An immune-based risk-stratification system for predicting prognosis in pulmonary sarcomatoid carcinoma (PSC)

Figures & data

Table 1. Univariable and multivariable Cox regression for DFS

Factor	Number	Univariable analysis		Multivariable analysis^a		Multivariable analysis^b
		HR (95% CI)	P value	HR (95% CI)	P value	HR (95% CI)	P value
Age (y)			0.061
≤70	81	1
>70	16	1.752 (0.996–3.176)
Gender			0.502
Female	82	1.259 (0.641–2.474)
Male	15	1
Smoking			0.896
No	52	1.033 (0.634–1.681)
Yes	45	1
Pathology			0.749
Pure PSC	89	1.180 (0.428–3.248)
PSC with other types	8	1
Chemotherapy			0.446
No	51	1.209 (0.741–1.973)
Yes	46	1
Radiotherapy			0.298
No	93	1.704 (0.618–4.703)
Yes	4	1
T-Stage			0.002		0.026
1–3	81	1		1
4	16	2.641 (1.442–4.905)		2.151 (1.098–4.214)
N-Stage			0.070
0–1	75	1
2	22	1.657 (0.959–2.864)
M-Stage			0.093
0	92	1
1	5	2.400 (0.864–6.666)
Stage			<0.0001				0.008
1–2	61	1				1
3–4	36	2.524 (1.539–4.137)				2.014 (1.204–3.368)
CD3			0.147
Low (≤25%)	46	1.436 (0.879–2.346)
High (>25%)	51	1
CD4			0.008		0.012		0.012
Low (≤10%)	63	2.034 (1.193–3.466)		1.998 (1.167–3.424)		1.997 (1.166–3.418)
High (>10%)	34	1		1		1
CD8			0.061
Low (≤45%)	76	1.889 (0.961–3.713)
High (>45%)	21	1
PD-1-TIL			0.003		<0.0001		<0.0001
Low (≤30%)	94	1		1		1
High (>30%)	3	6.182 (1.852–20.639)		9.397 (2.696–32.751)		10.782 (3.051–38.101)
PDL1-TIL			0.078
Low (≤10%)	93	1
High (>10%)	4	2.759 (0.850–8.949)
PDL1-TC			0.235
Low (≤75%)	70	1.406 (0.799–2.475)
High (>75%)	27	1
PDL2-TIL			0.961
Low (=0%)	96	1
High (>0%)	1	1.051 (0.145–7.616)
PDL2-TC			0.206
Low (≤20%)	96	21.172 (0.016–28141.140)
High (>20%)	1	1
GAL9-TIL			0.084
Low (=0%)	13	1.800 (0.916–3.539)
High (>0%)	84	1
GAL9-TC			0.021		0.015		0.021
Low (≤10%)	34	1.774 (1.084–2.901)		1.850 (1.128–3.034)		1.785 (1.090–2.923)
High (>10%)	63	1		1		1
OX40L-TIL			0.315
Low (≤20%)	76	1
High (>20%)	21	1.355 (0.747–2.456)
OX40L-TC			0.268
Low (≤45%)	83	1.554 (0.708–3.410)
High (>45%)	14	1
HLA-TIL			0.108
Low (≤10%)	62	1.530 (0.908–2.578)
High (>10%)	35	1
HLA-TC			0.085
Low (≤70%)	93	5.709 (0.787–41.436)
High (>70%)	4	1

Abbreviations: DFS, disease-free survival; HR, hazard ratio; PSC, pulmonary sarcomatoid carcinoma; NSCLCs, non-small cell lung cancers; TC, tumor cell; TIL, tumor-infiltrating lymphocyte; CD3, cluster of differentiation 3; CD4, cluster of differentiation 4; CD8, cluster of differentiation 8; PD-1, programmed death-1; PD-L1, programmed death-ligand 1; PD-L2, programmed death-ligand 2; Gal-9, galectin-9; OX40L, tumor necrosis factor ligand superfamily member 4; HLA, human leukocyte antigen.

Note: Considering the collinearity between T-stage and TNM-stage, we included them separately in two multivariable regression.

^aThe multivariable Cox regression included T-stage.

^bThe multivariable Cox regression included TNM-stage.

Figure 1. The expression status of nine detected immune-related proteins and their correlation

The representative of IHC images of high and low levels of CD4(a), PD-1(b), Gal-9 on TIL(c), Gal-9 on TC(d), and HLA on TIL (e). (f) The percentages of each type of positive TCs or TILs were demonstrated in the bar chart, and the P value was calculated by the analysis of variance (ANOVA). ***P < 0.001. (g) The correlation matrix of the percentages of all positive types of positive TCs or TILs, and the coefficients (>0.3) marked in red indicate a correlation between a pair of proteins. The scales of A to C were ×400. Abbreviations: IHC, immunohistochemistry; TC, tumor cell; TIL, tumor-infiltrating lymphocyte; CD3, cluster of differentiation 3; CD4, cluster of differentiation 4; CD8, cluster of differentiation 8; PD-1, programmed death-1; PD-L1, programmed death-ligand 1; PD-L2, programmed death-ligand 2; Gal-9, galectin-9; OX40L, tumor necrosis factor ligand superfamily member 4; HLA, human leukocyte antigen.

Figure 2. Kaplan–Meier survival curves with log-rank tests for DFS between patients with different levels of CD4 (a), PD-1 (b), Gal-9 on TC (c), and CD8 (d)

Abbreviations: DFS, disease-free survival; HR, hazard ratio; TC, tumor cell; CD4, cluster of differentiation 4; CD8, cluster of differentiation 8; PD-1, programmed death-1; Gal-9, galectin-9.

Figure 3. Kaplan–Meier survival curves with log-rank tests for OS between patients with different levels of CD4 (a), PD-1 (b), Gal-9 on TIL (c), HLA on TIL (d)

Abbreviations: OS, overall survival; HR, hazard ratio; TC, tumor cell; CD4, cluster of differentiation 4; PD-1, programmed death-1; Gal-9, galectin-9; HLA, human leukocyte antigen.

Figure 4. The performance of immune-based risk models for DFS. (a) The rank of relative importance of Gal-9 on TC, CD4, and PD-1 for DFS according to the random forest. The F1-score and AUC of CD4, PD-1, Gal-9, and the combination of the three proteins for 1 y- (b), 3-y (c), and 5-y DFS (d). The heights of the columns represent the average of 100 testing groups, and vertical lines represent the standard error of mean (SEM) of 100 testing groups. ***P < 0.001

Abbreviations: DFS, disease-free survival; HR, hazard ratio; TNM, tumor, nodes, and metastasis; TC, tumor cell; CD4, cluster of differentiation 4; PD-1, programmed death-1; Gal-9, galectin-9; AUC, areas under time-dependent receiver-operating characteristic curves.

Figure 5. The performance of immune-based risk models for OS. (a) The rank of relative importance of Gal-9 on TIL, HLA on TIL, CD4, and PD-1 for OS according to the random forest. The F1-score and AUC of CD4, PD-1, Gal-9 on TIL, HLA on TIL, and the combination of the four proteins for 1 y- (b), 3-y (c), and 5-y OS (d). The heights of the columns represent the average of 100 testing groups, and vertical lines represent the standard error of mean (SEM) of 100 testing groups. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant

Abbreviations: OS, overall survival; HR, hazard ratio; TIL, tumor-infiltrating lymphocyte; CD4, cluster of differentiation 4; PD-1, programmed death-1; Gal-9, galectin-9; HLA, human leukocyte antigen; AUC, areas under time-dependent receiver-operating characteristic curves.

Figure 6. The improvement brought by the immune-based models to the TNM-Stage for the F1-Score (a) and AUC (b) of DFS predictions, and for the F1-Score (c) and AUC (d) of OS predictions. The heights of the columns represent the average of 100 testing groups, and dots represent the standard error (SE) of 100 testing groups. ***P < 0.001; ns, not significant

Abbreviations: OS, overall survival; DFS, disease-free survival; TC, tumor cell; TNM, tumor, nodes, and metastasis; CD4, cluster of differentiation 4; PD-1, programmed death-1; Gal-9, galectin-9; AUC, areas under time-dependent receiver-operating characteristic curve.

Figure 7. The enriched pathways and genes in the samples with a high level of CD4, PDCD1, and LAGLS9 by GESA. (a) The top four significant enrichment plots in samples with a high level of CD4, PDCD1, and LGALS9, compared with those in a low expression. (b) The heatmap of the overlap between subsets: the darker the color, the greater the overlap between the subsets. (c) The volcano map of the genes enriched in the group with high expression of CD4, PDCD1, and LGALS9 (ES>0.6 or ES<-0.6). The red dots and blue present the genes significantly downgraded and upgraded in the PSC samples compared with normal tissues, respectively (FDR<0.05, log₂|fold change|>1). (d) The mRNA level of five DEGs between PSC and normal tissues. *P < 0.05

Abbreviations: GESA, Gene Set Enrichment Analysis; FDR, false discovery rate; RPM, reads of exon model per million mapped reads; DEGs, differentially expressed genes; ES, enriched score.

Figure 8. The landscapes of immune infiltration of the two groups with the low and high expression of CD4, PDCD1, LGALS9. (a) The stacked histogram of the proportion of 22 types of cells in ten PSC tissues with a P value<0.05 according to CIBERSORT. (b) The comparison of the percentage of each cell between the two groups in all samples. The black dots in the violins represent the mean of the group, and the vertical lines represent the standard of error (SE). *P < 0.05; ns, not significant

Abbreviations: PSC, pulmonary sarcomatoid carcinoma.