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BackgroundMore than 11,000 laboratories and companies developed their own next-generation sequencing (NGS) for screening and diagnosis of various diseases including cancer. Although inconsistencies of mutation calls as high as 43% in databases such as GDSC (Genomics of Drug Sensitivity in Cancer) and CCLE (Cancer Cell Line Encyclopedia) have been reported, not many studies on the reasons for the inconsistencies have been published. Methods: Targeted-NGS analysis of 151 genes in 35 cell lines common to GDSC and CCLE was performed, and the results were compared with those from GDSC and CCLE wherein whole-exome- or highly-multiplex NGS were employed.ResultsIn the comparison, GDSC and CCLE had a high rate (40-45%) of false-negative (FN) errors which would lead to high rate of inconsistent mutation calls, suggesting that highly-multiplex NGS may have high rate of FN errors. We also posited the possibility that targeted NGS, especially for the detection of low-level cancer cells in cancer tissues might suffer significant FN errors.ConclusionFN errors may be the most important errors in NGS testing for cancer; their evaluation in laboratory-developed NGS tests is needed.

The primary aim of this study was to investigate the genetic predisposition of Internet gaming disorder (IGD), and the secondary aim was to compare the results to those of alcohol dependence (AD). Two independent case-control studies were conducted. A total of 30 male participants with IGD, diagnosed according to the 5th edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) criteria, and 30 sex-matched controls participated in study 1. We designed targeted exome sequencing (TES) to test for 72 candidate genes that have been implicated in the pathogenesis of addiction. The genes included seven neurotransmitter (dopamine, serotonin, glutamate, r-aminobutyric acid (GABA), norepinephrine, acetylcholine, and opioid) system genes. A total of 31 male in-patients with AD and 29 normal male controls (NC) were enrolled in study 2. The same 72 genes included in study 1 and ten additional genes related to alcohol-metabolic enzyme were selected as the target genes, and we identified the genetic variants using the same method (TES). The IGD group had a lower frequency of the T allele of rs1044396 in the nicotinic acetylcholine receptor alpha 4 subunit (CHRNA4), and this variant represents a protective allele against IGD. However, we did not find a significant difference in the polymorphisms of the 72 genes that encode neurotransmitter systems between the AD and NC groups. This study demonstrated that rs1044396 of CHRNA4 was significantly associated with IGD.

The molecular mechanisms that mediate the ischemia-reperfusion (I/R) injury in kidney are not completely understood. It is also largely unknown whether such mechanisms overlap with those governing the early development of kidney.We performed gene expression analysis to investigate the transcriptome changes during regeneration after I/R injury in the rat (0 hr, 6 hr, 24 hr, and 120 hr after reperfusion) and early development of mouse kidney (embryonic day 16 p.c. and postnatal 1 and 7 day). Pathway analysis revealed a wide spectrum of molecular functions that may participate in the regeneration and developmental processes of kidney as well as the functional association between them. While the genes associated with cell cycle, immunity, inflammation, and apoptosis were globally activated during the regeneration after I/R injury, the genes encoding various transporters and metabolic enzymes were down-regulated. We also observed that these injury-associated molecular functions largely overlap with those of early kidney development. In particular, the up-regulation of kinases and kinesins with roles in cell division was common during regeneration and early developmental kidney as validated by real-time PCR and immunohistochemistry.In addition to the candidate genes whose up-regulation constitutes an overlapping expression signature between kidney regeneration and development, this study lays a foundation for studying the functional relationship between two biological processes.

To determine if the radiomic features on CT can predict progression-free survival (PFS) in epidermal growth factor receptor (EGFR) mutant adenocarcinoma patients treated with first-line EGFR tyrosine kinase inhibitors (TKIs) and to identify the incremental value of radiomic features over conventional clinical factors in PFS prediction.In this institutional review board-approved retrospective study, pretreatment contrast-enhanced CT and first follow-up CT after initiation of TKIs were analyzed in 48 patients (M:F = 23:25; median age: 61 years). Radiomic features at baseline, at 1st first follow-up, and the percentage change between the two were determined. A Cox regression model was used to predict PFS with nonredundant radiomic features and clinical factors, respectively. The incremental value of radiomic features over the clinical factors in PFS prediction was also assessed by way of a concordance index.Roundness (HR: 3.91; 95% CI: 1.72, 8.90; P = 0.001) and grey-level nonuniformity (HR: 3.60; 95% CI: 1.80, 7.18; P

To analyze the enhancement patterns and apparent diffusion coefficient (ADC) values of non-measurable surgical cavity wall enhancement pattern, newly appearing after completion of standard concurrent chemoradiotherapy (CCRT) with temozolomide in glioblastoma patients for the prognosis prediction.From January 2010 to April 2014, among 190 patients with histopathologically confirmed glioblastoma, a total of 33 patients with non-measurable wall enhancement on post-CCRT MR imaging were enrolled and divided into two subgroups: non-progression (n = 18) and progression groups (n = 15). We analyzed the wall enhancement patterns, which were categorized into three patterns: thin, thick and nodular enhancement. ADC values were measured in the enhancing portions of the walls. The progression-free survival (PFS) related to the wall enhancement was analyzed by Kaplan-Meier analysis, and survival curves were compared using the log-rank test.Statistically significant differences in the surgical cavity wall enhancement patterns was shown between the progression and non-progression groups (P = 0.0032). Thin wall enhancement was more frequently observed in the non-progression group, and thick or nodular wall enhancement were observed in the progression group (P = 0.0016). There was no statistically significant difference in the mean ADC values between the progression and non-progression groups. The mean PFS was longer in patients with thin wall enhancement than in those with nodular or thick wall enhancement (35.5 months vs. 15.8 months, P = 0.008).Pattern analysis of non-measurable surgical cavity wall enhancement on post-CCRT MR imaging might be useful tool for predicting prognosis of GBM patient before clear progression of non-measurable disease.

Preclinical and clinical studies have suggested that expression of ribonucleotide reductase regulatory subunit M1 (RRM1) and excision repair cross-complementation group 1 (ERCC1) is associated with resistance to gemcitabine and cisplatin, respectively. We evaluated the significance of RRM1 and ERCC1 expression to predict tumor response to gemcitabine plus platinum chemotherapy (GP) and survival in advanced UC. We retrospectively collected tumor samples and reviewed clinical data of 53 patients with unresectable or metastatic UC, who were treated with first-line GP. RRM1 and ERCC1 expression were measured by immunohistochemistry. Among 53 patients, 12 (22.6%) and 26 (49.1%) patients had tumors that demonstrated a high expression for RRM1 and ERCC1, respectively. Twenty-nine (70.7%) of 41 patients with low RRM1 expression achieved a clinical response (complete + partial responses), but only 3 (25.0%) of 12 patients with high RRM1 expression achieved a clinical response after GP (P=0.007). Nineteen (70.4%) of 27 patients with low ERCC1 expression achieved a clinical response, while 13 (50.0%) of 26 patients with high ERCC1 expression achieved a clinical response (P=0.130). High RRM1 expression was associated with shorter progression free survival and overall survival (PFS P=0.006, OS P=0.006). Multivariate analysis confirmed that patients with high RRM1 expression had a significantly greater risk of progression and death than those with low RRM1 expression. ERCC1 status was not a significant predictor for PFS and OS. RRM1 expression was predictive and prognostic of clinical outcome in advanced UC treated with gemcitabine plus platinum combination chemotherapy.

PurposeTo assess the prognosis predictability of a measurable enhancing lesion using histogram parameters produced by the normalized cerebral blood volume (nCBV) and normalized apparent diffusion coefficient (nADC) after completion of standard concomitant chemoradiotherapy (CCRT) and adjuvant temozolomide (TMZ) medication in glioblastoma multiforme (GBM) patients.Materials and methodsThis study was approved by the institutional review board (IRB), and the requirement for informed consent was waived. A total of 59 patients with newly diagnosed GBM who received standard CCRT with TMZ and adjuvant TMZ for six cycles underwent perfusion-weighted and diffusion-weighted imaging. Twenty-seven patients had a measurable enhancing lesion and 32 patients lacked a measurable enhancing lesion based on the Response Assessment in Neuro-Oncology (RANO) criteria in the follow-up MRI, which was performed within 3 months after adjuvant TMZ therapy was completed. We measured the nCBV and nADC histogram parameters based on the measurable enhancing lesion. The progression free survival (PFS) was analyzed by the Kaplan-Meier method with the use of the log-rank test.ResultsThe median PFS of patients lacking measurable enhancing lesion was longer than for those with measurable enhancing lesions (17.6 vs 3.3 months, PConclusionWe found that the nCBV value can be used for the prognosis prediction of a measurable enhancing lesion after the completion of standard treatment for GBM, wherein a high 99th percentile nCBV value (≧ 4.5) suggests a better PFS for GBM patients.

Ex vivo-expanded, allogeneic natural killer (NK) cells can be used for the treatment of various types of cancer. In allogeneic NK cell therapy, NK cells from healthy donors must be expanded in order to obtain a sufficient number of highly purified, activated NK cells. In the present study, we established a simplified and efficient method for the large-scale expansion and activation of NK cells from healthy donors under good manufacturing practice (GMP) conditions. After a single step of magnetic depletion of CD3(+) T cells, the depleted peripheral blood mononuclear cells (PBMCs) were stimulated and expanded with irradiated autologous PBMCs in the presence of OKT3 and IL-2 for 14 days, resulting in a highly pure population of CD3(-)CD16(+)CD56(+) NK cells which is desired for allogeneic purpose. Compared with freshly isolated NK cells, these expanded NK cells showed robust cytokine production and potent cytolytic activity against various cancer cell lines. Of note, expanded NK cells selectively killed cancer cells without demonstrating cytotoxicity against allogeneic non-tumor cells in coculture assays. The anti-tumor activity of expanded human NK cells was examined in SCID mice injected with human lymphoma cells. In this model, expanded NK cells efficiently controlled lymphoma progression. In conclusion, allogeneic NK cells were efficiently expanded in a GMP-compliant facility and demonstrated potent anti-tumor activity both in vitro and in vivo.

BACKGROUND: Long-term maintenance of avian primordial germ cells (PGCs) in vitro has tremendous potential because it can be used to deepen our understanding of the biology of PGCs. A transgenic bioreactor based on the unique migration of PGCs toward the recipients' sex cord via the bloodstream and thereby creating a germline chimeric bird has many potential applications. However, the growth factors and the signaling pathway essential for inducing proliferation of chicken PGCs are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Therefore, we conducted this study to investigate the effects of various combinations of growth factors on the survival and proliferation of PGCs under feeder-free conditions. We observed proliferation of PGCs in media containing bFGF. Subsequent characterization confirmed that the cultured PGCs maintained expression of PGC-specific markers, telomerase activity, normal migrational activity, and germline transmission. We also found that bFGF activates the mitogen-activated protein kinase kinase/extracellular-signal regulated kinase (MEK/ERK) signaling. Also, the expression of 133 transcripts was reversibly altered by bFGF withdrawal. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that chicken PGCs can be maintained in vitro without any differentiation or dedifferentiation in feeder free culture conditions, and subsequent analysis revealed that bFGF is one of the key factors that enable proliferation of chicken PGCs via MEK/ERK signaling regulating downstream genes that may be important for PGC proliferation and survival.