Immunolocalization of Cytoplasmic ER in ER-negative Breast Carcinoma as a Potent Favorable Prognostic Predictor

Abstract

It is known that estrogen receptor (ER) has extranuclear signaling functions in addition to classical genomic pathway, and estrogenic actions have been reported in ER-negative breast carcinoma cells. However, significance of cytoplasmic-ER immunoreactivity has not been reported in ER-negative breast carcinoma tissues. We immunolocalized cytoplasmic ER in 155 ER-negative breast carcinoma tissues and evaluated its clinicopathological significance including the prognosis. As a comparative cohort set, we also used 142 ER-positive breast carcinomas. Cytoplasmic-ER immunoreactivity was detected in the carcinoma cells, but not in the non-neoplastic mammary epithelium. Cytoplasmic-ER immunoreactivity was positive in the 35 out of 155 (23%) ER-negative breast carcinoma cases, whereas it was detected only in 2 out of 142 (1.4%) ER-positive cases. Cytoplasmic ER status was positively associated with cytoplasmic-PR status, but inversely associated with Ki67 labeling index or distant free-relapse survival rate. Moreover, cytoplasmic-ER status turned out to be an independent good prognostic factor for both distant relapse-free survival and breast cancer specific survival. These findings suggested that cytoplasmic ER plays important roles in the ER-negative breast carcinoma, and cytoplasmic ER is a potent good prognostic factor. Among the ER-negative breast cancer patients, clinical benefit of chemotherapy may be limited in the cytoplasmic-ER positive cases.

I.  Introduction

Breast cancer is the most commonly diagnosed cancer in women (25% in female cancer incidence) in the world, and one of major cause of cancer death (16% in female cancer mortality), followed by colorectal and lung cancer for incidence, and vice versa for mortality [23]. Estrogen receptor (ER) is the oldest and successful biomarker which presents in the breast carcinoma [7]. ER plays as a nuclear hormone receptor and function as transcription factors regulating gene expression referred to as genomic pathway [17, 22]. The current guidelines for measuring ER in a clinical setting assess nuclear staining using immunohistochemistry [1], and inhibition of ER signaling, such as anti-estrogen and aromatase inhibitors, has become one of the major strategies for the treatment of ER-positive breast cancer [12].

ER-negative breast cancer includes most aggressive subtypes of breast cancer such as triple negative breast cancer (TNBC) [10]. Recurrence rate was approximately 10% after 5-year endocrine therapy in ER-positive breast cancer [6], but approximately 30% in TNBC patients [5]. It is partly due to that TNBC patients were excluded from endocrine or targeted therapies which are effectively used for other subtypes of the breast cancer. Therefore, it is very important to examine new therapeutic targets and prognostic markers in the ER-negative breast cancer. It is known that ER also has nonnuclear signaling functions as a nongenomic pathway [2], in addition to the classical genomic pathway. For instance, Madak-Erdogan et al. figured out an ER/MAPK (mitogen-activated protein kinase)/cofilin network that specifies the degree of breast cancer cell aggressiveness through coupling of actin reorganization and hormone receptor-meditated transcription [13, 14]. However, to the best of our knowledge, cytoplasmic immunoreactivity of ER has not been reported in the ER-negative breast carcinoma tissues, which may be partly due to that pathologists evaluate only nuclear immunostaining of ER in the breast carcinoma. Therefore, in this study, we examined immunoreactivity of cytoplasmic ER and demonstrated its clinicopathological significance in the ER-negative breast cancers.

II.  Materials and Methods

Patients and tissues

We reviewed the data of 155 ER-negative breast cancer patients who received surgical treatment from 2016 to 2020 at Tohoku University Hospital (Sendai, Japan). The cut-off value of nuclear ER was 1% in this study according to a previous report [1], and all the cases were negative for nuclear-ER or nuclear-PR immunoreactivity. As a comparative cohort set, we also reviewed 142 ER-positive breast carcinomas in 2016 at Tohoku University Hospital. All the specimens used were primary lesions of the breast in this study. The specimens of breast carcinomas were fixed in 10% formalin and embedded in paraffin wax. 58 patients (37%) received neoadjuvant chemotherapy (NAC). The clinical outcome was evaluated by distant relapse-free survival and breast cancer specific survival. Stage IV cases were excluded in this study. Research protocol was approved by Ethics Committee at Tohoku University School of Medicine (2020-1-1162).

Immunohistochemistry

Immunohistochemistry for ER (CONFIRM anti-ER (SP1)) was automatedly performed with Ventana Benchmark XT (Roche Diagnostics Japan, Tokyo, Japan) according to the manufacturer’s instructions. The SP1 clone is a high affinity rabbit monoclonal antibody directed against an epitope of the C-terminus of the ER protein. Previously, Welsh et al. validated a panel of ER-specific antibodies by Western blot and quantitative immunofluorescent (QIF) analysis of cell lines and patient controls and demonstrated that SP1, 1D5, F10 and 60c antibodies were highly reproducible and specific [26]. Followingly, SP1 antibody was reported more sensitive than 1D5 by QIF ER assay [27]. Based on these results regarding the specificity of ER antibody, we selected SP1 antibody in this study. The brief procedure was as follow: deparaffinization (72°C), Cell Conditioning1 (CC1) for 64 min, antibody (SP1; ready-to-use) for 16 min, Hematoxylin II for 8 min and Bluing reagent for 4 min. A positive control tissue (ER-positive breast carcinoma) was included with every staining set. Negative Control Rabbit IgG was used as a negative control.

Immunohistochemistry for progesterone receptor (PR: CONFIRM anti-PR (1E2); Roche Diagnostics Japan) was also automatedly performed with Ventana Benchmark XT (Roche Diagnostics Japan). Immunohistochemistry for HER2 (Herceptest II; Agilent technologies Japan, Tokyo, Japan) and Ki-67 (FLEX Mouse monoclonal anti-Human Ki-67 Antigen (MIB1); Agilent technologies Japan) was automatedly performed with Autostainer Link 48 (Agilent technologies Japan). As a negative control, Negative Control Rabbit IgG (PR or HER2) or Mouse IgG (Ki67) was used in this study.

Scoring of immunoreactivity

ER or PR immunoreactivity was observed in the cytoplasm of breast carcinoma cells, and the cases which had more than 10% positive carcinoma cells were considered as cytoplasmic-ER or cytoplasmic-PR positive, since the cut off value was frequently used in the evaluation of the cytoplasmic immunoreactivity [16]. In this study, the cytoplasmic ER immunoreactivity was met 5 conditions proposed by Welsh et al. as follows [26], although no 4',6-diamidino-2-phenylindole (DAPI) staining performed in this study: (i) immunoreactivity was observed using 1 of 4 valid monoclonal antibodies (SP1, 1D5, SP1, 60c, and F10), (ii) immunoreactivity colocalized with cytokeratin but did not colocalize with DAPI, (iii) immunoreactivity was robust (at least 25% the intensity of nuclear staining, or greater than nuclear staining), (iv) immunoreactivity was not due to out-of-focus tissue or bleed-through from any other channel, (v) immunoreactivity was observed on the same slide as a positive (nuclear staining with no background) and negative (no staining) control cases. For each cytoplasmic case, conditions 1 to 5 were confirmed by 2 separate individuals (A. Ebata and T. Suzuki), including a certified pathologist (T. Suzuki).

Ki67 was immunolocalized in the nucleus, and the percentage of immunoreactivity (labeling index: LI) was determined. Ki67 LI was classified into two groups in the uni- and multi-variate analyses using 35% which is median of Ki67 LI as a cut-off value in this study.

HER2 immunostaining was scored according to the standardized HercepTest scoring system (score 0–3) (DAKO), and the score 3 was considered positive. HER2 gene amplification was also investigated by fluorescence in situ hybridization (FISH) in the score 2 invasive cases, using PathVysion HER-2 DNA probe kit (Abbott Japan, Tokyo, Japan), and the cases showed positive for FISH were considered positive for HER2 status [28]. We also assessed HER2 and chromosomal 17 signals in normal cells in each case as an internal control. The score 2 in DCIS was not investigated by FISH, and it was evaluated negative for HER2 status in this study.

Statistical analysis

Statistical analysis was performed with JMP Pro 15 software (SAS Institute, Inc., Cary, NC, USA). P value < 0.05 was considered significant. Association between immunohistochemical status of cytoplasmic ER and clinicopathological factors were evaluated using Mann-Whitney U test or a cross-table using the χ² test. Distant relapse-free survival and breast cancer-specific survival curves were generated according to the Kaplan-Meier method, and statistical significance was calculated using a proportional hazard model (Cox). Univariate and multivariate analyses were also evaluated using Cox.

III.  Results

Cytoplasmic-ER immunoreactivity in ER-negative breast carcinoma tissues

ER immunoreactivity was detected in the cytoplasm, but not in the nucleus, in ER-negative breast carcinoma cells (Fig. 1A–C). On the contrary, ER immunoreactivity was observed in the nucleus, but not in the cytoplasm, in the non-neoplastic mammary epithelium (Fig. 1D). ER immunoreactivity was negative in the stroma. The number of cytoplasmic-ER positive cases was 35 out of 155 (23%) ER-negative breast carcinoma cases. When we used the cut-off value of 1% instead of the 10%, the number of positive cases for cytoplasmic ER was not changed in this study. On the other hand, cytoplasmic ER was positive only in two out of 142 (1.4%) ER-positive breast carcinomas in this study (Fig. 1E, F). Representative photos of cytoplasmic-PR and HER2 in the ER-negative breast carcinoma tissues were summarized in Fig. 2.

Fig. 1.

Immunolocalization of cytoplasmic ER in ER-negative (A–D) and ER-positive (E, F) breast carcinoma tissues. A, B: Cytoplasmic-ER was diffusely (A) or focally (B) immunolocalized in the carcinoma cells, as granular scattered, in the ER-negative breast carcinomas (different cases). C: Cytoplasmic-ER was negative in the ER-negative case. D: ER immunoreactivity was only detected in the nucleus, but not in the cytoplasm, of the non-neoplastic mammary epithelium. E, F: cytoplasmic-ER was negative in a great majority of ER-positive breast carcinoma (E), but ER was immunolocalized both in the nucleus and cytoplasm in some ER-positive cases (F). Bar = 50 μm, respectively.

Fig. 2.

Cytoplasmic PR (A) and HER2 (B, C) in the ER-negative breast carcinoma. A: PR was immunolocalized in the cytoplasm of the carcinoma cells. B: HER2 immunoreactivity was detected in the membrane of carcinoma cells (score 3). C: A representative image of HER2 FISH. HER2 signals (orange; n = 9) and chromosome 17 signals (green; n = 4) were detected in the carcinoma cell. Bar = 50 μm (A, B) or 25 μm (C), respectively.

Associations between immunohistochemical cytoplasmic ER and clinicopathological parameters in the ER-negative breast carcinoma was summarized in Table 1. The cytoplasmic-ER status was inversely associated with Ki67 LI (P = 0.0013; Fig. 3) and positively associated with cytoplasmic-PR status (P < 0.0001), but no other significant association was detected in this study.

Table 1.  Association between immunohistochemical status of cytoplasmic ER and clinicopathological parameters in 155 ER-negative breast carcinomas

Parameter	Cytoplasmic ER status		P value
	+ (n = 35)	− (n = 120)
Patient age (years)†	63 (27–84)	59 (29–88)	0.75
Menopausal status
Premenopausal	10	35	1.00
Postmenopausal	25	85
NAC
Received	10	48	0.24
Not received	25	72
Adjuvant chemotherapy
Received	19	68	0.80
Not received	16	52
pT
is, 1††	24	93	0.28
2–4	11	27
pN
0	24	94	0.26
1, 2	11	26
pStage
0, 1	20	82	0.23
2, 3	15	38
Histological grade*
1, 2	22	50	0.11
3	12	54
N.E.	1	16
HER2 status
Positive	13	45	0.69
Negative	22	75
Ki67 labeling index†	23.5 (1.5–80)	41.5 (1–95)	0.0013
Cytoplasmic PR status
Positive	17	6	<0.0001
Negative	18	114

P values less than 0.05 were considered significant and described as boldface. NAC: neoadjuvant chemotherapy. ^†; Data are presented as median (min.–max.). All other values represent the number of cases. ^††; The patient number of pTis was 30. *; Van Nuys classification was used in the DCIS cases (n = 30). Histological grade was not estimated (N.E.) in 17 cases, because numbers of carcinoma cells were limited by receiving NAC.

Fig. 3.

Association between cytoplasmic-ER status and Ki67 LI in 155 ER-negative breast carcinomas. The median value was illustrated by a horizontal line in the box pot, and the box denotes the 75th (upper margin) and 25th percentiles of the values (lower margin), respectively. Dot indicated each case.

As shown in Table 2, cytoplasmic ER status was inversely associated with histological grade (P = 0.047) and Ki67 LI (P = 0.0011) in HER2-negative group, but not in HER2-positive group. In addition, inverse association between cytoplasmic ER and Ki67 LI was only detected in NAC-received group (P = 0.019). On the other hand, cytoplasmic-ER status was positively associated with cytoplasmic-PR status regardless of the HER2 or NAC status in this study.

Table 2.  Association between cytoplasmic-ER immunohistochemical status and clinicopathological parameters in 155 ER-negative breast carcinomas according to HER2 or NAC status

	Cytoplasmic ER status (positive/negative)
	HER2 positive group (n = 58)	HER2 negative group (n = 97)	NAC received (n = 58)	NAC Not received (n = 97)
Patient age (years)†	0.72	0.61	0.61	0.84
Menopausal status (premenopausal/postmenopausal)	0.68	0.80	0.73	1.00
NAC (received/not received)	1.00	0.23	N.E.	N.E.
pT (is,1/2–4)	0.21	0.79	0.71	0.095
pN (0/1,2)	1.00	0.11	0.26	0.56
pStage (0,1/2,3)	0.49	0.45	1.00	0.14
Histological grade (1,2/3)	1.00	0.047*	1.00	0.097
HER2 (positive/negative)	N.E.	N.E.	0.71	0.65
Ki67 labeling index†	0.44	0.0011*	0.019*	0.061
Cytoplasmic PR status (positive/negative)	0.0005	<0.0001	<0.0001	0.0001

Data are present as P values. P value < 0.05 was considered significant and was listed in bold. ^†; Data were used was continuous values. *; Inversely significant association. NAC: neoadjuvant chemotherapy, N.E.; not estimated.

Association between cytoplasmic-ER and clinical outcome of ER-negative breast cancer patients

As demonstrated in Fig. 4A, the rate of distant relapse-free survival was significantly higher in the cytoplasmic-ER positive group than in cytoplasmic-ER negative group in 155 ER-negative breast cancer patients (P = 0.0032). There was no patient who had distantly recurred in the cytoplasmic-ER positive group in this study. Similarly, the rate of breast cancer-specific survival was significantly higher in the cytoplasmic-ER positive group than in the cytoplasmic-ER negative group (P = 0.026, Fig. 4B).

Fig. 4.

Distant relapse-free survival (A, C–F) and breast cancer-specific survival (B) of 155 ER-negative breast cancer patients according to the cytoplasmic-ER status. A, B: cytoplasmic ER in whole cases (n = 155), C, D: HER2 positive (C; n = 58) or HER2 negative (D; n = 97) cases. E, F: NAC received (E; n = 58) or not received (F; n = 97) cases. The solid line shows cytoplasmic ER-positive group, and the dashed line shows cytoplasmic-negative group. P values < 0.05 was considered significant and listed in bold.

When we performed univariate analysis of distant relapse-free survival in 155 ER-negative breast cancer patients using Cox (Table 3, left side), pN (P < 0.0001) and cytoplasmic ER (P = 0.0032) were shown as significant prognostic factors. Following multivariate analysis demonstrated that both pN (P < 0.0001) and cytoplasmic ER (P = 0.0016) were independent prognostic factors for distant relapse-free survival.

Table 3.  Univariate and multivariate analyses of clinical outcome in 155 ER-negative breast cancer patients

	Distant relapse-free survival			Breast cancer-specific survival
	Univariate	Multivariate		Univariate	Multivariate
	P value	P value	Relative risk (95%CI)	P value	P value	Relative risk (95%CI)
pN (0/1, 2)	<0.0001	<0.0001	0.06 (0.01–0.22)	0.0074	0.014	0.18 (0.04–0.77)
Cytoplasmic ER (+/−)	0.0032	0.0016	N.E.	0.026	0.029	N.E.
pT (is, 1/2–4)	0.085	0.77	1.20 (0.36–4.03)	0.0029	0.23	0.42 (0.10–1.78)
Ki67 (<35%/35%≦)	0.085	0.65	0.75 (0.20–2.73)	0.028	0.78	0.78 (0.12–4.78)
Histological grade (1, 2/3)	0.68	0.89	0.91 (0.26–3.21)	0.22	0.59	0.62 (0.11–3.58)
Cytoplasmic PR (+/−)	0.77	0.33	3.57 (0.37–34.5)	0.63	0.58	2.10 (0.18–23.9)

Statistical analysis was evaluated by a proportional hazard model (Cox). P value < 0.05 were considered significant and listed in bold. All the factors were examined in the multivariate analysis. 95%CI; 95% confidence interval. N.E.; not estimated, because no patients had recurred or died in cytoplasmic ER positive group in this study.

On the other hand, as shown in the right side of Table 3, univariate analysis for breast cancer-specific survival revealed that pT (P = 0.0029), pN (P = 0.0074), cytoplasmic ER (P = 0.026) and Ki67 status (P = 0.028) were significant prognostic parameters. Subsequent multivariate analysis demonstrated that only pN (P = 0.014) and cytoplasmic ER (P = 0.029) were independent prognostic marker in this study.

IV.  Discussion

This is the first study that demonstrates clinicopathological significance of cytoplasmic ER in ER-negative breast cancer tissues, to the best of our knowledge. In this study, immunoreactivity of cytoplasmic ER was detected in 35 out of 155 (23%) ER-negative cases, but it was negative in the non-neoplastic mammary epithelium. On the other hand, cytoplasmic ER was only positive in 2 out of 142 (1.4%) ER-positive cases. Previously, cytoplasmic-ER immunoreactivity has been reported in the breast carcinoma by only two groups. Welsh et al. showed that the incidence of cytoplasmic ER staining only averaged 1.5% (ranging from 0–3.2%) in more over 3,000 breast carcinoma tissues although no information is available in the nuclear ER status of these cases [26]. On the other hand, Guo et al. reported that high ratio of extranuclear-to-nuclear ER in ER-positive breast carcinoma was associated with poor overall survival and disease-free survival by immunohistochemistry using phosphor-integrated dots [9].

There are three main mechanisms of ER action that include 1) nuclear genomic, direct DNA binding, 2) nuclear genomic, “tethered”-mediated protein-protein interactions, and 3) nonnuclear nongenomic, rapid action process [3, 15]. Intracellular ER localization is not static, but rather dynamic with continuous shuttling between the nucleus and cytoplasm [24]. For instance, ER was exclusively localized in the cytoplasm in MCF-7 breast carcinoma cells grown in the absence of estrogen [18] and overexpression of HER2 stimulated the translocation of ER from nucleus to the cytoplasm in MCF-7 cells [24]. In addition, nuclear translocation of ER was altered by mutation in the D-domain [3], and variants of ER (i.e., ER34 and ER46) were localized in the cytoplasm and/or membrane [24]. Considering that the incidence of cytoplasmic ER was much higher in the ER-negative breast carcinoma tissues (23%) compared to the ER-positive cases (1.4%) in our study, it is suggested that cytoplasmic-ER plays important roles especially in the ER-negative breast carcinoma.

In our present study, cytoplasmic-ER status was significantly associated with cytoplasmic-PR status in the ER-negative breast carcinoma, although PR was not immunolocalized in the nucleus. Transcription of PR gene is regulated by estrogenic actions through nuclear ER in the breast carcinoma. Therefore, it may be due to that PR synthesis is induced by a low or undetectable level of nuclear ER in these cases. In addition, it is also possibly to speculate that the PR expression is, at least in a part, induced by nongenomic signaling pathway through cytoplasmic ER, because Silva et al. [21] reported that transcription of PR by estrogen was partially suppressed by an ERK inhibitor (PD 98059) in MCF-7 cells. Interestingly, it is reported that crosstalk between PR and ER in the cytoplasm activates MAPK pathways in the breast carcinoma cells [25].

Our present study demonstrated that cytoplasmic ER was inversely associated to Ki67 LI in the ER-negative breast carcinoma. It is well recognized that ER signaling activation is lack in TNBC [29]. However, recently Contreras-Zarate et al. found that estradiol induced BDNF/TrkB signaling in TNBC to promote brain metastases [4], and Girgert et al. suggested that estradiol significantly increased cell proliferation of TNBC in vitro, along with the activation of Src, EGFR (epidermal growth factor receptor), Cyclin D1, and CREB (cAMP-responsive element binding) signaling [8]. Therefore, it is suggested that cytoplasmic ER involves in estrogen-induced proliferation in ER-negative breast carcinoma tissue. Inverse association between cytoplasmic ER and Ki67 LI may reflect the proliferative activity is relatively mild than other pathways in ER-negative breast carcinoma.

Our present results demonstrated that the rate of distant relapse-free survival is significantly higher in cytoplasmic ER positive patients than in cytoplasmic ER negative patients. All the patients who showed positive for cytoplasmic ER were alive without distant relapse regardless they received NAC or not. Moreover, cytoplasmic ER was independent favorable prognostic marker in ER-negative breast cancer patients. Currently, most ER-negative breast cancer patients receive chemotherapy. Considering that cytoplasmic-ER positive cases showed lower Ki67 LI in ER-negative breast carcinoma, benefit of chemotherapy is limited in these patients, and other treatments targeted cytoplasmic ER may be preferred. Biological functions of cytoplasmic ER remain unclear in the breast carcinoma, and further examinations are required.

There are some limitations in this study. First, this study is a descriptive study, and we could not fully guarantee the specificity of cytoplasmic ER, although SP1 is a highly reliable antibody [26, 27]. To obtain more precisely understanding of the cytoplasmic ER, further in vitro experiments are required, such as immunoprecipitation of ER protein using the cytoplasmic or nuclear protein extract. Second, this study lacks functional experiments. We have examined chemoresistance in the breast carcinoma [11, 19, 20], and following investigations are required to clarify significance of nucleo-cytoplasmic transport of ER and molecular functions of cytoplasmic-ER signaling in the ER-negative breast carcinoma cells.

In summary, we examined immunoreactivity of cytoplasmic ER in 155 ER-negative breast carcinoma tissues. Cytoplasmic ER immunoreactivity was detected in 23% of the ER-negative breast carcinoma tissues, but it was positive only in 1.4% of the ER-positive cases in a comparative cohort set. Cytoplasmic-ER status was positively associated with cytoplasmic-PR and inversely associated with Ki67 LI. Moreover, cytoplasmic-ER status was significantly associated with better prognosis of the ER-negative breast cancer patients, and it turned out an independent prognostic factor for distant relapse-free survival and breast cancer specific survival. These results suggest that cytoplasmic-ER status is a potent prognostic factor in ER-negative breast cancer patients, and benefit of chemotherapy may be limited in these patients.

V.  Conflicts of Interest

The authors have no conflict of interest to declare, in this study.

VI.  Acknowledgments

This work was supported in part by a Grant-in-Aid from KUROKAWA CANCER RESEARCH FOUNDATION.

VII.  Research Involving Human Participants and/or Animals

Statement of human rights

All procedures performed in studies involving human participants were in accordance with the ethical standards of the Ethics Committee at Tohoku University Graduate School of Medicine and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was received from all individual participants included in the study.

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