A 16-gene expression signature to distinguish stage I from stage II lung squamous carcinoma.

The present study aimed to perform screening of a gene signature for the discrimination and prognostic prediction of stage I and II lung squamous carcinoma. A microarray meta‑analysis was performed to identify differentially expressed genes (DEGs) between stage I and II lung squamous carcinoma samples in seven microarray datasets collected from the Gene Expression Omnibus database via the MetaQC and MetaDE package in R. The important DEGs were selected according to the betweenness centrality value of the protein‑protein interaction (PPI) network. Support vector machine (SVM) analysis was performed to screen the feature genes for discrimination and prognosis. One independent dataset downloaded from The Cancer Genome Atlas was used to validate the reliability. Pathway enrichment analysis was also performed for the feature genes. A total of 924 DEGs were identified to construct a PPI network consisting of 392 nodes and 686 edges. The top 100 of the 392 nodes were selected as crucial genes to construct an SVM classifier, and a 16‑gene signature (caveolin 1, eukaryotic translation elongation factor 1γ, casein kinase 2α1, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation η, tyrosine 3‑monooxygenase/tryptophan 5‑monooxygenase activation θ, pleiotrophin, insulin receptor, insulin receptor substrate 1, 3‑phosphoinositide‑dependent protein kinase‑1, specificity protein 1, COP9 signalosome subunit 6, N‑myc downstream regulated gene 1, retinoid X receptor α, heat shock protein 90α A1, karyopherin subunit β1 and erythrocyte membrane protein band 4.1) with high discrimination accuracy was identified. This 16‑gene signature had significant prognostic value, and patients with stage II lung squamous carcinoma exhibited shorter survival rates, compared with those with stage I disease. Seven DEGs of the 16-gene signature were significantly involved in the phosphoinositide 3‑kinase‑Akt signaling pathway. The 16‑gene signature identified in the present study may be useful for stratifying the patients with stage I or II lung squamous carcinoma and predicting prognosis.

Introduction

Lung cancer is the leading cause of cancer-associated mortality worldwide, with an estimated 224,390 newly diagnosed cases and a mortality rate of 158,080 in the United States in 2016 (1). The prognosis of patients with lung cancer has been demonstrated to be associated with stage at diagnosis, and the survival rate for patients at early stages I and II is generally substantially higher, compared with those at advanced stages III and IV (2,3). Therefore, several studies have been performed to attempt to identify diagnostic biomarkers, which may assist in distinguishing early-stage from advanced-stage lung cancer in order to plan suitable therapeutic strategies for improving prognosis (4,5). The discrimination of stage I and II lung cancer is also clinically meaningful as patients with stage I or II have different sucseptibilities to the development of distant metastases and chance of succumbing to mortality within 5 years (2,3). However, few studies have been performed on the further discrimination of stage I and II lung cancer.

Lung cancer consists of two major types, and non-small cell lung cancer (NSCLC) is the most common form, accounting for ~85% of lung cancer cases. NSCLC can be further divided into three subtypes: Adenocarcinoma (40%), squamous carcinoma (40%) and large-cell cancer (20%). Currently, the majority of studies on the identification of gene signatures for staging and prognosis have been focused on NSCLC (6–8) and its adenocarcinoma subtype (9,10). However, there have been fewer reports specific for lung squamous carcinoma (11,12).

The aim of the present study was to perform screening of a gene expression signature for the discrimination and predicting the prognosis of stage I and II lung squamous carcinoma by performing a meta-analysis of microarray datasets. The results of the meta-analysis may increase the statistical power and improve the reliability of investigations, compared with single study analysis (13).

Materials and methods

Microarray data collection and preprocessing

The Gene Expression Omnibus (GEO) database at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/geo/) was used to retrieve expression profile datasets of human lung squamous carcinoma. The inclusion criteria were as follows: i) mRNA expression profiles; ii) lung cancer tissues of patients with lung squamous carcinoma; iii) specific pathological stage I and II; and iv) number of samples >50. Finally, seven microarray datasets were obtained, including GSE43580, GSE50081, GSE42127, GSE41271, GSE17710, GSE68793 and GSE33532 (Table I).

Table I

General information of included expression profiles.

GEO accession	Chip	Probe number	Sample size	Stage I	Stage II
GSE43580	HG-U133_Plus_2	54,676	73	34	39
GSE50081	HG-U133_Plus_2	54,676	43	27	16
GSE42127	Illumina HumanWG-6	48,803	33	23	10
GSE41271	Illumina HumanWG-6	48,803	48	31	17
GSE17710	Agilent-UNC	33,421	53	34	19
GSE68793	HT_HG-U133A	22,277	101	70	31
GSE33532	HG-U133_Plus_2	25,906	16	8	8

GEO, Gene Expression Omnibus.

The raw data (CEL files) of GSE43580, GSE50081, GSE68793 and GSE33532 were downloaded from the Affymetrix Human Genome U133 Plus 2.0 Array platform (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL570). Background correction (MAS 5.0), normalization (quantile), log2-transformation and median polish summarization were then performed by using the robust multichip average algorithm (14) implemented in the affy package from R/Bioconductor (http://www.bioconductor.org/packages/release/bioc/html/affy.html). The expression value data (TXT files) of the other three microarray datasets (GSE42127, GSE41271 and GSE17710) were directly downloaded from their corresponding platforms. The probe IDs were converted into gene symbols according to the annotation files. The probes without gene symbols were filtered and the average expression value of the probes was calculated as the expression value for the gene with multiple probes, followed by data normalization using the Linear Models for Microarray Analysis package (15) in R (http://www.R-project.org).

Meta-analysis of multiple microarray datasetes for screening DEGs

In order to obtain more reliable genes associated with the stage of lung squamous carcinoma, a meta-analysis of the above seven microarray datasets was performed as previously described (16). The MetaQC package in R was used to eliminate the bias among datasets downloaded from different platforms by filtering out low-quality studies. This procedure provided six quantitative quality control measures, including the internal quality control (IQC, homogeneity of coexpression structure across studies), external quality control (EQC, consistency of coexpression information with pathway database), accuracy quality control (AQCg and AQCp, accuracy of differentially expressed gene and enriched pathway detection, respectively), and consistency quality control (CQCg and CQCp, consistency of differentially expressed gene and enriched pathway ranking, respectively) (17). In addition, the principal component analysis and standardized mean rank summary score were applied to assist in the identification of problematic studies (17). The datasets of high quality were screened for identification of DEGs using the MetaDE. ES algorithm of MetaDE package in R (18). The thresholds of homogeneity were set as tau2=0 and Qpval >0.05. A false discovery rate (FDR) of <0.05 was considered as the cut-off criterion for identifying DEGs between stage I and II of lung squamous carcinoma samples.

Protein-protein interaction (PPI) network-based prioritization of DEGs

Cytoscape is software for biological network visualization and analysis (19), in which the PPIs are downloaded from acknowledged Human Protein Reference Database (http://www.hprd.org) (20). The PPI network for the identified DEGs was constructed using Cytoscape software 3.5.0 to investigate the DEGs crucial for the development of lung squamous carcinoma. In addition, the non-differentially expressed genes, which interacted with at least 10 DEGs were included in this PPI network. The signaling pathways enrichment analysis was performed for the genes in the constructed PPI network with the criterion of P<0.05.

Betweenness centrality (BC) is one of the important topological characteristics of the PPI network for determining the highly connected nodes (hubs). BC is defined as the fraction of shortest paths, which pass through a node (v), and nodes with BC values closer to 1 have a higher hub degree. BC can be calculated using the following equation:

Where σst is the total number of shortest paths from node s to node t, and σst (v) is the number of those paths that pass through v.

Support vector machine (SVM) modeling and classification accuracy evaluation

Using the top 100 genes ranked by the BC value, a supervised SVM classifier was constructed with the e1071 package for R under the default parameters (radial basis function kernel, γ=0.5; cost=4; cross=10-fold) (21). Following screening of the optimal combination of genes, which completely distinguished between stage I or II lung squamous carcinoma samples by utilizing the dataset with the maximum sample size as the training sets, its predictive accuracy was computed using the other datasets as the test sets. The effects of classification were evaluated based on five parameters, including the accuracy, sensitivity, specificity, positive predictive value, negative predictive value, and area under the receiver operating curve (AUC).

Prognostic validation analysis

To further demonstrate the classification reliability and prognostic potential of the optimal combination of genes, an independent dataset, comprising 67 stage I lung squamous carcinoma samples, 27 stage II lung squamous carcinoma samples and survival rates was downloaded from The Cancer Genome Atlas (TCGA; http://tcgadata.nci.nih.gov/tcga/; TCGA_LUSC_exp_u133a level 3). Cox regression analysis was then performed for this independent dataset using the R survival package (22).

Pathway enrichment analysis

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (http://www.kegg.jp/) was performed for the gene signature with a two-tailed Fisher's exact test based on the hypergeometric distribution. The P-value was adjusted for multiple testing using the Benjamini and Hochberg approach (23), and adjusted P<0.05 was set as the threshold.

Results

Identification of DEGs with microarray meta-analysis

Among the seven microarray datasets downloaded from the GEO, five datasets (GSE43580, GSE50081, GSE42127, GSE41271 and GSE68793) were included in the meta-analysis for DEGs following MetaQC analysis. The other two datasets (GSE17710 and GSE33532) were excluded due to small sample size and lower quantitative quality control scores (Table II and Fig. 1). A total of 964 DEGs between the stage I and II lung squamous carcinoma samples were identified using the MetaDE.ES algorithm under the cut-off criterion of FDR <0.05. The top 10 DEGs ranked by the FDR value are listed in the Table III.

Table II

Results of QC measures and SMRs.

Study	IQC	EQC	CQCg	CQCp	AQCg	AQCp	SMR
GSE43580	4.52	2.00	307.65	133.86	32.71	82.01	2.17
GSE42127	4.96	1.70	307.65	101.51	30.42	29.94	2.58
GSE50081	5.42	2.00	307.65	60.02	29.49	29.18	3.33
GSE41271	4.58	2.00	19.10	90.31	8.24	58.61	3.25
GSE17710	0.23	0.37	0.09	1.89	0.05	0.36	6.83
GSE68793	5.92	1.10	276.14	35.54	19.03	10.66	3.92
GSE33532	1.79	5.00	0.59	1.69	0.52	3.21	5.67

QC, quality control; IQC, internal; EQC, external; CQCg, consistency QC of differentially expressed gene; CQCp, CQC of enriched pathway; AQCg, accuracy QC of differentially expressed gene; AQCp, AQC of enriched pathway; SMR, standardized mean rank.

Figure 1

Principal component analysis biplots of QC measures in seven microarray datasets. The numbers in circles represent the overall rank of a study based on the standardized mean rank score. Smaller numbers correspond to higher quality studies. QC, quality control; CQCg, consistency QC of differentially expressed gene; CQCp, CQC of enriched pathway; AQCg, accuracy QC of differentially expressed gene; AQCp, AQC of enriched pathway; IQC, internal QC; EQC, external QC.

Table III

Top 10 differentially expressed genes screened using microarray meta-analysis.

Symbol	P-value	FDR	tau2	Q-value	QP-value
PLP2	1.30E-10	1.59E-09	0	3.67E+00	4.52E-01
EEF1G	1.33E-10	1.62E-09	0	1.33E+00	8.56E-01
RPL19	1.33E-10	1.62E-09	0	3.62E+00	4.60E-01
C7	1.35E-10	1.64E-09	0	2.90E+00	5.75E-01
MDH2	1.36E-10	1.66E-09	0	8.43E-01	9.33E-01
IGFBP6	1.37E-10	1.67E-09	0	2.57E+00	6.33E-01
OR12D2	1.39E-10	1.69E-09	0	2.12E+00	7.14E-01
SERPINI1	1.44E-10	1.74E-09	0	3.88E+00	4.23E-01
TNS4	1.51E-10	1.81E-09	0	2.30E+00	6.82E-01
KDR	1.54E-10	1.84E-09	0	1.34E+00	8.54E-01

FDR, false discovery rate.

Prioritization of DEGs by PPI network analysis

The PPI network was constructed by mapping the 964 DEGs into PPI data, consisting of 392 nodes (proteins) and 686 edges (interactions). A total of five pathways were significantly enriched for genes in the constructed PPI network, including proteasome, Wnt signaling pathway, ribosome, apoptosis and focal adhesion (Fig. 2). The top 100 genes ranked by BC value were then selected as important genes in order to construct an SVM classifier for distinguishing between stage I and II lung squamous carcinoma samples (Table IV). Using the GSE68793 dataset as a training set, the classification accuracy was found to gradually improve with the increase in the number of DEGs. When the 16 DEGs, comprising 15 upregulated DEGs in stage II lung squamous carcinoma, including caveolin 1, (CAV1), eukaryotic translation elongation factor 1γ (EEF1G), casein kinase 2α1 (CSNK2A1), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation η (YWHAH), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation θ (YWHAQ), pleiotrophin (PTN), insulin receptor (INSR), insulin receptor substrate 1 (IRS1), 3-phosphoinositide-dependent protein kinase-1 (PDPK1), specificity protein 1 (Sp1), COP9 signalosome subunit 6 (COPS6), N-myc downstream regulated gene 1 (NDRG1), retinoid X receptor α (RXRA), heat shock protein 90α A1 (HSP90AA1) and karyopherin subunit β1 (KPNB1) and one downregulated DEG in stage II lung squamous carcinoma, namely erythrocyte membrane protein band 4.1 (EPB41) were included in the top 40 crucial genes, 100% accuracy was achieved (Fig. 3). It was also found that high accuracy was obtained using the other four datasets (GSE43580: 94.5%, 69/73; GSE50081: 97.7%, 42/43; GSE41271: 97.9%, 47/48; GSE42127: 100%, 33/33) as test sets (Table V).

Figure. 2

Protein-protein interaction network of critical genes. A total of 392 nodes and 686 edges were included. The nodes represented the differentially expressed genes. Purple represents upregulated genes in stage II; green represents downregulated genes in stage II; light pink represents non-differentially expressed genes.

Table IV

Top 10 genes with highest BC scores.

Gene	BC score	Expression	P-value	FDR	tau2	Q-value	QP-value
PORCN	0.6667	1	2.45E-02	3.26E-01	0	5.66E-01	9.67E-01
WNT1	0.5000	1	1.44E-02	2.90E-01	0	2.25E+00	6.89E-01
WNT4	0.5000	1	1.50E-02	2.95E-01	0	3.03E+00	5.54E-01
CAV1	0.1587	1	2.00E-03	1.45E-01	0	7.05E-01	9.51E-01
CSNK2A1	0.1452	1	4.00E-02	3.74E-01	0	2.39E+00	6.64E-01
HSP90AA1	0.1436	1	1.14E-02	2.66E-01	0	2.09E+00	7.20E-01
YWHAG	0.1087	–	9.95E-02	5.04E-01	7.70E-02	7.86E+00	9.70E-02
TP53	0.0993	–	8.75E-01	9.68E-01	2.15E-01	1.47E+01	5.40E-03
SP1	0.0966	1	1.70E-03	1.40E-01	0	3.41E+00	4.92E-01
YWHAQ	0.0861	1	1.01E-02	2.54E-01	0	2.08E+00	7.21E-01

Expression: 1, expression higher in stage II than stage I; –, not differentially expressed; BC, betweenness centrality; FDR, false discovery rate.

Figure 3

SVM modeling and classification accuracy evaluation. (A) Accurate rate using crucial genes (screened by the betweenness centrality score) as classifier; 100% accuracy was achieved in the top 40. (B) Visualization of classification result using the 16-gene signature as an SVM classifier and GSE68793 as a training dataset (orange, stage II sample; blue, stage I sample). (C) Hierarchical clustering analysis indicated samples of the same type, which were clustered using the 16-gene signature (yellow, higher expression; blue, lower expression). SVM, support vector machine.

Table V

Support vector machine classifier confirmation in test datasets.

Dataset	Sample size (n)	Correct rate, %	Sensitivity	Specificity	PPV	NPV	AUROC
GSE43580	73	94.5	0.941	0.949	0.941	0.949	0.989
GSE50081	43	97.7	1.000	0.936	0.964	1.000	0.991
GSE41271	48	97.9	1.000	0.941	0.969	1.000	0.994
GSE42127	33	100.0	1.000	1.000	1.000	1.000	1.000

PPV, positive predictive value; NPV, negative predictive value; AUROC, area under receiver operating curve.

The present study also examined the prognostic value of the 16 DEGs by performing survival analysis with the GSE42127, GSE50081 and GSE41271 (Fig. 4) datasets. The results indicated that patients with stage I lung squamous carcinoma exhibited significantly higher survival rates, compared with the patients with stage II lung squamous carcinoma in GSE50081 (P=0.000138) using these 16 genes (Fig. 4B). Therefore, this classification model may also be effective for clinical prognosis and treatment guidance.

Figure 4

Survival analysis. Survival analysis for dataset (A) GSE42171 and (B) GSE50081.

To further confirm the classification reliability of the above selected 16 genes, another independent dataset was downloaded from TCGA database. The results indicated that these 16 genes provided a predictive accuracy of 100% (67/67) for stage I lung squamous carcinoma samples, a predictive accuracy of 92.59% (25/27) for stage II lung squamous carcinoma, an overall predictive accuracy of 97.87% (92/94) and an AUC of 0.996 (Fig. 5A). The survival analysis also demonstrated that the survival rates of patients with stage II lung squamous carcinoma were significantly lower, compared with those of patients with stage I lung squamous carcinoma (P=0.004967) (Fig. 5B).

Figure 5

Support vector machine classifier confirmation and survival analysis using another independent dataset. (A) Receiver operating characteristic curve. (B) Kaplan-Meier curve. AUC, area under curve.

Only one significant KEGG pathway, the phosphoinositide 3-kinase (PI3K)-Akt signaling pathway (adjusted P=0.020268797), was enriched for seven DEGs (YWHAQ, IRS1, PDPK1, HSP90AA1, INSR, RXRA and YWHAH). No pathway was significantly enriched for the other nine DEGs.

Discussion

The present study identified 16 DEGs, which can be used as a gene signature to distinguish stage I from stage II lung squamous carcinoma and are of value for predicting prognosis. There were 15 upregulated DEGs (CAV1, EEF1G, CSNK2A1, YWHAH, YWHAQ, PTN, INSR, IRS1, PDPK1, Sp1, COPS6, NDRG1, RXRA, HSP90AA1 and KPNB1) and one downregulated DEG (EPB41) in stage II lung squamous carcinoma.

CAV1, encoding a 22–24 kDa structural protein component of caveolae in the plasma membrane, has been demonstrated to be upregulated in NSCLC (24). In addition, high expression of CAV1 has shown to be associated with poor prognosis in patients with NSCLC (25,26). The high expression level of EEF1G may promote the synthesis of several proteins to prevent oxidative stress and nutrient deprivation, resulting in the excessive proliferation of cells and development of cancer (27). However, direct investigations of EEF1G remain limited and further confirmation of EEF1G in lung squamous carcinoma remains essential. CSNK2A1, encoding an α catalytic subunit of protein kinase CK2, promotes tumor cell growth, adhesion and migration by activating the Janus kinase/signal transducer and activator of transcription, nuclear factor-κB, PI3K/AKT, Hsp90, Wnt and Hedgehog pathways (28). A study by O-charoenrat et al indicated that CSNK2A1 may serve as a potential predictor of poor prognosis for lung squamous carcinoma (29), which is consistent with the present study to a certain extent. PTN is a heparin-binding growth factor, with high secretion levels in NSCLC cells (30). PTN regulates cell proliferation and migration in NSCLC by activating its cell surface receptors and PI3K signaling (31,32), which may lead to a poor prognosis (33). Sp1, an oncogenic transcription factor for lung squamous carcinoma, may be involved in regulating the promoter activity of its target genes (34,35). Detecting the expression level of Sp1 is considered to be an effective approach for the diagnosis and prognosis of lung cancer (36).

COPS6, encoding one of the eight subunits of the COP9 signalosome, mediates the upregulation of E6AP by reducing E6AP poly-ubiquitination and targeting p53 for proteasome-mediated degradation, which in turn decreases the transcriptional activity of p53 target genes to induce cervical cell growth and motility (37). However, the underlying mechanism of COPS6 in lung squamous carcinoma remains to be fully elucidated. NDRG1, a member of the N-myc downstream regulated gene family, can be overexpressed in several types of lung cancer, including lung squamous cell carcinoma (38,39). The survival rate of patients negative for the nuclear expression of NDRG1 has been reported to be significantly higher, compared with that in patients who positive for NDRG1 (38). In addition, a high expression of NDRG1 may promote the progression of lung squamous cell carcinoma by upregulating vascular endothelial growth factor-A and interleukin-8/C-X-C-motif chemokine ligand 8 (38). EPB41, encoding the erythrocyte membrane protein band 4.1, has been shown to function as a tumor suppressor gene in meningioma cell lines (40). However, the role and mechanism of EPB41 in lung cancer remains to be fully elucidated and further experiments are essential.

According to the pathway enrichment analysis in the present study, seven DEGs (YWHAQ, IRS1, PDPK1, HSP90AA1, INSR, RXRA and YWHAH) among the 16 DEGs were significantly involved in the PI3K-Akt signaling pathway. Increasing evidence suggests that activation of the PI3K-Akt signaling pathway is correlated with poor prognosis in patients with NSCLC, as this pathway antagonizes apoptosis and contributes to the proliferation and metastasis of NSCLC cells (41,42). It has been reported that upregulated expression levels of HSP90AA1 and PDPK1 are significantly associated with shorter overall survival rates in patients with NSCLC (43,44). INSR can be activated by the insulin ligand to trigger the PI3K/AKT signaling pathway and enhance tumor growth, migration and angiogenesis (45,46). Previous multivariate analysis revealed patients with high expression levels of INSR had shorter overall survival rates, compared with those with low expression levels (47). A truncated version of RXR-α has been demonstrated to significantly contribute to the anchorage-independent growth of cancer cells by activation of the PI3K/AKT pathway (48). YWHAH and YWHAQ mediate oncogenic transformation by stimulating PI3K signaling, and these two genes may be correlated with a more advanced pathologic stage and lower overall survival rate (49,50). Therefore, it may be that these seven upregulated DEGs involved in the PI3K-Akt signaling pathway may also be associated with decreased survival rate in patients with stage II lung squamous carcinoma.

In conclusion, the 16-gene expression signature identified in the present study may be of value in the discrimination of patients with stage I or II lung squamous carcinoma and prediction of prognosis. Further investigations aim to analyze the association between the gene expression levels of this 16-gene signature and prognosis based on the prognosis of clinical patients.