Cancer panel analysis of circulating tumor cells in patients with breast cancer.

Liquid biopsy using circulating tumor cells (CTCs) is a noninvasive and repeatable procedure, and is therefore useful for molecular assays. However, the rarity of CTCs remains a challenge. To overcome this issue, our group developed a novel technology for the isolation of CTCs on the basis of cell size difference. The present study isolated CTCs from patients with breast cancer using this method, and then used these cells for cancer gene panel analysis. Blood samples from eight patients with breast cancer were collected, and CTCs were enriched using size-based filtration. Enriched CTCs were counted using immunofluorescent staining with an epithelial cell adhesion molecule (EpCAM) and CD45 antibodies. CTC genomic DNA was extracted, amplified, and screened for mutations in 400 genes using the Ion AmpliSeq Comprehensive Cancer Panel. White blood cells (WBCs) from the same patient served as a negative control, and mutations in CTCs and WBCs were compared. EpCAM+ cells were detected in seven out of eight patients, and the average number of EpCAM+ cells was 8.6. The average amount of amplified DNA was 32.7 µg, and the percentage of reads mapped to any targeted region relative to all reads mapped to the reference was 98.6%. The detection rate of CTC-specific mutations was 62.5%. The CTC-specific mutations were enhancer of zeste polycomb repressive complex 2 subunit, notch 1, AT-rich interaction domain 1A, serine/threonine kinase 11, fms related tyrosine kinase 3, MYCN proto-oncogene, bHLH transcription factor, APC, WNT signaling pathway regulator, and phosphatase and tensin homolog. The technique used by the present study was demonstrated to be effective at isolating CTCs at a sufficiently high purity for genomic analysis, and supported the use of comprehensive cancer panel analysis as a potential application for precision medicine.

Cancer gene panel analysis of cultured circulating tumor cells and primary tumor tissue from patients with breast cancer.

Although numerous effective therapies have improved the survival rate of patients with breast cancer, a number of patients present with tumor recurrence and metastasis. A liquid biopsy of circulating tumor cells (CTC) is a non-invasive method to obtain tumor cells and may be used as substitute for a tumor tissue biopsy. The present study focuses on determining whether CTC culture is an optimal method of obtaining sufficient amounts of CTCs for molecular analysis. The current study demonstrates a method of isolating and culturing CTCs from patients with breast cancer and the construction of a molecular profile of cultured cells using the Ion AmpliSeq Cancer Gene Panel V2. Gene mutations that were observed in cultured CTCs were compared with those observed in primary tumor tissues. CTCs were isolated and cultured from the blood of six patients with breast cancer. Mutations from the Catalogue Of Somatic Mutation In Cancer (COSMIC) were detected in Platelet-Derived Growth Factor Receptor Alpha, MET (also known as Hepatocyte Growth Factor Receptor), Phosphatase and Tensin Homolog, Harvey Rat Sarcoma Viral Oncogene Homolog, SWI/SNF Related, Matrix Associated, Actin Dependent Regulator of Chromatin Subfamily B Member 1, Cyclin Dependent Kinase Inhibitor 2A and MutL Homolog 1 genes in 5/6 samples. A comparison between mutations detected in cultured CTCs and mutations detected in primary tumor tissues demonstrated that a large number of mutations that were identified in CTCs were also detected in primary tumor tissues. The results from the current study describe a novel cell culture approach that may be used to obtain an optimal number of CTCs for molecular analysis. This novel approach is able to be used as a tool for liquid biopsy during breast cancer treatment.

Evaluation of a novel approach to circulating tumor cell isolation for cancer gene panel analysis in patients with breast cancer.

Liquid biopsy isolation of circulating tumor cells (CTCs) allows the genomic analysis of CTCs, which is useful in the determination of personalized cancer therapy. In the present study, CTCs from patients with breast cancer were enriched and successfully analyzed using cancer gene panel analysis. Blood samples from 11 patients with breast cancer were collected and CTCs enriched for using size-based filtration. The enriched CTCs were analyzed using immunofluorescence staining with antibodies directed against epithelial cell adhesion molecule (EpCAM) and cluster of differentiation 45. The genomic DNA of CTCs was extracted, amplified and 50 genes screened for mutations using the Ion AmpliSeq™ Cancer Hotspot Panel v2. EpCAM staining detected CTCs in 10/11 patients and the average CTC count was 3.9 in 5 ml blood. The average purity of enriched CTCs was 14.2±29.4% and the average amount of amplified DNA was 28.6±11.9 µg. Catalogue Of Somatic Mutations In Cancer mutations were detected in the CTCs and included IDH2, TP53, NRAS, IDH1, PDGFRA, HRAS, STK11, EGFR, PTEN, MLH1, PIK3CA, CDKN2A, KIT and SMARCB1. In conclusion, a novel size-based filtration approach for the isolation of CTCs was evaluated and successfully applied for the genomic analysis of CTCs from patients with breast cancer.

Identification of NUCKS1 as a putative oncogene and immunodiagnostic marker of hepatocellular carcinoma.

Although the molecular mechanisms underpinning hepatocellular carcinoma (HCC) are unknown, gene copy number and associated mRNA expression changes are frequently reported. Comparative genomic hybridization arrays spotted with 4041 bacterial artificial chromosome clones were used to assess copy number changes in 45 HCC tissues. Seventy more HCC tissues were used to validate candidate genes by using western blots and immunohistochemistry. A total of 259 clones were associated with copy number changes that significantly differed between normal liver and HCC samples. The chromosomal region 1q32.1 containing the nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) gene was associated with tumor vascular invasion. Western blot analysis demonstrated that NUCKS1 was up-regulated in 37 of 70 (52.8%) HCC tissues compared with adjacent non-tumor tissues, and over-expressed in a vast majority of HCCs (44/52, 84.6%) as determined by immunohistochemical staining. Furthermore, immunostaining of both NUCKS1 and glypican-3 improved the diagnostic prediction of HCC. Knock-down of NUCKS1 by siRNA implied the decrease in cell viability of the Hep3B cell line and reduced tumor formation in a xenograft mouse model. NUCKS1 was identified as a potential oncogene at chromosomal 1q32.1 in patients with HCC, and it might be a valuable immunodiagnostic marker for HCC.

Comparative whole genome transcriptome and metabolome analyses of five Klebsiella pneumonia strains.

The integration of transcriptomics and metabolomics can provide precise information on gene-to-metabolite networks for identifying the function of novel genes. The goal of this study was to identify novel gene functions involved in 2,3-butanediol (2,3-BDO) biosynthesis by a comprehensive analysis of the transcriptome and metabolome of five mutated Klebsiella pneumonia strains (∆wabG = SGSB100, ∆wabG∆budA = SGSB106, ∆wabG∆budB = SGSB107, ∆wabG∆budC = SGSB108, ∆wabG∆budABC = SGSB109). First, the transcriptomes of all five mutants were analyzed and the genes exhibiting reproducible changes in expression were determined. The transcriptome was well conserved among the five strains, and differences in gene expression occurred mainly in genes coding for 2,3-BDO biosynthesis (budA, budB, and budC) and the genes involved in the degradation of reactive oxygen, biosynthesis and transport of arginine, cysteine biosynthesis, sulfur metabolism, oxidoreductase reaction, and formate dehydrogenase reaction. Second, differences in the metabolome (estimated by carbon distribution, CO2 emission, and redox balance) among the five mutant strains due to gene alteration of the 2,3-BDO operon were detected. The functional genomics approach integrating metabolomics and transcriptomics in K. Pneumonia presented here provides an innovative means of identifying novel gene functions involved in 2,3-BDO biosynthesis metabolism and whole cell metabolism.

The influence of budA deletion on glucose metabolism related in 2,3-butanediol production by Klebsiella pneumoniae.

Klebsiella pneumoniae (K. pneumoniae), which is a promising microorganism for industrial bulk production of 2,3-butanediol (2,3-BDO), naturally converts glucose to 2,3-BDO. The 2,3-BDO biosynthesis from glucose is composed of three steps; α-acetolactate biosynthesis by α-acetolactate synthase (budB); acetoin biosynthesis by α-acetolactate decarboxylase (budA); and 2,3-BDO biosynthesis by acetoin reductase (budC). In an effort to understand the influence of blocked 2,3-BDO pathway on K. pneumoniae glucose metabolism by budA deletion, we constructed K. pneumoniaeΔwabGΔbudA (SGSB106). Carbon flux distribution analysis, transcriptome analysis and extracellular amino acid concentration analysis were carried out to understand the effects of the budA deletion, and K. pneumoniaeΔwabG (SGSB100) was used as a control strain. Approximately 50.3% decrease in CO2 emission; and approximately 3.8-fold increase in amino acid production was observed in SGSB106. In addition to, among the amino acids, valine production significantly increased, suggesting that the branched-chain amino acid biosynthesis (BACC) in SGSB106 was activated by deletion of budA. Furthermore, whole genome transcriptome analysis of SGSB106 and SGSB100, correlates with the results from carbon distribution and amino acids concentration analyses.

Complete genome sequence of Klebsiella oxytoca M1, isolated from Manripo area of South Korea.

Here we report the full genome sequence of Klesiella oxytoca M1, isolated from Manripo area of South Korea. The strain K. oxytoca M1 is able to produce either 2,3-butanediol or acetoin selectively by controlling the pH and temperature.

Gene expression defines natural changes in mammalian lifespan.

Mammals differ more than 100-fold in maximum lifespan, which can be altered in either direction during evolution, but the molecular basis for natural changes in longevity is not understood. Divergent evolution of mammals also led to extensive changes in gene expression within and between lineages. To understand the relationship between lifespan and variation in gene expression, we carried out RNA-seq-based gene expression analyses of liver, kidney, and brain of 33 diverse species of mammals. Our analysis uncovered parallel evolution of gene expression and lifespan, as well as the associated life-history traits, and identified the processes and pathways involved. These findings provide direct insights into how nature reversibly adjusts lifespan and other traits during adaptive radiation of lineages.

An epigenomic roadmap to induced pluripotency reveals DNA methylation as a reprogramming modulator.

Reprogramming of somatic cells to induced pluripotent stem cells involves a dynamic rearrangement of the epigenetic landscape. To characterize this epigenomic roadmap, we have performed MethylC-seq, ChIP-seq (H3K4/K27/K36me3) and RNA-Seq on samples taken at several time points during murine secondary reprogramming as part of Project Grandiose. We find that DNA methylation gain during reprogramming occurs gradually, while loss is achieved only at the ESC-like state. Binding sites of activated factors exhibit focal demethylation during reprogramming, while ESC-like pluripotent cells are distinguished by extension of demethylation to the wider neighbourhood. We observed that genes with CpG-rich promoters demonstrate stable low methylation and strong engagement of histone marks, whereas genes with CpG-poor promoters are safeguarded by methylation. Such DNA methylation-driven control is the key to the regulation of ESC-pluripotency genes, including Dppa4, Dppa5a and Esrrb. These results reveal the crucial role that DNA methylation plays as an epigenetic switch driving somatic cells to pluripotency.

Improvement of 2,3-butanediol yield in Klebsiella pneumoniae by deletion of the pyruvate formate-lyase gene.

Klebsiella pneumoniae is considered a good host strain for the production of 2,3-butanediol, which is a promising platform chemical with various industrial applications. In this study, three genes, including those encoding glucosyltransferase (wabG), lactate dehydrogenase (ldhA), and pyruvate formate-lyase (pflB), were disrupted in K. pneumoniae to reduce both its pathogenic characteristics and the production of several by-products. In flask cultivation with minimal medium, the yield of 2,3-butanediol from rationally engineered K. pneumoniae (ΔwabG ΔldhA ΔpflB) reached 0.461 g/g glucose, which was 92.2% of the theoretical maximum, with a significant reduction in by-product formation. However, the growth rate of the pflB mutant was slightly reduced compared to that of its parental strain. Comparison with similar mutants of Escherichia coli suggested that the growth defect of pflB-deficient K. pneumoniae was caused by redox imbalance rather than reduced level of intracellular acetyl coenzyme A (acetyl-CoA). From an analysis of the transcriptome, it was confirmed that the removal of pflB from K. pneumoniae significantly repressed the expression of genes involved in the formate hydrogen lyase (FHL) system.

Heterozygous mutations in cyclic AMP phosphodiesterase-4D (PDE4D) and protein kinase A (PKA) provide new insights into the molecular pathology of acrodysostosis.

Acrodysostosis without hormone resistance is a rare skeletal disorder characterized by brachydactyly, nasal hypoplasia, mental retardation and occasionally developmental delay. Recently, loss-of-function mutations in the gene encoding cAMP-hydrolyzing phosphodiesterase-4D (PDE4D) have been reported to cause this rare condition but the pathomechanism has not been fully elucidated. To understand the pathogenetic mechanism of PDE4D mutations, we conducted 3D modeling studies to predict changes in the binding efficacy of cAMP to the catalytic pocket in PDE4D mutants. Our results indicated diminished enzyme activity in the two mutants we analyzed (Gly673Asp and Ile678Thr; based on PDE4D4 residue numbering). Ectopic expression of PDE4D mutants in HEK293 cells demonstrated this reduction in activity, which was identified by increased cAMP levels. However, the cells from an acrodysostosis patient showed low cAMP accumulation, which resulted in a decrease in the phosphorylated cAMP Response Element-Binding Protein (pCREB)/CREB ratio. The reason for this discrepancy was due to a compensatory increase in expression levels of PDE4A and PDE4B isoforms, which accounted for the paradoxical decrease in cAMP levels in the patient cells expressing mutant isoforms with a lowered PDE4D activity. Skeletal radiographs of 10-week-old knockout (KO) rats showed that the distal part of the forelimb was shorter than in wild-type (WT) rats and that all the metacarpals and phalanges were also shorter in KO, as the name acrodysostosis implies. Like the G-protein α-stimulatory subunit and PRKAR1A, PDE4D critically regulates the cAMP signal transduction pathway and influences bone formation in a way that activity-compromising PDE4D mutations can result in skeletal dysplasia. We propose that specific inhibitory PDE4D mutations can lead to the molecular pathology of acrodysostosis without hormone resistance but that the pathological phenotype may well be dependent on an over-compensatory induction of other PDE4 isoforms that can be expected to be targeted to different signaling complexes and exert distinct effects on compartmentalized cAMP signaling.

Predictive efficacy of low burden EGFR mutation detected by next-generation sequencing on response to EGFR tyrosine kinase inhibitors in non-small-cell lung carcinoma.

Direct sequencing remains the most widely used method for the detection of epidermal growth factor receptor (EGFR) mutations in lung cancer; however, its relatively low sensitivity limits its clinical use. The objective of this study was to investigate the sensitivity of detecting an epidermal growth factor receptor (EGFR) mutation from peptide nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamp and Ion Torrent Personal Genome Machine (PGM) techniques compared to that by direct sequencing. Furthermore, the predictive efficacy of EGFR mutations detected by PNA-LNA PCR clamp was evaluated. EGFR mutational status was assessed by direct sequencing, PNA-LNA PCR clamp, and Ion Torrent PGM in 57 patients with non-small cell lung cancer (NSCLC). We evaluated the predictive efficacy of PNA-LNA PCR clamp on the EGFR-TKI treatment in 36 patients with advanced NSCLC retrospectively. Compared to direct sequencing (16/57, 28.1%), PNA-LNA PCR clamp (27/57, 47.4%) and Ion Torrent PGM (26/57, 45.6%) detected more EGFR mutations. EGFR mutant patients had significantly longer progressive free survival (14.31 vs. 21.61 months, P = 0.003) than that of EGFR wild patients when tested with PNA-LNA PCR clamp. However, no difference in response rate to EGFR TKIs (75.0% vs. 82.4%, P = 0.195) or overall survival (34.39 vs. 44.10 months, P = 0.422) was observed between the EGFR mutations by direct sequencing or PNA-LNA PCR clamp. Our results demonstrate firstly that patients with EGFR mutations were detected more frequently by PNA-LNA PCR clamp and Ion Torrent PGM than those by direct sequencing. EGFR mutations detected by PNA-LNA PCR clamp may be as a predicative factor for EGFR TKI response in patients with NSCLC.

Complete Genome Sequence of Raoultella ornithinolytica Strain B6, a 2,3-Butanediol-Producing Bacterium Isolated from Oil-Contaminated Soil.

Here we report the full genome sequence of Raoultella ornithinolytica strain B6, a Gram-negative aerobic bacillus belonging to the family Enterobacteriaceae. This 2,3-butanediol-producing bacterium was isolated from oil-contaminated soil on Backwoon Mountain in South Korea. Strain B6 contains 5,398,151 bp with 4,909 protein-coding genes, 104 structural RNAs, and 55.88% G+C content.

Complete genome sequence of the 2,3-butanediol-producing Klebsiella pneumoniae strain KCTC 2242.

Here we report the full genome sequence of Klebsiella pneumoniae KCTC 2242,consisting of a 5.26-Mb chromosome (57.6% GC%; 5,035 genes [4,923 encoding known proteins, 112 RNA genes]) and a 202-kb plasmid (50.2% GC%; 229 genes [229 encoding known proteins]).

Complete genome sequence of Klebsiella oxytoca KCTC 1686, used in production of 2,3-butanediol.

Here we report the full genome sequence of Klebsiella oxytoca KCTC 1686, which is used in production of 2,3-butanediol. The KCTC 1686 strain contains 5,974,109 bp with G+C content of 56.05 mol% and contains 5,488 protein-coding genes and 110 structural RNAs.

Complete genome sequence of Enterobacter aerogenes KCTC 2190.

This is the first complete genome sequence of the Enterobacter aerogenes species. Here we present the genome sequence of E. aerogenes KCTC 2190, which contains 5,280,350 bp with a G + C content of 54.8 mol%, 4,912 protein-coding genes, and 109 structural RNAs.

Genome sequence of Paenibacillus terrae HPL-003, a xylanase-producing bacterium isolated from soil found in forest residue.

This article reports on the full genome sequence of Paenibacillus terrae HPL-003, which is a gram-positive, endospore-forming, xylanase-producing bacterium isolated from soil found in forest residue on Gara Mountain. The strain HPL-003 contains 6,083,395 bp with a G+C content of 46.77 mol%, 2,633 protein-coding genes, and 117 structural RNAs.

FX: an RNA-Seq analysis tool on the cloud.

UNLABELLED: FX is an RNA-Seq analysis tool, which runs in parallel on cloud computing infrastructure, for the estimation of gene expression levels and genomic variant calling. In the mapping of short RNA-Seq reads, FX uses a transcriptome-based reference primarily, generated from ~160 000 mRNA sequences from RefSeq, UCSC and Ensembl databases. This approach reduces the misalignment of reads originating from splicing junctions. Unmapped reads not aligned on known transcripts are then mapped on the human genome reference. FX allows analysis of RNA-Seq data on cloud computing infrastructures, supporting access through a user-friendly web interface. AVAILABILITY: FX is freely available on the web at (http://fx.gmi.ac.kr), and can be installed on local Hadoop clusters. Guidance for the installation and operation of FX can be found under the 'Documentation' menu on the website. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Extensive genomic and transcriptional diversity identified through massively parallel DNA and RNA sequencing of eighteen Korean individuals.

Massively parallel sequencing technologies have identified a broad spectrum of human genome diversity. Here we deep sequenced and correlated 18 genomes and 17 transcriptomes of unrelated Korean individuals. This has allowed us to construct a genome-wide map of common and rare variants and also identify variants formed during DNA-RNA transcription. We identified 9.56 million genomic variants, 23.2% of which appear to be previously unidentified. From transcriptome sequencing, we discovered 4,414 transcripts not previously annotated. Finally, we revealed 1,809 sites of transcriptional base modification, where the transcriptional landscape is different from the corresponding genomic sequences, and 580 sites of allele-specific expression. Our findings suggest that a considerable number of unexplored genomic variants still remain to be identified in the human genome, and that the integrated analysis of genome and transcriptome sequencing is powerful for understanding the diversity and functional aspects of human genomic variants.

17p12 deletion in breast cancer predicts resistance to neoadjuvant chemotherapy.

Numerous studies have attempted to identify gene expression profiles which can be utilized to predict responses to neoadjuvant chemotherapy (NAC), but their findings are not clinically applicable at present. In the present study, we sought to determine DNA copy number alterations (CNAs) in breast cancer tissues which are associated with the response to NAC. Frozen tumor tissues from 63 breast cancer patients were obtained using core needle biopsy prior to NAC (3 cycles of docetaxel plus adriamycin) and were microdissected. Array comparative genomic hybridization (array CGH) with 4,045 bacterial artificial chromosome (BAC) probes was performed to identify the CNAs. Changes in tumor size in response to NAC were measured via magnetic resonance imaging. Fluorescence in situ hybridization (FISH) was conducted to verify array CGH results and for independent validation studies. CNAs at eight chromosomal loci encompassing 24 clones were correlated with changes in tumor size after NAC (p<0.05; t-test). Two CNAs were selected, 17p12 deletion and 17q21.32-33 gain, which were significantly associated with a smaller reduction in tumor size following NAC, via prioritization of the regions containing the candidate genes. In an independent validation set of samples from 39 patients, FISH assay further showed that the 17p12 deletion was markedly associated with smaller changes in tumor size (p=0.006), while the 17q21.32-33 gain was not significant (p=0.309). In conclusion, we successfully identified a 17p12 deletion in breast cancer tissue which can be applied in predicting tumor resistance to NAC.