Vimentin Expression in Stem Cell-like Subtypes of Triple Negative

Breast Cancer (TNBC)

Betty and Delyuzar

Department of Anatomic Pathology of Medical universitas Sumateraa Utara, Medan, Indonesia

Keywords: TNBC, CD44, CD24, Twist, Claudin7, Vimentin, Stemness

Abstract: Triple negative breast cancers (TNBC) tested phenotypically negative for estrogen (ER), progesterone

receptors (PR), and human epidermal growth factor receptor 2 (HER-2). This cross-sectional study was

performed on breast cancer patients in Haji Adam Malik Hospital Medan from 2013 to 2016. Data about

demographics were extracted from patients’ records and histopathologic features were obtained using

Hematoxylin Eosin (HE) and immunohistochemistry staining. By using CD44, CD24, Twist, Claudin7, and

Vimentin, a total 67 breast tumor samples with TNBC were classified as 19 cases of stem-cell like and 48

non stem-cells like subtypes. Postmenopause women with tumor size more than 5cm, higher stage and grade

histology were likely to have non stem-cells like subtypes. Women with mucinous carcinoma and

metaplastic carcinoma of no special type tended to have non stem-cells like subtypes. Both stem-cells like

and non stem-cells like subtypes commonly had low Vimentin. By using immunohistochemistry staining,

TNBC can be differentiated into stem-cell like and non-stem-cell like subtypes. Stemness in stem cell like

subtypes are resistant to therapy. Therefore, identification of stem-cells in TNBC needs special attention

assist in more optimal handling.

1 INTRODUCTION

Breast cancer is the second commonly found cancer

and one of the leading causes of death from cancer

worldwide for females.

Based on data from

Oncology Division in Haji Adam Malik Hospital in

Medan from 2011 to 2015, an estimated 600 new

cases of breast cancer were found each year in

Department of Oncology. Breast cancer has

heterogenous histopathological features and

molecular expressions. Breast cancers tested

phenotypically negative for ER, PR, and HER-2 will

be called as TNBC.

TNBC tends to be more

aggressive and until now there has been a

disagreement about the treatment because TNBC is

not effective to hormonal and targeted anti-HER2

therapy.

This subtype has variety of clinical

manifestations, histopathological features and

molecular expressions, some of which are high

grade, with high proliferation rates, grow

aggressively and have poor prognosis.

By gene expression profiling, TNBC is classified

into 2 major parts, such as basal-like and claudin

low.

Basal-like subtype is expressed with ER

HER2

, EGFR

and/or Ck5/6; meanwhile Claudin

low

is expressed with lacking of luminal epithelial

differentiation markers (claudin-3, claudin-4,

claudin-7, and E-cadherin) and increasing of

epithelial-mesenchymal transition markers (EMT)

and cancer stem cell (CSC) characteristics

(CD44

/CD24

-/low

5,6

This supports that claudin

low

is cell deriving from immature progenitor cells or

stem cells.

Although Claudin

low

and basal-like

subtype looks alike, but these two subtypes is

completely different.

EMT naturally occurs during

early embryogenesis phases. But this process also

occurs during formation, growth and tumour

metastases to distant area. In breast epithelial cells,

EMT is related to the invasion of breast cancer cell

and mesenchymal character, which is marked with

high expression of vimentin.

9,10

Breast cancer stem cells/BCSCs play a

significant role in the growth and development of

breast cancer, resistance to therapy, and metastasis.

To isolate and identify CSC from other tumous,

scientists can use various stem cell markers, such as

CD44 and CD24. CD24 is a little more expressed in

progenitor cells than in differentiated cells.

Therefore, for therapy to be effective, CSC must be

recognized and must be differentiated from normal

Betty, . and Delyuzar, .

Vimentin Expression in Stem Cell-like Subtypes of Triple Negative Breast Cancer (TNBC).

DOI: 10.5220/0010096408650870

In Proceedings of the International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches (ICOSTEERR 2018) - Research in Industry 4.0, pages

865-870

ISBN: 978-989-758-449-7

865

breast stem cells. Hence, we were interested to

identify vimentin expression in stem-cell-like

subtypes of TNBC.

2 METHODS

This descriptive cross-sectional study was carried

out in Department of Anatomical Pathology in Haji

Adam Malik Hospital and Medical Faculty USU

Medan and also in Department of Oncology/Surgical

Haji Adam Malik Hospital Medan from March to

October 2017. The research was done after

permission from Ethical Committee of Medical

Faculty USU Medan is granted. Studied population

are patients histopathologically diagnosed with

breast cancer. Clinical data such as age, tumour size

and clinical stage were obtained from medical

records and histopathological review of slide were

done based on Bloom and Richardson methods

modified by Elston Ellis (subtypes and grading

histology). ER (clone 6F11, dilution 1: 100, Dako),

PR (clone PgR 636, polyclonal Ab, dilution 1: 200,

Dako), and Her-2 immunohistochemically staining

(clone A0435, polyclonal Ab, dilution 1: 200, Dako)

were done. ER and PR were evaluated according to

ASCO/CAP guidelines. Tumours were scored as

positive for ER and PR (≥1% of nuclear tumour cells

stained).

Her-2 was considered positive if strongly

and homogenous membranous staining/chicken wire

pattern (score 3+). If weakly or negative (score 0 or

1+), and score of 2 if membrane cells were

incompletely homogenous stained

(borderline/moderate). Tumours were defined as

TNBC if ER (-), PR (-), and HER2 (-). TNBC

tumours were further stained with CD44 (DF1485,

dilution 1:100, Novocastra Laboratories Ltd.,

Newcastle upon Tyne, UK), CD24 (C-20, dilution

1:100, Santa Cruz Biotechnology, USA), TWIST-1

(H-81, dilution 1:100, Santa Cruz Biotechnology,

Santa Cruz CA), Claudin-7 (NBPI-35677, Rabbit

polyclonal antibody, dilution 1:100, Novus

Biological), and Vimentin (VIM 3B4, Mouse

monoclonal, 1:400, Dako).

Interpreting immunohistochemical stains of

CD44 and CD24 were based on Ricardo et al.

(2011). CD44, CD24, Twist-1, and Claudin-7 were

stained in membrane cells, but Vimentin in

cytoplasm. CD44, CD24 and Twist-1 were scored as

0 if no staining or only positive in <10% tumour

cells; 1 if 10-25% tumour cells; 2 if 25-50% tumour

cells; and 3 if >50% tumour cells.

Claudin-7

staining was scored as 0 if no membranous staining;

1+ (1-10% tumour cells); 2+ (10-30% tumour cells);

and 3+ (>30% tumour cells).

Meanwhile based on

percentage, Vimentin were scored as 0 if negative

staining; 1 if <30% tumour cells; 2 if 30-60%

tumour cells; and 3 if >60% tumour cells. All

intensity of staining was scored as 0 if unstained, 1 if

weakly stained, 2 if intermediate, and 3 if strong.

Interpretation of CD44, CD24, Twist-1, Claudin-7

and Vimentin staining were determined based on

multiplication of the percentage of positive cells and

the intensity of staining. CD44 and CD24 were

scored as 0 negative if (-), 1-3 (+1), 4-6 (+2), and 7-

9 (+3).

While Twist-1, Claudin-7 and Vimentin

was considered weak if total score <6 and strong if

total score >6.

TNBC was classified as Claudin

low

(

stem cell-like) when CD44

, but if CD44

, then

TNBC was classified as non- stem cell-like

subtypes. Then, stem cell-like and non-stem-cell like

subtypes of TNBC were assessed based on Vimentin

expression.

3 RESULTS

In order to determine ontogeny and differentiation of

TNBC subtypes in stem cells stages, we used CD44

immunohistochemistry stains. In this study,

researchers try to schematically illustrate ontogeny

and differentiation of breast epithelial from stem

cells to luminal cells with various TNBC molecular

markers (Figure 1).

Figure 1 : Ontogeny of stem-cells like, basal, baso-

luminal, and luminal subtypes.

From 67 TNBCs in this study, through CD44,

CD24, Twist-1 and Claudin-7

immunohistochemistry stains, TNBC was classified

as 19 cases of stem-cell like and 48 non stem-cells

like subtypes (Table 1). Postmenopause women with

tumor size more than 5cm, higher stage and grade

histology were likely to have non stem-cells like

subtypes of TNBC (86.7%, 71.6%, 71.7%, and 76%,

respectively). Women with mucinous carcinoma and

metaplastic carcinoma of no special type tended to

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

866

have non stem-cells like subtypes of TNBC (100%

and 100% respectively) (Table 2). Both stem-cells

like and non stem-cells like subtypes of TNBC

commonly had low Vimentin (84.2% and 87.5%,

respectively). Only 3 cases (12.5%) of stem-cells

like and 6 cases (14%) of non stem-cells like

subtypes were found with high Vimentin (Table 3).

4 DISCUSSION

The objective of this study is to identify stem cells in

breast cancer through immunohistochemistry stains.

CSCs or cancer initiating cells (CICs) is a minority

in cell populations derived from transformation of

self-renewing stem cells, which initiations and

maintains growth of cancer cells. There are various

of ‘stemness’ used in identifying BCSCs, for a

couple CD44 and CD24.

17,18

In this study we tried to

schematically illustrate the ontogeny of stem cells

using CD44 and CD24.

BCSCs expresses high

CD44 and negative/low CD24 (CD44

CD24

-/low

CD44

CD24

−/low

fenotype is often related to poor

prognosis.

CD44 is strong to expressed in

immature stem cell and will get weaker on

differentiation, whereas CD24 is strongly expressed

in more mature cells. The present study

demonstrated that the prevalence stem cell like

subtypes was 28,3% of all tumours. This result was

in opposite to Makki, et al. (2015). They found that

the prevalence is 73.7% of all tumours.

Twist displays ‘stemness’ and plays a role in

EMT transformation. Twist is a E-cadherin repressor

protein which stimulates EMT. EMT is

characterized by lack of keratin epithelial and E-

cadherin expression, but expressed Vimentin.

During EMT, epithelial cells lose cell-cell contacts,

and obtain a mesenchymal morphology.

On the

other hand, Claudin, an adhesion molecule found in

untransformed epithelial cells, is strongly positive in

mature differentiated epithelial cells. In this study,

CD44, CD24, Claudin-7 and Twist-1 were used as

molecular markers of TNBC stem cell-like subtypes.

Results from 67 TNBC showed marked

heterogenous and overlapping profiles. To

demonstrate EMT phenomenon, we also applied

vimentin in this study.

Identification of BCSCs gets special attention to

date because of having implications for its treatment.

Standard chemotherapy often fails because BCSCs

have low proliferation and are resistant to

chemotherapy which also cause the enhancement of

stem cells count. This is one important cause of

therapy failure and recurrence in TNBC. Therefore,

validation of stem cells in TNBC is mandatory. This

is one of the critical steps to develop an effective

targeted therapy in TNBC.

Several studies stated that CD44 expression was

related to higher histological grade, tumour growth,

lymph node invasion and visceral metastases.

This

results is in accordance with our study.

Mesenchymal-like CSCs with CD24

CD44

are

primarily quiescent and this condition tends to have

high invasive capacity. EMT may be regulated by

the tumor microenvironment, such as TGFβ and IL-

But in this study, researchers found that only 3

cases (12.5%) of stem-cells like and 6 cases (14%)

of non stem-cells like subtypes were found with high

vimentin.

Table 1 : Classification of stem-cells like subtypes based on immunohistochemistry staining.

No CD44 CD24 Claudin 7 Twist Vimentin Classification of stem cell like

subtype

1 +1 +2 0 3 0 SC

2 +1 0 0 0 2 SC

3 1+ +3 7 0 1 SC

4 1+ +3 8 2 1 SC

5 1+ 0 7 2 2 SC

6 1+ 0 4 3 4 SC

7 1+ 3+ 8 5 9 SC

8 1+ 3+ 8 3 3 SC

9 1+ 0 8 2 2 SC

10 1+ 0 8 2 6 SC

11 1+ 3+ 8 2 0 SC

12 1+ 0 8 0 0 SC

13 1+ 0 8 0 1 SC

14 1+ 3+ 8 2 2 SC

Vimentin Expression in Stem Cell-like Subtypes of Triple Negative Breast Cancer (TNBC)

867

15 1+ 3+ 5 0 4 SC

16 3+ 0 8 0 6 SC

17 2+ 0 8 4 0 SC

18 1+ 0 7 4 1 SC

19 1+ 3+ 8 4 2 SC

20 0 +3 8 4 2 NSC

21 0 +3 8 4 2 NSC

22 0 +1 0 3 2 NSC

23 0 +3 8 0 1 NSC

24 0 +3 8 3 0 NSC

25 0 +3 8 2 1 NSC

26 0 +3 7 0 0 NSC

27 0 0 7 0 1 NSC

28 0 +3 8 4 2 NSC

29 0 +3 6 0 1 NSC

30 0 0 7 0 1 NSC

31 0 0 8 0 2 NSC

32 0 +3 8 0 3 NSC

33 0 +3 0 0 0 NSC

34 0 +3 8 2 0 NSC

35 0 +3 8 0 2 NSC

36 0 +3 8 0 1 NSC

37 0 +3 4 0 3 NSC

38 0 +3 8 03 NSC

39 0 +3 0 0 0 NSC

40 0 2+ 0 0 0 NSC

41 0 3+ 8 2 2NSC

42 0 3+ 7 0 2 NSC

43 0 3+ 0 0 1 NSC

44 0 3+ 8 0 2 NSC

45 0 3+ 7 2 2 NSC

46 0 3+ 8 2 1 NSC

47 0 3+ 7 3 2 NSC

48 0 3+ 8 0 1 NSC

49 0 3+ 8 2 2 NSC

50 0 3+ 7 0 2 NSC

51 0 0 8 3 0 NSC

52 0 3+ 8 4 3 NSC

53 0 0 8 2 9 NSC

54 0 2+ 8 0 2 NSC

55 0 0 8 3 2 NSC

56 0 2+ 8 0 6 NSC

57 0 1+ 8 3 6 NSC

58 0 0 8 3 2 NSC

59 0 3+ 8 0 3 NSC

60 0 0 7 2 9 NSC

61 0 0 8 0 0 NSC

62 0 0 6 6 6 NSC

63 0 3+ 8 0 1 NSC

64 0 0 7 0 2 NSC

65 0 0 5 4 0 NSC

66 0 3+ 7 5 9 NSC

67 0 2+ 8 0 2 NSC

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

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Table 2 : Classification of stem-cells like subtypes based on clinicopathological characteristics.

Variables Classification of stem cell like Total

Stem cell like % Non stem cell like %

Status menopausal

 Premenopause



ostmeno

ause

32.7

13.3

67.3

86.7

Tumor size

 2-5cm

 >5c

33.3

28.1

66.7

71.6

Stage

 II

 III

28.6

28.3

71.4

71.7

Grade

 II

 III

Subtype histology

 IC-NST

30.9 38 69.1 55

 ILC 1 25 3 75 4

 Mucinous carcinoma 0 0 1 100 1

 Carcinoma with

medullary features 1 20 4 80 5

 Metaplastic

carcinoma of no

ecial t

e 0 0 2 100 2

Total 19 28.4 48 71.6 67

Table 3 : Classification of stem-cells like subtypes based

on Vimentin expression.

Classification

of stem cell

Vimentin Total

low % high %

Stem cell like

Non stem cell

84.2

87.5

15.8

12.5

Total 58 86.6 9 13.4 67

5 CONCLUSION

TNBC is a heterogenous breast cancer. By using

immunohistochemical staining panels, TNBC can be

classified as stem cell like and non-stem cell like

subtypes. Stem cell-like subtypes are resistant to

therapy. To classify non-stem cell like subtypes,

other immunohistochemistry stains are needed

Vimentin should be included in

immunohistochemistry staining panels because EMT

is correlated with the invasion of breast cancer cell

and mesenchymal character. The importance of this

study was to identify of stem-cell-ness/stemness

which will influence therapy.

ACKNOWLEDGEMENTS

This study was supported by the Ministry of

Research, Technology, and Higher Education

Republic of Indonesia, University of Sumatera

Utara, Research Institution, Number:

21/UN5.2.3.1/PPM/KP-TALENTA USU/2018.

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