Promoter hypermethylation of SHOX2 and SEPT9 is a potential biomarker for minimally invasive diagnosis in adenocarcinomas of the biliary tract.

BACKGROUND: Biliary tract carcinoma (BTC) is a fatal malignancy which aggressiveness contrasts sharply with its relatively mild and late clinical presentation. Novel molecular markers for early diagnosis and precise treatment are urgently needed. The purpose of this study was to evaluate the diagnostic and prognostic value of promoter hypermethylation of the SHOX2 and SEPT9 gene loci in BTC. METHODS: Relative DNA methylation of SHOX2 and SEPT9 was quantified in tumor specimens and matched normal adjacent tissue (NAT) from 71 BTC patients, as well as in plasma samples from an independent prospective cohort of 20 cholangiocarcinoma patients and 100 control patients. Receiver operating characteristic (ROC) curve analyses were performed to probe the diagnostic ability of both methylation markers. DNA methylation was correlated to clinicopathological data and to overall survival. RESULTS: SHOX2 methylation was significantly higher in tumor tissue than in NAT irrespective of tumor localization (p < 0.001) and correctly identified 71% of BTC specimens with 100% specificity (AUC = 0.918; 95% CI 0.865-0.971). SEPT9 hypermethylation was significantly more frequent in gallbladder carcinomas compared to cholangiocarcinomas (p = 0.01) and was associated with large primary tumors (p = 0.01) as well as age (p = 0.03). Cox proportional hazard analysis confirmed microscopic residual tumor at the surgical margin (R1-resection) as an independent prognostic factor, while SHOX2 and SEPT9 methylation showed no correlation with overall survival. Elevated DNA methylation levels were also found in plasma derived from cholangiocarcinoma patients. SHOX2 and SEPT9 methylation as a marker panel achieved a sensitivity of 45% and a specificity of 99% in differentiating between samples from patients with and without cholangiocarcinoma (AUC = 0.752; 95% CI 0.631-0.873). CONCLUSIONS: SHOX2 and SEPT9 are frequently methylated in biliary tract cancers. Promoter hypermethylation of SHOX2 and SEPT9 may therefore serve as a minimally invasive biomarker supporting diagnosis finding and therapy monitoring in clinical specimens.

Losses at chromosome 4q are associated with poor survival in operable ductal pancreatic adenocarcinoma.

Here we tested the prognostic impact of genomic alterations in operable localized pancreatic ductal adenocarcinoma (PDAC). Fifty-two formalin-fixed and paraffin-embedded primary PDAC were laser micro-dissected and were investigated by comparative genomic hybridization after whole genome amplification using an adapter-linker PCR. Chromosomal gains and losses were correlated to clinico-pathological parameters and clinical follow-up data. The most frequent aberration was loss on chromosome 17p (65%) while the most frequent gains were detected at 2q (41%) and 8q (41%), respectively. The concomitant occurrence of losses at 9p and 17p was found to be statistically significant. Higher rates of chromosomal losses were associated with a more advanced primary tumor stage and losses at 9p and 18q were significantly associated with presence of lymphatic metastasis (chi-square: p = 0.03, p = 0.05, respectively). Deletions on chromosome 4 were of prognostic significance for overall survival and tumor recurrence (Cox-multivariate analysis: p = 0.026 and p = 0.021, respectively). In conclusion our data suggest the common alterations at chromosome 8q, 9p, 17p and 18q as well as the prognostic relevant deletions on chromosome 4q as relevant for PDAC progression. Our comprehensive data from 52 PDAC should provide a basis for future studies with a higher resolution to discover the relevant genes located within the chromosomal aberrations identified.

Modern therapy of rectal carcinoma.

Throughout the past decade the treatment of rectal carcinoma has improved remarkably. Today, individualized multimodality treatment allows local and distant tumor freedom with preservation of anorectal and genitourinary function in a majority of patients. Radiotherapy is elementary in reducing the risk of local recurrence whereas chemotherapy including promising novel agents prevents or eliminates distant metastases. However, surgery revolutionized by TME (total mesorectal excision) remains the only curative treatment for rectal carcinoma. In this study the authors review the developments as well as the current status of modern treatment for rectal carcinoma.

Interdisciplinary treatment of primary hepatic angiosarcoma: emergency tumor embolization followed by elective surgery.

Among primary hepatic malignancies, sarcomas represent a minority of 2 %. Of those, primary hepatic angiosarcoma is the most common one. In the past its incidence has been related to the exposure of certain chemicals like thorotrast, vinyl-chloride or arsenic. - Patients suffering from this aggressive, highly vascular tumor have a poor prognosis in general. Without treatment most of them die after rapid tumor progression with multifocal dissemination. In case of tumor perforation, fatal abdominal hemorrhage has been observed. - We herein report the successful interdisciplinary treatment of an 81 year-old woman with a perforated primary hepatic angiosarcoma of the left hepatic lobe. Initially, tumor bleeding was stopped by emergency interventional coil embolization. After stabilization of the patient, we performed an elective tumor resection. The patient could eventually be discharged in a good clinical condition. - So far, no standard therapy has established for patients with primary hepatic angiosarcoma. Surgery seems to be the treatment of choice. In addition, preoperative interventional embolization of the tumor supplying vessels reduces the risk of pre- and intraoperative bleeding. The value of adjuvant chemotherapy is not yet clarified. - The outcome of most patients with primary hepatic angiosarcoma remains poor and there is a need for clinical studies.

Current status of radical systematic lymphadenectomy in pancreatic cancer--a review of the literature.

BACKGROUND: Pancreatic cancer is the fourth most common cause of death in malignancies with an incidence of 8-12 cases per 100000 in western world. In spite of numerous modifications in therapeutical approaches, prognosis has not improved. METHODS: In the last few years numerous studies have been performed to reduce tumor mortality with more radical surgical procedures. Several articles of the last 15 years have been investigated to objectivate the benefit of extended lymphadenectomy in pancreatic surgery. Staging of the cancers, prognostic factors, technique and interpretation of lymphadenectomy have been analysed RESULTS: All studies document a lowered perioperative mortality in pancreatic resections. The procedure is counted as a standardized and safe one. However, several controversies exist. The distinct staging systems in Japan and the western world aggravate the comparison in all studies. Japanese authors in mostly retrospective analyses seem to document a survival benefit by radical surgery. Similar results could not be achieved by western authors. CONCLUSION: Over all, a significant benefit in extreme radical surgery could not bee found. However, there are indications of subgroups of patients in whom extended lymphadenectomy might be beneficial. This subgroup should be defined only by large multicentric, prospective, randomized studies.

Stereoselectivity of noradrenaline uptake into synaptic vesicles of the rat brain.

To investigate the stereoselectivity of the ATP-Mg2+-dependent uptake of noradrenaline, synaptic vesicles were isolated from the rat brain by differential centrifugation and incubated with 3H-(+/-)-, 3H-(-)- or 14C-(+)-noradrenaline in the absence and in the presence of ATP-Mg2+. The Km values of the ATP-Mg2+-dependent uptake were found to be different for the two isomers (mumol/l): 3H-(+/-)-noradrenaline 14.9 +/- 2.2 x 10(-1), 3H-(-)-noradrenaline 7.7 +/- 0.5 x 10(-1), 14C-(+)-noradrenaline 17.3 +/- 3.7 x 10(-1), whereas the Vmax of the racemate was identical with those of the two isomers (pmol/mg protein/min): 3H-(+/-)-noradrenaline 5.5 +/- 0.4, 3H-(-)-noradrenaline 4.9 +/- 0.1, 14C-(+)-noradrenaline 5.1 +/- 0.4. Moreover, (+)-noradrenaline inhibited competitively the ATP-Mg2+-dependent uptake of 3H-(+/-)-noradrenaline (Ki 19.2 +/- 1.0 x 10(-1) mumol/l) and 3H-(-)-noradrenaline (Ki 17.7 +/- 1.8 x 10(-1) mumol/l), the Ki values being nearly identical with the Km of the ATP-Mg2+-dependent uptake of 14C-(+)-noradrenaline. It is concluded that the ATP Mg2+-dependent uptake of noradrenaline into synaptic vesicles of the rat brain is stereoselective and that both isomers share the same transport system.

Increase of circulating beta-endorphin-like immunoreactivity correlates with the change in feeling of pleasantness after running.

Twenty-one male regular long distance runners participated in two 10 km runs one week apart. Their beta-endorphin-like immunoreactivity (beta-EIR) was assayed in plasma before and immediately after running. Mood was monitored by an adjective check list (Eigenschaftswörterliste, EWL) pre- and post-run. beta-EIR was significantly elevated post-run. Self-reliance and good mood scored higher after running. Both mood elevation and plasma beta-EIR increase showed a considerable individual variability but there was a significant correlation in the mean values of the two runs between individual beta-EIR increases (delta beta-EIR) and the changes of ratings in feeling of pleasantness (delta FP). High delta beta-EIR corresponded to positive mood change post-run.

Endogenous benzodiazepine receptor agonist in human and mammalian plasma.

Using ultra-filtration steps and HPLC-separation, a low molecular weight ligand of the benzodiazepine receptor was isolated from plasma of various mammalian species including man. The endogenous ligand acts on benzodiazepine receptors agonistically and apparently has a receptor affinity similar to Diazepam. The ligand is not identical with Diazepam as indicated by HPLC and UV-spectroscopy.

Three types of acetylcholine-induced single channel currents in clonal rat pheochromocytoma cells.

Single channel currents induced by acetylcholine were recorded from nerve growth factor-differentiated clonal rat pheochromocytoma (PC12) cells by using the patch-clamp technique. Inward current amplitudes were heterogeneous and could be related to three classes of unitary current. The three current-voltage relations were linear for patch membrane potentials ranging from -50 mV to -120 mV. At 22 degrees C, conductances of 22 pS, 31 pS and 39 pS were obtained. The extrapolated reversal potential for the channels was 0 mV and the mean open time approximately 10-20 ms. These results may indicate that PC12 cells possess three types of acetylcholine receptor channels.

Single neuron cultivation of embryonic and perinatal rabbit or rat brains based on plasma clot technique.

Isolated neuronal cells dissociated from the brain of embryonic rabbits on the sixteenth day of gestation and of perinatal rats (eighteenth embryonic day, to E18, thirteenth day postnatum, p.n. 13) were selectively cultured using a plasma clot technique. The cells grown were shown to be neurons by means of the neuron-specific synaptosomal plasma membrane antibody (SPM). They differentiated at a very high frequency from rounded cells lacking processes into different shapes characteristic for several neuronal cell types. Morphological differences could be distinguished even after 24 h in culture. The neurons differentiated in vitro for up to 11 days, apparently without need of any direct intercellular contact. Cells caught inside the plasma clot were prevented from decreasing in number. This provides the opportunity to culture few neurons even from an extremely small area of a single brain. As an example, different cell types are shown originating from rat cerebella aged E18 to p.n. 13. Their appearance apparently corresponds to the genesis of cerebellar cell types, as is known from the in vivo situation. The high degree of characteristic neuronal differentiation and the prevention of direct intercellular contacts indicate that this culture method may serve as an in vitro assay for genetically fixed properties acquired in vivo.

A method for defined sectioning of fresh young brains and collection of small regions for cell and tissue culture.

A method for the collection of defined small regions from fresh brain under sterile conditions is described. Its reproducibility allows the cultivation of well-defined, corresponding regions from similar brains. After controlled orientation in agarose, the brain can be sectioned by means of a Vibratome in spite of its softness. The section level is defined by co-ordinates of a stereotaxic atlas, produced for this purpose from brains orientated in the same way. Areas desired for cultivation are punched out from tissue slices with 200-550 microns diameter needles according to the atlas pictures. The plugs can then be stored in cold buffer solution until preparation for culture. Exact locations of tissue samples collected can be determined histologically. Either whole or dissociated plugs cultivated by a plasma clot technique lead to morphologically differentiating neurons surviving for more than or up to 14 days, respectively.

Antagonistic effect of L-aspartic acid on decrease in body weight, and food and fluid intake, and naloxone reversible rectal temperature depression caused by D-aspartic acid.

Seventy-five rats, divided into five groups, were given D-aspartic acid (D-Asp), L-Asp and D + L-Asp in ratio 1/1 or 1/2 for one week. The body weight, food and fluid intakes, and rectal temperature of the rats received D-Asp or D + L-Asp in 1/1 ratio significantly decreased. The decrease in rectal temperature was antagonized by naloxone. L-Asp given together with D-Asp in 1/2 ratio prevented D-Asp-caused decrease in body weight, food and fluid intakes, and rectal temperature. Although D-amino acids, as antipeptidases have some effects through endorphinergic systems, D-Asp (an inhibitor of L-asparaginase) seems to act at the level of L-asparaginase presumably by increasing the level of endorphins since L-Asp antagonizes the inhibitory effect of both D-Asp and morphine.

The effects of D- and/or L-aspartic acids on the total weight of body, the weights of certain organs, and their protein, triglyceride and glycogen content.

125 rats which were divided into five groups were deprived of food or given orally D- (a potent inhibitor for L-asparaginase) and/or L-aspartic acids (Asp) for one week. The body weights before and at the end of the experiment were determined as well as post mortem the weights of brain, liver and kidneys, their protein contents, and the liver triglyceride and glycogen contents. D- and D+L-Asp caused significant decreases in the weights of body and liver, and in daily fluid intake; in addition liver and kidney protein, and liver triglyceride and glycogen contents were found to be lower than control. On the other hand, the food-deprived group which was subjected to more or less the same body weight loss due to food deprivation showed only a decrease in the liver triglyceride content. Since D-amino acids cause naloxone reversible analgesia which is, thus, considered as an involvement of endorphinergic system and of vasopressin, the effects of D-Asp were attributed to the changes in the availability of opioids and vasopressin, which simultaneously have an effect on each other as well as an effect of the release of ACTH. L-Asp appeared to antagonize the effects of D-Asp. Because L-Asp antagonizes the acute and chronic effects of morphine, including that on L-asparaginase activity, the hypothesis is proposed that the antagonizing effects of L-Asp observed may be caused at the level of L-asparaginase activity.

Immunochemical evidence for exocytosis in isolated chromaffin cells after stimulation with depolarizing agents.

After chemical stimulation with depolarizing agents (Ba2+ or Ca2+/carbachol) isolated living chromaffin cells display a drastically increased binding capacity for anti-DBH, distributed spotwise on or near the outer cell membrane. This effect is inhibited by noradrenaline; it is not evoked by the non-exocytotically releasing agents tyramine and reserpine. the effect of apparent externalization of DBH is paralleled by the observation of a DBH-dependent binding of 125I-labelled protein A upon the same depolarizing stimuli. These observations are discussed as possible evidence for exocytotic activities.

Post mortem changes in adenylate cyclase activity in rat brain striatum.

Adenylate cyclase activity in rat striatum decreased post mortem. Half-lives were about 2.7 h at 22 degrees C, 72 h at 4 degrees C. Differences in stability after death of adenylate cyclase in human brain and rat striatum, and possible heterogenity of the enzyme, are briefly discussed.

Tissue specificity of nonhistone proteins from human chromatin.

Nonhistone proteins were isolated from human placental and tonsillar chromatins. Antiserum was prepared against a complex from some nonhistone proteins and DNA (NP-DNA) from placental chromatin. With the help of polyacrylamide gel electrophoresis and immunological methods the tissue specificity of human chromatin nonhistone proteins was established. The described organ immunogenic specificity of the complex of DNA and nonhistone protein (NP-DNA) from human chromatin is in accordance with data published on similar complexes from different animal organs. Besides, it is shown that shearing of chromatin leads to large chifts in NP-DNA concentrations required for maximum complement fixation in the presence of the prepared antiserum. This may probably be due to a damage of certain chromatin super structures which involve some of the nonhistone proteins and DNA sequences from both the more condensed and less condensed parts of chromatin.