Introduction

The female hormone estradiol (E2) is a potent mitogen for estrogen receptor α (ERα)-positive breast cancers. Hence, ERα protein levels, as determined by immunohistochemistry (IHC), are strongly predictive for response to endocrine therapies [1]. 75 % of all breast cancers express ERα, but not all tumors that express this steroid receptor respond to hormonal therapies. ERα is a member of the nuclear hormone receptor gene family that regulates transcription in a hormone-dependent fashion through sequence-specific DNA binding [2]. Indeed, ERα binding sites are found proximal to many genes and consequently estrogen stimulation of breast cancer cells leads to significant changes in cellular gene expression [3, 4]. These responsive genes include the progesterone receptor (PR), one of the best-characterized ERα target genes. Hence, the PR is often co-expressed with ERα in breast cancers and PR testing is commonly performed in conjunction with ERα testing to assess hormone receptor status of a breast tumor. However, PR status is not a strong predictor of response to endocrine therapy, indicating that PR expression is not solely controlled by ERα activity [5].

Over a decade ago, the first large-scale gene expression profiling studies in breast cancer demonstrated that breast cancers consist of a number of “intrinsic” or “molecular” subtypes that are characterized by similarities in gene expression patterns [6]. Among these intrinsic subtypes are the “Luminal” and “Basal” tumors, which are thought to represent primarily ER-positive and -negative tumors, respectively. Consistent with this view, it was demonstrated that BluePrint™, an 80-gene mRNA expression signature that identifies Luminal and Basal tumors, is significantly enriched in bona fide ER target genes [7]. These data suggest that this intrinsic subtype signature primarily measures the functionality of the ER, as judged by expression of its downstream target genes. As such, this signature also has the potential to identify a subgroup of breast cancer patients who are ERα positive by IHC and/or mRNA expression, but fail to elicit the hormone-induced transcriptional responses that normally result from ER stimulation (ERα target genes “off”; Basal type). Such a scenario would imply that breast cancers having this phenotype express a dysfunctional ERα protein that can nevertheless be detected by IHC.

Several different ERα variant mRNAs have been described in human breast cancer. Almost all of these naturally occurring variants are mRNA splicing variants, in which one or more exons are absent from the ERα mRNA. In most ERα splicing variants, except for variants lacking exon 3 or 4, translation runs out of frame after the site of the splicing variation, leading to a truncated protein [8–12]. Since the antibodies for ERα used in IHC often include those that recognize an epitope encoded by the first exon of the ERα gene [13], such splice variants are likely detected as IHC positive for ERα, even though their function may be different from the normal ERα protein. The functional activity of these variant ERα proteins can be negative, dominant negative, or dominant active on ERα target genes. Dominant negative variants are not only inactive themselves but also inactivate wild-type ERα through heterodimerization. Two variants, the ERΔ3 and the ERΔ7 variants, have been described as dominant negative receptor forms in the presence of wild-type ERα [8–12]. The ERΔ7 mRNA has been reported to be the major alternatively spliced form in most human breast tumors and cancer cell lines [14]. The ERΔ7 is especially interesting because the hormone-binding domain, the transcription activation function-2 domain, and the dimerization domain are all partially located in exon 7 (Fig. 1). It has been shown that the ERΔ7 variant has the ability to suppress the E2-dependent transcriptional activation by both wild-type ERα and ERβ [14].

Fig. 1
figure 1

Organization of the ERα mRNA and functional domains. TAF-1 transcription activation function 1, TAF-2 transcription activation function 2, aa amino acid, bp base pair

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According to the guideline recommendations from the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) for IHC testing of ERα and PR in breast cancer, it is recommended that ERα assays should be considered positive if there are at least 1 % (weakly) positive tumor nuclei in the sample [13]. This threshold is based on a cut-point analysis correlating IHC scores with outcome in patients treated with adjuvant endocrine therapy alone, where patients with a score correlating to 1–10 % weakly positive cells had a statistically significant better prognosis than patients with scores correlating with <1 % positive cells [15]. However, Iwamoto et al. have shown recently that only a minority of the borderline (1–9 % positive nuclei) IHC ERα-positive tumors are of the Luminal subtype (as identified by the PAM50 classifier [16]) and that most of these borderline ERα-positive samples are of the Basal molecular subtype [17].

Here, we identify in a large cohort of molecular profiled breast cancers a subgroup of around 2 % of breast tumors that are ERα positive by mRNA expression analysis, but are of the Basal molecular subtype. These tumors express significantly lower levels of both ERα and PR mRNA than the Luminal-type tumors and have almost invariably (94.5 %) a high-risk MammaPrint™ profile. Furthermore, we show that these tumors have relatively high levels of the dominant negative ERΔ7 splice variant, in agreement with the notion that they may lack a functional response to estrogen and consequently may not respond to hormonal therapy.

Patients and methods

Patient samples and molecular profiling

A total of 3,527 breast cancer patient specimens were retrospectively analyzed. This selection was based on the availability of MammaPrint™, TargetPrint™, and BluePrint™ molecular profiling results as performed in the Agendia testing laboratories. The ERα status on mRNA levels was determined by TargetPrint, a microarray-based gene expression test, which offers a quantitative assessment of the patient’s level of ERα, PR, and HER2 expression [18]. The TargetPrint probe for ERα mRNA detection is located in the 3′ UTR region. The ERα, PR, and HER2 TargetPrint score is a value between −1 and 1, where the null cutoff value is calibrated to 1 % IHC ERα-positive cells, as identified in a reference laboratory according to ASCO/CAP guidelines. Tumors are reported ERα or PR positive when the TargetPrint score is above 0, corresponding to >1 % IHC-positive cells [18]. Molecular subtyping was performed using the 80-gene BluePrint™ molecular subtyping profile for the classification of breast cancer into Basal type, Luminal type, and ERBB2 type (HER2 positive) molecular subclasses [7]. In addition, the tumors were classified as low risk or high risk for distant recurrence using the 70-gene MammaPrint™ signature, a FDA-cleared breast cancer recurrence assay, performed by Agendia Inc. [19].

ERΔ7 variant analysis

We obtained RNA from 15 ERα-positive Luminal-type tumors and from 12 ERα-positive Basal-type tumors to analyze the relative ERΔ7 mRNA expression. cDNA was synthesized from 500 ng RNA using SuperScript II Reverse Transcriptase (Invitrogen) with random hexamer primers. The total ERα and ERΔ7 mRNA expression was determined by qRT-PCR. For total ERα expression, the forward primer was located in exon 1 and the reverse primer in exon 2. For ERΔ7 expression, the forward primer was located in exon 6 and the reverse primer was designed to specifically detect ERΔ7 and located partially in exon 6 (12 nucleotides) and partially in exon 8 (14 nucleotides) (Primer sequences in Supplementary Materials). All qRT-PCR reactions were performed in duplicates using SYBR Green reaction mix containing 5µl cDNA. The expression levels were quantified using a reference standard dilution curve. The relative expression of the ERΔ7 variant was calculated by dividing the ERΔ7 mRNA expression by the total ERα mRNA expression.

Identification of ERα target genes in the 70-gene MammaPrint™ breast cancer signature

The 70 MammaPrint genes were analyzed for ERα binding events within 20 kb from the transcription start site (TSS), representing the most commonly detected window for ER-mediated gene regulation [20]. ERα-binding sites were identified by ChIP-seq analyses [21], using available datasets for the Luminal breast cancer cell line MCF-7 [22] and 2 ER-positive Luminal breast tumor samples (paper in submission; GSE40867). Publically available data on E2-stimulated gene expression were used from [3], where Global Run-On sequencing was applied to assess gene transcription after 0-, 10-, 40-, and 160-min E2 treatment. Only genes with a differential expression as compared to control conditions with a false discovery rate of ~0.1 % were considered as E2 regulated.

Results

ERΔ7 splice variant expressed in an ERα-positive basal-type breast cancer

We have recently developed an 80-gene signature (BluePrint™) that identifies the three major intrinsic subtypes (Basal, Luminal, and HER2) of breast cancer [7]. Of these 80 genes, 58 are used to identify the Luminal subtype. Importantly, 32 out of these 58 Luminal subtype reporter genes have ERα-binding sites adjacent to the TSS [7]. This indicates that the genes that identify Luminal-type breast cancer are significantly enriched for bona fide ERα target genes and suggests that the Luminal subtype is characterized by tumors that have a functional ERα pathway. Conversely, BluePrint Basal-type tumors would be expected to have either no significant ERα expression or a non-functional ERα pathway; these same bona fide ERα target genes show an inverse expression pattern in Basal-type tumors [7].

Following argumentation as outlined above, one would expect that breast tumors that are ERα positive, but Basal type by BluePrint analysis, would either have a very low level of ERα protein or harbor a defective ERα protein. To test this hypothesis directly, we mined the Agendia database for patients who are ERα positive by TargetPrint, but Basal type by BluePrint molecular subtype analysis. We initially identified a patient (Table 1, patient 1; 60-year-old woman with 9 mm, moderately differentiated, HER2 negative, ER/PR > 90 % by IHC, invasive ductal carcinoma), who had undergone MammaPrint, TargetPrint, and BluePrint tests. She had MammaPrint high-risk result, was ER/PR positive by TargetPrint, but Basal subtype by BluePrint, suggesting that the ERα was present both at the protein (IHC > 90 %) and mRNA levels, but that ERα target genes were not expressed in this tumor (hence Basal type). The tumor was also analyzed using the OncotypeDX™ breast cancer assay (Genomic Health Inc.), classifying the tumor as low risk for distant recurrence (Recurrence Score 8, Table 1).

Table 1 Characteristics of ERα-positive Basal-type tumors for which the ERΔ7 expression was determined (N = 12)
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We used the same tumor mRNA sample as was used to perform the MammaPrint, TargetPrint, and BluePrint assays for detailed analysis of the ERα mRNA transcript in this patient. We first PCR amplified the coding sequence of ERα with specific oligonucleotides that span the start codon of ERα at the 5′ end and the stop codon at the 3′ end (Primer sequences in Supplementary Materials). Agarose gel electrophoresis of the PCR product revealed a smaller DNA fragment next to the expected DNA fragment coding for the open reading frame of ERα. Inspection of the DNA sequence of the smaller product revealed an ERα sequence-lacking exon 7 of the coding sequence (data not shown). This transcript corresponds to the previously reported ERΔ7, an ERα splice variant that inhibits the function of the normal (wild-type) ERα in a dominant fashion [14].

Frequency of ERα-positive basal-type tumors

To determine the frequency at which ERα-positive Basal-type breast tumors occur, we searched the Agendia database for additional cases. Out of 3,527 cases, we identified 2,658 ERα-positive, HER2-negative breast tumors, as judged by TargetPrint mRNA expression, for which BluePrint intrinsic subtyping data were available. From these 2,658 tumors, 2,603 (97.9 %) were classified as Luminal type and 55 (2.1 %) were classified as Basal type (Table 2). The mean ERα and PR TargetPrint indices for the ERα-positive Basal-type tumors were significantly lower than for the ERα-positive Luminal-type tumors (P < 0.0001).

Table 2 TargetPrint ERα/PR index, PR classification, and MammaPrint classification of 2,658 ERα-positive, HER2-negative tumors according to their BluePrint molecular subtype (Basal type vs Luminal type)
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ERΔ7 splice variant expression in ERα-positive basal-type breast cancers

We further analyzed an additional 11 of these 55 ERα-positive Basal-type tumors for expression of total ERα as well as the ERΔ7 variant by qRT-PCR. The specificity of the primer pairs was tested with cDNA from MCF7 breast cancer cells overexpressing either wild-type ERα or ERΔ7 and the calculated ERΔ7/total ERα ratio was correlated with ERα protein expression in these cells. The ERα antibody clone 1D5 (Dako) was used for western blot analysis, for which the epitope is located in the N-terminal domain of ERα and therefore recognizes both wild-type ERα and ERΔ7. We show in these cells that the relative ERΔ7 levels as measured by qRT-PCR are highly concordant with protein expression (Supplementary Fig. 1).

The average total ERα mRNA expression by qRT-PCR was significantly lower for the 12 analyzed ERα-positive Basal-type tumors compared to 15 randomly chosen ERα-positive Luminal-type tumors (Fig. 2a; P = 0.0019), consistent with the TargetPrint results (Table 2). There was no significant difference in average ERΔ7 mRNA expression between the ERα-positive Basal-type and Luminal-type samples (Fig. 2b; P = 0.4088). However, the relative ERΔ7 mRNA expression was significantly higher for the ERα-positive Basal-type group compared to the ERα-positive Luminal-type group (Fig. 2c; P < 0.0001), due to the lower overall ERα mRNA expression in the Basal-type tumors.

Fig. 2
figure 2

ERα-positive Basal-type tumors have a relatively high ERΔ7 expression compared to ERα-positive Luminal-type tumors. a Scatter plot of total ERα mRNA expression analysis by qRT-PCR in ER-positive Basal-type (n = 12) and ER-positive Luminal-type (n = 15) tumors. The qRT-PCR primers are located in exon 1 and exon 2. Points indicate individual tumors; lines indicate mean with SD. b Scatter plot of specific ERΔ7 mRNA expression analysis by qRT-PCR in ER-positive Basal-type (n = 12) and ER-positive Luminal-type (n = 15) tumors. The qRT-PCR Primers are located in exon 6 (forward) and over the exon 7 splice site (reverse). Points indicate individual tumors; lines indicate mean with SD. c Scatter plot of relative ERΔ7 expression calculated by dividing the ERΔ7 mRNA expression with the total ERα mRNA expression in ER-positive Basal-type (n = 12) and ER-positive Luminal-type (n = 15) tumors. Points indicate individual tumors; lines indicate mean with SD. P-values are calculated by unpaired t tests with Welch’s correction and are two-tailed

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The characteristics of the 12 ERα-positive Basal-type tumors, for which ERΔ7 splice variant expression was determined, are shown in Table 1. For 8 of the 12 patients, we were able to retrieve the ERα and PR IHC scoring. Based on the ERα IHC, six out of eight (75 %) patients were classified as ERα positive. In two patients, we found a discrepancy between TargetPrint and ERα IHC classification; in one of these patients, the TargetPrint ERα index was just above the ERα-positive threshold (patient 10). The PR IHC was in concordance with the PR classification based on TargetPrint in six of eight patients, and for two patients (patient 6 and 8), a small percentage of PR-positive cells was detected by IHC where the TargetPrint PR index was negative. The HER2 negative status was confirmed by fluorescence in situ hybridization (FISH) in all available cases. All patients (12/12) were stratified as high risk of distant recurrence by the MammaPrint prognostic gene signature.

MammaPrint measures ERα function independent of ERα expression

MammaPrint measures 70 genes that were selected from the entire complement of human genes, but ERα is not among the MammaPrint genes [23]. Nevertheless, we observed that 52 of the 55 (94.5 %) ERα-positive Basal-type tumors were MammaPrint high risk, while only 44.9 % of the ERα-positive Luminal-type tumors were classified as high risk of recurrence (Table 2; P < 0.0001). Since the MammaPrint assay identifies nearly all these ERα-positive Basal-type tumors as high risk, it suggests that the test measures ERα activity independent of the ERα mRNA expression level itself. To investigate this further, we determined how many of the 70 MammaPrint prognosis genes are directly responsive to E2 treatment. For this, a publically available dataset was used that assessed gene expression changes after 10, 40, and 180 min of E2 treatment [3]. We found that 16 MammaPrint reporter genes annotated in the most recent build of the human reference genome sequence are E2 regulated (Fig. 3a). Next, we tested whether these E2-responsive MammaPrint genes can be classified as direct ERα target genes. Using a publically available ChIP-seq dataset [22], the genome-wide chromatin-binding landscape of ERα in MCF7 cells was analyzed for the occurrence of an ERα binding event within 20,000 bp from the TSS of any of the MammaPrint genes. This window was chosen since most ERα-mediated gene regulation is found within this distance from a TSS [20]. Ten out of 16 genes had an ERα binding event within 20,000 bp from the TSS (Fig. 3a), as exemplified for the LPCAT1 locus (Fig. 3a). Importantly, the essential ERα coactivators AIB1 (also known as SRC3) and p300 were also present at this specific binding site, indicating that ERα is likely to be functional here [24]. Furthermore, we confirmed that ERα binding events in E2-regulated MammaPrint genes are also found in 2 ER-positive Luminal human breast tumor samples, for which ERα ChIP-seq data are available (Fig. 3a). In total, 12 out of 16 E2-regulated genes had an ERα-binding site in either MCF7 cells or in the two studied tumors (Fig 3a). Cumulatively, these data indicate that bona fide ERα target genes are enriched in the MammaPrint gene signature, providing a plausible explanation for why the MammaPrint can measure ERα functionality rather than its mere presence, in contrast to other available assays.

Fig. 3
figure 3

Functional ERα target genes in MammaPrint 70-gene set. a Pie chart, depicting the proportion of MammaPrint genes, which are affected by E2 treatment. Heat map (right panel) depicts proximal ERα ChIP-seq signal by tag count (blue) as well as relative gene expression values as measured by GRO-seq, after 10, 40, and 160 min of E2 treatment (green–black–red heat map). b Genome browser snapshot, depicting a shared ERα (red), AIB1 (green), and p300 (blue) proximal to the LPCAT1 transcription start site. Chromosome number, genomic coordinates, and tag count are indicated

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Discussion

The present study identifies approximately 1 in 50 ER-positive breast cancer patients as Basal molecular subtype. Basal-type breast tumors are characterized by an absence of expression of ERα target genes, which is generally thought to result from the absence of ERα expression [25]. However, the group of tumors identified here is ERα positive on the mRNA level, suggesting that their Basal phenotype is the result of a lack of ERα protein expression or a lack of functionality of the ERα protein present in these tumors. Indeed, we find that these tumors not only express relatively low levels of ERα mRNA but also express a splice variant of ERα-missing exon 7 (ERΔ7, Fig. 2a, b). This ERα variant has been shown previously to act in a dominant negative fashion, meaning that this variant can inhibit the function of the wild-type ERα protein when co-expressed in the same cell [14]. We note that the absolute levels of ERΔ7 are comparable in ERα-positive Basal-type versus ERα-positive Luminal-type tumors, but that the relative abundance of ERΔ7 is higher in the ERα-positive Basal-type tumors (Fig. 2c). We interpret these data as follows: When the levels of wild-type ERα in a breast tumor are high, the inhibitory effects of dominant negative ERΔ7 are by comparison minor, leaving the cell with considerable ERα activity and thus with a luminal phenotype (Fig. 4, right). In contrast, lower levels of wild-type ERα in the weakly ERα-positive breast tumors are inhibited to a greater extent by the presence of ERΔ7, leaving the tumor cells with insufficient ERα activity to regulate ERα target gene expression and thus with a Basal phenotype (Fig. 4, left). It remains to be explained why lower levels of ERα result in a relative increase in abundance of the ERΔ7 splice variant. It is possible that ERα also controls the expression of certain components of the splicing machinery and that low ERα activity therefore results in a different processing of the ERα (and potentially also other) precursor mRNAs.

Fig. 4
figure 4

Proposed model by which ERΔ7 mRNA expression can affect ERα activity in low ERα wild-type expressing tumors (left) and in high ERα wild-type expressing tumors (right)

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A clinically relevant question is whether this identified group of ERα-positive Basal-type tumors is likely to respond to hormonal therapy. The finding that ERα target genes are not expressed suggests that the mitogenic responses in such tumors are not driven by E2 and that such tumors would be unlikely to derive significant benefit from hormonal therapy. It was reported by Ellis et al. [26] in a cohort of postmenopausal women with clinical stage II to III ER-positive breast cancer that the single patient in their study with a basal-like intrinsic subtype was resistant to endocrine therapy. While it remains to be formally proven, there are other suggestions in the literature that the presence of ERΔ7 is associated with a lack of response to tamoxifen. Van Dijk [27] analyzed the relative ERΔ7 mRNA expression in a group of 21 primary breast tumors from postmenopausal early breast cancer patients treated with adjuvant tamoxifen. It was found that out of eleven ERα mRNA variants tested, only the ERΔ7 mRNA was significantly differentially expressed between primary breast tumors of patients who developed a tumor recurrence (13/21) and tumors of patients without recurrence (8/21). Tumors from patients with a recurrence expressed on average 24 % ERΔ7 mRNA (relative to wild-type ERα mRNA expression), while tumors from patients without recurrence expressed on average 9 % ERΔ7 mRNA [27]. While it may be premature to withhold hormonal therapy from this group of ERα-positive breast cancer patients, as this would require a large randomized outcome study, there are reasons to consider adding chemotherapy to the treatment regimen for these patients. We find that 94.5 % of the ERα-positive Basal-type breast cancer patients are high risk by the MammaPrint assay, making them potential candidates to benefit from chemotherapy based on their high recurrence risk. Moreover, Basal-type breast cancers have been shown to be significantly more responsive to neoadjuvant chemotherapy as compared to luminal breast cancers, again indicating that addition of chemotherapy could be effective in this patient group [7]. The St. Gallen consensus guidelines state that patients with an (borderline) ERα-positive Basal-type tumor are classified as incompletely endocrine responsive [28]. This relative lack of endocrine responsiveness together with a designation of “high risk” of relapse by MammaPrint will contribute to a clinician’s recommendation of whether endocrine therapy alone may be sufficient or supplementary chemotherapy may be beneficial for these patients.

Our finding that ERα-positive Basal-type tumors are in general borderline ERα positive on mRNA level is in agreement with the conclusions of Iwamoto et al. who found that most of the 1–9 % IHC ERα-positive tumors show molecular features similar to ERα-negative basal-like tumors [17]. The strength of our study is the high number of cases and therefore the better estimate we can make of the frequency of ERα-positive Basal-type tumors. In addition, we show that a majority of these tumors have a high-risk prognostic profile. One limitation of our study is that we do not have all the clinical information for the entire group of patients which was studied here. For instance, we did not have access to the IHC data for all the patients in this study and had to rely on TargetPrint to assess ERα levels. However, IHC data were available for 8 of the 12 ERα-positive Basal type for which ERΔ7 expression was determined (Table 1) and showed that 6 of 8 tumors scored clearly positive for ERα protein by IHC.

ERα-positive breast tumors have in general a better prognosis than ERα-negative tumors [29]. In spite of this, the group of ERα-positive Basal-type breast tumors consists nearly exclusively of high-risk patients as judged by the MammaPrint assay (Table 2). Our present data also provide a possible explanation for this finding. In contrast to the OncotypeDX™ prognostic signature, the 70-gene MammaPrint™ signature does not include ERα [23, 30]. We find that 16 MammaPrint genes are responsive to E2 treatment and that 12 of these are classified as direct ERα targets based on ERα/DNA associations in close proximity to the TSS, indicating that MammaPrint determines ERα activity rather than merely its expression. We believe that this likely explains why the first patient (Table 1, patient 1) having the ERα-positive basal phenotype was characterized by the OncotypeDX assay as “low risk”, but “high risk” by MammaPrint and patient 11 also had a discordant risk assessment in these two assays (Table 1). The ERα mRNA is expressed at a relatively high level in these patients, which is a “good prognosis” factor in the OncotypeDX assay. However, MammaPrint identified this tumor as lacking a functional ERα and came to a “high risk” reading.

In conclusion, by combining TargetPrint and BluePrint molecular subtyping analysis, we have identified a subgroup of some 2 % of breast cancer patients who lack ERα function while expressing ERα at the mRNA and protein level. Our data indicate that such patients are frequently at high recurrence risk and might benefit from adjuvant chemotherapy.