Consistency of the S5 DNA methylation classifier in formalin‐fixed biopsies versus corresponding exfoliated cells for the detection of pre‐cancerous cervical lesions

Abstract

Methylation biomarkers are promising tools for diagnosis and disease prevention. The S5 classifier is aimed at the prevention of cervical cancer by the early detection of cervical intraepithelial neoplasia (CIN). S5 is based on pyrosequencing a promoter region of EPB41L3 and five late regions of HPV types 16, 18, 31, and 33 following bisulfite conversion of DNA. Good biomarkers should perform well in a variety of sample types such as exfoliated cells, fresh frozen or formalin‐fixed paraffin‐embedded (FFPE) materials. Here, we tested the performance of S5 on 315 FFPE biopsies with paired exfoliated cervical samples using four different conversion kits (Epitect Bisulfite, Epitect Fast Bisulfite, EZ DNA Methylation, and EZ DNA Methylation‐Lightning). The S5 values from FFPE biopsies for all kits were significantly correlated with those obtained from their paired exfoliated cells. For the EZ DNA Methylation kit, we observed an average increased methylation of 4.4% in FFPE. This was due to incomplete conversion of DNA (73% for FFPE vs. 95% for cells). The other kits had a DNA conversion rate in FFPE similar to the cells (95%–97%). S5 performed well at discriminating <CIN2 lesions from CIN2+ lesions on the FFPE with all kits given optimized adjustments to the cut‐off. The area under the curve (AUC) for S5 on FFPE was not significantly different from the paired cells (0.74–0.79 vs. 0.81). The best sensitivity and specificity were obtained for EZ DNA Methylation after the adjustment of the cut‐off to reflect its lower conversion rate. Consistent methylation results can be obtained from FFPE material regardless of the conversion kit used. The S5 classifier performed as well on FFPE material as on exfoliated cells with adjusted cut‐off allowing easier clinical implementation.

Cytological DNA methylation for cervical cancer screening: a validation set

“…A number of studies have explored the role of a panel that includes EPB41L3 (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37), JAM3 (38), or both (7, 39-44) for the screening or triage of HSIL and/or cervical cancer. In this validation trial, the performance of a DNA methylation assay based on the EPB41L3 and JAM3 genes was similar to that of our training set (26), and the assessment showed favorable results in identifying CIN2+, including ADC.…”

HPV Infection Leaves a DNA Methylation Signature in Oropharyngeal Cancer Affecting Both Coding Genes and Transposable Elements

“…By assessing the methylation levels of a diagnostic signature in paired exfoliated cells and FFPE biopsies from women referred to colposcopy, Reuter and colleagues showed a correlation ranging from 0.379–0.550 between tests. FFPE cut-off adjustments were also necessary to improve the signature accuracy [ 45 ]. Therefore, the previously proposed HPV 5-CpG methylation signature is reproducible in OPSCC from a population with a low frequency of HPV infection and in FFPE, a more easily available source of tumor samples.…”

The Role of the IGF2 Methylation Score in Diagnosing Adrenocortical Tumors with Unclear Malignant Potential—Feasibility of Formalin-Fixed Paraffin-Embedded Tissue

“…Some studies show a promising correlation [17][18][19][20][21][22][23][24]. However, upon closer examination of the results, many studies experience high failure rates when using FFPE tissue [23][24][25][26][27], and both random and non-random deviations from the methylation percentage were found in frozen tissue [19][20][21][22][25][26][27][28][29]. Similar articles using other methods for methylation determination, such as Infinium Beadchip, Methylight, or methylation-specific PCR, show the same problems and limitations.…”

“…Similar articles using other methods for methylation determination, such as Infinium Beadchip, Methylight, or methylation-specific PCR, show the same problems and limitations. Some studies find a good correlation between FFPE and frozen tissues [28][29][30][31][32][33], while others experience problems with failure rates [27,28,[34][35][36] and poor correlation [28,29,36,37]. A possible explanation for the higher failure rate in FFPE tissue and the lack of replicability and correlation can be found in the degradation of DNA during storage [38].…”