- © 2008 by American Society of Clinical Oncology
Clinical Importance of Estrogen Receptor-β Evaluation in Breast Cancer Patients Treated With Adjuvant Tamoxifen Therapy
- Naoko Honma,
- Rie Horii,
- Takuji Iwase,
- Shigehira Saji,
- Mamoun Younes,
- Kaiyo Takubo,
- Masaaki Matsuura,
- Yoshinori Ito,
- Futoshi Akiyama and
- Goi Sakamoto
- From the Research Team for Geriatric Diseases, Tokyo Metropolitan Institute of Gerontology; Departments of Breast Pathology and Cancer Genomics, Cancer Institute; Departments of Breast Oncology and Medical Oncology, Cancer Institute Hospital; Division of Clinical Trials and Research, Tokyo Metropolitan Cancer and Infectious Diseases Center, Komagome Hospital, Tokyo, Japan; and the Department of Pathology, Baylor College of Medicine, Houston, TX
- Corresponding author: Naoko Honma, MD, Research Team for Geriatric Diseases, Tokyo Metropolitan Institute of Gerontology, Sakaecho 35-2, Itabashi-ku, Tokyo 173-0015, Japan; e-mail: nhonma{at}tmig.or.jp
Abstract
Purpose The clinicopathologic importance of a second estrogen receptor (ER), ER-β, in breast cancers has been intensely studied; however, there is still no real consensus regarding the clinical utility of an ER-β assay, probably because of the lack of standardized methodology, the presence of several ER-β isotypes (ER-β1-5, and so on), and, more importantly, the lack of convincing data on whether the ER-β status provides clinically useful information over what is already provided by the traditional ER-α/progesterone receptor (PR) assay. A large and systematic study is needed to address these important issues.
Patients and Methods Archival materials of 442 invasive breast cancers from women treated with adjuvant tamoxifen monotherapy and with a long follow-up period (median, 11.1 years) were subjected to immunohistochemical study using three commercially available anti–ER-β antibodies that detect ER-β1-3 (ER-βN), ER-β1, and ER-βcx (ER-β2).
Results Positive staining for ER-βN or ER-β1 was associated with significantly better survival. By contrast, ER-βcx status did not influence survival. In multivariate analysis, ER-β1 status emerged as an independent predictor of recurrence and mortality. ER-β1 status was significantly associated with survival in postmenopausal, but not premenopausal, women. Importantly, ER-β1 positivity was associated with significantly better survival in patients with ER-α–negative/PR-negative or ER-α–negative/PR–negative/human epidermal growth factor receptor 2–negative (triple-negative) tumors, which are widely believed to be hormone unresponsive, have poor prognosis, and require chemotherapy.
Conclusion Immunohistochemical examination of ER-β1 in addition to ER-α and PR is clinically important in patients with breast cancer treated with tamoxifen monotherapy. Further studies are needed to confirm our findings.
INTRODUCTION
Estrogen plays important roles in the pathogenesis and development of breast cancer.1 Tamoxifen, which inhibits estrogen-stimulated growth of breast cancer cells by competitively binding to and blocking estrogen receptor (ER), has been a standard endocrine therapy for breast cancer for decades.2 Breast cancers that express ER and/or progesterone receptor (PR)3 are expected to respond better to endocrine therapies and to exhibit a more favorable prognosis than those that lack ER and PR. Immunohistochemical examination of the ER/PR status on formalin-fixed, paraffin-embedded tissue sections has been standardized and used clinically for several years as a marker associated with prognosis and the response to tamoxifen or other endocrine therapies.2
Since the discovery of a second ER, ER-β, in 1996,4 its role in the pathogenesis of breast cancer and its clinical importance have been hotly debated.5-23 In earlier studies, mRNA assays were mainly used; however, it has been reported that mRNA levels of ER-β do not correlate with ER-β protein level.14 A number of immunohistochemical studies on formalin-fixed, paraffin-embedded tissue sections have been reported; however, the antibodies or isotypes (e.g., ER-β1-5, and so on, wild type is ER-β1)24-26 used in these studies were different, making it difficult to compare the results. Furthermore, these studies also differed in their study populations, including the number of patients, menopausal status, ethnic background, type of adjuvant therapy, consideration of the status of classic ER (ER-α), and the follow-up period. A large and systematic study with a detailed patient population and treatment characteristics and that takes into consideration the status of ER-α and PR is needed to determine the clinical utility of ER-β status. In the present study, archival materials of 442 invasive breast cancers from women treated with adjuvant tamoxifen monotherapy, with complete clinicopathologic data and a long follow-up period, were subjected to immunohistochemical study using three commercially available anti–ER-β antibodies that detect ER-β1-3 (ER-βN), ER-β1, and ER-βcx (ER-β2).
PATIENTS AND METHODS
Patients
Among 5,325 Japanese patients with primary invasive breast cancer who underwent curative surgery with lymph node dissection at the Cancer Institute Hospital between 1983 and 1993, adjuvant tamoxifen monotherapy was administered to 599 patients. Eliminating patients with noninfiltrating carcinoma, carcinoma with microinvasion, stage IV tumors, male sex, bilateral carcinomas, no residual carcinoma after biopsy, and tamoxifen treatment of less than 6 months, 442 patients (8.3% of the total) were entered onto the present study. This study population has the following characteristics: age range of 29 to 81 years (mean, 56 years); 281 postmenopausal patients, 149 premenopausal patients, and 12 artificially menopaused patients; and a follow-up period ranging from 0.5 to 19.4 years (median, 11.1 years). Among these patients, 377 (dextran-coated charcoal method, n = 32; and enzyme immunoassay, n = 345) were biochemically ER- and/or PR-positive, whereas the biochemical ER and/or PR status was not known in 65 patients. For patients with biochemically unknown status, tamoxifen was administered according to the doctors’ judgment considering many factors, including lack of effective chemotherapy at that time, patients’ request, menopausal status, and tumor grade. The duration of adjuvant tamoxifen monotherapy was 0.5 to 8.8 years (mean, 2.8 years; median, 2.0 years; tamoxifen administered for < 2.0 years, n = 51; and tamoxifen administered for ≥ 2.0 years, n = 391).27 Histologically, 403 invasive ductal carcinomas not otherwise specified (91%), 18 mucinous carcinomas (4.1%), 15 invasive lobular carcinomas (3.4%), and six other histologic types (1.4%) were included.28 Grading was performed according to the Japan National Surgical Adjuvant Study of Breast Cancer protocol, which has been confirmed to reflect the prognosis of Japanese breast cancer patients and is routinely used in Japan.29,30 As previously reported,29 the difference in the recurrence rate was most apparent when grade I tumors were compared with grade II and III tumors.
The study protocol was approved by the Ethics Committee of Tokyo Metropolitan Institute of Gerontology. Informed consent was obtained from each patient.
Immunohistochemistry
Sections of formalin-fixed and paraffin-embedded tissue were used. Representative slides were selected for immunohistochemistry. Immunostaining was performed according to routine methods using a DAKO Autostainer (DAKO, Carpinteria, CA). For ER-α, PR, and human epidermal growth factor receptor 2 (HER-2), immunohistochemistry was performed according to the manufacturer's instructions using anti–ER-α mouse monoclonal antibody (clone1D5; DAKO), anti-PR mouse monoclonal antibody (clone PgR636; DAKO), and a HercepTest kit (DAKO), respectively. For ER-β examination, the following three anti–ER-β antibodies, all of which are commercially available and confirmed to work well for automatic staining, were used: anti–ER-β1-3 rabbit polyclonal antibody specific for the N-terminal amino acid sequences shared by ER-β1-3 (MYEB, supplied by M.Y., and also available from Chemicon International Inc, Temecula, CA),8 anti–ER-β1 mouse monoclonal antibody specific for the C-terminal amino acid sequences of ER-β1 (clone PPG5/10; DAKO), and anti–ER-β2 (ER-βcx) mouse monoclonal antibody specific for the C-terminal amino acid sequences of ER-βcx (clone 57/3; Serotec Ltd, Oxford, United Kingdom). For antigen retrieval, autoclave treatment was performed for 7 minutes for anti–ER-β1-3 polyclonal in citrate buffer, 15 minutes for anti–ER-β1 monoclonal, and 7 minutes for anti–ER-βcx monoclonal in Target Retrieval Solution pH 9 (DAKO). After blocking nonspecific activity, the sections were incubated for 30 minutes at room temperature with each anti–ER-β antibody (anti–ER-β1-3 diluted to 1:500, anti–ER-β1 diluted to 1:10, and anti–ER-βcx diluted to 1:20). The bound antibodies were detected using the appropriate rabbit or mouse EnVision + HRP kit (DAKO). Appropriate negative and positive controls were included in each batch of immunostains. Hereafter, hormone receptors immunostained by anti–ER-β1-3 N-terminus polyclonal, anti–ER-β1 monoclonal, anti–ER-βcx monoclonal, anti–ER-α monoclonal, or anti-PR monoclonal antibodies are conventionally referred to as ER-βN, ER-β1, ER-βcx, ER-α, or PR, respectively. The results of immunohistochemistry were assessed by two pathologists (N.H. and R.H.) in a blinded fashion, independently examining the whole slide. In most cases, the estimations of the two pathologists were identical, and discrepancies were resolved by joint review of the slides. Nuclear immunoreactivity for each of the hormone receptors was scored independently by evaluating the percentage of positively stained cancer cells; the cutoff value was set to 10% according to conventional criteria or other reports.8,15,31 HER-2 scoring was according to the designated procedures. An HER-2 score of 3+ was considered positive according to conventional criteria.
Statistical Analysis
The χ2 test for independence using a contingency table was used to compare the immunohistochemical status and various clinicopathologic factors. The Kaplan-Meier method with log-rank test was used to establish the value of each hormone receptor status as a predictor of disease-free survival (DFS) or overall survival (OS). The association of various clinicopathologic factors with patient outcome was assessed in univariate and multivariate analysis using a Cox proportional hazards model. In all instances, StatView 5.0 (SAS Institute, Cary, NC) was used. P < .05, when necessary dividing with the number of factors examined (Bonferroni adjustment), was considered significant.
RESULTS
Data were obtained from 442 patients for ER-β1, ER-α, PR, and HER-2; 440 patients for ER-βN; and 419 patients for ER-βcx. Examples of typical nuclear staining of tumor cells for ER-βN, ER-β1, and ER-βcx are shown in Figure 1. Epithelial cell cytoplasm was often stained to various degrees.11,22 Nuclear staining was sometimes observed in myoepithelial cells, fibroblasts, endothelial cells, and lymphocytes.22
Photomicrograph showing typical staining for anti–estrogen receptor (ER)-β1-3 polyclonal (ER-βN), anti–ER-β1 monoclonal (ER-β1), and anti–ER-βcx monoclonal (ER-βcx; immunoperoxidase staining with hematoxylin counterstaining, ×100). In addition to the nuclei of cancer cells, epithelial cytoplasm or nonepithelial cells exhibit immunoreactivity to various degrees.
Relationship Between the Status of ER-βs and Various Clinicopathologic Factors
As shown in Table 1, the status of all ER-βs significantly correlated with ER-α status. The other clinicopathologic factors exhibited no significant relationship with the status of ER-βs after Bonferroni adjustment.
Relationship Between Status of ER-βs and Clinicopathologic Factors in Invasive Breast Cancers
Survival Analysis According to the Status of ER-βs
ER-βN positivity was associated with significantly better clinical outcome (Fig 2), validating the results of the previous report.8 Because anti–ER-βN detects the N-terminal amino acid sequences commonly expressed in ER-β1 and ER-βcx, the relationship between clinical outcome and the differential expression of ER-β1 and ER-βcx was further examined using the same cutoff value to know whether the clinical value of ER-βN status is a result of ER-β1 status or ER-βcx status. ER-β1 status exhibited distinct differences in both DFS and OS, whereas ER-βcx did not show significant differences after Bonferroni adjustment, with the result that ER-βN exhibited intermediate features of ER-β1 and ER-βcx (Fig 2). Considering these results, the power of ER-βN as a predictor of survival is most likely a result of the power of ER-β1. Given this finding, ER-β1 status was used in the following analysis regarding clinical outcome.
Kaplan-Meier (A) disease-free survival and (B) overall survival curves according to the status of each estrogen receptor (ER)-β. (——) represents positive patients, and (···) represents negative patients. Marks represent censored data. P value was determined using the log-rank test. (*) Significant, P < .05/3 (Bonferroni adjustment).
Hormone Receptors Status, Other Clinicopathologic Factors, and Risk of Recurrence and Mortality
Univariate and multivariate analyses were performed to explore the relationship between recurrence/mortality and tumor size, nodal status, grade, and HER-2, ER-α, PR, and ER-β1 status (Table 2). In multivariate analysis, tumor size, nodal status, and ER-β1 status remained independent predictors of both recurrence and mortality.
Cox Univariate and Multivariate Analysis of Recurrence and Mortality in Invasive Breast Cancers With Adjuvant Tamoxifen Therapy
Survival Analysis According to ER-β1/ER-α Status
DFS and OS significantly differed according to ER-β1/ER-α status (Fig 3). ER-β1–positive patients exhibited the most favorable survival irrespective of ER-α status.
Kaplan-Meier (A) disease-free survival and (B) overall survival curves according to the status of estrogen receptor (ER)-β1/ER-α (black lines, ER-β1 positive/ER-α positive; red lines, ER-β1 positive/ER-α negative; yellow lines, ER-β1 negative/ER-α positive; blue lines, ER-β1 negative/ER-α negative). Marks represent censored data. P value was determined using the log-rank test. (*) Significant, P < .05.
Clinical Outcome According to ER-β1 Status Among ER-α–Negative/PR-Negative or ER-α–Negative/PR-Negative/HER-2–Negative Patients
Of the 442 patients, there were 60 ER-α–negative/PR-negative patients, among whom 43 patients were biochemically ER-positive and/or PR-positive; the biochemical ER/PR status was unknown in the remaining 17 patients. Fifty (83%) of 60 ER-α–negative/PR-negative patients were ER-β1 positive. The difference in DFS between ER-β1–positive and ER-β1–negative patients was striking in this group (Fig 4); only 11 (22%) of 50 ER-β1–positive patients experienced recurrence during the whole follow-up period, whereas eight (80%) of 10 ER-β1–negative patients experienced recurrence within 5 years. Also among ER-α–negative/PR-negative patients, ER-β1 status remained an independent predictor of recurrence and mortality in multivariate analysis; the hazard ratio of ER-β1 (negative v positive) was 4.398 (95% CI, 1.692 to 11.872; P = .004) for recurrence and 3.648 (95% CI, 1.220 to 10.912; P = .021) for mortality (Appendix Table A1, online only).
Kaplan-Meier (A) disease-free survival and (B) overall survival curves according to the estrogen receptor (ER)-β1 status among ER-α–negative/progesterone receptor (PR)–negative patients and ER-α–negative/PR-negative/human epidermal growth factor receptor 2 (HER-2)–negative patients. (——) represent ER-β1–positive patients, and (···) represent ER-β1–negative patients. Marks represent censored data. P value was determined using the log-rank test. (*) Significant, P < .05.
Fifty of 60 ER-α–negative/PR-negative patients were HER-2 negative, resulting in so-called triple-negative cancers.32 These patients also exhibited significant difference in survival according to ER-β1 status (Fig 4). ER-β1 remained an independent predictor of recurrence in multivariate analysis, including tumor size, nodal status, and grade; the hazard ratio of ER-β1 (negative v positive) was 3.401 (95% CI, 1.221 to 9.474; P = .019) for recurrence and 2.747 (95% CI, 0.854 to 8.836; P = .0901) for mortality.
ER-β1 Status and Survival According to menopausal Status or Duration of Tamoxifen Treatment
ER-β1 status exhibited a clear difference in survival among postmenopausal patients (P < .001 for DFS and OS) but not among premenopausal patients (P = .129 for DFS, P = .069 for OS; Appendix Fig A1, online only). Survival according to ER-β1 status was compared between patients treated with tamoxifen for less than 2 years (n = 51) and patients treated for 2 years or more (n = 391; Appendix Fig A2, online only).27 In patients treated with tamoxifen for less than 2 years, ER-β1 status was not associated with survival (P = .381 for DFS, P = .904 for OS). By contrast, patients treated with tamoxifen for 2 years or more exhibited a significant difference in survival according to ER-β1 status (P < .001 for DFS and OS). In patients with ER-β1–positive tumors, the clinical outcome was distinctly different according to the duration of tamoxifen treatment (< 2 v ≥ 2 years, P < .001 for DFS and OS).
DISCUSSION
There have been many reports examining the clinicopathologic importance of ER-β expression in breast cancers.5-23 Although there is no real consensus, some common themes are emerging, including the association of ER-β expression with ER-α, PR, and low tumor grade, which are factors usually associated with a better clinical outcome.6,7,22 In the present study, expression of ER-β was positively correlated with ER-α expression; however, no association was observed between ER-β and PR. A favorable clinical outcome for patients with ER-βN–positive tumors has been reported in one study with a relatively small number of patients.8 Using the same antibody and cutoff values used in that study by Mann et al8 but a different, larger, and well-characterized set of patients, our results in this study are in agreement with, and thus validate, the previously published results.8 Because anti–ER-βN recognizes more than one ER-β isoform, it is important to identify the isoform associated with clinical outcome to facilitate future clinical and investigational studies. Our results strongly indicate that this isoform is ER-β1. Two studies have examined the clinical importance of ER-β1 in tamoxifen-treated patients with breast cancers using the same monoclonal antibody as in the present study (clone PPG5/10).15,16 One study, which used the same cutoff value as in the present study, reported that ER-β1–positive tumors exhibited favorable survival,15 as found in the present study. Survival according to ER-β1/ER-α status was also almost identical to our results.15 The other study, which used a much higher concentration of ER-β1 antibody and a different cutoff value, reported the opposite result.16 Two immunohistochemical studies have examined the clinical value of ER-βcx among tamoxifen-treated breast cancers using a homemade polyclonal antibody; similar to our present study, neither showed a significant difference in survival according to ER-βcx status.18,23
Obviously, the most important questions for clinicians is whether the ER-β status provides clinically useful information over what is already provided by the traditional ER-α/PR assay. One of the promising findings in our study is that ER-β1 status is a significant predictor of clinical outcome in ER-α–negative/PR-negative cancers in univariate and multivariate analysis. Most of these patients were biochemically ER positive and/or PR positive and treated with tamoxifen. The dextran-coated charcoal method, which detects both ER-α and ER-β as ER, is known to cause discrepancies between biochemical and immunohistochemical assays; however, the discrepancy can be explained by the presence of ER-β1 only in four patients. Triple-negative breast cancers,32 which are negative for ER, PR, and HER-2, are increasingly debated in the oncology literature because of the limited treatment options, poorer prognosis, and partial overlap with basal-like tumors classified by extensive genetic profiling assays.33 We found that patients with triple-negative tumors who are treated with adjuvant tamoxifen have significantly better survival when the tumors are positive for ER-β1. Because the present study included a relatively small number of ER-α–negative/PR-negative and triple-negative patients, the clinical importance of this finding awaits validation in a larger patient cohort by other investigators.
It is not known at present whether the present results reflect the predictive value of ER-β1 for tamoxifen treatment or the natural history of tumors according to the ER-β1 status because tamoxifen-untreated patients were not included in the present study. If a tamoxifen-untreated group exhibits similar results to the tamoxifen-treated group, ER-β1 reflects the natural history of the tumors. The tumor-suppressive effect of ER-β in vivo and in vitro has been suggested by several authors.34-37 If ER-β1 positivity is not associated with a favorable clinical outcome in tamoxifen-untreated patients, ER-β1 is of predictive value for the response to tamoxifen treatment; tamoxifen exerts its antitumor activity through ER-β1. In our study, contrary to patients treated with tamoxifen for 2 years or more, ER-β1 status did not affect the clinical outcome in patients treated with tamoxifen for less than 2 years. Patients with ER-β1–positive tumors exhibited a favorable clinical outcome if treated with tamoxifen for 2 years or more. However, because tamoxifen treatment was stopped in 17 (33%) of the 51 patients treated for less than 2 years as a result of early recurrence, it is difficult to determine whether the lack of association between ER-β1 status and clinical outcome in these patients is caused solely by the short duration of tamoxifen treatment. A similar study on tamoxifen-untreated cancers is strongly desired.
ER-β1 status exhibited a significant effect on survival among postmenopausal but not among premenopausal women. The reason for this finding is not known at present; however, this finding suggests a relatively more important role for ER-β1 in the pathobiology of breast cancer in postmenopausal than premenopausal women. Estrogen metabolism drastically changes with menopause, with a marked decrease of serum estrogens resulting in a relative increase of androgens. In postmenopausal women, estrogen-metabolizing enzymes such as aromatase, steroid sulfatase, and 17β-hydroxysteroid dehydrogenase play important roles in the pathobiology of breast cancer.38,39 In such conditions, for example, androstenediol is one of the major sex steroid hormones present in postmenopausal breast tissue.39,40 Androstenediol, which has estrogenic function, is known to preferentially bind to ER-β over ER-α.41 The relative importance of the action of ER-β–androstenediol (or other steroid hormones) may be increased in postmenopausal cancer compared with premenopausal cancer.
In the present study, ER-α status, PR status, and HER-2 status did not show a significant association with survival. Furthermore, only 23 (5.2%) of 442 patients were HER-2 positive. Patients included in the present study are patients who were treated with adjuvant tamoxifen monotherapy based on the results of the biochemical ER/PR assay (ER positive and/or PR positive in 377 patients and not known in 65 patients), menopausal status, grade, and so on. This selection may be partly responsible for the seemingly nonsignificant effect of ER-α, PR, and HER-2 status on patient survival and the low proportion of HER-2–positive patients in the present study. Because race-specific factors may also influence our findings, considering the relatively favorable clinical outcome and low prevalence of lobular carcinomas in the Japanese population compared with the white population,42 additional studies with patient cohorts of other ethnic groups are needed to determine whether our findings are applicable to other patient populations. It should also be noted that the patients enrolled onto this study represent only 8.3% of our whole breast cancer population during the entry period, which may be considered a limitation of this study.
In conclusion, the expression of ER-β, particularly ER-β1, in breast cancer tissue is associated with significantly better survival in postmenopausal women treated with tamoxifen, even in patients with ER-α–negative/PR-negative or ER-α–negative/PR-negative/HER-2–negative (triple-negative) tumors. Immunohistochemical determination of ER-β1 status in breast cancer tissue provides clinically useful information over what is already provided by the traditional ER-α/PR assay. Further studies are needed to confirm our findings.
AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
The author(s) indicated no potential conflicts of interest.
AUTHOR CONTRIBUTIONS
Conception and design: Naoko Honma
Administrative support: Kaiyo Takubo, Goi Sakamoto
Provision of study materials or patients: Rie Horii, Takuji Iwase, Mamoun Younes, Yoshinori Ito, Futoshi Akiyama, Goi Sakamoto
Collection and assembly of data: Naoko Honma, Rie Horii, Takuji Iwase, Yoshinori Ito, Futoshi Akiyama, Goi Sakamoto
Data analysis and interpretation: Naoko Honma, Shigehira Saji, Masaaki Matsuura
Manuscript writing: Naoko Honma, Shigehira Saji, Mamoun Younes
Final approval of manuscript: Naoko Honma, Rie Horii, Takuji Iwase, Shigehira Saji, Mamoun Younes, Kaiyo Takubo, Masaaki Matsuura, Yoshinori Ito, Futoshi Akiyama, Goi Sakamoto
Appendix
Kaplan-Meier (A) disease-free survival and (B) overall survival curves according to the estrogen receptor (ER)-β1 status among postmenopausal and premenopausal patients. (——) represent ER-β1–positive patients, and (···) represent ER-β1–negative patients. Marks represent censored data. P value was determined using the log-rank test. (*) Significant, P < .05/2 (Bonferroni adjustment).
Duration of tamoxifen treatment (< 2 v ≥ 2 years) and Kaplan-Meier (A) disease-free survival and (B) overall survival curves according to the estrogen receptor (ER)-β1 status. (——) represents ER-β1–positive patients, and (···) represent ER-β1–negative patients. Marks represent censored data. P value was determined using the log-rank test. (*) Significant, P < .05/2 (Bonferroni adjustment).
Cox Univariate and Multivariate Analysis of Recurrence and Mortality in ER-α–Negative and PR-Negative Invasive Breast Cancers With Adjuvant Tamoxifen Therapy
Acknowledgments
We thank Tomoyo Kakita, Genkichi Iwakoshi, Kazuko Yokokawa, Sachiko Nishimura, and the technical staff of the Department of Pathology, Cancer Institute for their excellent technical support. We also thank Nobuhiro Harada, PhD, Department of Biochemistry, and Toshiaki Utsumi, MD, Department of Breast Surgery, Fujita Health University School of Medicine, for their useful comments.
Footnotes
-
Supported by Grant-in-Aid for Scientific Research No. 17590324 from the Japan Society for the Promotion of Science and Grant-in-Aid for Cancer Research No. 17-7 from the Ministry of Health, Labour and Welfare of Japan.
-
Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article.
- Received September 4, 2007.
- Accepted April 2, 2008.
