Next-Generation Sequencing Analysis of Laser-Microdissected Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Specimens.

In recent years, next-generation sequencing (NGS) became widely used in molecular pathology. Comprehensive mutational profiling improved diagnosis and prognosis, as well as the identification of therapeutically relevant genetic alterations. However, the vast majority of studies analyzing tissue samples use DNA extracted from bulk tissue or only manually microdissected specimens. Laser-assisted microdissection offers the possibility of isolating morphologically defined small tissue compartments (like individual glands) or even of single cells for further molecular analysis. Even formalin-fixed paraffin-embedded (FFPE) tissue specimens can be used for laser-assisted microdissection. Combining these two innovative powerful methodological approaches provides invaluable insights into the genetic profile of any cell type and tissue compartment of interest, contributing to a better understanding of fundamental biological processes and disease-specific mechanisms.In this chapter, a detailed protocol is provided for microdissection of human mammary adenomyoepithelioma tissue specimens and subsequent targeted resequencing of a panel of cancer-related genes using IonTorrent/PGM technology.