Poor prognosis of single hormone receptor- positive breast cancer: similar outcome as triple-negative breast cancer

Response to endocrine therapy in breast cancer correlates with estrogen receptor (ER) and progesterone receptor (PR) status. Generally, hormone receptor-positive (HR+) breast cancers have favorable prognosis. In order to understand the exact clinical characteristics and prognosis of single HR-positive breast cancer (ER + PR- tumors and ER-PR+ tumors), we compared these tumors to double HR+ tumors as well as HR- negative tumors (ER-PR-). We examined the clinical and biological features of 6,980 women with invasive ductal carcinoma, and these patients were stratified according to ER and PR expression as double HR+ (ER + PR+), single HR+ (ER + PR- and ER-PR+) and double HR-negative (HR-, ER-PR-) tumors. In this study, 571 (8.2%) cases were single HR+ tumors, of which 90 (1.3%) were ER-PR+ tumors and 481 (6.9%) were ER + PR- tumors. Our multivariate analysis showed that in patients without HER2 overexpression ER + PR- tumors were associated with an increased risk of recurrence and death compared with ER + PR+ tumors, with a hazard ratio of 2.12 for disease-free survival (DFS) and 4.79 for overall survival (OS). In patients without HER2 overexpression ER-PR+ tumors had increased risk of recurrence and death compared with ER + PR+ tumor, with a hazard ratio of 4.19 for DFS and 7.22 for OS. In contrast, in patients with HER2 overexpression, the difference in survival between single HR+ tumors and double HR+ HR- tumors was not statistically significant. In patients without HER2 overexpression the DFS and OS of ER + PR- and ER-PR+ tumors were not significantly different from those of ER-PR- tumors. We have identified clinically and biologically distinct features of single HR+ tumors (ER–PR+ and ER + PR–) through comparison with both ER + PR+ and ER-PR- tumors. These differences were only significant in HER2- tumors, not in HER2+ tumors. Single HR+ tumors without HER2 overexpression (ER + PR-HER2- or ER-PR + HER2-) were associated with poorer survival than ER + PR + HER2- tumors, and had comparable poor survival to ER-PR-HER2- tumors (triple-negative breast cancer).


Background
In breast cancer, steroid hormone receptors (HRs; i.e., estrogen receptor [ER] or progesterone receptor [PR]) have been shown to be important prognostic factors and predictive markers for response to endocrine therapy in the treatment of breast cancer. About 70% of breast cancers are hormone receptor-positive tumors (HR+). HR+ breast cancers generally have a favorable prognosis, but HR-negative (HR-) breast cancers have a poor prognosis. PR is an estrogen-regulated gene; ER-positive (ER+) tumors are usually also PR positive (PR+), whereas ER-negative (ER-) tumors are usually PR negative (PR-). Therefore, single HR+ (i.e., ER+/PRor ER-/PR+) tumors represent a minority of breast cancers.
Clinical data have shown in both the metastatic and adjuvant treatment settings that tamoxifen is less efficacious in ER + PR− tumors than in ER + PR+ tumors [1][2][3], and single HR+ breast cancers, especially ER + PR-breast cancers, have aggressive features and poorer prognosis in comparison to double HR+ (ER + PR+) breast cancer [4,5]. However, to our knowledge, comparative studies of HR-(ER-PR-) breast cancers are very limited [6,7].
Previous studies have shown that ER + PR-tumors exhibit high expression of epidermal growth factor receptors [1,4,[7][8][9][10][11], but in most studies, the prognosis of ER + PR-tumors was determined without considering human epidermal growth factor receptor 2 (HER2) expression. Moreover, these studies had a common limitation, in that prognosis was evaluated without considering trastuzumab treatment.
Therefore, in order to understand the exact clinical characteristics and prognosis of single HR-positive breast cancer (ER + PR-tumors and ER-PR+ tumors), we compared these tumors to double HR+ tumors as well as HR-tumors (ER-PR-), and stratified these results according to HER2 overexpression.

Methods
Patients were selected from the clinical database of the Breast Cancer Center at Samsung Medical Center, Korea, between January 2003 and July 2013. A total of 7,010 women with invasive ductal carcinoma were identified. Of them, 6,980 patients were selected for this study excluding patients who were diagnosed with bilateral tumors or with distant metastases at preoperative work-up or underwent neoadjuvant chemotherapy.
We reviewed the clinicopathologic characteristics of patients, including biologic factors, such as ER, PR, HER2, epidermal growth factor receptors (EGFR), and Ki-67. The pathologic tumor stage was assessed according to the American Joint Committee on Cancer (AJCC) 6th Staging System. For ER and PR staging, nuclear (not cytoplasmic) staining was scored using the Allred score (AS) interpretation system, a method that provides semi-quantitative measurement of the proportion of positive cells (scored on a 0 to 5 scale) and staining intensity (scored on a 0 to 3 scale), with a maximum score of 8; an AS > 2 considered positive.
HER2 positivity was defined as an intensity of 3+ by IHC, a score of 2+ was interpreted as equivocal. A negative test was defined as staining with a score of 0/1+. For equivocal stating, silver in situ hybridization (SISH) or fluorescence in situ hybridization (FISH) were performed; the results were positive for HER2 amplification when the ratio of HER2 to CEP17 was > 2.2.
For EGFR and/or Ki-67, results were considered positive based on identification of the following criteria in at least one core. Immunostaining for EGFR was interpreted as positive when at least 10% of the tumor cells showed moderate to strong membrane staining. Ki-67 was considered positive when ≥ 14.0% of cells showed staining [17].
Differences in the frequencies of clinicopathological factors and subtypes were statistically analyzed using the chi-square test and Fisher's exact test. Disease-free survival (DFS) was defined as the time from surgery to the date of documentation of relapse, including locoregional recurrence and/or distant metastasis. Overall survival (OS) was defined as the number of months from surgery to the date of death. Survival curves were constructed using the Kaplan-Meier method. Hazard ratios were estimated using a Cox regression for DFS/OS in a multivariate analysis. Statistical significance was defined as P < 0.05. All statistical analyses were performed using SPSS Statistics 21.0 (IBM).
Study data were collected using a protocol approved by the Institutional Review Board of Samsung Medical Center, Korea (IRB number 2014-09-111). Specific patient consent was not required because we used retrospective data from medical records of patients who had previously signed information release documents.

Survival analysis of single hormone receptor-positive breast cancer
Approximately 97% of patients with ER + PR-tumors and 88% of patients with ER-PR+ tumors received endocrine therapy. More patients with ER + PR-(73.7%) and ER-PR+ (89.7%) tumors received chemotherapy than the group with ER + PR+ tumors (68.7%), but less than the group with ER-PR-tumors (91.9%, Table 1). Approximately 72% of patients with ER + PR-tumors received both endocrine therapy and chemotherapy, and 24.9% of patients received only endocrine therapy. In ER-PR+ tumors, 80% of patients received both chemotherapy and endocrine therapy, 8.2% of patients received only endocrine therapy and 9.4% of patients received only chemotherapy.
Among 1,376 patients with HER2 overexpression, there was no significant difference in DFS between four subgroups (P = 0.529), and patients with ER-PR-HER+ tumors had the worst OS (P = 0.010, Figure 2). However, the 790 patients who received trastuzumab therapy had similar OS (P = 0.113), as did the 586 patients who did not receive trastuzumab therapy (P = 0.147).
With multivariate analysis, in patients with HER2 overexpression, the single HR+ (ER + PR-and ER-PR+) tumors seem to increase risk of recurrence, but this difference was not significant (  (Table 3). ER + PR-tumors and ER-PR+ tumors were not significantly different in terms of DFS and OS compared ER-PR-tumors.

Discussion
We have evaluated in detail the biological characteristics and prognosis of single HR+ tumors through comparison with ER + PR+ tumors as well as ER-PR-tumors. In our series, 8.2% of cases were ER + PR-and 1.4% were ER-PR+. These numbers are somewhat smaller than those from previously published series where 10-15% of cases were ER + PR-and 2-4% were ER-PR+. Most previous studies included patients with breast cancer regardless of histologic type, but we analyzed patients with invasive ductal carcinoma [10,12,13,18]. However, the clinical and biological features of ER + PR-tumors were consistent with those found in previous studies, and there was a high incidence in postmenopausal women. In terms of NG and IHC of Ki-67 level, p53 and EGFR, ER + PR-tumors showed moderate characteristics between the levels of ER + PR+ and ER-PR-tumors, while ER-PR+ tumors were more similar to ER-PR-tumors than ER + PR+ tumors. In addition, on Kaplan-Meier analysis, the survival graph of the ER + PR-tumors was In previous studies, loss of PR has been suggested to be a marker of aberrant growth factor signaling and has been associated with one mechanism for endocrine resistance [11], and several studies have shown that ER + PR-tumors exhibit high expression of epidermal growth factor receptors [1,4,[7][8][9][10][11]. In our cases, HER2 overexpression was 34.5% in single HR+ tumors, and as high as 38.8% in ER-PR-tumors, but the rate of HER2 overexpression in ER + PR+ tumors was 11.4%. Therefore, we stratified our cases according to HER2 overexpression and we found that differences in clinicopathologic characteristics were not significantly different between the four subgroups (ER + PR+, ER-PR+, ER-PR-and ER + PR-) in patients with HER2 overexpression. In addition, there was no difference in survival between these four subgroups. However, in patients without HER2 overexpression, significant differences in biological characteristics were shown more distinctly; ER + PR-, ER-PR+ tumors and ER-PR-HER2-tumors (triple-negative breast cancer, TNBC) were both associated with poor survival.
As demonstrated in previous studies, PR negativity may be association with cross talk with epidermal growth factor receptor-i.e., HER2 or EGFR. In our study, ER + PR-tumors showed high HER2 overexpression. Nevertheless, PR negativity was not a significant prognostic factor in tumors with HER2 overexpression. This suggests that HER2 expression may be a more significant prognostic factor than PR loss in tumors with  HER2 overexpression (HER2+) or may be associated with the results of trastuzumab treatment. However, in tumors without HER2 overexpression, single HR positivity is a significant prognostic factor. The survival graph of ER + PR-tumors is between that of ER + PR+ tumors and ER-PR-tumors initially, but falls to as poor as TNBC at about the 10-year follow-up. Therefore, ER + PR-HER2-and TNBC tumors show no difference in terms of long-term survival. ER-PR + HER2-tumors show similar biological features to TNBC, including high Ki-67 level and high expression of EGFR (about 90%) and p53 (50%). Previous studies have shown incidence rate and clinicopathologic features, and ER-PR+ tumors have increased an incidence in premenopausal women and of an aggressive phenotype with higher tumor grade and HER2 overexpression [10,19,20]. Our results are consistent with those of previous studies. In our series, although there were only a few ER-PR + HER2-tumors, approximately 80% (43/53) of patients with ER-PR + HER2-tumors received chemotherapy and endocrine therapy. Nevertheless, the aggressive behavior of these tumors suggests that ER-PR+ tumors are very rare and represent a distinct biological subtype.  Our study is consistent with previous large studies demonstrating that single HR+ tumors have high expression of EGFR/HER2 and more aggressive features than ER + PR+ tumors. However, our results show that single HR positivity was not a significant prognostic factor in HER2+ breast cancer. Therefore, the aggressiveness of single HR+ tumors is not simply due to hyperactive growth factor signaling pathways. As in recent studies [7][8][9], our cases have shown that single HR+ tumors are associated with a high level of Ki-67 (≥14.0%), and, in multivariate analysis, Ki-67 was shown to have borderline significance (P = 0.068). When we additionally analyzed according to Ki-67 level, in cases with a high level of Ki-67 (≥14.0%), the differences among the four subtypes were still shown consistently. However, in patients with a low Ki-67 level (<14.0%), the prognosis of ER + PR-tumors was not different from that of ER + PR+ tumors. These results may suggest that PR is a significant prognostic factor in HR + HER2-tumors with a high level of Ki-67 expression, but not in HR + HER2+ tumors. These suggest that proliferation-related genes may be significantly associated with PR negativity. However, interestingly, ER-PR+ tumors have been shown to have the worst prognosis of the subtypes, regardless of Ki-67 level, suggesting that ER-PR+ tumors represent a distinct biological subtype.
This study had several limitations. It was a retrospective study, and adjuvant treatment was not determined on a randomized basis. Furthermore, we did not stratify patients according to treatment with tamoxifen or aromatase inhibitors. Although the use of aromatase inhibitors instead of selective estrogen receptor modulators improved the outcome of ER + PR− patients in the ATAC trial [21], the BIG 1-98 trial did not demonstrate a significant benefit of letrozole over tamoxifen in ER + PR− tumors [22]. In addition, at our center, most  postmenopausal patients with HR+ tumors received aromatase inhibitor, excluding patients with contraindications or adverse effects. Recent studies have emphasized the influence of PR, which provides highly significant stratification of ER+ breast cancer into luminal A and B types [7][8][9]. Prat et al. proposed that the IHC-based definition of luminal A tumors is HR+/HER2-/low Ki-67 (less than 14%), and high PR (more than 20%) [8] and Braun et al. also defined luminal B tumors by the presence of high-risk criteria (loss of PR expression or increased proliferation) [9]. Cancello et al. suggested that PR loss identifies luminal B breast cancer subgroups at higher risk of relapse and death, both with HER-2+ and HER-2-breast cancer [7]. The differences observed in HER2+ tumors in our study may be the result of differences in chemotherapy and trastuzumab treatment. In that study, about 30% of patients with ER + PR + HER2+ and ER + PR − HER2+ tumors received endocrine therapy alone, 65-70% received chemotherapy plus endocrine therapy as adjuvant treatments and about 1% in both the ER + PR + HER2+ and ER + PR − HER2+ subgroups received trastuzumab as an adjuvant therapy [7]. However, in our study, 89% of patients with HER2 overexpression received chemotherapy and 57.2% of patients received trastuzumab treatment.