The Role Of MicroRNA Detection In Cervical Exfoliated Cells In Cervical Cancer Screening

Cervical cancer ranks as the third most common malignancy in women worldwide with530,000new cases annually, and most of which occurrs in developing countries including China. From normal cervix to cervical intraepithelial neoplasia (CIN) and finally invasive cervical cancer (ICC), the development of cervical cancer experienced a long period (mean5-20years). Proper screenings and early diagnoses during this period can dramatically prevent the development of cervical cancer and reduce its morbidity and mortality.High-risk Human Papillomavirus (HR-HPV), as an necessary causal factor for cervical cancer, has been widely applied for cervical cancer screening. HPV testing bears the advantages of high sensitivity, high negative predictive value, no dependence on cytologists and is more impersonal than cytology. EUROGIN (European Research Organization on Genital Infection and Neoplasia) roadmap has recommended HPV testing as primary screening for cervical cancer. HPV testing has also been commonly applied as primary screening in less developed countries, where well trained cytologists are quite lacking. The main limitation of HPV testing is low specificity. The reported life-time risk of HPV infection for sex-active women is about80%, and of which,90%are transient infection, and will be eliminated spontaneously within two years. Only a small portion of HPV positive women will finally develop high-grade CIN, and even fewer will finally reach invasive cancer. Thus, HPV testing alone may result in unnecessary psychological burden and increase the risk of overtreatment. An available triage is necessary for HPV-positive population.Since the introduction of Pap (Papanicolaou) smear cytology for cervical cancer screening in1940’s, the incidence and mortality of cervical cancer has been reduced dramatically, especially in regions where Pap test is widely applied. Pap test is commonly performed as cytology alone or co-test with HPV testing. Recently, cytology triage has become a recommended option for HPV-positive women in EUROGIN roadmap. However, cytology triage still presents the disadvantages due to its limited sensitivity (38%-50%). Even the ThinPrep liquid-based cytology (LBC) technology was developed, no significant sensitivity improvement has been achieved. Further, cytology depends heavily on well-trained cytologists, which are quite lacking in less developed countries including China. An optimal triage test is still imperative for HPV-positive women.MicroRNAs (miRNAs), as important regulators of gene expression, are involved in many important intracellular pathways, such as apoptosis, proliferation and differentiation. Dysregulation of miRNA expression has been found in many human malignancies, including gastric cancer, rectal cancer, uterine cancer and so on. Recently, several researches focused on miRNAs as biomarkers for cancer diagnosis and obtained promising results. In those studies, most of miRNA detection were performed in serum or plasma samples, and some in other samples, such as urine (bladder cancer), saliva (esophagus cancer), and gastric juice (gastric cancer). However, miRNA detection in cervical exfoliated cells has not been reported yet. Dysregulation expression of miRNA has also been reported in cervical cancer and precancerous lesion tissues. For example, both miR218and miR34a had the role of tumor suppressor genes in cervical cancer. And in our previous study, we found14down-regulated miRNAs including miR-375and miR-424, and17up-regulated miRNAs including miR-92a and miR-93in cervical cancer and precancerous lesion tissues. We further found that miR-375via targeting Spl, miR-424via targeting Chkl, and miR-93via targeting RAB11FIP1participated in carcinogenesis or progression in cervical cancer. It was also reported that miR-34a was involved in HPV E6-p53degradation pathway and miR-218was involved in cervical carcinogenesis by targeting LAMB3. Dysregulated expression of these miRNAs implies their potential application as biomarkers for cervical cancer screening.Mainly according to the previous findings of their dysregulations in cervical cancer and precancerous tissues and their roles in cervical carcinogenesis, we here chose six dysregulated miRNAs (miR424, miR375, miR218, miR34a, miR92a, and miR93) as candidate biomarkers, and their relative expression levels in cervical exfoliated cells were detected by the method of stem loop RT-qPCR. We firstly confirmed the difference of expression levels between different cervical lesions. And further, compared with cytology, we evaluated the performance of miRNA detection as a triage for HPV-positive women when identifying high-grade CIN and ICC. PART Ⅰ miRNA expression in cervical exfoliated cells of normal cervix, cervical intraepithelial neoplasia, and invasive cervical cancerObjectivesTo compare the relative expression levels of candidate miRNAs between different cervical lesions, and to confirmed the difference of mRNAs relative expression levels between different lesions.MethodsThe inclusion criteria for the participants were:1) age:30-65years old;2) intact cervix;3) no previously confirmed CIN, cervical cancer or other malignancies;4) not pregnant. Cervical exfoliated cells were collected for miRNA detection. Participants were divided into groups by histological diagnoses. The method of Stem loop RT-qPCR was applied for miRNA detection, and U6was used as an endogenous normalization. Student t test and non-parametric Mann-Whitney U test were used for statistic analysis with SPSS17.0software. All statistic tests were two sided, and P<0.05was considered statistically significant.Results1. Total735eligible women were recruited, including240normal,100CIN1,111CIN2,117CIN3, and167ICC.2. There is no significant difference in age distributions between CIN1-and CIN2+, CIN2-and CIN3+, CIN3-and ICC groups (P>0.05).3. The relative expression levels of miR-218, miR-34a, miR-424, and miR-375in CIN2+, CIN3+and ICC groups were significantly lower than those in CIN1-, CIN2-and CIN3-groups, respectively (p<0.001). The relative expression levels of miR-92a and miR-93were not significantly different between these groups (p>0.05). Conclusions1. It is feasible for miRNA detect in cervical exfoliated cells.2. The relative expression levels of miR-424, miR-375, miR-34a, and miR-218in cervical exfoliated cells from high-grade CIN (CIN2-3) and ICC were significantly lower than those from CIN1and normal cervix.3. Four significantly lower expressed miRNAs (miR-424, miR-375, miR-34a, and miR-218) might be potential biomarkers for identification of high grade cervical intraepithelial neoplasia. PART Ⅱ miRNA detection versus cytology test as triages for Human Papillomavirus positive womenObjectivesTo compare the performance of cervical exfoliated cells miRNA detection and liquid-based cytology as triages for HPV-positive women, and evaluate the role of miRNA detection as a triage for HPV-positive women.MethodsThe inclusion criteria for the participants were:1) age:30-65years old;2) HPV positive;3) intact cervix;4) no previously confirmed CIN, cervical cancer or other malignancies;5) not pregnant. All eligible HPV-positive women underwent cytology and miRNA detection in cervical exfoliated cells, and further colposcopy examination. Those with normal cytology underwent colposcopy alone if no suspicious lesions were detected colposcopically, otherwise underwent biopsy further. For those with abnormal cytology (≥ASCUS), they underwent colpscopically directed multiple biopsies. Hybrid Capture2, liquid-based cytology, stem-loop reverse transcription and real time quantitative polymerase chain reaction (RT-qPCR) were applied for HPV testing, cytology test, and miRNA detection, respectively. miRNA relative expression level of each miRNA was compared between CIN1-and CIN2+, CIN2-and CIN3+. The receiver operating characteristic (ROC) curve was plotted, and the miRNA expression cut-off values were determined by the maxium Yonden index. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of miRNA detection were calculated for high-grade CIN identification. SPSS17.0software was used for statistic analysis. Non-parametric Mann-Whitney U test was used for miRNA level comparison. Pearson Chi-square test was used for proportions comparison. Logistic regression analysis was used to obtain multi-miRNA panels.Results 1. Total1021eligible women were recruited, and they were finally diagnosed as normal (n=579), CIN1(n=83), CIN2(n=144), CIN3(n=208),and ICC (n=7).2. There is no significant difference in age distributions between CIN1-and CIN2+, CIN2-and CIN3+groups (p>0.05). The relative expression levels of miR-424, miR-375, miR-34a, and miR-218in CIN2+and CIN3+groups were significantly lower than those in CIN1-and CIN2-groups respectively (p<0.001). The relative expression levels of miR-92a and miR-93were not significantly different between these groups (p>0.05).3. As triages for HPV-positive women to identify CIN2+, and compared with cytology:both miR-424and miR-375detections presented significantly higher sensitivity (76.4%and74.9%vs.63.8%; p<0.05), PPV (65.3%and66.3%vs.58.4%; p<0.05), NPV (85.7%and85.4%vs.79.3%, p<0.05), and comparable specificity. As triages for HPV-positive women to identify CIN3+, and compared with cytology:both miR-424and miR-375detections presented significantly higher sensitivity (82.3%and80.9%vs.69.8%, p<0.05), NPV (93.8%and93.4%vs.89.7%, p<0.05), and comparable specificity, PPV.4. Two multi-marker panels were identified by logistic regression ananlysis, miR-424/375/218and miR-424/375for CIN2+and CIN3+identification respectively. Both panels achieved significant higher sensitivity, specificity, PPV, and NPV than cytology. Compared with single miR-424or miR375detection, both panels achieved significant higher specificity, and comparable sensitivity, PPV, and NPV.Conclusions:1. To identify high-grade CIN in HPV-positive women, and compared with cytology, both miR-424and miR-375detections presented significantly higher sensitivity and NPV, without the cost of decreased specificity and PPV.2. Both multi-marker panels, miR-424/375/218and miR-424/375further improved the performances over single miR-424or miR-375detection, or cytology.3. miRNA detection in cervical exfoliated cells may be a triage for HPV positive women, and superior to cytology.