Original Study

High ASMAþ Fibroblasts and Low Cytoplasmic

HMGB1þ Breast Cancer Cells Predict
Poor Prognosis
Kamolporn Amornsupak,1 Pranisa Jamjuntra,2 Malee Warnnissorn,3
Pornchai O-Charoenrat,4 Doonyapat Sa-nguanraksa,4 Peti Thuwajit,2
Suzanne A. Eccles,5 Chanitra Thuwajit2
Abstract
Breast cancer is a major health problem in Thailand as the first-ranked cancer in Thai women. Using the
prognostic markers in both cancer cells and tumor stroma are of benefit for providing an accurate prediction of
disease progression. We found that high ASMAD fibroblasts and low HMGB1 in cancer cells are the most
reliable predictors of metastatic relapse.
Introduction: The influence of cancer-associated fibroblasts (CAFs) and high mobility group box 1 (HMGB1) has been
recognized in several cancers, although their roles in breast cancer are unclear. The present study aimed to determine
the levels and prognostic significance of a-smooth muscle actin-positive (ASMAþ) CAFs, plus HMGB1 and receptor
for advanced glycation end products (RAGE) in cancer cells. Materials and Methods: A total of 127 breast samples,
including 96 malignant and 31 benign, were examined for ASMA, HMGB1, and RAGE by immunohistochemistry. The
c2 test and Fisher’s exact test were used to test the association of each protein with clinicopathologic parameters. The
Kaplan-Meier method or log-rank test and Cox regression were used for survival analysis. Results: ASMAþ fibroblast
infiltration was significantly increased in the tumor stroma compared with that in benign breast tissue. The levels of
cytoplasmic HMGB1 and RAGE were significantly greater in the breast cancer tissue than in the benign breast tissues.
High ASMA expression correlated significantly with large tumor size, clinical stage III-IV, and angiolymphatic and
perinodal invasion. In contrast, increased cytoplasmic HMGB1 correlated significantly with small tumor size, pT stage,
early clinical stage, luminal subtype (but not triple-negative subtype), and estrogen receptor and progesterone
receptor expression. The levels of ASMA (hazard ratio, 14.162; P ¼ .010) and tumor cytoplasmic HMGB1 (hazard ratio,
0.221; P ¼ .005) could serve as independent prognostic markers for metastatic relapse in breast cancer patients. The
ASMA-high/HMGB1-low profile provided the most reliable prediction of metastatic relapse. Conclusion: We present
for the first time, to the best of our knowledge, the potential clinical implications of the combined assessment of
ASMAþ fibroblasts and cytoplasmic HMGB1 in breast cancer.

Clinical Breast Cancer, Vol. 17, No. 6, 441-52 ª 2017 The Authors. Published by Elsevier Inc. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: a-Smooth muscle actin, Breast cancer, High mobility group box 1, Metastasis-free survival, RAGE

Introduction proportion of cases remain resistant to treatment and patients develop

Breast cancer is the leading malignancy affecting women worldwide, disease relapse.3,4 Metastasis is still the major cause of treatment failure
including Thai women.1,2 Although early detection and innovative and death.3,5 Sensitive and specific prognostic markers that can predict
treatment regimens are leading to improved survival, a significant the risk of metastasis in patients with breast cancer are still needed.

1
Department of Immunology, Graduate Program in Immunology Submitted: Dec 28, 2016; Revised: Apr 4, 2017; Accepted: Apr 6, 2017; Epub: Apr
2
Department of Immunology 21, 2017
3
Department of Pathology
4
Division of Head, Neck and Breast Surgery, Department of Surgery, Faculty of Address for correspondence: Chanitra Thuwajit, PhD, Department of Immunology,
Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand Faculty of Medicine, Siriraj Hospital, Mahidol University, 2 Wanglang Road, Bangkok
5
Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, 10700, Thailand
Sutton, United Kingdom E-mail contact: cthuwajit@yahoo.com

- 441
1526-8209/ª 2017 The Authors. Published by Elsevier Inc. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
http://dx.doi.org/10.1016/j.clbc.2017.04.007 Clinical Breast Cancer October 2017
 ASMA and HMGB1 in Breast Cancer
The tumor microenvironment encompasses cellular and noncel-
lular components that significantly influence breast cancer cell
behavior through soluble and cell-mediated interactions.6 Fibro-
blasts constitute most stromal cells within breast carcinoma.7 The
effect of activated fibroblasts or cancer-associated fibroblasts (CAFs)
in breast cancer progression and metastasis has been increasingly
recognized.7,8 a-Smooth muscle actin-positive (ASMAþ) spindle-
shaped cells9 have been traditionally defined as CAFs.8 The
increased ASMA expression in stromal cells in breast cancer patients
was shown to correlate with a poor overall survival rate, although the
number of studied cases was low.10 Moreover, CAFs have been
proposed to promote drug resistance in breast cancer cells.11,12
Soluble molecules in the tumor stroma have also been established
as potential players in cancer progression and metastasis. High
mobility group box 1 (HMGB1), an abundant non-histone nuclear
protein produced by both tumor and stromal cells, has been
implicated in various intracellular biologic processes, including
transcription and DNA repair.13 HMGB1 can also be liberated into
the microenvironment by either active secretion from stressed cells
or passive release from damaged or dying cells.13 Secreted HMGB1
is considered as a prototypic “damage-associated molecular pattern”
molecule or as a danger signal eliciting immune responses.14 In
addition, HMGB1 has been increasingly recognized as an extra-
cellular component involved in several pathologic conditions
(including inflammation and cancer) by way of the receptor for
advanced glycation end products (RAGE).15,16 RAGEeHMGB1
interactions reportedly promoted tumor proliferation, metastatic
ability, and regrowth after chemotherapy in glioma, gastric cancer,
and colon cancer models.17-19
Although overexpression of HMGB1 has been detected in breast
cancer,20,21 its function remains controversial owing to its apparent
conflicting tumor-promoting and tumor-suppressive roles.13 For
example, nuclear HMGB1 has been shown to modulate telomere
homeostasis leading to a decrease in radiosensitivity.22 In contrast, it
has been reported that nuclear HMGB1 suppressed breast cancer
cell proliferation in vitro and reduced tumor growth in vivo in an
RB-dependent fashion.23 Moreover, HMGB1 released by hypoxic
stress induced tumor cell invasion by RAGE-induced nuclear factor-
kB nuclear accumulation, matrix metallopeptidase-2 and matrix
metallopeptidase-9 activation,24 and promoted doxorubicin resis-
tance by autophagy induction.11 Furthermore, extracellular
HMGB1 contributed to chemotherapy-induced antitumor immune
responses by binding to toll-like receptor 4 on dendritic cells,25
which was explained by the induction of antigen presentation
leading to activation of tumor-specific T cells.
The prognostic significance of HMGB1 expression in breast
cancer patients remains controversial. Sun et al20 showed that
increased nuclear HMGB1 expression in breast tissues correlated
significantly with differentiation grade, TNM stage, and lymphatic
metastasis in 56 breast cancer cases. In contrast, in a different study,
HMGB1þ breast cancer patients had a significantly better outcome
after adjuvant anthracycline-based chemotherapy.26 The combined
expression of nuclear HMGB1 and the autophagy marker LC3B
was significantly associated with prolonged metastasis-free survival
(MFS). The combination of high HMGB1 with other molecules
such as the nuclear cell cycle-associated protein geminin was linked
to significantly shorter MFS in patients with triple-negative breast
442 - Clinical Breast Cancer October 2017
cancer (TNBC) than in those with low expression.27 Recently, it has
been shown that HMGB1 expression in lung cancer cells can be
induced by diffusible factors from surrounding activated
fibroblasts.28
RAGE has been accepted as a key receptor for HMGB1 on
tumor cells.29 RAGE was upregulated in breast cancer tissues and
associated with poor prognostic parameters, including distant
metastasis.30 In support of these findings, RAGE expression was
associated with highly aggressive and metastatic breast cancer,
including the TNBC subtype.31 Using both HMGB1 and RAGE as
prognostic markers has been investigated in several malignancies
such as colorectal32 and prostate,33 but not in breast, cancer. In a
study of 51 breast cancer cases, patients with high HMGB1 levels
and low RAGE levels in the serum had no response to neoadjuvant
chemotherapy.34 According to our review of the published data, no
studies have been reported concerning the relationship between the
expression levels of HMGB1 and RAGE in breast cancer or stromal
tissues and their potential as prognostic markers.
The present study explored the clinical implications of ASMA
expression (as a marker of activated fibroblasts) and the expres-
sion of HMGB1 and RAGE and/or their combination in breast
tumor tissues. Thai breast cancer patients (n ¼ 96) were inves-
tigated for these 3 protein markers to establish whether any
individual protein or combination would correlate with the
clinicopathologic parameters and predict the probability of
metastatic relapse.
Materials and Methods
Patients and Specimens
A total of 96 cases of invasive ductal carcinoma, including 15
cases of inflammatory breast cancer (IBC) and others defined in the
present study as “non-IBC,” were included in the present study.
The mean patient age was 50.2  11.3 years. All paraffin-embedded
tissues were obtained from patients with a diagnosis of primary
invasive breast cancer undergoing surgery at Siriraj Hospital,
Mahidol University Bangkok from 2006 to 2011. All patients were
preoperatively chemotherapy and radiotherapy naive. We used 31
benign lesions, including tissue samples from 19 cases of breast
fibroadenoma and 12 cases of breast abscess, were used as negative
(nonmalignant) controls. The clinicopathologic data for each
patient were collected to analyze their correlation with HMGB1,
RAGE, and ASMA expression (Table 1). The MFS was observed by
following up the patients for 5 years or until death. All tissue
samples with their clinicohistopathologic records were obtained
from the pathology department (Faculty of Medicine, Siriraj
Hospital, Mahidol University) under approval of the Siriraj insti-
tutional review board (COA no. si274/2010).
Sample Size Calculation
The minimal number of cases for immunohistochemical (IHC)
staining of ASMA, HMGB1, and RAGE were calculated from the
sample size calculation formula according to the prevalence of each
protein in human breast cancer tissues previously reported
(ie, 28.15%,10 75%,35 and 76%,30 respectively). A simplified for-
mula was used: n ¼ Z2  P  (1  P)/d2. In the present study, a Z
score of 1.96, P values of .2815, .75, and .76, and d of 0.1, which
showed that 78, 72, and 71 samples were required for ASMA,
 Kamolporn Amornsupak et al
IHC Examination of ASMA, HMGB1, and RAGE
Table 1 Demographic Patient Data
Paraffin-embedded benign and malignant breast tissues were
Characteristic n (%) baked at 60 C overnight and then deparaffinized in xylene. The
IDC patients 96 (100) dewaxed tissues were rehydrated sequentially in decreasing concen-
Non-IBC 81 (84.4) trations of ethanol and rinsed in running tap water and distilled
IBC 15 (15.6) water according to a standard protocol. Heat-induced antigen
retrieval was performed in a 95 C water bath for 1 hour by incu-
Age (y)
bation with Tris/ethylenediaminetetraacetic acid buffer (pH 9.0) for
50 54 (56.3)
HMGB1 and RAGE staining and with citrate buffer (pH 6) for
>50 42 (43.8)
ASMA staining. Endogenous peroxidase was blocked by 1%
Tumor size (mm)
hydrogen peroxide in methanol for 30 minutes at room temperature
20 38 (39.6)
followed by blocking nonspecific-binding sites with 2% bovine
>20 58 (60.4) serum albumin for 30 minutes at room temperature. The sections
pT stage were subsequently incubated with 1:100 rabbit anti-HMGB1
T1-T2 88 (87.5) (Ab18256; Abcam, Cambridge, MA), 1:200 goat anti-RAGE anti-
T3-T4 12 (12.5) body (sc-8229; Santa Cruz Biotechnology, Santa Cruz, CA), or
pN stage 1:2000 mouse anti-ASMA (A5228; Sigma-Aldrich, St. Louis, MO)
N0 36 (37.5) overnight at 4 C. Secondary antibodies, including EnVisionþ
N1-N3 64 (62.5) System-horseradish peroxidase (HRP)-labeled polymer anti-rabbit
Histologic grade (K4003; Dako, Carpinteria, CA), EnVisionþ System HRP-labeled
WD/MD 57 (59.4) polymer anti-mouse (K4001; Dako), and HRP-conjugated rabbit
PD 39 (40.6) anti-goat IgG (HAF017; R&D Systems, Minneapolis, MN), were
Clinical stage used for HMGB1, RAGE, and ASMA, respectively. The immuno-
reactive signal was developed by Liquid DABþ Substrate Chromogen
I-II 52 (54.2)
System (K3467; Dako) and counterstained with hematoxylin. The
III-IV 44 (45.8)
tissue slides were dehydrated, mounted, and analyzed further using a
LN metastasis
Aperio ScanScope scanner (Leica Biosystems, Buffalo Grove, IL).
No 50 (52.1)
Yes 46 (47.9)
Evaluation of IHC Staining
Angiolymphatic invasion The sections were semiquantitatively evaluated and scored by 2
No 64 (66.7) investigators, 1 of whom was a board-certified pathologist; both
Yes 32 (33.3) were unaware of the clinical parameters. To grade the IHC results,
Perinodal invasion the staining intensity was recorded on a scale of 0 to 3 (0, negative;
No 79 (82.3) 1, weak; 2, moderate; and 3, strong). The proportion of positively
Yes 17 (17.7) stained cells in relation to the whole tumor-involved area was scored
Tumor subtype as 0, 1 (1%-25%), 2 (26%-50%), 3 (51%-75%), and 4 (> 75%).
TN 21 (21.9) The expression of ASMA was graded as the percentage of ASMA-
HER2þ 11 (11.5) positive fibroblasts in the stromal compartment of the lesion.
Luminal 64 (66.7) HMGB1 expression was scored as the intensity of cytoplasmic
ER
HMGB1 in cancer cells compared with that of mammary cells in
normal adjacent areas in breast cancer tissue or benign breast tissue.
Negative 33 (34.4)
RAGE expression in tumor cells was calculated as the sum of the
Positive 63 (65.6)
staining intensity and proportion of RAGEþ cancer cells compared
PR
with normal mammary cells in benign breast tissues. The total
Negative 39 (40.6)
RAGE expression score ranged from 0 to 7, and the total score for
Positive 57 (59.4)
ASMA and HMGB1 expression was 0 to 4 and 0 to 3, respectively.
HER2 Using these criteria, the final score was divided into low and high
Negative 53 (55.2) expression groups according to the cutoff of 5, 3, and 3 for RAGE,
Positive 33 (34.4) ASMA, and cytoplasmic HMGB1, respectively. These were the
Equivocal 10 (10.4) mid-values of the IHC score for each protein. The correlation of
each factor with the clinicopathologic parameters and the effect on
Abbreviations: ER ¼ estrogen receptor; IBC ¼ inflammatory breast cancer; IDC ¼ invasive
ductal carcinoma; LN ¼ lymph node; MD ¼ moderately differentiated; PD ¼ poorly differ- MFS of the patients was analyzed.
entiated; PR ¼ progesterone receptor; TN ¼ triple negative; WD ¼ well differentiated.
Statistical Analysis
HMGB1, and RAGE examination, respectively. Therefore, the The c2 test or Fisher’s exact test was used to analyze the clin-
sample size in the present study met the requirements and was icopathohistologic relevance of ASMA, RAGE, and cytoplasmic
powered to prove or disprove our hypothesis. HMGB1 in the breast cancer tissues. The effect of expression on

Clinical Breast Cancer October 2017 - 443

 ASMA and HMGB1 in Breast Cancer
MFS was plotted using the Kaplan-Meier method and log-rank test.
MFS was defined as the interval between the date of diagnosis and
the date of the first distant metastasis or death. Patients who were
alive and relapse free at the last contact with a follow-up period of 
5 years were censored at the last follow-up visit. Patients with a
follow-up period of < 5 years were excluded from the MFS analysis.
Univariate and multivariate Cox regression analyses of potential
factors were performed to identify the significant predictors for
metastatic relapse. For multivariate Cox regression analysis, different
methods for predictor selection (independent variable selection)
were used, including the forced-entry method and the forward-
stepwise method. All the statistical analyses were performed using
SPSS, version 17.0, statistical software. P < .05 was considered
statistically significant.
Results
ASMA, Cytoplasmic HMGB1, and RAGE Expression in
Benign and Malignant Breast Cancer Tissue
In the present study, ASMAþ spindle-shaped cells were taken to
represent activated CAFs in the breast cancer tissues. As a smooth

Figure 1 Immunohistochemical Staining of a-Smooth Muscle Actin (ASMA), Cytoplasmic High Mobility Group Box 1 (HMGB1) and
Receptor for Advanced Glycation End Products (RAGE) in Breast Cancer Patient Samples and Control Tissues. (A) ASMA
Expression in Normal Myoepithelial Cells in Benign Tissue. (B) Low and (C) High ASMA Expression of Activated Fibroblasts in
Breast Cancer Tissue. (D) Cytoplasmic HMGB1 Expression in Normal Mammary Cells in Benign Tissue. (E) Low and (F) High
Cytoplasmic HMGB1 Expression of Tumor Cells in Breast Cancer Tissue. (G) RAGE Expression in Normal Mammary Cells. (H)
Low and (I) High RAGE Expression of Tumor Cells in Breast Cancer Tissue. Original Magnification 3200. Bars Represent 200
mm. Graphs Show Distribution of (J) ASMA, (K) HMGB1, and (L) RAGE in Several Groups of Tissues. P < .05 and P < .001
Compared With Expression in Benign Breast Disease Tissue

444 - Clinical Breast Cancer October 2017

 Kamolporn Amornsupak et al
Table 2 Correlations of ASMA Expression With Clinicopathologic Characteristics in Breast Cancer (c2 Test)

96 With BC 81 With Non-IBC 15 With IBC

Characteristic Low High P Value Low High P Value Low High P Value
Age (y) .825 .882 .580
50 23 31 18 30 5 1
>50 16 26 11 22 5 4
Tumor size (mm) .010a .007a .600
20 22 16 18 15 4 1
>20 17 41 11 37 6 4
pT stage .889 .087 1.000
T1-T2 27 39 27 39 0 0
T3-T4 12 18 2 13 10 5
pN stage .217 .063 .258
N0 18 18 17 18 0 0
N1-N3 39 21 12 34 9 5
Histologic grade .321 .275 1.000
WD/MD 26 31 20 28 10 5
PD 13 26 9 24 0 0
Clinical stage .159 .004a 1.000
I-II 25 27 25 27 0 0
III-IV 14 30 4 25 10 5
LN metastasis .525 .535 .524
No 21 29 19 29 2 0
Yes 18 28 10 23 8 5
Angiolymphatic invasion .270 .034a 1.000
No 29 35 26 34 3 1
Yes 10 22 3 18 7 4
a
Perinodal invasion .190 .048 1.000
No 35 44 28 41 7 3
Yes 4 13 1 11 3 2
Tumor subtype
TN 8 13 .987 6 11 .814 2 2 .560
þ
HER2 3 8 .516 2 7 .478 1 1 1.000
Luminal 28 36 .508 21 34 .688 7 2 .329
ER .692 .688 .608
Negative 12 21 8 18 4 3
Positive 27 36 21 34 6 2
PR .781 .848 1.000
Negative 17 22 12 19 5 3
Positive 22 35 17 33 5 2
HER2 1.000
Negative 23 30 .686 23 18 .853 5 3
Positive 13 20 .967 13 7 .642 5 2 1.000
Equivocal 3 7 .735 3 3 .688 0 0 1.000

Abbreviations: ASMA ¼ a-smooth muscle actin; BC ¼ breast cancer; ER ¼ estrogen receptor; IBC ¼ inflammatory breast cancer; LN ¼ lymph node; MD ¼ moderately differentiated; PD ¼ poorly
differentiated; PR ¼ progesterone receptor; TN ¼ triple negative; WD ¼ well differentiated.
a
P < .05.

muscle actin protein, the expression of ASMA was mainly observed
in endothelial and myoepithelial cells in benign tissues (Figure 1A).
In contrast, ASMA was clearly present in fibroblasts in breast cancer
tissues with varying degrees of expression (Figure 1B, C). High
levels of ASMAþ fibroblasts were detected in 64.2% of non-IBC
tissues (52 of 81), 33.3% of IBC tissues (5 of 15), and 6.5% of
benign breast lesions (2 of 31; Figure 1J). Overexpression of
ASMAþ fibroblasts was significantly increased in breast cancer
tissues of both non-IBC and IBC subtypes compared with benign
breast tissues (P < .001 and P ¼ .029, respectively).

Clinical Breast Cancer October 2017 - 445

 ASMA and HMGB1 in Breast Cancer
As a nonhistone nuclear protein, the HMGB1 protein exhibited
nuclear localization in mammary epithelial, immune, and stromal
cells in benign breast tissues (Figure 1D). The expression of
HMGB1 protein was observed in both nuclei and cytoplasm of
breast cancer cells, with various staining intensities in the latter
(Figure 1E, F). Cells with high cytoplasmic HMGB1 levels were
detected in 56.8% of non-IBC cancers (46 of 81), 53.3% of IBC
cancers (8 of 15), and only 3.2% of benign breast lesions (1 of 31;
Figure 1K). Cytoplasmic HMGB1 levels were significantly
increased in breast cancer tissues in both non-IBC and IBC lesions
compared with benign breast tissues (P < .001 and P < .001,
respectively).
IHC staining showed membranous and cytoplasmic staining
patterns for RAGE expression. The immunoreactivity of RAGE in
benign breast samples was weak (Figure 1G). Only a few cells with
strong staining were seen, such as those in breast abscesses in which
inflammation was present. RAGE immunoreactivity in the cancer
tissue sections was stronger than in that in the benign disease tissues
(Figure 1H, I). High levels of RAGEþ cells were detected in 50.6%
of non-IBC (41 of 81), 53.3% of IBC (8 of 15), and 22.6% of
benign lesion samples (7 of 31; Figure 1L). Overexpression of
RAGE was significantly greater in non-IBC tissues compared with
that in normal breast tissues (P ¼ .011).
Correlation of ASMA, Cytoplasmic HMGB1, and RAGE
Expression With Clinicohistopathologic Characteristics in
Breast Cancer
The relationship of ASMA expression in fibroblasts with various
clinical and histopathologic features was analyzed using c2 tests. In
96 breast cancer patients, no statistically significant correlation was
found between ASMA expression and any clinicohistopathologic
features, except for large tumor size (P ¼ .01; Table 2). In a sub-
group analysis of 81 non-IBC cases, overexpression of ASMA was
significantly associated with large tumor size (P ¼ .007), high
clinical stage (P ¼ .004), angiolymphatic involvement (P ¼ .034),
and perinodal invasion (P ¼ .048; Table 2). However, no signifi-
cant association was found between ASMA expression and any
parameter in the 15 IBC cases. These results imply that ASMA
protein levels (as a surrogate for CAF infiltration) might be an
appropriate prognostic marker for predicting the metastatic poten-
tial of breast cancer, but only in non-IBC cases.
The cytoplasmic HMGB1 expression levels in breast cancer tissues
were significantly associated with small tumor size (P ¼ .031), low
clinical stage (P ¼ .01), luminal subtype (P < .001) but not the
TNBC subtype (P < .001) in accordance with the presence of
estrogen receptor (ER; P < .001), progesterone receptor (PR;
P ¼ .007), and HER2 expression (P ¼ .028; Table 3). In the
subgroup analysis, a similar pattern was detected in patients with non-
IBC. Increased cytoplasmic HMGB1 overexpression was significantly
associated with small tumor size (P ¼ .030), low pT stage (P ¼ .02),
low clinical stage (P ¼ .005), and the presence of ER (P < .001) and
PR (P ¼ .018) in 81 non-IBC cases. Elevated cytoplasmic HMGB1
expression in IBC tissues correlated significantly with TNBC
(P ¼ .026), luminal subtype (P ¼ .041), and the presence of ER
(P ¼ .041; Table 3). The significant correlation of tumor subtype
(TNBC and luminal subtypes) with cytoplasmic HMGB1 expression
was consistently found in both IBC and non-IBC cases.
446 - Clinical Breast Cancer October 2017
Increased RAGE expression in 96 breast cancer tissue specimens
showed no statistically significant association with any clinicopath-
ologic parameter (data not shown). In the subgroup analysis, similar
results (no statistical significance with any clinicopathologic
parameters) were found in the non-IBC and IBC cases. Therefore,
only the larger non-IBC subgroup was used in further analysis to
correlate with MFS of the patients.
Prognostic Significance of ASMA, Cytoplasmic HMGB1,
and RAGE Expression With MFS
We used the Kaplan-Meier method and log-rank test to identify
any prognostic implications of ASMA, cytoplasmic HMGB1, and
RAGE expression on the MFS of the patients with breast cancer.
The treatment regimens of the patients were analyzed and showed
no statistically significant differences between the patients with
low and high levels of each of these 3 proteins (Supplemental
Tables 1 and 2; available in the online version). The MFS of
patients with high ASMA expression was significantly lower than
that of patients with low ASMA expression (P ¼ .001; Figure 2A).
In contrast, patients with high cytoplasmic HMGB1 expression
had significantly better MFS than those with low HMGB1
expression (P ¼ .004; Figure 2B). However, no effect of RAGE
expression on MFS was detected in the present cohort study
(P ¼ .484; Figure 2C). We further assessed the potential utility of
these markers in combination. The results showed that patients
who had either high ASMA expression (P ¼ 1.37  105) or
RAGE expression (P ¼ .001) with low cytoplasmic HMGB1
expression had a significantly worse prognosis than those with only
1 positive marker (Figure 2D, E). Moreover, patients with high
ASMA and RAGE expression and low cytoplasmic HMGB1
expression had a rather poor prognosis, with high statistical sig-
nificance (P ¼ 1.87  106; Figure 2F). Patients with high ASMA
and RAGE expression and low cytoplasmic HMGB1 expression
had MFS (1159.67  291.57 days) that was significantly shorter
than that of the groups with different patterns of these 3 markers
(eg, high ASMA, RAGE, and cytoplasmic HMGB1 expression
with approximately 2403.65  94.48 days; P ¼ 1.87  106).
Other significant clinicopathologic characteristics, including
tumor size, pT stage, pN stage, clinical stage, perinodal invasion,
luminal subtype, ER expression, and PR expression, correlated
with MFS with statistical significance (Supplemental Figure 1;
available in the online version).
On univariate analysis, higher ASMA expression in fibroblasts
and an increased risk of metastasis was observed (hazard ratio [HR],
13.048; 95% confidence interval [CI], 1.721-98.892; P ¼ .013;
Table 4). In contrast, high cytoplasmic HMGB1 expression in
tumor cells was associated with a significantly decreased risk of
metastasis (HR, 0.243; 95% CI, 0.084-0.700; P ¼ .009; Table 4).
However, no association between RAGE expression and the risk of
breast cancer metastasis was observed (HR, 1.421; 95% CI, 0.529-
3.816; P ¼ .486; Table 4). In addition, the risk of metastatic relapse
was significantly increased in patients with specific expression
patterns, including (1) ASMA-high/HMGB1-low (HR, 7.049; 95%
CI, 2.540-19.592; P ¼ 1.77  104); (2) RAGE-high/HMGB1-
low (HR, 4.662; 95% CI, 1.745-12.460; P ¼ .002); and (3)
ASMA-high/RAGE-high/HMGB1-low (HR, 7.885; 95%
CI, 2.896-21.470; P ¼ 5.33  105; Table 4).
 Kamolporn Amornsupak et al
Table 3 Correlations of Cytoplasmic HMGB1 Expression With Clinicopathologic Characteristics (c2 Test)

96 With BC 81 With Non-IBC 15 With IBC

Characteristic Low High P Value Low High P Value Low High P Value
Age (y) .567 .422 1.000
50 25 29 23 25 2 4
>50 17 25 12 21 5 4
Tumor size (mm) .031a .030a 1.000
20 11 27 9 24 2 3
>20 31 27 26 22 5 5
pT stage .471 .020a 1.000
T1-T2 24 42 24 42 0 0
T3-T4 11 12 11 4 7 8
pN stage .071 .101 1.000
N0 11 25 11 24 0 1
N1-N3 31 29 24 22 7 7
Histologic grade .321 .306 .415
WD/MD 40 49 33 41 7 8
PD 2 5 2 5 2 5
Clinical stage .010a .005a 1.000
I-II 16 36 16 36 0 0
III-IV 26 18 19 10 7 8
LN metastasis .072 .139 1.000
No 17 33 17 31 0 2
Yes 25 21 18 15 1 6
Angiolymphatic invasion .827 .769 .569
No 28 36 27 33 1 3
Yes 14 18 8 13 6 5
Perinodal invasion .266 .406 .608
No 32 47 28 41 4 6
Yes 10 7 7 5 3 2
Tumor subtype
TN 19 2 <.001a 15 2 <.001a 4 0 .026a
HER2þ 5 6 1.000 4 5 1.000 1 1 1.000
Luminal 18 46 <.001a 16 39 <.001a 2 7 .041a
ER <.001 a
.041a
Negative 25 8 19 7 <.001 a
6 2
Positive 17 46 16 39 1 7
PR .007a .018a .315
Negative 24 15 19 12 5 3
Positive 18 39 16 34 2 5
HER2 .315
Negative 29 24 .028a 24 21 .067 5 3
Positive 10 23 .088 8 18 .189 2 5
Equivocal 3 7 .505 3 7 .502 0 0

Abbreviations: BC ¼ breast cancer; ER ¼ estrogen receptor; HMGB1 ¼ high mobility group box 1; IBC ¼ inflammatory breast cancer; LN ¼ lymph node; MD ¼ moderately differentiated;
PD ¼ poorly differentiated; PR ¼ progesterone receptor; TN ¼ triple negative; WD ¼ well differentiated.
a
P < .05.

Furthermore, the multivariate Cox proportional hazard regression
model with significant covariates from univariate analysis was used to
more accurately estimate the independent prognostic factors for MFS.
The results from the forced-entry method, in which all variables were
entered in a single step, showed that ASMA expression could be
considered an independent prognostic factor of metastatic relapse in
patients with breast cancer (HR, 9.891; 95% CI, 1.094-89.432;
P ¼ .041; Table 5). Moreover, the forward-stepwise method, in
which the variables were entered in a stepwise fashion, beginning with
the variable most strongly associated with the outcome variable, was

Clinical Breast Cancer October 2017 - 447

 ASMA and HMGB1 in Breast Cancer
Figure 2 Correlation of a-Smooth Muscle Actin (ASMA), Cytoplasmic High Mobility Group Box 1 (HMGB1) and Receptor for Advanced
Glycation End Products (RAGE) Expression on Metastasis-free Survival (MFS) of Patients With Breast Cancer. (A-C) Kaplan-
Meier Analysis (Log-rank Test) Showed an Association Between MFS and (A) ASMA Expression in Stromal Fibroblasts, (B)
Cytoplasmic HMGB1 Expression, and (C) RAGE Expression in Tumor Cells. The Following Combinations of ASMA,
Cytoplasmic HMGB1, and RAGE Receptor Staining Pattern Predicted for an Unfavorable Prognosis: (D) ASMA-high/
Cytoplasmic HMGB1-low, (E) RAGE-high/Cytoplasmic HMGB1-low, and (F) ASMA-high/RAGE-high/Cytoplasmic HMGB1-low

used to achieve the best set of predictors. The results indicated that
ASMA expression in activated fibroblasts and cytoplasmic HMGB1
expression in tumor cells constituted independent predictive factors of
metastasis in breast cancer (Table 5). The relative hazard of metastasis
in the high ASMA group was approximately 14.2-fold compared with
that of the low ASMA group (P ¼ .01; Table 5). The relative hazard
of metastasis in patients with high cytoplasmic HMGB1 expression
was approximately 0.2-fold compared with that for those with low
cytoplasmic HMGB1 expression (P ¼ .005; Table 5). The multi-
variate analysis further confirmed the results from the Kaplan-Meier
analysis and identified the truly significant effect of ASMA and
cytoplasmic HMGB1 (but not RAGE) expression as a predictive
marker of MFS in breast cancer (Figure 2). Therefore, the combined
expression of ASMA and HMGB1 could be the best set with which
to improve effectiveness of the prediction (Figure 2D). Moreover, this
finding indicates that ASMA expression could be considered a risk
factor for increasing the chance of metastatic relapse in breast cancer
patients, and cytoplasmic HMGB1 could be considered a protective
factor, decreasing the chance of metastasis.
Discussion
Metastasis remains the major cause of treatment failure and
cancer-related mortality in breast cancer patients.6 Although
advanced treatments and understanding of the molecular biology of
breast cancer have helped to improve patient survival, complete
tumor regression and cure are still limited.4,5 Therefore, markers to
predict a high possibility of metastatic relapse are required to tailor
treatment to those patients at greatest risk.
In the present study, we investigated the expression of activated
fibroblasts defined by ASMAþ spindle-shaped cells and the
expression of cytoplasmic HMGB1 and its receptor, RAGE, in 96
paraffin-embedded invasive ductal carcinoma specimens. The
translocation of nuclear HMGB1 to the cytoplasm and its secretion
were initially identified in colon and liver cancers.36 In addition,
positive correlations of nuclear and cytoplasmic HMGB1 expression
levels have been observed in breast cancer tissues.21 It has been
assumed that the level of HMGB1 in the cytoplasm of cancer cells
might represent the level of HMGB1 released to the extracellular
milieu. Thus, the grade of cytoplasmic HMGB1 intensity was
selected in the present study.
Our results revealed that ASMAþ fibroblast (CAF) infiltration
and the expression of cytoplasmic HMGB1 and RAGE were clearly
greater in breast tumor stroma in both non-IBC and IBC tissues
compared with noncancerous tissues, including fibroadenoma and
breast abscess. However, the prognostic significance of these 3
proteins was demonstrated only in non-IBC samples. This might be

448 - Clinical Breast Cancer October 2017

 Kamolporn Amornsupak et al
Table 4 Univariate Analysis (Cox Regression) of Factors Table 4 Continued
Associated With MFS
Univariate Analysis (Cox Regression)
Univariate Analysis (Cox Regression)
Characteristic n/Event HR 95% CI P Value
Characteristic n/Event HR 95% CI P Value Cytoplasmic .243 0.084-0.700 .009a
Age (y) 1.352 0.503-3.632 .550 HMGB1
50 41/9 Low 25/11
>50 25/7 High 41/5
Tumor size (mm) 4.138 1.177-14.540 .027a RAGE 1.421 0.529-3.816 .486
20 29/3 Low 34/7
>20 37/13 High 32/9
pT stage 3.35 1.159-9.684 .026 a
ASMA-high/ 7.049 2.540-19.592 1.77  104a
HMGB1-low
T1-T2 56/11
No 50/6
T3-T4 10/5
Yes 16/10
pN stage 7.491 1.700-33.007 .008a
RAGE-high/ 4.662 1.745-12.460 .002a
N0 31/2
HMGB1-low
N1-N3 35/14
No 52/8
Histologic grade 2.093 0.778-5.632 .144
Yes 14/8
WD/MD 39/7
ASMA-high/RAGE- 7.885 2.896-21.470 5.33  105a
PD 27/9 high/HMGB1-low
Clinical stage 3.638 1.361-9.723 .010a No 57/9
I-II 49/8 Yes 9/7
III-IV 17/8
Abbreviations: ASMA ¼ a-smooth muscle actin; CI ¼ confidence interval; ER ¼ estrogen
LN metastasis 1.509 0.566-4.023 .411 receptor; HMGB1 ¼ high mobility group box 1; HR ¼ hazard ratio; LN ¼ lymph node;
No 39/8 MD ¼ moderately differentiated; MFS ¼ metastasis-free survival; PD ¼ poorly differentiated;
PR ¼ progesterone receptor; RAGE ¼ receptor for advanced glycation end products;
Yes 27/8 TN ¼ triple negative; WD ¼ well differentiated.
a
P < .05.
Angiolymphatic 1.387 0.481-3.94 .545
invasion
No 49/11 explained by the inflammatory mediators and proteases found in the
Yes 17/5 microenvironment of IBC, which induce and facilitate tumor
Perinodal invasion 3.668 1.270-10.596 .016a
invasion and host immunosuppression.37 Thus, many factors might
be involved in determining the prognosis of IBC patients, far
No 57/11
beyond the 3 proteins evaluated in the present study.
Yes 9/5
Several studies have revealed the influence of CAFs on prolifer-
Luminal 0.260 0.094-0.718 .009a
ation, invasion, and drug resistance in breast cancer.11,38-40 More-
No 23/10
over, the potential use of some CAF molecular markers in clinical
Yes 43/6 diagnosis and prognosis of breast cancer have already been consid-
HER2þ 1.850 0.527-6.494 .337 ered.9 Recently, ASMA expression in tumor stromal cells was
No 58/13 quantified in 60 breast cancer patients using image analysis, and
Yes 8/3 increased ASMA expression was detected in patients with metastasis
Basal 3.279 1.215-8.849 .019a compared with the metastasis-free group and correlated with poorer
No 51/9 overall survival.10 In agreement with our findings from a larger
Yes 15/7 number of breast cancer cases, a significant correlation of increased
ER .260 0.094-0.718 .009a ASMAþ fibroblast infiltration with shorter MFS and greater disease
Negative 23/10 aggressiveness (eg, large tumor size, high clinical stage, and angio-
Positive 43/6 lymphatic or perinodal invasion) were demonstrated. We have
PR 0.356 0.129-0.980 .046a previously shown that substances secreted by primary cultures of
Negative 27/10
CAFs (but not normal fibroblasts) can induce breast cancer cell drug
resistance.11 Such findings support a correlation between the pres-
Positive 39/6
ence of ASMAþ fibroblasts and breast cancer progression. These
ASMA 13.048 1.721-98.892 .013a
data, taken together, suggest the feasibility of using ASMAþ fibro-
Low 27/1
blasts as a prognostic marker to aid in prediction of breast cancer
High 39/15
aggressiveness.

Clinical Breast Cancer October 2017 - 449

 ASMA and HMGB1 in Breast Cancer
Table 5 Multivariate Analysis (Cox Regression) for MFS Predictor

Multivariate Analysis (Forced-entry Method) Multivariate Analysis (Forward-stepwise Method)

Characteristic HR 95% CI P Value HR 95% CI P Value
Tumor size (mm)
20 1.976 0.464-8.417 .357
>20
pT stage
T1-T2 0.771 0.162-3.674 .744
T3-T4
pN stage
N0 3.122 0.519-18.792 .214
N1-N3
Clinical stage
I-II 0.575 0.133-2.486 .459
III-IV
Luminal type
No 0.653 0.104-4.114 .650
Yes
Basal type
No 2.283 0.439-11.877 .327
Yes
ASMA
Low 9.891 1.094-89.432 .041a 14.162 1.863-107.652 .010a
High
Cytoplasmic HMGB1
Low 0.430 0.096-1.930 .270 0.221 0.076-0.641 .005a
High
RAGE
Low 1.357 0.410-4.489 .617
High

Abbreviations: ASMA ¼ a-smooth muscle actin; CI ¼ confidence interval; ER ¼ estrogen receptor; HMGB1 ¼ high mobility group box 1; HR ¼ hazard ratio; LN ¼ lymph node; MD ¼ moderately
differentiated; MFS ¼ metastasis-free survival; PD ¼ poorly differentiated; PR ¼ progesterone receptor; RAGE ¼ receptor for advanced glycation end products; TN ¼ triple negative; WD ¼ well
differentiated.
a
P < .05.

Overexpression of HMGB1 is associated with several hallmarks
of cancer, including metastasis.16 However, the paradoxical roles of
HMGB1 as an anti- or pro-tumor protein in tumor development
and cancer therapy have been noted.13 In breast cancer, increased
nuclear or cytoplasmic HMGB1 expression has been correlated with
either a good or poor prognosis.20,21 Nuclear HMGB1 expression in
breast cancer tissues correlated negatively with the dense infiltration
of immunosuppressive cell types, in particular FOXP3þ regulatory
T cells.41 Cytoplasmic HMGB1 (which correlates directly with
levels in the nucleus) was shown to be significantly associated with
high levels of CD8þ effector cells.21 Hence, our findings that high
cytoplasmic HMGB1 was significantly associated with features
associated with a good prognosis (eg, small tumor size and low
clinical stage) and prolonged MFS in breast cancer patients might be
linked to high CD8þ cell and low regulatory T cell expression,
which could be tested in future studies.
Our present results also agree with the finding that over-
expression of HMGB1 might be a good prognostic marker, such as
was shown for patients with gastric cancer.42,43 It is interesting to
consider that HMGB1 released after cell death induced by
chemotherapy might have a role, which could stimulate immune-
mediated eradication of breast tumor cells. Hence, the level of
cytoplasmic HMGB1 in cancer cells might be considered a prog-
nostic marker to select the appropriate regimen of chemotherapeutic
agents. Breast cancer patients with low HMGB1 (intrinsic levels and
before chemotherapy) might need a more aggressive treatment
regimen than those with high HMGB1 expression.
Although both RAGE and toll-like receptor 4 (the 2 known
receptors for HMGB1) have been reported, interactions between
HMGB1 and RAGE have only been found in tumor cells and not
in normal tissue.29 In breast cancer, RAGE expression was upre-
gulated and associated with lymph node metastasis and poor
prognosis.30,31 Similar to our findings, RAGE overexpression was
detected in breast cancer tissues significantly more often than in
benign breast disease tissues. However, we found that RAGE levels
did not correlate with any clinical parameters or survival time. This
apparent controversial lack of an association between RAGE
expression and patient outcome has also been observed in prostate

450 - Clinical Breast Cancer October 2017


and colorectal cancers, in which only HMGB1, but not RAGE,
expression levels were associated with MFS.33,44 Co-expression of
HMGB1 and RAGE has been shown to have better potential than
either marker alone to predict patient outcomes in prostate cancer,33
which corresponds the findings from our study. We suggest that
using low HMGB1 and high RAGE levels might better predict for
short survival time than using either low HMGB1 or high RAGE
levels alone.
The expression of ASMAþ fibroblasts and cytoplasmic HMGB1
expression did not correlate with each other (data not shown), despite
previous observations that CAFs could induce HMGB1 expression in
cancer cells.11,28 This could be because in addition to the soluble
factors released from CAFs, HMGB1 induction could be stimulated
by several factors, including growth factors, cytokines, and cellular
stresses.45-47 Thus, HMGB1 is expressed intrinsically differently in
each patient owing to both genetic and epigenetic controls.14 In the
present study, the possible clinical value of ASMA expression and
cytoplasmic HMGB1 expression as an independent marker of good
prognosis was explored. HMGB1 has been considered to better
predict patient outcomes and to enable improved risk stratification of
patients with breast cancer when combined with other protein
markers. Ladoire et al26 showed that the combined positivity for an
autophagic marker (LC3B puncta) and nuclear HMGB1 was
significantly associated with prolonged MFS. Similarly, our findings
suggest that a combined score of low ASMA and high cytoplasmic
HMGB1 could be a candidate as a good prognostic indicator in
breast cancer patients, especially those with the non-IBC subtype.
Conclusion
Our findings highlight the prognostic potential of combined
cytoplasmic HMGB1 and CAF scores in breast cancer patients.
High levels of ASMAþ CAF infiltration plus low cytoplasmic
HMGB1 expression in cancer cells represent prognostic markers
that predict a greater risk of metastasis and suggest the need for
more aggressive treatment regimens for these patients. Furthermore,
the possibility of modulating CAF behavior and boosting HMGB1-
mediated anticancer immune responses could be an alternative
therapeutic approach for the effective treatment of breast cancer
patients.
Clinical Practice Points
þ inflammation and cancer. Annu Rev Immunol 2010; 28:367-88.
ASMA fibroblasts and low HMGB1 expression are prognostic
markers of poor outcomes for several carcinomas, including
breast cancer; however, the effect of their combination has not
been investigated. Moreover, the prognostic role of HMGB1 in
breast cancer is still controversial.
We found that high ASMAþ fibroblast and low HMGB1
expression in cancer cells are the most reliable predictors of
metastatic relapse.
Our findings suggest that using the ASMA-high/HMGB1-low
profile in breast cancer tissue can help predict disease aggres-
siveness, especially the risk of metastatic relapse.
Acknowledgments
This work was supported by a Mentorship Grant, Mahidol
University (grant R015420003) and the Royal Golden Jubilee-
Kamolporn Amornsupak et al
Thailand Research Fund (grant PHD/0051/2552). We thank Ms
Kanittar Srisook, Department of Pathology, Faculty of Medicine
Siriraj Hospital, for helpful advice on immunohistochemistry
staining, and Dr Carol Box and Ms Somaieh Hedayat (The Institute
of Cancer Research [ICR]) for critical reading and thoughtful
suggestions.
Disclosure
The authors declare that they have no competing interests.
Supplemental Data
Supplemental figure accompanying this article can be found in
the online version at http://dx.doi.org/10.1016/j.clbc.2017.04.007.
References
1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer
statistics, 2012. CA Cancer J Clin 2015; 65:87-108.
2. Attasara P, Buasom R. Hospital based cancer registry annual report 2012. Natl
Cancer Inst Thailand 2014:1-84.
3. Peto R, Davies C, Godwin J, et al. Comparisons between different polychemotherapy
regimens for early breast cancer: meta-analyses of long-term outcome among 100,
000 women in 123 randomised trials. Lancet 2012; 379:432-44.
4. Tang Y, Wang Y, Kiani MF, Wang B. Classification, treatment strategy, and
associated drug resistance in breast cancer. Clin Breast Cancer 2012; 16:335-43.
5. Chaffer CL, Weinberg RA. A perspective on cancer cell metastasis. Science 2011;
331:1559-64.
6. Andre F, Berrada N, Desmedt C. Implication of tumor microenvironment in the
resistance to chemotherapy in breast cancer patients. Curr Opin Oncol 2010; 22:
547-51.
7. Luo H, Tu G, Liu Z, Liu M. Cancer-associated fibroblasts: a multifaceted driver of
breast cancer progression. Cancer Lett 2015; 361:155-63.
8. Buchsbaum R, Oh S. Breast cancer-associated fibroblasts: where we are and where
we need to go. Cancers 2016; 8:E19.
9. Crawford Y, Kasman I, Yu L, et al. PDGF-C mediates the angiogenic and
tumorigenic properties of fibroblasts associated with tumors refractory to anti-
VEGF treatment. Cancer Cell 2009; 15:21-34.
10. Yamashita M, Ogawa T, Zhang X, et al. Role of stromal myofibroblasts in invasive
breast cancer: stromal expression of alpha-smooth muscle actin correlates with
worse clinical outcome. Breast Cancer 2012; 19:170-6.
11. Amornsupak K, Insawang T, Thuwajit P, O-Charoenrat P, Eccles SA, Thuwajit C.
Cancer-associated fibroblasts induce high mobility group box 1 and contribute to
resistance to doxorubicin in breast cancer cells. BMC Cancer 2014; 14:1-12.
12. Marusyk A, Tabassum DP, Janiszewska M, et al. Spatial proximity to fibroblasts
impacts molecular features and therapeutic sensitivity of breast cancer cells influ-
encing clinical outcomes. Cancer Res 2016; 76:6495-506.
13. Kang R, Zhang Q, Zeh HJ, Lotze MT, Tang D. HMGB1 in cancer: good, bad, or
both? Clin Cancer Res 2013; 19:4046-57.
14. Kang R, Chen R, Zhang Q, et al. HMGB1 in health and disease. Mol Aspects Med
2014; 40:1-116.
15. Sims GP, Rowe DC, Rietdijk ST, Herbst R, Coyle AJ. HMGB1 and RAGE in
16. Tang D, Kang R, Zeh HJ III, Lotze MT. High-mobility group box 1 and cancer.
Biochim Biophys Acta 2010; 1799:131-40.
17. Taguchi A, Blood DC, del Toro G, et al. Blockade of RAGE-amphoterin signalling
suppresses tumour growth and metastases. Nature 2000; 405:354-60.
18. Song B, Song WG, Li ZJ, et al. Effect of HMGB1 silencing on cell proliferation,
invasion and apoptosis of MGC-803 gastric cancer cells. Cell Biochem Funct 2011;
30:11-7.
19. Luo Y, Chihara Y, Fujimoto K, et al. High mobility group box 1 released from
necrotic cells enhances regrowth and metastasis of cancer cells that have survived
chemotherapy. Eur J Cancer 2013; 49:741-51.
20. Sun S, Zhang W, Cui Z, et al. High mobility group box-1 and its clinical value in
breast cancer. Onco Targets Ther 2015; 8:413-9.
21. Lee HJ, Kim A, Song IH, et al. Cytoplasmic expression of high mobility group B1
(HMGB1) is associated with tumor-infiltrating lymphocytes (TILs) in breast
cancer. Pathol Int 2016; 66:202-9.
22. Ke S, Zhou F, Yang H, et al. Downregulation of high mobility group box 1
modulates telomere homeostasis and increases the radiosensitivity of human breast
cancer cells. Int J Oncol 2015; 46:1051-8.
23. Jiao Y, Wang HC, Fan SJ. Growth suppression and radiosensitivity increase by
HMGB1 in breast cancer. Acta Pharmacol Sin 2007; 28:1957-67.
24. Tafani M, Schito L, Pellegrini L, et al. Hypoxia-increased RAGE and P2X7R
expression regulates tumor cell invasion through phosphorylation of Erk1/2 and
Akt and nuclear translocation of NF-kB. Carcinogenesis 2011; 32:1167-75.
Clinical Breast Cancer October 2017 - 451
 ASMA and HMGB1 in Breast Cancer
25. Gebremeskel S, Johnson B. Concepts and mechanisms underlying chemotherapy
induced immunogenic cell death: impact on clinical studies and considerations for
combined therapies. Oncotarget 2015; 6:41600-19.
26. Ladoire S, Penault-Llorca F, Senovilla L, et al. Combined evaluation of LC3B
puncta and HMGB1 expression predicts residual risk of relapse after adjuvant
chemotherapy in breast cancer. Autophagy 2015; 11:1878-90.
27. Ananthula S, Sinha A, El Gassim M, et al. Geminin overexpression-dependent
recruitment and crosstalk with mesenchymal stem cells enhance aggressiveness in
triple negative breast cancers. Oncotarget 2016; 7:20869-89.
28. Bartling B, Fuchs C, Silber RE, Simm A. Fibroblasts mediate induction of high
mobility group box protein 1 in lung epithelial cancer cells by diffusible factors. Int
J Mol Med 2007; 20:217-24.
29. Todorova J, Pasheva E. High mobility group B1 protein interacts with its receptor
RAGE in tumor cells but not in normal tissues. Oncol Lett 2012; 3:214-8.
30. Yin C, Li H, Zhang B, et al. RAGE-binding S100A8/A9 promotes the migration
and invasion of human breast cancer cells through actin polymerization and epi-
thelialemesenchymal transition. Breast Cancer Res Treat 2013; 142:297-309.
31. Nasser MW, Wani NA, Ahirwar DK, et al. RAGE mediates S100A7-induced
breast cancer growth and metastasis by modulating the tumor microenviron-
ment. Cancer Res 2015; 75:974-85.
32. Kuniyasu H, Chihara Y, Takahashi T. Co-expression of receptor for advanced
glycation end products and the ligand amphoterin associates closely with metastasis
of colorectal cancer. Oncol Rep 2003; 10:445-8.
33. Zhao C-B, Bao J-M, Lu Y-J, et al. Co-expression of RAGE and HMGB1 is
associated with cancer progression and poor patient outcome of prostate cancer.
Am J Cancer Res 2014; 4:369-77.
34. Stoetzer O, Fersching DI, Salat C, et al. Circulating immunogenic cell death
biomarkers HMGB1 and RAGE in breast cancer patients during neoadjuvant
chemotherapy. Tumour Biol 2013; 34:81-90.
35. Xiao-Wen Y, Ming C. Expression and clinical significances of HMGB1 and EGFr
v in Breast Cancer. JKMU 2008; 1:65-8.
36. Lee H, Shin N, Song M, et al. Analysis of nuclear high mobility group box 1
(HMGB1)-binding proteins in colon cancer cells: clustering with proteins involved
in secretion and extranuclear function. J Proteome Res 2010; 9:4661-70.
452 - Clinical Breast Cancer October 2017
37. Mohamed MM, Al-Raawi D, Sabet SF, El-Shinawi M. Inflammatory breast
cancer: new factors contribute to disease etiology: a review. J Adv Res 2014; 5:
525-36.
38. Busch S, Acar A, Magnusson Y, Gregersson P, Ryden L, Landberg G. TGF-beta
receptor type-2 expression in cancer-associated fibroblasts regulates breast cancer
cell growth and survival and is a prognostic marker in pre-menopausal breast
cancer. Oncogene 2013; 34:27-38.
39. Olsen CJ, Moreira J, Lukanidin EM, Ambartsumian NS. Human mammary
fibroblasts stimulate invasion of breast cancer cells in a three-dimensional cul-
ture and increase stroma development in mouse xenografts. BMC Cancer 2010;
10:444.
40. Mueller K, Madden J, Zoratti G, Kuperwasser C, List K, Boerner J. Fibroblast-
secreted hepatocyte growth factor mediates epidermal growth factor receptor
tyrosine kinase inhibitor resistance in triple-negative breast cancers through para-
crine activation of Met. Breast Cancer Res 2012; 14:R104.
41. Ladoire S, Enot D, Senovilla L, et al. The presence of LC3B puncta and HMGB1
expression in malignant cells correlate with the immune infiltrate in breast cancer.
Autophagy 2016; 12:864-75.
42. Akaike H, Kono K, Sugai H, et al. Expression of high mobility group box chro-
mosomal protein-1 (HMGB-1) in gastric cancer. Anticancer Res 2007; 27:449-57.
43. Bao G, Qiao Q, Zhao H, He X. Prognostic value of HMGB1 overexpression in
resectable gastric adenocarcinomas. World J Surg Oncol 2010; 8:52.
44. Fahmueller YN, Nagel D, Hoffmann R-T, et al. Immunogenic cell death bio-
markers HMGB1, RAGE, and DNAse indicate response to radioembolization
therapy and prognosis in colorectal cancer patients. Int J Cancer 2013; 132:2349-
58.
45. Ohmori H, Luo Y, Fujii K, et al. Dietary linoleic acid and glucose enhances
azoxymethane-induced colon cancer and metastases via the expression of high-
mobility group box 1. Pathobiology 2010; 77:210-7.
46. Ohmori H, Luo Y, Kuniyasu H. Non-histone nuclear factor HMGB1 as a ther-
apeutic target in colorectal cancer. Expert Opin Ther Targets 2011; 15:183-93.
47. Willenbrock S, Braun O, Baumgart J, et al. TNF-alpha induced secretion of
HMGB1 from non-immune canine mammary epithelial cells (MTH53A). Cyto-
kine 2012; 57:210-20.
 Kamolporn Amornsupak et al
Supplemental Figure 1 Kaplan-Meier Analysis (Log-rank Test) of Metastasis-free Survival (MFS) and Clinicopathologic Parameters:
(A) Tumor Size, (B) pT Stage, (C) pT Stage, (D) Clinical Stage, (E) Perinodal Invasion, (F) Luminal Subtype,
(G) Basal Subtype, (H) Estrogen Receptor (ER) Expression, and (I) Progesterone (PR) Expression

Clinical Breast Cancer October 2017 - 452.e1

 ASMA and HMGB1 in Breast Cancer
Supplemental Table 1 Treatment Regimens Stratified Into 2 Groups

Treatment Regimena
Marker Expression Level A B P Value
ASMA Low (n ¼ 27) 27 (100.0) 0 (0.0) .260
High (n ¼ 39)b 35 (89.7) 3 (7.7)
HMGB1 Low (n ¼ 25) 22 (88.0) 3 (12.0) .053
High (n ¼ 41)b 40 (97.0) 0 (0.0)
RAGE Low (n ¼ 34)b 32 (94.1) 1 (2.9) .613
High (n ¼ 32) 30 (93.8) 2 (6.2)

Data presented as n (%).

Abbreviations: ASMA ¼ a-smooth muscle actin; HMGB1 ¼ high mobility group box 1; RAGE ¼ receptor for advanced glycation end products.
a
Regimen A: surgery plus adjuvant chemotherapy and/or radiation and/or hormonal therapy; regimen B: neoadjuvant chemotherapy plus surgery plus radiation, and/or adjuvant chemotherapy and/or
hormonal therapy.
b
One patient did not undergo surgery because of lung metastasis found after completion of neoadjuvant chemotherapy.

Supplemental Table 2 Treatment Regimen Stratified Into 5 Groups

Treatment Regimena
Marker Expression Level I II III IV V P Value
ASMA Low (n ¼ 27) 23 (85.2) 0 (0.0) 0 (0.0) 4 (14.8) 0 (0.0) .429
High (n ¼ 39) 28 (71.8) 3 (7.7) 1 (2.6) 6 (15.4) 1 (2.6)
HMGB1 Low (n ¼ 25) 15 (60.0) 3 (12.0) 0 (0.0) 7 (28.0) 0 (0.0) .017b
High (n ¼ 41) 36 (87.8) 0 (0.0) 1 (2.4) 3 (7.3) 1 (2.4)
RAGE Low (n ¼ 34) 27 (79.4) 1 (2.9) 0 (0.0) 5 (14.7) 1 (2.9) .653
High (n ¼ 32) 24 (75.0) 2 (6.3) 1 (3.1) 5 (15.6) 0 (0.0)

Data presented as n (%).

Abbreviations: ASMA ¼ a-smooth muscle actin; HMGB1 ¼ high mobility group box 1; RAGE ¼ receptor for advanced glycation end products.
a
Regimen I: surgery plus chemotherapy plus radiotherapy; regimen II: neoadjuvant plus surgery plus chemotherapy plus radiotherapy; regimen III: surgery plus radiotherapy; regimen IV: surgery plus
chemotherapy; regimen V: chemotherapy plus radiotherapy.
b
P < .05.

452.e2 - Clinical Breast Cancer October 2017