PCR Clamp Method Research Articles

Characteristics and prognostic analysis of patients with detected KRAS mutations in resected lung adenocarcinomas by peptide nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamp method.

Kirsten rat sarcoma virus (KRAS) gene mutations are a type of driver mutation discovered in the 1980s, but for a long time no molecular targeted drugs were available for them. Recently, sotorasib was developed as a molecular targeted drug for KRAS mutations. It is therefore necessary to identify the characteristics of patients with KRAS mutations. This was the single-institution retrospective study. Surgically resected tumors from lung adenocarcinoma patients were collected at a single institution from June 2016 to September 2019. Peptide nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamp analysis of KRAS G12X mutations was compared with analysis by therascreen KRAS RGQ kit. The association between KRAS mutation status and patient characteristics and prognosis was assessed. Among 499 lung adenocarcinomas, KRAS mutations were evaluated in 197 cases, excluding stage IV lung cancer and tumors with epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) mutations. KRAS G12X mutations were detected in 59 cases (29.9%). The highest frequency by gene mutation subtype was G12V in 23 cases (39.0%), followed by G12C in 16 cases (27.1%), G12D in 12 cases (20.3%), G12S in 4 cases (6.8%) and G12A in 2 cases. For the G12C mutation, the PNA-LNA PCR clamp and therascreen methods were consistent, but for the G12D and G12S mutations, the PNA-LNA PCR clamp method showed higher detection rates. In operable tumors, G12C mutations were more frequent in males, smokers, and patients with high expression of programmed death-ligand 1 (PD-L1), and had no correlation with prognosis. By the PNA-LNA PCR clamp method, G12C mutation of surgical specimens was detected successfully. The PNA-LNA PCR clamp method is expected to be applied to the detection of druggable G12C mutations.

Monitoring of Plasma EGFR Mutations during Osimertinib Treatment for NSCLC Patients with Acquired T790M Mutation.

Osimertinib was first approved for the treatment of non-small cell lung cancer (NSCLC) in patients who have developed the epidermal growth factor receptor (EGFR) T790M mutation after treatment with EGFR tyrosine kinase inhibitors (TKIs). We routinely evaluated the plasma of NSCLC patients with the T790M mutation to more rapidly detect an increase in disease activity and resistance to treatment. Eligible patients received osimertinib after resistance to the first- or second-generation of EGFR-TKIs in NSCLC harboring T790M mutation detectable in tumor tissue or plasma. Plasma samples were collected every 8 weeks during osimertinib treatment. The plasma analysis was performed using an improved PNA-LNA PCR clamp method. We tested samples for a resistance mechanism, including EGFR-activating, T790M, and C797S mutations, and assessed the association between the mutations and osimertinib treatment. Of the 60 patients enrolled in the study, 58 were eligible for this analysis. In plasma collected before osimertinib treatment, activating mutations were detected in 47 of 58 patients (81.0%) and T790M was detected in 44 patients (75.9%). Activating mutations were cleared in 60.9% (28/46) and T790M was cleared in 93.0% (40/43). Of these, 71.4% (20/28) of activating mutations and 87.5% (35/40) of T790M mutation were cleared within 8 weeks of treatment. The total response rate (RR) was 53.4% (31/58). The median duration of treatment was 259 days, with a trend toward longer treatment duration in patients who experienced the clearance of activating mutations with osimertinib. At the time of disease progression during osimertinib treatment, C797S was detected in 3 of 37 patients (8.1%). Plasma EGFR mutation analysis was effective in predicting the effect of osimertinib treatment.

Development of a Detection System for ESR1 Mutations in Circulating Tumour DNA Using PNA-LNA-Mediated PCR Clamping.

Although circulating tumour DNA (ctDNA)-based next-generation sequencing (NGS) is a less invasive method for assessing ESR1 mutations that are essential mechanisms of endocrine therapy resistance in patients with oestrogen receptor-positive breast cancer, adequate amounts of DNA are required to assess polyclonal ESR1 mutations. By combining a peptide nucleic acid and locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamping assay, we have developed a novel detection system to screen for polyclonal ESR1 mutations in ctDNA. A validation assay was prospectively performed on clinical samples and compared with the NGS results. The PNA-LNA PCR clamp assay was validated using six and four blood samples in which ESR1 mutations were detected by NGS and no mutations were detected, respectively. The PNA-LNA assay results were comparable with those of NGS. We prospectively assessed the concordance between the PNA-LNA PCR clamp method and NGS. Using the PNA-LNA PCR clamp method, ESR1 mutations were detected in 5 out of 18 samples, including those in which mutations were not detected by NGS due to small amounts of ctDNA. The PNA-LNA PCR clamping method is a highly sensitive and minimally invasive assay for polyclonal ESR1 mutation detection in the ctDNA of patients with breast cancer.

Nanopore Filter: A Method for Counting and Extracting Single DNA Molecules Using a Biological Nanopore.

This paper describes a method for the real-time counting and extraction of DNA molecules at the single-molecule level by nanopore technology. As a powerful tool for electrochemical single-molecule detection, nanopore technology eliminates the need for labeling or partitioning sample solutions at the femtoliter level. Here, we attempt to develop a DNA filtering system utilizing an α-hemolysin (αHL) nanopore. This system comprises two droplets, one filling with and one emptying DNA molecules, separated by a planar lipid bilayer containing αHL nanopores. The translocation of DNA through the nanopores is observed by measuring the channel current, and the number of translocated molecules can also be verified by quantitative polymerase chain reaction (qPCR). However, we found that the issue of contamination seems to be an almost insolvable problem in single-molecule counting. To tackle this problem, we tried to optimize the experimental environment, reduce the volume of solution containing the target molecule, and use the PCR clamp method. Although further efforts are still needed to achieve a single-molecule filter with electrical counting, our proposed method shows a linear relationship between the electrical counting and qPCR estimation of the number of DNA molecules.

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

BackgroundBronchoscopy is a minimally invasive procedure for establishing the diagnosis of lung cancer. It sometimes fails to obtain tissue samples but readily collects cytological samples.MethodsWe 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.ResultsPeptide 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.ConclusionsPeptide 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.

Genetic alterations in epidermal growth factor receptor-tyrosine kinase inhibitor-naïve non-small cell lung carcinoma.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are an approved first-line therapy against unresectable or advanced non-small cell lung cancer (NSCLC) harboring EGFR gene activating mutations. However, the majority of tumors develop acquired resistance against EGFR-TKIs and some tumors exhibit natural resistance. A number of resistance mechanisms against the latest third-generation EGFR-TKIs have been reported, including tertiary EGFR C797S mutation and several gene alterations activating EGFR or other signaling pathways. The current study aimed to identify the frequency of natural EGFR-TKI resistance in pretreatment NSCLC and to predict the therapeutic effect of EGFR-TKIs. A total of 246 EGFR-TKI-naïve NSCLC patients harboring known EGFR gene mutations were identified. The presence of EGFR C797S and T790M mutations were determined using the peptide nucleic acid-locked nucleic acid PCR clamp method. ERBB2, MET, EGFR, ALK, BRAF, FGFR1, MYC, RET, CCND1, CCND2, CDK4, CDK6, MDM2 and MDM4 gene amplification, which can lead to resistance against any generation EGFR-TKIs, was determined using the multiplex ligation-dependent probe amplification assay. No concurrent C797S mutation with known EGFR mutations were identified. T790M mutation was identified in 12 patients (4.9%). ERBB2 or MET gene amplification was found in some patients (0.0-0.4%). MDM2 gene amplification was associated with tumor recurrence and shorter progression-free survival (PFS) for first- or second-generation EGFR-TKIs. De novo EGFR C797S mutation was not identified. Other resistance mechanisms against EGFR-TKIs were indicated in some patients with EGFR-TKI-naïve NSCLC. MDM2 gene amplification, which can lead to altered cell cycle, was associated with tumor recurrence and shorter PFS in EGFR-TKI therapy.

P2.14-52 The Results from Plasma EGFR Mutation Analysis in NEJ026 Study

EGFR mutation analysis of plasma circulating tumor DNA (ctDNA) has been reported to be useful to detect resistant mutations and to predict the efficacy of treatment. In NEJ026 study, we demonstrated the PFS of erlotinib plus bevacizumab (BE) treatment was significantly superior to the erlotinib alone (E) in NSCLC patients harboring EGFR mutation. Evaluation of plasma EGFR mutations included in NEJ026 study as preplanned analysis. At the time points of pretreated (P0), 6 weeks after study treatment started (P1), and confirmed progressive disease (P2), the plasma samples were collected from the patients enrolled to NEJ026 study. The number of enrolled patients were 112 in BE and 114 in E. Plasma ctDNA analysis for detection of the activating EGFR mutation and T790M mutation were performed by modified PNA-LNA PCR clamp method. The total numbers of collected plasma samples in BE and E were 108 (96.4%) and 107 (95.5%) at P0, 95 (84.8%) and 97 (86.6%) at P1, and 42 (37.5%) and 53 (47.3%) at P2, respectively. In eligible patients having EGFR activating mutation detected by cytohistological specimens, detection rate of plasma EGFR mutation at P0 was 68% (147/215). The detection ratio of T790M mutation at P2 were similar in both arms: 8 (19.0%) in BE and 11 (20.8%) in E. By detection pattern of activating EGFR mutation, PFS was evaluated among three groups: type A (P0 (-), P1 (-)), type B (P0 (+), P1(-)), and type C (P0 (+), P1(+)). Type A achieved the best response to both TKI [Type A BE: 18.1 M (n = 32, 95% CI, 11.5 to upper limit not reached (NR)), E: 16.7 M (n = 26, 95% CI, 11.2 to NR )]. Type B also had better PFS to TKI and BE is more favorable effect than E compared to other types [type B BE: 15.5 M (n = 48, 95% CI, 12.4 to 23.3), E: 11.1 M (n = 57, 95% CI, 8.5 to 13.7)]. Type C showed worse response to both treatment [type C BE: 6.0M. (n = 12, 95% CI 2.6 to NR), E: 4.3 M (n = 10, 95% CI, 2.8 to 20.2)]. BE had better PFS in all types. Frequency of T790M in P2 was similar among BE and E. When patients still had detectable activating EGFR mutation in plasma ctDNA after treatment for 6 weeks, you should consider that they might have poor response to both BE and E.

PS1325 IDENTIFICATION AND TARGETING OF MOLECULAR SIGNATURES OF HYPOMETHYLATING AGENT RESISTANCE IN MYELODYSPLASTIC SYNDROME AND CHRONIC MYELOMONOCYTIC LEUKEMIA

Background: Waldenström macroglobulinemia (WM) is a subset of lymphoplasmacytic lymphoma (LPL) with IgM paraprotein. Genetic hallmark of WM is MYD88 L265P mutation which is important for its diagnosis and pathophysiology of this disease. Other genetic changes including CXCR4 mutation, which is associated with WHIM syndrome, are reported to be common in WM. Whereas comprehensive information on genetic changes of LPL with other paraprotein like IgG is scarce. Formalin-fixed paraffin-embedded (FFPE) samples are available for genomic studies, but it contains abundant non-tumor cells. Aims: In this report, we aim to evaluate, first, genetic difference between WM and LPL with IgG paraprotein (IgG type) using targeted next-generation sequencing (NGS) from FFPE DNA and second, the usefulness of allele-specific oligonucleotide (ASO) PCR and peptide nucleic acid (PNA) PCR clamp method compared with next-generation sequencing. Methods: Twenty patients with pathologically confirmed LPL (10 of WMs and 10 of IgG type LPLs) were subjected to this study after informed consent. There were include 8 DNA extracted from bone marrow (BM) FFPE samples and 12 DNA extracted from BM cells. MYD88 L265P was detected by ASO-PCR. CXCR4 mutations were detected by PNA PCR clamp method with 18-mer PNA, which enables to detect the most frequently mutated amino acid: S338. Using the amplification products from that, detected mutations were confirmed by Sanger sequencing. We also tried detecting CXCR4 mutations by only Sanger sequencing. The mutations in 12 genes were detected by targeted sequencing on coding sequence with NGS. Mann-Whitney U test and Chi-squared test were used for statistical analysis. This study was approved by the research ethics committee of Gunma University Hospital. Results: Using NGS, we found the somatic mutations in WMs and the IgG type LPLs: MYD88 (4 vs. 5), CXCR4 (3 vs. 2), NOTCH2 (0 vs. 1), ARID1A (9 vs. 8), RAG2 (5 vs. 5), KMT2D (10 vs. 10), TP53 (0 vs. 0), MYBBP1A (0 vs. 1), CD79B (0 vs. 0), PRDM1 (0 vs. 1), CD274 (0 vs. 0), PDCD1LG2 (2 vs. 0); there were no statistical differences, suggesting genetic similarity between these two subsets of LPL, IgM type and IgG type. MYD88 L265P was found in 9/10 (90%) of WM cases and 6/10 (60%) of the IgG type by ASO-PCR (p = 0.40); However, it was found only in 4/10 (40%) of WM cases and 5/10 (50%) of the IgG type by NGS (p = 0.05). In addition, A260 V was found in 1 of IgG type LPL case. All MYD88 L265P mutations detected by NGS were also detected by ASO-PCR. CXCR4 mutations were found in 1/9 (11%) of WM cases (T318 frameshift) and 2/9 (22%) of IgG type (T318 frameshift and S338∗) by PNA PCR clamp method (p = 0.22). Whereas, these were found in 3/10 (30%) of WM cases and 2/10 (20%) of IgG type by NGS (p = 0.67). NGS enabled to detect the three CXCR4 mutations detected by PNA PCR clamp method and the mutations in outside of target area of PNA (E345 frameshift and L305F). Summary/Conclusion: Our NGS analysis suggest genetic similarity between two subsets of LPL, WM and IgG type. ASO-PCR is superior sensitive to NGS in detection of MYD88 L265P mutation, and PNA PCR clamp method enabled to detect the CXCR4 mutations in FFPE samples even if they were the frameshift mutations. Meanwhile, NGS analysis uncovered mutations other than ASO-PCR and PNA PCR clamp method targets.

Distinctive clinicopathological features of adenocarcinoma in situ and minimally invasive adenocarcinoma of the lung: A retrospective study

ObjectivesThe aim of this study was to investigate distinguishing clinicopathological features, in addition to histological invasiveness, in adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA) of the lung. Materials and methodsPatients with lung adenocarcinoma who underwent surgery at our hospital between 2007 and 2014 were reviewed, focusing on computed tomography (CT) images, operative procedures and clinical outcomes, histopathology, Ki-67 immunostaining, and EGFR-mutation status. EGFR mutations were examined using a peptide nucleic acid–locked nucleic acid PCR clamp method. Group comparisons were investigated by Mann–Whitney U or Fisher’s exact tests. ResultsOf 629 patients with lung adenocarcinoma who underwent surgery, 91 (14%) of 103 AIS (n = 34) or MIA (n = 69) tumors were reviewed. The ratio of male to female patients with MIA compared to AIS was significantly higher (p < 0.02). Of 103 tumors, 99 (96%) were non-mucinous. By CT, 74% of AIS appeared as pure ground-glass nodules and 75% of MIAs as part-solid ground-glass nodules. Pathological tumor diameters and Ki-67 labeling index (LI) values were significantly greater for MIAs compared to AIS (p < 0.001 for both). A Ki-67 LI of ≥2.8% indicated the presence of an MIA rather than an AIS. EGFR mutations were more frequently detected in MIAs (33/69, 48%) than AIS (9/34, 26%; p = 0.055). The ratio of exon 19 deletions to exon 21 missense mutations in MIAs tended to be higher than those in AIS (p = 0.06). Patients did not experience a local recurrence or metastasis after AIS and MIAs were removed by wedge resection, segmentectomy or lobectomy. Five-year recurrence-free survival rates were 100%. ConclusionDespite similar surgical outcomes for AIS and MIAs, we found differences in terms of gender, tumor diameters, CT findings, Ki-67 LI and a subset of EGFR mutations, highlighting the validity of classifying the two subtypes.

Comparison of detection methods of &lt;em&gt;EGFR&lt;/em&gt; T790M mutations using plasma, serum, and tumor tissue in EGFR-TKI-resistant non-small cell lung cancer

BackgroundOsimertinib, a third-generation epidermal growth factor receptor-tyrosine kinase inhibitor, exerts remarkable effects against EGFR T790M resistance mutation-positive non-small cell lung cancer. Identifying T790M mutation by re-biopsy is essential before prescribing osimertinib. Tissue biopsy is the golden standard for this purpose, but several factors limit its success rate. The liquid biopsy with blood, using circulating tumor DNA, has been an alternative method. However, the true biological meaning and equivalence of liquid biopsy and tumor biopsy are still under investigation. Especially, the usefulness of serum samples to detect T790M mutation is not yet been known.Patients and methodsWe prospectively evaluated the sensitivity, specificity, and parallelism of the detection of EGFR mutations in tissue re-biopsy and liquid biopsy (plasma and serum), simultaneously, from June 2016 to May 2017. EGFR mutations in tumor re-biopsy were evaluated by COBAS ver2 and PNA-LNA PCR clamp method, and those in liquid biopsy were evaluated with COBAS ver2.ResultsFifteen patients were enrolled. In 10 patients whose EGFR mutation was detected in liquid biopsy, the original EGFR mutation (exon 19 del or L858R) was detected in all patients. Detection of EGFR mutation by COBAS ver2 and by PNA-LNA method was almost the same in tissue re-biopsy. The detection rate of T790M was lower than that of the original EGFR mutation in liquid biopsy compared to that in tissue re-biopsy. The detection of T790M in serum exhibited a higher specificity (67%) and positive predictive value (50%) than that in plasma (50% and 40%, respectively). The detection sensitivity was similar in plasma and serum.ConclusionPlasma, serum, and tissue genotyping can have complementary roles for detecting EGFR-T790M using COBAS ver2. Repeated tests with different samples and different methods may improve accuracy of T790M detection and will lead to the maximum benefit for the patient.

HER3 expression is enhanced during progression of lung adenocarcinoma without EGFR mutation from stage 0 to IA1.

BackgroundActivating EGFR mutations, HER2, and HER3 are implicated in lung cancer; however, with the exception of EGFR gene amplification in lung adenocarcinoma harboring EGFR mutations, their involvement in disease progression during the early stages is poorly understood. In this paper, we focused on which receptor is correlated with lung adenocarcinoma progression in the presence or absence of EGFR mutation from stage 0 to IA1.MethodsHER2 and HER3 expression and activating EGFR mutations in surgically resected lung adenocarcinoma exhibiting ground glass nodules on chest computed tomography and re‐classified to stage 0 and IA1 were examined by immunohistochemistry and peptide nucleic acid‐locked nucleic acid PCR clamp method, respectively.ResultsHER2 and HER3 expression was detected in 22.2% and 86.1% of samples, respectively. The frequency of EGFR mutation was 45.7% and was not significantly different between stage 0 and IA1 (40.0% and 48.0%, respectively), suggesting that EGFR mutation does not correlate with cancer progression from stage 0 to IA1. HER2 expression also did not correlate to progression. However, not only the frequency, but also the intensity of HER3 expression was increased in stage IA1 lung adenocarcinoma, particularly in lung adenocarcinoma without EGFR mutation.ConclusionHER3 tends to be intensively expressed during the progression of lung adenocarcinoma without EGFR mutation from carcinoma in situ to invasive carcinoma.

Utility of Liquid Biopsy by Improved PNA-LNA PCR Clamp Method for Detecting EGFR Mutation at Initial Diagnosis of Non–Small-Cell Lung Cancer: Observational Study of 190 Consecutive Cases in Clinical Practice

BackgroundThe clinical benefit of liquid biopsy for unselected patients at initial diagnosis has thus far been unclear. We aimed to evaluate the utility of liquid biopsy at initial diagnosis, as well as the efficacy of epidermal growth factor receptor–tyrosine kinase inhibitor (EGFR-TKI) based on liquid biopsy results in clinical practice, using the improved peptide nucleic acid–locked nucleic acid (PNA-LNA) PCR clamp method. Patients and MethodsWe routinely performed liquid biopsy using the improved PNA-LNA PCR clamp method for all patients diagnosed with non–small-cell lung cancer (NSCLC) between June 2015 and October 2016. We retrospectively evaluated the reliability of liquid biopsy based either on clinical stage or between sensitizing EGFR mutation and T790M mutation, and the clinical benefit of EGFR-TKI based on the liquid biopsy results in practice. ResultsA total of 244 patients underwent liquid biopsies, with 168 patients tested at diagnosis and 22 tested for T790M after pretreatment of EGFR-TKI. For detecting a sensitizing EGFR mutation, the sensitivity, specificity, positive predictive value, and negative predictive value were 72.7%, 100%, 100%, and 93.7% in the group with advanced-stage NSCLC and 0, 100%, not evaluable, and 70.5% in the group with early-stage NSCLC. The positive predictive value and negative predictive value for T790M were 33.3% and 55.6%, respectively. Fourteen patients in the liquid-positive group and 16 patients in the tissue-positive group received EGFR-TKI. The objective response rates of first- and second-generation EGFR-TKI for the liquid-positive and tissue-positive groups were 90.0% and 90.9%, respectively. There was no significant difference in median progression-free survival between the liquid-positive and tissue-positive groups (P = .839). ConclusionPatients with early-stage NSCLC should not be candidates for this liquid biopsy method. We recommend tissue biopsy as the preferred initial method of molecular analysis, with the exception of patients who are T790M positive or patients who are unable to tolerate invasive biopsy.

P1.02-071 Detection of Multiple Low-Frequency Mutations by Molecular-Barcode Sequencing

Recent advantage of next-generation sequencing (NGS) in the field of cancer genome research has revealed extreme levels of genetic heterogeneity, suggesting the major roles of subclonal mutations in cancer relapse and in rapid emergence of acquired resistance. Unlike inherited mutations, somatic variants often occur at low allele frequencies that require sensitive methods for detection. Based on the results using another highly sensitive method, such as digital PCR, the study of cancer subclones requires to detect mutations that are present in <1% frequency, however, such a level of resolution cannot be obtained by conventional NGS approaches. In the current study, we performed molecular-barcode sequencing for primary lung adenocarcinoma cases in order to analyze mutations with <1% low frequency. Fresh frozen tumor samples from 58 primary lung adenocarcinoma patients whose tumors previously tested positive for EGFR by conventional method (the PNA-LNA PCR clamp method) were collected. All samples were obtained from surgically resected specimen. We used HaloPlex HS method, which is a high sensitivity amplicon-based targeted sequencing method incorporating molecular barcodes in the DNA library, allowing for the identification of duplicate reads to significantly improve the base calling accuracy even at low allelic frequencies compared to conventional NGS methods. We used a panel designed for 47 cancer-related genes including EGFR, and sequenced data was obtained by using Illumina Miseq Reagent v3 (600 Cycle). Normal tissue samples were also sequenced for threshold adjustment. Out of 58 samples, EGFR ‘major’ mutation (L858R, Exon19 deletion, G719A/S, T790M) profiles of 57 samples by molecular-barcode sequencing corresponded to those by clinical method (98.3%). The mean coverage of EGFR major mutation was 3078 (243-8285), and the minimal detectable frequency was 0.45%. Of note, we could detect the minor frequency of T790M mutation in addition to the major frequency of L858R mutation, at the allele frequency of 1.92% (20/1042) for T790M and 10.62% (155/1459) for L858R mutation, respectively. Minor genetic alterations, except major mutation, in EGFR were detected in 82.8% (48/58) cases, and the mean number was 2.3 (0-11). These results suggest the clinical applicability of this method. We could demonstrate good concordance rate between clinical examination and molecular barcode sequencing. Minor genetic alterations in EGFR were detected in 82.8% (48/58) cases. Further investigations are warranted to establish the confident detection of subclonal mutations with low frequency.

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.

Abstract 5248: Non-invasive EGFR T790M detection using droplet digital PCR system

Abstract Background: Non-small cell lung cancers (NSCLCs) harboring activating epidermal growth factor receptor (EGFR) mutations show significant response to EGFR-tyrosine kinase inhibitors (TKIs). However, most of them acquire resistance to EGFR-TKIs, and a half of the resistance is caused by EGFR T790M mutation. Recently, the third generation-TKIs which are effective to lung cancer harboring T790M mutation have been developed, thus the importance of T790M mutation detection is increasing in NSCLC treatment. Nowadays, the highly sensitive assays such as PNA-LNA PCR clamp, Scorpion ARMS have been developed to detect T790M mutation. In this study, we newly established non-invasive assay for T790M mutation detection from circulating cell free DNA (ccfDNA) using droplet digital PCR system (ddPCR). Materials and Methods: The sensitivity of ddPCR was determined using variable proportion of mixture DNAs of human bronchial epithelial cell with wild-type EGFR and NCI-H1975 with EGFR L858R and T790M mutations. We collected ccfDNA extracted from the 39 plasma samples of 22 NSCLC patients, who had been treated with EGFR-TKI and examined EGFR T790M mutational status by re-biopsy. The T790M mutational status of the re-biopsy samples were determined using PNA-LNA PCR clamp method. The circulating T790M alleles in ccfDNAs were detected using ddPCR, and compared with the T790M mutational status of re-biopsy samples. Results: In 8 of the 39 plasma samples, T790M alleles were detected by ddPCR. As 10 of 39 plasma samples were collected from the patient who determined T790M positive by re-biopsy after acquiring resistance to EGFR -TKI, the sensitivity of ddPCR was 80%. In 3 plasma samples, T790M alleles were detected by ddPCR although PNA-LNA PCR clamp method could not detect T790M mutation from the re-biopsy tissues of the patients who provided their plasmas. The specificity of ddPCR was determined as 89.7% although we have to consider about the difference between the timings of plasma collection and re-biopsy, and tumors’ heterogeneity. Conclusions: The result of circulating T790M allele detection using ddPCR was highly concordant with the result of T790M detection in re-biopsy samples using PNA-LNA PCR clamp method. The ddPCR is promising method for non-invasive T790M mutation detection. Citation Format: Shinsuke Hashida, Kadoaki Ohashi, Takehiro Matsubara, Tomoaki Ohtsuka, Mototsugu Watanabe, Ken Suzawa, Yuho Maki, Hiromasa Yamamoto, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Shinichiro Miyoshi, Katsuyuki Kiura, Shinichi Toyooka. Non-invasive EGFR T790M detection using droplet digital PCR system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5248. doi:10.1158/1538-7445.AM2015-5248

Abstract 4656: The novel mRNA in situ hybridization method for the detection of ALK, RET, ROS1 and NTRK1 mRNA in non-small cell lung cancer

Abstract [Background]In recent years, tumor genotyping is critical for making decisions of non-small cell lung cancer (NSCLC) treatment. However genetic rearrangement of ALK, RET, ROS1 and NTRK1 were identified in the subset of NSCLC patients, these clinical characteristics were still remained to be clear. Immunohistochemistry (IHC) is widely used as a simple and quick method for screening for protein expression of these oncogenes. Fluorescence in situ hybridization (FISH) or RT-PCR are performed consecutively to confirm the genotyping. However it is often taking time and expensive and it is needed to consolidate novel substitute methods. [Purpose]We performed the in situ hybridization (ISH) analysis to detect cellular mRNA in formalin-fixed paraffin-embedded (FFPE) tissue samples of lung cancer. We also conducted IHC with the use of ALK, RET, ROS1 and NTRK1 specific antibodies and investigate a comparative study to assess the usefulness of mRNA-ISH to detect expression of these mRNA. [Materials and Methods]We made tissue micro array (TMA) slides using 40 FFPE samples of resected lung adenocarcinoma with known epidermal growth factor receptor (EGFR) mutation status by PCR (PNA-LNA PCR clamp method). The mRNA in situ hybridization was employed using RNA scope R 2.0 Regant Kit with targeting probes. IHC staining was conducted by antibodies using 5A4 and D5F3 for ALK, RET01 for RET, D4D6 for ROS1, and 14G6 for NTRK1. [Results]All of the IHC stained cases were clearly detected by mRNA-ISH respectively. There were some cases which were only visualized by ISH, each case was partially positive and not totally of the tumor. ALK: 5% of cases (2 out of 40) were detected in mRNA-ISH and were strongly stained with both 5A4 and D5F3. RET: 20% of cases (8/40) were detected in mRNA-ISH. Two cases (5% of total) had positive RET01 staining, while 6 cases had negative staining. (3 had no other genetic alterations, 2 had EGFR-L858R confirmed by PCR, and 1 had ALK by IHC and FISH.) ROS1: 20% (8/40) were detected in mRNA-ISH. Three cases (7.5% of total) presented positive D4D6 staining, while 5 had negative staining. (3 had no other alterations, 1 had EGFR-E746-A750 and 1 had EGFR-L858R by PCR.) NTRK1: 17.5% of cases (7/40) were detected in mRNA-ISH. 1 case (2.5% of total) presented positive 14G6 staining, while 6 cases had negative staining. (2 had no other alterations, 2 had EGFR-L858R, 1 had EGFR-E746-A750, and 1 had ALK.) [Conclusion] The novel mRNA in situ hybridization method combined with traditional IHC could improve detection rate of ALK, RET, ROS1 and NTRK1 gene alterations in NSCLC FFPE samples. Citation Format: Noriko Hirai, Takaaki Sasaki, Yoshinori Minami, Yoshinobu Ohsaki. The novel mRNA in situ hybridization method for the detection of ALK, RET, ROS1 and NTRK1 mRNA in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4656. doi:10.1158/1538-7445.AM2014-4656

A phase II study of erlotinib monotherapy in pre-treated non-small cell lung cancer without EGFR gene mutation who have never/light smoking history: Re-evaluation of EGFR gene status (NEJ006/TCOG0903)

ObjectivesEpidermal growth factor receptor (EGFR) tyrosine kinase inhibitors are particularly effective in non-small cell lung cancer (NSCLC) patients harboring active EGFR mutations. However, some studies have reported survival benefits in NSCLC patients with wild-type EGFR upon erlotinib treatment. This trial was conducted to evaluate the efficacy of erlotinib monotherapy and investigate the predictive values of several biomarkers. Patients and methodsPatients with previously treated NSCLC but without EGFR gene mutations that had never or light smoked were eligible for this study. Gene status screening was performed using the PNA-LNA PCR clamp method. Erlotinib was administered until disease progression or unacceptable toxicities occurred. EGFR gene status was re-evaluated using the fragment method to detect exon 19 deletions and the Cycleave-PCR method to detect point mutations. Expression of hepatocyte growth factor (HGF), Met, and thymidylate synthase (TS) were evaluated using immunohistochemistry. ResultsForty-seven patients were enrolled in the study between March 2010 and November 2011. Objective response rate (ORR) and disease control rate (DCR) were 15.2% and 41.3%. Re-evaluations for EGFR gene were performed in 32 tumor samples. EGFR gene mutations were found in eight samples (5:exon 19 deletion, 2:G719X, 1:L858R). Six patients had PR and two had SD among these eight patients. A total of 24 patients were confirmed as wild-type EGFR using different methods. ORR and DCR were 4.2% and 41.7%. The median progression free survival (PFS) and median survival times were 2.0 and 6.0 months, respectively. Patients with tumors expressing HGF showed shorter PFS but not MET or TS. ConclusionsRe-examination of EGFR gene status using different detecting method or different sample should be considered to grasp a chance of erlotinib treatment after first line treatment. In confirmed EGFR wild NSCLC, negative HGF staining could be a biomarker for longer PFS by erlotonib treatment.

P2–082 - The Modified PNA-LNA PCR Clamp Method for the Detection of EGFR Mutations in Non-Small Cell Lung Cancer

Background: Survival benefit from erlotinib was observed in some cases with wild-type EGFR NSCLC. However, the mechanism remains unclear. We analyzed the cases with wild-type EGFR NSCLC who were effectively treated with erlotinib at our institution, and examined the possibility of EGFR mutation false negative.

P2-011 - A Case of Lung Adenocarcinoma with Wild-Type EGFR Malignant Pleural Effusion Well Controlled by Treatment with Gefitinib

ABSTRACT A 77-year-old male with a history of smoking (B.I. 800) was diagnosed with lung adenocarcinoma. DNA analysis revealed a mutation of the epidermal growth factor receptor (EGFR) gene in exon 19 deletion in his bronchoscopic transbronchial biopsy specimens by the Cycleave method. During observation only, as wished by the patient, the tumor grew gradually and pleural effusion occurred. He was diagnosed with malignant pleural effusion, but EGFR gene mutation was not observed in that specimens by the PNA-LNA PCR clamp method. Treatment with gefitinib was started and the tumor size and pleural effusion remarkably decreased. After 4 weeks of treatment with gefitinib, a re-biopsy was done. We could find adenocarcinoma by bronchoscopy, but cancer cells were not observed in pleural effusion. When analysis of the EGFR gene was conducted, mutation was not observed in the pulmonary tissue by the RT–PCR method. Nowadays, we can use several EGFR gene mutation detection methods using the PCR technique, and each is almost equal in quality. But some reports showed that different results were received from different methods. We probably thought the result of pretreatment specimen from pleural effusion was false negative. The EGFR tyrosine kinase inhibitor (EGFR-TKI) such as gefitinib is a key drug for mutated-EGFR non-small-cell lung cancer. Therefore, we should avoid the loss of treatment opportunity with EGFR-TKI because of false-negative results.

O2-029 - Rebiopsy of Non-Small-Cell Lung Cancer Patients with Acquired Resistance to EGFR-TKI: Comparison Between T790M Mutation-Positive and -Negative Populations

ABSTRACT Background The secondary EGFR mutation (MT) T790M accounts for approximately half of acquired resistances to EGFR-TKI. A recent report has demonstrated that the presence of T790M predicts a favorable prognosis and indolent progression, compared with the absence of T790M after TKI failure. However, rebiopsy to confirm the T790M status can be challenging due to limited tissue availability and procedural feasibility, and little is known about the differences among patients with or without T790M. Methods We investigated 73 patients harboring EGFR MTs who had undergone rebiopsy to confirm the emergence of T790M after TKI failure. The PNA-LNA PCR clamp method was used in EGFR MT analyses. Pt characteristics (age, gender, smoking history, performance status, EGFR MT site, initial TKI, response to initial TKI, line of initial TKI, PFS with initial TKI and biopsy site) and postprogression survivals (PPS) after initial TKI failure were retrospectively compared in patients with and without T790M. Results We identified T790M in 2 (10%) of 21 central nervous system (CNS) (19 cerebrospinal fluid and 2 brain tissue) specimens, and in 20 (38%) of 52 other lesions (25 lung tissue, 24 pleural effusion and 3 lymph node) (P = 0.0225). Other characteristics had no statistical association with the detection of T790M. Median PPS in patients with T790M was 34.0 months, and in those without T790M, 14.5 months (P = 0.0038). Although none of our patients received TKIs continuously after initial failure, 56 (77%) patients were re-administered TKIs. Regardless of T790M status, PPS in patients with TKI re-administration (23.4 months) was significantly longer than without re-administration (10.4 months) (P = 0.0085). Conclusions The emergence of T790M in CNS is rare compared with other lesions. Patients with T790M after TKI failure have significantly better prognosis than those without T790M. The effectiveness of TKI re-administration or continuous administration beyond progression is suggested after initial TKI failure.