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.

Background Immune‐related adverse events (irAEs) should be anticipated with treatment by immune checkpoint inhibitors (ICIs). Although the relationship between irAEs and efficacy of ICI has been reported, it has not yet been clarified whether the benefit from ICI outweighs the low frequency of proceeding to subsequent therapies after discontinuation due to irAEs. Methods The study comprised 61 patients with non‐small cell lung cancer who underwent treatment with ICIs (nivolumab or pembrolizumab monotherapy) at the Saga University Medical School Hospital from December 2015 to January 2018. Therapeutic effect and progression‐free survival (PFS) were compared between the irAEs discontinuation group (AEg) and the group with discontinuation due to all causes other than irAEs (Non‐AEg). Results A total of 30% patients(18/61) had therapy discontinued due to irAEs: 22.5% (9/40) with nivolumab and 42.9% (9/21) with pembrolizumab. The response rate was 50.0% in the AEg and 8.1% in the on‐AEg (P = 0.001). The median PFS was significantly longer in the AEg (9.3 months; 95% CI 2.1–12.1) than in the non‐AEg (1.9 months; 95% CI 0.9–3.6): HR 0.45 (95%CI 0.20–0.89; log‐rank test P = 0.026). The prevalence of drug‐induced interstitial lung disease (ILD) was 6.1% (3/49) in cases without interstitial pneumonia (IP) as the underlying disease, whereas it was 50% (6/12) in cases with IP (P = 0.001). Conclusion Discontinuation of treatment with ICIs due to irAEs predict a good response to ICIs and favorable outcome since their anti‐cancer effects continue even after discontinuation. However, the presence of IP as the underlying disease increases the risk of drug‐related ILD onset.

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.

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.

Liquid biopsy has become widely applied in clinical medicine along with the progress in innovative technologies, such as next generation sequencing, but the origin of circulating tumor DNA (ctDNA) has not yet been precisely established. We reported bimodal peaks of long fragment circulating free DNA (cfDNA) of 5 kb and short fragment cfDNA of 170 bp in patients with advanced lung cancer, and both contained ctDNA. In this paper, we demonstrate that the total amount of cfDNA is higher when patients with lung cancer have extrathoracic metastases, and the amount of long fragment cfDNA is significantly higher in those patients. To investigate the origin of long fragment cfDNA, conditioned media isolated from lung cancer cell lines was fractionated. Long fragment cfDNA was found concomitant with extracellular vesicles (EVs), but short fragment cfDNA was not observed in any fractions. However, in peripheral blood from a metastatic animal model both fragments were detected even with those same lung cancer cell lines. In human plasma samples, long fragment cfDNA was observed in the same fraction as that from conditioned media, and short fragment cfDNA existed in the supernatant after centrifugation at 100,000g. Concentration of ctDNA in the supernatant was two times higher than that in plasma isolated by the conventional procedure. Long fragment cfDNA associated with tumor progression might therefore be released into peripheral blood, and it is possible that the long fragment cfDNA escapes degradation by co-existing with EVs. Examination of the biological characteristics of long fragment cfDNA is a logical subject of further investigation.