Feasibility of using P16 methylation as a cytologic marker for esophageal squamous cell carcinoma screening: A pilot study

Abstract

Background Early diagnosis and treatment of esophageal squamous cell dysplasia (ESCdys) and esophageal squamous cell carcinoma (ESCC) could significantly reduce the incidence and mortality of ESCC. This pilot study aimed to investigate whether P16/CDKN2A methylation could serve as a cytologic biomarker for early detection of ESCdys and ESCC. Methods Paired esophageal biopsy and cytology specimens (exfoliated cells) were obtained from subjects at different stages of ESCC development. The methylation status of P16 gene in these two specimen types was determined using a 115‐bp MethyLight assay. Categorical data were compared by the Chi‐square test. Logistic regression was performed to assess adjusted odds ratios of P16 methylation associated with ESCC and ESCdys. Prediction models for identifying individuals at risk of ESCC and high‐grade ESCdys (high‐grade intraepithelial neoplasia, HGIN) were developed by multivariable logistic regression. Diagnostic performance was evaluated using receiver operating characteristic (ROC) analysis. Internal validation of the prediction models was performed using the 1000‐bootstrap resample. Results A total of 105 subjects with diagnoses ranging from normal mucosa through ESCC were included in this study. An increase in P16 methylation frequency was observed with increasing severity of esophageal lesions (p for trend <0.001). In the adjusted logistic regression models, P16 methylation in cytology specimens was positively associated with ESCC and ESCdys risk, whereas P16 methylation in biopsy specimens was only associated with a higher risk of developing ESCC. The predictive capacity of base model I (AUC, 0.816) for ESCC and HGIN was significantly increased by adding P16 methylation in cytology specimens (model III; AUC, 0.882; p = 0.043), but not P16 methylation in biopsy specimens (model II; AUC, 0.850; p = 0.225). Bootstrap validation showed optimism‐corrected AUC of 0.789 for model I, 0.822 for model II, and 0.854 for model III. Conclusion P16 methylation as a cytologic marker was associated with the ESCC development and has the potential for application in minimally invasive ESCC screening.

Genome-wide methylation profiling identify hypermethylated HOXL subclass genes as potential markers for esophageal squamous cell carcinoma detection

“…Growing evidence has demonstrated the complex relationship between gene methylation and expression. The methylated CDKN2A gene (also known as p16 locus), which encoded two genes, p14ARF and p16INK4a, has been attempted as a screening marker for ESCC [45]. Interestingly, its promoter hypermethylation only silenced the expression of p14ARF but not p16INK4a [20], which implied that hypermethylation in different locations showed various effects on gene expression.…”

Genome-wide methylation profiling identify hypermethylated HOXL subclass genes as potential markers for esophageal squamous cell carcinoma detection

“…Growing evidence has demonstrated the complex relationship between gene methylation and expression. The methylated CDKN2A gene (also known as p16 locus), which encoded two genes, p14ARF and p16INK4a , has been attempted as a screening marker for ESCC [ 46 ]. Interestingly, its promoter hypermethylation only silenced the expression of p14ARF but not p16INK4a [ 20 ], implying that hypermethylation in different locations has various effects on gene expression.…”

DNA methylation markers in esophageal cancer

“…In a study by Fan et al, a total of 52 samples of premalignant lesions, including LGD and HGD, were collected through endoscopic brushings to evaluate the detectability of methylated P16 . The sensitivity for LGD and HGD was reported as 25.0% and 37.5% respectively, with a specificity of 95.2% ( Fan et al, 2022 ). Yu et al investigated a panel of methylated markers, namely, Up10 , Up35-1 , Cg6522 , and YPEL3 , using a similar methodology as Fan et al, aiming to screen for early-stage EC.…”