Could the methylation of RASSF1A be the potential epigenetic biomarker for nasopharyngeal carcinoma? – Systematic review and meta-analysis

doi:10.21203/rs.3.rs-35655/v1

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Could the methylation of RASSF1A be the potential epigenetic biomarker for nasopharyngeal carcinoma? – Systematic review and meta-analysis

https://doi.org/10.21203/rs.3.rs-35655/v1

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Background: RASSF1A is a tumor suppressor gene. The methylation of RASSF1A has been reported to be associated with the nasopharyngeal tumorigenesis. Aiming to evaluate the association between the RASSF1A gene methylation and nasopharyngeal cancer, and its correlation could be used as an epigenetic biomarker for NPC cancer risk based on meta-analysis.

Methods: Relevant articles were identiﬁed by searching MEDLINE database. The frequency and Odds ratio (OR) were applied to estimate the effect of CDH-1 methylation based on random-/fix-effects models. Furthermore, subgroup analyses were performed by test-method, ethnicity, source of NPC samples.

Results: Total of 16 studies, included 1,766 samples: 1,178 samples from NPC samples, and 588 samples from non-cancerous samples, were enrolled in the meta-analysis. The overall frequency of RASSF1A methylation were 55.98% and 1.70% in case-group and control-group, respectively. By removing the poor relative studies, the heterogeneity was not observed among included studies. The association between the RASSF1A gene methylation and risk of NPC was also confirmed by calculating OR value of 51.43 (95%OR = 28.12-94.08) in fix-effects model (Q = 10.63, p = 0.99, I² = 0.00, 95% CI = 0.00-0.00). Additionally, the significant association was also found between the methylation of RASSF1A gene and subgroups.

Conclusion: this was the first meta-analysis provided scientific evidences to suggest the RASSF1A methylation was the potential biomarker for risk of NPC.

Cancer Biology

RASSF1A

nasopharyngeal carcinoma

meta-analysis

methylation

tumor suppressor gene

Nasopharyngeal carcinoma (NPC), a prevalent nasopharyngeal malignant tumor with the remarkable differences in distribution, gravitating toward Southern Asia [1–7]. According to the data from Global Cancer Observatory, updated to 2018, 129,079 new nasopharyngeal cases were recorded in the world, of which 72,987 nasopharyngeal death cases were occurred. Even though improvements in nasopharyngeal cancer treatment have been achieved, the diagnosis at an advanced stage led to reduce the success rate of treatment as well as the survival of patients. Thus, the early screening and diagnosis represents the beneficial opportunities to increase the survival of patients as well as the effects of nasopharyngeal cancer treatment. Because of the non-specific symptoms related to the early stage of NPC as well as the deeply seated location of nasopharynx, it leads to the major obstacle to early screening of NPC [8]. Therefore, effective biomarkers are truly needed [9]. Today, much efforts have been made for identification of early biomarkers based on focusing the etiological factors led to the nasopharyngeal tumorigenesis. The progression of NPC is a multiple steps associated with multiple factors, including the infection of Epstein-Barr Virus (EBV), environmental factors, as well as genetic and epigenetics alterations [10, 11]. Among them, DNA hypermethylation has been postulated as the best-studied epigenetic alterations in tumorigenesis [12]. The phenomenon of tumor suppressor gene’s (TSG) promoter cause the transcriptional silencing, led to gene inactivation, which inhibit the functions of those genes, resulting in the cancer development [13, 14].

Many TSGs have been recorded to be involved in the critical cancer-related cellular pathways. Thus, the patterns of TSG promoters’ methylation have been studied, and reported to be acted as the potential epigenetic biomarkers for NPC [9, 12]. RASSF1A (RAS-association domain family 1 isoform A), located 3p21.31, belonged to the family of RAS effectors, is a tumor suppressor gene coding protein. It functioned as the protein that modulates multiple apoptotic, cell cycle pathways and DNA repair process [15, 16]. Increasing evidences demonstrated that the inactivation of RASSF1A due to the hypermethylation, has been reported to associated with the pathogenesis of NPC. However, due to the different sensitivities and intra/inter-assay coefficients of variation of methods, the reported frequency of RASSF1A hypermethylation and its prognostic value is highly variable. Therefore, in current study, we performed the meta-analysis is to summarize the previously studies and identify the diagnosis and early screening value of RASSF1A for NPC.

Search strategy and inclusion/exclusion criteria

We conducted a comprehensive search strategy towards the guidelines of Preferred Reporting Items for Systematics Reviews and Meta-Analyses [17]. By using separation or combination of following keywords: “Nasopharyngeal carcinoma”, “methylation”, “RASSF1A”, “diagnosis”, “prognosis”, “epigenetic”, “hypermethylation”, were applied to search reveal published articles in MEDLINE database (Updated on December, 2019). Additional studies were also identiﬁed via the references listed in the articles.

Studies were deeply considered eligible only when they met all of the following inclusion criteria: i) The articles were limited to studies written in English; ii) case-control study designed; iii) provided that data about the frequency of RASSF1A methylation as well as the sample size in both case and control group. Exclusion criteria were as follows: i) The articles were written in other languages; ii) abstracts, case reports, letter to editor or unpublished articles were eliminated; iii) studies were related to other tumors and not specific for NPC; iv) studies lacked vital information for analysis.

Data extraction

The eligibility of each study, the relevant data from the eligible studies were independently retrieved by two authors. Disagreements were resolved through discussion within the third author or our research team. The relevant data were extracted from each study according to the data form, including first Author’s last name, year of publication, country where the study was performed, sample type, experimental methods to assess the methylation of RASSF1A, and number of cases and controls subjects.

Statistical analysis, publication bias and sensitivity analysis

MedCalc® software, by MedCalc Software Ltd, was applied to statistically analyze extracted data (https://www.medcalc.org/). The frequency of RASSF1A methylation was calculated in both case and control group. The strength of association between RASSF1A methylation and NPC was evaluated by Odds ratio (OR) with 95% confidence intervals (95%CI). In current study, the heterogeneity among the included studies was estimated by the Cochran Q test and I² statistics [18]. The cut-off point: p = 0.05 for the Q test and I² were used to test the heterogeneity between studies. The scale of I² value is classified as following: I² < 25%: no heterogeneity, 25% ≤ I² ≤ 50%: moderate heterogeneity, and I² > 50%: strong heterogeneity [18-20]. The random-effects model was applied if the heterogeneity among studies existed (p < 0.05 for Q test, I² > 50%). In the case of no between-study heterogeneity, a fixed-effects model was applied to compute the pooled ORs. In order to determine the presence of publication bias, the symmetry of the funnel plots in which ORs were plotted against their corresponding standard errors were assessed by the Begg’s funnel plot and Egger’s test (p < 0.05) indicates statistically signiﬁcant [21,22]. Additionally, the sensitivity analysis was also performed by sequential omission of individual studies to evaluate stability of the results.

Study characteristics

After exclusion of studies that not met the inclusion criteria, finally, 16 studies, published from 2001 to 2015, included 1,766 samples: 1,178 samples from NPC samples, and 588 samples from non-cancerous samples, were enrolled in the meta-analysis. The characteristics of included studies of RASSF1A methylation and risk of NPC were summarized in Table 1.

The number of NPC samples included studies ranged from 6 to 220 (mean: 42.07 ± 38.90), and number of control samples included studies ranged from 2 to 50 (mean: 21.78 ± 17.38). The patients’ ethnicity in 16 studies, comprised of 14 studies from Asian countries: China, Hong Kong, Singapore, Taiwan (counting for 87.50%), and 2 studies from African country: Tunisia (counting for 12.5%). Regarding to the test-method of RASSF1A methylation, twelve studies used MSP (counting for 75.00%), one study used COBRA (counting for 6.25%), one study used RT-qPCR (counting for 6.25%), one study used MMSP (counting for 6.25%), one study used MS-HRM counting for 6.25%).

Meta-analysis: The frequency of RASSF1A promoter methylation, and the association between RASSF1A gene methylation and NPC

The differences of RASSF1A gene methylation between the case-group and control-group in 16 studies, including 1,178 samples from NPC samples, and 588 samples from non-cancerous samples, were accessed. Concerning to the hetero heterogeneity between studies, as there was heterogeneity across studies in the case-group (Q = 443.5, p < 0.0001, I² = 93.91%, 95%CI for I² = 92.23-95.23), and no heterogeneity across studies in the control-group (Q = 9.67, p = 1.00, I² = 0.00%, 95%CI for I² = 0.00-0.00), thus, the random-effects model and fix-effects model were applied to calculate the frequency of RASSF1A gene in case-group and control-group, respectively (Fig. 1, Fig. 2). As the results, the frequency of RASSF1A gene methylation in case-group and control-group were 55.98% (ranged from 0.00% to 100.00%; 95% CI = 42.28-67.35) and 1.70% (ranged from 0.00 to 4.88%; 95% CI = 0.83-3.06), respectively. The meta-analysis result also indicated that the frequency of RASSF1A gene methylation in case-group was significant higher than control group.

The methylation of RASSF1A gene was significantly associated with an increased NPC risk with a pooled of Odds ratio (OR) of 33.40 (95%CI = 20.22-55.16) based on the fix-effect model (Q = 29.56, p = 0.29, I² = 12.05%, 95%CI = 0.00-44.45) (Fig. 3). The funnel plot of pooled analysis, which was small asymmetric, indicated that there was still small bias among included studies, therefore publication bias cannot be completely excluded as a factor of influence on the present meta-analysis. In additionally, the sensitivity analysis found that study by Chang et al. (2003) (OR = 1.54, 95%CI = 0.09-24.10), Chang et al. (2003) (OR = 0.46, 95%CI = 0.02-11.79) and Tian et al. (2013) (OR = 4.14, 95%CI = 0.80-21.29) were the relative poor-quality studies. Therefore, those studies were omitted to evaluate again the association between the methylation of RASSF1A gene and NPC risk through OR. As the result, the between heterogeneity decreased to I² = 0.00% (p = 0.99), indicated that no heterogeneity among enrolled studies was observed. Additionally, the association between RASSF1A methylation and risk of NPC increased, which was indicated by the increased OR of 51.43 (95%OR = 28.12-94.08) in fix-effects model (Q = 10.63, p = 0.99, I² = 0.00, 95% CI = 0.00-0.00) (Fig. 5) (compared to previously calculated OR of 33.40). Moreover, the funnel plot of pooled analysis, which was quite symmetric, indicated that there was no significant bias among the included studies (Fig. 6).

The subgroup analysis by ethnicity, test-method, as well as source of sample were performed. The results of subgroup analysis showed that the heterogeneity totally disappeared in all subgroup (Asian vs non-Asian countries, MPS vs other test-methods, tissue vs other source of samples, I² = 0.00, p > 0.05) (Table 2). With respect to the subgroups categorized by ethnicity, the significantly association between RASSF1A methylation and risk of NPC was observed among the Asian country and non-Asian countries in the fix-effect model (Asian countries: OR = 47.94, 95%CI = 25.61-89.75; non-Asian countries: OR = 135.96, 95%CI = 16.03-1,153.41). The subgroup analysis by source of NPC samples, there was a strong association between RASSF1A methylation and NPC among the NPC biopsy tissue group and non-biopsy group, including nasopharyngeal swab, mouth and rinsing fluid, plasma, blood in the fix-effect model (Biopsy tissue: OR = 55.29, 95%CI = 27.25-112.208; Non-biopsy: OR = 45.76, 95%CI = 15.56-134.60). Additionally, significant association between RASSF1A methylation and NPC risk among test-method subgroup was found in fix-effects model (MSP: OR = 40.86, 95%CI = 20.41-81.80; Other methods: OR = 76.24, 95%CI = 23.24-250.11).

Sensitivity analysis and publication bias

After removing the relative poor-quality study, the quite symmetric funnel plot, suggested there was no significant bias among included studies, was observed (Fig. 6). Additionally, the sensitivity analysis was performed to evaluate the stability and reliability of the conclusions according to the leave-one-out method by excluding one study. As the results, the pooled OR was ranged from 41.95 (95%CI = 22.13-79.49) to 55.06 (95%CI = 29.63-102.32) under the fix-effects model within the I² = 0.00 (p > 0.05) (Table 3), which confirmed the stability and reliability of the results. Therefore, the results, and conclusion of present meta-analysis, which was to evaluate the association between methylation of RASSF1A and NPC risk, were stable and reliable.

It is important to find out the effective biomarker for early screening of NPC. Based on the etiological factors of NPC, the methylation of TSGs promoter has been recognized as the common mechanism of the inactivation of TSG, leading to the tumorigenesis of NPC [13,39]. According to Xiong et al. (2004), they identified the susceptibility locus at the chromosome 3p21 linked to the NPC tumorigenesis based on the genome-wide linkage analyses of high-risk Chinese NPC patients [40]. RASSF1A, belonged this region, is reported as the tumor suppressor gene. The inactivation of RASSF1A via the methylation has been recognized as highly associated with the progression of NPC [41,42]. Notably that the methylation of TSGs promoter is reported as one of the earlier molecular modification during the human epithelial cells transformed to malignancy, and often occurred earlier than the changes of morphology of cancer [43]. Hence, the analysis of TSG hypermethylation, including RASSF1A, may be served as the potential epigenetic biomarker for early screening of NPC. As previous reports, the frequency of RASSF1A methylation in NPC was is highly variable, to determine whether RASSF1A methylation could be served as potential biomarkers for NPC, the meta-analysis and the subgroup analysis by ethnicity, source of samples, and test-methods were performed.

To our knowledge, this was the first meta-analysis of previous published studies, revealed that methylation of RASSF1A does the increasing of NPC, to evaluate the relationship between RASSF1A promoter and NPC tumorigenesis. In this meta-analysis, sixteen published studies included 1,766 samples: 1,178 samples from NPC samples, and 588 samples from non-cancerous samples, were enrolled. As the results, the overall frequency of RASSF1A methylation in the NPC and control-group were 55.98% (ranged from 0.00% to 100.00%; 95% CI = 42.28-67.35) and 1.70% (ranged from 0.00 to 4.88%; 95% CI = 0.83-3.06), respectively. It could be observed that the individuals with RASSF1A gene methylation was significantly associated with NPC based on the calculation of OR (OR = 33.40; 95%CI = 20.22-55.16) based on the fix-effect model (Fig. 3). However, the funnel plot as well as the sensitivity analysis found out that small bias among included studies still appeared. Therefore, the relative poor-quality studies, including study by Chang et al. (2003) (OR = 1.54, 95%CI = 0.09-24.10), Chang et al. (2003) (OR = 0.46, 95%CI = 0.02-11.79) and Tian et al. (2013) (OR = 4.14, 95%CI = 0.80-21.29) were removed. The heterogeneity between studies decreased to I2 of 0.00 in the fix-effects model. The resulting OR is 51.43 with a 95%CI ranged from 28.12 to 94.08, indicating a 51.43-fold increased odds of a positive outcome, and this increase was statistically significant at the 5% level. Therefore, the methylation of RASSF1A was significantly associated with the NPC tumorigenesis. It suggested the methylation of RASSF1A might play a crucial role in the pathogenesis of NPC. By subgroup analysis, the significant association between RASSF1A gene methylation and NPC risk were found among in all subgroups, including ethnicity, source of samples and test-method. Regarding to the ethnicity, a significant association between methylation of RASSF1A and NPC was found among the Asian region and the Non-Asia region. Additionally, most studies was performed in Asian region (14 of 16 studies), only two studies was done in Africa, thus, one again confirmed the nasopharyngeal cancer is native and posed to Asian region. The subgroup analysis by source of cancer samples revealed a significant correlation in both subgroup: biopsy samples and non-biopsy samples, and no heterogeneity was observed. It indicated that the type of biopsy was more suitable to apply to evaluate the methylation of RASSF1A gene. However, the source of non-biopsy samples, including nasopharyngeal swab, mouth and rinse, plasma – type of non/less-invasive source of sample, could be reflect alterations in the NPC and facility of collecting NPC samples led it a potential biomarker for early screening of NPC. Therefore, it should be focused on in the future to find out the non/less-invasive biomarker for NPC. The MSP method was used in twelve studies used MSP (counting for 75.00%). It could be explained MSP is the “gold standard method” of evaluation of methylation. The MSP shows the useful tool for the qualitative DNA methylation analysis within the ease of design and execution, sensitivity in the ability to detect small quantities of methylated DNA [44]. Moreover, in which MSP products are run on a gel, and the results are reported as methylated or unmethylated at the target DNA sequence [45]. These results are in accordance with that documented RASSF1A gene methylation to be the common and early epigenetic event in the progression of NPC tumorigenesis. So its epigenetic event might could be used as the potential epigenetic biomarker for risk of NPC. However, the current meta-analysis exhibited some limitations due to the number of current enrolled studies of 16, the data of non-English language studies may contribute to some bias, as well as the evaluation of the correlation between methylation of RASSF1A gene and clinicopathological features, age and the TNM stage which are differences in RASSF1A methylation between cases and controls.

This meta-analysis highlighted the significant association between the methylation of RASSF1A gene and risk of NPC based on the evaluation of frequency of both case-group, counting for 55.98% and control-group, counting for 1.70%, as well as OR value of 51.43. Additionally, our ﬁndings underscore the correlation among RASSF1A gene methylation and all subgroups, including region, source of samples, and test-method. The finding in present meta-analysis emphasized that the methylation of RASSF1A gene was recorded as the early epigenetic event in the progression of NPC tumorigenesis based on the literature-based meta-analysis.

Table 1. The characteristics of studies included in the meta-analysis of RASSF1A methylation and risk of NPC

Author, Reference	Year	Region	Case		Control		Method	Source of
Author, Reference	Year	Region	N	P	N	P	Method	Case	Control
Chow et al.	2004	Asia	26	20	-	-	COBRA	B	-
Chang et al.	2003	Asia	30	20	6	0	MSP	B	B
Chang et al.	2003	Asia	30	10	37	0	MSP	S	S
Chang et al.	2003	Asia	30	11	43	0	MSP	MT	MT
Chang et al.	2003	Asia	30	1	43	1	MSP	Pl	Pl
Chang et al.	2003	Asia	30	0	43	1	MSP	Bf	Bf
Kwong et al.	2002	Asia	29	24	6	0	MSP	B	B
Wong et al.	2004	Asia	41	2	43	0	RT-qPCR	Pl	Bl
Challouf et al.	2012	Africa	36	27	19	0	MSP	B	B
Zhou et al.	2005	Asia	28	23	8	0	MSP	B	B
Zhou et al.	2005	Asia	28	21	8	0	MSP	B	B
Zhou et al.	2005	Asia	28	13	8	0	MSP	B	B
Wang et al.	2009	Asia	38	27	14	0	MSP	B	B
Hutajulu et al.	2011	Asia	53	40	25	1	MSP	B	S
Fendri et ai.	2009	Africa	68	62	9	0	MSP	B	B
Tong et al.	2002	Asia	28	11	12	0	MSP	S	S
Tong et al.	2002	Asia	16	11	12	0	MSP	B	S
Zhang et al.	2012	Asia	49	39	20	0	MSP	B	S
Zhang et al.	2012	Asia	49	29	20	0	MSP	S	S
Wong et al.	2003	Asia	28	13	5	0	MSP	B	B
Wong et al.	2003	Asia	6	6	5	0	MSP	B	B
Qiu et al.	2004	Asia	27	20	3	0	MSP	B	B
Tian et al.	2013	Asia	40	7	41	2	MSP	Bl	Bl
Lo et al.	2001	Asia	21	14	6	0	MSP	B	B
Lo et al.	2001	Asia	21	14	2	0	MSP	B	B
Yang et al.	2015	Asia	52	36	50	0	MS-HRM	B	Pl
Yang et al.	2015	Asia	96	66	50	0	MS-HRM	S	Pl
Yang et al.	2015	Asia	220	53	50	0	MS-HRM	Pl	Pl

Note: MSP: Methylation-specific PCR; COBRA: combined bisulfite restriction analysis; MS-HRM: Methylation-sensitive high resolution melting; RT-qPCR: Real-Time Quantitative PCR;B: tumor biopsy; Pl: plasma, Bf: buffy coat; MT: Mouth-throat rinsing fluid; S: nasopharyngeal swab; Bl: blood; -: not recorded; P: positive; N: total samples

Table 2. Summary of subgroup analysis in meta-analysis of RASSF1A methylation and NPC risk

Group	Case		Control		Model, OR, 95% CI (Fix-effects model)	Heterogeneity
Group	N	P	N	P	Model, OR, 95% CI (Fix-effects model)	I² (%)	p
Total	1,052	592	461	1	51.43, 28.12-94.08	0.00	0.99
Ethinicity
Asia	948	503	433	1	47.94, 25.61-89.75	0.00	0.99
Non-Asia	104	89	28	0	135.96, 16.03-1,153.41	0.00	0.82
Sources of sample
Biopsy sample	558	410	206	1	55.29, 27.25-112.208	0.00	0.98
Non-biopsy	494	182	255	0	45.76, 15.56-134.60	0.00	0.73
Test-methods
MSP	545	367	228	1	40.86, 20.41-81.80	0.00	1.00
Other test-method	507	225	233	0	76.24, 23.24-250.11	0.00	0.48

Table 3. Sensitivity analysis of methylation of RASSF1A and NPC risk by the fix-effects model

	OR, 95% CI	Heterogeneity
	OR, 95% CI	I², 95% CI	p
Omitting Lo et al. 2001	54.81, 29.12-103.17	0.00, 0.00-0.00	0.99
Omitting Kwong et al. 2002	51.28, 27.74-94.77	0.00, 0.00-0.00	0.98
Omitting Tong et al. 2002	53.98, 28.72-101.45	0.00, 0.00-0.00	0.98
Omitting Chang et al. 2003	53.42, 27.96-102.28	0.00, 0.00-0.00	0.96
Omitting Wong et al. 2003	54.02, 28.96-100.77	0.00, 0.00-0.00	0.99
Omitting Wong et al. 2004	55.06, 29.63-102.32	0.00, 0.00-0.00	1.00
Omitting Qiu et al. 2004	52.69, 28.44-97.60	0.00, 0.00-0.00	0.98
Omitting Zhou et al. 2005	53.68, 28.19-102.20	0.00, 0.00-0.00	0.97
Omitting Wang et al. 2009	50.80, 27.42-94.14	0.00, 0.00-0.00	0.98
Omitting Fendri et al. 2009	49.69, 26.90-91.78	0.00, 0.00-0.00	0.99
Omitting Hutajulu et al. 2011	50.18, 26.79-93.99	0.00, 0.00-0.00	0.98
Omitting Challouf et al. 2012	49.76, 26.86-92.18	0.00, 0.00-0.00	0.98
Omitting Zhang et al. 2012	48.46, 25.76-91.14	0.00, 0.00-0.00	0.98
Omitting Yang et al. 2015	41.95, 22.13-79.49	0.00, 0.00-0.00	0.99

COBRA: Combined bisulfite restriction analysis

MMSP: Multiplex methylation-specific PCR

MS-HRM: Methylation-Sensitive High Resolution Melting

MSP: Methylation-specific PCR

NPC: Nasopharyngeal carcinoma

OR: Odds ratio

RASSF1A: RAS-association domain family 1 isoform A

RT-qPCR: Real-time quantitative PCR

TSG: tumor suppressor gene

Ethics approval and consent to participate

This is an observation and review study, no ethics and consent applied

Funding Information

This study was funded by Ho Chi Minh City Department of Science and Technology, Ho Chi Minh City, Vietnam (Grant No. 42/2017/HD-SKHCN), and Ho Chi Minh City Open University, Ho Chi Minh City, Vietnam.

Author contribution

Lao DT conceived, designed, performed, analyzed data, wrote the draft manuscript and acted as primary author for the project, Thieu HH, Nguyen HD contributed to perform and analyze data. Le HAT designed, performed, analyzed data, oversaw the research and edited the article.

Conﬂicts of Interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article

Acknowledgements

We wish to express our thanks to the research project sponsored by Ho Chi Minh City Department of Science and Technology, Ho Chi Minh City, Vietnam and Ho Chi Minh City Open University, Ho Chi Minh City, Vietnam

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Could the methylation of RASSF1A be the potential epigenetic biomarker for nasopharyngeal carcinoma? – Systematic review and meta-analysis

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