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ABSTRACT: A plethora of data is accumulating from high throughput methods on metabolites, coenzymes, proteins, and nucleic acids and their interactions as well as the signalling and regulatory functions and pathways of the cellular network. The frozen moment viewed in a single discrete time sample requires frequent repetition and updating before any appreciation of the dynamics of component interaction becomes possible. Even then in a sample derived from a cell population, time-averaging of processes and events that occur in out-of-phase individuals blur the detailed complexity of single cell organization. Continuously-grown cultures of yeast can become spontaneously self-synchronized, thereby enabling resolution of far more detailed temporal structure. Continuous on-line monitoring by rapidly responding sensors (O2 electrode and membrane-inlet mass spectrometry for O2, CO2 and H2S; direct fluorimetry for NAD(P)H and flavins) gives dynamic information from time-scales of minutes to hours. Supplemented with capillary electophoresis and gas chromatography mass spectrometry and transcriptomics the predominantly oscillatory behaviour of network components becomes evident, with a 40 min cycle between a phase of increased respiration (oxidative phase) and decreased respiration (reductive phase). Highly pervasive, this ultradian clock provides a coordinating function that links mitochondrial energetics and redox balance to transcriptional regulation, mitochondrial structure and organelle remodelling, DNA duplication and cell division events. Ultimately, this leads to a global partitioning of anabolism and catabolism and the enzymes involved, mediated by a relatively simple ATP feedback loop on chromatin architecture.
35th Annual Conference of the IEEE Engineering in Medicine and Biology Society, Osaka, Japan; 07/2013
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ABSTRACT: Early embryonic rodent ventricular cells exhibit spontaneous action potential (AP), which disappears in later developmental stages. Here, we used 3 mathematical models-the Kyoto, Ten Tusscher-Panfilov, and Luo-Rudy models-to present an overview of the functional landscape of developmental changes in embryonic ventricular cells. We switched the relative current densities of 9 ionic components in the Kyoto model, and 160 of 512 representative combinations were predicted to result in regular spontaneous APs, in which the quantitative changes in Na(+) current (I Na) and funny current (I f) made large contributions to a wide range of basic cycle lengths. In all three models, the increase in inward rectifier current (I K1) before the disappearance of I f was predicted to result in abnormally high intracellular Ca(2+) concentrations. Thus, we demonstrated that the developmental changes in APs were well represented, as I Na increased before the disappearance of I f, followed by a 10-fold increase in I K1.
The Journal of Physiological Sciences 06/2013; · 1.61 Impact Factor
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Yu Hisano,
Satoshi Ota,
Kazuharu Arakawa,
Michiko Muraki,
Nobuaki Kono,
Kazuki Oshita,
Tetsushi Sakuma, Masaru Tomita,
Takashi Yamamoto,
Yasushi Okada,
Atsuo Kawahara
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ABSTRACT: Artificially designed nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can induce a targeted DNA double-strand break at the specific target genomic locus, leading to the frameshift-mediated gene disruption. However, the assays for their activity on the endogenous genomic loci remain limited. Herein, we describe a versatile modified lacZ assay to detect frameshifts in the nuclease target site. Short fragments of the genome DNA at the target or putative off-target loci were amplified from the genomic DNA of TALEN-treated or control embryos, and were inserted into the lacZα sequence for the conventional blue-white selection. The frequency of the frameshifts in the fragment can be estimated from the numbers of blue and white colonies. Insertions and/or deletions were easily determined by sequencing the plasmid DNAs recovered from the positive colonies. Our technique should offer broad application to the artificial nucleases for genome editing in various types of model organisms.
Biology open. 04/2013; 2(4):363-7.
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Kenjiro Kami,
Tamaki Fujimori,
Hajime Sato,
Mutsuko Sato,
Hiroyuki Yamamoto,
Yoshiaki Ohashi,
Naoyuki Sugiyama,
Yasushi Ishihama,
Hiroko Onozuka,
Atsushi Ochiai,
Hiroyasu Esumi,
Tomoyoshi Soga, Masaru Tomita
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ABSTRACT: Metabolic microenvironment of tumor cells is influenced by oncogenic signaling and tissue-specific metabolic demands, blood supply, and enzyme expression. To elucidate tumor-specific metabolism, we compared the metabolomics of normal and tumor tissues surgically resected pairwise from nine lung and seven prostate cancer patients, using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Phosphorylation levels of enzymes involved in central carbon metabolism were also quantified. Metabolomic profiles of lung and prostate tissues comprised 114 and 86 metabolites, respectively, and the profiles not only well distinguished tumor from normal tissues, but also squamous cell carcinoma from the other tumor types in lung cancer and poorly differentiated tumors from moderately differentiated tumors in prostate cancer. Concentrations of most amino acids, especially branched-chain amino acids, were significantly higher in tumor tissues, independent of organ type, but of essential amino acids were particularly higher in poorly differentiated than moderately differentiated prostate cancers. Organ-dependent differences were prominent at the levels of glycolytic and tricarboxylic acid cycle intermediates and associated energy status. Significantly high lactate concentrations and elevated activating phosphorylation levels of phosphofructokinase and pyruvate kinase in lung tumors confirmed hyperactive glycolysis. We highlighted the potential of CE-TOFMS-based metabolomics combined with phosphorylated enzyme analysis for understanding tissue-specific tumor microenvironments, which may lead to the development of more effective and specific anticancer therapeutics.
Metabolomics 04/2013; 9(2):444-453. · 4.51 Impact Factor
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ABSTRACT: Aphid infection reduces soybean (Glycine max [L.] Merr.) yield. Consequently, cultivation of aphid-resistant strains is a promising approach to pest control, and understanding the resistance mechanism is of importance. Here, we characterized the resistance of soybeans to foxglove aphid, Aulacorthum solani Kaltenbach, at the metabolite level. First, we evaluated aphid mortality and settlement rates on the leaves of two soybean strains, 'Tohoku149' and 'Suzuyutaka', and found that the former had strong resistance soon after introduction of the aphids. The metabolomic response to aphid introduction was analyzed using capillary electrophoresis-time-of-flight mass spectrometry. We found the following three features in the profiles: (1) concentrations of citrate, amino acids, and their intermediates were intrinsically higher for Tohoku149 than Suzuyutaka, (2) concentrations of several metabolites producing secondary metabolites, such as flavonoids and alkaloids, drastically changed 6h after aphid introduction, and (3) concentrations of TCA cycle metabolites increased in Tohoku149 48h after aphid introduction. We also profiled free amino acids in aphids reared on both soybean strains and under starvation, and found that the profile of the aphids on Tohoku149 was similar to that of the starved aphids, but different to that of aphids on Suzuyutaka. These tests confirmed that aphids suck phloem sap even from Tohoku149. This study demonstrates the metabolomic profiles of both soybean strains and aphids, which will contribute to the molecular level understanding of mechanisms of soybean resistance to aphids.
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 03/2013; 925C:95-103. · 2.78 Impact Factor
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ABSTRACT: We have recently reported that eugenol exerted indiscriminate cytotoxicity towards normal oral cells and oral squamous cell carcinoma (OSCC) cell lines without induction of apoptosis markers. In order to investigate the underlying mechanisms of cytotoxicity induction, we investigated the effect of short-term treatment with eugenol on the metabolic profiles of a human OSCC cell line (HSC-2).
The viable cell number was determined by direct cell counting with a hemocytometer after trypsinization. After washing with 5% D-mannitol solution (found to retain the highest amounts of intracellular metabolites among several washing conditions), cellular metabolites were extracted with methanol with internal markers and then subjected to metabolomic analysis.
Cytotoxic concentrations of eugenol induced the reduction of ATP utilization (assessed by a significant reduction of the AMP/ATP and ADP/ATP ratio), of oxidative stress (assessed by the increase in oxidized form of glutathione, cysteine-glutathione disulfide and methionine sulfoxide), and an increase in the polyamines and glycolytic metabolites.
The metabolic changes observed in this study suggest the induction of non-apoptotic cell death by eugenol.
In vivo (Athens, Greece) 03/2013; 27(2):233-43. · 1.17 Impact Factor
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Science 02/2013; 339(6120):646. · 31.20 Impact Factor
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ABSTRACT: Pre-mRNA splicing is a complex process involving combinatorial effects of cis- and trans- elements. Here, we focused on histone modifications as typical trans- regulatory elements and performed systematic analyses of associations between splicing patterns and histone modifications by using publicly available ChIP-Seq, mRNA-Seq, and exon-array data obtained in two human cell lines. We found that several types of histone modifications including H3K36me3 were associated with the inclusion or exclusion of alternative exons. Furthermore, we observed that the levels of H3K36me3 and H3K79me1 in the cell lines were well correlated with the differences in alternative splicing patterns between the cell lines.
FEBS letters 01/2013; · 3.54 Impact Factor
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ABSTRACT: The CRP/FNR-type transcription factors (TFs) are members of a well-characterized global TF family in bacteria and have two conserved domains: the N-terminal ligand-binding domain for small molecules (e.g., cAMP, NO, or O(2)) and the C-terminal DNA-binding domain. Although the CRP/FNR-type TFs recognize very similar consensus DNA target sequences, they can regulate different sets of genes in response to environmental signals. To clarify the evolution of the CRP/FNR-type TFs throughout the bacterial kingdom, we undertook a comprehensive computational analysis of a large number of annotated CRP/FNR-type TFs and the corresponding bacterial genomes. Based on the amino acid sequence similarities among 1,455 annotated CRP/FNR-type TFs, spectral clustering classified the TFs into 12 representative groups, and stepwise clustering allowed us to propose a possible process of protein evolution. Although each cluster mainly consists of functionally distinct members (e.g., CRP, NTC, FLP, and FixK), FNR-related TFs are found in several groups and are distributed in a wide range of bacterial phyla in the sequence similarity network. This result suggests that the CRP/FNR-type TFs originated from an ancestral FNR protein, involved in nitrogen fixation. Furthermore, a phylogenetic profiling analysis showed that combinations of TFs and their target genes have fluctuated dynamically during bacterial evolution. A genome-wide analysis of TF-binding sites also suggested that the diversity of the transcriptional regulatory system was derived by the stepwise adaptation of TF-binding sites to the evolution of TFs.
Genome Biology and Evolution 01/2013; · 4.62 Impact Factor
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ABSTRACT: By the combinations of high-throughput analytical technologies in the fields of transcriptomics, proteomics, and metabolomics, we are now able to gain comprehensive and quantitative snapshots of the intracellular processes. Dynamic intracellular activities and their regulations can be elucidated by systematic observation of these multi-omics data. On the other hand, careful statistical analysis is necessary for such integration, since each of the omics layers as well as the specific analytical methodologies harbor different levels of noise and variations. Moreover, interpretation of such multitude of data requires an intuitive pathway context. Here we describe such statistical methods for the integration and comparison of multi-omics data, as well as the computational methods for pathway reconstruction, ID conversion, mapping, and visualization that play key roles for the efficient study of multi-omics information.
Methods in molecular biology (Clifton, N.J.) 01/2013; 985:459-70.
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ABSTRACT: The central dogma of molecular biology has come under scrutiny in recent years. Here, we reviewed high-throughput mRNA and protein expression data of Escherichia coli, Saccharomyces cerevisiae, and several mammalian cells. At both single cell and population scales, the statistical comparisons between the entire transcriptomes and proteomes show clear correlation structures. In contrast, the pair-wise correlations of single transcripts to proteins show nullity. These data suggest that the organizing structure guiding cellular processes is observed at omics-wide scale, and not at single molecule level. The central dogma, thus, globally emerges as an average integrated flow of cellular information.
Frontiers in Physiology. 11/2012; 3:349.
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ABSTRACT: Recently, the extremely thermophilic bacterium Thermus thermophilus HB8 has been demonstrated to harbor a circular plasmid designated by pVV8 in addition to two well-known plasmids, pTT8 and pTT27, and its entire sequence has been determined. The absence of any obvious replication initiation gene in the 81.2 kb plasmid prompted us to isolate its minimum replicon. By in vivo replication assays with fragments deleted in a stepwise manner, a minimum replicon containing a single ORF, TTHV001, was identified. A protein encoded by TTHV001 showed no amino acid sequence similarity to other function-known proteins. As the results of in vivo and in vitro experiments strongly suggested that the TTHV001 protein was involved in the replication initiation of pVV8, the protein and the gene were referred to as RepV and repV, respectively. The RepV protein binds to an inverted repeat sequence within its own repV gene and then triggers the unwinding of the DNA duplex in an A + T-rich region located just downstream from the inverted repeat. The in vivo replication assays with minimum replicon mutants in the RepV binding site or the unwinding region demonstrated that the unwinding in the region by the RepV binding was essential for pVV8 replication initiation.
Extremophiles 11/2012; · 2.94 Impact Factor
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Shoji Tanaka,
Hitoshi Taga,
Kiyoshi Maehara,
Azusa Kaneshima,
Mamoru Machino,
Hiromi Onuma,
Miku Kaneko,
Hiroshi Sakagami,
Masahiro Sugimoto,
Tomoyoshi Soga, Masaru Tomita
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ABSTRACT: Occlusal raising method (so-called 'Template therapy') has been reported to alleviate various diseases and symptoms, but the underlying mechanism is not clear. We searched the low-molecular weight metabolite(s) in the saliva, the concentration of which is significantly changed by the template therapy.
One female patient with headache underwent the template therapy for 12 days, and her total saliva was subjected to non-targeted analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS).
One hundred and thirteen substances were identified in the saliva. Glycine was the most abundant amino acid in the saliva, followed by alanine, serine and proline. After the start of the template therapy, her headache was alleviated, accompanied by a significant (p=0.042) increase of salivary concentration of glycine, as compared with total amino acids whereas that of other amino acids was not significantly changed. In the metabolomics profile, salivary concentration of large number of metabolites as compared with total metabolite concentration decreased, including N-acetylneuraminate (p=0.025) and p-hydroxyphenylacetate (p=0.039).
This pilot study demonstrated, to our knowledge for the first time, that only glycine exhibited unique changes among total metabolites, suggesting its significant role in template therapy.
In vivo (Athens, Greece) 11/2012; 26(6):1015-20. · 1.17 Impact Factor
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ABSTRACT: Escherichia coli lyses by lambda phage propagation. Circular plasmid DNA was present during E. coli lysis as an extracellular plasmid DNA (excpDNA) that was stable enough to transform coexisting competent Bacillus subtilis cells. Detailed investigations unveiled that excpDNA is transient in both quality and quantity, with stability lasting no more than several h. A survey using E. coli lambda lysogens with various genetic backgrounds demonstrated that the loss of Endonuclease I (ΔendA::kan) conferred extraordinary stability upon excpDNA for as long as 48 h. Studies on endA mutants suggested that excpDNA remained localized in cell debris, in contrast to E. coli genome DNA, which diffused into medium at an early point in lysis. Lambda lysogens constructed on endA recA mutants are presented for potential pipelines in delivery to other competent proficient microbes.
Journal of biochemistry 10/2012; · 1.95 Impact Factor
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ABSTRACT: Capillary electrophoresis coupled with time-of-flight mass spectrometry was used to explore new serum biomarkers with high sensitivity and specificity for diabetic nephropathy (DN) diagnosis, through comprehensive analysis of serum metabolites with 78 diabetic patients. Multivariate analyses were used for identification of marker candidates and development of discriminative models. Of the 289 profiled metabolites, orthogonal partial least-squares discriminant analysis identified 19 metabolites that could distinguish between DN with macroalbuminuria and diabetic patients without albuminuria. These identified metabolites included creatinine, aspartic acid, γ-butyrobetaine, citrulline, symmetric dimethylarginine (SDMA), kynurenine, azelaic acid, and galactaric acid. Significant correlations between all these metabolites and urinary albumin-to-creatinine ratios (p < 0.009, Spearman's rank test) were observed. When five metabolites (including γ-butyrobetaine, SDMA, azelaic acid and two unknowns) were selected from 19 metabolites and applied for multiple logistic regression model, AUC value for diagnosing DN was 0.927 using the whole dataset, and 0.880 in a cross-validation test. In addition, when four known metabolites (aspartic acid, SDMA, azelaic acid and galactaric acid) were applied, the resulting AUC was still high at 0.844 with the whole dataset and 0.792 with cross-validation. Combination of serum metabolomics with multivariate analyses enabled accurate discrimination of DN patients. The results suggest that capillary electrophoresis-mass spectrometry based metabolome analysis could be used for DN diagnosis.
Analytical and Bioanalytical Chemistry 09/2012; · 3.78 Impact Factor
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ABSTRACT: Lapatinib is a clinically potent kinase inhibitor for breast cancer patients due to its outstanding selectivity for epidermal growth factor receptor (EGFR) and EGFR2 (also known as HER2). However, there is only limited information about the in vivo effects of lapatinib on EGFR/HER2 and downstream signaling targets. Here, we profiled the lapatinib-induced time- and dose-dependent phosphorylation dynamics in SKBR3 breast cancer cells by means of quantitative phosphoproteomics. Among 4,953 identified phosphopeptides from 1,548 proteins, a small proportion (5-7%) was regulated at least 2-fold by 0.001-0.01 mM lapatinib. We obtained a comprehensive phosphorylation map of 21 sites on EGFR/HER2, including 9 novel sites on HER2. Among them, serine/threonine phosphosites located in a small region of HER2 (amino acid residues 1049-1083) were up-regulated by the drug, whereas all other sites were down-regulated. We show that cAMP-dependent protein kinase is involved in phosphorylation of this particular region of HER2 and regulates HER2 tyrosine kinase activity. Computational analyses of quantitative phosphoproteome data indicated for the first time that protein-protein networks related to cytoskeletal organization and transcriptional/translational regulation, such as RNP complexes (i.e., hnRNP, snRNP, telomerase, ribosome), are linked to EGFR/HER2 signaling networks. To our knowledge, this is the first report to profile the temporal response of phosphorylation dynamics to a kinase inhibitor. The results provide new insights into EGFR/HER2 regulation through region-specific phosphorylation, as well as a global view of the cellular signaling networks associated with the anti-breast cancer action of lapatinib.
Molecular & Cellular Proteomics 09/2012; · 7.40 Impact Factor
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Kenjiro Kami,
Yasunori Fujita,
Saori Igarashi,
Sayaka Koike,
Shoko Sugawara,
Satsuki Ikeda,
Naomi Sato,
Masafumi Ito,
Masashi Tanaka, Masaru Tomita,
Tomoyoshi Soga
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ABSTRACT: Pyruvate treatment was found to alleviate clinical symptoms of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome and is highly promising therapeutic. Using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), we measured time-changes of 161 intracellular and 85 medium metabolites to elucidate metabolic effects of pyruvate treatment on cybrid human 143B osteosarcoma cells harboring normal (2SA) and MELAS mutant (2SD) mitochondria. The results demonstrated dramatic and sustainable effects of pyruvate administration on the energy metabolism of 2SD cells, corroborating pyruvate as a metabolically rational treatment regimen for improving symptoms associated with MELAS and possibly other mitochondrial diseases.
Mitochondrion 08/2012; · 3.62 Impact Factor
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ABSTRACT: It is of practical interest to investigate the effect of nitrates on bacterial metabolic regulation of both fermentation and energy generation, as compared to aerobic and anaerobic growth without nitrates. Although gene level regulation has previously been studied for nitrate assimilation, it is important to understand this metabolic regulation in terms of global regulators. In the present study, therefore, we measured gene expression using DNA microarrays, intracellular metabolite concentrations using CE-TOFMS, and metabolic fluxes using the (13)C-labeling technique for wild-type E. coli and the ΔarcA (a global regulatory gene for anoxic response control, ArcA) mutant to compare the metabolic state under nitrate conditions to that under aerobic and anaerobic conditions without nitrates in continuous culture conditions at a dilution rate of 0.2 h(-1). In wild-type, although the measured metabolite concentrations changed very little among the three culture conditions, the TCA cycle and the pentose phosphate pathway fluxes were significantly different under each condition. These results suggested that the ATP production rate was 29% higher under nitrate conditions than that under anaerobic conditions, whereas the ATP production rate was 10% lower than that under aerobic conditions. The flux changes in the TCA cycle were caused by changes in control at the gene expression level. In ΔarcA mutant, the TCA cycle flux was significantly increased (4.4 times higher than that of the wild type) under nitrate conditions. Similarly, the intracellular ATP/ADP ratio increased approximately two-fold compared to that of the wild-type strain.
Molecular BioSystems 07/2012; 8(10):2593-604. · 3.53 Impact Factor
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ABSTRACT: In cancer, apoptosis or programmed cell death has been demonstrated through the tumor necrosis factor related apoptosis-inducing ligand (TRAIL) signal transduction. As a result, TRAIL-based therapies have been widely investigated to fight cancers. However, several malignant cancer types still remain resistant to TRAIL. Recently, we developed a dynamic computational model to investigate the resistance mechanisms in TRAIL-stimulated human fibrosarcoma (HT1080) cells. The macroscopic average-cell response model, based on the law of mass action and signaling flux conservation, successfully simulates the semi-quantitative temporal profiles of cell survival (IκB, JNK, p38) and apoptotic (caspase-8 and -3) molecules in wildtype and several mutants (FADD, RIP1 and TRAF2 knockdowns or KD). However, cancer populations are known to be highly heterogeneous, and various studies have demonstrated the importance of stochasticity and variability for phenotypic diversity between identical cells. Here, we extend our original model to investigate the effect of such fluctuations on TRAIL signaling response by adopting probabilities of signaling reactions through the Gillespie algorithm. Notably, when we stimulated the model 1000 times to indicate the variability of 1000 single cell responses in all 4 experimental conditions with different levels of stochasticity, we notice that TRAF2 KD produced the most variable signaling response. This variance subsequently affected the level of cellular apoptosis analysed through the cell-survival metric (CSM). Our work highlights the necessity to understand variable responses of cell signaling reactions to different levels of stochasticity. Thus, prior to the actual development of potential drug targets for killing cancer cells, the effect of stochastic variance could be investigated through dynamic models.
IEEE/ICME Int. Conf. on Complex Med. Eng.; 07/2012
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Masahiro Takada,
Masahiro Sugimoto,
Yasuhiro Naito,
Hyeong-Gon Moon,
Wonshik Han,
Dong-Young Noh,
Masahide Kondo,
Katsumasa Kuroi,
Hironobu Sasano,
Takashi Inamoto, Masaru Tomita,
Masakazu Toi
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ABSTRACT: The aim of this study was to develop a new data-mining model to predict axillary lymph node (AxLN) metastasis in primary breast cancer. To achieve this, we used a decision tree-based prediction method-the alternating decision tree (ADTree).
Clinical datasets for primary breast cancer patients who underwent sentinel lymph node biopsy or AxLN dissection without prior treatment were collected from three institutes (institute A, n = 148; institute B, n = 143; institute C, n = 174) and were used for variable selection, model training and external validation, respectively. The models were evaluated using area under the receiver operating characteristics (ROC) curve analysis to discriminate node-positive patients from node-negative patients.
The ADTree model selected 15 of 24 clinicopathological variables in the variable selection dataset. The resulting area under the ROC curve values were 0.770 [95% confidence interval (CI), 0.689-0.850] for the model training dataset and 0.772 (95% CI: 0.689-0.856) for the validation dataset, demonstrating high accuracy and generalization ability of the model. The bootstrap value of the validation dataset was 0.768 (95% CI: 0.763-0.774).
Our prediction model showed high accuracy for predicting nodal metastasis in patients with breast cancer using commonly recorded clinical variables. Therefore, our model might help oncologists in the decision-making process for primary breast cancer patients before starting treatment.
BMC Medical Informatics and Decision Making 06/2012; 12:54. · 1.48 Impact Factor
