High Expression of H2BC12 Predicts Poor Survival Outcome of Low-Grade Gliomas: A Study Based on TCGA Data


 Purpose

Low-grade gliomas (LGG) have highly variable clinical behaviors, with a high incidence of disease progression as 70% within ten years. Regardless of treatment combining surgery and radiotherapy or chemotherapy, LGG is still associated with adverse survival outcomes. Therefore, our study was performed to satisfy the increasing demand of novel sensitive biomarkers and therapeutic targets in treatment and diagnosis of LGG.
Methods

The TCGA data set was used to examine the relationship between H2BC12 expression and clinical pathologic characteristics. The significance of H2BC12 expression in prognosis was also investigated. In addition, H2BC12 expression-related pathways were enriched by gene set enrichment analysis (GSEA). Association analysis of H2BC12 gene expression and immune infiltration was performed by single sample gene set enrichment analysis (ssGSEA).
Results

Significantly up-regulated expression of H2BC12 mRNA was found in LGG tissue when compared to normal tissue and was proven to be diagnostic (have diagnostic significance) for LGG. In the meantime, high H2BC12 levels were associated with WHO grade, IDH status, 1p/19q codeletion, primary therapy outcome and histological type of LGG, and additionally, prognostic for adverse survival outcomes. In the multivariate analysis, high H2BC12 levels were identified to be an independent predictor for poor survival outcomes of LGG patients. Pathways in cancer, signaling by Wnt or PI3K-AKT signaling pathway, DNA repair, cellular senescence and DNA double strand break repair were differentially activated in the phenotype that positively associated with H2BC12.
Conclusion

H2BC12 is a promising biomarker for the diagnosis and prognosis of LGG.


Introduction
Gliomas are tumors occurred at glial cells, which are important for the function of cerebral nerve cells, and the most prevalent primary malignancy in brain tumors (1). According to the World Health Organization (WHO) classi cation, pathologically con rmed gliomas can be categorized into four grades: I, II, III, and IV. Low-grade gliomas (LGG) contain grades II-III gliomas characterized by highly variable clinical behaviors (2). It is known that LGG, as compared to high-grade gliomas (grade IV), correlates to a more favorable survival outcome, while it was reported to have a 70% risk of disease progression in 10 years (1). Aggressive high-grade gliomas have poor outcomes, even when managed by surgical resection and radiotherapy or chemotherapy (3,4). Due to the highly offensive property, it is impossible to achieve a complete cure in LGG. In this context, delaying tumor onset and reducing tumor progression are the most challenging issues, urgently prompting us to search for new sensitive biomarkers and new targets for treatments and diagnosis of LGG.
It is known that genetic abnormality predisposing to tumorigenesis is always accompanied by epigenetic alterations. Aberrant histone modi cations, for example, can potentially enhance the effects of oncogenic drivers in disease progression, metastatic potential, and resistance to therapy (5). Structurally, histone modi cation-related proteins are responsible for the compact structure of chromatin in nucleosomes and can be modi ed via diverse enzymes, including histone family genes (H2A, H2B, H3 and H4), two heterodimers (H2A, H2B), and one DNA-associated H3/H4 tetramer. Heterodimers H2A/H2B are important in chromatinrelated processes involved in transcription, DNA replication and repair. It is established that H2B monoubiquitination (H2Bub1) at lysine 120 is vitally signi cant in proper DNA repair, and the absence of H2Bub1 is associated with abnormal H2AX phosphorylation resulting in durable DNA damage response (6, 7). Notably, RNF20/40, ubiquitin ligases indispensable for H2Bub1, were also reported to be a part of tumorigenesis. Recent research indicated that low levels of H2Bub1 were prognostic for disease progression, which supported the role of H2Bub1 as a tumor suppressor (8). Combining the above ndings, histone genes play a crucial role in tumorigenesis and progression.
Here, H2B Clustered Histone 12 (H2BC12) was studied from aspects of expression in LGG tissue, associations with clinical characteristics, and the most involved biological pathways. Our ndings suggested that H2BC12 can be recognized as a promising biomarker for LGG diagnosis and prognosis.

Data acquisition
Target RNA-seq data in TPM format, which were documented in TCGA and GTEx databases, were jointly processed by the Toil work ow software (9)  Correlations between the H2BC12 expression and all genes were characterized by R (v.3.6.3), followed by GSEA analysis using the R package clusterPro ler (10). Signi cance threshold was set to |ES| > 1, p.adjust < 0.05, and FDR < 0.25.

Analysis Of Immune In ltration
From Bindea's investigation (11), the marker gene of 24 immune cells was retrieved. Based on LGG mRNA TPM data, single-sample GSEA (ssGSEA) (12) was utilized to quantify the number of tumor-in ltrating immune cells.
Spearman correlation was used to determine the relationship between H2BC12 and these 24 types of cells. The ggplot2 package was used to create the gures.
Statistical analysis R (v.3.6.3) was run to complete all statistical analyses of the study. Diagnostic receiver operating characteristic (ROC) curve was generated using package pROC, while time-dependent ROC (tROC) curve was plotted assisted by package timeROC. Differential expression of H2BC12 in tumor versus normal was statistically analyzed via Wilcoxon rank sum tests. For correlational analysis between the H2BC12 mRNA and clinicopathologic characteristics of LGG, the tumor samples were assigned into two cohorts representative of high and low H2BC12 expression, respectively, with the cutoff value as the median H2BC12 expression of all samples. Chisquare test was implemented to identify the signi cance. Comparisons between two sets of data were completed by Wilcoxon rank sum test upon two groups or Kruskal-Wallis test when there were three groups or more. Prognostic signi cance of H2BC12 mRNA expression and clinicopathologic characteristics for overall survival (OS) of LGG patients were identi ed by univariate and multivariate Cox regression analysis. The survival signi cance of H2BC12 mRNA expression in subgroups of clinicopathologic characteristics was investigated by strati cation and Kaplan-Meier analysis.

Clinical characteristics
In total 523 primary tumor samples were obtained from the TCGA-LGG dataset. Matched clinical data were retrieved, involving WHO grade, IDH status, 1p/19q codeletion, primary therapy outcome, gender, race, age, histological type, laterality and OS event (Table 1), which revealed that LGG is much common among the youth and the elderly.  Figure 1A, p<0.001). Besides, H2BC12 protein expression was also detected by immunohistochemistry using the HPA database (https://www.proteinatlas.org/), indicating that the H2BC12 proteins were in high levels in both LGG tissue and normal tissue ( Figure 1B). Together, H2BC12 might be an oncogene in LGG and play a vital role in the normal development of the brain.

Roc Analysis For H2bc12 In The Diagnosis Of Lgg
ROC curve was plotted to evaluate the diagnostic signi cance of H2BC12 mRNA expression for LGG. The area under the curve (AUC) was 0.823 ( Figure 2A), indicating signi cance in distinguishing between normal and tumor samples with certain accuracy. Besides, tROC curves were drawn to identify the predictive ability of H2BC12 mRNA for OS of LGG patients. The AUC values for 1-, 2-, and 3-year OS were 0.766, 0.702, and 0.677, respectively ( Figure 2B). It was suggested that H2BC12 is equipped with a good prognostic performance.

Correlations Between H2bc12 Mrna And Clinicopathologic Characteristics Of Lgg
The correlational analysis demonstrated that there were signi cant associations between the H2BC12 mRNA and clinicopathologic characteristics including WHO grade, IDH status, 1p/19q codeletion, primary therapy outcome and histological type ( Figure 3A-E

Role Of H2bc12 In Lgg Patient Survival
In survival analysis, the OS of patients with high H2BC12 expression was much poorer as compared to those with low H2BC12 expression ( Figure 4A, p<0.001), and similar results were observed as regards DSS and PFI ( Figure 4B-

Clinical Strati cation
As proven in the multivariate Cox regression analysis, WHO grade, primary therapy outcome, IDH status, age and histological type were independent prognostic factors for the OS in LGG. In subsequent work, clinical strati cation was conducted based on the TCGA-LGG dataset, and it was found that in subgroups of WHO grade: G2, WHO grade: G3, primary therapy outcome: PD&SD, primary therapy outcome: PR&CR, IDH status: Mut, age<=40 and age>40, patients with low H2BC12 expression had better survival outcomes than those with highly expressing H2BC12 ( Figure 5, p<0.001). This re ected that H2BC12 had independent prognostic signi cance for OS of LGG patients and increased H2BC12 level is associated with poorer OS.

H2bc12 -Related Signaling Pathways Based On Gsea
GSEA was performed to nd the activated signaling pathways related to H2BC12 in LGG. Based on the Curated collection, there were six signaling pathways activated in H2BC12 overexpressed phenotype, including pathways in cancer, signaling by Wnt or PI3K-AKT signaling pathway, DNA repair, cellular senescence, and DNA double strand break repair ( Figure 6). Based on the Hallmarks collection de ned by MSigDB, other than the six pathways above, KRAS signaling up, TNFA signaling via NFKB, G2M checkpoint, glycolysis, hypoxia and p53 pathway also presented with signi cant enrichment in H2BC12 overexpressed phenotype (Figure 7).
Collectively, the H2BC12 mRNA expression may serve as an important player in the initiation and development of LGG.

H2BC12 expression is linked to the level of immune in ltration
Tumor-in ltrating lymphocytes are independent indicators of cancer survival. As a result, we evaluated whether H2BC12 expression is related to the level of immune in ltrate in LGG. According to our ndings, H2BC12 showed a strong positive correlation with macrophages, eosinophils, neutrophils, and T cells; H2BC12 exhibited a strong inverse relationship with pDC, NK CD56bright cells, TReg, and DC ( Figure 8A). Further analysis showed that compared with the low-H2BC12 group, the in ltration level of Neutrophils and T cells in the high-H2BC12 group was signi cantly increased ( Figure 8B). The in ltration levels of pDC, NK CD56bright cells, TReg and DC was signi cantly reduced in the high-H2BC12 group ( Figure 8C).

Discussion
Gliomas are fetal tumors most prevalent in the central nervous system (CNS).
LGG (WHO II, III) are low-grade tumors that grow slowly with lesser malignant properties than high-grade tumors. However, there is a high risk of disease progression to advanced glioma in most LGG patients (1). Besides, there was research reporting that patients with LGG receiving chemotherapy would experience a poor outcome (13). According to bioinformatics, the WHO included several molecular markers, such as IDH mutation status, chromosome 1p or 19q codeletion (1p/19q codeletion) status, into the guideline for diagnosis of glioma, to raise the accuracy in disease diagnosis and further treatment (14). In this context, the demand of biomarkers with early diagnostic value is increasing, which will be of vital signi cance for the treatment and prognosis of LGG patients.
In this study, we rstly obtained RNA-seq data documented in TCGA-LGG and matched normal samples from GTEx in the UCSC XENA database, demonstrating that H2BC12 mRNA signi cantly increased in tumor tissue compared to the normal control, suggesting that H2BC12 might be active in promoting LGG initiation. Kim et al. (15) reported the top six most highly expressed genes in breast cancer, including HIST1H2BK (H2BC12), STAT3, CTSD, SREBF1, IGFBP5, and DDR1, from 49 signature genes of tumor dormancy based on cancer cell line data and microarray data, which further veri ed the role of H2BC12 as a potential tumor dormancy marker (15).
Dormant cells are highly adaptable in chemotherapy since they can rapidly target proliferating cells. In the meantime, they can still survive for a long time and even reproduce after chemotherapy is terminated. Han (21). It was studied that DNA repair genes are associated with the LGG (22). DNA repair damage is a main cause for the development of radio-resistance and chemo-resistance in gliomas (23).
All these ndings indicate the potential important role of H2BC12 in LGG progression.
In all, this research veri ed the signi cance of H2BC12 in the diagnosis and prognosis of LGG patients.
Inevitably, limitations still exist which remain to be resolved. First, the study was carried out only with bioinformatics analysis, requiring further validation in clinical samples. Second, there is a need to clarify the H2BC12-mechanism of action in LGG.

Conclusion
This study identi ed the differentially up-regulated expression of H2BC12 in LGG tissue and proved its signi cant ability in predicting adverse overall survival of LGG patients. H2BC12, therefore, is promising to be applied for the diagnosis and prognosis of LGG.

Abbreviations
LGG      Clinical strati cation analysis of the survival difference between patients with LGG in the high-and low-H2BC12 groups by the WHO grade, primary therapy outcome, IDH status, age and histological type.