BACKGROUND
cancer is not only complex, in that many genetic variations can contribute to malignant transformation, but also wildly heterogeneous, in that genetic mechanisms can vary between patients of same pathological type.
kidney renal clear cell carcinoma  is the eighth most common cancer and is known to be the most lethal of all the genitourinary tumours with an estimation of approximately  <dig>  new cases and approximately  <dig>  deaths annually in united states  <cit> .
this disease is known resistant to radiotherapy and chemotherapy  <cit> , and there are very few cases that have been reported to respond immunotherapy  <cit> .
if kirc can be detected in very early stages, it is potentially curable by surgical resection, while adjuvant therapies have not been proven beneficial.
the recurrence rate is not very high, although still considered not uncommon.
nevertheless, there is no curative treatment for late stage kirc.
the 2-year survival rate of patients with metastatic kirc is less than 20%  <cit> .
therefore, further investigations of the genomic alterations and underlying molecular mechanisms are essential for early diagnosis and treatment.
as cancer is a consequence of the accumulation of genetic alterations and dysregulation of pathways, identification of differentially expressed genes and pathways is important.
we aimed to develop integrative approaches to identify differentially expressed genes and pathways in combination with gene network analysis for finding effective early cancer biomarkers and drug targets.
in this study, we designed computational approaches to identify differentially expressed genes from the rna-seq data provided by the tcga data portal.
we further performed gene ontology  analysis and categorized expression patterns.
categorization of differentially expressed genes and expression patterns suggested distinct disease subtypes that are associated with distinct biological processes.
many studies have indicated that same type of cancer can have different subtypes with different genetic mechanisms and treatment responses.
bannon et.al.
discovered two distinct kirc subtypes using gene microarray expression data  <cit> , whereas four stable subtypes of kirc were detected using both mrna and mirna expression data sets  <cit> .
despite of discoveries of differentially expressed genes and genetic mutations, the knowledge of biological pathways involved in the disease is limited.
to facilitate the effective biomarker identification, we therefore further analysed pathways and gene networks related to the differentially expressed genes.
kegg  pathway analysis revealed that differentially expressed genes are significantly enriched in a number of biological pathways that are known in cancer, as well as previously unreported pathways.
this study provided new insights into the regulatory mechanisms of kirc through comprehensive differential gene expression, pathway and network analyses.
RESULTS
differentially expressed genes in kirc
analysis of  <dig> differentially expressed genes was performed using rna-seq data of  <dig> kirc patients from the tcga data portal, including both matched and unmatched samples.
matched samples have sequencing data from both normal kidney and kidney cancer tissues in the same kirc patients, whereas unmatched samples only have sequencing data from either disease or no disease tissues.
tcga data not only contain  <dig> matched kirc tumour and normal kidney tissue paired sequencing data, but also provide  <dig> unpaired tumour and  <dig> normal kidney tissue sequencing data.
we applied multidimensional scaling  on the  <dig> paired kirc samples and found that almost all tumour and normal samples were separated well from each other .
the mds result suggested that tcga data quality was acceptable.
we performed differential gene expression analysis using edger  <cit>  package.
using statistical p-values and fold changes  as selection criteria, we identified  <dig> differentially expressed genes with p <  <dig> , and |log| >  <dig> .
these differentially expressed genes not only confirmed some previous research findings but also provided new findings.
for example, tnfaip, a known tumour α-induced protein that acts as a natural brake on inflammation  <cit> , was up-regulated in kirc in the analysis.
another differentially expressed gene, slc6a <dig>  which is implicated in lung and breast cancer  <cit> , has not yet been well known in kirc.
based on the expression levels of differentially expressed genes, we performed hierarchical clustering analysis and revealed homogenous tumour and normal  clusters .
only one normal sample and one tumour sample were mis-clustered, but both samples came from the same patient .
this suggested that they are more likely mislabelled rather outliers.
the expression levels of the differential genes showed distinct patterns between tumour and normal kidney tissue samples.
we clustered both overly expressed genes and genes with diminished expression in tumour samples .
we performed go   <cit>  analysis on four groups of genes defined as over-expressed, under-expressed, weakly over-expressed and weakly under-expressed.
fisher exact test and benjamini-hochberg multiple testing corrections were performed to obtain significance with go terms.
the significant go terms associated with each group are listed in table  <dig>  the over-expressed genes are significantly associated with biological processes of defence responses and responses to environmental stimulus.
the under-expressed genes are associated with organismal physiological processes, system development and organ development.
furthermore, we performed hierarchical clustering of all samples including  <dig> unpaired kirc and  <dig> normal samples using the differentially expressed genes detected from the matched samples.
in consistence with the results of the matched sample analysis, we obtained two homogeneous clusters; only  <dig> including the mis-labelled sample out of all  <dig> samples  were mis-clustered, demonstrating that overall, the identified differentially expressed genes can effectively distinguish kirc from normal kidney tissue samples.
furthermore, we have combined the pathway and network analyses to enhance overall classification performance.
from the comprehensive analyses, the up-regulated genes were strongly associated with cellular responses to the tumour development, and the down-regulated genes implicated compromises of kidney function as result of the cancer.
kidney functional damages are common among cancer patients.
in addition to biological function association, the distinct gene groups lead to the identification of subtypes of kirc.
system development
lip metabolism
signal transmission across a synapse
secretion
biological pathways associated with kirc
we employed hypergeometric test to assess the differentially expressed genes in kegg  <cit>  pathway analysis to identify significant pathways involved in the cancer.
significantly affected pathways in kirc include taurine and hypotaurine metabolism , neuroactive ligand-receptor interaction, glycosaminoglycan biosynthesis - heparin sulphate, peroxisome proliferator-activated receptor  signalling pathway , and hepatitis c, gastric acid secretion pathway .
methylation of the genes in taurine and hypotaurine metabolism has been associated with the worst prognosis in renal cell carcinoma  <cit> .
hepatitis c pathway has been implicated in kidney tumour  <cit> .
some of those pathways are known related to cancer, for example, ppar signalling pathway has been shown related to renal cell carcinoma  <cit> .
in addition, ingenuity  pathway analysis revealed most significant networks , including network of molecular transport, hereditary disorder, metabolic disease , and network of renal and urological disease , respectively.
in the figures, nodes in red colour represent genes that are up-regulated, while notes in green colour represent genes that are down-regulated in the disease.
nodes circled in yellow colour represent genes that have been known implicated in cancer.
interestingly, most differentially expressed genes located at the periphery of the networks are more likely downstream targets rather than regulators themselves.
the central node of molecular transport, hereditary disorder, and metabolic disease network is nf-κb , which is known active in kidney carcinoma and plays roles in tumour development as well as therapeutic resistance  <cit> .
ubc , a gene associated with protein degradation, dna repair and cell cycle regulation, is located at the centre of the renal and urological disease network, and interconnects many other genes in the network as a hub protein.
ubc has been implicated in breast cancer and contributes to cancer metastasis  <cit> .
in cancer studies, we often firstly identify differentially expressed genes to help find the biomarkers for tumour early detection, and then use the results to identify upstream disease causal genes through further network analysis.
in this study, we combined the investigation of differentially expressed genes with pathway and network analyses.
combining gene expression profiles with pathway and network analyses has helped the identification of regulators for downstream genes, regardless that the expression levels were significantly altered or not.
construction of a classifier for kirc prediction
we built a predictive classifier using support vector machine  algorithm to classify unknown tissue samples.
the expression levels of differential genes were utilized as input features.
to achieve robust performance, we have used boosting with bootstrapping and aggregation algorithm and randomly selected 4/ <dig> of samples for training, while the remaining 1/ <dig> of samples were used for testing.
we repeated the process for  <dig> times.
sensitivity and specificity were used to assess the classifier performance.
as there is always a trade-off between specificity and sensitivity, we used receiver operating characteristic  curve to measure the overall performance of the classifier.
if the area under roc curve is  <dig>  or 100%, it means the classifier is perfect.
the svm-based classifier achieved average sensitivity, specificity and area under roc curve from  <dig> trials as  <dig> %,  <dig> % and  <dig> % respectively.
the performances of the classifier were summarized in table  <dig>  the results demonstrated that the classifier has achieved accurate and robust classification performance with low standard deviations.
the intelligent machine can be used successfully for categorizing disease samples with high accuracy.
discussion
for identification of differentially expressed genes, we used  <dig> paired tumour and normal kidney tissue samples.
in each paired samples, one sample was from kirc tumour tissue and the paired sample was from the pathologically normal kidney tissue from the same patient.
using paired sequencing data obtained from the same patient can reduce false positives due to genetic difference or other confounding factors that are not necessarily related to the cancer.
this is particularly advantageous in avoiding genetically natural polymorphisms among the population.
although only the matched sets of samples were used in the identification of differentially expressed genes, we have obtained highly homogenous clusters using expression levels of differentially expressed genes in clustering all samples including  <dig> unmatched kirc and  <dig> normal kidney tissue samples.
moreover, the svm-based classifier utilizing the expression levels of differentially expressed genes was shown capable of predicting unknown tissue samples with high accuracy, suggesting that the differentially expressed genes can serve as expression signatures of the disease.
analyses of gene expression profiles and differently expressed genes suggested four different subtypes of the cancer.
this was further supported by the pathway and network analyses that have revealed differentially disturbed pathways and networks influenced by the subtypes of the disease.
analyses of cancer related pathways and networks not only confirmed some of discoveries that have been already reported in literature, but also provided new findings.
amongst the significant networks that we identified, nf-κb and ubc are examples of central nodes for molecular transport, hereditary disorder, metabolic disease network and network of renal or urological disease.
interestingly, the expression levels of both nf-κb and ubc were not significantly altered in kirc, instead they highly interconnect with other genes in the network structure, suggesting their important regulatory roles in the cancer.
though they can be considered as markers of the disease, the differentially expressed gene set shall be further utilized to infer upstream disease causal genes by combining pathway and network analyses with the systems biology approaches.
this research was part of investigations of integrative systems biology approaches to identify disrupted pathways in disease development .
integrating the disease-associated networks with differentially expressed genes and pathways can lead to the identification of useful biomarkers and effective drug targets.
CONCLUSIONS
our differentially expressed genes and pathway analyses have utilized large-scale rna-seq data and have provided new insights into the molecular mechanisms in the cancer.
in the study, the expression levels of differentially expressed genes detected in the paired tumour samples were used as input features for the machine learning classifier.
we were able to identify a set of genes associated with molecular perturbations in the disease development, and obtain highly homogenous clusters.
the intelligent machine built for this study was able to achieve high accuracy in clustering and classifying the tumour samples.
indeed, pathway and network analyses based on significant genes not only confirmed pathways implicated in the disease, but also identified new roles of unreported pathways in the cancer.
combing differentially expressed genes with pathway and network analyses not only provided an unprecedented opportunity to reveal subtypes of the disease, but also better understanding of underlying molecular mechanisms related the cancer development.

 cancer is not only complex, in that many genetic variations can contribute to malignant transformation, but also wildly heterogeneous, in that genetic mechanisms can vary between patients of same pathological type. kidney renal clear cell carcinoma is the eighth most common cancer and is known to be the most lethal of all the genitourinary tumours with an estimation of approximately new cases and approximately deaths annually in united states. this disease is known resistant to radiotherapy and chemotherapy, and there are very few cases that have been reported to respond immunotherapy. if kirc can be detected in very early stages, it is potentially curable by surgical resection, while adjuvant therapies have not been proven beneficial. the recurrence rate is not very high, although still considered not uncommon. nevertheless, there is no curative treatment for late stage kirc. the 2-year survival rate of patients with metastatic kirc is less than 20%. therefore, further investigations of the genomic alterations and underlying molecular mechanisms are essential for early diagnosis and treatment. as cancer is a consequence of the accumulation of genetic alterations and dysregulation of pathways, identification of differentially expressed genes and pathways is important. we aimed to develop integrative approaches to identify differentially expressed genes and pathways in combination with gene network analysis for finding effective early cancer biomarkers and drug targets. in this study, we designed computational approaches to identify differentially expressed genes from the rna-seq data provided by the tcga data portal. we further performed gene ontology analysis and categorized expression patterns. categorization of differentially expressed genes and expression patterns suggested distinct disease subtypes that are associated with distinct biological processes. many studies have indicated that same type of cancer can have different subtypes with different genetic mechanisms and treatment responses. bannon et.al. discovered two distinct kirc subtypes using gene microarray expression data, whereas four stable subtypes of kirc were detected using both mrna and mirna expression data sets. despite of discoveries of differentially expressed genes and genetic mutations, the knowledge of biological pathways involved in the disease is limited. to facilitate the effective biomarker identification, we therefore further analysed pathways and gene networks related to the differentially expressed genes. kegg pathway analysis revealed that differentially expressed genes are significantly enriched in a number of biological pathways that are known in cancer, as well as previously unreported pathways. this study provided new insights into the regulatory mechanisms of kirc through comprehensive differential gene expression, pathway and network analyses.  differentially expressed genes in kirc analysis of differentially expressed genes was performed using rna-seq data of kirc patients from the tcga data portal, including both matched and unmatched samples. matched samples have sequencing data from both normal kidney and kidney cancer tissues in the same kirc patients, whereas unmatched samples only have sequencing data from either disease or no disease tissues. tcga data not only contain matched kirc tumour and normal kidney tissue paired sequencing data, but also provide unpaired tumour and normal kidney tissue sequencing data. we applied multidimensional scaling on the paired kirc samples and found that almost all tumour and normal samples were separated well from each other. the mds result suggested that tcga data quality was acceptable. we performed differential gene expression analysis using edger package. using statistical p-values and fold changes as selection criteria, we identified differentially expressed genes with p <, and |log| >. these differentially expressed genes not only confirmed some previous research findings but also provided new findings. for example, tnfaip, a known tumour α-induced protein that acts as a natural brake on inflammation, was up-regulated in kirc in the analysis. another differentially expressed gene, slc6a which is implicated in lung and breast cancer, has not yet been well known in kirc. based on the expression levels of differentially expressed genes, we performed hierarchical clustering analysis and revealed homogenous tumour and normal clusters. only one normal sample and one tumour sample were mis-clustered, but both samples came from the same patient. this suggested that they are more likely mislabelled rather outliers. the expression levels of the differential genes showed distinct patterns between tumour and normal kidney tissue samples. we clustered both overly expressed genes and genes with diminished expression in tumour samples. we performed go analysis on four groups of genes defined as over-expressed, under-expressed, weakly over-expressed and weakly under-expressed. fisher exact test and benjamini-hochberg multiple testing corrections were performed to obtain significance with go terms. the significant go terms associated with each group are listed in table the over-expressed genes are significantly associated with biological processes of defence responses and responses to environmental stimulus. the under-expressed genes are associated with organismal physiological processes, system development and organ development. furthermore, we performed hierarchical clustering of all samples including unpaired kirc and normal samples using the differentially expressed genes detected from the matched samples. in consistence with the results of the matched sample analysis, we obtained two homogeneous clusters; only including the mis-labelled sample out of all samples were mis-clustered, demonstrating that overall, the identified differentially expressed genes can effectively distinguish kirc from normal kidney tissue samples. furthermore, we have combined the pathway and network analyses to enhance overall classification performance. from the comprehensive analyses, the up-regulated genes were strongly associated with cellular responses to the tumour development, and the down-regulated genes implicated compromises of kidney function as result of the cancer. kidney functional damages are common among cancer patients. in addition to biological function association, the distinct gene groups lead to the identification of subtypes of kirc. system development lip metabolism signal transmission across a synapse secretion biological pathways associated with kirc we employed hypergeometric test to assess the differentially expressed genes in kegg pathway analysis to identify significant pathways involved in the cancer. significantly affected pathways in kirc include taurine and hypotaurine metabolism, neuroactive ligand-receptor interaction, glycosaminoglycan biosynthesis - heparin sulphate, peroxisome proliferator-activated receptor signalling pathway, and hepatitis c, gastric acid secretion pathway. methylation of the genes in taurine and hypotaurine metabolism has been associated with the worst prognosis in renal cell carcinoma. hepatitis c pathway has been implicated in kidney tumour. some of those pathways are known related to cancer, for example, ppar signalling pathway has been shown related to renal cell carcinoma. in addition, ingenuity pathway analysis revealed most significant networks, including network of molecular transport, hereditary disorder, metabolic disease, and network of renal and urological disease, respectively. in the figures, nodes in red colour represent genes that are up-regulated, while notes in green colour represent genes that are down-regulated in the disease. nodes circled in yellow colour represent genes that have been known implicated in cancer. interestingly, most differentially expressed genes located at the periphery of the networks are more likely downstream targets rather than regulators themselves. the central node of molecular transport, hereditary disorder, and metabolic disease network is nf-κb, which is known active in kidney carcinoma and plays roles in tumour development as well as therapeutic resistance. ubc, a gene associated with protein degradation, dna repair and cell cycle regulation, is located at the centre of the renal and urological disease network, and interconnects many other genes in the network as a hub protein. ubc has been implicated in breast cancer and contributes to cancer metastasis. in cancer studies, we often firstly identify differentially expressed genes to help find the biomarkers for tumour early detection, and then use the results to identify upstream disease causal genes through further network analysis. in this study, we combined the investigation of differentially expressed genes with pathway and network analyses. combining gene expression profiles with pathway and network analyses has helped the identification of regulators for downstream genes, regardless that the expression levels were significantly altered or not. construction of a classifier for kirc prediction we built a predictive classifier using support vector machine algorithm to classify unknown tissue samples. the expression levels of differential genes were utilized as input features. to achieve robust performance, we have used boosting with bootstrapping and aggregation algorithm and randomly selected 4/ of samples for training, while the remaining 1/ of samples were used for testing. we repeated the process for times. sensitivity and specificity were used to assess the classifier performance. as there is always a trade-off between specificity and sensitivity, we used receiver operating characteristic curve to measure the overall performance of the classifier. if the area under roc curve is or 100%, it means the classifier is perfect. the svm-based classifier achieved average sensitivity, specificity and area under roc curve from trials as %, % and % respectively. the performances of the classifier were summarized in table the results demonstrated that the classifier has achieved accurate and robust classification performance with low standard deviations. the intelligent machine can be used successfully for categorizing disease samples with high accuracy. discussion for identification of differentially expressed genes, we used paired tumour and normal kidney tissue samples. in each paired samples, one sample was from kirc tumour tissue and the paired sample was from the pathologically normal kidney tissue from the same patient. using paired sequencing data obtained from the same patient can reduce false positives due to genetic difference or other confounding factors that are not necessarily related to the cancer. this is particularly advantageous in avoiding genetically natural polymorphisms among the population. although only the matched sets of samples were used in the identification of differentially expressed genes, we have obtained highly homogenous clusters using expression levels of differentially expressed genes in clustering all samples including unmatched kirc and normal kidney tissue samples. moreover, the svm-based classifier utilizing the expression levels of differentially expressed genes was shown capable of predicting unknown tissue samples with high accuracy, suggesting that the differentially expressed genes can serve as expression signatures of the disease. analyses of gene expression profiles and differently expressed genes suggested four different subtypes of the cancer. this was further supported by the pathway and network analyses that have revealed differentially disturbed pathways and networks influenced by the subtypes of the disease. analyses of cancer related pathways and networks not only confirmed some of discoveries that have been already reported in literature, but also provided new findings. amongst the significant networks that we identified, nf-κb and ubc are examples of central nodes for molecular transport, hereditary disorder, metabolic disease network and network of renal or urological disease. interestingly, the expression levels of both nf-κb and ubc were not significantly altered in kirc, instead they highly interconnect with other genes in the network structure, suggesting their important regulatory roles in the cancer. though they can be considered as markers of the disease, the differentially expressed gene set shall be further utilized to infer upstream disease causal genes by combining pathway and network analyses with the systems biology approaches. this research was part of investigations of integrative systems biology approaches to identify disrupted pathways in disease development. integrating the disease-associated networks with differentially expressed genes and pathways can lead to the identification of useful biomarkers and effective drug targets.  our differentially expressed genes and pathway analyses have utilized large-scale rna-seq data and have provided new insights into the molecular mechanisms in the cancer. in the study, the expression levels of differentially expressed genes detected in the paired tumour samples were used as input features for the machine learning classifier. we were able to identify a set of genes associated with molecular perturbations in the disease development, and obtain highly homogenous clusters. the intelligent machine built for this study was able to achieve high accuracy in clustering and classifying the tumour samples. indeed, pathway and network analyses based on significant genes not only confirmed pathways implicated in the disease, but also identified new roles of unreported pathways in the cancer. combing differentially expressed genes with pathway and network analyses not only provided an unprecedented opportunity to reveal subtypes of the disease, but also better understanding of underlying molecular mechanisms related the cancer development. 
kidney renal clear cell carcinoma is one of fatal genitourinary diseases and accounts for most malignant kidney tumours. our study identified a set of differentially expressed genes and pathways in kidney renal clear cell carcinoma, and represents a comprehensive computational approach to analysis large-scale next-generation sequencing data. kidney renal clear cell carcinomatcgarna-seqdifferentially expressed genespathwaysgene network analysismachine learning classifier21- july international conference on bioinformatics and computational biology las vegas, nv, usa combining differentially expressed genes with pathway and network analyses using intelligent computational approaches provide an unprecedented opportunity to identify upstream disease causal genes and effective drug targets. like many types of cancers, there is no curative treatment for metastatic kirc. the study not only confirmed a number of identified differentially expressed genes in literature reports, but also provided new findings. based on these findings, we built a support vector machine based supervised-learning classifier to predict unknown samples, and the classifier achieved high accuracy and robust classification results. identification of distinctly expressed genes and altered pathways are important in effective biomarker identification for early cancer diagnosis and treatment planning. differentially expressed genes were obtained from the rna-seq data. we combined differentially expressed genes, pathways and network analyses to gain new insights into the underlying molecular mechanisms of the disease development. using advanced sequencing technologies, the cancer genome atlas project of nih/nci-nhgri has produced large-scale sequencing data, which provide unprecedented opportunities to reveal new molecular mechanisms of cancer. followed by the experimental design for obtaining significant genes and pathways, comprehensive analysis of kirc patients' sequencing data provided by tcga was performed. furthermore, this study can provide a viable alternative in identifying effective drug targets. pathway and network analyses were performed. the pathway and network analyses suggested that information from distinctly expressed genes can be utilized in the identification of aberrant upstream regulators. the network analysis leads to the identification of significantly disrupted pathways and associated genes involved in the disease development.
 kidney renal clear cell carcinoma is one of fatal genitourinary diseases and accounts for most malignant kidney tumours. kirc has been shown resistance to radiotherapy and chemotherapy. like many types of cancers, there is no curative treatment for metastatic kirc. using advanced sequencing technologies, the cancer genome atlas project of nih/nci-nhgri has produced large-scale sequencing data, which provide unprecedented opportunities to reveal new molecular mechanisms of cancer. we combined differentially expressed genes, pathways and network analyses to gain new insights into the underlying molecular mechanisms of the disease development.  followed by the experimental design for obtaining significant genes and pathways, comprehensive analysis of kirc patients' sequencing data provided by tcga was performed. differentially expressed genes were obtained from the rna-seq data. pathway and network analyses were performed. we identified differentially expressed genes with significant p-value and large fold changes | > 5). the study not only confirmed a number of identified differentially expressed genes in literature reports, but also provided new findings. we performed hierarchical clustering analysis utilizing the whole genome-wide gene expressions and differentially expressed genes that were identified in this study. we revealed distinct groups of differentially expressed genes that can aid to the identification of subtypes of the cancer. the hierarchical clustering analysis based on gene expression profile and differentially expressed genes suggested four subtypes of the cancer. we found enriched distinct gene ontology terms associated with these groups of genes. based on these findings, we built a support vector machine based supervised-learning classifier to predict unknown samples, and the classifier achieved high accuracy and robust classification results. in addition, we identified a number of pathways that were significantly influenced by the disease. we found that some of the identified pathways have been implicated in cancers from literatures, while others have not been reported in the cancer before. the network analysis leads to the identification of significantly disrupted pathways and associated genes involved in the disease development. furthermore, this study can provide a viable alternative in identifying effective drug targets.  our study identified a set of differentially expressed genes and pathways in kidney renal clear cell carcinoma, and represents a comprehensive computational approach to analysis large-scale next-generation sequencing data. the pathway and network analyses suggested that information from distinctly expressed genes can be utilized in the identification of aberrant upstream regulators. identification of distinctly expressed genes and altered pathways are important in effective biomarker identification for early cancer diagnosis and treatment planning. combining differentially expressed genes with pathway and network analyses using intelligent computational approaches provide an unprecedented opportunity to identify upstream disease causal genes and effective drug targets. kidney renal clear cell carcinomatcgarna-seqdifferentially expressed genespathwaysgene network analysismachine learning classifier21- july international conference on bioinformatics and computational biology las vegas, nv, usa 