Signal transduction and modulation represent central mechanisms in cellular processes such as cell-cycle regulation, oncogenesis, and apoptosis. The aim of this study was to determine the prognostic relevance of two kinases important in the regulation of cell proliferation and apoptosis in 135 colorectal cancer cases: AKT and extracellular regulated kinases (ERK1/2). We investigated the relationship of phospho-ERK1/2 (pERK1/2) and phospho-AKT (pAKT) with associated parameters (EGFR, COX-2, cyclin-D1), proliferative activity (Ki-67), and apoptosis (TUNEL) using immunohistochemistry. Additionally, the k-ras gene was screened for mutations to determine its putative association with ERK1/2 activation. Activation of ERK1/2 but not AKT correlated statistically with the presence of k-ras mutations (P = 0.015). Survival analysis of phospho-ERK1/2 immunoexpression showed a significant correlation with decreased overall survival (OS). The multivariate Cox regression analysis identified pERK1/2 as an independent prognostic parameter (P = 0.005). Activation of ERK1/2 in colorectal cancer may indicate aggressive tumor behavior and may constitute an independent prognostic factor. Furthermore, our data suggest that mutations of the k-ras oncogene may induce activation of ERK1/2. We propose immunohistochemical determination of pERK1/2 status as a promising candidate for the identification of high-risk patients who would benefit from new anticancer drugs targeting the ERK pathway.

Purpose: Signaling via the G protein G alpha s pathway is linked to proapoptotic processes in cancer cell lines. We have recently shown an association between the GNAS1 T393C polymorphism and disease progression in patients with bladder cancer with homozygous TT genotypes displaying increased transcription of Gas and a more favorable clinical course compared with C-allele carriers.Experimental Design: In the present study, 151 patients with sporadic colorectal cancer were retrospectively genotyped to examine a potential association between T393C genotypes and survival. Moreover, two other single-nucleoticle polymorphisms in common haplotype blocks within the gene GNAS1 and their interaction with the T393C polymorphism were investigated.Results: The allele frequency in the patients group was not significantly different from that of healthy blood donors. Kaplan-Meier curves for overall survival (mean follow-up, 43 months) showed that in International Union Against Cancer (UICC) stages I to 11, the 5-year survival rate was significantly higher in TT genotypes (87.8%) compared with TC (71.0%) and CC genotypes (50.0%; P = 0.009), whereas no genotype effect could be observed for UICC stages III to IV. In multivariate Cox proportional analysis the T393C polymorphism was an independent prognostic factor for survival. Homozygous CC patients were at highest risk for death (hazard ratio, 12.1; P = 0.006) compared with TT genotypes. Heterozygous patients had an intermediate risk compatible with a gene-dose effect. The two haplotype blocks investigated were not associated with clinical outcome.Conclusions: The results support the role of the T393C polymorphism as a marker for survival in patients with colorectal cancer stages I to 11 and in the identification of patients who may benefit from adjuvant chemotherapy.

Current processing of renal biopsy samples provides limited information about immune mechanisms causing kidney injury and disease activity. We used flow cytometry with transplanted kidney biopsy samples to provide more information on the immune status of the kidney.To enhance the information available from a biopsy, we developed a technique for reducing a fraction of a renal biopsy sample to single cells for multicolor flow cytometry and quantitation of secreted cytokines present within the biopsy sample. As proof of concept, we used our technique with transplant kidney biopsy samples to provide examples of clinically relevant immune information obtainable with cytometry.A ratio of CD8+ to CD4+ lymphocytes greater than or equal to 1.2 in transplanted allografts is associated with rejection, even before it is apparent by microscopy. Elevated numbers of CD45 leukocytes and higher levels of interleukin (IL)−6, IL-8, and IL-10 indicate more severe injury. Antibody binding to renal microvascular endothelial cells can be measured and corresponds to antibody-mediated forms of allograft rejection. Eculizumab binding to endothelial cells suggests complement activation, which may be independent of bound antibody. We compared intrarenal leukocyte subsets and activation states to those of peripheral blood from the same donor at the time of biopsy and found significant differences; thus the need for new techniques to evaluate immune responses within the kidney.Assessment of leukocyte subsets, renal microvascular endothelial properties, and measurement of cytokines within a renal biopsy by flow cytometry enhance understanding of pathogenesis, indicate disease activity, and identify potential targets for therapy.

BACKGROUND:Patients with extensive, long-standing ulcerative colitis have increased risk of colorectal cancer. AIMS:To improve the detection of high-risk patients, using a combination of colonic cytology, histology, and DNA image cytometry after segmental colonic lavage. PATIENTS:A series of 16 patients (8 high-risk patients) with ulcerative colitis were investigated. METHODS:After segmental lavage step, biopsies were obtained. Gradient centrifugation of the colonic fluid was performed for isolation and purification of epithelial cells. The smears and biopsy specimens obtained were stained for routine interpretation and for DNA image cytometry. RESULTS:Segmental lavage could be performed in all patients. Specimens from two high-risk patients showed low grade dysplasia and atypia by means of histology and cytology, respectively. In one patient, without increased colorectal cancer risk, atypia was detected. Three patients in the high-risk group, two of those diagnosed as positive for dysplasia and atypia, showed aneuploidy histologically and cytologically. DNA aneuploidy, in cytological material, was found exclusively in three low-risk patients, one of those had atypia cytologically. CONCLUSIONS:Isolation and purification of epithelial cells after segmental colonic lavage using density gradient centrifugation can be performed as part of routine endoscopy. It provides information about atypical cells and DNA aneuploidy as additional markers of malignant transformation. The combination of cytologic examination and DNA image cytometry might improve the detection of high-risk ulcerative colitis patients.

BACKGROUND AND STUDY AIMS:Various methods of fluorescence excitation and detection have been developed in gastrointestinal endoscopy. This study reports an endoscopic technique using locally applied fluorescein-labeled antibodies for in-vivo detection of colorectal dysplasia and carcinoma. PATIENTS AND METHODS:Fluorescence endoscopy with a fluorescein-labeled monoclonal antibody against carcinoembryonic antigen (CEA) was carried out in 27 patients with colonic polypoid lesions. During conventional colonoscopy, the monoclonal antibody was applied directly onto the mucosal surface. After an incubation time of 10 min, specific fluorescence was visualized with a conventional endoscope whose optical range was increased via two narrow-band filters. RESULTS:Fluorescence in vivo was present in 19 out of 25 carcinomas and in three of eight adenomas. The technique failed in the presence of mucosal ulceration or bleeding. One fluorescence-positive villous adenoma showed high-grade dysplasia, and another fluorescence-positive polypoid lesion was diagnosed as carcinoma in adenoma. Normal-appearing mucosa was fluorescence-negative in all cases. Endoscopic fluorescence significantly correlated with the CEA expression of luminal epithelial cells as determined immunohistochemically (Wilcoxon-Mann-Whitney U-test, P < 0.01). In all cases without ulceration or bleeding, the specificity of fluorescence endoscopy was 100%, the sensitivity was 78.6%, and the accuracy was 89.3%. CONCLUSIONS:Fluorescence endoscopy using fluorescein-labeled monoclonal antibody against CEA was shown to be positive in most cancers and some adenomas. Further and larger studies will be needed to demonstrate the value of this technique for differential diagnosis.

The detection of gastrointestinal signet-ring cell carcinoma by endoscopy can be a diagnostic challenge. The main clinical features include atypical metastasis and a poor prognosis. We present a case of a metastasizing signet-ring cell carcinoma with unknown primary location arising in 71-year-old female. Following 6 cycles of a routine intravenous FU/FA chemotherapy, an almost complete remission could be observed. After 2 years of follow up, metastatic recurrence was detected to the lower back musculature. This case report emphasizes the difficulties in diagnosing signet-ring cell carcinoma by endoscopy and demonstrates an unusual clinical course.

We report the case of a 42-year-old male who was admitted to our hospital after an acute hypertensive crisis despite four-way anti-hypertensive therapy. The renal scintigraphy, the excretory urogram and the biochemical profile performed two years before were unremarkable, except for slightly elevated serum creatinine and plasma aldosterone, in presence of normal aldosterone/ renin ratio. The renal arterial angiography that was performed despite a second unremarkable scintigraphy revealed high-grade bilateral arterial stenosis in the presence of multiple renal arteries. Following dilatation of the left stenosis, the aldosterone/renin ratio was pathologic. Recumbent and orthostatic aldosterone values were 830 pg/ml and 1824 pg/ml, respectively, and recumbent and orthostatic renin values were 0.82 and 1.21 ng angiotensin I/ml/h, respectively. The abdominal computed tomography performed to investigate a possible concomitant Conn’s syndrome resulted in the detection of a left adrenal tumor. After resection of the lesion, plasma-aldosterone levels normalized and a pronounced rise in serum potassium levels was observed. Following angioplasty of the right renal artery stenosis, blood pressure could easily be managed with combined gB and calcium channel blocker therapy. Particularly in cases of bilateral (but also in the presence of unilateral) renal artery stenosis in association with Conn’s syndrome, all the available screening methods for these disorders can fail. In cases of poor response to combination hypertensive therapies, renal arteriography and a fludrocortisone- suppression test should be performed in order to rule out both renal arterial stenosis and Conn’s syndrome, even in the absence of clinical and biochemical findings suspicious for either disorder.

In more than 20% of subjects, at least one kidney is found to be supplied by more than one artery arising from the aorta. This aberrant renovascular anatomy has been reported in the literature to occur in up to 80% of patients who suffer from essential hypertension. Predominant numbers of the so-called 'accessory' vessels are longer and narrower than the segmental arteries arising in the main renal artery. As a result (in accordance with Poiseuille's law of fluid flow), the renal segments supplied by these 'accessory' vessels may have lower levels of blood pressure than the remainder of the parenchyma, thereby increasing the renin secretion. This hypothesis could be significant in terms of finding a causal treatment for a disorder induced by such a mechanism. We first review the literature in which the frequency of these vascular anomalies in normotensive and hypertensive patients is described, and then advance a hypothesis explaining the frequent incidence of essential hypertension in these subjects, as well as the ramifications of this phenomenon.

Most pheochromocytomas can be cured by resection. In view of the unfavourable prognosis for surgical therapy in cases of late tumour detection and malignant tumours, the aim of the present study is to differentiate between typical signs and symptoms of malignant versus benign pheochromocytomas. We investigated the records of 133 patients retrospectively (1967-1998). In cases of benign tumours (104 of 133, mean age 42+/-15.8 years) tumour size was 5.9+/-3.4 cm, and history was 47.4+/-75.4 months. 7.7% of the tumours were extraadrenal, and 77% had paroxysmal manifestations. The other 29 patients (mean age: 39.2+/-21.9 years) had malignant lesions (tumour size: 9.4+/-5.9 cm (p=0.0022); history: 7.4+/-5.6 months (p=0.0137); extraadrenal: 24.1% (p=0.0219); paroxysmal: 37.9% (p=0.0012)). Symptoms of patients with benign tumours were hypertension (80%), headaches (42.3%), sweating (30.8%), tachycardia (26%) and pallor (24%) (Malignant: Hypertension 46%, p=0.0873; headaches 11%, p=0.0008; sweating 11%, p=0.0196; tachycardia 14%, p=0.1961 and pallor 0%, p=0.0010). Abdominal pain and dorsalgia occurred more frequently in malignant pheochromocytomas (26% versus 7%, p=0.0014). Unusually short histories and extraadrenal localization appear to be suspicious for malignancy. The "typical" clinical signs and symptoms occur more frequently in patients with benign tumours and can therefore be regarded as typical signs of benign pheochromocytomas.

Nonsteroidal anti-inflammatory drugs (NSAIDs) can regress adenomas in patients with familial adenomatous polyposis (FAP), and the mechanism involves inhibition of cyclooxygenases (COX). Reactive intermediates formed during the arachidonic acid cascade, notably by COX-2, which is upregulated in polyps of FAP patients, may promote various stages of the polyp --> adenoma --> carcinoma sequence. Etheno-DNA adducts can be derived from reactive intermediates generated during arachidonic acid metabolism and lipid peroxidation. We tested this hypothesis in colonic polyps from FAP patients acid colorectal tissue from cancer patients to see whether increased formation of etheno-DNA adducts occurs. Using an ultra-sensitive and specific immunoaffinity/P-32-postlabelling method, I,N-6-ethenodeoxyadenosine (epsilon dA) and 3,N-4-ethenodeoxycytidine (epsilon dC) were quantitated in epithelial cell DNA from asymptomatic colon, FAP polyps and colon tumor tissues. Mean adduct levels in FAP polyps were 65 epsilon dA/10(9) and 59 epsilon dC/10(9) parent nucleotides, being 2 to 3 times higher than in unaffected colon tissue (p < 0.02 for epsilon dA; p < 0.05 for epsilon dC), Adduct levels in colonic epithelia decreased in the order: FAP polyps > tumor-adjacent tissue > tumor, normal and tumor-distal tissue. Based on this study, requiring confirmation in a larger number of patients and in experimental models, we have demonstrated the formation of promutagenic etheno-DNA adducts in adenomatous polyps of FAP patients that may contribute to genetic instability and cancer progression. Int. J. Cancer 87: 1-4, 2000. (C) 2000 Wiley-Liss, Inc.

The goal of this study was to improve assessment of diagnostic measures for lateral localization of aldosterone-producing adrenal adenomas preparatory to retroperitoneoscopic removal, in view of the fact that this technique allows for only unilateral access. A retrospective study was carried out of the medical records of 64 patients (38 women, 26 men, average age 46.8+/-11.2) who underwent surgery at University Hospital, Münster, between 1969 and 1998. Seventeen of the 64 patients presented with hyperplasia and 47 had adrenal adenoma. In cases of hyperplasia, computerized tomography imaged a false-positive unilateral tumor 10 times, a false-negative 3 times, and a unilateral hyperplasia 1 time (ultrasonography: tumor 2 times, false-negative 3 times; 131I-Iodomethylnorcholesterol scintigraphy: tumor 5 times, false-negative 1 time, correct 1 time). In cases of adenoma, computerized tomography yielded accurate results 40 times, imaged a false-negative 2 times, and indicated the incorrect side 1 time (Ultrasonography: false-negative 12 times, correct side 9 times, incorrect side 1 time; 131I-Iodomethylnorcholesterol scintigraphy: correct side 19 times, false-positive (both sides) 5 times, negative 3 times, incorrect side 2 times). Venous sampling, which was carried out seven times, yielded accurate results six times, and failed technically one time. Venous sampling appears to be the method of choice for preoperative lateral localization. Thus, retroperitoneoscopic treatment of Conn's syndrome should not be carried out unless venous sampling is carried out first.

BACKGROUND & AIMS:Glutamine is recognized as a conditionally essential amino acid. Recent studies indicate that glutamine-containing total parenteral nutrition improves nitrogen economy, enhances gastrointestinal and immune functions and shortens hospital stay.METHODS:Thirty-seven patients (19 w and 18 m; age 61. 4+/-10.4 years; BMI 23.7+/-2.8 kg/m(2)) following major abdominal surgery receiving an isonitrogenous isoenergetic TPN with or without alanyl-glutamine supplementation (0.5 g/kg BW/day), were evaluated in a double-blind, randomized, controlled trial over a five-day period by measuring nitrogen balance, selected biochemical parameters and length of hospital stay.RESULTS:Supplemental alanyl-glutamine improved the overall mean (-3.5+/-1.6 vs. -5.5+/-1. 4 g N;P<0.05) and cumulative nitrogen balance (-14.1+/-9.1 vs. -21.7+/-11.4 g N;P<0.05) compared with the isonitrogenous, isoenergetic standard regimen. Alanyl-glutamine normalized plasma glutamine concentration and reduced the length of hospital stay (12.8+/-2.6 vs. 17.5+/-6.4 days;P<0.05).CONCLUSIONS:The results of the study confirm that supplementation with synthetic alanyl-glutamine dipeptide is associated with cost containment due to shortened hospitalization and improved nitrogen economy.

OBJECTIVE:Subjects with multiple renal arteries have been shown to suffer more frequently from hypertension and to have higher blood pressures than subjects whose kidneys are supplied by single renal arteries. This study was carried out to determine whether subjects with multiple renal arteries also have higher renin activity. METHODS:We studied 62 consecutive patients who had undergone angiography for various reasons. They were divided into two groups. Group A comprised 29 patients whose kidneys were supplied by single arteries (male :female ratio 1.63, mean age 51.8 +/- 1.9 years) while Group B comprised 33 patients with multiple renal arteries (male:female ratio 2, mean age 47.3 +/- 2.3 years). RESULTS:Before stimulation with frusemide, the plasma renin in Group A was 0.79 +/- 0.13 ng angiotensin l/ml per h, while in Group B the corresponding figure was 1.73 +/- 0.38 ng angiotensin l/ml per h. This difference was statistically significant (P= 0.0127). Thirty minutes later the plasma renin level in Group A was 2.43 +/- 0.37 ng angiotensin l/ml per h versus a level of 3.86 +/- 0.53 ng angiotensin l/ml per h in Group B (P= 0.0169). Again, 30 minutes later the level was 2.59 +/- 0.4 ng angiotensin l/ ml per h in Group A, versus 3.79 +/- 0.59 ng angiotensin l/ ml per h in Group B (P= 0.0495). CONCLUSIONS:We conclude that patients with multiple renal arteries constitute a group who have high plasma renin activity and may therefore be prone to develop arterial hypertension.

Background: Evaluations of the tumor antigens CA 19-9 and CEA in gallbladder bile, even without analysing Lewis-blood-antigenitiy and the degree of jaundice, are superior to any serum and pancreatic juice examination with respect to sensitivity, specificity and positive predictive value of malignant lesions of the subhepatic pancreato-biliary system. Methods and Results: The combination of CA 19-9 and CEA proved sensitivities of 100% for bile duct carcinomas, 100 % for carcinomas of the papilla and 82.3 % for pancreatic carcinoma at a specificity of 100 %. Conclusion: CA 19-9 showed significant differences for the local tumor burden and for the degree of lymph node metastasis. Examination of tumor antigens in gallbladder bile results in a high degree of discrimination for malignant and benign lesions of the subhepatic pancreato-biliary system.

Patienten mit langjähriger Colitis ulcerosa (CU) haben ein erhöhtes Risiko, an einem kolorektalen Karzinom zu erkranken. Als Risikofaktoren gelten insbesondere das Vorhandensein einer Pancolitis, eine Erkrankungsdauer von mehr als 10 Jahren und das Auftreten von Dysplasien. Im Gegensatz zum sporadischen kolorektalen Karzinom (CRC), das sich aus Adenomen entwickelt, tritt das Colitis ulcerosa assoziierte Karzinom (CAC) häufig in jüngerem Alter auf, die präkanzerösen Läsionen bei CU sind sowohl endoskopisch als auch histologisch schwer zu erkennen. Zahlreiche molekulargenetische Veränderungen wurden sowohl in der Karzinomentstehung als auch in der Prognose des CRC und CAC beschrieben. Dabei besitzt das p53-Tumorsuppressorgen eine Schlüsselrolle [1]. So finden sich in beiden Karzinomentitäten häufig Mutationen des p53-Tumorsuppressorgens, wobei in der Karzinogenese des CAC die p53-Mutation zu einem früheren Zeitpunkt auftreten soll als beim CRC [2,3]. Eine Überexpression des bcl-2 Protoonkogens, welches der Apoptose entgegenwirkt und direkt durch p53 herunterreguliert werden kann, wurde sowohl in kolorektalen Adenomen als auch in Karzinomen häufig nachgewiesen [4], wogegen die Inaktivierung des p53 durch Komplexbildung mit dem Protoonkogen mdm-2 beim CRC eine eher untergeordnete Rolle spielt [5]. Die Induktion von waf-1 durch p53 spielt eine wichtige Rolle in der Zellzyklusregulation, wobei beim CRC eine veränderte waf-1 Expression unabhängig von einer p53-Inaktivierung zu sein scheint [6]. Ziel der vorliegenden Studie war es, ist die Bedeutung des p53 und der Protoonkogene mdm-2, waf-1 und bcl-2 in der Entwicklung des CAC zu untersuchen.

OBJECTIVES:Patients with extensive, long-standing ulcerative colitis (UC) have an increased risk for developing colorectal cancer. In this study, we wanted to establish a method for retrieving cytological material after segmental colonic lavage for further cytopathological investigations and for performing DNA image cytometry. METHODS:Ten patients with long-standing and extensive ulcerative colitis and 10 patients without macroscopic abnormalities were investigated. After segmental colonic lavage during routine colonoscopy a three-layer (1.146, 1.075, and 1.046 g/ml, respectively) density gradient centrifugation of the retrieved colonic fluid was performed for isolation and purification of the epithelial cells. For identification of the epithelial cells flow cytometry with monoclonal antibody against cytokeratin and counterstaining with propidium iodine was performed. The smears obtained were stained for routine cytopathological interpretation and for DNA image cytometry. RESULTS:In eight of 10 UC patients and in nine of 10 control group patients adequate cytological material could be obtained. The band on top of the density gradient at 1.046 g/ml could be identified as the epithelial cells. Atypical cells were found in smears of three UC patients. In these patients and in one additional patient aneuploid stemlines could be detected. In smears of control group patients neither atypical cells nor aneuploidy were present. CONCLUSIONS:Isolation and purification of epithelial cells after segmental colonic lavage by using density gradient centrifugation was performed. This cytological material is adequate for cytopathological interpretation and for DNA image cytometry. Information about atypical cells and DNA aneuploidy as an additional marker of malignant transformation in UC patients was obtained. The combination of cytological examination and DNA image cytometry might improve the detection of UC patients with high risk for colorectal cancer.

Background: The role of the tumorsuppressorgene p53 and the protooncogenes mdm-2, waf-1 and bcl-2 both in the development and prognostic outcome of sporadic colorectal carcinoma (CRC) has been well investigated. However,little is known about the role of these genes in the development of ulcerative colitis-associated colorectal carcinoma (CAC). Methods: Colectomy specimens of patients with CAC, patients with UC and dysplasia, patients with long-standing UC without carcinoma or dysplasia and patients with CRC were investigated by immunohistochemistry with antibodies against p53, mdm 2, waf 1 and bcl 2. Normal colon specimens (NC) from patients with diverticulosis without histological signs of inflammation were examined as controls. Expression of more than 20% of tumor cell nuclei or epithelial cell nuclei in nontumor specimens was considered positive for each antibody. Results: No substantial differences were observed between CAC and CRC. P53 was positive in about 50% of CRC and 60% of CAC. In dysplasia a positive expression of p53 was found in most HGD, but not in LGD. While mdm-2 and bcl-2 were only sporadically expressed, waf-1 was observed in the majority of specimens with high prevalence in UC without carcinoma or dysplasia. Conclusion: CAC is developing the same line as CRC in terms of expression of p53, mdm-2, waf-1 and bcl-2. The malignant potential of dysplasia in UC is partially confirmed by the investigated oncogenes. High expression of waf-1 in non malignant long-standing UC has to be proved in long-term course in it's role as an independent cancer risk factor in UC.