LncRNA ADAMTS9-AS2 is a Prognostic Biomarker and Correlated with Immune Infiltrates in Lung Adenocarcinoma

Figures & data

Table 1 Characteristics of Patients with LUAD Based on TCGA

Characteristic	Levels	Overall
n		535
T stage, n (%)	T1	175 (32.9%)
	T2	289 (54.3%)
	T3	49 (9.2%)
	T4	19 (3.6%)
N stage, n (%)	N0	348 (67.1%)
	N1	95 (18.3%)
	N2	74 (14.3%)
	N3	2 (0.4%)
M stage, n (%)	M0	361 (93.5%)
	M1	25 (6.5%)
Primary therapy outcome, n (%)	PD	71 (15.9%)
	SD	37 (8.3%)
	PR	6 (1.3%)
	CR	332 (74.4%)
Pathologic stage, n (%)	Stage I	294 (55.8%)
	Stage II	123 (23.3%)
	Stage III	84 (15.9%)
	Stage IV	26 (4.9%)
Gender, n (%)	Female	286 (53.5%)
	Male	249 (46.5%)
Race, n (%)	Asian	7 (1.5%)
	Black or African American	55 (11.8%)
	White	406 (86.8%)
Age, n (%)	≤65	255 (49.4%)
	>65	261 (50.6%)
Residual tumor, n (%)	R0	355 (95.4%)
	R1	13 (3.5%)
	R2	4 (1.1%)
Anatomic neoplasm subdivision, n (%)	Left	205 (39.4%)
	Right	315 (60.6%)
Anatomic neoplasm subdivision 2, n (%)	Central Lung	62 (32.8%)
	Peripheral Lung	127 (67.2%)
Number_pack_years_smoked, n (%)	<40	188 (50.9%)
	≥40	181 (49.1%)
Smoker, n (%)	No	75 (14.4%)
	Yes	446 (85.6%)
Age, median (IQR)		66 (59, 72)

Table 2 Correlation Between ADAMTS9-AS2 Expression and Clinical Characteristics in LUAD

Characteristic	Low Expression of ADAMTS9-AS2	High Expression of ADAMTS9-AS2	p
n	267	268
T stage, n (%)			0.162
T1	79 (14.8%)	96 (18%)
T2	157 (29.5%)	132 (24.8%)
T3	24 (4.5%)	25 (4.7%)
T4	7 (1.3%)	12 (2.3%)
N stage, n (%)			0.044
N0	164 (31.6%)	184 (35.5%)
N1	54 (10.4%)	41 (7.9%)
N2	46 (8.9%)	28 (5.4%)
N3	1 (0.2%)	1 (0.2%)
M stage, n (%)			1.000
M0	182 (47.2%)	179 (46.4%)
M1	13 (3.4%)	12 (3.1%)
Primary therapy outcome, n (%)			0.388
PD	40 (9%)	31 (7%)
SD	20 (4.5%)	17 (3.8%)
PR	2 (0.4%)	4 (0.9%)
CR	156 (35%)	176 (39.5%)
Pathologic stage, n (%)			0.102
Stage I	135 (25.6%)	159 (30.2%)
Stage II	65 (12.3%)	58 (11%)
Stage III	51 (9.7%)	33 (6.3%)
Stage IV	13 (2.5%)	13 (2.5%)
Gender, n (%)			0.003
Female	125 (23.4%)	161 (30.1%)
Male	142 (26.5%)	107 (20%)
Race, n (%)			0.233
Asian	4 (0.9%)	3 (0.6%)
Black or African American	33 (7.1%)	22 (4.7%)
White	194 (41.5%)	212 (45.3%)
Age, n (%)			0.726
≤65	129 (25%)	126 (24.4%)
>65	127 (24.6%)	134 (26%)
Residual tumor, n (%)			0.315
R0	186 (50%)	169 (45.4%)
R1	4 (1.1%)	9 (2.4%)
R2	2 (0.5%)	2 (0.5%)
Anatomic neoplasm subdivision, n (%)			0.748
Left	104 (20%)	101 (19.4%)
Right	154 (29.6%)	161 (31%)
Anatomic neoplasm subdivision 2, n (%)			0.356
Central Lung	29 (15.3%)	33 (17.5%)
Peripheral Lung	70 (37%)	57 (30.2%)
Number_pack_years_smoked, n (%)			0.031
<40	89 (24.1%)	99 (26.8%)
≥40	107 (29%)	74 (20.1%)
Smoker, n (%)			< 0.001
No	22 (4.2%)	53 (10.2%)
Yes	239 (45.9%)	207 (39.7%)
Age, median (IQR)	65 (57, 72)	66 (60, 72)	0.211

Table 3 Correlation of ADAMTS9-AS2 Expression with Clinical Features of LUAD (Logistic Regression)

Characteristics	Total (N)	Odds Ratio (OR)	P value
T stage (T2&T3&T4 vs T1)	532	0.740 (0.514–1.063)	0.104
N stage (N1&N2&N3 vs N0)	519	0.618 (0.425–0.893)	0.011
M stage (M1 vs M0)	386	0.939 (0.412–2.124)	0.878
Pathologic stage (Stage III&Stage IV vs Stage I&Stage II)	527	0.662 (0.431–1.011)	0.057
Primary therapy outcome (CR vs PD&SD&PR)	446	1.345 (0.879–2.066)	0.173
Gender (Male vs Female)	535	0.585 (0.415–0.823)	0.002
Race (White vs Asian&Black or African American)	468	1.617 (0.944–2.812)	0.083
Age (>65 vs ≤65)	516	1.080 (0.765–1.526)	0.661
Residual tumor (R1&R2 vs R0)	372	2.018 (0.751–5.967)	0.176
Anatomic neoplasm subdivision (Right vs Left)	520	1.077 (0.757–1.531)	0.681
Anatomic neoplasm subdivision 2 (Peripheral Lung vs Central Lung)	189	0.716 (0.388–1.315)	0.282
Number_pack_years_smoked (≥40 vs <40)	369	0.622 (0.411–0.937)	0.024
Smoker (Yes vs No)	521	0.360 (0.208–0.604)	<0.001

Figure 1 ADAMTS9-AS2 expression in LUAD is significantly lower than in normal or adjacent normal tissues. (A) Differential expression of ADAMTS9-AS2 in LUAD tissues and adjacent lung tissues. (B) Differential expression of ADAMTS9-AS2 in LUAD tissues and paired adjacent lung tissues. (C) Effectiveness of ADAMTS9-AS2 expression in distinguishing LUAD tissues from nontumor tissues (ROC curve). Significance markers: ***P<0.001.

Figure 2 Relationship between ADAMTS9-AS2 expression and clinical features of LUAD. (A) Relationship between ADAMTS9-AS2 expression and N stage. (B) Relationship between ADAMTS9-AS2 expression and gender. (C) Relationship between expression of ADAMTS9-AS2 and number pack years smoked. (D) Relationship between expression of ADAMTS9-AS2 and smoker. Significance markers: *P<0.05; **P<0.01; ***P<0.001.

Table 4 Association Analysis of OS and Clinical Characteristics of LUAD Patients (Cox Regression)

Characteristics	Total (N)	Univariate Analysis		Multivariate Analysis
		Hazard Ratio (95% CI)	P value	Hazard Ratio (95% CI)	P value
T stage (T2&T3&T4 vs T1)	523	1.728 (1.229–2.431)	0.002	1.247 (0.728–2.136)	0.422
N stage (N1&N2&N3 vs N0)	510	2.601 (1.944–3.480)	<0.001	1.457 (0.904–2.347)	0.122
M stage (M1 vs M0)	377	2.136 (1.248–3.653)	0.006	1.177 (0.451–3.074)	0.739
Pathologic stage (Stage III &Stage IV vs Stage I & Stage II)	518	2.664 (1.960–3.621)	<0.001	1.925 (1.063–3.485)	0.031
Primary therapy outcome (CR vs PD&SD&PR)	439	0.372 (0.265–0.521)	<0.001	0.298 (0.181–0.490)	<0.001
Gender (Male vs Female)	526	1.070 (0.803–1.426)	0.642
Race (White vs Asian & Black or African American)	468	1.475 (0.902–2.411)	0.121
Age (>65 vs ≤65)	516	1.223 (0.916–1.635)	0.172
Residual tumor (R1&R2 vs R0)	363	3.879 (2.169–6.936)	<0.001	4.736 (1.959–11.451)	<0.001
Anatomic neoplasm subdivision (Right vs Left)	512	1.037 (0.770–1.397)	0.810
Anatomic neoplasm subdivision 2 (Peripheral Lung vs Central Lung)	182	0.913 (0.570–1.463)	0.706
Number_pack_years_smoked (≥40 vs <40)	363	1.073 (0.753–1.528)	0.697
Smoker (Yes vs No)	512	0.894 (0.592–1.348)	0.591
ADAMTS9-AS2 (High vs Low)	526	0.683 (0.511–0.914)	0.010	0.626 (0.397–0.986)	0.043

Figure 3 Low expression of ADAMTS9-AS2 is associated with poor OS and DSS in LUAD patients. (A) Kaplan–Meier curves and number at risk of OS in LUAD patients. (B) Kaplan–Meier curves and number at risk of DSS in LUAD patients.

Abbreviations: OS, overall survival; DSS, disease-specific survival.

Figure 4 Forest plot of the multivariate Cox regression analysis for LGG.

Figure 5 Nomogram for predicting the probability of patients with 1-, 3- and 5-year overall survival.

Table 5 Gene Enrichment of ADAMTS9-AS2 High and Low Expression Groups (GSEA)

Gene Set Name	NES	P Adjust	FDR
REACTOME_UNWINDING_OF_DNA	−2.039	0.042	0.033
BIOCARTA_EXTRINSIC_PATHWAY	−2.049	0.042	0.033
REACTOME_POLO_LIKE_KINASE_MEDIATED_EVENTS	−2.254	0.042	0.033
WP_CORI_CYCLE	−2.200	0.042	0.033
BIOCARTA_MCM_PATHWAY	−2.143	0.042	0.033
BIOCARTA_PROTEASOME_PATHWAY	−2.019	0.042	0.033
REACTOME_LAGGING_STRAND_SYNTHESIS	−2.082	0.042	0.033
REACTOME_PCNA_DEPENDENT_LONG_PATCH_BASE_EXCISION_REPAIR	−2.158	0.042	0.033
REACTOME_REGULATION_OF_GENE_EXPRESSION_IN_BETA_CELLS	−2.059	0.042	0.033

Abbreviations: NES, normalized enrichment score; FDR, false discovery rate.

Figure 6 Enrichment plots from GSEA. (A) Unwinding of DNA, (B) extrinsic pathway, (C) polo-like kinase mediated events, (D) cori cycle, (E) MCM pathway, (F) PCNA-dependent long complement base excision repair, (G) proteasome pathway, (H) lagging strand synthesis, (I) regulation of gene expression in beta cells.

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

Figure 7 Expression of ADAMTS9-AS2 correlated with immune cells in LUAD patients (grouped comparison chart). (A) DC, (B) Eosinophils, (C) iDC, (D) Macrophages, (E) Mast cells, (F) NK CD56bright cells, (G) NK CD56dim cells, (H) NK cells, (I) pDC, (J) T cells, (K) T helper cells, (L) Tcm, (M) Tem, (N) TFH, (O) Th1 cells, (P) Th2 cells, and (Q) TReg. Significance markers: *P<0.05; **P<0.01; ***P<0.001.

Figure 8 Expression of ADAMTS9-AS2 correlated with immune cells in LUAD patients (scatter plot). (A) DC, (B) Eosinophils, (C) iDC, (D) Macrophages, (E) Mast cells, (F) NK CD56bright cells, (G) NK CD56dim cells, (H) NK cells, (I) pDC, (J) T cells, (K) T helper cells, (L) Tcm, (M) Tem, (N) TFH, (O) Th1 cells, (P) Th2 cells, and (Q) TReg.

Figure 9 ADAMTS9-AS2 expression correlated with 24 immune cells in LUAD patients (lollipop chart).