Heiko Fensterer

Expression profiling of the influence of RAS mutants on the TGFB1-induced phenotype of the pancreatic cancer cell line PANC-1

Expression profiling analyses were used to elucidate the functional relevance of RAS proteins in mediating the effect of TGFB1 on the transcriptional phenotype of the pancreatic cancer cell line PANC‐1. Despite the presence of one mutated KRAS2 allele in parental PANC‐1 pancreatic cancer cells, RAS‐dependent signal transduction remained susceptible to stimulation by EGF and TGFB1. To analyze the impact of RAS proteins on the TGFB1‐induced transcriptional phenotype, we used PANC‐1 cells stably transfected with a dominant negative HRAS(S17N) mutant or with a constitutively active KRAS2(G12V) mutant. TGFB1 treatment of mock‐transfected PANC‐1 cells led to an expression profile suggestive of epithelial‐mesenchymal transdifferentiation (EMT). Profiling of the HRAS(S17N)‐expressing clone demonstrated that induction of endogenous RAS activity by TGFB1 is required for the development of the TGFB1‐induced transcriptional phenotype of PANC‐1 cells. The expression of the KRAS2(G12V) mutant by itself repressed transcription of markers of epithelial differentiation and induced transcription of several extracellular matrix‐associated genes. This effect was not enhanced further by TGFB1 treatment. In contrast, transcript levels of genes associated with proliferation and cell cycle progression did not appear to be the primary targets of the synergism between the RAS‐ and TGFB1‐dependent cascades. The introduction of the dominant negative and the constitutively active RAS mutants induced partly overlapping and partly inverse effects on the TGFB1‐induced expression profile of PANC‐1 cells. Additional mechanisms such as the induction of autocrine loops and the use of different RAS isoforms or alternate, ERK‐independent signaling pathways may be involved in the interaction between the RAS‐ and the TGFB1‐dependent signaling cascades.

Multicenter phase II trial to investigate safety and efficacy of gemcitabine combined with cetuximab as adjuvant therapy in pancreatic cancer (ATIP)

BACKGROUND To investigate whether addition of cetuximab to standard adjuvant chemotherapy with gemcitabine improves outcome in pancreatic cancer, specifically whether the rate of disease-free survival (DFS) at 18 months (primary end point) exceeds the previously reported 35% of gemcitabine alone. PATIENTS AND METHODS Prospective, open-label, multicenter, nonrandomized phase II study in 76 patients with R0- or R1-resected ductal adenocarcinoma of the pancreas included between October 2006 and November 2008. Gemcitabine and cetuximab were administered for 24 weeks. Secondary end points included overall survival (OS) and toxic effect. RESULTS Seventy-three patients received cetuximab. Median DFS was 10.0 [95% confidence interval (CI) 8.9-13.6] months and the DFS rate at month 18 of 27.1% (16.7%-37.6%) was inferior to 35%. Median OS was 22.4 (18.2-27.9) months. Subgroup analyses revealed a nonsignificant increase in DFS for patients with versus without skin toxic effect ≥ grade 2 (median 14.7 versus 8.3 months, P = 0.073) and wild-type versus mutated K-Ras (median 11.5 versus 9.3 months, P = 0.57). Grade 3/4 toxic effects included neutropenia (11.0%), thrombopenia (7%), skin toxic effect (7%) and allergic reactions (7%). CONCLUSION Addition of cetuximab to adjuvant gemcitabine does not seem to improve DFS or OS of unstratified pancreatic cancer patients. Trends for improved DFS in patients with wild-type K-Ras and skin toxic effect remain to be confirmed.

Matrix-comparative genomic hybridization from multicenter formalin-fixed paraffin-embedded colorectal cancer tissue blocks

BackgroundThe identification of genomic signatures of colorectal cancer for risk stratification requires the study of large series of cancer patients with an extensive clinical follow-up. Multicentric clinical studies represent an ideal source of well documented archived material for this type of analyses.MethodsTo verify if this material is technically suitable to perform matrix-CGH, we performed a pilot study using macrodissected 29 formalin-fixed, paraffin-embedded tissue samples collected within the framework of the EORTC-GI/PETACC-2 trial for colorectal cancer. The scientific aim was to identify prognostic genomic signatures differentiating locally restricted (UICC stages II-III) from systemically advanced (UICC stage IV) colorectal tumours.ResultsThe majority of archived tissue samples collected in the different centers was suitable to perform matrix-CGH. 5/7 advanced tumours displayed 13q-gain and 18q-loss. In locally restricted tumours, only 6/12 tumours showed a gain on 13q and 7/12 tumours showed a loss on 18q. Interphase-FISH and high-resolution array-mapping of the gain on 13q confirmed the validity of the array-data and narrowed the chromosomal interval containing potential oncogenes.ConclusionArchival, paraffin-embedded tissue samples collected in multicentric clinical trials are suitable for matrix-CGH analyses and allow the identification of prognostic signatures and aberrations harbouring potential new oncogenes.

Use of DNA Arrays/Microarrays in Pancreatic Research

In recent years, enormous technical advances in experimental protocols as well as robotic and bioinformatic techniques have allowed DNA array/microarray technology to emerge as the leading technology in the field of functional, disease-related genome analysis. Multiple applications exist for DNA arrays/microarrays including comparative genomic analysis to identify chromosomal imbalances (Matrix-CGH), the study of mutations and genetic polymorphisms, and the study of gene expression (expression profiling). Expression profiling is the most widely used application of DNA array/microarray technology and allows to measure gene expression of thousands of genes simultaneously. The present review describes the basic principles of expression profiling analyses and outlines some applications in pancreatic cancer research.

Impact of Pantoprazole on Duodeno-Gastro-Esophageal Reflux (DGER)

BACKGROUND Duodeno-gastro-esophageal reflux (DGER) is considered as an independent risk factor for complicated reflux disease (GERD). Patients with Barretts esophagus have significantly higher levels of DGER than patients with uncomplicated GERD. However, the clinical response to conventional high-dose PPI therapy in patients with uncomplicated GERD and DGER is largely unknown. METHODS 30 patients with uncomplicated GERD and combined pathological reflux (acid and bile) were enrolled in the study. Clinical work-up included evaluation of clinical symptoms, esophageal manometry and upper endoscopy. After 6 - 8 weeks of treatment with Pantoprazole 80 mg/d pH measurement and Bilitec 2000 were repeated, and the pattern of symptoms was re-evaluated. RESULTS Under treatment with Pantoprazole 80 mg/d acid reflux was normalised in 28 patients (93 %). Similarly the mean percentage of DGER (time with an absorption greater than 0.14) was significantly reduced from 19.6 % (+/- 13.7) to 5.7 % (+/- 7.7, p < 0.05). In 15 patients (50 %) an elevated DGER persisted under treatment with Pantoprazole (DGER-NR group) whereas in 15 cases (50 %) a normalisation could be achieved (DGER-R group). The DGER-NR group had significantly higher levels of bile reflux before (and under) treatment compared to the DGER-R group: 22.9 % (9.98 %) vs. 15.6 % (0.72 %), respectively. Overall, the median quality of life index (QLI) improved from 4.78 (+/- 0.86) before to 8.04 +/- 1.84) under therapy. The clinical response under treatment was marikedly reduced in the DGER-NR group compared to the DGER-R group: QLI 7.3 vs. 8.9. Particularly heartburn and nocturnal coughing persisted. CONCLUSIONS Our data confirm that high-dose pantoprazole therapy effectively exerts acid suppression in GERD patients with combined pathological reflux. However, DGER could only normalised in 50 % of patients. High levels of DGER at diagnosis enhance the risk of persistent DGER under PPI therapy and are associated with a reduced clinical outcome.