Efficiency of CIN2+ Detection by Thyrotropin-Releasing Hormone (TRH) Site-Specific Methylation

Abstract

Cervical cancer screening typically involves a Pap smear combined with high-risk human papillomavirus (hr-HPV) detection. Women with hr-HPV positivity but normal cytology, as well as those with precancerous abnormal cytology, such as low-grade squamous intraepithelial lesions (LSIL) and high-grade SIL (HSIL), are referred for colposcopy and histology examination to identify abnormal lesions, such as cervical intraepithelial neoplasia (CIN) and cervical cancer. However, in order to enhance the accuracy of detection, bioinformatics analysis of a microarray database was performed, which identified cg01009664, a methylation marker of the thyrotropin-releasing hormone (TRH). Consequently, a real-time PCR assay was developed to distinguish CIN2+ (CIN2, CIN3, and cervical cancer) from CIN2- (CIN1 and normal cervical epithelia). The real-time PCR assay utilized specific primers targeting methylated cg01009664 sites, whereas an unmethylated reaction was used to check the DNA quality. A cut-off value for the methylated reaction of Ct < 33 was established, resulting in improved precision in identifying CIN2+. In the first cohort group, the assay demonstrated a sensitivity of 93.7% and a specificity of 98.6%. In the cytology samples identified as atypical squamous cells of undetermined significance (ASC-US) and LSIL, the sensitivity and specificity for detecting CIN2+ were 95.0% and 98.9%, respectively. However, when self-collected samples from women with confirmed histology were tested, the sensitivity for CIN2+ detection dropped to 49.15%, while maintaining a specificity of 100%. Notably, the use of clinician-collected samples increased the sensitivity of TRH methylation testing. TRH methylation analysis can effectively identify women who require referral for colposcopy examinations, aiding in the detection of CIN2+.

mRNA-lncRNA gene expression signature in HPV-associated neoplasia and cervical cancer

“…TRH has been well investigated in the type of cancer such as acute myeloid leukemia, and a correlation between risk groups and TRH expression was found, and it was discovered that patients with higher TRH expression were more sensitive to chemotherapy (Gao et al, 2022). Regarding CIN and cervical cancer, site-specific assessment of TRH gene methylation (cg01009664) was investigated for the detection of CIN2+ and demonstrated high sensitivity and specificity with clinician-collected samples, but not with the self-collected ones (Chaiwongkot et al, 2023). A similar analysis was also performed using screening of TRH cg01009664 methylation for prediction of oral squamous cell carcinoma and oropharyngeal squamous cell carcinoma (Puttipanyalears et al, 2018).…”