High-throughput sequencing and copy number variation detection using formalin fixed embedded tissue in metastatic gastric cancer.
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.
Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Seokhwi
S
Kim
Department of Medicine, Division of Hematology-Oncology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Jeeyun
J
Lee
Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea; Cancer Companion Diagnostics Center, Samsung Medical Center, Seoul, Korea.
Min Eui
ME
Hong
Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea; Cancer Companion Diagnostics Center, Samsung Medical Center, Seoul, Korea.
In-Gu
IG
Do
Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
So Young
SY
Kang
Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Sang Yun
SY
Ha
Department of Medicine, Division of Hematology-Oncology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Seung Tae
ST
Kim
Department of Medicine, Division of Hematology-Oncology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Se Hoon
SH
Park
Department of Medicine, Division of Hematology-Oncology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Won Ki
WK
Kang
Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Min-Gew
MG
Choi
Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Jun Ho
JH
Lee
Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Tae Sung
TS
Sohn
Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Jae Moon
JM
Bae
Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Sung
S
Kim
Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Korea.
Duk-Hwan
DH
Kim
Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea; Cancer Companion Diagnostics Center, Samsung Medical Center, Seoul, Korea.
Kyoung-Mee
KM
Kim
D000328:Adult; D000368:Aged; D000483:Alleles; D019943:Amino Acid Substitution; D056915:DNA Copy Number Variations; D005260:Female; D005500:Follow-Up Studies; D005784:Gene Amplification; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D008875:Middle Aged; D059645:Mutation Rate; D009362:Neoplasm Metastasis; D009367:Neoplasm Staging; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D013274:Stomach Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*