Abstract
Background/Aim
Head and neck cancer (HNC) is the sixth most common type of cancer worldwide. While it is widely accepted that human papillomavirus (HPV) infection is a crucial risk factor for the development of this type of cancer, there is still a lack of support from large-scale studies. This article addresses this gap by comprehensively analyzing the causal relationship between HPV infection and head and neck cancer using Mendelian randomization methods.
Patients and Methods
The HPV database was utilized to identify instrumental variables via the most enormous GWAS database search tool. The study examined five cancer groups of data of interest from the UK Biobank and three groups of cancer data from the GAME-ON network, all obtained from public databases. The causal relationship was estimated using various approaches, including IVW, weighted median, MR-Egger, simple mode, and weighted mode.
Results
The results indicated no causal relationship between HPV16/18 E7 proteins and head and neck cancer, oral cancer, oropharyngeal cancer, and laryngeal cancer, including UKB database and GEME-ON network. Furthermore, there was no heterogeneity or horizontal pleiotropy in the data.
Conclusion
The analysis does not support a causal relationship between HPV infection and HNC, as indicated by the lack of evidence linking HPV16/18 E7 proteins to the development of this cancer.
Keywords: Head and neck cancer, human papillomaviruses, Mendelian randomization, causal effect
Introduction
Head and neck cancer (HNC) is the sixth most prevalent malignant tumor worldwide, with complex subtypes and particular anatomical locations, including the oral cavity, pharynx, and larynx (1,2). Approximately 90% of HNC cases are classified as squamous cell carcinoma (SCC), the most common histological subtype of these malignancies (3). Reports in 2020 demonstrated that the global incidence of head and neck squamous cell carcinoma (HNSCC) was about 900,000, with a death toll of about 450,000 and a 5-year survival rate of about 50% (4). HNC originates from epithelial cells and preferentially occurs in the oral cavity, pharynx, and larynx. These neoplasms include oral squamous cell carcinoma (OSCC), oro- and nasopharyngeal carcinoma (NPC), and laryngeal cancer (LC) (5). HNC is a type of cancer that causes high functional, aesthetic and social burdens on patients. Furthermore, the tumor treatment is expensive and brings heavy economic burdens to society and individuals (6). The National Institutes of Health reported that the US government spent approximately $4 billion on HNC treatment in 2016. Research has also shown that factors such as smoking, exposure to specific compounds in the workplace (including aromatic amines and arsenic), and genetics may increase the likelihood of developing HNC (7).
Human papillomavirus (HPV) is a mucosal epithelium, non-enveloped, circular double-stranded DNA virus with a diameter of approximately 55 nm belonging to the Papillomaviridae family (8). Its genome is approximately 8,000 base pairs long, and the virus is primarily transmitted through close physical contact, including sexual behavior. HPV is associated with the formation of proliferative lesions on human mucous membranes, such as those lining the anogenital tract, oral cavity, and respiratory tract (9). Based on their oncogenic potential, HPV types are classified into high-risk HPV (such as types 16 and 18) and low-risk HPV (e.g., types 6 and 11) (10). High-risk HPV strains, such as HPV-16, 18, 31, and 33, are responsible for approximately 10% of all cancers worldwide (11). This includes over 90% of cervical cancer cases, most anal cancer cases, and some cases of vulvar, vaginal, and penile cancer (12). The genome of HPV can be divided into three regions based on function: 1) Early control area, which encodes proteins involved in the virus cycle and plays a crucial role in malignant cell transformation. It includes E1, E2, E4, E5, E6, and E7 proteins, with some HPV subtypes having E3 and E8; 2) Late control area, which encodes the capsid protein of the virus particle, including the main capsid protein L1 and the secondary capsid protein L2; and 3) Long control area, also known as the upstream regulatory region, which encodes a valine-containing protein p97 that regulates virus replication and transcription. This region has the highest degree of DNA variation in the HPV virus (13).
Globally, it is estimated that there are 690,000 cases of HPV infection annually. In the United States, approximately 45.2% of men aged 18 to 59 are infected with HPV, while about 80% of women of childbearing age have a lifetime risk of HPV infection (14).
Previous studies have reported an increasing trend in the number of HPV-related HNC in some developed countries due to changes in sexual behavior, such as early onset, lack of condom use, multiple sexual partners, and increased sexual activity (15). Villagomez-Ortiz et al. detected HPV positivity in 62 out of 253 head and neck tumors (24.5%), with 90.0% of them being HPV16 (16). However, some studies have found different results, with some suggesting that patients with HPV-positive oropharyngeal cancer yielded a better prognosis than those with HPV-negative cancer (17). A study has shown that individuals with HPV-positive HNC showed a better response to radiotherapy and chemotherapy and had higher 3-year survival rates compared to those with HPV-negative HNC (18). Multicenter joint statistics have found regional differences in the increasing incidence of HNC caused by HPV, with current data indicating a significant increase in North America, Europe, and Australia. However, similar trends have not been well-documented or remain unclear in regions such as South America, Africa, and Asia (19,20). Although many studies have been conducted, scientists have not fully clarified whether HPV increases the risk of HNC. The relationship between HPV positivity and HNC remains controversial, possibly due to the limited number of studies reporting the relationship between HPV positivity and prognosis, as well as conflicting results among studies and potential biases due to small sample sizes. Mendelian randomization (MR) is a technique that employs genetic variation as a tool to assess the causal relationship between exposure and outcomes while eliminating the influence of confounding factors present in observational studies. Two-sample MR analysis, however, utilizes summary data from genome-wide association studies (GWAS) instead of individual-level data. To address the knowledge gap between HPV infection and head and neck carcinoma, a systematic review and meta-analysis, as well as a two-sample MR analysis, were conducted to re-examine the association between HPV infection and HNC risk and to explore critical genes related to HPV using bioinformatics methods (21).
In the present study, we first utilized the two-sample MR to explore the potential impact of HPV16/18 E7 protein on HNC (Figure 1). This is the largest study conducted thus far to investigate the role of HPV infection on HNC through Mendelian randomization. We analyzed five groups of HNCs from the UK Biobank, including HNC, laryngeal cancer (LC), oral and oropharyngeal cancer, oral cavity cancer, and oropharyngeal cancer, as well as three groups from the GAME-ON project, including oral and oropharyngeal cancer, oral cavity cancer, and oropharyngeal cancer.
Figure 1.
The flowchart of the current two-sample MR study. HPV: Human Papillomavirus; GWAS: genome-wide association study; MR: Mendelian randomization.
Patients and Methods
Selection of data sources and IVs for HPV infection. The analysis was conducted using previously published genome-wide association study (GWAS) datasets related to HPV. The study protocol was approved by the Ethics Committees of the Guiyang Hospital of Stomatology. Sun et al. conducted the largest GWAS related to HPV infection to date, involving data from 3,080 European participants collected through medical history, questionnaires, and blood protein tests. This study included specific genetic information on the HPV16 E7 and HPV18 E7 proteins. This dataset can also be found in MRC IEU OpenGWAS (version: v6.5.2-2022-04-11). Instrumental variables (IVs) were selected based on the following criteria (p-value less than 5×10–5, r-squared value less than 0.001, and cluster distance greater than 10,000 kb), which are commonly used screening criteria in HPV research. The strength of IVs was evaluated by calculating the F-statistic, with F<10 considered weak IVs (22).
Data sources for HNC. The summary-level data from GAME-ON. Lesseur et al. conducted a GWAS on 6,034 cases of HNC and 6,585 controls from 12 studies in the GAME-ON network. The data included oral and oropharyngeal, oral cavity, and oropharyngeal cancer. The GAME-ON study population consisted of participants from Europe (45.3%), North America (43.9%), and South America (10.8%) (23). Our study selected data from Europeans in the GAME-ON database for further evaluation.
Data from UK Biobank. The UK Biobank is currently the world’s largest GWAS database, with a study population of volunteers from various regions of the UK. The following datasets were downloaded from UKB: HNC, LC, oral and oropharyngeal cancer, oral cavity cancer, and oropharyngeal cancer (24).
Statistical analysis of MR. The five main Mendelian randomization (MR) methods, including inverse-variance weighted estimator (IVW), weighted median, MR-Egger, simple mode, and weighted mode, were used to study the causal relationship between HPV16 and HPV18 infections and HNC. Cochran’s Q statistics were used to test heterogeneity, and a random-effects model was used if heterogeneity was present. In contrast, a fixed-effects model was used if there was no heterogeneity. Funnel plots were also used to assist in heterogeneity testing. Since many IVs are associated with multiple traits (pleiotropy), it is essential to conduct a sensitivity analysis of the results. MR-PRESSO was employed to identify and correct for outliers among the single nucleotide polymorphisms (SNPs). The process involved iterative removal of outlier SNPs until the global test p-value was no longer significant, ensuring that pleiotropic effects did not bias the causal estimates (25). All MR analyses in this study were conducted using the Two Sample MR package and MR-PRESSO package in R (4.2.1).
Results
Selection of IVs. IV satisfies three key assumptions: 1) IV is closely related to HPV; 2) IV is unrelated to confounding factors; 3) IV only affects the outcome of head and neck cancer (HNC) through HPV, not through other pathways. After the screening, 23 and 13 SNPs related to HPV16 E7 protein and HPV18 E7 protein were obtained, respectively. The information on the selected IVs for HPV16 and HPV18 can be found in Table I. The F-statistic values of all SNPs were greater than 10, indicating no weak instrumental variable bias.
Table I. Information of human papilloma virus (HPV) single nucleotide polymorphisms (SNPs) selected for genotyping.
CHR: Chromosome number; POS: position; N: sample size; beta: effect size; se: standard error; ALT: alternate allele; REF: reference allele; F: F-statistics
A causal connection between HPV16 infection and HNC. A total of 8 datasets related to HNC were included in the analysis. When performing MR-PRESSO testing on oral and oropharyngeal cancer in the GAME-ON data, an outlier value for rs7339361 was found and removed. After removal, the overall p-value was significantly higher than 0.05, and no outlier values were found in the other datasets, with overall p-values greater than 0.05. MR-Egger regression analysis showed that the p-values of each group were higher than 0.05, indicating no pleiotropy in this part of the study (Table II). Table III and Figure 2 show the correlation between HPV16 infection and HNC datasets. The inverse-variance weighted estimator (IVW) results of each dataset showed no correlation between HPV16 and the outcome (p>0.05), and the other four MR methods, including weighted median, MR-Egger, simple mode, and weighted mode, also did not show significant associations (p>0.05). The scatter plot visualizes the causal relationship between HPV16 infection and HNC analyzed by the five MR methods. If the slope is more significant than zero, it indicates a positive correlation; if it is less than zero, it means a negative correlation. However, the images show that the slopes of each group did not show a significant positive or negative correlation. In addition, the Cochran Q p-values in IVW and MR-Egger testing showed no heterogeneity in each group (p>0.05) (Table II), and the funnel plots of each group showed good symmetry (Supplementary Figure 1). Even after deleting a single SNP in the leave-one-out analysis, the results did not change significantly, indicating the stability of the analysis results (Supplementary Figure 2).
Table II. Results of the sensitivity analysis.
Q: Cochran's Q statistic.
Table III. HPV16 infection and its association with head and neck cancer in the Mendelian randomization (MR) analyses.
Exposure N: Number of exposure samples; N SNPs: number of IVs; OR: odds ratio; CI: confidence interval.
Figure 2.
Scatter plots for the causal association between HPV16 infection and head and neck cancer (HNC). A) HNC from UK Biobank; B) LC from UK Biobank; C) Oral and oropharyngeal cancer from UK Biobank; D) Oral cavity cancer from UK Biobank; E) Oropharyngeal cancer from UK Biobank; F) Oral and oropharyngeal cancer from GAME-ON; G) Oral cavity cancer from GAME-ON; H) Oropharyngeal cancer from GAME-ON.
A causal association between HPV18 infection and HNC. The MR analysis results of HPV18 infection and eight datasets of HNC are shown in Table IV, and the scatter plot is shown in Figure 3. In LC, the MR PRESSO overall p-value was less than 0.05. After removing the outlier value of rs4849449, the overall p-value was higher than 0.05, and subsequent analysis was performed. No outlier values were found in the other datasets in MR-PRESSO testing, and the global p-values were higher than 0.05 (Table II). The intercepts in the MR-Egger regression analysis were close to 0 (Table II), indicating no statistical significance, and no pleiotropy. In-depth information on the MR analysis results of HPV18 infection and various datasets of HNC is shown in Table IV, including the number of participants in the study and the number of SNPs analyzed for each dataset and the results of the five MR methods. The p-values of each group were higher than 0.05, indicating no significant correlation between HPV18 infection and each group of cancer data (p>0.05), which is visualized in the scatter plot in Figure 2. The Cochran Q p-values were all greater than 0.05 (Table II), and the funnel plots showed good symmetry in each group (Supplementary Figure 3). The leave-one-out analysis showed that the SNP points were continuously smooth, indicating that the results were not driven by a single SNP causal effect (Supplementary Figure 4).
Table IV. HPV18 infection and its association with head and neck cancer in the Mendelian randomization (MR) analyses.
Exposure N: Number of exposure samples; N SNPs: number of IVs; OR: odds ratio; CI: confidence interval.
Figure 3.
Scatter plots for the causal association between HPV18 infection and head and neck cancer (HNC). A) HNC from UK Biobank; B) LC from UK Biobank; C) Oral and oropharyngeal cancer from UK Biobank; D) Oral cavity cancer from UK Biobank; E) Oropharyngeal cancer from UK Biobank; F) Oral and oropharyngeal cancer from GAME-ON; G) Oral cavity cancer from GAME-ON; H) Oropharyngeal cancer from GAME-ON.
Discussion
Worldwide, HPV infection results in 630,000 new cancer cases yearly, with 83% of patients being cervical cancer, followed by HNC (26). The HPV positivity rate for oropharyngeal cancer is 50.46%-84.3%, while the HPV infection rates for oral and LC are 4.3%-11% and 4.6%-11%, respectively. Previous observational studies have shown that HPV infection is a considerable risk factor for HNC (27). Based on the relationship between HPV and tumors, HPV is classified into high-risk and low-risk types. The high-risk types include 16, 18, 26, 31, 35, 39, 45, 51-53, and 56. High-risk HPV can transform the infected mucosal epithelium into cancer by mediating the degradation of p53 and Rb proteins through E6 and E7 proteins. HPV16 accounts for 85% of HPV-positive HNC cases (28).
So far, no other MR studies have investigated the causal relationship between HPV infection and HNC. This study utilized the largest GWAS database on HPV infection, which includes data on HPV16 E7 and HPV18 E7 proteins. These viral oncoproteins play a crucial role in the pathogenesis of high-risk HPV infections, as they are directly involved in disrupting cell cycle regulation and contributing to carcinogenesis (29,30). The data on HNC were obtained from UK Biobank and GAME-ON, including HNSC, LC, oral and oropharyngeal cancer, oral cavity cancer, and oropharyngeal cancer, which are significant types of HNC and the largest GWAS databases related to HNC (31). A common application of MR is evaluating causal inferences between disease risk factors and exposure outcomes by utilizing genetic variation as a statistical tool. To avoid issues with heterogeneity, we restricted our main MR analyses to Europeans. This study broadly investigated the relationship between HPV infection and HNC through two-sample MR methods and found no significant association between HPV16 or HPV18 E7 protein and any HNC data (32). This contrasts with previous research, suggesting that HPV may act primarily as a co-factor or co-infection in the development of HNC, rather than as a direct causative agent. Nonetheless, results of the present study should be interpreted cautiously. HPV is a non-enveloped, double-stranded DNA virus, whose genome primarily encodes early oncoproteins, such as E6 and E7, which are involved in cellular transformation, as well as late-stage structural proteins, including the capsid protein L1 (33,34). Over-expression of these oncogenes is thought to stimulate the proliferation and expansion of epithelial basal cells, which develop a malignant phenotype. Cell cycle checkpoints are deregulated when HPV E6 forms a complex with tumor suppressor p53, which leads to rapid degradation of the protein. E7 binds to a complex that ubiquitinates another tumor suppressor protein, retinoblastoma (pRb), again leading to loss of control of the G1/S phase of the cell cycle (35). This results in an uncontrolled G1/S phase of the cell cycle (36). In this study, only E7 proteins of HPV16/18 were included, data on other HPV phenotypes and other related proteins such as L1 are lacking. Despite these limitations, we can conclude that there is no causal relation between the E7 protein of HPV16/18 and the development of HNC.
Compared with traditional observations, the most significant advantage of the present research is that the causal estimation obtained by MR avoids reverse causality and confounding bias (37). At the same time, our study used Cochran’s Q, MR-Egger regression, MR-PRESSO, and other methods to detect and control the heterogeneity and pleiotropy of IVs (38). However, there are still limitations. In this study, the threshold for screening IVs for HPV16/18 was 5×10–5, higher than the commonly used screening condition of 1×10–5 for MR IVs. This is because lower thresholds will yield very few SNPs that cannot meet the requirements of MR analysis. Still, the F values of the selected IVs are all greater than 10, indicating no weak IVs (39). In addition, there are multiple types of HPV infection, and up to this point, there is only GWAS data on HPV16/18 E7 protein. Although these are generally considered the two types most closely related to HNC, it cannot be ruled out that other types of HPV are associated with HNC. Moreover, the study population included only Europeans, limiting the results’ generalizability to different ethnicities. The reverse MR could not be performed due to lack of enough SNPs matched between head and neck cancer and HPV16/18 E7 protein.
Conclusion
The consequential relationship between human papillomavirus (HPV) infection and head and neck cancer (HNC) was thoroughly measured using inverse-variance weighted estimator (IVW), weighted median, MR-Egger, simple, and weighted modes. No significant association was found between HPV16/18 E7 protein and the occurrence of HNC.
Supplementary Material
Supplementary Figures 1-4 are available at DOI: 10.6084/m9.figshare.28009160
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
Authors’ Contributions
YF: Conceived the study, supervised the experiments and drafted the manuscript. JZ: conceived the study, supervised the experiments, and drafted the manuscript. TV: data evaluation, manuscript preparation. MG, RS, RR and REF: analyzed the data and revised the manuscript. JHW and LYX: conceived the study, performed the data evaluation, and prepared the manuscript.
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