We investigated the potential of a nanofiber-based poly(DL-lactide-co-glycolide) (PLGA) scaffold to be used for cartilage reconstruction. The mechanical properties of the nanofiber scaffold, degradation of the scaffold and cellular responses to the scaffold under mechanical stimulation were studied. Three different types of scaffold (lactic acid/glycolic acid content ratio = 75 : 25, 50 : 50, or a blend of 75 : 25 and 50 : 50) were tested. The tensile modulus, ultimate tensile stress and corresponding strain of the scaffolds were similar to those of skin and were slightly lower than those of human cartilage. This suggested that the nanofiber scaffold was sufficiently mechanically stable to withstand implantation and to support regenerated cartilage. The 50 : 50 PLGA scaffold was degraded faster than 75 : 25 PLGA, probably due to the higher hydrophilic glycolic acid content in the former. The nanofiber scaffold was degraded faster than a block-type scaffold that had a similar molecular weight. Therefore, degradation of the scaffold depended on the lactic acid/glycolic acid content ratio and might be controlled by mixing ratio of blend PLGA. Cellular responses were evaluated by examining toxicity, cell proliferation and extracellular matrix (ECM) formation using freshly isolated chondrocytes from porcine articular cartilage. The scaffolds were non-toxic, and cell proliferation and ECM formation in nanofiber scaffolds were superior to those in membrane-type scaffolds. Intermittent hydrostatic pressure applied to cell-seeded nanofiber scaffolds increased chondrocyte proliferation and ECM formation. In conclusion, our nanofiber-based PLGA scaffold has the potential to be used for cartilage reconstruction.

BackgroundEGFR genotyping in pulmonary adenocarcinoma patients who develop pleural effusions is mostly performed using cytology or cell block slides with low sensitivity. Liquid biopsy using the supernatant of pleural effusions may be more effective because they contain many components released by cancer cells. Extracellular vesicles (EVs) are known to carry oncogenic double-stranded DNA that is considered a notable biomarker. Here, we investigate the efficiency of liquid biopsy using cell-free DNA (cfDNA) and extracellular vesicle-derived DNA (EV-derived DNA) from the supernatant of pleural effusions for EGFR genotyping in patients with pulmonary adenocarcinoma.MethodsFifty pleural effusion samples from patients with pulmonary adenocarcinoma were evaluated. The supernatant, after removing the cell pellet by centrifugation, was used for liquid biopsy, and EVs were isolated from the pleural effusion by ultracentrifugation. EV-derived DNA and cfDNA were extracted separately, and EGFR genotyping was performed by the PNA clamping method.ResultsAmong 32 patients who were EGFR-tyrosine kinase inhibitor (TKI) naïve with a known tissue EGFR genotype, liquid biopsy using EV-derived DNA from the pleural effusion supernatant showed 100% matching results with tissue EGFR genotyping in 19 EGFR mutant cases and detected three additional EGFR mutations in patients with wild-type (WT) tissue. Liquid biopsy using cfDNA from pleural effusion supernatants missed two cases of tissue-based EGFR mutations and found two additional EGFR mutation cases. In 18 patients who acquired resistance to EGFR-TKI, EGFR genotyping using EV-derived DNA from the pleural effusion supernatant detected the T790 M mutation in 13 of 18 (72.2%) patients, and this mutation was detected in 11 (61.1%) patients using cfDNA. By contrast, only three patients were found to present the T790 M mutation when using cell block or cytology slides.ConclusionsLiquid biopsy using the supernatant of pleural effusions showed significantly improved results for EGFR genotyping compared to those using conventional cell block or cytology samples. Liquid biopsy using EV-derived DNA is promising for EGFR genotyping, including T790 M detection in pulmonary adenocarcinoma patients who develop pleural effusions.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-5138-3) contains supplementary material, which is available to authorized users.

Nuclear B-catenin forms a transcription complex with TCF-4, which is implicated in colon cancer development and progression. Recently, we and others have shown that B-catenin could be a regulator of RNA splicing and it also stabilizes the cyclooxygenase-2 (COX-2) mRNA. Here, we further explored the role of B-catenin in the RNA metabolism in colon cancer cells. To specifically modulate the subcellular functions of B-catenin, we expressed the RNA aptamer in the form of RNA intramers with unique cellular localizations. The nucleus-expressed RNA intramer proved to be effective in reducing the protein-protein interaction between B-catenin and TCF-4, thus shown to be a specific regulator of B-cateninactivated transcription. It could also regulate the alternative splicing of E1A minigene in diverse colon cancer cell lines. In addition, we tested whether B-catenin could stabilize any other mRNAs and found that cyclin D1 mRNA was also bound and stabilized by B-catenin. Significantly, the cytoplasmexpressed RNA intramer reverted the B-catenin-induced COX-2 and cyclin D1 mRNA stabilization. We show here that B-catenin regulated multiple steps of RNA metabolism in colon cancer cells and might be the protein factor coordinating RNA metabolism. We suggest that the RNA intramers could provide useful ways for inhibiting B-catenin-mediated transcription and RNA metabolism, which might further enhance the antitumorigenic effects of these molecules in colon cancer cells. [Cancer Res 2007;67(19):9315-21]

Background: Extracellular vesicles (EV) have been proven to contain double-stranded DNA reflecting the mutational status of the parental tumor cells in non-small cell lung cancer (NSCLC), which can be translated into clinically useful EV-based liquid biopsy for Epidermal growth factor receptor (EGFR) genotyping using bronchoalveolar lavage fluid (BALF) obtained from tumor site.Methods: Patients subjected for an initial lung cancer work-up underwent bronchoscopy and BALF was obtained from tumor site. After isolating EVs from BALF by ultracentrifugation, EV-derived DNA (EV DNA) was extracted for subsequent EGFR genotyping performed through peptide nucleic acid (PNA)mediated Real-Time PCR. The sensitivity, specificity, and concordance rate of BALF EV-based EGFR genotyping were calculated in comparison to tissue genotyping. Results:The average sensitivity and specificity of BALF EV-based EGFR genotyping were 76% and 87%, respectively, while the sensitivity significantly increased as the stage progressed. Especially, in stage IV, BALF EV-based EGFR typing identified all tissue-proven EGFR mutant cases (n=31) and detected 6 additional mutant cases. The concordance rate was 79% in stage I, 100% in stage II, 74% in stage III, and 92% in stage IV. As TNM stage advanced, especially in the presence of metastasis, concordance rate significantly increased (P<0.05). Conclusions:The use of BALF for the collection of EV DNA in lung cancer patients resulted in a highly accurate diagnosis. The establishment of a fast and reliable method to identify target genes using EV DNA illustrated that it can overcome the problems of low sensitivity and instability in using cell-free DNA (cfDNA).

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Background Despite surgical resection, early lung adenocarcinoma has a recurrence rate of 20–50%. No clear predictive markers for recurrence of early lung adenocarcinoma are available. Targeted next-generation sequencing (NGS) is rarely used to identify recurrence-related genes. We aimed to identify genetic alterations that can predict recurrence, by comparing the molecular profiles of patient groups with and without recurrence. Methods Tissues from 230 patients with resected stage I–II lung adenocarcinoma (median follow-up: 49 months) were analyzed via targeted NGS for 207 cancer-related genes. The recurrence-free survival according to the number and type of mutation was estimated using the Kaplan–Meier method. Independent predictive biomarkers related to recurrence were identified using the Cox proportional hazards model. Results Recurrence was observed in 64 patients (27.8%). In multivariate analysis adjusted for age, sex, smoking history, stage, surgical mode, and visceral pleural invasion, the CTNNB1 mutation and fusion genes (ALK, ROS1, RET) were negative prognostic factors for recurrence in early-stage lung adenocarcinoma (HR 4.47, p = 0.001; HR 2.73, p = 0.009). EGFR mutation was a favorable factor (HR 0.51, p = 0.016), but the CTNNB1/EGFR co-mutations were negative predictors (HR 19.2, p < 0.001). TP53 mutation was a negative predictor compared with EGFR mutation for recurrence (HR 5.24, p = 0.02). Conclusions: Targeted NGS can provide valuable information to predict recurrence and identify patients at high recurrence risk, facilitating selection of the treatment strategy among close monitoring and adjuvant-targeted therapy. Larger datasets are required to validate these findings.

A growing trend in identifying biomarkers in diseases has brought on a dramatic rise in the number of tissue biopsies performed in the era of precision medicine. With advanced non-small cell lung cancer (NSCLC) patients often relying on small biopsies, the clinical need for an established liquid biopsy protocol has become an urgent necessity. Unlike gastrointestinal cancer, where tumor lesions in all locations can generally be visualized, lung cancers can only be approached by bronchoscopy, which limits visualization to central tumors. Even with the use of endobronchial ultrasound, peripheral lung cancers, such as adenocarcinoma before metastasis to the mediastinal lymph node, must rely on the invasive percutaneous needle biopsy (PCNB). Recent trends have shown a decrease in the incidence of central lung cancers, such as squamous cell carcinoma and small cell carcinoma, with a concomitant increase in the incidence adenocarcinoma, where testing to identify driver oncogenic mutations is essentially useful [1-3]. With the development of target therapy drugs, the clinical need for rebiopsy or even repeated biopsies is increasing in order to identify drug resistance mechanisms in the targeted tissues [4,5]. Lung cancer tumor lesions are often small in size or positioned in a way that makes them difficult to target. In the case of ground-glass nodules (GGNs), a biopsy is not possible until enough of a solid portion develops to obtain tumor tissue by PCNB. For these reasons, "tissue is the issue" is a challenge faced by many clinicians, especially by those treating lung cancers. Liquid biopsy using blood was introduced to over

BackgroundAlthough lung adenocarcinoma with activating epidermal growth factor receptor (EGFR) mutations is common in never smokers, one-third of the patients are ever-smokers. We aimed to investigate the effect of cumulative smoking dose(CSD) on clinical outcomes, including progression-free survival (PFS) and overall survival (OS), in patients with EGFR-mutated lung adenocarcinoma receiving EGFR-tyrosine kinase inhibitors (TKIs).MethodsWe retrospectively analyzed 142 patients with EGFR-mutation positive advanced or recurrent lung adenocarcinoma who were administered gefitinib, erlotinib, afatinib, and osimertinib. These patients were classified based on their CSD as never smokers, light smokers (≤10 pack-years [PYs]), moderate smokers (11–30 PYs), and heavy smokers (> 30 PYs). PFS and OS were analyzed according to smoking subgroups via Kaplan-Meier curves.ResultsAmong the 142 patients, 91 (64.1%), 12 (8.5%), 22 (15.5%), and 17 (12%) were never, light, moderate, and heavy smokers, respectively. CSD was inversely associated with median PFS in a statistically significant dose-dependent manner (11.8 months (mo), 11.0 mo, 7.4 mo, and 3.9 mo; p < 0.001). Statistically significant negative association was observed between CSD and median OS (33.6 mo, 26.3 mo, 20 mo, and 8.9 mo; p < 0.001). In the multivariate analysis adjusted for age, sex, performance status, stage, and timing of EGFR-TKIs, CSD was an independent predictive factor for disease progression (hazard ratio [HR], 4.00; 95% confidence interval [CI], 1.95–8.23; p = 0.012) and OS (HR, 3.9; 95% CI, 1.84–8.28; p < 0.001).ConclusionCSD is an important predictive and prognostic factor in patients with EGFR-mutated lung adenocarcinoma, and associated smoking-related gene signatures might affect the outcomes.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4691-0) contains supplementary material, which is available to authorized users.