DNA methylation biomarkers accurately detect esophageal cancer prior and post neoadjuvant chemoradiation

Abstract

Background: Esophageal cancer (ECa) is associated with high mortality, mostly due to late diagnosis, precluding curativeintent surgery. Hence, neoadjuvant chemoradiation (ChRT) is recommended in most patients regardless of histological subtype. A proportion of these patients, however, achieve complete disease remission and might be spared of radical surgery. The lack of reliable, minimally invasive biomarkers able to detect post-ChRT disease persistence is, nonetheless, a major drawback. We have previously shown that miRNA promotor methylation enables accurate cancer detection in tissues and liquid biopsies but has been seldom explored in ECa patients.

Aims: Herein, we sought to unveil and validate novel candidate biomarkers able to detect ECa prior and post ChRT.

Materials and methods: Promoter methylation of miR129-2, miR124-3 and ZNF569 was assessed, using quantitative methylation-specific PCR (qMSP), in tissue samples from normal esophagus, treatment-naïve and post-ChRT ECa, as well as in liquid biopsies from ECa patients.

Results: All genes disclosed significantly different promoter methylation levels between ECa and normal esophagus, accurately detecting post-ChRT disease, especially for adenocarcinoma. Remarkably, miR129-2_me /ZNF569_me methylation panel identified ECa in liquid samples with 53% sensitivity and 87% specificity.

Discussion: MiR129-2_me , miR124-3_me and ZNF569_me accurately discriminate ECa, either pre- or post-ChRT, from normal tissue, enabling ECa detection. Furthermore, circulalting methylation-based biomarkers are promising minimally invasive tools to detect post-ChRT residual ECa.

Conclusion: Overall, our results encourage the use of miRNA methylation biomarkers as accurate ECa detection tools as a novel approach for ChRT response monitoring.

Keywords: esophageal cancer; liquid biopsies; methylation biomarkers; miRNAs; neoadjuvant chemoradiation.

FIGURE 1

Relative miRNAs promoter methylation levels in naïve ECa tissue samples. (A) miR129‐2 _me and (B) miR124‐3 _me levels were compared between 85 naïve ECa and 56 normal esophagus tissue samples. Both graphs are represented by violin plots with individual dots. Red line represents median values. Median ranks between two groups were compared using the Mann–Whitney test, where **** represents a p value lower than 0.0001. ECa, esophageal cancer.

FIGURE 2

Tissue biomarker performance. Receiver‐operating characteristic (ROC) curve of the (A) miR129‐2 _me and (B) miR124‐3 _me in naïve ECa tissues. ECa, esophageal cancer.

FIGURE 3

Relative miRNAs promoter methylation levels in post‐ChRT tissue samples. (A) miR129‐2 _me and (B) miR124‐3 _me levels were compared between 16 non‐responders (5 grade 1, 8 grade 2, and 3 grade 3) and 20 complete responders to neoadjuvant ChRT treatment. Furthermore, methylation levels of complete responders with no clinical evidence of disease were compared with normal esophagus (n = 56). Both graphs are represented by violin plots with individual dots. Red line represents median values. Median ranks between two groups were compared using the Mann–Whitney test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, non‐significant. ChRT, chemoradiation.

FIGURE 4

Relative miRNAs promoter methylation levels in plasma samples. (A) miR129‐2 _me, (B) miR124‐3 _me, and (C) ZNF569 _me levels were compared between 32 naïve ECa and 30 healthy donors, plasma samples. All graphs are represented by violin plots with individual dots. Red line represents median values. Median ranks between two groups were compared using the Mann–Whitney test. *p < 0.05; ns, non‐significant. ECa, esophageal cancer.