Mutation Detection of Epidermal Growth Factor Receptor and KRAS Genes Using the Smart Amplification Process Version 2 from Formalin-Fixed, Paraffin-Embedded Lung Cancer Tissue

Miyamae, Yohei; Shimizu, Kimihiro; Mitani, Yuichiro; Araki, Takuya; Kawai, Yosuke; Baba, Masaru; Kakegawa, Seiichi; Sugano, Masayuki; Kaira, Kyoichi; Lezhava, Alexander; Hayashizaki, Yoshihide; Yamamoto, Keiji; Takeyoshi, Izumi

doi:10.2353/jmoldx.2010.090105

Cited by 39 publications

(33 citation statements)

References 38 publications

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“…We previously reported that the SmartAmp2 assay effectively detects mutations in DNA extracted from formalinfixed paraffin-embedded (FFPE) tissue because of this method's ability to amplify quite short sequences (30). We believe that the advantages of PNA-clamp SmartAmp2, its rapid detection time and ability to detect mutations in FFPE tissue, will enable this method to be particularly useful in the treatment of outpatients.…”

Section: Discussionmentioning

confidence: 99%

Clinical screening assay for EGFR exon 19 mutations using PNA-clamp smart amplification process version 2 in lung adenocarcinoma

et al. 2011

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Abstract. The presence of EGFR mutations is correlated with a positive therapeutic response to tyrosine kinase inhibitors; therefore, the accurate detection of EGFR mutations is crucial when deciding appropriate therapeutic strategies. Recently, the rapid and sensitive assay smart amplification process version 2 (SmartAmp2) was developed. However, this method can only detect one type of mutation in EGFR exon 19; therefore, we applied the PNA technology to the SmartAmp2 assay to develop PNA-clamp SmartAmp2 for the detection of many types of deletions in EGFR exon 19, in a single reaction. This new assay was evaluated using 172 clinical samples. Thirty-nine (22.7%) samples were found to have deletions by PNA-clamp SmartAmp2; whereas 30 (17.4%) and 38 (22.1%) tumors were found to have deletions by direct sequencing and PNA-enriched sequencing, respectively. Three cases, in which we detected mutations with PNA-clamp SmartAmp2, but not with direct sequencing, were treated with gefitinib, and all cases showed a partial therapeutic response. Using clinical samples, we demonstrated that PNA-clamp SmartAmp2 can detect various types of mutations in EGFR exon 19 in a relatively short time and with high sensitivity. This method detected small amounts of mutant DNA and identified patients for whom clinical information was previously unavailable from other tests. This test may contribute to the administration of efficient therapeutic strategies.

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Section: Discussionmentioning

confidence: 99%

Clinical screening assay for EGFR exon 19 mutations using PNA-clamp smart amplification process version 2 in lung adenocarcinoma

et al. 2011

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“…SmartAmp2 assays can detect deletions in the EGFR exon 19, and a mutation (L858R) in the EGFR exon 21. Although various deletions have been reported in the EGFR exon 19, PNAclamp methods allow the detection of almost all types of known deletions in a single assay (16,17). The assay principles of SmartAmp2, including PNA-clamp methods, have been described previously (18).…”

Section: Methodsmentioning

confidence: 99%

“…Rabbit monoclonal primary antibodies with specificity for the 15-bp deletion in EGFR exon 19; EGFReceptor (E746-A750del Specific) (6B6) XP™ Rabbit mAb (Cell Signaling Technology, Inc., Beverly, MA) were used (1:100) and IHC was performed according to the manufacturer's recommendations. For positive samples from IHC, a section slide of the tumor was divided into several pieces (the adenocarcinomatous and squamous cell carcinomatous components) and macrodissected for DNA extraction using the QIAamp DNA FFPE tissue kit (Qiagen) as described previously (16). Mutational analyses were performed on extracted DNA samples using SmartAmp2 and PNA-enriched sequencing.…”

Section: Mutant-enriched Assay For Egfr Exon 19 (Pna-enriched Sequencmentioning

confidence: 99%

Significance of epidermal growth factor receptor gene mutations in squamous cell lung carcinoma

Miyamae

Shimizu

Hirato³

et al. 2011

Oncol Rep

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Abstract. Epidermal growth factor receptor (EGFR) gene mutations have been reported to be clinically significant in non-small cell lung cancer (NSCLC). However, because most previous studies focused only on adenocarcinomas, EGFR mutations in other histotypes are poorly investigated. We evaluated the frequency of EGFR gene mutations in squamous cell carcinoma (SCC) and its clinicopathological features. In total, 89 frozen tumor specimens that had been first diagnosed as SCCs, were examined for EGFR mutations in exons 19 and 21 using direct sequencing, PNA-enriched sequencing and SmartAmp2. Additionally, pathological investigation, including immunostaining for p63 and TTF-1, alcian blue staining and EGFR mutation-specific immunohistochemistry in mutation-positive samples was also performed. The frequency of EGFR mutations was 5.6% (5/89); all mutations were deletions in EGFR exon 19. Immunohistological investigation of these samples revealed that two of five were positive for p63 and TTF-1 staining, and showed production of mucin, as evidenced by alcian blue staining. Consequently, three of the samples were considered to be true SCC at final pathological diagnosis, while the remaining two samples were revised to adenosquamous carcinoma and adenocarcinoma. The final frequency of the EGFR mutations in true SCC was 3.4% (3/87). In conclusion, EGFR mutations were found in a small, but significant, number of SCC tumor samples and thus EGFR mutational analysis was useful in the accurate diagnosis of SCC. Our data demonstrate that EGFR mutational analysis should be performed not only in adenocarcinoma, but also in SCC to allow accurate diagnosis and treatment.

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“…Genomic DNA was extracted from a 3-5 mm cube of tumor tissue using a DNA Mini kit (Qiagen, Hilden, Germany) and subsequently diluted to a concentration of 20 ng/µl. KRAS and EGFR mutations in lung cancer tissue were analyzed by peptide nucleic acid-enriched sequencing, as previously described (15)(16)(17).…”

Section: Methodsmentioning

confidence: 99%

Postrecurrence survival of surgically resected pulmonary adenocarcinoma patients according to EGFR and KRAS mutation status

Ohtaki

Shimizu

Kakegawa

et al. 2013

Molecular and Clinical Oncology

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Abstract. The aim of this study was to investigate the prognosis of pulmonary adenocarcinoma patients following postoperative recurrence, according to epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) gene mutation status and recurrence site. In total 58 adenocarcinoma patients with recurrence following surgical resection were retrospectively evaluated between 2002 and 2011. The patients were divided into groups according to the presence or absence of EGFR and KRAS mutations and the clinicopathological characteristics, recurrence sites and postrecurrence survival were compared. EGFR and KRAS mutations were detected in 26 (45%) and 11 patients (19%), respectively. Initial recurrence was distant in 25 (43%), local in 17 (29%) and both distant and local in 16 cases (28%). In EGFR-mutant (EGFR+) cases, bilateral/contralateral lung recurrence was a frequent finding. EGFR+ cases exhibited significantly better outcomes compared to KRAS+ and EGFR-KRAS-(wild-type) cases. The 2-year post-recurrence survival rates were 81, 18 and 47% in EGFR+, KRAS+ and wild-type cases, respectively. The patients with distant organ recurrence exhibited significantly worse survival compared with those without distant recurrence in wild-type, but not in the EGFR+ cases or the entire cohort. Multivariate analysis revealed that EGFR mutations and a number of recurrent lesions were the only statistically significant independent predictors of postrecurrence prognosis. Our results indicated distinct survival differences in recurrent adenocarcinoma patients according to driver mutations. Patients with EGFR-mutated tumors exhibited increased survival, regardless of recurrence at distant sites, whereas patients with KRAS-mutated adenocarcinoma exhibited poor outcome following postoperative recurrence. Therefore, the assessment of driver mutations is essential for predicting postrecurrence survival following surgical resection.

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Mutation Detection of Epidermal Growth Factor Receptor and KRAS Genes Using the Smart Amplification Process Version 2 from Formalin-Fixed, Paraffin-Embedded Lung Cancer Tissue

Cited by 39 publications

References 38 publications

Clinical screening assay for EGFR exon 19 mutations using PNA-clamp smart amplification process version 2 in lung adenocarcinoma

Clinical screening assay for EGFR exon 19 mutations using PNA-clamp smart amplification process version 2 in lung adenocarcinoma

Significance of epidermal growth factor receptor gene mutations in squamous cell lung carcinoma

Postrecurrence survival of surgically resected pulmonary adenocarcinoma patients according to EGFR and KRAS mutation status

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