Abstract

Purpose: To assess the prognostic value of co-expression of estrogen receptor (ER)-beta and human epidermal growth factor receptor 2 (HER2) in primary breast cancer patients in China.

Methods: Tumour specimens from 308 patients undergoing surgery for primary breast cancer were evaluated. Expression of ER-beta and HER-2 was investigated by the immunohistochemistry.

Results: 123 patients (40%) were ER-beta positive and 58 (18.5 %) were HER2 positive. Among the 58 HER2 positive patients, 44 were ER-beta positive and 14 were ER-beta negative. ER-beta positive was associated with HER2 positive (75.9%, P=0.018) as well as ER-alpha positive (79.7%, P=0.023), poor cell differentiation (77.2% grade 2 or 3, P=0.010) and menopause age <45 yr (55.3%, P=0.031). HER2 positive was associated with poor cell differentiation (93.1%, P=0.001), ≥3cm tumour size (67.2%, P=0.011).

Conclusion: Both ER-beta positive and HER2 positive status was associated with poorer overall survival (OS) by univariate analysis. In both HER2 positive and HER2 negative subgroups, ER-beta positive was associated with poorer distant disease free survival (DDFS) but not OS, which implied that ER-beta might relate to metastasis in breast cancer.

 

 

Estrogens are potent mitogens in the mammary gland in which they regulate the growth, development, and function of normal as well as cancerous breasts.^1,2 Epidemiological evidence and numerous animal studies have indicated that estrogens play a role in the proliferation and progression of breast cancer: removal of the ovaries or treatment with antiestrogens opposes their deleterious effects.^3,4

 Since cloning of the estrogen receptor (ER) in 1986,⁵ it was believed that only a single receptor (now termed ER-alpha) was responsible for mediating the effects of estrogens on target tissues. In 1996, a second ER, referred to as ER-beta, was identified.^6-8 Although the molecular mechanisms of ER-beta function are poorly understood, it is becoming increasingly clear that the two receptor types are responsible for different biological functions, as indicated by their specific expression patterns.^9,10 ER-beta is expressed in both human breast tumours¹¹ and chemically transformed human breast epithelial cells.¹² ER-beta splice variants have also been discovered in some breast tumours.^13,14 Besides their different physiological functions, Jarvinen et al. suggested that ER-beta was expressed in 75% of ER-alpha-positive and PR-positive breast cancer tumours, and was likely to respond to hormonal therapy.¹⁵ Some studies suggested that wild-type ER-beta tended to be a good prognostic indicator,^16,17 whereas othersr eached contrary conclusions. ¹⁸ These studies illustrated that the relationship between ER-beta and ER-alpha was complicated and demanded further research.

Human epidermal growth factor receptor 2 (HER2, neu or c-erb-B2) is a ligand orphan receptor. HER2 activates several downstream signaling cascades and new downstream HER2 signaling proteins remain to be identified.¹⁹ In preclinical studies, HER2 overexpression was found to be associated with a variety of features that make tumour cells more aggressive.^20,21 In clinical studies, amplification or overexpression of HER2 in tumour cells was associated with poor clinical outcome.^22,23

Recent studies demonstrated that HER2 could regulate the function of ER through the Ras/MAPK signaling pathway. HER2 overexpression activates the Ras/MAPK signaling pathway in breast tumour cell lines and carcinomas.^24,25 Clinical studies also suggested that HER2/neu were related to the ER status in human breast cancer.^26-30

However, few studies have reported the relationship between ER-beta and HER2, especially in oriental people. In this study, we investigated the expression of ER-beta and HER2 in 308 cases of human breast cancer in China, evaluated their prognostic values in the breast cancer patients and indicated the possible clinical relationship between ER-beta and HER2 expression.

 

Patients and Methods

Patients

Tumour specimens from 308 patients (mean age, 58 yr; range, 30–79 yr) with no sign of distant metastasis and who underwent surgery for primary breast cancer from 1993–1998 [modified mastectomy, 217 patients, breast-conserving lumpectomy, 91 patients] at Shanghai Cancer Hospital in Shanghai, China were evaluated. Tumour specimens were submitted to the Laboratory of Hormone Receptors for steroid receptor analysis. Women with positive lymph nodes generally received adjuvant chemotherapy (164 patients); 109 patients received hormonal therapy (tamoxifen), and 73 patients were irradiated. The median follow-up for patients was 48 months. A computerized database containing updated clinicopathological information was established for statistical analysis. The study received Institutional Ethics Committee approval.

 

Immunohistochemistry (IHC)

The following primary antibodies and dilutions were used: anti-ERß (Ab-1, Oncogene), rabbit polyclonal antibody; (Novocastra); an affinity-purified rabbit polyclonal antiserum raised against an intracellular epitope of the human HER-2 molecule (specification sheet K5205, Dako). Both antibodies were diluted at 1:5000. Five-µm -thick sections were cut and mounted on SuperFrost Plus slides. Sections were left unbaked until immediately before use when they were baked for 1 hour at 60°C. After baking, sections were deparaffinized by three changes of xylene and rehydrated through graded alcohols into water. Heat-induced epitope retrieval was performed by boiling sections in citrate buffer pH 6.0 (pH 6.2 for ERß) for 15 min on a laboratory hotplate. After boiling, sections were removed from the hotplate, allowed to cool at room temperature for 20 min, and were then rinsed thoroughly with water. Sections were then placed in 3% hydrogen peroxide for 15 min at room temperature to block endogenous peroxidase, washed with water, and placed in PBS (Sigma). Sections were incubated with Power Block (Biogenex) nonspecific blocking reagent for another 10 min at room temperature to reduce nonspecific staining, washed with water, and placed in PBS. After that, sections were incubated with normal goat serum at 1:50 (Vector, Burlingame, CA) for 15 min at room temperature. The goat serum was then shaken off and sections were incubated with primary antibodies overnight at 4°C. After overnight incubation, each section received 20 sec washing with PBS, 20 sec washing with Biogenex Optimax detergent wash solution, followed by 10 min washing in PBS on a rotator. Solutions were changed for every eight slides. After washing, sections were incubated with Biogenex Multilink secondary antibody at a dilution of 1:20 for 20 min at room temperature. Sections were again washed according to the protocol described above. Slides were stained using the ABC elite kit (Vector Laboratories) with diaminobenzidine according to the manufacturer’s protocol. All ER-beta and PR immunoreactive cells were considered positive regardless of the staining intensity, and the fraction of positive cells was recorded. A case was considered positive if > 10% cells were stained. Immunostaining was analyzed with an Olympus Corp. BX-50 microscope, and pictures were taken with an Olympus Corp. Oly 760 video camera.^31,32 

Each HER2 immunohistochemical staining experiment included a set of control slides. For the negative control section the primary antibody was replaced with an irrelevant, isotype-matched antibody, to control for nonspecific binding of the secondary antibody reagent. The positive control slide consisted of sections of cell blocks of the three breast cancer cell lines SKBR3, MDA-MB-175 and MDA-MB-231, which express 2.4 million, 92,000 and 22,000 HER-2 receptor molecules, by Scatchard analysis, respectively. These receptor numbers correspond to IHC HER-2 scores of +++, + and 0.³³ A score of ≥ + or above was counted as positive.

 The staining results were read by a group of senior pathologists. In each case, 2,000 tumuor cells were counted from 10 randomly selected areas, ensuring that the whole section was scanned. All negative cases were repeated for eliminating false negative and confirming true negative.

 

Statistical Analysis

Life tables were calculated according to the Kaplan-Meier method. DDFS was computed from the date of diagnosis to the occurrence of metastases outside the locoregional area or to death from breast cancer, whichever came first. Breast cancer-specific survival was calculated from the date of diagnosis until death from breast cancer. Patients who died from an intercurrent cause were censored at the date of death. Survival curves were compared using the Log-rank test. Multivariate survival analyses were performed using the following covariates: (a) grade (well differentiated, 0; moderately or poorly differentiated, 1, because moderately and poorly differentiated cancers had similar outcome); (b) ER and PgR status (positive or borderline, 1; negative, 0); (c) HER2 positive status (negative, 0; positive, 1); (e) histological type (lobular or special, 0; ductal, 1; the lobular and special types were associated with similar outcome); and (f) tumour size in millimeters as a continuous variable. The final multivariate model was constructed using backward Cox stepwise proportional hazards regression, and a P <  0.05 was adopted as the limit for inclusion of a covariate. All P values are two sided.

 

Results

Demographics

308 patients were studied. The basic demographics of this group and the pathological characteristics are shown in Tables 1 and 2, respectively. The median age was 58 yr (range 30-79 yr).

 

Expression of ER-beta and HER2

The tumours were divided into three main histological types: invasive ductal carcinoma, invasive lobular carcinoma and adenocarcinoma. ER-beta positive cells were stained brown by IHC staining in the nuclei, while HER2 positive cells were stained brown by IHC staining on the cytomembrane. (Fig. 1).

 

Association of ER-beta expression with other prognostic parameters

123 patients (40%) were ER-beta positive by IHC. ER-beta positive status was associated with ER-alpha (P=0.023), poor cell differentiation, grade 3 (P=0.010) and menopause age < 45 yr (P=0.031). ER-beta positive status has no association with PR status, lymph node or tumour size. (Table 3).

 

Association of HER2 positive with other prognostic parameters except estrogen receptors

58 patients (18.5 %) were HER2 positive by IHC. HER2 positive associates with poor cell differentiation of grade 3 (93.1%, P=0.001) and ≥3cm tumour size (63.2%, P=0.011).  However, it has no association with PR status lymph node, or menopausal status (Table 4).

 

Association of estrogen receptors with HER2 positive

ER-beta expression was associated with the HER2 positive (P=0.018). In 58 HER2 positive patients, 44 were ER-beta positive, while only 14 were ER-beta negative. ER-alpha positive was not associated with HER2 positive. In addition, subgroups of ER-beta positive and ER-alpha negative (ER-beta +/ER-alpha -) were associated with HER2 positive (P<0.001) (Table 4).

 

ER-beta expression and HER2 positive are associated with poorer survival

Univariate analysis of ER-beta positive status indicated its association with poorer OS compared with ER-beta negative sub-group (P=0.003) (Fig. 2A). HER2 positive showed association with  poorer OS compared with HER2 negative sub-group (P<0.001) in univariate analysis (Fig. 2B).

 

Outcome Survival of subgroups of ER-beta and HER2

We then analyzed the OS of four subgroups: ER-beta positive and HER2 negative (ER-beta +/HER2 -); ER-beta positive and HER2 positive (ER-beta +/HER2 +); ER-beta negative and HER2 negative (ER-beta -/HER2 -); ER-beta negative and HER2 positive (ER-beta -/HER2 +). In the subgroup of ER-beta positive patients, the OS of HER2 negative patients was longer than that of HER2 positive patients (P<0.001) (Fig. 3A) and, in the subgroup of ER-beta negative patients, the OS of HER2 negative patients was longer than that of HER2 positive patients (P<0.001) (Fig. 3B). However, in the subgroup of HER2 positive patients, the OS of ER-beta negative patients was not different from that of ER-beta positive patients (Fig. 3C). In the subgroup of HER2 negative patients, the OS of ER-beta negative patients was not different from that of ER-beta positive patients (P=0.541) (Fig. 3D).

 

DDFS of subgroups of ER-beta and HER2

The DDFSs of the above four subgroups were also analyzed. In the subgroup of ER-beta positive patients, the DDFS of HER2 negative patients was longer than that of HER2 positive patients (P<0.001) (Fig. 4A). In the subgroup of ER-beta negative patients, the DDFS of HER2 negative patients was longer than that of HER2 positive patients (P<0.001) (Fig. 4B). In the subgroup of HER2 positive patients, the DDFS of ER-beta negative patients was no longer than that of ER-beta positive (Fig. 4C). In the subgroup of HER2 negative patients, the DDFS of ER-beta negative patients was longer than that of ER-beta positive patients (P=0.005) (Fig. 4D).

 

Discussion

We found that ER-beta positivity was associated with ER-alpha positivity, poor cell differentiation and menopausal age < 45 yr, but was not associated with PR We investigated ER-beta status and HER2 positive in 308 patients of breast cancer. ER-beta positivity was associated with HER2 positivity in breast cancer (Tables 3 and 4). HER2 positivity was a prognostic factor in breast cancer, and ER-beta positivity showed poorer outcome than ER-beta negativity in univariate analysis (Fig. 2A). ER-beta was not an independent prognostic indicator of breast cancer but was associated with a shorter survival time by univariate analysis (Fig. 2A). Considering that ER-beta positivity was likely accompanied with HER2 positivity, we hypothesized that the poorer survival of ER-beta positive patients would be due to HER2 positivity. We then analyzed the survival of subgroups of patients: ER-beta +/HER2 -; ER-beta +/HER2 +; ER-beta -/HER2 -; ER-beta -/HER2 +. HER2 positive was s associated with poorer OS in either ER-beta positive or ER-beta negative subgroups (Figs. 3A and 3B). In either HER2 positive or HER2 negative subgroups, however, ER-beta positive was not associated with poorer OS (Figs. 3C and 3D). These results implied that ER-beta positive would not be associated with poorer OS when the interference of HER2 positive was ruled out. Analysis of DDFS figured similar results for HER2 positive in either ER-beta positive or ER-beta negative subgroups (Figs. 4A and 4B). ER-beta positive was associated with poorer DDFS in HER2 negative subgroup (Fig. 4D), implying that ER-beta might be related to metastasis of breast cancer. In addition, the subgroup of ER-beta positive accompanied with ER-alpha negative (ER-beta +/ER-alpha -) was associated with HER2 positive.

ER-alpha and ER-beta are the major mediators of a variety of biological functions of estrogens.^34,35 There are many reports of ER-beta in basic and clinical research. Multiple biological functions as well as the prognostic value of ER-beta have been reported.^17,36 Jarvinen et al. reported that ER-beta positive associated with both ER-alpha positive and PR positive in breast cancer tumours.¹⁵ The fact that their results are partly different from ours may be due to racial diversity. Several recent papers have suggested that HER2 expression in breast carcinomas may be an indicator of resistance to hormonal (predominantly tamoxifen) therapy.^37,38 This unfortunate resistance to endocrine therapy could be partly due to crosstalk between the ER and EGFR/HER2 and/or their downstream effectors. HER2 gene amplified in breast cancer cells partially restored the ability of tamoxifen to inhibit estradiol-stimulated ER reporter activity.^39,40 Since ER-beta exerts multiple effects in the estrogen signaling pathway, the relation between ER-beta and HER2 should attract researchers. However, there are few papers concerning ER-beta together with HER2.

In 2001, ER-beta positive in the absence of ER-alpha associated with high expression of Ki67 was found in breast cancer.^41,42 This result and the present study imply that, in the absence of ER-alpha, ER-beta plays a different role in breast cancer from that in the presence of ER-alpha, and this role might relate to HER2 and Ki67.

In conclusion, we have shown that ER-beta positive status is associated with HER2 positivity, poor cell differentiation and menopause age ≤45 years. HER2 positive status is associated with poor cell differentiation, ≥3cm tumour size and ER-beta positive status. ER-beta positive status and HER2 positivity showed association with poorer OS. When interference of HER2 positivity was ruled out, ER-beta positive status was associated with poorer DDFS but not OS, which implied that ER-beta might be related to the metastasis of breast cancer.

 

 

References

1.     Haslam SZ, Counterman LJ, Nummy KA: Effects of epidermal growth factor, estrogen, and progestin on DNA synthesis in mammary cells in vivo are determined by the developmental state of the gland. J Cell Physiol 1993;155:72–8.

2.     Key T, Appleby P, Barnes I, Reeves G.: Endogenous sex hormones and breast cancer in postmenopausal women: reanalysis of nine prospective studies. J Natl Cancer Inst 2002;94:606-16.

3.     Hilakivi-Clarke L, Clarke R, Lippman M: The influence of maternal diet on breast cancer risk among female offspring. Nutrition 1999;15:392–401.

4.     Yager JD, Davidson NE: Estrogen carcinogenesis in breast cancer. N Engl J Med 2006;354:270–82.

5.     Greene GL, Gilna P, Waterfield M, Baker A, Hort Y, Shine J: Sequence and expression of human estrogen receptor complementary DNA. Science 1986;231:1150-54.

6.     Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustaffson JA: Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci 1996;93:5925-30.

7.     Mosselman S, Polman J, Dijkema R: ER-ß: identification and characterisation of a novel human estrogen receptor. FEBS Lett.1996;392:49-53.

8.     Pettersson K, Gustafsson JA: Role of estrogen receptor β in estrogen action. Annu Rev Physiol 2001;63:165-92.

9.     O’Lone R, Frith MC, Karlsson EK. Hansen U: Genomic targets of nuclear estrogen receptors. Mol Endocrinol 2004;18:1859–75.

10.  Acconcia F, Totta P, Ogawa S et al: Survival versus apoptotic 17β-estradiol effect: role of ER-alpha and ER-beta activated non-genomic signaling. J Cell Physiol  2005;203:193-201.

11.  Enmark E, Pelto-Huikko M, Grien K et al: Human estrogen receptor ß-gene structure, chromosomal localisation and expression pattern. J Clin Endocrinol Metab 1997;82:4258-65.

12.  Hu YF, Lau KM, Ho S-M, Russo J: Increased expression of estrogen receptor ß in chemically transformed human breast epithelial cells. Int J Oncol 1998;12:1225-8.

13.  Vladusic EA, Hornby AE, Guerra-Vladusic FK, Lupu R: Expression of estrogen receptor ß messenger RNA variant in breast cancer. Cancer Res 1998;58:210-4.

14.  Lu B, Leygue E, Dotslaw H, Murphy LJ, Murphy LC, Watson PH: Estrogen receptor-ß mRNA variants in human and murine tissues. Mol Cell Endocrinol 1998;138:199-203.

15.  Jarvinen TA, Pelto-Huikko M, Holli K, Isola J: Estrogen receptor beta is coexpressed with ERalpha and PR and associated with nodal status, grade, and proliferation rate in breast cancer. Am J Pathol 2000;156:29-35.

16.  Murphy LC, Watson P: Steroid receptors in human breast tumorigenesis and breast cancer progression. Biomed Pharmacother 2002;56:65-77.

17.  Omoto Y, Kobayashi S, Inoue S et al: Evaluation of oestrogen receptor beta wild-type and variant protein expression, and relationship with clinicopathological factors in breast cancers. Eur J Cancer 2002;38:380-6.

18.  Wen XF, Shen Z, Shen ZZ, Nguyen M, Shao ZM: The expression of ER-beta protein correlates with vascular endothelial growth factor and its prognostic significance in human breast cancer. Oncol Rep 2002;9:937-44.

19.  Bose R, Molina H, Patterson AS et al: Phosphoproteomic analysis of Her2/neu signaling and inhibition. Proc Natl Acad Sci 2006;103:9773-8.

20.  Li YM, Pan Y, Wei Y et al: Upregulation of CXCR4 is essential for HER2-mediated tumor metastasis. Cancer Cell 2004;6:459-69.

21.  Subbaramaiah K, Howe LR, Port ER et al: HER2/neu status is a determinant of mammary aromatase activity in vivo: evidence for a cyclooxygenase-2-dependent mechanism. Cancer Res 2006;66:5504-11.

22.  Vinatzer U, Dampier B, Streubel B et al: Expression of HER2 and the coamplified genes GRB7 and MLN64 in human breast cancer: quantitative real-time reverse transcription-PCR as a diagnostic alternative to immunohistochemistry and fluorescence in situ hybridization. Clin Cancer Res 2050;11:8348-57.

23.  Lopez-Guerrero JA, Llombart-Cussac A, Noguera R et al: HER2 positive in recurrent breast cancer following breast-conserving therapy correlates with distant metastasis and poor survival. Int J Cancer 2006;118:1743-9.

24.  Tzahar E and Yarden Y: The ErbB-2/Her2 oncogenic receptor of adenocarcinomas: from orphanhood to multiple stromal ligands. Biochim Biophys Acta 1998;1377:25-37.

25.  Hirokazu K, Anne EG, Jean F et al: Forbes and Carlos L. Arteaga Inhibition of Her2/neu (erbB-2) and Mitogen-activated Protein Kinases Enhances Tamoxifen Action against Her2-overexpressing, Tamoxifen-resistant Breast Cancer Cells. Cancer Res 2000;60:5887-94.

26.  Massarweh S, Osborne CK, Jiang S et al: Mechanisms of Tumor Regression and Resistance to Estrogen Deprivation and Fulvestrant in a Model of Estrogen Receptor-Positive, HER2/neu-Positive Breast Cancer. Cancer Res. 66:8266-73, 2006.

27.  Tokunaga E, Kimura Y, Oki E et al: Akt is frequently activated in HER2/neu-positive breast cancers and associated with poor prognosis among hormone-treated patients. Int J Cancer 2006;118:284-9.

28.  Huang HJ, Neven P, Drijkoningen M et al: Association between tumour characteristics and HER2/neu by immunohistochemistry in 1362 women with primary operable breast cancer. J Clin Pathol 2005;58:611-6.

29.  Weitsman GE, Li L, Skliris GP et al: Estrogen receptor-alpha phosphorylated at Ser118 is present at the promoters of estrogen-regulated genes and is not altered due to HER2 overexpression. Cancer Res 2006;66:10162-70.

30.  Wang LH, Yang XY, Zhang X et al: Disruption of estrogen receptor DNA-binding domain and related intramolecular communication restores tamoxifen sensitivity in resistant breast cancer. Cancer Cell 2006;10:487-99.

31.  Roger P, Sahla ME, Makela S, Gustafsson JA, Baldet P, Rochefort H: Decreased expression of estrogen receptor β protein in proliferative preinvasive mammary tumors. Cancer Res 2001;61:2537-41.

32.  Saji S, Jensen EV, Nilsson S, Rylander T, Warner M, Gustafsson JA: Estrogen receptors

and ß in the rodent mammary gland. Proc Natl Acad Sci 2000;97:337-42.

33.  Lottner C, Schwarz S, Diermeier S et al: Simultaneous detection of HER2/neu gene amplification and protein overexpression in paraffin-embedded breast cancer. J Pathol 2005; 205:577-84.

34.  Masood S, Bui MM, Yung JF et al: Reproducibility of LSI HER2/neu SpectrumOrange and CEP 17 SpectrumGreen Dual Color deoxyribonucleic acid probe kit. For enumeration of gene amplification in paraffin-embedded specimens: a multicenter clinical validation study. Ann Clin Lab Sci 1998;28:215–23.

35.  Pettersson K, Gustafsson JA: Role of estrogen receptor β in estrogen action. Annu Rev Physiol 2001;63:165-92.

36.  Osborne CK, Schiff R: Estrogen-receptor biology: continuing progress and therapeutic implications. J Clin Oncol 2005;23:1616-22.

37.  Cheng EJ, Madison LD, Lazennec G: Expression of estrogen receptor β in prostate carcinoma cells inhibits invasion and proliferation and triggers apoptosis, FEBS Lett 2004;566:169–72.

38.  Schiff R, Massarweh S, Shou J, Osborne CK: Breast cancer endocrine resistance: how growth factor signaling and estrogen receptor coregulators modulate response. Clin Cancer Res 2003;9:447-54.

39.  Elledge RM, Green S, Ciocca D et al: HER2 expression and response to tamoxifen in estrogen receptor-positive breast cancer: a Southwest Oncology Group Study. Clin Cancer Res1998;4:7-12.

40.  Yamauchi H, Stearns V and Hayes DF: When is a tumour marker ready for prime time? A case study of c-erbB-2 as a predictive factor in breast cancer. J Clin Oncol 2001;8:2334–56.

41.  Kurokawa H, Arteaga CL: ErbB (HER) Receptors can abrogate antiestrogen action in human breast cancer by multiple signaling mechanism. Clin Cancer Res  2003;9:511–5.

42.  Jensen EV, Cheng G, Palmieri C et al: Estrogen receptors and proliferation markers in primary and recurrent breast cancer. Proc Natl Acad Sci U S A.2001;98:15197-202.

 

 

 

Correspondence to:

 

Dr. Fan Li-hong, Cancer Center and Dr. Shen Zan,

Department of Oncology,

People’s 6th Hospital,

Shanghai Jiaotong University,

Shanghai 200233, P. R. China.

E-mail: sshenzzan@gmail.com

 

FIGURE 1. ER-beta expression and HER2 expression. First row - IHC staining for ER-beta expression in three different histologic types (brown). Second row are IHC staining for HER2 expression (brown).

 

FIGURE 2. Overall survival (OS) rates of different ER-beta status and HER2 status of 308 breast carcinoma patients. ER-beta - (ER-beta negative), ER-beta + (ER-beta positive); HER2 - (HER2 negative); HER2 + (HER2 positive). (A) Real line indicates the survival curve of ER-beta negative patients. Broken line indicates the survival curve of ER-beta positive patients. (B) Real line indicates the survival curve of HER2 negative patients. Broken line indicates the survival curve of HER2 positive patients.

 

FIGURE 3. Overall survival (OS) of the subgroups: ER-beta +/HER2 -; ER-beta +/HER2 +; ER-beta -/HER2 -; ER-beta -/HER2 +. ER-beta - (ER-beta negative), ER-beta + (ER-beta positive); HER2 - (HER2 negative); HER2 + (HER2 positive). (A) Subgroup ER-beta positive patients, the OS of HER2 negative patients is longer than that of HER2 positive patients (P<0.001). Real line indicates the survival curve of ER-beta positive and HER2 negative patients. Broken line indicates the survival curve of ER-beta positive and HER2 positive patients. (B) Subgroup ER-beta negative patients, OS of HER2 negative patients is longer than that of HER2 positive patients (P<0.001). Real line - survival curve of ER-beta negative and HER2 negative patients. Broken line - survival curve of ER-beta negative and HER2 positive patients. (C) Subgroup HER2 positive patients, the OS of ER-beta negative patients is not different from that of ER-beta positive patients Real line indicates the survival curve of HER2 positive patients and ER-beta negative patients. Broken line indicates the survival curve of HER2 positive patients and ER-beta positive. (D) Subgroup HER2 negative patients, the OS of ER-beta negative patients is not different from that of ER-beta positive patients. Real line indicates the survival curve of HER2 negative patients and ER-beta negative patients. Broken line indicates the survival curve of HER2 negative and ER-beta positive patients.

 

FIGURE 4. Distance disease free survival (DDFS) curves of the subgroups: ER-beta +/HER2 -; ER-beta +/HER2 +; ER-beta -/HER2 -; ER-beta -/HER2 +. ER-beta - (ER-beta negative), ER-beta + (ER-beta positive); HER2 - (HER2 negative); HER2 + (HER2 positive).  (A) Subgroup ER-beta positive patients, DDFS of HER2 negative patients is longer than that of HER2 positive patients (P<0.001). Real line indicates the survival curve of ER-beta positive and HER2 negative patients. Broken line indicates the survival curve of ER-beta positive and HER2 positive patients. (B) Subgroup of ER-beta negative patients, the DDFS of HER2 negative patients is longer than that of HER2 positive patients (P<0.001). Real line indicates the survival curve of ER-beta negative and HER2 negative patients. Broken line indicates the survival curve of ER-beta negative and HER2 positive patients. (C) Subgroup of HER2 positive patients, the DDFS of ER-beta negative patients is not different that of ER-beta positive patients. Real line indicates the survival curve of HER2 positive patients and ER-beta negative patients. Broken line indicates the survival curve of HER2 positive patients and ER-beta positive. (D) Subgroup of HER2 negative patients, the DDFS of ER-beta negative patients is longer than that of ER-beta positive patients (P=0.005). Real line indicates the survival curve of HER2 negative patients and ER-beta negative patients. Broken line indicates the survival curve of HER2 negative and ER-beta positive patients.

 

TABLE 1. Patient Demographics
Characteristic		No. of Patients (n=308)
Age, yr
Median	58
Range	30-79
< 50 yr		115
≥ 50 yr		193
Menstrual status
Premenopause		117
menopause≥45		146
menopause<45		45
Surgical treatment
Lumpectormy		18
Lumpectormy + RT		73
Mastectormy		217
Tumour size, cm
< 3 cm		130
≥ 3 cm		178
Node status
Negative		115
Positive		193
Grade
1		43
2		143
3		122
ER status
Positive		198
Negative		110
PR status
Positive		133
Negative		175
Adjuvant therapy
Chemotheropy		164
Hormonal therapy (tamoxifen)		109
Duration follow-up, months
Median	48
Range	10-74
Relapses		78
Deaths		38
ER, estrogen receptor; PR, progesterone receptor.

 

TABLE 2. Histology
	n	%
Invasive ductal carcinoma	247	81
Papillary carcinoma	11	3.5
Medullary carcinoma	9	3
Paget's disease	3	1
Adenocarcinoma	10	3.3
Invasive lobular carcinoma	19	6.2
Other types	6	3
Total	308	100

 

TABLE 3.  Association Between ER-β Expression and Other Prognostic Parameters
	ER-β		P
	positive	negative
ER-alpha
negative	25	85
positive	98	100	.023
PR
negative	75	100
positive	48	85	.662
Grade
1	28	15
2	43	100	.010
3	52	70
Node
negative	72	121
positive	51	64	.113
Size
＜3cm	50	80
≥3cm	73	105	.441
Menstrual status
Premenopause	30	87
Menopause ≥45	25	20	.031
Menopause <45	68	78

 

TABLE 4. Association Between HER-2 Expression and Other Prognostic Parameters
	HER-2		P
	Positive	Negative
PR
negative	32	143
positive	26	107	.862
Grade
1	4	39
2	14	129	.001
3	40	82
Node
negative	20	95
positive	38	155	.213
Size
＜3cm	19	111
≥3cm	39	139	.011
ER alpha
negative	26	84
positive	32	166	.104
Mensis status†
Premenopause	16	101
Menopause ≥45	23	123	.327
Menopause <45	7	38
ER-beta
negative	14	171
positive	44	79	.018
Estrogen receptors ‡
ER-beta +/ER-alpha -	21	4
ER-beta +/ER-alpha +	12	86	<.001
† Menopause age younger than 45 years Tables 1, 3 4 not due to previous chemotherapy.  ‡ ER-beta +/ER-alpha –subgroups of ER-beta positive in the absence of ER-alpha.  ER-beta +/ER-alpha + subgroup of ER-beta positive in the presence of ER-alpha.