Citations

Abstract High throughput sequencing technologies open up a new dimension in cancer genomics by enabling the characterization of cancer genomes at base-pair resolution. To date, the majority of large-scale genomics projects rely on fresh-frozen biospecimens in their studies, which are difficult to obtain and often lack long-term clinical data. Unlike fresh-frozen samples, archived formalin-fixed paraffin-embedded (FFPE) tissues are more readily accessible, and are often associated with known clinical outcomes and more complete clinical annotations. However, sequencing library preparation methods need to be further optimized with regard to applicability to FFPE samples. To test whether next-generation sequencing technologies could overcome previously reported artifacts associated with formalin fixation and report accurate sequencing results, we compared whole-genome and targeted enrichment DNA sequencing data obtained from five FFPE tumor samples for which matching frozen tissues were available. We successfully generated sequencing libraries using 1 µg of genomic DNA as input material and a modified TruSeq™ sample preparation protocol. We designed a custom enrichment pool targeting exons from 215 cancer-related genes and utilized this pool to pull-down and sequence approximately 1.3 Mb region of the genome with an average 400x coverage depth to test if deep sequencing would improve validation of tumor-specific somatic mutations. We used the Illumina sequence analysis pipeline and the Windows-based second generation DNA sequencing software NextGENe® to analyze the data and identify sequence variations that are different from the human reference genome. CNV detection was overall higher among FFPE samples compared with fresh-frozen samples, demonstrating that tissue processing impacts sequencing data quality. We obtained good concordance in variant calls between matched FFPE and fresh-frozen samples. Discordant variant calls were mainly due to low depth of coverage in the regions where variant calls were made. Improvements to DNA sequencing methods for archived samples will significantly enhance cancer research and will result in more reliable prediction of individual cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3179. doi:1538-7445.AM2012-3179

Abstract During early metastatic events in cancer, reorganization of the actin cytoskeleton must occur to allow for cell invasion and migration. A key enzyme initiating actin remodeling at the leading edge is Cofilin, a small actin binding protein of the ADF/cofilin family with actin severing activities. Cofilin activity is tightly regulated by phosphorylation and dephosphorylation events that are mediated by LIM Kinase (LIMK) and the phosphatase Slingshot (SSH), respectively. Protein kinase D (PKD) is a serine/threonine kinase that has previously been shown to regulate actin driven directed cell migration through phosphorylation and thus inactivation of Slingshot. Here we show that PKD can also regulate cofilin activity via activation of LIMK. We further show that PKD-induced regulation of LIMK and the resulting diminished cofilin activity translates to altered cell motility. Our data suggest that PKD inactivates Cofilin through modulation of both of its regulatory pathways. Further elucidation of the mechanisms by which PKD regulates directed cell migration could enable development of a potential treatment strategy for invasive cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2364. doi:10.1158/1538-7445.AM2011-2364