Fractionated evaluation of immunohistochemical hormone receptor expression enhances prognostic prediction in breast cancer patients treated with tamoxifen as adjuvant therapy

Fractionated evaluation of immunohistochemical hormone receptor expression enhances prognostic prediction in breast cancer patients treated with tamoxifen as adjuvant therapy 1 first-author 2 3 1 1 3 4 1 5 5 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:huangcs@ntu.edu.tw †E-mail:huangcs@ntu.edu.tw huangcs@ntu.edu.tw Department of Oncology, National Taiwan University Hospital, Taiwan, Taipei 10016 Department of Oncology, National Taiwan University Hospital, Taiwan, Taipei 10016 Department of Pathology, National Taiwan University Hospital, Taiwan, Taipei 10016 Department of Pathology, National Taiwan University Hospital, Taiwan, Taipei 10016 National Center of Excellence for General Clinical Trial and Research, National Taiwan University Hospital, Taiwan, Taipei 10016 National Center of Excellence for General Clinical Trial and Research, National Taiwan University Hospital, Taiwan, Taipei 10016 Graduate Institute of Epidemiology, College of Public Health, National Taiwan University, Taiwan, Taipei 10055 Graduate Institute of Epidemiology, College of Public Health, National Taiwan University, Taiwan, Taipei 10055 Department of Surgery, National Taiwan University Hospital, Taiwan, Taipei 10016 Department of Surgery, National Taiwan University Hospital, Taiwan, Taipei 10016 Objective: 2 To compare the prognostic prediction between dichotomized and fractionated evaluations of hormone receptor expressions. Methods: 2 Patients with stages I–III breast cancers, who received adjuvant tamoxifen, were enrolled. The expression of estrogen receptor (ER) and progesterone receptor (PR) was evaluated by immunohistochemistry (IHC). A fractionated score (F score), the percentage of positive-staining nuclei (0=none, 1=1%–10%, 2=11%–30%, 3=31%–50%, 4=51%–70%, and 5=71%–100%), was assigned to each case. The dichotomized scoring method defines an F score >1 as positive. The prognostic values of both scores were compared by multiple Cox’s proportional hazard models of disease-free survival (DFS) and overall survival (OS). Results: 2 Four hundred and sixteen patients with a median follow-up of 78.0 months were included. F scores for ER and PR correlated directly with DFS and OS. Although both the dichotomized and fractionated ER and PR scores were significantly associated with DFS and OS in univariate analyses, only fractionated ER and PR scores remained as independent prognostic factors of DFS and OS in the final multiple Cox’s proportional hazard models. Conclusion: 2 Fractionated IHC hormone receptor expression evaluation enhances the prognostic prediction compared with a dichotomized assessment. Breast cancer Fractionated evaluation Estrogen receptor Progesterone receptor Introduction 1 In breast cancer patients, both the estrogen receptor (ER) and progesterone receptor (PR) are important prognostic and predictive markers (Ravdin et al., 1992 1992 ; Elledge et al., 2000 2000 ; Esteva and Hortobagyi, 2004 2004 ). Over the past decade, immunohistochemistry (IHC) has replaced the ligand-binding assay (LBA) for measuring ER and PR. Some studies comparing the predictive values of these two methods found that IHC is an equal or a superior method (Reiner et al., 1986 1986 ; Stierer et al., 1993 1993 ; Barnes et al., 1996 1996 ; Harvey et al., 1999 1999 ; Mohsin et al., 2004 2004 ; Regan et al., 2006 2006 ). However, the definitions of ER and PR positivity by IHC were not consistent among the different studies. Using a definition of positivity as the presence of any level of staining by IHC, the Early Breast Cancer Trialists’ Collaborative Group (EBCTCG, 2005 2005 ) showed in a meta-analysis that tamoxifen significantly improved the prognosis of patients with ER positive tumors. However, in clinical practice and in some clinical trials, a cut-off of ≥10% ER and/or PR positive nuclei is often used for determining that a tumor is hormone receptor-positive and should be treated with adjuvant hormonal therapy (Ellis et al., 2001 2001 ; Jonat et al., 2002 2002 ; Thurlimann et al., 2005 2005 ). The commonly applied 10% cut-off was established by Pertschuk et al. ( 1996 1996 ) who demonstrated a response to treatment in patients with metastatic breast cancer. Two recent reports by Viale et al. ( 2008 2008 ) and Stendahl et al. ( 2006 2006 ) showed that fractionated scoring of ER and PR, defined as assigning scores based on the fraction of cells that were stained positively for ER and PR by IHC, could enhance the prediction of a benefit from adjuvant chemotherapy and/or hormonal therapy. The study by Viale et al. ( 2008 2008 ) demonstrated a gradually decreasing benefit to adding chemotherapy to endocrine therapy with increasing ER and PR expression fractions when compared with endocrine therapy alone in patients with node-negative early breast cancer. Stendahl et al. ( 2006 2006 ) showed that adjuvant tamoxifen could significantly improve survival in premenopausal patients with tumors showing > 75% PR positive nuclei. These two studies highlighted the predictive roles of fractionated evaluation of hormone receptors. In the present study, we assessed whether the hormone receptor status determined by fractionated IHC evaluation could enhance the prognostic significance of the predictive value compared with the traditional dichotomized evaluation. Materials and methods 1 Patients and procedures 2 In this study, women with stage I, II, or III breast cancer who received tamoxifen as adjuvant therapy at the National Taiwan University Hospital (NTUH) between 1992 and 2000 were recruited. Breast cancer staging was conducted according to the American Joint Committee on Cancer (AJCC) criteria (the 6th Ed.). Patients’ demographic information was collected from a genetic epidemiology study on the association between selected genetic polymorphisms and breast cancer in 482 patients, which was carried out at the same hospital (Huang et al., 2008 2008 ). To avoid bias caused by the uncertain treatment effects of tamoxifen in tumors with weak ER and/or PR expression, 43 patients who did not receive adjuvant tamoxifen were excluded. In addition, 23 patients whose archival tumor tissues were not available for fractionated scoring of ER and PR expression were also excluded. Therefore, a total of 416 patients were enrolled in the present analysis. Clinical data, including staging, type of surgery, adjuvant chemotherapy, and hormone therapy, were extracted from medical charts. Disease status and survival outcome were obtained from medical charts, hospital cancer registry records, and the National Death Certificate Registry system if a patient was lost to follow-up. The data managers of our hospital cancer registry usually contact each patient every 6 months. Additionally, all deaths in Taiwan are registered through the household registration offices. These death certificates are coded by the Department of Health, Executive Yuan, and have been computerized since 1979 (Lu et al., 2000 2000 ). The survival data in this study were updated to Feb. 5, 2005. Histological slides were reviewed by one pathologist, Dr. H.C. Lien, who was blinded to the patient information. Histological grade was categorized as grades I, II, and III according to the Nottingham modification of the Scarff-Bloom-Richardson criteria (Elston and Ellis, 1991 1991 ), except for invasive lobular and mucinous carcinomas. The status of ER and PR in tumors was determined by IHC performed on formalin-fixed (fixation time 6–8 h), paraffin-embedded tissue sections (thickness 4 μm) in the Central Pathology Laboratory at NTUH. Ventana Benchmark system (Ventana Medical Systems Inc., Tucson, AZ, USA) and prediluted antibodies (anti-ER Clone 6F11 and anti-PgR Clone 16) were used. The reviewing pathologist assigned a six-point fractionated score called “F score” to each case after estimating the percentage of positive-staining tumor cells (0=none, 1=1%–10%, 2=11%–30%, 3=31%–50%, 4=51%–70%, and 5=71%–100%). A percentage of 10% (i.e., F score=1) was chosen as the cut-off value to dichotomize the assessments of ER and PR into positive versus negative. Statistical analysis 2 Statistical analyses were performed with SAS software (version 9.1.3, SAS Institute Inc., Cary, NC, USA) and S-PLUS software (version 7.0.4, Insightful Corporation, Seattle, WA, USA). A two-sided P ≤0.05 was considered statistically significant. The dichotomized ER and PR scores are binary variables, whereas the fractionated ER and PR scores (i.e., F scores) are ordinal variables. The recruited subjects were followed from the date of diagnosis to the one of death or last contact. The disease-free survival (DFS) time was defined as the duration from diagnosis to either the confirmation of disease recurrence, including local, regional, and distant recurrences, or death from breast cancer (if the time of recurrence was missed), except for contralateral breast cancer. The overall survival (OS) time was defined as the duration between diagnosis and death from any cause. The dependent variables evaluated were age, histological type, histological grade, tumor size, axillary lymph node status, AJCC stage, dichotomized ER, dichotomized PR, fractionated ER, fractionated PR, and neoadjuvant and/or adjuvant chemotherapy. Survival curves were estimated with the Kaplan-Meier method. The association between each of the categorical variables and survival was analyzed by log-rank test. Multivariate analysis was performed using Cox’s proportional hazard models to predict the hazard rates of DFS and OS, respectively. The time lag from the time of recurrence to each time at which a death from any cause occurred was computed for each patient and was included in the Cox’s proportional hazard model of OS as a time-dependent covariate to falsify the hypothesis that earlier recurrence leads to earlier death. For patients whose breast cancer had not recurred at the time of death, this time lag was set to zero. Basic model-fitting techniques for (1) variable selection, (2) assessment of goodness-of-fit (GOF), and (3) regression diagnostics were used in our regression analysis to assure the quality of the analysis results. In the stepwise variable selection procedure, all the significant univariates and non-significant covariates were considered and both the significance levels for entry (SLE) and for stay (SLS) were set to 0.15 or larger. The adjusted generalized R 2 and the Grønnesby-Borgan GOF test were examined to assess the GOF of the fitted Cox’s proportional hazard model. However, the value of the adjusted generalized R 2 for Cox’s proportional hazard model is usually low. Larger P values in the Grønnesby-Borgan GOF test indicate better fit. Regression diagnostics, including verification of proportional hazard assumption, residual analysis, detection of influential cases, and check for multi-collinearity, were performed. Based on the fitted final Cox’s regression models for DFS and OS, estimated covariate-adjusted survival curves were plotted for personalized prediction of the patient’s prognosis. Results 1 A total of 416 patients with a median age of 49 (range: 25–81) years were included. The representative immunohistochemical image of each F score of ER is shown in Fig. 1 1 . The distribution of each F score of ER and PR expression is shown in Table 1 1 . Using the dichotomized definition of an F score > 1 as positive, 216 (63%) and 257 (62%) cases were ER + and PR + , respectively. The clinical and pathological characteristics of the patients are summarized in Table 2 2 by dichotomized ER and PR status. The tumor histology consisted of infiltrating ductal carcinoma (95%), infiltrating lobular carcinoma (2%), mucinous carcinoma (3%), and others (1%) including 2 medullary carcinomas and 2 invasive papillary carcinomas. The frequency of ER + and/or PR + status was heterogeneous among tumors of different sizes, axillary lymph node statuses, histologic types, and grades. Specifically, ER + was associated with a higher probability of a tumor size ≤2 cm and PR + was associated with higher probability of absence of axillary lymph node involvement. All of the lobular carcinomas and mucinous carcinomas were ER + . Tumors with histologic grades I and II were more likely to be ER + or PR + , whereas tumors with histologic grade III were more likely to be ER − or PR − . The expression of ER and PR was not significantly associated with the use of neoadjuvant and/or adjuvant chemotherapy. 2 ER score 6 PR score 2 Total 0 1 2 3 4 5 0 77 15 6 4 5 21 128 1 10 15 2 0 0 0 27 2 0 0 6 1 5 6 18 3 7 1 4 3 2 4 21 4 8 1 0 4 10 5 28 5 22 3 10 11 20 128 194 Total 124 35 28 23 42 164 416 2 2 All cases ( n =416) 3 ER, No. (%) 3 PR, No. (%) Positive ( n =261) Negative ( n =155) P -value Positive ( n =257) Negative ( n =159) P -value Age 0.0922 0.0926  <36 years 26 (6%) 15 (6%) 15 (10%) 14 (6%) 16 (10%)  36–50 years 204 (49%) 135 (52%) 65 (42%) 132 (51%) 68 (43%)  >50 years 186 (45%) 111 (43%) 75 (48%) 111 (43%) 75 (47%) Tumor size 0.0057 0.1190  ≤2 cm 167 (40%) 120 (46%) 47 (30%) 111 (43%) 56 (35%)  3–5 cm 216 (52%) 124 (48%) 92 (59%) 130 (51%) 86 (54%)  >5 cm 33 (8%) 17 (7%) 16 (10%) 16 (6%) 17 (11%) Axillary lymph node 0.9399 0.0271  None 232 (56%) 148 (57%) 84 (54%) 158 (61%) 74 (47%)  1–3 96 (23%) 59 (23%) 37 (24%) 53 (21%) 43 (27%)  4–9 54 (13%) 34 (13%) 20 (13%) 29 (11%) 25 (16%)  ≥10 34 (8%) 20 (8%) 14 (9%) 17 (7%) 17 (11%) AJCC stage 0.0445 0.0198  I 114 (27%) 82 (31%) 32 (21%) 82 (32%) 32 (20%)  II 209 (50%) 121 (46%) 88 (57%) 125 (49%) 84 (53%)  III 93 (22%) 58 (22%) 35 (23%) 50 (19%) 43 (27%) Histologic subtype 0.0006 0.3523  Ductal 391 (94%) 237 (91%) 154 (99%) 238 (93%) 153 (96%)  Lobular 8 (2%) 8 (3%) 0 (0%) 5 (2%) 3 (2%)  Mucinous 13 (3%) 13 (5%) 0 (0%) 11 (4%) 2 (1%)  Others 4 (1%) 3 (1%) 1 (1%) 3 (1%) 1 (1%) Histologic grade <0.0001 <0.0001  I 103 (25%) 78 (30%) 25 (16%) 79 (31%) 24 (15%)  II 164 (39%) 115 (44%) 49 (32%) 113 (44%) 51 (32%)  III 98 (24%) 33 (13%) 65 (42%) 37 (14%) 61 (38%)  Not available 51 (12%) 35 (13%) 16 (10%) 28 (11%) 23 (15%) 4 Neoadjuvant or adjuvant chemotherapy 0.3577 0.3098  Yes 262 (63%) 160 (61%) 102 (66%) 157 (61%) 105 (66%)  No 154 (37%) 101 (39%) 53 (34%) 100 (39%) 54 (34%) Univariate survival analyses of prognostic factors 2 The median follow-up time was 78.0 (range: 74.7–81.3) months. As listed in Table 3 3 , the univariate analysis showed that age, tumor size, axillary lymph node status, AJCC stage, dichotomized ER and PR, histologic grade, and use of neoadjuvant or adjuvant chemotherapy were significantly associated with DFS and OS. Specifically, age < 36 years, larger tumor size, higher AJCC stage, negative ER, negative PR, more axillary lymph node involvement, higher tumor grade, and use of neoadjuvant and/or adjuvant chemotherapy were prognostic factors for a poor outcome (Table 3 3 ). The estimated DFS and OS curves according to the F scores of hormone receptors for all patients are shown in Figs. 2a and 2b 2a and 2b ), respectively. Univariate linear trend analysis also revealed that higher ER and PR IHC scores were significantly associated with favorable DFS ( P =0.003 and P < 0.001, respectively) and OS ( P < 0.001 and P < 0.001, respectively). HR: hazard ratio; CI: confidence interval; NA: not available 2 2 N (%) 3 DFS 3 OS HR 95% CI P -value HR 95% CI P -value Age 0.0129 0.0191  <36 years 26 (6%) 1.00 1.00  36–50 years 204 (49%) 0.41 0.23–0.76 0.40 0.20–0.77  >50 years 186 (45%) 0.50 0.28–0.91 0.48 0.25–0.92 Tumor size <0.0001 <0.0001  ≤2 cm 167 (40%) 1.00 1.00  3–5 cm 216 (52%) 1.86 1.18–2.92 2.27 1.28–4.02  >5 cm 33 (8%) 5.43 3.02–9.79 7.99 4.04–15.84 Axillary lymph nodes <0.0001 <0.0001  None 232 (56%) 1.00 1.00  1–3 96 (23%) 1.98 1.17–3.35 1.92 0.99–3.70  4–9 54 (13%) 3.81 2.22–6.53 4.67 2.49–8.75  ≥10 34 (8%) 12.08 7.19–20.30 11.81 6.47–21.55 AJCC stage <0.0001 <0.0001  I 114 (27%) 1.00 1.00  II 209 (50%) 3.23 1.53–6.84 4.10 1.45–11.62  III 93 (22%) 11.11 5.27–23.40 16.43 5.90–45.80 ER expression 0.0090 0.0001  Positive 261 (63%) 1.00 1.00  Negative 155 (37%) 1.65 1.13–2.42 2.35 1.50–3.69 PR expression <0.0001 <0.0001  Positive 257 (62%) 1.00 1.00  Negative 159 (38%) 2.12 1.44–3.13 2.67 1.68–4.22 Histologic subtype 0.7717 0.8421  Ductal 391 (94%) 1.00 1.00  Lobular 8 (2%) 1.09 0.27–4.44 0.75 0.10–5.40  Mucinous 13 (3%) 1.03 0.33–3.23 0.87 0.21–3.55  Others 4 (1%) NA NA NA NA Histologic grade 0.0072 0.0010  I 103 (25%) 1.00 1.00  II 164 (39%) 2.07 1.14–3.77 1.88 0.89–3.99  III 98 (24%) 2.88 1.55–5.36 3.75 1.78–7.88  Not available 51 (12%) 1.84 0.85–3.98 1.98 0.79–5.00 4 Neoadjuvant or adjuvant chemotherapy 0.0010 0.0013  Yes 262 (63%) 1.00 1.00  No 154 (37%) 0.49 0.31–0.75 0.43 0.25–0.73 Multivariate survival analyses of prognostic factors 2 The results of multivariate analyses by fitting Cox’s proportional hazard models for DFS and OS are listed in Table 4 4 . Fractionated ER, age < 36 years, ≥10 positive axillary lymph nodes, histologic grade III, and AJCC stage were independent prognostic factors for DFS. Fractionated PR, 1–9 positive axillary lymph nodes, ≥10 positive axillary lymph nodes, tumor size, histologic grade III, and the time lag between recurrence and the time of death from any cause were independent prognostic factors for OS. Longer lag time implied an earlier recurrence. Neither dichotomized ER nor dichotomized PR was an independent prognostic factor for DFS or OS at the completion of the stepwise variable selection procedure. Moreover, we could draw covariate-adjusted survival curves for the prognostic prediction of patient’s DFS or OS under any given condition. As an illustration, the estimated covariate-adjusted DFS curves by six-point fractionated ER scores for a young woman (aged < 36 years, AJCC stage II breast cancer, histologic grade I or II, fractionated PR score 0, and ≥10 involved axillary lymph nodes) are shown in Fig. 3 3 . Cox’s proportional hazard model: n =416, adjusted generalized R 2 =0.2486 Cox’s proportional hazard model: n =416, adjusted generalized R 2 =0.3971 Covariate Estimate Standard error Wald chi-square Hazard ratio P value 6 Disease-free survival (month) 1 1  Fractionated ER −0.13960 0.05628 6.1530 0.870 0.0131  Fractionated PR −0.10107 0.05869 2.9655 0.904 0.0851  Age <36 years 0.67577 0.29191 5.3592 1.966 0.0206  Lymph node ≥10 1.35863 0.27997 23.5496 3.891 <0.0001  Histology grade III 1.32154 0.61828 4.5687 3.749 0.0326  AJCC stage 0.91674 0.18495 24.5675 2.501 <0.0001 6 Overall survival (month) 2 2  Fractionated PR −0.21830 0.06066 12.9512 0.804 0.0003  Lymph node 1–9 0.81272 0.30782 6.9707 2.254 0.0083  Lymph node ≥10 1.56278 0.33739 21.4549 4.772 <0.0001  Histology grade III 0.68063 0.25213 7.2875 1.975 0.0069  Tumor size (cm) 0.32670 0.16657 3.8469 1.386 0.0498  Neoadjuvant or adjuvant chemotherapy 0.61693 0.35181 3.0750 1.853 0.0795  Time lag since recurrence 0.07444 0.00775 92.2451 1.077 <0.0001 Discussion 1 In this study, we found that fractionated ER and PR were predictors of DFS and OS, respectively, both independently and after competing with the dichotomized ER and dichotomized PR in a stepwise variable selection procedure. We concluded that implementing a fractionated evaluation of ER and PR enhances the prognostic correlation when compared with the dichotomized assessment. All of the patients included in this analysis received adjuvant tamoxifen, including a substantial proportion (26%) of patients with ER − /PR − breast cancer. This provided us with a suitable platform to examine the effect of ER and PR expression on survival. By examining the Kaplan-Meier estimates of DFS curves stratified by fractionated ER and PR scores (Fig. 2a 2a ), the best cut-off points of dichotomized evaluation of ER and PR positivity were more than 10% positive nuclei (F score > 1). Dichotomized ER and PR expression was also highly associated with favorable DFS and OS in the present study, which provides evidence to support the criteria commonly used in clinical practice and some clinical trials in the adjuvant setting (Ellis et al., 2001 2001 ; Jonat et al., 2002 2002 ; Thurlimann et al., 2005 2005 ). In addition to the prognostic factors identified by our univariate analyses for DFS and OS, which include age, tumor size, number of axillary lymph nodes, and AJCC stage, dichotomized ER and PR are well-recognized prognostic factors for breast cancer survival (Cianfrocca and Goldstein, 2004 2004 ). The present study provides empirical evidence to show that earlier recurrence leads to an earlier death in breast cancer patients. Although this result is biologically sound, most of the previous studies analyzed time to recurrence (DFS) and death (OS) separately. The causal relationship between recurrence and death should be pursued further in the future, because it may shed light on improvements of the prognostic predictions in such patients. Moreover, we found that fractionated ER was an independent prognostic factor of DFS, and fractionated PR was an independent prognostic factor of OS after adjusting for the effect of time lag since recurrence. Since time to recurrence was also a prognostic factor for death, both fractionated ER and fractionated PR could predict OS, but fractionated PR had a direct prognostic effect on OS. The finding that PR added significant prognostic value beyond that obtained with ER alone was consistent with recent studies (Banerjee et al., 2004 2004 ; Arpino et al., 2005 2005 ; Liu et al., 2010 2010 ). Fractionated ER and PR remained in the final multiple Cox’s proportional hazard models after competing with the dichotomized ER and PR in the stepwise variable selection procedure. Thus, a fractionated evaluation of ER and PR enhanced the prognostic prediction when compared with the dichotomized assessment. ER analysis using LBA (as fmol/mg of protein) revealed a broad range of values for ER, so there has been expectation that immunohistochemical assay for ER analysis should result in a similar distribution. However, both fractionated ER and PR in the present study were shown to display a bimodal distribution, with substantial proportions at F scores of 0 (ER: 30.8%; PR: 29.8%) and 5 (ER: 46.6%; PR: 39.4%) (Table 1 1 ). The distribution was consistent with several previous studies of ER and PR expression measured by immunohistochemical assay (Collins et al., 2005 2005 ; Nadji et al., 2005 2005 ; Stendahl et al., 2006 2006 ; Badve et al., 2008 2008 ; Viale et al., 2008 2008 ). Despite the bimodal distribution of ER and PR expression, the present study clearly demonstrates that fractionated ER and PR scores predict a patient’s prognosis more accurately than a dichotomized assessment. The Allred, Quick and H scores, which include the evaluation of both the proportion and staining intensity of positive cells, have been used to assess IHC staining of breast tumors (Huang et al., 1996 1996 ; Harvey et al., 1999 1999 ; Ferrero-Pous et al., 2001 2001 ). The Allred scores for both of ER and PR expression were reported to correlate highly with patient’s survival outcome, and the predictive value of Allred score was superior to the LBA (Harvey et al., 1999 1999 ; Mohsin et al., 2004 2004 ). The Quick and H scores for ER and/or PR expression were shown to correlate with LBA (Huang et al., 1996 1996 ; Ferrero-Pous et al., 2001 2001 ) and possess similar predictive values as LBA for response to hormonal therapy (Elston and Ellis, 1998 1998 ). Nevertheless, many clinical practices simply adopt a proportion score rather than the semi-quantitative scoring systems for predictive and prognostic evaluations, because the intensity interpretation is relatively subjective and some tumors have heterogeneous intensity (Horii et al., 2007 2007 ). The superiority of prognostic value of fractionated assessment to dichotomized assessment may have important clinical implications. For example, the information could be used to improve risk estimation by Adjuvant! Online ( https://www.adjuvantonline.com/breastnew.jsp https://www.adjuvantonline.com/breastnew.jsp ), which is one of the most popular and ambitious tools to estimate the risk of breast cancer relapse and death. One of the limitations inherent in the current Adjuvant! Online version is that only dichotomized ER is used to estimate the risk of relapse and mortality. As illustrated in Fig. 3 3 , the covariate-adjusted survival curves may provide some fine tuning according to the level of ER positivity and PR status to adjust the risk estimation. Of course, further population-based studies with larger sample sizes and a comparison between pure fractionated (F score) and intensity incorporated (Allred, Quick or H score) evaluations are necessary to prove that the “F score” is a simple and powerful method for risk adjustment. Acknowledgement 1 We thank the members of the Office of Medical Records at National Taiwan University Hospital for their help in assessing the prospectively collected data. References G Arpino , H Weiss , AV Lee , R Schiff , S de Placido , CK Osborne , RM Elledge . Estrogen receptor-positive, progesterone receptor-negative breast cancer: association with growth factor receptor expression and tamoxifen resistance . J Natl Cancer Inst , 2005 . 97 ( 17 ): 1254 - 1261 . 16145046 . Estrogen receptor-positive, progesterone receptor-negative breast cancer: association with growth factor receptor expression and tamoxifen resistance SS Badve , FL Baehner , RP Gray , BH Childs , T Maddala , ML Liu , SC Rowley , S Shak , EA Perez , LJ Shulman , 等 . Estrogen- and progesterone-receptor status in ECOG 2197: comparison of immunohistochemistry by local and central laboratories and quantitative reverse transcription polymerase chain reaction by central laboratory . J Clin Oncol , 2008 . 26 ( 15 ): 2473 - 2481 . 18487567 DOI: 10.1200/JCO.2007.13.6424 http://doi.org/10.1200/JCO.2007.13.6424 . Estrogen- and progesterone-receptor status in ECOG 2197: comparison of immunohistochemistry by local and central laboratories and quantitative reverse transcription polymerase chain reaction by central laboratory M Banerjee , J George , EY Song , A Roy , W Hryniuk . Tree-based model for breast cancer prognostication . J Clin Oncol , 2004 . 22 ( 13 ): 2567 - 2575 . 15226324 DOI: 10.1200/JCO.2004.11.141 http://doi.org/10.1200/JCO.2004.11.141 . Tree-based model for breast cancer prognostication DM Barnes , WH Harris , P Smith , RR Millis , RD Rubens . Immunohistochemical determination of oestrogen receptor: comparison of different methods of assessment of staining and correlation with clinical outcome of breast cancer patients . Br J Cancer , 1996 . 74 ( 9 ): 1445 - 1451 . 8912543 . Immunohistochemical determination of oestrogen receptor: comparison of different methods of assessment of staining and correlation with clinical outcome of breast cancer patients M Cianfrocca , LJ Goldstein . Prognostic and predictive factors in early-stage breast cancer . Oncologist , 2004 . 9 ( 6 ): 606 - 616 . 15561805 DOI: 10.1634/theoncologist.9-6-606 http://doi.org/10.1634/theoncologist.9-6-606 . Prognostic and predictive factors in early-stage breast cancer LC Collins , ML Botero , SJ Schnitt . Bimodal frequency distribution of estrogen receptor immunohistochemical staining results in breast cancer: an analysis of 825 cases . Am J Clin Pathol , 2005 . 123 ( 1 ): 16 - 20 . 15762275 DOI: 10.1309/HCF035N9WK40ETJ0 http://doi.org/10.1309/HCF035N9WK40ETJ0 . Bimodal frequency distribution of estrogen receptor immunohistochemical staining results in breast cancer: an analysis of 825 cases EBCTCG (Early Breast Cancer Trialists’ Collaborative Group) Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials . Lancet , 2005 . 365 ( 9472 ): 1687 - 1717 . 15894097 DOI: 10.1016/S0140-6736(05)66544-0 http://doi.org/10.1016/S0140-6736(05)66544-0 . Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials RM Elledge , S Green , R Pugh , DC Allred , GM Clark , J Hill , P Ravdin , S Martino , CK Osborne . Estrogen receptor (ER) and progesterone receptor (PgR), by ligand-binding assay compared with ER, PgR and pS2, by immunohistochemistry in predicting response to tamoxifen in metastatic breast cancer: a Southwest Oncology Group Study . Int J Cancer , 2000 . 89 ( 2 ): 111 - 117 . 10754487 DOI: 10.1002/(SICI)1097-0215(20000320)89:2<111::AID-IJC2>3.0.CO;2-W http://doi.org/10.1002/(SICI)1097-0215(20000320)89:2<111::AID-IJC2>3.0.CO;2-W . Estrogen receptor (ER) and progesterone receptor (PgR), by ligand-binding assay compared with ER, PgR and pS2, by immunohistochemistry in predicting response to tamoxifen in metastatic breast cancer: a Southwest Oncology Group Study MJ Ellis , A Coop , B Singh , L Mauriac , A Llombert-Cussac , F Janicke , WR Miller , DB Evans , M Dugan , C Brady , 等 . Letrozole is more effective neoadjuvant endocrine therapy than tamoxifen for ErbB-1- and/or ErbB-2-positive, estrogen receptor-positive primary breast cancer: evidence from a phase III randomized trial . J Clin Oncol , 2001 . 19 ( 18 ): 3808 - 3816 . 11559718 . Letrozole is more effective neoadjuvant endocrine therapy than tamoxifen for ErbB-1- and/or ErbB-2-positive, estrogen receptor-positive primary breast cancer: evidence from a phase III randomized trial CW Elston , IO Ellis . Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up . Histopathology , 1991 . 19 ( 5 ): 403 - 410 . 1757079 DOI: 10.1111/j.1365-2559.1991.tb00229.x http://doi.org/10.1111/j.1365-2559.1991.tb00229.x . Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up CW Elston , IO Ellis . The Breast , 1998 . : Edinburgh Churchill Livingston , 673 . The Breast FJ Esteva , GN Hortobagyi . Prognostic molecular markers in early breast cancer . Breast Cancer Res , 2004 . 6 ( 3 ): 109 - 118 . 15084231 DOI: 10.1186/bcr777 http://doi.org/10.1186/bcr777 . Prognostic molecular markers in early breast cancer M Ferrero-Pous , M Trassard , V Le Doussal , K Hacene , M Tubiana-Hulin , F Spyratos . Comparison of enzyme immunoassay and immunohistochemical measurements of estrogen and progesterone receptors in breast cancer patients . Appl Immunohistochem Mol Morphol , 2001 . 9 ( 3 ): 267 - 275 . 11556756 DOI: 10.1097/00022744-200109000-00012 http://doi.org/10.1097/00022744-200109000-00012 . Comparison of enzyme immunoassay and immunohistochemical measurements of estrogen and progesterone receptors in breast cancer patients JM Harvey , GM Clark , CK Osborne , DC Allred . Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer . J Clin Oncol , 1999 . 17 ( 5 ): 1474 - 1481 . 10334533 . Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer R Horii , F Akiyama , Y Ito , T Iwase . Assessment of hormone receptor status in breast cancer . Pathol Int , 2007 . 57 ( 12 ): 784 - 790 . 17988279 DOI: 10.1111/j.1440-1827.2007.02174.x http://doi.org/10.1111/j.1440-1827.2007.02174.x . Assessment of hormone receptor status in breast cancer A Huang , NM Pettigrew , PH Watson . Immunohistochemical assay for oestrogen receptors in paraffin wax sections of breast carcinoma using a new monoclonal antibody . J Pathol , 1996 . 180 ( 2 ): 223 - 237 . 8976884 DOI: 10.1002/(SICI)1096-9896(199610)180:2<223::AID-PATH635>3.0.CO;2-H http://doi.org/10.1002/(SICI)1096-9896(199610)180:2<223::AID-PATH635>3.0.CO;2-H . Immunohistochemical assay for oestrogen receptors in paraffin wax sections of breast carcinoma using a new monoclonal antibody CS Huang , SH Kuo , HC Lien , SY Yang , SL You , CY Shen , CH Lin , YS Lu , KJ Chang . The CYP19 TTTA repeat polymorphism is related to the prognosis of premenopausal stage I–II and operable stage III breast cancers . Oncologist , 2008 . 13 ( 7 ): 751 - 760 . 18614591 DOI: 10.1634/theoncologist.2007-0246 http://doi.org/10.1634/theoncologist.2007-0246 . The CYP19 TTTA repeat polymorphism is related to the prognosis of premenopausal stage I–II and operable stage III breast cancers W Jonat , M Kaufmann , W Sauerbrei , R Blamey , J Cuzick , M Namer , I Fogelman , JC de Haes , A de Matteis , A Stewart , 等 . Goserelin versus cyclophosphamide, methotrexate, and fluorouracil as adjuvant therapy in premenopausal patients with node-positive breast cancer: the Zoladex Early Breast Cancer Research Association Study . J Clin Oncol , 2002 . 20 ( 24 ): 4628 - 4635 . 12488406 DOI: 10.1200/JCO.2002.05.042 http://doi.org/10.1200/JCO.2002.05.042 . Goserelin versus cyclophosphamide, methotrexate, and fluorouracil as adjuvant therapy in premenopausal patients with node-positive breast cancer: the Zoladex Early Breast Cancer Research Association Study S Liu , SK Chia , E Mehl , S Leung , A Rajput , MC Cheang , TO Nielsen . Progesterone receptor is a significant factor associated with clinical outcomes and effect of adjuvant tamoxifen therapy in breast cancer patients . Breast Cancer Res Treat , 2010 . 119 ( 1 ): 53 - 61 . 19205877 DOI: 10.1007/s10549-009-0318-0 http://doi.org/10.1007/s10549-009-0318-0 . Progesterone receptor is a significant factor associated with clinical outcomes and effect of adjuvant tamoxifen therapy in breast cancer patients TH Lu , MC Lee , MC Chou . Accuracy of cause-of-death coding in Taiwan: types of miscoding and effects on mortality statistics . Int J Epidemiol , 2000 . 29 ( 2 ): 336 - 343 . 10817134 DOI: 10.1093/ije/29.2.336 http://doi.org/10.1093/ije/29.2.336 . Accuracy of cause-of-death coding in Taiwan: types of miscoding and effects on mortality statistics SK Mohsin , H Weiss , T Havighurst , GM Clark , M Berardo , D Roanhle , TV To , Z Qian , RR Love , DC Allred . Progesterone receptor by immunohistochemistry and clinical outcome in breast cancer: a validation study . Mod Pathol , 2004 . 17 ( 12 ): 1545 - 1554 . 15272277 DOI: 10.1038/modpathol.3800229 http://doi.org/10.1038/modpathol.3800229 . Progesterone receptor by immunohistochemistry and clinical outcome in breast cancer: a validation study M Nadji , C Gomez-Fernandez , P Ganjei-Azar , AR Morales . Immunohistochemistry of estrogen and progesterone receptors reconsidered: experience with 5993 breast cancers . Am J Clin Pathol , 2005 . 123 ( 1 ): 21 - 27 . 15762276 DOI: 10.1309/4WV79N2GHJ3X1841 http://doi.org/10.1309/4WV79N2GHJ3X1841 . Immunohistochemistry of estrogen and progesterone receptors reconsidered: experience with 5993 breast cancers LP Pertschuk , JG Feldman , YD Kim , L Braithwaite , F Schneider , AS Braverman , C Axiotis . Estrogen receptor immunocytochemistry in paraffin embedded tissues with ER1D5 predicts breast cancer endocrine response more accurately than H222Sp gamma in frozen sections or cytosol-based ligand-binding assays . Cancer , 1996 . 77 ( 12 ): 2514 - 2519 . 8640700 DOI: 10.1002/(SICI)1097-0142(19960615)77:12<2514::AID-CNCR14>3.3.CO;2-T http://doi.org/10.1002/(SICI)1097-0142(19960615)77:12<2514::AID-CNCR14>3.3.CO;2-T . Estrogen receptor immunocytochemistry in paraffin embedded tissues with ER1D5 predicts breast cancer endocrine response more accurately than H222Sp gamma in frozen sections or cytosol-based ligand-binding assays PM Ravdin , S Green , TM Dorr , WL McGuire , C Fabian , RP Pugh , RD Carter , SE Rivkin , JR Borst , RJ Belt , 等 . Prognostic significance of progesterone receptor levels in estrogen receptor-positive patients with metastatic breast cancer treated with tamoxifen: results of a prospective Southwest Oncology Group study . J Clin Oncol , 1992 . 10 ( 8 ): 1284 - 1291 . 1634918 . Prognostic significance of progesterone receptor levels in estrogen receptor-positive patients with metastatic breast cancer treated with tamoxifen: results of a prospective Southwest Oncology Group study MM Regan , G Viale , MG Mastropasqua , E Maiorano , R Golouh , A Carbone , B Brown , M Suurkula , G Langman , L Mazzucchelli , 等 . Re-evaluating adjuvant breast cancer trials: assessing hormone receptor status by immunohistochemical versus extraction assays . J Natl Cancer Inst , 2006 . 98 ( 21 ): 1571 - 1581 . 17077359 . Re-evaluating adjuvant breast cancer trials: assessing hormone receptor status by immunohistochemical versus extraction assays A Reiner , J Spona , G Reiner , M Schemper , R Kolb , W Kwasny , R Fugger , R Jakesz , JH Holzner . Estrogen receptor analysis on biopsies and fine-needle aspirates from human breast carcinoma. Correlation of biochemical and immunohistochemical methods using monoclonal antireceptor antibodies . Am J Pathol , 1986 . 125 ( 3 ): 443 - 449 . 3541638 . Estrogen receptor analysis on biopsies and fine-needle aspirates from human breast carcinoma. Correlation of biochemical and immunohistochemical methods using monoclonal antireceptor antibodies M Stendahl , L Ryden , B Nordenskjold , PE Jonsson , G Landberg , K Jirstrom . High progesterone receptor expression correlates to the effect of adjuvant tamoxifen in premenopausal breast cancer patients . Clin Cancer Res , 2006 . 12 ( 15 ): 4614 - 4618 . 16899609 DOI: 10.1158/1078-0432.CCR-06-0248 http://doi.org/10.1158/1078-0432.CCR-06-0248 . High progesterone receptor expression correlates to the effect of adjuvant tamoxifen in premenopausal breast cancer patients M Stierer , H Rosen , R Weber , H Hanak , J Spona , H Tuchler . Immunohistochemical and biochemical measurement of estrogen and progesterone receptors in primary breast cancer. Correlation of histopathology and prognostic factors . Ann Surg , 1993 . 218 ( 1 ): 13 - 21 . 8328824 DOI: 10.1097/00000658-199307000-00004 http://doi.org/10.1097/00000658-199307000-00004 . Immunohistochemical and biochemical measurement of estrogen and progesterone receptors in primary breast cancer. Correlation of histopathology and prognostic factors B Thurlimann , A Keshaviah , AS Coates , H Mouridsen , L Mauriac , JF Forbes , R Paridaens , M Castiglione-Gertsch , RD Gelber , M Rabaglio , 等 . A comparison of letrozole and tamoxifen in postmenopausal women with early breast cancer . N Engl J Med , 2005 . 353 ( 26 ): 2747 - 2757 . 16382061 . A comparison of letrozole and tamoxifen in postmenopausal women with early breast cancer G Viale , MM Regan , E Maiorano , MG Mastropasqua , R Golouh , T Perin , RW Brown , A Kovacs , K Pillay , C Ohlschlegel , 等 . Chemoendocrine compared with endocrine adjuvant therapies for node-negative breast cancer: predictive value of centrally reviewed expression of estrogen and progesterone receptors–International Breast Cancer Study Group . J Clin Oncol , 2008 . 26 ( 9 ): 1404 - 1410 . 18349391 DOI: 10.1200/JCO.2007.10.6393 http://doi.org/10.1200/JCO.2007.10.6393 . Chemoendocrine compared with endocrine adjuvant therapies for node-negative breast cancer: predictive value of centrally reviewed expression of estrogen and progesterone receptors–International Breast Cancer Study Group 10.1631/jzus.B0900295 Project supported by the National Center of Excellence for Clinical Trial and Research of National Taiwan University Hospital (No. DOH97-TD-B-111-001), Taiwan, Taipei 2009-09-24 2009-12-04 2009-11-23 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2010 2010 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 01 11