Pretreatment serum levels of matrix metalloproteinase-9 and vascular endothelial growth factor in non-small-cell lung cancer.

BACKGROUND: Matrix metalloproteinase (MMP)-9 and vascular endothelial growth factor (VEGF) are two proteins involved in angiogenesis. In the present study we investigated the association of pretreatment MMP-9 and VEGF serum levels with clinicopathological parameters and outcome in patients with non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: From February 1998 to October 1999, pretreatment serum levels of MMP-9 and VEGF were analysed in 118 patients with enzyme-linked immunoassays. At diagnosis 50 patients (42%) were staged as early disease (I/II), 27 patients (23%) as locally advanced (IIIA/IIIB), and 41 patients (35%) had metastatic disease (IV). In 72 of the 118 patients tumours were resected and 46 patients received combination chemotherapy with gemcitabine and vinorelbine. RESULTS: The median survival of all 118 patients was 602 days. The 72 patients who had undergone surgery had a median survival of 972 days and the 46 patients who were treated with chemotherapy had a median survival of 298 days (P <0.001). Resected patients with stage I/II disease and an MMP-9 serum level

Tumor markers in the diagnosis and follow-up of head and neck cancer: role of CEA, CA 19-9, SCC, TK, and dTTPase.

The clinical relevance of the carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9, squamous cell carcinoma antigen (SCC), thymidine kinase (TK), and deoxythymidine-5'-triphosphatase (dTTPase) as tumor markers in the diagnosis and follow-up treatment of 26 patients with head and neck cancer is evaluated. Serum levels prior to treatment were found elevated just above the upper limit of normal in 46% (SCC), 15% (CEA), 12% (CA 19-9), 27% (TK), and 39% (dTTPase) of all patients. If all markers were taken into account, they were elevated in 73% of the untreated patients. However, only in a few cases were the tumor marker values elevated significantly (8%-12%). No significant correlation was detected between serum levels and tumor localization, staging, grading, or performance status for any of the markers. In the follow-up none of the markers tested revealed any disease-related information despite therapy variation. Patients with originally elevated marker levels showed decreasing and in some cases increasing values after primary therapy, although no tumor recurrence was detected. Even considering the results as preliminary due to the rather small sample size, they suggest that the routine assessment of CEA, CA 19-9, SCC, TK, and dTTPase serum levels is of limited practical value.

[The "false positive" tumor marker in malignant testicular tumor]. / Der "falsch-positive"Tumormarker beim malignen Hodentumor.

In addition to the histological diagnosis, alpha fetoprotein (AFP) and chorion gonadotropin (HCG) are used in clinical staging, therapy monitoring, and follow-up. Elevated markers without localization of metastases by imaging procedures are generally classified as progressive disease. However, other causes may be responsible for the elevated tumor markers: other malignant or benign diseases such as hepatocellular carcinomas, gastrointestinal tumors, bronchial carcinomas and benign diseases of the liver for AFP, and vesicular mole, hepatocellular, stomach, pancreatic and urothelial carcinomas for HCG. Moreover, technical disturbances in the modern sandwich assays with monoclonal antibodies are possible by heterophilic antibodies. These human anti-animal antibodies are built after immunoscintigraphy, immunostimulation and oral immunization by macromolecules. As a result, if progressive disease of a malignant germ cell tumor is unlikely, several steps have to be taken to determine the true causes for the elevated tumor markers before chemotherapy can be applied.

Purification and characterization of two different thymidine-5'-triphosphosphate-hydrolysing enzymes in human serum.

Two different enzymes capable of hydrolysing dTTP to the corresponding diphosphate were purified from human serum in order to investigate their enzymatic properties. A specific dTTPase was purified to apparent homogeneity with a purification factor of ca. 10,000 and showed a molecular mass of 46,000 Da, consisting of two identical subunits. This enzyme revealed an isoelectric point of 5.8 and a Km value of 38 microM. The other enzyme showed substrate specificity for dTTP and dCTP and was purified with a factor of ca. 5,000. It seems to be a multifunctional enzyme of one subunit (96,000 Da) with two different catalytic sites for dTTP and dCTP. The isoelectric point was 5.2, the Km values were 20 microM for dTTP and 17 microM for dCTP, respectively. Both enzymes were sensitive to inorganic phosphate, but the dTTPase to a minor extent. In contrast to the dCTPase-dTTPase, the dTTPase was strongly inhibited by ZnSO4. Physico-chemical and biochemical data suggest the purification of two different enzymes.

Luminography--a new, highly sensitive visualization method for electrophoresis.

A highly sensitive method for protein visualization following electrophoresis and protein blotting was developed. The method is based on the light-emitting reaction of luminol and hydrogen peroxide catalyzed by horseradish peroxidase. The luminescent assay can be applied either to the native gel or after protein blotting, and it has a sensitivity two orders of magnitude higher than that achieved with chromogenic detection systems. Analogous to autoradiography the luminescent signal is recorded on an X-ray film with similar sensitivity. We present several examples of application emphasizing the general versatility of this innovative method.

Serum deoxythymidine-5'-triphosphatase activity in lymphoproliferative disorders of men and mice.

A recently discovered enzyme of the pyrimidine pathway, deoxythymidine-5'-triphosphatase (dTTPase), was estimated in sera from leukemic mice and 64 untreated patients with non-Hodgkin's lymphoma (NHL) as well as 30 patients with plasmocytoma. At the age of 19 weeks the Mov-9 substrain of 129 mice developed leukemia in contrast to the congenic controls. Patient lymph node biopsies were classified according to the Kiel classification. The results showed a significant correlation between dTTPase activity and the onset of proliferation (studied in mice), as well as the grade of malignancy (studied in men). The more advanced the disease or the less aggressive the tumor, the higher the dTTPase activity. This gives rise to the speculation that dTTPase might be part of a control in the proliferation process.

Homogeneous preparation of human thymidine-5'-triphosphatase by electroelution from SDS/PAGE with subsequent renaturation.

This paper describes a rapid and inexpensive method for homogeneous enzyme preparation from SDS/polyacrylamide gels with subsequent renaturation. The method was optimized for an enzyme of pyrimidine metabolism, thymidine-5'-triphosphatase (dTTPase), present in human serum in small amounts. After gel electrophoresis, the enzyme was eluted from gel pieces in an elution chamber based on a tube gel electrophoresis system. Renaturation conditions were optimized in preliminary tests. The best results were obtained with an initial acetone precipitation to remove sodium dodecyl sulfate. The precipitate was then dissolved in 8 M guanidine hydrochloride and diluted 50-fold for renaturation. Adding 1.5 mg/ml lauryl maltoside to the renaturation buffer, followed by subsequent dialysis of the renaturating samples, improved the renaturation yield up to 95%. This method was used to purify dTTPase to homogeneity from a partially purified sample, and to determine the molecular mass of the subunits. The procedure can also be applied to other enzymes and could give rise to a general strategy for enzyme purification.

A colorimetric assay for the determination of acid nucleoside triphosphatase activity.

A photometric method for the determination of the acid nucleoside triphosphatase (EC 3.6.1.-) is described, in which inorganic phosphate is liberated from ATP or other nucleoside triphosphates. Colorimetric determination of liberated phosphate is based on the formation of a green complex of phosphomolybdate and malachite green hydrochloride. Optimal test conditions were evaluated as well as the sample preparation. The enzyme activities measured in 100 normal human sera are in the range of 0.5 to 9.0 U/l with an average of 4.0 U/l for men and 3.8 U/l for women.

[Tumor markers in patients with head-neck carcinomas]. / Tumormarker bei Patienten mit Kopf-Hals-Karzinomen.

The clinical relevance of the tumor-associated antigens SCC (squamous-cell carcinoma), CEA (carcinoembryonic antigen), and CA (carbohydrate antigen) 19-9 as tumor markers is evaluated. Twenty-six patients with squamous-cell carcinoma of the head and neck region were studied in a six-month period. Concentrations above 2 ng/ml (SCC), 5 ng/ml (CEA), and 37 U/ml (CA 19-9) are regarded as markers of abnormal activity. Elevated tumor markers were found only in 12-15%. No correlation between the serum levels and tumor localization, staging, grading, or general condition was detected for any of the markers. In the follow-up, they revealed no disease-related information despite treatment variation. The results obtained suggest that, given the present state of biochemical possibilities and considering the rather low sensitivity for head and neck cancer, the routine assessment of SCC, CEA, and CA 19-9 serum levels is of no account.

Acid nucleoside triphosphatase: partial purification and characterization of a new enzyme from human serum.

Acid nucleoside triphosphatase (Acid NTPase), an enzyme which catalyzes the hydrolysis of all nucleoside triphosphates to the corresponding diphosphates was purified from human serum with a purification factor of 190 and a recovery of 31%. The molecular weight was 75,000 as estimated by gel filtration. Gel-electrophoresis revealed an Rf-value of 0.11, and the isoelectric point was determined at pH 4.4. It exhibited a temperature optimum of 44 degrees C and the activation energy was estimated to be 41.6 kJ/mol. The enzyme was active in the absence of divalent cations, since activity was not inhibited by EDTA. The presence of this chelator reduced the Km-value from 70 to 40 microM. Inhibitor experiments revealed that tartrate was a weak mixed-type noncompetitive inhibitor, Ki = 88 mM. The enzyme was specific for the hydrolysis of nucleoside triphosphates. P-nitrophenyl phosphate was not accepted as a substrate. The enzyme revealed optimum activity at the exceptionally acid pH of 3.0. These unique characteristics indicate the presence of a novel enzyme.

Chemotherapy of a patient because of spuriously elevated alpha-fetoprotein levels. Identification of the responsible factor.

Because of spuriously elevated alpha-fetoprotein levels, a course of polychemotherapy was given to a patient. We purified and identified the serum factor responsible for falsely high AFP levels as an IgG directed against mouse IgG. On gel filtration it behaves differently from true AFP, exhibiting a molecular weight of around 150,000. Chromatography on 'Protein A-Sepharose' revealed properties indistinguishable from those of IgG. The patient had never been treated with mouse antibodies, neither for diagnostic nor therapeutic purposes and he had had no contact with animals professionally.

FILEMAN41C--an interactive data management system for biomedical data selected from a free definable file by a pocket calculator.

An interactive system for the storage, retrieval and analysis of numerous clinical data is described. Due to the simple structure of the dialogue, no special knowledge of computer handling is required by the experimenter. The program is written for the Hewlett-Packard HP-41C pocket calculator and is suitable for a number of moderately sized experiments.

A one-step liquid-chromatographic technique for the estimation of the deoxythymidine-5'-triphosphatase in human serum.

Deoxythymidine-5'-triphosphatase (dTTPase) from human serum was separated by DEAE-cellulose chromatography from unspecific hydrolases in a single step. The method was adapted to a microscale and the enzymatic activity was determined for five different groups of patients including epithelial carcinoma, leukemia and three control groups. The result is that the purified dTTPase preparations of these groups reflect the situation obtained in the whole serum.