Utilization Of DNA Hypermethylation In Primary Screening Of Cervical Cancer

BackgroundCervical cancer is the fourth most common cancer and the fourth most common cause of cancer death in women worldwide. It is the second most prevalent cancer in woman reproductive system. According to the cancer epidemiological data released by the International Cancer Research Institute (IRAC) of World Health Organization in 2012, cervical cancer is with an estimated 528,000 new cases and 266,000 deaths annually. The incidence among countries varies quite greatly; 70% occur in developing countries and China alone is expected to account for 11.75% of all new cancers and 11.1% of the cancer deaths. The National Central Cancer Registry of China released the population-based data to estimate the new cases of cervical cancer and cancer deaths for 2015 were 98,900 and 30,500, respectively. In addition, significant upward trends of incidience and mortility were observed for cervical cancer in China, which is totally different from that of the developed countries. By 2030, cervical cancer is expected to be responsible for the death of 474,000 women annually, with more than 95% of these deaths are anticipated to occur in low-and middle-income countries. IRAC points out that the reason for such great difference is that those countries lack of effective screening strategies and early treatment. Therefore, establishing a proper screening strategy to find cervical preneoplasm and neoplasm is of great importance in China.The most widely used screening methods for cervical cancer are the cytology-based Pap smear and high-risk human papillomavirus (hrHPV) testing. However, both methods have drawbacks. The drawbacks of cytology test lie in that first of all, the sensitivity is so low that one study covering more than 60,000 women, cytology-based Pap smear screening did not detect almost half of the cases of cervical intraepithelial neoplasia 2 (CIN2), CIN3 and cancer (CIN2+). Moreover, cytology testing is based on morphological changes and too subjective to call for a great number of professional cytologists. HrHPV testing of cervical scrapings can improve the sensitivity of cervical screening; however, the lifetime risk of hrHPV infection amounts to be as high as 80% and the screening hrHPV test cannot discriminate between persistent infections and transient infections. Such a less specific screening test may lead to a substantially heavy burden on health care resources, such as unnecessary referrals to colposcopy. To avoid missed diagnoses and over-diagnoses, other triage and/or complementary biomarkers that are molecularly based and not morphology based are urgently needed.DNA methylation is the methylation of unmethylated cytosine phosphate guanine (Cytosine-phosphate-Guanosine, CpG). Methylation is an important epigenetic modification mechanism; the characteristic of tumor is the coexistence of ubiquitous genomic hypomethylation and regional hypermethylation. Loss of heterozygosity (LOH), gene mutation and/or hypermethylation cause the loss of functions in tumor suppressor gene. Thereafter, uncontrolled proliferation of malignant tumor cells promotes the development of tumor. DNA methylation is one of the key mechanisms of tumor suppressor gene inactivation and inhibition of gene transcription’. In some cases, it may be the only mechanism. DNA methylation is involved in the early phase of cervical carcinogenesis. Epigenetic changes occur during each stage of cervical cancer and various genes are silenced by promoter methylation at distinct stages in the transformation process. The accumulation of epigenetic alterations in the host genome promotes the progression to invasive cervical cancer. Therefore, as an early event in cancer, detection of aberrant DNA methylation makes molecular diagnosis of cervical preneoplasm and neoplasm possible, finding out people who are in need of immediate clinical interventions, which is helpful for early screening, early diagnosis and early treatment of cervical cancer.Studies on the hypermethylation of tumor suppressor gene have made exciting progress in the primary screening of various tumors. Hypermethylation of tumor suppressor gene also showed a promising prospect of clinical application in the primary screening of cervical cancer. However, there are still some serious problems listed as follows:firstly, the unsatisfactory diagnostic efficacy shows that significant drop of sensitivity compared to hrHPV and poor stability presented by strong heterogeneity, methylation frequencies vary widely among studies even for the same gene; what is more, the majority of researches are without in-depth analysis of the feasibility of various clinical utilizations; the sample size of most researches is not large enough to be representative.Based on it, there are two parts in this paper:Part I Selection and Validation of Methylation Biomarkers for Primary Screening of Cervical CancerBackground and PurposeCervical cancer is one leading cause of cancer deaths in women all over the world, especially in the developing countries, such as China. As the early event of carcinogenesis, DNA methylation is stage-specific and accumulative in the transforming stages during the development of cancer. To select methylation biomarkers specific for high grade lesions of cervix, the relationship between methylation level of various loci of some genes and the severity of lesions was evaluated.Materials and Methods1. Based on the review of papers and data from The Cancer Genome Atlas (TCGA), some hypermethylated genes which are specific for cervical cancer were selected and different loci of one gene were designed for detection.2. Methylation-sensitive PCR (MSP) was performed on normal cervical (n=27) and cervical cancer tissue (n=43) to select a few loci with significant difference between the two groups.3. Quantitative MSP (QMSP) of cervical scrapings as screening biomarkers(1) In the train set, QMSP was performed on cervical scrapings (n=267) to further select and validate the methylation biomarkers selected by MSP.(2) In the test set, QMSP was performed on cervical scrapings (n=224) to further validate the selected methylation biomarkers and compare the consistency between the train set and test set.4. Validation of the methylation status of selected methylation biomarker by pyrosequencing.Results1. Genes CCNA1、CADM1、DAPK1、JAM3 were selected and 27 loci of those 4 genes were to be detected after the specificity of those primers was validated.2. The top 5 gene loci (CADM1-M2, CADM1-M8, DAPK1-M2, DAPK1-M3, and JAM3-M4) with significant difference between the two groups were selected from the initial 27 markers. The positive rate of methylation in those 5 loci of cervical cancer tissue was 65.1%(28/43),74.4%(32/43),69.8%(30/43),76.7%(33/43) and 86.0%(37/43), respectively. The positive rate of methylation in those 5 loci of normal cervical tissue was 22.2%(6/27),0.0%(0/27),11.1%(3/27),7.4%(2/27) and 7.4%(2/27), respectively. The difference of those five loci between two groups were all significant (P<0.001).3. To further investigate the data, we used different classifications for diagnostic groups. CIN2+/CIN1-and CIN3+/CIN2-were the classifications when the end point was CIN2 and CIN3, respectively. Because of the controversy with CIN2 itself, we evaluated the classification CIN3+/CIN1-.QMSP of cervical scrapings as screening biomarkers in the train set(1) The relationship between methylation level of various loci of some genes and the severity of lesions:①Except for DAPK1-M3, the differences between CIN3+ and CIN1-were all significant for the other four biomarkers.② The differences between CIN2 and CIN1-were significant for DAPK1-M1, DAPK1-M3、JAM3-M4. ③ The differences between every two groups of CIN3+、CIN2 and CIN1-were significant for JAM3-M4.(2) A logistic regression model was used to explore the predictive power of methylation of the 5 loci with different diagnostic classifications: ① For the CIN3+/CIN1-and CIN3+/CIN2-classifications, JAM3-M4 had adequate predictive power. Other genes did not add substantial information for discrimination. ② For the CIN2+/CIN1-classification, CADM1-M8 (P=0.001), DAPK1-M3 (P=0.038) and JAM3-M4 (P=0.011) showed the best discriminating power. However, the area under the receiver operating characteristic curve (AUC) for the 3 genes combined (AUC=0.806) was only slightly higher than that with JAM3-M4 alone (AUC=0.793). Therefore, in the following analysis, we evaluated JAM3-M4 alone with the diagnostic groups.QMSP of cervical scrapings as screening biomarkers in the test set:The AUC for JAM3-M4 for CIN3+/CIN1-, CIN3+/CIN2-and CIN2+/CIN1-classifications in the train set was 0.907,0.860 and 0.793, respectively. The AUC for JAM3-M4 was reproducible in the two sets, with similar AUC of 0.900,0.870 and 0.765 for CIN3+/CIN1-, CIN3+/CIN2-and CIN2+/CIN1-classifications in the test set.4. Pyrosequencing validation of the methylation status of JAM3-M4 indicated that it was significantly discriminative among relevant diagnostic groups of CIN1-, CIN2 and CIN3+.ConclusionsIn general, the methylation ratio increased with increasing lesion severity. Particularly, JAM3-M4 was the most discriminative marker. It was significantly different between every two classifications of CIN1-, CIN2 and CIN3+, which indicated that JAM3-M4 was promising to detect the high grade lesions of cervix and could be used as a screening biomarker.Part II Utilization of Methylation Biomarker JAM3-M4 in the Primary Screening of Cervical CancerBackground and PurposeThe etiology of cervical cancer is relatively clear. It is the only malignancy that WHO recommend worldwide screening. It lasts almost 10 to 20 years from HPV infection progresses to cervical cancer. Thus, early detection and early treatment of cervical cancer is available. The most widely used screening strategies in clinical practice are the cytology-based Pap smear and hrHPV testing. Taken from the data of Part Ⅰ, we could conclude that JAM3-M4 is of promising utilization in the primary screening of cervical cancer. Based on this, compared to the cytology and hrHPV testing, its diagnostic efficacy and triage performance of CIN2 was analyzed.Materials and Methods1. Clinicopathological data and available hrHPV and cytology results for cervical scrapings were collected. The diagnostic efficacy of JAM3-M4 was compared to that of cytology and hrHPV testing.2. In the train set, the diagnostic efficacy (sensitivity, specificity, positive predictive value, negative predictive value) of JAM3-M4 and clinical practice were combined to evaluate and select the application schemes in primary screening for JAM3-M4. The schemes were further validated in the test set.3. Immunohistochemistry staining of P16 was performed on sections from patients histologically confirmed to be CIN2. The coincidence between P16 staining and JAM3-M4 was analyzed to evaluated the feasibility of JAM3-M4 to triage CIN2 patients into high-grade squamous intraepithelial lesion (HSIL) and LSIL.Results1. Compared to cytology and hrHPV testing, the specificity and PPV of JAM3-M4 was increased significantly for all classification groups (CIN3+/CIN1-, CIN3+/CIN2-and CIN2+/CIN1-).2. The most promising utilization of JAM3-M4 in clinical practice(1) Triage performance ①With JAM3-M4 used as a triage marker for hrHPV-positive patients, JAM3-M4 increased specificity, PPV, and NPV as compared with triage performance of cytology testing for all classification groups.② Compared the triage performance of JAM3-M4 to that of hrHPV testing, the specificity and PPV of JAM3-M4 was increased significantly for all classification groups in both ASCUS and LSIL subgroups.③ In the train set and test set, the triage performance of JAM3-M4 was with good consistency.④ Patients with ASCUS who were<30 years old (n=19) were all hrHPV-positive; 15 had CIN1-, all negative for the JAM3-M4 biomarker.(2) Complementary performance ① With JAM3-M4 used as a complementary biomarker in cytology testing, the specificity and PPV was increased greatly as compared with the combination of cytology and hrHPV testing. ② In the train set and test set, the complementary performance of JAM3-M4 was with good consistency.3. The rate of positive staining for P16 was 69.9% and the rate of positive methylation for JAM3-M4 was 48.8%. The coincidence rate was 60.5%.ConclusionsCompared to the widely used screening methods, JAM3-M4 improved the diagnostic efficacy significantly. Based on the ROC analysis and clinical practice, JAM3-M4 was most promising as the triage biomarker of hrHPV-positive patients and those patients whose cytology testing were with LSIL, and with ASCUS, especially those who are<30 years old. Meantime, it could be used as a complementary biomarker to cytology. In addition, JAM3-M4 was helpful for differentiating CIN2 patients into LSIL and HSIL.