Promoter hypermethylation analysis of host genes in cervical intraepithelial neoplasia and cervical cancers on histological cervical specimens

Abstract

Background DNA methylation is an essential factor in the progression of cervical intraepithelial neoplasia (CIN) to cervical cancer. The aim was to investigate the diagnostic value provided by methylation biomarkers of six tumor suppressor genes (ASTN1, DLX1, ITGA4, RXFP3, SOX17 and ZNF671) for cervical precancerous lesions and cervical cancer. Methods The histological cervical specimens of 396 cases including 93 CIN1, 99 CIN2, 93 CIN3 and 111 cervical cancers were tested for methylation-specific PCR assay (GynTect®) of score and positive rate. Among them, 66 CIN1, 93 CIN2, 87 CIN3 and 72 cervical cancers were further used for paired analysis. A chi-square test was used to analyze the difference of methylation score and positive rate in cervical specimens. The paired t-test and paired chi-square test were for analyzing the methylation score and positive rate in paired CIN and cervical cancer cases. The specificity, sensitivity, odds ratio (OR) and 95% confidence interval (95% CI) of the GynTect® assay for CIN2 or worse (CIN2 +) and CIN3 or worse (CIN3 +) were evaluated. Results According to the chi-square test trend, hypermethylation increased with severity of the lesions as defined by histological grading (P = 0.000). The methylation score above 1.1 was more common in CIN2 + than in CIN1. The DNA methylation scores in the paired groups of CIN1, CIN3 and cervical cancer were significant differences (P = 0.033, 0.000 and 0.000, respectively), except for CIN2 (P = 0.171). While the positive rate of GynTect® in each paired group had no difference (all P > 0.05). The positive rate of every methylation marker in the GynTect® assay showed differences in four cervical lesion groups (all P < 0.05). The specificity of GynTect® assay for detection of CIN2 + /CIN3 + were higher than high-risk human papillomavirus test. With CIN1 as a reference, the positive status of GynTect®/ZNF671 were significantly higher in CIN2 + : odds ratio (OR) 5.271/OR 13.909, and in CIN3 + : OR 11.022/OR 39.150, (all P < 0.001). Conclusion The promoter methylation of six tumor suppressor genes is related to the severity of cervical lesions. The GynTect® assay based on cervical specimens provides diagnostic values for detecting CIN2 + and CIN3 + .

The construction and preliminary validation of methylation haplotype load markers of multiple genes for cervical cancer screenings

“…However, this strategy still has a relatively high rate of unnecessary colposcopy [31]. On the other hand, p16/Ki67 dual staining [32], gene methylation detection [33], and E6 protein detection have shown promise in reducing unnecessary colposcopic referrals. Given the advantages of objective and highthroughput gene methylation detection, coupled with the rich biological information contained in gene methylation results, this study constructed three machine learning models based on multistep screening of multigene methylation information.…”

Discovery and technical validation of high-performance methylated DNA markers for the detection of cervical lesions at risk of malignant progression in low- and middle-income countries

“…Others have tested methylated gene markers alone, or combined with HPV-testing and cytology to determine whether the three-pronged approach would improve detection and risk stratification of low-grade lesions [ 26 ]. A number of commercial methylation tests have used single-host gene methylation of POU4F3 (sensitivity 74% and specificity 89%) [ 48 ], PAX1 (sensitivity 78%, and specificity 92%) [ 49 ], or panels of two, PAX1/ZNF582 (sensitivity 78.85% and specificity 73.55%) [ 50 ] to six markers [ 17 – 19 , 51 ]. A number of studies have used the 6-marker GynTect® assay.…”

“…A number of studies have used the 6-marker GynTect® assay. Sensitivity for CIN3 detection in these studies ranged from 64 to 91% and specificity ranged from 42 to 95% [ 17 – 19 , 51 ]. The S5-classifier consists of markers of HPV gene methylation combined with a host gene marker, EPB41L3, showed a high sensitivity of 93.2% but a low specificity of 41.8% [ 20 ].…”

“…A follow-up paper by the same group on 280 Thin Prep liquid-based cervical scrapes showed similar sensitivity of 64.8% with an improved specificity of 94.8% [ 18 ]. A recent paper reported that among 396 cervical smears of women in a colposcopy clinic, the sensitivity and specificity of GynTect® assay for CIN3 detection was 91.2% and 42.2%, respectively, which was a significant improvement over hrHPV detection which showed 95.6% sensitivity and 14.1% specificity in the same study [ 19 ]. Another test, the S5 DNA-methylation classifier, based on markers from both host and virus genomes, detected CIN2/3 and cervical carcinoma but not CIN1 or normal cytology [ 20 – 23 ].…”

Detection of DNA Methylation in Gene Loci ASTN1, DLX1, ITGA4, RXFP3, SOX17, and ZNF671 for Diagnosis of Cervical Cancer

“… 16 However, the overall number of clinical observational studies on DNA gene methylation is relatively small and most of them mention the use of a six-gene kit (ASTN1, DLX1, ITGA4, RXFP3, SOX17 and ZNF671) for combined methylation detection, which deserves our special attention. For example, Shi et al 17 found that it showed excellent diagnostic efficiency in screening samples with CIN2+ and above in the diagnostic value of methylation biomarkers using this kit for cervical precancerous lesions and cervical cancer. Zhang et al 18 found that using methylation screening can reduce the referral rate of colposcopy by 67.2%, with a sensitivity and specificity of 83.0% and 69.9% for detecting CIN2+.…”