A highly specific and sensitive massive parallel sequencer-based test for somatic mutations in non-small cell lung cancer

Inoue, Yoshiaki; Shiihara, Jun; Miyazawa, Hitoshi; Ohta, Hiromitsu; Higo, Megumi; Nagai, Yoshinori; Kobayashi, Kunihiko; Saijo, Yasuo; Tsuchida, Masanori; Nakayama, Mitsuo; Hagiwara, Koichi

doi:10.1371/journal.pone.0176525

Cited by 12 publications

(18 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Next‐generation sequencing (NGS) was first used to analyze the biology of cancers . It has since been rapidly implemented in clinical oncology to guide therapy . EGFR, ALK, ROS1 and BRAF mutations account for approximately 30% and 60% of adenocarcinomas in the United States and Japan, respectively, and treatment targeting these gene alterations has been approved globally .…”

Section: Introductionmentioning

confidence: 99%

Small lung tumor biopsy samples are feasible for high quality targeted next generation sequencing

et al. 2019

View full text Add to dashboard Cite

Next‐generation sequencing (NGS) has been implemented in clinical oncology to analyze multiple genes and to guide therapy. In patients with advanced lung cancer, small biopsies such as computed tomography‐guided needle biopsy (CTNB), endobronchial ultrasound‐guided transbronchial needle aspiration (EBUS‐TBNA) and transbronchial biopsy (TBB) are less invasive and are preferable to resection to make a pathological diagnosis. However, the quality of DNA/RNA and NGS from small lung tumor biopsy samples is unknown. Between April 2017 and March 2018, 107 consecutive samples were obtained from thoracic tumors or metastatic sites for targeted NGS analysis. Fifteen samples were obtained through CTNB, 11 through EBUS‐TBNA, 11 through TBB and 70 through surgical resection. All samples were formalin‐fixed and paraffin‐embedded. DNA and RNA quality was measured using the ddCq method and the percentage of RNA fragments above 200 nucleotides (DV200), respectively. Our custommade probes were designed to capture exon sequences of 464 cancer‐related genes and transcripts of 463 genes. DNA and RNA yield from the 3 biopsy methods were similar, and less than the yield obtained from resected samples. The quality of DNA and RNA was similar across all methods. Overall, 12 of 15 CTNB samples (80%), all 11 EBUS‐TBNA samples, and 9 of 11 TBB samples (82%) underwent successful NGS assays from DNA. NGS analysis from RNA was successful in all 12 CTNB samples, 9 of 11 EBUS‐TBNA samples (82%), and 8 of 11 TBB samples (73%). CTNB, EBUS‐TBNA and TBB mostly resulted in adequate DNA and RNA quality and enabled high‐quality targeted NGS analysis.

show abstract

Section: Introductionmentioning

confidence: 99%

Small lung tumor biopsy samples are feasible for high quality targeted next generation sequencing

et al. 2019

View full text Add to dashboard Cite

show abstract

“…Various PCR-based methods have been developed to detect mutations of interest. For example, blocking PCR using LNAs or PNAs has been used to detect EGFR mutations for clinical diagnosis [25,26,27]. In bronchoscopic specimens, this approach can individually distinguish more than ten types of mutations in the EGFR gene [28,29].…”

Section: Discussionmentioning

confidence: 99%

Simultaneous Detection of the T790M and L858R Mutations in the EGFR Gene by Oligoribonucleotide Interference-PCR

Baba

Fujita

Tasaka

et al. 2019

IJMS

View full text Add to dashboard Cite

A de novo single-nucleotide mutation in the EGFR gene can cause the development of lung cancer. EGFR tyrosine kinase inhibitors (EGFR-TKIs) are used for clinical treatment of such lung cancers, but acquired resistance often mitigates their efficacy. Accordingly, monitoring of de novo and acquired nucleotide mutations is essential for clinical treatment of lung cancers with EGFR-TKIs. Previously, we reported that oligoribonucleotide interference-PCR (ORNi-PCR) can accurately and cost-effectively detect single-nucleotide mutations. In this study, we applied ORNi-PCR to simultaneous detection of the de novo L858R and acquired T790M mutations in the EGFR gene in lung cancer cells. First, we established optimal experimental conditions for ORNi-PCR to simultaneously detect the two single-nucleotide mutations in genomic DNA from lung cancer cells. The conditions we established could also be used for ORNi-PCR using complementary DNA reverse-transcribed from extracted RNA. We found that ORNi-PCR could detect lung cancer cells possessing both single-nucleotide mutations among a large number of cells harboring wild-type sequences, even when the cancer cells constituted less than ~0.2% of all cells. Our findings demonstrate that ORNi-PCR can simultaneously detect multiple single-nucleotide mutations in a gene of interest and might therefore be useful for simultaneous detection of EGFR mutations in clinical examinations.

show abstract

“…Mutations in some of the cytological samples were exploratorily investigated using the MINtS system that employs the MiSeq sequencer (Illumina K.K.) as previously reported 32 …”

Section: Methodsmentioning

confidence: 99%

Highly sensitive detection of driver mutations from cytological samples and cfDNA in lung cancer

et al. 2021

Self Cite

View full text Add to dashboard Cite

Background Bronchoscopy is a minimally invasive procedure for establishing the diagnosis of lung cancer. It sometimes fails to obtain tissue samples but readily collects cytological samples. Methods We developed PNA‐LNA dual‐PCR (PLDP), which amplified mutant sequences by a high‐fidelity DNA polymerase in the presence of a peptide nucleic acid (PNA) oligomer having a wild‐type sequence. Mutations are detected either by locked nucleic acid (LNA) probes for quick detection of a limited number of mutations, which are EGFR, KRAS, and BRAF mutations in the current study, or by direct sequencing for a comprehensive screening. In a total of 233 lung cancer samples, the results for cytological samples by PLDP were compared with those for tissue samples by cobas® EGFR mutation test (cobas) or by the PNA‐LNA PCR clamp method (P‐LPC). Moreover, the performance of PLDP using cell‐free DNA (cfDNA) was investigated. Results Peptide nucleic acid‐LNA dual‐PCR was able to detect each synthesized mutant sequence with high sensitivity. PLDP detected EGFR mutations in 80 out of 149 clinical samples, while the cobas or the P‐LPC detected in 66 matched. The correctness of PLDP was confirmed both by clinical response and by the results of sequencing using a next‐generation sequencer. PLDP detected mutations from cfDNA in approximately 70% of patients who harbors mutations in the tumor. Conclusions Peptide nucleic acid‐LNA dual‐PCR exhibited an excellent performance, even using cytological samples. PLDP is applicable for the investigation of cfDNA. The combination of bronchoscopy and PLDP is attractive and will expand the utility of bronchoscopy in clinical practice.

show abstract

A highly specific and sensitive massive parallel sequencer-based test for somatic mutations in non-small cell lung cancer

Cited by 12 publications

References 33 publications

Small lung tumor biopsy samples are feasible for high quality targeted next generation sequencing

Small lung tumor biopsy samples are feasible for high quality targeted next generation sequencing

Simultaneous Detection of the T790M and L858R Mutations in the EGFR Gene by Oligoribonucleotide Interference-PCR

Highly sensitive detection of driver mutations from cytological samples and cfDNA in lung cancer

Contact Info

Product

Resources

About