Skip to main content

Re-assessing ZNF331 as a DNA methylation biomarker for colorectal cancer

Abstract

We have previously shown that aberrant promoter methylation of ZNF331 is a potential biomarker for colorectal cancer detection with high sensitivity (71%) and specificity (98%). This finding was recently confirmed by others, and it was additionally suggested that promoter methylation of ZNF331 was an independent prognostic biomarker for colorectal cancer (n = 146). In the current study, our initial colorectal cancer sample series was extended to include a total of 423 cancer tissue samples. Aberrant promoter methylation was found in 71% of the samples, thus repeatedly suggesting the biomarker potential of ZNF331 for detection of colorectal cancer. Furthermore, multivariate Cox’s analysis indicated a trend towards inferior overall survival for colorectal cancer patients with aberrant methylation of ZNF331.

Introduction

In cancer, increased promoter DNA methylation is a frequent event commonly occurring early in tumor development. Methylated DNA sequences may serve as tumor biomarkers in liquid biopsies for detecting cancer and for predicting patient prognosis [1].

In 2011, we filed a patent application covering methylation of ZNF331 (Zinc finger protein 331) as a biomarker for gastrointestinal cancers [2]. ZNF331 was shown by Yu et al. to be inactivated by promoter methylation in gastric cancer, providing the cancer cells with increased growth potential and invasiveness [3]. We also found a high methylation frequency in patients with gastric cancer (80%) and to a lesser extent in patients with pancreatic cancer (40%) and cholangiocarcinomas (26%) [4]. Most importantly, we reported high sensitivity (71%) and specificity (98%) for ZNF331 methylation in colorectal cancer early 2015, strengthening the potential of ZNF331 as a biomarker for colorectal cancer detection [4]. Interestingly, these findings were recently confirmed, further supporting the biomarker potential of ZNF331 in colorectal cancer [5]. The same study also suggested aberrant promoter methylation of ZNF331 as an independent prognostic marker for colorectal cancer, analyzing 146 samples [5]. In the present study, we analyzed the effect of ZNF331 methylation on overall survival, including altogether 423 colorectal tissue samples.

Results and discussion

Methylation of the ZNF331 promoter was found in 71% (301/423) of the patients with colorectal cancer and was associated with localization in the right colon, microsatellite instability (MSI), and the BRAFV600E mutation. Furthermore, ZNF331 methylation was strongly associated with CpG island methylator phenotype (CIMP) and MLH1 methylation (Table 1). Wang et al. [5] reported a similar methylation frequency of ZNF331 in colorectal cancer (67%; 98/146). However, in contrast to our data Wang et al. did not find associations between methylated ZNF331 and BRAF mutation, CIMP nor MLH1 methylation, which may be explained by differences in sample size (Wang et al., n = 146; current study, n = 423), marker panels to define CIMP, method to identify methylation, age (median age Wang et al. 60; current study 72), and/or ethnicity (Wang et al.: Asian; current study: Caucasian).

Table 1 Associations between ZNF331 methylation and clinical and molecular features

Wang et al. [5] further reported that patients with ZNF331 promoter methylation had a worse prognosis than patients with unmethylated promoters. Our results were in accordance with their study, although statistical significance was not reached in the multivariate Cox regression model adjusting for age and stage (HR = 1.44 (0.97–2.14), P = 0.069; Table 2). The univariate model is presented in Fig. 1 (P = 0.143).

Table 2 Multivariate Cox proportional hazard analysis with overall survival as endpoint
Fig. 1
figure1

Effect of ZNF331 promoter methylation on overall survival modeled by the Kaplan-Meier method and compared using the log-rank test

In conclusion, in an extended series of colorectal cancer samples, we have showed the potential of promoter methylation of ZNF331 as a biomarker for colorectal cancer detection. We have further provided data indicating a trend towards poorer prognosis for patients with ZNF331 methylation.

Material and methods

Colorectal cancer tissue samples

This study included 423 colorectal cancer tissue samples. Fifty-nine of the samples were obtained from several different hospitals in the southeast region of Norway in the period 1987–1989 (Oslo 3 series; described in [6]), and 364 of the samples were obtained from patients undergoing surgical resection at the Oslo University Hospital–Aker from 2005 to 2011 (Oslo 2 series; described in [7, 8]). Survival data was available for 419 patients (Oslo 3, n = 59; Oslo 2, n = 360).

Bisulfite treatment and quantitative methylation-specific PCR (qMSP)

DNA from cancer tissue samples were bisulfite treated using the EpiTect Bisulfite Kit (Qiagen), and the samples were purified using the QIAcube (Qiagen).

Quantitative methylation-specific PCR (qMSP) was used to analyze the methylation of the ZNF331 promoter (NM_018555), with primers and probe sequences as reported earlier [4]. The method was performed as previously described [4, 9], with the ALU-C4 element as a normalization control [10]. As described in ref. [4], samples with percent methylated reference (PMR) values ≥ 1 were considered methylated. Information about MSI, CIMP, MLH1 methylation, and BRAF mutation status were available from previous studies [11, 12].

Statistical analyses

Associations between ZNF331 methylation and clinicopathological data were analyzed by Pearson chi-square or Fisher’s exact tests. For all analyses, patients were divided into three age groups (< 60 years, 60–74 years, and ≥ 75 years). Breakpoints were chosen as previously described [11]. Overall survival was used as endpoint in the survival analyses and was calculated from time of surgery until death of any cause. Cases were censored at last follow-up. The univariate effect of ZNF331 on survival was modeled by the Kaplan-Meier method and compared using the log-rank test. A multivariate Cox’s proportional hazard model was generated by a stepwise selection procedure (backward likelihood model) in order to identify a subset of relevant predictor variables from the set of available clinicopathological data (series, age, stage, gender, CIMP-, MSI-, BRAF-, and ZNF331 methylation status). Hazard ratios (HRs) and 95% confidence intervals (CIs) were derived from the model, and significance of the parameters was assessed using Wald’s test. To evaluate the assumption of proportionality, a chi-square test was performed. A P value < 0.05 was considered statistically significant. The analyses were performed using IBM SPSS Statistics 21 and R version 3.4.1.

Abbreviations

CIMP:

CpG island methylator phenotype

References

  1. 1.

    Vedeld HM, Goel A, Lind GE. Epigenetic biomarkers in gastrointestinal cancers: the current state and clinical perspectives. Semin Cancer Biol. 2017; https://doi.org/10.1016/j.semcancer.2017.12.004.

  2. 2.

    Lothe RA, Ahmed D, Andresen K et al. Methods and biomarkers for detection of gastrointestinal cancers. US provisional application filed 61/451,198, INVEN-31899/US-1/PRO 2011.

  3. 3.

    Yu J, Liang QY, Wang J, et al. Zinc-finger protein 331, a novel putative tumor suppressor, suppresses growth and invasiveness of gastric cancer. Oncogene. 2012;32:307–17.

    Article  PubMed  CAS  Google Scholar 

  4. 4.

    Vedeld HM, Andresen K, Eilertsen IA, et al. The novel colorectal cancer biomarkers CDO1, ZSCAN18 and ZNF331 are frequently methylated across gastrointestinal cancers. Int J Cancer. 2015;136:844–53.

    Article  PubMed  CAS  Google Scholar 

  5. 5.

    Wang Y, He T, Herman JG, et al. Methylation of ZNF331 is an independent prognostic marker of colorectal cancer and promotes colorectal cancer growth. Clin Epigenetics. 2017;9:115.

    Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Lothe RA, Peltomaki P, Meling GI, et al. Genomic instability in colorectal cancer: relationship to clinicopathological variables and family history. Cancer Res. 1993;53:5849–52.

    PubMed  CAS  Google Scholar 

  7. 7.

    Sveen A, Agesen TH, Nesbakken A, et al. ColoGuidePro: a prognostic 7-gene expression signature for stage III colorectal cancer patients. Clin Cancer Res. 2012;18:6001–10.

    Article  PubMed  CAS  Google Scholar 

  8. 8.

    Berg M, Danielsen SA, Ahlquist T, et al. DNA sequence profiles of the colorectal cancer critical gene set KRAS-BRAF-PIK3CA-PTEN-TP53 related to age at disease onset. PLoS One. 2010;5:e13978.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. 9.

    Lind GE, Danielsen SA, Ahlquist T, et al. Identification of an epigenetic biomarker panel with high sensitivity and specificity for colorectal cancer and adenomas. MolCancer. 2011;10:85.

    CAS  Google Scholar 

  10. 10.

    Weisenberger DJ, Campan M, Long TI, et al. Analysis of repetitive element DNA methylation by MethyLight. Nucleic Acids Res. 2005;33:6823–36.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. 11.

    Vedeld HM, Merok M, Jeanmougin M, et al. CpG island methylator phenotype identifies high risk patients among microsatellite stable BRAF mutated colorectal cancers. Int J Cancer. 2017;141:967–76.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. 12.

    Merok MA, Ahlquist T, Royrvik EC, et al. Microsatellite instability has a positive prognostic impact on stage II colorectal cancer after complete resection: results from a large, consecutive Norwegian series. Ann Oncol. 2013;24:1274–82.

    Article  PubMed  CAS  Google Scholar 

Download references

Funding

This work was supported by grants from the South-Eastern Norway Regional Health Authority (project number 2016071 to G.E. Lind, funding HM Vedeld as a postdoc).

Availability of data and materials

The datasets generated and analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.

Author information

Affiliations

Authors

Contributions

GEL contributed to the conception and design. HMV, AN, and RAL contributed to the acquisition of data. HMV, AN, RAL, and GEL contributed to the analyses and interpretation of the data. HMV contributed to the drafting of the manuscript. All authors were involved in the revision of the manuscript and have approved the final version.

Corresponding author

Correspondence to Guro E. Lind.

Ethics declarations

Ethics approval and consent to participate

The research biobanks have been registered according to national legislation (numbers 2781 and 236-2005-16141). The study is part of a project approved by the Regional Committee (REC) for Medical and Health Research Ethics (numbers 1.2005.1629 and S-09282c 2009/4958).

Competing interests

RAL and GEL are inventors of a US provisional patent application filed in 2011, describing methylation of ZNF331 and five additional genes as biomarkers for detection of gastrointestinal cancers (61/451,198, INVEN-31899/US-1/PRO). The rest of the authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Vedeld, H.M., Nesbakken, A., Lothe, R.A. et al. Re-assessing ZNF331 as a DNA methylation biomarker for colorectal cancer. Clin Epigenet 10, 70 (2018). https://doi.org/10.1186/s13148-018-0503-2

Download citation

Keywords

  • Colorectal cancer
  • Diagnosis
  • DNA methylation
  • Prognosis
  • ZNF331