The detection of disseminated tumor cells in peripheral blood from colorectal cancer patients by RT-PCR could be an attractive method for selecting patients for adjuvant therapy. We here report on real-time RT-PCR assays (LightCycler) to quantitate potential mRNA markers. We investigated specimens from colon carcinoma and normal colon mucosa tissues, cell lines, blood samples from 129 patients with colorectal cancer (all stages) and 58 reference blood samples (healthy donors, persons suffering from inflammatory bowel or infectious diseases). The expression profile in tissues showed high values for CEA and CK20, whereas in cell lines ProtM was predominant. All markers were detected in reference and patient blood samples (ProtM, 22, 17%; CEA, 84, 86%; CK20, 85, 88%). After quantitative analysis, the definition of cutoff values for each marker and the combination of markers, 13% of patients were judged to have elevated marker concentrations in their blood, from which only 6 had values significantly differing from cutoff value. There were no differences between stages of disease. In the case of 19 patients, investigated prior to and 1 week after surgery, 2 samples revealed a significant postoperative increase in CEA or CK20 mRNA concentration. In spite of high expression levels in tissues and cell lines, we were not able to differentiate satisfyingly mRNA markers originating from tumor cells and those from illegitimate transcription in hematopoetic cells in blood. We conclude that either copy numbers of analyzed markers in circulating tumor cells are not sufficient for detection or, more probably, peripheral blood is not a suitable compartment for detection of tumor cells in colorectal cancer. © 2003 Wiley-Liss, Inc. Key words: colorectal cancer; real-time RT-PCR; minimal residual disease; carcinoembryonic antigene; cytokeratin 20; protease MThe molecular monitoring of circulating tumor cells by RT-PCR, routinely applied in patients with certain leukemias and lymphomas, 1-4 is still under debate for patients with solid malignancies. In colorectal cancer, where indication for adjuvant therapy without metastasis is yet performed by histologic investigation of lymph nodes, 5 the immunocytochemical identification of epithelial cells in bone marrow 6 encouraged the detection of epithelial mRNA markers in blood and bone marrow by RT-PCR. 7 However, a series of subsequent investigations by a number of groups with conventional nested PCR led to conflicting reports on both the frequency of gene transcripts in blood of patients and the specificity of the method. 8 -20 The recent availability of real-time PCR equipment has obviously changed the situation. 21-23 The quantification of low-level background transcription allows the definition of cutoff values for marker expression in blood and thus improves specificity. 16,22,24,25 Furthermore, the reliable quantification of housekeeping gene expression allows excellent quality control on a per-sample basis and relates absolute marker concentration to sample quality.We now devel...

There are numerous examples showing that the metabolism of cells can be severely impaired if the activity of only one of the participating enzymes undergoes large-scale alterations, resulting, for example, from spontaneous mutations (inherited or aquired enzymopathies), the administration of toxic drugs or self-inactivation of enzymes during cell aging. However, a quantitative relationship between the degree of enzyme deficiency and the extent of metabolic dysfunction is very difficult to establish by experimental means. An alternative is to tackle this problem by mathematical modelling. Our approach is based on a comprehensive mathematical model of the energy and redox metabolism for human erythrocytes. We calculate stationary states of the cell metabolism, varying the activity of each of the participating enzymes by several orders of magnitude. The metabolic states are then evaluated in terms of a performance function which relates the metabolic variables to the overall functional fitness of the cell. The performance function for the erythrocyte takes into account the homeostasis of three essential metabolic variables : the energetic state (ATP), the reductive capacity (reduced glutathione), and the osmotic state. Based on the behaviour of the performance function at varying enzyme activities, we estimate those ranges of enzyme activities, in which the metabolic alterations should be either tolerable, associated with non-chronic or chronic diseases, or letal. For most enzymopathies, the experimental and clinical observations can be satisfactorily rationalized by the computational results. Moreover, a surprisingly high correlation is found between the range of the activity range where disease is predicted by the model and the observed number of diseased probands.Another objective of our study was to contribute to the theory of metabolic control. The well-elaborated concept of the metabolic control theory is restricted to (infinitely) small activity alterations. In order to quantify the metabolic effect of finite (large-scale) changes in the activity of an enzyme, we propose, as a control measure, the effective activity Em, defined as the relative activity of an enzyme (with respect to the activity in a reference state) required to bring about a change in the stationary value of a metabolic variable by the (finite) factor a. We demonstrate that none of the existing extrapolation methods using the conventional control coefficient is capable to provide reliable predictions of the effective activities for all enzymes of erythrocyte metabolism.

A mathematical model of mammalian cell intermediary metabolism is presented. It describes the distribution of the carbon-13 isotope ("C) at the different carbon positions of metabolites in cells fed with "C-enriched substrates. The model allows the determination of fluxes through different metabolic pathways from and 'H-NMR spectroscopy and mass spectrometry data. The considered metabolic network includes glycolysis, gluconeogenesis, the citric acid cycle and a number of reactions corresponding to protein or fatty acid metabolism. The model was used for calculating metabolic fluxes in a rat tumor cell line, the C6 glioma, incubated with [l-'T]glucose.After evolution to metabolic and isotopic steady states, the intracellular metabolites were extracted with perchloric acid. The specific enrichments of glutamate, aspartate and alanine carbons were determined from ' T -, 'H-NMR spectroscopy, or mass spectrometry data. Taking into account the rate of glucose consumption and of lactate formation, determined from thc evolution of glucose and lactate contents in the cell medium, and knowing the activity of the hexose monophosphate shunt, it was possible to estimate the absolute values of all the considered fluxes. From the analysis the following results were obtained. The use of substrates enriched with carbon-13 ("C) in conjunction with "C-NMR spectroscopy has proved to be a valuable method for investigating the metabolism of living systems by analyzing the label distribution in their metabolites [l, 21. The approach has been successfully applied to the metabolic studies of normal and pathological biological systems, irz vitro [3] and in vivo [ l , 21. "C-NMR spectroscopy is therefore a potent technique for monitoring the regulation of the intermediary metabolism, or metabolic disfunctions under pathological situations. The prerequisite of such studies is to develop methods allowing the evaluation of metabolic fluxes from the NMR data, that requires, as for radioactive tracer experiments [4, 51, mathematical modelling of metabolism [6]. The ability of modelling to provide accurate yuantitativc information on metabolic pathways has already been proved in IT-NMR studies on microorganisms [7] cellular metabolism [lo, 12, 15 -171 and therefore, they are related to restricted metabolic networks. Other models were based on the analysis of the NMR data from only one metabolite, i.e. glutamate [13,14, 181. Despite its low sensitivity, an advantage of the IT-NMR technique is its non-specificity in metabolite detection, giving access to much information from a single spectrum recorded from a complex mixture. In experiments using "C-enriched substrates, each detected resonance may give further details on the fate of the label through the metabolism. Therefore, it is likely that the metabolic network of the model designed to analyze the experimental data should include as many pathways as possible, in connection with the wide range of the NMR-detected resonances.The purpose of the present work was to determine whether it is possi...

There are numerous examples showing that the metabolism of cells can be severely impaired if the activity of only one of the participating enzymes undergoes large-scale alterations, resulting, for example, from spontaneous mutations (inherited or aquired enzymopathies), the administration of toxic drugs or self-inactivation of enzymes during cell aging. However, a quantitative relationship between the degree of enzyme deficiency and the extent of metabolic dysfunction is very difficult to establish by experimental means. An alternative is to tackle this problem by mathematical modelling. Our approach is based on a comprehensive mathematical model of the energy and redox metabolism for human erythrocytes. We calculate stationary states of the cell metabolism, varying the activity of each of the participating enzymes by several orders of magnitude. The metabolic states are then evaluated in terms of a performance function which relates the metabolic variables to the overall functional fitness of the cell. The performance function for the erythrocyte takes into account the homeostasis of three essential metabolic variables : the energetic state (ATP), the reductive capacity (reduced glutathione), and the osmotic state. Based on the behaviour of the performance function at varying enzyme activities, we estimate those ranges of enzyme activities, in which the metabolic alterations should be either tolerable, associated with non-chronic or chronic diseases, or letal. For most enzymopathies, the experimental and clinical observations can be satisfactorily rationalized by the computational results. Moreover, a surprisingly high correlation is found between the range of the activity range where disease is predicted by the model and the observed number of diseased probands.Another objective of our study was to contribute to the theory of metabolic control. The well-elaborated concept of the metabolic control theory is restricted to (infinitely) small activity alterations. In order to quantify the metabolic effect of finite (large-scale) changes in the activity of an enzyme, we propose, as a control measure, the effective activity Em, defined as the relative activity of an enzyme (with respect to the activity in a reference state) required to bring about a change in the stationary value of a metabolic variable by the (finite) factor a. We demonstrate that none of the existing extrapolation methods using the conventional control coefficient is capable to provide reliable predictions of the effective activities for all enzymes of erythrocyte metabolism.

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Purpose: The aim of this study was to determine in patients with high-risk primary uveal melanoma whether the detection of circulating tumor cells by quantitative reverse transcription-PCR (RT-PCR) is of prognostic relevance. Experimental Design: Blood samples from 110 patients with high-risk nonmetastatic uveal melanoma were collected on the occasion of primary treatment or follow-up visit. mRNA expression of tyrosinase and MelanA/MART1 were analyzed by real-time RT-PCR and compared with clinical data at presentation and follow-up by univariate and multivariate analyses. Results: The RT-PCR assay yielded a positive result in 11 of 110 patients, with five positive findings for tyrosinase and five for MelanA/MART1, and one sample positive for both markers. At a median follow-up of 22 months, 25% of patients had developed metastases and 15% had died. Univariate statistical analysis revealed RT-PCR and the largest tumor diameter as important prognostic factors for the development of metastases and for survival. In a Cox proportional hazard model, RT-PCR result and largest tumor diameter predicted metastases (hazard ratios 7.3 and 2.6, respectively), whereas PCR result, largest tumor diameter, and Karnofsky performance status were significant variables for disease-specific survival (hazard ratios 22.6, 4.7, and 6.0, respectively). Analysis of individual RT-PCR results revealed both tyrosinase and MelanA/ MART1 transcripts as independent prognostic factors. Conclusion: The presence of tyrosinase or MelanA/MART1 transcripts is an independent prognostic factor in patients with high-risk primary uveal melanoma for subsequent development of metastases and for survival and can be used to select patients for adjuvant treatment studies.A major problem with uveal melanoma is the development of systemic metastases, which occur in up to 35% of cases even after successful treatment of the primary tumor (1 -6). The survival rate of patients with metastatic uveal melanoma remains poor with a median of between 2 and 9 months in spite of a variety of systemic therapeutic approaches (5, 7 -9). There is an urgent need to define risk factors for systemic metastasis to evaluate novel adjuvant treatments for high-risk patients.Several clinical, histologic, and genetic factors (e.g., tumor size and monosomy 3) have been identified as important risk factors for local recurrence, development of metastases, and survival (5, 6, 10 -15). However, histologic and genetic predictive factors are not usually identified because most patients with uveal melanoma are treated with radiotherapy (6,16,17). It is therefore necessary to rely on clinical features, such as largest basal tumor diameter, ciliary body involvement, and extraocular growth, which have firmly been established as important clinical prognostic factors by the Collaborative Ocular Melanoma Study (COMS; ref. 6).The detection of disseminated tumor cells in the blood by reverse transcription-PCR (RT-PCR) is particularly relevant to uveal melanoma, which metastasizes early and ex...

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