Oestrogen receptor low positive breast cancer: associations with prognosis

Purpose In this study of oestrogen receptor (ER) Low Positive breast cancers (BC) in three large cohorts of BC patients, we assess associations between levels of ER expression and tumour characteristics and prognosis. Methods Cases were stratified into patients unlikely to have received adjuvant therapy according to treatment guidelines at time of diagnosis (before 1995), and those who could have received adjuvant therapy (diagnosed in 1995 or later). ER status was divided into < 1%; ≥ 1 < 10%; ≥ 10%. Results were correlated with time of diagnosis, histopathological grade, proliferation status, and molecular subtypes, using Pearson’s Chi-square test. For prognosis, hazard ratios and cumulative incidence of death from BC were used. Results Of the 1955 tumours, 65 (3.3%) were ER Low Positive (ER ≥ 1 < 10%). Overall, the highest proportion of ER Low Positive tumours was observed among Luminal B (HER2 +) subtype (9.4%) and grade 3 tumours (4.3%). The risk of death from BC was lower in ER Low Positive and ER ≥ 10% compared to ER-negative cases. Compared to patients diagnosed before 1995, women diagnosed in 1995 or later showed a higher proportion of ER Low Positive BCs, and their tumours were of smaller size, lower grade, and lower proliferative status. There was no significant difference in prognosis compared to those with ER ≥ 10% tumours. Conclusion Women with ER Low Positive tumours diagnosed in a time period when adjuvant therapy was available had tumours of smaller size, lower grade, and lower proliferative status, and similar prognosis to those with ER ≥ 10% compared to women diagnosed earlier.


Introduction
Oestrogen receptor (ER) status plays an essential role in clinical decision-making and predicting outcome and treatment response for breast cancer (BC) patients [1]. According to current guidelines [2], patients with ER-positive tumours are considered eligible for endocrine therapy. Patients with ER-negative tumours are more likely to benefit from chemotherapy and generally have a poorer outcome than patients with ER-positive (ER +) tumours [3,4].
Breast cancer differs from most tumours because of its dependence on female sex hormones for development and growth [5]. Expression of ER by immunohistochemistry (IHC) is seen in more than 70% of BC tumours [6]. The ASCO/CAP and current national BC guidelines state that BC tumours with ≥ 1% positive staining tumour cell nuclei should be interpreted as ER + , and negative if < 1% of tumour cell nuclei express ER [2,7]. However, the ASCO/ CAP Expert Panel states that data on the effect of endocrine therapy for cancers with ER ≥ 1 < 10% are limited. They suggest that samples with ER ≥ 1 < 10% should be reported as ER Low Positive, with a comment mentioning the limited data available on the therapeutic benefit of anti-hormonal treatment for this group of patients [2]. According to the St. Gallen 2019 Consensus Discussion on The Optimal Primary Breast Cancer Treatment, there is a need for better evaluation of ideal cut-off levels for the prescription of endocrine therapy for ER + tumours, particularly for ER Low Positive cases [8][9][10].
In this study we examined expression levels of ER in BC tumours and associations between ER status and time of diagnosis, and tumour characteristics such as histopathological grade, molecular subtypes, proliferation and prognosis, with emphasis on ER Low Positive tumours.

Study population
This study comprises women from three population-based surveys conducted in Trøndelag County, Norway. Information on breast cancer incidence was obtained from the Cancer Registry of Norway. Date of death, and/or emigration was obtained from the National Population Register and causes of death from the Norwegian Cause of Death Registry. Formalin-fixed, paraffin embedded (FFPE) tumour tissue from the primary tumours and corresponding pathology reports were retrieved from the Department of Pathology at St. Olav's Hospital, Trondheim University Hospital, Norway (Fig. 1).
Cohort 1: The cohort includes 25,727 women born 1886-1928 [11] invited to attend a population-based survey for the early detection of breast cancer conducted in Nord-Trøndelag County, Norway, between 1956 and 1959. During 47 years of follow-up (1961 to end of 2008), 1379 new BCs were registered among these women. In a previous study 909 of these tumours were classified according to histopathological type and grade and divided into molecular subtypes [12]. For one case ER status was missing, and this case was excluded from the present study, leaving 908 cases. After diagnosis, patients were followed until time of death from BC or death from other causes, or until December 31st, 2015.
Cohort 2: The second cohort comprises 34,221 women born between 1897 and 1977 and derives from the HUNT2 Study conducted between 1995 and 1997 in Nord-Trøndelag County, Norway [13]. From attendance until December 31st, 2009, 728 women were diagnosed with BC. Of these, 157 cases were already included in Cohort 1 and 57 were unavailable for subtyping. The remaining tumours (n = 514) from Cohort 2 were assigned histopathological type and grade and reclassified into molecular subtypes [14]. ER status was available for all 514 cases. After diagnosis, these patients were followed until time of death from BC or death from other causes, or until December 31 st , 2015.
Cohort 3: The third cohort includes 22,931 women born at E.C. Dahl's Foundation, Trondheim, Norway between 1920 and 1966. During 52 years of follow-up (1961 to the end of 2012), a total of 870 women were diagnosed with BC. Among them, 598 were diagnosed at St Olav's Hospital, and histopathological typing, grading and molecular subtyping were successful for 537 of these cases [15]. ER status was available for 533 of these cases. After diagnosis, patients were followed until time of death from BC or death from other causes, or until December 31st, 2015.

Specimen characteristics
Tissue Microarray (TMA) paraffin blocks were made from the archival tumour tissue using the TissueArrayer Minicore with TMA Designer2 software (Alphelys). Three 1 mm in diameter tissue cylinders from the periphery of the FFPE primary tumours were transferred to TMA recipient blocks. TMA Sections (4 µm) were cut and IHC-staining for ER was carried out within four weeks after sectioning. Between cutting and staining, sections were stored at − 20 °C. Staining intensity was not quantified in this study. Molecular subtypes for all cases in all three cohorts were determined using IHC and in situ hybridization in lieu of gene expression analyses, and have been published previously [12,14,15]. The IHC markers including ER are shown in Table 1.
Pearson's chi square test was used to compare patient and tumour characteristics across categories of ER. In analyses of prognosis, we distinguished between women diagnosed before 1995 and women diagnosed in 1995 or later. This cut-off was used to approximate the gradual implementation of adjuvant treatment in Norway [14,16]. Cumulative incidence of death from BC was estimated, with death from other causes as competing events. Gray's test was used to compare equality between cumulative incidence curves. Cox proportional hazard analyses were used to estimate hazard ratios (HR) of BC death with 95% confidence intervals (CI) within each diagnostic period, censoring at time of death from other causes. We adjusted for age, stage, histopathological grade, and for these variables combined. No clear violations of proportionality were found in log-minus-log plots. Statistical analyses were performed using Stata/MP version 17 (StataCorp LP, College Station, Texas, USA).

Results
Patient and tumour characteristics for the 1955 patients included in the present study are shown in Table 2. Mean age at diagnosis was 67.3 years (SD: 12.8) and mean follow-up after diagnosis was 9.9 years (SD: 7.

Comparisons between women diagnosed before 1995 and women diagnosed in 1995 or later
A total of 774 cases were diagnosed before 1995, and 1181 were diagnosed in 1995 or later. The distribution of cases according to time of diagnosis are shown in Table 3

Characteristics of ER low positive tumours
The distribution of tumour characteristics in patients with ER Low tumours are shown in Table 4. There was a total of 65 (3.3%) ER Low Positive tumours in this study. Of these, 16 were diagnosed before 1995, and 49 was diagnosed in 1995 or later. Among the ER Low Positive tumours diagnosed before 1995, 8/16 (50%) died from BC during follow-up, as opposed to 8/49 (16.3%) of those diagnosed in 1995 or later. Among ER Low tumours, the proportion of tumours < 2 cm, rose from 31% in patients diagnosed before 1995 to 57% in those diagnosed in 1995 or later (p < 0.0001). For all cases, there was a higher proportion of grade 1 tumours (17.2%), and a lower proportion of tumours with grade 3 (29.6%) among women diagnosed in 1995 or later, compared to women diagnosed before 1995 (Grade 1: 10.9%, Grade 3: 39.1% (p < 0.0001)). Among ER Low Positive cases, there was a higher proportion of grade 1 (12.2%) and 2 (53.1%) tumours among women diagnosed in 1995 or later, compared to the women diagnosed before 1995 (grade 1: 0%, grade 2: 31.2%). For grade 3 tumours the proportion of ER low tumours was lower when diagnosed in 1995 or later (p = 0.04) ( Table 4).
For all cases, the proportion of Luminal A subtype was higher for women diagnosed in 1995 or later (52.5%) compared to those diagnosed before 1995 (41.0%). The proportion of Luminal B (HER2-) and HER2 subtypes was lower for women diagnosed in 1995 or later (p < 0.0001) ( Table 3), compared to those diagnosed before 1995. Among ER Low Positive tumours, the proportion of Luminal A subtype rose from 25% in ER Low tumours diagnosed before 1995, to 51% when diagnosed in 1995 or later. The proportion of Luminal B (HER2 +) tumours was lower among the women diagnosed in 1995 or later (18.4%), than the women diagnosed before 1995 (50%) (p = 0.037) ( Table 4).

ER categories and prognosis
Cumulative incidence of death by BC according to ER status is shown in Fig. 2. The risk of death from BC for all categories of ER expression was lower for women diagnosed in 1995 or later compared to women diagnosed before 1995 ( Table 5). The cumulative risk of death from BC after 5 years, for women diagnosed before 1995, was 47.4% among cases with ER < 1%, 37.5% for cases with ER Low Positive and 20.8% for cases with ER ≥ 10%. Among women diagnosed with breast cancer in 1995 or later the cumulative risk of death from BC was 22.3% after 5 years for ER < 1%, and 8.3% for both the ER Low Positive and ER ≥ 10% group (Table 5). Thus, among patients diagnosed in 1995 or later, there was no clear difference in risk of death from BC between cases with ER Low Positive and ER > 10%.
Cox regression analyses showed that the risk of death was lower among patients with ER ≥ 10%, compared to those with ER < 1%, both among patients diagnosed before 1995, and among patients diagnosed in 1995 or later. The Cox analysis shows a lower relative risk of death from BC among patients with ER ≥ 10% tumours, compared to ER < 1% both before and after 1995. We observed a tendency towards a lower relative risk of death from BC among ER Low Positive, compared to ER < 1%. However, these findings were not statistically significant (Table 5).

Discussion
In this study of 1955 primary BC tumours, we found that 65 (3.3%) tumours fell under the ER Low Positive category. We found the highest proportion of ER Low Positive among Luminal B (HER2 +) tumours (9.4%). Among cases diagnosed before 1995, 2.1% were ER Low Positive rising to 4.2% among cases diagnosed in 1995 or later. We found an association between ER Low Positive and high histopathological grade, high Ki-67 levels and high mitotic count. However, the results did not show a significant association with prognosis.
Breast cancer survival in Norway has increased since the mid-1990s as seen in the present and other studies [17]. This may be ascribed to earlier detection [18,19] and improved treatment [6,20]. The reduced risk of death observed between the two time-periods for all categories of ER expression, probably reflects earlier diagnosis with the introduction of mammography screening and the introduction of adjuvant treatment therapies in the mid-1990s. The change in prognosis observed across time for patients with ER Low Positive tumours may also be attributed to adjuvant therapy other than antihormonal treatment in addition to changing tumour characteristics such as smaller tumour size and lower histopathological grade. However, a drawback of the present study was lack of availability of diseasefree survival data. ER status is an important indicator of prognosis and a predictor of the effect of endocrine treatment. ER signalling is a main driver of proliferation in ER Positive BCs, and inhibition of ER signalling has improved survival among ER Positive BC patients [6,21]. Studies suggest that selection of patients for endocrine therapy may need to be further personalized [9,22,23]. While most ER + BCs have high IHC scores, about 2-3% of cases are ER Low Positive [10,24,25]. In the present study, 3.3% of the total number of cases were ER Low Positive. While these tumours are classified within the ER + category, their risk profile appears to be more like that of ER-negative breast cancers [24]. A recent study found no benefit of endocrine therapy in the ER < 10% group compared to the ER > 10% group [25]. The lack of benefit of endocrine therapy in patients with low ER expression has recently been shown in a meta-analysis, including more than 16,000 patients [26]. The meta-analysis indicated that primary BC patients with ER 1-9% gained no significant survival benefit from endocrine therapy, but manifested better overall prognosis than patients with cancers expressing ER < 1% [26]. In the present study, among patients diagnosed in 1995 or later, the ER Low Positive patient group had similar survival to those with ER ≥ 10%. The patients included in this study were diagnosed with BC between 1961 and 2012, and the ER > 1% cut-off level for endocrine treatment was first introduced in Norway in 2011 after recommendations from ASCO/CAP [27]. Therefore, the improved prognosis seen among ER Low Positive patients diagnosed in 1995 or later, can most likely not be attributed to endocrine treatment [28]. Among women diagnosed in 1995 or later, we found a greater proportion of ER Low Positive tumours with smaller size, lower grade, and lower proliferation compared to ER Low Positive tumours diagnosed before 1995. Thus, the improved prognosis may be attributed to factors other than endocrine treatment, such as earlier diagnosis due to the introduction of mammography screening and greater BC awareness among women. Determining endocrine treatment for patients with a diagnosis of ER Low Positive BC should be carefully considered in light of the potential risks and benefits of the treatment [24].
In the present study, the proportion of Luminal A tumours was higher among women diagnosed in the time period during which adjuvant treatment and earlier diagnosis became available, a finding previously observed by our group in an analysis of cohorts 1 and 2 [14]. It has been suggested that BC patients with ER Low Positive are more similar to the ER-negative group, and therefore may not profit from endocrine therapy [9]. Thus, it has been suggested that cut-off levels should be further investigated in order to offer BC patients personalized endocrine treatment [22,29,30]. In the present study we found that among cases diagnosed in 1995 or later, ER Low Positive cases showed a prognosis similar to that of ER ≥ 10% cases. However, the impact of hormonal therapy could not be assessed in this study, due to lack of individual information on treatment.
Similar to our findings, a recent study showed that ER Low Positive tumours were more frequently grade 3 and had a higher expression of Ki-67, compared to BCs with intermediate or high expression of ER [31]. Furthermore, they found that the expression of immune-related biomarkers in ER Low Positive was similar to that of ER-negative tumours. We observed four cases of medullary carcinoma and one metaplastic carcinoma among the ER Low Positive cases. When determining treatment for patients with ER Low Positive BC, it may be useful to consider including a panel of immune-related biomarkers.
The FFPE tumour tissue included in this study covered a diagnostic timespan of several decades, and preanalytical conditions may have varied over time. Many of the tumours were diagnosed at a time when ER IHC was not done in the diagnostic setting. However, valuable information can be drawn from archival tissue blocks [32,33]. It has been shown that antigenicity is, for the most part, preserved in NOS = Not otherwise specified, HPF = High Power Field paraffin blocks over decades but may decrease in sections stored over time, resulting in weaker staining [33][34][35]. We observed no apparent trend towards a negative result among the older specimens but felt it would be unwise to attempt to quantify staining intensity due to the varying preanalytical conditions over which we had no control. Other strengths of this study include reliable information on BC incidence and follow-up data that were available from high-quality national registries like the Cancer Registry of Norway, the Cause of Death Registry and the Norwegian Patient register [36,37] thus enabling comparability within the study population across time.
Using TMA sections enables us to stain hundreds of tumour samples at the same time, under the same conditions. The samples comprise a small amount of the original tumour tissue samples, compared to full-face sections. Thus, some important information from the tumour may be lost. However, it has been shown that IHC for ER carried out  of Science and Technology, Trondheim, Norway. Data included in this study received financial support from the Liaison Committee between the Central Norway Regional Health Authority and the Norwegian University of Science and Technology and The Research Council of Norway.

Data availability
The datasets generated and/or analysed during this study are not publicly available due to issues of sensitivity and limitations determined in the conditions for approval by the Regional Committee for Medical and Health Research Ethics. However, the data may be made available from the corresponding author on reasonable request.

Declarations
Competing interest The authors declare that they have no competing interests.
Ethical approval This study and publication of its results was granted approval by the Regional Committee for Medical and Health Research Ethics, Central Norway (REK 836-09). The approval includes dispensation from the usual requirement of patient consent.
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