Pan-Cancer Profiling and Digital Pathology Analysis Reveal Negative Prognostic Biomarker ZPR1 Associated with Immune Infiltration and Treatment Response in Hepatocellular Carcinoma

Figures & data

Table 1 The Detailed Information About antibodies in This Research

Antibody	Product Code	Company	IHC Dilution	Immune Cell Markers	QuPath Identification
ZPR1	ab134970	Abcam	1:100		Cytoplasm
CD3	ZM-0417	ZSGB-BIO	Working fluid	T cell	Cytoplasm
CD4	GT219107	Gene Tech	Working fluid	T helper	Cytoplasm
CD8	GT211207	Gene Tech	Working fluid	Cytotoxic T	Cytoplasm
CD20	GM075504	Gene Tech	Working fluid	B cell	Cytoplasm
CD38	GT212907	Gene Tech	Working fluid	Plasma cell	Cytoplasm
CD56	GT200507	Gene Tech	Working fluid	NK cell	Cytoplasm
CD68	GM087607	Gene Tech	Working fluid	Macrophage	Cytoplasm
PD-L1	22C3 pharmDx	Agilent	Working fluid		Membrane

Figure 1 The ZPR1 expression in pan-cancer. (A) ZPR1 expression in pan-cancer tissues from TCGA database. (B) ZPR1 expression in pan-cancer tissues based on TCGA and GTEx databases. (C) ZPR1 expression in paired tumor and adjacent normal tissues from TCGA database. (D) ZPR1 protein expression in pan-cancer tissues from the UALCAN-CPTAC database. ZPR1 mRNA expression in various tissues (E) and cancer cell lines (F) from the HPA database. (G) Cellular localization of ZPR1 from the HPA database. *p < 0.05; **p < 0.01; and ***p < 0.001.

Abbreviation: Ns, no significance.

Figure 2 DNA methylation analysis of ZPR1. (A) Chromosomal distribution of the 14 methylation probes associated with ZPR1 on chromosome 11. (B)Detailed location of 14 probes in CpG Island. (C) ZPR1 methylation levels in tumor and normal samples cross different types of cancers. p > 0.05; *p <= 0.05; **p <= 0.01; ***p <= 0.001; ****p <= 0.0001.

Abbreviation: Ns, no significance.

Figure 3 Clinical correlation analysis between the ZPR1 expression and patient’s age (A), gender (B), tumor status (C), pathological stage (D), TMB (E), MSI (F), and stemness (G) in pan-cancer. *p < 0.05; **p < 0.01; ***p < 0.001.

Table 2 The Prognostic Values of ZPR1 (RNAseq ID: 8882) Expression Stratified by Different Clinicopathologic Features from Kaplan-Meier Plotter Databases

Clinical Features	OS (n = 364)			PFS (n = 370)
	N	HR	p	N	HR	p
Total	364	1.61 (1.12–2.33)	0.0096*	370	1.66 (1.04–2.65)	0.0731
Gender
Female	118	2.04 (1.06–3.91)	0.0288*	121	0.77 (0.46–1.28)	0.3083
Male	246	2.32 (1.41–3.82)	0.0007*	249	1.43 (0.99–2.05)	0.0547
Race
White	181	1.76 (1.04–2.99)	0.0345*	184	1.51 (1.01–2.25)	0.0422*
Asian	155	2.3 (1.27–4.17)	0.0047*	157	1.44 (0.87–2.37)	0.1539
Sorafenib treatment
Treated	29	4.02 (1.05–15.39)	0.0296*	30	2.42 (0.89–6.53)	0.0737
Alcohol consumption
None	202	2.05 (1.28–3.27)	0.0022*	205	1.66 (1.07–2.57)	0.1628
Yes	115	2.27 (0.99–5.17)	0.0461*	117	1.35 (0.81–2.26)	0.2454
Hepatitis virus
None	167	1.71 (1.09–2.68)	0.0192*	169	1.66 (1.07–2.57)	0.0222*
Yes	150	2.67 (1.4–5.1)	0.002*	153	0.76 (0.48–41.22)	0.2536
Stage
Stage 1	170	1.94 (1.04–3.61)	0.0335*	171	0.6 (0.36–1)	0.047*
Stage 1+2	253	1.71 (1.04–2.81)	0.0339*	256	0.76 (0.51–1.12)	0.1631
Stage 2	83	0.64 (0.29–1.45)	0.2854	85	1.72 (0.92–73.21)	0.0862
Stage 2+3	166	1.32 (0.82–2.15)	0.2538	170	1.44 (0.96–2.15)	0.0776
Stage 3	83	1.4 (0.75–2.62)	0.2853	85	1.86 (1.06–3.25)	0.0277*
Stage 3+4	87	1.4 (0.76–2.55)	0.2754	90	1.85 (1.04–3.29)	0.0336*
Grade
G1	55	2.48 (0.96–6.42)	0.0518	55	4.05 (1.84–8.92)	0.0002*
G2	174	0.66 (0.37–1.17)	0.1506	177	0.71 (0.43–1.16)	0.1713
G3	118	3.15 (1.72–5.75)	8.70E-05*	121	1.67 (0.99–2.82)	0.0531
AJCC stage T
Stage T1	180	1.92 (1.06–3.47)	0.028*	181	0.64 (0.39–1.05)	0.0727
Stage T2	90	1.44 (0.67–3.09)	0.3501	93	1.64 (0.92–2.92)	0.0899
Stage T3	78	1.3 (0.7–2.39)	0.4052	80	1.61 (0.89–2.91)	0.112
Vascular invasion
None	203	1.58 (0.94–2.66)	0.0825	205	0.61 (0.38–0.98)	0.0406*
Micro	90	0.58 (0.27–1.25)	0.1599	92	0.56 (0.3–1.06)	0.0711

Note: *Statistically significant P values.

Figure 4 ZPR1 could be considered as an independent predictor for HCC patient survival. (A) The difference of ZPR1 expression in HCC retrieved from the GENT2 database. (B) The heatmap showing the clinical correlation analysis of ZPR1 expression in HCC by the Chi-square test. (C) The overall survival analysis of ZPR1 in TCGA HCC samples. Univariate Cox analysis (D) and multivariate Cox analysis (E) of ZPR1 expression and clinicopathological characteristics. (F) The ROC curves of 1-year and 3-year OS of ZPR1. (G) Nomogram of ZPR1 expression and clinicopathological features. (H) The calibration curves of the nomogram for 1-year and 3-year OS.

Figure 5 The functional prediction of the ZPR1 gene in HCC. (A) Co-expression analysis of ZPR1. (B) Target gene prediction through CHEA, ENCODE, and JASPAR databases. (C) Pearson correlation analysis between ZPR1 and predicted target gene. (D) Differential expression genes in high- and low-ZPR1 expression groups. (E and F) GO and KEGG analyses showing biological processes and molecular functions involved in ZPR1-related DEGs. (G) GSEA analysis showing cancer- or metabolism-related pathways enriched in high-ZPR1 groups.

Figure 6 Immune landscape analysis of the ZPR1 gene. (A) TME score comparison in low- and high-ZPR1 groups. (B) The immune cell infiltration correlation analysis of the ZPR1 gene using the CIRBSORT algorithm. (C) The immune cell infiltration correlation analysis of the ZPR1 gene by multiple methods. (D) Correlation analysis between ZPR1 gene and immune checkpoint genes. (E and F) The correlation between the ZPR1 gene and immune cell infiltration scores or immune-related functions. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 7 ZPR1 expression in TME-associated cells. Annotation of all cell types, ZPR1 expression percentages and differential expression in GSE125449 (A-C), GSE140228 (D-F), and GSE145409 (G-I) datasets. *p < 0.05; **p < 0.01; and ***p < 0.001.

Abbreviation: Ns, no significance.

Figure 8 ZPR1 immunostaining in HCC tissue samples. ZPR1 immunostaining showing low expression in the cytoplasm of normal hepatocytes, and prominent positive expression in the cytoplasm of HCC cells. (A) The ZPR1 expression between HCC cells and adjacent normal hepatocytes in the same visual field (200 ×). (B) The H-score difference of ZPR1 expression in HCC tissues. (C and D) Correlation between ZPR1 expression and common immune cell markers, as well as PD-L1. (E) The AUC value for tumor diagnosis of ZPR1. (F) Correlation analysis of ZPR1 expression with clinicopathological characteristics.

Figure 9 Treatment response prediction of ZPR1. (A) TIDE score prediction (B) The IC50 difference of common chemotherapeutic or targeted drugs in low- and high-ZPR1 expression groups. *p < 0.05; **p < 0.01; ***p < 0.001.