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Whole Exome Sequencing Revealed Putative Driver Mutations in Esophageal Cancer
Peter P. Grimminger*1, Martin Peifer2,4, Roman Thomas4, Martin K. Maus1, Jan Brabender3, Arnulf H. HöLscher1, Reinhard BüTtner5, Margarete Odenthal5
1Department of General-, Visceral- and Tumor surgery, University Clinic Cologne, Cologne, Germany; 2Department of Translational Genomics,, University of Cologne, Cologne, Germany; 3General- and Visceral Surgery, St. Antonius Hospital, Cologne, Germany; 4Cologne Center for Genomics, University of Cologne, Cologne, Germany; 5Pathology, University of Cologne, Cologne, Germany
Esophageal cancer is one of the most common malignancies in the Western world with increasing incidence of esophageal adenocarcinoma (EAC). Despite improvements in staging, surgical procedures, and post-operative treatments, the overall survival of patients with esophageal cancer remains low. In order to evaluate the mutation status of EAC and squamous cell cancer of the esophagus (SCC) we performed next generation sequencing (NGS) approaches on a wide set of tumor-derived DNA from histological classified EAC and SCC biopsies.
Whole exome analysis was performed on 16 DNA samples from histological characterised esophageal cancer (n=8) and the corresponding non-tumor biopsies (n=8). Extracted DNA was applied to NimbleGen capture exon hybridisation, adapter ligation and subsequent deep sequencing on an Illumina HiSeq platform. After tumor macrodissection, DNA from additional 147 formalin-fixed and paraffin-embedded (FFPE) EAC and SEC biopsies was extracted using the Qiagen M48 robotic system. After DNA quality control, multiplex PCR libraries, representing tumor-relevant genetic loci, were prepared from 50 quality controlled EAC and SCC DNA samples. Multiplex libraries were analyzed for more than 2000 putative driver mutations by next generation sequencing on the MiSeq Illumina platform.
745 putative driver mutations in 657 genetic loci were found in a first whole exome screening step. p53 hot spot mutations occurred in two third of the esophageal cancers. In addition to the p53 mutations, whole exome analysis identified more than two mutation hits in genes for the regulatory phosphatase unit, an adhesion P-cadherin and cycline kinase 12. These mutations were also addressed by conventional Sanger sequencing. Subsequently, DNA samples from 147 SCC and EAC were studied. Analyses of a hot spot cancer panel in 50 samples, that had passed the quality control, confirmed high frequency of p53 mutations, but a lack of K-Ras mutations. In addition, a set of further mutations such as in PIC3CA, PP2R1B, and PPP1R1B were shown, whose clinical relevance has to be addressed in future studies.
NGS is a sensitive method in evaluation of the mutation status of esophageal cancer, providing the opportunity to detect a wide range of genetic alterations, which have to be linked to cancer progression, therapeutic outcome and personalized treatment options in future studies.
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