Hypermethylated PCDHGB7 as a universal cancer only marker and its application in early cervical cancer screening

医学 生物标志物 DNA甲基化 甲基化 癌症 宫颈癌 肿瘤科 癌症研究 生物 基因 内科学 遗传学 基因表达
作者
Shihua Dong,Qi Lu,Peng Xu,Li‐Mei Chen,Xiaoling Duan,Zhanrui Mao,Baolong Zhang,Long Sui,Yudong Wang,Wenqiang Yu
出处
期刊:Clinical and translational medicine [Springer Science+Business Media]
卷期号:11 (6) 被引量:14
标识
DOI:10.1002/ctm2.457
摘要

Dear Editor, We identified hypermethylated PCDHGB7 as a novel cancer marker and applied it to early cervical cancer (CC) screening. It outperforms the widely implemented high-risk human papillomavirus (hrHPV) test and ThinPrep cytologic test (TCT) and even can be used in the self-sampled vaginal secretions, proving itself as a much more convenient yet highly effective screening method. DNA methylation aberration occurs during cancer progression. DNA methylation has emerged as a promising diagnostic, prognostic, and predictive biomarker of various types of cancer.1 However, the common biomarker of cancers has been rarely explored. Previously, we provided the concept of Universal Cancer Only Marker (UCOM) and identified hypermethylated HIST1H4F as the first UCOM marker.2 In our genome-wide methylation analysis, we found PCDH family genes were cancer cell-differentially methylated genes (CC-DMG).2 In the current study, we focused on PCDHGB7, a member of the protocadherin gamma gene cluster, which plays critical roles in the establishment and function of specific neuronal connections,3 and investigated whether it could be a novel UCOM marker. As CC is one of the most common female malignancies4 and the widely implemented hrHPV and TCT yield a high false-positive rate,5, 6 we aimed to applied PCDHGB7 in the early CC screening. We compared the methylation status of PCDHGB7 in 17 cancer types with their corresponding normal tissues in TCGA and GEO database (n = 7114). It turned out PCDHGB7 was hypermethylated in all cancer types (Figure 1A). When analyzing FIGO staging, we found that PCDHGB7 was already hypermethylated in stage I of all cancer types analyzed (Figure S1), suggesting hypermethylated PCDHGB7 could be an early-stage cancer indicator. Additionally, in different histological types, keratinizing squamous cell carcinoma, lymphovascular invasion, or histologic grades, there was no methylation difference of PCDHGB7 (Figure S2). To verify these analytical results, we collected 13 types of clinical cancer samples (n = 727), in which PCDHGB7 was hypermethylated accordingly (Figure 1B). Hypermethylation may account for the downregulated expression of PCDHGB7 (Figure S3) and the lower frequency of CTCF peaks located on PCDHGB7 promoter (Figure S4). Additionally, we assessed the performance of PCDHGB7 hypermethylation as a biomarker for distinguishing between cancer and normal samples. The area under the curve (AUC) values were obtained for distinguishing 13 types of clinical cancer and control tissues with pyrosequencing data (Figure 1C and Table S1). It showed that all the AUC was larger than 0.85 (Table S1), especially in biliary cancer (AUC = 0.98) and esophagus cancer (AUC = 0.99). These results highly suggested that hypermethylated PCDHGB7 can serve as a novel UCOM marker and play vital roles in CC progression. The management strategies for high- and low-grade squamous intraepithelial lesion (HSIL, LSIL) are distinct; hence, there is an urgent demand for distinguishing HSIL from LSIL. We found the methylation level of PCDHGB7 in HSIL or CC (defined as “≥HSIL”) was significantly higher than that in LSIL and normal samples (defined as “≤LSIL”) (Figure 2A), implying it could act as a stage divider to classify ≥HSIL from ≤LSIL stage and an early cervical precancerous lesion biomarker. To avoid bisulfite treatment in bisulfite-PCR pyrosequencing, we modified methylation-sensitive restriction enzyme combined real-time fluorescent quantitative PCR (MSRE-qPCR) to quantify methylation status. In samples with lower methylation levels (10%–20%), the value of ΔCt dropped dramatically (Figure 2B), indicating MSRE-qPCR was superior for early cancer screening since less cancerous DNA existed alongside relatively lower methylation level. In 404 cervical smears, ΔCt for quantified PCDHGB7 methylation was significantly lower in ≥HSIL compared with that in ≤LSIL (Figure 2C). Furthermore, the ROC curve showed that MSRE-qPCR quantification of PCDHGB7 methylation could be used for classifying CC and distinguishing HSIL from ≤LSIL samples. The AUC was 0.97 for CC, 0.87 for HSIL, and 0.88 for ≥HSIL (Figure 2D). With the methylation cutoff ΔCt = 4.0 when the Youden index is maximized (ΔCt ≤ 4.0 indicates ≥HSIL; ΔCt > 4.0 indicates ≤ LSIL), the specificity was 94.3%, and the sensitivity was 96.0% for CC (Figure 2E). Next, we comprehensively evaluated the performances of PCDHGB7 hypermethylation, hrHPV test, and TCT in CC screening (Table 1). For CC, the sensitivity of PCDHGB7 and hrHPV was similar (96% vs. 95.7%), while the specificity was improved dramatically (94.3% vs. 20.3%). It was also the case in HSIL. As for TCT, its specificity (51.2%) is much lower than that of PCDHGB7 in CC and HSIL samples. Furthermore, we evaluated the combined effect of PCDHGB7 hypermethylation, hrHPV test, and TCT. For screening clinical samples with ≥HSIL, if we define “positive” as both positive diagnosis for CC, PCDHGB7 combined with either hrHPV or TCT increased the specificity to 95.7% and 96.2%, which is higher than either of hrHPV (20.3%) or TCT (51.2%), or the combination of hrHPV and TCT (57.8%). However, the sensitivity of PCDHGB7 decreased due to these combinations. Similar results were found in three-method combinations. These results demonstrated that hypermethylated PCDHGB7 by itself is an ideal alternative tool for CC screening, and there is no need for combining it with either hrPHV test or TCT. Additionally, the robustness of PCDHGB7 hypermethylation was also testified in the validation set, yielding 82.1% sensitivity and 88.7% specificity for ≥HSIL (Figure 2F); while the sensitivity could reach 100% with 88.7% specificity for identifying CC (Figure 2G). Despite vaginal secretion being much easier to collect than cervical smears, its capacity in CC screening has long been ignored. In 273 vaginal secretions, we found the methylation level of PCDHGB7 represented by the lowering ΔCt of MSRE-qPCR was significantly higher in ≥HSIL than in ≤LSIL (Figure 3A). When used for distinguishing patients with CC or HSIL, the AUC were 0.92 and 0.71, respectively (Figure 3B); with 90.4% specificity and 90.9% sensitivity for identifying CC (Figure 3C), these results demonstrated that vaginal secretion is an encouraging sample type for early CC screening by applying PCDHGB7 methylation detection. Collectively, hypermethylated PCDHGB7 is identified as a novel UCOM marker and an ideal biomarker for distinguishing HSIL from LSIL. The introduction of PCDHGB7 makes vaginal secretions feasible for CC screening, which will allow testing to be more easily applied and adopted. We thank Yue Yu for editorial help and comments on the manuscript. We thank Jiangjing Yuan at International Peace Maternity and Child Health Hospital for assistance in collecting clinical samples. This work was supported by the National Key R&D Program of China (Grant No. 2018YFC1005004), the Science and Technology Innovation Action Plan of Shanghai (Grant No. 17411950900), the National Natural Science Foundation of China (Grant No. 31671308, 31872814, 81172477, 81272295, 81402135, 81701398), Major Special Projects of Basic Research of Shanghai Science and Technology Commission (Grant No. 18JC1411101, 18JC1411104), Science and Technology Commission of Shanghai Municipality (Grant No. 12ZR1402200, 17441907400, 18411963600), the Ministry of Education of the People's Republic of China (Grant No. 2009CB825600), and the Innovation Group Project of Shanghai Municipal Health Commission (Grant No. 2019CXJQ03), Shanghai Municipal Key Clinical Specialty (Grant No. shslczdzk06302), and Shanghai Jiao Tong University Medicine-Engineering Fund (Grant No. YG2017MS41). Wenqiang Yu and Shihua Dong report having a pending patent application. The other authors disclosed no potential conflicts of interest. Samples were collected from Xijing Hospital of Air Force Military Medical University, Jinshan Hospital of Fudan University, and International Peace Maternity and Child Health Hospital. Written informed consent was provided to all patients before sample collection. Institutional Review Board approval for research on human subjects was obtained from hospitals. D. S. H., Y. W. Q., and L. Q. designed and initiated the project. D. S. H. and Y. W. Q. supervised the project. D. S. H., X. P., L. Q., C. L. M., D. X. L., M. Z. R., Z. B. L., Y. W. Q., and S. L. generated the data, acquired and managed patients, and provided facilities. D. S. H., X. P., and M. Z. R. performed analysis and interpretation of data. X. P., D. S. H. and Y. W. Q. wrote the manuscript. X. P. and D. S. H. drew the graphical abstract. All the authors read and approved the final manuscript. The DNA methylation data are available from UCSC Xena browser (https://xenabrowser.net/), and the expression data are downloaded from TCGA Hub (https://tcga.xenahubs.net). CTCF ChIP-Seq data were downloaded from ENCODE database. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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