|Ahead of print publication
Role of estrogen receptor-a and estrogen receptor-β expression in the disease outcome of invasive luminal type A breast carcinoma patients: A pilot study
Abhay Vilas Deshmukh1, Anupama Gupta1, Vandna Yadav2, Shubhangi Mangam1, Nitin M Gangane1
1 Department of Pathology, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha, Maharashtra, India
2 Department of Pathology, Sawai Man Singh Medical College, Jaipur, Rajasthan, India
|Date of Submission||20-Feb-2020|
|Date of Decision||13-Apr-2020|
|Date of Acceptance||22-Apr-2020|
Mahatma Gandhi Institute of Medical Sciences, Sevagram - 442 102, Wardha, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Estrogen receptor (ER)-α and ERβ, members of family of ERs are expressed in many breast tumors. However, their role in carcinogenesis and their association with regard to the prognosis are still under the investigation and unclear because of limited data. Aims: The present cross-sectional, observational pilot study was aimed to study the immunohistochemical expression and prognostic significance of ERα and ERβ in invasive luminal Type A breast carcinoma patients. Materials and Methods: Twenty-five old diagnosed cases of luminal Type A breast carcinoma diagnosed between 2012 and 2015 were included in the study. The expression of markers ERα and ERβ was correlated with the clinical characteristics and disease outcome in 2–7 years' follow-up period. Statistical analysis was performed by SPSS version 17.0 and graph pad PRISM 5.0 version. Results: Eleven cases out of 25 were both (ERα and ERβ) positive, whereas 1 case was both (ERα and ERβ) negative. Positivity with single marker was 12 for ERα and 20 for ERβ. Adverse outcomes, i.e., recurrence, distant metastasis, and death were reported in 10 cases (40%), 8 out of which were ERα negative cases. While ERα-positive expression alone or along with ERβ expression was shown to be associated with less adverse outcomes (2 out of 10, P = 0.005). Conclusions: When both ERα and ERβ expression is present, they appear to act as allies and together presents with the better prognosis in disease outcomes of breast cancer in our study cases.
Keywords: Breast cancer adverse outcomes, breast cancer prognosis, estrogen receptor-alpha, estrogen receptor-beta, luminal type a breast carcinoma
|How to cite this URL:|
Deshmukh AV, Gupta A, Yadav V, Mangam S, Gangane NM. Role of estrogen receptor-a and estrogen receptor-β expression in the disease outcome of invasive luminal type A breast carcinoma patients: A pilot study. Med J DY Patil Vidyapeeth [Epub ahead of print] [cited 2021 Mar 7]. Available from: https://www.mjdrdypv.org/preprintarticle.asp?id=309190
| Introduction|| |
One in ten of all new cancers diagnosed worldwide each year are a cancer of the female breast, and it has become the most common cancer in women in both developing and developed countries. It is also the principal cause of cancer deaths among women globally as well as in India., Breast cancer is the most common cancer in urban and some rural areas in India also and accounts for about 27.75% of all cancers in women in Wardha district, Maharashtra, India.
According to the molecular classification of breast cancers, luminal Type A breast carcinoma is defined as cancers having estrogen receptor (ER), progesterone receptor (PR) positivity and HER2/neu negativity. It is a known fact that the dominant ER in the mammary gland is one factor that is associated comparatively better prognosis in luminal Type A breast carcinoma in comparison to other molecular types., However, ER is not one but a family of receptors, i.e., its three types; ERα, ERβ, and ERγ exists. Among the family, ERα expression has been reported to be an important prognostic marker in recent past which is associated with low histological grade and less aggressive phenotypes of breast cancer, whereas the role of ERβ is still under investigation. Five ERβ isoforms (β1–β5) have been cloned till date., It has also been postulated that the expression of ERβ indicates an alteration in the expression pattern of different ER variants during mammary carcinogenesis and so if the role of ERβ can be ascertained, it could well be the next candidate for targeted hormone replacement therapy of breast cancer.
There are very few studies conducted on the prognostic importance of ER subfamilies in India as per our knowledge. Our research question was why some of the luminal Type A breast cancers behave aggressively and to get the answer, we evaluated ERα and ERβ receptor expression in the tissue samples of diagnosed invasive luminal Type A breast carcinoma patients in our rural tertiary care hospital and correlated it with the disease course. The project was taken as a pilot study to get the initial results before planning for a bigger study.
| Materials and Methods|| |
It was a cross-sectional, observational pilot study. In this study, 25 old consecutive patients of luminal Type A breast cancer with reported ER, PR positivity, and Her2/neu negativity were selected as study cases which were diagnosed 0–60 months back from the initiating point of the study. The cases that were lost to follow-up or patients who were died soon after initial diagnosis were excluded from the study. The clinical details of cases were retrieved from the hospital records. The paraffin blocks of breast tumor tissue obtained from breast modified radical mastectomy (MRM) or from lumpectomy specimens along with all available slides were also retrieved and screened. Out of 25 selected cases, 88% (n = 22) cases were histological Grade III carcinoma and more than half (n = 15, 60%) were at Tumour, Nodes and Metastases (TNM) Stage III at the time of the first diagnosis. Fifteen cases (68.18%) out of 22 cases with primary tumor size >2 cm had positive axillary lymph nodes and out of these 15 cases, 11 cases (61%) were also having positive skin changes at primary site. After case selection, patients were contacted on their follow-up visits, informal consent was taken and they were followed up for a period of minimum for 2 years for the course of their disease.
Haematoxylin and Eosin stained slides were screened to obtain the best section for immunohistochemistry (IHC). Immunohistochemical stain for ERα and ERβ was performed by the standard technique according to IHC system manual. Sections of 3–5μ in thickness were obtained. The tissue was fixed in formalin for 12 hours. Antigen retrieval, i.e., unmasking of antigen was done with the help of microwave oven (operated at 5 min at 750 watts and then 15 min at 200 watts). Inactivation of endogenous peroxidase was done by 3% hydrogen peroxidase. Then power blocking, i.e., removal of nonspecific antibodies to highly charged sites was done. Cases along with appropriate positive and negative controls were stained with ERα (6 mL ready to use the rabbit monoclonal IgG primary antibody Anti ER-Alpha, BioGenex, Fremont, California, USA) and ERβ (ready to use polyclonal rabbit primary antibody Anti ER -Beta, BioGenex, Fremont, California, USA). The polyclonal ERβ antibody was used to assess the expression for all the isoforms of the ER beta (β1–β5). Evaluation of staining was done by using Allred score system which consisted of addition of score for intensity and score for proportion in all the cases., The scores were added together to obtain a total score that ranged from 0 to 8. Patients with tumors scoring 2 or less were considered as negative for the immune marker expression, whereas more than 2 were considered as positive [Figure 1].
|Figure 1: (a) Estrogen receptorα, staining with Alred score 8 (ERα positive), (b) Estrogen receptor-α, staining with Alred score 1 (ERα negative), (c) Estrogen receptor-β staining with Alred score 3 (ERβ positive), (d) Estrogen receptorβ staining with Alred score 6 (ERβ positive) (×400, IHC)|
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All outdoor and indoor visits of study cases to the hospital after primary management were recorded using (1) records of department of surgery, (2) department of radiation oncology, (3) district cancer registry working from our department of pathology, and (4) computerized hospital information system. During follow-up period, the patients were evaluated for any adverse outcome in particular.
The adverse outcome in our study was defined as the recurrence of cancer at primary site, distant metastasis or death, after completion of primary treatment.
The study protocol was approved by the Institute Ethics committee of Mahatma Gandhi Institute of Medical Sciences, Sevagram, letter number MGIMS/IEC/ PATH/192/2012, dated 05-10-2012. Informed consent was obtained from the patients if we interviewed them for any details of disease course. Patient confidentiality was maintained during all research procedures.
Statistical analysis was performed by using descriptive and inferential statistics, Chi-square test, and fisher's exact test. Software used was SPSS17.0 (IBM Corp. Released 2011. IBM Statistics for Windows, Version 20.0: Armonk, New York, United States) and graph pad PRISM 5.0 version and P < 0.05 was considered as the minimum level of significance.
| Results|| |
Perimenopausal age group (41–60 years) was the most common age group of luminal type A breast carcinoma presentation, comprising 64% (n = 16) in the present study. A total of 10 cases (40%) were reported to have adverse outcomes during the follow-up period of 2–7 years. It was found that the incidence of unfortunate events was more if disease presents itself in early age. Most of the recurrence cases (44%) occurred in 41–50 years of age and 90% occurred in patients of 21–50 years age. If looked proportion wise within the age group, most of adverse outcomes were again found in younger age group accounting for 66.67% and 75% in 21–30 and 31–40 age groups, respectively [Table 1].
Recurrence at primary scar site was found to be the most common complication (n = 5). Deaths due to cancer were reported in three cases. All the live patients (n = 22) are still under follow-up [Table 2].
|Table 2: Follow-up findings in cases with adverse outcomes: Sites of adverse outcomes versus follow-up period|
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Treatment plan in our study was divided in Plan I and Plan II. Plan I consisted of surgery + adjuvant chemotherapy + radiotherapy, whereas Plan II consisted of neoadjuvant chemotherapy + surgery + radiotherapy. For most of the patients, treatment plan I was advised (80%). However, more than half (n = 14) (56%) patients did not complete the treatment plan due to personal or financial reasons. Among all study cases, four patients ended up with adverse outcomes despite completing the treatment, whereas six patients did not complete their treatment and reported with complications later [Table 3].
Whether we look at ERα and ERβ positivity individually [lower half of [Table 4] or combining them [upper half of [Table 4], we find that ERα positivity is having statistically significant inverse association with the adverse outcomes (P ≤ 0.0001). ERα expression alone or along with ERβ was associated with significantly less number of adverse outcomes (2 out of 12 positive tumors), whereas ERα negative tumors were associated with more adverse outcomes (8 out of 13 negative tumors) even if these were ERβ positive (n = 9). This finding was statistically highly significant (P = 0.005). ERβ expression did not show any such association to disease outcome on its own in any significant way, but if expressed combined with ERα, it also contributed to better prognosis [Table 4].
|Table 4: Correlation of estrogen receptor-a and estrogen receptor-β positivity with adverse outcomes in study cases|
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| Discussion|| |
During our follow-up period of 2–7 years in post primary treatment phase, the prognosis was good with ERα positivity while adverse outcomes were associated with ERα negativity, both findings were highly significant (P < 0.0001 and P = 0.005, respectively). There is some evidence that ERα expression is positively correlated with low-grade tumors and negatively correlated with tumor stage (P = 0.028) and with lymph node status (P = 0.168).
It was also seen that combined ERα and ERβ positivity proved to have good prognostic importance (P = 0.005) [Table 4]. Only 2 out of 11 twin positive tumors were reported to have adverse outcome. We also found that adverse outcome was less in group of all ERα positive in comparison to the group of all ERβ-negative cases (P = 0.022) [Table 5]. Many studies found the similar results such as Shaaban et al. (ERβ2 correlated positively with ERα and predicted better overall survival and disease-free survival), Maehle et al. (ERα +ve and ERβ high and medium levels has more survival than ERα +ve and ERβ low). And so, ERβ expression was seen to be a good prognostic indicator but only when coexpressed with ERα. Han et al. also reported negative correlation of nodal status with ERα and ERβ coexpression in their series of 84 patients (P = 0.028). When present alone, ERβ offered no protection from adverse outcomes as far as our study cases are concerned.
It is suggested that some isoforms of ERβ are related to better outcome while others are not. However, rare studies done on this subject failed to show any direction. Sugiura et al. studied real-time polymerase chain reaction and IHC on 150 breast carcinoma tissues which showed that the high expression of ERβ1 or ERβ2 had a significantly better disease-free survival than those with low expression. On the other hand, Shaaban et al. who studied nuclear and cytoplasmic expression of ERβ1, ERβ2, and ERβ5 in 842 cases of breast cancer patients found that ERβ2 overexpression is associated with significantly worse outcome (P = 0.00014).
Our study population presented an overall advanced disease already: 60% presented at TNM III, 88% had histological Grade III tumors, and moreover, 56% patients could not complete their treatment plan. Though these aspects were confounding factors in knowing the true worth of ERα and ERβ in the prognosis of breast cancer, but these factors are bound to be present even in a bigger study in our geographical region because of many genetic, social, cultural, and gender issues still prevalent in the society. Any ambition to curb the menace of this disease or any other public health problem must be prepared to address all these issues also.
Madeira et al. in 2013 in their study to find role of ER alpha/beta ratio and ER beta as the predictors of endocrine therapy responsiveness found that the frequency of ER-α expression did not change after treatment with anastrozole and tamoxifen groups (P = 0.33). However, in the ER-β-positive cases, the anastrozole (P = 0.01) and tamoxifen groups (P = 0.04) presented a significant reduction in posttreatment Ki67 scores. Only patients with an ER-α/ER-β expression ratio between 1 and 1.5 demonstrated significant differences in Ki67 levels after treatment with anastrozole (P = 0.005) and tamoxifen (P = 0.026). Roger et al. found that in the spectrum of benign breast disease to frank carcinoma breast ERα/ERβ ratio is gradually increasing. They found that in the histologically normal resting mammary gland, ER β-positive cells (median, 85%) greatly exceeded ER α-positive cells (median, 4%), whereas the ERα level progressively supersede the ER β level in benign breast disease with atypia and in carcinoma in situ.
It seems that ERβ presents a parent and stable subfamily of ERs and it is ERα that changes its expression in the mammary tissue during benign and malignant transformation and is responsive to the chemotherapy. Thus, it helps in the prognosis of patients.
We did not find any Indian study related to the prognosis in relation to ERα and ERβ expression in breast carcinoma patients till date as per our knowledge. This is probably first pilot study in the Indian literature on this aspect. Thus, we recommend a larger study sample to study the role of ERα and ERβ markers in the disease course of breast carcinoma patients in India.
| Conclusions|| |
ERα expression alone as well as combined with ERβ expression appears to be a sign of good prognosis in our study cases.
We would like to acknowledge Dr Dilip Gupta; Professor&Head, Department of Surgery and Dr Virendra Vyas; Professor&Head, Department of Radiation Oncology, MGIMS, Sevagram for sharing their patient data for this study. We would also like to acknowledge Kasturba Health Society, Sevagram, Wardha, Maharashtra, for funding this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]