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Next‐generation sequencing (NGS) and digital PCR technologies allow analysis of the mutational profile of circulating cell‐free DNA (cfDNA) in individuals with advanced lung cancer. We have now evaluated the feasibility of cfDNA sequencing for mutation detection in patients with non‐small cell lung cancer at earlier stages. A total of 150 matched tumor and serum samples were collected from non‐small cell lung cancer patients at stages IA–IIIA. Amplicon sequencing with DNA extracted from tumor tissue detected frequent mutations in EGFR (37% of patients), TP53 (39%), and KRAS (10%), consistent with previous findings. In contrast, NGS of cfDNA identified only EGFR, TP53, and PIK3CA mutations in three, five, and one patient, respectively, even though adequate amounts of cfDNA were extracted (median of 4936 copies/mL serum). Next‐generation sequencing showed a high accuracy (98.8%) compared with droplet digital PCR for cfDNA mutation detection, suggesting that the low frequency of mutations in cfDNA was not due to a low assay sensitivity. Whereas the yield of cfDNA did not differ among tumor stages, the cfDNA mutations were detected in seven patients at stages IIA–IIIA and at T2b or T3. Tumor volume was significantly higher in the cfDNA mutation‐positive patients than in the negative patients at stages T2b–T4 (159.1 ± 58.0 vs. 52.5 ± 9.9 cm3, P = 0.014). Our results thus suggest that tumor volume is a determinant of the feasibility of mutation detection with cfDNA as the analyte. The detectability of somatic mutations in cfDNA obtained from lung cancer patients at earlier stages. Detection of the mutations relies on the tumor burden in the patients. Deep sequencing as well as digital PCR is reliable technology for detection of somatic mutations in serum.

Fibroblast growth factor receptor (FGFR) gene alterations are relatively frequent in lung squamous cell carcinoma (LSCC) and are a potential targets for therapy with FGFR inhibitors. However, little is known regarding the clinicopathologic features associated with FGFR alterations. The angiokinase inhibitor nintedanib has shown promising activity in clinical trials for non‐small cell lung cancer. We have now applied next‐generation sequencing (NGS) to characterize FGFR alterations in LSCC patients as well as examined the antitumor activity of nintedanib in LSCC cell lines positive for FGFR1 copy number gain (CNG). The effects of nintedanib on the proliferation of and FGFR signaling in LSCC cell lines were examined in vitro, and its effects on tumor formation were examined in vivo. A total of 75 clinical LSCC specimens were screened for FGFR alterations by NGS. Nintedanib inhibited the proliferation of FGFR1 CNG‐positive LSCC cell lines in association with attenuation of the FGFR1–ERK signaling pathway in vitro and in vivo. FGFR1 CNG (10.7%), FGFR1 mutation (2.7%), FGFR2 mutation (2.7%), FGFR4 mutation (5.3%), and FGFR3 fusion (1.3%) were detected in LSCC specimens by NGS. Clinicopathologic features did not differ between LSCC patients positive or negative for FGFR alterations. However, among the 36 patients with disease recurrence after surgery, prognosis was significantly worse for those harboring FGFR alterations. Screening for FGFR alterations by NGS warrants further study as a means to identify patients with LSCC recurrence after surgery who might benefit from nintedanib therapy. The angiokinase inhibitor nintedanib is a potential targeted agent for lung squamous cell carcinoma (LSCC) positive for genetic alterations affecting FGFR genes. We screened 75 LSCC specimens for FGFR alterations by next‐generation sequencing and thereby identified various such genetic changes including copy number gain, mutations, and a fusion in 15 of the 75 (20.0%) cases. Among the patients with relapse after surgery, the presence of FGFR alterations was associated with a significantly shorter overall survival.

Comprehensive genomic profiling (CGP) is increasingly used as a clinical laboratory test and being applied to cancer treatment; however, standardization and external quality assessments (EQA) have not been fully developed. This study performed cost-effective EQA and proficiency tests (PT) for CGP testing among multiple institutions those belong to the EQA working group of Japan Association for Clinical Laboratory Science (JACLS). This study revealed that preanalytical processes, such as derived nucleic acids (NA) extraction from formalin fixed paraffine embedded (FFPE) samples, are critical. First, EQA with extracted DNA from cell lines showed a detection rate of 100% (9 out of 9) in KRAS (c.38G > A; p.G13D), PIK3CA (p.H1047R), and B-Raf proto-oncogene, serine/threonine kinase ( BRAF ) (c.1799 T > A; p.V600E) in cases of > 10% variant allele frequency (VAF). However, BRAF (c.1799 T > A; p.V600E) detection decreased to 67% (6 out of 9) for a VAF of 4.9%. Second, when DNA was extracted from FFPE samples, pathogenic variants and variants with companion diagnostic indications were detected in all 10 participating laboratories. Each variant had < 20% VAFs on average (8.1–19.1%) and wide variability among laboratories was observed (relative standard deviation, 13–60%). Nonetheless, BRAF (c.1798_1799delinsAA; p.V600K) of 8.1% VAF, EGFR (c.2235_2249del; p.E746_A750del) of 9.7% VAF, and EGFR (c.2254_2277del; p.S752_I759del) of 9.8% VAF were detected with 70% (7/10), 70% (7/10), and 60% (6/10) frequency, respectively. Therefore, 10% VAF in pre-analytic processing for DNA extraction from FFPE was critical for variant detection in CGP analysis. Further, incorrect results were reported in case independent variant calling of BRAF; c.1798_1799delinsAA (p.V600K) was mistakenly interpreted as c.1798G > A, and c.1799 T > A was on the other strand. In conclusion, the EQA/PT among 10 institutes with common samples revealed the importance of VAF in pre-analysis and helped us understand the significance of the pipeline and common pitfalls usually ignored by the internal quality control in a single institute.

Despite its recommendation by the current guidelines, the role of long-term oral beta-blocker therapy has never been evaluated by randomized trials in uncomplicated ST-segment elevation myocardial infarction (STEMI) patients without heart failure, left ventricular dysfunction or ventricular arrhythmia who underwent primary percutaneous coronary intervention (PCI). In a multi-center, open-label, randomized controlled trial, STEMI patients with successful primary PCI within 24 hours from the onset and with left ventricular ejection fraction (LVEF) ≥40% were randomly assigned in a 1-to-1 fashion either to the carvedilol group or to the no beta-blocker group within 7 days after primary PCI. The primary endpoint is a composite of all-cause death, myocardial infarction, hospitalization for heart failure, and hospitalization for acute coronary syndrome. Between August 2010 and May 2014, 801 patients were randomly assigned to the carvedilol group (N = 399) or the no beta-blocker group (N = 402) at 67 centers in Japan. The carvedilol dose was up-titrated from 3.4±2.1 mg at baseline to 6.3±4.3 mg at 1-year. During median follow-up of 3.9 years with 96.4% follow-up, the cumulative 3-year incidences of both the primary endpoint and any coronary revascularization were not significantly different between the carvedilol and no beta-blocker groups (6.8% and 7.9%, P = 0.20, and 20.3% and 17.7%, P = 0.65, respectively). There also was no significant difference in LVEF at 1-year between the 2 groups (60.9±8.4% and 59.6±8.8%, P = 0.06). Long-term carvedilol therapy added on the contemporary evidence-based medications did not seem beneficial in selected STEMI patients treated with primary PCI. CAPITAL-RCT (Carvedilol Post-Intervention Long-Term Administration in Large-scale Randomized Controlled Trial) ClinicalTrials.gov.number, NCT 01155635.

Background Neuropathic pain is caused by sensory nerve injury, but effective treatments are currently lacking. Microglia are activated in the spinal dorsal horn after sensory nerve injury and contribute to neuropathic pain. Accordingly, molecules expressed by these cells are considered potential targets for therapeutic strategies. Our previous gene screening study using a mouse model of motor nerve injury showed that the G-protein-coupled receptor 34 gene (GPR34) is induced by nerve injury. Because GPR34 is now considered a microglia-enriched gene, we explored the possibility that it might be involved in microglial activation in the dorsal horn in a mouse model of neuropathic pain. Methods mRNA expression of GPR34 and pro-inflammatory molecules was determined by quantitative real-time PCR in wild-type and GPR34-deficient mice with L4 spinal nerve injury. In situ hybridization was used to identify GPR34 expression in microglia, and immunohistochemistry with the microglial marker Iba1 was performed to examine microglial numbers and morphology. Mechanical sensitivity was evaluated by the von Frey hair test. Liquid chromatography–tandem mass spectrometry quantified expression of the ligand for GPR34, lysophosphatidylserine (LysoPS), in the dorsal horn, and a GPR34 antagonist was intrathecally administrated to examine the effect of inhibiting LysoPS-GPR34 signaling on mechanical sensitivity. Results GPR34 was predominantly expressed by microglia in the dorsal horn after L4 nerve injury. There were no histological differences in microglial numbers or morphology between WT and GPR34-deficient mice. However, nerve injury-induced pro-inflammatory cytokine expression levels in microglia and pain behaviors were significantly attenuated in GPR34-deficient mice. Furthermore, the intrathecal administration of the GPR34 antagonist reduced neuropathic pain. Conclusions Inhibition of GPR34-mediated signal by GPR34 gene deletion reduced nerve injury-induced neuropathic pain by suppressing pro-inflammatory responses of microglia without affecting their morphology. Therefore, the suppression of GPR34 activity may have therapeutic potential for alleviating neuropathic pain.

Naomichi Matsumoto and colleagues report mutations in the SWI/SNF chromatin remodeling complex in Coffin-Siris syndrome. Twenty affected individuals (87%) harbored mutations in one of six SWI/SNF subunit genes: SMARCB1 , SMARCA4 , SMARCA2 , SMARCE1 , ARID1A or ARID1B . By exome sequencing, we found de novo SMARCB1 mutations in two of five individuals with typical Coffin-Siris syndrome (CSS), a rare autosomal dominant anomaly syndrome. As SMARCB1 encodes a subunit of the SWItch/Sucrose NonFermenting (SWI/SNF) complex, we screened 15 other genes encoding subunits of this complex in 23 individuals with CSS. Twenty affected individuals (87%) each had a germline mutation in one of six SWI/SNF subunit genes, including SMARCB1 , SMARCA4 , SMARCA2 , SMARCE1 , ARID1A and ARID1B .

Trimethylamine N-oxide (TMAO), a metabolite derived from the gut microbiota, is proatherogenic and associated with cardiovascular events. However, the change in TMAO with secondary prevention therapies for ST-segment elevation acute myocardial infarction (STEMI) remains unclear. The purpose of this study was to investigate the sequential change in TMAO levels in response to the current secondary prevention therapies in patients with STEMI and the clinical impact of TMAO levels on cardiovascular events We included 112 STEMI patients and measured plasma TMAO levels at the onset of STEMI and 10 months later (chronic phase). After the chronic-phase assessment, patients were followed up for cardiovascular events. Plasma TMAO levels significantly increased from the acute phase to the chronic phase of STEMI (median: 5.63 to 6.76 μM, P = 0.048). During a median period of 5.4 years, 17 patients experienced events. The chronic-phase TMAO level independently predicted future cardiovascular events (adjusted hazard ratio for 0.1 increase in log chronic-phase TMAO level: 1.343, 95% confidence interval 1.122–1.636, P = 0.001), but the acute-phase TMAO level did not. This study demonstrated the clinical importance of the chronic-phase TMAO levels on future cardiovascular events in patients after STEMI.

Balloon pulmonary angioplasty (BPA) is an innovative treatment for chronic thromboembolic pulmonary hypertension (CTEPH). We retrospectively examined the clinical outcomes and complications of BPA at Yokohama City University Hospital (YCUH) between 2012 and 2018. In 2012, we began to conduct BPA sessions in 46 patients with inoperable CTEPH; 34 completed the BPA scheme and the follow-up plan. A longitudinal sub-analysis was performed with cohorts 1 and 2 receiving BPA before and after April 2015. Significant improvements in the mean pulmonary arterial pressure, pulmonary vascular resistance, and other parameters were detected after BPA. The total rate of thoracic complications was 25%. Specifically, the increase in SaO 2 and home oxygen therapy discontinuation rate, and oral riociguat discontinuation rate was significantly higher in cohort 2 (+ 7.7, 75, and 59%) compared to cohort 1 (+ 3.1, 27, and 10%) ( P  < 0.05). Moreover, the need for non-invasive positive pressure ventilation was significantly lower: 0% (cohort 2) vs. 7% (cohort 1) ( P  < 0.05). The BPA sessions conducted at the YCUH resulted in significant improvements in patients with CTEPH. This study demonstrates a clear learning curve regarding the effectiveness of BPA both in normalizing SaO 2 and facilitating the cessation of home oxygen therapy, as well as in reducing the incidence of severe complications. Graphical abstract

Background Impaired glucose metabolism is an established risk factor for coronary artery disease. Previous studies revealed that glycemic variability (GV) is also important for glucose metabolism in patients with acute coronary syndrome (ACS). We explored the association between GV and prognosis in patients with ACS. Methods A total of 417 patients with ACS who received reperfusion wore a continuous glucose monitoring system (CGMS) in a stable phase after admission and were monitored for at least 24 consecutive h. The mean amplitude of glycemic excursion (MAGE) was calculated as a marker of GV. We divided into two groups based on the highest tertile levels of MAGE (MAGE = 52 mg/dl). The groups were followed up for a median of 39 months [IQR 24–50 months]. The primary endpoint was the incidence of major adverse cardiovascular and cerebrovascular events (MACCE). Result During follow-up, 66 patients experienced MACCE (5 patients had cardiovascular death, 14 had recurrence of ACS, 27 had angina requiring revascularization, 8 had acute decompensated heart failure, and 16 had a stroke). MACCE was more frequently observed in the high MAGE group (23.5% vs. 11.6%, p  = 0.002). In multivariate analysis, high MAGE was an independent predictive factor of poor prognosis for MACCE (odds ratio, 1.84; 95% confidence interval, 1.01–3.36; p  = 0.045). Conclusion Glycemic variability determined with a CGMS is a predictor of prognosis in patients with ACS without severe DM. Trial registration UMIN 000010620. Registered April 1st 2012

Background Cachexia, characterized by loss of muscle with or without loss of fat mass, is a poor prognostic factor in patients with heart failure (HF). However, there is limited investigation on the prognostic impact of muscle and fat mass separately in HF. We hypothesized that muscle and fat mass have different effects on the prognosis of HF. Methods This was an observational cohort study of 418 patients (59% were men) admitted with a diagnosis of HF (71 ± 13 years [mean ± standard deviation]), with left ventricular ejection fraction (LVEF) of 39 ± 16%, including 31.3%, 14.8%, and 53.8% of patients with preserved LVEF (LVEF ≥ 50%), mid‐range LVEF (40–50%), and reduced (<40%) LVEF, respectively. Dual‐energy X‐ray absorptiometry was performed with the patients in the stable state after decongestion therapy. Results The mean body mass index of patients was 22.1 ± 4.6 kg/m2, and the mean appendicular skeletal mass (ASM) index was 6.88 ± 1.23 kg/m2 in men and 5.59 ± 0.92 in women; 54.1% of the patients showed reduced muscle mass defined by the international cut‐off value (7.0 kg/m2 for men and 5.4 for women). The mean fat mass was 20.4 ± 7.2% in men and 27.2 ± 8.6% in women. During a median follow‐up of 37 months, 92 (22.0%) of 418 patients with HF died (1 and 3 year mortality: 8.4% and 17.3%, respectively). Lower values of both skeletal muscle and fat mass were independently associated with increased risk of mortality adjusted for age, sex, haemoglobin, New York Heart Association functional class, and height squared (hazard ratio with 95% confidence interval of 0.825 [0.747–0.908] per 1 kg increase of ASM, P < 0.001, and 0.954 [0.916–0.993] per 1 kg increase of fat mass, P = 0.018, respectively). Conclusions More than half of the patients with HF showed reduced muscle mass. Lower values of both muscle and fat mass were associated with higher mortality in HF.