Original article
Whole exome sequencing and transcriptome-wide profiling identify potentially subtype-relevant genes of nasopharyngeal carcinoma

https://doi.org/10.1016/j.prp.2020.153244Get rights and content

Abstract

Background

To date, no targeted therapy has been approved for nasopharyngeal carcinoma (NPC), suggesting that comprehensive understanding of genomic changes turns out to be an urgent need to break through the calm of currently known therapies of NPC.

Methods

Whole exome sequencing (WES) was performed for 14 NPC patients, including 6 NPC-IIA cases, 8 NPC-IIB cases. The cancer chip expression data named GSE12452 was downloaded from the Gene Expression Omnibus (GEO) database, and differentially expressed genes (DEGs) of each subtype were obtained using the Lima R package. Then gene ontology (GO) function enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed. Protein-protein interaction (PPI) network and Gene Set Enrichment Analysis (GSEA) were performed. Finally 7 potentially subtype relevant genes (PSRGs)1 were obtained.

Results

In total, 37 clinically relevant mutations (CRMs)2 were obtained from WES. The 2 NPC subtypes exhibited different mutational landscapes, indicating that different NPC subtypes harbor different CRMs. Notably, we discovered that mutations of CCND1 and FGF family appeared simultaneously in 3 NPC-IIB cases, but 0 in NPC-IIA. In addition, 1395 DEGs were identified from GSE12452. PI3K-Akt signaling pathway showed significant enrichment in both the pathway distribution of CRMs and KEGG analysis of DEGs, suggesting that it is a key pathway in the development of NPC. Through PPI analysis of genes involved in the PI3K-Akt pathways and expression significance analysis of DEGs co-expressed by the 2 subtypes, 54 genes finally were screened for expression significance analysis. The GSEA analysis between patients with high and low expression of 11 candidate genes were performed. As a result, 7 PSRGs were selected, including COL4A1, ASB9, RDH10, TNFRSF21, BACE2, EVA1C and LHX2.

Conclusions

These results indicate that different NPC subtypes have different genetic changes, suggesting that they may be potential targets for the diagnosis and treatment of NPC, and ultimately point to new strategies for intelligence.

Introduction

Nasopharyngeal carcinoma is a special head and neck tumor that originates from the lining of the nasopharyngeal epithelium, which has obvious epidemiological characteristics, ethnicity, regionality and family aggregation. There are 3 pathological types of nasopharyngeal carcinoma, including keratinizing squamous cell carcinoma (type I), nonkeratinizing differentiated carcinoma (type IIA), and nonkeratinizing undifferentiated carcinoma (type IIB), are closely related to the degree of cell differentiation.

At present, the main treatment method is comprehensive treatment based on TNM staging system, mainly radiotherapy. According to the guideline, there is no difference in treatment for the three histologic subtypes of nasopharyngeal carcinoma. However, studies have shown that different types of nasopharyngeal carcinoma have different survival periods, and the death rate of undifferentiated NPC is much higher than that of differentiated NPC. At present, with the rapid development of molecular medicine, more and more molecular targets are discovered, and their corresponding targeted drugs are used in the clinical treatment of cancer, which has significantly improved the survival rate of some solid tumors and reduced toxic side effects of treatment, such as lung cancer [1], breast cancer [2], and colorectal cancer [3]. However only a few clinical trials against EGFR and VEGF were conducted in NPC in the past decades. Unfortunately these targeted drugs have shown little effect and not all patients could benefit from targeted therapy [4], suggesting that comprehensive understanding of genomic changes turns out to be an urgent need to break through the calm of currently known therapies of NPC.

With the technology development of gene sequencing, computer analysis and bioinformatics analysis, more and more information hidden behind the tumor genomic come to the surface, including key mutations or pathway changes of tumor and drug-resistant mechanisms, which could play an important auxiliary role in tumor classification in addition to histopathological changes. As opposed to conventional assays (e.g. PCR, FISH, SNP), which only could detect known mutations, next generation sequencing could detect those potentially important mutations that are presented in very low prevalence or not been reported. Until now, some research has focused on the genome-wide information of NPC. Lin [5] et al. focused on the genomic landscape of NPC through whole exome sequencing and reveled some mutations that had not been implicated previously. Ali [6] et al. reported that different subtypes of NPC have different genomic changes. Wang [7] et al. developed a molecular classifier that could provide a reference for prognosis analysis.

In present study, the whole exome sequencing was performed for 14 NPC samples and 37 clinically relevant mutations (CRMs) were obtained, including 6 NPC-IIA cases, 8 NPC-IIB cases. In addition, we downloaded the cancer chip expression data named GSE12452 from GEO database, GO and KEGG enrichment analyses of the differentials expression genes (DEGs) obtained were performed. The protein-protein interaction (PPI) network of these DEGs was constructed and candidate genes were obtained. Finally, Gene Set Enrichment Analysis (GSEA) was performed and 7 potentially subtype relevant genes were obtained.

Section snippets

Materials and methods

Patient samples: We retrospectively screened patients with pathologically proved NPC in the Department of Radiation Oncology of the Affiliated Hospital of Qingdao University (Qingdao, China) from September 2016 to September 2019, and collected 14 formalin-fixed, paraffin-embedded (FFPE) tissue sections and matched blood specimens from the same patient. All samples were collected before treatment (radiotherapy or chemotherapy). At least two senior pathologists performed pathological diagnosis on

Results

Clinicopathological information: The clinicopathological characteristics of 14 NPC (including 6 WHO IIA type patients, 8 WHO IIB type patients) are summarized in Table1. The median age was 52years (range 18−68years), while the media age was 49.5 years in NPC-IIB, 53.5 years in NPC-IIA. No significant differences in age were detected across patients within the 2 subtypes (P = 0.855[t test]). The male-to-female ratio was 2.5(10 males and 4 females), which agreed with previous reports, and this

Discussion

In the present study, the whole exome sequencing was performed for 14 NPC samples and comprehensive genomic information were obtained. No obvious differences in the clinicopathological characters or mutation numbers or TMB were identified across patients within 2 subtypes. However, significant differences in the CRMs combinations were detected in 2 subtypes, which proved the wide heterogeneity of tumors and highlighted the potential significance of molecular therapies targeted specific

Author contributions

HL designed the experiments. JL conceived the study and performed the bioinformatics analyses. XL prepared figures and tables. SY collected the samples. JL and MJ wrote the manuscript. All authors read and approved the final manuscript.

Data availability statement

The data included in the current study are available in the GEO database (https://www.ncbi.nlm.nih.gov/geo).

CRediT authorship contribution statement

Ji Liu: Investigation, Writing - original draft. Xu Li: Formal analysis, Visualization. Shanshan Yang: Resources, Data curation. Junjun Mou: Writing - review & editing. Haijun Lu: Conceptualization, Methodology.

Declaration of Competing Interest

The authors declare that there is no conflict of interests.

Acknowledgments

The authors thanks all the individuals who donated their FFPE tumor tissue sections and blood specimens for this project. Without their support, this study would not be feasible.

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  • 1

    PSRG: potentially subtype relevant genes.

    2

    CRMs: clinically relevant mutations.

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