CKS2 (CDC28 protein kinase regulatory subunit 2) is a prognostic biomarker in lower grade glioma: a study based on bioinformatic analysis and immunohistochemistry

Figures & data

Table 1. Details of the two GEO datasets used in this study

Dataset	Normal brain samples	Lower grade glioma		Platform
		II	III
GSE4290	23	45	31	GPL570[HGU133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array
GSE16011	8	24	85	GPL8542Affymetrix GeneChip Human Genome U133 Plus 2.0 Array [CDF: Hs133P_Hs_ENTREZG.cdf]
Total	31	69	116

Figure 1. The differential expression of CKS2 in LGG tissues and non-tumor tissues. (a-c) Differential expression of CKS2 mRNA in LGG tissues and normal brain tissues in GEPIA2, GSE4290 and GSE16011 (*P < 0.05). (d) Differential expression of CKS2 protein in LGG tissues and adjacent normal brain tissues. (e) Representative images of the CKS2 protein expression in LGG tissues and adjacent normal brain tissues

Figure 2. The differential expression of CKS2 in grade II and grade III gliomas. (a-d) Differential expression of CKS2 mRNA in grade II and grade III gliomas in TCGA, GSE4290, GSE16011 and CGGA. (e) Differential expression of CKS2 protein in grade II and grade III gliomas. (d) Representative images of the CKS2 protein expression in grade II and grade III gliomas

Figure 3. The correlation between the CKS2 mRNA expression and IDH1 mutation status in (a) TCGA database, (b) CGGA database and (c) TIMER2.0 database

Figure 4. The association between the CKS2 and copy number alteration (CNA), or methylation in LGG. (a) CKS2 mRNA expression in different CNA groups. (b) Correlation analysis between CKS2 mRNA expression and CKS2 DNA methylation

Figure 5. Survival prediction by the CKS2 gene in the TCGA-LGG and CGGA cohorts. (a-b) Kaplan-Meier (KM) survival analysis and time-dependent ROC analysis of CKS2 in LGG based on the TCGA-LGG cohort. (c-d) Kaplan-Meier (KM) survival analysis and time-dependent ROC analysis of CKS2 in LGG based on the CGGA cohort

Figure 6. Time-dependent ROC analysis of CKS2, LGG grade and IDH1 mutation status in (a) the TCGA-LGG cohort and (b) the CGGA cohort

Table 2. Association between CKS2 expression and the clinical parameters in patients with LGG in TCGA-LGG cohort

Characteristics	CKS2		P value
	Low (n = 253)	High (n = 253)
Age			0.026
≤40	137	112
>40	116	141
Gender			1.000
Male	140	140
Female	113	113
Grade			<0.001
Unknown	1
G2	165	80
G3	87	173
IDH mutation status			0.076
Unknown	189	192
Mutant	51	40
Wildtype	13	21
Radiotherapy			0.654
Unknown	181	171
No	49	53
Yes	23	29
Chemotherapy			0.005
Unknown	182	170
No	40	28
Yes	31	55

Bold values indicate P < 0.05.

Table 3. Association between CKS2 expression and the clinical parameters in patients with LGG in CGGA cohort

Characteristics	CKS2		P value
	Low (n = 86)	High (n = 86)
Age			0.219
≤40	52	44
>40	34	42
Gender
Male	48	58	0.117
Female	38	28
Grade			<0.001
G2	68	30
G3	18	56
IDH mutation status			0.118
Unknown	1	0
Mutant	68	59
Wildtype	17	27
Radiotherapy			0.540
Unknown	2	2
No	16	13
Yes	68	71
Chemotherapy
Unknown	3	5	0.027
No	45	30
Yes	38	51

Bold values indicate P < 0.05.

Table 4. Univariate analysis and multivariate analysis of the correlation of CKS2 expression with OS among LGG patients in TCGA-LGG cohort and CGGA cohort

	Univariable analysis					Multivariable analysis
Variables		HR	95% CI of HR		P	HR	95% CI of HR		P
			Lower	Upper			Lower	Upper
TCGA (n = 506)
Age	Continuous	1.06	1.04	1.07	<0.001	1.06	1.04	1.08	<0.001
Sex	Male vs. female	1.11	0.78	1.59	0.56	1.32	0.91	1.92	0.14
IDH status	Wildtype vs. mutant	5.29	2.08	13.45	<0.001	2.30	0.89	5.97	0.09
LGG Grade	G3 vs. G2	3.43	2.32	5.06	<0.001	2.24	1.46	3.44	<0.001
Radiotherapy	Yes vs. No	0.97	0.61	1.54	0.89	1.19	0.74	1.93	0.47
Chemotherapy	Yes vs. No	1.76	1.06	2.92	0.03	2.08	1.20	3.60	0.01
CKS2	Continuous	1.57	1.35	1.84	<0.001	1.34	1.11	1.60	0.002
CGGA (n = 172)
Age	Continuous	1.03	1.01	1.06	0.005	1.01	0.99	1.04	0.17
Sex	Male vs. female	0.64	0.43	0.97	0.03	0.45	0.28	0.71	0.001
IDH status	Wildtype vs. mutant	2.64	1.70	4.09	<0.001	2.49	1.56	3.97	<0.001
LGG Grade	G3 vs. G2	3.58	2.33	5.48	<0.001	2.36	1.38	4.03	0.002
Radiotherapy	Yes vs. No	0.56	0.34	0.94	0.03	0.62	0.36	1.06	0.08
Chemotherapy	Yes vs. No	1.62	1.05	2.51	0.03	0.76	0.45	1.27	0.29
CKS2	Continuous	1.99	1.62	2.46	<0.001	1.96	1.54	2.49	<0.001

Bold values indicate P < 0.05. HR, hazard ratio; CI, confidence interval.

Figure 7. Functional enrichment analysis of the differential expressed genes (DEGs) in the TCGA database. (a) Heatmap showing differentially expressed genes in the low and high CKS2 expression groups in LGG. (b-c) GO and KEGG enrichment analysis of the DEGs

Table 5. Gene sets enriched in the high CKS2 expression group

Gene set name	NES	NOM p-value	FDR q-value
HALLMARK_DNA_REPAIR	2.26	0.000	0.000
HALLMARK_MYC_TARGETS_V1	2.19	0.000	0.000
HALLMARK_E2F_TARGETS	2.14	0.000	0.000
HALLMARK_G2M_CHECKPOINT	2.11	0.000	0.001
HALLMARK_MTORC1_SIGNALING	2.05	0.000	0.002
HALLMARK_MYC_TARGETS_V2	2.03	0.000	0.002
HALLMARK_MITOTIC_SPINDLE	1.95	0.006	0.006
HALLMARK_P53_PATHWAY	1.84	0.002	0.017
HALLMARK_WNT_BETA_CATENIN_SIGNALING	1.70	0.012	0.048
HALLMARK_ANGIOGENESIS	1.69	0.025	0.046

NES, normalized enrichment score; NOM, nominal; FDR, false discovery rate.

Figure 8. Gene Set Enrichment Analysis (GSEA) showing the signaling pathways enriched in phenotypes with overexpressed CKS2 in LGG

Data availability statement

All the data was downloaded from UCSC Xena (http://xena.ucsc.edu/public/), CGGA (http://www.cgga.org.cn/index.jsp), GEO (https://www.ncbi.nlm.nih.gov/geo/) and cBioPortal web server (https://www.cbioportal.org/study/summary?id=lgg_tcga). Besides, all the codes we used are available from the corresponding author for noncommercial purposes.