Interaction Between Rare Variants in NOTCH1 and Betel Quid Chewing in Oral Squamous Cell Carcinoma

Abstract

Background:

In this study, we investigated rare variants of the NOTCH1 gene located near somatic mutations as surrogate markers, as well as the relationship of these rare variants with betel quid (BQ) chewing and the occurrence of oral squamous cell carcinoma (OSCC).

Materials and Methods:

A total of 410 patients diagnosed with OSCC and 282 unrelated, healthy subjects without cancer were recruited from two medical centers in Taiwan. Odds ratios (OR) and 95% confidence intervals (CI) were assessed by logistic regression. The Cox proportional hazard model was used to assess the interaction between rare NOTCH1 variants and BQ chewing in OSCC.

Results:

The genetic variant rs139994842 in exon15 of NOTCH1 was significantly associated with an increased risk of OSCC (OR = 2.88 95% CI: 1.07-7.79), and the association between rs202133782 in exon13 of NOTCH1 with OSCC was borderline significant (p = 0.0627). Moreover, a combination of four rare variants was significantly associated with OSCC (p = 0.012). Patients who carried these NOTCH1 variants were at a higher risk of recurrence (OR = 18.95; 95% CI, 1.01-326.74; p = 0.0428). Furthermore, the OSCC incidence rate was significantly higher in patients who had chewed BQ (for a mean of 24 years) and had a NOTCH1 variant (p < 0.0001).

Conclusion:

This information is applicable to prevention; the surveillance of patients at risk; and for early detection to reduce morbidity and mortality from OSCC.

Introduction

Oral squamous cell carcinoma (OSCC) is one of the most common malignancies worldwide, with 400,000 new cases and 350,000 related deaths worldwide each year (Parkin et al., 1999; Argiris et al., 2008). However, the incidence and mortality of OSCC have geographical variation. Melanesia, South-Central Asia, and Central/Eastern Europe have been reported to have a higher incidence of OSCC, whereas that in Africa and Central America are lower (Henderson et al., 2012). The reasons for this geographical and/or ethnic variation may reflect diverse etiologic factors and inherited genetic backgrounds.

OSC carcinogenesis includes multiple genetic alterations and environmental influences. Its development and progression involve genetic and epigenetic alterations within signaling pathways, such as those involving TP53, NOTCH1, CDKN2A, CCND1, CASP8, and FAT1 (Agrawal et al., 2011; Stransky et al., 2011; Pickering et al., 2013). The NOTCH signaling pathway is critical for cell proliferation, differentiation, and apoptosis (Artavanis-Tsakonas et al., 1999). NOTCH proteins regulate various oncogenes and tumor suppressor genes. Therefore, aberrant activation of NOTCH1 signaling may play a dual role in tumorigenesis. NOTCH1 signaling seems to play an important role in the development of OSCC. In Caucasians, NOTCH1 mutations occur in 11-15% of OSCC patients, with NOTCH1 and TP53 being the most frequently mutated genes found in OSCC tumors (Agrawal et al., 2011; Stransky et al., 2011; Pickering et al., 2013). Recently, studies have reported that the NOTCH1 mutation frequency was greater than 40% in Chinese patients with OSCC. In particular, Chinese patients with NOTCH1 mutations had a significantly shorter overall and disease-free survival rate (Song et al., 2014). These data indicate that NOTCH signaling is involved in oral tumorigenesis of both Asian/Chinese and Caucasian populations.

Betel quid (BQ) chewing is prevalent in Central Asia. It is estimated that currently 10% of the world's population, or nearly 700 million individuals, chew BQ regularly (Sharan et al., 2012). Research conducted in Taiwan has confirmed a correlation between BQ use and oral, pharyngeal, and laryngeal cancers. In addition to BQ chewing, smoking and alcohol consumption are also major risk factors for OSCC (Lazarus et al., 1996). The high frequency of NOTCH1 mutations in head and neck squamous cell carcinoma (HNSCC) has been reported to be associated with tobacco and alcohol consumption in Caucasians (Sun and Califano, 2014). However, somatic mutations in NOTCH1 were not associated with either smoking or alcohol consumption in Chinese populations (Izumchenko et al., 2015; Mao, 2015). These conflicting results may be due to variations in prevalence of substance use and habit. BQ chewing is prevalent in Central Asia and Taiwan, whereas smoking and excessive alcohol consumption are prevalent in Central/Eastern European countries, such as France, Germany, and Hungary (Ko et al., 1995; Humans, 2004). Oral cancer is primarily caused by tobacco and alcohol use, and accounts for 2-3% of all cases of cancer in Western countries. However, BQ consumption reportedly only accounts for 7-8% of oral cancer cases in Taiwan (Lee et al., 2012).

Frequent NOTCH1 somatic mutations have been reported to be associated with OSCC (Agrawal et al., 2011; Stransky et al., 2011; Pickering et al., 2013). However, these somatic mutations occurred sporadically throughout in the genome and cannot be applied as prognostic markers of the disease, and no reliable markers of clinical utility have been identified to date. Herein, we investigated rare variants of NOTCH1 near somatic mutations as surrogate markers and the relationship between these variants, BQ chewing, and OSCC occurrence.

Materials and Methods

Subjects

A total of 410 patients diagnosed with OSCC and 282 unrelated, healthy control subjects were recruited from the Department of Dentistry and the Department of Otorhinolaryngology at Kaohsiung Medical University Hospital China, Medical University Hospital, and Changhua Christian Hospital (Taiwan). Data on social, demographic, and anthropometric factors; smoking, drinking, and BQ chewing habits; medical history; and current medications were obtained by interviewing subjects. An individual without a history of alcohol, BQ, or cigarette use was defined as a nondrinker, nonchewer, or nonsmoker, respectively. Details regarding BQ, alcohol, and tobacco included types consumed, age of initial use, daily consumption, use frequency, years of use, and achievement of abstinence (Chiang et al., 2008). This study was approved by the institutional review boards of Kaohsiung Medical University Hospital, China Medical University Hospital, and Changhua Christian Hospital, as well as the informed consent committee on human subjects and biospecimen unitization committee.

Genotyping methods

Based on previous sequence screening results (Liu et al., 2016), we used single-nucleotide polymorphisms (SNPs) close to somatic variants or polymorphisms for clinical samples. In this regard, a total of four SNPs (rs201174576, rs202133782, rs139994842, and rs200699541) were selected. Genomic DNA was extracted from peripheral blood samples of subjects using a Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN). Genotyping of the four SNPs from OSCC and normal subjects was performed using the Sequenom MassARRAY System (San Diego, CA) by the Academia Sinica National Genotyping Center (Taipei, Taiwan).

Statistical analysis

All genotype frequencies of the control population were tested for Hardy-Weinberg equilibrium. The difference between the practical and expected number of each genotype was compared by χ² test. Hardy-Weinberg equilibrium was assumed for p-values >0.05. A Student's t-test was applied to compare age differences, while Pearson χ² or Fisher's exact tests were used to determine difference in gender distribution and SNP genotype frequencies between OSCC and control subjects. The odds ratio (OR) and corresponding 95% confidence interval (CI) were assessed using logistic regression. All tests are two-tailed and a p < 0.05 was considered to be statistically significant. Interactions between BQ chewing and NOTCH1 variants were analyzed with a Cox proportional hazards regression model. The cumulative incidence for the rate of different groups of the OSCC endpoint was calculated according to Kaplan-Meier estimates. Statistical analyses were performed using SAS 9.2 software, and results are reported as the mean ± standard deviation, unless specified otherwise.

Results

The mean age of OSCC cases and controls was 53.8 ± 10.4 years and 50.8 ± 10.4 years, respectively (p = 0.004).The majority of participants were males; 96.3% and 97.5% of OSCC and healthy subjects were males, respectively (p = 0.386). Among the 410 cases, 72.4% drank alcohol compared to 29.1% of the 282 controls (p < 0.0001). Of OSCC subjects, 82.2% consumed BQ compared to 13.5% of the controls (p < 0.0001), and 84.1% of OSCC cases smoked cigarettes versus 52.8% of controls (p < 0.0001) (Table 1). The sample of 1000 genome project included more than 2500 individuals from 26 global populations (Genomes Project et al., 2015). The 1000 Genomes Project provided a comprehensive description of common human genetic variation by applying whole-genome sequencing. We used a sample of 1000 genome project as normal controls to compare allelic difference to OSCC patients.

Table 1.

Characteristics of Oral Squamous Cell Carcinoma and Control Subjects

	Case	Control
Variable	N = 410	N = 282	p
Male, N (%)	395 (96.3)	275 (97.5)	0.386
Age, year	53.8 (10.4)	50.8 (14.3)	0.004
Alcohol, N (%)
None	113 (27.6)	200 (70.9)
Ever	93 (22.7)	20 (7.1)
Current	204 (49.7)	62 (22.0)	<0.0001
BQ Chewing, N (%)
None	73 (17.8)	244 (86.5)
Ever	194 (47.3)	30 (10.6)
Current	143 (34.9)	8 (2.9)	<0.0001
Cigarette, N (%)
None	65 (15.9)	133 (47.2)
Ever	110 (26.8)	66 (23.4)
Current	235 (57.3)	83 (29.4)	<0.0001

Data are presented as the mean (standard deviation).

BQ, betel quid.

Table 2 lists main results of the association between the four NOTCH1 variants and OSCC risk. Variant rs139994842 in exon 15 of NOTCH1 was significantly associated with an increased risk for OSCC (OR = 2.88; 95% CI: 1.07-7.79; Table 2), while the association between rs202133782 in exon 13 and OSCC showed borderline significance (p = 0.0627). Due to a lower frequency, single variants rs201174576 and rs200699541 were not significantly associated with OSCC occurrence. Then, we combined these three rare variants to examine their association with OSCC occurrence and found a significant association (p = 0.012; Table 2). The allelic frequency of the three variants was also obtained from the 1000 Genomes Project (http://browser.1000genomes.org.) as control, and again significantly associated with OSCC occurrence (p < 0.0001). Patients who carried NOTCH1 variants had a higher risk of recurrence (OR = 18.95; 95% CI, 1.01-326.74; p = 0.0428). The penetrance rate for the combined variants was 100%. Subjects carrying these combined NOTCH1 variants had OSCC.

Table 2.

Associations Between Coding Single-Nucleotide Polymorphisms on NOTCH1 and Oral Squamous Cell Carcinoma

dbSNP rs#	Variation types	Function	Genotype	Case (n = 410)	Control (n = 282)	1000 genome^a (n = 5000)	OR (95%CI)	p^b	p^c
rs139994842	Common	Synonymous	A/G	28	8	24	2.88 (1.07-7.79)	0.0268	<0.0001
			G/G	379	269	4976	1
rs201174576	Rare	Missense	G/T	3	0	1	4.85(0.25-94.32)	0.2746	0.0008
			T/T	407	282	4999	1
rs202133782	Rare	Synonymous	A/G	5	0	3	7.66(0.42-139.15)	0.0627	<0.0001
			G/G	405	282	4997	1
rs200699541	Rare	Missense	C/T	1	0	1	2.07(0.08-50.98)	0.4066	0.1154
			C/C	409	282	4999	1
Combined rare	—	—	Mutation	9	0	5	18.95(1.01-326.74)	0.0123	<0.0001
variants			None	401	282	4995	1

Fisher's exact test was used to make comparisons between the oral cancer cases and normal controls.

p-value and genotyping frequencies of normal controls obtained from the 1000 Genomes Project.

p-value for genotype test.

p-value for allele test.

CI, confidence interval; OR, odds ratio; SNPs, single-nucleotide polymorphisms.

Interaction between rare NOTCH1 variants and BQ chewing

The mean follow-up years for OSCC patients who chewed BQ was 24.53 ± 10.59 years. Alcohol consumption, BQ chewing, and cigarette smoking were significantly associated with risk of OSCC (Table 3). Due to collinearity, only BQ chewing was associated with OSCC after adjusting for age and substance use (adjusted OR = 22.94; 95% CI = 13.58-38.7; Table 3). We further examined the interaction between rare NOTCH1 variants and BQ chewing using the Cox proportional hazard model and found that the adjusted hazard ratio (HR) increased with BQ chewing and presence of rare NOTCH1 variants compared to nonchewer controls without a NOTCH1 variant. OSCC cases carrying a rare NOTCH1 variant had a greater risk of OSCC [HR = 4.01; 95% CI: 1.25-12.85; p = 0.0194]. OSCC patients who chewed BQ, but did not carry a NOTCH1 variant also had an increased risk for OSCC occurrence (HR = 10.95; 95% CI, 8.16-14.69; p < 0.0001). Furthermore, habitual BQ chewing and presence of a NOTCH1 variant had a significant synergistic effect on OSCC occurrence (HR = 14.35; 95% CI, 7.66-26.89; p < 0.0001; Table 3). Of the 24-year mean BQ exposure period, the OSCC incidence rate was significantly higher in OSCC patients who chewed BQ and had a NOTCH1 variant (p < 0.0001; Fig. 1).

FIG. 1.

Kaplan-Meier curve of the cumulative oral squamous cell carcinoma occurrence rate stratified with BQ chewing and NOTCH1 mutations. BQ, betel quid.

Table 3.

Interaction Between Rare NOTCH1 Mutations and Multiple Substance Use

Variables	OR	95% CI	aOR	95% CI	HR	95% CI
Alcohol drinking	5.00	3.49-7.16	1.38	0.83-2.30
BQ chewing	30.13	18.96-47.90	22.94	13.58-38.77
Cigarette smoking	5.58	3.69-8.45	1.41	0.80-2.47
0 + N					1.00
0 + M					4.01	1.25-12.85
1 + N					10.95	8.16-14.69
1 + M					14.35	7.66-26.89

0, BQ nonchewer; 1, BQ chewer; N, no rare NOTCH1 mutations; M, presence of rare NOTCH1 mutations.

aOR, adjusted odds ratio; HR, hazard ratio.

Discussion

In this study, we identified four rare variants of NOTCH1 that associate with risk of OSCC. Although somatic mutations of NOTCH1 associated with HNSCC have been published in several previous studies (Agrawal et al., 2011; Stransky et al., 2011; Keysar et al., 2013; Song et al., 2014; Sun et al., 2014), we further assessed rare variants that serve as surrogate markers, which may predict risk of OSCC. The penetrance rate for the combined rare variants was 100% (Table 2). These results could be further applied to clinical screens for detecting high-risk patients earlier.

For NOTCH1, exposure to different carcinogens and variations in inherited genomic background likely contributes to the differences observed between Asian and Caucasian populations (Song et al., 2014). Because exposure to tobacco and alcohol has been associated with a high frequency of NOTCH1 mutations in OSCC in Asians and Caucasians (Brennan et al., 1995; Aoyama et al., 2014; Song et al., 2014; Mao, 2015), it was important to determine their potential impact on NOTCH1 mutation. Our study demonstrated that the interaction between BQ chewing and NOTCH1 mutation resulted in a high risk for OSCC. However, it is still unclear whether cultural and genetic differences play a role in OSC carcinogenesis. However, we cannot rule out the possibility that rare NOTCH1 variants exposed to these risk factors might be a result of OSCC occurrence. Further investigation of the NOTCH1 variant signatures in OSCC tumors from distinct ethnic and geographic areas will be needed to clarify the precise role of NOTCH signaling in development and progression of OSCC.

The etiology of OSCC is a multistep process requiring the accumulation of a wide range of genetic and epigenetic alterations, influenced by the patient's genetic predisposition and environmental factors (Hashibe et al., 2009). These predisposing factors may lead to a wide range of genetic and epigenetic events, including inactivation of tumor suppressor genes and activation of oncogenes and promoter methylation (Brinkman and Wong, 2006). Previous studies reported that P16 methylation was detected in 63% of OSCCs in betel-chewing individuals with oral cancer (Tran et al., 2005), related to higher-grade dysplasia (Ruesga et al., 2007) and associated with the malignant transformation of leukoplakia (Lingen et al., 2011), especially in relationship to tobacco use. In addition to P16, DAPK and MGMT promoter hypermethylation have been reported in association with OSCC (Supic et al., 2011; Don et al., 2014). Promoter hypermethylation can be detected in tumors and histologically healthy tissue adjacent to the tumor. These genes may be involved in oral carcinogenesis and might serve as an early marker for early detection of oral cancer (Don et al., 2014).

The strengths of our study include comprehensive environmental exposure information and utilization of multivariate logistic regression analysis to assess the combinatory effect of rare genetic variants on the risk of developing OSCC. We recognize the complexity of the NOTCH1 variants identified in our study and acknowledge several limitations, which will require further investigation. In particular, our study cohort had incomplete social/behavioral information, such as tobacco and alcohol consumption/use and follow-up years, which prevented us from analyzing effects on the use of multiple substances among our ethnic group. However, taken together, our findings provide new insight into the role of NOTCH1 in OSCC development and help explain its progression.

Conclusions

These results can be applied to prevention, careful surveillance of patients at risk, and early detection for reducing morbidity and mortality related to OSCC. However, larger studies are required to further characterize the contribution of genetic variations and epigenetic effects to predict OSCC risk.

Footnotes

Acknowledgments

We thank the National Center for Genome Medicine for their technical support. This study was supported, in part, by the Taiwan Ministry of Health and Welfare, Clinical Trial, and Research Centre of Excellence (MOHW 106-TDU-B-212-113004), the Health and Welfare surcharge of tobacco products (MOHW 106-TDU-B-212-144003 Taiwan), and the China Medical University Hospital (DMR-106-032, 132).

Author Disclosure Statement

No competing financial interests exist.

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