Correlation of gene methylation in surgical margin imprints with locoregional recurrence in head and neck squamous cell carcinoma

Abstract

Background Securing negative surgical margins is a critical goal for head and neck surgery. Local recurrence occurs even in some cases with histologically negative surgical margins. Minimal residual tumor cells may lead to locoregional recurrence despite clear histologic margins reported at the time of resection of head and neck squamous cell carcinoma (HNSCC). In order to identify subclinical residual disease, we analyzed deep margin imprint samples collected on one layer nitrocellulose sheets. Methods Bisulfite treated DNA from 73 eligible cases was amplified by quantitative methylation-specific polymerase chain reaction (QMSP) targeting 6 genes (DCC, EDNRB, HOXA9, KIF1A, NID2, NR2B). QMSP values were dichotomized as positive or negative. Associations between the QMSP status of deep margin samples and clinical outcomes were evaluated. Results Two gene methylation combinations among DCC, EDNRB and HOXA9 were associated with decreased locoregional recurrence-free survival (LRFS), recurrence-free survival (RFS) and overall survival (OS). The methylated gene combination EDNRB and HOXA9 in margin imprints was the most powerful predictor of poor LRFS (HR=3.31, 95% CI=1.30-8.46; P=0.012), independent of standard histological factors. In addition, methylation of EDNRB/HOXA9 showed a trend toward reduced RFS (HR=2.74, 95% CI=0.90-8.33; P=0.075) and OS (HR=5.78, 95% CI=0.75-44.7; P=0.093) by multivariable analysis. Conclusion A panel of gene methylation targets in deep surgical margin imprints provides a potential predictive marker of post-operative locoregional recurrence. Intra-operative use of molecular margin-imprint analysis might assist surgeons to obtain rigorously negative surgical margins and improve the outcome of head and neck surgery.

Paired Box 5 Methylation Detection by Droplet Digital PCR for Ultra-Sensitive Deep Surgical Margins Analysis of Head and Neck Squamous Cell Carcinoma

“…We hypothesized that margin imprints might assess the molecular margin status more accurately than collecting tissue samples from margins in the standard manner. We confirmed this hypothesis in a recent study(10) showing that the methylation marker combination of HOXA9 and EDNRB was a more powerful predictor of outcome than conventional tumor factors (TNM stage, differentiation). Locoregional recurrence free survival (LRFS) was more highly associated with molecular margin status than was recurrence free survival or overall survival.…”

“…QMSP is a well-established technique for detecting low levels of tumor DNA in various body fluids such as saliva(27, 28) and urine(29). Practically, the adaptation of this approach to HNSCC surgical margin analysis using imprint methods also has been shown to have acceptable quality in our recent study(10). In order to realize general clinical adoption of imprint molecular assessment, several refinements are needed.…”

Expression profiles of selected genes in tumors and matched surgical margins in oral cavity cancer: Do we have to pay attention to the molecular analysis of the surgical margins?

“…53 Moreover, epigenetic events, such as aberrant promoter gene hypermethylation, are often observed not only in tumor tissue, but also in surgical margins. 39,54,55 Abnormal DNA methylation patterns in promoter regions can inactivate genes and facilitate tumor formation and progression. 56 Environmental factors like exposure to alcohol and cigarettes can influence aberrant methylation patterns, too.…”

Discovery and development of differentially methylated regions in human papillomavirus‐related oropharyngeal squamous cell carcinoma

“…Given these barriers of HPV DNA‐based population screening for HPV‐positive OPSCC, it is attractive to define other measurable DNA alterations characteristic of HPV‐positive OPSCC. Extensive number of studies have demonstrated that abnormal epigenetic regulation is closely related to tumorigenesis . The dominant paradigm includes that methylation of CpG island promoter results in transcriptional repression of tumor suppressor gene.…”