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...

Purpose: Inconsistent reports on the detection of melanoma cells in peripheral blood by reverse transcriptase-PCR (RT-PCR) have resulted in uncertainty on the prognostic value of circulating melanoma cells.Experimental Design: We developed real-time RT-PCR assays for quantitation of tyrosinase, MelanA/MART1, and gp100 and for porphobilinogen deaminase housekeeping gene. Melanoma tissue (n ‫؍‬ 18), peripheral blood samples from healthy donors (n ‫؍‬ 21), and patients with cutaneous (n ‫؍‬ 122) and uveal (n ‫؍‬ 64) melanoma from our institution were analyzed. For quality control, an additional 251 samples from ongoing multicenter studies were compared with in-house samples.Results: Tyrosinase was not detected in healthy donor blood samples. For the two other markers, cutoff values had to be defined to distinct patient samples from controls. Patients with stage IV uveal and cutaneous melanoma expressed all three markers more frequently and at higher levels in peripheral blood as compared with earlier stages. The variation of expression was 4 logs and correlated with tumor load and serum lactate dehydrogenase. In 2 of 3 uveal melanoma patients, detection of circulating tumor cells preceded the development of liver metastases. The diagnostic sensitivity was optimal in blood samples containing >0.1pg/l porphobilinogen deaminase (95.7% of in-house samples and 57.4% of multicenter samples).Conclusions: Real-time RT-PCR is able to quantitatively define the quality of a sample and provides quantitative data for melanoma markers. Disparities in the results of previous studies may be attributable to undetected differences in sample quality. The prognostic relevance of this assay is currently under evaluation in several prospective randomized trials.

1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (MeQDO) was purified from quinaldine-grown Arthrobacter sp. Ru6la. It was enriched %fold in a yield of 22%, and its properties were compared with 1 H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO) purified from Pseudomonas putida 3311. The enzyme-catalyzed conversions were performed in an ('80)0,/('60)0, atmosphere. Two oxygen atoms of either ('*O)O, or (l60)O, were incorporated at C2 and C4 of the respective substrates, indicating that these unusual enzymes, which catalyze the cleavage of two carbon-carbon bonds concomitant with CO formation, indeed are 2,4-dioxygenases. Both enzymes are small monomeric proteins of 32 kDa (MeQDO) and 30 kDa (QDO). The apparent K,, values of MeQDO for 1 H-3-hydroxy-4-oxoquinaldine and QDO for lH-3-hydroxy-4-oxoquinoline were 30 pM and 24 pM, respectively. In both 2,4-dioxygenases, there was no spectral evidence for the presence of a chromophoric cofactor. EPR analyses of MeQDO did not reveal any signal that could be assigned to an organic radical species or to a metal, and Xray fluorescence spectrometry of both enzymes did not show any metal present in stoichiometric amounts. Ethylxanthate, metal-chelating agents (tiron, n,d-bipyridyl, 8-hydroxyquinoline, o-phenanthroline, EDTA, diphenylthiocarbazone, diethyldithiocarbamate), reagents that modify sulfhydryl groups (iodoacetamide, N-ethylmaleimide, p-hydroxymercuribenzoate), and reducing agents (sodium dithionite, dithiothreitol, mercaptoethanol j either did not affect 2,4-dioxygenolytic activities at all or inhibited at high concentrations only. With respect to the supposed lack of any cofactor and with respect to the inhibitors of dioxygenolytic activities, MeQDO and QDO resemble aci-reductone oxidase (CO-forming) from Klebsiellu pneumoniae, which catalyzes 1,3-dioxygenolytic cleavage of 1,2-dihydroxy-3-keto-S-methylthiopentene anion ( Chem. 270,[3147][3148][3149][3150][3151][3152][3153]. 1H-3-Hydroxy-4-oxoquinaldine and 1H-3-hydroxy-4-oxoquinoline were reactive towards molecular oxygen in the presence of the base catalyst potassiumtert.-butoxide in the aprotic solvent N,N-dimethylformamide. Base-catalyzed oxidation, yielding the same products as the enzyme-catalyzed conversions, provides a non-enzymic model reaction for 2,4-dioxygenolytic release of CO from 1 H-3-hydroxy-4-oxoquinaldine and 1 H-3-hydroxy-4-oxoquinoline.Keywords: carbon monoxide ; heterocyclic ring cleavage; 3-hydroxy-4( 1H)-quinolone(derivatives) ; 1 H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (decyclizing, CO-forming) ; 1 H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (decyclizing, CO-forming).Arfhrobacfer sp. strain Rii61 a and Pseudomonas putida strain 33/1 utilize quinaldine (2-methylquinoline) and 1H-4-0~0-quinoline, respectively, as sole source of carbon, nitrogen, and energy. In the gram-positive strain Rii61 a, initial hydroxylation

The fibroblast growth factor-binding protein (FGF-BP) binds and activates FGF-1 and FGF-2, thereby contributing to tumor angiogenesis. In this study, we identified novel binding partners of FGF-BP, and we provide evidence for a role of this protein in epithelial repair processes. We show that expression of FGF-BP increases after injury to murine and human skin, in particular in keratinocytes. This upregulation is most likely achieved by major keratinocyte mitogens present at the wound site.Most importantly, we demonstrate that FGF-BP interacts with FGF-7, FGF-10, and with the recently identified FGF-22, and enhances the activity of low concentrations of ligand. Due to the important functions of FGF-7 and FGF-10 for repair of injured epithelia, our findings suggest that upregulation of FGF-BP expression after injury stimulates FGF activity at the wound site, thus enhancing the process of epithelial repair.

Background:Tumour budding is an adverse prognostic indicator in colorectal cancer (CRC). Marked overall peritumoural inflammation has been associated with favourable outcome and may lead to the presence of isolated cancer cells due to destruction of invading cancer cell islets.Methods:We assessed the prognostic significance of tumour budding and peritumoural inflammation in a cohort of 381 patients with CRC applying univariate and multivariate analyses.Results:Patients with high-grade budding and marked inflammation had a significantly better outcome compared with patients with high-grade budding and only mild inflammation. Outcome in these cases, however, was still worse compared with cases with low-grade budding, in which the extent of peritumoural inflammation had no further prognostic effect.Conclusions:Tumour budding proved to be a powerful prognostic variable in patients with CRC. Scattering of invading cancer cell islets by marked overall peritumoural inflammation seems to have a minor role.

Abstract. Mixed adenoneuroendocrine carcinomas (MANECs) are rare biphasic tumour types, which are morphologically recognisable as both gland-forming and neuroendocrine neoplasms. Within the gastrointestinal tract, MANECs occur predominantly in the stomach or colorectum. The present study described a case of a MANEC originating from the ampullary region. The patient presented with widespread metastatic disease. Biopsy samples obtained from the ampullary primary tumour disclosed a complex lesion with adenocarcinoma and neuroendocrine small cell carcinoma components, positive for the intestinal transcription factor caudal type homeobox-2 and for neuroendocrine markers, including chromogranin A, synaptophysin, cluster of differentiation 56/neural cell adhesion molecule. By contrast, biopsy samples obtained from metastatic tissue revealed pure neuroendocrine carcinoma. As exemplified by this true mixed tumour, tumour heterogeneity evolves as the major challenge in oncology today, with potentially severe implications for the choice of chemotherapy. The assessment of metastatic sites may render valuable diagnostic information that is crucial for clinical decision-making and patient management.

For the molecular detection of rare tumour cells in clinical samples, real-time reverse transcription-polymerase chain reaction (RT-PCR) offers two important advantages over conventional RT-PCR assays: the results are quantitative and, perhaps more importantly, it facilitates exact sensitivity controls on a per sample basis as well as exact comparison of different assay protocols. We report here on quantitative results obtained with different protocols for RNA isolation and cDNA synthesis for amplification of beta2-microglobulin transcripts using the light cycler system. Furthermore, housekeeping gene-specific PCRs were compared with PCRs specific for an artificial transcript (internal standard) detected simultaneously at a level comparable to the wild-type sequence. Artificial tyrosinase transcripts derived from a vector construct stably transfected into a human lymphoma cell line were used as a model to test the usefulness of artificial internal standards as an alternative to housekeeping genes. The highest RNA yields were obtained using a combination of phenol-chloroform extraction and the High Pure RNA Isolation Kit. Analysing beta2-microglobulin transcript-specific RT-PCRs, the highest sensitivity was obtained for cDNAs generated with Omniscript reverse transcriptase and oligo-p(dT)15 primer. Regarding patient blood samples, RT-PCRs specific for beta2-microglobulin, porphobilinogen deaminase and artificial tyrosinase transcripts provided quantitative data for all, for 18 out of 21, and for 10 out of 21 samples, respectively. Quantification of beta2-microglobulin transcripts by the light cycler system defined the protocol revealing the highest cDNA quality. Comparisons of quantitative data from RT-PCRs specific for beta2-microglobulin, porphobilinogen deaminase and artificial tyrosinase transcripts enabled us to determine a close range for crossing points within which sufficient cDNA quality can be guaranteed, even for the detection of rare transcripts. PCRs specific for the artificial internal standard are ideally suited for cDNA quality assessment on a per sample basis.

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