AIM: To study the dimensional analysis CD44high CD24low and Ki67 in triple negative breast cancer (TNBC).
METHODS: This cross-sectional study was performed on patients with breast cancer in Haji Adam Malik Hospital Medan from 2013 to 2016 to determine the frequency and pathologic features of TNBC by immunohistochemistry stained.
RESULTS: By using immunohistochemistry staining panel of CD44, CD24, Twist, Claudin 7, CK5, CK8/18, EMA, E-Cadherin, Ki-67, a total 67 breast tumour samples with TNBC were classified as 9 stem-cells like 1 basal, 22 baso-luminal, and 23 luminal subtypes
CONCLUSION: By using immunohistochemical staining panel, TNBC can be differentiated into stem cells like basal, baso-luminal and luminal subtypes. Didifferentiation and EMT can produce heterogeneity in TNBC subtypes and this will affect in handling TNBC. Stemness in stem cells- like subtypes are resistant to therapy. Therefore, TNBC needs special attention in order to assist in more optimal handling

Bachtiar, Adang

Kanoko, Mpu

Munir, Delfitri

Tan, Betty

Tan, Gino

Open Access Macedonian Journal of Medical Sciences 

 
Open Access Maced J Med Sci electronic publication ahead of print,  
published on February 27, 2019 as https://doi.org/10.3889/oamjms.2019.182 
_______________________________________________________________________________________________________________________________ 
Open Access Maced J Med Sci.                                                                                                                                                                                                          1 
 
ID Design Press, Skopje, Republic of Macedonia 
Open Access Macedonian Journal of Medical Sciences. 
https://doi.org/10.3889/oamjms.2019.182 
eISSN: 1857-9655 
Basic Science 
 
 
  
 
Dimensional Analysis of CD44
High
 CD24
Low
 and Ki67 in Triple 
Negative Breast Cancer 
 
 
Betty Tan
1*
, Mpu Kanoko
2
, Gino Tan
3
, Adang Bachtiar
4
, Delfitri Munir
5
 
 
1
Department of Anatomic Pathology of Medical Faculty, University of Sumatera Utara, Jl. Dr Mansyur No 5, Medan 20155, 
Indonesia; 
2
Department of Anatomic Pathology of Medical Faculty, University of Indonesia, Jakarta, Indonesia; 
3
Department 
of Clinical Pathology of Medical Faculty, University of Sumatera Utara, Medan, Indonesia; 
4
Faculty of Public Health, 
University of Indonesia, Jakarta, Indonesia; 
5
Department of Otorhinolaryngology of Medical Faculty, University of Sumatera 
Utara, Medan, Indonesia 
 
Citation: Tan B, Kanoko M, Tan G, Bachtiar A, Munir D. 
Dimensional Analysis of CD44High CD24Low and Ki67 in 
TNBC. Open Access Maced J Med Sci. 
https://doi.org/10.3889/oamjms.2019.182 
Keywords: Triple-negative breast cancer; Stemness; 
Differentiation; EMT; CD44; CD24 
*Correspondence: Betty Tan. Department of Anatomic 
Pathology of Medical Faculty, University of Sumatera 
Utara, Jl. Dr Mansyur No 5 Medan 20155, Indonesia. E-
mail: andbethgo@yahoo.com 
Received: 15-Jan-2019; Revised: 18-Feb-2019; 
Accepted: 19-Feb-2019; Online first: 27-Feb-2019 
Copyright: © 2019 Betty Tan, Mpu Kanoko, Gino Tan, 
Adang Bachtiar, Delfitri Munir. This is an open-access 
article distributed under the terms of the Creative 
Commons Attribution-NonCommercial 4.0 International 
License (CC BY-NC 4.0) 
Funding: This research did not receive any financial 
support 
Competing Interests: The authors have declared that no 
competing interests exist 
 
 
 
Abstract 
AIM: To study the dimensional analysis CD44
high
 CD24
low
 and Ki67 in triple negative breast cancer (TNBC).  
METHODS: This cross-sectional study was performed on patients with breast cancer in Haji Adam Malik Hospital 
Medan from 2013 to 2016 to determine the frequency and pathologic features of TNBC by immunohistochemistry 
stained.  
RESULTS: By using immunohistochemistry staining panel of CD44, CD24, Twist, Claudin 7, CK5, CK8/18, EMA, 
E-Cadherin, Ki-67, a total 67 breast tumour samples with TNBC were classified as 9 stem-cells like 1 basal, 22 
baso-luminal, and 23 luminal subtypes 
CONCLUSION: By using immunohistochemical staining panel, TNBC can be differentiated into stem cells like 
basal, baso-luminal and luminal subtypes. Didifferentiation and EMT can produce heterogeneity in TNBC 
subtypes and this will affect in handling TNBC. Stemness in stem cells- like subtypes are resistant to therapy. 
Therefore, TNBC needs special attention in order to assist in more optimal handling. 
 
 
 
 
 
 
Introduction 
 
Breast cancer is thought to derive from the 
stem or progenitor cells having abnormalities in the 
self-renewal process [1]. Mammary stem cells 
(MaSCs) play an important role in the growth and 
development of breast cancer, resistance to therapy, 
and metastasis [2]. Various stem cell markers are 
used to identify and isolate CSC from various solid 
tumours, such as CD44 and CD24 [3]. CD24 is a little 
more expressed in progenitor cells compared to 
differentiated cells [4]. Therefore, for therapy to be 
effective, CSC must be recognised and must be 
differentiated from normal breast stem cells.  
The increasing level of Ki-67 shows 
aggressiveness of tumour growth and indicates a poor 
prognosis. Triple-negative breast cancer (TNBC) is 
also correlated with high Ki-67 level [5]. Hence, we 
were interested in studying about the dimensional 
analysis CD44
high
 CD24
low
 and Ki67 TNBC. 
We aimed to study the dimensional analysis 
CD44
high
 CD24
low
 and Ki67 in triple negative breast 
cancer. 
 
 
Material and Method 
 
This descriptive study with the cross-sectional 
design was conducted from March to October 2017 
and was carried out after getting permission from the 
Ethical Committee of Medical Faculty USU Medan.  
The population were patients diagnosed as 
breast cancer based on histopathology 
(mastectomy/biopsy) at RSUP Haji Adam 
Basic Science 
_______________________________________________________________________________________________________________________________ 
 
_______________________________________________________________________________________________________________________________ 
2                                                                                                                                                                                                   https://www.id-press.eu/mjms/index 
 
Malik/Departement of Anatomical Pathology Medical 
Faculty of USU Medan. TNBC tumours were further 
stained with CD44 (DF1485, Novocastra Laboratories 
Ltd., dilution 1:100), CD24 (C-20, Santa Cruz 
Biotechnology, dilution 1:100), TWIST-1 (H-81, Santa 
Cruz Biotechnology, dilution 1:100), CK5 (XM26, 
Novocastra-Vision Biosystems, dilution 1 : 25), 
CK8/18 (5D3, Lab Vision, dilution 1:300), Claudin-7 
(NBPI-35677, Rabbit polyclonal antibody, Novus 
Biological, dilution 1:100), E-Cadherin (NCH-38; 
M3612, monoclonal primary antibody, 
DakoCytomation, Denmark, dilution 1:50), EMA (E29, 
monoclonal antibody, DAKO, dilution 1:400), and Ki-
67 (clone SP6, biomarkers, dilution 1:100).  
CD44, CD24 and Twist were stained in 
membrane cells, with score 0 if < 10% positive tumour 
cells; 1 if 10-25%; 2 if 25-50%; and 3 if > 50%. 
Intensity was scored as 0 if unstained, 1 if weakly 
stained, 2 if intermediate, and 3 if strong. 
Interpretation of CD44, CD24, and Twist staining was 
determined based on multiplication of the percentage 
of positive cells and the intensity of staining. CD44 
and CD24, were scored as 0 if (-), 1-3 (+1), 4-6 (+2), 
and 7-9 (+3) [6]. While Twist was considered weak if 
total score < 6 and strong ≥ 6 [7]. Claudin-7 staining 
was scored as 0 if no membranous staining; 1+ (1-
10% tumour cells); 2+ (10-30%); and 3+ (> 30%) [6].  
For E-cadherin staining, the interpretation of 
staining is divided into 0 if (-); +1 if weakly and 
heterogenous stained; +2 if weakly but homogenous 
stained; +3 if moderately stained, or if strongly but 
heterogenous stained; and +4 if strongly and 
homogenous stained. Percentage of tumour cells 
were scored as 0 if (-); 1 if < 10% membranous 
stained; 2 if 10-50%; and 3 if > 50%. Interpretation of 
E-cadherin was determined based on multiplication of 
percentage of positive cells and intensity of staining, 
which is negative (scored as 0); weakly stained (total 
score 1-4); moderately stained (5-8); strongly stained 
(9-12) [8]. CK5 and CK8/18 were stained in the 
cytoplasm and positive if ≥ 10% tumour cells. EMA 
was stained positive in membranous/ cytoplasm cells.  
Intensity of staining was scored as 0 if < 25%, 
1 if 26-50%, and 2 if 51-100%. Score 0 and 1 was 
considered low and scored 2 as high [9]. For Ki-67, 
300 cells were counted (include proliferating and non-
proliferating cells), and the percentage of proliferation 
were counted with cut-off point 10% positively nuclear 
cells [10]. After that, molecular classification of TNBC 
was done and classified as Claudin low (stem cell-
like) subtypes if CD44
+ 
CD24
-
, Claudin-low, Twist-
1
high
; basal-like subtypes if CK5
+
 and EMA
+
, and 
luminal subtypes if CK8/18
+ 
and E-cadherin
+
.  
The results of this study were processed 
using statistical software and displayed in frequency 
distribution in tables.  
 
 
Results 
 
To determine the ontogeny and differentiation 
of TNBC subtypes in stem cells stages, we used 
CD44 and CD24 immunohistochemical stains. The 
classification of stem cells-like type into SC-1 to SC-3 
were arbitrary (Figure 1). 
 
Figure 1: Schema of ontogeny differentiation breast epithelial from 
stem cells to luminal cells associated with a panel of various 
molecular markers for TNBC 
 
From 67 TNBCs in this study, there were 10 
cases of CD44
+
CD24
-
, 36 cases of CD44
-
CD24
+
, 9 
cases of CD44
+
CD24
+
, and 12 cases of CD44
-
CD24
-
. 
With Twist, Claudin 7, Cytokeratin 5 dan 8/18 (CK5 
dan CK8/18), EMA, E-Cadherin, and Ki67 
immunohistochemical stain panel, TNBC was 
classified as 9 cases of stem-cell-like, 1 basal, 22 
baso-luminal and 23 luminal subtypes. After classified, 
Tumours would then be categorised as low and high 
proliferation based on Ki67 staining. Both low and 
high Ki67 were more commonly found in TNBC with 
CD44
-
CD24
+
 (both 18 cases, 50%).  
 
 
Discussion 
 
MaSCs are marked with high CD44 and 
negative/low CD24 (CD44
+
CD24
-/low
) adhesion 
molecule expressions. CD44
+
CD24
−/low
 phenotype is 
often related to poor prognosis [11]. In this study, 
CD44, CD24, Claudin-7 and Twist-1 were used as 
molecular markers of TNBC stem cell-like subtypes; 
CK5, EMA for basal-like sub-types; and CK8/18 for 
luminal sub-types. Results from 67 TNBC cases 
showed marked heterogeneous and overlapping 
profiles. 
After immunohistochemistry staining of 67 
TNBC cases was seen, we concluded that the clinical 
application of dividing stem cell-like subtypes into SC-
1, SC-2, and SC-3 would not be useful. The 
importance of this study was the identification of 
Tan et al. Dimensional Analysis of CD44High CD24Low and Ki67 in TNBC 
_______________________________________________________________________________________________________________________________ 
_______________________________________________________________________________________________________________________________ 
Open Access Maced J Med Sci.                                                                                                                                                                                                         3 
 
stemness which will influence therapy. The 
classification of SC-1 to SC-3 can only help to 
facilitate understanding these complicated problems 
of ontogeny. In this study, CD44
-
CD24
+
 groups were 
very heterogeneous.  
In conclusion, by using immunohistochemical 
staining panels, TNBC can be classified into stem cell-
like basal, baso-luminal, and luminal subtypes. 
Differentiation signs (EMT) in basal, baso-luminal and 
luminal subtypes can be recognised with CD44, CD24 
and Twist. Differentiation and EMT can cause 
heterogeneity in TNBC subtypes, and this influence in 
TNBC therapy. Stemness behaviour in stem cell-like 
subtypes is resistant to therapy. Besides that, Ki-67 
expression shows the aggressiveness of tumours. In 
controlling the aggressiveness of tumours, effective 
medicines must be used to manage the cell cycle. 
Therefore, TNBC needs special attention to assist in 
more optimal handling.  
 
 
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Dimensional Analysis of CD44High CD24Low and Ki67 in Triple Negative Breast Cancer

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Dimensional Analysis of CD44High CD24Low and Ki67 in Triple Negative Breast Cancer

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