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

	ABSTRACT

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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.

	INTRODUCTION

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Current guidelines recommend adjuvant chemotherapy followed by endocrine therapy for a substantial number of patients with node-negative, steroid hormone receptor–positive breast cancer.^1,2 Yet, most of these patients have a rather good prognosis and would derive sufficient benefit from endocrine treatment alone, suggesting that they could be spared potential overtreatment by adjuvant chemotherapy. Unfortunately, such low-risk patients cannot be reliably identified by using only traditional prognostic factors such as tumor size, grade, or age.³

DNA methylation of cytosine phosphoguanine dinucleotides within gene regulatory regions is a common and early event in cancer.^4,5 Frequently, hypermethylation within gene promoter regions is associated with suppression of gene expression.⁶ We have recently correlated DNA methylation of 117 candidate genes with outcome of breast cancer patients after adjuvant tamoxifen therapy. Among all genes analyzed, DNA methylation of the paired-like homeodomain transcription factor 2 (PITX2) gene showed the strongest association with metastasis-free survival and was selected for further analysis.⁷ PITX2 is required for morphogenesis of anterior structures, such as eyes, teeth, and anterior pituitary, and plays a role in left/right patterning.⁸ Regulation by the WNT/DVL/beta-catenin and hedgehog/transforming growth factor β pathways⁹ and differential expression in pituitary adenomas were reported.^10-12 In our previous study, into which PITX2 had been included based on reported methylation of the gene in acute myeloid leukemia,¹³ a potential link between PITX2 and breast cancer was established.⁷

In the present study, we aimed to test the clinical relevance of PITX2 as observed in fresh frozen breast cancer specimens⁷ for formalin-fixed paraffin-embedded (FFPE) tissue. For this purpose, a real-time polymerase chain reaction (PCR) –based quantitative DNA methylation assay⁷ was adapted for FFPE breast cancer specimens. We then used this assay to analyze the DNA methylation score of PITX2 in FFPE tissue obtained from 427 patients with steroid hormone receptor–positive, node-negative primary breast cancer treated by adjuvant tamoxifen alone. None of these patients had been included in the previous study.⁷ Our results substantiate the evidence that PITX2 DNA methylation may be a clinically relevant biomarker for outcome prediction in hormone receptor–positive, node-negative breast cancer.

	PATIENTS AND METHODS

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Technical Assay Validation
Paraffin blocks from 30 breast cancer patients were obtained from a commercial provider and used to establish the analytic performance of the PITX2 quantitative methylation PCR (QM-PCR) assay. From each block, 15 sections (10 µm) were prepared. Three consecutive sections were pooled and used for DNA extraction, thus resulting in five different preparations of genomic DNA per tumor block. In addition, 89 pairs of frozen and FFPE specimens obtained from the same tumor were provided by the Department of Obstetrics and Gynecology, Technical University of Munich, Munich, Germany; University Hospital Eppendorf, Hamburg, Germany; and Clinical Experimental Oncology Laboratory, National Cancer Institute, Bari, Italy.

Clincial Testing Study
Consecutive breast cancer patients (n = 427), who had not been part of our previous study,⁷ were identified in 10 clinical centers (Department of Obstetrics and Gynecology, Technical University of Munich, Munich; Institute of Pathology, Charite University Hospital, Berlin; Institute of Pathology, University of Regensburg, Regensburg; Department of Visceral, Thoraic, and Vascular Surgery, University Hospital Carl Gustav Carus, Technical University Dresden, Dresden; Department of Gynecology, University Hospital Hamburg Eppendorf, Hamburg, Germany; 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 NY). Inclusion criteria were availability of FFPE specimens with an invasive tumor component, stage pT1-3 disease, estrogen receptor (ER) and/or progesterone receptor (PgR) expression (either 10 fmol/mg of cytosolic protein determined by enzyme immunoassay or dextran-coated charcoal or positive Remmele score¹⁴), no pathologic axillary lymph node involvement, age more than 35 years at time of diagnosis, surgery in or before 1998, adjuvant tamoxifen monotherapy (indicated duration of 5 years), availability of follow-up data, and appropriate patient consent. Patients treated using neoadjuvant or adjuvant chemotherapy were excluded. Primary therapy and regular follow-up were performed according to local guidelines. Patient characteristics are listed in Table 1. Approval for the study was obtained from the local ethics committee of each participating center.

DNA Extraction and Bisulfite Conversion
FFPE tissue sections were incubated in limonene (Fluka, Deisenhofen, Germany) for 10 to 30 minutes to remove paraffin. For DNA extraction, the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) was used according to the manufacturer's recommendations (extended lysis for 2 days at 50°C). The DNA Tissue Mini Kit (Qiagen) was used to isolate DNA from fresh frozen tumor specimens. Protocols for bisulfite treatment of genomic DNA isolated from FFPE specimens¹⁷ and fresh frozen specimens⁷ were used as previously described.

Quantitative Real-Time PCR
Quantitative real-time PCR (QM-PCR) to assess PITX2 methylation was conducted as previously described.⁷ Primers excluding cytosine phosphoguanine dinucleotides and TaqMan probes specific for methylated and unmethylated states were designed to target the PITX2 gene promoter region (for details, see Appendix, online only). Threshold cycles (Ct) were used to calculate methylation scores: methylation score = 100/[1 + 2^(Ct_m – Ct_u)]; Ct_m and Ct_u denote threshold cycles of the probes specific for the methylated and unmethylated state, respectively. The median of at least three replicate measurements was calculated for each sample and used for statistical analysis. Predefined quality criteria were set such that measurements with both probes greater than 38 cycles were excluded.

Statistical Analyses
The relationship between PITX2 DNA methylation score and established clinical factors to the primary end point, time to distant metastasis (TDM), was analyzed using univariate and multivariate Cox proportional hazard models.^18,19 Contralateral disease, other second primary cancers, death before distant recurrence, and locoregional recurrence were considered censoring events. Hazard ratios (HRs) for continuous variables were calculated relative to an increment of the interquartile range (25% to 75% quantile).¹⁹ Wald tests were used to test for significance of HRs. Survival curves were calculated according to the Kaplan-Meier method.²⁰

	RESULTS

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PITX2 DNA Methylation Measurements in FFPE Primary Breast Cancer Tissue
At first, we aimed to verify that DNA methylation measurements can be reliably obtained from routinely archived specimens. For this purpose, replicate measurements of 150 tissue preparations from 30 paraffin blocks originating from different breast cancer patients were performed. We observed high correlation of repeated measurements of DNA isolated from the same tissue preparation (Spearman correlation, r_s = 0.95; n = 150; Fig 1A), indicating that PITX2 DNA methylation scores can be reproducibly assessed using FFPE breast cancer tissue specimens.

View larger version (13K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Technical validation of paired-like homeodomain transcription factor 2 (PITX2) DNA methylation measurements in formalin-fixed paraffin-embedded breast cancer tissue specimens. (A) Repeatability. Quantitative methylation polymerase chain reaction was used to measure PITX2 DNA methylation scores in 150 DNA preparations from 30 different breast carcinomas in three replicates each. Highly concordant results between replicate measurements were obtained (rs 0.95). (B) Intra- and intersample variability. PITX2 DNA methylation scores were determined in consecutive preparations (n = 5) from the same paraffin block. Results for 30 different tumors, ranked by the median methylation score for each tumor, are shown. The variability of PITX2 DNA methylation scores among different tumors, assessed by averaging over all measurements from the same tumor, were significantly higher (standard deviation, 25.7%; range, 0% to 88%) compared with the variability among different preparations from the same tumor (average standard deviation, 4.3%; range 0.3% to 17%).

Clinicial Testing Study
FFPE specimens obtained from 427 primary breast cancer patients from nine clinical centers were analyzed for PITX2 DNA methylation scores using QM-PCR. All patients included in this retrospective clinical study had steroid hormone receptor–positive, node-negative breast cancers and had received tamoxifen as sole adjuvant systemic therapy. Valid DNA methylation measurements passing the predefined quality control criteria were obtained for 399 patients (93.4%; Table 1).

TDM was defined as the primary end point, and the proportion of patients without evidence of metastasis after 10 years was estimated to be 88.8%. Among the established prognostic factors, only tumor size was a significant predictor of outcome by univariate analysis (P = .001; Table 2).

View larger version (15K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 2. Time to distant metastasis in steroid hormone receptor–positive, node-negative patients (n = 399) treated for 5 years with tamoxifen as their sole adjuvant systemic therapy. Patients were stratified into two groups according to the paired-like homeodomain transcription factor 2 (PITX2) DNA methylation score as measured by quantitative methylation polymerase chain reaction assay. The methylation score cutoff was derived using the patient group (n = 236) from our previous study7 as an independent training set. To account for a potential bias in assay performance over time, we compared measurements of 84 patient samples analyzed in both the present and the previous study7 and determined the numerical value of the cutoff for the present validation study (methylation score = 22.9; see Appendix Fig A2 for details). Statistically significant separation of the two groups was observed (log-rank test, P = .032; hazard ratio = 2.69 [PITX2 dichotomized]; 95% CI, 1.05 to 6.89). Patients in the low-risk group were characterized by PITX2 methylation scores less than the cutoff.

	DISCUSSION

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To successfully establish a tumor biomarker for routine clinical use, not only does relevance to clinical management have to be demonstrated, but also the technology to detect a marker needs to be applicable in clinical routine. Aberrant DNA methylation has been well established as an early, frequent, and stable molecular event during tumor progression.^21-28 Using fresh frozen tumor specimens, we previously reported PITX2 DNA methylation to be a strong marker for outcome prediction in patients with steroid hormone receptor–positive, node-negative primary breast cancer treated with adjuvant tamoxifen monotherapy.⁷

Compared with fresh frozen tissue, nucleic acids isolated from FFPE tissue have been reported to be more often degraded, thus representing potentially less than ideal tissue for molecular analysis.²⁹ Nevertheless, even on FFPE samples, we were able to demonstrate high repeatability of measurements for our workflow (Fig 1A). Moreover, we observed high correlation of measurements between FFPE and fresh frozen tissue (r_s = 0.81) when analyzing matched tissue pairs obtained from the same tumor. Taking into account that fresh frozen and FFPE samples not only differed in preparation techniques but also were subject to tumor heterogeneity within each tumor, we considered this high correlation adequate to exclude systematic differences in methylation measurements between fresh frozen and FFPE specimens. To further explore the impact of tumor heterogeneity on methylation analysis, we determined PITX2 methylation scores of different samples from the same tumor tissue (Fig 1B). The fact that we found relatively low variability of methylation measurements within individual tumors compared with variability between tumors provides further evidence for the potential of PITX2 methylation analysis for routine molecular diagnostics. Our robust and reliable workflow for DNA methylation measurement was further exemplified in our multicenter clinical study, where we obtained successful measurements in 93.4% of the patients (399 of 427 patients) analyzed.

For further clinical testing of the PITX2 biomarker, 427 FFPE specimens from node-negative, steroid hormone receptor–positive breast cancer patients treated with adjuvant tamoxifen monotherapy were collected from nine clinical centers. Our results confirm the potential of PITX2 DNA methylation as a marker for risk of recurrence (HR = 2.75; 95% CI, 1.40 to 5.41). We used a previously analyzed patient cohort⁷ as a training set to define the methylation score cut point for Kaplan-Meier analysis. Applying this cut point to stratify our study population (n = 399) resulted in a significant risk group separation regarding TDM (P = .03), with 52% of patients being classified as low risk with an estimated 10-year metastasis-free survival rate of 95%. These results suggest that PITX2 DNA methylation analysis might be used to identify node-negative, steroid hormone receptor–positive breast cancer patients who may be sufficiently treated using adjuvant tamoxifen alone. Moreover, these low-risk patients, if postmenopausal, may not require aromatase inhibitor therapy. For patients in the higher risk group, the additional benefit from aromatase inhibitors and/or adjuvant chemotherapy needs to be evaluated. The relatively late separation of survival curves could suggest that patients with a high PITX2 methylation score might not derive benefit from the well-established tamoxifen carryover effect.³⁰ However, whether PITX2 methylation predicts tamoxifen response or is prognostic independent of therapy cannot be distinguished at present. In this context, it is interesting to mention that PITX2 DNA methylation was also found to be a strong prognostic factor for distant metastasis–free survival and overall survival in a cohort of 412 primary breast cancer patients enrolled in the Netherlands who did not receive any adjuvant systemic therapy.³¹ The latter study suggests that performance of PITX2 in tamoxifen-treated breast cancer patients might, in part, rely on a prognostic component. In a further study, we found that PITX2 methylation predicted outcome for distant metastasis–free survival and overall survival in a patient group of 241 ER-positive, node-positive, HER-2/neu–negative patients treated with adjuvant chemotherapy (Hartmann O et al, submitted for publication).³² In our present study, however, we were not able to demonstrate a significant association of PITX2 DNA methylation with overall survival, likely because of the relatively low number of events in this patient group with good overall prognosis.

We analyzed the clinical impact of PITX2 DNA methylation taking into account the established prognostic factors of tumor size, grade, and age. In the multivariate model, PITX2 DNA methylation contributed statistically significant independent outcome information. Univariate analysis showed that only tumor size was significantly associated with outcome, a result that is in line with previous observations.³² Interestingly, we found different proportions for T stage when comparing our patient training set of 236 patients⁷ (35% with stage T1) with our current testing set (60% with stage T1). A possible explanation for this difference could be that the specimens obtained from the patient training group (n = 236) were in large part collected for routine ER and PgR determination by biochemical methods, which, at the time, was not routinely performed for the smaller T1 tumors. Recently, ER status and PgR status have been routinely assessed by immunohistochemistry in FFPE tissue, which is feasible even for the smallest T1 tumors. Furthermore, because the training set patients were diagnosed earlier in time compared with the testing set, over-representation of larger tumors in the training set might also be explained in part by the general trend toward smaller tumors in more recent times, mostly as a result of mammography screening.³³ It seems important to consider that, in our group of patients, most tumors (72%) were classified as grade 2 (moderately differentiated) and that, therefore, the remaining groups of grade 1 and grade 3 tumors were relatively small for meaningful subgroup analysis. Although, in addition to ER status, quantitative measurements of ER levels were only available for a subgroup of patients (n = 286), our finding that the actual ER level is not a significant predictor of TDM (data not shown) in this ER-positive group is in line with reports on similar treatment groups.³⁴ As is expected for an ER-positive population, only 15% of the patients for whom PgR status was reported were PgR negative. No statistically significant association of PgR status with patient outcome was observed. In our patient group, prognostic significance of HER-2/neu status could not be demonstrated (HR = 2.60; 95% CI, 0.75 to 8.98; P = .13) likely because of the small proportion of HER-2/neu–positive patients (n = 28; 8.8%); however, this is in line with previous observations in similar patient groups.³⁵ According to current guidelines,² the majority of HER-2/neu–positive patients will receive trastuzumab as adjuvant therapy, and only HER-2/neu–negative patients qualify for tamoxifen monotherapy. Therefore, it is important to note that PITX2 methylation predicted outcome in the HER-2/neu–negative patient subgroup in univariate (P = .039) and multivariate analyses (P = .019; Appendix Fig A1, online only). Patient age has been associated with risk of disease recurrence.³⁵ However, in our cohort, the majority of patients were older than 50 years at time of diagnosis, indicating that, at the time our patients were treated for primary breast cancer ( 1998), most premenopausal patients did not receive adjuvant tamoxifen. As a consequence, younger patients were not present in significant numbers in our cohort, which may explain the lack of correlation of age with outcome. In summary, we conclude that the distributions of covariates as well as their performance for outcome prediction in our patient cohort are within expectations from similar studies in the literature.

Although our results represent, to our knowledge, the first large-scale testing of a DNA methylation marker for outcome prediction in primary breast cancer, other groups have identified respective gene expression signatures.^36-38 In fresh frozen tumor tissue, prognostic signatures have been reported for breast cancer patients who had not received any adjuvant systemic therapy.^37,38 So far, logistics for preservation of fresh frozen tissue are only established in specialized centers. Moreover, tumor sizes at primary diagnosis are decreasing as a result of improved early detection. Thus, the amount of fresh breast cancer tissue remaining after routine histopathology examination may be limited. Using RNA isolated from FFPE specimens, a recurrence score was developed for tamoxifen-treated patients based on reverse transcription PCR analysis of 21 genes.³⁶ With regard to establishing a new biomarker for clinical decision making, the key criterion will most likely be the outcome in the low-risk group; the predicted proportion of patients without evidence for distant metastasis will have to be low enough as to safely allow avoiding adjuvant chemotherapy. In addition, this low-risk group has to include a substantial proportion of patients to justify testing the whole population. For both criteria, the recurrence score (low-risk group size, 51%; 10-year metastasis-free survival rate, 93.2%)³⁶ and our single DNA methylation marker PITX2 (low-risk group size, 52%; 10-year metastasis-free survival rate, 95%; Fig 2) seem to render comparable results.

Our studies provide strong evidence that, based on measurement of PITX2 DNA methylation, a relevant proportion of patients with steroid hormone receptor–positive, node-negative breast cancer may be considered to derive sufficient benefit from adjuvant endocrine therapy with tamoxifen alone. The results are encouraging to initiate further large-scale studies in well-characterized patient groups, ultimately aiming at a prospective clinical validation study in a similar set up as described for other breast cancer gene signatures.^39,40 Measurement of a single stable DNA-based marker from FFPE tissue holds promise as a practical, robust, and cost-effective method for routine clinical application.

	AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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Although all authors completed the disclosure declaration, the following author(s) indicated a financial or other interest that is relevant to the subject matter under consideration in this article. Certain relationships marked with a "U" are those for which no compensation was received; those relationships marked with a "C" were compensated. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.

Employment or Leadership Position: Inko Nimmrich, Epigenomics AG (C); Oliver Hartmann, Epigenomics AG (C); Ina Schwope, Epigenomics AG (C); Antje Lukas, Epigenomics AG (C); Sabine Maier, Epigenomics AG (C); Ralf Lesche, Epigenomics AG (C) Consultant or Advisory Role: None Stock Ownership: Inko Nimmrich, Epigenomics AG; Ina Schwope, Epigenomics AG; Antje Lukas, Epigenomics AG; Sabine Maier, Epigenomics AG; Ralf Lesche, Epigenomics AG Honoraria: None Research Funding: Nadia Harbeck, Epigenomics AG; Arndt Hartmann, Epigenomics AG; Jeffrey S. Ross, Millenium, Veridex, Roche; Robert Grützmann, Epigenomics AG; Glen Kristiansen, Epigenomics AG; Angelo Paradiso, Epigenomics AG; Astrid Margossian, Epigenomics AG; Volkmar Müller, Epigenomics AG; John Foekens, Epigenomics AG, Veridex Expert Testimony: None Other Remuneration: None

	AUTHOR CONTRIBUTIONS

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Conception and design: Nadia Harbeck, Inko Nimmrich, Tanja Cufer, Robert Grützmann, John Martens, Volkmar Müller, John Foekens, Sabine Maier, Manfred Schmitt, Ralf Lesche

Provision of study materials or patients: Nadia Harbeck, Arndt Hartmann, Jeffrey S. Ross, Tanja Cufer, Robert Grützmann, Glen Kristiansen, Angelo Paradiso, Astrid Margossian, Antje Lukas, Volkmar Müller, Karin Milde-Langosch, Jörg Nährig, John A. Foekens, Manfred Schmitt

Collection and assembly of data: Nadia Harbeck, Inko Nimmrich, Arndt Hartmann, Jeffrey S. Ross, Tanja Cufer, Robert Grützmann, Glen Kristiansen, Angelo Paradiso, Ina Schwope, Antje Lukas, Volkmar Müller, Karin Milde-Langosch, Jörg Nährig, Sabine Maier, Manfred Schmitt, Ralf Lesche

Data analysis and interpretation: Nadia Harbeck, Inko Nimmrich, Angelo Paradiso, Oliver Hartmann, John Martens, Ina Schwope, Sabine Maier, Ralf Lesche

Manuscript writing: Nadia Harbeck, Arndt Hartmann, Tanja Cufer, Oliver Hartmann, John Martens, John Foekens, Sabine Maier, Manfred Schmitt, Ralf Lesche

Final approval of manuscript: 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

	Appendix

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View larger version (14K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig A1. Estrogen receptor–positive, node-negative, human epidermal growth factor receptor 2 (HER-2)/neu–negative patient subgroup. Time to distant metastasis in steroid hormone receptor–positive, node-negative, HER-2/neu–negative patients (n = 292) treated for 5 years with tamoxifen as their sole adjuvant systemic therapy. Patients were stratified into two groups according to the paired-like homeodomain transcription factor 2 (PITX2) DNA methylation score as measured by quantitative methylation polymerase chain reaction assay. The methylation score cutoff was derived using the patient group (n = 236) from our previous study7 as an independent training set. To account for a potential bias in assay performance over time, we compared measurements of 84 tumor samples analyzed in both the present and the previous study,7 and determined the numerical value of the cutoff for the present validation study (methylation score = 22.9; see Fig A2 for details). Statistically significant separation of the two groups was observed (log-rank test, P = .039). Patients in the low-risk group were characterized by PITX2 methylation scores less than the cutoff.

View larger version (9K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig A2. Determination of cutoff value from previously published 236 patient cohort.7 To account for a potential bias in assay performance over time, we compared measurements of 84 patient samples analyzed in both the present and the previous study and determined, based on the median cut point from the previous study, the numerical value of the cut point for the present validation study (methylation score [MS] = 22.9). QM, quantitative methylation assay.

	NOTES

 
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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Submitted September 3, 2007; accepted June 10, 2008.

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