Abnormal methylation characteristics predict chemoresistance and poor prognosis in advanced high-grade serous ovarian cancer

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 specific methylation characteristics could distinguish chemoresistant and chemosensitive ovarian cancer patients.

Methods: In this study, we used 450 K Infinium Methylation BeadChip to detect the different methylation CpGs between ovarian cancer patients. The differential methylation genes were analyzed by GO and KEGG Pathway bioinformatics analysis. The candidate CpGs were confirmed by pyrosequencing. The expression of abnormal methylation gene was identified by QRT-PCR and IHC. ROC analysis confirmed 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, specificity and AUC of 8 CpGs (ITGB6:cg21105318, cg07896068, cg18437633; NCALD: cg27637873, cg26782361, cg16265707; LAMA3: cg20937934, cg13270625) methylation to predict chemotherapy 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 significantly shorter. The expression of NCALD and LAMA3 in chemoresistant patients was lower than that of chemosensitive 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 stratification of demethylation therapy.

Keywords: 450 K Infinium Methylation BeadChip; Chemotherapy resistance; DNA methylation; Ovarian cancer; Prognosis.

Fig. 1

Genome-wide DNA methylation in epithelial ovarian cancer: a heat map of differential methylated genes. b The biological process of differential methylation genes (GO). c The signal pathways of differential methylation genes (KEGG)

Fig. 2

ROC curve of candidate CpGs to predict chemotherapy sensitivity in advanced HGSOC

Fig. 3

CpGs hypermethylation associated with poor PFS in advanced HGSOC (a ITGB6/cg21105318, b ITGB6/cg07896068, c NCALD/cg27637873, d NCALD/cg26782361, e NCALD/cg16265707 and f LAMA3/cg20937934)

Fig. 4

CpGs hypermethylation associated with poor PFS in advanced HGSOC with complete debulking (a ITGB6/cg07896068, b NCALD/cg27637873 and c NCALD/ cg16265707)

Fig. 5

a The methylation rate of CpGs in advanced HGSOC (pyrosequencing). b Expressions of hypermethylated genes (ITGB6, NCALD and LAMA3) in advanced HGSOC (QRT-PCR). c Immunohistochemical stain (400 ×)

Fig. 6

The relationship between NCALD and LAMA3 methylation and mRNA expression in advanced HGSOC (a cg27637873 methylation and NCALD expression. b cg26782361 methylation and NCALD expression. c cg16265707 methylation and NCALD expression. d cg20937934 methylation and LAMA3 expression. e cg13270625 methylation and LAMA3 expression)