Cellular Oncology

, Volume 36, Issue 5, pp 411–419 | Cite as

Regulation of MLH1 mRNA and protein expression by promoter methylation in primary colorectal cancer: a descriptive and prognostic cancer marker study

  • Lars Henrik Jensen
  • Anders Aamann Rasmussen
  • Lene Byriel
  • Hidekazu Kuramochi
  • Dorthe Gylling Crüger
  • Jan Lindebjerg
  • Peter V. Danenberg
  • Anders Jakobsen
  • Kathleen Danenberg
Original Paper

Abstract

Background

In colorectal cancer MLH1 deficiency causes microsatellite instability, which is relevant for the patient’s prognosis and treatment, and its putative heredity. Dysfunction of MLH1 is caused by sporadic gene promoter hypermethylation or by hereditary mutations as seen in Lynch Syndrome. The aim of this study was to determine in detail how DNA methylation regulates MLH1 expression and impacts clinical management.

Methods

Colorectal cancer samples were collected from 210 patients. The laboratory methods used to study these samples included methylation specific multiplex ligation-dependent probe amplification (MS-MLPA), real-time quantitative PCR (qPCR), and immunohistochemistry (IHC).

Results

We found that the MLH1 mRNA and protein expression levels were highly related. MS-MLPA was successful in tumors from 195 patients. In these tumors, hypermethylation was observed in promoter regions A (n = 57), B (n = 30), C (n = 28), and D (n = 47), and in intron 1 (n = 25). The promoter region C and intron 1 methylation levels were found to be excellently suited for discriminating between low and high gene expression levels, whereas those of promoter regions A, B and D were less specific. Hypermethylation in any region (n = 77) served as an independent prognostic factor (hazard ratio 0.56, 95 % confidence interval 0.36–0.89, p = 0.01).

Conclusions

MLH1 inactivation through hypermethylation was found to be related to improved survival. Hypermethylation in promoter region C and intron 1 served as the most specific markers for this inactivation.

Keywords

Colorectal cancer Mismatch repair MLH1 Gene expression Promoter hypermethylation qPCR Methylation specific MLPA 

Notes

Acknowledgments

The methylation analyses were funded by the Vejle Hospital and The Region of Southern Denmark. We are grateful for the technical assistance by Birgit Sørensen, for comments by Steen Koelvraa, and for proofreading by Karin Larsen. Response Genetics, Inc, performed the mRNA expression analyses.

Competing interests

Kathleen D. Danenberg, Response Genetics, Inc, leadership role (CEO), stock ownership. Peter V. Danenberg, Response Genetics, Inc, advisory role, stock ownership. None of the other authors have relevant conflicts to declare.

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Copyright information

© International Society for Cellular Oncology 2013

Authors and Affiliations

  • Lars Henrik Jensen
    • 1
    • 4
  • Anders Aamann Rasmussen
    • 1
  • Lene Byriel
    • 1
  • Hidekazu Kuramochi
    • 2
  • Dorthe Gylling Crüger
    • 1
  • Jan Lindebjerg
    • 1
  • Peter V. Danenberg
    • 2
  • Anders Jakobsen
    • 1
  • Kathleen Danenberg
    • 3
  1. 1.Danish Colorectal Cancer Group SouthUniversity of Southern Denmark and Vejle HospitalVejleDenmark
  2. 2.Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer CenterUniversity of Southern CaliforniaLos AngelesUSA
  3. 3.Response GeneticsLos AngelesUSA
  4. 4.Department of OncologyLillebaelt HospitalVejleDenmark

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