Association Between Monocyte Levels and Breast Lump Detected By Ultrasound Among Chinese Women: A Longitudinal Study

Few studies have focused on the relationship between monocyte and breast lump. To explore whether absolute monocyte count (AMC) or monocyte percent (%MONO) could be used as a new circulation tumor marker for breast lump detection by ultrasonography among Chinese women. Methods A total of 3,231 women who had at least two breast ultrasonography examinations were followed up from January 2014 to December 2019. Adjusted Cox proportional hazards regression models were used to evaluate the relationships between AMC and %MONO and the incidence of breast lump. Results a total of of participants developed a breast the nal and (1.02, for in the 2nd, 3rd, and 4th quartiles (P trend < 0.001). The corresponding HRs (95%CIs) for %MONO in the 2nd, 3rd, and 4th quartiles were 1.03 (0.83, 1.28), 1.28 (1.03, 1.29), and 1.62 (1.30, 2.02, P trend < 0.001), respectively. The per unit of and were 7.38; P 0.007) and (1.08, 1.21; P < 0.001), respectively. The effect of high monocyte levels on increased risks of breast lump were more remarkable in younger women. absolute basophil count (ABC), percentage of basophil (%BASO), absolute lymphocyte count (ALC), percentage of lymphocyte (%LY), AMC, %MONO, red blood cell (RBC) count, hemoglobin (Hb), and platelet (PLT) count. Data regarding self-reported marital status, smoking status, drinking status, history of chronic disease, and family disease were also collected using a detailed questionnaire.


Introduction
Worldwide, breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death among females [1]. In 2015, it was estimated that there were about 268,000 newly diagnosed invasive breast cancer cases among Chinese women [2], with 68,500 deaths [2], making breast cancer a major threat to women's health in China. Breast lump are the most dreaded signs in women; even though most breast lump are benign, breast lump have a relatively high probability of being malignant [3,4]. In addition, even in women with proven benign breast disease (BBD) (e.g., broadenosis and broadenoma), the benign breast lump indicates an increased risk of developing breast cancer compared with women without BBD [3,5]. Therefore, the presence of breast lump can be an indicator of breast cancer or an early indicator of an increased risk of breast cancer. Mammography and core needle biopsy are the most reliable detection methods for breast lump, but these techniques are not su ciently sensitive for use in dense breasts or for detection of small breast lump, and they are uncomfortable and invasive for women to select as routine examinations [6,7]. In contrast, ultrasound is more accurate at detecting small tumors, even in dense breasts, and repetitive evaluation over a short interval is more feasible because it does not involve radiations and is non-invasive [8]. Owing to the increasing development and frequency of use of breast ultrasound in breast cancer screening, the incidence of breast lump is rising [9].
Since Rudolf Virchow noted "lymphoreticular in ltrate" in neoplastic tissues in 1863, in ammation has been associated with cancer [10][11][12][13]. As a major component of the in ltrates of most tumors, tumorassociated macrophages (TAMs) are derived from monocytes and recruited to the tumor microenvironment, where they play an important role in the development of tumors [14,15]. In solid tumors, TAMs have seemingly contradictory roles. On the one hand, they can kill tumor cells and, when appropriately activated, elicit tumor tissue destructive reactions centered on the vascular endothelium; on the other hand, TAMs can stimulate tumor-cell proliferation and tumor growth, promote angiogenesis, and favor invasion and metastasis [16,17]. Proin ammatory processes in adipose tissue contribute to breast cancer development [18]. In turn, many studies have previously reported that malignant neoplasms adversely affect monocyte function and could hinder the immune-mediated destruction of malignant cells (i.e., the infamous immune escape) [19][20][21]. Many studies have previously reported monocyte levels or monocyte-related markers of in ammation as predictive and prognostic biomarkers of breast cancer [22][23][24][25][26][27] or malignant neoplasms adversely affect monocyte function and could hinder immunologically mediated destruction of malignant cells [19][20][21]. However, the relationship between absolute monocyte count (AMC) or monocyte percentage (%MONO), as systemic indicators of monocyte activity and breast lump, is not well understood [3,28]. Therefore, this longitudinal study aimed to explore the association between AMC and %MONO with the breast lump detected by ultrasound in a large sample of Chinese with a long follow-up period. The results could provide additional markers for the risk management of women undergoing breast screening.

Study design and population
This was a retrospective study of female patients who underwent comprehensive health examinations at Tianjin Medical University General Hospital from January 2014 to December 2019. The protocol of this study was approved by the institutional review board of Tianjin Medical University General Hospital. The need for individual consent was waived by the board.
The inclusion criteria were 1) >18 years of age, and 2) no abnormality in the rst breast ultrasound examination, and 3) had at least two subsequent breast ultrasound examinations. The repeat examinations were recognized by their individual health examination ID. The exclusion criteria were 1) with history of breast cancer, or 2) with history of breast surgery, or 3) with abnormal AMC or %MONO, or 4) with incomplete blood routine examination data from the rst health examination, or 5) with a chronic in ammatory disease such as cholecystitis, chronic nephritis, rhinitis, pharyngitis, bronchitis, gastritis, rheumatoid arthritis, myocarditis, or others, at the rst health examination.

Assessment of breast lump
For the routine breast examination, all participants underwent a whole breast ultrasound scan in the supine position. The presence of a breast lump was de ned as the detection of a three-dimensional lesion, such as a breast cyst, solid or mixed lump, or intraductal lump, that occupied space within the breast (Fig. 1) [29]. Experienced board-certi ed radiologists performed all breast ultrasound examinations using a 5-12 MHz linear probe (OXANA 2, Siemens, Erlangen, Germany) or 5-12 MHz linear probe (XARIO, Toshiba Corp., Tokyo, Japan); they also performed the subsequent evaluations.

Data collection
This study used data from the cohort of Tianjin Chronic Disease Risk and Health Management. Basic information, including age and sex, was checked before health examinations. Blood pressure and anthropometric parameters (height, weight, and waist circumference) were measured by experienced medical staff following standard procedures. Body mass index (BMI) was calculated as weight (kg) divided by the square of height (m 2 ). All blood samples were taken after at least an 8-h fast and analyzed at the Department of Laboratory Medicine. The following blood values were collected: total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), fasting blood glucose (FBG), uric acid (UA), white blood cell (WBC) count, absolute neutrophil count (ANC), percentage of neutrophil (%NEUT), absolute eosinophil count (AEC), percentage of eosinophil (%EO), absolute basophil count (ABC), percentage of basophil (%BASO), absolute lymphocyte count (ALC), percentage of lymphocyte (%LY), AMC, %MONO, red blood cell (RBC) count, hemoglobin (Hb), and platelet (PLT) count. Data regarding self-reported marital status, smoking status, drinking status, history of chronic disease, and family disease were also collected using a detailed questionnaire.

Follow-up
All patients were followed by ultrasound examinations in the context of annual physical examinations until December 31, 2019. All examinations were conducted by doctors and nurses and breast ultrasound scan were performed by radiologists in the ultrasound imaging department. Based on the results of the ultrasound scan, the patients were grouped according to whether a breast lump was found during followup.

Statistical analysis
Patient baseline characteristics are presented as median (interquartile range) for continuous variables and as number (percentage) for categorical variables. All continuous variables were examined for normality using the Shapiro-Wilk test; for non-normal distribution variables, the differences among breast lump categories were examined using the Mann-Whitney U test, and categorical variables were examined using the chi-squared test. The incidence of breast lump was used as the dependent variable, and AMC and %MONO were used as the independent variable. The Cox proportional hazards regression model was used to examine the association between AMC or %MONO and the presence of breast lump initially, using the lowest quartile as the reference group. We also analyzed AMC and %MONO as continuous variables in all regression models. In model 1, crude hazard ratios (HRs) with corresponding 95% con dence intervals (CIs) were calculated. Model 2 was further adjusted for age and BMI as potential confounding variables. Model 3 was adjusted for age, BMI, WC, SBP, DBP, TC, TG, LDL-C, HDL-C, FBG, UA, marital status, smoking status, drinking status, family history of cancer, hypertension, diabetes, history of cancer, history of hypertension, and history of diabetes. Tests for trend across quartiles of AMC and %MONO were examined using the median value of each quartile as an ordinal variable in the Cox proportional hazards regression models. The cumulative incidence of breast lump strati ed by age was analyzed using the Kaplan-Meier method, and the differences between groups were compared using the log-rank test. STATA 13.0 (StataCorp LP, College Station, TX, USA) was used in all analyses. All P-values are twosided, and P<0.05 is de ned as statistically signi cant.

Results
Characteristics of the patients A total of 5760 patients were screened for eligibility, and 2602 were excluded: 48 with a history of breast cancer, 879 with a history of breast surgery, 401 with chronic in ammation at baseline, 1141 with incomplete blood routine data at the rst health examination, and 133 with abnormal absolute monocyte counts or percentage of monocyte. Therefore, the nal study population includes 3158 participants. During a total of 6037 person-years of follow-up (median follow-up time, 2.35 years), breast lumps were found in 803 patients (Fig. 2). Table 1    <0.001 Con dence intervals (CIs) not including 1.00 are considered statistically signi cant.
Model 2: adjusted for age and BMI as potential confounding variables.
Model 3: adjusted for age, BMI, WC, SBP, DBP, TC, TG, LDL-C, HDL-C, FBG, UA, marital status, smoking status, drinking status, family history of cancer, hypertension, diabetes, history of cancer, history of hypertension, and history of diabetes.
Cumulative incidence of breast lump strati ed by age Figure 3 shows the cumulative incidence of breast lump strati ed by age and quartile of AMC and %MONO; these data indicated that the effect of high monocyte levels on increased risks of a breast lump was more remarkable in women <60 years of age (both P<0.05), while no signi cant differences were observed for women >60 years of age (both P 0.05).

Discussion
The relationship between monocytes and breast cancer is well-established [22][23][24][25][26][27], but their relationship with BDD is not well understood [3,28], and fewer studies examined those markers in relation to breast lumps, irrespective of their malignancy status. This study suggests that increased monocyte levels were independently associated with breast lump and might be used as an indicator of the incidence of breast lump, especially for women <60 years. Those results suggest additional markers for the risk management of women undergoing breast screening.
Previous studies reported a relationship between monocyte-related markers of in ammation and monocyte activation in BBD tissue. For instance, in an age-matched control study, Degnim et al. [3] found that a higher density of macrophages is observed in BBD tissues compares with that in normal mammary gland tissue. Moreover, BBD stromal tissues are far more frequently in amed than normal breast tissues [30]. Charles et al. [31] demonstrated that the re-calci cation time, an indicator of monocyte' generation of pro-coagulant products, was signi cantly shorter in breast cancer patients than in patients with cystic hyperplasia, which in turn was signi cantly shorter than in healthy women. However, unlike risk factors for breast cancer, risk factors for breast lump (irrespective of their malignancy status) development are not clear. This study revealed that increased monocyte levels were independently associated with breast lump. Taken together, those results suggest that breast lump, irrespective of their benign/malignant status, are more likely to display elevated monocyte-related markers. Those results could help the management of women with breast lump under ultrasound. Women with higher monocyte-related markers could bene t from shorter screening intervals without harm. Of course, additional cohort studies are necessary to examine this hypothesis. Furthermore, additional studies are necessary to verify whether monocyte-related markers at baseline could help predict the development of benign vs. malignant breast lump.
We also analyzed the association of AMC and %MONO with the incidence of breast lump strati ed by age and found high monocyte levels display a more pronounced effect on breast lump risk in women <60 years of age. A previous study had also found that a high density of TAM in ltration was signi cantly related to younger age (≤ 50 years) [32]. Furthermore, data suggested that the total number of immune cells does not change between younger (< 40 years) and middle-aged (40-60 years) groups, but it signi cantly decreased at an older age (>60 years) [33]. The decreased levels of immune cells, especially platelet-derived beta-2 microglobulin, shifts monocytes and macrophages to a pro-reparative phenotype and reduce the risk of pro-brotic responses [34].
Notable strengths of this study include its large sample size and the initial report regarding the causal associations between monocytes and breast lump, including standardized assessment of outcomes over a 5-year follow-up period. Nevertheless, there are some potential limitations to this study. First, the participants were patients from the Health Management Center rather than from the general population; thus, the representativeness is limited. Moreover, the prevalence of breast lump in the general population could not be determined. Second, all enrolled subjects were Chinese women from Tianjin city, and racial and epidemiologic factors should be considered when interpreting our ndings. Third, the AMC and %MONO are easily available markers obtained from a simple blood draw, but they might not necessarily represent the exact monocyte in ltration of the breast tissue. Future studies should examine the correlations between blood AMC/%MONO and monocytes in breast biopsies. Last, although there is a lack of information concerning reproductive factors in the present study, we adjusted for a variety of other potential confounders and achieved robust results. These limitations can be addressed in future studies.

Conclusions
In summary, increased AMC and %MONO increase the risk of developing a breast lump. And we encourage the implementation of prevention measures as breast lump can be either malignant or be a marker of increased breast cancer risk. Future studies are needed to explore monocytes as a circulation marker for the incidence of BBD and breast cancer.

Declarations
Ethics approval and consent to participate The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Tianjin Medical University General Hospital (IRB2020-WZ-201) and informed consent was waived. All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval and consent to participate.

Consent for publication
Not applicable.
Availability of data and materials The datasets generated and/or analyzed during the current study are not publicly available due ethical/privacy reasons. Upon request the analyses generated from the data are available from the corresponding author.   Cumulative curves of incidence of breast lump strati ed by age and quartile of AMC and %MONO. A/C: incidence of breast lump according to the quartiles of absolute monocyte count for age ≤60 years (A) or for age >60 years (C); B/D: incidence of breast lump according to the quartile of the percentage of monocyte for age ≤60 years (B) or for age >60 years (D). P-values <0.05 are considered statistically signi cant.