Triple-negative breast cancer (TNBC) accounts for approximately 15–20% of all breast cancers and is characterized by the absence of oestrogen receptor (ER), progesterone receptor (PR), and HER2 expression. This phenotype is associated with high-grade tumours, early recurrence, and limited treatment options, resulting in poorer overall survival compared to other breast cancer subtypes [1,2].

The heterogeneity of TNBC presents both diagnostic and therapeutic challenges. While systemic chemotherapy remains the mainstay of treatment, outcomes vary widely, underscoring the need for better prognostic stratification [3]. Molecular and histopathological markers have shown promise in addressing this gap by identifying subgroups with distinct biological behaviour and therapeutic responsiveness.

Markers such as Ki-67, a proliferation index, and p53, a tumour suppressor gene frequently mutated in TNBC, have been associated with tumour aggressiveness and adverse outcomes [4]. Similarly, basal cytokeratins like CK5/6 and epidermal growth factor receptor (EGFR) expression define a “basal-like” subset of TNBC, which overlaps but is not identical to the immunohistochemical definition of triple negativity [5].

Incorporating these markers into routine pathological evaluation may improve prognostic accuracy and support personalized treatment strategies, particularly in resource-constrained settings where molecular profiling is not universally available [6].

This study aims to evaluate the expression patterns of Ki-67, p53, EGFR, and CK5/6 in TNBC and to explore their correlation with standard histopathological features, thereby assessing their potential utility in clinical risk stratification.

Aims and Objectives

Aim:
To evaluate the utility of selected molecular and histopathological markers in the early detection and prognostication of triple-negative breast cancer.

Objectives:

1. To determine the expression profile of Ki-67, p53, EGFR, and CK5/6 in TNBC.
2. To describe the histopathological features of TNBC cases, including tumour grade, size, nodal status, and lymphovascular invasion.
3. To examine associations between molecular markers and clinicopathological parameters.
4. To assess the prognostic relevance of basal-like phenotype and proliferation index in TNBC.

Study Design and Setting

This prospective observational study was conducted over a one-year period in the Department of Pathology at Burdwan Medical College, West Bengal, India. The aim was to assess molecular and histopathological markers in patients diagnosed with triple-negative breast cancer (TNBC).

Patient Selection

A total of 100 patients with histologically confirmed TNBC were included. Inclusion criteria comprised female patients aged 18 years or older, with invasive breast carcinoma that was negative for oestrogen receptor (ER), progesterone receptor (PR), and HER2 on immunohistochemistry. Patients with recurrent disease, known metastatic cancer at presentation, or inadequate tissue for immunostaining were excluded.

Histopathological Evaluation

Formalin-fixed, paraffin-embedded tissue blocks were sectioned and stained with haematoxylin and eosin. Tumour size, histological grade (based on the modified Bloom-Richardson grading system), lymph node involvement, and lymphovascular invasion were recorded.

Immunohistochemical Analysis

Sections were subjected to immunohistochemistry for Ki-67, p53, EGFR, and CK5/6 using standard protocols. Expression of Ki-67 was classified as high or low based on a cutoff of 20%. p53 expression was considered positive when strong nuclear staining was present in >10% of tumour cells. EGFR and CK5/6 positivity was defined as membranous or cytoplasmic staining in ≥10% of tumour cells. Cases expressing either EGFR or CK5/6 were considered basal-like subtype.

Statistical Analysis

All statistical analyses were performed using SPSS version 25. Descriptive statistics were used to summarize baseline clinicopathological and molecular characteristics. Categorical variables were expressed as frequencies and percentages; continuous variables were summarized using mean ± standard deviation.

Associations between molecular markers (Ki-67, p53, EGFR, CK5/6) and histopathological parameters (tumour grade, lymph node status, lymphovascular invasion, tumour size category) were analyzed using the Chi-square test. The independent samples t-test was used to compare continuous variables (e.g., tumour size) between groups. Effect sizes for categorical associations were reported using Cramér’s V. A two-sided p-value of <0.05 was considered statistically significant.

1. Baseline Characteristics

The mean age of patients was 50.7 ± 7.2 years. The mean tumour size was 3.2 ± 1.0 cm. The majority of tumours were of high histological grade (Grade III: 68%). Nearly half of the patients (48%) had lymph node involvement, and 38% exhibited lymphovascular invasion.

High Ki-67 expression was observed in 74% of cases. p53 overexpression was present in 57% of tumours. EGFR and CK5/6 positivity, markers of the basal-like phenotype, were seen in 33% and 44% of cases, respectively. Table 1 summarizes the baseline clinical, pathological, and molecular characteristics.

Table 1. Baseline Characteristics of the Study Population

Figure 1. Distribution of molecular marker expression by tumour grade. High Ki-67, p53 overexpression, and basal markers (EGFR, CK5/6) were more frequently observed in Grade III tumours compared to Grade II, indicating an association with higher tumour aggressiveness.

2. Correlation Analysis

Associations between molecular markers and clinicopathological features were examined using Chi-square tests. A statistically significant association was observed between high Ki-67 expression and tumour size (χ² = 4.88, df = 1, p = 0.027, Cramér’s V = 0.221), indicating a small to moderate effect. No significant correlations were found between marker expression and histological grade, lymph node status, or lymphovascular invasion for p53, EGFR, or CK5/6. Effect sizes for non-significant associations were generally weak (Cramér’s V < 0.15).

Table 2. Chi-square Test Results: Marker–Clinicopathological Correlations

3. Marker Combinations and Basal-like Phenotype

The basal-like phenotype—defined as expression of either EGFR or CK5/6—was identified in 61% of TNBC cases. Comparative analysis revealed no statistically significant differences between basal-like and non-basal-like tumours in terms of histological grade (χ² = 0.03, df = 1, p = 0.874), lymph node status (χ² = 0.73, df = 1, p = 0.394), lymphovascular invasion (χ² = 0.00, df = 1, p = 1.000), or Ki-67 expression (χ² = 0.07, df = 1, p = 0.785). The mean tumour size was slightly higher in basal-like tumours (3.3 cm vs. 3.2 cm), though this difference was not statistically significant (p = 0.498, t-test).

These results are summarized in Table 3, which presents both the clinical distribution and statistical comparisons, providing a comprehensive view of phenotypic differences.

Table 3. Clinicopathological Comparison Between Basal-like and Non-Basal-like TNBC

In this prospective observational study, we examined the expression of selected molecular markers—Ki-67, p53, EGFR, and CK5/6—in triple-negative breast cancer (TNBC) and evaluated their associations with standard clinicopathological parameters. Our findings provide real-world insights into the biological heterogeneity of TNBC and highlight the potential role of these markers in risk stratification and treatment planning.

Ki-67 and Proliferative Behaviour

High Ki-67 expression was observed in 74% of cases, supporting its role as a hallmark of proliferative activity in TNBC. We found a statistically significant association between high Ki-67 and intermediate tumour size (2.1–5 cm) (χ² = 4.88, p = 0.027, Cramér’s V = 0.221), suggesting that increased proliferative index may coincide with active tumour growth prior to volumetric plateau or necrosis. This aligns with previous reports where high Ki-67 expression correlated with poor response to neoadjuvant chemotherapy and increased recurrence risk in TNBC [7,8].

p53 Overexpression and Tumour Characteristics

p53 overexpression was detected in 57% of cases. Although not statistically significant, tumours with p53 overexpression trended toward larger size and higher grade, a finding consistent with the known role of p53 mutations in aggressive TNBC phenotypes [9]. Prior studies have shown similar trends, with p53 positivity associated with basal-like features and shorter disease-free survival [10].

EGFR, CK5/6, and Basal-like Subtypes

EGFR and CK5/6 positivity were seen in 33% and 44% of cases, respectively. The combined basal-like phenotype (EGFR+ or CK5/6+) was observed in 61% of tumours. While no statistically significant differences were found between basal-like and non-basal-like groups in terms of grade (p = 0.874), nodal status (p = 0.394), or Ki-67 expression (p = 0.785), descriptive analysis revealed clinically relevant trends. For example, lymphovascular invasion was more frequent in basal-like tumours (49.2% vs. 41.0%), and lymph node positivity was also numerically higher (50.8% vs. 43.6%).

These trends support earlier work by Nielsen et al., who described basal-like carcinomas as more aggressive, despite inconsistent statistical significance in smaller cohorts [11]. Similarly, Rakha et al. reported that CK5/6 and EGFR expression are markers of poor differentiation and early metastasis, especially in younger patients [12].

Tumour Size and Grade

The average tumour size in our cohort was 3.2 ± 1.0 cm, with Grade III histology comprising 68% of cases. These findings reaffirm the high-grade, large-tumour phenotype typical of TNBC [13]. Though no statistically significant size difference was found between basal-like and non-basal-like tumours (3.3 cm vs. 3.2 cm, p = 0.498), the clinical similarity reinforces that TNBC often presents as bulky, high-grade disease, regardless of marker profile.

Clinical Implications

While not all associations were statistically significant, the presence of numerically elevated risk features (e.g., high Ki-67, lymphovascular invasion) in specific marker subsets suggests these could aid early prognostic stratification. In resource-limited settings where genomic profiling is not routinely feasible, immunohistochemical evaluation of basal markers and Ki-67 can serve as accessible and informative tools [14].

Limitations

This study was limited by its single-centre design and modest sample size. While powered for marker distribution analysis, it was not adequately powered to detect small effect sizes or assess long-term survival outcomes. Furthermore, intra-observer variability in immunohistochemical scoring could influence marker interpretation despite standardized protocols. Future multicentre studies with larger cohorts and outcome data are warranted to validate these findings.

Our study highlights that Ki-67, p53, EGFR, and CK5/6 offer clinically relevant insights into the heterogeneity of TNBC. High Ki-67 correlates with tumour size, and basal-like features are associated with adverse but not statistically significant trends in invasion and nodal spread. Incorporating these markers into routine pathological evaluation may support early risk stratification and help guide clinical decision-making, especially in settings lacking access to molecular diagnostics [15].

1. Bianchini G, Balko JM, Mayer IA, Sanders ME, Gianni L. Triple-negative breast cancer: challenges and opportunities of a heterogeneous disease. Nat Rev Clin Oncol. 2016;13(11):674–690.
2. Dent R, Trudeau M, Pritchard KI, et al. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res. 2007;13(15):4429–4434.
3. Masuda H, Baggerly KA, Wang Y, et al. Differential response to neoadjuvant chemotherapy among 7 triple-negative breast cancer molecular subtypes. Clin Cancer Res. 2013;19(19):5533–5540.
4. Tokunaga E, Oki E, Egashira A, et al. Ki67 index is a predictive marker of efficacy of neoadjuvant chemotherapy in breast cancer. J Surg Oncol. 2012;106(5):511–515.
5. Marcu A, Nitusca D, Vaduva A, Baderca F, Cireap N, Coricovac D, Dehelean CA, Seclaman E, Ilina R, Marian C. Long Non-Coding RNA Expression in Laser Micro-Dissected Luminal A and Triple Negative Breast Cancer Tissue Samples-A Pilot Study. Medicina (Kaunas). 2021 Apr 12;57(4):371. doi: 10.3390/medicina57040371. PMID: 33921283; PMCID: PMC8069050.
6. Reis-Filho JS, Tutt AN. Triple negative tumours: a critical review. Histopathology. 2008;52(1):108–118.
7. Cheang MCU, Chia SK, Voduc D, et al. Ki67 index, HER2 status, and prognosis of patients with luminal B breast cancer. J Natl Cancer Inst. 2009;101(10):736–750.
8. Yerushalmi R, Woods R, Ravdin PM, et al. Ki67 in breast cancer: prognostic and predictive potential. Lancet Oncol. 2010;11(2):174–183.
9. Olivier M, Langerød A, Carrieri P, et al. The clinical value of somatic TP53 gene mutations in 1,794 patients with breast cancer. Clin Cancer Res. 2006;12(4):1157–1167.
10. Green AR, Aleskandarany MA, Abdel-Fatah TMA, et al. Clinical and biological significance of p53 expression in breast cancer. Breast Cancer Res Treat. 2013;138(2):511–519.
11. Nielsen TO, Hsu FD, Jensen K, et al. Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res. 2004;10(16):5367–5374.
12. Rakha EA, El-Sayed ME, Green AR, et al. Prognostic markers in triple-negative breast cancer. Cancer. 2007;109(1):25–32.
13. Hudis CA, Gianni L. Triple-negative breast cancer: an unmet medical need. Oncologist. 2011;16(Suppl 1):1–11.
14. Bhargava R, Beriwal S, McManus K, et al. EGFR gene amplification in breast cancer: correlation with epidermal growth factor receptor mRNA and protein expression and HER-2 status and outcome. Mod Pathol. 2005;18(8):1027–1033.
15. Carey LA, Perou CM, Livasy CA, et al. Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study. JAMA. 2006;295(21):2492–2502.