Microsatellite instability analysis: a multicenter study for reliability and quality control.

The molecular biology section of the Hereditary Non-Polyposis Colorectal Cancer study group-Germany, instituted a multicenter study to test the reliability and quality of microsatellite instability (MSI) analysis. Eight laboratories compared MSI analyses performed on 10 matched pairs of normal and tumor DNA from patients with colorectal carcinomas. A variety of techniques were applied to the detection of microsatellite changes: (a) silver and ethidium bromide staining of polyacrylamide gels; (b) radioactive labeling; and (c) automated fluorescence detection. The identification of highly unstable tumors and tumors without MSI was achieved in high concordance. However, the interpretation of the band patterns resulted in divergent classifications at several microsatellite marker loci for a large fraction of this tumor/normal panel. The data on more than 30 primers per case suggest that the enlargement of the microsatellite panel to more than 10 loci does not influence the results. In this study, cases with MSI in less than 10% of loci were classified as microsatellite stable, whereas MSI was diagnosed in cases with more than 40% of all markers unstable. We propose that a panel of five microsatellite loci consisting of repeats with different lengths should be analyzed in an initial analysis. When less than two marker loci display shifts in the microsatellite bands from tumor DNA, the panel should be enlarged to include an additional set of five marker loci. The number of marker loci analyzed as well as the number of unstable marker loci found should always be identified. These criteria should result in reports of MSI that are more comparable between studies.

Glutathione depletion in fibroblasts is the basis for apoptosis-induction by endogenous reactive oxygen species.

Our study aimed at clarifying the role of the intracellular concentration of reduced glutathione for induction of apoptosis in fibroblasts. Treatment of fibroblasts with buthionine sulfoximine (BSO) caused efficient depletion of intracellular reduced glutathione which was followed by substantial cell death. Based on the induction of membrane blebbing, chromatin condensation and DNA strand breaks, cell death was characterized as apoptosis. Apoptosis after glutathione depletion seemed to be induced by endogenous reactive oxygen species (ROS), as it was antagonized by the antioxidant catechol and the hydroxyl radical scavenger DMSO. Paracrine interaction between cells prevented ROS-induced apoptosis and therefore points to the existence of extracellular survival factors. Our data show that the apoptosis-inducing potential of endogenous ROS is controlled by the intracellular glutathione concentration and by paracrine survival factors.

Cells obtained from colorectal microadenomas mirror early premalignant growth patterns in vitro.

LT97, a permanent cell line consisting of epithelial cells with an early premalignant genotype was established from small colorectal polyps. LT97 cells have lost both alleles of the APC tumour suppressor gene. In addition, they carry a mutated Ki-ras oncogene, while TP53 is normal. LT97 growth characteristics are thus representative of early adenomas. They had to be passaged as multicellular aggregates indicating a dependency of survival on cell-cell contact and in accordance with their premalignant genotype were not capable of growth in soft agar. LT97 cells did express both the EGF-receptor and small amounts of TGF(alpha) establishing an autocrine growth or survival pathway. However, in spite of autocrine TGF(alpha) production, growth was strongly dependent on exogenous growth factors--mainly EGF, insulin and HGF. Inhibition of the EGF-receptor kinase induced apoptosis at an IC(50) concentration of 4 micromolar indicating that TGF(alpha) activated survival pathways in the early adenoma cell.

The importance of the effect of underlying disease on rejection outcomes following orthotopic liver transplantation.

Despite major advances in immunopharmacology, virtually all patients receive the same center-specific immunosuppressive regimen following orthotopic liver transplantation (OLT). The present analysis was performed on the hypothesis that the original disease representing the indication for OLT leads to a different initial immunological situation of the patient. The type of original disease might therefore be a predisposing factor for acute rejection episodes and influence graft and patient survival. From January 1988 to July 1994, 34 patients received OLT at our institution for end-stage primary biliary cirrhosis (group 1) and 66 patients for end-stage alcoholic cirrhosis (group 2). Overall survivals at 1 and 5 years in group 1 versus group 2 were 67% versus 80% and 50% versus 68%, respectively (P<0.04). Retransplantation was performed in 21% of patients from group 1 and in 6% from group 2. The estimated risk for freedom from acute rejection amounts to 38% in group 1 compared with 59% in group 2 (P<0.02). Multivariate regression analysis of potential risk factors identified only the underlying disease as independent predictor. Analysis of cumulative rates of clinically relevant rejection episodes stratified by group revealed 0.29 and 0.05 episodes per patient at one month and 0.80 and 0.06 at six months (P<0.009) respectively. In our clinical experience it was possible to confirm the hypothesis that the underlying disease is the reason for a significantly different incidence of acute rejection episodes and that it subsequently influences graft and patient survival. This approach to an individually adapted immunosuppressive therapy should be taken into consideration and other appropriate parameters investigated.

Oncogenic transformation increases the sensitivity for apoptosis induction by inhibitors of macromolecular synthesis.

Inhibition of RNA or protein synthesis causes apoptosis in fibroblasts. This points to the constitutive expression of a long-lived apoptosis machinery which is controlled by shortlived negative regulatory proteins, termed endogenous survival factors. The length of time between addition of the inhibitor of macromolecular synthesis and the onset of apoptosis can be used as an estimation of the effective survival factor concentration. Transformation of rat fibroblasts by a constitutively expressed src oncogene or an inducible ras oncogene increases the sensitivity for apoptosis induction by inhibitors of macromolecular synthesis, indicating that their endogenous survival factor pool has been decreased.

Ophthalmic and genetic screening in pedigrees with familial adenomatous polyposis.

PURPOSE: To study the possible association between ophthalmic findings, genetic status, and clinical course of the disease in Austrian pedigrees with familial adenomatous polyposis (FAP). METHODS: Thirty-nine members of 16 consecutive FAP families with 20 affected patients and 19 relatives with a 50% a priori risk to develop the disease were examined ophthalmologically. The intestinal status of all persons was established by colonoscopy. Direct or indirect molecular genetic analysis, or both, was possible in eight of the 16 FAP families. RESULTS: Congenital hypertrophy of the retinal pigment epithelium (CHRPE) was discovered in 15 (75%) of the 20 persons affected by familial adenomatous polyposis. Five (25%) of the patients with an established FAP were CHRPE-negative. Four of the 19 at-risk individuals were CHRPE-positive. According to DNA analysis, five of the 19 at-risk relatives had a high risk to develop a manifest disease. The ophthalmoscopic tests were in complete agreement with the molecular risk estimation. Furthermore, the combined results of endoscopy and ophthalmoscopy suggested a relationship between a positive CHRPE status and the severity of FAP. CONCLUSIONS: Ophthalmic examinations facilitate predictive diagnosis in FAP patients and first-degree relatives, permitting a noninvasive, highly reliable risk assessment. When present, CHRPE lesions are a reliable clinical marker for FAP in CHRPE-positive families. In CHRPE negative families, negative ophthalmic examinations are of no predictive value. The CHRPE status can add information about the location of the genetic mutation. The combination of an ophthalmic examination with DNA analysis and endoscopy improves the risk assessment of FAP carriers.

Three distinct roles for TGF-beta during intercellular induction of apoptosis: a review.

During intercellular induction of apoptosis, transformed fibroblasts are eliminated through the action of neighbouring nontransformed cells. TGF-beta thereby plays three distinct and central roles. a) TGF-beta released by transformed cells or added exogenously to the assay system triggers nontransformed cells to release a shortlived apoptosis inducing factor, which is specifically directed against transformed cells. b) TGF-beta is involved in the maintenance of the transformed state, which is required for expression of sensitivity for intercellular induction of apoptosis. c) TGF-beta further sensitizes transformed cells through downmodulation of their endogenous survival factors, which control a constitutively expressed apoptosis machinery. These data demonstrate that TGF-beta which is utilized by transformed fibroblasts for the maintenance of their transformed state, causes recognition of transformed cells by their nontransformed neighbours and triggers and enhances an apoptosis-inducing response which finally causes elimination of potential tumor cells.

Intercellular induction of apoptosis through modulation of endogenous survival factor concentration: a review.

Fibroblasts constitutively express a functional apoptosis machinery which is under negative control by operationally defined endogenous survival factors. Oncogenic transformation causes a marked downmodulation of endogenous survival factor concentration which renders transformed cells more sensitive to various apoptosis stimuli compared to their nontransformed counterparts. Endogenous survival factors can be inactivated by reactive oxygen species (ROS). Endogenous survival factors are the ultimate targets for apoptosis-inducing factors derived from TGF-beta-triggered nontransformed cells during intercellular induction of apoptosis. During this control step of oncogenesis, endogenous survival factors in transformed cells are inactivated by ROS and the apoptosis machinery is released from negative control. This mechanism leads to the specific elimination of transformed cells. Our data show that the transformed state causes both the ability of the cells to perceive the apoptosis-inducing signal and a decrease in the concentration of endogenous survival factors. These two mechanisms are of central importance for the regulation of intercellular induction of apoptosis.

Vegetable oil as an MR contrast agent for rectal applications.

The purpose of this study was to evaluate the feasibility of pure vegetable oil as an MR contrast agent for rectal applications. The hypothesis was that vegetable oil highlights the lumen of the rectum after rectal application as a positive contrast medium and offers additional contrast qualities using fat suppression techniques. Eleven MRI examinations were performed on 11 subjects (five healthy volunteers, all males, mean age 35 yr; and six patients, three males, three females, mean age 49 yr). Peanut oil, 200 ml, was applied rectally. In addition, 0.1 mmol/kg GD-DTPA was administered intravenously to the six patients only. Conventional T1-weighted SE sequences and T1-weighted SE images with fat suppression were obtained. Criteria for image evaluation were: overall image quality; uniformity of contrast distribution; chemical shift artifact; and delineation of the rectal wall. Side effects were assessed. There were no complaints reported by the 11 subjects. The image quality was sufficient in all studies. In all five of the volunteers and five of the six patients, the distribution of oil was uniform. Chemical shift artifacts did not deteriorate image quality. After rectal application of vegetable oil, the delineation of the rectal wall was sufficient with and without fat suppression techniques. Vegetable oil highlights the lumen of the rectum in MRI studies and offers additional contrast qualities with fat suppression techniques, acting as a positive as well as a negative contrast agent, depending on the chosen sequence.

Effects of nutrition on plasma, liver and muscle amino acids in scalded rats.

This study set out to investigate the effect of three different parenterally administered diets on the free amino acid (AA) levels in the plasma, muscle, and liver of scalded rats. Diet I consisted of AA (1.4 g/100 g weight) and a high glucose dose (6 g/100 g weight), diet II consisted of AA and a low glucose dose (1.4 g/100 g weight) and in diet III only a low glucose dose was infused. Parenteral nutrition was started on the 3rd day posttraumatically. Sampling was performed on the 7th day posttraumatically. Nitrogen balances were significantly different in all three groups, being lowest in group III. Scalded rats fed isonitrogenously, but with different amounts of glucose showed only minor changes in AA concentrations. However scalded rats fed with a nitrogen-free diet exhibited significantly reduced total muscle and liver AA levels. These decreased AA levels were due to a drop of glycine in the muscle tissue (74%) and liver (49%). Contrary to the clinical catabolic situation in scalded and starved rats, it was not intracellular glutamine but glycine which was considerably influenced by catabolism and starvation.

[Significance of emergency endoscopy in severe upper gastrointestinal hemorrhage]. / Die Bedeutung der Notfallendoskopie bei schwerer oberer gastrointestinaler Blutung.

312 of a total of 543 emergency endoscopies were carried out in patients with severe haemorrhage of the upper gastrointestinal tract. This was defined as a haemorrhage of such severity that at least 2 of the following 3 criteria were present: a shock index greater than 1, an erythrocyte count of less than 3 million/mm3, and a transfusion requirement of three or more 500 ml bags of blood. The source of the bleeding was exactly located in 247 endoscopies, and accurate diagnoses were established in 94.4% of the cases examined. The most frequent source of bleeding was oesophageal varices, followed by duodenal ulcers. In 20.2% of these cases, further sources of potential haemorrhage were found in the upper gastrointestinal tract. Endoscopic diagnosis resulted in immediate, specific therapy in 286 cases. Treatment was given within the first 24 hours in every case. 24.7% of our patients had to undergo laparotomy immediately after endoscopy. 30.4% were given H2 receptor inhibitors, and 35.6% underwent endoscopic haemostasis. The mortality rate in these patients was 29.5%. These results indicate that emergency endoscopy is an important aid to decision-making in cases of severe haemorrhage of the upper gastrointestinal tract.

[Incidence and regional distribution of colorectal cancers in Austria]. / Häufigkeit und regionale Verteilung der kolorektalen Karzinome in Osterreich.

Based on data obtained from the official Austrian cancer registry, we evaluated the incidence of, and death rate from cancer of the colon and rectum. The increase in the incidence of colorectal cancer in Austria is comparable to that of other western industrial countries. There is an estimated rise in the annual figures of newly registered patients from 3174 in 1971 to 3981 cases in 1981. Regional distribution within Austria is not homogeneous: the highest risk of disease was observed in Vienna (60 new cases per year per 100000 inhabitants), the lowest in Tyrol with 40 new cases per 100000. The marked east-west slope in the incidence of colorectal cancer in Austria is a good basis for the investigation of pathogenetic factors.

[Familial colon cancer]. / Das familiäre Kolonkarzinom.

5 families with autosomal dominant inherited colonic cancer were analyzed with reference to the characteristics of familial colonic cancer: 45% out of 54 family members had colorectal carcinomas, with a mean of 4.5 affected members per family. The mean age on diagnosis of colonic cancer was 43.9 years. 50% of the colorectal carcinomas were located in the right colon; 11 out of 14 carcinomas were Dukes C, with a predominance of poorly differentiated adenocarcinomas and mucin-producing cancers. 22% had synchronous cancers and 13% developed metachronous cancers. Cancer at another location was found in 8.7% of cases. Prospective screening of relatives (siblings, children) revealed a colonic neoplasm in 3 out of 6 asymptomatic subjects. The typical characteristics of familial colonic cancers were demonstrable in our patients. The elucidation of an exact family history in all patients with colorectal carcinoma and a strategy of meticulous surveillance are absolutely necessary for the diagnosis and management of this inherited trait.

[Results of molecular diagnosis in 30 Austrian families with familial adenomatous polyposis]. / Ergebnisse der molekularen Diagnostik bei 30 österreichischen Familien mit familiär adenomatöser Polyposis.

Familial adenomatous polyposis is a dominantly inherited precancerous condition of the colorectum. The isolation of the responsible gene has facilitated the search for mutation in affected individuals and risk estimation for family members. The aim of our study was the assessment of the disease by molecular biological methods in order to estimate the risk for family members. Blood probes from 30 non-related Austrian families (44 persons affected, 61 at risk) were examined for detection of a defect in the adenomatous polyposis gene by means of the protein truncation test and, if necessary, by linkage analysis. The protein truncation test led to successful identification of the defect gene in 66.7% (20/30 families). In 3 families, the presymptomatic difference between mutation carriers and healthy subjects could only be assessed by linkage analysis. Genetic diagnosis enabled us to detect the disease before the onset of clinical symptoms in 16 persons at risk, 37 could be identified as genetically healthy. In 8 persons at risk out of 5/30 families we were unable to identify a defect gene by the methods used until now. In conclusion, we have succeeded in establishing genetic diagnosis of familial adenomatous polyposis using the protein truncation test in Austria. Our method of genetic risk estimation is an important step in Austria towards earlier diagnosis and well-timed therapy management, and helps to exclude persons at risk who are genetically healthy from the laborious screening program.

[Management of severe infection complicating aortofemoral grafts]. / Management der tiefen Infektion als Komplikation nach Bifurkationsprothese.

OBJECTIVE: Infection of the retroperitoneum after implantation of an abdomino-femoral aortic graft remains one of the main problems in vascular surgery. On basis of a critical review of own experiences we evaluated the management of this difficult clinical situation. PATIENTS: From 1970-1996 1500 aortofemoral graft operations (aneurysmal disease: 512, aortoiliacal occlusive disease: 988) were performed. Abdominal infection occurred in 12 patients (0.8%) (12 men, median age 60.5 [48-80] years). RESULTS: The median interval between operation and infection was 17.7 (0.5-108) months. The port of infection was in 50% the groin, 25% suffered from abdominal infection, in 3 cases it was not to identify. Clinical manifestation of infection was aortoduodenal fistula in 2 patients, false aneurysms in 2 cases, and a paraprosthetic abscess in another 4 patients. Operative therapy comprised (partial) removal of infected material in 10 patients with consecutive extraanatomical reconstruction in 8 of these. Mortality of graft infection was 50%. Causes of death were untreatable sepsis in 4 patients, another 2 died from hemorrhagic shock. 3 out of 6 surviving patients finally lost their limbs following multiple vascular procedures. CONCLUSION: Adequate surgical therapy of infected aortofemoral grafts remains an unsolved problem. Lack of knowledge of suitable parameters for the best treatment leaves the outcome of prosthetic infection unpredictable. Removal of the infected graft with extraanatomic reconstruction seems to be the standard of surgical treatment, which is recommended in these cases.

[Determinants of pouch function after continence-preserving proctocolectomy]. / Determinanten der Pouchfunktion nach kontinenzerhaltender Proktokolektomie.

A prospective study was undertaken in 17 patients undergoing restorative proctocolectomy for ulcerative colitis (13 patients) or familial adenomatous polyposis (4 patients) to determine relationship between pre- and postoperative anal sphincter function, pouch characteristics and functional results. Postoperatively all manometric parameters were significantly reduced and remained so permanently. Only squeeze pressure rose to normal values again. The most important factor for a favourable functional outcome was pouch volume. A capacious reservoir was associated with a low stool frequency, low risk of incontinence and general success of the operation, as assessed subjectively. Perianal soreness with considerable skin problems occurred frequently when resting and squeeze pressures were markedly reduced postoperatively.

Preoperative short-term radiation therapy (25 Gy, 2.5 Gy twice daily) for primary resectable rectal cancer (phase II).

To evaluate the feasibility, effectiveness, and long-term bowel function of preoperative hyperfractionated accelerated radiotherapy in primary resectable rectal cancer. A total of 184 consecutive patients (median age 65 years, male : female=2 : 1) with clinical T3Nx rectal adenocarcinoma received preoperative pelvic radiation therapy with single fractions of 2.5 Gy twice daily (interval 6 h between fractions) to a total dose of 25 Gy within 1 week. Surgery was conducted the following week. Postoperative histology revealed UICC stage I in 33%, stage II in 26%, stage III in 34%, and stage IV in 7% of the patients. Median follow-up was 43 months (53 months for surviving patients). The actuarial 4-year-local-recurrence rate was 2.1%, overall recurrence 23%. Disease-specific and disease-free survivals at 4 years (excluding stage IV) were 82 and 69%, respectively. Overall survival for 4 years was 68%. Postoperative mortality was 0.5% (one patient), early anastomotic leakage occurred in 11.4%, and anastomotic stenosis requiring treatment in 6%, of 132 patients with primary anastomosis. Seven of 184 patients (3.8%) died of abdominal complications, all within the first year. Bowel function was satisfactory after more than 5 years. Local control in primarily resectable rectal cancer after 10 x 2.5 Gy is excellent, warranting further evaluation of this treatment.

ras oncogene expression determines sensitivity for intercellular induction of apoptosis.

Fibroblasts carrying an inducible ras oncogene acquire the transformed phenotype after oncogene induction. As a consequence, the transformed cells become sensitive to intercellular induction of apoptosis, a novel regulatory process directed by non-transformed fibroblasts against their transformed descendants. The causal relationship between oncogene expression and sensitivity to intercellular induction of apoptosis is based on extracellular superoxide anion production by oncogene-expressing cells. Superoxide anions (after dismutation to hydrogen peroxide) thereby foster HOCl synthesis and at the same time direct the selectivity of apoptosis induction through hydroxyl generation from HOCl. In parallel, ras expression enhances the sensitivity of fibroblasts for apoptosis-inducing stimuli like cycloheximide, ceramide and mitomycin C. This sensitization seems to be based on a decreased concentration of short lived endogenous apoptosis inhibitors. TGF-beta, like ras induction, decreases the concentration of endogenous apoptosis inhibitors, but does not induce the transformed phenotype. Therefore, TGF-beta treatment alone is not sufficient to render fibroblasts sensitive for intercellular induction of apoptosis, but TGF-beta treatment in parallel with ras activation enhances intercellular induction of apoptosis. Our findings demonstrate that Ras-mediated superoxide anion production determines sensitivity to intercellular induction of apoptosis, whereas the parallel decrease in endogenous apoptosis inhibitors modulates the kinetics of apoptosis induction.

Family history as a marker of risk for colorectal cancer: Austrian experience.

A family history of colorectal cancer is a known risk factor for the disease. As a result of different study designs from different populations, the strength of this association varies in the literature. We intended to define the incidence and the risk predictors in first-degree relatives of patients with colorectal cancer in the Austrian population. A family history was obtained from first-degree relatives of 100 consecutive patients with sporadic colorectal cancer. Life-table methods were used to compare the observed and expected incidence of colorectal cancer and the influence of differences in risk for first-degree family members. The calculated lifetime risk for colorectal cancer in Austria is 1:16. Individuals with a positive family history had a 4.6-fold risk (p