Hans Konrad Schackert

Meta-analysis of microarray data on pancreatic cancer defines a set of commonly dysregulated genes.

Pancreatic ductal adenocarcinoma is the eighth most common cancer with the lowest overall 5-year relative survival rate of any tumor type today. Expression profiling using microarrays has been widely used to identify genes associated with pancreatic cancer development. To extract maximum value from the available gene expression data, we applied a meta-analysis to search for commonly differentially expressed genes in pancreatic ductal adenocarcinoma. We obtained data sets from four different gene expression studies on pancreatic cancer. We selected a consensus set of 2984 genes measured in all four studies and applied a meta-analysis approach to evaluate the combined data. Of the genes identified as differentially expressed, several were validated using RT–PCR and immunohistochemistry. Additionally, we used a class discovery algorithm to identify a gene expression signature. Our meta-analysis revealed that the pancreatic cancer gene expression data sets shared a significant number of up- and downregulated genes, independent of the technology used. This interstudy crossvalidation approach generated a set of 568 genes that were consistently and significantly dysregulated in pancreatic cancer. Of these, 364 (64.1%) were upregulated and 204 (35.9%) were downregulated in pancreatic cancer. Only 127 (22%) were described in the published individual analyses. Functional annotation of the genes revealed that genes presumably associated with the cell adhesion-mediated drug resistance pathway are frequently overexpressed in pancreatic cancer. Meta-analysis is an important tool for the identification and validation of differentially expressed genes. These could represent good candidates for novel diagnostic and therapeutic approaches to pancreatic cancer.

Frequent loss of the CDKN2C (p18INK4c) gene product in pituitary adenomas

Genomic alterations of cyclin‐dependent kinase inhibitors have been demonstrated in a variety of tumor types including brain tumors. Among them, the cyclin‐dependent kinase inhibitor 2A (CDKN2A or p16INK4a) gene has been shown to be frequently deleted or inactivated in astrocytic tumors. The CDKN2C (p18INK4c) gene is functionally related to CDKN2A. Moreover, mice with targeted disruption of CDKN2C alone or combined CDKN2C and cyclin‐dependent kinase inhibitor 1B (CDKN1B or p27Kip1), or CDKN2C and TP53 gene disruption develop pituitary adenomas (PA) at high frequencies. The purpose of our study was to investigate genetic alterations of the CDKN2C gene by analysis of loss of heterozygosity (LOH), screening for mutations, analysis of promoter methylation, and protein expression in 38 PAs. In addition, genomic alterations and protein expression of the cell cycle genes CDKN2A and its alternatively spliced form, p14ARF, as well as the retinoblastoma RB1 gene were investigated. LOH at the CDKN2C gene locus was detected in 25% of pituitary adenomas, whereas the RB1 and CDKN2A loci were altered in only 10%. No mutations were detected within the coding regions of the CDKN2C gene. However, 39.5% of adenomas displayed CDKN2C promoter methylation. The absence of CDKN2C protein was correlated with LOH of the CDKN2C locus on chromosome 1 and with methylation of the CDKN2C promoter. This is the first report to describe that the tumor suppressor gene CDKN2C is frequently targeted by genomic alterations in pituitary adenoma. The most common genetic alteration was promoter methylation suggesting that inactivation of CDKN2C by this mechanism may play an important role in pituitary adenoma development. Additional Supporting Information may be found in the online version of this article.

An intronic germline transition in the HNPCC gene hMSH2 is associated with sporadic colorectal cancer

The aim of this study was to determine whether an intronic germline substitution in the hereditary non-polyposis colorectal cancer (HNPCC) gene hMSH2 represents a genetic risk factor for sporadic CRC. Possible effects of this substitution were investigated by assessment of microsatellite instability and hMSH2 cDNA sequencing. Constitutional DNA from patients with sporadic CRC and healthy controls from the same region in Germany was analysed for the intronic germline T-->C transition six bases upstream of exon 13 of hMSH2. 29 of 106 patients (27%) were found to harbour the germline T-->C transition as opposed to only 13 of 125 controls (10%; P < 0.001; OR 3.2, CI 1.58-6.63). CRCs from patients with the substitution displayed neither clinical HNPCC-like features nor an increased rate of microsatellite instability. No abnormal cDNA sequence was found at the exon 12-13 border. These data suggest a 3.2-fold increased risk of sporadic CRC for individuals with the intronic hMSH2 transition. However, this substitution might not be pathogenic itself, but may be linked to a locus nearby that is.

TLR4 and IL-18 Gene Variants in Chronic Periodontitis: Impact on Disease Susceptibility and Severity

The aim of the study was to assess whether genotypes in the Toll-like receptor 4 gene and in the promoter of the interleukin-18 gene are associated with the susceptibility to chronic periodontitis. 108 chronic periodontitis patients and 76 controls were genotyped for c.896A>G/1196C>T (TLR4 gene) and for c.-368G>C/ c.-838C>A (IL-18 promoter). There were no significant differences in genotype and allele distributions between the study groups. Periodontitis severity in patients with TLR4 c.896AG/1196CT genotype was significantly higher than wildtype carriers. The percentage of teeth with clinical attachment loss ≥ 5 mm was 77.3% and 58.8%, respectively (p ≤ 0.006, t-test). All subjects were further classified into carriers and non-carriers of at least one variant of each gene. A logistic regression analysis adjusted for gender, smoking, and age showed no association between gene variant carrier status and periodontitis (OR = 1.98, 95% CI 0.61–6.39). The results did not show that IL-18 and TLR4 variants have an effect on the susceptibility to chronic periodontitis. Considering the low number of periodontitis patients carrying TLR4 variants (11%), a comparison of the periodontitis severity depending on the genotype has to be interpreted cautiously.

Identifizierung und Validierung von differentiell exprimierten Genen im Stroma von duktalen Pankreaskarzinomen und chronischer Pankreatitis

Background: Pancreatic ductal adenocarcinoma is characterized by an abundant desmoplastic stroma. Interactions between cancer and stromal cells play a critical role in tumour invasion, metastasis and chemoresistance. Therefore, we hypothesised that the gene expression profile of the stromal components of pancreatic carcinoma is different from chronic pancreatitis and reflects the interaction with the tumour. Methods: We investigated the gene expression of eleven stromal tissue from pancreatic ductal adenocarcinoma, nine from chronic pancreatitis and cell lines of stromal origin using the Affymetrix U133 GeneChip set. The tissue samples were microdissected, the RNA was extracted, amplified, and labelled using a repetitive in vitro transcription protocol. Differentially expressed genes were identified using the significance analysis of microarrays program. Results: We found 255 genes to be over-expressed and 61 genes to be under-expressed within the stroma of pancreatic carcinoma compared to the stroma of chronic pancreatitis. Analysis of the involved signal transduction pathways revealed a number of genes associated with the Wnt pathway. Therefore, we confirmed the differential expression of SFRP1 and WNT5a using immunohistochemistry. Moreover, we could demonstrate that WNT5a expression was induced in fibroblasts during co-cultivation with a pancreatic carcinoma cell line. Conclusion: The identified differences in the expression profile of stroma cells derived from tumour compared to cells of inflammatory origin suggest a specific response of the tissue surrounding malignant cells. The over-expression of WNT5a, a gene involved in the non canonical Wnt signalling and chondrocyte development might contribute to the strong desmoplastic reaction seen in pancreatic cancer.

Hereditäre kolorektale Karzinome – Überlegung zu präventiven chirurgischen Maßnahmen

Hereditary Colorectal Carcinomas – Reflection on Preventive Surgery Hereditary Nonpolyposis Colorectal Cancer (HNPCC) accounts for about 5% of all colorectal cancers and is the most frequent familial form; familial adenomatous polyposis coli accounts for about 1%. Prerequisitive for individually tailored surveillance is the identification of the pathogenic germline mutation. In classical FAP, surgical standard is a restorative proctocolectomy while in HNPCC there is no surgical standard other than standard oncological resection due to missing evidence. In HNPCC, prophylactic colectomy before the onset of the first colorectal cancer is not recommended. Main arguments for the extension of the resection in the case of the first colorectal carcinoma in HNPCC are the rate of metachronous colorectal carcinomas of 40–45% in a 10-year interval and rapid tumor progression. In HNPCC, in the case of first colon cancer a subtotal colectomy seems to be indicated. A proctocolectomy or, if indicated, a restorative proctocolectomy may be considered in the case of carcinomas in the lower rectum. These considerations should be evaluated in a prospective clinical trial. Counselling, molecular diagnosis and surgery in patients with hereditary colorectal cancers should only be performed in interdisciplinary centers.

SEQUENZANALYSE DES BRCA1 GENS BEI BRUSTKREBSPATIENTINNEN MIT JUNGEM ERKRANKUNGSALTER UND/ODER POSITIVER FAMILIENANAMNESE

Das BRCA1 Gen ist eines der beiden bislang identifizierten Brustkrebsgene, die vermutlich in hohem Mase zum Auftreten von hereditaren Brust- und Ovarialkarzinomen pradisponieren. Aufgrund von Gen-Kopplungsanalysen wurde zunachst angenommen, das ca. 45% aller hereditaren Mammakarzinomfalle sowie die Mehrzahl der hereditaren Mamma-/Ovarialkarzinomfalle durch Mutationen in BRCA1 bedingt sind [1]. Umfangreiche Untersuchungen zum Nachweis von BRCA1 Keimbahnmutationen in Familien mit mehreren Mamma-/Ovarialkarzinomfallen ergaben jedoch deutlich niedrigere Mutationsraten von 12%-32% [2, 3, 4]. Bei sporadischen Brust-/Ovarialtumoren spielen BRCA1 Keimbahnmutationen nur eine untergeordnete Rolle [5, 6]. Es gibt Hinweise darauf, das haufig vorkommende niedrigpenetrante Varianten oder seltene Allele die Krebspradisposition beeinflussen konnen [7, 8, 9]. Untersuchungen von solchen Polymorphismen konnen somit wichtige Hinweise auf eine Genotyp-Phanotyp Korrelation der Erkrankung geben.

MUTATIONSSCREENING MITTELS RESTRIKTIONSFRAGMENTLANGENANALYSE (RFLA) AM BEISPIEL DES GENS FUR DIE FAMILIARE ADENOMATOSE POLYPOSIS (APC GEN)

In grosen Genen, wie dem APC Gen fur die familiare adenomatose Polyposis (FAP), ist die DNA Sequenzanalyse nach Sanger des gesamten kodierenden Abschnittes mit erheblichem Zeit- und Arbeitsaufwand verbunden. Hier stellen wir eine Screeningmethode (Restriktionsfragmentlangenanalyse, RFLA) vor, die es uns erlaubt, Deletionen und Insertionen mit hoher Sensitivitat, schnell und einfach mit einem automatischen Laser-Fluoreszenz-Sequenzierer (A.L.F.) uber einen grosen Genabschnitt (3,2 kb) nachzuweisen. Die RFLA kombiniert die Vorteile zweier bekannter Techniken. Grose DNA Abschnitte konnen mit Restriktionsenzymen in kleinere Fragmente aufgespalten werden [1, 2, 3]. Die Laufeigenschaften mutierter und nicht mutierter DNA Fragmente auf Polyacrylamidgelen sind unterschiedlich und konnen durch Grosenbestimmung der auffalligen Fragmente einem umschriebenen Genbereich zugeordnet werden [4, 5]. Um die RFLA Screeningmethode zu evaluieren, analysierten wir genomische DNA von FAP Patienten, bei denen bereits eine Deletion bzw. Insertion in der ersten Halfte des Exon 15 im APC Gen durch DNA Sequenzierung nachgewiesen worden war.

Monocyte- and Lymphocyte-Mediated Cytotoxicity After Gene Transfer and Expression of Human Interleukin-2 in a Human Glioblastoma Cell Line

Failing defense mechanisms against malignant tumors are thought to be caused by low immunogenicity of tumor cells and defective T helper cell functions. The concept of associate recognition by Lake and Mitchison [5] posits the necessity of at least two antigens being expressed to achieve a sufficient immune response against the cellular target. In vivo experiments show that expression of a viral antigen together with the putative tumor-associated antigen(s) not only triggers a strong immune response against itself but also remarkably improves immune response mechanisms against the tumor-associated antigen [6]. Local cytokine expression by tumor cells, on the other hand, bypasses a defective T helper function [2] and elicits an effective immune response against tumor cells in animal models [2, 3].

Charakterisierung von Keimbahnmutationen bei Patienten mit familiärer adenomatöser Polyposis (FAP) durch DNA-Sequenzanalyse

Die familiare adenomatose Polyposis ist eine autosomal dominant vererbte Erkrankung mit hoher Penetranz. Im symptomatischen Stadium der Erkrankung treten Hunderte von adenomatosen Polypen in Kolon und Rektum auf, die von verschiedenartigen Tumoren in anderen Organen begleitet werden konnen. Ohne chirurgische Behandlung entwickeln sich bei betroffenen Patienten meist kolorektale Karzinome [1]. Verschiedene Untersuchungen deuten darauf hin, das Mutationen im APC-Gen, das auf dem langen Arm des Chromosoms 5 lokalisiert ist, ursachlich an der Entwicklung der FAP beteiligt sind [2]. Die uberwiegende Zahl dieser Mutationen, von denen mehr als 60% als Deletionen bzw. Insertionen auftreten, fuhren zur Synthese eines verkurzten APC-Proteins. Fast alle (97%) Mutationen sind in der ersten Halfte des APC-Gens lokalisiert [3]. Eine prasymptomatische Diagnostik ist bei Risikopersonen durch DNA-Sequenzanalyse moglich. Die indirekte Genotypanalyse (Kopplungsuntersuchung) erlaubt die Abschatzung des Erkrankungsrisikos in einer Familie nur dann, wenn mindestens zwei Familienangehorige die Krankheit ausgepragt haben [4]. Ziel war es, eine direkte Genotypanalyse (Sequenzanalyse) im APC-Gen bei FAP-Patienten durchzufuhren. Mutationen sind die Grundlage einer praklinischen Diagnostik in betroffenen Familien und ermoglichen die Korrelation von Genotyp (Art der Mutation) und Phanotyp (Auspragung der Erkrankung).