While the precise oncogenic pathways of HPV in OPSCC remain incompletely characterized, accumulating evidence from translational studies strongly implicates HPV16 as the predominant etiological agent in oropharyngeal carcinogenesis. The high-risk HPV E6 and E7 genes are pivotal drivers of cell transformation, especially in basal squamous epithelial cells. For HPV types 16 and 18, both E6 and E7 proteins play essential roles in preventing senescence in human primary keratinocytes, potentially activating oncogenes and inducing carcinogenic cell proliferation (9, 10). E6 forms a complex as E6-AP, binding to p53 and promoting its degradation, thereby accelerating cell division and malignant transformation (11). Meanwhile, the E7 oncoprotein interacts with the retinoblastoma protein (pRb), disrupting its association with E2F transcription factors and facilitating unregulated progression into the S-phase of the cell cycle (12). The limited oncogenic potential demonstrated by HPV16/18 E2-E4-E5 genomic segments, which becomes further attenuated upon viral genome integration, may mechanistically explain their predominant association with non-malignant epithelial transformations rather than invasive carcinomas (13).

Some researchers propose that the cis-activating effect of HPV DNA on adjacent host genes has been postulated by investigators as a potential mechanism driving oncogenic transformation processes (11, 14). Hu et al. found that the E6/E7 proteins regulate the activity of key enzymes in the aerobic glycolysis pathway by influencing the binding of IGF2BP2 to the MYC m6A site (overexpression of IGF2BP2 in E6/E7-knockout CC cells). Ultimately, this reduces the glycolytic flux, leading to a decrease in cancer cell proliferation (15). Besides, hu et al. suggest that oncogenic human papillomaviruses (HPVs) could generate cir-cRNAs, some of which encompass the E7 oncogene (circE7), which is N6-methyladenosine (m6A) modified, preferentially localized to the cytoplasm, associated with polysomes. CircE7 can be translated and produce E7 oncoprotein (16). Other studies have noted that the integration of HPV DNA into chromosomes is a significant inducer of p53 gene mutations and may also lead to its overexpression (17). Palefsky et al. discovered that HPV16 infection is a cause of cellular transformation, likely due to alterations in the p53 and retinoblastoma proteins(pRb) triggered by HPV E6 and E7 (18). The cumulative evidence establishes HPV E6/E7’s pivotal function in p53 and pRb pathway disruption, which initiates cellular transformation and oncogenic signaling cascades, thereby mechanistically underpinning OPSCC pathogenesis.

Although HPV alone can induce malignant cell transformation, its carcinogenicity is often enhanced by synergistic interactions with other factors (14, 17). Oral tissues, exposed to various physical, chemical, and microbial agents, are particularly susceptible to the combined effects of HPV with smoking, alcohol, trauma, fungal infections, or other viruses, contributing to oral malignancies (17). Animal models confirm HPV’s role as a cofactor in carcinogenesis, with smoking facilitating HPV invasion and colonization in oral tissues (19). Additionally, HPV, smoking, and alcohol collectively induce oral cancer, suggesting a synergistic interaction. HPV’s carcinogenicity may also be influenced by hormone levels and immune status. For instance, The observed elevation in HPV prevalence among gravid populations, combined with augmented cervical carcinogenesis risk in prolonged contraceptive users, implies endocrine-mediated potentiation of viral genome duplication and mitotic activity (17, 19). Elevated hormone levels may enhance viral DNA expression, favoring carcinogenesis. Furthermore, higher HPV infection rates and tumor incidence in immunocompromised individuals, such as kidney transplant and acquired immunodeficiency syndromes (AIDS) patients, indicate latent HPV reactivation under such conditions (19).

Compared with HPV-negative OPSCC, HPV-OPSCC typically presents with smaller primary lesions but is more likely to develop early lymphatic metastasis, often manifesting as cystic changes in metastatic neck lymph nodes (cystic metastases). HPV-OPSCC also exhibits a distinctive pattern of lymph node invasion: the involved lymph nodes tend to be large and prone to extracapsular spread (2). Although HPV-OPSCC and HPV-negative OPSCC differ substantially in terms of their lymphatic metastasis rate and time to metastasis, they share similar locations and numbers of invaded lymph nodes, suggesting a fundamentally similar route of lymphatic invasion (22). Given the different pathogenic mechanisms and biological behaviors of HPV-OPSCC and HPV-negative OPSCC, the 8th edition of the American Joint Committee on Cancer (AJCC) staging manual has established an independent TNM staging system for HPV-OPSCC (21).

Positron emission tomography-computed tomography (PET-CT) is currently one of the most employed clinical methods for detecting lymphatic metastasis in head and neck tumors (23). However, a clinical study by Snyder et al. reported a misdiagnosis rate of 36–43% in detecting lymphatic metastasis in 49 HPV-OPSCC cases using PET-CT, with challenges in accurately determining the number and size of affected lymph nodes (24). Some researchers have established predictive models based on clinical presentation and imaging results to evaluate the risk of lymphatic metastasis in HPV-OPSCC patients; however, these models require validation through prospective studies (25). Fine-needle aspiration (FNA) biopsy is also insufficient for accurate detection, as aspirates from cystic metastatic lymph nodes in HPV-OPSCC resemble fluid from benign cystic lesions, potentially leading to diagnostic omissions (26). High-risk HPV testing on FNA samples is recommended for patients presenting with unexplained neck masses to determine whether these lesions might represent HPV-OPSCC metastases (26). HPV-RNA in situ hybridization has shown promise, with an 88.9% concordance rate with p16 immunohistochemical staining in detecting HPV in neck FNA samples (27).

Although HPV-associated oropharyngeal squamous cell carcinoma (HPV-OPSCC) is categorized as a distinct disease entity in the AJCC 8th edition staging system, current clinical management remains largely similar to that for HPV-negative OPSCC because of limited high-quality clinical evidence justifying disease-specific therapeutic strategies. (28). Patients with early HPV-OPSCC can undergo surgical monotherapy; however, most patients present with lymphatic metastasis and extracapsular spread at diagnosis, necessitating combined surgery, radiotherapy, and chemotherapy (28). It is important to emphasize that patients with lymphatic metastasis have a poorer prognosis compared to those without nodal involvement, underscoring the need to further elucidate the mechanisms of lymphatic metastasis and develop targeted interventions. Despite early and high rates of lymphatic metastasis, the overall prognosis of HPV-OPSCC is markedly better than that of HPV-negative OPSCC. A recent clinical study found that the 3-year overall survival rate was significantly higher (93%) among HPV-OPSCC patients than among HPV-negative OPSCC patients with a smoking history (46.2%) (28). The number of invaded lymph nodes exerts minimal effect on overall survival in HPV-OPSCC, contrasting sharply with other head and neck malignancies (29). The distinct pathobiological profile of HPV-OPSCC necessitates urgent elucidation of molecular mediators governing its preferential lymphatic dissemination, a prerequisite for optimizing nodal disease management protocols.

Despite HPV-OPSCC’s sensitivity to radiotherapy and chemotherapy, its high lymphatic metastasis incidence remains paradoxical. EMT is critical for lymphatic metastasis (30). HPV E6/E7 upregulates EMT transcription factors (Slug, Twist, ZEB1, ZEB2), enhancing tumor proliferation and invasiveness (31). Nevertheless, PRKCZ, an oncogenic factor, promotes HPV-OPSCC progression but is inhibited by HPV E6-induced hypermethylation, suppressing EMT (32). Recent studies indicate that miRNA 34a significantly inhibits tumor stem cell proliferation, invasiveness, and EMT in HNSCC, with reduced miRNA 34a levels correlating with increased tumor invasiveness (33). The oncogenic proteins E6 and E7 of HPV can also enhance the tendency of tumor cells to metastasize to lymph nodes by activating the pathways related to epithelial-mesenchymal transition (EMT), such as the Wnt/β-catenin pathway (33). Cancer stem cells (CSCs) are pivotal for EMT and tumor metastasis, with markers like ALDH1A1, CD44, CD98, BMI1, and OCT4. Gunduz et al. reported lower CD44 and CD98 expression in HPV+ OPSCC specimens (34), while another study found reduced BMI1 expression in HPV+ OPSCC (35). Zhang et al. found that HPV-OPSCC contains more CSCs than HPV-negative OPSCC, with HPV E6 degrading p53 or blocking its acetylation, increasing CSC numbers and tumor invasiveness (36). Hufbauer et al. showed that HPV E6/E7 mediates the transition from stationary to migratory CSCs by regulating CD44 and EpCAM, promoting lymphatic metastasis in HPV-OPSCC (37). It shows that the intricate interplay between cancer stem cell plasticity and microRNA-mediated regulatory networks in HPV-driven oncogenesis is compellingly demonstrated by these experimental observations.

The VEGF family, particularly VEGF-C, VEGF-D, and their receptor VEGFR-3, plays a critical role in lymphangiogenesis and lymphatic metastasis in various malignancies, including HNSCC (38). Elevated VEGF-C/D expression in HNSCC correlates with increased peritumoral lymphatic vessel density (LVD), lymphatic metastasis, and poor prognosis, with VEGF-C serving as a predictive marker for metastasis (39, 40). In HPV-related tumors, HPV16-E6 seems to induce VEGF expression independently of TP53 inactivation, using the SP1 transcription factor for E6-mediated induction of the VEGF promoter (41). However, Baruah et al. found that circulating VEGF levels in HPV-OPSCC were similar to healthy controls, with no significant difference in VEGF-D expression among HPV-OPSCC, HPV-negative OPSCC, and healthy groups (42). This low VEGF expression contrasts with the lymphatic invasiveness of HPV-OPSCC, raising questions about whether HPV-OPSCC forms specific lymphatic metastases via VEGF-C-mediated lymphangiogenesis ( Table 1 ).

A hypoxic tumor microenvironment leads to elevated levels of hypoxia-inducible factor (HIF), which promotes EMT and increased secretion of matrix metalloproteinases by inducing downstream target genes, thereby enhancing tumor invasiveness (43). HPV-associated oropharyngeal squamous cell carcinoma (HPV-OPSCC) tumor cells may demonstrate hypoxia-inducible factor (HIF)-mediated suppression of the activation of E-cadherin and concurrent EMT, promoting increased invasive potential. Under hypoxic conditions, CSCs can maintain stemness and self-renewal capacity under the stimulation of HIF-1, further enhancing tumor invasiveness (43). However, another study showed that the tumor microenvironment of HPV-OPSCC lacks significant hypoxia, with minimal HIF expression, but displays significantly increased neovascular density around the cancer cells (44). This finding suggests that HPV-OPSCC may possess a unique mechanism of hypoxia.

TLS form from lymphoid and stromal cell aggregation in non-secondary lymphoid organs under pathological conditions, including autoimmune diseases, infections, transplant rejection, and malignancies (56). Persistent inflammation upregulates chemokines (e.g., CXCL13, IL-7), attracting lymphoid tissue inducer (LTi) cells (e.g., Th17, B cells, M1 macrophages) to inflamed sites (57, 58). LTα1β2 on LTi cells interacts with LTβR and IL-17 receptors on stromal cells, inducing VEGF-C release, high endothelial venule (HEV) formation, and adhesion molecule expression (VCAM-1, ICAM-1) (59). Macrophages and endothelial cells secrete IL-36γ, further enhancing VCAM-1, ICAM-1, and chemokines (IL-8, CCL2, CCL20), promoting HEV recruitment of lymphocytes and TLS maturation (60).

The role of TLS in HPV-positive OPSCC has garnered increasing attention. A study found that the presence of TLS in HPV-positive OPSCC is associated with higher CD8+ T cell infiltration and improved clinical outcomes (61). Tertiary lymphoid structures (TLS), residing within the tumor microenvironment, represent ectopic lymphoid formations that actively promote localized anti-tumor immune activity. In HPV-positive OPSCC, B cells and T cells within TLS collaborate to generate HPV-specific antibodies and cytotoxic T cell (CTL) responses, thereby enhancing anti-tumor immunity. The formation and maintenance of TLS are closely linked to the expression of chemokines such as CXCL13 and CCL21. High CXCL13 expression is associated with TLS formation and function in HPV-positive OPSCC, providing a theoretical basis for targeting chemokine networks to enhance TLS activity (56). Additionally, follicular helper T cells (Tfh) within TLS play a critical role in B cell differentiation and antibody production, further amplifying anti-tumor immune responses (62). However, TLS functionality is not uniformly effective across all patients, as some exhibit dysfunctional TLS characterized by B cell exhaustion or T cell impairment. Therefore, it is meaningful to further explore and focus on elucidating the mechanisms underlying the dysfunction of tertiary lymphoid structures (TLS), and to develop targeted strategies to restore their anti-tumor activity.

Immune checkpoint blockade (ICB) targeting PD-1/PD-L1 is a prominent immunotherapy. It has demonstrated significant efficacy in treating OPSCC. However, the overall response rate to PD-1/PD-L1 blockade in OPSCC remains below 20% (63). Studies have revealed that CD20+ B cells predominantly localize within TLS (64). Moreover, the presence of CD20+ B cells is associated with a more favorable prognosis for patients with OPSCC (65). The induction of TLS formation enhances the response to PD-1 blockade treatment in HPV-OPSCC mouse models. Therefore, promoting TLS formation may improve the response rates of HPV-OPSCC patients to ICB therapy (66). A study found that by regulating chemokines such as CXCL13 or lymphoid tissue inducers like LTα/β, the maturation and function of TLS can be promoted, and the anti-tumor immunity can be enhanced (67). Another study found that the PD-1 inhibitor pembrolizumab is effective in enhancing the response to immunotherapy. It demonstrated clinically meaningful anti-tumour activity in recurrent or metastatic squamous cell carcinoma of the head and neck (68).

In HPV-OPSCC, the maturity of TLS is closely linked to their functional capacity. Immature TLS may fail to effectively facilitate antigen presentation and lymphocyte activation, thereby compromising immunotherapy efficacy. In contrast, mature TLS are enriched with memory B cells, plasma cells, and CD4+ T cells, accompanied by upregulated expression of B-cell activation-related genes. Consequently, promoting TLS maturation could enhance the therapeutic response to immunotherapy in HPV-OPSCC (69).

Besides, in HPV-associated oropharyngeal squamous cell carcinoma (HPV-OPSCC), dysfunction of TLS may be linked to a significant reduction in CD8+ T cells and B lymphocytes within the tumor microenvironment. This association is particularly pronounced in cases of disease recurrence following chemotherapy or radiotherapy (70). Further research revealed T cell hat conventional chemoradiotherapy may disrupt TLS integrity, leading to deterioration of the local immune microenvironment. Consequently, this impairment reduces response rates to immunotherapy. Given favorable prognosis of HPV-OPSCC patients, decreasing therapeutic intensity-such as reducing radiation doses and avoiding chemotherapy, and minimizing overtreatment-could preserve TLS functionality and sustain antitumor immunity (71). A recent study found that neoadjuvant chemotherapy (NAC) can enhance the HPV-specific T cell response of the primary tumor and improve the survival rate of patients (72) ( Figure 1 ).