Prognostic Evaluation of Human Papillomavirus and p16 in Esophageal Squamous Cell Carcinoma
Esophageal squamous cell carcinoma (ESCC) ranks as the sixth most common cancer worldwide, with a five-year survival rate below 20% due to its aggressive progression and limited therapeutic advances. This study investigated the prognostic significance of human papillomavirus (HPV) infection and p16 expression in ESCC, alongside alterations in tumor suppressor genes p53 and retinoblastoma protein (Rb), to elucidate their roles in disease outcomes.
Background and Clinical Context
HPV, a double-stranded DNA virus infecting mucosal and cutaneous keratinocytes, has been implicated in various cancers, including cervical and oropharyngeal squamous cell carcinomas. In ESCC, reported HPV infection rates range from 11.7% to 38.9% globally. The p16 protein, encoded by the CDKN2A gene, serves as a surrogate marker for HPV-related carcinogenesis in certain cancers. This study aimed to clarify the association between HPV, p16, and survival outcomes in ESCC, while exploring interactions with p53 and Rb pathways.
Study Design and Methods
A cohort of 73 ESCC patients from Henan Cancer Hospital was analyzed, alongside 20 normal esophageal tissue samples from healthy individuals. Immunohistochemical staining was performed using antibodies against HPV, p16, p53, and Rb. HPV 16/18 RNA was detected using the RNAscope 2.5 Assay Reagent Kit. Statistical analyses included Fisher’s exact test for categorical variables and Kaplan-Meier survival curves with log-rank tests for five-year survival comparisons.
Patient Characteristics and Biomarker Profiles
The 73 ESCC patients exhibited no significant differences in age, tumor stage (TNM classification), differentiation grade, smoking history, or alcohol consumption between groups. HPV positivity was detected in 45.2% (33/73) of tumors, while p16 expression was observed in only 6.8% (5/73). P53 and Rb alterations occurred in 54.8% (40/73) and 43.8% (32/73) of cases, respectively. Notably, all 20 normal esophageal samples tested negative for HPV.
Co-expression analyses revealed minimal overlap between HPV and p16 positivity: Only 4.1% (3/73) of cases were dual-positive for HPV and p16. Conversely, HPV-positive tumors frequently coexisted with p53 mutations (30.1%, 22/73) and Rb alterations (16.4%, 12/73). RNAscope testing confirmed the absence of HPV 16/18 RNA in all samples, suggesting transcriptional inactivity of the virus in ESCC.
Survival Outcomes
With a median follow-up of five years and 24 recorded deaths among 68 patients with complete survival data, the study found no significant association between biomarker status and survival. The five-year survival rates were comparable across groups:
- HPV-positive vs. HPV-negative: 9 vs. 15 deaths (P = 0.415)
- p16-positive vs. p16-negative: 1 vs. 23 deaths (P = 0.479)
- p53-altered vs. p53-normal: 14 vs. 10 deaths (P = 0.929)
- Rb-altered vs. Rb-normal: 12 vs. 12 deaths (P = 0.609)
Kaplan-Meier curves illustrated no survival advantage or detriment linked to HPV infection or p16 expression. These findings contrast with HPV-driven cancers like oropharyngeal squamous cell carcinoma, where p16 overexpression strongly correlates with improved prognosis.
Mechanistic and Clinical Implications
The discordance between HPV infection and p16 expression in ESCC challenges the utility of p16 as a reliable HPV biomarker in this cancer type. While p16 typically inhibits cyclin-dependent kinases to arrest the cell cycle in HPV-associated malignancies, its low expression in ESCC suggests alternative mechanisms of tumorigenesis. The high prevalence of HPV DNA without transcriptional activity raises questions about its etiological role—whether HPV acts as a passenger virus or contributes to carcinogenesis through latent mechanisms.
Alterations in p53 and Rb, key regulators of cell cycle checkpoints, were frequent but unrelated to survival. Mutant p53 disrupts apoptosis and DNA repair, while Rb inactivation promotes uncontrolled cell proliferation. However, their prognostic neutrality in this cohort implies that ESCC progression may depend on other molecular drivers, such as epidermal growth factor receptor (EGFR) or vascular endothelial growth factor (VEGF) pathways, which were not examined here.
Limitations and Future Directions
The study’s sample size (73 patients) limits statistical power to detect subtle survival differences. Regional factors, such as Henan Province’s high ESCC incidence, may influence HPV prevalence compared to global populations. Further research with larger cohorts, standardized HPV detection protocols, and genomic profiling is needed to clarify HPV’s role in ESCC. Investigating viral integration sites, epigenetic modifications, and immune microenvironment interactions could unravel mechanisms linking HPV to esophageal carcinogenesis.
Conclusions
This comprehensive analysis demonstrates that HPV infection and p16 expression lack prognostic significance in ESCC, despite HPV’s prevalence in nearly half of the tumors. The absence of HPV 16/18 RNA and minimal p16 co-expression challenges the hypothesis that HPV directly drives ESCC progression. While p53 and Rb alterations are common, their lack of correlation with survival underscores the complexity of ESCC biology. These findings highlight the need for biomarker discovery beyond traditional candidates, focusing on novel molecular targets to improve risk stratification and therapeutic outcomes.
doi.org/10.1097/CM9.0000000000000662
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