Tomonori Abe

A Noninvasive System for Monitoring Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors with Plasma DNA

Introduction: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors are widely used to treat lung adenocarcinomas with EGFR-activating mutations. However, half of the patients acquire resistance because of the gatekeeper T790M mutation. Noninvasive mutation detection system is desired considering the difficulty in obtaining tissue specimens during disease progression. Methods: Sixty-seven plasma DNA samples from 49 patients with lung adenocarcinoma and 30 healthy volunteers were evaluated. T790M in plasma DNA was determined using the mutation-biased polymerase chain reaction (PCR) quenching probe (MBP-QP) method. The method combines MBP and genotyping, the latter based on analysis of the melting curve of the probe DNA binding the target mutated site using a fluorescence QP system. Results: The detection limit was two copies of control plasmid and 0.2 ng of genomic DNA. The mutant plasmid could be detected when it accounted for as little as 0.3% of a mixture of plasmids carrying EGFR exon 20 with or without T790M. The T790M mutation was detected in plasma DNA from 10 of 19 patients (53%) who acquired resistance, but not in nonresponders, patients responding to treatment, or those not treated with EGFR tyrosine kinase inhibitor. Other mutation detection systems, such as the nucleic acid-locked nucleic acid PCR clamp, the cycleave PCR technique, and allele-specific oligonucleotide PCR, detected T790M in three, four, and six patients, respectively, among 10 in which T790M was detected by the MBP-QP method. Conclusions: The MBP-QP method is simple, sensitive, and—intriguingly—reflective of clinical course, compared with the other three mutation-detection systems. Thus, the MBP-QP method is an ideal noninvasive monitoring system for detecting T790M in plasma samples.

Functional analysis of Discoidin domain receptor 2 mutation and expression in squamous cell lung cancer

OBJECTIVES Discoidin domain receptor (DDR) 2 mutations have recently been reported to be candidate targets of molecular therapy in lung squamous cell carcinoma (SQCC). However, the status of DDR2 expression and mutations, as well as their precise roles in lung SQCC, have not been clarified. We here report DDR2 mutation and expression status in clinical samples and its role of lung SQCC. MATERIALS AND METHODS We investigated DDR2 expression and mutation status in 44 human clinical samples and 7 cell lines. Biological functions of DDR2 were assessed by in vitro cell invasion assay and animal model experiments. RESULTS Endogenous DDR2 protein expression levels were high in one cell line, PC-1, and immunohistochemistry of lung cancer tissue array showed high levels of DDR2 protein in 29% of lung SQCC patients. A mutation (T681I) identified in lung SQCC and the cell line EBC-1 was detected among 44 primary lung SQCC samples and 7 lung SQCC cell lines. Although Forced expression of DDR2 and its mutant (T681I) led to induce SQCC cell invasion in vitro, only wild type DDR2 enhanced lung metastasis in an animal model. We also found that ectopic expression of DDR2 induced MMP-1 mRNA expression accompanied by phosphorylation of c-Jun after treatment with its ligand, collagen type I, but DDR2 with the T681I mutation did not, suggesting that T681I mutation is an inactivating mutation. CONCLUSION Overexpression of DDR2 might contribute to tumor progression in lung SQCC. The overexpression of DDR2 could be potential molecular target of lung SQCC.

Automated DNA extraction using cellulose magnetic beads can improve EGFR point mutation detection with liquid biopsy by efficiently recovering short and long DNA fragments

The clinical utility of plasma DNA for detecting cancer-specific mutations has rapidly achieved recognition, but reliability has not been established because of relatively low mutation-detection rates compared with those from tissue re-biopsy. To address this shortcoming we examined efficiency, in terms of mutation detection, of an automated DNA extraction system that uses cellulose magnetic beads. A fully automated, highly sensitive point-mutation-detection method, mutation-biased PCR and quenching probe (MBP-QP) system, was used for this study. Plasma DNA was extracted from 61 plasma samples collected from patients with advanced non-small cell lung cancer. Extraction was performed manually with 200 μl plasma (200-M) by using a silica membrane spin column system or an automated system using 200 μl (200-A) or 1000 μl (1000-A) plasma. Median DNA yield quantified by real-time PCR was 4.4, 4.5, and 17.3 ng with the three methods, respectively. Sensitivity for detecting epidermal growth factor receptor (EGFR) L858R point mutation was 36.6%, 58.5%, and 77.5%, and specificity was 93.3%, 100%, and 96.7%, respectively. Concordance rates were 60.6%, 76.1%, and 85.7%. The size distribution of plasma DNA with automated extraction was bimodal with modes at about 170 bp and 5 Kb, and plasma DNA of both sizes included tumor-derived DNA. In this report, we demonstrate that automated DNA extraction using cellulose magnetic beads can improve mutation-detection rates with plasma DNA in association with two overall sizes of DNA fragments recovered by this DNA isolation system. Examining the biological characteristics of these fragments will be the subject of further investigation.

Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy

Liquid biopsy with circulating free DNA (cfDNA) is a recommended alternative method of re-biopsy. Quality control with cfDNA is indispensable for precise examinations, and it is desirable to achieve high-quality cfDNA separation. We investigated two issues: the influence of pre-analytical procedures on cfDNA analysis performed as a routine procedure in a standard clinical laboratory, and the extent of deterioration of cfDNA quality due to long-term storage. Comparisons among blood collection tube types, storage temperatures, and periods of blood separation were performed in terms of cfDNA quantification, cfDNA size distribution, and detection of EGFR mutations. Quality of cfDNA was better with collection tubes containing 3.2% sodium citrate than with those containing EDTA 2K, and was maintained with storage at 4° C for up to 72 h after blood collection, equivalent to results with cell-stabilizing blood collection tubes. Analysis of cfDNA stored for 7 years showed that samples with low allele frequency (AF) deteriorated more readily than samples with high AF. Despite the same storage period and extraction method, AF of plasma stored for 7 years was remarkably lower than that of cfDNA. However, deterioration due to long-term plasma storage was overcome by changing the DNA extraction method from a silica membrane spin column to a cellulose magnetic beads system. These results can guide the establishment of standardized pre-analytical procedures for liquid biopsy with cfDNA.

Current Status and Problems of T790M Detection, a Molecular Biomarker of Acquired Resistance to EGFR Tyrosine Kinase Inhibitors, with Liquid Biopsy and Re-biopsy

Background/Aim: The purpose of this study was to consider appropriate application of liquid and re-biopsy through analysis of current status in practice. Patients and Methods: We performed a retrospective analysis of 22 patients with epidermal growth factor receptor (EGFR) mutation-positive non-small cell lung cancer who exhibited 1st/2nd generation EGFR-tyrosine kinase inhibitors resistance. The cobas® method was used to detect T790M with re-biopsy and the mutation-biased PCR and quenched probe method was used with liquid biopsy. Results: T790M detection rate was 52% with re-biopsy and 58% with liquid biopsy. The concordance between tissue and plasma was 58%. One patient who was T790M-positive with liquid biopsy showed heterogeneity among metastatic lesions in terms of osimertinib efficacy, as revealed by T790M detection with re-biopsy. Conclusion: Liquid biopsy reflects the whole body, whereas re-biopsy is useful for spatial diagnosis. Considering these characteristics, a combination of liquid and re-biopsy contribute to enhanced treatment.

Abstract 2751: Automatic DNA extraction system can improve the EGFR point mutation detection rate of liquid biopsy

The usefulness of liquid biopsy to detect mutations from cancer patients has been well recognized today. However, because the mutation detection rates from plasma DNA were relatively lower than those of tissue re-biopsy, its clinical utility has not been confirmed yet. As previously we reported, we have developed fully automatic high-sensitive point mutation detecting system named mutation-biased PCR and quenched probe (MBP-QP) system for liquid biopsy. Recently, the importance of pre-analytical procedures for plasma DNA anazysis has been highlighted. In this study, we examined whether the automatic DNA extraction system can improve the mutation detection rate in our MBP-QP system. Sixty-one plasma samples were obtained from advanced non-small cell lung cancer patients, and plasma DNA extraction was performed from 200μl plasma by manually (200-M), and 200μl (200-A), 1000μl (1000-A) plasma by automatically. We used silica membrane spin column system for manual DNA extraction, and magnet beads system for automatic DNA extraction procedure. The median DNA concentrations quantified by quantitative real-time PCR of 200-M, 200-A, 1000-A were 4.92, 6.00, 20.1 ng/mL plasma, respectively. In terms of the epidermal growth factor receptor (EGFR) L858R point mutation detection, the sensitivity of 200-M, 200-A, 1000-A were 36.6%, 58.5%, 77.5%, that of the specificity were 93.3%, 100%, 96.7%, and the concordance rates were 60.6%, 76.1%, 85.7%, respectively. The size distribution of automatically extracted plasma DNA represented two peaks characteristics at 170 bp and 5 kb. In this study, we indicate the automatic DNA extraction can improve mutation detection rates in plasma DNA. Citation Format: Chiho Nakashima, Akemi Sato, Tomonori Abe, Tomomi Nakamura, Kazutoshi Komiya, Eisaburo Sueoka, Shinya Kimura, Naoko Sueoka-Aragane, Junichi Kato, Mitsuharu Hirai. Automatic DNA extraction system can improve the EGFR point mutation detection rate of liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2751. doi:10.1158/1538-7445.AM2017-2751