Clinical value of peripheral blood M2/M1 like monocyte ratio in the diagnosis of breast cancer and the differentiation between benign and malignant breast tumors

Background: During tumor progression, circulating monocytes and macrophages are actively recruited into tumors where they alter the tumor microenvironment to accelerate tumor progression. In response to multiple microenvironmental signals from the tumor and stromal cells, macrophages change their functional phenotypes. Based on their function, macrophages are commonly classified into both, classical M1 and alternative M2 macrophages. M2-like tumor-associated macrophages promote breast tumor growth and survival, and may migrate into the peripheral blood. However, the level of circulating M2/M1-like monocyte ratio in the peripheral blood of breast cancer patients has not been yet clarified. Aim: To compare peripheral blood M2/M1 monocyte ratio among breast cancer patients, benign breast tumor patients and healthy subjects. Also, to investigate the role of peripheral blood M2/M1 monocyte ratio as a circulating breast cancer tumor marker and to asses the validity of this marker in differentiation between benign and malignant breast tumors. Methods: Flow cytometry technique was used to determine the peripheral blood M2/M1 monocyte ratio in three groups of subjects, i.e. 45 patients with breast cancer, 40 patients with benign breast tumor, and 40 healthy subjects as a control group. The results of carbohydrate antigen15-3 (CA15-3) determination were analyzed comparatively. Results: The peripheral blood M2/M1 monocyte ratio in patients with breast cancer (0.27±0.1) was significantly higher (P<0.001) than that in healthy subjects (0.07±0.05) and than in benign tumor subjects (0.08±0.04). The area under the receiver operating characteristic (ROC) curve of peripheral blood M2/M1 monocyte ratio determination was significantly higher (P≤0.001) than that of CA15-3 levels. Conclusion: M2/M1-like monocyte ratio is of a high diagnostic value for breast cancer and is a promising differentiating marker between benign and breast cancer tumor groups.


INTRODUCTION
Breast cancer is the most common cancer diagnosed in women, accounting for more than 1 in 10 new cancer diagnoses each year. It is the second most common cause of death from cancer among women in the world (Bray et al., 2018). Breast cancer has become a major threat to female health in Iraq, where it is the leading cause of death after cardiovascular diseases among women, with a cancer-related mortality rate of 23% (Alwan, 2016). Since 1986, it has been the highest-ranked malignancy among the Iraqi population in general (Alwan, 2016). Breast tumors are broadly classified as a benign or malignant mass. The main fear of many women presenting with breast tumor is the likelihood of cancer, but reassuringly, most breast masses are benign (Degnim et al., 2017). Malignant breast mass encompasses many histological types, but is not limited, such as in situ ductal or lobular carcinoma, infiltrating ductal or lobular carcinoma and inflammatory carcinoma (Degnim et al., 2017). Breast cancer is known as cancer that developed from breast tissues. Breast cancer most commonly develops in cells from the lining of milk ducts and the lobules that supply the ducts with milk (Foster, Jr, 2003). Cancers developing from the ducts are known as ductal carcinomas, while those developing from lobules are known as lobular carcinomas (Calhoun & Anderson, 2017). About 5-10% of cases are due to genes inherited from a person's parents (Kuchenbaecker et al., 2017). Breast cancer is the most prevalent type of cancer among women around the world and has the highest fatality rate (Parkin et al., 2005). The most reliable detection methods are mammography and core needle biopsy. However, these methods are not sensitive or comfortable enough for women to select as routine exami nations. For the liquid biopsy, the existing markers, such as carcino embryonic antigen or carbohydrate antigen 15-3, are not recommended for screening or diagnosis of breast cancer be cause of their low sensitivity in early detection (Harris et al., 2007). Therefore, a convenient, effective method for early detection is urgently needed. Monocytes are released from the bone marrow into the blood, and after 3 to 4 days in circulation they enter the tissues and serosal cavity where they are considered as macrophages (Sica et al., 2008). Tumor related macrophage (tumor-associated macrophage TAM) plays an important role in the development of tumors (Sica et al., 2008). Studies have shown that TAM usually has 2 states in the tumor tissue which are characterized by a completely different polarization ,  (Sica et al., 2008). Previous studies have confirmed that TAM can allow cancer cells to pass through the capillary wall in the tumor tissue and enter the blood circulation (Roussos et al., 2011;Adams et al., 2014). Wyckoff et al. have shown that mammary tumors exhibit a paracrine loop between TAMs and cancer cells (Wyckoff et al., 2004). TAMs express monocyte colony-stimulating factor receptor (M-CSFR, also known as CSF-1R or cFMS), which binds the monocyte colony-stimulating factor (M-CSF, also known as CSF-1) secreted by cancer cells. Conversely, TAMs secrete epidermal growth factor (EGF) and activate the EGF receptor (EGFR) on the cancer cells. This allows co-migration of the two cell types, and thus enhancing motility and subsequent invasion of healthy surrounding tissue and intravasation (Sousa et al., 2015). The immune system is a major player in the cancer cell/tumor microenvironment crosstalk. In solid tumors, 5-40% of the tumor mass consists of tumor-associated macrophages (TAMs). Approximately 80% of the publications in this field report an association between TAMs and poor prognosis (Pollard, 2008). The new nomenclature that groups monocytes into three subsets, based on expression of the surface markers CD14 and CD16, has been approved by the Nomenclature Committee of the International Union of Immunologic Societies (Ziegler-Heitbrock et al., 2010). Based on this nomenclature, the major population of human monocytes (90%) with high CD14 but no CD16 expression (CD14 ++ CD16 − or CD14 + CD16 − ) are termed classical monocytes, whereas the minor population of human monocytes (10%) is further subdivided into the intermediate subset, with low CD16 and high CD14 (CD14 ++ CD16 + or CD14 + CD16 + ), and the non-classical subset, with high CD16 but with relatively lower CD14 expression (CD14 + CD16 ++ orCD14 dimCD16 + ) (Italiani & Boraschi, 2014). This study was aimed to explore whether the circulating M2/M1-like monocyte ratio might be used as a new circulating tumor marker for Breast Cancer (BC) diagnosis and differentiating between BC and benign breast tumors.

Patients and ethics.
A case-control study was conducted at the Department of Chemistry and Biochemistry at the College of Medicine, Al-Nahrain University, during the period from November 2018 to April 2019. The study involved 125 women who are consulting at the specialized tumor hospital in medical city at Baghdad governorate.
The patients were women having breast tumors. They were divided into two groups. Group 1 included a total of 45 cases of malignant breast tumor patients whose age range was between 18-65 years.
Group 2 included 40 cases of benign breast tumor patients whose age range was between 18-65 years.
Control group included 40 healthy volunteer women with matched age range.
Exclusion criteria of the study included patients with breast cancer after surgical removal, patients after chem-otherapy, patients with viral hepatitis, infectious mononucleosis, atherosclerosis, positive human immunodeficiency virus (HIV) patients, as well as patients with any autoimmune disorder.
All patients that were included were interviewed as having breast tumors by using triple assessment, mammography, clinical examination, ultrasonography and some of them by using fine-needle aspiration cytology.
The study was approved by the Institutional Review Board (IRB) and was performed in accordance with the revised Helsinki Declaration of the World Medical Association. Informed consent was acquired from all individuals.
The data were collected using a short structured questionnaire that included information on age, weight, height, family history of breast cancer or other malignancies, and history of other diseases (Table 1).
Assay principle. Proportion of monocytes was defined by the flow cytometry method. The basic principles of flow cytometry are the passage of cells in single file in front of a laser, so they can be detected, counted and sorted. Flow cytometry measures optical and fluorescence characteristics of a single cell. Physical properties, such as size (represented by forward scatter FSC) and inner complexity (represented by side scatter SSC), resolve certain cell populations (Bain & Dacie, 2011). The dyes are bound with different cellular components, such as DNA or RNA. Furthermore, antibodies conjugated to fluorescent dye can bind specific proteins on cell membranes or inside cells. When labeled cells are passed by a light source, the fluorescent molecules are excited to a higher energy state. Upon returning to resting states, the fluorochromes emit light energy at higher wavelengths. The use of multiple fluorochromes, each with similar excitation and different emission wavelengths allows several cell properties to be measured at the same time. The common dyes include phycoerythrin, propidium iodide, and fluorescein. Tandem dyes with internal fluorescence resonance energy transfer can create more colors and longer wavelengths (Bain & Dacie, 2011). Inside a flow cytometer, cells in suspension are drawn into a stream created by a surrounding sheath of isotonic fluid that creates laminar flow, allowing the cells to pass individually through an interrogation point. A laser beam intersects the cells. The emitted light is given off in all directions and collected via optics that direct the light to a series of filters and mirrors that isolate particular wavelength bands. The light signals are detected by photomultiplier tubes and digitized for computer analysis. The resulting information is displayed in a histogram. The entire operation is fully automated: from washing to final reading (Bain & Dacie, 2011).
Determination of M2 monocytes was done by CD16 antibody and of M1 by CD14 antibody, and the gating was done by CD45 antibody. Gating refers to selecting a particular region of data to be analyzed andallows to look at parameters that are specific to only that subset.
The cell group was gated with CD45. CD16 was used as an M2-like surface marker and CD14 was used as M1 macrophage surface marker.
Immunohistochemistry. IHC methods involve special tissue-staining techniques with labeling antibodies, which requires pathology laboratory infrastructure; quality control is quite important to testing accuracy (Gelband et al., 2015). Immunohistochemistry for ER and PR-formalin (10%) fixed, paraffin-embedded sections of cell blocks were stained for ER and PR using the primary monoclonal antibodies to ER (Clone ID 5; DAKO, Glostrup, Denmark, Cat No. M 7047), and PR (Clone PgR 636; DAKO, Carpinteria -CA, Cat No. M 3569) at a dilution of 1:50, according to the manufacturer's specification. The ASCO/CAP guideline recommendations were used for assessment of the hormone receptors. The tumor cells were considered positive for ER or PR status if more than or equal to 1% of tumor cells demonstrated nuclear staining; less than 1% is negative. Normal breast tissue was used as a positive external control in all cell block preparations (Francis et al., 2019). Immunohis-tochemistry for HER-2 invloved firstly fixed by formalin, paraffin-embedded sections of cell blocks, which were stained for HER-2 using HER-2/neu antibodies (polyclonal; DAKO, Glostrup, Denmark, Cat No. A 0485) at a dilution of 1:50, according to the manufacturer's specification. Stains were assessed by using the ASCO/CAP 2013 guidelines, HER-2-IHC-3+ was considered when >10% of tumor cells had shown a homogeneous dark circumferential (chicken wire) pattern. Incomplete and/ or weak/moderate membrane staining and within more than 10% of tumor cells, or complete membrane staining that is intense and within less than or equal to 10% of the tumor cells, was interpreted as equivocal (HER-2-IHC-2+). Incomplete membrane staining that was faint/ barely perceptible and within >10% of the tumor cells was classified as HER-2-IHC-1+, and HER-2-IHC-0 was defined by no staining observed, or membrane staining that is incomplete and is faint/barely perceptible and within ≤10% of the tumor cells. HER-2-IHC-1+ and HER-2-IHC-0 were interpreted as HER-2 negative (Wolff et al., 2013).
Quantification of plasma CA15-3 level. The level of plasma cancer antigen 15-3 was measured by using an ELISA kit (CALBIOTECH, U.S.A) for quantitative measurement of serum CA 15-3.
Statistical analysis. Stasistical analyses were performed using SPSS 17.0. Results were expressed as mean ± standard deviation (S.D.) and all statistical comparisons were carried out by means of independent t-test and analysis of variance (ANOVA) test. All statistical tests' differences with a P level of <0.05 were considered to be significant. In addition, areas under the curve (AUC) of receiver operating characteristics (ROC) curves were calculated, as well as specificity and sensitivity were also estimated (Perkins & Schisterman, 2006).

RESULTS
The Hematological and biochemical characteristics of the three groups (45 breast cancer patients, 40 benign breast disease patients and 40 controls) are summarized in (Table 2).
As shown in Table 2, there is no significant difference among the three groups in the WBC count, monocytes count, neutrophil count, and platelets level. The ratio of M2/M1 monocytes was examined by evaluation of M2like and M1 percentages.
A generally accepted definition of M2 like macrophages is CD16 ++ and CD14 + , and M1 type definition is CD14 ++ and CD16 -, while intermediate monocytes are those cells which express CD14 + CD16 + as shown in (Fig. 1).
As expected, the percentages of M2-like macrophages were significantly elevated (P≤0.05) in BC, and are higher than those in benign and healthy controls (Table 3).
However, there was no significant difference (P<0.005) in the percentage of M2 between the benign and healthy subjects.
A preliminary analysis was then performed to identify whether the percentage of M2 and M1-like macrophages were associated with patient's characteristics as shown in Tables 4 and 5. ROC curve was used to evaluate the diagnostic value of M2 and M2/M1 monocyte ratio and then a comparison with CA15-3 diagnostic value was performed by assessing the sensitivity, specificity, and accuracy of markers.
Each of M2, M2/M1 ratio and CA15-3 had significantly higher values in BC women when compared to BBT. Accordingly, these parameters were evaluated for their diagnostic value in the context of discrimination between breast cancer and benign breast tumor group (Fig. 2).

DISCUSSION
Macrophages continuously infiltrate into the microenvironment of solid tumors and are induced to form TAMs by cancer cells and other factors (Heusinkveld & van der Burg, 2011). Several studies have reported that the polarized states and levels of TAMs in pretreatment biopsies are altered in BC (Tang, 2013). TAMs resemble M2-like macrophages, and M2-like TAMs facilitate tumor progression, angiogenesis, metastasis, matrix remodeling, and treatment resistance (Sica et al., 2008;Obeid et al., 2013). TAMs derived from the tumor tissue disseminate into the peripheral blood circulation in large numbers, in conjunction with circulating tumor cell (CTCs) via transendothelial migration (Adams et al., 2014). Recently, circulating M2-like monocytes have been reported as a useful marker for diagnosis of many cancers, such as non-small-cell lung cancer and colorectal cancer (Zhang et al., 2017). However, until now, few studies have reported the use of circulating M2-like monocytes as a diagnostic marker for breast cancer. Furthermore, data on this cell type are limited, and there have been contradictory results regarding the value of circulating tumorassociated monocytes as potential biomarkers in cancer   patients (Jóźwik et al., 2015).Thus, this study focuses on evaluating the diagnostic value of circulating M2/M1-like monocytes in breast cancer. Our data indicated that the ratio of circulating M2/ M1-like monocytes was increased in the peripheral blood of BC patients and this ratio was promising in differentiating malignant tumor from benign breast tumor patients. Zhang and coworkers (Zhang et al., 2017) stated that the number of M2-like macrophages was increased in histological BC sections.
Accordingly, this research investigated whether M2like monocytes could be detected in the circulating blood of BC patients. We found that the levels of circulating M2-like monocytes and M2/M1 monocyte ratio were significantly elevated in patients with breast cancer when compared with healthy controls and benign patients. This result confirmed previous findings that M2like TAMs may leak into circulation from tumor sites, and the subsequent increase in circulating M2/M1-like monocyte ratio may serve as a tumor marker (Jóźwik et al., 2015).
Most of the currently used tumor markers are not suitable as diagnostic tools for the early detection of cancers. CA15-3 are a common marker of BC; however, it lacks sensitivity for early diagnosis (Mirabelli & Incoronato, 2013). Additionally, the American Society of Clinical Oncology (ASCO) guidelines do not recommend the use of CA15-3 for BC screening or directing treatment (Harris et al., 2007).
In this study, plasma CA15-3 levels were significantly higher in BC patients than in healthy controls and benign patients. In accordance with findings from previous studies, our results showed that CA15-3 had a relatively low sensitivity in detecting BC. When patients with BC were compared with the healthy population, the ratio of circulating M2/M1-like monocytes had a higher AUC and sensitivity. Furthermore, when comparing BC patients with benign patients, we found that the sensitivity of circulating M2/M1-like monocyte ratio was higher than CA15-3. Accordingly, in terms of diagnostic value in BC, circulating M2/M1-like monocytes were superior to CA15-3. Furthermore, there were no associations between M2/ M1-like monocyte ratio, and cancer characteristics or hormonal receptor statuses in our study. Therefore, it is reasonable to state that there are no possible interactions between circulating M2-like monocytes and BC progression.
In conclusion, our data showed that circulating M2/ M1-like monocyte ratio may represent a novel bloodbased biomarker for breast cancer and may have a potential utility as a diagnostic tool. The high sensitivity of circulating M2/M1-like monocyte ratio for detecting BC patients indicates that this ratio may be helpful to the currently used biomarkers in breast cancer diagnosis and differentiating malignancy from benign cases. It should be noted, however, that our analyses only focused on monocytes and the sample size was relatively small. Therefore, further studies with a larger population and the collection of matching histological specimens are necessary to confirm our results.