Introduction
Cervical cancer is one of the most common cancers among women globally, with an estimated 660,000 new cases and 350,000 deaths in 2022. Despite a decrease in incidence in well-developed countries due to effective preventive screening programs and vaccination, an estimated 13,360 new cases and 4,320 deaths from cervical cancer were reported in the U.S. in 2025 (1). Approximately 80–90% of cervical cancers are squamous cell carcinomas (SCCs), and more than 90–95% of these tumors are related to high-risk human papillomaviruses (HPVs), most commonly HPV16 and HPV18 (2,3). In the majority of cases, high-risk HPV DNA integrates into the host cell genome, resulting in persistent overexpression of the viral oncoproteins E6 and E7, which subsequently inactivate tumor suppressors p53 and RB1, respectively, and promote uncontrolled cell growth, genomic instability, and tumor development (4).
The evolution of invasive SCCs from their precursor lesions, namely, low-grade and high-grade squamous intraepithelial lesions (LSIL and HSIL), occurs in a stepwise fashion, as demonstrated in model systems (5). While HPV-related inactivation of p53 and RB1 represents the initial step of oncogenesis, somatic driver mutations accumulate as the disease progresses from precancerous stages to invasive carcinoma. Genome-wide analyses have revealed recurrent pathogenic mutations in cervical carcinomas, including PIK3CA, EP300, FBXW7, PTEN, HLA-A/B, ARID1A, NFE2L2, KRAS, ERBB2/3, and other less common mutations (6,7). Among these genes, PIK3CA mutations represent the most frequent genetic alteration in cervical SCCs (6,7).
The PIK3CA gene encodes the p110α protein, which is the catalytic subunit of the phosphoinositide 3-kinase (PI3K) enzyme. PI3K is a lipid kinase generating lipid second messengers, which subsequently activate AKT and mTOR signaling cascades. This pathway is involved in numerous essential and pathogenic cellular processes, including cell proliferation and growth, survival, apoptosis, DNA damage repair, migration and motility, angiogenesis, and cancer metastasis (8,9). Mutations in PIK3CA lead to an overactive PI3K enzyme, potentially contributing to cancer development, metastasis, and therapeutic resistance. Notably, PIK3CA is one of the most frequently mutated genes in cancer (9), with most mutations occurring in the helical domain (exon 9) and the kinase domain (exon 20) (10). In cervical cancers, the reported PIK3CA mutation rate varies widely, ranging from 8.15% to 60.0% (11–32), underscoring its potential role in cervical cancer development and progression. While some studies suggest that PIK3CA mutation confers favorable survival outcomes, others have reported an association with poorer prognosis. In a meta-analysis comprising 12 articles and 2,196 women with cervical cancer, the authors demonstrated that the impact of PIK3CA mutations on survival outcomes remains inconclusive (33). To minimize confounding factors, we investigated the prevalence of PIK3CA hotspot mutations in pathology-confirmed SCCs in a single institution and correlated these mutations with clinicopathological features.
Discussion
In this study, we characterized the clinicopathological features of cervical SCC with PIK3CA mutations at a single institution, focusing primarily on the most common PIK3CA mutations in the helical domain (exon 9, E542K and E545K) and the kinase domain (exon 20, H1047R) (10,33). We found that the prevalence of PIK3CA hotspot mutations in this cohort was 13%, consistent with findings from several previous studies (6,11,20,24). However, our reported mutation rate is relatively lower than that observed in most other studies. The reasons for these differences remain unclear; however, Sanger sequencing of PIK3CA hotspot mutations, unlike next-generation sequencing, may underestimate the overall mutation rate, as these hotspot mutations account for approximately 80% of all mutations in the gene (10,24,38,39). Other possible contributing factors include age distribution, ethnic heterogeneity, differences in tumor stage, and degradation of genomic material in archival formalin-fixed, paraffin-embedded (FFPE) tissues (11).
As an oncogene regulating the PI3K/AKT/mTOR pathway, PIK3CA is one of the most frequently mutated genes in human cancers. Consistent with previous investigations (11,16–18,24,38), we found that 96.3% (26/27) of mutations were clustered in the helical domain (exon 9), whereas only one case (3.7%) harbored H1047R, a mutation in the kinase domain (exon 20). Our findings in cervical cancer, as well as the findings in previous investigations (7), are similar to results reported in bladder cancer (40) and HPV-associated head and neck cancers (41) but differ from observations in breast (42), endometrial (43), and many other cancers, which predominantly harbor PIK3CA mutations in the kinase domain. This distinct mutation profile may reflect the difference in etiology, cell-of-origin, and oncogenic mechanisms across different cancers (44,45). The helical domain of the PIK3CA protein (p110α) has been shown to function as a scaffold that biochemically interacts with the inhibitory p85α protein. Somatic mutations in this domain disrupt the inhibitory interface between the p110α catalytic subunit and the p85α regulatory subunit, releasing the enzyme from normal inhibitory control and resulting in enzymatic overactivity. This overactivity mimics a growth-factor-activated state and is highly dependent on the Ras-binding domain. In contrast, mutations in the kinase domain alter the protein's conformation, enhancing its affinity for substrates on the cell membrane, which increases kinase activity. This pathway functions independently of Ras. Regardless of the mutation sites, these variants directly affect the PI3K enzyme, leading to overactive AKT and subsequent mTOR activity, which promotes tumor growth, proliferation, and resistance to certain therapies. Given their different oncogenic potential and context-dependent crosstalk, mutations in the helical domain and kinase domain may respond differently to inhibitors (46–48), a factor that should be considered when designing targeted therapies for cervical SCC.
In this cohort, patients with mutant PIK3CA tended to be older, and the mutation rate was significantly higher among those aged ≥50 years compared with those <50 years (18.4% vs. 8.3%, P=0.031). Because more than half of the patients were younger than 50 years, this age distribution may partly explain the relatively low PIK3CA mutation rate observed in this study. A similar correlation between age and PIK3CA mutational status has been consistently reported in several studies (11,24), although the reason for this association remains unclear. Interestingly, although less common than the H1047R kinase domain mutation, helical domain hotspot mutations in breast cancer have also been associated with older age at diagnosis (P=0.004) (49). These observations in cervical SCC and breast cancer may highlight an age-associated link in the pathogenesis of PIK3CA helical domain hotspot mutations.
Although most studies have reported no correlation between PIK3CA mutations and tumor stage (16,20,24), our results showed that patients with higher-stage disease tended to harbor mutant PIK3CA (P=0.0575). When stratified by stage, patients with stage II or higher tumors had a significantly higher frequency of PIK3CA mutations than those with stage I tumors (P=0.0284). Consistent with their higher stage, nearly all PIK3CA-mutant patients (96%) in this cohort underwent chemotherapy and/or radiation therapy (P=0.0089). Another study also observed a similar trend between PIK3CA mutations and pT2-T3 staging, although the difference did not reach statistical significance (P=0.078) (23). PIK3CA somatic mutations have been postulated to represent a late event in cervical carcinogenesis, as these mutations are rarely detected in cervical intraepithelial neoplasia (38).
Some studies have reported an association between PIK3CA mutations and HPV16 infection (50). Consistent with previous studies (7), HPV16 was the most common HPV subtype among cervical SCCs in this cohort. However, our results indicated that HPV status was not associated with PIK3CA mutations. Although one study reported that American Indian and Alaska Native populations have the highest rate of PIK3CA mutations in cervical cancer (32), statistical analyses have shown no significant differences in PIK3CA mutation frequency by ethnicity, consistent with our findings.
Compared with single-agent chemotherapy, PD-1-blocking antibody-based immunotherapy significantly improves survival among patients with recurrent cervical cancer following first-line platinum-containing treatment (51). One study reported that PIK3CA mutations lead to increased mRNA and protein expression of PD-L1 in cervical cancer (52). To investigate the relationship between PD-L1 expression and PIK3CA mutation, we analyzed 59 cases of cervical SCC (32 PIK3CA wild-type and 27 PIK3CA-mutant). Consistent with previous studies (51), PD-L1 expression (CPS ≥1) was observed in 76.3% of cervical SCCs. However, immunohistochemical analysis did not reveal a statistically significant association between PD-L1 expression and PIK3CA mutation. These findings suggest that in vivo PD-L1 expression may be regulated by complex mechanisms involving diverse genetic and epigenetic alterations. Notably, although PD-L1 expression was observed in more than 75% of samples in our series, the lack of correlation with PIK3CA mutational status may reflect the limited sample size (n=59), resulting in an underpowered subset analysis. Future studies involving all cases, with evaluation of PD-L1 expression and assessment of its relationship with clinical outcomes, are warranted.
In our study, PIK3CA mutations were not associated with the prognosis of cervical SCCs. The only factor significantly correlated with the CSS and OS in this cohort was clinical stage. PIK3CA plays a well-established role in carcinogenesis; although its prognostic significance in cervical SCC has been extensively investigated, the results remain controversial. Some studies, including the largest to date (771 cases), have reported that the presence of PIK3CA mutations is associated with significantly better clinical outcomes (11–15), whereas others have identified PIK3CA mutations as a poor prognostic factor linked to unfavorable survival (16–20). In contrast, several investigations, including the present study, have demonstrated no association between PIK3CA mutations and survival outcomes (21–25). Multiple factors, including tumor stage, patient demographics, mutation subtype, and treatment modalities, may contribute to these discrepancies. Interestingly, in a phase II study evaluating the long-term efficacy and survival outcomes of sintilimab (a monoclonal antibody targeting PD-1) in combination with anlotinib (a multikinase inhibitor with broad inhibitory effects on tumor angiogenesis and growth) in patients with PD-L1-positive recurrent or metastatic cervical cancer, PIK3CA mutation was identified as an independent favorable prognostic factor for OS (15). Similarly, another phase II study demonstrated that patients with PIK3CA mutations treated with camrelizumab (a PD-1 inhibitor) in combination with apatinib (a vascular endothelial growth factor receptor-2 inhibitor) experienced significantly improved progression-free survival and OS (12,14). These clinical trial-based studies suggest that activation of the PI3K/AKT/mTOR pathway through PIK3CA mutation may represent a promising indicator for response to immunotherapy.
However, our study has certain limitations. First, as mentioned above, targeted Sanger sequencing of PIK3CA hotspot mutations may underestimate the overall mutation rate. This Sanger sequencing-based analysis is limited by its sensitivity in detecting mutant allele frequencies of approximately 80 to 85%, which may miss a subset of low-level mosaic mutations below 15 to 20% (53,54). Ideally, next-generation sequencing should be used to investigate the mutational profile in these tumors. The application of advanced molecular techniques not only allows for a thorough investigation of all possible pathogenic mutations across the entire PIK3CA gene but also helps identify a mutational landscape that may involve other key genes in the PI3K/AKT/mTOR pathway or crosstalk with other carcinogenic pathways (21,26). Second, although a single-institution study with a sizable case number and centralized pathology review helps minimize heterogeneity in patient population and tumor characteristics, our findings may still be influenced by local variations in patient demographics and treatment approaches, thereby reducing generalizability. Regional and demographic differences may affect mutation frequencies and clinical outcomes. Notably, 41.3% of patients in this cohort had stage I disease, and most were cured after treatment at our tertiary care center. The high proportion of stage I patients and the relatively low frequency of PIK3CA mutations in this group may have confounded the analysis of survival outcomes. Third, in addition to somatic mutations, PIK3CA gene expression and copy number variation may also affect the downstream pathway. These alterations were not investigated in our cases. Although the relationship between PIK3CA gene expression and mutation has been reported, the findings remain inconclusive (55,56). Given its clinicopathological focus, the functional validation of PIK3CA gene expression and mutations was not performed in this study. Future studies incorporating these analyses may provide a more comprehensive understanding of the role of the PI3K/AKT/mTOR pathway in cervical cancer development, progression, and treatment. Finally, as with other retrospective studies, selection bias may be present in our cohort due to reliance on existing clinical data. In particular, therapeutic strategy may have varied over time, contributing to treatment heterogeneity.
In summary, despite certain limitations, our study highlights and expands upon the clinicopathological characteristics of cervical SCCs with PIK3CA hotspot mutations within a single-institution cohort. We further demonstrate that PIK3CA helical domain mutations are more frequently observed in tumors from older patients and are associated with higher tumor stage. Nearly all patients harboring PIK3CA mutations underwent chemoradiation therapy; however, the mutation was not an independent prognostic factor in this cohort. Additionally, PD-L1 expression appears to be unrelated to PIK3CA mutational status. Future studies should focus on evaluating the efficacy of PI3K inhibitors alone or in combination with immunotherapy and other targeted treatments in selected patients, based on molecular alterations that may predict therapeutic benefit.