LINE1 mean methylation levels among OSCC, HGSIL, normal healthy donors, and contralateral normal mucosa. Asterisks indicate a statistical significance as calculated by the Kruskal-Wallis test. (PDF 22Â kb

Achille Tarsitano (4358260)

Andrea Gabusi (4358263)

Claudio Marchetti (4358266)

Davide Gissi (4358257)

Luca Morandi (324431)

Lucio Montebugnoli (4358269)

Maria Foschini (4358272)

Sofia Asioli (4358275)

Clinical Epigenetics

Heatmap from 325 CpG methylation data points (rows) and 130 samples (column). Annotation labels refer to histology and smoking status. Rows are centered; unit variance scaling is applied to rows. Both rows and columns are clustered using correlation distance and average linkage. Smoking status and histology are highlighted in color. Three clusters are marked: left cluster: 55 normal donors, 21 contralateral mucosa, one OSCC, and the OSCC with sarcomatoid features; center cluster: three HGSIL, 11 OSCC, five contralateral mucosa, and one normal donor; right cluster: 16 OSCC, three HGSIL, four contralateral mucosa, and nine normal donors. (PDF 376Â kb

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Additional file 4: of 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

PCA for validation dataset: Unit variance scaling is applied to rows; SVD with imputation is used to calculate principal components. X and Y axes show principal component 1 and principal component 2 that explain 53.9 and 9.8% of the total variance, respectively. Prediction ellipses are such that with probability 0.95, a new observation from the same group will fall inside the ellipse. N = 40 data points. (PDF 29 kb

Additional file 6: of 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

Heatmap for validation dataset. Rows are centered; unit variance scaling is applied to rows. Imputation is used for missing value estimation. Both rows and columns are clustered using correlation distance and average linkage; 54 rows, 40 columns. (PDF 45Â kb

Additional file 7: of 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 Background Oral 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. Methods With 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. Results Comparing 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. Conclusions Our data highlight the importance of CpG location and correct estimation of DNA methylation level for highly accurate early diagnosis of OSCC

Luca Morandi

Davide Gissi

Achille Tarsitano

Sofia Asioli

Andrea Gabusi

Claudio Marchetti

Lucio Montebugnoli

Maria Pia Foschini

Directory of Open Access Journals

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

Crossref

Background: Oral 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. Methods: With 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. Results: Comparing 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 &lt; 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. Conclusions: Our data highlight the importance of CpG location and correct estimation of DNA methylation level for highly accurate early diagnosis of OSCC

MORANDI, LUCA

GISSI, DAVIDE BARTOLOMEO

TARSITANO, ACHILLE

ASIOLI, SOFIA

GABUSI, ANDREA

MARCHETTI, CLAUDIO

MONTEBUGNOLI, LUCIO

FOSCHINI, MARIA PIA

Archivio istituzionale della ricerca - Alma Mater Studiorum Università di Bologna

English

Summary tables of each gene targets with the mean, the standard deviation, the minimum, the maximum, and the number of missing data for each position and group of samples. This test is the non-parametric version of the ANOVA (one-way analysis of variance) and tests whether samples originate from the same distribution. If the test is statistically significant (P value less than 0.05), it means that at least one of the samples is different from the other samples. (XLSX 122Â kb

Additional file 2: of 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

ROC analysis discriminating OSCC vs normal healthy donors using easyROC as a webtool, showing the three best performing CpGs from each gene of 18 evaluated. Comparing OSCC vs normal healthy donors in 355 CpGs, the following epigenetically altered genes revealed high discrimination power: ZAP70, ITGA4, KIF1A, PARP15, EPHX3, NTM, LRRTM1, FLI1, MIR193, LINC00599, PAX1, and MIR137HG showing hypermethylation and MIR296, TERT, and GP1BB showing hypomethylation. (PDF 255Â kb

Additional file 3: of 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