Abstract
Breast cancer is a heterogeneous disorder with various subcategories of different cellular compositions, molecular alterations, and clinical behaviour and is dependent on numerous factors like grade, size, and hormonal receptors which influence the overall prognosis and response to the treatment. This study was done to determine the frequency of estrogen receptor (ER), progesterone receptor (PR), and Her2 neu positivity in breast cancer patients, further classifying them into their molecular types (luminal A, B, Her2 neu, and triple-negative) and studying their association with histological subtypes, lymph node status, and other epidemiological parameters. This was a 5-year retrospective study done on 314 patients. All relevant clinical data including the age, sex, and lymph node status along with the histological type and grade of the tumour were recorded, and immunohistochemical evaluation of Her2 neu, ER, and PR receptors was done. The results showed that ER was the most predominant immunomarker, followed by PR, with an inverse relationship between ER, PR, and Her2 neu. Among the molecular subtypes, luminal B showed the maximum prevalence followed by triple-negative and Her2 neu subtype. The least frequency was shown by luminal A. Our study concluded that molecular subtyping of breast carcinoma plays an important role in determining the prognosis, recurrence, and treatment. Increased expression of luminal B subtype can be correlated due to the advancing age of the patients.
Keywords: Breast carcinoma, Immunohistochemistry, Molecular subtypes
Introduction
Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in females in India and worldwide [1]. Breast cancer is a multifaceted disease that shows a diverse natural history and presents with a varied spectrum of clinical, pathologic, and molecular features with different prognostic and therapeutic implications [2]. Breast tumours are classified histologically according to the location of origin. Tumours developing from the breast duct are the ductal tumours and represent 80% of tumours while the lobular tumours develop inside the lobes and account for 10 to 15% of cases. Other subtypes represent less than 10% of cases diagnosed per year [3]. The pathogenesis of breast carcinoma starts with ductal hyperproliferation which leads to the development of carcinoma in situ, invasive carcinoma, and finally into metastatic disease [4]. Since breast carcinoma shows variation in its clinical progression, identification of markers that could predict tumour behaviour is quite essential. Moreover, such tumour markers play an important tool in the diagnosis, clinical management, staging, detection of recurrence, prognosis, and in the development of new treatment modalities [5]. The status of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor type 2 (Her2) has been used as predictive markers for identifying a high-risk phenotype and for selection of the most efficient therapies [6]. The ER and PR are highly associated with patient age at diagnosis, rising continuously with age [7]. Tamoxifen binds to the ligand-binding domain of ER, blocking the potential for estrogen stimulation. It further prevents critical ER conformational changes that are required for the association of coactivators thus helping in producing clinical remission in patients with breast cancer positive for ER due to a better response to hormone therapy and a higher survival [8].
The Her2 gene, also known as ERB B2 or Her2 neu, is located on chromosome 17q11. Its amplification or over-expression of erbb2 protein has been detected in 10 to 30% of breast cancers and shows resistance to tamoxifen [9]. Trastuzumab which is a humanized monoclonal antibody is directed against the extracellular domains of Her2 and is currently indicated for the treatment of Her2 positive breast cancer cases [10]. The majority of breast malignancies are luminal tumours in which breast cancer cells line the inner (luminal) layer. Among various molecular subtypes, luminal A tumours have the best prognosis, relatively higher survival, and low recurrence rates [10]. However, luminal B subtypes are diagnosed at a younger age, show p53 mutation, and are associated with a poorer prognosis [11]. Triple-negative or basal-like tumours constitute 15–20% of breast malignancies and have cells similar to the outer (basal) cells surrounding the mammary ducts. These are aggressive tumours with poor prognosis and associated with BRCA 1 mutation [12]. Therefore, understanding the hormonal receptors which impact the prognosis is very necessary for further planning investigation and deciding treatment strategies [13]. This study emphasizes the expression of estrogen, progesterone, and Her2 neu receptors among various histological variants of breast carcinoma along with molecular subtyping into luminal (estrogen receptor/progesterone receptor [ER/PR] positive and Her2 negative), luminal B (ER/PR and Her2 positive), Her2 (ER/PR negative and Her2 positive), and triple-negative (ER/PR and Her2 negative) subtypes and correlating with various epidemiological and prognostic parameters.
Materials and Methods
This study was a 5-year retrospective study from 2017 to 2021 which was done on a total of 314 cases. All histologically (tissue or paraffin block) diagnosed breast cancer cases that were referred to the Department of Pathology, Jawaharlal Nehru Medical College, AMU, Aligarh were included. All the relevant clinical and histological parameters were recorded wherever available including age, gender along with histological type, lymph node status, grade, and distant metastasis. Immunohistochemistry for Her2 neu, ER, and PR receptors were applied on all the 314 cases. A 3 to 4 μm thick paraffin-embedded tissue sections were taken and were placed on poly-L-lysine coated slides. After deparaffinization, antigen retrieval, and blocking of endogenous peroxidase, immunostaining was performed using a primary antibody at 1:100 dilution. The binding of the primary antibody was done and diaminobenzidine (DAB) as a chromogen was added. The slides were then examined microscopically and different histological variants of breast carcinoma were identified and categorized into four main groups according to their hormone receptor positivity status: ER/PR + , Her2 − was classified under luminal A category; ER/PR + , Her2 + under luminal B category; ER/PR − , Her2 + under Her2 enriched; and ER/PR − , Her2 − under triple-negative/basal-like category. ER, PR receptor evaluation was done by Allred scoring under which the percentage of positive cells and the intensity of the reaction product was combined. These two scores were added together for a final score with 8 possible values. Scores of 0 and 2 were considered negative. Scores of 3–8 were considered positive. Human epidermal growth factor receptor 2 scoring of IHC slides was done on light microscopy as per the recommended American Society of Clinical Oncology (ASCO) guidelines 2007. Scores 0 and 1 were considered as negative while scores 2 and 3 were considered as equivocal and positive, respectively. Sections that showed strong membrane staining of normal epithelia of the breast were rejected and subjected to a repeat IHC.
Results and Discussion
A 5-year retrospective study involving 314 cases of female patients with breast cancer was done and the expression of various immunohistochemical markers on each case was studied. A similar retrospective study was conducted by Jain et al. and Ambroise et al. on 203 and 321 females, respectively [14, 15]. The maximum number of cases occurred between 40 and 60 years of age with the mean age of presentation being 50 years, accounting for a total of 48.6% of all the total cases followed by 19.7% of cases occurring in the 3rd to 4th decade (Fig. 1). A similar peak of cases in the 4th to 6th decade was noted in a study done by Balekouzou et al., emphasizing the increased incidence with the advancing age [16]. However, study by Jain et al. and Nikhra et al. found the predominant presentation in the younger age group [14, 17].
Fig. 1.
Age distribution of total cases
The most common histological subtype was found to be infiltrating ductal carcinoma (non-specific type) consisting of 84.3% of all the cases (Table 1). This was per findings in a similar study done by Nikhra et al. and Ghosh et al. [17, 18]. The total cases of IDC-NST were divided based on Scarff-Richardson-Bloom (SBR) grading system including tubule formation, mitotic activity, and pleomorphism into grades I, II, and III. A maximum number of cases fell under the category of grade II (68.6%) followed by grade III (19.2%) and grade I (12.1%). This was similar to findings by Jain et al. and Nikhra et al. [14, 17]. IDC-NST was followed by the invasive lobular carcinoma as the second most common histological type (6.36%). Least common histological type was the metaplastic carcinoma (2%). The total number of cases was further divided into the total number of lymph nodes involved as lymph node involvement is an important prognostic marker. Maximum cases showed no nodal involvement (42.6%) followed by 1–3 lymph nodes involvement (33.5%). The least cases showed > 9 lymph node involvement (7%) (Fig. 2).
Table 1.
Distribution of cases according to the histopathological type of carcinoma
| Histological type | Number of cases | Percentage | |
|---|---|---|---|
| Infiltrating ductal carcinoma, non-specific type (NST) | Grade I | 32 | 12.1 |
| Grade II | 182 | 68.6 | |
| Grade III | 51 | 19.2 | |
| Total | 265 | 84.3 | |
| Invasive lobular carcinoma | 20 | 6.36 | |
| With medullary pattern | 15 | 4.8 | |
| Mucinous carcinoma | 8 | 2.6 | |
| Metaplastic carcinoma | 6 | 2.0 | |
| 314 | 100 | ||
Fig. 2.
Distribution of cases according to the lymph node status
When immunomarkers were applied to the total 314 cases, the most commonly expressed immunomarker was estrogen (65.2%) followed by progesterone (53.8%). The least commonly expressed marker was the Her2 neu (22.9%). When the expression of this immunomarker was studied according to the histological type, invasive ductal carcinoma (NST) showed maximum expression of ER (67.5%), followed by PR (57.7%). A similar pattern of immunoexpression of ER (85%) and PR (55%) was shown by invasive lobular carcinoma. IBC with medullary pattern however showed equal expression for ER (15%) and Her2 neu (15%) and least for PR (13.3%). Mucinous carcinoma showed a higher expression for ER (50%) while metaplastic carcinoma showed equal expression for ER, PR, and Her2 neu (16.6%) (Table 2). Higher expression of ER, PR was also reported by Jain et al. and Munjal et al. [14, 19]. Nikhar et al. noted a higher expression of PR than ER and Her2 neu [17]. Higher expression of ER, PR as compared to Her2 neu can be attributed to the higher number of middle to elderly women in our study.
Table 2.
Distribution of cases according to the hormone receptors
| Histological type of breast carcinoma | Estrogen receptor (ER) | Progesterone receptor (PR) | Herceptin receptor 2 (Her2 neu) | Total |
|---|---|---|---|---|
| IBS (NST) | 180 (67.9%) | 153 (57.7%) | 64 (24.1%) | 265 |
| Invasive lobular carcinoma | 17 (85%) | 11 (55%) | 3 (15%) | 20 |
| With medullary pattern | 3 (15%) | 2 (13.3%) | 3 (15%) | 15 |
| Mucinous | 4 (50%) | 2 (25%) | 1(12.5%) | 8 |
| Metaplastic | 1 (16.6%) | 1(16.6%) | 1 (16.6%) | 6 |
| Total | 205 (65.2%) | 169 (53.8%) | 72 (22.9%) |
When the total number of cases was divided into various molecular subtypes based on ER, PR, and Her2 neu status, IBC-NST and invasive lobular carcinoma showed maximum cases under luminal B category (57.3%, 60%). IBC with medullary pattern showed maximum cases under the triple-negative category (66.6%). Mucinous carcinoma showed equal distribution under luminal B and triple-negative category (37.5%). Metaplastic carcinoma showed maximum cases under the triple-negative category (66.6%). Overall maximum number of cases was of the luminal B category (54.4%) followed by triple-negative category (22.9%) (Table 3). A similar study was done in Indonesia by Paramita et al. also found the highest percentage of luminal B cases [20]. Ambroise et al., Sharif et al., and Lund et al. reported a similar higher percentage of 25%, 20%, and 29% cases respectively under the triple-negative category [15, 21, 22]. An overall inverse relation was seen between the ER, PR, and Her2 neu in our study with a considerable percentage of triple-negative cases (22.9%).
Table 3.
Distribution and correlation of histological and molecular types of breast carcinoma
| Histological types | Molecular types | ||||
|---|---|---|---|---|---|
| Luminal A | Luminal B | Her2 neu | Triple-negative | Total | |
| IBS (NST) | 41 (15.4%) | 152 (57.3%) | 21 (7.9%) | 51 (19.2%) | 265 |
| Invasive lobular carcinoma | 3 (15%) | 12 (60%) | 1 (15%) | 4 (20%) | 20 |
| With medullary pattern | 1 (6.6%) | 2 (13.3%) | 2 (13.3%) | 10 (66.6%) | 15 |
| Mucinous | 1 (12.5%) | 3 (37.5%) | 1 (12.5%) | 3 (37.5%) | 8 |
| Metaplastic | 0 | 1 (25%) | 1 (25%) | 4 (66.6%) | 6 |
| Total | 46 (14.6%) | 170 (54.4%) | 26 (8.2%) | 72 (22.9%) | 314 |
Conclusion
Our study concluded that the most common tumour subtype is the luminal B tumours, followed by triple-negative tumours, while invasive breast carcinoma (no specific type) is the most common histological type. ER, PR showed a positive correlation and an inverse correlation with Her2 neu. The predominance of ER expression was seen in the majority of breast carcinoma cases in our study probably because of the predominance of the middle to the elderly aged population.
As breast cancer is a heterogeneous disease that shows marked diversities not only in its morphology, clinical presentation, prognosis, and response to treatment but also varies according to ethnicity and geographic distribution, the use of immunomarkers plays a substantial role in further understanding in terms of prognosis, treatment, and recurrence.
Author Contribution
First author 1: Dr BS: conceived and designed the analysis, contributed in data analysis, wrote the paper.
Author 2: Dr SA: conceived and designed the analysis, contributed in data analysis, wrote the paper.
Author 3: Dr DS: collected data, performed the analysis, and wrote the paper.
Author 4: Dr AVLS: collected the data and contributed in data analysis.
Declarations
Conflict of Interest
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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