Estrogen receptor α gene ESR1 amplification may predict endocrine therapy responsiveness in breast cancer patients (2024)

Abstract

Estrogen receptor (ER) α plays a crucial role in normal breast development and has also been linked to mammary carcinogenesis and clinical outcome in breast cancer patients. However, the molecular mechanisms controlling the expression of ERα are as yet not fully understood. Gene amplification is one of the important factors regulating protein expression. Recent studies on the amplification of the ESR1 gene, which encodes ERα, have presented conflicting data. Using fluorescence insitu hybridization and real‐time quantitative polymerase chain reaction analysis, we examined the ESR1 status in a series of breast cancer tissues and analyzed its clinical importance. ESR1 gene amplification and gain were found in 22.6 and 11.3% of samples, respectively, as determined by three‐dimensional fluorescence insitu hybridization assay. Moreover, ESR1 amplification and amplification plus gain were significantly negatively correlated with tumor size, number of positive lymph nodes, negative ERα, and positive human epidermal growth factor receptor 2 status. It has also been shown that ESR1 amplification strongly correlates with higher expression levels of ER protein and that patients with ESR1 amplification in their tumors apparently experience longer disease‐free survival than those without. Our data suggest that ESR1 amplification might prove to be helpful in selecting patients who may potentially benefit from endocrine therapy. (Cancer Sci 2009; 100: 1012–1017)

Estrogen receptor (ER) α signaling is known to be necessary for the growth and differentiation of normal breast epithelium, as well as for the initiation and progression of estrogen‐dependent breast cancer. It was reported that the percentage of normal breast epithelial cells staining positive for ERα is generally low,⁽¹⁾ and that expression of the ERα protein fluctuates throughout the menstrual cycle.⁽²⁾ On the other hand, significantly higher expression has been reported in premalignant breast disease,⁽³⁾ and the level of ERα expression changes with progression of the disease in individual patients. Clinically, the ERα status of breast cancer patients is widely used both as an indicator for endocrine therapy responsiveness and also for prognosis prediction. However, despite its importance, the molecular mechanisms controlling the expression of ERα are not fully understood.

Nevertheless, it is known that ERα expression is regulated by numerous mechanisms. The silencing of ERα has been identified in breast tumor, including via a mutation within the open reading frame of the ESR1 gene,⁽⁴⁾ and via epigenetic changes such as DNA methylation of the promoter‐proximal CpG island in the ESR1 gene.^{(5, 6)} Moreover, it was recently reported that micro‐ribonucleic acid (miRNA)‐206^{(7, 8)} and miRNA‐221/222⁽⁹⁾ negatively regulate ERα at the post‐transcriptional level. These abnormalities act alone or in combination to alter the functions or expression levels of ERα.

Gene amplification is one of the important factors regulating protein expression. Many copy number aberrations have been shown to recur across multiple cancer samples. These recurrent copy number aberrations frequently contain oncogenes and tumor‐suppressor genes, and are associated with tumor progression, the clinical course of the cancer, or its responsiveness to therapy.⁽¹⁰⁾ Knowledge of copy number aberration can have an immediate clinical use; for example, an effective diagnosis of the amplification or overexpression of human epidermal growth factor receptor (HER) 2/neu in breast tumors will allow such patients to benefit from treatment with the monoclonal antibody trastuzumab (Herceptin).⁽¹¹⁾ With respect to ERα, we previously described that loss of heterozygosity of the ER gene does not play an important role in the lack of ER function in breast cancer tissues.⁽¹²⁾ On the other hand, some studies have reported amplification of ESR1.^{(13, 14, 15, 16, 17, 18, 19, 20)} For example, Holst and colleagues recently reported that more than 20% of breast cancers harbor genomic amplification of the ESR1 gene. They also found that ESR1 amplification is an indicator for defining a subtype of primary breast cancers that had particularly high ER expression levels and that this subtype might be optimally suited for hormonal therapy.⁽¹⁵⁾ However, contradictory findings on the clinical importance of ESR1 amplification for breast cancer patients have also been reported.^{(16, 17, 18, 19, 20)}

Given the clinical importance of ERα and its gene ESR1 in relation to ERα expression levels, we investigated the status of ESR1 in a series of breast cancer tissues by means of fluorescence insitu hybridization (FISH) and real‐time quantitative polymerase chain reaction (qPCR) analysis, in an attempt to elucidate the relationship between ESR1 status and clinicopathological factors and prognosis of the patients.

Materials and Methods

Patients and breast cancer tissues.  Primary invasive breast carcinoma specimens were obtained by surgical excision from 147 cases of patients at the Department of Breast and Endocrine Surgery, Kumamoto University Hospital (Kumamoto, Japan) between June 2001 and August 2006. FISH staining of ESR1 status was from 133 available cases of paired tissues. These patients were from a consecutive series, and no exclusion criteria were applied. Informed consent was obtained from all patients before surgery. The Ethics Committee of Kumamoto University Graduate School of Medicine (Kumamoto, Japan) approved the study protocol.

The median age of the patients was 62years (range, 30–86years). Postoperative treatment was done in accordance with the recommendations of St Gallen's international consensus meeting for primary breast cancer.^{(21, 22, 23)} On recurrence, patients with ERα‐negative and progesterone receptor‐(PgR) negative tumors were treated with cyclophosphamide+methotrexate+ fluorouracile, fluorouracil+epirubicin+cyclophosphamide, and/or taxanes. Patients with hormone receptor‐positive tumors and non‐visceral metastases were treated with endocrine therapy, such as antiestrogens and aromatase inhibitors. Patients were followed postoperatively every 3months for the first 5years, and every 6months thereafter. The median follow‐up period was 40months (range, 4–87months).

Immunohistochemistry.  Immunohistochemical staining was carried out as previously described,⁽²⁴⁾ on 4‐µm thick tumor sections. Serial sections were prepared from selected blocks and float‐mounted on adhesive‐coated glass slides for ERα, PgR, or HER2 staining. Primary antibodies were monoclonal mouse antihuman ERα antibody (1D5; Dako) at 1:100 dilution, PgR antibody (636; DAKO) at 1:100 dilution, and rabbit antihuman c‐erbB‐2 oncoprotein antibody (Dako) at 1:200 dilution for HER2. The Envision system (EnVision labeled polymer, peroxidase; Dako) was used as the detection system for ERα, PgR, and HER2.

Immunohistochemical scoring.  Immunostained slides were scored after the entire slide had been evaluated by light microscopy. The expression of ERα and PgR was scored by assigning a proportion score and an intensity score according to Allred's procedure.⁽²⁵⁾ In brief, a proportion score represented the estimated proportion of tumor cells staining positive, as follows: 0, none; 1, <1/100; 2, 1/100–1/10; 3, 1/10–1/3; 4, 1/3–2/3; and 5, >2/3. Any brown nuclear staining in invasive breast epithelium counted toward the proportion score. An intensity score represented the average intensity of the positive cells, as follows: 0, none; 1, weak; 2, intermediate; and 3, strong. The proportion and intensity scores were then added to obtain a total score, which could range from 0 to 8. Tumors with a score of 3 or more were considered to be positive for ERα expression. HER2 immunostaining was evaluated using the same method as used by the HercepTest (Dako). To determine the score of HER2 expression, the membrane staining pattern was estimated and scored on a scale of 0 to 3+. Tumors with scores of 2 or more were considered to be positive for HER2 overexpression.

ESR1 gene amplification detection by real‐time polymerase chain reaction.  Patient and control genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. The concentration and purity of the genomic DNA preparations were measured and genomic DNA was stored at 4°C until use.

Probes and primers.  We used the primers and probe for ESR1 as reported by Holst etal.⁽¹⁵⁾ The sequence of the primers and probe were: ESR1 forward primer 5′‐GCCAACGCGCAGGTCTA‐3′ (562–578); ESR1 reverse primer 5′‐GCCGCAGCCTCAGA‐3′ (623–610); and ESR1 TaqMan probe FAM‐CTCCCCTACGGCCCC‐NFQ. The polymerase chain reaction (PCR) product size for ESR1 was 62bp. The final optimized concentration for the ESR1 TaqMan probe and each pair of primers was 250 and 900nM, respectively. RNaseP was chosen as a reference for gene dosage because of its single copy number. Primers and probe for RNaseP were obtained from TaqMan endogenous control kits (4316844; Applied Biosystems).

Real‐time qPCR.  Real‐time qPCR was carried out in a 96‐well optical plate on a 7500 Real‐Time PCR System (Applied Biosystems, Foster City, CA, USA) in a total volume of 20µL, adjusted with sterile water: 4.5µL of DNA (~50ng), 900nM of each ESR1 primer, 10µL of Master Mix (2×), 250nM of ESR1 probe, and 1µL of RNaseP kit (20×). All reactions were carried out in triplicate. Thermal cycling conditions included an initial denaturation at 95°C for 10min, followed by 40 cycles of 15s at 95°C and 60s at 60°C.

Statistical analysis.  The χ²‐test for independence was adopted for statistical analysis of associations between ESR1 gene status and clinicopathological factors. A disease‐free survival curve was generated by the Kaplan–Meier method and verified by the log‐rank (Mantel–Cox) test. Differences were considered significant when a P‐value <0.05 was obtained.

Results

ESR1 amplification and gain in relation to clinicopathological factors.  We analyzed the ESR1 gene copy number ratio in 133 primary breast tumors (109 ER‐positive tumors and 24 ER‐negative tumors) by FISH. Of the 133 cases, 30 (22.6%) cases showed amplification for ESR1, and 15 (11.3%) demonstrated ESR1 gene gain.

ESR1 amplification and amplification plus gain were significantly negatively correlated with tumor size (P=0.0025 and P=0.0175, respectively) and the number of positive lymph nodes (P=0.0238 and P=0.0252, respectively), and positively correlated with ERα status (P=0.0035 and P=0.0001, respectively), whereas ESR1 gene amplification plus gain was also significantly negatively correlated with HER2 status (P=0.0256) (Table1).

Discussion

In the present study, using FISH, we demonstrated that 30/133 (22.6%) breast cancer samples showed amplification and 15/133 (11.3%) showed gain of ESR1, which is consistent with the findings of Holst etal. that ESR1 gene amplification was frequent in breast cancer.⁽¹⁵⁾ Gene amplification, in general, occurs frequently in breast cancer. Multiple different oncogenes have been described previously as being amplified in breast cancer, including HER2, epidermal growth factor receptor, c‐Myc gene (MYC), cyclin D1 gene (CCND1), and murine double minute 2 gene (MDM2).^{(26, 27, 28, 29, 30)} However, there have been few reports about the frequency of ESR1 gene amplification, even though increased ERα expression levels were found to be associated with breast cancer tumorigenesis and progression. The increased frequency of ESR1 gene amplification reported by Holst etal. has prompted investigations about ESR1 amplification. However, concerning the frequency of ESR1 gene amplification in breast cancer, no consistent conclusions have been drawn.^{(16, 17, 18, 19, 20)} Now, a variety of assays are available for detecting copy number variation (CNV), including Southern blotting, FISH, array‐based comparative genomic hybridization (aCGH) and real‐time qPCR. Southern blotting requires a large amount of genomic DNA and is costly in terms of reagents, work, and time. FISH is considered to be the most precise method for amplification detection,⁽³¹⁾ as it is unaffected by the problem of tumor cell heterogeneity (see discussion below). aCGH is a powerful screening tool for detecting DNA copy changes and hence is used to identify cancer‐associated chromosomal aberrations.⁽³²⁾ However, it is more expensive, time‐consuming, and requires special equipment with software. Additionally, aCGH reflects the pattern of changes observed in the dominant clonal populations of a given tumor, which means that changes found in the less prevalent clonal populations are less likely to be identified with this technique.⁽¹⁰⁾ The same applies to real‐time qPCR. However, real‐time qPCR offers several advantages, including speed, sensitivity, reproducibility, and low cost. Furthermore, it is informative due to a large dynamic range of quantification, and the need for very small amounts of starting material. Therefore, we analyzed DNA copy number ratios using real‐time qPCR to confirm the FISH results. We observed, however, a discrepancy between FISH and real‐time qPCR results. We found that only 2/147 (1.4%) samples showed amplification of the ESR1 gene 30/133 (22.6%) using FISH. With regard to ESR1 gene amplification, several studies,^{(16, 17, 18, 19, 20)} have contested the frequency of the phenomenon as reported by Holst etal.⁽¹⁵⁾ The findings from these other studies on ESR1 amplification reported a frequency ranging from only 0 to 10% as detected by a variety of methods, including FISH, aCGH, and real‐time qPCR.^{(16, 17, 18, 19, 20)} The differences between FISH data obtained by various research groups could mainly result from whether manual or automated methodology was applied for scoring hybridization signals. It may also be possible that a proportion of low‐level amplification was missed by automated scoring of FISH signals. Therefore, we carried out manual scoring by taking the image stacks containing three‐dimensional data sets through the z‐axis and analyzed the projection image in order to detect small‐sized amplicons (Fig.3). Analysis using three‐dimensional FISH has proved to be a useful and reliable way to evaluate small nuclear components, such as chromatin arrangement and telomere clustering, as reported previously.^{(33, 34, 35)} Most of the ESR1‐amplified tumors had tiny confluent or small gene clusters of ESR1 (3–10 copies), whereas only 6/30 (20%) cases had large clusters (>10 copies), as observed in the present study (Fig.4). If we had not taken the image stacks into account as described above, the detection rate of ESR1 amplification in our patient population would have been 18/133 (13.5%). On the other hand, it is likely that the inability of array‐CGH and real‐time qPCR to detect CNV was caused by breast tumor heterogeneity. This might have led the normal cells, or the tumor cells without CNV, to dilute the population of mutated tumor cells and thereby lower the average copy number of the population of cells detected. Therefore, further studies using microdissected breast cancer tissue are urgently needed.

We observed that ESR1 amplification was significantly correlated with small tumor size and negative lymph nodes. These data suggest that although ESR1 amplification has a role in the proliferative mechanism of early stage breast cancer, it is not involved in tumor progression. Therefore, it is possible that breast cancer cells with ESR1 amplification have a lower growth potential than those without amplification (which have acquired growth potential via other means), and that such tumor cells with ESR1 amplification will gradually die or be outnumbered by the cells without amplification.

Although the expression status of ERα is a primary determinant in the hormone therapy of breast cancer, it can not reliably predict the responsiveness of the tumors to hormone therapy; not all ERα‐positive breast cancers respond to hormone therapy. This discrepancy between ERα expression status and response to hormonal therapy probably derives from the different estrogen signaling pathway conditions.⁽³⁶⁾

Therefore, a new diagnostic marker to screen patients who may potentially benefit from hormone therapy is required. Our results showed that ESR1 amplification was significantly correlated with higher expression levels of ER protein and better disease‐free survival. Considering that most patients with ERα‐positive tumors will have adjuvant antiestrogen treatment, it is likely that ESR1 amplification could be helpful in identifying a subgroup of individuals with breast cancer who would benefit most from the hormone therapy. Further studies in large cohorts of patients are needed to evaluate the predictive value of ESR1 amplification for hormone therapy.

In conclusion, FISH assays appear to be highly sensitive and specific for detecting ESR1 gene amplification, and obtaining images taken through the z‐axis. Our present study has demonstrated ESR1 gene amplification and gain in 22.6 and 11.3% of breast cancer samples, respectively, as determined by FISH assay. ESR1 amplification and amplification plus gain were significant negatively correlated with tumor size, number of positive lymph nodes, and HER2 status, and positively correlated with ERα status. Moreover, it has been shown that ESR1 amplification is strongly correlated with higher expression levels of ER protein and has possibilities as a predictive marker. Our data suggest that ESR1 amplification might prove helpful in selecting patients who may potentially benefit from endocrine therapy.

Acknowledgments

We thank Mrs Noriko Saitoh and Dr Mitsuyoshi Nakao (Department of Regeneration Medicine, Institute of Molecular Embryology and Genetics, Kumamoto University, Japan) for helpful discussions and Ms Yuu Oogaki for her excellent technical support.

References