DNA methylation-mediated low expression of ZNF582 promotes the proliferation, migration, and invasion of clear cell renal cell carcinoma.

OBJECTIVE: The methylation of DNA promoter region mediates the low expression of many tumor suppressor genes and plays an essential part in cancer progression. We investigated methylation and expression of ZNF582 in clear cell renal cell carcinoma (ccRCC), and to study the function of ZNF582 in ccRCC cells. METHODS: Methylation data and mRNA expression data of TCGA-KIRC were obtained from TCGA database to screen methylation-driven genes. Survival analysis and gene set enrichment analysis (GSEA) were done for the target gene. The methylation degree and mRNA level of ZNF582 in ccRCC cell line were detected by methylation-specific PCR (MSP) and qRT-PCR, respectively. Effects of overexpression of ZNF582 on ccRCC cells were assessed via CCK-8, flow cytometry, wound healing, Transwell, and cell adhesion assays. RESULTS: Eighteen methylation-driven genes were identified via bioinformatics methods. Among them, ZNF582 was noticeably hypermethylated and lowly expressed in tumor tissue, and ZNF582 methylation and expression levels were pronouncedly associated with prognosis and clinical stage. MSP also displayed that the ZNF582 DNA promoter region was hypermethylated in ccRCC cells, and the mRNA expression of ZNF582 was dramatically elevated after demethylation. In vitro cell experiments disclosed that overexpression of ZNF582 markedly hindered cell proliferation, invasion, migration, and fostered cell apoptosis and adhesion of ccRCC. CONCLUSION: ZNF582 was hypermethylated in ccRCC, which mediated its low level. Overexpression of ZNF582 inhibited tumor cell proliferation, migration and invasion. This study generates novel ideas for ccRCC diagnosis and treatment.

ZNF582 hypermethylation as a prognostic biomarker for malignant transformation of oral lesions.

OBJECTIVES: This hospital-based cohort study evaluated whether ZNF582 and PAX1 methylation levels at baseline can be used as biomarkers to identify lesions with a high potential for malignant transformation in patients with normal mucosa and oral potentially malignant disorders. PATIENTS AND METHODS: We recruited 171 adult patients with normal mucosa and oral potentially malignant disorders in 2012-2014. They were followed until 2017. Outcomes, including advanced histopathological findings and oral cancer occurrence, were obtained from medical charts, the Taiwan Cancer Registry, and cause-of-death data. Kaplan-Meier analysis and Cox proportional hazards regression models were used to examine the association of ZNF582 and PAX1 methylation levels at baseline with subsequent outcome occurrences. RESULTS: After 260,192 days of follow-up, 11 cases of oral cancer and 4 cases of advanced histopathological progression occurred. Patients with higher ZNF582 and PAX1 methylation levels at baseline had a higher incidence of disease progression. After adjustment for all studied factors using Cox proportional hazards regression models, ZNF582m level (adjusted hazard ratio, 11.41; 95% CI, 2.05-63.36; p = 0.005) was the only significant and independent predictor of disease progression. CONCLUSIONS: ZNF582 hypermethylation can be an effective and noninvasive biomarker for identifying oral lesions with a high potential for malignant transformation.

Methylated ZNF582 as a triage marker for occult cervical cancer and advanced cervical intraepithelial neoplasia.

Aim: To explore the appropriate triage methods for women infected with high-risk human papillomavirus (hrHPV). Materials & methods: A total of 424 out of 872 hrHPV-infected women were divided into cervicitis (n = 123), cervical intraepithelial neoplasia grade 1 (CIN1; n = 89), CIN2 (n = 72), CIN3 (n = 87) and cervical cancer (n = 53) groups. Results: The sensitivity/specificity of ZNF582m, PAX1m and liquid-based cytology (LBC) for hrHPV-infected women with transformation zone 3 CIN3+ was 83.9/93.1, 77.4/90.6 and 80.6/58.5%, respectively. The ZNF582m/PAX1m test had a higher specificity than LBC (p < 0.001) and similar sensitivity to that observed for LBC (p > 0.05). ZNF582m/PAX1m improved the positive predictive value of CIN3+ (64.7/60.0%) in low-grade LBC (negative predictive value: 91.7/88.7%). Conclusion: ZNF582m was superior to PAX1m and LBC tests in detecting CIN3+ in hrHPV-infected women.

Human papillomavirus (HPV) testing is the main method for cervical cancer screening. Although most HPV infections are transient and can be cleared by the body, persistent infection with HPV can lead to cervical cancer. In this study, 424 HPV-infected women were divided into normal, cervical intraepithelial neoplasia grade 1 (CIN1), CIN2, CIN3 and cervical cancer groups according to the grade of cervical lesion (low to high). Women with CIN3 or cervical cancer need treatment. ZNF582^m, PAX1^m and liquid-based cytology detected 83.9, 77.4 and 80.6% of women with CIN3+ and 93.1, 90.6 and 58.5% of women without CIN3+. However, the ZNF582^m test was superior to the PAX1^m and liquid-based cytology tests.

High methylation of the same promoter of lncRNA ZNF582-AS1/ZNF582 promotes malignant progression of esophageal cancer.

Aim: This study aimed to investigate the functions of ZNF582-AS1 and ZNF582 in esophageal cancer (EC). Materials & methods: Bioinformatics analysis, qRT-PCR and western blot were used to analyze the expression levels. Biological functions were evaluated using cell-counting kit 8, colony formation, Transwell assays and flow cytometry. FISH was used to detect subcellular localization, and methylation-specific PCR determined gene methylation levels. Animal experiments validated the impact on tumor progression. Results: ZNF582-AS1 and ZNF582 were highly methylated and downregulated in EC. Overexpression of ZNF582-AS1 up-regulated the expression of ZNF582, thereby inhibiting EC cell viability and metastasis, promoting apoptosis and inhibiting tumor growth. Conclusion: Low expression of ZNF582-AS1/ZNF582 mediated by DNA hypermethylation facilitates the malignant progression of EC.

Promoter hypermethylation silences ZNF582-AS1 and ZNF582, driving esophageal cancer progression, which has the potential for novel therapeutic strategies. # Methylation # Esophageal Cancer.

ZNF582 promoter methylation predicts cervical cancer radiosensitivity and ZNF582 protein overexpression reduces radiosensitivity by cell cycle arrest in S phase.

This study aimed to investigate the relationship between ZNF582 promoter methylation (ZNF582m) level and radiosensitivity of cervical cancer and its biological basis. This was a prospective multicenter clinical study, comprising two independent cohorts of locally advanced cervical cancer patients. Exfoliated cervical cells were collected at 0, 24, 30, 36, 48, and 64 Gy to test ZNF582m levels. Radiotherapy response was evaluated according to RECIST Version. RT-PCR and WT were used to detect the mRNA and protein expression levels; MTT and flow cytometry were used to detect the cell viability and cell cycle, respectively. While clone formation and subcutaneous tumorigenesis in nude mice were used to detect the growth of HeLa cells with/without ZNF582 overexpression. In the first cohort, 22 cases achieved complete remission (CR) or partial response (PR), and the other 28 cases exhibited stable disease (SD). Radiotherapy reduced ZNF582m levels among all patients. Initial lever of ZNF582m was significantly higher in the Responder (CR + PR) group than in the SD group. Also, patients with higher initial lever ZNF582m were more sensitive towards radiotherapy than ZNF582m-low patients. The second cohort confirmed the above results. The amplitude of ZNF582m levels were related to the radiotherapeutic response; some patients of ZNF582m-low showed a transient increase in ZNF582m, and present greater radiosensitivity than other ZNF582m-low patients. In vitro, ZNF582 protein overexpression promoted cell cycle arrest in S phase. These results suggested that higher ZNF582m levels predicted greater radiosensitivity in clinical cervical cancer cases. Overexpressed ZNF582 conferred radioresistance by cell cycle arrest in vitro.

Methylated ZNF582: a therapeutic target in oral cancer.

Tweetable abstract Zinc finger proteins control the transcription of downstream genes that are implicated in migration, invasion, cell death and proliferation. More mechanistic research on ZNF582 is needed to ascertain how this protein's methylation regulates the inflammatory pathway in oral cancer.

Improving the Diagnostic Performance by Adding Methylation Marker to Conventional Visual Examination in Identifying Oral Cancer.

BACKGROUND: Visual oral examination (VOE) is a conventional oral cancer screening method. This study aimed to evaluate the value of methylation marker to assist VOE in identifying oral epithelial dysplasia and oral squamous cell carcinoma (OED/OSCC) from non-cancerous lesions in a real-world situation. METHODS: 201 patients with high-risk personal habits who self-perceived oral anomaly were VOE examined, ZNF582 methylation (ZNF582m) tested, and histologically diagnosed. RESULTS: Among them, 132 patients (65.7%) were histologically diagnosed OED/OSCC. Using VOE, 56.1% OED/OSCC patients had possible oral cancer, whereas 37.7% non-OED/OSCC patients had leukoplakia. ZNF582m-positive was detected in 90.2% OED/OSCC patients and 44.9% non-OED/OSCC patients. Various logistic regression models were postulated to evaluate the diagnostic performance of conventional VOE and new strategies using ZNF582m. ROC analysis and its corresponding C-index demonstrated that either triage or co-testing models of VOE and ZNF582m could improve diagnostic performance and discriminative abilities compared with the VOE only approach. CONCLUSIONS: In conclusion, methylation marker test shows equivalent performance to an experienced judgment by oral maxillofacial surgeons and plays a significantly supplementary role in increasing the efficacy in identifying oral malignant lesions. ZNF582m may be an especially important tool for family physicians or general dentists to properly diagnose suspicious oral lesions.

Triage by PAX1 and ZNF582 Methylation in Women With Cervical Intraepithelial Neoplasia Grade 3: A Multicenter Case-Control Study.

Background: The colposcopy-conization inconsistency is common in women with cervical intraepithelial neoplasia grade 3 (CIN3). No adequate method has been reported to identify the final pathology of conization. In this study, we explored the ability of PAX1 and ZNF582 methylation to predict the pathological outcome of conization in advance. Methods: This was a multicenter study and included 277 histologically confirmed CIN3 women who underwent cold knife conization (CKC) from January 2019 to December 2020. The methylation levels of PAX1 (PAX1m) and ZNF582 (ZNF582m) were determined by quantitative methylation-specific polymerase chain reaction (qMSP) and expressed in ΔCp. Receiver operating characteristic curves were used to evaluate predictive accuracy. Results: The final pathological results in 48 (17.33%) patients were inflammation or low-grade squamous intraepithelial lesion (LSIL), 190 (68.59%) were high-grade squamous intraepithelial lesion (HSIL), and 39 (14.08%) were squamous cervical cancer (SCC). PAX1m and ZNF582m increased as lesions progressed from inflammation/LSIL, HSIL, to SCC. PAX1 and ZNF582 methylation yielded better prediction performance compared with common screening strategies, whether individually or combined. A 4.33-fold increase in the probability of inflammation/LSIL was observed in patients with lower ZNF582 methylation levels (ΔCpZNF582 ≥ 19.18). A 6.53-fold increase in SCC risk was observed in patients with elevated ZNF582 methylation (ΔCpZNF582 < 7.09). Conclusions: DNA methylation would be an alternative screening method to triage and predict the final outcome of conization in CIN3 cases.

LncRNA ZNF582-AS1 Expression and Methylation in Breast Cancer and Its Biological and Clinical Implications.

Background: Long non-coding RNAs (lncRNAs) play an important role in cellular activities and functions, but our understanding of their involvement in cancer is limited. Methods: TCGA data on RNA expression and DNA methylation were analyzed for lncRNAs' association with breast cancer survival, using the Cox proportional hazard regression model. Fresh tumor samples and clinical information from 361 breast cancer patients in our study were used to confirm the TCGA finding on ZNF582-AS1. A RT-qPCR method was developed to measure ZNF582-AS1 expression. Survival associations with ZNF582-AS1 were verified with a meta-analysis. In silico predictions of molecular targets and cellular functions of ZNF582-AS1 were performed based on its molecular signatures and nucleotide sequences. Results:ZNF582-AS1 expression was lower in breast tumors than adjacent normal tissues. Low ZNF582-AS1 was associated with high-grade or ER-negative tumors. Patients with high ZNF582-AS1 had a lower risk of relapse and death. These survival associations were confirmed in a meta-analysis and remained significant after adjustment for tumor grade, disease stage, patient age, and hormone receptor status. Correlation analysis indicated the possible suppression of ZNF582-AS1 expression by promoter methylation. Bioinformatics interrogation of molecular signatures suggested that ZNF582-AS1 could suppress tumor cell proliferation via downregulating the HER2-mediated signaling pathway. Analysis of online data also suggested that HIF-1-related transcription factors could suppress ZNF582-AS1 expression, and the lncRNA might bind to hsa-miR-940, a known oncogenic miRNA in breast cancer. Conclusions: ZNF582-AS1 may play a role in suppressing breast cancer progression. Elucidating the lncRNA's function and regulation may improve our understanding of the disease.

The Value and Clinical Significance of ZNF582 Gene Methylation in the Diagnosis of Cervical Cancer.

INTRODUCTION: The aim of this study was to determine whether ZNF582 gene methylation and tissue protein expression can be used as a tool with high sensitivity and specificity for cervical cancer screening. We analyzed the correlation between promoter methylation of ZNF582 gene and cervical cancer and high risk HPV16/18 infection. METHODS: Tissue samples of normal cervical or chronic cervicitis (n=51), CIN (cervical intraepithelial neoplasia) (n=35), and cervical carcinoma (n=68) were tested for HPV16/18 infection by polymerase chain reaction (PCR). We also detected the methylation status of the ZNF582 gene promoter in the same tissues by methylation-specific PCR (MSP), then analyzed the correlation between ZNF582 promoter methylation and HPV16/18 infection. Immunohistochemistry was used to analyze ZNF582 gene expression in 152 cervical tissues. We detected ZNF582 mRNA expression in cervical tissues (including cancer and non-cancer) by real-time fluorescence quantitative PCR (qPCR). RESULTS: Among 93 high-grade cervical lesions (CINII and above) and cervical cancer samples, 57 cases were positive for HPV16/18 infection and 36 cases were negative. ZNF582 gene methylation occurred in 9 out of 51 cases in normal cervical tissues (17.6%), 16 of 35 cases in CIN tissues (45.7%), and 50 of 68 cases in cervical cancer (73.5%). The differences in methylation rate of the three groups were statistically significant (P<0.05). The ZNF582 methylation rate in the positive HPV16/18 infection group was 73.7%, while the negative group was 63.9%. Compared with normal tissues, ZNF582 protein was highly expressed in cervical cancer tissues, but mRNA expression was low. CONCLUSION: While ZNF582 protein is highly expressed in cervical cancer tissues, it was not sufficient for use as a standard for cervical cancer staging. On the other hand, ZNF582 promoter methylation had high specificity and sensitivity in detecting CINII and highly diseased cervical lesions and could be used as a diagnostic marker for cervical cancer of women.

The application value of PAX1 and ZNF582 gene methylation in high grade intraepithelial lesion and cervical cancer.

PURPOSE: To investigate the possibility of using the methylation level of PAX1/ZNF582 gene as molecular marker to differentiate the progression of cervical cancer. METHODS: From January 2016 to March 2018, 150 patients, who were admitted to Cervical Disease Diagnosis and Treatment Center of Xuzhu Maternity and Child Care Hospital, were enrolled in this study. Patients were classified into chronic cervicitis (for 19 cases), low-grade squamous intraepithelial lesion (LSIL) (18 cases), high-grade squamous intraepithelial lesion (HSIL) (37 cases) and squamous cell carcinoma (SCC) (31 cases). All patients underwent several tests including Thin-prep cytology test (TCT), HPV DNA detection and detection of methylation level of PAX1/ZNF582 genes. RESULTS: For diagnosis of HSIL, the area under curve (AUC) was 0.878 (95% CI 0.806 ~ 0.950); the threshold for PAX1 was 12.285, the sensitivity and specificity were 91.9% and 72.8%, respectively. The AUC of ZNF582 gene detection was 0.900 (95% CI 0.842 ~ 0.959), the threshold was 11.56, while the sensitivity and specificity were 97.3% and 76.7%, respectively. Among various tests we conducted, PAX gene detection methods showed the highest specificity (97.30%). PAX1/ZNF582 gene detection method demonstrated the highest accuracy. CONCLUSIONS: For patients with high-grade cervical lesion and cervical cancer, the methylation level of PAX1/ZNF582 gene could be applied as a noteworthy biomarker for diagnosis and for cervical cancer classification.

Downregulation of lncRNA ZNF582-AS1 due to DNA hypermethylation promotes clear cell renal cell carcinoma growth and metastasis by regulating the N(6)-methyladenosine modification of MT-RNR1.

BACKGROUND: Emerging evidence confirms that lncRNAs (long non-coding RNAs) are potential biomarkers that play vital roles in tumors. ZNF582-AS1 is a novel lncRNA that serves as a potential prognostic marker of cancers. However, the specific clinical significance and molecular mechanism of ZNF582-AS1 in ccRCC (clear cell renal cell carcinoma) are unclear. METHODS: Expression level and clinical significance of ZNF582-AS1 were determined by TCGA-KIRC data and qRT-PCR results of 62 ccRCCs. DNA methylation status of ZNF582-AS1 promoter was examined by MSP, MassARRAY methylation and demethylation analysis. Gain-of-function experiments were conducted to investigate the biological roles of ZNF582-AS1 in the phenotype of ccRCC. The subcellular localization of ZNF582-AS1 was detected by RNA FISH. iTRAQ, RNA pull-down and RIP-qRT-PCR were used to identify the downstream targets of ZNF582-AS1. rRNA MeRIP-seq and MeRIP-qRT-PCR were utilized to examine the N(6)-methyladenosine modification status. Western blot and immunohistochemistry assays were used to determine the protein expression level. RESULTS: ZNF582-AS1 was downregulated in ccRCC, and decreased ZNF582-AS1 expression was significantly correlated with advanced tumor stage, higher pathological stage, distant metastasis and poor prognosis. Decreased ZNF582-AS1 expression was caused by DNA methylation at the CpG islands within its promoter. ZNF582-AS1 overexpression inhibited cell proliferative, migratory and invasive ability, and increased cell apoptotic rate in vitro and in vivo. Mechanistically, we found that ZNF582-AS1 overexpression suppressed the N(6)-methyladenosine modification of MT-RNR1 by reducing rRNA adenine N(6)-methyltransferase A8K0B9 protein level, resulting in the decrease of MT-RNR1 expression, followed by the inhibition of MT-CO2 protein expression. Furthermore, MT-RNR1 overexpression reversed the decreased MT-CO2 expression and phenotype inhibition of ccRCC induced by increased ZNF582-AS1 expression. CONCLUSIONS: This study demonstrates for the first time that ZNF582-AS1 functions as a tumor suppressor gene in ccRCC and ZNF582-AS1 may serve as a potential biomarker and therapeutic target of ccRCC.

ZNF582 hypermethylation promotes metastasis of nasopharyngeal carcinoma by regulating the transcription of adhesion molecules Nectin-3 and NRXN3.

BACKGROUND: Epigenetic regulation plays an important role in the development and progression of nasopharyngeal carcinoma (NPC). However, the epigenetic mechanisms underlying NPC metastasis remains poorly understood. We aimed to find functional genes which regulate the metastasis of NPC and identify therapeutic targets for NPC treatment. METHODS: Bisulfite pyrosequencing was used to analyze zinc finger protein 582 (ZNF582) methylation in NPC tissues and cell lines. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting were used to determine the expression of ZNF582. In vitro and in vivo experiments were performed to evaluate the biological function of ZNF582 in NPC. ZNF582-targeting genes were identified by chromatin immunoprecipitation sequencing (ChIP-seq) and were confirmed by ChIP-qPCR and luciferase assay. RESULTS: ZNF582 promoter was hypermethylated in NPC, and both the mRNA and protein levels of ZNF582 were down-regulated in NPC tissues and cell lines. The restoration of ZNF582 inhibited NPC migration, invasion, and metastasis, while the knockdown of ZNF582 promoted NPC migration, invasion, and metastasis in vitro and in vivo. ZNF582 directly regulated the transcription and expression of adhesion molecules Nectin-3 and NRXN3. Both Nectin-3 and NRXN3 were identified as functional targets of ZNF582, and the restoration or abrogation of these genes reversed the tumor suppressor effect of ZNF582 in NPC metastasis. CONCLUSIONS: ZNF582 acts as a tumor suppressor gene in NPC by regulating the transcription and expression of adhesion molecules Nectin-3 and NRXN3, which may provide novel therapeutic targets for NPC treatment.

High methylation of ZNF582 in cervical adenocarcinoma affects radiosensitivity and prognosis.

BACKGROUND: Our previous study demonstrated hypermethylation of the ZNF582 gene in cervical cancer, but its prognostic value in cervical cancer, especially in cervical adenocarcinoma (CAC), remains unclear. The present study aimed to investigate the value of ZNF582 gene methylation for diagnosis and prediction of radiochemotherapy sensitivity and prognosis in CAC. METHODS: We first determined ZNF582 methylation levels using quantitative methylation-specific PCR in a training set. Disease-free survival and overall survival (DFS and OS) rates were estimated using the Kaplan-Meier method. A Cox regression model was used to assess the prognostic significance of ZNF582 gene methylation in CAC patients. Immunohistochemistry was used to test ZNF582 protein expression in CAC tissues, and an MTT assay evaluated the sensitivity of Hela cells (with or without ZNF582 transfection) to radiation and chemotherapy. RESULTS: The ZNF582 gene showed a higher level of methylation in the CAC group than in the noncancer group, and patients negative for ZNF582 methylation had worse prognoses. We also found that ZNF582 methylation levels were reduced in concomitant chemo-radio-therapy (NCRT) patients compared with that in non-NCRT patients. Methylation-negative status was correlated with high ZNF582 protein expression, and ZNF582 protein overexpression could increase resistance to radiation and chemotherapy in Hela cells. CONCLUSIONS: Aberrant high methylation of ZNF582 may be a potential biomarker for CAC detection and prognosis monitoring. Overexpression of ZNF582 protein could increase CAC chemoradiotherapy resistance.

Aberrant DNA methylation of PAX1, SOX1 and ZNF582 genes as potential biomarkers for esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a highly invasive malignant tumor and the majority of patients have advanced stage of ESCC at diagnosis with poor outcome. Identification of sensitive and specific biomarkers for early screening of ESCC is critical for improving patient overall survival. METHODS: Pyrosequencing was used to determine the magnitude of DNA methylation on the selected regions PAX1 (paired box gene1), SOX1(sex determining region Y-box-1), and ZNF582 (zinc finger protein 582) in ESCC. RESULTS: The methylation levels ofPAX1, SOX1, and ZNF582 genes were significantly higher in the cancerous tissues compared to those in the non-cancerous (all P < 0.0001). The accuracy, sensitivity and specificity of PAX1 methylation for the detection of cancer were respectively 0.754, 96.0% and 51.4%; for SOX1 which were 0.781, 89.2% and 59.5%; for ZNF582 which were 0.898, 93.2% and 75.7%. Most importantly, both the sensitivity and specificity of ZNF582 methylation testing achieved 100% in female ESCC patients. Hypermethylation of PAX1/SOX1/ZNF582 exhibited as an independent risk factor for ESCC development. In addition, ZNF582 methylation level in tumor tissue from the female patients was higher than that from male patients, and it was higher in the moderate and poor differentiated tumors compared to that in well-differentiated tumors. SOX1 methylation level was significantly higher in tumors located in the upper region than those located in the middle or lower regions. CONCLUSION: The methylation levels ofPAX1, SOX1 and ZNF582 genes were all higher in cancer tissues than in paired-adjacent and normal tissues, suggesting that detection of PAX1/SOX1/ZNF582 methylation status may serve as a promising biomarker for ESCC screening and diagnosis.

Hypermethylated ZNF582 and PAX1 genes in mouth rinse samples as biomarkers for oral dysplasia and oral cancer detection.

BACKGROUND: Effective biomarkers for oral cancer screening are important for early diagnosis and treatment of oral cancer. METHODS: Oral epithelial cell samples collected by mouth rinse were obtained from 65 normal control subjects, 108 patients with oral potentially malignant disorders, and 94 patients with oral squamous cell carcinoma (OSCC). Methylation levels of zinc-finger protein 582 (ZNF582) and paired-box 1 (PAX1) genes were quantified by real-time methylation-specific polymerase chain reaction after bisulfite conversion. RESULTS: An abrupt increase in methylated ZNF582 (ZNF582m ) and PAX1 (PAX1m ) levels and positive rates from mild dysplasia to moderate/severe dysplasia, indicating that both ZNF582m and PAX1m are effective biomarkers for differentiating moderate dysplasia or worse (MODY+) oral lesions. When ZNF582m /PAX1m tests were used for identifying MODY+ oral lesions, the sensitivity, specificity, and odds ratio (OR) were 0.65/0.64, 0.75/0.82, and 5.6/8.0, respectively. CONCLUSION: Hypermethylated ZNF582 and PAX1 genes in oral epithelial cells collected by mouth rinse are effective biomarkers for the detection of oral dysplasia and oral cancer.

DNA Methylation Status of PAX1 and ZNF582 in Esophageal Squamous Cell Carcinoma.

Hypermethylation of specific gene promoters is an important mechanism of carcinogenesis. A high frequency of promoter methylation of PAX1 and ZNF582 genes has been detected in cervical cancer. In the present study, we investigated the methylation status of PAX1 and ZNF582 genes in esophageal squamous cell carcinoma (ESCC) tissues. Tumor and paracancerous tissues were obtained from 14 ESCC patients. Genomic DNA was extracted from both tumor and paracancerous tissues, and the concentration of DNA were determined. DNA methylation analysis of PAX1 and ZNF582 genes was carried out using quantitative methylation-specific PCR. To assess the diagnostic performance of the two methylated genes for cancer detection, receiver operating characteristic (ROC) curves were generated. Sensitivities and specificities were tested at cut-offs obtained from the ROC curves. The methylation levels of both PAX1 and ZNF582 genes were significantly higher in tumor tissues compared to non-tumor paracancerous tissues. The methylation rates of PAX1 and ZNF582 in ESCC tumor and paracancerous tissues were 100% and 21.4% (p = 0.006), 85.7% and 0% (p < 0.001), respectively. The sensitivities and specificities of PAX1 and ZNF582 methylation for the detection of cancer were 100% and 85.7%, and 78.6% and 100%, respectively. The DNA methylation levels and frequencies of PAX1 and ZNF582 genes were markedly higher in ESCC tumor tissues compared to those in paracancerous tissues. Moreover, the conclusions were verified by using The Cancer Genome Atlas (TCGA) datasets. DNA methylation status of these two genes showed a relatively good sensitivity and specificity for the detection of ESCC tumors. This data suggests that DNA methylation testing holds a great promise for ESCC screening and warrants further prospective population-based studies.

Utility of gene methylation analysis, cytological examination, and HPV-16/18 genotyping in triage of high-risk human papilloma virus-positive women.

In 2015, the American Society for Colposcopy and Cervical Pathology and the Society of Gynecologic Oncology issued interim guidance for the use of a human papillomavirus (HPV) test for primary screening, suggesting triage of women positive for high-risk human papillomavirus (hrHPV) by HPV-16/18 genotyping and cytology for women positive for non-16/18 hrHPV. The design of the present study was based on this interim guidance and analysis of the methylation status of specific candidate genes, which has been proposed as a tool to reduce unnecessary referral following primary HPV screening for cervical cancer. We performed a hospital-based case-control study including 312 hrHPV-positive women. hrHPV genotyping was performed by nested multiplex PCR assay with type-specific primers.Residual cervical cells from liquid-based cytology were used for extraction of genomic DNA for assessment of the methylation status of PAX1, ZNF582, SOX1, and NKX6-1 and HPV genotyping. Combined with HPV-16/18 genotyping, both a dual methylation test for PAX1/ZNF582 and testing for ZNF582 methylation demonstrated 100% association of methylation with pathology results, indicating carcinoma in situ or squamous cell carcinoma. The sensitivity and specificity of the dual methylation test for PAX1/ZNF582 as a reflex test for identification of CIN3+ lesions were 78.85% and 73.55% (odds ratio = 10.37, 95% confidence interval = 4.76-22.58), respectively. This strategy could reduce the number of patients referred for colposcopic examination by 31.3% compared with cytology, and thus provide a feasible follow-up solution in regions where colposcopy is not readily available. This strategy could also prevent unnecessary anxiety in women with hrHPV infection.

Hypermethylated ZNF582 and PAX1 genes in oral scrapings collected from cancer-adjacent normal oral mucosal sites are associated with aggressive progression and poor prognosis of oral cancer.

OBJECTIVE: This study assessed whether hypermethylated ZNF582 and PAX1 genes in oral scrapings are correlated with the progression and prognosis of oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: Methylation levels of ZNF582 and PAX1 genes in oral scrapings, collected from the cancer and adjacent normal oral mucosal sites of 80 OSCC patients before surgical cancer excision, were quantified using real-time methylation-specific PCR after bisulfite conversion. RESULTS: Both the mean methylation (M)-indices of ZNF582 and PAX1 genes in oral scrapings were significantly higher at the cancer sites than at the adjacent normal oral mucosal sites (both P < .001). In the oral scrapings collected from the adjacent normal oral mucosal sites, the higher M-index of methylated ZNF582 (ZNF582m) was significantly correlated with a more advanced clinical stage (P = .04). Moreover, the higher M-index of methylated PAX1 (PAX1m) was significantly related to larger tumor size (P = .046). When the 80 OSCC patients were classified based on gene methylation tests, using the oral scrapings collected from the adjacent normal oral mucosal sites, we found a significantly shorter 3-year overall survival in ZNF582m-positive, PAX1m-positive, and ZNF582m/PAX1m-positive OSCC patients than in ZNF582m-negative (P = .02), PAX1m-negative (P = .04), and ZNF582m/PAX1m-negative OSCC patients (P = .02), respectively. Multivariate Cox regression analyses identified ZNF582m and ZNF582m/PAX1m as independent unfavorable prognostic factors. CONCLUSION: Hypermethylated ZNF582 and PAX1 genes in the oral scrapings collected from adjacent normal oral mucosal sites rather than cancer sites are associated with aggressive progression and poor prognosis of OSCC.

Hypermethylated ZNF582 and PAX1 are effective biomarkers for detection of oral dysplasia and oral cancer.

OBJECTIVES: This study investigated whether the methylation of ZNF582, PAX1, SOX1, NKX6.1, and PTPRR genes in oral scrapings could be used to detect oral dysplasia and oral cancer and to predict oral cancer recurrence. MATERIALS AND METHODS: Oral scrapings were collected from 65 normal oral mucosa subjects, 107 oral precancer patients, and 95 oral squamous cell carcinoma patients. Methylation levels of the five genes were quantified by real-time methylation-specific PCR after bisulfite conversion. RESULTS: Among the five tested genes, methylated ZNF582 (ZNF582m) and PAX1 (PAX1m) were found to be appropriate biomarkers for oral dysplasia and oral cancers. ZNF582m could detect mild dysplasia or worse oral lesions with the sensitivity and specificity being 0.85 and 0.87, respectively. PAX1m performed better in identifying moderate dysplasia or worse oral lesions with the sensitivity and specificity being 0.72 and 0.86, respectively. Moreover, the methylation levels and positive rates for ZNF582m and PAX1m were increased when disease severity increased. Thus, they may be applicable as a triage tool for patients with abnormal visual oral examinations. After cancer excision, both ZNF582m and PAX1m levels decreased. However, their levels increased again at the subsequently recurrent sites in some patients approximately 3-4 months before cancer recurrence. Finally, areca-quid chewing alone and in combination with cigarette smoking or alcohol drinking were found to be correlated with ZNF582 and PAX1 hypermethylation. CONCLUSION: We conclude that hypermethylated ZNF582 and PAX1 are effective biomarkers for the detection of oral dysplasia and oral cancer and for the prediction of oral cancer recurrence.