Ki67 Changes and Its Association with Pathological Response According To Molecular Sub Type after Neoadjuvant Chemotherapy

Ki67 Changes and Its Association with Pathological Response According To Molecular Sub Type after Neoadjuvant Chemotherapy

Rufina Soomro^1*, Afsheen Javaid Khoker¹, Zahid Habib¹, Naila Zahid²¹Department of Surgery, Liaquat National Hospital and Medical College, Karachi, Pakistan
²Department of Oncology, Liaquat National Hospital and Medical College, Karachi, Pakistan

Received Date: 11 August, 2019; Accepted Date: 20 August, 2019; Published Date: 30 August, 2019
^*Corresponding Author: Rufina Soomro, Department of Surgery, Liaquat National Hospital and Medical College, Karachi, Pakistan. +9200923008255117; Email: rufina.soomro@hotmail.com
Citation: Soomro R, Khoker AJ, Habib Z, Zahid N (2019) Ki67 Changes and Its Association with Pathological Response According To Molecular Sub Type after Neoadjuvant Chemotherapy. Adva Gen and Molbio: AGMB-101.

Abstract
Objective: This study evaluated the significance of proliferating marker Ki-67 as a prognostic marker and the relationship of Ki-67 with different clinic-pathological factors and molecular subtypes The purpose of this study is to assess the potential value of Ki67 for the prediction of therapeutic response after neo-adjuvant chemotherapy in breast cancer.

Methods: This observational study enrolled 303 patients with invasive breast cancer who had neoadjuvant chemotherapy from year 2013 to 2018. Age, clinical stage, T, N, ER, PR, HER2 status, and grade were recorded. Ki67 was correlated with all these markers and molecular subtypes. Ki67 was recorded on the first biopsy and after chemotherapy. Change in Ki67 was compared with prognostic markers and molecular types.

Results: There was statistically significant correlation between Ki67 and T, N, Histopathology, grade, ER, PR, HER2, and luminal subtypes (p<0.001). There was a significant reduction in the mean Ki67 after neo-adjuvant therapy (p <0.001). Significant reduction in Ki67 was seen in all molecular subtypes except Luminal A (p<0.001).

Conclusions: Ki-67 was associated with common histo-pathological parameters. Post NAC change in Ki67 correlates with clinico-pathological response.These ﬁndings underline the importance of Ki-67 as a prognostic parameter.

Keywords: Breast cancer; Ki-67; Molecular subtypes; Post Neo-adjuvant Ki67; Prognosis

Introduction
The breast cancer is a heterogeneous disease and outcome varies in patients with the same stage of the disease. The aim in modern, evidence based medicine is to identify patients who have an unfavorable prognosis and to select patients who can improve prognosis associated with a specific form of treatment. In addition to traditional clinico-pathological markers, various other Biomarkers [1] and genetic tests [2] are available to subtype them to predict prognosis and treatment response. Along with conventional histopathological parameters, the assessment of tumor proliferation is one of the major factors for prediction of risk of recurrence and the treatment decisions in breast cancer patients [3]. Ki67 is one of the major markers of tumor proliferation, assessed by immunohistochemistry (IHC) with the anti-Ki67 antibody called MIB-1 [4]. Ki-67 is a nuclear protein with a nuclear function that is expressed in all phases of the cell cycle, except G0 [5].

The St. Gallen Consensus meeting determined that the Ki67 labelling index is the main distinguishing feature between “luminal A” and “luminal B” breast cancer subtypes (1), and genetic test Oncotype DX has Ki67 is one of the 21 variables tested showing significant value of this marker.

Several breast cancer trials have adopted pathological complete response (pCR) as a surrogate marker for long term treatment efficacy and disease free survival. However, the rate of pCR achievement is usually small, generally reported in only 10-30 % of patients, with standard Neo-adjuvant regimens. For residual patient with non PCR a useful marker for prognosis is highly desirable.

Clinical aim of this marker is to identifying patients with unfavorable prognosis and is aiming to improve treatment according to individual risk.

Various studies have been done to explore the Value of Ki67 and show that high ki67 is associated with bad prognosis [7]. International working group worked hard and have found clinical utility of Ki67 as prognostic and predictive marker, but because of lack of standardization there is inter laboratory variability. To avoid that they suggested guidelines for assessment of ki67 in breast cancer [8].Ki67 expression showed a positive correlation with known adverse clinico-pathological parameters like lymph node status, lympho-vascular invasion, and tumor grade and tumor size [9-11]. The detailed relationship between Ki67 and other clinico-pathological parameters requires further investigation.

In this part of the world, we generally see patients at a locally advanced stage [12] and apparently with aggressive biology [13], so it would be interesting to see the correlation of Ki67 with clinico-pathological features in our population.

Neo-adjuvant chemotherapy (NAC) has been established as a standard treatment strategy for patients with not only locally advanced but also operable breast cancer [14].This strategy allows patients to benefit a reduction in the extent of surgery and provides information on the efficacy of chemotherapy. It has been demonstrated that patients who achieve a pathologic complete response (pCR) to NAC are likely to have a favorable long-term outcome [15, 16]. Clinical and molecular biomarkers capable of predicting pCR have been assessed following neo-adjuvant treatment in breast cancer patients [17, 18]. In patients receiving neo-adjuvant therapy the value of Ki67 as a predictive and prognostic marker can be better understood.

Looking at Ki67 values in different molecular subtypes, Ki67 is a well-established prognostic marker in ER positive breast cancer [19], there is little evidence to support a role for Ki67 in hormone Receptor negative breast cancer prognosis [20]. It is true that there are some patients with negative hormone receptors, whose prognosis remains good. Therefore, a better understanding of the molecular and histo-pathological features of HR-negative breast cancers and its heterogeneity is important for the development of a new therapeutic strategy and to improve the prognosis of HR-negative breast cancers. So, the benefit of Ki67 assessment in HR-negative breast cancers needs further workup.

The patients who have residual disease after NAC, we may asses Ki67 on the residual disease and it seems to be promising in terms of prognosis [21, 22]. We aim to study the correlation of Ki67 with known clinico-pathological markers and also to see the prognostic and predictive value of Ki67 in patients receiving NAC and its effect on each intrinsic sub types. By this we can also select high risk patient and can plan further adjuvant chemo or radiotherapy. Thus, assessment of Ki-67 may be used in daily practice to discriminate breast cancer subtypes, predict oncological outcomes, or decide on indications for adjuvant treatment

Material and Methods
Methods

Settings: Department of General and Breast surgery, Liaquat National hospital and Medical College, Karachi, Pakistan.

Duration of study: Prospective study from Jan 2013 -July 2018.

Inclusion Criteria: All female patients with the diagnosis of breast cancer, who received neo-adjuvant therapy.

Exclusion Criteria:

Patients who did not have neo-adjuvant therapy.
Patients who did not undergo surgery after neo-adjuvant therapy.
Patients’ lost to follow up after chemotherapy.
Patients whose pre chemo Ki- 67 were not available.

Study Design: Observational study.

Patients and Methods

All patients with biopsy proven breast cancer requiring Neo-adjuvant therapy were included in this study. Clinical data and original pathological information was collected including, patient’s age, clinical tumor size, nodal status, initial tumor stage according to TNM classification, Estrogen& Progesterone receptor, HER 2-neu status and Ki-67 levels. Neo-adjuvant and targeted therapy given was noted. Along with this postneo-adjuvant therapy tumor size (ypT) and nodal status (ypN) and post therapy Ki-67 levels were recorded.

Pathological complete response (pCR) was defined by the absence of invasive carcinoma in the primary breast tumor. Presence of residual ductal carcinoma in situ was included in the pCR.

Partial responders were those when tumor size (ypT) and lymph node (yPN) status decreased in response to chemotherapy, but still there was disease, Disease was considered stable if there was no response to chemotherapy and Disease Progression means disease worsened despite of chemotherapy. Ki67 has been categorized in to pre and post ki 67 with reference to neo-adjuvant chemotherapy. The immunohistochemistry assessment of Ki67 was consistent during the study period.Ki67 was quantiﬁed using a visual scoring system, which included an external control for validation. Stained cells were counted and expressed as a percentage. Only nuclear staining was incorporated into the Ki67 score, which was deﬁned as the percentage of positively stained cells among the total number of malignant cells scored. If staining was homogeneous, at least 500 cells within ten randomly selected high-power ﬁelds selected. The documentation of the percentage of Ki67 positivity were recorded.

Ki 67 was considered low if it was = or less than 14, 15-30 was borderline and it was grouped as high when it was more than 30.

The patients were classified into 5 subtypes on the basis of immunohistochemistry (IHC) according to St. Galen’s Guidelines as follows (1):

Luminal A-like (ER and/or PR Positive, HER2negative, and Ki67 < 14%),
Luminal B-like (ER and/or PR Positive, HER2-negative, and Ki67 > 14%)
B Her (ER and/or PR Positive and Her2- Positive)
Her2 Positive ((ERnegative, PR-negative, and HER2-positive)
Triple-negative (ER negative, PR-negative, and HER2-negative)

All chemotherapy regimens were allowed for this analysis, commonest being 4 cycles of AC (dose dense) followed by Paclitaxel weekly for 12 weeks. In HER2 positive patients either confirmed by IHC 3+ or gene amplification by fluorescence in situ hybridization (FISH), trastuzumab was administered as neo-adjuvant treatment to those who could afford it. After NAC surgery was done, patients who achieved pCR were recorded and who had residual disease (Deﬁned as evidence of invasive residual tumor in the breast or axilla) Ki67 was again done and pathological pyT, pyN, Stage and Ki67 values were recorded. The documentation of the percentage of Ki67 positivity before and after NAC was mandatory for inclusion in the study group. All patients with positive expression of ER and/or PR received adjuvant endocrine therapy.

None of the patients received additional chemotherapy after surgery (adjuvant chemotherapy). But those who had received Trastuzumab prior to Surgery continue to receive it in the adjuvant setting. Adjuvant radiotherapy was given as per guidelines depending on the stage that they presented with and all those who underwent Breast conservation surgery. Mean change in the Ki67 was noted along with the change in Ki67 in each Sub-type separately to see if molecular subtype has any effect.

Statistical Analysis
The data was entered and analyzed in SPSS version 24.0. T, N, M, grade, stage, histopathology type, nature of surgery, ER, PR, Her2status and intrinsic subtypes were recorded in percentage. Each of these variables were compared to see association with three Ki67 groups Low, Moderate and high ANOVA was used to compare pre and post op Ki67 means between groups Paired t test was used to compare Pre and post op Mean Ki67 in each intrinsic sub-type. For all categorical variables frequencies and percentages were computed and Chi square test was used to determine the proportion difference. All statistical tests were two sided and had a 95 % confidence interval (CI), with the level of significance established at p value of less than 0.05.

Results
A total of 303 patients were eligible and analyzed in this investigation. (Table 1) shows the baseline characteristics of all patients. The mean age was 42.6±11.6 years. Most common tumor status T4 was found in 45% (n=137) patients, 58% (n=175) patients had palpable mobile nodes, the data revealed stage III in 56% (n=174) patients. Mastectomy was performed in 79.6% (n=241) patients, 61% (n=185) patients were grade 2, 60.7% (n=184) patients were ER positive. Half of the patients were PR negative, while 12.8% (n=72) were Her2 positive and in 5.3 %( n=16) Her 2 was undetermined as they were Her2 2+, but Fish could not be done. Luminal B was present in 35% (n= 106) patients. Postoperative histo-pathology (ypT) 31.4% (n=95) patients wereT1, ypN0 was present in 46.2% (n=140) patients, (Table 2) shows statistically significant reduction in mean Ki67 between pre and post NAC groups (P value<0.001).

There was a statistically significant difference between Pre and post NAC in Ki67 in each intrinsic subtypes and mean difference in each group was statically significant (p value < 0.001) shown in (Table3). In (Table 4) Ki67 increased in 55 patients after the NAC and maximum increase was seen in Triple negatives (40%)(Table 5,6,7)shows that when the tumor was down staged with NAC from stage III to II there was statistically significant decrease in Ki67 (p value < 0.001) and even when the stage did not change there was significant reduction in Ki67.The paired significant mean reduction in Ki-67 was found between preKI-67 and post KI-67 (38.9±23.6 vs. 20.75±22.2, p<0.001).

Variables	Mean	SD
Age in years	42.6	11.6
Clinical tumor status	n	(%)
T0	1	0.3
T1	5	1.7
T2	83	27.4
T3	76	25.1
T4	137	45.2
Clinical Nodal Status
0	90	29.7
1	175	57.8
2	35	11.6
3	2	0.7
Missing	1	0.3
Clinical Stages
I	8	2.6
II	71	27.5
III	174	56
IV	41	13.5
Surgery
Breast Conservation	62	20.4
Mastectomy	241	79.6
Grade
1	24	7.9
2	185	61.1
3	85	28.1
Missing Information	9	3
ER
Negative	119	39.2
Positive	184	60.7
PR
Negative	150	49.5
Positive	153	50.4
HER2
Negative	214	71
Her 2+ ( Fish not done)	16	5.3
Positive	72	12.8
Missing information	1	0.3

Table 1 (a): Baseline Characteristic of patients (N =303).

Variables	Number	(%)
Intrinsic Sub Types
Luminal A	23	4.6
Luminal B	112	36.9
Luminal B(HER 2+)	58	17.1
HER2+	18	5.9
Triple Negative	78	25.7
ypT
pCR	44	14.5
T0	1	0.3
T1	98	31.4
T2	83	27.4
T3	64	21.1
T4	4	1.3
Missing information	9	2.9
ypN
N0	140	46.2
N1	81	26.7
N2	34	11.2
N3	39	12.9
Missing information	9	2.9
Y Stage
pCR	35	11.6
I	45	14.9
II	129	42.7
III	86	27.5
Missing information	8	2.6

Table 1(b): Baseline Characteristic of patients (N =303).

Variable

Mean

S.D

Mean Difference

p-value

Pre Ki-67

38.9

23.6

18.67

<0.001

Post Ki-67

20.75

22.2

Table 2: Comparison of Ki-67 before and after (N=303).

Variables	Luminal A(n=24)	Luminal B(n=114)	B Her(n=45)	Her2 +ve(n=35)	Triple negative(n=81)	P-value
Pre ki 67	10.1(±3.6)	43.3(±18.7)	36.2(±28.1)	41.1(±23.6)	43.14(±23.9)	<0.001
Post ki 67	7.1(±6.3)	18.2(20.5)	15.4(19.67)	20.1(21.8)	27.4(±26.2)	<0.001
Mean difference of pre and post ki67	3	25.2	21	20.9	14.9	-
P- value	0.067	<0.001	<0.001	<0.001	<0.001	-

Table 3: Comparison of baseline characteristics (N=303).

Group	Luminal A n=8	Luminal B n=13	Luminal B (HER2+) n=8	Her2+ve n=5	Triple negative n=22
Post op ki67 increase	8(14.54%)	13(23.63%)	8(14.54%)	5(9.09%)	22 (40%)

Table 4: Increase in Post op ki67 (n=55).

Variables	Clinical stage	Path stage	No.	Pre Ki-67 Mean (SD)	Post Ki-67 Mean (SD)	Mean difference	p-value
Ki-67	III	III	44	34.6 (23.9)	22.3-22.9	12.3	0.002
	III	II	73	40.2(22.2)	19.9 (24.2)	20.3	<0.001
	II	III	23	34.8(20.1)	24.7(21.9)	10.1	0.023
	II	II	37	44.8(32.4)	21.73(20.8)	23.1	<0.001

Table 5: Change in stages after NAC.

No of Patients	Luminal A	Luminal B	B Her	Her2 +ve	Triple -ve
N=55	n=8	n=13	n=8	n=5	n=22
%	14.54	23.63	14.54	9.09	40

Table 6: Increase in post NAC Ki 67 in relation to intrinsic sub-types.

No of patients	Luminal A	Luminal B	Luminal B(HER 2+)	Her2 +ve	Triple -ve
N= 35	n=2	n= 7	n= 8	n= 4	n= 14
%	5.71	20	22.8	11.4	40

Table 7: Post Neo-adjuvant complete pathological response in relation to intrinsic sub-types.

Discussion
Breast cancer, most prevalent cancer, prognosis has been greatly improved by early diagnosis and effective treatment. Currently prognostic factors have changed treatment regimens drastically. This was a single center prospective study to look at the utility of Ki 67 in the clinical setting. There has been significant evidence in the literature showing proliferation being associated with clinical outcome [23], but even than Ki67 is still not there in any Guidelines like NCCN, St. Gallen, reasons being lack of standardization among the laboratories and consensus on what level of percentage positivity should be significant [24].

Our study demonstrated that Ki67 is associated with known clinic-pathological prognostic markers like T, N, stage, and Grade, PR, and Her2 neu status. The similar results have been observed in other studies [25, 26].

Although genomic tests are available, but cost always remains an issue for the developing world. Cuzik[27]suggested that on IHC 4 (ER, PR, Her2 and Ki 67) can give the similar results and it is further supported by Goldrish in St. Gallen meeting, where they also advised that Grade can be used in Lieu of Ki67 to differentiate between Luminal A and B. Grade being essentially the same showing proliferation. Our study shows significant association with Grade which has been proved to be a significant prognostic marker [28]. The association of Ki67 is seen in other studies as well [29]. Node positivity is obviously a known bad prognostic marker with possible worse outcome. Ki67 can have a correlation with that, as shown in our study that node positivity was associated with higher Ki levels, also seen in study of Sahin AA [30]. We sub-divided our patients according to St. Gallen recommendation in to 5 sub types and classification into Luminal A and B is on the basis of Ki67 according to St. Gallen criteria. There has not been consensus on the cutoff point of Ki67 to differentiate between Luminal A and B. We used 14%, but other have used different levels with equally reliable prognostic index [31].

Hormone receptor positivity is generally associated with good prognosis and Ki67 may not be a useful marker of prognosis in these patients [32], others have found ER positivity associated with lower Ki67 and same results are seen in our study. In hormone positives Viale, et al. [33] use Ki 67 11% cut off, they showed high ki67 was associated with worse outcome and in post-menopausal patients Letrazole is a better choice in these patients. We found a significant association according to each sub group. Luminal A as expected had low ki67, but aggressive tumors like Her2 positive and triple negatives were mainly seen in high Ki67 group suggesting that Ki67 can predict the tumor aggressiveness and can be a useful marker according to molecular sub-types [34]. In our patients, where patients were given NAC and Ki67 was checked before and after the treatment. 24 patients (7.92%) had complete pathological response. In patients with residual disease there was a statistically significant reduction in mean Ki67 scores after Neo-adjuvant therapy and reduction in Ki67 is associated with good prognosis [35]. Looking at sub types in patients with Luminal A where ki67 is low, the change in Ki67 was insignificant, while in all other sub-types including Luminal B, Her2 positive and triple negatives showed a statistically significant decrease in the marker.( p value < 0.001). Pathological complete response is associated with good prognosis and pCR is expected more in triple negative patients (TN) [36] as seen in our study where 40% who had pCR were triple negative followed by luminal B. However others have not observed higher pCR in TN [37]. In TN patients with high Ki67 may achieve high pCR, in spite of that they tend to have more aggressive disease, this may help in classifying TN into 2 prognostic groups and can predict chemo sensitivity[38].

This change in Ki67 after NAC relates well in Her2 positive patients, we found a statistically significant decrease in Her2 positive patients after the treatment. Kwan II kim in their study suggested that Ki-67 is a useful predictive factor for pCR, especially in patients with ER-negative and HER2-positive breast cancer [39].There has been a change in the Ki67 estimates after the surgery. We wanted to see whether the change in Pathological stage correlates with the change in Ki67 status. (Table5) shows that there was a statistically significant drop in Ki67 when clinical stage changed from Stage III to pathological stage II. There was the significant drop in Ki67 when stage remained the same stage II. However we were unable to see the statistical significance in other groups. Acs B showed that though Ki67 is a controversial predictive and prognostic marker but they showed a significant association with pathological response and they also concluded that if a tumor is non-responder to NAC, increased Ki-67 is a poor prognostic marker [40]. As discussed that there is association of Ki67 with known clinico-pathological features. But after NAC when there is no pCR and we have other Ki67 estimation on the residual disease, we need to know which one has better prognosis? Robin L. Jones et al. [41] and Bottini [42] concluded that Post-chemotherapy Ki67 is a strong predictor of outcome for patients not achieving a pathological complete response. Dowsett M [43] in this respect has shown very important therapeutic concept that if short term use of hormonal treatment (Tamoxifen vs Anastrozole) can bring the proliferation down as seen in our stage II patient, one will be confident in continuing with the same hormonal treatment in adjuvant setting. .These findings could lead to a profound change in approaches to drug development in breast cancer.

Our data shows that Post NAC Ki67 increased in 55 patients (18.1%) majority of them being in the triple negative group (40%). Miral Mashhour [44] found increase in Ki67 in 36% of their patients and they also noticed increase Ki67 in Triple negative patients. In conclusion this single center study demonstrated that Ki-67 can be applied in clinical practice. These findings underline the importance of Ki-67 as a prognostic parameter. Post NAC change in Ki67 correlates to some extent with clinico-pathological response and may turn out to be a better prognostic test.

Future Direction
As we know that patients with pCR has better survival, we need a large prospective study to see survival in patients where Ki67 increases after NAC and also correlate survival with pre and post NAC KI67.

Conflict of Interest: The authors declare that they have no conflict of interest.

References