Fall in Ki67 Index After Short-Term Preoperative Letrozole: a Gateway to Assess the Response in Hormone-Positive Early Breast Cancers

Abstract

Endocrine treatment for breast cancer acts largely by inhibiting tumor cell proliferation. The study aimed to explore the fall in proliferative marker Ki67 in patients receiving preoperative endocrine therapy and the factors associated with it. A prospective series of hormone receptor-positive postmenopausal women with early N0/N1 breast cancer were enrolled. Patients were requested to take letrozole OD while they await surgery. The fall in Ki67 after the endocrine therapy was defined as the percentage of the difference between the pre-and postoperative Ki67 value with the preoperative Ki67. Sixty cases matched the criteria of which 41 (68.3%) of women showed a good response to preoperative letrozole (fall in Ki67 > 50%; p-value < 0.001). The average mean fall in Ki67 was 57.083 ± 37.97. Postoperative Ki67 after the therapy was less than 10% in 39 (65%) patients. Ten patients (16.6%) had a low Ki67 index at baseline, which continued to remain low after preoperative endocrine therapy. The duration of the therapy did not affect the percentage of Ki67 fall in our study. Short-term changes in the Ki67 index in the neoadjuvant settings may predict outcomes during adjuvant use of the same treatment. Proliferation index on residual tumor holds prognostic importance, and our results reflect that greater attention should be given to the percentage of reduction of Ki67, rather than focusing purely on a fixed value. This could help predict patients who respond well to endocrine therapy, while those who respond poorly may require further adjuvant treatment.

Introduction

Breast cancer (BC) is the most common cancer among women in the world, accounting for a significant disease burden. According to WHO, by the end of 2020, 7.8 million women were living with breast cancer, whereas 685,000 women succumbed to the disease [1]. The widespread prevalence of this disease highlights the importance of early detection and adequate treatment. Hormone receptor-positive (HR +) early BC patients make up a subset of patients with good prognosis and survival. Early BC treatment usually includes primary surgery, followed by adjuvant treatments with chemotherapy, endocrine therapy (ET), radiotherapy, and antihuman epidermal growth factor receptor 2 (HER2)-targeted therapies in appropriate patients [2, 3]. ET is the mainstay of systemic therapy for HR + BC. Endocrine treatment for BC appears to act largely by inhibiting tumor cell proliferation [4]. Preclinical studies have shown that cancer surgery has a growth-stimulating effect that could promote metastasis in HR + BC, and preoperative endocrine therapy (PoET) could inhibit the stimulatory effect [5, 6].

The proliferation of BC can be predicted by using biomarkers. Among predictive biomarkers, the Ki67 labeling index is a reliable measure of tumor proliferation fraction that has been shown to have independent prognostic significance in clinical trials of adjuvant ET [7, 8]. Ki67 protein is found in the cell cycle’s G1, S, G2, and M phases. During the G1 and early S phase of the cell cycle, Ki67 levels are low and gradually reach a maximum during mitosis [9]. Therefore, Ki67 protein expression acts as a useful predictive marker of cell proliferation [10]. High expression of Ki67 protein has been associated with an increased risk of BC recurrence and death [11].

Surgery remains the mainstay in treatment for hormone-positive early breast cancer. However, from the time of diagnosis, there is often a short interval before definitive treatment is initiated, which could range from a few weeks to a few months. This period provided us with a window of opportunity, during which we attempted to assess the response of patients to short-term PoET. This also enabled us to objectively assess the in situ response of the tumor to ET, thereby identifying the subset of patients who may respond poorly to ET. In India, no studies have been conducted to evaluate the effect of PoET in HR + HER2 − ve BC. Therefore, this study aimed to determine the response in patients receiving PoET by studying the fall in the Ki67 index.

Materials and Methods

This prospectively ascertained pre-post interventional study was a single site study aimed to determine the fall in Ki67 in HR + HER2 − ve BC patients receiving short-term preoperative letrozole therapy. Postmenopausal women diagnosed with strong estrogen receptor-positive (ER +  > 50%) HER2 − ve invasive BC by core needle biopsy were eligible for the study. All patients were judged by their primary physicians as having a good performance status (0–2). For the analysis of proliferative markers, the eligible participants were required to have their paraffin-fixed core needle tissue block available. Exclusion criteria included pregnant or lactating women, patients who received chemotherapy or ET for breast or ovarian cancer in the last 5 years, and those who were not willing to participate in the study. The study was approved by the Amrita Institute of Medical Science institutional ethics committee.

Women were approached about the trial after their primary diagnosis of the disease was confirmed through tissue diagnosis by core biopsy and their immunohistochemistry (IHC) profile was established. Eligible women who fulfilled the study inclusion criteria were enrolled in the study. After collecting the baseline data, enrolled patients were requested to take letrozole 2.5 mg tablet once daily during the preoperative window period from the primary diagnosis until the day of surgery. This roughly corresponded to 2–4 weeks.

Ki67 proliferative index in the tumor was measured in the pretreatment biopsy (Baseline Ki67–Ki67_B) and on the postsurgical specimen (post surgery Ki67–Ki67_S). IHC was performed in deparaffinized tissue, using a predefined rabbit monoclonal antibody against human Ki67 (Clone MIB-1, Dako). The percentage of the invasive cancer cells expressing Ki67 before and after preoperative letrozole therapy was measured in hotspots by a single expert pathologist specialized in BC pathology. Nuclear staining of any intensity was considered positive. The fall in Ki67 after the preoperative letrozole was defined as below:

$$\mathrm{Fall}\mathrm{in}\mathrm{Ki}67=\frac{\mathrm{Preoperative}\mathrm{Ki}67\mathrm{index}-\mathrm{Post}\mathrm{operative}\mathrm{Ki}67\mathrm{index}}{\mathrm{Preoperative}\mathrm{Ki}6\mathrm{index}}\times 100$$

The prespecified Ki67 10% cutoff to distinguish high and low Ki67 was chosen for consistency with previous clinical trials (ALTERNATE trial, POETIC trial). According to the change in Ki67 value observed in patients receiving short-term PoET, they were categorized as low-low (Ki67_B and Ki67_s < 10%), high-low (Ki67_B > 10%, and Ki67_s < 10%), and high-high (Ki67_B and Ki67_S > 10%) [12, 13].

Statistical Analysis

Statistical analysis was done using IBM SPSS 20 (SPSS Inc., Chicago, USA). Mean ± SD of all the continuous variables were calculated, and categorical variables were presented as frequency and percentage. To test the statistical significance of the association of categorical variables with fall in Ki67, chi-square was applied. To test the statistical significance of the change in the median of the findings of pre and post Ki67, Wilcoxon-signed rank was applied. A p-value < 0.05 was considered as statistically significant.

Result

Between October 2021 and March 2022, 65 newly diagnosed breast cancer women who fulfilled the study criteria were identified, and 60 were eligible for the final analysis. The mean age of the study population was 64.44 ± 7.79. Analyzing the tumor characteristics revealed that pT1 tumors represented 26.7%, and pT2 represented 66.7% of the study cohort. A total of 80% of the patients had a grade 2 tumor, whereas only 4 (6.6%) patients had a grade 3 tumor and 8 (13.3%) patients had a grade 1 tumor. Our study cohort included only patients with strong ER-positive tumors (ER > 50%), with nearly 92% (n = 55) of the patients having ER positivity > 80%. The women were representative of the population with operable early-stage hormone-positive HER2 − ve BC in postmenopausal women. Twenty-one (35%) patients received letrozole for less than 2 weeks, while 27 (45%) received letrozole for 2 to 4 weeks and 12 (20%) received it for more than 4 weeks. Characteristics of the study population are presented in Table 1.

Table 1.

Patient characteristics

Characteristics	Number (%)
Age (in years)
< 60   60–70    > 70	20 (33.3) 27 (45) 13 (21.7)
Breastfeeding history
≤ 1 year each   1–2 years each    ≥ 3 years each	10 (16.7) 40 (66.7) 10 (16.7)
Family history
Breast cancer in a first-degree relative   Breast cancer in second-degree relative   Ovarian/endometrial cancer   Some other cancer	5 (8.3) 1 (1.7) 1 (1.7) 11 (18.3)
History of hysterectomy + oophorectomy	15 (25)
Tumor size
T1   T2   T3	16 (26.7) 40 (66.7) 4 (6.7)
Nodal status
No   N1	32 (53.3) 28 (46.7)
Tumor grade
G1   G2   G3	8 (13.3) 48 (80) 4 (6.7)
ER positive
50–80   80–90    > 90	5 (8.3) 44 (73.3) 11 (18.3)
PR positive
< 50   50–90    > 90	25 (41.7) 33 (55) 2 (3.3)
Duration of HT
< 14 days   14–28 days    > 28 days	21 (35) 27 (45) 12 (20)

ER estrogen receptor, HT hormone therapy, PR progesterone receptor

Tissue sections of both the biopsy sample and surgical specimen to estimate the Ki67 index was available for all 60 enrolled subjects. The large majority of patients (85%) showed at least 10% of fall in Ki67 with short-term preoperative letrozole. In addition, 9 (15%) patients showed a fall of > 90%, and 8 (13.3%) patients showed a fall of 80–90%. Only six (10%) women had an increase/no change in Ki67 after the short-term preoperative endocrine period. Among the study cohort, 10 (16.6%) of them presented with low initial Ki67 value (Ki67_B). Following preoperative endocrine therapy, 8 of these patients had a reduction in the postoperative Ki67 value, whereas 2 of them remained unchanged. Fifty (83.3%) patients had tumors that had a high Ki67_B. After subjecting them to preoperative letrozole therapy, 42 (84%) patients with high Ki67_B showed a reduction by 10% or more in their Ki67_S value. Moreover, 29 (48.3%) patients with high Ki67_B were noted to have a Ki67s value < 10% after PoET. However, 21 (42%) of them continued to have a high Ki67_S value after therapy (Fig. 1, Fig. 2). The number of patients in the high-high, high-low, and low-low category was 10 (16.6%), 29 (48.3%), and 21 (35%), respectively. The overall mean fall in Ki67 index after PoET in our study population was 57.083 ± 37.97. This was found to have a statistical significance of p < 0.001. The fall in Ki67 index in patients who received preoperative letrozole is presented in Table 2.

Fig. 1.

Ki67 expression pattern. Average postoperative Ki67 (Ki67_S) value corresponding to average preoperative Ki67 value (Ki67_B)

Fig. 2.

Ki67 expression pattern. Average preoperative Ki67 (Ki67_B) and average postoperative Ki67 (Ki67_S) value corresponding to the fall in Ki67 index

Table 2.

Change in Ki67 index

Variable	Median (IQR)	p-value
Average pre-Ki67 Average post-Ki67	22.5 (12.5–35.0) 6.5 (3.5–16.250)	< 0.001
Change in Ki67 index
Low-low (n (%))	10 (16.6%)
High-low (n (%))	29 (48.3%)
High-high (n (%))	21 (35%)
Average fall in Ki67 (mean ± SD)	57.083 ± 37.97

IQR interquartile range, Ki67_B Ki67 at baseline, Ki67_S Ki67 after surgery, n number of patients

In our analysis, there was a statistically significant correlation between the fall in Ki67 after preoperative letrozole with the progesterone receptor status (p < 0.001). However, the statistical analysis revealed no correlation between the tumor size, grade or nodal status of the patients, and the fall in Ki67 index. The decrease in proliferation seen with 2 weeks of letrozole was similar to that seen with a duration of 4 weeks or more of preoperative therapy. Results are shown in detail in Table 3.

Table 3.

Factors affecting fall in Ki67 index

Characteristics	Fall in Ki67 < 50%	Fall in Ki67 50–80%	Fall in Ki67 > 80%	p-value
	n (%)	n (%)	n (%)
Age (in years)
< 60	7 (35)	8 (40)	5 (25)
60–70    > 70	8 (29.6) 4 (30.8)	11 (40.7) 5 (38.5)	8 (29.6) 4 (30.8)	0.993
Breastfeeding history
≤ 1 year each   1–2 years each    ≥ 3 years each	2 (20) 14 (35) 3 (30)	6 (60) 14 (35) 4 (40)	2 (20) 12 (30) 3 (30)	0.713
Tumor status
T1   T2   T3	3 (18.8) 14 (35) 2 (50)	8 (50) 14 (35) 2 (50)	5 (31.3) 12 (30) 0 (0)	0.497
Nodal status
No   N1	11 (34.4) 8 (28.6)	15 (46.9) 9 (32.1)	6 (18.8) 11 (39.3)	0.203
Tumor grade
G1   G2   G3	3 (37.5) 14 (29.2) 2 (50)	3 (37.5) 19 (39.6) 2 (50)	2 (25) 15 (31.3) 0 (0)	0.730
ER positive
50–80%	2 (40)	2 (40)	1 (20)
80–90%    > 90	15 (34.1) 2 (18.2)	17 (38.6) 5 (45.5)	12 (27.3) 4 (36.4)	0.859
PR positive
< 50%   50–90    > 90%	12 (48) 6 (18.2) 1 (50)	13 (52) 10 (30.3) 1 (50)	0 (0) 17 (51.5) 0 (17)	0.001*
Duration of HT
< 14 days	6 (28.6)	10 (47.6)	5 (23.8)
14–28 days    > 28 days	9 (33.3) 4 (33.3)	11 (40.7) 3 (25)	7 (25.8) 5 (41.7)	0.728

^*Statistically significant, ER estrogen receptor, HT hormone therapy, n number of subjects, PR progesterone receptor

Discussion

The present study analyzed the effect of short-term preoperative letrozole therapy on BC proliferation, as assessed by the change in the percentage of cells expressing the Ki67 index. We also assessed the factors that influenced the change in the Ki67 index. A review of existing literature revealed that PoET resulted in a significant reduction in Ki67 in the postoperative surgical specimen [13, 14]. Our study aimed at analyzing this trend among HR + postmenopausal early BC patients in the Indian context.

The study evaluated the effect of short-term preoperative letrozole on Ki67 in postmenopausal women with strong HR + early breast cancer. In addition to gauging the in situ response of the tumor to ET, we also wanted to find an objective method to quantify the degree of response, which prompted us to determine the percentage fall in Ki67 in the pre-and post-intervention specimen. This may provide the clinician with more insight regarding the proliferative potential of the tumor and its response to letrozole, when compared to a single Ki67 value. Our goal was to document an objective and reproducible method of assessing the degree of response to preoperative Letrozole in postmenopausal women with strong HR + early breast cancer, which can pave the way for future studies with a larger number of patients.

The proliferative marker, Ki67, has a predictive and prognostic significance in early-stage BC. The right Ki67 cutoff is currently being debated. The established threshold value according to the latest suggestions from the St. Gallen expert panel is 20%. Beyond this value, the tumor is considered to be highly proliferative and consequently more aggressive [15]. However, review of literature showed that similar preoperative clinical trials use a 10% cutoff to distinguish between low and high Ki67 index as a way of limiting the interobserver bias in the 10–30% group. The study evaluated the proliferative index at baseline (Ki67_B) and in the postsurgical specimen (Ki67_s) to evaluate the response to endocrine therapy. Preoperative treatment had significantly lowered the proliferation marker, and the majority (39, 65%) had a Ki67 index lesser than 10% at the end of the therapy. The establishment of a decreasing trend in Ki67 in response to preoperative endocrine therapy may predict improved outcomes in the adjuvant setting. This observation reflects the findings of the study done by Ianza A. et al., which concluded that a higher percentage fall in proliferation index was strongly associated with a longer 5-year disease-free survival (92%). They concluded that the greater the Ki67 variation, the greater the probability of clinical complete response after treatment [16]. Similarly, Ian Smith et al., have reported that postmenopausal HER2 − ve BC patients receiving 2 weeks of preoperative aromatase inhibitors decreases Ki67 index in breast cancer by a mean change of 76.8% as compared to the control group (5.3%) [13].

Our study recorded a significant reduction in the expression of Ki67 index after short-term preoperative letrozole in postmenopausal women with strong HR + early breast cancer, and the results were similar to previous studies.

Proliferation index on residual tumor holds prognostic importance. A high Ki67 value on residual tumor after treatment, rather than at baseline, has a negative prognostic value, and Ianza et al. have recorded a poorer DFS and overall survival (OS) among these patients [16]. Similarly, our results reflect that greater attention should be given to the percentage decrease in Ki67, rather than focusing merely on a fixed value. In the era of precision medicine, considering the possibility of tumor heterogeneity in patients, this would indeed be a more comprehensive and patient-oriented approach. A reduction in the proliferative index after preoperative endocrine therapy could indicate a less aggressive tumor, a more stable response over time with adjuvant endocrine therapy and longer survival. Thus, it may provide the basis of a simple and inexpensive test to personalize adjuvant treatment in patients with HR + HER2 − ve early-stage BC [13].

Our study also aimed to determine the clinical and pathological factors influencing fall in the proliferative index. A study by Madani S. H. et al. concluded that a higher Ki67 index had a direct significant correlation with higher grade, P-53 positivity, and HER2 positivity [17]. Although these factors did not influence the fall in Ki67 index with PoET in our study, we found that the progesterone receptor status showed statistical significance with the fall in Ki67.

In addition, we found that the duration of the therapy did not influence the fall in the Ki67 index. The decrease in proliferation observed within 2 weeks of letrozole was similar to that seen in 4 weeks or more duration of preoperative therapy. This was in concordance with the inferences of the POWERPIINC trial. They concluded that the decrease in the Ki67 after 7 days of PoET was similar to those reported for longer courses [18]. Thus, our study would suggest that rather than extending the duration of PoET in HR + HER2 − ve patients, it would suffice to treat with a shorter duration to identify the endocrine-sensitive patients, which can then help individualize the extent of adjuvant therapy.

The largest multi-institutional trial studying the significance of fall in Ki67 following preoperative endocrine therapy in postmenopausal (age < 70 years) breast cancer patients is the POETIC trial, which analyzed the recurrence risk following perioperative therapy with aromatase inhibitors. Using a cutoff point of 10%, the POETIC trial showed that 18% of the patients fell in the high-high category, and 49% fell in the high-low category. This was similar to our study, where the percentage of patients in the high-high category was 16.6% and in the high-low category was 48.3%. The POETIC trial also showed that there was a 5-year risk of recurrence of 13% in the high-high category, whereas the 5-year recurrence risk in the high-low category was 5.6% [13]. This highlights the importance of categorizing HR + ve early breast cancer patients into high-high and high-low categories following preoperative endocrine therapy, which can help gauge the response to therapy as well as assess risk of recurrence. While there are several commercially available genomic platforms to provide prognostic and predictive information for the individual patient, these tests are expensive and beyond the means for patients from a developing nation like India. Our study concluded that the majority of early-stage hormone-positive BC patients respond well to PoET and had a significant fall in proliferative markers. However, 10% of the study cohort did not show any significant change in their proliferative index, and it is possible that this represents a subset of HR + patients who are inherently poor responders to endocrine therapy. This represents the patients that fall in the high-high category, who may be at a higher risk of recurrence despite completing treatment according to current standard of care, and therefore require closer follow-up. Analyzing the concordance of fall in the Ki67 index in patients receiving preoperative endocrine therapy with a multigene prognostic test like Oncotype DX could in turn help predict patients who are likely to develop disease recurrence. A larger study will allow us to verify the significance of fall in Ki67 index in patients receiving preoperative endocrine therapy, with a prime focus on validating the fall in Ki67 with established multigene prognostic assays.

Declarations

Conflict of Interest

The authors declare no competing interests.