Abstract

Background: Ki-67 is a key proliferative marker in breast cancer, often associated with tumor aggressiveness and grade. Despite its established role, the correlation between Ki-67% and histological grade remains inconsistent across studies.

Objective: This study aims to explore the Ki-67% expression and its association with tumor grades in breast cancer patients.

Methods: A cross-sectional study was conducted at the Department of Medical Oncology, Jinnah Postgraduate Medical Centre, Karachi, over a six-month period. A sample of 114 breast cancer patients was included, divided into groups based on Ki-67% expression (<15% and >15%). Histological grading was assessed using the Scarff-Bloom-Richardson grading system, while demographic and clinical data, including age, marital status, and BMI, were collected and analyzed using SPSS version 24.0.

Results: High Ki-67 expression (>15%) was observed predominantly in Grade III tumors, with 33 cases (91.7%) compared to Grade II with 52 cases (66.7%) and Grade I with 3 cases (50%) (p=0.008). No significant associations were found between Ki-67% and other variables, including tumor size, axillary lymph node involvement, and TNM stage.

Conclusion: This study demonstrates a strong association between higher Ki-67% and advanced histological grades in breast cancer, suggesting that Ki-67 may serve as a valuable prognostic indicator. However, further research is needed to clarify its role in predicting clinical outcomes across diverse patient populations.

Keywords: Ki-67, breast cancer, tumor grade, histological grade, prognosis, proliferation marker.

INTRODUCTION

Breast cancer is the most frequently diagnosed cancer in women, representing about one-third of all female malignancies. It possesses a high potential for metastasis, which contributes significantly to its mortality rate. Early detection of this disease leads to improved outcomes and increased survival rate [1]. In Asian countries, breast cancer is the most prevalent malignancy, with the highest incidence occurring between the ages of

50 and 64 [2]. Women living in urban areas have a higher risk of developing breast cancer compared to those in rural settings [3]. The clinical features and behavior of the disease vary widely among individuals and across racial groups, largely due to genetic diversity [4]. Several clinicopathological parameters assist in assessing prognosis and guiding treatment decisions in breast cancer patients. These include age, tumor size, lymph node involvement, histological type and grade, lymphovascular invasion, hormone receptor status, HER2 expression, and the Ki-67 index [3-5]. Molecular techniques are employed to classify breast cancer into molecular subtypes, which aid in selecting appropriate, targeted, and personalized therapies [6].

Ki-67 is a non-histone nuclear protein discovered in the early 1980s, known for its instability and strong association with cell proliferation. As a crucial regulator

of the cell cycle, it is the most commonly used marker to evaluate proliferative activity [7]. Research has demonstrated that Ki-67 is present in all active phases of the cell cycle but is absent during the quiescent G0 phase. Additionally, it serves as a valuable biomarker for determining the growth fraction of a cell population [8, 9]. The biology of breast cancer with high ki67% and grade looks similar but whether there is a definite and clear correlation between high grade and high ki67% and vice versa is not yet clear [10, 11].

Although elevated Ki-67 levels have been linked to poorer prognosis and reduced survival in early breast cancer patients [12], this marker is not yet routinely used in clinical practice. The College of American Pathologists (CAP) advises against the regular use of Ki-67 for prognostic assessment in breast cancer due to concerns over data reliability and insufficient quality control measures [13]. Nonetheless, the St. Gallen Consensus Conferences in 2011 and 2013 endorsed the use of Ki-67 to evaluate cellular proliferation and to help distinguish between luminal A and B tumor subtypes [14, 15]. A study by Nigam JS et al. [16] reported the positive vs. negative expression rate of Ki-67 in 39.13% vs. 10.87% among patients for grade 2 breast cancer, respectively. In this study [16], the investigator evaluated the expression of Ki-67 in various histologic grades of breast cancer. In patients with grade 2 tumors, 39.13% showed positive Ki-67 expression, while 10.87% had negative Ki-67 expression. This suggests that a substantial proportion of grade 2 tumors exhibit

proliferative activity, as indicated by Ki-67 positivity. These findings highlight a potential association between tumor grade and proliferative index. While there is still no agreement on the ideal cutoff value of Ki-67 for guiding chemotherapy decisions, multiple studies have shown that a high Ki-67 index is linked to increased relapse rates and poorer breast cancer survival outcomes [17].

We anticipated that a high Ki-67% would correspond to a higher grade of breast cancer, and vice versa; however, some studies did not demonstrate a clear association between these parameters. Therefore, we conducted this study to explore the Ki-67% expression and its association with tumor grades in breast cancer patients.

MATERIALS AND METHODS

This cross-sectional study was carried out in the Department of Medical Oncology at Jinnah Postgraduate Medical Centre (JPMC) Hospital in Karachi, over six months from 1st July 2024 to 31st December 2024 following approval. A sample size of 114 patients was calculated using the Open-Epi calculator, based on a Ki- 67 <15% frequency of 39.2% in breast cancer patients, with a 95% confidence interval and a 9% margin of error. Non-probability consecutive sampling was employed to select participants.

The inclusion criteria for the study were female patients aged 18 to 75 years with histologically confirmed breast cancer, where both tumor grade and Ki-67% were assessed. All stages of breast cancer were included. Exclusion criteria comprised individuals unwilling to participate, patients under 18 years of age, and male patients.

After obtaining Institutional Review Board (IRB) approval, eligible women with breast cancer were enrolled in the outpatient department. Study details were thoroughly explained to participants, and written informed consent was obtained. Baseline demographic and clinical data, including age, residence, education, ethnicity, marital status, height (measured using a wall-mounted scale in cm), weight (measured with a digital scale in light clothing), and BMI (calculated as weight in kg divided by height in m²), were recorded in a predesigned proforma. Each participant underwent breast cancer screening, immunohistochemical staining, and histological grading based on their histopathology report.

For immunohistochemical analysis, breast tissue samples were fixed in 10% formaldehyde for 24 hours, dehydrated, cleared, embedded in paraffin, and sectioned into 5 μm slices. The slices were baked at 65°C, dewaxed with xylene, hydrated with graded ethanol, treated with 3% hydrogen peroxide, and incubated at 37°C for 10 minutes to inactivate endogenous peroxidase. After antigen retrieval through microwave heating and blocking with normal goat serum, the slices were incubated at 4°C overnight with a primary Ki-67 antibody. The following day, biotin-labeled secondary antibody incubation was

performed at room temperature for 30 minutes, followed by development with diaminobenzidine, counterstaining with hematoxylin, differentiation with hydrochloric acid ethanol, dehydration with graded ethanol, clearing with xylene, and mounting with neutral gum for microscopic examination. Phosphate-buffered saline was used in place of the primary antibody as a negative control. The Ki-67 marker index was considered positive if 15% or more of the tumor cells exhibited nuclear staining, categorizing participants into Group A (Ki-67% positive) and Group B (Ki-67% negative). Histological grading of breast cancer was performed according to the Scarff- Bloom-Richardson grading system.

Data were entered and analyzed using SPSS version

24.0. Continuous variables were summarized as mean

± SD or median (IQR), while categorical variables, were presented as frequencies and percentages. The association between breast cancer grades and Ki- 67% was assessed using the Chi-square or Fisher’s Exact test. A p-value ≤ 0.05 was considered statistically significant.

RESULTS

A total of 114 breast cancer patients were included. The mean age at the time of diagnosis was 48±12.5 years. The mean age at menarche was 12.7±1.6 years. The

Table 1: Sociodemographic variables of participants.

age distribution of participants was as follows: <30 years, 7 (6.1%); 30-50 years, 71 (62.3%); 51-70 years,

34 (29.8%); and >70 years, 2 (1.8%) (Table 1).

The clinical characteristics of patients were as follows: cancer was located on the left side in 39 (34.2%), right side in 69 (60.5%), and bilateral in 6 (5.3%) patients.

Histologically,   95    (83.3%)    had    infiltrating   ductal

carcinoma, 17 (14.9%) had lobular carcinoma, and 2

(1.8%) had other types. Grading revealed that 6 (5.3%)

had grade I, 76 (66.7%) had grade II, and 32 (28.1%)

Table 2: Clinical characteristics of patients.

Table 3: Association between Ki-67% and clinical variables in breast cancer patients.

had grade III cancer. The Ki67 index was <15% in 13 (11.4 %). Tumor size was <2 cm in 6 (5%). Multifocal/ multicentric tumors were present in 26 (22.8%) patients. Axillary lymph node involvement was observed in 85 (74.6%) patients (Table 2).

The distribution of histological grade, tumor type, and nuclear stage showed significant differences between patients with Ki-67 <15% and those with Ki-67 ≥15%. However, tumor size, presence of multifocal/multicentric lesions, axillary lymph node involvement, TNM stage, and HER2 status were not significantly different between the two Ki-67 categories (Table 3).

DISCUSSION

In our study, Ki-67 was categorized into two groups: Ki67 <15% and Ki67 >15%. Mostly grade II and grade III cancers are associated with high Ki67 which is more than 15%. Our findings are consistent with Trihia et al., who reported Ki-67 >20% in 85% of Grade III tumors, reinforcing its role as a proliferation marker. Conversely, Kanyılmaz et al. found Ki-67 expression above 14% in

only 47% of high-grade tumors, suggesting variability due to population differences or Ki-67 assessment methods.

Our study confirms a significant association between Ki-67% and the histological grade of breast cancer, suggesting that higher Ki-67 levels are predominantly observed in higher-grade tumors. This finding aligns with prior studies, such as Trihia et al., where high Ki- 67 expression was also correlated with increased tumor grade, specifically in Grades II and III [18]. Similarly, Kanyılmaz et al. identified a clear link between high Ki- 67 and more aggressive tumor grades, reinforcing the role of Ki-67 as an indicator of tumor proliferation and aggressiveness [5].

The relationship between Ki-67 and cancer grade in our findings also mirrors observations in other international studies, which have reported a trend of higher Ki- 67 expression in tumors of higher histological grade. For instance, Brown et al. demonstrated that in early hormone receptor-positive breast cancer, patients with Grade-3 tumors had significantly higher Ki-67 levels (over 20%), supporting its utility as a prognostic marker for aggressive disease [19]. Moreover, in a retrospective cohort by Stathopoulos et al., patients classified under the basal-like subtype, typically associated with higher grade, frequently exhibited elevated Ki-67 levels [20].

The significance of Ki-67 as a proliferation marker is further underscored in studies focusing on molecular subtypes of breast cancer. Yip et al. and Pai et al. reported that in luminal subtypes, particularly in luminal B, Ki-67 was frequently elevated, contrasting with lower levels in luminal A cases, which tend to have a better prognosis [21, 22]. This aligns with our study, where high-grade tumors with elevated Ki-67 likely represent more aggressive molecular subtypes such as luminal B or HER2-enriched types.

Additionally, Stathopoulos et al. highlighted that the triple-negative subtype also presented with consistently high Ki-67, further linking it with poor prognosis [20].

Beyond histological grading, our study found no statistically significant association between Ki-67 and histological type, tumor size, axillary lymph node involvement, or TNM staging. This observation aligns with certain studies but diverges from others, such as the work by Kanyılmaz et al., which reported a correlation between tumor size and Ki-67 levels [5]. These discrepancies may stem from population heterogeneity, sample size variations, or differences in assessment methods for Ki-67. The diversity in findings reinforces the need for standardized assessment protocols in Ki-67 testing, as advocated by Brown et al. [19].

Other studies have also illustrated the variability in Ki-67 association with TNM stage and nodal involvement. For example, Fasching et al. demonstrated that while Ki-67

could predict five-year disease-free survival in specific subgroups, it had limited prognostic value in isolation [23]. This underscores the importance of combining Ki-67 with other biomarkers to enhance prognostic accuracy, particularly for cases with intermediate risk.

Finally, HER2 status did not exhibit a significant association with Ki-67 in our study, a finding consistent with Yip et al., who noted that HER2 overexpression does not necessarily correspond to higher Ki-67 levels [21]. However, this is somewhat contradictory to studies like Trihia et al., which showed a positive relationship between HER2 positivity and elevated Ki-67, emphasizing that the influence of HER2 on proliferation may vary based on additional tumor characteristics [18].

CONCLUSION

In conclusion, our study highlights the strong association between Ki-67 and histological grade in breast cancer, supporting its role as a marker of tumor proliferation and aggressiveness. While Ki-67’s relationship with other clinical parameters remains less definitive, this study reinforces its utility as a complementary biomarker in evaluating breast cancer prognosis, particularly when used alongside other established indicators.

ETHICS APPROVAL

The ethical exemption was obtained from the Institutional Review Board of Jinnah Hospital, JPMC Karachi (REF Letter no. F.2-81/2024-GENL/29/jpmc). All procedures performed in studies involving human participants followed the ethical standards of the institutional and/ or national research committee.

CONSENT FOR PUBLICATION

Written informed consent was taken from the participants.

AVAILABILITY OF DATA

The data set may be acquired from the corresponding author upon a reasonable request.

FUNDING

None.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ACKNOWLEDGEMENTS

Declared none.

AUTHORS’ CONTRIBUTION

SNK: study concept and designing, critical reviewing. GH: result interpretation, manuscript drafting, critical review and revision of initial draft, KF: result analysis and interpretation, manuscript drafting, SZ: critical review and revision of initial draft, AS: critical review and revision of initial draft, AR: critical review and revision of initial draft, MZ: data collection, MJ: data collection.

REFERENCES