Breast Cancer Outcomes as Defined by the Estrogen Receptor, Progesterone Receptor, and Human Growth Factor Receptor-2 in a Multi-ethnic Asian Country

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1 DOI /s ORIGINAL SCIENTIFIC REPORT Breast Cancer Outcomes as Defined by the Estrogen Receptor, Progesterone Receptor, and Human Growth Factor Receptor-2 in a Multi-ethnic Asian Country S. Subramaniam 1 N. Bhoo-Pathy 2 N. A. Taib 3 G. H. Tan 3 M. H. See 3 S. Jamaris 3 G. F. Ho 4 L. M. Looi 5 C. H. Yip 3 Société Internationale de Chirurgie 2015 Abstract Introduction Breast cancer can be divided into four subtypes based on the expressions of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor-2 (HER2). Each subtype has different clinicopathological features and outcomes. Objective To compare the clinicopathological features and survival of ER and/or PR positive HER2 negative (ER?PR?HER2-, ER?PR-HER2- or ER-PR?HER2-), ER and/or PR positive HER2 positive (ER?PR?- HER2?, ER?PR-HER2? or ER-PR?HER2?), ER negative PR negative HER2 positive (ER-PR-HER2?), and ER negative PR negative HER2 negative (ER-PR-HER2-) subtypes. Methods 1957 patients with Stage 1 3 breast carcinoma diagnosed between Jan 2005 and Dec 2011 were categorized into the four subtypes. The clinicopathological features between the subtypes were compared using v 2 test. Kaplan Meier analysis was performed to estimate 5-year overall survival. Multivariate Cox regression was used to determine the association between subtypes and mortality adjusted for age, ethnicity, stage, pathological features, and treatment. Results ER-PR-HER2? and ER-PR-HER2- subtypes were associated with younger age, larger tumors, and higher grade. There was no difference in the 5-year survival of the ER-PR-HER2? and ER-PR-HER2- subtypes (75.1 and 74.4 %, respectively) and survival was poorer than in the ER and/or PR positive HER2 negative and ER and/or PR positive HER2 positive subtypes (87.1 and 83.1 %, respectively). Only 9.5 % of women with HER2 positive breast cancer had access to trastuzumab. Conclusion In a low resource setting with limited access to trastuzumab, there is no difference in survival between the ER-PR-HER2? and ER-PR-HER2- subtypes of breast cancer. & C. H. Yip chenghar.yip@gmail.com S. Subramaniam shridevi@crc.gov.my N. Bhoo-Pathy ovenjjay@gmail.com N. A. Taib nuraish@gmail.com G. H. Tan giehooi@yahoo.com M. H. See smhoong76@gmail.com S. Jamaris ahshun82@gmail.com G. F. Ho gwofuang@gmail.com

2 Introduction Breast cancer is a heterogeneous disease that is made up of several distinct entities with different biological characteristics and clinical behaviors. In 2000, gene expression using DNA microarray identified 4 subtypes of breast cancer, namely, Luminal A, Luminal B, HER2 (Human epidermal growth factor 2) overexpressing, and Basal-like, with differences in survival, demographics, and tumor characteristics. [1] However because gene expression using microarray is not used in routine clinical practice, studies were limited to small numbers. Immunohistochemical (IHC) expression of estrogen receptor (ER) protein, progesterone receptor (PR) protein, and human epidermal growth factor-2 (HER2) oncoprotein is routinely available, and often used as a practical substitute for the more expensive molecular subtyping, which are not available for population-based or cohort studies. While the ER?PR?HER2- tumor is a surrogate for Luminal A breast cancer, the IHC surrogate for Luminal B is imprecise. While some groups consider any ER?PR?HER2? as Luminal B [2], others have added a Luminal B HER2- group where ER is positive, HER2 negative and at least one of: Ki67 high (defined as [14 %) and PR negative or low (defined as \20 %) [3]. Some have also used Grade 3 as a surrogate for Ki67 which is not readily available [4]. Objectives The objective of this study is to determine the proportions of the 4 subtypes of breast cancer based on immunohistochemical assessment of ER, PR, and HER2 in women presenting with breast cancer in a multiethnic Malaysian setting, and to investigate the differences in patients demography, tumor characteristics, and overall survival between the four subtypes L. M. Looi looilaimeng@gmail.com National Clinical Research Centre, Kuala Lumpur Hospital, Kuala Lumpur, Malaysia Department of Social and Preventive Medicine, Julius Center University of Malaya, University of Malaya, Kuala Lumpur, Malaysia Department of Surgery, University Malaya Medical Centre, Kuala Lumpur, Malaysia Department of Clinical Oncology, University Malaya Medical Centre, Kuala Lumpur, Malaysia Department of Pathology, University Malaya Medical Centre, Kuala Lumpur, Malaysia Methodology Patient population The University Malaya Medical Centre (UMMC) Breast Cancer Registry, which prospectively collects clinicopathological data from women newly diagnosed with breast cancer since 1993 was used for this study. The registry was approved by the Ethical Review Committee of UMMC. Because PR and HER2 were only routinely done from 2004, 2821 consecutive Malaysian patients who were newly diagnosed with invasive breast cancer in University Malaya Medical Centre (UMMC), Malaysia between 1st Jan 2005 and 31st Dec 2011 were included in this study. The patients age at onset, self-reported ethnicity, stage of disease, size, tumour grade, ER, PR, and HER2 status were evaluated from pathological and clinical reports. The patients were staged according to the American Joint Commission on Cancer (AJCC) Cancer Staging Manual 6th Ed. [5]. Patients survival status was confirmed by obtaining the data from National Registration Department. Of the 2821 patients, 324 (11.5 %) had de novo metastatic breast cancer. In the metastatic breast cancer group, 15.1 % were not tested for HER2, while 7.7 and 10.5 % did not have known ER or PR status, respectively. Hence, metastatic breast cancer was excluded from further analysis since this would hamper interpretation. Of the remaining 2497 patients with Stage 1 3 breast cancer, only 2.4, 2.9, and 4.3 % had unknown ER, PR, and HER2 status, respectively. 426 patients (17.8 %) had an equivocal HER2 status. Given that fluorescence in situ hybridization (FISH) was not done for a large proportion of patients in this group, they were excluded leaving 1957 patients for further analysis. Immunohistochemistry for ER, PR, and HER2 Immunohistochemistry using standard Envision methodology (DAKO kit, performed manually) for estrogen and progesterone receptor protein expression, and c-erbb-2/ HER-2 overexpression was performed on 4-micron-thick microtomed sections of formalin-fixed paraffin-embedded tumor biopsies or excised tumor tissue. The primary antibodies were obtained commercially and dilutions used were ER clone SP1 (Neomarkers, USA) at 1:100 dilution, PR clone PgR636 (DAKO, Denmark), at 1:200 dilution; c-erbb- 2 (HER2) (DAKO, Denmark Code A0485) at 1:600 dilution. The antigen retrieval solution was Tri-EDTA at ph9. The tumor was deemed ER or PR positive when 10 % or more of the invasive tumor nuclei were stained for the respective antibody, regardless of staining intensity. The c-erbb2/her-2 expression was categorized according to ASCO CAP guidelines 2007 [6] as 0 (no

3 staining), 1? (weak incomplete membrane staining in any proportion or weak, complete membrane staining in\10 % of cells), 2? (weak, complete membrane staining in[10 % of cells, or complete intense membrane staining of [10 % but \30 % of invasive tumor cells), or 3? (complete intense membrane staining of [30 % of invasive tumor cells). Expressions of 0 or 1? were regarded as negative. Only tumors with 3? expressions were regarded as HER2 overexpressed (positive). Expressions of 2? were regarded as equivocal for overexpression and would require further in situ hybridization testing for HER2 gene amplification. However, since no in situ hybridization testing (with FISH or SISH) was available during the period of this study, this category was excluded from analysis. Statistical analysis From the three biomarkers, patients were grouped into 4 IHC subtypes i.e., ER and/or PR positive HER2 negative (ER?PR?HER2-, ER?PR-HER2-, or ER-PR?- HER2-), ER and/or PR positive HER2 positive (ER?PR?HER2?, ER?PR-HER2?, or ER-PR?- HER2?), ER negative PR negative HER2 positive (ER-PR-HER2?), and ER negative PR negative HER2 negative (ER-PR-HER2-, or triple negative). The demography and tumor characteristics between the subtypes were compared using v 2 test. Patients were followed up from the date of diagnosis, to date of death (all-cause), or censored on the date of last mortality update with the National Registration Department (31st March 2014). Overall survival of patients was compared between different IHC subtypes using Kaplan Meier method [7]. To adjust for baseline differences in demography, tumor characteristics, and treatment between the IHC groups, we used Cox regression analysis [8]. Prognostic factors that were included in the multivariable Cox model was selected based on a priori knowledge of their associations with breast cancer survival; age at diagnosis, ethnicity, tumor size, number of pathologically positive axillary lymph nodes, tumor grade, lymphovascular invasion, locoregional treatment, neoadjuvant chemotherapy, and adjuvant chemotherapy. Two-tailed p value of less than 0.05 and 95 % confidence intervals (CI) for HR not including 1 was considered statistically significant. All statistical analyses were performed using SPSS for Windows version 20.0 Table 1 Breast cancer clinicopathological features: Jan 2007 Dec 2011 Characteristics Overall n = 1957 Age, n (%) \ (6.1) (37.5) (41.1) [ (15.2) Race, n (%) Chinese 949 (48.5) Malay 747 (38.2) Indian 245 (12.5) Others 16 (0.8) Stage, n (%) (28.6) (44.6) (27.4) Lymph node, n (%) Negative 1079 (57.4) Positive 801 (42.6) Unknown 77 Grade, n (%) (9.7) (49.1) (41.2) Unknown 355 Tumour Size, n (%) B 2 cm 756 (39.4) [ 2 5 cm 921 (47.9) [ 5 cm 244 (12.7) Unknown 36 Estrogen Receptor, n (%) Positive 1159 (59.2) Negative 798 (40.8) Progesterone Receptor, n (%) Positive 1022 (52.2) Negative 935 (47.8) Human Epidermal Growth Receptor, n (%) Positive 644 (32.9) Negative 1313 (67.1) IHC subtypes, n (%) ER and/or PR positive HER2 negative 929 (47.5) ER and/or PR positive HER2 positive 293 (15.0) ER negative PR negative HER2 positive 351 (17.9) ER negative PR negative HER2 negative 384 (19.6) Results Table 1 shows the demographic features of the 1957 patients with Stage 1 3 breast cancer. The majority were Chinese (48.5 %). The median age was 53 years old. Of those who had axillary dissection done, 40.9 % were lymph node positive. Approximately 50 and 40 % were Grade 2 and 3, respectively. The majority of patients (55.7 %) had tumors measuring more than 2 cm, while 44.6 % had Stage 2 disease.

4 Table 2 Comparison of four subtypes of breast cancer: Jan 2007 Dec 2011 ER and/or PR positive HER2 negative (%) ER and/or PR positive HER2 positive (%) ER negative PR negative HER2 positive (%) ER negative PR negative HER2 negative (%) p Value Race Chinese 466 (49.1) 140 (14.8) 164 (17.3) 179 (18.9) Malays 328 (43.9) (16.5) 135 (18.1) 161 (21.6) Indians 126 (51.4) 029 (11.8) 048 (19.6) 042 (17.1) Others 009 (56.2) 001 (06.2) 004 (25.0) 002 (12.5) Age \ (04.0) 019 (06.5) 023 (06.6) 041 (10.7) \ (37.6) 118 (40.3) 135 (38.5) 132 (34.4) (39.7) 116 (39.6) 161 (45.9) 159 (41.4) [ (18.7) 040 (13.7) 032 (09.1) 052 (13.5) Stage (32.8) 86 (29.4) 67 (19.1) 101 (26.3) \ (42.1) 119 (40.6) 170 (48.4) 182 (47.4) (25.1) 88 (30) 114 (32.5) 101 (26.3) Size B 2 cm 406 (44.4) (42.7) 96 (28.2) 131 (34.7) \0.001 [ 2 5 cm 426 (46.6) 122 (42.4) 178 (52.4) 195 (51.6) [ 5 cm 083 (09.1) 043 (14.9) 066 (19.4) 052 (13.8) Lymph node Negative 513 (57.1) 152 (55.1) 180 (54.1) 234 (62.7) Positive 385 (42.9) 124 (44.9) 153 (45.9) 139 (37.3) Grade 1 (16.3) 17 (6.9) 6 (2.1) 10 (3.1) \ (61.2) 135 (54.4) 104 (37) 86 (26.9) (22.4) 96 (38.7) 171 (60.9) 224 (70) Type of Surgery No surgery 31 (3.3) 17 (5.8) 21 (6) 18 (4.7) Mastectomy 645 (69.4) 198 (67.6) 269 (76.6) 270 (70.3) Lumpectomy 253 (27.2) 78 (26.6) 61 (17.4) 25 (25) Chemotherapy No 366 (42.3) 96 (35.7) 83 (26.1) 78 (22.7) \0.001 Yes 500 (57.7) 173 (64.3) 235 (73.9) 266 (77.3) Hormone therapy No 51 (5.8) 15 (5.5) 275 (93.9) 279 (91.2) \0.001 Yes 829 (94.2) 260 (94.5) 18 (6.1) 27 (8.8) The majority of patients had ER and/or PR positive HER2 negative (47.5 %) tumors, whereas 19.6 % had ER negative PR negative HER2 negative. The remainder (32.9 %) was HER2 positive, either ER and/or PR positive HER2 positive, (15 %) or ER negative PR negative HER2 positive (17.9 %). Table 2 shows the association of ethnicity, age, stage, size, lymph node involvement, and grade with the four IHC subtypes. In univariable analysis, ethnicity and lymph node involvement was not significantly associated with IHC subtypes. However, patients with ER negative PR negative HER2 negative (triple negative breast cancer) and ER negative PR negative HER2 positive tumors were younger, and had tumors, which were larger, of higher grade, and of more advanced stages compared to the other subtypes. Patients with ER negative PR negative HER2 positive cancers had larger tumors and later stages than their counterparts with triple negative breast cancer (TNBC), whereas TNBC were younger and had higher grade tumors. Overall, 62.1 % of patients received chemotherapy; ER negative PR negative HER2 positive

5 and TNBC were more likely to receive chemotherapy % of women who were ER and/or PR positive received tamoxifen. Only 62 out of 646 HER2 positive patients (9.6 %) received trastuzumab. Median survival in the overall cohort of patients with stage I to III breast cancer was 7.7 years (95 % CI years). Figure 1 shows the cumulative overall survival of the four subtypes of breast cancer. The 5-year overall survival in ER and/or PR positive HER2 negative, ER and/or PR positive HER2 positive, ER negative PR negative HER2 positive, and ER negative PR negative HER2 negative subtype were 87.1 %(95 % CI %), 83.1 %(95 %CI %), 75.1 %(95 %CI %), and 74.4 %(95 %CI %), respectively. Following multivariable Cox regression as in Table 3, the difference in overall survival of the ER and/or PR positive HER2 positive subtypes against the ER and/or PR positive HER2 negative subtypes was not found to be significant (HR 1.26 (95 %CI 0.91,1.74). However, compared to ER and/or PR positive HER2 negative subtype, both the ER negative PR negative HER2 positive and ER negative PR negative HER2 negative subtypes were associated with substantially higher risk of mortality ranging between 2- and 2.5-fold; HR 1.97 (95 %CI 1.47,2.63), and 2.51(95 %CI 1.88, 3.35), respectively. Discussion Testing for ER, PR, and HER2 is considered critical to the management of breast cancer in this current day and age. In low- and middle-income countries, testing may still be not routine and quality assurance remains a problem. Resource limitations may impact tissue handling and processing that can result in falsely negative results in the receptors [9]. It has been noted that inter-laboratory variations in assessment of ER and PR exist even in developed countries [10]. Another issue is that the definition of ER and PR positivity has changed with the recent guideline stating that 1 % rather than 10 % should be the cutoff used to define ER and Fig. 1 Comparison of overall survival in four subtypes of breast cancer

6 Table 3 Factor associated with overall survival of breast cancer patients in Asian setting Variable Univariable odds ratio (95 % CI) a Multivariable odd ratio (95 %CI) b Molecular subtype ER and/or PR positive HER2 negative ER and/or PR positive HER2 positive 1.43 ( ) c 1.26 (0.91, 1.74) ER negative PR negative HER2 positive 1.95 (1.49, 2.56) c 1.97 (1.47, 2.63) c ER negative PR negative HER2 negative 2.04 (1.57, 2.65) c 2.51 (1.88, 3.35) c Age, years 1.02 (1.01, 1.02) c 1.01 (1.00, 1.02) c Race Chinese Malay 1.12 (0.89, 1.40) 1.01 (0.80, 1.28) Indian 1.36 (1.00, 1.85) c 1.25 (0.91, 1.71) Others 1.52 (0.62, 3.72) 1.21 (0.49, 3.00) Tumor size, cm 1.16 (1.13, 1.18) c 1.09 (1.06, 1.13) Axillary lymph nodes (2.26, 4.01) c 3.14 (2.30, 4.29) c (3.05, 5.76) c 4.04 (2.85, 5.74) c 10 or more 7.38 (5.46, 9.98) c 5.84 (4.11, 8.30) c Tumor grade (1.01, 2.69) c 1.20 (0.72, 2.00) (1.32, 3.50) c 1.19 (0.70, 2.01) Lymphovascular invasion Absent Present 2.09 (1.66, 2.64) c 1.52 (1.17, 1.96) c Neoadjuvant chemotherapy Yes 3.06 (2.23, 4.21) c 1.13 (0.71, 1.80) No Locoregional treatment No Mastectomy 2.34 (0.17, 0.32) c 0.64 (0.39, 1.03) Breast conserving therapy 0.12 (0.08, 0.17) c 0.49 (0.29, 0.83) c Chemotherapy Yes 1.00 (0.80, 1.25) 0.52 (0.39, 0.67) c No a Derived using a enter univariable logistic regression model b Derived using a enter multivariable logistic regression model c Statistically significant PR positivity [11]. However for this study, a cut-off point of 10 % was used, as this was the standard criteria during the years of the study. Guidelines have been set for the standardized testing and reporting of HER [2, 12] but these are in use in high income countries. Data on the prevalence of HER2 positive

7 breast cancer in Asia is limited. Most studies use immunohistochemistry to assess HER2 status but vague definitions of positivity and the lack of a standard reliable HER2 assessment method available to patients across Asia has been reported, leading to a rather wide variation of positive rates from 6 to 65 % [13]. In the current study, 17.8 % had an equivocal HER2 result, which in high income countries would lead to reflexive testing by FISH. Unfortunately, FISH was not available in UMMC for a large proportion of patients during the study period. Hence the dilemma would be where to place these equivocal cases. The proportion of equivocal cases should not exceed 15 % [14] and can be reduced further with the use of more specific antibodies [15]. Because we were unable to resolve where these equivocal cases actually belong to, equivocal HER2 results were excluded from further analysis. In the metastatic setting, where surgery is not carried out or the tumor is inoperable, all three molecular markers may not be carried out, especially HER2 because of the extra costs incurred. Although hormonal therapy with tamoxifen for hormone positive breast cancer is fairly cheap and affordable, the same is not true of treatment with the anti-her2 therapy such as trastuzumab, which is not given to patients in the metastatic setting unless the patients are able to pay for the treatment themselves. Trastuzumab was not provided by the health services in Malaysia both in the adjuvant or metastatic setting during the years of the study because of the high cost incurred. However trastuzumab was available to those who could afford to pay for the treatment themselves. Since knowing the HER2 status will not change the management of the patient, the need to know is more for academic rather than practical reasons. Because the HER2 status was not available for a significant proportion of the metastatic breast cancers (15.1 %) in our study, metastatic breast cancers were excluded from the analysis. Since all the three markers in our study were done by a single laboratory which subscribes to the UK National External Quality Assessment Scheme (UK NEQAS) for immunocytochemistry, we believe that the results of the markers are accurate especially as the rates of HER2 positive cancers (32.9 % after excluding the equivocal HER2) and triple negative breast cancer (19.6 %) is similar to other reports on Asian women [16, 17]. HER2? breast cancers have been reported to be higher in Asians compared to Caucasians. The HER2 positive rate was reported to be 36 % in Koreans in the California Breast Cancer Registry. (odds ratio (OR) 1.8, 95 % confidence interval (CI) , compared to non-hispanic White women) [16]. In China, a triple negative breast cancer incidence of 17 % has been reported in a large series of Chinese women with breast cancer [17]. A study in Sarawak, Malaysia showed that HER2 positive and triple negative breast cancer predominate in the Asian region, with significant differences among the different ethnic groups, with Malays having significantly more HER2 positive tumors than Chinese [18]. However in the current study, there were no significant differences in the proportions of the IHC subtypes among the Malay, Chinese, and Indian women. It has been hypothesized that risk factors for different subtypes vary markedly, and Westernized populations are more likely to have factors that increase the risk of ER and/or PR positive HER2 negative breast cancer [19]. Because of the controversies regarding the IHC surrogates for the molecular subtypes, for this study, to avoid any confusion, we decided to study the different categories of breast cancer according to the expression of ER, PR, and HER2 by IHC. While HER2 overexpressing and triple negative are clearly represented by the IHC subgroups of ER negative PR negative HER2 positive and ER negative PR negative HER2 negative respectively, the IHC surrogates of Luminal A and Luminal B are still under debate. To add to the confusion, the St Gallens guidelines suggest using Ki67 or the percentage of PR positivity to differentiate between Luminal A and Luminal B, both of which are not practical in a low resource setting [3]. The IHC subtypes are associated with age and grade, with HER2 overexpressing and triple negative breast cancer being significantly associated with high grade [20, 21]. In the current study, as well as being younger and higher grade, ER negative PR negative HER2 positive, and triple negative breast cancers were also associated with larger size, but there is no significant difference in involved lymph nodes among the different subgroups. In fact, triple negative breast was the least likely subtype to have involved lymph nodes in the current study. The paradox of larger size and less lymph node involvement in triple negative breast cancer has been reported before [22]. IHC subtypes are also significantly related to stage of disease, with the ER negative PR negative HER2 positive subtype presenting with more advanced stages in this study. Because ER negative PR negative HER2 positive and triple negative breast cancer are more likely to be associated with poor prognostic factors, survival has been reported to be poorer in these subtypes in several studies. A study on 496 incident cases of breast cancer from the Carolina Breast Cancer Study (between May 1993 and Dec 1996), where the IHC surrogates for each subtype is similar to the current study, showed that breast cancer specific survival differed by subtype with the poorest survival in the ER negative PR negative HER2 positive subtype and the triple negative subtype. This was before the era of anti- HER2 therapy [2]. With wider use of trastuzumab, the survival of HER2 overexpressing breast cancer has improved significantly [23]. The current study shows no difference in survival between the ER negative PR negative

8 HER2 positive and the ER negative PR negative HER2 negative subtype, which is expected since there was very limited access to trastuzumab during this period. Without targeted therapy for the HER2 positive group, the prognosis would be similar to the ER negative PR negative HER2 negative subtype where there is no targeted therapy available. A trial to investigate the effect of adding a targeted therapy, bevacizumab, which is a monoclonal antibody against the vascular endothelial growth factor (EGFR), showed no effect on survival [24]. It is also of interest that the survival of hormone receptor negative breast cancer is poorer than that of hormone receptor positive breast cancer regardless of HER2 status, which means that hormone receptor status rather than HER2 status has more impact on survival. This is also seen in another study where ER negativity was a stronger predictor of poor survival than HER2 positivity [25]. Conclusion Breast cancer subtypes as defined by ER, PR, and HER2 showed that the ER negative PR negative HER2 positive and ER negative PR negative HER2 negative presented with more aggressive clinicopathological features and poorer overall survival compared to the ER and/or PR positive HER2 negative and ER and/or PR positive HER2 positive subtypes. There was no difference in overall survival between the ER negative PR negative HER2 positive and ER negative PR negative HER2 negative subtypes, in the absence of targeted therapy with trastuzumab in the ER negative PR negative HER2 positive subtype. Survival from breast cancer depends on optimal access to treatment [26], and in low- and middle-income countries, targeted therapy is limited by financial constraints, and hence survival of women with ER negative PR negative and HER2 positive breast cancer will not be better than those with triple negative breast cancer. Acknowledgments This paper is supported by HIR Grant UM.C/ HIR/MOHE/06 from the Ministry of Education Malaysia. Compliance with Ethical Standards The authors declare that they have no com- Conflict of interest peting interests. Ethics Approval This study was approved by the Ethics Review Committee of the University Malaya Medical Centre. References 1. Perou CM, Sorlie T, Eisen MB et al (2000) Molecular portraits of human breast tumours. Nature 406(747 75):2 2. Carey LA, Perou CM, Livasy CA et al (2006) Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study. 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