Utility of Ki67 in predicting response to neoadjuvant chemotherapy in women with locally advanced breast cancer at the Kenyatta national hospital
Mogere, E.O 1, Githaiga J. W1, Owilla F1, Mungania M 2, Ojuka D. K1.
1 Department of surgery, University of Nairobi.
2 Department of Pathology, Kenyatta National Hospital.
Correspondence to: Dr. Mogere Edwin Obiri, Department of Surgery, University of Nairobi. P.O. Box 2715-40200 Kisii. Email:
Background: Ki67 levels have been shown to have good predictive value in breast cancer treatment. There is paucity of data on Ki67 levels in predicting response to neoadjuvant chemotherapy (NACT) in Kenya. This study evaluated the utility of Ki67 in predicting response to NACT.
Methods: This was a prospective observational study carried out at the Kenyatta National Hospital between December 2017 to January 2019 on patients with locally advanced breast cancer. Informed written consent was obtained from the patients following approval by institutional review board. We recruited 61 through consecutive sampling technique. Data collected included patients’ demographics, pre-treatment tumour size, Ki67 levels and tumour biology. After 3 cycles of first line chemotherapy, determination of response was assessed by ultrasonography. Data was analyzed by SPSS for proportion of change in tumour size. The response was correlated with tumor biology and pretreatment levels of Ki67 using Chi-square at a 95% confidence interval and a p-value <0.05 was considered statistically significant.
Results: The percentage response rate after 3 cycles of NACT was 39.4%, sensitivity and specificity of Ki67 levels was 70.8% and 43.2% respectively with a cut-off value of 32.5%. Conclusions: Ki67 was found to predict response in our context at a rate of 39.4% at 20% cut off after three cycles.
Key words: Ki67, breast cancer, neoadjuvant chemotherapy.
Ann Afr Surg. 2020; **(*):*** DOI:
Conflicts of Interest: None
© 2020 Author. This work is licensed under the Creative Commons Attribution 4.0 International License.
Breast cancer contributes a significant proportion of morbidity and mortality globally; Data from GLOBACAN indicates it is the 2nd in incidence and 5th cause of mortality globally (1). Data from Nairobi Cancer Registry reveals a high burden of breast cancer it is the leading malignancy with a prevalence of 51.7 per 100,000 of all cancer in women in Kenya(2)
Locally advanced Breast Cancer (LABC) is commonest stage accounting for between 50.7% to 60% of the breast cancer cases at first presentation (3). Currently in Kenyatta national hospital approximately 3 to 4 patients receive neoadjuvant chemotherapy weekly, this led to an average of 12 to 16 patients per month and 144 to192 patients per year (unpublished data). Treatment of LABC involves a multidisciplinary approach with neoadjuvant chemotherapy (NACT), surgery i.e. breast conserving surgery or modified radical mastectomy for the operable patients, plus radiation therapy depending on patient selection (4).
LABC poses difficulties in achieving tumour free resection margins, low chance of breast conserving surgeries, higher recurrence rate and poses challenges in wound closure (5). NACT therefore enables the surgeon to achieve the acceptable resection margins, increases operability and increases the chances of wound closure (6). DNA microarrays have shown that some breast cancers are resistant to chemotherapy this could result is some patients receiving unnecessary chemotherapy. DNA microarrays have been used to predict response to chemotherapy but they are expensive and its utility is not universally adopted (7).The methods that have been used to monitor treatment are physical examination, imaging studies, pathologic response, NACT and biomarkers (8). Clinical methods have been shown to be inadequate on their own in predicting response to therapy (9).
Ki67 is a nuclear antigen which is expressed in actively dividing cells. It is a marker of cellular proliferation (10). It is produced in large amounts in all cancerous tissues and therefore can be used to predict response to treatment (11). Measurement of levels of Ki67 has been recommended in Breast cancer prior to initiating neoadjuvant therapy (12). Values of Ki67 levels of more than 25% have been shown to have a favorable response to NACT (13). There is however, paucity of data on its utility in locally advanced breast cancer in Kenya and the data available lacks uniformity of Ki67 cut-off levels. Ki67 applied with good accuracy has been shown to have good prediction values. This study therefore aims at determining the utility of Ki67 in predicting response to neoadjuvant chemotherapy in LABC.
This was a prospective observational study carried out at Kenyatta National Hospital surgical wards, surgical outpatient clinic, oncology clinics, radiology department and histopathology laboratory. The study was conducted between December 2017 and January 2019. Inclusion criteria were women with locally advanced breast cancer, who had no systemic metastasis, T3 disease on ultrasonography and chemotherapy naïve patients. Staging was done by clinical examination and radiological assessment using CT scan chest or plain chest x-ray, abdominal ultrasound. Only patients who were negative for systemic metastases and fully staged were included in the study. Ultrasonography was performed by qualified radiologist as per hospital policy.
Patients were recruited through convenient sampling procedure after an informed consent was administered. Data was collected using the data collection sheet. Data collected included patients’ demographics, pretreatment Ki67 levels, pretreatment tumor size, tumour biology (tumor grade, lymphovascular invasion and Immunohistochemistry: ER, PR and HER2) as per the attached data sheet. Tumor size was determined by ultrasound prior to therapy and at the end of 3 cycles. Patients were assessed using Ultrasound Machine Aplio 400 with a high frequency linear probe of 12MHz to measure the longest tumour diameter. Standard neoadjuvant first line chemotherapy was given for 3 cycles. Every third week the regime given was Adriamycin 60mg/m2 and cyclophosphamide 600mg/m2.
Sample size was calculated using Daniel’s formula with finite population correction for standard distribution with 95% confidence interval and a desired precision of 0.05. A repeat breast ultrasound was performed after the three cycles of chemotherapy and sizes recorded. The evaluation of response to NACT was performed using the response evaluation criteria in solid tumours (RECIST).
Data was entered into MS excel sheet, cleaned and transferred to SPSS (Version 21.0, Chicago-Illinois) for analysis. Analysis was performed for mean, and range of age, mean tumour size change from pretreatment to after 3 cycles of NACT, proportions of patient with high or low Ki67 according to St Galen classification which used 20% as the cut-off, and proportion of patient who had response as per RECIST. Chi-square was used to analyse the association between Ki67 level to response and other aspects of tumour biology (molecular subtype, tumor grade, and perineural and lymphovascular invasion).
Receiver operator curve was drawn using the Ki67 levels against response to NACT. This was then used to determine the sensitivity, specificity, cut offs, and area under the curve of Ki67 for our population. p-values and 95% confidence intervals (CIs) were calculated as applicable and p- value <0.05 was considered statistically significant.
This study commenced upon approval from the Department of surgery and the UoN-KNH ERC under the approval number P247/05/2017.
Sixty-one patients were recruited for this study. The mean age of the patients was 45.9 (SD=10.4) years, while the minimum age was 28 years, and maximum 73 years. 27 (44.3%) patients were 41-50 years of age. All the patients in this study had invasive ductal carcinoma and the majority had tumor grade II at 63.9% and Luminal A molecular subtype at 34.4% (Table 1).
The correlation between Ki67 levels and response to NACT based on sensitivity and specificity, cut off and area under the curve for KI67 is as shown in Figure 1 and Table 2. Point marked pink on the graph is the cut-off point, giving sensitivity and specificity for prediction of response.
The paired sample mean change in tumour size on ultrasonography from pretreatment to post treatment was 1.24 (SD=2.19) with Confidence Interval of 0.68-1.80, t (60) = 4.420, p<0.001. To test the hypothesis that the size of the breast mass (cm) by ultrasonography at week 0 (M=5.95, SD=4.34) and the size of the breast mass (cm) greatest dimensions following 3 cycles of NACT (M=4.71, SD=4.07) were equal, a dependent samples t-test was performed. It is worth noting that the correlation between the two conditions was estimated at r=0.866, p<0.001, suggesting that the dependent samples t-test is appropriate in this case. The null hypothesis of equal breast mass means was rejected t (60) = 4.42, p<0.001. Thus, the mean of size of breast mass (cm) greatest dimensions at the end of 3 cycles of NACT was statistically significant lower than the mean of size of breast mass (cm) by ultrasonography at week 0.
The Correlation of Ki67 levels with tumor biological characteristics
The pathological response after three cycles of chemotherapy was seen in 24 (39.4%), majority 31 (50.8%) remained stable while only 6(9.8%) had progression of their disease while on chemotherapy. When considered against the cut off agreed at Galen for Ki67, there was no significance difference between those above or below 20%.
A chi-square test for proportion was conducted between the Ki67 levels and response. There were no statistically significant differences in the proportions between Ki67 levels and Responders and non-responders, χ2 (1) = 0.884, p = .347. (Table 3 ).
Response rates in relation to receptor status.
HER2+ had a higher proportion of responders at 37.0%), followed by ER+ at 31.4% and PR+ at 20.7%. Results are as shown in table 4 below (Table 4).
Relationship between Ki67 and perineural and lymphovascular invasion
The Perineural invasion after three cycles of chemotherapy was seen in 24 (39.4%), majority 31 (50.8%) remained stable while only 6(9.8%) had progression of their disease while on chemotherapy. When considered against the cut off agreed at Galen for Ki67, there was no significance difference between those above or below 20%.
A chi-square test for association was conducted between the lymphovascular invasion and perineural invasion with the KI67 levels. There was no statistically significant association between lymphovascular invasion and KI67 levels, χ2 (1) = 2.198, p = .138, but there was statistically significant association between perineural invasion and higher KI67 levels, χ2 (1) = 10.509, p = .005.
This study included 61 women all with locally advanced ductal carcinoma. The mean age at diagnosis 45.9±10.4years, the median age at diagnosis is 48.5years in Kenya and 64.1 years among the US born Americans(14). A study conducted in Aga Khan University, Nairobi concluded that the median age at diagnosis was 47.5years(15).The mean age of breast cancer diagnosis in African population is 46years ± 6.2 S.D with age range of 35-49 years (16)Our study findings on age and commonest age group at diagnosis are within the findings of these studies.
Levels of Ki67 have been documented to correlate with both clinical and pathologic response to NACT and can help in selecting patients who will benefit from the NACT. However, the Cut off values vary widely with different patient populations and the type of NACT used(17). The value in our study is close to that reported Jain et al who conducted a prospective observational study with 134 patients with Stage II/III breast cancer between February 2014 and March 2016, in our study all the patients were stage III. The study reported Ki67 as an independent predictive factor for response and suggested 35% as best cut off for Ki67 expression in predicting response to NACT and achievement of pathologic complete response. The sensitivity was 68.7% and specificity 71.6%. While the sensitivity is comparable to our study, they had a higher specificity. However, in Jain’s study, the clinical complete response rate was lower at 26.1% compared to this study (39.4%). Jain et al study in the Indian population , had twice our sample size and patients completed all the cycles of NACT(18).
Kim Ki et al in Gachon University in South Korea, conducted a nine-year retrospective study of three large centres with 9,321 records retrieved; the study demonstrated a cut off value of 25% (Sensitivity 71%, specificity 77%) especially in ER- and HER2+ tumors with ki67 being the only independent predictor (13). A Cohort study conducted by Acs B et al in Hungary between 2002 to 2013 with a sample size of 120 patients with invasive ductal carcinoma, reported that NACT is more efficient in tumors with at least Ki67 20% with a 95.7% sensitivity and 54.3% specificity, a cut off value lower than our study. A similar cut off value of 20% was reported by another study (19). Ki67 cut-off values therefore vary in different studies a study by Denkert et al suggested that Ki67 levels are a continuum variable and proposed that cut-off points are in the context dependent on three groups of tumors: Tumors with low levels of Ki67 have good outcome and those with high levels of Ki67 have good outcomes only when the tumor responded to NACT(20).
This study also looked at other biological characteristics of the tumour in relation to Ki67 levels. There was no statistically significant association between lymphovascular invasion and Ki67 levels(21). Contrary to these findings, a study by Kilickap et al reported that there was no relationship between perineural invasion and Ki67 positivity. However, they reported that higher ki67 levels are associated with unfavorable prognostic factors including higher grade, ER negativity, Her2 positivity and axillary lymph node involvement(22). The differences in our study could be as a result of a smaller sample size hence further studies will be needed. This study showed no statistically significant association between Ki67 levels and tumor grade. A study by Awadelkarim KD and co-workers in Sudan suggested positive correlation between ki67 and tumor differentiation but it wasn’t linked to any other variables tested including receptor type and tumor size(23). A similar study by Haroon S et al among 194 cases of newly diagnosed breast cancer with an aim of correlating Ki67 expression with other prognostic markers including tumor grade, the study reported that Ki67 is positively correlated with histological grade, the study also elucidated that high ki67>30% shows a lower HER2neu expression and lymph node metastasis hence may have worse prognosis(24).
Inter-observer variability during ultrasonography may introduce limitations in the study but this was delimited by use of standard machine and use of two radiologists in the reading of the ultrasonographic report as part of quality assurance.
Ki67 was found to predict response in out context with a response rate of 39.4% using the St. Galen’s cut off of 20%. Using ROC, sensitivity was found to be 70.8% and specificity of 43.2% at cut-off of 32.5%.
Kenyatta National Hospital for providing funding this research, Mr. Gabriel Omondi, a laboratory technician at Kenyatta National Hospital for helping in collecting data and the statistician Mr. Wickliffe Ayieko.
Conflict of interest
There is no conflict of interest of either the principal author or co-authors to declare. The funder had no role in the design, collection, analysis and interpretation of the data.
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