recommended as diagnostic tool in breast cancer. Since 1978, radiolabeled poly- or monoclonal tumor antibodies and their fragments have gained widespread application. Especially the Tc-99m 225.28S melanoma antibody, I-131 or Tc-99m CEA and In-111/I-131 labeled OC-125 antibodies have proven to be of clinical significance in melanoma, colorectal and ovarian cancer.

[Photodynamic laser therapy with antibody-bound dyes. A new procedure in therapy of gynecologic malignancies]. / Photodynamische Lasertherapie mit antikörpergebundenen Farbstoffen. Ein neues Verfahren zur Therapie gynäkologischer Malignome.

In the present paper, a new therapeutic concept of photodynamic laser therapy using antibody-linked dyes for the treatment of gynecological malignancies is described. So far, HPD (hematoporphyrin derivative) has been employed in this area, but is associated with toxic systemic reactions. We see a solution to this problem in the linking of a systemically non-toxic dye--known to induce photodynamic reactions while not itself being selectively accumulated within tumor cells--to an antibody directed against a selective tumor-associated antigen. The results of our study demonstrate the efficacy of this therapeutic concept as exemplified by the selective destruction of dye-labeled ovarian carcinoma cells by laser light of a defined wavelength (675 nm). The potential of this form of photodynamic therapy extends far beyond its use in ovarian carcinoma.

Immunotherapy of advanced ovarian carcinomas by activation of the idiotypic network.

The positive effect of an immunotherapy using tumor-associated antigens or tumor cells of ovarian carcinomas has not yet been proven. Although many unique tumor-associated antigens have been described and a tumor rejection could be seen in occasional cases, the failure of the immune system to destroy tumor cells is not clearly understood. An alternative approach is to initiate the idiotypic network utilizing antibodies (Ab1 or 2) against a tumor-associated antigen, which induces the production of anti-idiotypic-antibodies (Ab2 beta), mimicking the "internal image" of the tumor-associated antigen. These antibodies are able to induce a specific antitumor immunity in two ways: (1) the Ab2 can present the critical epitope in a different way and so modulates the immune system, or (2) it can induce the production of an Ab3, which by itself binds to the tumor antigen. Our first results on 22 patients with advanced ovarian carcinomas show that the induction of an anti-idiotypic antibody (Ab2 beta) against OC 125 mimicking the TAA Class III CA 125 leads to a prolongation of the survival rate also for extended stages. We see a beneficial role of the induction of the idiotypic network against a tumor-associated antigen showing delayed clinical courses of the disease after vaccination of the patients with antibody fragments of the OC 125.

[Clinical use of photodynamic therapy in gynecologic tumor patients--antibody-targeted photodynamic laser therapy as a new oncologic treatment procedure]. / Klinischer Einsatz der photodynamischen Therapie bei gynäkologischen Tumorpatienten--Antikörper-vermittelte photodynamische Lasertherapie als neues onkologisches Behandlungsverfahren.

The clinical report addresses the first application of a antibody-targeted photodynamic laser therapy with Phthalocyanines. Photodynamic therapy (PDT) has been proposed as a further treatment modality in oncology. The concept of PDT is based on the interaction between a dye which is accumulated in the target and laserlight. The phototoxic effect is achieved by the augmentation of oxygen radicals. The improvement of the selectivity and the reduction of side-effects is achieved by our concept of using an antibody-targeted phthalocyane induced PDT. The clinical application of the antibody-targeted Phthalocyanine was performed in 3 patients suffering from an advanced ovarian carcinoma (FIGO III). By means of an ultramicroscopical analysis a selective devitalisation of tumor cells was demonstrated. The perspective of PDT reaches far beyond the application for the ovarian carcinomas and relates to all tumor types, where the presence of tumor-associated antigens implicates a treatment in a similar way.

Photodynamic laser therapy of carcinomas--effects of five different photosensitizers in the colony-forming assay.

Photodynamic therapy (PDT) with laser radiation opens a new field in the treatment of malignancies. We evaluated the phototoxic effects of five different substances for photodynamic therapy in concentrations showing no systemic toxicity. We used gynecologic tumor cells and evaluated the photodynamic effects for cell growth in a colony-forming assay. For Indigocarmin we found a reduction in the colony-forming assay compared to the control group from 87% to 66% after irradiation at 50 mumol/l and 608 nm wavelength. "IR 132" showed a reduction in the colony-forming assay from 73% to 72% 50 mumol/l (590 nm). For the incubation of the HeLa cells with 50 mumol/l of "Sulfo-Phthalocyanin" we found a reduction of the colony-forming potential from 81% to 67% (595 nm). The most strikingly differences were found for the incubation and irradiation of Methylene Blue, showing a reduction from 77% to 16% caused by a toxic effect of the substance itself (5 mumol/l, 660 nm) and "Sulfo-Aluminium-Phthalocyanin" stained cells, which shows a reduction from 86% to 17% (50 mumol/l, 675 nm). We see the possibility of a photoactivation and cell devitalisation by "Sulfo-Aluminium-Phthalocyanin" and laser radiation. A future clinical trial would seem justified.