Feng et al. Clin Epigenet (2021) 13:141 https://doi.org/10.1186/s13148-021-01133-2 RESEARCH Open Access Abnormal methylation characteristics predict chemoresistance and poor prognosis in advanced high-grade serous ovarian cancer Li‑yuan Feng , Bing‑bing Yan, Yong‑zhi Huang and Li Li* Abstract Background: Primary or acquired chemoresistance is a key link in the high mortality rate of ovarian cancer. There is no reliable method to predict chemoresistance in ovarian cancer. We hypothesized that specifc methylation charac‑ teristics could distinguish chemoresistant and chemosensitive ovarian cancer patients. Methods: In this study, we used 450 K Infnium Methylation BeadChip to detect the diferent methylation CpGs between ovarian cancer patients. The diferential methylation genes were analyzed by GO and KEGG Pathway bioin‑ formatics analysis. The candidate CpGs were confrmed by pyrosequencing. The expression of abnormal methylation gene was identifed by QRT‑PCR and IHC. ROC analysis confrmed the ability to predict chemotherapy outcomes. Prognosis was evaluated using Kaplan–Meier. Results: In advanced high‑grade serous ovarian cancer, 8 CpGs (ITGB6:cg21105318, cg07896068, cg18437633; NCALD: cg27637873, cg26782361, cg16265707; LAMA3: cg20937934, cg13270625) remained hypermethylated in chemoresistant patients. The sensitivity, specifcity and AUC of 8 CpGs (ITGB6:cg21105318, cg07896068, cg18437633; NCALD: cg27637873, cg26782361, cg16265707; LAMA3: cg20937934, cg13270625) methylation to predict chemo‑ therapy sensitivity were 63.60–97.00%, 46.40–89.30% and 0.774–0.846. PFS of 6 candidate genes (ITGB6:cg21105318, cg07896068; NCALD: cg27637873, cg26782361, cg16265707; LAMA3: cg20937934) hypermethylation patients was signifcantly shorter. The expression of NCALD and LAMA3 in chemoresistant patients was lower than that of chemo‑ sensitive patients. Spearman analysis showed that NCALD and LAMA3 methylations were negatively correlated with their expression. Conclusions: As a new biomarker of chemotherapy sensitivity, hypermethylation of NCALD and LAMA3 is associated with poor PFS in advanced high‑grade serous ovarian cancer. In the future, further research on NCALD and LAMA3 will be needed to provide guidance for clinical stratifcation of demethylation therapy. Keywords: Ovarian cancer, Chemotherapy resistance, DNA methylation, 450 K Infnium Methylation BeadChip, Prognosis Background Ovarian cancer is the most lethal cancer of the female reproductive system. In 2019, 22,530 new cases and 13,980 death occurred in the USA [1]. For the frst diag- *Correspondence: [email protected] Department of Gynecology and Oncology, Guangxi Medical University nosis of ovarian cancer patients, the standard treatment Cancer Hospital and Key Laboratory of Early Prevention and Treatment is the optimal surgical cytoreduction combined with plat- for Regional High Frequency Tumor, Ministry of Education, 71 Hedi Road, inum-based chemotherapy [2]. In the past decade, the Nanning 530021, Guangxi, People’s Republic of China survival rate of ovarian cancer has changed only a little, © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Feng et al. Clin Epigenet (2021) 13:141 Page 2 of 18 and the 5-year survival rate has remained below 30% [3, pathological diagnosis. All patients provided written 4]. Primary or acquired chemoresistance is a key link informed consent and were approved by the institutional in the high mortality rate of ovarian cancer [5, 6]. If the review committee of Guangxi Medical University Cancer patient’s sensitivity to traditional chemotherapy can be Hospital. assessed before treatment, doctors can guide chemore- sistant patients to undergo clinical trials to seek survival opportunities. Terefore, it is necessary to use robust and Follow‑up sensitive biomarkers to predict chemotherapy outcome OS (overall survival) was defned as the time from the in ovarian cancer patients. diagnosis to the death from ovarian cancer, and the sur- At present, there is no reliable method to predict chem- vival data of the last follow-up survivors were recorded oresistance in ovarian cancer. DNA methylation as a bio- as censored data. PFS (progression-free survival) was marker has the following advantages: chemical stability, defned as the time from initial treatment to tumor pro- quantitative detection, chemoresistance-related methyla- gression. Response to treatment was evaluated using the tion changes usually occur before the start of chemore- Response Evaluation Criteria In Solid Tumors (RECIST sistance, noninvasive detection (can be detected in the 1.1 criteria) [19]. Platinum-resistant and refractory ovar- patient’s body fuids) [7]. With the update of DNA meth- ian cancer was defned as those whose disease had pro- ylation detection platform and technology, more and gressed during frst-line platinum-based chemotherapy more genes involved in ovarian cancer chemoresistance or relapsed within 6 months after the last platinum treat- have been reported. Except BRCA1, DNA damage repair ment [20]. Platinum-sensitive ovarian cancer was defned pathway-related genes, PTEN, RASSF1, MDR1 and as relapse more than 6 months after platinum-based FANCF gene hypermethylation were positively corre- chemotherapy [21]. lated with chemotherapy sensitivity. In recent years, new DNA methylation studies related to chemotherapy resist- ance of ovarian cancer include at least MLH1 [8], SER- Study design PINE1 [9], TRIB2 [10], KLF4 [11], FZD10 [12], ZNF671 In this study, we included two stages to identify and [13], ABCB1 [14], hMSH2 [15] and other genes. Tis validate chemoresistance-related CpGs in ovarian can- shows that the regulatory mechanism of DNA methyla- cer. In the discovery stage, 450 K Infnium Methylation tion in ovarian cancer chemotherapy resistance is com- BeadChip was used for screening, and enrichment anal- plex and diverse. A variety of methylated genes interact ysis was used to select biologically meaningful CpGs. with each other, which together leads to chemotherapy Te relationship between abnormal methylation and resistance in ovarian cancer. chemotherapy resistance and prognosis was analyzed. Relative to genetic mutations, DNA methylation can In the verifcation stage, candidate CpGs were verifed be reversed. Demethylation drugs can reverse abnormal by pyrosequencing. To clarify whether diferential CpGs methylation, improve the sensitivity of ovarian cancer play a biological function, we used QRT-PCR and immu- patients to chemotherapy drugs, improve efcacy and nohistochemistry to detect gene expression. prolong survival [16–18]. Currently, there are few results on genome-wide methylation in chemoresistant ovarian cancer patients. Here, we used 450 K Infnium Methyla- 450K Infnium Methylation BeadChip tion BeadChip to study the genome-wide methylation Qualifed DNA was extracted from 108 samples. DNA characteristics of chemotherapy resistance in ovarian was modifed by Epitect bisulfte kit (Qiagen, Cat. No. cancer. 59110, Germany) and analyzed by 450 K Infnium Meth- ylation BeadChip (Illumina, San Diego, CA, USA) in Materials and methods Shanghai Jingneng company. Te methylation level was Patients scored with standardized beta score values ranging from We collected the initial surgical samples of ovarian can- 0 (unmethylated) to 1 (fully methylated). Limma pack- cer patients (only carcinomas and not borderline tumors) age (R) calculates diferential methylation sites between from the Guangxi Medical University Cancer Hospital. chemotherapy-resistant and chemotherapy-sensitive A total of 108 frozen samples (epithelial ovarian cancer) patients (P ≤ 0.01, |Dif Beta Score| ≥ 0.1). Go (http:// were used for 450 K Infnium Methylation BeadChip, geneo ntolo gy. org/) and KEGG (http:// www. genome. pyrosequencing and QRT-PCR. 132 parafn samples jp/ kegg/) databases were used to enrich the diferential (advanced high-grade serous ovarian carcinoma) were methylation genes. Te candidate CpGs were selected used for immunohistochemistry. All patients had com- according to the Dif Beta Score value and biological plete chemotherapy outcome records and postoperative function. Feng et al. Clin Epigenet (2021) 13:141 Page 3 of 18 Literature search strategy low expression, while > 6 points were classifed as a high Gene search strategies were based on Yan’s article [22]. expression [23]. Used ‘ovarian cancer’ or ‘ovarian carcinoma,’ ‘DNA methylation’ or ‘methylation,’ ‘resistant’ or ‘resistance’ Statistical analysis or ‘chemoresistance’ as keywords, we screened methyl-