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

Kim S, Lee J, Hong ME, Do IG, Kang SY, Ha SY, Kim ST, Park SH, Kang WK, Choi MG, Lee JH, Sohn TS, Bae JM, Kim S, Kim DH, Kim KM - PLoS ONE (2014)

Bottom Line: We validated the results with Sanger sequencing, real-time quantitative PCR, fluorescence in situ hybridization and immunohistochemistry.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.

ABSTRACT
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.

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Immunohistochemical staining for EGFR, CCNE1 and HER2, and FISH for HER2.Cases with copy number increases showed strong positive for EGFR (A), CCNE1 (B) and HER2 (C). A case with HER2 2+ by immunohistochemistry reveals amplification of HER2 genes in FISH (D).
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pone-0111693-g002: Immunohistochemical staining for EGFR, CCNE1 and HER2, and FISH for HER2.Cases with copy number increases showed strong positive for EGFR (A), CCNE1 (B) and HER2 (C). A case with HER2 2+ by immunohistochemistry reveals amplification of HER2 genes in FISH (D).

Mentions: 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 (Table 3). We did not observe amplification of MET, FGFR2, CDK4 and CDK6 in any of the cases. In cases with amplification, IHC for HER2, EGFR and CCNE1 showed overexpression of proteins in the tumor cells (Figure 2A, B and C). In one case with HER2 2+ by HercepTest, FISH showed heterogeneous amplification of HER2 genes (Figure 2D).


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

Kim S, Lee J, Hong ME, Do IG, Kang SY, Ha SY, Kim ST, Park SH, Kang WK, Choi MG, Lee JH, Sohn TS, Bae JM, Kim S, Kim DH, Kim KM - PLoS ONE (2014)

Immunohistochemical staining for EGFR, CCNE1 and HER2, and FISH for HER2.Cases with copy number increases showed strong positive for EGFR (A), CCNE1 (B) and HER2 (C). A case with HER2 2+ by immunohistochemistry reveals amplification of HER2 genes in FISH (D).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4221102&req=5

pone-0111693-g002: Immunohistochemical staining for EGFR, CCNE1 and HER2, and FISH for HER2.Cases with copy number increases showed strong positive for EGFR (A), CCNE1 (B) and HER2 (C). A case with HER2 2+ by immunohistochemistry reveals amplification of HER2 genes in FISH (D).
Mentions: 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 (Table 3). We did not observe amplification of MET, FGFR2, CDK4 and CDK6 in any of the cases. In cases with amplification, IHC for HER2, EGFR and CCNE1 showed overexpression of proteins in the tumor cells (Figure 2A, B and C). In one case with HER2 2+ by HercepTest, FISH showed heterogeneous amplification of HER2 genes (Figure 2D).

Bottom Line: We validated the results with Sanger sequencing, real-time quantitative PCR, fluorescence in situ hybridization and immunohistochemistry.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus