CpG location and methylation level are crucial factors for the early detection of oral squamous cell carcinoma in brushing samples using bisulfite sequencing of a 13-gene panel

Abstract

BackgroundOral squamous cell carcinoma (OSCC) is usually diagnosed at an advanced stage and is commonly preceded by oral premalignant lesions. The mortality rates have remained unchanged (50% within 5 years after diagnosis), and it is related to tobacco smoking and alcohol intake. Novel molecular markers for early diagnosis are urgently needed. The purpose of this study was to evaluate the diagnostic value of methylation level in a set of 18 genes by bisulfite next-generation sequencing.MethodsWith minimally invasive oral brushing, 28 consecutive OSCC, one squamous cell carcinoma with sarcomatoid features, six high-grade squamous intraepithelial lesions (HGSIL), 30 normal contralateral mucosa from the same patients, and 65 healthy donors were evaluated for DNA methylation analyzing 18 target genes by quantitative bisulfite next-generation sequencing. We further evaluated an independent cohort (validation dataset) made of 20 normal donors, one oral fibroma, 14 oral lichen planus (OLP), three proliferative verrucous leukoplakia (PVL), and two OSCC.ResultsComparing OSCC with normal healthy donors and contralateral mucosa in 355 CpGs, we identified the following epigenetically altered genes: ZAP70, ITGA4, KIF1A, PARP15, EPHX3, NTM, LRRTM1, FLI1, MIR193, LINC00599, PAX1, and MIR137HG showing hypermethylation and MIR296, TERT, and GP1BB showing hypomethylation. The behavior of ZAP70, GP1BB, H19, EPHX3, and MIR193 fluctuated among different interrogated CpGs. The gap between normal and OSCC samples remained mostly the same (Kruskal-Wallis P values < 0.05), but the absolute values changed conspicuously. ROC curve analysis identified the most informative CpGs, and we correctly stratified OSCC and HGSIL from normal donors using a multiclass linear discriminant analysis in a 13-gene panel (AUC 0.981). Only the OSCC with sarcomatoid features was negative. Three contralateral mucosa were positive, a sign of a possible field cancerization. Among imprinted genes, only MIR296 showed loss of imprinting. DNMT1, TERC, and H19 together with the global methylation of long interspersed element 1 were unchanged. In the validation dataset, values over the threshold were detected in 2/2 OSCC, in 3/3 PVL, and in 2/14 OLP.ConclusionsOur data highlight the importance of CpG location and correct estimation of DNA methylation level for highly accurate early diagnosis of OSCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-017-0386-7) contains supplementary material, which is available to authorized users.

Peculiar pathological, radiological and clinical features of skull‐base de‐differentiated chordomas. Results from a referral centre case–series and literature review

“…Bisulphite treatment of genomic DNA was performed using the EZ DNA Methylation‐Lightning™ Kit (Zymo Research, Irvine, CA, USA; code D5031), according to the manufacturer's protocol. DNA methylation analysis was performed by bisulphite‐NGS for BRCA1 , BRCA2 , PDCD1 , PD‐L1 , CDH1 , PRC1 , MAGEA2 , MAGEA11 , TERT and UXT genes, as previously described . Briefly, a two‐step library was prepared following the same protocol described above for mutation analysis.…”

Whole-Blood DNA Methylation Markers in Early Detection of Breast Cancer: A Systematic Literature Review

“…Rigorous independent validation is crucial to derive reliable genetic and epigenetic makers or marker panels, particularly for candidates discovered in genome-wide approaches (57,60). In only eight of the studies validations in independent samples were performed.…”

Prognostic Role of DNA Methylation Analysis from Oral Brushing in Oral Squamous Cell Carcinoma

“…Brushing cell collection was performed prior to any cancer treatment in the tumor mass. Two hundred forty-five CpG islands from a set of 13 previously described methylated genes in OSCC (ZAP70, KIF1A, LRRTM1, PARP15, FLI1, NTM, LINC0059, EPHX3, ITGA4, MIR193, GP1BB, MIR296, TERT) [1] were investigated by bisulfite-Target Next Generation Sequencing (NGS) using MiSEQ platform (Illumina, San Diego, CA, USA).…”