ARL14 as a Prognostic Biomarker in Non-Small Cell Lung Cancer

Figures & data

Table 1 Correlation Between ARL14 Expression and Clinicopathological Characteristics in LUAD and LUSC

Clinical Characteristics	Expression of ARL14 in LUAD		P	Expression of ARL14 in LUSC		P
	Low	High		Low	High
Number	267	268		251	251
Age (years)			0.596			0.003
≤65	132 (25.6%)	123 (23.8%)		111 (22.5%)	80 (16.2%)
>65	128 (24.8%)	133 (25.8%)		133 (27%)	169 (34.3%)
Gender			0.280			0.416
Female	136 (25.4%)	150 (28%)		70 (13.9%)	61 (12.2%)
Male	131 (24.5%)	118 (22.1%)		181 (36.1%)	190 (37.8%)
Race			0.358			0.135
Asian	3 (0.6%)	4 (0.9%)		3 (0.8%)	6 (1.5%)
Black or African American	32 (6.8%)	23 (4.9%)		10 (2.6%)	20 (5.1%)
White	196 (41.9%)	210 (44.9%)		176(45.2%)	174 (44.7%)
Smoker			0.121			0.797
No	31 (6%)	44 (8.4%)		10 (2%)	8 (1.6%)
Yes	231 (44.3%)	215 (41.3%)		234 (47.8%)	238 (48.6%)
T stage			0.227			0.867
T1	85 (16%)	90 (16.9%)		60 (12%)	54 (10.8%)
T2	154 (28.9%)	135 (25.4%)		146 (29.1%)	148 (29.5%)
T3	19 (3.6%)	30 (5.6%)		33 (6.6%)	38 (7.6%)
T4	8 (1.5%)	11 (2.1%)		12 (2.4%)	11 (2.2%)
N stage			0.385			0.009
N0	181 (34.9%)	167 (32.2%)		146 (29.4%)	174 (35.1%)
N1	45 (8.7%)	50 (9.6%)		80 (16.1%)	51 (10.3%)
N2	31 (6%)	43 (8.3%)		22 (4.4%)	18 (3.6%)
N3	1 (0.2%)	1 (0.2%)		1 (0.2%)	4 (0.8%)
M stage			0.979			0.273
M0	179 (46.4%)	182 (47.2%)		213 (50.8%)	199 (47.5%)
M1	13 (3.4%)	12 (3.1%)		2 (0.5%)	5 (1.2%)
Pathologic stage			0.314			0.330
I	155 (29.4%)	139 (26.4%)		115(23.1%)	130 (26.1%)
II	58 (11%)	65 (12.3%)		86 (17.3%)	76 (15.3%)
III	35 (6.6%)	49 (9.3%)		46 (9.2%)	38 (7.6%)
IV	13 (2.5%)	13 (2.5%)		2 (0.4%)	5 (1%)
Residual tumor			0.017			0.708
R0	178 (47.8%)	177 (47.6%)		205 (49.4%)	194 (46.7%)
R1	10 (2.7%)	3 (0.8%)		6 (1.4%)	6 (1.4%)
R2	0 (0%)	4 (1.1%)		1 (0.2%)	3 (0.7%)

Table 2 Correlation Between ARL14 Expression and Clinicopathological Characteristics in 120 Patients with NSCLC

Characteristics	All Patients (n=120)	ARL14 Expression		P
		Low (n=74)	High (n=46)
Age (years)				0.222
<55	44	24	20
≥55	76	50	26
Gender				0.288
Male	80	52	28
Female	40	22	18
Smoke				0.125
Never	42	22	20
Ever	78	52	26
Type of operation				0.125
Lobectomy	111	68	43
Sleeve lobectomy	2	0	2
Pneumonectomy	4	4	0
Wedge resection or segmentectomy	3	2	1
Maximum diameter of tumor				0.98
≤4cm	68	42	26
>4cm	52	32	20
Pathological type				0.253
Adenocarcinoma	56	34	22
Squamous cell carcinoma	52	30	22
Other	12	10	2
T stage				0.345
T1	51	28	23
T2	42	30	12
T3	17	11	6
T4	10	5	5
N stage				0.944
N0	88	55	33
N1	14	9	5
N2	16	9	7
N3	2	1	1
M stage				0.625
M0	116	72	44
M1	4	2	2
Pathologic stage				0.488
I	66	39	27
II	27	20	7
III	23	13	10
IV	4	2	2
Differentiation				0.011
Well	25	18	7
Moderate	50	36	14
Poor	45	20	25
Treatment after surgery				0.155
None	72	43	29
Chemotherapy	20	16	4
Targeted therapy	28	15	13

Figure 1 ARL14 expression in LUAD and LUSC. (A) ARL14 expression in LUAD tissues and normal samples. (B) ARL14 expression in LUAD tissues and paired normal samples. (C) ARL14 expression in LUSC tissues and normal samples. (D) ARL14 expression in LUSC tissues and paired normal samples.

Figure 2 Correlation between ARL14 expression and clinicopathologic features in patients with LUAD and LUSC in TCGA database (P < 0.05). (A and B) The correlation between ARL14 expression and T stage (A) and pathologic stage (B) of patients with LUAD. (C–E) The correlation between the expression of ARL14 and age (C), N stage (D) and race (E) of patients with LUSC.

Figure 3 Kaplan–Meier curve for OS in LUAD and LUSC. (A) Kaplan–Meier analysis for ARL14 in LUAD; (B–F) Subgroup analysis for T3/T4, N1/N2/N3, M0, female, and R0 in LUAD. (G) Kaplan–Meier analysis for ARL14 in LUSC; (H–M) Subgroup analysis for age ≤65 years, smoker, T1/T2, N1/N2/N3, pathologic stage II/III/IV, and R0 in LUSC.

Table 3 Correlations Between Overall Survival and Expression of ARL14 Analyzed by Univariate and Multivariate Cox Regression in LUAD and LUSC

Clinical Characteristics	Univariate Analysis		Multivariate Analysis
	Hazard Ratio (95% CI)	P	Hazard Ratio (95% CI)	P
LUAD
ARL14 (High vs Low)	1.375 (1.031–1.834)	0.03	1.496 (1.029–2.173)	0.035
T stage (T3 & T4 vs T1 & T2)	1.728 (1.229–2.431)	0.002	1.664 (1.027–2.695)	0.038
N stage (N1 & N2 & N3 vs N0)	2.321 (1.631–3.303)	<0.001	1.503 (0.625–3.616)	0.363
M stage (M1 vs M0)	2.136 (1.248–3.653)	0.006	0.937 (0.364–2.410)	0.892
Pathologic stage (Stage II & Stage III & Stage IV vs Stage I)	2.664 (1.960–3.621)	<0.001	1.802 (0.761–4.268)	0.18
Residual tumor (R1 & R2 vs R0)	3.879 (2.169–6.936)	<0.001	3.317 (1.558–7.062)	0.002
LUSC
ARL14 (High vs Low)	1.373 (1.046–1.802)	0.022	1.341 (1.017–1.770)	0.038
T stage (T3 & T4 vs T1 & T2)	1.658 (1.200–2.291)	0.002	1.457 (0.994–2.135)	0.054
Pathologic stage (Stage II & Stage III & Stage IV vs Stage I)	1.321 (1.006–1.735)	0.046	1.170 (0.851–1.609)	0.334

Figure 4 Nomogram for predicting probability of patients with 1-, 3- and 5-year OS. (A) Nomogram for predicting probability of patients with LUAD in TCGA database. (B) Nomogram for predicting probability of patients with LUSC in TCGA database.

Figure 5 IHC staining and survival analysis of ARL14 in human NSCLC. (A) ARL14 expression of IHC on 120 human NSCLC tissues and 120 adjacent normal lung tissues. (B) Immunohistochemical staining for ARL14 expression in adjacent normal lung tissues (×400). (C) Immunohistochemical staining for low ARL14 expression in NSCLC tissues (×400). (D) Immunohistochemical staining for high ARL14 expression in NSCLC tissues (×400). (E) Kaplan–Meier plots of OS according to ARL14 expression. (F) Kaplan–Meier plots of PFS according to ARL14 expression.

Table 4 Univariate and Multivariate Analyses of OS and PFS in 120 Patients with NSCLC

Clinical Characteristics	OS				PFS
	Univariate Analysis		Multivariate Analysis		Univariate Analysis		Multivariate Analysis
	Hazard Ratio (95% CI)	P	Hazard Ratio (95% CI)	P	Hazard Ratio (95% CI)	P	Hazard Ratio (95% CI)	P
Age (years)	2.388 (1.427–3.996)	0.001	1.651 (0.961–2.835)	0.069	2.529 (1.509–4.24)	<0.001	1.929 (1.127–3.302)	0.017
<55
≥55
Gender	1.245 (0.734–2.112)	0.417	–	–	1.354 (0.798–2.297)	0.262	–	–
Male
Female
Smoke	1.285 (0.761–2.171)	0.349	–	–	1.357 (0.803–2.293)	0.253	–	–
Never
Ever
Type of operation	1.36 (0.691–2.676)	0.374	–	–	1.329 (0.688–2.566)	0.397	–	–
Lobectomy
Sleeve lobectomy
Pneumonectomy
Wedge resection or segmentectomy
Maximum diameter of tumor	1.634 (0.979–2.727)	0.060	–	–	1.579 (0.947–2.633)	0.080	–	–
≤4cm
>4cm
Pathological type	1.149 (0.788–1.675)	0.469	–	–	1.174 (0.803–1.717)	0.407	–	–
Adenocarcinoma
Squamous cell carcinoma
Other
T stage	1.148 (0.873–1.510)	0.323	–	–	1.103 (0.841–1.447)	0.478	–	–
T1
T2
T3
T4
N stage	1.319 (0.977–1.780)	0.070	–	–	1.348 (1.000–1.817)	0.050	–	–
N0
N1
N2
N3
M stage	5.611 (1.952–16.126)	0.001	2.350 (0.628–8.796)	0.205	4.177 (1.458–11.973)	0.008	1.361 (0.375–4.934)	0.639
M0
M1
Pathologic stage	1.419 (1.064–1.894)	0.017	1.307 (0.933–1.830)	0.120	1.403 (1.054–1.866)	0.020	1.325 (0.948–1.852)	0.100
I
II
III
IV
Differentiation	0.558 (0.382–0.814)	0.002	0.861 (0.578–1.283)	0.462	0.574 (0.394–0.835)	0.004	0.874 (0.588–1.299)	0.506
Well
Moderate
Poor
Treatment after surgery	0.897 (0.669–1.202)	0.466	–	–	0.899 (0.672–1.202)	0.472	–	–
None
Chemotherapy
Targeted therapy
ARL14 expression	0.213 (0.125–0.364)	<0.001	0.248 (0.140–0.440)	< 0.001	0.230 (0.135–0.392)	<0.001	0.279 (0.158–0.491)	<0.001
Low
High

Figure 6 Correlation analysis of ARL14 in LUAD and LUSC. (A) Top 50 genes most positively associated with ARL14 in patients with LUAD were shown in a heatmap. (B) Top 50 genes most negatively associated with ARL14 in patients with LUAD were shown in a heatmap. (C) Top 50 genes most positively associated with ARL14 in patients with LUSC were shown in a heatmap. (D) Top 50 genes most negatively associated with ARL14 in patients with LUSC were shown in a heatmap. *p< 0.05; **p<0.001.

Figure 7 Enrichment analysis of ARL14 in LUAD and LUSC. (A–C) Significant Gene Ontology terms of the top 1200 genes most associated with ARL14 in LUAD, including BP, MF, and CC. (D) Significant KEGG pathways of the top 1200 genes most associated with ARL14 in LUAD. (E–G) Significant Gene Ontology terms of the top 1200 genes most associated with ARL14 in LUSC, including BP, MF, and CC. (H) Significant KEGG pathways of the top 1200 genes most associated with ARL14 in LUSC.

Figure 8 Enrichment plot from the GSEA. Enrichment plots of (A) keratinization, (B) systemic lupus erythematosus, (C) formation of the cornified envelope, (D) positive epigenetic regulation of RRNA expression, and (E) HDACS deacetylate histones in LUAD cases with high ARL14 expression. Gene set enrichment plots of (F) neutrophil degranulation, (G) signaling by interleukins, (H) NABA secreted factors, (I) class A1 rhodopsin like receptors, and (J) extracellular matrix organization in LUAD or LUSC cases with high ARL14 expression.

Abbreviations: NES, normalized NS; FDR, false discovery rate.

Figure 9 Correlation between immune cell infiltration and ARL14 in LUAD and LUSC. The forest plots showed the association between ARL14 expression and 24 immune cells in LUAD (A) and LUSC (E), respectively. (B) The relationship between ARL14 expression and TFH in LUAD. (C) The relationship between ARL14 expression and aDC in LUAD. (D) The relationship between ARL14 expression and Th1 in LUAD. (F) The relationship between ARL14 expression and TFH in LUSC. (G) The relationship between ARL14 expression and neutrophils in LUSC. (H) The relationship between ARL14 expression and Th1 in LUSC.