Advertisement

Amplification Testing in Breast Cancer by Multiplex Ligation-Dependent Probe Amplification of Microdissected Tissue

  • Cathy B. MoelansEmail author
  • Roel A. de Weger
  • Paul J. van Diest
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 755)

Abstract

This chapter describes a method for the rapid assessment of gene copy numbers in laser-microdissected materials using multiplex ligation-dependent probe amplification (MLPA). An MLPA is a powerful multiplex PCR technique that can identify gains, amplification, or losses of up to 50 genes in a single experiment, thereby requiring only minute quantities of DNA extracted from frozen or paraffin-embedded materials. A previous study in breast cancer has shown that MLPA can detect amplifications in cases with a tumor percentage lower than 10%, but still a low tumor percentage in the tissue tested could obscure low levels of amplification due to dilution of the tumor cell population by normal cells. Laser capture microdissection allows enrichment of tumor cells by eliminating background noise from normal and preinvasive cells, thereby increasing specificity and sensitivity.

This chapter describes a method for MLPA analysis using invasive breast tumor cells acquired by laser capture microdissection. This protocol can also be applied to MLPA analysis of preinvasive lesions and metastases.

Key words

Multiplex ligation-dependent probe amplification MLPA Coffalyser Laser microdissection Cancer 

References

  1. 1.
    Ross JS, Fletcher JA, Bloom KJ et al (2004) Targeted therapy in breast cancer: the HER-2/neu gene and protein. Mol Cell Proteomics 3, 379–398.Google Scholar
  2. 2.
    Slamon DJ, Godolphin W, Jones LA et al (1989) Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244, 707–712.Google Scholar
  3. 3.
    Baak JPA, Chin D, Van Diest PJ et al (1991) Comparative long term prognostic value of quantitative Her2/Neu protein expression, DNA ploidy, morphometric and clinical features in paraffin-embedded invasive breast cancer. Lab Invest 64, 215–222.Google Scholar
  4. 4.
    Slamon DJ, Leyland-Jones B, Shak S et al (2001) Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 344, 783–792.Google Scholar
  5. 5.
    Hudis CA (2007) Trastuzumab, mechanism of action and use in clinical practice. N Engl J Med 357, 39–51.Google Scholar
  6. 6.
    Borg A, Baldetorp B, Ferno M et al (1994) ERBB2 amplification is associated with tamoxifen resistance in steroid-receptor positive breast cancer. Cancer Lett 81, 137–144.Google Scholar
  7. 7.
    Tetu B, Brisson J, Plante V et al (1998) p53 and c-erbB-2 as markers of resistance to adjuvant chemotherapy in breast cancer. Mod Pathol 11, 823–830.Google Scholar
  8. 8.
    Schouten JP, McElgunn CJ, Waaijer R et al (2002) Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 30, e57.Google Scholar
  9. 9.
    Moelans CB, de Weger RA, van Blokland MT et al (2010) Simultaneous detection of TOP2A and HER2 gene amplification by multiplex ligation-dependent probe amplification in breast cancer. Mod Pathol 23, 62–70.Google Scholar
  10. 10.
    Moelans CB, de Weger RA, and van Diest PJ (2010) Absence of chromosome 17 polysomy in breast cancer: analysis by CEP17 chromogenic in situ hybridization and multiplex ligation-dependent probe amplification. Breast Cancer Res Treat 120, 1–7.Google Scholar
  11. 11.
    Vorstman JA, Jalali GR, Rappaport EF et al (2006) MLPA: a rapid, reliable, and sensitive method for detection and analysis of abnormalities of 22q. Hum Mutat 27, 814–821.Google Scholar
  12. 12.
    Eldering E, Spek CA, Aberson HL et al (2003) Expression profiling via novel multiplex assay allows rapid assessment of gene regulation in defined signalling pathways. Nucleic Acids Res 31, e153.Google Scholar
  13. 13.
    Hess CJ, Denkers F, Ossenkoppele GJ et al (2004) Gene expression profiling of minimal residual disease in acute myeloid leukaemia by novel multiplex-PCR-based method. Leukemia 18, 1981–1988.Google Scholar
  14. 14.
    Dikow N, Nygren AO, Schouten JP et al (2007) Quantification of the methylation status of the PWS/AS imprinted region: comparison of two approaches based on bisulfite sequencing and methylation-sensitive MLPA. Mol Cell Probes 21, 208–215.Google Scholar
  15. 15.
    Nygren AO, Ameziane N, Duarte HM et al (2005) Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences. Nucleic Acids Res 33, e128.Google Scholar
  16. 16.
    Procter M, Chou LS, Tang W et al (2006) Molecular diagnosis of Prader-Willi and Angelman syndromes by methylation-specific melting analysis and methylation-specific multiplex ligation-dependent probe amplification. Clin Chem 52, 1276–1283.Google Scholar
  17. 17.
    Purnomosari D, Aryandono T, Setiaji K et al (2006) Comparison of multiplex ligation dependent probe amplification to immunohistochemistry for assessing HER-2/neu amplification in invasive breast cancer. Biotech Histochem 81, 79–85.Google Scholar
  18. 18.
    Moelans CB, de Weger RA, van Blokland MT et al (2009) HER-2/neu amplification testing in breast cancer by multiplex ligation-dependent probe amplification in comparison with immunohistochemistry and in situ hybridization. Cell Oncol 31, 1–10.Google Scholar
  19. 19.
    Moelans CB, de Weger RA, Ezendam C et al (2009) HER-2/neu amplification testing in breast cancer by Multiplex Ligation-dependent Probe Amplification: influence of manual- and laser microdissection. BMC Cancer 9, 4.Google Scholar
  20. 20.
    Micke P, Bjornsen T, Scheidl S et al (2004) A fluid cover medium provides superior morphology and preserves RNA integrity in tissue sections for laser microdissec-tion and pressure catapulting. J Pathol 202, 130–138.Google Scholar
  21. 21.
    Coffa J, van de Wiel MA, Diosdado B et al (2008) MLPAnalyzer: data analysis tool for reliable automated normalization of MLPA fragment data. Cell Oncol 30, 323–335.Google Scholar
  22. 22.
    Bunyan DJ, Eccles DM, Sillibourne J et al (2004) Dosage analysis of cancer predisposition genes by multiplex ligation-dependent probe amplification. Br J Cancer 91, 1155–1159.Google Scholar
  23. 23.
    Murase T, Inagaki H, and Eimoto T (2000) Influence of histochemical and immunohistochemical stains on polymerase chain reaction. Mod Pathol 13, 147–151.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Cathy B. Moelans
    • 1
    Email author
  • Roel A. de Weger
    • 1
  • Paul J. van Diest
    • 1
  1. 1.Department of PathologyUniversity Medical Centre UtrechtUtrechtThe Netherlands

Personalised recommendations