[Gastrointestinal stromal tumors of the stomach and precursor lesions]. / Gastrointestinale Stromatumoren des Magens und ihre Vorstufen.

Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors in the gastrointestinal tract although they are much less frequent than epithelial tumors. In more than 60% of cases they occur in the stomach. Especially small lesions measuring ≤1 cm in diameter, so-called microscopic GIST can occur multifocally, frequently in the proximal stomach wall and sometimes as an incidental finding in a gastrectomy specimen resected for gastric cancer. The multicentricity of GIST alone is not proof of a metastatic behavior or a syndromal or hereditary disease. Multiple sporadic synchronous and metachronous GIST are characterized by different primary mutations mostly in the KIT or PDGFRA genes and are often less aggressive. It is speculative whether a field effect is responsible or whether still unknown GIST-promoting factors may facilitate the development of several independent lesions. If KIT or PDGFRA mutations are lacking, a succinate dehydrogenase (SDH) deficient GIST has to be considered, either hereditary as Carney-Stratakis syndrome or syndromal as part of a Carney triad.

[Will molecular diagnostics become established in pancreatic pathology?]. / Findet die molekulare Diagnostik Einzug in die Pankreaspathologie?

Genetic alterations of solid and cystic tumors of the pancreas have been increasingly more characterized over the last few years. Pancreatic ductal adenocarcinoma (PDAC) carries numerous point mutations and, to a lesser extent, deletions and amplifications of genes that are associated with at least 13 tumor relevant signalling pathways and processes. Besides the four common driver mutations in the KRAS, p53, CDKN2a and SMAD4 genes there are a number of low frequency driver mutations. The classification of PDAC subtypes has benefited from recent analyses of transcriptional profiles that revealed a classical KRAS driven and a KRAS independent quasi-mesenchymal subtype. The analyses of mRNA and miRNA expression profiles of fine needle aspirates serve as a basis for reliable preoperative diagnosis of pancreatic masses.The four most common cystic pancreatic tumors bear tumor-specific genetic alterations, such as GNAS mutations in intraductal papillary mucinous neoplasms, ß-catenin mutations in solid pseudopapillary neoplasms and VHL mutations or loss of heterozygosity in serous cystadenoma. Recovery of DNA from aspirates of cyst fluids enables an improved preoperative management of cystic pancreatic tumors by mutational analysis. In addition to the analysis of DNA there are promising approaches in distinguishing benign and premalignant/malignant cystic tumors by evaluating miRNA profiles.In recent years much progress has been made in molecular genetic characterization and preoperative evaluation of pancreatic tumors. Hopefully these results will contribute to prognostic and therapeutic stratification of PDAC and to a reliable preoperative diagnostics of benign cystic pancreatic tumors in the future.

Multicenter validation of cancer gene panel-based next-generation sequencing for translational research and molecular diagnostics.

The simultaneous detection of multiple somatic mutations in the context of molecular diagnostics of cancer is frequently performed by means of amplicon-based targeted next-generation sequencing (NGS). However, only few studies are available comparing multicenter testing of different NGS platforms and gene panels. Therefore, seven partner sites of the German Cancer Consortium (DKTK) performed a multicenter interlaboratory trial for targeted NGS using the same formalin-fixed, paraffin-embedded (FFPE) specimen of molecularly pre-characterized tumors (n = 15; each n = 5 cases of Breast, Lung, and Colon carcinoma) and a colorectal cancer cell line DNA dilution series. Detailed information regarding pre-characterized mutations was not disclosed to the partners. Commercially available and custom-designed cancer gene panels were used for library preparation and subsequent sequencing on several devices of two NGS different platforms. For every case, centrally extracted DNA and FFPE tissue sections for local processing were delivered to each partner site to be sequenced with the commercial gene panel and local bioinformatics. For cancer-specific panel-based sequencing, only centrally extracted DNA was analyzed at seven sequencing sites. Subsequently, local data were compiled and bioinformatics was performed centrally. We were able to demonstrate that all pre-characterized mutations were re-identified correctly, irrespective of NGS platform or gene panel used. However, locally processed FFPE tissue sections disclosed that the DNA extraction method can affect the detection of mutations with a trend in favor of magnetic bead-based DNA extraction methods. In conclusion, targeted NGS is a very robust method for simultaneous detection of various mutations in FFPE tissue specimens if certain pre-analytical conditions are carefully considered.

Establishment of robust controls for the normalization of miRNA expression in neuroendocrine tumors of the ileum and pancreas.

There is need to determine tissue-specific robust controls for normalization of microRNA expression to avoid false results and misinterpretation. The aim of this study was to evaluate the expression of different small RNAs in neuroendocrine tumors (NETs) and their suitability as normalizers in miRNA real-time PCR experiments. We investigated the expression of the nine small RNAs miR-93, miR-191, SNORD48, SNORD61, SNORD68, SNORD72, SNORD95, SNORD96a, and RNU6-2 in formalin-fixed, paraffin-embedded tissue samples of 25 ileal NETs by real-time PCR determining the most stable controls for expression normalization using four different algorithms. This analysis was expended to ten pancreatic NETs. Finally, five small RNAs were further tested as normalizers for miRNA-133a expression, which is known to be downregulated in metastases of ileal NETs, in ten matched pairs of ileal NETs and their metastases. Ranking of the expression results revealed the following order of stability from high to low: SNORD61 < SNORD95 < SNORD72 < SNORD96a < SNORD68 < miR-191 < miR-93 < RNU6-2 < SNORD48 for ileal NETs and SNORD95 < miR-93 < SNORD96a < SNORD61 < SNORD68 < SNORD72 < RNU6-2 < miR-191 < SNORD48 for pancreatic NETs. The determination of SNORD61 and SNORD95 for ileal NETs and SNORD95 and miR-93 for pancreatic NETs as good normalizers presents a useful tool for experiments involving the analysis of miRNA expression.