Biomarker For Cervical Cancer Screening Research Articles

Current role of viral biomarkers in cervical carcinogenesis

Viral biomarkers for cervical cancer screening are valuable in improving timely diagnosis and detection at early stage. The various biomarkers currently there is evidence include high-risk human papillomavirus (hrHPV) deoxyribonucleic acid (DNA) testing, there is place for further research for biomarkers such as hrHPV genotyping hrHPV transcriptional status, and host DNA methylation. Though the evidence is conclusive for hrHPV DNA testing, there is place for further research for biomarkers such as hrHPV genotyping hrHPV transcriptional status, and host DNA methylation. This application of these biomarkers in context of low resource settings has its limitations despite high prevalence of the disease. Improved women participation can be achieved by use of self-samples thereby improving diagnoses rate of the disease and higher cost effectiveness of the screening programme. In due course, biomarkers will be made available in the screening programmes together with self-sampling that will detect hrHPV infections and low-grade squamous intraepithelial lesions (LSIL) before they progress to high-grade squamous intraepithelial lesions (HSIL), and from there to malignancy. This would facilitate prudent timely clinical decisions, reduced patient anxiety, reduction in over referral and unnecessary treatments in women, especially in developing countries.

Expression and hypermethylation of JAM and EPB41L3 in cervical squamous cell carcinoma: Clinical significance and applications.

This study aimed to explore the expression and hypermethylation of EPB41L3 and JAM3 in cervical squamous cell carcinoma (CSCC) and to investigate their clinical significance. JAM3 and EPB41L3 mRNA expression was analyzed using a public database, and protein expression was detected using immunohistochemistry. The methylation status of JAM3 and EPB41L3 was detected in CSCC tissues and cervical cytological specimens using a quantitative methylation-specific PCR (qMSP). JAM3 and EPB41L3 mRNA were downregulated in CSCC. The JAM3 protein was positively detected in 39.4% of CSCC tissues and frequently expressed in those with lower FIGO stage and no lymph node metastasis. EPB41L3 was expressed in 18.9% of CSCC tissues. The hypermethylation of JAM3 was detected in 52.3% of CSCC tissues and related to higher FIGO stage and lymph node metastasis. EPB41L3 hypermethylation was detected in 72.7% of CSCC tissues and related to older ages and lymph node metastasis. In cervical cytological specimens, no methylation of JAM3 and EPB41L3 was found in normal or inflamed cervical epithelial cells. The methylation of JAM3 was detected in 0%, 8.3%, and 6.3% of ASCUS, LSIL, and HSIL samples, while EPB41L3 was detected in 12.5%, 42.9%, and 71.4%, respectively. The sensitivity of the combination of JAM3 and EPB41L3 methylation detection in ASCUS, LSIL, and HSIL was 8.3%, 15.6%, and 85.7%, respectively. The specificity of the combination of JAM3 and EPB41L3 methylation detection was 100%. Downregulation of JAM3 and EPB41L3 by hypermethylation was detected in CSCC. JAM3 and EPB41L3 hypermethylation are potential biomarkers for cervical cancer screening.

Research Progress in the Relationship Between P2X7R and Cervical Cancer.

Cervical cancer is one of the most common and serious tumors in women. Finding new biomarkers and therapeutic targets plays an important role in the diagnosis, prognosis, and treatment of cervical cancer. Purinergic ligand-gated ion channel 7 receptor (P2X7R) is a purine ligand cation channel, activated by adenosine triphosphate (ATP). Studies have shown that P2X7R plays an important role in a variety of diseases and cancers. More and more studies have shown that P2X7R is also closely related to cervical cancer; therefore, the role of P2X7R in the development of cervical cancer deserves further discussion. The expression level of P2X7R in uterine epithelial cancer tissues was lower than that of the corresponding normal tissues. P2X7R plays an important role in the apoptotic process of cervical cancer through various mechanisms of action, and both antagonists and agonists of P2X7R can inhibit the proliferation of cervical cancer cells, while P2X7R is involved in the antitumor effect of Atr-I on cervical cancer cells. This review evaluates the current role of P2X7R in cervical cancer in order to develop more specific therapies for cervical cancer. In conclusion, P2X7R may become a biomarker for cervical cancer screening, and even a new target for clinical treatment of cervical cancer.

Impedimetric sensing platform for human papillomavirus and p53 tumor suppressor gene in cervical samples

The oncogenic human papillomavirus (HPV) is an excellent biomarker for cervical cancer screening. The present research reports the construction of an integrated sensor platform (biosensor MY11 and biosensor p53 ) for the impedimetric detection of the HPV L1 sequence and p53 tumor suppressor gene. The nanostructured biosensor is composed of electrosynthesized polypyrrole (PPy) and gold nanoparticles (AuNPs). Methodological strategies based on cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM), and atomic force microscopy (AFM) were used. The exposure of the genetic biosensors to molecular targets triggered changes in the analytical signals. Calibration plots for the biosensor MY11 showed a linear range between 0.001 to 100 pg μL −1 for all analyzed genotypes (HPV6, 11, 16, 18, 45, 53, 54, and 58). The detection limits were measured between 0.11 to 0.61 pg μL −1 and limits of quantification between 0.33 to 1.86 pg μL −1 . The sensors exhibited reproducibility with a relative standard deviation of 4.21% and high repeatability (less than 2.5%). Two electrochemical profiles were obtained for high and low-risk HPV groups, suggesting the differential diagnosis using a degenerate oligonucleotide probe. Our results allow correlating the p53 gene expression in clinical samples infected with papillomavirus. The proposed biosensor represents an innovative device for the integrated monitoring of biomarkers related to cervical cancer with ultra-sensitive analytical performance. • A nanointegrated system was designed for the differential diagnosis of papillomavirus. • Polypyrrole conjugated to gold particles acted as an ultrasensitive signal transducer. • High and low-risk HPV groups were identified using a degenerate oligonucleotide probe. • Molecular screening for oncogenic HPV and p53 gene was performed in clinical samples. • The biosensor showed high specificity with a linear range from 0.001 to 100 pg μL −1 .

Abstract 76: Global LINE-1 Hypomethylation as Novel Biomarker for Cervical Cancer in Nigerian Women Living with HIV

Abstract Purpose: Nigeria faces a high health burden of cervical cancer (CC), which is worsened by high prevalence of human immunodeficiency virus (HIV) infection. Repetitive elements (RE) are DNA sequences that occur in multiple copies throughout the human genome. HIV infection can lead to RE hypomethylation that causes genome instability, an event often seen in the early phase of tumorigenesis. We aim to examine global RE hypomethylation as a novel epigenetic biomarker for CC among HIV-positive women in Nigeria. Methods: This study involved three groups of women: a) HIV-positive with CC (n=39); b) HIV-positive and cancer-free (n=52); and c) HIV-negative with CC (n=23). We estimated three types of global RE methylation in cervical tissue using genome-wide methylation data: long interspersed nuclear elements (LINE-1), Alu, and human endogenous retrovirus (HERV). We used multiple linear regression adjusting for age, education, parity, employment, cancer stage, body mass index, and sample batch to compare the biomarkers across the HIV/ICC groups and paired t-test to compare 26 pairs of tumor vs. surrounding normal tissues, stratified by HIV status. Receiver operating characteristic curve (ROC) and area under the ROC (AUC) were used to examine the diagnostic value. Results: Among HIV-positive women, all 3 global RE methylation biomarkers were hypomethylated in CC compared to cancer-free (LINE-1: mean difference [MD]=-0.049, p-value=2.9e-8; Alu: MD=-0.011, p-value=2.0e-4; HERV: MD=-0.013, p-value=1.7e-6). Paired analyses showed a larger, more significant MD in HIV-positive stratum than HIV-negative, especially LINE-1 (MD=-0.048 vs. -0.020, p-value=0.004 vs. 0.254). LINE-1 achieved the highest AUC (0.85, 95% CI: 0.76-0.95) in distinguishing tumor tissue from normal tissue among HIV-positive women, followed by HERV (0.82, 95% CI: 0.73-0.92) and Alu (0.60, 95% CI: 0.47-0.72). Conclusion: Global LINE-1 hypomethylation may serve as a novel biomarker for CC screening and early detection for women living with HIV in low- and middle-income countries. Citation Format: Yinan Zheng, Jonah Musa, Kyeezu Kim, Yishu Qu, Brian Joyce, Jun Wang, Demirkan Gursel, Olugbenga Silas, Abdulkareem Fatimah, Godwin Imade, Alani Akanmu, Jian-Jun Wei, Masha Kocherginsky, Kwang-Youn Kim, Firas Wehbe, Chad Achenbach, Rose Anorlu, Melissa Simon, Atiene Sagay, Folasade Ogunsola, Robert Murphy, Lifang Hou. Global LINE-1 Hypomethylation as Novel Biomarker for Cervical Cancer in Nigerian Women Living with HIV [abstract]. In: Proceedings of the 9th Annual Symposium on Global Cancer Research; Global Cancer Research and Control: Looking Back and Charting a Path Forward; 2021 Mar 10-11. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2021;30(7 Suppl):Abstract nr 76.

Evaluation of Cervical Cancer Screening Tools; INNO-LiPA® HPV Genotyping Extra-II Assay versus E7/E6 oncoproteins, How is reliable and practical?

Background: High-Risk Human papillomavirus (HR-HPV) has been well established as the cervical cancer (CC) risk factor. In recent years, various diagnostic methods of human papillomaviruses (HPV) have been developed to promote sensitivity and specificity of CC screening which leads to a low mortality rate. This study aimed to compare diagnostic test metrics of two HPV diagnostic techniques, including Western blot and INNO-LiPA HPV Genotyping Extra II assay methods in asymptomatic or subclinical patients, among the South-Eastern Iranian women. Methods: 323 women were referred to the Pathology and Stem Cell Research Center, from February 2018 to January 2020. HPV-DNA with the INNO-LiPA HPV Genotyping Extra-II Assay kit and the western blot assays for HPV E7 and E6 assessment were employed. Results: Overall, 163 (50.4%) samples were dysplastic pap smear, the specificity of the HPV DNA test by INNO-LiPA HPV Genotyping Extra-II Assay test was significantly higher than the E7/E6 oncoproteins finding (67.3 vs. 49.9%), and the sensitivity was lower (96.6 vs. 74.8%), respectively. Conclusions: HR-HPV E7/E6 oncoproteins expression was evaluated as a possible novel biomarker for CC screening in pap smear as the preliminary test with satisfactory diagnostic values for HR-HPV types 16 and 18. The corresponding diagnostic values may be further improved by combining HPV DNA tests with the INNO-LiPA HPV Genotyping Extra-II test. Also, they may prove helpful for HR-HPV infection diagnosis in cases that the patients are asymptomatic or subclinical. Keywords: Cervical Cancer; Human Papillomavirus (HPV); Diagnostic Screening Programs; Oncogene Proteins

Global line-1 hypomethylation as novel biomarker for cervical cancer in Nigerian women living with HIV.

e17513 Background: Nigeria faces a high health burden of cervical cancer (CC), which is worsened by high prevalence of human immunodeficiency virus (HIV) infection. Repetitive elements (RE) are DNA sequences that occur in multiple copies throughout the human genome. HIV infection can lead to RE hypomethylation that causes genome instability, an event often seen in the early phase of tumorigenesis. We aim to examine global RE hypomethylation as a novel epigenetic biomarker for CC among HIV-positive women in Nigeria. Methods: This study involved three groups of women: a) HIV-positive with CC (n=39); b) HIV-positive and cancer-free (n=52); and c) HIV-negative with CC (n=23). We estimated three types of global RE methylation in cervical tissue using genome-wide methylation data: long interspersed nuclear elements (LINE-1), Alu, and human endogenous retrovirus (HERV). We used multiple linear regression adjusting for age, education, parity, employment, cancer stage, body mass index, and sample batch to compare the biomarkers across the HIV/ICC groups and paired t-test to compare 26 pairs of tumor vs. surrounding normal tissues, stratified by HIV status. Receiver operating characteristic curve (ROC) and area under the ROC (AUC) were used to examine the diagnostic value. Results: Among HIV-positive women, all 3 global RE methylation biomarkers were hypomethylated in CC compared to cancer-free (LINE-1: mean difference [MD]=-0.049, p-value=2.9e-8; Alu: MD=-0.011, p-value=2.0e-4; HERV: MD=-0.013, p-value=1.7e-6). Paired analyses showed a larger, more significant MD in HIV-positive stratum than HIV-negative, especially LINE-1 (MD=-0.048 vs. -0.020, p-value=0.004 vs. 0.254). LINE-1 achieved the highest AUC (0.85, 95% CI: 0.76-0.95) in distinguishing tumor tissue from normal tissue among HIV-positive women, followed by HERV (0.82, 95% CI: 0.73-0.92) and Alu (0.60, 95% CI: 0.47-0.72). Conclusions: Global LINE-1 hypomethylation may serve as a novel biomarker for CC screening and early detection for women living with HIV in low- and middle-income countries.

The Association of Molecular Biomarkers in the Diagnosis of Cervical Pre-Cancer and Cancer and Risk Factors in Senegalese.

Background:Cervical intraepithelial neoplasia (CIN) grading is subjective and affected by substantial rates of discordance among pathologists. Although recent studies have suggested that p16INK4a may be a useful surrogate biomarker of cervical neoplasia, Ki-67 and human papillomavirus testing have also been shown to be useful in detecting neoplasia. The purpose of this study was to determine the expression of p16INK4a and Ki-67 in cervical neoplasia and its correlations with cofactors. Methods: The study involved 69 patients with and without cervical neoplasia who underwent colposcopic directed biopsy. On each patient, two samples were taken; the first was used for immunohistochemistry and the second for molecular testing, using HPV16and18 genotyping Real-Time PCR Kit. Results: The study revealed the expression level of p16INK4a and Ki-67 in a descending order, from invasive squamous cell carcinoma (SCC), CIN2/3, CIN1 and non-dysplastic lesions. Correlations showed an association between the staining of p16NK4a and Ki-67 with the increase of age (OR: 1.79 (95%IC: 0.49 – 6.55), p = 0.037) and marital status (OR: 0.17 (95%IC: 0.04 – 0.68), p = 0.003). We found that the expressions of p16INK4a and Ki-67 were significantly different between invasive SCC vs non-dysplasia (OR: 44.57 (95%IC: 4.91 – 403.91), p<0.0001). The study showed significant correlation between HPV 16and18 infection with p16 INK4a and Ki-67 expression (OR: 0.13 (95%IC: 0.03 – 0.52), p<0.0001). Strong expression of p16INK4a and Ki-67 were observed in invasive squamous cell carcinoma, moderate staining was found in CIN2/3, weak staining in CIN1 and normal histology. Conclusion:Our findings indicate that p16INK4a and Ki-67 expressions associated strongly with cervical pathology. Therefore, p16/Ki-67 could be considered as a suitable biomarker for cervical cancer screening, particularly in HPV-based screening programs.

Serum anti-GAPDH autoantibody levels reflect the severity of cervical lesions: A potential serum biomarker for cervical cancer screening.

Recent studies have indicated that a certain level of autoantibodies may be essential for maintaining good health as well as preventing cancer development, and that the levels of serum autoantibodies can decline during malignant progression. The aim of the present study was to identify such an autoantibody-based biomarker for screening cervical lesions. An autoantigen reactive with healthy female sera was detected in the cytosolic fraction of HeLa cells, a cervical cancer cell line, and identified. Serum immunoglobulin (Ig)-G and IgM levels against the purified autoantigen in normal, cervical intraepithelial neoplasias (CINs) I, II and III, and cervical cancer were compared using ELISAs. The autoantigen in HeLa cells was identified to be GAPDH. The serum levels of anti-HeLa-GAPDH IgG decreased with increasing severity of cervical lesions, and similar decreases in IgM levels were revealed. Notably, the anti-HeLa-GAPDH IgG level was discovered to discriminate cervical cancer from normal samples with 80.0% sensitivity and 96.6% specificity. The serum anti-HeLa-GAPDH autoantibody level, as a single parameter, is a promising serum biomarker for screening cervical lesions.

ZNF582 methylation as a potential biomarker to predict cervical intraepithelial neoplasia type III/worse: A meta-analysis of related studies in Chinese population.

DNA methylation markers have been assessed as potential biomarkers for early cervical cancer detection. Herein, we evaluated the diagnostic performance of zinc finger protein 582 (ZNF582) methylation for cervical cancer detection. Eligible studies were systematically searched from the electronic databases. The quality of enrolled studies was evaluated using the second version of the check list for Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2). The bivariate meta-analysis model was employed to plot the summary receiver operator characteristic (SROC) curve using Stata 14.0 software. Cochran's Q test and I statistics were applied to assess heterogeneity among studies. Publication bias was evaluated by the Deeks' funnel plot asymmetry test. Seven studies composed of 1749 patients were eventually included. The pooled sensitivity of ZNF582 methylation was estimated to be 0.71 [95% confidence interval (CI): 0.67-0.75] in differentiating patients with cervical intraepithelial neoplasia type III/worse (CIN3+), corresponding to a specificity of 0.81 (95% CI: 0.79-0.83) and area under the curve (AUC) of 0.85. Our stratified analysis suggested that sequential combined of HPV DNA and ZNF582 methylation test (AUC, sensitivity, and specificity of 0.876, 0.75, and 0.87, respectively) achieved higher diagnostic accuracy than single HPV DNA testing test (AUC, sensitivity and specificity of 0.669, 0.96, and 0.41, respectively). ZNF582 methylation has a prospect to be an auxiliary biomarker for cervical cancer screening. A new strategy of co-testing HPV DNA and ZNF582 methylation test in cervical scrapings confers an improved diagnostic accuracy than single HPV DNA testing.

DNA methylation of imprinted gene control regions in the regression of low-grade cervical lesions.

The role of host epigenetic mechanisms in the natural history of low-grade cervical intraepithelial neoplasia (CIN1) is not well characterized. We explored differential methylation of imprinted gene regulatory regions as predictors of the risk of CIN1 regression. A total of 164 patients with CIN1 were recruited from 10 Duke University clinics for the CIN Cohort Study. Participants had colposcopies at enrollment and up to five follow-up visits over 3 years. DNA was extracted from exfoliated cervical cells for methylation quantitation at CpG (cytosine-phosphate-guanine) sites and human papillomavirus (HPV) genotyping. Hazard ratios (HR) and 95% confidence intervals (CI) were estimated using Cox regression to quantify the effect of methylation on CIN1 regression over two consecutive visits, compared to non-regression (persistent CIN1; progression to CIN2+; or CIN1 regression at a single time-point), adjusting for age, race, high-risk HPV (hrHPV), parity, oral contraceptive and smoking status. Median participant age was 26.6 years (range: 21.0-64.4 years), 39% were African-American, and 11% were current smokers. Most participants were hrHPV-positive at enrollment (80.5%). Over one-third of cases regressed (n = 53, 35.1%). Median time-to-regression was 12.6 months (range: 4.5-24.0 months). Probability of CIN1 regression was negatively correlated with methylation at IGF2AS CpG 5 (HR = 0.41; 95% CI = 0.23-0.77) and PEG10 DMR (HR = 0.80; 95% CI = 0.65-0.98). Altered methylation of imprinted IGF2AS and PEG10 DMRs may play a role in the natural history of CIN1. If confirmed in larger studies, further research on imprinted gene DMR methylation is warranted to determine its efficacy as a biomarker for cervical cancer screening.

PAX1 and SOX1 methylation as an initial screening method for cervical cancer: a meta-analysis of individual studies in Asians.

Epigenetic alterations of gene or DNA methylation have been highlighted as promising biomarkers for early cervical cancer screening. Herein, we evaluated the diagnostic performance of paired boxed gene 1 (PAX1) and sex determining region Y-box 1 (SOX1) methylation for cervical cancer detection. Eligible studies were retrieved by searching the electronic databases. Study quality was assessed according to the Quality Assessment of Diagnostic Accuracy Studies (QUADAS) checklist. The bivariate meta-analysis model was employed to plot the summary receiver operator characteristic (SROC) curve using Stata 12.0 software. The pooled sensitivity of PAX1 methylation was estimated to be 0.73 [95% confidence interval (CI): 0.70-0.75] in differentiating patients with HSIL (high-grade squamous intraepithelial lesion) or CIN3+ (cervical intraepithelial neoplasia type III/worse) or cervical cancer from normal individuals, corresponding to a specificity of 0.87 (95% CI: 0.85-0.89) and area under the curve (AUC) of 0.91. The SOX1 methylation test yielded an AUC of 0.82, under which, the pooled sensitivity was 0.71 (95% CI: 0.67-0.74) and specificity was 0.64 (95% CI: 0.61-0.67). Notably, the stratified analysis suggested that combing parallel testing of PAX1 methylation and human papillomavirus (HPV) DNA (AUC, sensitivity, and specificity of 0.89, 0.75, and 0.81, respectively) achieved higher accuracy than single HPV DNA testing (AUC, sensitivity, and specificity of 0.77, 0.81, and 0.70, respectively). PAX1 or SOX1 methylation has a prospect to be an auxiliary biomarker for cervical cancer screening, and parallel testing of PAX1 methylation and HPV DNA in cervical swabs confers an improved diagnostic accuracy than single HPV DNA testing.

P16/Ki-67 co-expression associates high risk human papillomavirus persistence and cervical histopathology: a 3-year cohort study in China.

PurposeTo evaluate the association of p16/Ki-67 co-expression and persistence of high-risk human papillomavirus (HR-HPV) infection as well as cervical abnormalities.MethodsWe performed a 3-year cohort study among which 2498 Chinese women aged 25 to 65 years were screened by different HPV tests in 2011. 690 women who were positive at any of the tests and a random sample of 164 women with all negative results received colposcopy, cervical specimens for cobas HPV test (Roche diagnostics) were collected before colposcopy; of this group, 737 cervical specimens were collected to perform cobas, Liquid-based cytology, HPV E6 test (Arbor Vita Corporation) and p16/Ki-67 dual staining (Roche diagnostics) in 2014. Colposcopy and biopsies was performed on women with any abnormal result.ResultsCompared to women without HR-HPV persistent infection, women in the HR-HPV persistence group had a higher risk of p16/Ki-67 positive, with an adjusted Odds Ratio(OR) and 95% confidence interval (CI) of 6.29 (4.07-9.72); moreover, adjusted odds ratio for women who had HPV16/18 persistent infection was nearly 4-folder higher than women with other 12 HR-HPV persistent infection (adjusted OR = 17.15, 95% CI: 7.11-41.33 vs adjusted OR = 4.68, 95% CI: 2.89-7.58). Additionally, p16/Ki-67 positivity rate significantly increased with the severity of the cytological and histological abnormalities, and resulted strongly associated with a CIN2+ diagnosis (OR = 16.03, 95% CI: 4.46-57.59).Conclusionsp16/Ki-67 co-expressions associated strongly with HR-HPV persistence, especially with HPV16/18, and the presence of a CIN2+ lesion. Therefore, p16/Ki-67 could be considered as a suitable biomarker for cervical cancer screening, particularly in HPV-based screening programs.

PAX1 methylation as an auxiliary biomarker for cervical cancer screening: A meta-analysis

ObjectiveSeveral studies have implicated PAX1 epigenetic regulation in cervical neoplasia. The aim of this meta-analysis was to assess PAX1 gene methylation as a potential biomarker in cervical cancer screening. MethodsA systematical search of all major databases was performed, in order to include all relevant publications in English until December 31st 2014. Studies with insufficient data, conducted in experimental models or associated with other comorbidities were excluded from the meta-analysis. Summary receiver operating characteristics (SROC) for Cervical Intraepithelial Neoplasia grade 2 or worse (CIN2+) versus normal, and CIN grade 3 or worse (CIN3+) versus normal, were estimated using the bivariate model. ResultsOut of the 20 initially included studies, finally 7 (comprising of 1385 subjects with various stages of CIN and normal cervical pathology) met the inclusion criteria. The sensitivity of CIN2+ versus normal was estimated to be 0.66 (CI 95%, 0.46-0.81) and the specificity 0.92 (CI 95%, 0.88-0.95). On the other hand, the sensitivity of CIN3+ versus normal was 0.77 (CI 95%, 0.58-0.89) and the specificity 0.92 (CI 95%, 0.88-0.94). Moreover, the area under the curve (AUC) in the former case was 0.923, and in the latter 0.931. ConclusionThe results of this meta-analysis support the utility of PAX1 methylation as an auxiliary biomarker in cervical cancer screening. PAX1 could be used effectively to increase the specificity of HPV DNA by detecting women with more advanced cervical abnormalities.

PAX1 methylation as a potential biomarker for cervical cancer screening.

DNA methylation is a potential biomarker for early cancer detection. Previous studies suggested that the methylations of several genes are promising markers for the detection of cervical intraepithelial neoplasia at grade III or worse (CIN3+). The purpose of the present study was to explore the feasibility of these DNA methylation testing in cervical cancer screening. A total of 443 women were recruited from the Yuan's General Hospital. Cervical scrapings were collected for Papanicolaou (Pap) test by using cervical brushes, and the cytological data were used for analysis. The residual cells on the brush were preserved in phosphate-buffered saline solution at 4°C until DNA extraction. Then, the extracted DNA were used for molecular tests, which included human papillomavirus typing and quantification of the methylation levels for PAX1, SOX1, and NKX6-1 genes. Subjects who had abnormal Pap test results underwent colposcopy or biopsy with subsequent conization or major surgery when biopsy results revealed CIN2+. The final diagnosis for this group was confirmed by colposcopy or pathological examination. The study was approved by the institutional review board of Yuan's General Hospital, and all the molecular tests were performed by ISO17025 certified laboratories. The sensitivity of PAX1 and SOX1 was greater than 80%, and the specificity of PAX1 and NXK6-1 was greater than 80% for the detection of CIN3+ lesions. PAX1 detection alone had a sensitivity and specificity of 86% and 85%, respectively, whereas when used as a cotest with the Pap test, the sensitivity and specificity were 89% and 83%, respectively. PAX1 showed great potential as a biomarker for cervical cancer screening. When incorporating PAX1 detection into current screening protocol, the efficacy of screening could be greatly improved. Moreover, unnecessary referral for colposcopy and biopsy could be reduced up to 60%. However, prospective population-based studies are necessary for further implementation of this screening program.

Methylomics analysis identifies epigenetically silenced genes and implies an activation of β‐catenin signaling in cervical cancer

Using DNA methylation biomarkers in cancer detection is a potential direction in clinical testing. Some methylated genes have been proposed for cervical cancer detection; however, more reliable methylation markers are needed. To identify new hypermethylated genes in the discovery phase, we compared the methylome between a pool of DNA from normal cervical epithelium (n = 19) and a pool of DNA from cervical cancer tissues (n = 38) using a methylation bead array. We integrated the differentially methylated genes with public gene expression databases, which resulted in 91 candidate genes. Based on gene expression after demethylation treatment in cell lines, we confirmed 61 genes for further validation. In the validation phase, quantitative MSP and bisulfite pyrosequencing were used to examine their methylation level in an independent set of clinical samples. Fourteen genes, including ADRA1D, AJAP1, COL6A2, EDN3, EPO, HS3ST2, MAGI2, POU4F3, PTGDR, SOX8, SOX17, ST6GAL2, SYT9, and ZNF614, were significantly hypermethylated in CIN3+ lesions. The sensitivity, specificity, and accuracy of POU4F3 for detecting CIN3+ lesions were 0.88, 0.82, and 0.85, respectively. A bioinformatics function analysis revealed that AJAP1, EDN3, EPO, MAGI2, and SOX17 were potentially implicated in β-catenin signaling, suggesting the epigenetic dysregulation of this signaling pathway during cervical cancer development. The concurrent methylation of multiple genes in cancers and in subsets of precancerous lesions suggests the presence of a driver of methylation phenotype in cervical carcinogenesis. Further validation of these new genes as biomarkers for cervical cancer screening in a larger population-based study is warranted.

Molecular biomarkers in cervical cancer diagnosis: a critical appraisal

It is expected that in the near future high-risk human papillomavirus (hr-HPV) testing will be implemented as the primary cervical cancer screening method in some countries. However, only a fraction of hr-HPV positive women will have a clinically relevant lesion. As a result, there is an urgent need for additional biomarkers that can detect these lesions and that can at the same time be applied to cytological specimens. This overview evaluates the most promising cytological biomarkers. Cytological biomarkers that can be used are being discussed in view of their molecular background. The most promising biomarkers are p16(INK4a)/Ki-67 dual immunostaining; methylation of the promoter region of the cell adhesion molecule 1 (CADM1) gene and the T-lymphocyte maturation associated protein (MAL) gene and viral integration. Their sensitivity, specificity and limitations are discussed in detail and their diagnostic accuracy is evaluated. The most promising cytological biomarkers for cervical cancer screening are p16(INK4a)/Ki-67 dual immunostaining, methylation of CADM1 and MAL and viral integration. Although some of the biomarkers are very promising for this purpose, no studies have evaluated how accurately these biomarkers classify or predict the outcome. Additional clinical trials are needed to determine the true clinical value of these promising cytological biomarkers.

Biomarkers for cervical cancer screening: the role of p16INK4a to highlight transforming HPV infections

Biomarkers indicating the initiation of neoplastic transformation processes in human papillomavirus (HPV)-infected epithelial cells are moving into the focus of cancer prevention research, particularly for anogenital cancer, including cancer of the uterine cervix. Based on the in-depth understanding of the molecular events leading to neoplastic transformation of HPV-infected human cells, the cyclin-dependent kinase inhibitor p16INK4a turned out to be substantially overexpressed in virtually all HPV-transformed cells. This finding opened novel avenues in diagnostic histopathology to substantially improve the diagnostic accuracy of cervical cancer and its precursor lesions. Furthermore, it provides a novel technical platform to substantially improve the accuracy of cytology-based cancer early-detection programs. Here, we review the molecular background and the current evidence for the clinical utility of the p16INK4a biomarker for HPV-related cancers, and cervical cancer prevention in particular.

A unified sample preparation protocol for proteomic and genomic profiling of cervical swabs to identify biomarkers for cervical cancer screening

Cervical cancer screening is ideally suited for the development of biomarkers due to the ease of tissue acquisition and the well-established histological transitions. Furthermore, cell and biologic fluid obtained from cervix samples undergo specific molecular changes that can be profiled. However, the ideal manner and techniques for preparing cervical samples remains to be determined. To address this critical issue a patient screening protein and nucleic acid collection protocol was established. RNAlater was used to collect the samples followed by proteomic methods to identify proteins that were differentially expressed in normal cervical epithelial versus cervical cancer cells. Three hundred ninety spots were identified via 2-D DIGE that were expressed at either higher or lower levels (>three-fold) in cervical cancer samples. These proteomic results were compared to genes in a cDNA microarray analysis of microdissected neoplastic cervical specimens to identify overlapping patterns of expression. The most frequent pathways represented by the combined dataset were: cell cycle: G2/M DNA damage checkpoint regulation; aryl hydrocarbon receptor signaling; p53 signaling; cell cycle: G1/S checkpoint regulation; and the ER stress pathway. HNRPA2B1 was identified as a biomarker candidate with increased expression in cancer compared to normal cervix and validated by Western blot.

Biomarkers in Cervical Cancer Screening

In industrialized countries, population wide cytological screening programs using the Pap test have led to a substantial reduction of the incidence of cervical cancer. Despite this evident success, screening programs that rely on Pap-stained cytological samples have several limitations. First, a number of equivocal or mildly abnormal test results require costly work up by either repeated retesting or direct colposcopy and biopsy, since a certain percentage of high grade lesions that require immediate treatment hide among these unclear test results. This work up of mildly abnormal or equivocal cytological tests consumes a large amount of the overall costs spent for cervical cancer screening. Improved triage of these samples might substantially reduce the costs. Cervical cancer is induced by persistent infections with oncogenic human papilloma viruses (HPV). While HPV infection is an indispensable factor, it is not sufficient to cause cancer. The majority of acute HPV infections induce low grade precursor lesions that are cleared spontaneously after several months in more than 90% of cases, and less than 10% eventually progress to high grade lesions or invasive cancer. Progression is characterized by the deregulated expression of the viral oncogenes E6 and E7 in infected basal and parabasal cells. Novel biomarkers that allow monitoring these essential molecular events in histological or cytological specimens are likely to improve the detection of lesions that have a high risk of progression in both primary screening and triage settings. In this review, we will discuss potential biomarkers for cervical cancer screening with a focus on the level of clinical evidence that supports their application as novel markers in refined cervical cancer screening programs.