Genome-wide association analysis of diverticular disease points towards neuromuscular, connective tissue and epithelial pathomechanisms.

OBJECTIVE: Diverticular disease is a common complex disorder characterised by mucosal outpouchings of the colonic wall that manifests through complications such as diverticulitis, perforation and bleeding. We report the to date largest genome-wide association study (GWAS) to identify genetic risk factors for diverticular disease. DESIGN: Discovery GWAS analysis was performed on UK Biobank imputed genotypes using 31 964 cases and 419 135 controls of European descent. Associations were replicated in a European sample of 3893 cases and 2829 diverticula-free controls and evaluated for risk contribution to diverticulitis and uncomplicated diverticulosis. Transcripts at top 20 replicating loci were analysed by real-time quatitative PCR in preparations of the mucosal, submucosal and muscular layer of colon. The localisation of expressed protein at selected loci was investigated by immunohistochemistry. RESULTS: We discovered 48 risk loci, of which 12 are novel, with genome-wide significance and consistent OR in the replication sample. Nominal replication (p<0.05) was observed for 27 loci, and additional 8 in meta-analysis with a population-based cohort. The most significant novel risk variant rs9960286 is located near CTAGE1 with a p value of 2.3×10-10 and 0.002 (ORallelic=1.14 (95% CI 1.05 to 1.24)) in the replication analysis. Four loci showed stronger effects for diverticulitis, PHGR1 (OR 1.32, 95% CI 1.12 to 1.56), FAM155A-2 (OR 1.21, 95% CI 1.04 to 1.42), CALCB (OR 1.17, 95% CI 1.03 to 1.33) and S100A10 (OR 1.17, 95% CI 1.03 to 1.33). CONCLUSION: In silico analyses point to diverticulosis primarily as a disorder of intestinal neuromuscular function and of impaired connective fibre support, while an additional diverticulitis risk might be conferred by epithelial dysfunction.

Metabolic biomarker signature to differentiate pancreatic ductal adenocarcinoma from chronic pancreatitis.

OBJECTIVE: Current non-invasive diagnostic tests can distinguish between pancreatic cancer (pancreatic ductal adenocarcinoma (PDAC)) and chronic pancreatitis (CP) in only about two thirds of patients. We have searched for blood-derived metabolite biomarkers for this diagnostic purpose. DESIGN: For a case-control study in three tertiary referral centres, 914 subjects were prospectively recruited with PDAC (n=271), CP (n=282), liver cirrhosis (n=100) or healthy as well as non-pancreatic disease controls (n=261) in three consecutive studies. Metabolomic profiles of plasma and serum samples were generated from 477 metabolites identified by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. RESULTS: A biomarker signature (nine metabolites and additionally CA19-9) was identified for the differential diagnosis between PDAC and CP. The biomarker signature distinguished PDAC from CP in the training set with an area under the curve (AUC) of 0.96 (95% CI 0.93-0.98). The biomarker signature cut-off of 0.384 at 85% fixed specificity showed a sensitivity of 94.9% (95% CI 87.0%-97.0%). In the test set, an AUC of 0.94 (95% CI 0.91-0.97) and, using the same cut-off, a sensitivity of 89.9% (95% CI 81.0%-95.5%) and a specificity of 91.3% (95% CI 82.8%-96.4%) were achieved, successfully validating the biomarker signature. CONCLUSIONS: In patients with CP with an increased risk for pancreatic cancer (cumulative incidence 1.95%), the performance of this biomarker signature results in a negative predictive value of 99.9% (95% CI 99.7%-99.9%) (training set) and 99.8% (95% CI 99.6%-99.9%) (test set). In one third of our patients, the clinical use of this biomarker signature would have improved diagnosis and treatment stratification in comparison to CA19-9.

Treatment of Complicated Anal Fistula by an Endofistular Polyurethane-Sponge Vacuum Therapy: A Pilot Study.

BACKGROUND: Treatment of supra- and transsphincteric anal fistulas remains a clinical challenge because current treatment results are variable and potentially endanger sphincter function. OBJECTIVE: Based on positive results of endoluminal polyurethane-sponge vacuum therapy in the upper and lower GI tract, a new system for endofistular vacuum therapy was developed for anal fistulas to utilize vacuum therapy to remove the endofistular pseudoepithelium and to induce granulation in the fistula tract. DESIGN: This study is based on a prospective case series. PATIENTS: Seven patients with complicated anal fistulas (3 associated with Crohn's disease and 4 of cryptoglandular origin) longer than 4 cm were treated. Initially, the fistula was curettaged and the first endofistular vacuum therapy sponge was positioned in the fistula tract. The inner fistula opening was closed by suture. A 125 mm Hg constant vacuum was applied to the sponge, and the endofistular vacuum therapy sponge was changed a median of 3 (3-5) times after each 48 to 72 hours of constant vacuum therapy. After final removal, the fistulas were reevaluated every other week for 3 months. MAIN OUTCOME MEASURE: The main outcome measured was the closure of the fistula. RESULTS: All patients tolerated the therapy well and no adverse events were observed. Fistula tract closure was demonstrated within 4 weeks after the termination of vacuum therapy. One patient with cryptoglandular fistula developed a recurrence within the follow-up of 3 months. LIMITATIONS: This was an observational study that had no control arm. CONCLUSION: In this pilot case series, the results are encouraging. Because endoluminal vacuum therapy would be a new and sphincter-sparing therapy, this concept warrants further investigation in controlled trials.

Genetic and functional identification of the likely causative variant for cholesterol gallstone disease at the ABCG5/8 lithogenic locus.

UNLABELLED: The sterolin locus (ABCG5/ABCG8) confers susceptibility for cholesterol gallstone disease in humans. Both the responsible variant and the molecular mechanism causing an increased incidence of gallstones in these patients have as yet not been identified. Genetic mapping utilized patient samples from Germany (2,808 cases, 2,089 controls), Chile (680 cases, 442 controls), Denmark (366 cases, 766 controls), India (247 cases, 224 controls), and China (280 cases, 244 controls). Analysis of allelic imbalance in complementary DNA (cDNA) samples from human liver (n = 22) was performed using pyrosequencing. Transiently transfected HEK293 cells were used for [(3) H]-cholesterol export assays, analysis of protein expression, and localization of allelic constructs. Through fine mapping in German and Chilean samples, an ∼250 kB disease-associated interval could be defined for this locus. Lack of allelic imbalance or allelic splicing of the ABCG5 and ABCG8 transcripts in human liver limited the search to coding single nucleotide polymorphisms. Subsequent mutation detection and genotyping yielded two disease-associated variants: ABCG5-R50C (P = 4.94 × 10(-9) ) and ABCG8-D19H (P = 1.74 × 10(-10) ) in high pairwise linkage disequilibrium (r(2) = 0.95). [(3) H]-cholesterol export assays of allelic constructs harboring these genetic candidate variants demonstrated increased transport activity (3.2-fold, P = 0.003) only for the ABCG8-19H variant, which was also superior in nested logistic regression models in German (P = 0.018), Chilean (P = 0.030), and Chinese (P = 0.040) patient samples. CONCLUSION: This variant thus provides a molecular basis for biliary cholesterol hypersecretion as the mechanism for cholesterol gallstone formation, thereby drawing a link between "postgenomic" and "pregenomic" pathophysiological knowledge about this common complex disorder. (HEPATOLOGY 2012).

Endoscopic endoluminal vacuum therapy is superior to other regimens in managing anastomotic leakage after esophagectomy: a comparative retrospective study.

BACKGROUND: Anastomotic leakage after esophagectomy is a life-threatening complication. No comparative outcome analyses for the different treatment regimens are yet available. METHODS: In a single-center study, data from all esophagectomy patients from January 1995 to January 2012, including tumor characteristics, surgical procedure, postoperative anastomotic leakage, leakage therapy regimens, APACHE II scores, and mortality, were collected, and predictors of patient survival after anastomotic leakage were analyzed. RESULTS: Among 366 resected patients, 62 patients (16 %) developed an anastomotic leak, 16 (26 %) of whom died. Therapy regimens included surgical revision (n = 18), endoscopic endoluminal vacuum therapy (n = 17), endoscopic stent application (n = 12), and conservative management (n = 15). APACHE II score at the initiation of treatment for leakage was the strongest predictor of in-hospital mortality (p < 0.0017). Conservatively managed patients showed mild systemic illness (mean APACHE II score 5) and no mortality. In systemically ill patients matched for APACHE II scores (mean, 14.4), endoscopic endoluminal vacuum therapy patients had lower mortality (12 %) compared to surgically treated (50 %, p = 0.01) cases and patients managed by stent placement (83 %, p = 00014, log rank test). No other clinical or laboratory parameters significantly influenced patient survival. CONCLUSIONS: Endoscopic endoluminal vacuum therapy was the best treatment of anastomotic leakage in systemically ill patients after esophagectomy in this retrospective analysis. It should therefore be considered an important instrument in the management of this disorder.

Lack of CCR7 expression is rate limiting for lymphatic spread of pancreatic ductal adenocarcinoma.

CCR7 expression on tumor cells promotes lymphatic spread in several malignant tumors. However, a comprehensive characterization of the CCL19/CCL21-CCR7 axis in pancreatic ductal adenocarcinoma (PDAC), which is known for its high rates of lymph-node metastases, is still lacking. CCR7 mRNA and CCR7 protein were found to be expressed in spheroid cultures of all six examined PDAC cell lines. In migration assays, CCR7 expressing PDAC cells showed enhanced migration toward CCL19 and CCL21, the two ligands of CCR7. In an orthotopic nude mouse model, CCR7-transfected PT45P1 cells gave rise to significantly larger tumors and showed a higher frequency of lymph vessel invasion and lymph-node metastases than mock-transfected cells. In an analysis using quantitative real-time PCR, CCR7 showed fourfold overexpression in microdissected PDAC cells compared to normal duct cells. Moderate-to-strong immunohistochemical CCR7 expression, found in 58 of 121 well-characterized human PDACs, correlated with high rates of lymph vessel invasion. Conversely, PDACs completely lacking CCR7 expression showed only low rates of lymph vessel invasion and lymph-node metastases. The evaluation of CCL21 expression by immunofluorescence staining revealed a significant upregulation of CCL21 in peritumoral and intratumoral lymph vessels compared to lymph vessels in disease-free pancreata. In conclusion, our study revealed strong evidence that lack of CCR7 impairs the metastatic potential of PDAC. Lymph vessel invasion by CCR7 expressing PDAC cells may be additionally enhanced by upregulation of CCL21 in tumor-associated lymph vessels, representing a previously unknown factor of lymphatic spread.

Distinct DNA methylation patterns in cirrhotic liver and hepatocellular carcinoma.

Abberrant DNA methylation is one of the hallmarks of cancerogenesis. Our study aims to delineate differential DNA methylation in cirrhosis and hepatic cancerogenesis. Patterns of methylation of 27,578 individual CpG loci in 12 hepatocellular carcinomas (HCCs), 15 cirrhotic controls and 12 normal liver samples were investigated using an array-based technology. A supervised principal component analysis (PCA) revealed 167 hypomethylated loci and 100 hypermethylated loci in cirrhosis and HCC as compared to normal controls. Thus, these loci show a "cirrhotic" methylation pattern that is maintained in HCC. In pairwise supervised PCAs between normal liver, cirrhosis and HCC, eight loci were significantly changed in all analyses differentiating the three groups (p < 0.0001). Of these, five loci showed highest methylation levels in HCC and lowest in control tissue (LOC55908, CELSR1, CRMP1, GNRH2, ALOX12 and ANGPTL7), whereas two loci showed the opposite direction of change (SPRR3 and TNFSF15). Genes hypermethylated between normal liver to cirrhosis, which maintain this methylation pattern during the development of HCC, are depleted for CpG islands, high CpG content promoters and polycomb repressive complex 2 (PRC2) targets in embryonic stem cells. In contrast, genes selectively hypermethylated in HCC as compared to nonmalignant samples showed an enrichment of CpG islands, high CpG content promoters and PRC2 target genes (p < 0.0001). Cirrhosis and HCC show distinct patterns of differential methylation with regards to promoter structure, PRC2 targets and CpG islands.

Eso-Sponge® for anastomotic leakage after oesophageal resection or perforation: outcomes from a national, prospective multicentre registry.

BACKGROUND: Anastomotic leakage (AL) after oesophagectomy and oesophageal perforations are associated with significant morbidity and mortality. Minimally invasive endoscopy is often used as first-line treatment, particularly endoluminal vacuum therapy (EVT). The aim was to assess the performance of the first commercially available endoluminal vacuum device (Eso-Sponge®) in the management of AL and perforation of the upper gastrointestinal tract (GIT). METHODS: The Eso-Sponge® registry was designed in 2014 as a prospective, observational, national, multicentre registry. Patients were recruited with either AL or perforation within the upper GIT. Data were collected with a standardized form and transferred into a web-based platform. Twenty hospitals were enrolled at the beginning of the study (registration number NCT02662777; http://www.clinicaltrials.gov). The primary endpoint was successful closure of the oesophageal defect. RESULTS: Eleven out of 20 centres recruited patients. A total of 102 patients were included in this interim analysis; 69 patients with AL and 33 with a perforation were treated by EVT. In the AL group, a closure of 91 per cent was observed and 76 per cent was observed in the perforation group. The occurrence of mediastinitis (P = 0.002) and the location of the defect (P = 0.008) were identified as significant predictors of defect closure. CONCLUSIONS: The Eso-Sponge® registry offers the opportunity to collate data on EVT with a uniform, commercially available product to improve standardization. Our data show that EVT with the Eso-Sponge® is an option for the management of AL and perforation within the upper GIT.

Preoperative risk evaluation of postoperative morbidity in IBD patients--impact of the POSSUM score.

BACKGROUND: The aim of this study was to assess the morbidity and mortality of patients undergoing surgery for inflammatory bowel disease (IBD) with special focus of the predictive value of the Physiological and Operative Severity Score for the Enumeration of Mortality and Morbidity (POSSUM) scoring system for preoperative risk adjustment of postoperative morbidity. METHODS: The operative notes and hospital files of 191 patients with IBD were analyzed. The POSSUM scoring system was used to predict morbidity rates after surgery. The physiological sub-score of the POSSUM score was analyzed with regard to its ability to predict postoperative complications. RESULTS: The overall complication rate was 27.7%, and the mortality was 0.5%. The morbidity rate predicted by POSSUM was 28.4% and the mortality rate 7.2%. The mean POSSUM-phys sub-score in patients without the major complications (anastomotic leakages, peritonitis, bleeding) was significant lower compared to patients with at least one of these complications (14.7 vs. 18.6; p < 0.001). Regarding the major complications separately, there were significant differences in the POSSUM-phys scores in patients developing a sepsis (14.1 vs. 23.4; p < 0.001) and/or a peritonitis (14.8 vs. 19.2; p = 0.05), whereas patients developing an anastomotic leakage/suture dehiscence or a postoperative bleeding did not differ significantly. CONCLUSION: POSSUM was an accurate predictor of morbidity in IBD patients and overpredicted mortality. The POSSUM-phys score is a promising instrument for identifying patients at increased risk of developing major postoperative complications after surgery for IBD.

A new technique for bedside placement of enteral feeding tubes: a prospective cohort study.

INTRODUCTION: To accomplish early enteral feeding in the critically ill patient a new transnasal endoscopic approach to the placement of postpyloric feeding tubes by intensive care physicians was evaluated. METHODS: This was a prospective cohort study in 27 critically ill patients subjected to transnasal endoscopy and intubation of the pylorus. Attending intensive care physicians were trained in the handling of the new endoscope for transnasal gastroenteroscopy for two days. A jejunal feeding tube was advanced via the instrument channel and the correct position assessed by contrast radiography. The primary outcome measure was successful postpyloric placement of the tube. Secondary outcome measures were time needed for the placement, complications such as bleeding and formation of loops, and the score of the placement difficulty graded from 1 (easy) to 4 (difficult). Data are given as mean values and standard deviation. RESULTS: Out of 34 attempted jejunal tube placements, 28 tubes (82%) were placed correctly in the jejunum. The duration of the procedure was 28 ± 12 minutes. The difficulty of the tube placement was judged as follows: grade 1: 17 patients, grade 2: 8 patients, grade 3: 7 patients, grade 4: 2 patients. In three cases, the tube position was incorrect, and in another three cases, the procedure had to be aborted. In one patient bleeding occurred that required no further treatment. CONCLUSIONS: Fast and reliable transnasal insertion of postpyloric feeding tubes can be accomplished by trained intensive care physicians at the bedside using the presented procedure. This new technique may facilitate early initiation of enteral feeding in intensive care patients.

Evaluation of the POSSUM score in surgical treatment of cholangiocarcinoma.

BACKGROUND/AIMS: The surgical treatment for cholangiocarcinoma (CCC) is still a challenge. The aim of this study was to evaluate the POSSUM scoring system for preoperative physiological risk adjustment and for the operative risk of postoperative morbidity and mortality. METHODOLOGY: The operative notes and hospital files of 171 patients with CCC were analyzed retrospectively. The POSSUM scoring system was used to predict morbidity and mortality rates after surgery. The physiological sub score and the operative sub score of the POSSUM score were analyzed with regard to their ability to predict major postoperative complications. RESULTS: The overall complication rate was 40.9% and the mortality was 11.2%. The morbidity predicted by POSSUM was 63.5% and the prediction for postoperative mortality was 23.7%. Both rates are much higher than the observed morbidity and mortality. High operative severity sub scores correlate with the occurrence of major complications. CONCLUSION: The POSSUM scoring system over predicts morbidity and mortality in surgery for CCC. Operative severity and intraoperative parameters have a greater influence on postoperative mortality and morbidity rates than the physiological parameters represented in the POSSUM physiological score. Prospective studies will have to show whether POSSUM can be modified or whether it is necessary to develop a new score for assessing morbidity and mortality in surgery for CCC.

Investigation of the colorectal cancer susceptibility region on chromosome 8q24.21 in a large German case-control sample.

Human chromosome 8q24.21 has been implicated as a susceptibility region for colorectal cancer (CRC) as a result of genome-wide association and candidate gene studies. To assess the impact of molecular variants at 8q24.21 upon the CRC risk of German individuals and to refine the disease-associated region, a total of 2,713 patients with operated CRC (median age at diagnosis: 63 years) were compared with 2,718 sex-matched control individuals (median age at inclusion: 65 years). Information on microsatellite instability in tumors was available for 901 patients. Association analysis of SNPs rs10505477 and rs6983267 yielded allelic p-values of 1.42 x 10(-7) and 2.57 x 10(-7), respectively. For both polymorphisms, the odds ratio was estimated to be 1.50 (95% CI: 1.29-1.75) under a recessive disease model. The strongest candidate interval, outside of which significance dropped by more than 4 orders of magnitude, was delineated by SNPs rs10505477 and rs7014346 and comprised 17 kb. In a subgroup analysis, the disease association was found to be more pronounced in MSI-stable tumors (odds ratio: 1.71). Our study confirms the role of genetic variation at 8q24.21 as a risk factor for CRC and localizes the corresponding susceptibility gene to a 17 kb candidate region.

Impact of the site of anastomosis after oncologic esophagectomy on quality of life--a prospective, longitudinal outcome study.

BACKGROUND: For patients undergoing oncologic surgery, the quality of life (QoL) is generally accepted as an important outcome parameter in addition to long-term survival, mortality, and complication rates. Our study focused on outcome in terms of QoL in patients with esophageal cancer, comparing the sites of anastomosis (cervical versus thoracic anastomosis). METHODS: In a prospective longitudinal single-center study from 1998 to 2005, 105 patients underwent surgery for esophageal cancer. To assess QoL the EORTC-QLQ-C-30 and a tumor-specific module were administered before surgery, at discharge, and three, six, 12, and 24 months after surgery. Clinical data were collected prospectively and follow-up was performed every six months. RESULTS: The histological type was squamous cell carcinoma in 51.4% of the cases, adenocarcinoma in 41.9%, and some other type in 6.7%. There was no significant difference between cervical and thoracic anastomosis with regard to morbidity, mortality, and survival rates (30% five-year survival rate), whereas tumor stage was a significant (p < 0.001) prognostic factor. Most QoL scores dropped significantly below baseline in the early postoperative period. Even though they recovered slowly during the follow-up period, they never reached preoperative levels again. There was no statistically significant difference in any of the QoL scales between patients with a cervical or a thoracic anastomosis. CONCLUSIONS: Esophageal resections are associated with significant deterioration of QoL, which persists during the follow-up period. The surgical technique and position of the esophagogastrostomy did not affect QoL deterioration.

Long-term quality of life after endovac-therapy in anastomotic leakages after esophagectomy.

BACKGROUND: Endoluminal vacuum therapy (EVT) has been successfully established with promising survival rates in the treatment of anastomotic leakages after esophagectomy. It is still unclear how this therapy affects health related quality of life (HRQOL). METHODS: HRQOL was prospectively assessed using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-C30 (EORTC QLQ-C30) questionnaire. Assessment was carried out prior to surgery, after discharge, 6 months and 12 months after surgery. We compared HRQOL after EVT (n=23) to patients without anastomotic leakages as a control group (n=50). Investigated parameters included age, sex, and localization of anastomosis, number of EVT sessions, length of ICU and hospital stay, therapy failure, anastomotic stricture, tumour stage, neoadjuvant and adjuvant treatment, sepsis. RESULTS: After esophagectomy HRQOL increased within 12 months. Compared to patients without leakages the EVT-group showed significantly better HRQOL-scores for pain, social and emotional functioning after discharge and 6 months after surgery. In the long-term follow up HRQOL was comparable between the groups. After EVT age, advanced tumour stage, tumour recurrence, anastomotic strictures, length of ICU and hospital stay and length of EVT had a significant influence on HRQOL. CONCLUSIONS: EVT is a promising therapeutic option in leakages after esophagectomy. In the long-term, HRQOL of EVT-treated patients is comparable to patients, who did not suffer from postsurgical leakages.

Establishment of a novel orthotopic xenograft model of human gallbladder carcinoma.

OBJECTIVE: Gallbladder cancer is characterized by high morbidity and mortality. An appropriate human xenograft animal model could serve as a research tool to investigate new therapeutic strategies. SUMMARY BACKGROUND DATA: To date, the few reports describing a xenograft animal model showed significant limitations. We improved a murine orthotopic human xenotransplantation model by implanting human gallbladder carcinoma cells directly into the lumen of the gallbladder. METHODS: Mz-ChA-1 cells were orthotopically injected into the gallbladder of Severe Combined Immune Deficiency (SCID) beige mice inducing the growth of solid tumors. The natural course of the disease, tumor growth, and metastases were analyzed. The cytotoxic drug gemcitabine was tested in vitro and in vitro. RESULTS: All animals revealed solid tumors in the inoculated area with liver infiltration. The median tumor volume in the untreated group was significantly higher than in the gemcitabine-treated group. Immunohistochemical staining revealed expression of human cytokeratin 7 and cytokeratin 8. To analyze tumor cell proliferation, the tumors were stained for the antigen Ki-67, and labeling indices were calculated for both groups. Animals receiving gemcitabine treatment showed significantly lower mean labeling indices. In vitro investigation revealed a significant reduction of DNA synthesis. DNA fragmentation, as a measure of apoptosis, was elevated by roughly 20% within 24 h of treatment. With this, we successfully established an orthotopic xenotransplant animal model and investigated the in vitro and in vivo effects of gemcitabine in human xenografted Mz-ChA-1 gallbladder adenocarcinoma. CONCLUSION: This model resembles the clinical situation as closely as possible and offers a relevant option for the preclinical testing of new therapeutic strategies.

Combination phenylbutyrate/gemcitabine therapy effectively inhibits in vitro and in vivo growth of NSCLC by intrinsic apoptotic pathways.

BACKGROUND: Standard chemotherapy protocols in NSCLC are of limited clinical benefit. Histone deacetylase (HDAC) inhibitors represent a new strategy in human cancer therapy. In this study the combination of the HDAC inhibitor phenylbutyrate (PB) and the nucleoside analogue gemcitabine (GEM) was evaluated and the mechanisms underlying increased cell death were analyzed. METHODS: Dose escalation studies evaluating the cytotoxicity of PB (0.01-100 mM), GEM (0.01-100 microg/ml) and a combination of the two were performed on two NSCLC cell lines (BEN and KNS62). Apoptotic cell death was quantified. The involvement of caspase-dependent cell death and MAP-kinase activation was analyzed. Additionally, mitochondrial damage was determined. In an orthotopic animal model the combined effect of PB and GEM on therapy was analyzed. RESULTS: Applied as a single drug both GEM and PB revealed limited potential to induce apoptosis in KNS62 and Ben cells. Combination therapy was 50-80% (p = 0.012) more effective than either agent alone. On the caspase level, combination therapy significantly increased cleavage of the pro-forms compared to single chemotherapy. The broad spectrum caspase-inhibitor zVAD was able to inhibit caspase cleavage completely, but reduced the frequency of apoptotic cells only by 30%. Combination therapy significantly increased changes in MTP and the release of cyto-c, AIF and Smac/Diabolo into the cytoplasm. Furthermore, the inhibitors of apoptosis c-IAP1 and c-IAP2 were downregulated and it was shown that in combination therapy JNK activation contributed significantly to induction of apoptosis. The size of the primary tumors growing orthotopically in SCID mice treated for 4 weeks with GEM and PB was significantly reduced (2.2-2.7 fold) compared to GEM therapy alone. The Ki-67 (KNS62: p = 0.015; Ben: p = 0.093) and topoisomerase IIalpha (KNS62: p = 0.008; Ben: p = 0.064) proliferation indices were clearly reduced in tumors treated by combination therapy, whereas the apoptotic index was comparably low in all groups. CONCLUSION: Therapy combining GEM and the HDAC inhibitor PB initiates a spectrum of apoptosis-inducing mitochondrial and further JNK-dependent events, thereby overcoming the therapeutic resistance of NSCLC tumor cells. In vivo, the combination therapy substantially reduced tumor cell proliferation, suggesting that the well tolerated PB is a useful supplemental therapeutic agent in NSCLC.

Paraneoplastic leukemoid reaction and rapid progression in a patient with malignant melanoma: establishment of KT293, a novel G-CSF-secreting melanoma cell line.

Paraneoplastic leukemoid reaction (PLR) is a rare condition of leucocytosis in cancer patients. Here we report the rapid progression of a patient suffering from a metastasized malignant melanoma and PLR. The patient's white blood cell count exceeded 200,000 cells per mul and the serum level of Granulocyte Colony-Stimulating Factor (G-CSF) was elevated up to 780 pg/mul. A Tc-m99-labeled anti-NCA90/95 based granulocyte scan demonstrated reactive bone marrow expansion, splenomegaly and granulocyte infiltration into the tumor. KT293, a S100, gp100 and CD68 positive melanoma cell line derived from an axillary metastasis, produced large amounts of G-CSF in vitro and induced rapidly growing tumors and PLR after subcutaneous inoculation in SCID mice. In contrast to G-CSF-secreting cancer cells of other tissue origin, G-CSF-neutralizing antibodies failed to inhibit the growth of KT293 cells. In addition, KT293 cells did not express G-CSF-receptor. These observations suggest that paracrine effects of G-CSF-secretion and PLR might promote an aggressive melanoma phenotype, as seen in this patient.

Gemcitabine-mediated apoptosis is associated with increased CD95 surface expression but is not inhibited by DN-FADD in Colo357 pancreatic cancer cells.

This study investigates the role of caspase-8 and DN-FADD, an inhibitor of CD95-dependent caspase-8 activation, in gemcitabine-induced apoptosis of Colo357 pancreatic cancer cells. Gemcitabine-mediated apoptosis was monitored by the kinetics of caspase-8 activation and cytochrome c release. Gemcitabine treatment of Colo357 cells increased CD95 surface expression, raising the possibility of the involvement of CD95 in gemcitabine-mediated caspase-8 activation. However, ectopic expression of DN-FADD and treatment of cells with the antagonistic anti-CD95 antibody ZB4 both failed to suppress gemcitabine-induced apoptosis but substantially inhibited CD95-mediated apoptosis. DN-FADD, which surprisingly accumulated in nuclei of Colo357 cells, was unable to block caspase-8 activation mediated by either gemcitabine or CD95. These observations argue against a role of CD95 in gemcitabine-induced caspase-8 activation and reveal that the anti-apoptotic function of DN-FADD differs from caspase-8 inhibition in Colo357 cells.

Detection of tumor cell dissemination in pancreatic ductal carcinoma patients by CK 20 RT-PCR indicates poor survival.

PURPOSE: This prospective study evaluates the diagnostic potential of Cytokeratin 20 (CK 20) RT-PCR for the detection of disseminated tumor cells in bone marrow and blood of a large cohort of patients with ductal adenocarcinoma of the pancreas and the prognostic value on overall survival prediction. METHODS: Between 1994 and 2003, 172 patients (83 male, 89 female; 13-82 years) with pancreatic ductal adenocarcinoma underwent surgery. Bone marrow samples and venous blood were taken preoperatively and analyzed for disseminated tumor cells by nested CK 20 RT-PCR. RESULTS: Disseminated tumor cells were detected in 81 (47.1%) of the 172 patients in the bone marrow and/or the venous blood. Overall, in 45 of the 135 (33.3%) bone marrow samples and in 52 of the 154 (33.8%) blood samples, CK 20 positive cells were detected. Detection rates increased with the UICC-tumor stage. According to Kaplan-Meier, univariate survival analysis of all 172 patients (n = 78 R0-; n = 18 R1- and n = 5 R2-resected; n = 71 palliative surgery) showed a statistically significant relationship of overall survival to radicality of the operation (P < 0.0001), the UICC-stage of the tumors (P = 0.0011) and the detection of disseminated tumor cells in bone marrow and/or venous blood (P = 0.05). Patients with well- and moderately- differentiated tumors (G1 and G2) had a significantly longer survival (P = 0.045) than patients suffering from poorly differentiated tumors (G3). A positive CK 20 status in the bone marrow and/or blood within the group of patients with G1 and G2 tumors had a significantly negative prognostic impact on their survival (P = 0.046). CONCLUSIONS: Disseminated tumor cells can be detected in patients with pancreatic ductal adenocarcinoma by CK 20 RT-PCR. Detection rates are stage dependent, and survival analysis demonstrated statistically relevant data. From a clinical point of view, this finding is especially noteworthy for the group of well- and moderately-differentiated tumors.

Efficient dose-dependent and time-dependent protein transduction of pancreatic carcinoma cells in vitro and in vivo using purified VP22-EGFP fusion protein.

We constructed a prokaryotic vector expressing a truncated VP22-EGFP gene and purified this fusion protein from Escherichia coli cultures using nickel resin. Application of purified VP22-EGFP protein to human pancreatic carcinoma cells showed a highly efficient time-dependent and dose-dependent uptake and resulted in green fluorescence predominantly located in the nuclei of treated cells. Purified VP22-EGFP efficiently translocated into deeper layers of pancreatic tumor cell spheroids. Homogeneous uptake into the whole tumor was observed after peritumoral injection in human pancreatic tumors in SCID mice. We conclude that the direct application of purified VP22 fusion proteins offers a new, peptide-mediated and potentially systemic therapy for pancreatic cancer. This opens the possibility of achieving specific antitumor effects induced by fused apoptosis-enhancing proteins.