ORIGINAL ARTICLE

J Adv Biotechnol Exp Ther. 2024 May; 7(2): 433-441

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Association of neutrophils, monocytes, and lymphocytes with

CA15-3 as a predictor of breast cancer in female patients
Raed Madhi1, * , Hussein Ali Jumaah2 , Hasan Hanoon Hasan2
1Department of Biology, Collage of Science, University of Misan, 62001 Maysan, Iraq
2Al-Hussein Teaching Hospital, Thi-Qar Health Directorate, 64001 Thi-Qar, Iraq

*Corresponding author ABSTRACT

Raed Madhi, PhD
Department of Biology, Collage of
Infiltrated leukocytes, such as neutrophils, monocytes, and lymphocytes can be involved in a
Science, University of Misan, variety of tumor development. The present study aimed to investigate the linkage of
Maysan, Iraq neutrophils, monocytes, and lymphocytes with CA15-3 as a predictor of breast cancer.ICLE The
E‑mail: [email protected] study included 116 patients (58 patients before chemotherapy and 58 patients after
chemotherapy. CA15-3 antigen levels were measured, and the grading of breast cancer was
Academic editor
Md Jamal Uddin, PhD
identified. In addition, neutrophils, monocytes, and lymphocytes percentage and their
ABEx Bio-Research Center, Dhaka, correlation to CA15-3 antigen were determined. The results observed a significant increase in
Bangladesh the CA15-3 antigen levels. Moreover, the study also showed that CA15-3 antigen levels have a
great correlation with grades of breast cancer particularly in grade 3 before treatment. The
Article info results also found that neutrophils and monocyte percentages were significantly increased in
Received: 07 March 2024
patients before treatment. However, lymphocyte percentage decreased before treatment and
Accepted: 25 April 2024
Published: 15 May 2024 greatly increased after treatment. Additionally, the results showed a positive correlation
between neutrophil and monocytes percentage with CA15-3 antigen levels before and after
Keywords treatment. However, the correlation was negative between lymphocyte percentage and CA15-
Female patients, Neutrophil, 3 antigen levels. The present findings observed that neutrophils and monocytes have a great
Monocytes, Lymphocytes, Breast
cancer, CA15-3
association with CA15-3 antigen levels, therefore, targeting these cells could protect against
breast cancer development. However, targeting these cells can negatively affect the patient's
immune response, thus further studies are needed to study the exact mechanism by which
neutrophils and monocytes could enhance breast cancer development. Additionally, increased
lymphocytes after treatment might be a good strategy to treat breast cancer.

INTRODUCTION

Breast cancer is considered one of the most prevalent cancers and the second most
common cause of cancer death in women. It is indeed one type of cancer that is
Copyright: © by the authors. This initiated and developed in breast tissue. Histopathological findings have shown that
article is an open-access article breast cancer may begin in either the left or right breast [1-3]. Age and breast cancer
distributed under the terms and incidence are strongly correlated, with incidence rising in the age range above 45 years
conditions of the Creative Commons
[1, 4]. There are various methods for identifying and tracking the incidence and
Attribution (CC BY) license.
development of breast cancer. For example, blood tumor indicators like CA15-3 antigen
or genetic profiles can be used to diagnose breast cancer. CA15-3 has indeed been
shown to be assured for detecting breast cancer [5-7].
There is mounting evidence that the immune system and its constituent parts play an
essential role in the initiation and growth of malignant tumors. Cancer indeed triggers
an immune response in its microenvironment, but these responses are typically
inefficient at eliminating cancerous cells [8, 9]. In both bone marrow and peripheral
blood, neutrophils are known to be the most prevalent innate immune cells [10, 11].
They have considerable flexibility and a potent effector response and are
instantaneously infiltrated into sterile or infected inflammatory sites. Neutrophils have

www.bsmiab.org/jabet Madhi et al., J Adv Biotechnol Exp Ther. 2024 May; 7(2): 433-441
433
 a brief lifespan [12], possibly to head off unneeded tissue injury. Therefore, the quantity
of neutrophils depends on ongoing granulopoiesis in the bone marrow replenishment.
Numerous ways explain how neutrophils are engaged in pathogenesis. In the
beginning, neutrophils attracted to the inflamed site produce proteolytic enzymes and
generate a lot of reactive oxygen species (ROS), which damages the tissue and makes it
more vulnerable to pathogens and can even lead to the emergence of chronic
inflammation [13-15]. Numerous cases of severe coronavirus disease 2019 (COVID-19)
cases as well as other viral and pulmonary disorders have shown this pathogenic
consequence [15, 16]. Neutrophils are frequently found in the tumor microenvironment,
but little is known about their function in the development of cancer, and they have
typically been viewed as bystanders [17, 18]. Neutrophils are an essential part of
developing cancer by causing inflammation and damaging tissues. Neutrophils
therefore establish a connection between inflammation and malignancy [19, 20].
Moreover, it has been demonstrated that monocytes and lymphocytes play a key role in
systemic inflammatory response [21, 22]. These cells indeed vary in their function with
respect to cancer development and metastasis. For instance, lymphocytes have been
shown to work as tumor suppressors. In contrast, monocytes can positively participate
in tumor cell invasion and metastasis by constructing the microenvironment of the
tumor [23, 24]. Thus, the current study aimed to estimate the linkage of neutrophils,
monocytes, and lymphocytes with CA15-3 in breast cancer.

MATERIALS AND METHODS

Study design
The current study was carried out in Maysan City, Iraq. Samples were collected from
116 patients, including 58 patients before getting the chemotherapy treatment and 58
patients after getting the chemotherapy treatment who suffered from breast cancer and
attended the oncology center in Al-Sader Hospital, Maysan, Iraq. Specialist pathologist
staff were diagnosed with breast cancer and based on different approaches; clinical
examinations, estimation of CA15-3 levels, and biopsy of the breast. In addition, 40
blood samples from healthy women were also collected as a control.

Ethical approval
The ethics committee of Trigis Medical Center affiliated Maysan Health Department
has approved the protocol of the present study (approval number: 2021-02).

Measurement of CA15-3
A commercial kit (EIA-3941, DRG, Germany) was used to measure CA15-3 levels of
patients and healthy groups according to the manufacturer’s instructions. Briefly, 10 μL
of standard, samples, and controls were dispensed into corresponding wells. The
amount of assay buffer used for each well was 250 μL. The wells were mixed smoothly
and incubated at room temperature for 60min. Then, each well was rinsed with a 400
μL wash solution three times. After washing, each well was incubated with 100 μL of
enzyme conjugate at room temperature for 60 min. Thereafter, each well was rinsed
with 400 μL wash solution three times and incubated with 100 μL of solution of
substrate at room temperature for 30 minutes. Finally, 100 μL the reaction was stopped
in each well by adding a stop solution (100 μL). The reaction was read at wavelength
450 nm after ten min of stopping the reaction.

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 Histological examinations
A biopsy of the breast was collected to confirm the diagnosis of breast cancer in
suspected women. Normal breast tissues were obtained from the oncology center/Al-
Sader hospital/Maysan and used for comparison by specialist pathologist staff. Breast
tissue samples were subjected to fixation in 4% formaldehyde and then dehydration
and embedding with paraffin. After, hematoxylin and eosin (H&E) stains were used for
staining the tissue samples. Examinations of tissue samples were done under light
microscopy. Breast cancer grading was performed according to a standard protocol [25].

Measurement of leukocytes in blood samples

To count and determine leukocytes in blood samples of control and patients, a CELL-
DYN (Abbott, Abbott Park, Illinois, U.S.A.) was used by using Multi-Angle Polarized
Scatter Separation (MAPSS) technology. Briefly, the identification and differentiation of
neutrophils, monocytes, and lymphocytes were performed by using four light scatter
detectors. Cell passes through a tiny opening that separates the two electrodes while
whole blood is transferred between them. The cell count and volume were performed
quickly by measuring the changes in electrical conductance when cells floating in a
conductive fluid passed through a tiny opening, which is proportional to the cell
volume, and the final results were presented as percentages.

Statistical analysis
SigmaStat 3.5 program was used to perform statistical analysis. A T-test was used to
compare two different means. In addition, the LDS method was also used for further
comparison. Data are introduced as mean values ± standard error of the mean. P < 0.05
is considered as a statistical significance between the groups.

RESULTS
CA15-3 antigen levels in breast cancer patients
It is believed that the CA15-3 is used frequently as a diagnostic biomarker for breast
cancer [26]. Serum CA15-3 was measured in breast cancer patients and control groups
(Figure 1). Our results demonstrated that serum CA15-3 antigen levels were
significantly increased by 3.7-fold and 1.5-fold in patients with breast cancer before
getting chemotherapy and patients with breast cancer after getting chemotherapy,
respectively (Figure 1).

Breast cancer grading and CA15-3 antigen levels

Histological examinations were performed for further confirmation of breast cancer
diagnosis in suspected women with breast cancer (Figure 2). The histological
examinations observed that the cells and tissue architecture of grade 1 breast cancer are
more similar to normal cells (Figure 2A). However, in grade 2, histological
examinations showed that cells and tissue architecture look quite different from normal
cells (Figure 2B). Moreover, grade 3 showed a very different look in the cells and tissue
architecture in breast tissue (Figure 2C). It was interesting to evaluate the linkage
between CA15-3 antigen levels and the grading of breast cancer. CA15-3 antigen levels

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 were divided into three groups (10-20, 21-29, and ≤30, respectively) (Figures 2D and E).
Our results revealed that the levels of CA15-3 antigen were significantly increased
(P˂0.05) in grade 3 as compared to grade 1 in patients with breast cancer before getting
chemotherapy (Figures 2D and E). However, the statistical analysis showed no
significant differences (P = 0.461) among the grading of breast cancer in CA15-3 antigen
levels after treatment (Figure 2E).

Figure 1. CA15-3 antigen levels. ELISA method was used to measure the levels of CA15-3 antigen in control
and patients with breast cancer before and after getting the chemotherapy. Patients with breast cancer are
introduced by the black box. Control groups are introduced by the grey box. Data introduced as means ±
SEM. #P <0.05 versus control.

Figure 2. Breast cancer grading and CA15-3 antigen correlation. Sectioning of (H&E) of breast tissue. Scale bar
= 50 μm. A) Normal breast tissue, B) Grade 1, C) Grade 2, and D) Grade 3. CA15-3 antigen association into
breast cancer grading, E) before getting chemotherapy and F) after getting chemotherapy. Data represented
as mean of percentage; the LDS method was used to compare between groups. *P-value <0.05 is considered as
a significant difference between the groups.

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 Estimation of neutrophils, monocytes, and lymphocytes in breast cancer
Neutrophils play a great role in cancer cell development and metastasis. In this study,
we examined the percentage of neutrophils before and after treatment in patients with
breast cancer (Figure 3A). Our results found that levels of neutrophils were
significantly increased (P˂0.05) in patients with breast cancer before getting the
treatment as compared with control (Figure 3A). Moreover, the present results show
that the levels of monocytes were substantially increased (P˂0.01) in breast cancer
patients before chemotherapy compared to the control group (Figure 3B). However, the
results also found that the levels of lymphocytes were increased by 1.5-fold as
compared to the control group after treatment (Figure 3C).

Figure 3. Estimation of neutrophils, monocytes, and lymphocytes in the blood. Measurement percentage of A)
neutrophils, B) monocytes, and C) lymphocytes in both control and patient groups. Control group (gray box).
Patients with breast cancer before and after getting the chemotherapy (black box). Data represent means ±
SEM. *A p-value of <0.05 is considered a significant difference between the groups.

Correlation of neutrophil, monocytes, and lymphocytes with CA15-3 antigen levels

It was important to investigate the relationship among neutrophils, monocytes, and
lymphocytes with serum CA15-3 antigen levels in breast cancer patients. Linear
regression was used to analyze the relationship of neutrophils, monocytes, and
lymphocytes with serum levels of CA15-3 antigen (Figure 4). Interestingly, the
statistical analysis showed a strong correlation (y = 0.981x + 52.352 R² = 0.7204 and y =
3.2024x - 22.109 R² = 0.7512) between neutrophils and CA15-3 antigen levels before and
after getting the chemotherapy, respectively (Figure 4A and B). In addition, our results
revealed that there is a significant correlation (y = 0.2342x + 2.3657 R² = 0.5628 and y = -
0.6086x + 16.817 R² = 0.7486) between monocytes and CA15-3 antigen levels before and
after getting the chemotherapy, respectively (Figure 4C and D). In contrast, the
statistical analysis found a negative correlation (y = 0.1195x + 11.458 R² = 0.0502 and y =
-0.0776x + 48.993 R² = 0.0011) between lymphocytes and CA15-3 antigen levels before
and after getting the chemotherapy, respectively (Figure 4E and F).

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Figure 4. Linkage of neutrophils, monocytes, and lymphocytes with CA15-3 antigen levels. Correlation of CA15-3 antigen levels into neutrophil A)
before getting chemotherapy (y = 0.981x + 52.352, R² 0.7204 ) and B) after getting chemotherapy (y = 3.2024x - 22.109, R² = 0.7512), monocytes C)
before getting chemotherapy (y = 0.2342x + 2.3657, R² = 0.5628) and D) after getting chemotherapy (y = -0.6086x + 16.817, R² = 0.7486), and
lymphocytes E) before getting chemotherapy (y = 0.1195x + 11.458, R² = 0.0502) and F) after getting chemotherapy (y = -0.0776x + 48.993, R² =
0.0011). Linear Regression was used to compare between groups.

DISCUSSION
In the current study, the data demonstrated that CA15-3 levels have an association with
the diagnosis of breast cancer. Furthermore, neutrophils and monocytes were greatly
increased in the blood of patients with breast cancer before treatment and have an
association with levels of serum CA15-3 antigen. Additionally, lymphocytes were
increased after treatment and have a negative correlation with levels of serum CA15-3
antigen. Therefore, an increased percentage of neutrophils and monocytes could be
used as an indicator for breast cancer diagnosis and increasing lymphocyte levels after
chemotherapy might provide a successful strategy to treat patients with breast cancer.
Moreover, breast cancer cells have been found to express high levels of CA15-3 antigen
compared to the normal [26]. In the current study, our results revealed that the CA15-3
levels significantly increased in breast cancer patients. In addition, CA15-3 antigen
levels showed an increase in different grades of breast cancer, however, the increase of
CA15-3 antigen levels was significant particularly in grade 3 before chemotherapy
treatment. This could explain why the CA15-3 be utilized as a biomarker for the
diagnosis of breast cancer.
It is well known that the most abundant leukocytes in the blood are represented by
neutrophils, which are regarded as the first line of defense when the body is exposed to

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 infection and inflammation [27-29]. An inflammatory response caused by invasive
bacteria draws neutrophils from the bloodstream into the tissues. The pathogen is then
eliminated by neutrophils using a variety of methods, chiefly phagocytosis, the release
of microbicidal agents, and the development of neutrophil extracellular traps (NETs)
[13, 14, 30]. Additionally, proteinases are released by activated neutrophils into the
tissue around them, harming the host [31, 32]. Moreover, neutrophils and monocytes
were shown to play a significant role in regulating cancer cell development [33]. In the
present study, it was important to estimate the percentage of neutrophils and
monocytes in breast cancer patients and their correlation to the levels of CA15-3 antigen.
Interestingly, the current results demonstrated that neutrophils and monocytes were
significantly increased in the blood of patients with breast cancer before getting the
treatment. Moreover, we investigated the association between the percentage of
neutrophils and monocytes with CA15-3 levels. Our findings showed a great
correlation between the percentage of neutrophils and monocytes CA15-3 antigen
levels. Notably, neutrophils and monocytes have been reported to be infiltrated into
different kinds of cancers. It was difficult to envision those neutrophils, because of their
short-lived, could have an impact on chronic and progressive diseases like cancer in the
early investigations. However, it has lately become evident that tumor-related
neutrophils play important roles in malignant illness. This newfound interest is partly
due to the realization that cancer-associated inflammation is a crucial component for
the growth of many tumors [34, 35] and is a distinguishing feature of cancer. In
addition, we have also examined the percentage of lymphocytes in patients with breast
cancer. Our results found that the percentage of lymphocytes greatly increased after
treatment. It is thought that chemotherapy destroys the immune cells, particularly
innate immune cells that represent the first defense lines. However, cell debris that
results from the cytotoxic effects of chemotherapy releases signals that can lead to the
actuation of dendritic cells (DCs) to engulf the dying cells. Subsequently, DC, as
antigen-presenting cells, stimulate lymphocyte recruitment such as helper T cells.

CONCLUSIONS
In conclusion, the current findings demonstrate a great association between CA15-3
levels and the grading of breast cancer. Moreover, neutrophils were highly elevated in
breast cancer, and therefore, an increased percentage of neutrophils besides the levels
of CA15-3 antigen might be a good strategy to be used as an indicator for breast cancer
diagnosis. However, the limitation presented in the current study is that the conclusion
was based on one diagnostic marker. Thus, further studies are needed to study the role
of neutrophils in breast cancer development based on different diagnostic markers. In
addition, according to the current findings, neutrophils might be involved in the
development and spread of breast cancer. Thus, the present results emphasize the role
of the immune system in the development of cancer and the possibility of
immunotherapies to treat breast cancer, even though the exact mechanisms are still
being investigated.

ACKNOWLEDGMENTS
We thank the Health Department in Maysan for authorizing the present study. We are
also thankful to Aqil Ifrih Jazza and Mntather Hatem Frhan for their help in blood and
tissue sample collection.

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 AUTHORS CONTRIBUTIONS
RM, HAJ, and HHH assisted in conducting the experiments, performed the statistical
analysis and data visualization, and wrote the manuscript. RM designed and conducted
all of the experiments and wrote the manuscript. All authors have read and approved
of the final manuscript.

CONFLICTS OF INTEREST
There is no conflict of interest among the authors.

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