Molecular Subtypes of Breast Carcinoma and Their Association with Clinicopathological Features

By Ambreen Bari¹, Summaya Shawana²

Doi: https://doi.org/10.36283/ziun-pjmd14-1/005

How to cite: Bari A, Shawana S Molecular Subtypes of Breast Carcinoma and Their Association with Clinicopathological Features. Pak J Med Dent. 2025 Jan ;14(1): 24-31. Doi: https://doi.org/10.36283/ziun-pjmd14-1/005.

Received: Fri, September 27, 2024  Accepted: Sat, November 30, 2024 Published: Fri, January 10, 2025

Background: Breast cancer is categorized into four primary molecular subtypes based on gene expression profiles and hormone receptor status: Luminal A & B subtypes, Basal-like, and HER2-enriched. This study assessed the association of molecular categories of breast carcinoma with the clinicopathological features.

Methods: A cross-sectional study was conducted between January 2022 and August 2024; 200 mastectomy specimens were collected with a non-probability consecutive sampling technique from Ziauddin Hospital Histopathology Department in Karachi, Pakistan. Immunohistochemical analysis was done for ER, PR, HER 2 Neu, and Ki-67 and the findings were statistically analyzed for association with clinicopathological parameters.

Results: Luminal B subtype was found to be the most common subtype. The study identified pT2 and pT3 as the most frequent pathological stages, with 74 cases (38.1%) and 71 cases (34.4%), respectively. Low-grade tumors were less frequent as compared to intermediate and high-grade tumors with 11 (5.5%), 73(36.5%), and 116 cases (58.0%), respectively. A significant association was observed between molecular subtypes and tumor grade, stage, and histological subtypes. However, insignificant association was seen with age, laterality, and tumor site.

Conclusion: Luminal B tumor was the most frequent subtype, while luminal A was the least prevalent. Luminal B tumors were primarily associated with invasive ductal carcinoma invasive ductal carcinoma (NOS). These tumors were commonly found in women aged 41-50. Furthermore, a strong association of molecular subtypes was observed with histological grading and staging.

Keywords: Molecular Subtypes, Breast Cancer, Clinicopathological Features

Cancer remains a leading cause of death globally¹. Breast cancer is the most often diagnosed cancer in women and the second leading cause of cancer-related mortality². It was reported that, in 2022, a total of 670,000 patients had died due to breast carcinoma worldwide. Breast cancer has been identified as the most common cancer among women in 157 out of 185 countries³. It had been estimated that 2.3 million new cases of BC were diagnosed globally each year⁴. The international landscape had revealed varying prevalence across different regions, with Western European countries exhibiting a higher incidence rate of breast cancer compared to Eastern Asian or African countries⁵. According to estimates, breast cancer may affect approximately one out of every nine women in Pakistan during their lifetime⁶. A total of 30,682 cases of breast carcinoma were recorded in both genders for the year 2022⁷.

Advancing age is a paramount risk factor for breast cancer, with incidence escalating significantly among older women⁸. However, recent researches show that females in their younger years are more prone to develop breast cancer⁹. Breast cancer is treatable if managed effectively early in the disease; however, treatment strategies for breast cancer are highly dependent on various physiological and anatomical factors, such as age, stage of cancer, size of the tumor, menopausal status, hormone receptors, and status of axillary lymph nodes⁹. The cDNA microarray technique was used to categorize breast cancer into subgroups by analyzing gene expression profiles ^10,11.

Carcinoma breasts were thus categorized into various molecular subtypes, including Luminal A & B, HER2-enriched, and basal-like, which were determined by mRNA gene expression levels¹². Luminal-like breast cancer is the predominant subtype, accounting for 60%-70% of all breast tumors¹². Luminal A, making up 14.81% of breast cancer cases, is hormone receptor-positive and HER2-negative with Ki67 < 14%, a favorable prognosis, and strong responsiveness to hormone therapy. Luminal B is typically hormone receptor-positive, sometimes expressing HER2 (if HER 2 negatives with Ki67 > 14%), and is more aggressive than Luminal A^13,14,20,22. It was stated that Triple Negative Breast Cancer (TNBC) was characterized by the absence of estrogen, progesterone, and HER2 receptors, leading to a more aggressive subtype and limited to nonspecific treatment options, primarily chemotherapy. The molecular distribution underscored the importance of individualized treatment plans, as each subtype presented distinct biological features and therapeutic responses. ER-positive tumors indicated a favorable response to hormonal therapies like tamoxifen or aromatase inhibitors, while ER-negative breast carcinomas warrant other therapeutic approaches¹⁴. Similarly, with this near-equal split, PR-positive and PR-negative cases suggested that PR status added complexity to treatment decisions, mainly when considered alongside ER status. HER2-negative tumors do not overexpress the HER2 gene, while HER2-positive, which is typically linked to more aggressive disease, may benefit from HER2-targeted therapies like trastuzumab (Herceptin)¹⁵.

Thus, receptor status emphasizes the importance of a tailored treatment approach to optimize outcomes in breast cancer management. The research had been designed to analyze the association of molecular classification of breast carcinoma with clinicopathological features, which are crucial for treatment options and predicting prognosis.

The Ethical Review Committee ZU approved the study with the reference code 7310623ABPAT. A Cross-sectional study was done utilizing mastectomy specimens of primary breast carcinoma cases received at the Histopathology Department of Ziauddin Hospital Karachi between January 2022 and August 2024. A sample size of 191 was calculated using Open Epi version 3, assuming a 95% confidence level, taking a 5% margin of error, and 14.5 % prevalence of total breast cancer cases (WHO, Globocan 2020)⁷.

Two hundred cases (n = 200) were taken with a non-probability consecutive sampling technique. Grossing and reporting were done according to the CAP protocol (Invasive Carcinoma of the Breast 4.5.0.0, 2021)¹⁶. Primary, treatment-naive breast carcinomas, including all invasive and in situ carcinomas, were included in the study. Metastatic breast cancer, insufficient tissue, and autolyzed specimens or specimens showing extensive necrosis were excluded from the study. After formalin fixation of the specimen, paraffin-embedded block formation, sectioning, and Haematoxylin and Eosin staining, these cases were subjected to histopathological examination for confirmation of diagnosis. Relevant parameters were recorded, including age, tumor laterality, tumor size (on gross and ultrasound), tumor location, tumor extent, histologic type, and lymph node status for every case. The original request cards were collected, and the proforma was filled for the required variables. Immunohistochemistry was performed, and stained slides were analyzed for ER, PR, HER 2 neu, and Ki67 using the Allred scoring method (based on the percentage and intensity of positive staining). The Allred score assesses hormone receptor expression by adding proportion score (0-5) and intensity score (0-3). A total score of 3 and above was positive. HER2/neu staining was graded from 0 to 3+. Cases that showed equivocal HER 2 neu staining were reanalyzed using fluorescent in situ hybridization (FISH). The Modified Bloom Richardson grading system was used to evaluate histological grades, and staging was done according to TNM Classification by AJCC¹⁷. Cases were categorized into Luminal A, luminal B, Triple Negative, and HER2 positive subtypes. The data was tabulated and analyzed using IBM SPSS version 27. Pearson Chi-square was used to assess the association between qualitative factors. A p-value less than 0.05 was regarded as significant.

A total of 200 cases of breast carcinoma were received during the study period, and the mean age at diagnosis was 52.98 +/- 13.96 years. Luminal B and infiltrating ductal Carcinoma (IDC) were found to be the most common molecular and histological subtypes. The biomarker distribution shows that most breast cancer cases were ER-positive and HER2-negative, which aligned with subtypes that respond well to hormonal therapy.

Fig: 1 illustrates the distribution of breast cancer cases according to receptor status for Estrogen Receptor (ER), Progesterone Receptor (PR), and HER2neu. Most patients, 59.8%, were ER-positive, while 40.2% were ER-negative. Similarly, 51% of the patients were PR-positive, and 49% were PR-negative. Regarding HER2 status, 74.5% of cases were HER2-negative, while 25.5% were HER2-positive.

Figure 1; Distribution of Breast Cancer Cases Based on Hormone Receptor Status

Fig:2 illustrates the distribution of breast cancer cases according to molecular subtypes, i.e., Luminal A, Luminal B, HER2 enriched, and Triple-Negative Breast Cancer (TNBC).

Figure 2: Molecular Subtypes of Breast Carcinoma Distribution

Table 1: Age distribution in Different Molecular Subtypes of Breast Carcinoma

Age grouping showed that the majority of the cases were in the 31-60 years of age group. TNBC appeared to be more prevalent in younger patients, particularly those between 31-60 years old. Across all age groups, HER2neu-negative cases outnumbered HER2neu-positive ones. Despite these patterns, the p-value for molecular subtypes (0.266) indicated no statistically significant difference in how these subtypes were distributed across age groups. The high p-values suggested that variations were likely due to random chance rather than a significant age-related trend. This finding indicated that age did not strongly influence this dataset’s distribution of molecular subtypes or HER2 status.

Table 2: Association of The Molecular Subtypes of Breast Cancer Cases with Study Parameters

Table 2 provides a detailed breakdown of the association of subtypes of breast carcinomas with histological subtypes, tumor size, grade, and stage. A key observation was the predominance of IDC, NOS (Invasive Ductal Carcinoma, Not Otherwise Specified), the most frequent histological subtype, particularly within the Luminal B category. In contrast, rarer histological subtypes like Medullary IDC and Invasive Secretory Carcinoma were far less common. The p-value for the histological subtype (0.002) pointed to a statistically significant relationship between molecular subtypes and histological subtypes, suggesting that the histological subtype of breast cancer was closely linked to how it was classified at the molecular level. On the other hand, factors like tumor laterality (left vs. right breast) and specific tumor sites showed non-significant association with molecular subtypes.

Breast cancer is the most commonly diagnosed malignant tumor among women worldwide and is the leading cause of cancer-related deaths in this population. Its incidence continues to rise in all regions across the globe. This disease presents a diverse range of morphological characteristics, distinct immunohistochemical profiles, and various histopathological subtypes, each associated with specific clinical courses and outcomes. The molecular classification of breast carcinoma based on hormone receptor expression stands out as the most widely accepted and utilized approach in understanding and categorizing different breast cancer subtypes.

Molecular Subtype distribution indicated that most cases were luminal B type, followed by Triple Negative, Luminal A, and HER2-enriched subtypes in descending order of frequency. This finding agreed with the study by Mohammed et al. and Paramita et al^{. 18,19}. This consistency suggested that Luminal B might be a pervasive subtype across many populations, making it significantly relevant in clinical practice. Three studies show a contrasting observation, with Luminal A type breast cancers being the most prevalent type with a frequency of 58.5%, 37%, and 41.1% of all cases, respectively^20,21,22. When taking into account previous studies, a comparatively higher proportion of triple negative breast cancers was observed comprising 26.5% of all cases. It was noted that this difference in subtype prevalence underlined the relevance of regional and population-specific factors for interpreting receptor profiles and planning treatment strategies.

Molecular subtypes of breast carcinoma indicated significant association with histopathological subtypes. The most common histological subtype in the current study was Invasive Ductal Carcinoma (IDC NOS), and this finding agreed with other studies conducted worldwide^18,20,21,23. This study had a relatively lower frequency of lobular carcinomas compared to other studies ^22,23. Al-Thoubaity et al. documented that lobular carcinomas comprise 11.4% of all cases, most of which were of Luminal A subtype^22,23. This observation was supported by the present study, which observed the Luminal A subtype as the most common variant in lobular carcinomas. Thus, while lobular carcinomas were uncommon, they usually present with a specific molecular subtype, i.e., Luminal A.

Molecular subtypes of breast carcinomas were strongly correlated to the Bloom Richardsons grades and revealed a significant association. This strong statistical correlation underscores the importance of understanding how molecular characteristics relate to tumor differentiation and aggressiveness. In agreement with that finding, another related study noted a considerable p-value of <0.001 for the association between grade and molecular subtype of breast cancer²³. It was observed in the current study that luminal A subtype tumors were predominantly Grade II tumors, and this observation has been confirmed by other studies ^23,25. It is also consistent with the known biology of luminal A breast cancers, which are typically well-differentiated and associated with a better prognosis. This study agreed with previous studies that usually showed higher histological grades in Luminal B, HER2-positive, and triple-negative tumors. Specifically, TNBC presented as grade 3 tumors^18,22,24. This finding highlights the aggressive nature of these subtypes, characterized by poorer differentiation, higher proliferation rates, and, often, a more challenging clinical course.

T staging indicated a significant association with the molecular subtypes. This finding aligns with existing literature that also reports a robust relationship between molecular subtypes and the extent of disease at presentation²³. It was further observed that Luminal A tumors were associated with an earlier stage (T1 and T2) at diagnosis, and their findings were supported by various studies, where the majority of cases with a Luminal A subtype presented with stage T2 disease²⁵. In comparison, stage T4 tumors were predominant in triple-negative patients in the present study. This trend agreed with the findings in other studies, where triple-negative and HER2-positive patients were more commonly diagnosed at an advanced stage²⁵, confirming the aggressive nature of these molecular subtypes. Mohammad et al. did not find a statistically significant association with tumor grade however, they reported stages II and III as the most common clinical stages in patients with breast cancer¹⁸. Significant association of molecular subtypes with the T stage reinforces the significance of staging in assessing disease progression and guiding treatment strategies.

The study demonstrated the predominance of the Luminal B subtype amongst the studied cases. Molecular subtypes of breast carcinoma showed a significant relationship with histological subtype, grading, and t-staging. The current study confirmed some of the established patterns while identifying unique trends; a higher frequency of triple-negative breast cancers and concordance of molecular and histopathological subtypes of breast carcinoma. These findings must be incorporated into molecular and histopathological data to guide effective clinical decisions and treatment strategies. Further studies using more extensive and diverse cohorts will be needed to further understand factors influencing breast cancer subtypes and for appropriate treatment strategies.

ER: Estrogen Receptor

PR: Progesterone Receptor

HER2 neu: Human Epidermal growth factor Receptor 2

FISH: Fluorescence in Situ Hybridization

AJCC: American Joint Committee on Cancer

TNBC: Triple Negative Breast Cancer

IDC NOS: Invasive Ductal Carcinoma, not otherwise specified (NOS)

CAP: College of American Pathologists

The author acknowledged her supervisory team and technologists for their immense contribution.

All writers affirmed that they have no conflicts of interest.

On September 4, 2023, the institution granted ethical approval under reference number 7310623ABPAT.

This study was based on tissue samples, so participants were not subjected to any extra investigation or interventions. Their details were kept confidential.

AB: Major contribution to writing the manuscript and collecting data. SS: Data Analysis and Editing.

1. Momenimovahed Z, Salehiniya H. Epidemiological characteristics of and risk factors for breast cancer worldwide. Breast Cancer: Targets and Therapy. 2019 April;11, 151-64. https://doi.org/10.2147/BCTT.S176070
2. Fahad Ullah M. Breast cancer: current perspectives on the disease status. Breast Cancer Metastasis and Drug Resistance: Challenges and Progress. 2019 August; 51-64. https://doi.org/10.1007/978-3-030-20301-6_4
3. World Health Organization: WHO, https://www.who.int/news-room/fact-sheets/detail/breast-cancer?gad_source=1&gclid=CjwKCAjw9eO3BhBNEiwAoc0-jZThd7r0xs_QGzTqOfsbFb8sTTYOpkqkc9ZPYw8Gg4zwizvLqxaBaxoCB6wQAvD_BwE (assessed on 29/10/2024)
4. Łukasiewicz S, Czeczelewski M, Forma A, Baj J, Sitarz R, Stanisławek A. Breast cancer—epidemiology, risk factors, classification, prognostic markers, and current treatment strategies—an updated review. Cancers. 2021 August;13(17):4287, https://doi.org/10.3390/cancers13174287
5. Khan R, Javed U, Rehman AU. Comparison between the Young and Elderly Diagnosed Patients of Carcinoma of the Breast: Breast Carcinoma in Young and Elderly Patients. Pakistan Journal of Health Sciences. 2024 October:32-7. https://doi.org/10.1634/theoncologist.2019-0982
6. Jabeen S, Zakar R, Zakar MZ, Fischer F. Experiences of family caregivers in dealing with cases of advanced breast cancer: a qualitative study of the sociocultural context in Punjab, Pakistan. BMC Public Health. 2024 August;24(1):1030. https://doi.org/10.1186/s12889-024-18404-1

7. Globocan Pakistan; https://canscreen5.iarc.fr/?page=countryfactsheet&q=PAK(assesed on 29/09/2024)
8. Iftikhar B, Zeeshan S, Shareef S, Haider Z, Naqvi DMAFA IM. Socio-Demographic, Physical and Clinical Risk Factors of Breast Cancer Patients in Pakistan. History of Medicine. 2024 October;10(2):177-89. DOI: 10.17720/2409-5834.v10.2.2024.019
9. Naz S, Siddiqui M, Memon AI, Bhatti AM, Hussain ZI. Analysis of Breast Cancer Receptors Status and Molecular Subtypes Among Female Population. Pakistan Journal of Medical & Health Sciences. 2023;17(01):656-. DOI: https://doi.org/10.53350/pjmhs2023171656
10. Sørlie T, Tibshirani R, Parker J, Hastie T, Marron JS, Nobel A, et al. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proceedings of the national academy of sciences. 2003 June;100(14):8418-23. https://doi.org/10.1073/pnas.0932692100
11. Perou CM, Sørlie T, Eisen MB, Van De Rijn M, Jeffrey SS, Rees CA, et al. Molecular portraits of human breast tumors. Nature. 2000 june;406(6797):747-52. https://doi.org/10.1073/pnas.0932692100
12. W. Ma, Y. Zhao, Y. Ji, X. Guo, X. Jian, P. Liu, et al. Breast cancer molecular subtype prediction by mammographic radiomic features, Academic radiology 2019 February Vol. 26 Issue 2 Pages 196-201, https://doi.org/10.1016/j.acra.2018.01.023
13. A. Atoui, M. Bou Zerdan, A. El Mahmoud, N. Chamseddine, L. Hamad and H. I. Assi Clinical significance of breast cancer molecular subtypes and ki67 expression as a predictive value for pathological complete response following neoadjuvant chemotherapy: experience from a tertiary care center in Lebanon International Journal of Breast Cancer 2022 Feb Vol. 2022 Issue 1 Pages 1218128. https://doi.org/10.1155/2022/1218128
14. L. Yin, J.-J. Duan, X.-W. Bian and S.-c. Yu, Triple-negative breast cancer molecular subtyping and treatment progress, Breast Cancer Research 2020 June Vol. 22 Pages 1-13 https://doi.org/10.1186/s13058-020-01296-5

15. S. Zhao, D. Ma, Y. Xiao, X. M. Li, J. L. Ma, H. Zhang, et al. Molecular subtyping of triple‐negative breast cancers by immunohistochemistry: molecular basis and clinical relevance The oncologist 2020 April Vol. 25 Issue 10 Pages e1481-e1491 doi: 10.21037/atm.2020.03.194

16. Protocol for Examining Resection Specimens from Patients with Invasive Carcinoma of the Breast; College of American Pathologists [Protocol Posting Date: June 2021]. Available from: http ://documents.cap.org/protocols/Breast.Invasive_4.5.0.0.REL_CAPCP.pdf
17. Seventh Edition © 2010 American Joint Committee on Cancer. All rights reserved. The AJCC Cancer Staging Manual is the Official Publication of the American Joint Committee on Cancer
18. Mohammed AA. The clinical behavior of different molecular subtypes of breast cancer. Cancer Treatment and Research Communications. 2021Oct;29:100469. https://doi.org/10.1016/j.ctarc.2021.100469
19. S. Paramita, E. N. Raharjo, M. Niasari, F. Azizah and N. A. Hanifah. Luminal B is the most common intrinsic molecular subtype of invasive ductal breast carcinoma patients in East Kalimantan, Indonesia. Asian Pacific journal of cancer prevention: APJCP 2019 Aug Vol. 20 Issue 8 Pages 2247doi: 10.31557/APJCP.2019.20.8.2247
20. Pandit P, Patil R, Palwe V, Gandhi S, Patil R, Nagarkar R. Prevalence of molecular subtypes of breast cancer: a single institutional experience of 2062 patients. European Journal of Breast Health. 2020 Nov;16(1):39. doi: 10.5152/ejbh.2019.4997
21. Soliman NA, Yussif SM. Ki-67 as a prognostic marker according to breast cancer molecular subtype. Cancer biology & medicine. 2016;13(4):496. doi: 10.20892/j.issn.2095-3941.2016.0066
22. Al-Thoubaity FK. Molecular classification of breast cancer: A retrospective cohort study. Annals of medicine and surgery. 2020 Dec;49:44-8. https://doi.org/10.1016/j.amsu.2019.11.021
23. Park S, Koo JS, Kim MS, Park HS, Lee JS, Lee JS, et al. Characteristics and outcomes according to molecular subtypes of breast cancer as classified by a panel of four biomarkers using immunohistochemistry. The Breast. 2012 Aug;21(1):50-7. https://doi.org/10.1016/j.breast.2011.07.008
24. Elidrissi Errahhali M, Elidrissi Errahhali M, Ouarzane M, El Harroudi T, Afqir S, Bellaoui M. First report on molecular breast cancer subtypes and their clinicopathological characteristics in Eastern Morocco: series of 2260 cases. BMC women’s health. 2017 Jan;17:1-11. https://doi.org/10.1186/s12905-016-0361-z
25. Henna N, Shafqat F, Rehman MU, Fakhr SA, Anjum S, Sameen S, et al. Clinicopathological characteristics of molecularly classified groups of invasive ductal breast carcinoma in Pakistani women. Pakistan Journal of Medicine and Dentistry. 2022;11(2):3-8 DOI: https://doi.org/10.36283/PJMD11-2/002

This is an open-access article distributed under the terms of the CreativeCommons Attribution License (CC BY) 4.0 https://creativecommons.org/licenses/by/4.0/