Metastasis Predictors in Luminal Breast Cancer (Kendarini NPY, et al.)
Indones Biomed J. 2025; 17(3): 263-72

DOI: 10.18585/inabj.v17i3.3586

RESEARCH ARTICLE

Serum CD44 Variant 6 to CD44 Ratio and Vascular Endothelial Growth Factor
Levels as Predictors of Metastasis in Luminal Subtype Breast Cancer
Ni Putu Yenny Kendarini1,, Ida Bagus Tjakra Wibawa Manuaba2, I Wayan Sudarsa3,
Tjokorda Gde Bagus Mahadewa4, Ni Nyoman Ayu Dewi5, Prayuda6, Putu Febry Krisna Pertiwi7
1
Division of General Surgery, Balimed Hospital, Jl. Mahendradatta No.57 X, Denpasar, Bali 80119, Indonesia
Division of Surgical Oncology, Prima Medika Hospital, Jl. Raya Sesetan No.10, Denpasar, Bali 80223, Indonesia
3
Division of Surgical Oncology, Department of Surgery, Faculty of Medicine, Universitas Udayana/Prof. dr. I.G.N.G. Ngoerah General Hospital,
Jl. P.B. Sudirman, Denpasar, Bali 80232, Indonesia
4
Department of Neurosurgery, Faculty of Medicine, Universitas Udayana/Prof. dr. I.G.N.G. Ngoerah General Hospital, Jl. P.B. Sudirman, Denpasar,
Bali 80232, Indonesia
5
Department of Biochemistry, Faculty of Medicine, Universitas Udayana/Prof. dr. I.G.N.G. Ngoerah General Hospital, Jl. P.B. Sudirman, Denpasar,
Bali 80232, Indonesia
6
Division of Internal Medicine, Balimed Hospital, Jl. Mahendradatta No.57 X, Denpasar, Bali 80119, Indonesia
7
Faculty of Medicine, Universitas Udayana, Jl. P.B. Sudirman, Denpasar, Bali 80232, Indonesia
2

*Corresponding author. Email: yennykendarinibedah@gmail.com
Received date: Feb 22, 2025; Revised date: May 7, 2025; Accepted date: May 9, 2025

Abstract

B

ACKGROUND: Metastasis is the leading cause of mortality in luminal subtype breast cancer. While cluster of
differentiation (CD)44 has been widely studied, the prognostic relevance of its isoforms particularly the CD44v6/
CD44s ratio remains unclear. This study evaluates CD44v6, CD44s, vascular endothelial growth factor (VEGF), and
the CD44v6/CD44s ratio as potential prognostic biomarkers for metastasis in luminal breast cancer.
METHODS: This case-control study included 38 luminal subtype breast cancer patients (18 with metastasis, 20 without
metastasis). Serum levels of CD44v6, CD44s, VEGF, and the CD44v6/CD44s ratio were measured using Enzyme-Linked
Immunosorbent Assay (ELISA). Statistical analyses included ROC analysis to determine optimal cut-off points, logistic
regression to assess risk factors, and correlation analysis for biomarker relationships.
RESULTS: A low CD44v6/CD44s ratio (<0.03) was identified as a significant independent factor for metastasis (adjusted
OR 7.0, 95% CI: 1.2–40.6, p=0.03). While serum levels of CD44v6, CD44s, and VEGF were higher in the metastasis group,
these individual markers showed a non-significant trend toward association with metastasis. A strong positive correlation was
observed between CD44s and VEGF levels (r=0.7, p<0.01).
CONCLUSION: The CD44v6/CD44s ratio showed a significant association with metastasis and may have potential as a
prognostic marker in luminal breast cancer. Further studies with larger sample sizes are needed to confirm these findings.
KEYWORDS: CD44v6, CD44s, VEGF, CD44v6/CD44s ratio, luminal breast cancer, metastasis
Indones Biomed J. 2025; 17(3): 263-72

Introduction

Breast cancer is the most frequently diagnosed cancer
in women worldwide, with metastasis being the primary
cause of cancer-related mortality. Despite advances in
early detection and treatment, approximately 5-10% of

breast cancer cases present with metastasis at diagnosis.(1)
The luminal subtype, characterized by hormone receptor
positivity, accounts for the majority of breast cancer cases
in Indonesia.(2) During 2015 to 2017, in geriatric clinics at
Prof. Ngoerah General Hospital, it was found that 25% of
breast cancer patients had experienced metastasis. The study
revealed that 25% of breast cancer patients were diagnosed

Copyright © 2025 The Prodia Education and Research Institute.
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International (CC-BY-NC) License.

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at stage IV, indicating a substantial proportion present with
advanced disease.(3) While luminal breast cancers are
generally sensitive to hormonal therapy, their prognosis
remains unpredictable due to the risk of micrometastases.
Liquid biopsy allows for early detection, even at the
micrometastasis level, before macrometastasis occurs.
This non-invasive and minimally invasive diagnostic
methods are preferred over invasive procedures due to their
proven advantages.(4) Plasma-based biomarkers have been
investigated in luminal breast cancer subtypes to detect
molecular and metabolic differences, including amino
acid profiles.(5) This highlights the potential of circulating
markers, such as cluster of differentiation (CD)44 isoforms
and vascular endothelial growth factor (VEGF), as well as
other non-traditional serum components, including mucins,
which have shown promise in distinguishing malignant
from benign breast lesions.(6) Biomarkers such as CD44
variants have emerged as potential tools for detecting
early micrometastasis.(7) The human CD44 gene, located
on chromosome 11p13, comprises 19 exons. Through
alternative splicing of its pre-mRNA, different combinations
of exons are included or excluded, resulting in structurally
distinct protein isoforms. The standard isoform (CD44s)
includes only the constant exons (1–5 and 16–20), whereas
variant isoforms (CD44v) incorporate one or more variable
exons (v1–v10), giving rise to diverse protein forms with
different functional properties. CD44 is considered a
marker associated with cancer stem cells (CSCs), indicating
stemness, or the ability of a cell to self-renew and initiate
tumor growth.(8) CD44v6, a variant isoform of CD44
produced through alternative splicing, is mainly expressed
by epithelial and tumor cells. Its expression can be induced
by inflammatory cytokines, hypoxia, and growth factors
within the tumor microenvironment.(9) CD44v6 may enter
the circulation via proteolytic shedding or extracellular
vesicles. Among CD44 variants, CD44v6 has been most
strongly associated with tumor aggressiveness, including
higher histological grade, lymph node metastasis, and poor
prognosis in breast cancer.(10) VEGF, a pro-angiogenic
factor and prognostic marker in various cancers, including
breast cancer.(11) VEGF is a key angiogenic factor that
plays a crucial role in stimulating angiogenesis and tumor
growth, together with VEGF receptors, activates signaling
pathways essential for tumor-associated blood vessel
formation.(12) In addition to CD44 and VEGF pathways,
pro-inflammatory cytokines such as tumor necrosis factor
(TNF)-α and interferon (IFN)-γ have been shown to induce
apoptosis in breast cancer cells through upregulation of proapoptotic genes such as Bax and p53, and downregulation of

anti-apoptotic gene B-cell lymphoma (Bcl)-2, highlighting
the critical role of immune-mediated mechanisms in tumor
progression.(13)
CD44 isoforms and VEGF play key roles in tumor
progression, but their utility as circulating biomarkers for
micrometastasis detection in luminal breast cancer remains
unclear. Liquid biopsy offers a promising non-invasive tool,
yet no specific serum biomarkers have been validated for
this purpose. Although several studies have evaluated CD44
expression in breast cancer, most focused on either total
CD44 or individual isoforms. The prognostic significance
of the CD44v6/CD44s ratio, which may reflect a shift in
isoform expression linked to tumor aggressiveness, has
not been well studied. Previous research suggests CD44v6
is elevated in metastatic tumors, whereas CD44s may be
more prevalent in less aggressive phenotypes. This study
investigates CD44v6, CD44s, their ratio, and VEGF
to explore their potential as prognostic biomarkers for
metastasis in luminal breast cancer.

264

Methods

Study Design
This study employed a case-control design to investigate
serum biomarkers associated with metastasis in luminal
subtype breast cancer. The research was conducted at
Prof. Ngoerah General Hospital Denpasar, Bali, and
involved a total of 38 subjects, consisting of 18 patients
with confirmed metastasis (cases) and 20 patients without
metastasis (controls). This study was conducted following
the ethical principles outlined in the Declaration of
Helsinki and had been approved by the Ethics Committee
of Prof. Dr. I.G.N.G. Ngoerah General Hospital (No. 2195/
UN14.2.2.VII.14/LT/2022). All subejcts provided written
informed consent before enrolling in the study. They were
given comprehensive information regarding the study’s
objectives, procedures, and their right to withdraw at any
point without consequences to their medical care. To ensure
confidentiality, all patient identifiers were removed.
Inclusion and Exclusion Criteria
The inclusion criteria for cases were female patients aged
30-70 years diagnosed with luminal metastatic subtype
breast cancer who developed metastases during therapy
and received surgical, chemotherapy, and/or hormonal
treatments. Control group patients were those diagnosed
with stage II luminal subtype breast cancer according
to the 8th edition merican Joint Committee on Cancer

DOI: 10.18585/inabj.v17i3.3586

Metastasis Predictors in Luminal Breast Cancer (Kendarini NPY, et al.)
Indones Biomed J. 2025; 17(3): 263-72

(AJCC) tumor, nodes, and metastasis (TNM) criteria
and had undergone surgery, followed by chemotherapy
and/or hormonal therapy.(14) Case group defined as
patients diagnosed with metastatic luminal subtype breast
cancer, where, during the course of treatment, metastasis
occurred, and the patient has undergone therapeutic
modalities, including surgery, chemotherapy, targeted
therapy, or hormonal therapy. Exclusion criteria included
pregnancy, non-luminal subtypes, history of other cancers,
unwillingness to participate, and hemolyzed blood samples.

kit (Cat. No. E0080Hu; BT Lab) with a sensitivity of 10.42
pg/mL and a detection range of 20–6000 pg/mL were used
according to the manufacturer’s instructions. The plates
were first pre-coated with capture antibodies, followed by
sequential incubation with the sample, biotinylated detection
antibodies, and streptavidin-HRP. Colorimetric detection
was performed using substrate solution and measured at
450 nm. All samples were analyzed in duplicate. The intraassay and inter-assay coefficients of variation (CV) for all
kits were <8% and <10%, respectively.

Sample and Data Collection
Three mL of blood samples were collected from each
subjects using standard venipuncture procedures. After
centrifugation, the serum was aliquoted into 0.5 mL
Eppendorf tubes and stored at −80°C until analysis. In addition
to blood collection, relevant clinical and pathological
data were obtained from the patients’ medical records.
Cancer stage was determined according to the 8th edition
of the AJCC TNM classification system. Histopathological
grading data was retrieved from the Pathology Anatomy
Laboratory, based on standard histologic criteria assessing
tubule formation, nuclear pleomorphism, and mitotic count.
Estrogen receptor (ER), progesterone receptor (PR),
human epidermal growth factor receptor 2 (HER2)
status, and Ki-67 index were previously assessed using
immunohistochemistry (IHC) by the Pathology Anatomy
Laboratory. ER and PR positivity were defined as ≥1%
nuclear staining. HER2 status was scored as 0 to 3+; a score
of 3+ was considered positive, and 2+ was followed by
confirmatory testing if available. Ki-67 was reported as a
percentage of positively stained tumor cell nuclei.
Breast cancer subtype was classified as luminal based
on positive ER and/or PR status, with or without HER2
overexpression. The location of metastases was determined
through radiology reports, including ultrasound (USG),
computed tomography (CT), and/or magnetic resonance
imaging (MRI). These findings were confirmed by clinicians
based on imaging results and documented in the clinical
records.

Statistical Analysis
Data analysis included descriptive analysis, correlation tests,
receiver operating characteristic (ROC) analysis, proportion
difference tests, and logistic regression, all conducted using
SPSS version 25.0 (IBM Corporation, Armonk, NY, USA).
Descriptive analysis was used to characterize subjects by
case and control groups, including variables such as breast
cancer status (metastasis or no metastasis), age (≤40 years
or >40 years), histopathological grading (Grade I, II,
III), and CD44v6 expression (high or low). Correlations
between CD44v6, CD44s, and VEGF were evaluated using
Pearson’s test for normally distributed data or Spearman’s
test for non-normal distributions. ROC analysis determined
optimal cutoff points for CD44v6, CD44s, and VEGF by
identifying the farthest point from the diagonal line on the
ROC curve. Subjects were grouped based on metastatic
status, and odds ratios (ORs) were calculated to assess
associations between biomarkers and metastasis. Proportion
difference tests compared risk factor distributions between
case and control groups using cross-tabulations and Chisquare tests to calculate the OR or Crude OR. Logistic
regression assessed independent associations of variables
while controlling for confounders, with results reported
as adjusted OR and significance set at p≤0.05. Correlation
analysis was performed to evaluate the strength and direction
of relationships between variables

ELISA Analysis of CD44, CD44v6, and VEGF
Serum levels of CD44v6, CD44s, and VEGF were measured
using sandwich Enzyme-Linked Immunosorbent Assay
(ELISA) methods. The CD44v6 kit (Cat. No. E7578Hu; BT
Lab, Jiaxing, China) with sensitivity of 0.12 ng/mL and a
standard curve range of 0.25–16 ng/mL, the CD44s kit (Cat.
No. E0239Hu; BT Lab) with a sensitivity of 2.5 ng/mL and
a standard curve range of 5–1500 ng/mL, and the VEGF

Baseline Characteristics
This study involved 38 subjects with luminal subtype breast
cancer, including 18 cases (metastasis, clinical stage IV)
and 20 controls (non-metastasis, clinical stage II). The
mean age of participants was 50 years, and most tumors
were classified as histopathological grade II. ER and PR
positivity were prevalent, while HER2-negative status was
dominant in both groups. Higher Ki67 levels (≥20%) were

Results

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observed more frequently in cases, with Luminal B subtype
being more common. The detailed characteristics were
presented in Table 1.

with higher levels (≥4 ng/mL) (OR 2.4, 95% CI: 0.65–
9.13, p=0.19). However, this result was not statistically
significant.

Cut-off Points for Study Variables
Before conducting bivariate analysis, the variables
CD44v6, CD44s, VEGF, and the CD44v6/CD44s ratio were
categorized based on cut-off points determined using ROC
curves. Optimal cut-off points were identified using ROC
analysis: 4 ng/mL for CD44v6, 119.2 ng/mL for CD44s,
340 ng/mL for VEGF, and 0.034 for CD44v6/CD44s ratio
(Figure 1). The distribution of baseline characteristics and
biomarker were provided in Table 2.

Serum CD44s Levels and the Risk of Metastasis
Subjects with high serum CD44s levels (≥119.2 ng/mL)
had a 2.4 times higher risk of metastasis compared to
those with low levels (<119.2 ng/mL) (OR 2.4, 95% CI:
0.6–8.6, p=0.19). However, this result was not statistically
significant.

Serum CD44v6 Levels and the Risk of Metastasis
Subjects with low serum CD44v6 levels (<4 ng/mL) had
a 2.4 times higher risk of metastasis compared to those

Serum CD44v6/CD44s Ratio and the Risk of Metastasis
Participants with a low CD44v6/CD44s ratio (<0.03) had a
significantly higher rate of metastasis (88.9%) compared to
those with a high ratio (≥0.03), who had a metastasis rate of
11.1% (p<0.05). A low CD44v6/CD44s ratio was associated
with a 6.5 times greater risk of metastasis compared to a

Table 1. Baseline haracteristics of study subjects.
Variable
Age (year), mean±SD
Grade, n (%)

Control
(n=20)

50.8±11.1

50.5±9.0

Grade 1

0

3 (15)

Grade 2

12 (66.7)

12 (60)

Grade 3
Estrogen Receptor, n (%)

6 (33.3)

5 (25)

Positive
Negative
Progesterone Receptor, n (%)

18 (100)
0

19 (95)
1 (5)

Positive
Negative
HER2, n (%)

15 (83.3)
3 (16.7)

15 (75)
5 (25)

Positive
Negative
Ki67, n (%)

4 (22.2)
14 (77.8)

5 (25)
15 (75)

<20
≥20
Subtype, n (%)

6 (33.3)
12 (66.7)

9 (45)
11 (55)

Luminal A
Luminal B
Metastasis Location, n (%)

3 (16.7)
15 (83.3)

7 (35)
13 (65)

Lung

7 (38.9)

Bone
Liver
Lung and Bone
Liver and Brain
Lung and Liver
CD44v6 (ng/mL), median (min-max)

2 (11.1)
2 (11.1)
4 (22.2)
1 (5.6)
2 (11.1)
3.9 (2.3-12)

4.3 (2.5-18.8)

CD44s (ng/mL), median (min-max)
VEGF (ng/mL), median (min-max)

127.3 (86.9-893.6)

116.4 (94.1-976.81)

431(270.9-800)
0.0291±0.0066

381(243-792.6)
0.0305±0.0083

CD44v6/CD44s ratio, mean±SD

266

Case
(n=18)

Metastasis Predictors in Luminal Breast Cancer (Kendarini NPY, et al.)
Indones Biomed J. 2025; 17(3): 263-72

DOI: 10.18585/inabj.v17i3.3586

1.0

Source of the curve
CD44V6
VEGF
CD44s
CD44V6/CD44s Ratio
CD44s/CD44V6 Ratio
Reference Line

Sensitivity

0.8
0.6
0.4
0.2

Figure 1. ROC curve to determine the cut-off points
for CD44v6, CD44s, VEGF, CD44v6/CD44s ratio,
and CD44s/CD446v ratio.

0

0.2

0.4

0.6

0.8

1.0

1 - Specificity

high ratio (OR 6.5, 95% CI: 1.2–36.2, p=0.02). These
findings are statistically significant and reflect a realworld association, as indicated by the confidence interval
not crossing 1. This study was the first ones to specifically
identify the CD44v6/CD44s ratio as a significant risk factor
for metastasis in breast cancer. The results suggested that
the lower the CD44v6/CD44s ratio, the greater the risk of
metastasis in luminal subtype breast cancer. This highlights
the potential clinical relevance of the CD44v6/CD44s ratio
as a biomarker for predicting metastasis.

Table 2. Baseline characteristics and
distribution of biomarker levels in study
subjects.

Serum VEGF Levels and the Risk of Metastasis
Subjects with high serum VEGF levels (≥340 ng/mL) had
a 4.3 times higher risk of metastasis compared to those
with low VEGF levels (<340 ng/mL) (OR 4.3, 95% CI:
0.8–24.4, p=0.13). However, this result was not statistically
significant.
Association of CD44v6, VEGF, CD44s, CD44v6/CD44s
Ratio and Clinicopathological Characteristics
CD44v6 expression showed no statistically significant
difference between tumor grade groups or ER status.
However, a trend towards higher CD44v6 expression was
observed in HER2-positive tumors compared to HER2negative tumors. Similarly, CD44v6 expression was higher
in Luminal B tumors compared to Luminal A, but this
difference was not statistically significant (Table 3).
CD44s expression showed no significant association
with tumor grade, ER status, PR status, or HER2 status. The
expression of CD44s between Luminal A and Luminal B
subtypes also showed no significant difference (Table 3).
A significant difference in CD44v6/CD44s ratio was
observed between tumor stages (p=0.02), suggesting a

Variable

n (%)

Stage
Stage II
Stage IV
Grade

20 (52.6)
18 (47.4)

Grade 1
Grade 2
Grade 3
Estrogen Receptor

3 (7.9)
24 (63.2)
11 (28.9)

Positive
Negative
Progesterone Receptor

37 (97.4)
1 (2.6)

Positive

30 (78.9)

Negative
HER2

8 (21.1)

Positive
Negative
Ki67

9 (23.7)
29 (76.3)

<20
≥20
Subtype

15 (39.5)
23 (60.5)

Luminal A
Luminal B
CD44v6 (ng/mL)

10 (26.3)
28 (73.7)

<4

21 (73.7)

≥4
CD44s (ng/mL)

17 (26.3)

≥119.2
<119.2
VEGF (ng/mL)

19 (50)
19 (50)

≥340
<340
CD44v6/CD44s ratio

29 (76.3)
9 (23.7)

<0.034
≥0.034

27 (71.1)
11 (28.9)

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Table 3. Association between CD44v6, VEGF, CD44s, and CD44v6/CD44s ratio with clinicopathological characteristics.
CD44v6

Characteristic

CD44s

p -value

<4

≥4

Stadium
Std. 2
Std. 4

9
12

11
6

Grade
Grade 1
Grade 2
Grade 3

2
14
5

1
10
6

ER Status
Positive
Negative

20
1

PR Status
Positive

p -value

≥119.2

<119.2

8
11

12
7

0.71

1
11
7

17
0

0.36

0.25

0.18

CD44v6/CD44s
Ratio
<0.03

≥0.03

0.19

11
9

9
2

2
13
4

0.51

2
18
7

19
0

18
1

0.31

15

15

1.00

4

4

VEGF

p -value

p -value

≥340

< 340

0.02*

13
16

7
2

0.08

1
6
4

0.77

2
17
10

1
7
1

0.39

27
0

10
1

0.11

28
1

9
0

0.57

22

8

0.54

23

7

0.92

5

3

6

2

18

12

Negative
HER2 Status

3

5

Positive
Negative

3
18

6
11

0.13

6
13

3
16

0.25

7
20

2
9

0.61

7
22

2
7

0.90

10
11

5
12

0.25

6
13

9
10

0.31

11
16

4
7

0.80

12
17

3
6

0.66

5
16

5
12

0.69

5
14

5
14

1.00

7
20

3
8

0.93

7
22

3
6

0.58

Ki67%
<20%
≥20%
Subtype
Luminal A
Luminal B

*Significant if p<0.05, analyzed with Chi-square tests.

potential role of this ratio in tumor progression. However,
no significant differences were found in relation to tumor
grade, ER status, PR status, HER2 status, or Ki67 levels.
The CD44v6/CD44s ratio did not significantly differ
between Luminal A and Luminal B subtypes (Table 3).
VEGF levels (≥340 vs. <340) were not significantly
different across tumor grade, ER status, PR status, or HER2
status. A borderline association was noted with tumor stage,
where VEGF expression was slightly higher in Stadium 2
tumors compared to Stadium 4, eventhough not statistically
significant (Table 3).
These findings suggest that CD44v6/CD44s ratio may
have prognostic value in tumor staging, whereas CD44v6
and VEGF show trends in association with HER2 status
and tumor stage, respectively. However, no significant
associations were observed with other clinicopathological
parameters.
Multivariate Analysis
Following bivariate analysis of the independent and
dependent variables, multivariate analysis was performed
to evaluate the relationship between independent variables

268

and confounders with the dependent variable. The analysis
identified a low CD44v6/CD44s ratio (<0.03) as an
independent risk factor for metastasis after controlling
for the confounding effect of the breast cancer subtype
(Table 4). The adjusted OR was 7.0, with a statistically
significant result (p=0.03) and a confidence interval (CI)
that did not cross 1. These findings indicate that patients
with a low CD44v6/CD44s ratio have a 7-fold higher risk
of developing metastasis compared to those with a higher
ratio. This highlights the significant role of the CD44v6/
CD44s ratio as a predictor of metastasis in luminal subtype
breast cancer.
Correlations between CD44v6, CD44s, CD44v6/CD44s
ratio and VEGF
The correlation analysis between CD44v6, CD44s, VEGF,
and the CD44v6/CD44s ratio was assessed using Pearson’s
(r) and Spearman’s (ρ) correlation tests, depending on data
normality (Table 5).
A strong positive correlation was observed between
CD44v6 and CD44s (ρ=0.7, p<0.01). Similarly, CD44s was
strongly correlated with VEGF (ρ=0.7, p<0.01), indicating a

Metastasis Predictors in Luminal Breast Cancer (Kendarini NPY, et al.)
Indones Biomed J. 2025; 17(3): 263-72

DOI: 10.18585/inabj.v17i3.3586

Table 4. Association of serum CD44v6, CD44s, VEGF levels, and CD44v6/
CD44s ratio with metastasis risk in luminal subtype breast cancer.
Variable

Adjusted OR

95%CI

Adjusted P

Age

1.0

0.93 – 1.1

0.78

Subtype

0.3

0.06 – 1.7

0.19

Grade

0.7

0.12 – 4.3

0.72

VEGF

2.4

0.32 – 18

0.39

CD44v6/CD44s ratio

7.0

1.22 – 40.6

0.03*

*Significant if p<0.05, analyzed with logistic regression.

possible role for CD44s in angiogenesis-related processes. A
moderate correlation was also found between CD44v6 and
VEGF (ρ=0.4, p=0.01), further supporting the involvement
of CD44 variants in tumor vascularization. Conversely, the
CD44v6/CD44s ratio exhibited negative correlations with
all tested markers. A moderate negative correlation was
observed between the CD44v6/CD44s ratio and CD44s (r=0.6, p<0.01), indicating that a higher CD44v6/CD44s ratio
was associated with lower CD44s expression. Additionally,
a weak to moderate negative correlation was found between
the CD44v6/CD44s ratio and VEGF (r=-0.4, p<0.01),
suggesting that tumors with a higher CD44v6/CD44s ratio
may exhibit lower VEGF expression and possibly rely
on non-angiogenic mechanisms for tumor progression.
A similar weak negative correlation was noted between
the CD44v6/CD44s ratio and CD44v6 (r=-0.4, p=0.02),
which may indicate a shift in the balance of CD44 isoform
expression.
No previous studies have specifically examined the
correlation between CD44s and VEGF in luminal subtype
breast cancer. This study identified a strong positive
correlation between these biomarkers, suggesting a potential
interaction in tumor progression. Higher VEGF levels were

accompanied by an increase in CD44s levels, supporting
their role in metastasis.

Discussion

The release of soluble CD44 is thought to regulate CD44
surface expression and is associated with lymphocytes.(15)
Previous studies have shown a positive correlation between
CD44 expression in primary tumors and its soluble form
in serum.(16) In this study, it was found that low serum
CD44v6 levels might indicate a risk of metastasis, though the
association was not statistically significant. Contradictory
findings in earlier studies report elevated serum CD44v6
levels in metastatic patients, potentially due to differences
in sample distribution or the cut-off points used. For
example, previous reported study identified elevated serum
CD44v6 in cases with liver and bone metastasis, while our
study primarily involved patients with lung metastasis.(17)
Additionally, the low cut-off point of 4 ng/mL in this study
differs from other studies reporting higher cut-off points.
High serum CD44s levels were associated with breast
cancer metastasis in this study, though not statistically

Table 5. Correlations between CD44v6, CD44s, CD44v6/CD44s ratio and
VEGF.
ρ

p -value

CD44v6 and CD44s

0.7

0.00*

CD44v6 and VEGF

0.4

0.01*

CD44s and VEGF

0.7

0.00*

Compared Variables

r

CD44v6/CD44s ratio and CD44v6

-0.4

0.02*

CD44v6/CD44s ratio and CD44s

-0.6

0.00*

CD44v6/CD44s ratio and VEGF

-0.4

0.00*

*Significant if p<0.05, r was analyzed with Pearson’s, while ρ was analyzed with
Spearman’s Correlation test.

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Print ISSN: 2085-3297, Online ISSN: 2355-9179

significant. While CD44s is known to act as a tumor
suppressor in certain cancers like fibrosarcoma and
prostate cancer, some studies have shown increased CD44s
expression in higher-grade breast tumors.(18) CD44s
expression increased significantly in grade 2 and 3 breast
tumors compared to grade 1 tumors.(18) This contextdependent role of CD44s might explain its association with
metastasis in this study.
This study is the first to identify the CD44v6/CD44s
ratio as a statistically significant risk factor for metastasis
in luminal subtype breast cancer (adjusted OR=7, p<0.05).
The lower the ratio, the higher the risk of metastasis. The
low CD44v6/CD44s ratio observed in this study can be
explained through the isoform splicing mechanisms of
CD44. In a study investigating isoform switching of CD44
during epithelial-mesenchymal transition (EMT) in human
and mouse epithelial cells, as well as in breast cancer
progression, it was shown that epithelial-mesenchymal
transition (EMT) induces a shift in CD44 expression,
transitioning from variant isoforms (CD44v) to the standard
isoform (CD44s) in both in vitro and in vivo models.(18)
This isoform switching to CD44s is critical for cells to
undergo EMT and is essential for breast tumor formation.
The transition is regulated by epithelial splicing regulatory
protein 1 (ESRP1), a splicing factor that promotes
alternative splicing of CD44 to produce CD44v isoforms.
During EMT, ESRP1 mRNA levels significantly decrease,
leading to the downregulation of CD44v and upregulation
of CD44s. This finding establishes a strong link between
isoform switching and EMT, highlighting the critical role
of ESRP1 in regulating CD44 isoform expression and its
impact on breast cancer development.(18) This mechanism
supports the significance of the low CD44v6/CD44s ratio
as a potential biomarker for breast cancer metastasis and its
association with tumor progression.
Our finding shows high VEGF levels (≥340 ng/mL)
were associated with a 4.3-fold increased risk of metastasis,
but the result was not statistically significant. Several
studies have confirmed that serum VEGF is associated with
breast cancer, with higher concentrations generally detected
in advanced-stage or metastatic disease compared to earlystage breast cancer.(19–23) Previous study demonstrated
that higher VEGF levels correlate with poorer clinical
outcomes and shorter progression-free survival in metastatic
breast cancer patients.(23) The non-significant finding in
this study may be due to the small sample size.
This study identified a strong positive correlation
between CD44s and VEGF (r=0.7, p<0.01). Previous
research supports this strong positive correlation, showing

that CD44s enhances VEGF expression through the HIF1α pathway, thereby promoting tumor angiogenesis.(24)
CD44s also modulates cancer invasion and proliferation via
the PI3K/AKT pathway, which can independently regulate
VEGF secretion.(25,26) This interplay between CD44s
and VEGF underscores their combined role in cancer
progression and metastasis.
This study introduces two novel findings that
contribute to the understanding of metastasis in luminal
subtype breast cancer. First, it identifies the low CD44v6/
CD44s ratio as a significant risk factor for metastasis, a
discovery not previously reported in breast cancer research.
This finding highlights the potential utility of the CD44v6/
CD44s ratio as a biomarker for metastasis, particularly in
luminal subtypes. Second, the study establishes a strong
positive correlation between CD44s and VEGF levels in
luminal breast cancer cases, a relationship that has not been
documented in earlier studies.
Despite these novel contributions, this study has
several limitations. The sample consisted of patients who
had undergone surgery and no longer had primary tumors,
contrasting with previous studies that included samples with
primary tumors, which may explain differences in findings.
Additionally, the study focused solely on two CD44 isoforms,
CD44s and CD44v6, while other studies have investigated a
broader range of CD44 isoforms for a more comprehensive
analysis. Genomic Expression Profiling (GEP), a critical
tool for prognostic evaluation, was not utilized in this
study. The timing of sample collection, which occurred
after surgery or adjuvant therapy, may have influenced the
findings; a cohort study assessing variables before and after
therapy could provide more clinically relevant insights.
Furthermore, conventional diagnostic methods were
used to determine metastasis, whereas advanced imaging
techniques like positron emission tomography (PET) scans,
unavailable at the research site, would have provided more
accurate assessments.

270

Conclusion

The findings suggest that a lower CD44v6/CD44s ratio
may be associated with an increased risk of metastasis,
indicating its potential utility as a prognostic marker. A
strong positive correlation between CD44s and VEGF
further supports the interplay between stemness markers and
angiogenic signaling in tumor progression. However, given
the relatively small sample size and the absence of tissue
validation or external cohort replication, these findings

DOI: 10.18585/inabj.v17i3.3586

should be interpreted with caution. Further studies with
larger, independent populations and integrated molecular
analyses are needed to validate the prognostic relevance
of these markers and their possible role in guiding clinical
management.

Acknowledgments

The authors would like to thank the Faculty of Medicine,
Udayana University, and Prof. Ngoerah General Hospital,
Denpasar, Bali, for providing the facilities and resources
necessary for this study. We are also grateful to the laboratory
staff for their assistance in histopathological preparations
and technical support.

Authors Contribution

NPYK and TGBM were involved in concepting and
planning the research, IBTWM and IWS performed the data
acquisition/collection, NPYK and NNAD calculated the
experimental data and performed the analysis, NPYK and
IBTWM drafted the manuscript and designed the figures,
IWS aided in interpreting the results. P contributed to
literature review and final data verification. PFKP assisted
in coordinating the project workflow and administrative
tasks. All authors took part in giving critical revision of the
manuscript.

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