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In recent years, the advent of massively parallel next-generation sequencing technologies has enabled substantial advances in the study of human diseases. Combined with targeted DNA enrichment methods, high sequence coverage can be obtained for different genes simultaneously at a reduced cost per sample, creating unique opportunities for clinical cancer diagnostics. However, the formalin-fixed, paraffin-embedded (FFPE) process of tissue samples, routinely used in pathology departments, results in DNA fragmentation and nucleotide modifications that introduce a number of technical challenges for downstream biomolecular analyses. We evaluated the HaloPlex target enrichment system for somatic mutation detection in 80 tissue fractions derived from 20 clinical cancer cases with paired tumor and normal tissue available in both FFPE and fresh-frozen format. Several modifications to the standard method were introduced, including a reduced target fragment length and two strand capturing. We found that FFPE material can be used for HaloPlex-based target enrichment and next-generation sequencing, even when starting from small amounts of DNA. By specifically capturing both strands for each target fragment, we were able to reduce the number of false-positive errors caused by FFPE-induced artifacts and lower the detection limit for somatic mutations. We believe that the HaloPlex method presented here will be broadly applicable as a tool for somatic mutation detection in clinical cancer settings., (Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

The potential of a variety of xenobiotic compounds to modulate or disrupt the endocrine system of humans and wildlife is now widely recognized. In the present study, we developed a molecular tool for the evaluation of endocrine disruption in common carp (Cyprinus carpio). Suppression Subtractive Hybridization PCR was applied for the isolation of a relevant gene set, consisting of gender- and hormone-responsive gene fragments. This resulted in 398 different gene fragments that were most related to endocrine functioning. To investigate the applicability of this gene collection for studying endocrine disruption in fish, the gender-related genes were spotted on a cDNA macroarray, and expression profiles were generated for 17beta-estradiol (E2) and cortisol. Therefore, fish were injected with these hormones, and after 24 h and 96 h RNA was extracted and used for macroarray hybridizations. E2 exposure resulted in a total of 35 differentially expressed genes, whereas cortisol only affected 3 genes spotted on the macroarray. These results indicate the discriminating power of the developed array, and its usefulness to describe the toxicological mode of action of endocrine disruptive chemicals.