Sebastian Hinz

Foxp3 Expression in Pancreatic Carcinoma Cells as a Novel Mechanism of Immune Evasion in Cancer

The forkhead transcription factor Foxp3 is highly expressed in CD4+CD25+ regulatory T cells (Treg) and was recently identified as a key player in mediating their inhibitory functions. Here, we describe for the first time the expression and function of Foxp3 in pancreatic ductal adenocarcinoma cells and tumors. Foxp3 expression was induced by transforming growth factor-beta2 (TGF-beta2), but not TGF-beta1 stimulation in these cells, and was partially suppressed following antibody-mediated neutralization of TGF-beta2. The TGF-beta2 effect could be mimicked by ectopic expression of a constitutively active TGF-beta type I receptor/ALK5 mutant. Down-regulation of Foxp3 with small interfering RNA (siRNA) in pancreatic carcinoma cells resulted in the up-regulation of interleukin 6 (IL-6) and IL-8 expression, providing evidence for a negative transcriptional activity of Foxp3 also in these epithelial cells. Coculture of Foxp3-expressing tumor cells with naive T cells completely inhibited T-cell proliferation, but not activation, and this antiproliferative effect was partially abrogated following specific inhibition of Foxp3 expression. These findings indicate that pancreatic carcinoma cells share growth-suppressive effects with Treg and suggest that mimicking Treg function may represent a new mechanism of immune evasion in pancreatic cancer.

Bcl-XL protects pancreatic adenocarcinoma cells against CD95- and TRAIL-receptor-mediated apoptosis

In this study we sought to clarify the role of the pro-apoptotic potential of mitochondria in the death pathway emanating from the TRAIL (APO-2L) and CD95 receptors in pancreatic carcinoma cells. We focused on the role of the Bcl-2 family member Bcl-XL, using three pancreatic carcinoma cell lines as a model system, two of which have high (Panc-1, PancTuI) and one has low (Colo357) Bcl-XL expression. In these cell lines, the expression of Bcl-XL correlated with sensitivity to apoptosis induced by TRAIL or anti-CD95. Flow cytometric analysis revealed cell surface expression of TRAIL-R1 and TRAIL-R2 on PancTuI and Colo357, and TRAIL-R2 on Panc-1 cells. In Colo357 cells retrovirally transduced with Bcl-XL, caspase-8 activation in response to treatment with TRAIL or anti-CD95 antibody was not different from parental cells and EGFP-transfected controls, however, apoptosis was completely suppressed as measured by the mitochondrial transmembrane potential Δψm, caspase-3 activity (PARP cleavage) and DNA-fragmentation. Inhibition of Bcl-XL function by overexpression of Bax or administration of antisense oligonucleotides against Bcl-XL mRNA resulted in sensitization of Panc-1 cells to TRAIL and PancTuI cells to anti-CD95 antibody-induced cell death. The results show that Bcl-XL can protect pancreatic cancer cells from CD95- and TRAIL-mediated apoptosis. Thus, in these epithelial tumour cells the mitochondrially mediated ‘type II’ pathway of apoptosis induction is not only operative regarding the CD95 system but also regarding the TRAIL system.

Investigation of innate immunity genes CARD4, CARD8 and CARD15 as germline susceptibility factors for colorectal cancer.

BackgroundVariation in genes involved in the innate immune response may play a role in the predisposition to colorectal cancer (CRC). Several polymorphisms of the CARD15 gene (caspase activating recruitment domain, member 15) have been reported to be associated with an increased susceptibility to Crohn disease. Since the CARD15 gene product and other CARD proteins function in innate immunity, we investigated the impact of germline variation at the CARD4, CARD8 and CARD15 loci on the risk for sporadic CRC, using a large patient sample from Northern Germany.MethodsA total of 1044 patients who had been operated with sporadic colorectal carcinoma (median age at diagnosis: 59 years) were recruited and compared to 724 sex-matched, population-based control individuals (median age: 68 years). Genetic investigation was carried out following both a coding SNP and haplotype tagging approach. Subgroup analyses for N = 143 patients with early manifestation of CRC (≤50 age at diagnosis) were performed for all CARD loci and subgroup analyses for diverse age strata were carried out for CARD15 mutations R702W, G908R and L1007fs. In addition, all SNPs were tested for association with disease presentation and family history of CRC.ResultsNo significant differences were observed between the patient and control allelic or haplotypic spectra of the three genes under study for the total cohort (N = 1044 patients). None of the analysed SNPs was significantly associated with either tumour location or yielded significant association in the familial or non-familial CRC patient subgroups. However, in a patient subgroup (≤45 age at diagnosis) with early disease manifestation the mutant allele of CARD15 R702W was found to be significantly associated with disease susceptibility (9.7% in cases vs 4.6% in controls; Pallelic = 0.008, Pgenotypic = 0.0008, ORallelic = 2.22 (1.21-4.05) ORressessive = 21.9 (1.96-245.4).ConclusionVariation in the innate immunity genes CARD4, CARD8 and CARD15 is unlikely to play a major role in the susceptibility to CRC in the German population. But, we report a significant disease contribution of CARD15 for CRC patients with very early disease manifestation, mainly driven by variant R702W.

Functional TLR5 genetic variants affect human colorectal cancer survival

Toll-like receptors (TLR) are overexpressed on many types of cancer cells, including colorectal cancer cells, but little is known about the functional relevance of these immune regulatory molecules in malignant settings. Here, we report frequent single-nucleotide polymorphisms (SNP) in the flagellin receptor TLR5 and the TLR downstream effector molecules MyD88 and TIRAP that are associated with altered survival in a large cohort of Caucasian patients with colorectal cancer (n = 613). MYD88 rs4988453, a SNP that maps to a promoter region shared with the acetyl coenzyme-A acyl-transferase-1 (ACAA1), was associated with decreased survival of patients with colorectal cancer and altered transcriptional activity of the proximal genes. In the TLR5 gene, rs5744174/F616L was associated with increased survival, whereas rs2072493/N592S was associated with decreased survival. Both rs2072493/N592S and rs5744174/F616L modulated TLR5 signaling in response to flagellin or to different commensal and pathogenic intestinal bacteria. Notably, we observed a reduction in flagellin-induced p38 phosphorylation, CD62L shedding, and elevated expression of interleukin (IL)-6 and IL-1β mRNA in human primary immune cells from TLR5 616LL homozygote carriers, as compared with 616FF carriers. This finding suggested that the well-documented effect of cytokines like IL-6 on colorectal cancer progression might be mediated by TLR5 genotype-dependent flagellin sensing. Our results establish an important link between TLR signaling and human colorectal cancer with relevance for biomarker and therapy development.

Impact of Preoperative Physiological Risk Profile on Postoperative Morbidity and Mortality After Emergency Operation of Complicated Peptic Ulcer Disease

AimThe aim of this study was to evaluate the preoperative physiological risk profile for postoperative morbidity and mortality after emergency treatment of complicated peptic ulcer disease (PUD).MethodsOperative notes and hospital files of 261 patients—111 female, 150 male; median age 67 years (range 17–100 years)—undergoing an emergency operation from 1993 to 2005 were analyzed retrospectively. The physiologic subscore of the POSSUM score (POSSUM-phys) was analyzed with regard to predicting postoperative complications. Follow-up was obtained from questionnaires sent to family practitioners or by patient interviews.ResultsThe overall complication rate was 44%, and mortality was 24%. Among risk factors studied (e.g., sex, patient’s age, duration of symptoms, type of surgery), a high POSSUM-phys score was the strongest predictor for postoperative sepsis, anastomotic/suture dehiscence, postoperative bleeding, and mortality. Cut points for patients at risk could be calculated. Surgical procedures (organ-preserving versus resection) had no influence when matched for POSSUM-phys score. Nevertheless, organ resections were associated with higher scores. Recurrent PUD was a rare event (7.6%).ConclusionThe preoperative physiologic POSSUM score is a promising instrument for identifying patients at increased risk to develop major postoperative complications after emergency surgery for complicated PUD. Prospective studies are needed to prove its applicability for adjusting treatment to individual patients.

Electrolytic ablation is as effective as radiofrequency ablation in the treatment of artificial liver metastases in a pig model

The best treatment option for liver metastases is complete surgical resection. Unfortunately, at the time of diagnosis, not all patients are candidates for complete resection. Electrolytic therapy (ECT) is a novel non‐thermal method of tissue destruction. We evaluated its safety and effectiveness in comparison with radiofrequency ablation (RFA).

Investigation of the Lith6 candidate genes APOBEC1 and PPARG in human gallstone disease

Background: Genetic susceptibility contributes to the aetiology of gallbladder diseases as shown by multiple epidemiological studies. A major gallstone susceptibility locus (Lith6) was identified in 2003 by quantitative trait locus mapping in mice. Two attractive positional and functional candidate genes in apolipoprotein B mRNA‐editing protein (APOBEC1) and peroxisome proliferator‐activated receptor γ (PPARG) are located in this interval.

Detection of circulating tumor cells with CK20 RT-PCR is an independent negative prognostic marker in colon cancer patients – a prospective study

BackgroundDetection of circulating (CTC) or disseminated tumor cells (DTC) has been associated with negative prognosis and outcome in patients with colorectal cancer, though testing for these cells is not yet part of clinical routine. There are several different methodological approaches to detect tumor cells but standardized detection assays are not implemented so far.MethodsIn this prospective monocentric study 299 patients with colon cancer were included. CTC and DTC were detected using CK20 RT-PCR as well as immunocytochemistry staining with anti-pan-keratin and anti-EpCAM antibodies. The primary endpoints were: Evaluation of CTC and DTC at the time of surgery and correlation with main tumor characteristics and overall (OS) and disease free survival (DFS).ResultsPatients with detectable CTC had a 5-year OS rate of 68% compared to a 5-year OS rate of 85% in patients without detectable CTC in the blood (p = 0.002). Detection of DTC in the bone marrow with CK20 RT-PCR was not associated with a worse OS or DFS. Detection of pan-cytokeratin positive DTC in the bone marrow correlated with a significantly reduced 5-year OS rate (p = 0.048), but detection of DTC in the bone marrow with the anti-EpCAM antibody did not significantly influence the 5-year OS rate (p = 0.958). By multivariate analyses only detection of CTC with CK20 RT-PCR in the blood was revealed to be an independent predictor of worse OS (HR1.94; 95% CI 1.0–3.7; p = 0.04) and DFS (HR 1.94; 95% CI 1.1–3.7; p = 0.044).ConclusionsDetection of CTC with CK20 RT-PCR is a highly specific and independent prognostic marker in colon cancer patients. Detection of DTC in the bone marrow with CK20 RT-PCR or immunohistochemistry with anti-EpCAM antibody is not associated with a negative prognostic influence.

Gastrointestinal stromal tumors with pseudocystic change mimicking a pancreatic tumor: two case reports

IntroductionCystic lesions of the upper abdomen normally develop from pancreatic tissue. The differential diagnoses include neoplastic and non-neoplastic lesions. Pseudocystic tumors that secondarily involve the pancreas are very rare and may lead to diagnostic pitfalls.Case presentationA 51-year-old woman and a 65-year-old man, both German, presented with abdominal cystic lesions suspected to be pancreatic pseudocysts. Both tumors were classified as gastrointestinal stromal tumors, epithelioid subtype. In one case, tumor origin in the gastric wall was confirmed by relaparotomy. In the other case, a point mutation in PDGFRalpha gene, exon 18 proved the diagnosis of gastrointestinal stromal tumor. The tumors were resected and both patients are still alive and disease-free.ConclusionsThe differential diagnosis of cystic lesions in the upper abdomen must include gastrointestinal stromal tumors with pseudocystic change. The origin of a large cystic gastrointestinal stromal tumor may be difficult to determine. Epithelioid tumor pattern, weak or absent KIT expression and detection of PDGFRalpha mutation are typical diagnostic parameters of gastric gastrointestinal stromal tumors.

Heterozygous carriage of the alpha1-antitrypsin Pi*Z variant increases the risk to develop liver cirrhosis

Objective Homozygous alpha1-antitrypsin (AAT) deficiency increases the risk for developing cirrhosis, whereas the relevance of heterozygous carriage remains unclear. Hence, we evaluated the impact of the two most relevant AAT variants (‘Pi*Z’ and ‘Pi*S’), present in up to 10% of Caucasians, on subjects with non-alcoholic fatty liver disease (NAFLD) or alcohol misuse. Design We analysed multicentric case–control cohorts consisting of 1184 people with biopsy-proven NAFLD and of 2462 people with chronic alcohol misuse, both cohorts comprising cases with cirrhosis and controls without cirrhosis. Genotyping for the Pi*Z and Pi*S variants was performed. Results The Pi*Z variant presented in 13.8% of patients with cirrhotic NAFLD but only in 2.4% of counterparts without liver fibrosis (p<0.0001). Accordingly, the Pi*Z variant increased the risk of NAFLD subjects to develop cirrhosis (adjusted OR=7.3 (95% CI 2.2 to 24.8)). Likewise, the Pi*Z variant presented in 6.2% of alcohol misusers with cirrhosis but only in 2.2% of alcohol misusers without significant liver injury (p<0.0001). Correspondingly, alcohol misusers carrying the Pi*Z variant were prone to develop cirrhosis (adjusted OR=5.8 (95% CI 2.9 to 11.7)). In contrast, the Pi*S variant was not associated with NAFLD-related cirrhosis and only borderline with alcohol-related cirrhosis (adjusted OR=1.47 (95% CI 0.99 to 2.19)). Conclusion The Pi*Z variant is the hitherto strongest single nucleotide polymorphism-based risk factor for cirrhosis in NAFLD and alcohol misuse, whereas the Pi*S variant confers only a weak risk in alcohol misusers. As 2%–4% of Caucasians are Pi*Z carriers, this finding should be considered in genetic counselling of affected individuals.