High-throughput sequencing and copy number variation detection using formalin fixed embedded tissue in metastatic gastric cancer

• Published in: PloS one 2014-11, Vol.9 (11), p.e111693
• Main Authors: Kim, Seokhwi, Lee, Jeeyun, Hong, Min Eui, Do, In-Gu, Kang, So Young, Ha, Sang Yun, Kim, Seung Tae, Park, Se Hoon, Kang, Won Ki, Choi, Min-Gew, Lee, Jun Ho, Sohn, Tae Sung, Bae, Jae Moon, Kim, Sung, Kim, Duk-Hwan, Kim, Kyoung-Mee
• Format: Article
• Language: English
• Subjects: Adenomatous polyposis coli; Adult; Aged; Alleles; Amino Acid Substitution; Amplification; Analysis; Cancer; Cancer metastasis; Cancer screening; Cancer therapies; Cell adhesion & migration; Chemotherapy; Copy number; Deoxyribonucleic acid; DNA; DNA Copy Number Variations; DNA sequencing; Epidermal growth factor receptors; ErbB-2 protein; Female; Fluorescence; Fluorescence in situ hybridization; Follow-Up Studies; Formaldehyde; Gastric cancer; Gene Amplification; Genes; Health aspects; Hematology; High-Throughput Nucleotide Sequencing; High-throughput screening; Hot spots; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; K-Ras protein; Male; Medical prognosis; Medicine; Medicine and Health Sciences; Metastases; Metastasis; Middle Aged; Mutation; Mutation hot spots; Mutation Rate; Myc protein; Neoplasm Metastasis; Neoplasm Staging; Next-generation sequencing; Oncology; p53 Protein; Paraffin; Pathology; Patients; Physicians; Proteins; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Sequences; Smad4 protein; Stomach cancer; Stomach Neoplasms - genetics; Stomach Neoplasms - metabolism; Stomach Neoplasms - pathology; Studies; Surgery; Tumor cells; Tumor proteins; Variation
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0111693

In the era of targeted therapy, mutation profiling of cancer is a crucial aspect of making therapeutic decisions. To characterize cancer at a molecular level, the use of formalin-fixed paraffin-embedded tissue is important. We tested the Ion AmpliSeq Cancer Hotspot Panel v2 and nCounter Copy Number Variation Assay in 89 formalin-fixed paraffin-embedded gastric cancer samples to determine whether they are applicable in archival clinical samples for personalized targeted therapies. We validated the results with Sanger sequencing, real-time quantitative PCR, fluorescence in situ hybridization and immunohistochemistry. Frequently detected somatic mutations included TP53 (28.17%), APC (10.1%), PIK3CA (5.6%), KRAS (4.5%), SMO (3.4%), STK11 (3.4%), CDKN2A (3.4%) and SMAD4 (3.4%). Amplifications of HER2, CCNE1, MYC, KRAS and EGFR genes were observed in 8 (8.9%), 4 (4.5%), 2 (2.2%), 1 (1.1%) and 1 (1.1%) cases, respectively. In the cases with amplification, fluorescence in situ hybridization for HER2 verified gene amplification and immunohistochemistry for HER2, EGFR and CCNE1 verified the overexpression of proteins in tumor cells. In conclusion, we successfully performed semiconductor-based sequencing and nCounter copy number variation analyses in formalin-fixed paraffin-embedded gastric cancer samples. High-throughput screening in archival clinical samples enables faster, more accurate and cost-effective detection of hotspot mutations or amplification in genes.