Circulating nucleosomes in serum.

In the nucleus of eukaryotic cells, DNA is associated with several protein components and forms complexes known as nucleosomes. During cell death, particularly during apoptosis, endonucleases are activated that cleave the chromatin into multiple oligo- and mononucleosomes. Subsequently, these nucleosomes are packed into apoptotic bodies and are engulfed by macrophages or neighboring cells. In cases of high rates of cellular turnover and cell death, they also are released into the circulation and can be detected in serum or plasma. As enhanced cell death occurs under various pathologic conditions, elevated amounts of circulating nucleosomes are not specific for any benign or malignant disorder. However, the course of change in the nucleosomal levels in circulation of patients with malignant tumors during chemotherapy or radiotherapy is associated with the clinical outcome and can be useful for the therapeutic monitoring and the prediction of the therapeutic efficacy.

Serum tenascin-C is an indicator of inflammatory bowel disease activity.

Tenascin-C is a multifunctional matrix protein that is induced in inflammation and neoplasia. In the colonic mucosa of ulcerative colitis patients tenascin-C indicates tissue repair, and mucosal concentrations are correlated with local disease activity. We prospectively examined the relationship between serum concentrations of tenascin-C parameters of disease activity in surgically treated patients with ulcerative colitis and patients with inflammatory bowel disease (IBD). Perioperative serum concentrations were quantified by ELISA in 58 patients admitted for restorative proctocolectomy; controls were 37 patients with familial adenomatous polyposis receiving the same treatment. We also measured tenascin-C serum levels in 47 patients with ulcerative colitis and Crohn's disease who were receiving nonsurgical treatment. Preoperative serum tenascin-C levels were significantly higher in ulcerative colitis patients than in controls (17.2 +/- 14.6 microg/ml vs. 3.2 +/- 1.7 microg/ml) and were significantly correlated with clinical and histological parameters of disease activity; levels decreased significantly after restorative proctocolectomy. Serum tenascin-C levels were also correlated with the course of disease activity in conservatively treated IBD patients. Tenascin-C is thus not disease-specific. However, it does indicate the activity of IBD and may reflect the degree of tissue remodeling. The tenascin-C levels therefore offers a novel serum parameter for assessing disease activity and monitoring therapy in patients with IBD.

Nucleosomes in serum as a marker for cell death.

The concentration of nucleosomes is elevated in blood of patients with diseases which are associated with enhanced cell death. In order to detect these circulating nucleosomes, we used the Cell Death Detection-ELISAplus (CDDE) from Roche Diagnostics (Mannheim, Germany) (details at http:\\biochem.roche.com). For its application in liquid materials we performed various modifications: we introduced a standard curve with nucleosome-rich material, which enabled direct quantification and improved comparability of the values within (CVintraassay:3.0-4.11%) and between several runs (CVinterassay:8.6-13.5%), and tested the analytical specificity of the ELISA. Because of the fast elimination of nucleosomes from circulation and their limited stability, we compared plasma and serum matrix and investigated in detail the pre-analytical handling of serum samples which can considerably influence the test results. Careless venipuncture producing hemolysis, delayed centrifugation and bacterial contamination of the blood samples led to false-positive results; delayed stabilization with EDTA and insufficient storage conditions resulted in false-negative values. At temperatures of -20 degrees C, serum samples which were treated with 10 mM EDTA were stable for at least 6 months. In order to avoid possible interfering factors, we recommend a schedule for the pre-analytical handling of the samples. As the first stage, the possible clinical application was investigated in the sera of 310 persons. Patients with solid tumors (n=220; mean=361 Arbitrary Units (AU)) had considerably higher values than healthy persons (n=50; mean=30 AU; p=0.0001) and patients with inflammatory diseases (n=40; mean= 296 AU; p=0.096). Within the group of patients with tumors, those in advanced stages (UICC 4) showed significantly higher values than those in early stages (UICC 1-3) (p=0.0004).

Nucleosomes in serum of patients with benign and malignant diseases.

High quantities of mono- and oligonucleosomes circulate in the blood of patients with malignant tumors. For their direct quantification in serum, we modified the Cell Death Detection(plus)-ELISA for its application in liquid materials. We examined sera samples from 590 persons, including 418 patients with malignant tumors, 109 patients with benign diseases and 63 healthy persons. We also observed the kinetics of the concentration of nucleosomes in serum samples from 20 patients undergoing chemotherapy and from 16 patients undergoing radiotherapy. Sera of patients with malignant tumors contained considerably higher concentrations of nucleosomes (mean = 350 arbitrary units [AU], median = 190 AU) compared with those of healthy persons (mean = 36 AU, median = 24 AU; p = 0.0001) and patients with benign diseases (mean = 264 AU, median = 146 AU; p = 0.072). Concerning the follow-up investigations, the concentration of nucleosomes in serum increased 24-72 hr after the first application of chemotherapy and 6-24 hr after the start of radiotherapy. A subsequent decrease was often correlated with regression of the tumor. In patients undergoing chemotherapy, an increase in the baseline values of circulating nucleosomes >50%, which were determined before each new therapeutic cycle, was correlated with progression of disease; all patients with disease regression showed a decrease >50% of the baseline values. In patients undergoing radiotherapy, an early decrease of the nucleosomal concentration (< or = 1 day after the initial peak during therapy) to low minimum levels (< or = 100 AU) correlated with good clinical outcome; a late decrease (>1 day) to higher minimum levels (>100 AU) was associated with a worse clinical outcome. Thus, the concentration of nucleosomes in serum might be a useful tool for monitoring the biochemical response during antitumor therapy, especially for the early estimation of therapeutic efficacy.

Immunohistochemical profiles of 30 monoclonal antibodies against cytokeratins 8, 18 and 19. Second report of the TD5 workshop.

In the first report of the TD5 workshop (TD5-1), the epitope specificities of 30 different monoclonal antibodies against cytokeratins 8, 18 and 19 were determined. This second report presents the immunohistochemical profiles of these antibodies using human appendix and normal skin for evaluation. Each antibody was tested by one or two different laboratories recruited from the Dutch Working Group on Immunohistochemistry and Cytochemistry. Eight different laboratories participated. The histological specimens were pretreated by the participants in three different ways for immunohistochemistry: microwave antigen retrieval in citrate buffer, enzymatic digestion to restore epitope exposure, no specific treatment (untreated paraffin-embedded samples), and tested blindly without knowledge of cytokeratin or epitope specificity of the antibodies at three different concentrations of 50, 10 and 1 microg/ml. Most of the tested antibodies (29/30) were useful in at least one pretreatment method, with microwave antigen retrieval being the most sensitive approach. For some antibodies, very high backgrounds were observed. Furthermore, it can be concluded that 11 MAbs performed well using all three staining protocols, including untreated paraffin-embedded sections. Interestingly, all the antibodies with documented selected specificity towards cytokeratin 8 (i.e. 178, 191, 199, 202 and 206) are reactive with an immunodominant region corresponding to amino acids 340-365 on cytokeratin 8, which evidently is well-suited as target for immunohistochemical interactions. Similarly, three antibodies with the same capacity to react with untreated samples had specificity against cytokeratin 19 (i.e. 179, 197 and 204) in the corresponding region in this filament, i.e. amino acids 311-335, or the KS 19.1 epitope. None of the six antibodies against the other major cytokeratin 19 epitope (BM 19.21) were found useful for immunohistochemistry on untreated samples. The overall conclusions from the present investigation are that all cytokeratin-8-specific antibodies with defined epitope specificities were very useful. Only one of the major two epitopes on cytokeratin 19 seems to be available for efficient immunohistochemistry. Cytokeratin 18 exposes some epitopes outside the immunodominant region reactive with the antibodies 190, 203 and 205 which can be used for untreated samples. The implications of these findings are of significance both for diagnostic histopathology and for the biology of tumor marker epitope expression in tissues.

Epitope specificity of 30 monoclonal antibodies against cytokeratin antigens: the ISOBM TD5-1 Workshop.

The epitope specificities of 30 monoclonal antibodies (MAbs) against the most common human cytokeratins. i.e., Nos. 8, 18, and 19, in epithelial cells were investigated in the ISOBM TD-5 Workshop. Seven research groups from universities or companies participated independently in the evaluation of the antibody specificities. The complex assembly of cytokeratins in vivo, with obligatory heterologous dimeric combinations of different cytokeratins from each of the two major groups, comprising together more than 20 different individual cytokeratins, made analysis of the antibody reactivity patterns with isolated single cytokeratins necessary. The concordance of the evaluations was striking and independent of the technologies used. As antigens purified individual cytokeratins, chemically degraded purified cytokeratins, recombinant intact and truncated cytokeratins, as well as specific synthesized shorter peptides were used. In order to elucidate the epitope specificity, reactivity patterns in ELISA assays and immunoblots with partial enzymatic degradation of the antigens were performed. Competitive cross-inhibition experiments between antibodies using antigens and antibodies in all possible combinations were performed with radioimmunometric assays, BIAcore, and ELISA technology. All 30 antibodies could convincingly be classified with regard to target cytokeratin. One MAb (192) had to be deleted due to dual specificities in both isotype and epitope specificity against its target. Six antibodies bound selectively to cytokeratin 8, 14 to cytokeratin 18, and 10 to cytokeratin 19, as demonstrated by using native, recombinant, and synthesized antigens. The immunodominant part of the molecule for all three types of cytokeratins was located in the region of amino acid (aa) 270-400. Out of the six MAbs reactive with cytokeratin 8, four MAbs, i.e., 178, 199, 202, and 206, were reactive with a sequence in the interval aa 340-365, and MAb 191 reacted with a closely related epitope. The remaining antibody, 192, presented dual specificities. At least two closely related major immunogenic epitopes could be identified in cytokeratin 8. In cytokeratin 18 four distinct epitopes could be documented, again with the dominating sequence region 270-429 as target for 10 (181, 184, 186, 188, 189, 190, 193, 196, 198, and 200) out of 14 antibodies. Since MAb 193 is known to react with the M3 epitope, aa 322-342 in cytokeratin 18, this entire group is reactive in the region close to the charge shift, in the middle of the rod 2B region, as shown by competitive binding. The remaining four anticytokeratin 18 antibodies (180, 185, 203, and 205) displayed unique, noncompetitive binding to this filament. Cytokeratin 19, reactive with altogether ten antibodies, displayed two major epitopes, all of them also within the large immunodominant region. MAbs 179, 195, 197, and 204 were reactive with the peptides aa 311-335 also known as the KS 19.1 epitope, and MAbs 182, 183, 187, 194, and 201 bound to peptide aa 346-367, known as the BM 19.21 epitope. One antibody, 231, was selectively reactive with aa 356-370 in cytokeratin 19. A complex pattern of binding specificities comprising at least ten different, noncompetitive epitopes, mainly situated in the rod portion, 2A and 2B, situated close to the charge shift in the rod of all three cytokeratins was documented. Out of the 29 classifiable antibodies, altogether 22 were reactive in this very short region, i.e., from aa 311 to 370 in all cytokeratin filaments. The remaining seven antibodies displayed unique binding properties. The implications of the findings are of significance both for immunohistochemistry and for assaying circulating heterodimeric, partially degraded complexes in patients' blood for tumor marker evaluation.

Occult epithelial tumor cells detected in bone marrow by an enzyme immunoassay specific for cytokeratin 19.

The presence of isolated carcinoma cells detected immunocytochemically in bone marrow has been shown to be of prognostic relevance for cancer patients. Unfortunately, the immunocytochemical method (ICC) is laborious and depends on the subjective interpretation of the individual investigator. Therefore, an immunoassay was designed for detection of cytokeratin 19 (CK19). By analyzing blood samples from 52 healthy volunteers and 40 bone-marrow aspirates from control patients, a cut-off point of 250 pg/ml CK19 was determined. Application of this cut-off point enabled a specificity of 95% to be shown for bone marrow and of nearly 100% for venous blood. The assay detected 10 HT-29 colon-carcinoma cells among 5 x 10(6) peripheral-blood leukocytes. In comparison with controls, bone-marrow samples of cancer patients were found to have significantly elevated levels of CK19 (p < 0.05). In the analysis of 386 marrow aspirates of cancer patients, a significant concordance of ELISA and ICC was observed (chi 2 = 18.3; p < 0.001). Both procedures, nevertheless, differed in 147 (38%) samples, of which more than two thirds (101) were only ELISA-positive. The CK status detected by ELISA did not correlate with the TNM stage and the histological grading. The established immunoassay allowed sensitive and specific detection of disseminated epithelial tumor cells and appeared to be faster, less laborious and more objective than ICC. Follow-up studies are required to assess the prognostic relevance of this ELISA before it can be applied as a routine method for monitoring of minimal residual epithelial cancer.

Significance of tenascin serum level as tumor marker in primary colorectal carcinoma.

Tenascin serum levels were evaluated in 118 patients with primary colorectal carcinoma and in a control group of 51 healthy persons in a double-sided sandwich ELISA. The data were correlated with post-operative TNM-staging. Patients with colorectal carcinomas had significantly higher serum levels of tenascin than the control group. At the 95% level of specificity, sensitivity was 25%. Tumor grading obviously had no influence on the level of tenascin in serum. With increasing pT-category, tenascin levels increased as well. In patients with distant metastatic disease, serum tenascin levels were significantly higher than in patients without distant metastases. These data suggest that, in colorectal carcinoma, the preoperative level of serum tenasin reflects the total tumor burden and correlates with metastatic disease. Our observation warrants a prospective study of the relevance of tenascin serum levels with regard to prognosis and as an indicator of relapse.

The biochemistry of CYFRA 21-1 and other cytokeratin-tests.

Soluble forms of keratins in human sera seem to be useful analytes for the monitoring of cancer patients. CYFRA 21-1 is a new test measuring keratin 19 in human blood. The test was developed as sandwich ELISA based on two monoclonal antibodies, both reacting with keratin 19. The epitopes recognised by the antibodies are located on the rod region of the molecule. In sera from malignant patients CYFRA 21-1 detects immunoreactive compounds which appear to be larger than keratin 19 itself, indicating the presence of oligomers in these sera. In a comparison study the reactivity of other keratin tests (TPA, TPS, TPAcyk) with the keratins 8, 18 and 19 were measured both in solution and on immunoblots. The reactivity pattern was found to be different what may explain the different diagnostic properties of the individual tests.

CYFRA 21-1--clinical applications and analytical requirements.

CYFRA 21-1 has proved to be a useful marker for non-small-cell lung cancer (NSCLC), which is the major form of lung cancer. Its most effective application is in monitoring. CYFRA 21-1 provides diagnostic information about the success of primary surgery, the response to chemotherapy and the detection of relapse. It is also an independent prognostic factor. The diagnostic potential may not be fully used because decision-making is currently based on group reference ranges. It seems useful to carry out systematic studies to investigate if the application can be improved and extended by using individual reference ranges for decision-making. In contrast to most of the other tumour markers the comparability of the commercial CYFRA 21-1- assays currently available on the market is good. The high degree of comparability should be maintained by international standardization. The analytical performance of the currently available commercial CYFRA 21-1 tests meets requirements derived from its current clinical applications. However, there are no data available about the analytical performance under field conditions. CYFRA 21-1, an established tumour marker for lung cancer, should be included in external quality assurance schemes.

Lung cancer-associated keratin 19 fragments: development and biochemical characterisation of the new serum assay Enzymun-Test CYFRA 21-1.

From a panel of 4 murine monoclonal antibodies directed against keratin 19 various antibody combinations were evaluated in solid-phase enzyme-linked sandwich immunoassays for detection of soluble keratin 19 fragments in patient sera. One of these antibody combinations, comprised of the monoclonal antibodies Ks 19.1 and BM 19.21, was selected for further development to a routine test (Enzymun-Test CYFRA 21-1) because of its high diagnostic sensitivity and specificity for non-small cell lung carcinoma (NSCLC). Both antibodies are specific for keratin 19, no reactivity could be observed with cytokeratin 8 or 18. The epitopes of the two antibodies were determined to be within helix 2B of the rod romain. The epitope sequences lie within the sequence 311-335 for the catcher antibody Ks 19.1 and 346-367 for the detector antibody BM 19.21. These sequences are unique, as could be confirmed from sequence databases. The standard material for the assay was prepared from a cytoskeleton fraction of cultivated MCF-7 cells. Subsequent digestion of this fraction with chymotrypsin yielded a soluble and stable standard material. Both the standard material and the serum analyte appeared as oligomers when analysed on gel chromatography: the serum analyte appeared exclusively at a M(r) of 100 +/- 10 kD, whereas the standard material eluted in fractions corresponding to 100 +/- 10 kD and 450 kD. Due to the precise definition of the antigen and the localisation of the antibody binding sequences, Enzymun-Test CYFRA 21-1 is one of the best characterised tumor markers so far.

The tumor markers TPA, TPS, TPACYK and CYFRA 21-1 react differently with the keratins 8, 18 and 19.

The commercially available tumor marker tests TPA, TPS, TPACYK and CYFRA 21-1 react with simple epithelium keratins. From clinical studies it can be deduced that the pattern of keratin recognition must be different for each of these tests. We therefore studied the reactivity of the keratin fragment combinations K8/K18 and K8/K19 in the different tests and determined the reactivity of the corresponding soluble antibodies with purified keratin 8, 18 and 19 in immunoblots. TPS and CYFRA 21-1 were found to distinguish clearly between the keratin fragment combinations K8/K18 (TPS) and K8/K19 (CYFRA 21-1). TPA and TPACYK reacted with both combinations, however, with different intensities. On immunoblots the CYFRA 21-1 antibodies reacted exclusively with K19, whereas the antibodies of the other assays reacted with at least 2 of the keratins investigated.

CYFRA 21-1. A new marker in lung cancer.

BACKGROUND: It is known that cytokeratin 19 is particularly abundant in carcinoma of the lung. METHODS: A sandwich enzyme-linked immunosorbent assay called CYFRA 21-1 was, therefore, developed to detect soluble cytokeratin 19 fragments in serum using two specific monoclonal antibodies (Ks 19.1 and BM 19.21). The authors investigated the clinical significance of this new marker compared with the established markers carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC), neuron-specific enolase (NSE), carbohydrate antigen (CA) 19-9, CA 125, CA 15-3, CA 72-4, alpha-fetoprotein, and prostate-specific antigen in a pilot study on 1741 serum samples from patients with various benign and malignant diseases. RESULTS: Postulating a specificity of 95% versus benign diseases of the lung, the diagnostic sensitivity of CYFRA 21-1 in lung cancer (independent of histologic type) at primary diagnosis was superior (47%) to CEA (27%), SCC (15%), and NSE (16%). Especially in squamous cell carcinomas of the lung, the true-positive test results were much higher for CYFRA 21-1 (60%) than for CEA (18%) or SCC (31%). CONCLUSIONS: In small cell lung carcinomas, NSE was confirmed as the marker of first choice. For all of the other solid tumors investigated, CYFRA 21-1 showed no better profile of specificity and sensitivity than the established markers.

Cytokeratin 19 fragments: a new marker for non-small-cell lung cancer.

We developed a new and automated assay for the detection of lung cancer associated cytokeratin 19 fragments in patients' sera/plasma. This new tumour marker assay CYFRA 21-1 was evaluated in technical and clinical studies using the multibatch analysers ES 300 and ES 600 from Boehringer Mannheim GmbH. The analytical performance was shown to be excellent. The clinical data from 2,037 patients demonstrate that for non-small-cell lung carcinoma CYFRA 21-1 has a higher diagnostic sensitivity compared to the established markers. Mainly for squamous cell carcinoma CYFRA 21-1 was superior (60%) to CEA (18%) or SCC (31%).

Multicenter evaluation of Iso-ALP test kit for measurement of bone alkaline phosphatase activity in serum and plasma.

A test kit (Iso-ALP, Boehringer Mannheim) for measuring human bone alkaline phosphatase activity in serum or plasma was evaluated in five laboratories in three countries. The assay is based on the principle described by Rosalki and Foo (Clin Chem 1984;30:1182-6) and uses wheat germ lectin to precipitate bone alkaline phosphatase. Residual ALP in the supernate in comparison with total ALP is used to quantify the bone fraction. The imprecision of residual ALP measurement was low (median between-run CV 4.9%) and comparable with that of total ALP. Linearity of precipitation was demonstrable up to a bone ALP activity (diethanolamine buffer 37 degrees C) of 2000 U/L, though a matrix effect was observed for dilutions of high-activity sera in saline or bovine serum albumin. For assaying patients' samples, different batches of lectin demonstrated excellent comparability. Taking electrophoresis as a basis for standardization, we determined that the lectin precipitated approximately 90% of bone ALP, but < 5% of nonbone ALP. From this we derived serum/plasma upper reference limits for bone ALP activity in adults and children.

1H-nuclear magnetic resonance studies of the neuropeptide head activator.

The 1H-NMR spectrum of the neuropeptide head activator in aqueous solution has been completely assigned by two-dimensional NMR spectroscopy and selective deuteration. The apparent pseudo-first-order exchange rate, kex, of the backbone amide protons and the correspondent activation enthalpies, delta H not equal to, were determined. The exchange rates decrease and the activation enthalpies increase from the N-terminal to the C-terminal part of the peptide. The exchange rates vary from 21 to 0.3 s-1 at 274 K, the activation enthalpies from 60 to 75 kJ.mol-1. The pK values of the terminal carboxyl group and of the lysine amino group have been estimated as 3.3 and 10.3, respectively. The NMR results are in line with a dimeric structure in an antisymmetric arrangement of the subunits, forming an antiparallel beta-pleated sheet between C-terminal segments. The peptide bonds between pGlu-1, Pro-2 and Pro-3 are predominantly in trans-configuration, in fact no cis-isomers can be observed spectroscopically. The structure appears to be very stable; in the temperature and pH range studied, i.e., from 274 to 338 K and from pH 0.8 to pH 11.6, there are no spectroscopic indications for a global structural change.

Elevated levels of head activator in human brain tumors and in serum of patients with brain and other neurally derived tumors.

In normal human tissue high concentrations of the neuropeptide head activator are found in the hypothalamus, in the retina, and in the gastro-intestinal tract. Up to 100-fold elevated levels of head activator over neighbouring tissue were found in tumors of the brain, especially in tumors of neural origin like astrocytoma and glioblastoma, but also in meningioma. Coincident with elevated tissue levels, an increased secretion into the general circulation was observed. Elevated levels of head activator in the blood were also observed in patients with tumors in peripheral locations, especially in tumors of gastrointestinal tract and/or of neuroendocrine origin. After tumor removal, the head activator levels in the blood dropped to normal values suggesting a possible role of head activator in neuroendocrine tumorigenesis.

Synthesis of new head-activator analogues and their application for improved radioimmunoassays.

New analogues of head activator were produced for receptor and radioimmunoassay studies. The precursor molecules [(4'-I)Phe11] head activator and [Tyr11] head activator were synthesised for catalytic tritiation and iodination, respectively. With the tracer [(3,5-125 I2)Tyr11] head activator the sensitivity range of the radioimmunoassay was 5-100 fmol.

The neuropeptide head activator loses its biological acitivity by dimerization.

On molecular sieve columns the neuropeptide head activator elutes at two distinct positions corresponding to apparent mol. wts of 700 and 1400 daltons. The low mol. wt component is stable only under high ionic conditions and represents the monomeric state of the head activator. Only this form is biologically active. The higher mol. wt component, which is reapidly formed under physiological conditions, is the dimeric head activator and is biologically inactive. We suggest that this dimerization is of biological relevance as a mechanism for inactivation of neuropeptides.