Mutant-enriched PCR Assay Research Articles

Abstract 2249: Detection of EGFR mutations in circulating cell-free DNA of non-small cell lung cancer patients by next-generation sequencing

Abstract [Background] Mutational analysis is essential for recent personalized therapeutic approaches of non-small cell lung cancer (NSCLC) patients. Circulating cell-free DNA (cfDNA) from plasma is an emerging topic as a tool for liquid biopsy and possibly enable repetitive genetic diagnosis or monitoring of cancer status. However, detection of cfDNA from the circulation still remains challenging because of its highly fragmented and low concentration nature. In this study, we analyzed cfDNA samples by targeted deep sequencing at high coverage. [Materials and Methods] Twenty matched DNA samples were collected from metastatic NSCLC patients with EGFR mutation, which was detected by a clinical EGFR mutant enriched PCR assay using PNA-LNA PCR clamp method. These samples were extracted from tissue samples of fresh tumor tissue except one formalin fixed paraffin embedded (FFPE) sample (tDNA), and plasma samples (cfDNA). Mutation hotspots were enriched by multiplex PCR using the GeneRead DNAseq Targeted Panels V2 Human Tumor Actionable Mutations panel (QIAGEN, Hilden, Germany), followed by library construction, and sequenced on the MiSeq sequencer. Resulting data was analyzed using CLC Genomics Workbench and Genomics Server and mapped to the full human genome. [Results and discussion] The mean read depth in target region was 59,000 reads. Matched EGFR driver mutations between cfDNA and tDNA were detected with an overall concordance of 25% (5 of 20 samples). The concordance rate of exon21 L858R point mutation, exon19 deletions and exon20 T790M point mutation were 32.5% (3 of 8 samples), 16.7% (2 of 12 samples), and 66.7% (2 of 3 samples), respectively. The mutant allele frequencies in cfDNA ranged from 0.1% to 44.5% (median, 0.3%). While further improvement is mandatory, these results suggest that targeted deep sequencing of cfDNA at high coverage may have a potential utility as a non-invasive mutational analysis. In addition, there seemed to be a tendency of better detection rate for point mutations (45.5%) than deletions (16.7%), while the difference didn't reach statistical significance (p = 0.19), which were corresponding to previous reports. Citation Format: Ken Suzawa, Shuta Tomida, Takahiro Matsubara, Kadoaki Ohashi, Yuho Maki, Hiromasa Yamamoto, Mizuki Morita, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Katuyuki Kiura, Shinichiro Miyoshi, Shinichi Toyooka. Detection of EGFR mutations in circulating cell-free DNA of non-small cell lung cancer patients by next-generation sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2249.

Predictive factors associated with gefitinib response in patients with advanced non-small-cell lung cancer (NSCLC).

A number of different clinical characteristics have been reported to singly correlate with therapeutic activity of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in advanced non-small-cell lung cancer (NSCLC). This study aimed to identify predictive factors associated with prognostic benefits of gefitinib. EGFR gene typing in 33 advanced NSCLC patients received gefitinib (250 mg/day) were analyzed with mutant-enriched PCR assay. Gefitinib response was evaluated with potential predictive factors retrospectively. The overall objective response rate (ORR) and median progression-free survival (PFS) in the 33 patients treated by gefitinib were 45.5% and 3.0 (2.0-4.0) months. The ORR and median PFS in EGFR gene mutation patients were significantly higher/longer than those in EGFR gene wild-type patients (P<0.01). Similarly, the ORR and median PFS in non-smoker patients were significantly higher/longer than those in smoker patients (P<0.05, P<0.01, respectively). However, no difference for ORR and median PFS occurred between male and female patients. Logistic multivariate analysis showed that only EGFR mutated gene was significantly associated with the ORR (P<0.01). Both EGFR mutated gene and non-smoker were the major factors that contributed to PFS (P<0.05). EGFR mutated gene and non-smoker status are potential predictors for gefitinib response in NSCLC patients.

Detection of KRAS codon 12 and 13 mutations by mutant-enriched PCR assay

BackgroundThe identification of KRAS mutations before the administration of anti-epidermal growth factor receptor (EGFR) therapy of metastatic colorectal cancer (mCRC) has become important. The aim of the present study was to develop a novel technology that can increase detection sensitivity for KRAS mutations. MethodsDNAs were extracted from colorectal cancer tissues and formalin-fixed, paraffin-embedded (FFPE) colorectal cancer samples. Mutant-enriched PCR assay utilizes the exceptionally thermostable endonucleases, PspGI for codon 12 and PhoI for codon 13, for specific amplifying KRAS mutations from mixed samples. The amplified PCR products were subjected to single-base primer extension or sequencing. Digital PCR was used to evaluate some of the results. ResultsWe compared the results with that from direct sequencing. In the FFPE samples, thirteen discordant samples were found. We showed that the mutant-enriched PCR assay can identify the codons 12 and 13 mutation in a mixed population of mutant and wild type DNA sequences at 1:1000 and 1:400, respectively. The sensitivity of this method is lower than the digital PCR. ConclusionsWe developed a rapid and highly sensitive method to detect codons 12 and 13 mutations of the KRAS gene. This method is a powerful tool for finding low-abundance variations in genomic DNA.

Abstract A36: High T790M detection rate in TKI-naïve NSCLC with EGFR sensitive mutation: Truth or artifact?.

Abstract T790M in epidermal growth factor receptor (EGFR) accounts for about 50% of the acquired resistance to EGFR tyrosine kinase inhibitor (TKI) in patients with non-small cell lung cancer (NSCLC) carrying sensitive EGFR mutations. Earlier studies suggested that T790M mutation was also detected in about 2% of TKI-naive NSCLCs. Recently, three groups reported that, by using highly sensitive assays, T790M mutation was detected in about 40% of TKI-naive NSCLC with sensitive EGFR mutations. When we carefully studied these reports, we realized that all of those data were generated from formalin-fixed paraffin embedded (FFPE) tumor tissue samples, which raised a concern that the high mutation positivity could be consequence of formalin fixation. To address this, we assessed the T790M mutation in 36 pairs of frozen and FFPE tumor tissues of TKI-naïve NSCLC with sensitive EGFR mutations using an enzyme-based mutant-enriched PCR assay (ME-PCR) with 0.1% sensitivity. In addition, frozen and FFPE adjacent normal tissues from the same patients were also assessed for a further comparison. While 41.7% (15/36) T790M positive rate was detected in the tumor FFPE samples which was consistent with previous reports, only 1 of the 36 (2.8%) frozen counterparts was T790M positive. As suspected, 48.5% (16/33) of T790M mutation rate was also identified in FFPE adjacent normal tissues but none of the 35 frozen adjacent normal tissues was T790M positive. Our results indicate that the high T790M positivity detected in TKI-naïve NSCLC using some highly sensitive methods may in some cases be FFPE-derived artifacts. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A36. Citation Format: Xin Ye, Zhong-zheng Zhu, Lei Zhong, Yachao Lu, Yun Sun, Xiaolu Yin, Zhenfan Yang, Guanshan ZHU, Qunsheng Ji. High T790M detection rate in TKI-naïve NSCLC with EGFR sensitive mutation: Truth or artifact?. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A36.

MIS4-4 - Predictive Factors Associated with Gefitinib Response in Patients with Advanced Non-Small-Cell Lung Cancer (NSCLC)

ABSTRACT Background A number of different clinical characteristics have been reported to singly correlate with therapeutic activity of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in advanced non-small-cell lung cancer (NSCLC). This study aimed to identify predictive factors associated with prognostic benefits of gefitinib. Methods EGFR gene typing in 33 advanced NSCLC patients received gefitinib (250 mg/day) were analyzed with mutant-enriched PCR assay. Gefitinib response was evaluated with potential predictive factors retrospectively. Results The overall objective response rate (ORR) and median progression-free survival (PFS) in the 33 patients treated by gefitinib were 45.5% and 3.0(2.0–4.0) months. The ORR and median PFS in EGFR gene mutation patients were significantly higher/longer than those in EGFR gene wild-type patients (P Conclusions EGFR-mutated gene and non-smoker status are potential predictors for gefitinib response in NSCLC patients.

Abstract 3988: Aberrant NFKBIA expression in lung adenocarcinomas arising from never smokers

Abstract [Background] Lung cancer in never-smoker (NS) is known as a seventh cause of cancer-related death. Activating mutation of epidermal growth factor receptor (EGFR) gene is frequently observed in non-small-cell lung cancers (NSCLCs) with never smoking history and it is significantly associated with the sensitivity of EGFR tyrosine kinase inhibitor (EGFR-TKI). Recently, NFKBIA gene, a tumor suppressor gene, has been reported to be silenced by deletion in glioblastomas, and in NSCLCs, its expression level is associated with clinical outcome of EGFR-TKI therapy. In this study, we examined NFKBIA expression and the genetic alterations of EGFR, K-ras, EML4-ALK to investigate the inter-relationship between these genetic alterations and clinicopathological factors among lung adenocarcinomas in NS. [Material and Method] We obtained ninety seven lung adenocarcinomas samples in NS which were resected at our institution. Four NSCLC cell lines (NCI-H1299, NCI-H1650, HCC827, NCI-H1819) were also investigated. We examined NFKBIA expression using immunohistochemistory (IHC) in tumor samples and real time reverse transcriptional PCR (RT-PCR) in cell lines, respectively. Mutational statuses of EGFR and K-ras genes were determined using mutant-enriched PCR assay and direct sequencing, respectively. EML4-ALK fusion was screened by ALK IHC and confirmed using RT-PCR assay. The methylation status of NFKBIA gene is investigated by methylation specific PCR assay and bisulfite sequencing. [Result] NFKBIA expression was silenced in 36 out of 97 samples (37.1%). EGFR mutation, K-ras mutation, and EML4-ALK fusion were detected in 61.8%, 2.1%, and 1.0% of 97 samples, respectively. Of note, the silencing of NFKBIA expression was significantly frequent in EGFR wild-type patients than in EGFR mutant patients (p=0.0024), while there was no correlation between NFKBIA expression level and K-ras mutation or EML4-ALK fusion. Although NFKBIA expression was silenced in NCI-H1299 and NCI-H1650, NFKBIA expression was not restored after 5-Aza treatment in these cell lines. In addition, NFKBIA methylation was found in these two cell lines. [Conclusion] Our findings suggest that the degradation of NFKBIA expression is a frequent event in lung adenocarcinomas of NS and may mediate an important non-tobacco carcinogenic pathway especially among lung adenocarcinoma with wild-type EGFR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3988. doi:1538-7445.AM2012-3988

Presence of EGFR mutation in pathologically non-malignant specimens from computed tomography-guided lung needle biopsies

Activating mutations of the epidermal growth factor receptor (EGFR) gene are characteristic of non-small cell lung cancer (NSCLC). EGFR mutations were previously detected in histologically normal lung tissue around NSCLC tumors. Computed tomography-guided lung needle biopsy (CTNB) is an accurate and useful technique for the diagnosis of lung tumors. However, pathologically non-malignant cases occasionally become apparent following lung tumor resection. In this study, we determined the EGFR mutational status of lung tumors diagnosed as non-malignant in CTNB specimens, but diagnosed as NSCLC following surgical resection. Between 2000 and 2008, 1,109 CTNBs were performed at Okayama University Hospital. Among them, 15 cases were initially diagnosed as non-malignant by CTNB, but diagnosed as NSCLC following surgical resection as a result of a high likelihood of malignancy by clinical findings. Twelve paired DNAs of CTNB and corresponding resected specimens were available to examine the EGFR mutational status using a mutant-enriched PCR assay. EGFR mutations were detected in one out of 12 CTNB specimens and three of the corresponding resected tumors. This case harbored the same EGFR mutation in the CTNB specimen and resected tumor, but not in the distant corresponding non-malignant lung tissue. Our results indicated that the detection of EGFR mutations may therefore aid the diagnosis of NSCLC in pathologically non-malignant CTNB specimens.

EGFR mutation status in pleural fluid predicts tumor responsiveness and resistance to gefitinib

It has been reported that the threonine-to-methionine substitution at amino acid position 790 (T790M) of the epidermal growth factor receptor (EGFR) gene is correlated with acquired resistance to gefitinib. We previously reported that there was some population that harbored the EGFR T790M mutation as a minor clone of tumor cells prior to drug treatment, may be causing resistance to gefitinib during treatment. This fact also suggests that the detection of the EGFR T790M mutation prior to treatment may predict the development of resistance. We also showed that pleural fluid is a useful specimen for detection of EGFR mutation using sensitive assays. In this study, we reported a female patient who was treated with gefitinib because an EGFR L858R mutation was found in her pleural fluid. Our patient showed partial response to gefitinib, but she had progressive disease only 4 months after the start of treatment. Furthermore, the EGFR T790M mutation was detected in the pleural fluid before gefitinib treatment by the mutant-enriched PCR assay. Our findings confirmed that the EGFR T790M mutation was occasionally present as a minor population in tumor cells before treatment and caused resistance after gefitinib administration. The detection of a small fraction of T790M-positive alleles may be useful to predict the clinical course of the gefitinib-treated non-small-cell lung cancer patients.

Usefulness of EGFR mutation screening in pleural fluid to predict the clinical outcome of gefitinib treated patients with lung cancer

The importance of epidermal growth factor receptor (EGFR) gene mutation has been recognized in nonsmall cell lung cancer (NSCLC), requiring the standardization of mutation screening system including the kind of samples. Here, we examined the EGFR mutation status in 61 pleural fluid samples from NSCLC cases using direct sequencing, nonenriched PCR, mutant-enriched PCR and peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp assay. The mutant-enriched PCR assay detected 16 mutant cases. Among them, the nonenriched PCR assay failed to detect 3 mutant cases. Regarding the discrepancy between mutant-enriched PCR and PNA-LNA PCR clamp assays, 3 cases of exon19-deletions were detected only by mutant-enriched PCR assay and no difference at the L858R mutation. There was no difference in results between direct sequencing and nonenriched PCR assay. We also correlated the EGFR mutation with clinical outcome of gefitinib-treated 29 cases. EGFR mutations were present in 10 cases, revealing 7 partial response and 3 no change (NC). In EGFR wild-type cases, 10 revealed NC and 9 progressive disease. The responders were significantly more frequent among the EGFR mutant cases than among the wild-type (p < 0.0001). Overall survival (p = 0.0092) and progression-free survival (p = 0.018) were significantly longer among the EGFR mutant cases than among the wild-type. In summary, we evaluated the utility of EGFR mutation screening in pleural fluid using 4 assays that showed some discrepancies arising from the designs of the assays. As clinical importance, the EGFR mutation status in pleural fluid can be a biomarker for the favorable outcome of gefitinib-treated NSCLC cases.

Presence ofEpidermal Growth Factor ReceptorGene T790M Mutation as a Minor Clone in Non–Small Cell Lung Cancer

The threonine-to-methionine substitution at amino acid position 790 (T790M) of the epidermal growth factor receptor (EGFR) gene has been reported in progressing lesions after gefitinib treatment in non-small cell lung cancer (NSCLC) that causes sensitive tumors to become resistant to gefitinib. Alternatively, the EGFR T790M mutation might be present in small fractions of tumor cells before drug treatment, and the tumor cells harboring the T790M mutation might be enriched during the proliferation after drug treatment. We developed a mutant-enriched PCR assay to detect small fractions of cells with T790M mutation and used this technique to detect mutations in 280 NSCLCs, including gefitinib-treated 95 cases. Although the direct sequencing detected only 1 T790M mutant case, the mutant-enriched PCR (confirmed to enrich one mutant out of 1 x 10(3) wild-type alleles) detected 9 additional cases among 280 cases. As linkage to clinicopathologic factors, the T790M mutation showed no bias for sex, smoking status, or histology but was significantly more frequent in advanced tumors (9 of 111 cases) than in early-stage tumors (1 of 169 cases; P = 0.0013). Among gefitinib-treated cases, gefitinib-sensitive mutations were found in 30 cases. The T790M mutation was present in 3 of 7 no-responders with the gefitinib-sensitive mutation and was not present in 19 responders (P = 0.014). Our results indicate that the T790M mutation is sometimes present in a minor population of tumor cells during the development of NSCLC and suggest that the detection of small fractions of T790M mutant alleles may be useful for predicting gefitinib resistance of NSCLCs with sensitive EGFR mutations.

Detection ofEGFRGene Mutation in Lung Cancer by Mutant-Enriched Polymerase Chain Reaction Assay

Mutations in the epidermal growth factor receptor (EGFR) gene have been reported to be present in non-small cell lung cancer (NSCLC) and related to the responsiveness of tumors to EGFR tyrosine kinase inhibitors, suggesting its usefulness as a biomarker. Because clinical samples contain tumor and normal cells or genes, a highly sensitive assay for detecting mutation is critical for clinical applications. The mutant-enriched PCR is a rapid and sensitive assay with selective restriction enzyme digestion. We developed the mutant-enriched PCR assay targeting exons 19 and 21 of EGFR and applied the developed assay to detect mutations in 108 cases of surgically resected specimens of NSCLCs, 18 samples of computed tomography (CT)-guided needle lung biopsies, and 20 samples of pleural fluid. In addition, results were then compared with those from direct sequencing and a nonenriched PCR assay. The mutant-enriched PCR that was proved to enrich one mutant of 2 x 10(3) normal genes detected mutations in 37 cases of 108 resected tumors, seven samples of CT-guided lung biopsies, and seven samples of pleural fluid. Among mutant cases, four resected tumors, two CT-guided lung biopsies, and two pleural fluid were identified as additional mutant cases by the mutant-enriched PCR, which were considered normal based on nonenriched assays. Our results indicate that EGFR mutations are readily detectable by mutant-enriched PCR in various clinical samples. Thus, mutant-enriched PCR may provide a valuable method of potentially detecting a small fraction of mutant genes in heterogeneous specimens, indicating its possible use in clinical application for NSCLC.