Potential impact of ADAM- 10 genetic variants with the clinical features of oral squamous cell carcinoma

A disintegrin and metalloproteinase domain- containing protein 10 (ADAM- 10) involves in the tumour progression, but the impacts of single- nucleotide polymorphism (SNP) of ADAM- 10 on oral squamous cell carcinoma (OSCC) remain unclear. The aim of this study was to investigate the influence of SNP of ADAM- 10 on the clinical features of OSCC in male Taiwanese. Five loci of ADAM- 10 SNPs including rs653765 (C/T), rs2305421 (A/G), rs514049 (A/C), rs383902 (T/C) and rs2054096 (A/T) were geno-typed by TaqMan allelic discrimination in 1138 OSCC patients and 1199 non- OSCC individuals. The ADAM- 10 SNP rs2305421 GG (AOR: 1.399, 95% CI: 1.045– 1.874, p = 0.024) and G allele (AOR: 1.170, 95% CI: 1.012– 1.351, p = 0.034) illustrated a significantly higher genotypic frequencies in the OSCC group compared to the distribution of the ADAM- 10 SNP rs2305421 AA wild type. In the subgroup analysis, the ADAM- 10 SNP rs383902 TC + CC was significantly correlated to tumour size larger than T2 in betel quid chewer (AOR: 1.375, 95% CI: 1.010– 1.872, p = 0.043), while the ADAM- 10 SNP rs653765 CT + TT was significantly associated with tumour size larger than T2 in cigarette smoker (AOR: 1.346, 95% CI: 1.023– 1.772, p = 0.034). The results from


| INTRODUC TI ON
The oral squamous cell carcinoma (OSCC) is a common cancer which belongs to the group of head and neck squamous carcinoma. 1 The initial presentation of OSCC may be a plaque in the oral cavity and oral lesion including oral potentially malignant disorders can progress to OSCC. [2][3][4] The common treatment option for OSCC including the surgical incision and radiotherapy, 1,5 and the immunotherapy for the OSCC have been developed in recent years. 6 The overall 5-year survival rate of OSCC had improved from 59% to 70% in the past decades in Taiwan. 7 The presence or progression of OSCC is related to certain predisposing factors in previous publications including cigarette smoking, betel quid chewing and alcohol drinking. 1,8,9 On the other hand, the existence of human papillomavirus (HPV) is a risk factor for OSCC and the HPV 16 species contributes to the majority of HPV-related OSCC. 10 About the field of genetics, numerous studies have shown that the single-nucleotide polymorphism (SNP) may involve in the OSCC development. [11][12][13][14][15] A disintegrin and metalloproteinase domain-containing protein 10 (ADAM-10) belong to a group of transmembrane and secreted proteins and involves in the pathway of cell migration, cell adhesion, proteolysis and cell signalling. 16 In previous studies, the ADAM-10 is also associated with certain type of malignancies such as the prostate cancer and pancreatic cancer. 17,18 Besides, the silencing of ADAM-10 can influence the invasion, migration and proliferation of specific human tongue squamous cell carcinoma cell line, 19 and the up-regulation of ADAM-10 was observed in patients with OSCC. 20 Moreover, the SNPs rs514049 and rs514049 of ADAM-10 are involved in the hepatocellular carcinoma progression. 21 Still, whether the genetic polymorphism of ADAM-10 will affect the clinical condition of OSCC is unknown.
Because the SNP of ADAM-10 could influence the clinical course of other disease and ADAM-10 itself is related to OSCC development, 20,22 such possibility may exist which need additional elucidation.
In this study, we hypothesized that ADAM-10 SNPs (rs653765, rs2305421, rs514049, rs383902 and rs2054096) might influence the susceptibility of OSCC in an Asian male population. The effects of several risk factors of OSCC were also considered in our analytic model.

| Subject selection
This study was executed in the Chung Shan Medical University Hospital which locates in the central Taiwan region. After obtaining the informed consents, 1138 men with the diagnosis of OSCC participated this study. The diagnosis standard of OSCC in our study is according to the result of incisional biopsy which was judged by an experienced oncologist/otolaryngologist. For the comparison, another 1199 non-cancer male constituted the control group. We obtained the demographic data such as the age, betel quid chewing, cigarette smoking and alcohol drinking from the medical documents.
In addition, the clinical stage, tumour-node-metastasis (TNM) stage and degree of cell differentiation were judged with the assistance of AJCC 7th chart. Our study was approved by the Institutional Review Boards of Chung Shan Medical University Hospital (project identification code: CS1-21151) and written informed consents were signed by all the patients in our study before the start of our project. For the processing of ADAM-10 genes and associated SNPs, we draw venous blood in the study population and the venous blood samples were preserved in ethylenediaminetetraacetic acid-containing tubes. Immediately after the sample acquisition, those venous blood were centrifuged and placed in a laboratory refrigerator at −80°C.

| DNA extraction and analysed ADAM-10 SNP with real-time PCR
Five ADAM-10 SNPs like the rs653765 (C/T), rs2305421 (A/G), rs514049 (A/C), rs383902 (T/C) and rs2054096 (A/T) were enrolled in the analysis model since the previous studies illustrated the potential correlation between these ADAM-10 SNPs and other carcinoma. 21 The DNA extraction and analyses procedures in our study were similar to the methods in our previous research. 23 We separated genome and DNA from leukocytes in venous blood samples

| Statistical analysis
We used the SAS version 9.4 (SAS Institute Inc, NC, USA) for the analyses included in our study. Firstly, the descriptive analyses were used to present the demographic data between the OSCC group and control groups, and Mann-Whitney U test or Fisher's exact test was used to evaluate the difference of demography between the two groups. Then, the genotypic frequencies of each ADAM-10 SNP was compared between the OSCC group and control group via the application of multiple logistic regression models after controlling for age, betel quid chewing, cigarette smoking and alcohol drinking. The adjusted odds ratio (AOR) and confidence intervals (CI) were produced in this process. We divided the OSCC patients into those with betel quid chewing and those without such habit, and we estimated the AOR and 95% CI between OSCC clinical characters and genotypic frequencies of ADAM-10 SNP rs383902 in each subgroup by multiple logistic regression models after controlling for age, alcohol drinking and cigarette smoking. Similarly, the AOR and 95% CI of ADAM-10 SNP rs653765 genotypic frequencies for OSCC clinical statuses were estimated by multiple logistic regression models after controlling for age, betel quid chewing and alcohol drinking in cigarette smokers and non-cigarette smokers of OSCC. A p value lower than 0.05 was defined as statistical significance.

| Basic information between the two groups
The demographic data between the two groups are presented in Table 1. The age distribution was similar between the two groups (p = 0.670), while the ratio of betel quid chewing, cigarette smoking and alcohol drinking were all significantly higher in the OSCC group than the control group (all p < 0.001). The detail of OSCC characters are also presented in Table 1.

| Genotypic frequencies of ADAM-10 SNPs in OSCC and non-OSCC male population
The distribution frequencies of the five ADAM-10 SNPs between OSCC patients and control group are demonstrated in Table 2. In the control group, the genotypic frequencies of ADAM-10 rs653765, rs2305421, rs514049 and rs2054096 were in Hardy-Weinberg equilibrium (p > 0.05). The ADAM-10 SNP rs2305421 GG revealed a significantly higher genotypic frequencies (AOR: 1.399, 95% CI:

| The relationship between ADAM-10 mRNA levels and clinical characters of head and neck squamous cell carcinoma in The Cancer Genome Atlas program
Concerning the results from TCGA, the ADAM-10 mRNA level was significantly higher in the HNSCC group than the control group (p < 0.0001) ( Figure 1A) and associated with lower 5-year survival rate of HNSCC (p = 0.008) ( Figure 1B). The ADAM-10 mRNA level was significantly higher in the clinical stage II of HNSCC compared to other clinical stages (all p < 0.05) ( Figure 1C) but did not associate with the N statuses ( Figure 1D). About the T statuses of HNSCC group, the ADAM-10 mRNA expression was not correlated to the T status in all HNSCC group (Figure 2A) and lifelong non-smoker ( Figure 2B), while the ADAM-10 mRNA expression was significantly higher the T2 status than other T statuses in current smoker (all p < 0.05) ( Figure 2C).

| DISCUSS ION
Briefly, our study demonstrated the higher distribution frequency The ADAM-10 and its genetic polymorphism had been proven to influence various diseases including neoplasms according to previous literatures. [25][26][27] The main function of ADAM-10 is the cell reaction like the cell migration and cellular signal pathways. 16 In a previous study, the ADAM-10 can alter the CD44 function at different levels and especially in the malignancies which with high CD44 level. 28 Also, the ADAM-10 can regulate the E-cadherin pathway which controls the cell adhesion, cell differentiation and tissue development. 29 On the other hand, the ADAM-10 is associated with the disease course of some diseases. The Alzheimer's disease may be retarded by ADAM-10 which can reduce the generation of amyloidβ peptides, 26 and the ADAM-10 SNP rs2305421 is related to late-onset Alzheimer's disease. 22 In another study, the mutation of ADAM-10 which causes loss-of-function can be a predisposing factor for the development of Alzheimer disease. 30 In addition, the ADAM-10 SNP TA B L E 3 Odds ratio and 95% confidence intervals of clinical statuses associated with genotypic frequencies of ADAM-10 rs383902 in male oral squamous cell carcinoma patients.

TA B L E 4
Odds ratio and 95% confidence intervals of clinical statuses associated with genotypic frequencies of ADAM-10 rs653765 in male oral squamous cell carcinoma patients.  rs653765 was correlated to the formation of atherosclerotic cerebral infarction, thus may own therapeutic implications. 31 About the relationship between ADAM-10 and malignancy, the higher expression of ADAM-10 may trigger the metastasis of prostate cancer via the ephrin-A5 shedding. 17 And the patients carry ADAM-10 SNPs rs514049 and rs514049 have a higher chance to develop hepatocellular carcinoma in more advanced TNM stage. 21 On the other side, the clinical manifestations of OSCC are influenced by several genetic factors. 32 For example, the ADAM-10 expression is significantly correlated to the presence of OSCC. 20 Besides, the fibroblast growth factor receptor 4 with Arg allele carrier polymorphism can attribute to higher chance of nodal metastasis patients with OSCC than the wild type. 33  and another study also found the possible relationship between the ADAM-10 SNP rs2305421 G and Alzheimer's disease. 34 Thus, the ADAM-10 SNP rs2305421 G allele may be a risk factor for many diseases. According to the data from the TCGA, the mRNA expression of ADAM-10 was significantly higher in the HNSCC group than that in the non-HNSCC individuals. In general, the OSCC is a subtype of HNSCC and both cancers share similar predisposing factors and histological features. 35 Consequently, we assumed that the ADAM-10 writing -original draft (equal); writing -review and editing (equal).

This study was supported by Chung Shan Medical University
Hospital, Taiwan (CSH-2021-C-016).

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare that there is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.