Abstract
Background
Efforts have been made to improve the performance of nasopharyngeal carcinoma screening strategies, which include EBV related biomarkers. However, the advances achieved still remain imperfection. DNA methylation occurs early in cancer development and can be used as potential diagnostic biomarker. This study aimed to investigate the diagnostic performance of three methylated genes in nasopharyngeal carcinoma (NPC) patients.
Methods
A total of 255 nasopharyngeal swabs and 35 plasma samples were collected from patients with newly diagnosed or treated NPC and healthy controls. Methylation-specific PCR (MSP) was used to evaluate the methylation levels of three genes (SEPTIN9, RASSF1A, and H4C6) in swabs and plasma samples. The methylation rates, sensitivity, and specificity of the candidate genes were calculated. Furthermore, the detectability of methylated genes in paired swabs and plasma was compared.
Results
The detection rate of methylated SEPTIN9, RASSF1A, and H4C6 in nasopharyngeal swabs of patients with newly diagnosed NPC was 88.2%, 92.9% and 71.8%, respectively, while it reduced to 54.3%, 42.9% and 45.7% in blood plasma. The sensitivity of detecting methylated SEPTIN9, RASSF1A, and H4C6 to distinguish between untreated NPC and healthy controls was 88%, 93%, and 72%, respectively. Methylated RASSF1A showed the highest classification accuracy (AUC = 0.956). The detection rate of the methylated target genes was considerably lower in paired plasma samples.
Conclusion
The detection of RASSF1A methylation through non-invasive nasopharyngeal cavity swab sampling demonstrates significant potential for NPC diagnosis.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12885-025-14508-y.
Keywords: Nasopharyngeal carcinoma, Non-invasive diagnostic method, Methylation-specific PCR
Novelty and impact
Nasopharyngeal carcinoma (NPC) had more frequently methylated genes than normal individuals, suggesting their utility for NPC identification. There are few noninvasive tests for NPC that have been reported, and diagnostic performance is usually not very satisfactory. Nasopharyngeal swabs were collected conveniently by noninvasive means. Then we analyzed the performance of three genes methylation testing for NPC diagnosis, and the RASSF1A methylation assay performed best, with a sensitivity of 92.9% and specificity of 100.0%. Good findings were obtained by independently detecting RASSF1A methylation, which can be an independent diagnostic biomarker or even a nasopharyngeal cancer screening tool, which is inexpensive and automated.
Posting statement
The paper is being posted on a non-commercial preprint server, including the DOI and a link to the article.
Introduction
Nasopharyngeal carcinoma (NPC) is a malignancy originating from the nasopharyngeal epithelium. The stage of the tumor is a significant influencing factor of prognosis, with higher-stage NPC having poorer outcomes [1]. Early detection and diagnosis are particularly important in improving the cure rate and reducing recurrence of NPC.
Since infection with the highly pathogenic Epstein-Barr virus (EBV) is one of the most important etiologic factors of NPC, plasma EBV antibody and EBV DNA testing have been considered for the early detection of NPC. Two main approaches are based on enzyme linked immunosorbent assay (ELISA) EBV antibody tests and quantitative polymerase chain reaction (qPCR), detecting the antibodies responses to EBV antigens and circulating free EBV DNA. Both methods have been proven to be effective for NPC screening in high-incidence populations. However, these biomarkers have unsatisfactory positive predictive values (PPV), leading unnecessary procedures and anxiety for subjects with false positive results. Additionally, the level of circulating free DNA fragments in the blood is influenced by tumor stage and biological factors [2] which may lead to limited value of plasma EBV DNA in detecting patients with early clinical stage disease. New modalities and effective biomarkers for NPC screening is needed to reduce false positives and improve diagnostic accuracy.
DNA methylation is an epigenetic mechanism of gene silencing of tumor suppressors, which has been recognized as promising targets for the creation of robust diagnostic and predictive biomarkers. Alterations in DNA methylation usually occur in the early stages of cancer, even before changes in cell morphology and tissue structure. Abnormal DNA methylation patterns are an important feature of cancer [3, 4]. Studies have shown that methylation markers have prognostic and diagnostic significance in a variety of cancers and precancerous tissues [5, 6]. It was reported that DNA methylation is a highly stable molecular feature in NPC [7]. Compared to the plasma, nasopharyngeal swab sampling can possibly bring higher DNA concentrations, improving the sensitivity of tumor marker tests. In fact, some genes hypermethylated in NPC tissues have been reported. For example, our previous study found that Septin9, which is commonly used in the diagnosis of colorectal cancer, has significant difference in methylation levels between NPC tissues and benign ones [8]. In a meta-analysis, RASSF1A methylation is closely associated with the development of NPC [9]. H4C6, also known as HIST1H4F, is considered as a pan-oncogene and has been shown to be highly methylated in a variety of cancerous tissues including head/neck tumors but hypomethylated in benign tissues [10]. Therefore, this study investigated the clinical value of three methylated genes (SEPTIN9, RASSF1A, and H4C6) in nasopharyngeal swabs for NPC diagnosis.
Materials and methods
Study design and samples
This is a case control study with samples retrospectively collected from June 2023 till September 2023. The study flowchart is shown in Fig. 2. Three groups of subjects were screened: patients with newly diagnosed NPC (85 participants), patients with complete regression of tumor (97 participants) and healthy controls (73 participants). Patients with NPC were recruited at Sun Yat-sen University Cancer Center (SYSUCC), and the healthy controls were recruited at physical examination center. This study was approved by the Human Ethics Committee of the SYSUCC, and all participants provided informed consent.
Fig. 2.
Study flow gram. Abbreviations: NPC, nasopharyngeal carcinoma; PCR, polymerase chain reaction
Nasopharyngeal swabs were collected from all subjects via blind brushing method using nasopharyngeal swabs and accompanying preservation solution containing guanidine salt (Guangdong Jingquan Medical Technology Co., Ltd). In fact, this kind of swab has been successfully used for sampling during the pandemic of Covid-19. The flocked swab was inserted via the nose until the nasopharyngeal cavity was reached. Subsequently, the brush was rotated several times and quickly removed. Immediately after sampling, the brush tip was cut and placed in tubes containing 3 mL of preservation solution. Bilateral sampling was performed and swabs from the same patient were put in one tube. Furthermore, 5 mL of blood samples were collected from 35 patients and the plasma was isolated.
DNA extraction, bisulfite conversion and methylation-specific PCR (MSP)
Due to the relatively large sample size of 3 mL of preservation solution containing cells from nasopharyngeal swabs, as well as the tedious process of methylation detection, we here applied the AutoPure-12 (Guangdong Bright-Innovation BioMed Co., Ltd., Shunde, China) system to process cell preservation solution. As shown in Fig. 1, the cassettes within AutoPure-12 contain various reagents that are used for DNA extraction and bisulfite modification. This platform can maximally process 3.5 mL of liquid sample and 12 specimens at a time. The whole procedure including DNA extraction, bisulfite treatment, and purification/recovery of bisDNA (bisulfite-converted DNA) can be done automatically within 3 h, resulting in a 100 uL of bisDNA solution. Since plasma contains abundant proteins, the high viscosity leads to the aggregation of magnetic bead even use of proteinase K for protein digestion. Therefore, for plasma samples, the process of DNA isolation, bisulfite conversion and purification/recovery of bisDNA remains manually.
Fig. 1.
Schematic diagram of sample processing cassettes in the AutoPure-12 platform
Methylation fluorescence quantitative kit (Guangdong Bright-Innovation BioMed Co., Ltd.) was two-channel assay consisting of each target gene along with an internal control ACTB gene. The ACTB gene served as a reference gene and samples with a cycle threshold (Ct) value of ACTB above 35 were deemed “low quality” and excluded. Ct value of methylated gene less than 40 was judged as positive. The methylation level of the target gene was determined using the difference between two Ct values (ΔCt = Ct ACTB - Ct target gene), and a methylation score was calculated as 2ΔCt x 100.
Epstein–Barr virus DNA load in plasma
DNA from plasma samples was extracted using the Nucleic Acid Extraction Kit (Sansure Biotech Inc.) following the protocol recommended by the manufacturer. The EBV DNA load was measured by real-time qPCR using the EBV DNA PCR Reagents Kit (Sansure Biotech Inc.).
Sample size calculation
This study is a diagnostic test for evaluating the accuracy of methylated genes in diagnosing NPC. Based on our preliminary experiments, the anticipated sensitivity and specificity of methylated RASSF1A is 80% and 90%, respectively. With an allowable error of 0.1 and a two-sided α of 0.05, the sample size is calculated with PASS software to be N = 70 cases. The study goal was to recruit at least 78 patients with NPC, anticipating a 10% sampling or assay failure. Actually 85 untreated NPC were recruited in this study.
Statistical analysis
Descriptive statistical analysis was conducted for clinical and demographic characteristics. The Kruskal-Wallis test and χ2 tests were used to analyze continuous and categorical variables, respectively. The sensitivity, specificity, Youden index, positive predictive value, and negative predictive value were calculated to evaluate the performance of methylation status in NPC diagnosis. The receiver-operating-characteristic (ROC) curves were plotted based on the gene methylation score and the area under the curve (AUC) was calculated. The Mc Nemar test was used to compare the methylation status of genes in paired plasma and swab samples. Statistical analyses were performed using R Studio (publicly available). Statistical significance was defined as the conventional P value of < 0.05 (two-sided test).
Results
Patients characteristics
After excluding samples with failed quality control or PCR assays, 255 swabs were collected from 85 patients with untreated NPC, 97 patients with complete responded NPC, and 73 healthy subjects without tumor disease (Fig. 2). Paired plasma samples were obtained from 35 patients with untreated NPC. More than half of the patients had advanced stage (Stage III-IVB) (AJCC 9th) [11]. Age, gender and tumor stage were balanced between the untreated and treated group. The plasma EBV DNA was highest in patients with newly diagnosed NPC, while it was undetectable in all of the healthy controls (Table 1).
Table 1.
Characteristics of the subjects with untreated NPC, treated NPC and healthy controls
| Characteristics | Subjects | p value* | ||
|---|---|---|---|---|
| No.(%)/Median(Range) | Untreated NPC (n = 85) | Treated NPC (n = 97) | Healthy CTL (n = 73) | |
| Gender | 0.653 | |||
| Male | 56 (65.9) | 68 (70.1) | 29 (39.7) | |
| Female | 29 (34.1) | 29 (29.9) | 44 (60.3) | |
| Age (mean (SD)) | 47.29 (12.72) | 46.53 (11.64) | 29.29 (5.70) | 0.947 |
| T stage | 0.058 | |||
| T1-2 | 10 (11.8) | 23 (23.7) | NA | |
| T3-4 | 75 (88.2) | 74 (76.3) | NA | |
| N stage | 0.922 | |||
| N0-1 | 37 (43.5) | 44 (45.4) | NA | |
| N2-3 | 48 (56.5) | 53 (54.6) | NA | |
| M stage | 1 | |||
| M0 | 79 (92.9) | 91 (93.8) | NA | |
| M1a-1b | 6 (7.1) | 6 (6.2) | NA | |
| Stage (AJCC 9th) | 0.323 | |||
| Stage IA-II | 50 (58.8) | 65 (67.0) | NA | |
| Stage III-IVB | 35 (41.2) | 32 (33.0) | NA | |
| Plasma EBV DNA (cp/ml) (median (IQR)) | 238.5 (1267.5) | 0 (0) | 0 (0) | < 0.001 |
| Plasma EBV DNA | < 0.001 | |||
| =0 | 23 (27.4) | 88 (96.7) | 21 (100) | |
| > 0 | 61 (72.6) | 3 (3.3) | 0 (0.0) | |
| SEPTIN9 methylation | < 0.001 | |||
| No | 10 (11.8) | 55 (56.7) | 45 (61.6) | |
| Yes | 75 (88.2) | 42 (43.3) | 28 (38.4) | |
| RASSF1A methylation | < 0.001 | |||
| No | 6 (7.1) | 94 (96.9) | 73 (100.0) | |
| Yes | 79 (92.9) | 3 (3.1) | 0 (0.0) | |
| H4C6 methylation | < 0.001 | |||
| No | 24 (28.2) | 91 (93.8) | 60 (82.2) | |
| Yes | 61 (71.8) | 6 (6.2) | 13 (17.8) | |
| SEPTIN9 methylation score | 0.39 | 0.00 | 0.00 | < 0.001 |
| RASSF1A methylation score | 0.22 | 0.00 | 0.00 | < 0.001 |
| H4C6 methylation score | 0.06 | 0.00 | 0.00 | < 0.001 |
*The p-value is used to determine whether the differences between untreated NPC and treated NPC are statistically significant
Diagnostic performance of SEPTIN9, RASSF1A and H4C6 methylation
The detection rate and methylation scores are shown in Table 1. In swabs of newly diagnosed NPC, the highest methylated gene detection rate was 92.9% for RASSF1A, and the lowest for H4C6 (71.8%). Methylated RASSF1A (mRASSF1A) was detected in 3 (3.1%) patients after treatment, and was undetectable in all of healthy controls. Sensitivity and specificity of detectable mRASSF1A distinguishing between untreated NPC and healthy controls were 93% and 100%, respectively (Table 2). The sensitivity of the mSEPTIN9 and mH4C6 was 88% and 72%, respectively, and the specificity was 62% and 82%. Besides, Table S1 shows the performance of these methylated markers distinguishing patients with untreated NPC from treated NPC patients plus healthy controls. Both of the sensitivity and specificity of the mRASSF1A were the highest.
Table 2.
The performance of variables for distinguishing between untreated NPC and heathy controls
| Variable | Sensitivity | Specificity | Youden index | NPV | PPV |
|---|---|---|---|---|---|
| SEPTIN9 methylation | 0.88 | 0.62 | 0.50 | 0.82 | 0.73 |
| RASSF1A methylation | 0.93 | 1.00 | 0.93 | 0.92 | 1.00 |
| H4C6 methylation | 0.72 | 0.82 | 0.54 | 0.71 | 0.82 |
| plasma EBV DNA | 0.73 | 1.00 | 0.73 | 0.48 | 1.00 |
ROC curves were constructed by the result of methylated gene detection (Fig. 3). ROC curve analysis showed AUCs of 74.9%, 96.5% and 77.0% by detecting methylated SEPTIN9, RASSF1A and H4C6 to differentiate between untreated NPC and healthy controls. And it also showed AUCs of 73.5%, 95.6% and 80.3% to distinguish between untreated NPC, and treated NPC plus healthy controls by detecting those genes. Additionally, RASSF1A methylation showed the highest NPV (92%) and PPV (100%). The plasma EBV DNA was negative in 23 (27.4%) patients with untreated NPC, 88 (96.7%) patients with treated NPC and 21 (100%) healthy controls. The sensitivity and specificity of qPCR detectable plasma EBV DNA for distinguishing between untreated NPC and healthy subjects were 73% and 100%, respectively.
Fig. 3.
The receiver operating characteristic (ROC) curves of methylated gene detection. A ROC curves showing the diagnostic accuracy of methylated gene detection in differentiating between patients with NPC (N = 85) and healthy controls (N = 73). The optimal cut-off point on the ROC curve was plotted with the confidence intervals. B ROC curves showing the diagnostic accuracy of methylated gene detection in distinguishing between patients with NPC (N = 85), and patients with complete responded NPC plus healthy controls (N = 170). The optimal cut-off point on the ROC curve was plotted with the confidence intervals
Methylation difference in paired nasopharyngeal swabs and plasma
Paired plasma and swab samples were obtained from 35 patients. The Mc Nemar test showed significantly lower detection rate of methylated genes in the plasma samples (Table 3). Methylated SEPTIN9, RASSF1A and H4C6 was detected in 19 (54.3%), 15 (42.9%) and 16 (45.7%) plasma samples, respectively. Moreover, the methylation score was compared between the plasma and swab samples. Significant higher scores of methylated SEPTIN9 (P = 0.04) and RASSF1A (P = 0.02) were found in the swabs (Fig. 4).
Table 3.
Identification of methylated genes in the matched swab or blood samples
| Detection of methylated SEPTIN9 | |||||
|---|---|---|---|---|---|
| blood | |||||
| negative | positive | overall | P value | ||
| swab | negative | 0 | 2 | 2 | <0.01 |
| positive | 16 | 17 | 33 | ||
| overall | 16 | 19 | 35 | ||
| Detection of methylated RASSF1A | |||||
|---|---|---|---|---|---|
| blood | |||||
| negative | positive | overall | P value | ||
| swab | negative | 3 | 0 | 3 | <0.001 |
| positive | 17 | 15 | 32 | ||
| overall | 20 | 15 | 35 | ||
| Detection of methylated H4C6 | |||||
|---|---|---|---|---|---|
| blood | |||||
| negative | positive | overall | P value | ||
| swab | negative | 3 | 3 | 6 | <0.01 |
| positive | 16 | 13 | 29 | ||
| overall | 19 | 16 | 35 | ||
Fig. 4.
Methylation score in paired swab and blood samples. The methylation score of SEPTIN9 (A) and RASSF1A (B) was significantly higher in the nasopharyngeal swab than that in the plasma sample, while no significant difference of the methylation score of H4C6 (C) were found between the sample types
Discussion
Early diagnosis of NPC is quite challenging due to its anatomical peculiarity and the lack of obvious clinical signs in the early stages. At present, nasopharyngeal endoscopy combined with pathological examination of suspected cases remains the gold standard for the diagnosis of NPC, although assessment of clinical symptoms and family history provides good clues which sometimes are not specific. However, this way is mainly applicable to patients with suspected NPC and is not suitable for early screening and especially for large-scale screening due to it is costive, invasive and inconvenient. Although some reports suggest that plasma EBV DNA has high sensitivity and specificity in detecting NPC, many studies have not reached very good conclusions. For example, two studies concluded that plasma EBV DNA had a sensitivity of 84.9% and 68.8%, and a specificity of 89.7% and 88.2%, respectively, in distinguishing NPC patients from healthy individuals [12, 13]. Our study showed that plasma EBV DNA had a sensitivity of 72.6% but a specificity of 100%, which possibly due to the fact that only 21 of the healthy controls had plasma EBV DNA tested. Those false-positive results lead to repeated nasal endoscopic examination, biopsy and long-term follow-up. DNA methylation is an epigenetic mechanism of gene silencing of tumor suppressors, which has been recognized as promising targets for the creation of robust diagnostic and predictive biomarkers, more important was that the sample can be collected with noninvasive nasopharyngeal swabs.
In this study, nasopharyngeal brush sampling was performed in outpatient for newly diagnosed NPC patients (87 patients), without controlling artificially for the proportion between early NPC (Stage IA-II) and advanced NPC (Stage III-IVB). The TNM stage distribution of the patients in this study was consistent with that of initial diagnosis NPC in South China [14]. Therefore, the diagnosis performance of these newly diagnosed patients that we sampled together is reflective of the real-world situation. In this study, 115 patients with early stage NPC and 67 patients with advanced stage NPC were enrolled. We found that there was no statistical difference in methylation rate of three genes between early NPC and advanced NPC. As well, SEPTIN9 and RASSF1A methylation scores were not statistically different between early and advanced NPC. (Table S2). Further, we calculated the diagnostic performance of methylation detection rates for 3 genes for early and advanced NPC, respectively. Sensitivity of detecting SEPTIN9, RASSF1A and H4C6 methylation in early NPC was 86%, 92% and 66%, respectively, compared to 91%, 94% and 80% in advanced NPC. Especially RASSF1A methylation test indicated good performance in detecting early NPC (Table S3).
In the present study, we investigated the value of methylated SEPTIN9, RASSF1A and H4C6 in distinguishing NPC patients from normal controls. The three genes indicated a good potential to distinguish NPC from nasopharyngitis and health controls in a previous study, mainly using FFPE specimens [15]. The present study focused on real-world nasopharyngeal swab methylation detection. In the present study, the size of samples that were tested by our nasopharyngeal swabs were significantly larger, and additional analyses were performed (e.g. compared to plasma methylation tests). In the present study, we used commercial kits containing the qMS-PCR assays of the three genes. We did not quantify the nucleic acids extracted from the nasopharyngeal swabs of each participant because the process from nasopharyngeal swab DNA extraction to sulfite conversion is fully automated. However, in the final qPCR reaction, the three target genes were reacted simultaneously with the internal reference gene, ACTB gene, and the Ct value of ACTB represented the amount of DNA extracted from nasopharyngeal swabs [5, 6]. In addition, we indicated the relative content of methylated fragments of the three target genes using a methylation score formula, allowing the methylation content of the target genes in different specimens to be compared [16, 17]. We found that the methylation status and score of RASSF1A have high sensitivity and specificity in diagnosing NPC. Moreover, the methylation level in nasopharyngeal swab samples was higher than that in the plasma, suggesting that swab might be a better modality in NPC screening. In our pilot study, the sensitivity of bilateral nasal swab sampling was significantly higher than that of unilateral nasal swab sampling(data not shown). Because if nasopharyngeal malignancy is one-sided, and unilateral sampling will miss the contralateral tumor nidus.
Downregulated expression of RASSF1A has been associated with pathogenesis of cancer [18, 19]. Introducing RASSF1A exogenously has been shown to induce growth inhibition and apoptosis in NPC cell lines, suggesting its tumor suppressor function [20]. Previous studies with NPC tissue have implied that hypermethylation may contribute to the transcriptional inactivation of the RASSF1A gene [21]. In addition, hypermethylation of RASSF1A was correlated with advanced tumor stage and the presence of lymph node metastasis in patients with NPC [22]. Therefore, methylation tests of RASSF1A are of potentially valuable in improving the sensitivity for NPC detection. Our results also showed that the diagnostic sensitivity of mRASSF1A was over 90%, suggesting that it may aid in the screening of NPC. mRASSF1A was detected in 3 of 97 patients with complete responded NPC. These 3 patients in complete remission were with Stage II or III NPC, and completed treatment at least 1 year ago. As of preparing this manuscript, there were no signs of recurrence or metastasis over 15 months after the methylation tests. We will continue following these patients.
Swab sampling involves applying a brush to collect specimens of NPC in a painless and non-invasive manner, which is a relatively simple process and can be completed quickly and easily. In comparison to plasma, nasal swab can be collected without specialized training from a diverse range of populations, making it an excellent option for self-sampling in NPC screening. After the sample has been collected, it can be sent to a lab for further testing and analysis. Swabs can be used to detect the same biomarkers that were usually obtained from plasma specimens, such as EBV DNAand microRNAs [9, 23, 24]. A study in 2024 collected samples by noninvasive means such as oropharyngeal swabs, oral brushes, saliva, and gargles, and then detected their EBV DNA methylation [25]. The Youden index for detecting methylation (via the W promoter of EBV DNA) in samples collected with oropharyngeal swabs was the highest among multiple methods (sensitivity 79.7% and specificity 96.5%). Because the test required detection of EBV DNA load and EBV DNA methylation, 50.4% patients in the case group of this assay has failed to receive a diagnosis of EBV DNA methylation detection and were not included in the diagnostic performance analysis. In contrast, 97.7% of untreated NPC patients successfully underwent testing in our present study, because we detected methylation of human nasopharyngeal cells. Viral infection is only a high risk factor for cellular carcinogenesis. Medical studies have shown that EBV, HPV, and HBV are high risk factors for NPC, cervical cancer, and hepatocellular carcinoma, respectively. But, after all, cancer occurs in human cells, and these genes we tested for methylation are genes that are associated with the cancerous process in human cells [3, 4]. Therefore, if we test the methylation of human genes, it will be more indicative of cellular carcinogenesis than viruses, similar to how testing the methylation of cervical cells is closer to the reality of cellular carcinogenesis than HPV testing [26]. Methylation of nasopharyngeal cells can more directly reflect the carcinogenesis of cells compared with methylation of EBV DNA, therefore the diagnosis performance of our present study is better (sensitivity 92.9%, specificity 100.0%). Our findings indicated that the detection rate of methylated genes is significantly higher than that in the plasma for patients with NPC, probably because swab samples are close to the tumor lesion. In addition, the ingredients is relatively simple after elution of nasal swabs with preservation solution compared to plasma samples that containing abundant proteins. Thus, the nasopharyngeal epithelial cells within preservation buffer can be processed automatically for DNA extraction, bisulfite treatment, and purification/recovery of bisDNA, otherwise, manual operation would be very tedious.
Conclusion
NPC showed more frequent methylated genes compared to normal individuals, suggesting their utility for NPC identification. RASSF1A methylation may be a diagnostic biomarker in swab samples that could be used for the clinical diagnosis of NPC and even for NPC screening from the perspective of cost and automation.
Limitation
We must admit that the present study was a single-center study with a limited sample size and was retrospective, and therefore was lack of robust power to clarify the practice value in clinical situation. We conducted a multicenter prospective study starting from January 2025, including three hospitals in two provinces in China, and it was also registered on clinicaltrials.gov (NCT06367049).
Supplementary Information
Acknowledgements
Not applicable.
Abbreviations
- AUC
Area under the curve
- EBV
Epstein-Barr virus
- ELISA
Enzyme linked immunosorbent assay
- mH4C6
Methylated H4C6
- mRASSF1A
Methylated RASSF1A
- mSEPTIN9
Methylated SEPTIN9
- MSP
Methylation-specific PCR
- NPC
Nasopharyngeal carcinoma
- NPV
Negative predictive values
- PPV
Positive predictive values
- qPCR
Quantitative polymerase chain reaction
- ROC
Receiver-operating-characteristic
Authors’ contributions
All the authors contributed in the preparation of this work. Q. Z. H., D. H. and Z. X. were responsible for the collection of nasopharyngeal swabs. Q. Z. H., L. L. X. and G. Q. L. collected clinical data from healthy controls and nasopharyngeal cancer patients. Z. S. took charge of the methylation detection. Q. Z. H. and C. S. Y. were responsible for data analysis, writing, edit and revision of the body text. H. Y. J., Z. L., and Q. Z. H. were drafted and revised the article. H. Y. J., Z. L., Q. Z. H., C. S. Y. and Z. S. were responsible for the theme, final editing, and preparation of the manuscript for submission. H. Y. J., Z. L., C. M. Y. and H. P. Y. critically revised the manuscript. All authors read and approved the final manuscript.
Funding
This study was supported by Basic and Applied Basic Research Foundation of Guangdong Province (2021A1515220021).
Data availability
The data of this research was submitted to RDD (Research Data Deposit) platform (http://www.researchdata.org.cn), and was currently under review. A unique RDD identification code will be provided later.
Declarations
Ethics approval and consent to participate
All procedures performed in studies involving human participants were in accordance with the ethical standards of the Ethics Committee of Sun Yat-sen University Cancer Center, and National Health Commission of the PRC and with the 1964 Helsinki Declaration and its later amendments. We have initiated a prospective clinical study that has been registered on clinicaltrials.gov (NCT06367049) (first posted at 2024-04-15) and approved by Ethics Committee of Sun Yat-sen University Cancer Center (B2023-716-01).
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Zi-Han Qin, Si-Yuan Chen and Shuai Zhou contributed equally to this work.
Contributor Information
Liang Zhang, Email: zhangliang1999@tsinghua.org.cn.
Yi-Jun Hua, Email: huayj@sysucc.org.cn.
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Data Availability Statement
The data of this research was submitted to RDD (Research Data Deposit) platform (http://www.researchdata.org.cn), and was currently under review. A unique RDD identification code will be provided later.