Clinical Cancer Research 14, 3494, June 1, 2008. doi: 10.1158/1078-0432.CCR-07-4557
© 2008 American Association for Cancer Research
Methylated NEUROD1 Promoter is a Marker for Chemosensitivity in Breast Cancer
Heidi Fiegl1,2,
Allison Jones1,
Cornelia Hauser-Kronberger6,
Georg Hutarew6,
Roland Reitsamer7,
Robin L. Jones8,
Mitch Dowsett8,
Elisabeth Mueller-Holzner3,
Gudrun Windbichler3,
Günter Daxenbichler3,
Georg Goebel4,
Christian Ensinger5,
Ian Jacobs1 and
Martin Widschwendter1
Authors' Affiliations: 1 Department of Gynecological Oncology, UCL Institute for Women's Health, University College London, London, United Kingdom; 2 Tyrolean Cancer Research Institute, Departments of 3 Gynecology, 4 Medical Statistics, Informatics, and Health Economics, and 5 Pathology, Medical University, Innsbruck, Austria; Departments of 6 Pathology and 7 Gynecology, General Hospital and Paracelsus University Salzburg, Salzburg, Austria; and 8 Department of Medicine, Royal Marsden Hospital, London, United Kingdom
Requests for reprints: Martin Widschwendter, Department of Gynecological Oncology, Institute for Women's Health, University College London, EGA Hospital, 2nd Floor Huntley Street, London WC1E 6DH, United Kingdom. Phone: 44-20-7380-9140; Fax: 44-20-7380-9033; E-mail: M.Widschwendter{at}ucl.ac.uk.
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Abstract
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Purpose: Chemotherapy can be an integral component of the adjuvant management strategy for women with early stage breast cancer. To date, no tool is available to predict or monitor the efficacy of these therapies. The aim of this proof-of-principle study was to assess whether NEUROD1 DNA methylation is able to predict the response to neoadjuvant and adjuvant chemotherapy.
Experimental Design: Recently, we showed that NEUROD1 DNA is differentially methylated in neoplastic versus nonneoplastic breast tissue samples. In this study, we used MethyLight and analyzed NEUROD1 methylation in (a) 74 breast cancer tissue samples, (b) two independent sets of pretreatment core biopsies of 23 (training set) and 21 (test set) neoadjuvantly treated breast cancer patients, and (c) pretherapeutic and posttherapeutic serum samples from 107 breast cancer patients treated with adjuvant chemotherapy.
Results: High-grade tumors showed higher NEUROD1 methylation levels. Estrogen receptor–negative breast cancers with high NEUROD1 methylation were 10.8-fold more likely to respond with a complete pathologic response following neoadjuvant chemotherapy. Patients with positive serum pretreatment NEUROD1 methylation, which persisted after chemotherapy, indicated poor relapse-free and overall survival in univariate and multivariate analyses (relative risk for relapse, 6.2; 95% confidence interval, 1.6-24; P = 0.008, and relative risk for death, 14; 95% confidence interval, 1.6-120; P = 0.02).
Conclusions: These data support the view that NEUROD1 methylation is a chemosensitivity marker in estrogen receptor–negative breast cancer.
Breast cancer is the most frequent malignancy among women in the industrialized world. To date, the presence or absence of metastatic involvement in the axillary lymph nodes is still the most powerful prognostic factor available for patients with primary breast cancer (1), although this is just an indirect measure reflecting the tendency of the tumor to spread. Recently, we showed that RASSF1A DNA methylation in serum is a marker of poor prognosis in women with breast cancer (2) and that this cancer-specific DNA alteration allows the monitoring of adjuvant tamoxifen therapy (3), which is applied mainly in estrogen receptor (ER)–positive tumors. To date, no tool is available to sufficiently predict or monitor the efficacy of neoadjuvant or adjuvant systemic chemotherapy which is frequently applied in ER-negative breast cancer.
We recently reported that stem cell polycomb group targets (PCGT) are up to 12-fold more likely to have cancer-specific promoter DNA hypermethylation compared with nontargets (4). This supports the idea of a stem cell origin of cancer whereby reversible gene repression is replaced by permanent silencing, forcing the cell into a perpetual state of self-renewal and therefore increasing the possibility for subsequent malignant transformation (4). A large number of PCGT genes have not yet been described to play a role in cancer and this could explain why non–tumor suppressor genes are found to be frequently hypermethylated in adult epithelial cancers. In a recent study, we analyzed the methylation status of 61 genes in breast cancer and nonneoplastic breast tissues of 15 patients and 15 healthy controls, respectively. NEUROD1 DNA methylation was the best discriminator between these different groups (4). In this proof-of-principle study, we focused on the role of NEUROD1 methylation in breast cancer biology. We analyzed tumor samples, pretreatment core biopsies, and pretherapeutic and posttherapeutic serum samples by means of MethyLight, a sensitive fluorescence-based real-time PCR technique (5). We found that NEUROD1 methylation is a marker for chemosensitivity in breast cancer.
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Materials and Methods
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Patients and samples. The following samples were analyzed:- Frozen breast tissue samples from 74 patients with breast cancer. All samples were collected during surgery at the Department of Obstetrics and Gynecology of the Innsbruck Medical University, Austria in compliance with and approved by the Institutional Review Board. Breast cancer specimens were obtained immediately after resection of the breast or lumpectomy. Specimens were brought to our pathologist, and a part of the tissue was pulverized under cooling with liquid nitrogen and stored at –70°C. Patients were 35 to 90 years old (mean age at diagnosis, 62 years). Other clinicopathologic features are shown in Table 1
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- Paraffin-embedded pretreatment core biopsies (formalin-fixed 16-gauge cores) from patients with breast cancer. Samples were obtained from the Departments of Pathology and Gynecology, General Hospital and Paracelsus University Salzburg (training set samples); the Department of Obstetrics and Gynecology, Medical University Innsbruck, Austria; and the Royal Marsden Hospital, London, United Kingdom (test set samples). All samples were collected at diagnosis prior to chemotherapy in compliance with and approved by the Institutional Review Boards. In the training set, we analyzed samples from 23 patients who received six cycles of anthracycline-based therapy. Twenty-one of 23 samples yielded sufficient amounts of DNA. Seven of 21 patients showed a complete pathologic response (CR; disappearance of the invasive cancer in the breast). Clinicopathologic features are shown in Table 2A
. For further evaluation, we analyzed samples from an independent test set from 21 patients. One patient received three cycles of a combination of cyclophosphamide, methotrexate, and 5-fluorouracil, 10 patients received four cycles, 9 patients received six cycles, and 1 patient received three cycles of an anthracycline-based therapy. Clinicopathologic features are shown in Table 2B.
- Pretherapeutic and posttherapeutic serum samples from 107 patients with breast cancer, treated at the Department of Gynecology and Obstetrics, Medical University Innsbruck, Innsbruck, Austria, with primary non–metastatic breast cancer. Serum samples were recruited from all patients diagnosed with breast cancer between September 1992 and February 2002 who met all the following criteria: (a) primary breast cancer without metastasis at diagnosis, (b) adjuvant treatment with chemotherapy (41 patients received an anthracycline-based therapy, 64 patients received a combination of cyclophosphamide, methotrexate, and 5-fluorouracil, and 2 patients received another kind of chemotherapy), (c) availability of serum samples at diagnosis and 1 year after treatment (a time when the patient has completed her chemotherapy), and (d) alive after 1 year. Hormone receptor status was determined by either radioligand binding assay or immunohistochemistry. Clinicopathologic features are shown in Table 3
. Patients' blood samples were drawn before or 1 year after therapeutic intervention. Blood was centrifuged at 2,000 x g for 10 min at room temperature and 1 mL aliquots of serum samples were stored at –30°C.
DNA isolation, bisulfite modification, and MethyLight analysis. Genomic DNA from fresh-frozen tissue samples or paraffin-embedded tissue samples were isolated using the DNeasy Tissue Kit (Qiagen) according to the manufacturer's protocol.
DNA isolation from serum samples, bisulfite modification, and MethyLight analysis was done as described previously (2). Briefly, two sets of primers and probes, designed specifically for bisulfite-converted DNA, were used; a methylated set for the gene of interest and a reference set, actin β (ACTB), to normalize for input DNA. The specificity of the reactions for methylated DNA was confirmed separately using SssI-treated (New England Biolabs) human WBC DNA (heavily methylated). The percentage of fully methylated molecules at a specific locus was calculated by dividing the GENE/ACTB ratio of a sample by the GENE/ACTB ratio of SssI-treated human WBC DNA and multiplying by 100. Primers and probes for NEUROD1 have been described recently (6).
Statistics. Descriptive analysis of obtained data was done and the median as well as interquartile ranges were given. Data from parametric distributed variables were shown as mean and SD. Differences in the percentage of methylated reference values between groups were analyzed by means of a two-sided Mann-Whitney U test. Survival analysis was done by using univariate Kaplan-Meier curves and Cox regression models. All statistical analyses were done with SPSS Software 10.0.
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Results
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Based on our recent study, NEUROD1 methylation is the best discriminator between breast cancer and nonneoplastic tissue samples (ref. 4; Supplementary Table S1). To further explore the role of NEUROD1 methylation in primary breast cancer, in this study, we first analyzed NEUROD1 methylation in 74 frozen primary breast cancer specimens. High-grade tumors showed higher NEUROD1 methylation levels (P = 0.03), whereas no other clinicopathologic feature was associated with NEUROD1 methylation (Table 1). The promoter of NEUROD1 is occupied by repressive regulators in human embryonic stem cells (7), which would be consistent with NEUROD1 DNA methylation marking cancer stem cells in the tumor. Although there is a highly significant increase in NEUROD1 methylation from nonneoplastic to breast cancer tissue (Supplementary Table S1), with higher levels in high-grade tumors (Table 1), surprisingly, NEUROD1 methylation in breast cancer is not an indicator of tumor aggressiveness, which is shown in a lack of association of NEUROD1 methylation and lymph node metastasis (Table 1) or survival (Table 4
). This rather surprising finding led us to the hypothesis that NEUROD1 methylation may be associated with other tumor features like responsiveness to systemic treatment in breast cancer.
To test this hypothesis, we used two in vivo experiments: NEUROD1 methylation analysis in core breast cancer biopsies taken prior to preoperative chemotherapy with complete pathologic response as the end point (model 1) and seroconversion of NEUROD1 methylation in serum DNA during adjuvant chemotherapy with survival as the end point (model 2). For model 1, we first analyzed DNA from pretreatment core biopsies from 23 patients with breast cancer (training set). Twenty-one of 23 samples yielded sufficient DNA and 7 of 21 patients showed a CR (Table 2A). Patients with a CR showed significantly higher NEUROD1 methylation levels in their pretreatment cancer cores (Fig. 1A
). To exclude the possibility that this association was merely a reflection of cellularity in the core, we adjusted for the percentage of tumor cells (reviewed by G. Hutarew, a pathologist who was blinded for the chemotherapy response) and still observed a significant (P = 0.006) association between pretreatment core NEUROD1 methylation and response to neoadjuvant chemotherapy.
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Fig. 1. NEUROD1 DNA methylation in the pretreatment breast cancer core biopsies of the training set. A, samples stratified by response (PMR, percentage of methylated reference; CR, complete pathologic response; PR, partial response; Mann-Whitney U test, P = 0.025). B, samples stratified by ER status (Mann-Whitney U test, P = 0.024 for ER-negative samples, P = 0.28 for ER-positive samples).
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As ER-negative tumors are more likely to respond to neoadjuvant chemotherapy (8–10), we analyzed the association of CR and NEUROD1 methylation separately in ER-negative and ER-positive tumor samples. Although the numbers are small, the association between NEUROD1 methylation and response to neoadjuvant chemotherapy was retained in ER-negative cancers (Mann-Whitney U test; P = 0.02; Fig. 1B).
In order to validate these finding and to calculate the predictive potential of NEUROD1 methylation, we analyzed an independent test set of 21 core biopsies taken prior to the start of neoadjuvant chemotherapy from patients with ER-negative breast cancer (Table 2B). We classified NEUROD1 methylation as low (n = 11) or high (n = 10) using the median percentage of methylated reference value (PMR = 2.18) as the cutoff. Eight of 10 (80%) women with high NEUROD1 methylation and 3 of 11 (27%) women with low NEUROD1 methylation in their core biopsy had a CR. Using a logistic regression model, and adjusting for age and HER2 status, high NEUROD1 methylation in ER-negative pretreatment breast cancer biopsies was associated with a 10.8-fold increased likelihood for a CR following neoadjuvant chemotherapy [95% confidence interval (95% CI), 1.1-106.4; P = 0.042]. This means that NEUROD1 methylation had a sensitivity of 80% (44.4-96.3) and a specificity of 72% (39.0-92.0) to predict complete pathologic response in women treated with neoadjuvant chemotherapy. In our second model, we assessed whether serum NEUROD1 methylation was able to predict the response to adjuvant chemotherapy in patients with primary breast cancer. In previous articles, we have shown that DNA methylation of specific genes in circulating serum DNA is a marker for poor prognosis (2) and a tool to monitor adjuvant tamoxifen treatment (3). If our hypothesis is true that NEUROD1 methylation is a marker for chemosensitivity in breast cancer, we would expect that women whose serum NEUROD1 methylation was positive before but not detectable after adjuvant chemotherapy have an improved relapse-free and overall survival as their chemosensitive tumor cells have been eliminated. We identified 107 patients who received adjuvant chemotherapy due to primary non–metastatic breast cancer and from whom we have stored both pretreatment and postchemotherapy serum samples. The characteristics of these patients are shown in Table 3A. Pretreatment NEUROD1 serum DNA methylation was more prevalent in postmenopausal women, whereas no difference in any of the other clinicopathologic features could be observed (data not shown). In the group of 21 ER-negative patients with positive pretreatment NEUROD1 methylation in their serum, the persistence of NEUROD1 DNA methylation after chemotherapy indicated poor overall and relapse-free survival in the univariate analysis (Fig. 2
; Table 5
). The characteristics of these patients are shown in Table 3B. Using a Cox multiple-regression analysis which included tumor size, grade, lymph node metastasis, and menopausal status, the persistence of methylated NEUROD1 serum DNA was the only predictor of poor outcome (relative risk for relapse, 6.2; 95% CI, 1.6-24; P = 0.008; relative risk for death, 14; 95% CI, 1.6-120; P = 0.02). No association between serum NEUROD1 DNA methylation and response to adjuvant chemotherapy could be observed for patients with ER-positive breast cancer (data not shown).
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Fig. 2. Kaplan-Meier survival curves and NEUROD1 DNA methylation status in serum samples. Overall (A) and relapse-free survival (B) of 21 ER-negative primary breast cancer patients with positive NEUROD1 methylation in pretreatment serum. Broken lines, negative serum NEUROD1 methylation after chemotherapy; continuous lines, positive serum NEUROD1 methylation after chemotherapy.
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Table 5. Univariate analysis of 21 patients with ER-negative primary breast cancer with positive NEUROD1 methylation in pretreatment serum
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Discussion
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Neoadjuvant chemotherapy has been widely used prior to surgery for locally advanced breast cancer (11, 12). Response to this kind of therapy has been shown to be a valid surrogate marker of survival and facilitates breast-conserving surgery (13–15). However, current clinical and pathologic markers poorly predict response to neoadjuvant chemotherapy. In our study, ER-negative breast cancers with high NEUROD1 methylation were more likely to respond with a complete pathologic response following neoadjuvant chemotherapy.
Predictive factors in adjuvant breast cancer therapy are limited to ER, progesterone receptor, and HER-2/neu. These markers are used to predict response to hormonal treatment and herceptin, respectively (16, 17). Recently, HER-2/neu in serum was shown to be a significant predictor of response to neoadjuvant anthracycline-based chemotherapy for breast cancer, whereas the HER-2/neu status of tumor tissue did not correlate with response to treatment (18). Furthermore, HER-2/neu overexpression was identified as a major prognostic factor in patients with stage II and III breast cancer treated with a neoadjuvant docetaxel and epirubicin combination (19). Despite these findings, a more extensive range of predictive markers is highly needed in order to extend the range of individualized therapies for patients with breast cancer.
The biological characteristics of circulating tumor cells are poorly understood despite their potential contribution towards the formation of distant metastases. Up until recently, only a limited number of reports examined the occurrence of circulating tumor cells in the context of systemic therapy for primary or metastatic breast cancer. It has been shown that circulating tumor cells are present in a substantial fraction of breast cancer patients undergoing systemic therapy (20). These circulating tumor cells are usually nonproliferative, and a fraction of these cells seem to be resistant to chemotherapy (20). Only very limited data is available regarding specific characterization of these circulating tumor cells. In our proof-of-principle study, we described NEUROD1 methylation, a marker which may act as a surrogate for breast cancer cells which are responsive to chemotherapy. Expression of cyclooxygenase-2 has recently been shown to be a marker of doxorubicin-resistant breast cancer (21). In addition, inhibitors of cyclooxygenase-2 increase doxorubicin-induced cytotoxicity (22), and this is at least in part due to cyclooxygenase-2–mediated up-regulation of MDR1/P-glycoprotein (MDR1/P-gp; refs. 23, 24), an energy-dependent pump that participates in multidrug resistance. In addition, cyclooxygenase-2–derived prostaglandin E2 protects embryonic stem cells from apoptosis (25). Interestingly, we observed a strong inverse correlation of cyclooxygenase-2 expression and NEUROD1 methylation in ER-negative breast cancer specimens (correlation coefficient r = –0.4; P = 0.03; Supplementary Fig. S1), which supports our hypothesis that NEUROD1 methylation is a surrogate for the status of the cell associated with chemosensitivity. Further studies will be needed to show whether NEUROD1 upstream regulators (e.g., members of the polycomb repressor complex) have a particular effect on the responsiveness of tumor cells to chemotherapy.
In summary, this is the first study describing a DNA-based marker which is able to predict the response to neoadjuvant as well as adjuvant chemotherapy in a solid tumor independent of gene transcription and the source of DNA analyzed.
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Disclosure of Potential Conflicts of Interest
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No potential conflicts of interest were disclosed.
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Acknowledgments
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We thank I. Gaugg, A. Wiedemair, E. Perkmann, M. Chamson, and V. Stivic for their excellent technical assistance, and S. Taucher and C. Marth for providing clinical samples and patient data.
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Footnotes
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Grant support: European Union (FP6-016467, Biognosis) and FWF Austrian Science Fund (L69-B05 and P15995-B05; M. Widschwendter), and the Jubiläumsfonds der Österreichischen Nationalbank project no. 11178 (H. Fiegl).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).
Received 10/ 5/07;
revised 1/29/08;
accepted 2/ 4/08.
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Abstract
Materials and Methods
