Research Paper Volume 12, Issue 20 pp 20561—20586

Colorectal cancer patients with CASK promotor heterogeneous and homogeneous methylation display different prognosis

Ying Liu1, , Hao Huang1, , Jinming Fu1, , Yuanyuan Zhang1, , Jing Xu1, , Lei Zhang1, , Simin Sun1, , Liyuan Zhao1, , Ding Zhang1, , Justina Ucheojor Onwuka1, , Hongru Sun1, , Binbin Cui2, , Yashuang Zhao1, ,

  • 1 Department of Epidemiology, Public Health College, Harbin Medical University, Nangang District, Harbin 150086, Heilongjiang Province, The People’s Republic of China
  • 2 Department of Colorectal Surgery, The Affiliated Tumor Hospital of Harbin Medical University, Harbin 150086, Heilongjiang Province, The People’s Republic of China

Received: April 30, 2020       Accepted: July 30, 2020       Published: October 28, 2020
How to Cite

Copyright: © 2020 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Homogenous DNA methylation clearly affects clinical outcomes. However, less is known about the effects of heterogeneous methylation. We aimed to investigate the different effects between CASK promoter methylation heterogeneity and homogeneity on colorectal cancer (CRC) patients' prognosis. The methylation status of CASK in 296 tumor tissues and 255 adjacent normal tissues were evaluated using Methylation-sensitive high-resolution melting (MS-HRM). Digital MS-HRM (dMS-HRM) visualized heterogeneous methylation and subsequent sequencing provided exact patterns. Log-rank test and Cox regression model were adopted to assess the association between CASK methylation status and CRC prognosis with propensity score (PS) method to control confounding biases. Heterogeneous methylation was detected in both tumor (52.2%) and non-neoplastic tissue surrounding the tumor (62.4%). It occurred more frequently in lower levels of tumor invasion (P = 0.002) and male patients (P < 0.001). Compared with heterogeneous methylation, patients with CASK homogeneous methylation presented poorer overall survival (OS) (HR: 1.919, 95% CI: 1.146-3.212, P = 0.013) and disease-free survival (DFS) (HR: 1.913, 95% CI: 1.146-3.194, P = 0.013). This unfavorable effect still existed among older (≥ 50), Dukes staging C/D, and rectal cancer patients. MS-HRM and dMS-HRM when combined can assess the degree and complexity of heterogeneous methylation with a visible pattern.


CA19-9: Carbohydrate antigen 19-9; CASK: Calcium/calmodulin-dependent serine protein kinase; CEA: Carcinoembryonic antigen; CRC: Colorectal cancer; ddPCR: Droplet digital PCR; DFS: Disease-free survival; dMS-HRM: Digital methylation-sensitive high-resolution melting; Hem 1-1: Early melting heterogeneous methylation 1; Hem 1-2: Early melting heterogeneous methylation 2; Hem 2-1: Cross peak heterogeneous methylation 1; Hem 2-2: Cross peak heterogeneous methylation 2; HM450K: Infinium HumanMethylation450 beadchip array; Hom: Homogeneous methylation; HR: Hazard ratio; MAGUK: Membrane-associated guanylate kinase; MS-HRM: Methylation-sensitive high-resolution melting; NTC: Non-template control; OS: Overall survival; PS: Propensity score; SD: Standard deviation; TCGA: The Cancer Genome Atlas; Thm: Total heterogeneous methylation; Tm: Melting temperature; Tpm: Total positive methylation; TTT: time to treatment; Unm: Unmethylation; WGA: Whole genome amplified.