94 Chapter 4 method. The first and last sections were stained with haematoxylin and eosin (H&E) to check for the presence of lesions. In-between sections were collected in sterile PCR tubes for DNA isolation. DNA was isolated using the QIAamp DNA FFPE tissue kit (QIAgen, Hilden, Germany) according to the manufacturer’s instructions and eluted with easyMAG 3 elution buffer (bioMérieux, Boxtel, The Netherlands). DNA concentrations were measured using a Qubit fluorometer (Qubit, ThermoFisher Scientific, Waltham, MA, USA). HOST-CELL DNA METHYLATION ANALYSIS ASCL1/LHX8 methylation analysis was performed, blinded for cytology and histology outcomes, by quantitative methylation specific PCR (qMSP) on bisulphite-converted DNA essentially as described by Snellenberg et al. 27. DNA was subjected to sodium bisulphite treatment using the EZ DNA Methylation Kit (D5002, Zymo Research, Irvine, CA, USA), according to the manufacturer’s instructions. Bisulphite-converted DNA was subsequently used as input for qMSP analysis of the ASCL1 and LHX8 genes. In the multiplex qMSP, the housekeeping gene β-actin (ACTB) was used as a reference to ensure successful bisulphite conversion and sample quality. The methylation levels of ASCL1 and LHX8 were normalised to ACTB using the quantification cycle (Cq) value (2-ΔΔCq ×100) to obtain ΔΔCq ratios 28. DATA AND STATISTICAL ANALYSIS The original cytology and histology results were retrieved from pathology laboratories through the Dutch Nationwide Pathology Databank (PALGA) 29. For analysis, the CISOE-A classification was translated into the Bethesda nomenclature 26. Histology was categorised as no CIN, LSIL/CIN1, HSIL/CIN2, HSIL/CIN3 (further referred to as CIN1, CIN2 and CIN3), or invasive cervical cancer, according to the latest WHO classifications 30. Adenocarcinoma in situ (AIS) and carcinoma in situ (CIS) were included in the group of CIN3 lesions. Data on HPV were retrieved from the study database 5, with HPV genotype information available for HPV16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66 and -68. All statistical analyses and visualisations were performed using the square roottransformed ΔΔCq ratios of ASCL1 and LHX8. Methylation levels of each marker were categorised in sextiles and visualised in relative frequency histograms per disease category (2x NILM, no CIN, CIN1, CIN2, CIN3 and cervical cancer). The Kruskal-Wallis omnibus test was applied to calculate differences in continuous DNA methylation levels among disease categories, with post hoc testing using the Mann-Whitney U test. Spearman’s rank correlation coefficient was used to analyse correlations between methylation levels in paired self-collected samples, clinician-collected cervical samples
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