Investigation of Diagnostic and Prognostic Value of CLEC4M of Non-Small Cell Lung Carcinoma Associated with Immune Microenvironment

Figures & data

Figure 1 The CLEC4M expression was down-regulated in NSCLC. (a) The expression level of CLEC4M in normal tissue and NSCLC (LUAD and LUSC) in GSE30219. (b) The CLEC4M expression of LUAD and LUSC versus normal tissue in GEPIA2 database. (c) The expression of CLEC4M in matched LUAD adjacent tissue. (d) The expression of CLEC4M in matched LUSC adjacent tissue. (e) Representative images of immunohistochemistry detection of LUAD and LUSC patients (magnification 200x, 400x). (g) Histochemistry staining score of lung tumor and paired adjacent normal tissue derived from clinical lung tumor samples. (h and i) Correlation between CLEC4M expression and clinicopathological characteristics of NSCLC patients in TCGA and GSE30219.

Notes: *p value <0.05; **p value <0.01; ***p value <0.001.

Abbreviations: LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; IHC, immunohistochemistry.

Figure 2 Diagnostic value of CLEC4M in NSCLC. (a and b) ROC curves of LUAD and LUSC patients in TCGA. (c and d) ROC curves of LUAD and LUSC patients in GSE30219. (e) ROC curves of all stage T1 patients in GSE30219. (f) CLEC4M expression between non-tumor and stage T1 NSCLC tumor tissue of GSE30219. *p value <0.05.

Figure 3 Prognostic value of CLEC4M in NSCLC. (a) OS curves of all NSCLC samples in GSE30219. (b and c) OS curves of LUAD and LUSC samples in GSE30219. (d and e) OS and PFS curves of NSCLC patients in CAarray database. (f and g) OS and PFS curves of NSCLC patients in GSE31210. (h) Nomogram plot of independent clinicopathological features in GSE30219.

Abbreviations: OS, overall survival; PFS, progression-free survival.

Table 1 The Impact of CLEC4M on NSCLC Prognosis with Clinicopathological Factors in the Kaplan-Meier Plotter Database

Clinical Features	Overall Survival (n = 1928)			Progression-Free Survival (n = 982)
	No.	HR (95% CI)	p-value	No.	HR (95% CI)	p-value
Age (years)
>60	1298	0.81 (0.61–1.08)	0.16	607	1.43 (1.04–1.98)	0.029
≤60	593	0.69 (0.47–1.02)	0.059	351	0.77 (0.51–1.18)	0.23
Gender
Male	1100	1.17 (1–1.38)	0.045	514	1.54 (1.13–2.11)	0.0065
Female	714	1.14 (0.89–1.45)	0.3	468	1.8 (1.24–2.62)	0.0019
Stage
1	577	0.52 (0.3–0.92)	0.022	325	1.47 (0.91–2.35)	0.11
2	244	1.79 (1.24–2.58)	0.0017	130	0.61 (0.36–1.04)	0.068
3	70	0.87 (0.67–1.15)	0.33	19	na	na
4	4	na	na	0	na	na
T stage
1	437	0.69 (0.51–0.93)	0.014	177	1.95 (1.17–3.23)	0.0085
2	589	0.79 (0.61–1.03)	0.085	351	2.17 (1.48–3.18)	4.2e-05
3	81	0.62 (0.38–1.02)	0.06	21	0.53 (0.19–1.47)	0.22
4	46	0.54 (0.27–1.1)	0.086	7	na	na
N stage
0	781	0.78 (0.62–0.98)	0.033	374	1.96 (1.29–2.97)	0.0013
1	252	0.61 (0.42–0.89)	000099	130	1.94 (1.23–3.07)	0.004
2	111	1.37 (0.91–2.06)	0.13	51	0.6 (0.28–1.26)	0.17
M stage
0	681	0.85 (0.69–1.06)	0.14	195	1.52 (0.91–2.55)	0.11
1	10	na	na	0	na	na
Grade
I	201	0.72 (0.49–0.91)	0.027	140	0.59 (0.37–0.96)	0.033
II	310	0.71 (0.51–1)	0.047	165	0.46 (0.27–0.8)	0.0043
III	77	0.57 (0.25–1.3)	0.17	51	0.36 (0.15–0.85)	0.16
Smoke
Ever	820	1.3 (1.02–1.65)	0.035	603	1.63 (1.25–2.12)	0.00024
Never	205	2.93 (1.67–5.15)	8.4e-05	193	2.06 (1.27–3.33)	0.0027

Abbreviations: HR, hazard ratio; CI, confidence interval.

Table 2 Univariate and Multivariate Cox Regression Analyses of LUAD Patients in GSE30219 Regarding Overall Survival

Variable	Univariate Analysis		Multivariate Analysis
	HR (95% CI)	p-value	HR (95% CI)	p-value
Age (>60 vs <60 years)	1.38 (0.73–2.61)	0.322	1.29 (0.68–2.49)	0.432
Gender
Male VS female	1.18 (0.56–2.49)	0.662	1.14 (0.53–2.43)	0.734
T stage
T1 vs T2 vs T3 vs T4	1.0(0.42–2.42)	1.0	1.06 (0.36–3.09)	0.921
N stage
N0 vs N1	0.57(0.138–2.41)	0.45	0.43 (0.08–2.39)	0.339
CLEC4M expression
High vs low	0.51 (0.27–0.99)	0.047	0.51 (0.25–0.99)	0.048

Table 3 Univariate and Multivariate Cox Regression Analyses of LUSC Patients in GSE30219 Regarding Overall Survival

Variable	Univariate Analysis		Multivariate Analysis
	HR (95% CI)	p-value	HR (95% CI)	p-value
Age (>60 vs <60 years)	0.99 (0.96–1.03)	0.734	0.98 (0.95–1.02)	0.518
Gender
Male VS female	1.75 (0.53–5.78)	0.36	1.51 (0.45–5.06)	0.504
T stage
T1 vs T2 vs T3 vs T4	0.92 (0.44–1.95)	0.834	1.25 (0.42–3.68)	0.680
N stage
N0 vs N1	0.75 (0.34–1.64)	0.469	0.84 (0.26–2.69)	0.7675
CLEC4M expression
High vs low	0.25 (0.07–0.80)	0.018	0.23 (0.07–0.75)	0.013

Figure 4 Association of CLEC4M expression with the infiltration level of immune cells in NSCLC. (a and b) TheCLEC4M expression of LUAD and LUSC in low immune score and high immune score groups. (c–p) The correlation of CLEC4M expression with tumor purity and immune infiltration level of LUAD and LUSC (TIMER). (q) The connection of CLEC4M copy number variations (CNVs) and immune infiltration levels in LUAD and LUSC. ***p value <0.001.

Table 4 The Correlations Between CLEC4M with Immune-Related Markers

Description	Gene Markers	LUAD				LUSC
		None		Purity		None		Purity
		Cor	p-value	Cor	p-value	Cor	p-value	Cor	p-value
T cell	CD3D	0.247	***	0.140	**	0.232	***	0.165	***
	CD3E	0.249	***	0.139	**	0.245	***	0.18	***
	CD2	0.256	***	0.149	***	0.238	***	0.173	***
CD8^+ T cell	CD8A	0.244	***	0.156	***	0.213	***	0.158	***
	CD8B	0.206	***	0.134	**	0.127	**	0.103	*
	CD45	0.308	***	0.212	***	0.311	***	0.255	***
Monocyte	CD14	0.206	***	0.127	**	0.252	***	0.19	***
	CSF1R	0.233	***	0.154	***	0.295	***	0.243	***
TAM	CCL2	0.19	***	0.113	*	0.196	***	0.142	**
	CD68	0.268	***	0.198	***	0.312	***	0.275	***
	IL10	0.28	***	0.192	***	0.279	***	0.234	***
M1 macrophage	NOS2	0.174	***	0.126	**	0.083	0.06	0.087	0.057
	CD80	0.318	***	0.241	***	0.305	***	0.262	***
	IRF5	0.219	***	0.138	**	0.092	*	0.073	0.109
	IL6	0.234	***	0.179	***	0.194	***	0.175	***
	FCGR1A	0.228	***	0.143	**	0.278	***	0.236	***
M2 macrophage	CD163	0.309	***	0.239	***	0.339	***	0.303	***
	CD206	0.312	***	0.251	***	0.335	***	0.3	***
	VSIG4	0.264	***	0.194	***	0.306	***	0.271	***
	MS4A4A	0.298	***	0.221	***	0.349	***	0.316	***
Neutrophils	CEACAM8	0.154	***	0.147	**	0.174	***	0.182	***
	ITGAM	0.2	***	0.124	**	0.237	***	0.183	***
	CD15	0.093	*	0.06	0.183	0.137	**	0.104	*
NK cell	KIR2DL1	0.154	***	0.123	**	0.138	**	0.096	*
	KIR2DL3	0.213	***	0.161	***	0.134	**	0.104	*
	KIR3DL1	0.208	***	0.172	***	0.158	***	0.127	**
	KIR3DL2	0.12	**	0.065	0.147	0.172	***	0.132	**
	NCAM1	0.07	0.111	0.028	0.528	0.028	0.526	0.032	0.481
	NCR1	0.228	***	0.169	***	0.229	***	0.192	***
Dendrotic cell	CD1C	0.179	***	0.117	**	0.262	***	0.208	***
	THBD	0.296	***	0.252	***	0.077	0.08	0.053	0.245
	NRP1	0.035	0.431	0.015	0.739	0.198	***	0.142	**
	IL3RA	0.294	***	0.215	***	0.3	***	0.248	***
	ITGAX	0.257	***	0.176	***	0.308	***	0.253	***
Th1	CXCR3	0.186	***	0.079	0.081	0.231	***	0.162	***
	STAT4	0.229	***	0.137	**	0.294	***	0.244	***
	STAT1	0.177	***	0.117	**	0.109	*	0.055	0.228
Th2	GATA3	0.189	***	0.112	*	0.056	0.213	0.005	0.912
	STAT6	0.105	*	0.096	*	0.056	0.214	0.052	0.256
	IL13	0.115	**	0.065	0.152	0.157	***	0.113	*
Tfh	BCL6	−0.005	0.903	−0.005	0.917	0.075	0.094	0.105	*
	IL21	0.131	**	0.077	0.087	0.131	**	0.101	*
Th17	STAT3	0.007	0.876	0.008	0.852	0.051	0.253	0.014	0.766
	IL17A	0.099	*	0.046	0.309	0.033	0.458	0.003	0.955
	RORyt	−0.077	0.079	−0.037	0.416	0.259	***	0.225	***
Treg	FOXP3	0.18	***	0.074	0.099	0.2	***	0.127	**
	CD25	0.251	***	0.172	***	0.252	***	0.201	***
	CCR8	0.206	***	0.115	*	0.231	***	0.168	***
	STAT5B	0.168	***	0.138	**	0.117	**	0.113	*
T cell exhaustion	PDCD1	0.2	***	0.105	*	0.247	***	0.187	***
	CTLA4	0.235	***	0.135	**	0.217	***	0.145	**
	LAG3	0.154	***	0.078	0.082	0.154	***	0.1	*
	HAVCR2	0.26	***	0.171	***	0.317	***	0.271	***
	GZMB	0.241	***	0.171	***	0.164	***	0.1	*

Notes: *p < 0.05; **p < 0.01; ***p < 0.001.

Abbreviations: LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; None, correlation without adjustment; Purity, correlation adjusted by Tumor purity; TAM, tumor-associated macrophage; Th, T helper cell; Tfh, Follicular helper T cell; Treg, regulatory T cell; Cor, Spearman correlation value.

Figure 5 The functional enrichment analysis of CLEC4M in NSCLC. (a) Enriched GO terms of the CLEC4M-coexpressed genes. (b) Enriched KEGG pathway of the CLEC4M-coexpressed genes. (c and d) The enriched biological process of immune activation by GSEA according to CLEC4M expression of LUAD and LUSC patients. (e and f) The enriched cell proliferation-related functions using GSEA based on CLEC4M expression of LUAD and LUSC patients.