Detecting Aberrantly Methylated Genes in HNSCC Saliva

Abstract

Problem Aberrant promoter hypermethylation of multiple tumor suppressor genes is an early event in carcinogenesis of HNSCC. Saliva is a non-invasive sample which has immense potential for use in molecular screening and early diagnosis of head and neck cancers due to ease of collection, patient compliance allowing repeatabililty. We evaluated saliva DNA for simultaneous detection of promoter hypermethylation in multiple genes in HNSCC cases and compared them with normal controls. Methods Saliva was collected in a prospective cohort of 37 study subjects: 27 HNSCC patients and 10 normal controls. Saliva DNA was interrogated for aberrant methylation status using a multicandidate gene probe panel. 35 unique genes related to HNSCC were simultaneously examined of which 22 were tumor suppressor genes. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay was used. Results Of the 22 tumor suppressor genes examined simultaneously in the saliva DNA of the study subjects, four HNSCC cases showed aberrant promoter hypermethylation in one or more genes. Aberrantly methylated genes included TIMP3, APC, MLH1, RARB and CDKN1B, CDKN2B, GSTP1, CD44, and ESR. aberrant methylation of CDKN2B was observed in 3/4 cases and GSTP1 in 2/4 cases. Only one of the 10 controls had aberrant methylation of CDKN2B. Conclusion In this exploratory study, MS-MLPA identified multiple aberrantly methylated genes simultaneously in the same assay run. The most important advantage is its utility as a high throughput multi-gene screening approach requiring very small amounts of DNA, unlike more laborious single gene methods such as MSP. Significance Epigenetic signatures from MS-MLPA profiling, upon subsequent validation as diagnostic or prognostic biomarkers, can become reduced to a more definitive candidate gene panel of only a few key genes for early detection and screening of HNSCC. Support HFHS A10236; NIH R01 DE15990.