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Targeted sequencing with enrichment PCR: a novel diagnostic method for the detection of EGFR mutations.

ABSTRACT: Epidermal growth factor receptor (EGFR) is an important mediator of tumor cell survival and proliferation. The detection of EGFR mutations can predict prognoses and indicate when treatment with EGFR tyrosine kinase inhibitors should be used. As such, the development of highly sensitive methods for detecting EGFR mutations is important. Targeted next-generation sequencing is an effective method for diagnosing mutations. We compared the abilities of enrichment PCR followed by ultra-deep pyrosequencing (UDP), UDP alone, and PNA-mediated RT-PCR clamping to detect low-frequency EGFR mutations in tumor cell lines and tissue samples. Using enrichment PCR-UDP, we were able to detect the E19del and L858R mutations at minimum frequencies of 0.01% and 0.05%, respectively, in the PC-9 and H197 tumor cell lines. We also confirmed the sensitivity of detecting the E19del mutation by performing a titration analysis in FFPE tumor samples. The lowest mutation frequency detected was 0.0692% in tissue samples. EGFR mutations with frequencies as low as 0.01% were detected using enrichment PCR-UDP, suggesting that this method is a valuable tool for detecting rare mutations, especially in scarce tissue samples or those with small quantities of DNA.

SUBMITTER: Kang S

PROVIDER: S-EPMC4537046 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Targeted sequencing with enrichment PCR: a novel diagnostic method for the detection of EGFR mutations.

Kang Suki S Kim Baek Gil BG Han Hyun Ho HH Lee Joo Hyun JH Kim Ji Eun JE Shim Hyo Sup HS Cho Nam Hoon NH

Oncotarget 20150501 15

Epidermal growth factor receptor (EGFR) is an important mediator of tumor cell survival and proliferation. The detection of EGFR mutations can predict prognoses and indicate when treatment with EGFR tyrosine kinase inhibitors should be used. As such, the development of highly sensitive methods for detecting EGFR mutations is important. Targeted next-generation sequencing is an effective method for diagnosing mutations. We compared the abilities of enrichment PCR followed by ultra-deep pyrosequen ...[more]

PMID: 25915533

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Project description:Despite widespread interest in next-generation sequencing (NGS), the adoption of personalized clinical genomics and mutation profiling of cancer specimens is lagging, in part because of technical limitations. Tumors are genetically heterogeneous and often contain normal/stromal cells, features that lead to low-abundance somatic mutations that generate ambiguous results or reside below NGS detection limits, thus hindering the clinical sensitivity/specificity standards of mutation calling. We applied COLD-PCR (coamplification at lower denaturation temperature PCR), a PCR methodology that selectively enriches variants, to improve the detection of unknown mutations before NGS-based amplicon resequencing.We used both COLD-PCR and conventional PCR (for comparison) to amplify serially diluted mutation-containing cell-line DNA diluted into wild-type DNA, as well as DNA from lung adenocarcinoma and colorectal cancer samples. After amplification of TP53 (tumor protein p53), KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog), IDH1 [isocitrate dehydrogenase 1 (NADP(+)), soluble], and EGFR (epidermal growth factor receptor) gene regions, PCR products were pooled for library preparation, bar-coded, and sequenced on the Illumina HiSeq 2000.In agreement with recent findings, sequencing errors by conventional targeted-amplicon approaches dictated a mutation-detection limit of approximately 1%-2%. Conversely, COLD-PCR amplicons enriched mutations above the error-related noise, enabling reliable identification of mutation abundances of approximately 0.04%. Sequencing depth was not a large factor in the identification of COLD-PCR-enriched mutations. For the clinical samples, several missense mutations were not called with conventional amplicons, yet they were clearly detectable with COLD-PCR amplicons. Tumor heterogeneity for the TP53 gene was apparent.As cancer care shifts toward personalized intervention based on each patient's unique genetic abnormalities and tumor genome, we anticipate that COLD-PCR combined with NGS will elucidate the role of mutations in tumor progression, enabling NGS-based analysis of diverse clinical specimens within clinical practice.

Project description:BACKGROUND:Germline mutations in BRCA1 or BRCA2 lead to a high lifetime probability of developing ovarian or breast cancer. These genes can also be involved in the development of non-hereditary tumours as somatic BRCA1/2 pathogenic variants are found in some of these cancers. Since patients with somatic BRCA pathogenic variants may benefit from treatment with poly ADP ribose polymerase inhibitors, it is important to be able to test for somatic changes in routinely available tumour samples. Such samples are typically formalin-fixed paraffin-embedded (FFPE) tissue, where the extracted DNA tends to be highly fragmented and of limited quantity, making analysis of large genes such as BRCA1 and BRCA2 challenging. This is made more difficult as somatic changes may be evident in only part of the sample, due to the presence of normal tissue. METHODS:We examined the feasibility of analysing DNA extracted from FFPE ovarian and breast tumour tissue to identify significant DNA variants in BRCA1/ BRCA2 using next generation sequencing methods that were sensitive enough to detect low level mutations, multiplexed to reduce the amount of DNA required and had short amplicon design. The utility of two GeneRead DNAseq Targeted Exon Enrichment Panels with different designs targeting only BRCA1/2 exons, and the Ion AmpliSeq BRCA community panel, followed by library preparation and adaptor ligation using the TruSeq DNA PCR-Free HT Sample Preparation Kit and NGS analysis on the MiSeq were investigated. RESULTS:Using the GeneRead method, we successfully analysed over 76% of samples, with >95% coverage of BRCA1/2 coding regions and a mean average read depth of >1000-fold. All mutations identified were confirmed where possible by Sanger sequencing or replication to eliminate the risk of false positive results due to artefacts within FFPE material. Admixture experiments demonstrated that BRCA1/2 variants could be detected if present in >10% of the sample. A sample subset was evaluated using the Ion AmpliSeq BRCA panel, achieving >99% coverage and sufficient read depth for a proportion of the samples. CONCLUSIONS:Detection of BRCA1/2 variants in fixed tissue is feasible, and could be performed prospectively to facilitate optimum treatment decisions for ovarian or breast cancer patients.

Dataset Information

Targeted sequencing with enrichment PCR: a novel diagnostic method for the detection of EGFR mutations.

Publications

Targeted sequencing with enrichment PCR: a novel diagnostic method for the detection of EGFR mutations.

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