Genome-wide methylation screen in low-grade breast cancer identifies novel epigenetically altered genes as potential biomarkers for tumor diagnosis

Genome-wide methylation screen in low-grade breast cancer identifies novel epigenetically altered genes as potential biomarkers for tumor diagnosis

1. Marta Faryna*,¹,
2. Carolin Konermann*,^‡,¹,
3. Sebastian Aulmann^‡,
4. Justo Lorenzo Bermejo^§,¹,
5. Markus Brugger^§,
6. Sven Diederichs^†‡,
7. Joachim Rom^‖,
8. Dieter Weichenhan*,
9. Rainer Claus*,^#,
10. Michael Rehli^¶,
11. Peter Schirmacher^‡,
12. Hans-Peter Sinn^‡,
13. Christoph Plass* and
14. Clarissa Gerhauser*,²

+ Author Affiliations

1. ^*Division of Epigenomics and Cancer Risk Factors and
2. ^†Helmholtz University Group Molecular RNA Biology and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany;
3. ^‡Institute of Pathology,
4. ^§Institute of Medical Biometry and Informatics, and
5. ^‖Department of Gynecology and Obstetrics, University Hospital Heidelberg, Heidelberg, Germany;
6. ^#Department of Medicine, Division of Hematology and Oncology, University of Freiburg Medical Center, Freiburg, Germany; and
7. ^¶Department of Hematology and Oncology, University Hospital Regensburg, Regensburg, Germany

1. ↵2Correspondence: German Cancer Research Center (DKFZ), Division of Epigenomics and Cancer Risk Factors, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. E-mail: c.gerhauser@dkfz-heidelberg.de

1. ↵1 These authors contributed equally to this work.

Abstract

Aberrant DNA methylation constitutes a well-established epigenetic marker for breast cancer. Changes in methylation early in cancer development may be clinically relevant for cancer detection and prognosis-based therapeutic decisions. In the present study, a combination of methyl-CpG immunoprecipitation (MCIp) and human CpG island (CGI) arrays was applied to compare genome-wide DNA methylation profiles in 10 low-grade in situ and invasive breast cancers against 10 normal breast samples. In total, 214 CGIs were found to be hypermethylated in ≥6 of 10 tumors. Functional term enrichment analyses revealed an overrepresentation of homeobox genes and genes involved in transcription and regulation of transcription. Significant hypermethylation of 11 selected genes in tumor vs. normal tissue was validated in two independent sample sets (45 tumors and 11 controls, 43 tumors and 8 controls) using quantitative EpiTyper technology. In tumors, median methylation levels of BCAN, HOXD1, KCTD8, KLF11, NXPH1, POU4F1, SIM1, and TCF7L1 were ≥30% higher than in normal samples, representing potential biomarkers for tumor diagnosis. Using the 90th percentile of methylation levels in normal tissue as cutoff value, 62–92% of in situ samples (n=13), 72–97% of invasive samples from the first validation set (n=32), and 86–100% of invasive samples from the second validation set (n=43) were classified as hypermethylated. Hypermethylation of KLF11 and SIM1 might also be associated with increased risk of developing metastases. In summary, early methylation changes are frequent in the low-grade pathway of breast cancer and may be useful in the development of differential diagnostic and possibly also prognostic markers.—Faryna, M., Konermann, C., Aulmann, S., Bermejo, J. L., Brugger, M., Diederichs, S., Rom, J., Weichenhan, D., Claus, R., Rehli, M., Schirmacher, P., Sinn, H.-P., Plass, C., Gerhauser, C. Genome-wide methylation screen in low-grade breast cancer identifies novel epigenetically altered genes as potential biomarkers for tumor diagnosis.

• estrogen receptor-positive
• EpiTyper MassArray
• CpG island array
• methyl-CpG immunoprecipitation
• DMR
• ductal carcinoma in situ

Footnotes

• This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.
• Abbreviations:
  

  ACTB

  β-actin

  AUC

  area under curve

  BCAN

  brevican

  CGI

  CpG island

  CI

  confidence interval

  DAC

  2′-deoxy-5-azacytidine (decitabine)

  DCIS

  ductal carcinoma in situ

  DMR

  differentially methylated region

  DMSO

  dimethyl sulfoxide

  ER

  estrogen receptor

  ESC

  embryonic stem cell

  FBS

  fetal bovine serum

  FFPE

  formalin-fixed paraffin-embedded

  gDNA

  genomic DNA

  HOXD1

  homoeobox D1

  HR

  hazard ratio

  IDC

  invasive ductal carcinoma

  ILC

  invasive lobular carcinoma

  KCTD8

  potassium channel tetramerization domain containing 8

  KLF11

  Kruppel-like factor 11

  LCIS

  lobular carcinoma in situ

  MCIp

  methyl-CpG immunoprecipitation

  NXPH1

  neurexophilin 1

  PCDH10

  protocadherin 10

  PcG

  polycomb group

  POU4F1

  POU class 4 homeobox 1

  PR

  progesterone receptor

  ROC

  receiver operating characteristic

  qPCR

  quantitative PCR

  RYR2

  ryanodine receptor 2

  SIM1

  single-minded homolog 1

  TAC1

  tachykinin precursor 1

  TCF7L1

  transcription factor 7-like 1

  TMA

  tissue microarray

  TUB

  tubular carcinoma

  TUB-LOB

  tubulolobular carcinoma

• Received June 27, 2012.
• Accepted August 13, 2012.