Study Of FAM19A4 Methylation In Diagnosis Of Cercical Cancer And Its Precancerous Lesions

IntroductionCervical cancer is the second-largest female malignancy,with the mortality rates ranking the third,and nearly 90% of cervical cancer deaths occur in developing countries.Although the prevalence of cervical cancer screening and the use of Human Papillomavirus(HPV)vaccines have made cervical cancer almost a preventable malignancy,there is still a high risk of missed diagnosis of HPV and cytology screening,leading to a lack of effective early diagnosis of cervical cancer.Therefore,it is of great significance to find specific molecular markers that can effectively identify cervical cancer and its precancerous lesions,which can make up for the current screening methods for cervical cancer.Studies have shown that DNA methylation is an early event of tumorigenesis,which is closely related to the occurrence and development of cervical cancer.DNA methylation can be used as a specific molecular biomarker for the early diagnosis of cervical cancer,increasing the detection rates of cervical lesions.FAM19A4(family with sequence similarity 19(chemokine(C–C motif)-like)member A4),a member of the TAFA gene family,has been found that the promoter methylation of this gene can effectively identify cervical cancer and its precancerous lesions,which is expected to become a novel tumor biomarker.However,there are no related studies on the relationship between FAM19A4 methylation with cervical lesions in Chinese women,and about the association between FAM19A4 methylation and clinicopathological features of cervical cancer as well as HPV genotyping.In order to explore the diagnostic value of FAM19A4 methylation in cervical disease of Chinese women,a taqman probe-based quantitative PCR(qPCR)method based on previous studies was used in this study to detect FAM19A4 methylation in different severity of cervical lesions,using samples of cervical formalin-fixed and parrffin-embedded(FFPE)and cervical scrapes.The differences of FAM19A4 methylation in different clinicopathological characteristics of cervical cancer and HPV genotyping were also analyzed,to assess the diagnostic value of FAM19A4 methylation in cervical cancer and its precancerous lesions.Chapter 1: Clinical significance of FAM19A4 methylation in FFPE tissue of cervical cancer and its precancerous lesionsObjectiveTo detect the expression in cervical cancer,cervical precancer and healthy cervix of FFPE samples,and explore the clinical significance of FAM19A4 methylation Methods31 cervical cancers,22 HSILs(CIN?)and 23 healthy cervical tissues of FFPE specimen diagnosed by pathology were randomly selected.qPCR was used to detect FAM19A4 methylation in different cervical lesions,and estimate the clinical value of this gene in the diagnosis of cervical cancer.Result1.Statistical differences of FAM19A4 methylation detection rates were observed in the specimen of healthy cervix/cervicitis,HSIL and cervical cancer,respectively 8.70%(2/23),72.73%(16/22)and 96.77%(30/31)(P<0.05).The detection rates of FAM19A4 methylation increased with severity of cervical lesion(Cochran-Armitage trend test,P<0.05).2.Pairwise comparison showed that the methylation rates of FAM19A4 in cervical cancer were senior to HSIL(96.77% vs 72.73%,P<0.05);and the methylation rates of FAM19A4 in cervical cancer were senior to healthy cervix(96.77% vs 8.70%,P<0.05);and the methylation rates of FAM19A4 in HSIL were senior to healthy cervix(72.72% vs 8.70%,P<0.05).3.The detection rates of FAM19A4 methylation were not statistically different in different age groups,pathological types,clinical stages,tumor sizes and lymph node metastasis of cervical cancer(P>0.05).ConclusionFAM19A4 is probably a specific biomarker of cervical cancer.With the progress of healthy cervix to cervical precancer finally to invasive carcinoma,FAM19A4 methylation rates gradually increase,suggesting that FAM19A4 may play a role in the development and progress of cervical cancer.Chapter 2: Clinical significance of FAM19A4 methylation in high-risk HPV-positive cervical scrapes samples for the detection of cervical cancer and its precancerous lesions ObjectiveTo investigate the differences of FAM19A4 methylation in high-risk types of human papillomavirus(hr-HPV)positive cervical scrapes samples with different progression of cervical lesions,and explore the differences of FAM19A4 methylation rates in different clinicopathological features of cervical cancer and different genotyping,and compare them with cytology and HPV16/18 genotyping in the diagnostic value of cervical cancer and its precancerous lesions.MethodsA total of 264 cases of hr-HPV-positive(including HP16,18,31,33,35,39,45,51,52,56,58,59,66 and 68)cervical scrapes were selected,they were all diagnosed by histopathology,including 72 patients with cervical cancer,70 with HSIL,45 with LSIL and 77 with healthy cervix/ cervicitis.The technology of qPCR was used to detect the differences FAM19A4 methylation in this five groups of cervical scrapes,and SPSS 23.0 software was used for statistical analysis.The differences of FAM19A4 methylation rates in different clinicopathological features of cervical cancer and different HPV genotyping were also examined.Then compared the three methods of HPV16/18 genotyping,cytology and FAM19A4 methylation in sensitivity,specificity,positive predictive value(PPV),negative predictive value(NPV),and Youden index(YI)for detecting HSIL or the above lesions(?HSIL/ HSIL+),to evaluate the clinical value of FAM19A4 methylation in early diagnosis of cervical cancer.Result1.The detection rates of hr-HPV positive samples of FAM19A4 methylation in different severity cervical lesions were statistically significant(P<0.05),respectively 9.09%(7/77)in healthy cervix/ cervicitis,24.44%(11/45)in LSIL,58.57%(41/70)in HSIL and 94.44%(68/72)in cervical cancer.The detection rates of FAM19A4 methylation gradually increased with severity of cervical lesions(Cochran-Armitage trend test,P<0.05).2.Pairwise comparison showed that there were statistically significant differences of FAM19A4 methylation rates among any other groups(P<0.05).3.Compared with healthy cervix/ cervicitis,the odds ratio(OR)of FAM19A4 methylation detection rates in cervical cancer,HSIL and LSIL were 170.00(95% CI: 47.60-607.16),14.138(95%CI:5.685-35.156)and3.24(95% CI: 1.15-9.08)respectively,with statistically significant differences.4.There were no significant differences of FAM19A4 methylation in hr-HPV positive scrapes samples between different age groups,pathological types,clinical stages,tumor sizes and lymph node metastasis(P>0.05).5.There was a correlation between FAM19A4 methylation and HPV genotyping(P<0.05).The detection rates of FAM19A4 methylation in group of HPV16/18 were higher than non-HPV16/18 group(66.15% vs 30.60%,P<0.05).When cancerous cases were excluded,the detection rates of FAM19A4 methylation in group of HPV16/18 were also higher than non-HPV16/18 group(43.84% vs 22.69%,P<0.05).6.The sensitivity of FAM19A4 methylation in detecting ?HSIL lesions was slightly lower than that of HPV16/18 genotyping combined with cytology(85.84% vs 93.81%),but the specificity,PPV,NPV and YI were all higher than the combined tests.Compared with the FAM19A4 methylation test alone,the sensitivity and NPV were slightly higher than FAM19A4 methylation combined with HPV 16/18 genotyping in detecting ?HSIL,but the specificity,PPV,and YI all significantly decrease.Conclusion1.FAM19A4 in hr-HPV scrapes samples may be a specific biomarker for cervical cancer.With the progression of cervical lesions,the detection rates of FAM19A4 methylation is gradually increased,indicating that FAM19A4 methylation is closely related to the occurrence and development of cervical cancer.2.Compared with traditional screening methods(HPV genotyping and cytological tests)for cervical cancer,FAM19A4 methylation has great advantages in the diagnosing ?HSIL,which can improve the detection rates of cervical cancer and its precancerous lesions,and reduce the false positive rates.