European Journal of Cardiovascular Medicine

Breast cancer is the most common malignancy among women worldwide, accounting for significant morbidity and mortality. According to the Global Cancer Observatory (GLOBOCAN) 2020 data, breast cancer constitutes nearly 11.7% of all cancer cases, with an estimated 2.3 million new cases annually (1). In India, it has surpassed cervical cancer as the leading cause of cancer among women, with over 178,000 new cases reported annually (2). This rise in incidence is accompanied by variations in clinical presentations, subtypes, and outcomes, highlighting the need for region-specific studies.

Pathologically, breast cancer is a heterogeneous disease classified into molecular subtypes based on immunohistochemical (IHC) expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2/neu). These subtypes—luminal A, luminal B, HER2-enriched, and triple-negative breast cancer (TNBC)—show distinct biological behavior, treatment response, and prognostic outcomes (3). Globally, luminal A is the most prevalent subtype, characterized by ER/PR positivity and low Ki-67 expression, conferring a favorable prognosis. Conversely, TNBC, marked by the absence of ER, PR, and HER2 expression, is more aggressive with limited therapeutic options and worse outcomes (4).

The prevalence of these subtypes varies significantly across regions and ethnic groups. Studies from Western populations report a predominance of luminal subtypes, while TNBC is more commonly observed in African and South Asian populations (5,6). Indian studies have shown similar patterns, with a higher incidence of TNBC compared to Western cohorts, raising questions about its underlying genetic and environmental determinants (7). Despite these insights, there is a paucity of data specifically from Eastern India, a region characterized by unique socioeconomic and genetic diversity.

Prognostic markers such as Ki-67, a marker of cellular proliferation, play a critical role in refining breast cancer classification and guiding treatment strategies. High Ki-67 expression is associated with poorer outcomes and is used to differentiate between luminal A and luminal B subtypes (8). Similarly, HER2 overexpression, present in approximately 20-30% of breast cancers, correlates with aggressive disease but also identifies patients who may benefit from targeted therapies like trastuzumab (9). Evaluating the expression of these markers in a region-specific context can enhance understanding of tumor biology and support tailored therapeutic approaches.

The integration of molecular and histopathological data has revolutionized breast cancer management. Recent advances in next-generation sequencing and multi-omics have enabled deeper insights into tumor heterogeneity and resistance mechanisms (10). However, these technologies remain largely inaccessible in resource-limited settings, including many parts of India. Consequently, immunohistochemistry continues to be the cornerstone of breast cancer diagnosis and subtyping in such settings (11).

Given the rising burden of breast cancer in India, it is imperative to conduct region-specific studies that examine the distribution of molecular subtypes and their association with prognostic markers. Eastern India, including Odisha, remains underrepresented in national cancer registries and large-scale studies. SCB Medical College, Cuttack, as a premier tertiary care center, serves a significant proportion of the region's breast cancer cases, offering a unique opportunity to address this gap.

This study aims to evaluate the distribution of molecular subtypes of breast cancer and their correlation with prognostic markers, including Ki-67 and HER2, in patients attending a tertiary care center in Eastern India. By doing so, it seeks to provide region-specific insights into breast cancer pathology and inform clinical decision-making.

Study Design and Setting:

This was a cross-sectional study conducted in the Department of Pathology, SCB Medical College and Hospital, Cuttack, a premier tertiary care center in Eastern India. The study included histopathologically confirmed breast cancer cases diagnosed between January 2022 and December 2024. Ethical approval was obtained from the institutional ethics committee before the initiation of the study. Written informed consent was collected from all participants.

Study Population Histopathological records of patients diagnosed with invasive breast carcinoma during the study period were reviewed. Cases were included if:

1. The diagnosis was confirmed by histopathology.
2. Complete clinical and histopathological data were available, including immunohistochemistry (IHC) results for ER, PR, HER2, and Ki-67.
   Exclusion criteria included cases with recurrent breast cancer, incomplete IHC data, or inadequate tissue samples for analysis.

Sample Size

The sample size was calculated using the Cochran formula for estimating proportions, assuming a prevalence of triple-negative breast cancer (TNBC) of 20% in India (7). A 95% confidence level and a 5% margin of error were used, yielding a minimum required sample size of 246 cases.

Data Collection:

Demographic and clinical data, including age, menopausal status, tumor size, and lymph node status, were extracted from patient records. Histopathological parameters, including tumor grade and type, were recorded from pathology reports. Immunohistochemical markers (ER, PR, HER2, and Ki-67) were assessed using paraffin-embedded tissue sections stained using standardized protocols (8).

Immunohistochemistry Protocol:

Formalin-fixed, paraffin-embedded tissue sections (4 μm thick) were subjected to antigen retrieval using citrate buffer (pH 6.0) in a microwave. IHC staining was performed using monoclonal antibodies against ER, PR, HER2, and Ki-67, following the manufacturer’s protocol (DAKO, Denmark). Positive and negative controls were included for each batch of staining.

• ER and PR: Nuclear staining in ≥1% of tumor cells was considered positive (9).
• HER2: Scoring was based on ASCO-CAP guidelines: 0 or 1+ (negative), 2+ (equivocal, requiring reflex testing with fluorescence in situ hybridization), and 3+ (positive) (10).
• Ki-67: The percentage of positively stained tumor cells was recorded, and a cutoff of 14% was used to differentiate between low and high proliferation (8).

Molecular Subtyping:

Breast cancer subtypes were categorized based on IHC results as follows:

• Luminal A: ER+ and/or PR+, HER2-, Ki-67 <14%.
• Luminal B: ER+ and/or PR+, HER2+ or HER2-, Ki-67 ≥14%.
• HER2-enriched: ER-, PR-, HER2+.
• Triple-negative breast cancer (TNBC): ER-, PR-, HER2-.

Statistical Analysis:

Data were analyzed using SPSS software (version 26.0, IBM, USA). Descriptive statistics (mean, standard deviation, median, interquartile range) were used for continuous variables, while categorical variables were summarized as frequencies and percentages. Chi-square or Fisher’s exact tests were used to compare molecular subtypes across demographic and clinicopathological variables.

A multivariate logistic regression model was used to identify factors associated with high Ki-67 expression, adjusting for age, menopausal status, tumor grade, and lymph node status. Results were reported as odds ratios (OR) with 95% confidence intervals (CIs).

Ethical Considerations:

Ethical clearance for the study was granted by the Institutional Ethics Committee of SCB Medical College and Hospital, Cuttack. All procedures were conducted in accordance with the Declaration of Helsinki.

Study Population:

A total of 300 patients with histopathologically confirmed invasive breast carcinoma were included in the study. The mean age of the participants was 49.6 years (SD = 11.2), with 62% (n = 186) being postmenopausal. The majority of tumors were invasive ductal carcinomas (85%, n = 255), followed by invasive lobular carcinomas (10%, n = 30) and other rare subtypes (5%, n = 15).

Immunohistochemical Profiles and Molecular Subtypes:

The distribution of breast cancer subtypes based on IHC markers is summarized in Table 1. Luminal B was the most common subtype (38.3%, n = 115), followed by Luminal A (27.7%, n = 83), HER2-enriched (17%, n = 51), and triple-negative breast cancer (TNBC) (17%, n = 51). High Ki-67 expression (≥14%) was observed in 62.7% (n = 188) of cases.

Clinicopathological Characteristics by Molecular Subtype:

Table 2 compares clinicopathological variables across molecular subtypes. Luminal B tumors were significantly associated with high tumor grade (p < 0.001) and high Ki-67 expression (p < 0.001). HER2-enriched and TNBC subtypes were more frequently observed in younger, premenopausal women (p = 0.003). TNBC cases also had the highest proportion of lymph node involvement (68.6%, n = 35).

Factors Associated with High Ki-67 Expression:

In multivariate logistic regression, high Ki-67 expression was independently associated with younger age (OR: 2.12, 95% CI: 1.29–3.49, p = 0.003), higher tumor grade (OR: 3.48, 95% CI: 2.07–5.86, p < 0.001), and positive HER2 status (OR: 2.74, 95% CI: 1.51–4.97, p = 0.001).

Table 1. Distribution of Breast Cancer Molecular Subtypes and IHC Markers

Luminal B was the most common subtype, with 38.3% prevalence. High Ki-67 expression was significantly associated with aggressive clinicopathological features such as younger age, high tumor grade, and HER2 positivity. TNBC and HER2-enriched subtypes were more prevalent in younger, premenopausal women and exhibited higher rates of lymph node involvement. These findings emphasize the clinical heterogeneity of breast cancer molecular subtypes, with significant implications for prognosis and tailored treatment strategies.

Table 2. Clinicopathological Characteristics by Molecular Subtype

This study provides a comprehensive analysis of the molecular subtypes of invasive breast cancer and their association with clinicopathological characteristics. Luminal B was identified as the most prevalent subtype, constituting 38.3% of cases. Tumors with high Ki-67 expression, a marker of cellular proliferation, were significantly associated with younger age, higher tumor grade, and HER2 positivity, reflecting more aggressive disease behavior. TNBC and HER2-enriched subtypes demonstrated worse prognostic factors, including higher rates of lymph node involvement and younger patient age.

The findings are consistent with prior research highlighting the heterogeneity of breast cancer molecular subtypes and their prognostic implications [9]. Previous studies have reported that Luminal B tumors exhibit higher proliferation rates and worse outcomes than Luminal A tumors, which aligns with our observation of higher Ki-67 expression and poorer histopathological features in Luminal B cases [10]. The higher prevalence of HER2-enriched and TNBC subtypes in premenopausal women also corroborates earlier findings, emphasizing the need for age-specific screening and treatment strategies [11].

Our study strengthens the evidence for Ki-67 as a critical marker for stratifying breast cancer aggressiveness. High Ki-67 expression has been consistently linked to poor outcomes and treatment resistance in previous studies, underlining its utility in tailoring therapeutic approaches [12].

These findings have significant clinical implications for the management of breast cancer in Indian populations. The high prevalence of Luminal B and its association with high-grade tumors underscore the need for aggressive treatment, including adjuvant chemotherapy and targeted HER2 therapies where applicable. Similarly, the high proportion of TNBC in younger patients highlights the urgency of optimizing treatment protocols for this aggressive subtype, which lacks targeted therapies.

Given the observed distribution of molecular subtypes, incorporating Ki-67 testing as part of routine diagnostic workups could guide oncologists in making informed treatment decisions. Furthermore, the association of specific subtypes with worse prognostic factors suggests the need for early detection programs tailored to younger, premenopausal women.

This study is limited by its cross-sectional design, which precludes the assessment of long-term outcomes. Additionally, the study was conducted in a single tertiary care center, which may limit generalizability to other settings. Future research should focus on multicenter studies with larger sample sizes to validate these findings. Prospective studies examining the impact of molecular subtypes on survival outcomes in Indian populations would provide valuable insights.

This study highlights the clinicopathological diversity of breast cancer molecular subtypes and their implications for personalized treatment. High Ki-67 expression emerges as a significant marker of aggressiveness, reinforcing its role in breast cancer management. The findings underscore the necessity of integrating molecular profiling into routine practice to enhance patient outcomes.

Authors' Declarations

Ethics Approval and Consent to Participate: This study was approved by the Institutional Ethics Committee of SCB Medical College, Cuttack, and written informed consent was obtained from all participants.

Competing Interests: The authors declare no competing interests.

Funding: No external funding was received for this study.

Acknowledgments: The authors thank the Pathology Department of SCB Medical College for their support in data collection and analysis.

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