Originally published as JCO Early Release 10.1200/JCO.2007.14.1697 on August 18 2008

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Multicenter Study Using Paraffin-Embedded Tumor Tissue Testing PITX2 DNA Methylation As a Marker for Outcome Prediction in Tamoxifen-Treated, Node-Negative Breast Cancer Patients

Nadia Harbeck, Inko Nimmrich, Arndt Hartmann, Jeffrey S. Ross, Tanja Cufer, Robert Grützmann, Glen Kristiansen, Angelo Paradiso, Oliver Hartmann, Astrid Margossian, John Martens, Ina Schwope, Antje Lukas, Volkmar Müller, Karin Milde-Langosch, Jörg Nährig, John Foekens, Sabine Maier, Manfred Schmitt, Ralf Lesche

From the Departments of Obstetrics and Gynecology and Pathology, Technical University of Munich, Munich; Epigenomics AG; Charite Hospital, Humboldt University, Berlin; Institute of Pathology, University of Regensburg, Regensburg; Department of Visceral, Thoracic, and Vascular Surgery, University Hospital Carl Gustav Carus, Technical University Dresden, Dresden; Department of Gynecology, University Hospital Hamburg Eppendorf, Hamburg; Institute of Pathology, University of Erlangen, Erlangen, Germany; Institute of Clinical Pathology; University Hospital, Zurich, Switzerland; Institute of Oncology, Ljubljana, Slovenia; Clinical Experimental Oncology Laboratory, National Cancer Institute, Bari, Italy; Halitus Instituto Medico, Buenos Aires, Argentina; Department of Medical Oncology, Erasmus Medical Center, Rotterdam, the Netherlands; and Albany Medical College, Albany, New York

Corresponding author: Nadia Harbeck, MD, Frauenklinik der Technischen Universität München, Ismaninger Strasse 22, 81675 München, Germany; e-mail: nadia.harbeck{at}lrz.tu-muenchen.de

Purpose We recently reported DNA methylation of the paired-like homeodomain transcription factor 2 (PITX2) gene to be strongly correlated with increased risk of recurrence in node-negative, hormone receptor–positive, tamoxifen-treated breast cancer patients using fresh frozen specimens. Aims of the present study were to establish determination of PITX2 methylation for routine analysis in formalin-fixed paraffin-embedded (FFPE) breast cancer tissue and to test PITX2 DNA methylation as a biomarker for outcome prediction in an independent patient cohort.

Patients and Methods Real-time polymerase chain reaction (PCR) technology was validated for FFPE tissue by comparing methylation measurements in FFPE specimens with those in fresh frozen specimens from the same tumor. The impact of PITX2 methylation on time to distant metastasis was then evaluated in FFPE specimens from hormone receptor–positive, node-negative breast cancer patients (n = 399, adjuvant tamoxifen monotherapy).

Results Reproducibility of the PCR assay in replicate measurements (r_s 0.95; n = 150) and concordant measurements between fresh frozen and FFPE tissues (r_s = 0.81; n = 89) were demonstrated. In a multivariate model, PITX2 methylation added significant information (hazard ratio = 2.35; 95% CI, 1.20 to 4.60) to established prognostic factors (tumor size, grade, and age).

Conclusion PITX2 methylation can be reliably assessed by real-time PCR technology in FFPE tissue. Together with our earlier studies, we have accumulated substantial evidence that PITX2 methylation analysis holds promise as a practical assay for routine clinical use to predict outcome in node-negative, tamoxifen-treated breast cancer, which might allow, based on future validation studies, the identification of low-risk patients who may be treated by tamoxifen alone.

published online ahead of print at www.jco.org on August 18, 2008

Supported by Grant No. LSHC-CT-2003-504586 from the European Union Sixth Framework Program (J.M., J.F., M.S., T.C., N.H.). This work was performed in collaboration with Roche Diagnostics GmbH/Hoffmann-LaRoche Basel and funded in part by Epigenomics and Roche Diagnostics.

Presented at the 41st Annual Meeting of the American Society of Clinical Oncology, May 13-17, 2005, Orlando, FL.

Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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