Oropharyngeal Helicobacter pylori colonization increases risk and worsens prognosis of head and neck squamous cell carcinoma

Abstract

Helicobacter pylori (H. pylori) colonization in the oropharynx has been suggested to contribute to the development of head and neck squamous cell carcinoma (HNSCC), but prospective evidence remains limited. This prospective cohort study aimed to investigate the association between oropharyngeal H. pylori colonization, including its virulence gene types, and the risk and prognosis of HNSCC. A total of 220 high-risk individuals and 220 diagnosed HNSCC patients were enrolled. Oropharyngeal samples were collected for H. pylori detection by culture and quantitative PCR (qPCR). Virulence genes cagA and vacA were genotyped. Cox proportional hazards models assessed HNSCC incidence and overall survival, with Kaplan–Meier and log-rank tests evaluating survival differences. H. pylori positivity was significantly higher in the HNSCC group compared to the high-risk group (43.6% vs. 32.7%, P = 0.033). In high-risk individuals, H. pylori colonization was an independent risk factor for HNSCC onset (HR = 1.50, 95% CI: 1.02–2.21, P = 0.039), with higher bacterial loads and presence of high-virulence genotypes (e.g., cagA+/vacA s1/m1) linked to increased risk (P < 0.05). Among HNSCC patients, H. pylori-positive individuals showed higher recurrence (26.0% vs. 16.9%, P = 0.018) and mortality rates (13.5% vs. 8.1%, P = 0.043), with poorer overall survival (HR = 1.57, P = 0.035). Co-infection with HPV may further complicate the clinical profile, potentially involving the local immune microenvironment. Oropharyngeal H. pylori colonization, particularly by high-virulence strains, significantly elevates the risk of HNSCC development and worsens clinical outcomes. The interplay between H. pylori and HPV suggests the need for integrated pathogen screening and targeted interventions to improve early detection and individualized treatment strategies for HNSCC.

Introduction

Head and neck squamous cell carcinoma (HNSCC) is a prevalent malignant tumor worldwide, particularly affecting high-risk populations such as smokers, drinkers, and individuals with occupational exposures¹. Prolonged exposure to carcinogens often leads to chronic inflammation and genetic damage in the oropharyngeal mucosa, creating a conducive environment for tumor development. In addition to well-established risk factors, infectious agents such as Helicobacter pylori and human papillomavirus (HPV) have attracted increasing attention for their potential roles in HNSCC pathogenesis^2,3.

Helicobacter pylori is a well-known gastric pathogen strongly linked to gastric cancer; however, emerging evidence indicates its ability to colonize the oropharyngeal mucosa and modify the local microenvironment^4,5. Although several small-scale studies have suggested an association between oropharyngeal H. pylori colonization and the development of squamous cell carcinoma, robust and large-scale prospective data remain scarce⁶. Similarly, high-risk HPV infection is recognized as an important etiological factor for HNSCC, sharing potential overlapping pathogenic pathways with H. pylori⁷. Nevertheless, the interplay between H. pylori colonization and HPV infection within the oropharynx and their combined impact on HNSCC remains poorly understood.

Given that traditional carcinogenic factors such as smoking and alcohol remain predominant contributors to HNSCC, clarifying the specific role of H. pylori is crucial for understanding the multifactorial etiology of this disease and optimizing prevention and therapeutic strategies⁸. While previous studies have suggested an association, they were often limited by retrospective designs or small sample sizes. Current epidemiological evidence remains scarce, particularly from prospective cohorts that simultaneously evaluate the impact of H. pylori virulence factors and adjust for HPV status, which are critical gaps this study aims to address. Importantly, the pathogenicity of H. pylori is largely influenced by specific virulence genes, notably cagA and vacA, which modulate the bacterium’s inflammatory and carcinogenic potential⁹. However, few longitudinal studies have incorporated virulence gene typing to evaluate clinical outcomes in HNSCC. Furthermore, HPV infection significantly affects HNSCC prognosis, and co-infection with H. pylori may complicate disease progression and treatment response¹⁰.

To address these gaps, we designed a prospective cohort study involving both high-risk individuals and confirmed HNSCC patients, aiming to systematically monitor H. pylori colonization, virulence gene profiles, and HPV status. This study intends to elucidate the impact of oropharyngeal H. pylori colonization on HNSCC incidence, recurrence, and survival, while exploring the potential interactions among bacterial virulence, viral infection, and traditional risk factors. Our integrative approach seeks to establish a comprehensive framework for individualized prevention and management of HNSCC.

Materials and methods

Study design

A prospective cohort design was adopted, comprising two prospective cohorts: a high-risk population cohort (Cohort 1) and a confirmed head and neck squamous cell carcinoma cohort (Cohort 2). The study was conducted from January 2021 to December 2024 in the Departments of Oral and Maxillofacial Surgery, Otorhinolaryngology–Head and Neck Surgery, and Oncology of this hospital. Baseline oropharyngeal Helicobacter pylori test results, genotype results, and other relevant clinical or lifestyle information were recorded for each participant, and incidence and prognosis were observed during follow-up.

The study protocol was approved by the Medical Ethics Committee of this hospital (approval IRB-2022-067). All participants signed written informed consent after fully understanding the study purpose and potential risks. All methods were performed in accordance with the relevant guidelines and regulations, including the Declaration of Helsinki.

Study subjects and sample size estimation

Cohort 1 (high-risk population cohort): Participants were recruited through a combination of hospital outpatient and health examination centers, community health service centers, occupational health monitoring cohorts, and online recruitment. Inclusion criteria: ① age 40–75 years; ② according to the NCCN (2023) Head and Neck Cancer Clinical Guidelines⁹ and the IARC (2020) report¹⁰, meeting any one of the following high-risk exposure factors at the specified cumulative dose or duration: cumulative smoking ≥ 20 pack-years and currently smoking or quit < 5 years; daily ethanol intake ≥ 40 g for ≥ 10 years; daily betel-nut chewing for ≥ 10 years; long-term (≥ 10 years) exposure to nickel, chromium, formaldehyde, or wood dust recognized by IARC as Group 1 carcinogens; ③ physical examination and medical history confirming no diagnosis of head and neck squamous cell carcinoma or other malignancies; ④ voluntary written informed consent and ability to complete scheduled follow-up. Exclusion criteria: ① previous diagnosis of any malignant tumor; ② inability to undergo oropharyngeal H. pylori testing due to severe hepatic or renal dysfunction or other medical reasons; ③ severe mental illness or cognitive impairment seriously affecting study compliance.

Cohort 2 (confirmed head and neck squamous cell carcinoma cohort): Inclusion criteria: ① pathologically confirmed head and neck squamous cell carcinoma; ② stage I–IVa; ③ planned to receive surgery or chemoradiotherapy; ④ age 18–80 years; ⑤ ability to cooperate with testing and sign written informed consent. Exclusion criteria: ① prior oropharyngeal H. pylori eradication intervention before head and neck cancer treatment; ② presence of other active malignancies; ③ acute severe cardio-cerebrovascular events; ④ mental disorders or cognitive dysfunction preventing cooperation.

Cohort 1 was primarily used to observe the occurrence of new head and neck squamous cell carcinoma cases, whereas Cohort 2 was used to observe recurrence and survival outcomes in confirmed patients.

Based on the binomial distribution formula for cohort study sample size and literature-reported three-year cumulative incidence of head and neck squamous cell carcinoma, oropharyngeal H. pylori colonization rate, and proportion of confounding factors, the required sample size for each cohort was calculated. With a two-sided α of 0.05 and power of 0.80, and considering loss to follow-up, at least 200 cases were required for each cohort. To enhance statistical power and account for unforeseen dropouts, 220 participants were enrolled in both Cohort 1 and Cohort 2.

Baseline data collection

After screening and enrollment, all participants underwent a detailed questionnaire and physical examination. The survey covered basic demographic information such as age, sex, ethnicity, and education level; personal lifestyle habits including tobacco use, alcohol consumption and duration; family history of head and neck or other tumors in first- and second-degree relatives; occupational exposure to dust, chemicals, high temperatures, or radioactive materials; and comorbidities such as hypertension, diabetes, and chronic hepatic or renal diseases. Cohort 1 participants provided oropharyngeal swab samples for generic PCR detection of high-risk HPV DNA, whereas Cohort 2 patients had tumor paraffin sections tested by p16 immunohistochemistry, with ≥ 70% diffuse nuclear-cytoplasmic positivity considered HPV positive. After completing the questionnaire, study staff collected oropharyngeal swabs from all participants and recorded basic vital signs such as blood pressure and heart rate.

Sample collection and detection methods

Oropharyngeal Helicobacter pylori sampling method

Before oropharyngeal swab collection, each participant rinsed the mouth twice with clean water to remove visible debris. Using a disposable sterile cotton swab, the researcher asked the participant to open the mouth and pronounce “ah” to fully expose the posterior oropharyngeal wall and tonsillar surfaces. The swab was gently rotated and wiped back and forth across the posterior pharyngeal wall and both tonsillar regions for at least 10 s to obtain sufficient epithelial cells and potential bacteria. The swab was then immediately placed into a transport tube containing a microaerophilic protective agent, labeled with the participant’s unique identifier, delivered to the microbiology laboratory within 1 h, and stored at − 80 ℃. If the subject was scheduled for head and neck squamous cell carcinoma surgery, a mucosal biopsy adjacent to the tumor was taken under general or local anesthesia, labeled identically, and stored in the same manner to ensure comparability with the swab sample.

H. pylori culture and molecular detection

Culture conditions: In a dedicated laboratory room, staff inoculated swab samples onto selective Helicobacter pylori agar plates and incubated them at 37 ℃ in a microaerophilic incubator with an atmosphere of 5% O₂, 10% CO₂, and 85% N₂. The cultures were maintained for 7 days, with colony growth observed every 24 h; suspicious colonies were picked for Gram staining and rapid urease testing. Colony counts were determined by visually inspecting the agar plates after 7 days of incubation, and the results were recorded as colony-forming units (CFU) per swab.

Molecular detection: Genomic DNA was extracted from suspicious colonies, and polymerase chain reaction (PCR) was performed targeting H. pylori–specific regions (ureA gene or 16 S rRNA–specific sequence). The reaction mixture contained 2 × PCR Mix, forward and reverse primers, and an appropriate amount of template DNA. PCR conditions were 94 ℃ for 3 min (pre-denaturation); 35 cycles of 94 ℃ for 30 s (denaturation), 58 ℃ for 30 s (annealing), and 72 ℃ for 30 s (extension); followed by a final extension at 72 ℃ for 5 min. Amplified products were subjected to 1.5% agarose gel electrophoresis, and the presence of the target band indicated H. pylori positivity.

Secondary verification: For samples with inconsistent culture and PCR results, a repeat swab was collected for retesting; only concordant repeat results were accepted as the final determination.

Helicobacter pylori virulence gene typing

Target gene selection: cagA and vacA are the main virulence-associated genes of H. pylori. Specific primers for cagA were designed based on published sequences, and vacA was tested for the signal region (s1 or s2) and the middle region (m1 or m2) fragments¹¹.

Typing procedure: Genomic DNA extracted from bacteria or directly from swab samples was amplified by PCR using cagA and vacA primers, respectively. The PCR reaction components and cycling parameters were similar to those described above for H. pylori identification, except for primer sequences and annealing temperatures. A band size identical to the positive control was interpreted as positive for the corresponding gene.

Result interpretation: If the vacA signal region product was type s1 and the middle region was type m1, the strain was classified as having high virulence; other combinations were considered to have progressively lower virulence. When cagA was positive, the vacA typing result was combined to assess a higher level of pathogenic risk.

Human papillomavirus (HPV) testing

Cohort 1 (high-risk population) used baseline oropharyngeal swab sampling; Cohort 2 (confirmed patients) used formalin-fixed, paraffin-embedded (FFPE) tissue from surgically resected tumors, and patients receiving chemoradiotherapy alone used forceps or fine-needle biopsy FFPE specimens taken at diagnosis; if only superficial lesions were obtained, liquid-based cytology slides or cell-block preparations were made. In Cohort 2, HPV status was primarily determined using p16 immunohistochemistry with the E6H4 monoclonal antibody, a standard surrogate for transcriptionally active high-risk HPV infection in clinical practice. Tumors were deemed HPV-positive when ≥ 70% of tumor cells showed strong and diffuse nuclear-cytoplasmic staining; otherwise, they were considered negative. Positive HPV DNA samples were genotyped for types 16, 18, 31, 33, and 45 using a commercial reverse dot blot hybridization assay (Hybribio Limited, China) according to the manufacturer’s instructions. Cohort 1 swab samples were amplified for the HPV DNA L1 region with universal primers GP5⁺/GP6⁺, and positive samples were further genotyped as types 16, 18, 31, 33, and 45; in Cohort 2, when the FFPE tissue amount was insufficient or p16 staining was atypical, DNA was extracted from the same tissue for the same PCR amplification and genotyping.

Quality control and assay validation

For H. pylori culture, uninoculated plates served as negative controls. For PCR assays (H. pylori and HPV), positive and negative controls were included in every run. Positive controls consisted of genomic DNA from H. pylori strain ATCC 43504 and HPV-16 positive SiHa cell lines, respectively. Nuclease-free water was used as the negative control. The primer sequences used for the H. pylori-specific 16S rRNA gene were Forward: 5’- GCGACCTGCTGGAACATTAC-3’ and Reverse: 5’- CGTTAGCTGCATTACTGGAGA-3’. For HPV detection, the universal primers GP5+/GP6 + were used as described in the literature¹².”

Clinical data and prognostic information collection

Tumor diagnosis and staging

All confirmed head and neck squamous cell carcinoma patients were pathologically diagnosed by two independent pathologists. Tumor sites included the oral cavity, pharynx, and larynx, and specific subsites were confirmed by surgical records or findings from fiberoptic laryngoscopy or electronic nasopharyngoscopy. Tumor staging followed the TNM classification system of the Union for International Cancer Control (UICC)¹³, recording T stage, N stage, and the presence or absence of distant metastasis.

Treatment regimen and compliance record

After multidisciplinary team discussion, confirmed patients received surgery, radiotherapy, or concurrent chemoradiotherapy. Researchers recorded the surgery date, surgical extent, margin status, radiation dose and schedule, chemotherapy drug names, and cycles. After each treatment, compliance interviews were conducted in the ward or outpatient clinic to document whether the patient completed the treatment as planned, whether dose reductions occurred, and whether treatment was terminated early due to severe adverse effects.

Follow-up and prognostic evaluation indices

Researchers performed regular follow-up through outpatient reviews, telephone calls, and electronic medical record systems. Follow-up intervals were every 6 months at 6, 12, 18, 24, 30, and 36 months after baseline and at the final cut-off date. For patients with limited mobility, home visits by family physicians or professional nurses were arranged in advance to minimize loss to follow-up. The primary evaluation index for Cohort 1 (high-risk population cohort) was the time to first occurrence of head and neck squamous cell carcinoma, with all cases requiring clinical confirmation and pathological verification; the primary evaluation indices for Cohort 2 (confirmed head and neck squamous cell carcinoma cohort) were the time and cause of patient recurrence or death, with recurrence classified as local, regional, or distant metastasis, and death classified as tumor-related or due to other causes. Follow-up was terminated when a predefined primary endpoint occurred or when the preset follow-up deadline was exceeded for a participant.

Statistical analysis

All data were entered into the electronic data capture system by double entry. Researchers established logic checks, and any values outside preset reasonable ranges were promptly verified against original case records and corrected. If a sample had serious missing data, the individual was excluded after confirmation.

Analyses were performed using R software (version 4.1 or above). Continuous variables were expressed as mean ± standard deviation or median with interquartile range, and the choice between independent-samples t-test and nonparametric tests was based on normality tests. Categorical variables were expressed as percentages, and differences were compared using the chi-square test or Fisher’s exact test. All tests were two-sided. For head and neck squamous cell carcinoma occurrence and recurrence, Kaplan–Meier survival curves were plotted, and log-rank tests compared survival differences among different oropharyngeal Helicobacter pylori colonization statuses and virulence gene types. The Cox proportional hazards regression model was used to calculate hazard ratios and 95% confidence intervals, assessing differences in incidence or recurrence risk between H. pylori-positive and H. pylori-negative groups. Based on the Cox model, potential confounders—including age, sex, smoking history, alcohol history, HPV status (positive/negative), clinical tumor stage, and treatment modality—were simultaneously included to obtain adjusted hazard ratios (HRs) with their 95% confidence intervals, thereby excluding interference from non-H. pylori factors. All Cox models employed the Schoenfeld residual test for the proportional hazards assumption, with a global test P > 0.05 indicating the assumption was satisfied. A P < 0.05 was considered statistically significant.

Results

Participant enrollment and baseline characteristics

A total of 440 participants were enrolled, comprising 220 high-risk individuals (Cohort 1) and 220 patients diagnosed with head and neck squamous cell carcinoma (HNSCC; Cohort 2). Independent-samples t tests and χ² tests revealed significant differences in age, smoking duration, and HPV positivity between the two cohorts (all P < 0.05), while no significant differences were observed in sex, current smoking status, alcohol use duration, comorbidities, or occupational exposure (all P > 0.05) (Table 1). These results establish a comparable baseline while identifying specific covariates that may contribute to HNSCC risk and were adjusted for in subsequent models.

Table 1.

Comparison of baseline characteristics between the two cohorts.

Variable	Cohort 1 (n = 220)	Cohort 2 (n = 220)	Test value	P value
Age (years)	55.23 ± 6.28	57.48 ± 6.91	t = − 2.044	0.042
Sex (male)	138 (62.7%)	145 (65.9%)	χ² = 0.468	0.494
Smoking history (years)	21.34 ± 3.16	22.97 ± 3.26	t = − 2.575	0.011
Current smoker (yes)	104 (47.3%)	111 (50.5%)	χ² = 0.493	0.483
Alcohol history (years)	17.26 ± 2.48	16.84 ± 2.23	t = 1.610	0.108
Comorbidities (present)	58 (26.4%)	76 (34.5%)	χ² = 3.083	0.079
Occupational exposure (present)	42 (19.1%)	37 (16.8%)	χ² = 0.363	0.547
HPV positive (n, %)	46 (20.9%)	98 (44.5%)	χ² = 19.602	< 0.001
Tumor stage (Cohort 2 only)	N/A	Stage I 56 (25.5%), Stage II 74 (33.6%), Stage III 52 (23.6%), Stage IV 38 (17.3%)	–	–

Oropharyngeal Helicobacter pylori colonization and virulence gene typing

Compared with Cohort 1, participants in Cohort 2 exhibited significantly higher oropharyngeal H. pylori colonization rates (43.6% vs. 32.7%), culture-based colony counts, and qPCR bacterial loads (all P < 0.05) (Table 2). A subsite analysis within Cohort 2 revealed the highest H. pylori positivity rates in tumors of the oropharynx (50.0%) and larynx (45.2%), followed by the oral cavity (35.9%). Stratified analysis of virulence gene profiles indicated that strains harboring the high-virulence combination cagA(+)/vacA s1/m1 were associated with the highest bacterial loads in both cohorts. Kruskal–Wallis H tests confirmed significant differences in bacterial burden across genotypes (Cohort 1: P = 0.014; Cohort 2: P = 0.004) (Table 3), suggesting that strain virulence may influence colonization intensity and potentially impact carcinogenesis.

Table 2.

Detection rate and bacterial load of oropharyngeal H. pylori colonization (qPCR copy number).

Index	Cohort 1 (n = 220)	Cohort 2 (n = 220)	Test value	P value
H. pylori-positive cases, n (%)	72 (32.73%)	96 (43.64%)	χ² = 4.561	0.033
Culture count (CFU), M (IQR)	3385.21 (1642.27–4997.52)	3752.59 (1704.31–5864.78)	Z = 2.231	0.026
qPCR (copies/swab), M (IQR)	25346.73 (10852.44–42631.59)	31254.89 (12475.38–50510.26)	Z = 2.548	0.011

Table 3.

Distribution of virulence genotypes (cagA, vacA) and corresponding bacterial loads.

Virulence-gene combination	Cases detected	Proportion (%)	qPCR copies (M [IQR])
Cohort 1
cagA(+)/vacA s1/m1	20	27.78	38247.16 (23456.32–52462.19)
cagA(+)/vacA s1/m2	13	18.06	32675.89 (20123.75–43842.07)
cagA(+)/vacA s2/m1	10	13.89	29864.73 (16754.38–39214.82)
cagA(+)/vacA s2/m2	8	11.11	27653.48 (14024.57–36518.66)
cagA(–)/vacA s1/m1	15	20.83	23147.59 (12856.37–31425.44)
cagA(–)/vacA s2/m1	6	8.33	20874.66 (11248.63–27946.81)
	Overall (Kruskal–Wallis): H = 14.237, P = 0.014
Cohort 2
cagA(+)/vacA s1/m1	28	29.17	46382.17 (26342.56–60847.33)
cagA(+)/vacA s1/m2	18	18.75	39648.25 (23576.81–50012.74)
cagA(+)/vacA s2/m1	12	12.5	34218.92 (18344.59–47536.27)
cagA(+)/vacA s2/m2	10	10.42	30587.64 (16453.19–42475.52)
cagA(–)/vacA s1/m1	18	18.75	28963.78 (15328.47–38816.62)
cagA(–)/vacA s2/m1	10	10.42	25194.55 (13044.73–34508.96)
	Overall (Kruskal–Wallis): H = 17.486, P = 0.004

Follow-up results of cohort 1 (high-risk population)

During the 36-month follow-up of Cohort 1 (n = 220), a total of 24 participants developed HNSCC. The incidence was significantly higher in the H. pylori-positive group (15 of 72 individuals; 20.8%) compared to the H. pylori-negative group (9 of 148 individuals; 6.1%). Kaplan-Meier survival analysis confirmed a significantly lower disease-free survival (DFS) rate in H. pylori-positive individuals (P = 0.028) (Fig. 1). In Cox regression models, H. pylori positivity (HR = 1.50, P = 0.039), HPV positivity (HR = 1.85, P = 0.011), and longer smoking history (HR = 1.06, P = 0.042) were identified as independent risk factors for HNSCC onset (Table 4). These findings suggest that oropharyngeal H. pylori colonization is an important modifiable factor associated with cancer development in at-risk individuals. Notably, among the 15 incident HNSCC cases in Cohort 1 who were positive for oropharyngeal H. pylori at baseline, 12 (80.0%) were also found to have H. pylori present in their tumor-adjacent mucosal biopsies upon diagnosis, as confirmed by PCR. Analysis of these 12 cases revealed that the primary tumor sites were the oropharynx (n = 7), larynx (n = 3), and oral cavity (n = 2). All were diagnosed at an early stage (Stage I or II), highlighting the potential role of H. pylori in the initial phases of carcinogenesis.

Fig. 1.

Kaplan-Meier curve of disease-free survival in Cohort 1, stratified by H. pylori status. Please note that the y-axis is truncated (70–100%) to better visualize the differences between the curves. The log-rank test indicates a significant difference (p = 0.028).

Table 4.

Cox regression model analysis (Cohort 1).

Variable	Univariate HR (95% CI)	P value	Multivariate HR (95% CI)	P value
H. pylori colonization (positive vs. negative)	1.71 (1.14–2.56)	0.009	1.50 (1.02–2.21)	0.039
HPV status (positive vs. negative)	1.98 (1.27–3.08)	0.003	1.85 (1.15–2.97)	0.011
H. pylori load (high vs. low)	1.56 (1.03–2.36)	0.034	1.42 (0.94–2.15)	0.094
Age (per + 1 year)	1.04 (1.01–1.07)	0.016	1.03 (0.99–1.06)	0.078
Sex (male vs. female)	0.95 (0.63–1.43)	0.803	0.98 (0.65–1.49)	0.929
Years of smoking (per + 1 year)	1.08 (1.02–1.15)	0.007	1.06 (1.00–1.13)	0.042
Years of drinking (per + 1 year)	1.02 (0.97–1.06)	0.361	1.01 (0.96–1.07)	0.623
Occupational exposure (yes vs. no)	1.17 (0.78–1.77)	0.447	1.12 (0.72–1.75)	0.609

Note: The proportional hazards assumption of the Cox model was satisfied; global test P = 0.271.

Follow-up results of cohort 2 (diagnosed head and neck squamous cell carcinoma patients)

Among diagnosed HNSCC patients, those with H. pylori colonization had significantly higher recurrence rates (26.04% vs. 16.94%, P = 0.018) and overall mortality rates (13.54% vs. 8.06%, P = 0.043) than H. pylori-negative patients (Table 5). Kaplan–Meier analysis further confirmed a poorer overall survival (OS) in H. pylori-positive individuals (P = 0.031) (Fig. 2). In multivariate Cox regression (Table 6), H. pylori positivity (HR = 1.57, P = 0.035) and advanced tumor stage (HR = 2.12, P = 0.002) were associated with worse prognosis, while HPV positivity remained a favorable prognostic factor (HR = 0.63, P = 0.046). These findings indicate that H. pylori colonization may contribute to tumor progression and adverse outcomes, especially in individuals lacking protective HPV-associated immune responses.

Table 5.

Recurrence and survival outcomes in cohort 2.

Follow-up outcome	H. pylori-negative group (n = 124)	H. pylori-positive group (n = 96)	χ² value	P value
Recurrence — local (n, %)	9 (7.26)	11 (11.46)	–	–
Recurrence — regional (n, %)	7 (5.65)	8 (8.33)	–	–
Recurrence — distant (n, %)	5 (4.03)	6 (6.25)	–	–
Total recurrence (n, %)	21 (16.94)	25 (26.04)	5.572	0.018
Tumor-related death (n, %)	8 (6.45)	11 (11.46)	–	–
Other-cause death (n, %)	2 (1.61)	2 (2.08)	–	–
Total death (n, %)	10 (8.06)	13 (13.54)	4.093	0.043

Fig. 2.

Kaplan-Meier curve of overall survival (OS) in Cohort 2, stratified by H. pylori status. Please note that the y-axis is truncated (80–100%) to better visualize the differences between the curves. The log-rank test indicates a significant difference in survival (p = 0.031).

Table 6.

Multivariate Cox regression model (Cohort 2).

Variable	Univariate HR (95% CI)	P value	Multivariate HR (95% CI)	P value
H. pylori colonization (positive vs. negative)	1.68 (1.11–2.54)	0.014	1.57 (1.03–2.41)	0.035
Virulence genotype (high vs. low)	1.52 (1.02–2.27)	0.040	1.41 (0.94–2.13)	0.092
HPV positivity (positive vs. negative)	0.59 (0.38–0.92)	0.019	0.63 (0.40–0.99)	0.046
Tumor stage (Ⅲ–Ⅳ vs. Ⅰ–Ⅱ)	2.28 (1.46–3.57)	< 0.001	2.12 (1.32–3.41)	0.002
Treatment modality (non-surgery vs. surgery)	1.35 (0.88–2.06)	0.171	1.28 (0.82–1.98)	0.281
Age (per + 1 year)	1.03 (1.00–1.06)	0.049	1.02 (0.99–1.05)	0.128
Sex (male vs. female)	1.08 (0.71–1.63)	0.724	1.06 (0.69–1.63)	0.789
Years of smoking (per + 1 year)	1.06 (1.01–1.12)	0.019	1.05 (0.99–1.11)	0.111
Years of drinking (per + 1 year)	1.04 (0.99–1.09)	0.114	1.03 (0.97–1.09)	0.280
Severe comorbidities (present vs. absent)	1.36 (0.89–2.07)	0.156	1.32 (0.85–2.04)	0.212

Note: The proportional hazards assumption of the Cox model was satisfied; global test P = 0.183. The high-virulence genotype is defined as cagA(+)/vacA s1/m1.

Discussion

This prospective cohort study provides robust evidence that oropharyngeal colonization by Helicobacter pylori is significantly associated with both the development and prognosis of head and neck squamous cell carcinoma (HNSCC). In high-risk individuals, H. pylori colonization—particularly at higher bacterial loads—was associated with increased cancer incidence. Among HNSCC patients, H. pylori positivity correlated with a higher risk of recurrence and mortality, highlighting its potential role in both tumor initiation and disease progression.

Previous research has largely focused on H. pylori in gastric pathology, with limited and inconclusive studies investigating its presence in the oropharynx¹⁴. The oropharyngeal H. pylori colonization rate in our high-risk cohort (32.7%) and HNSCC patient cohort (43.6%) is a key finding of this study. A cross-sectional study by Mohtasham et al. using PCR on tumor tissue reported a 32.25% H. pylori prevalence in HNSCC patients⁷. This figure is remarkably consistent with the rate in our high-risk, cancer-free cohort, suggesting a high prevalence of colonization in the at-risk population. Our higher rate of 43.6% in diagnosed HNSCC patients, detected via oropharyngeal swabs, likely reflects a more comprehensive assessment of “field colonization” across the mucosal surface, as compared to a localized tumor biopsy. Taken together, despite methodological differences, both our prospective findings and previous cross-sectional data underscore a substantial association between H. pylori and the HNSCC disease continuum. Our findings not only confirm oropharyngeal colonization but also suggest a pathogenic contribution to mucosal dysregulation, chronic inflammation, and carcinogenesis. Our finding that a high proportion of incident tumors were also positive for H. pylori in the adjacent mucosa provides compelling evidence supporting a direct pathogenic role. This suggests that persistent colonization in the oropharynx may directly promote carcinogenesis by inducing chronic inflammation, disrupting the epithelial barrier, and creating a pro-carcinogenic microenvironment at the site of tumor development, rather than acting solely through indirect systemic effects. The epithelial barrier of the oropharynx, already vulnerable due to environmental insults (e.g., smoking, alcohol), may become more susceptible to bacterial colonization. This persistent microbial exposure may induce chronic inflammation, promote epithelial hyperplasia, and impair local immune surveillance—thereby creating a pro-carcinogenic environment^15,16.

Strikingly, colonization by high-virulence H. pylori strains (cagA(+)/vacA s1/m1) was associated with the greatest bacterial loads, suggesting enhanced mucosal pathogenicity. These strains are known to secrete virulence factors that disrupt epithelial integrity, modulate host immune responses, and potentially accelerate malignant transformation¹⁷. Importantly, our study observed a combined impact of H. pylori and HPV, with co-infected individuals exhibiting higher cancer risk and potentially more aggressive clinical profiles^18,19. This highlights a multi-pathogen interaction model, where bacterial and viral agents may collaboratively drive carcinogenesis through immune modulation, barrier dysfunction, and inflammatory pathways^15,18.

In diagnosed HNSCC patients, H. pylori colonization was independently associated with poor prognosis, even after adjusting for tumor stage and HPV status. The immunosuppressive microenvironment caused by H. pylori may weaken anti-tumor immune responses and promote tumor progression²⁰. Although high-virulence genotypes demonstrated stronger associations in univariate analysis, their independent impact diminished in multivariate models, indicating complex interactions between microbial, viral, and clinical factors.

This study also raises important questions regarding clinical management. While HPV screening is widely adopted for risk stratification, our data suggest that assessing oropharyngeal H. pylori colonization—especially high-virulence genotypes—may offer additional prognostic value²¹. The possibility of incorporating bacterial eradication strategies into treatment or prevention plans warrants further exploration²². Notably, whether H. pylori eradication could influence radiotherapy sensitivity or reduce recurrence risk in HNSCC patients remains unknown and merits investigation through randomized controlled trials.

The HPV positivity rate in our HNSCC cohort (44.5%, determined primarily by p16 status) and high-risk cohort (20.9%, by PCR) warrants discussion. This rate in HNSCC patients is largely driven by the substantial proportion of oropharyngeal cancers (40.9%) in our cohort, a subgroup known for high HPV prevalence. While our overall rates may seem higher than some historical data from Asia, they are consistent with recent reports indicating a rising prevalence of HPV-positive HNSCC in Southern China²³. Furthermore, our study population consists of high-risk individuals with significant exposure to tobacco and alcohol, which may independently increase the risk of persistent oral HPV infection, contributing to the elevated prevalence observed in both cohorts. Further large-scale epidemiological studies are needed to confirm these regional trends and risk-factor interactions.

Our study has several limitations. First, being a single-center study, our findings may have limited generalizability due to a lack of regional and ethnic diversity. Future multicenter studies are necessary to validate these conclusions. Second, our virulence gene screening was limited to cagA and vacA, and we did not sufficiently stratify by HPV genotypes or anatomical tumor sites.Future multicenter studies with broader molecular profiling are needed to validate and expand upon our findings.

In summary, this study highlights H. pylori as a novel microbial factor in the pathogenesis and progression of HNSCC. Its interaction with HPV, virulence gene expression, and impact on immune and inflammatory pathways suggest it may be a critical target in individualized risk prediction and treatment planning. Integrating bacterial profiling into current screening and management strategies may significantly enhance early detection and improve clinical outcomes for HNSCC patients.

Conclusion

This prospective cohort study provides compelling evidence that oropharyngeal colonization by Helicobacter pylori (H. pylori) significantly contributes to both the development and adverse prognosis of head and neck squamous cell carcinoma (HNSCC). Individuals with H. pylori colonization—particularly those harboring high-virulence genotypes (cagA(+)/vacA s1/m1)—are at increased risk of HNSCC onset, recurrence, and mortality. The complex relationship between H. pylori and HPV infection suggests a potential interplay in promoting tumorigenesis and modulating clinical outcomes through immune microenvironment disruption.These findings underscore the need to integrate microbial biomarkers into HNSCC risk stratification, early screening, and personalized treatment strategies. In addition to traditional factors such as smoking and HPV status, assessing H. pylori colonization—especially virulence profiles—may enhance the identification of high-risk individuals and inform targeted interventions. Further multicenter, longitudinal, and mechanistic studies are warranted to validate these associations and explore whether eradication therapy may improve clinical outcomes in H. pylori-positive HNSCC patients.

Author contributions

Author Contributions: Xianyao Jiang, Yongjin Huang and Changwu Li: Investigation, Writing - original draft, Conceptualization, Methodology, Formal analysis, Data curation, Writing - review & editing. Hongyan Jiang: Methodology, Writing - original draft, Resources, Writing - review & editing, Supervision, Project administration, Funding acquisition.

Funding

This study was supported by the Major Science and Technology Program of Hainan Province (ZDKJ2021040).

Data availability

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to confidentiality.

Declarations

Competing interests

The authors declare no competing interests.