Innovative rapid gene methylation analysis of surgical margin tissues in head and neck cancer

Abstract

Background: Securing the negative surgical margin is the first step in surgical cancer treatment. However, tumor recurrence sometimes occurs even with histologically negative surgical margins. To detect minimal residual cancer cells in the deep margin intraoperatively, a time-efficient molecular approach is required.

Methods: We established an innovative rapid quantitative methylation PCR (QMSP) assay, which consists of substantially time-minimized DNA extraction, bisulfite treatment, and QMSP assays. To demonstrate the feasibility of this procedure, 10 serial surgical specimens of primary head and neck squamous cell carcinoma (HNSCC) were evaluated by both rapid and conventional QMSP. Two frequently methylated genes in head and neck cancer, homeobox A9 (HOXA9) and endothelin receptor type B (EDNRB) were analyzed in 10 HNSCCs and surgical margin tissues, as well as normal muscle and oral mucosa samples.

Results: The product quality of DNA extraction and bisulfite treatment using the time-saving procedure was comparable to the conventional procedure. In the QMSP assay, target gene methylation and reference gene methylation were equally detected by both the rapid and conventional method. Finally, relative results of rapid and conventional QMSP were quite similar to each other in tumors, margins, and normal tissues. The average total time required for the rapid QMSP procedure was less than 3 h and could be accomplished by a single person.

Conclusion: From the viewpoint of accuracy, cost, and time consumption, the innovative rapid QMSP maintains highly sensitive methylation detection accomplished within the time frame of a major ablative and reconstructive procedure.

FIG. 1

Sequencing results of four types of bisulfite treatment are displayed. There were seven CpGs in the promoter region of HOXA9 (−680 ~ −632 bp). All four types (#5–8) showed almost the same extent of methylation in every CpG site, which were not completely methylated

FIG. 2

Standard curves of both rapid (a) and conventional (b) QMSP of ACTB gene are shown. Five serial dilutions of positive controls were examined. Amplification curves are illustrated on the left, and dot-plot standard curves on the right by Sequence Detector System (SDS) 2.4 software (Applied Biosystems). Values of slope and Y-intercept were very similar

FIG. 3

Relative QMSP values of tumors (a-1), normal mucosa tissues (a-2), margin tissues (b-1), and normal muscle tissues (b-2) are shown (average ± standard error). Values from rapid QMSP (blue bar) and conventional QMSP (yellow bar) are compared. Ten sequential samples are displayed on the x axis, and relative QMSP values are on the y axis in a logarithmic manner