Introduction
Breast Cancer (BC) is the most frequently diagnosed
cancer and the leading cause of cancer-related death
in women worldwide.[] Non-metastatic BC is treated
with a multidisciplinary approach, including breast surgery, radiation oncology, and medical oncology.
[] The surgical option is decided on a patient basis,
considering tumor location, size, cancer stage, and patient-
related factors. Adjuvant treatments are added
to tumor-free patients with proven non-metastatic to
prevent local and systemic recurrences after breast and axillary surgery because of the possibility of cellular
micro-tumors. Adjuvant systemic treatment options in
BC are endocrine therapy, chemotherapy, or biological
therapies. Radiotherapy (RT) is added for adjuvant regional
and lymphatic therapy.[,]
Patients whose adjuvant treatment process is completed
are followed up.[] While full screening is performed
every 3 months for the first 2 years, then the frequency
of follow-up is reduced. Although BC recurrences
are common in the first 1-2 years, they may occur within 5
years after diagnosis, especially in hormone receptor-negative
disease[6]. Unfortunately, it has been reported that
10-20% recurrence is observed in early-stage BC even
after 5-10 years, despite complete surgical resection (R0)
and successful adjuvant treatments.[] Recurrence or
metastases developing after 5 years in these patients who
received adjuvant treatments following R0 tumor surgery
and were followed up completely tumor-free draw attention
to the presence of cellular micro-tumors.[]
BC is not only caused by neoplastic cells but also
caused by the stroma surrounding the tumor or
changes in the tumor microenvironment. The BC microenvironment
encompasses fibroblasts, leukocytes,
adipocytes, and myoepithelial and endothelial cells, as
well as the extracellular matrix, cytokines, hormones,
proteins, and enzymes. The tumor microenvironment
is regarded as a critical factor for tumor growth, progression,
and therapy response.[]
Many studies on cancer biology have revealed many
potential biomarkers for tumor development, progression,
treatment, and follow-up. Two of these biomarkers
associated with malignancy, metastasis, and survival
are calcium-binding protein B (S100B) and Heat shock
proteins (HSP). S100 calcium-binding protein family,
of which S100B is a member, is a protein group that
contains a multigene group consisting of 21 low molecular
weight proteins. S100B plays a role in cell proliferation
and is secreted from various inflammatory cells,
neurons, adipocytes, melanocytes, and chondrocytes.
There is a direct relationship between S100B expression
and the degree of malignancy and survival time.[]
HSP are mainly involved in protein folding, protein
transport, and protein targeting for lysosomal degradation.[] HSP27, HSP70, and HSP90 have been previously
reported in BC.[] Serum HSP70 is expressed
through the plasma membrane and released into the
bloodstream in many different tumor types such as
pancreatic cancer, colorectal cancer, BC, brain, and
lung cancer. Recent studies have indicated that HSP70
plays a role in cancer development, tumor cell proliferation,
differentiation, metastases, and death.[]
Nevertheless, in the selected population with nonmetastatic,
Luminal-A disease there is no clear information
regarding the difference of serum levels of
S100B and HSP70 from the healthy population and the
effects of post-operative RT on early serum markers.
This is the first study to present the acute impact of RT
on S100B and HSP70 change in selected BC patients.
Our study was designed to compare biomarker levels
and investigate the effect of RT. We compared the
biomarkers in patients with non-metastatic, Luminal-A
BC who completed adjuvant systemic therapy with the
healthy population. In addition, we have detailed the
impact of RT on early biomarker changes below.
Methods
Twenty-one BC patients who received chestwall/
breast±axilla RT in our clinic between February 2017
and December 2019 were included in the study group.
Twenty-one healthy individuals whose ages and body
mass index (BMI) was compatible with the study group
were randomly recruited into the control group. The
study group consisted of patients who received adjuvant
chemotherapy after R0 breast surgery and localregional
50 Gy RT. The age, height, weight, BMI, performance
status (PS), comorbid diseases, surgery type,
histopathological tumor type, tumor and lymph node
stage, tumor grade, and hormone receptor group data
of the patients were recorded. The patients had no comorbid
kidney disease, cardiovascular disease, inflammatory
disease, diabetes, and none of them had a previous
cancer diagnosis.
The pathological tumor stage was defined according
to the eighth edition of the International Union
Against Cancer"s tumor-lymph node-metastasis classification.
Both tumor size and lymph node metastasis
status were evaluated separately. Estrogen receptor
(ER), progesterone receptor (PR), and human epidermal
growth factor receptor (HER2) data were obtained
from the pathology records of the patients. Biological
subclassification was made using ER, PR, HER2. Patients
with Luminal A disease (ER-positive, PR-positive,
and HER2-negative) were included in the study.
Performance scoring was performed according to the
Eastern Cooperative Oncology Group PS (ECOG-PS).
Blood samples of tumor-free patients whose adjuvant
systemic treatments were completed after surgery
were taken into biochemistry tubes on the 1st day of
RT and in the 1st week after RT, centrifuged and separated
into serum and stored at -80°C until the day of evaluation. S100B and HSP70 levels were measured in
the Biotek instruments USA ELISA device using Abbkine
Human HSP70 and Cusabio S100B ELISA kits.
Briefly, the microtiter plate provided in these kits has
been pre-coated with an antibody. Standards or samples
are added to the appropriate microtiter plate wells
with Horseradish Peroxidase conjugated detection antibody
and incubated. Then, chromogen solutions are
added to each well, respectively. The enzyme-substrate
reaction is terminated by adding a stop solution, and
the color change is measured spectrophotometrically
at a wavelength of 450 nm±2 nm. The concentration of
the samples is then determined by comparing the O.D.
of the samples to the standard curve.
Statistical Analysis
Number Cruncher Statistical System 2007 (Kaysville,
Utah, USA) program was used for statistical analysis.
Descriptive statistical methods (mean, standard
deviation, median, frequency, rate, minimum, and
maximum values) evaluated the study data. The suitability
of quantitative data for normal distribution was
tested by Kolmogorov-Smirnov, Shapiro-Wilk test,
and graphical evaluations. Student's t-test compared
two groups" data with normal distribution, while the
Mann Whitney U test was used to compare two groups
of non-normally distributed data. Pearson Chi-square
test evaluated qualitative data. A paired sample t-test
compared the data measured before and after RT. The
Kruskal-Wallis test was used for comparisons of three
or more groups of data that did not show normal distribution.
Wilcoxon Signed Ranks Test evaluated the data
measured before and after RT. Statistical significance
level was considered as a p<0.05.
Ethics
This study was conducted with permission from Local
Institutional Ethics Committee (Ethics Committee Decision
Number: 2020-06-18).
Results
A total of 42 participants, 21 patients and 21 healthy,
were included in this study. The ECOG PS was 0-1. The
mean age of the study group was 57.43±12.54 years,
and the control group was 48.81±16.27. There was no
statistically significant difference in mean age between
the two groups (p=0.062). Both groups had a BMI between
20 and 30, and there was no obese person with a
BMI of >30. When the histology types were examined, it was determined that 76.2% (n=16) were invasive
ductal carcinoma, and 23.8% (n = 5) were invasive lobular
carcinoma. Tumor differentiation (grades) of the
9.5% (n=2) were Grade 1, 76.1% (n=16) were Grade 2
and 14.4% (n=3) were Grade 3. All patients diagnosed
with BC in the study group were from the Luminal A
biological subgroup. There were not any T4 patients
and N3 diseases (Table 1).
Table 1: Disease characteristics distribution regarding
the patient group
A statistically significant difference was found between
the HSP70 measurements of the study and control
groups before and after RT (p=0.001 and p<0.01,
respectively). At the same time, the increase in HSP70
measurements after RT was found to be statistically
significant in the study group patients compared to
before RT (p=0.025 and p<0.05, respectively). A statistically
significant difference was found between the
S100B measurements of the study and control groups
before and after RT (p=0.001 and p<0.01, respectively).
However, the change in S100B measurements after
RT was not statistically significant in the study group
patients compared to before RT (p>0.627) (Table 2).
Both HSP70 and S100B measurements were significantly
higher in the study group than in the control
group (Figs. 1, 2).
Table 2: Evaluation of HSP70 and S100B measurements by groups
Fig 1: The distribution of HSP70 measurements.
RT: Radiotherapy.
Fig 2: The distribution of S100B measurements.
RT: Radiotherapy.
The relationship between serum levels of S100B
and HSP70 and pathological features of BC was also
examined. HSP70 and S100B measurements of the
patients according to tumor differentiation Grade 1,
2, and 3 levels were not statistically evaluated because
73% of the patients were Grade 2. HSP70 and S100B
measurements before and after RT did not show statistically
significant differences according to T and N
stages (p>0.05). As a result, no relationship was observed
between pathological features of BC and S100B
and HSP70 measurements.
Discussion
Ionizing radiation can indirectly cause deoxyribonucleic
acid (DNA) damage by generating reactive oxygen
species. Chromosomal deletions, translocations, or
inversions, as well as single and double-stranded DNA
breakage and base-pair mismatch during replication,
are potential mechanisms of radiation-induced cellular
damage. If genomic damage is too great, p53 initiates
programmed cell death by activating the apoptotic cascade.
In the apoptosis pathway, p53 activates calciumdependent
endonucleases and proteases such as interleukin
1 converting enzyme, DNAse I, and caspases.
Activation of these enzymes leads to sequential DNA
cleavage, an irreversible step in apoptosis. The genetic
determinants and molecular mechanisms of therapeutic
radiation sensitivity are not fully understood. Few
reports document endogenous inhibitors of radiationinduced
apoptosis.[] Considering the radiation-induced
apoptosis mechanism, we examined acute biomarker
changes after RT. When the HSP70 and S100B
results before and after BC RT were compared in our
study, HSP70 levels were significantly higher in the
acute period after RT. However, there was no significant
change in S100B.
Moore described S100B as a calcium-binding protein
from brain tissue in 1965.[] S100 proteins show
a certain tissue and cellular distribution, and S100B is
secreted from nervous system glial cells, melanocytes,
adipocytes, and chondrocytes. Serum S100B protein
plays a role in cell proliferation. S100B levels have also
been found to be elevated in malignant melanoma,
glioma and neuroblastoma, progressive BC, and many
other cancers. The strength of S100B expression was directly related to the degree of malignancy.[,]
Many articles in the literature aimed to use S100B as a
biomarker in the diagnosis, treatment, or follow-up of
BC.[-] Charmsaz et al.[] investigated the inhibition
of the S100B signaling network in endocrine-resistant
BC patients as a biomarker. They examined the
S100B expression in tissue and serum and determined
that tissue and serum S100B levels predicted poor disease-
free survival in patients receiving endocrine therapy.
The marker was proven to have the potential to
be a new surveillance tool for monitoring the ongoing
response to endocrine therapy for ER-positive BC patients.
There was no relationship between serum S100B
levels and clinical-pathological data. However, when
they evaluated S100B levels before and after surgery,
elevated serum S100B levels returned to normal following
surgical resection of the tumor. These data suggest
that high S100B levels may indicate the presence
of tumor burden in BC patients and have the potential
to predict disease progression. Nonetheless, even in the
selected tumor-free group with good prognostic factors
who completed adjuvant systemic therapy in our study,
the S100B values before and after RT were significantly
higher than in the healthy population.
HSPs are among the target molecular chaperonesin
cancer treatment. HSP70 is actively released by
viable, intact tumor cells and also at a lower level by
dying tumor cells. It functions to induce mitotic signals,
suppress stress-induced and apoptosis, as well
as oncogene-induced aging.[] The mechanisms by
which HSPs regulate cancer cell proliferation, invasion,
metastasis, and avoidance of apoptosis have also been
investigated, and they have also been found to increase
resistance to anti-cancer treatments such as chemotherapy
and RT.[-] It has been shown that HSP70
is frequently overexpressed in many different tumor
types such as brain, breast, prostate, colon, and lung
cancer compared to the healthy group and will cause
metastasis development through upregulation of mesenchymal
markers.[,]
Hurwitz et al.[] reported on increased levels of
circulating sHsp70 up to several days after whole-body
irradiation of mice bearing xenograft prostate tumors
that might be explained by dying cells. The slight increase
in sHSP70 levels after radiation therapy might
account for sHSP70 which is released by dying cells.
Apart from dying cells viable tumor cells actively secrete
large amounts of HSP70 in vesicles.
Gehrmann et al.[] measured HSP70 levels in biopsy
and serum parameters of 21 head and neck cancer
patients. HSP70 levels were significantly higher in patients compared to healthy volunteers. They also
found that HSP70 levels decreased in patients without
tumor recurrence during the follow-up period
after surgery and RT. Moreover, in this study, the reduction
of HSP70 levels in post-surgery tumor-free
patients suggests that HSP70 levels may be beneficial
not only for detecting tumors but also for monitoring
the therapeutic response to RT. Similarly, in our study,
HSP70 levels in the group that completed the adjuvant
systemic therapy were significantly higher than in the
healthy population. In addition, RT caused a significant
increase in HSP70 levels in the early period that
might be explained by dying cells.
In our study, the patients were admitted to our clinic
with post-operative adjuvant chemotherapy completed
and at the post-operative tumor-free 7th month
on average. Numerous biomarker studies have been
conducted to measure the presence of microtumors.
In our study, we demonstrated the significant S100B
and HSP70 difference between the healthy group and
the study group and evaluated the clinical use of these
markers Luminal-A non-metastatic BC.
The limitations of this study are the small number
of patients, the absence of S100B and HSP70 levels
measured in the long-term after RT, the absence of accompanying
pathological and immunohistochemical
studies, and the short duration of follow-up. Therefore,
a survival analysis could not be performed. In
our subsequent study, we aim to demonstrate the
long-term follow-up of our patient group and the
chronic period marker change after RT, along with
the correlation between biomarkers with progression
and/or overall survival.
Conclusion
In summary, even if there is no macroscopic tumor
focus, S100B and HSP70 levels are higher than the
healthy population before and after RT in non-metastatic,
Luminal-A BC. Moreover, a significant increase
in HSP70 levels was determined in the acute period after
RT. The significant increase in HSP70 after RT may
be due to the release from dying tumor cells in the microenvironment.
Therefore, HSP70 levels in the blood
may be useful for microscopic tumor focus detection
or evaluation of treatment response.
Peer-review: Externally peer-reviewed.
Conflict of Interest: All authors declared no conflict of interest.
Ethics Committee Approval: The study was approved by
The Bakirkoy Dr. Sadi Konuk Training and Research Hospital
Clinical Research Ethics Committee (No: 2020-06-18,
Date: 16/03/2020).
Financial Support: This study has received no financial
support.
Authorship contributions: Concept - E.E.Ö., N.I., G.P.S.,
G.Ş.E., A.G., M.K.B.; Design - E.E.Ö., N.I., G.P.S., G.Ş.E.,
A.G., M.K.B.; Supervision - E.E.Ö., N.I.; Funding - None;
Materials - E.E.Ö., N.I., G.P.S., G.Ş.E., A.G., M.K.B.; Data
collection and/or processing - E.E.Ö., N.I., G.P.S., M.K.B.;
Data analysis and/or interpretation - E.E.Ö., N.I., G.P.S.,
M.K.B.; Literature search - E.E.Ö., N.I., G.P.S., G.Ş.E., A.G.,
M.K.B.; Writing - E.E.Ö., G.Ş.E., M.K.B.; Critical review -
E.E.Ö., M.K.B.
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