The Association between Leukocyte and Its Subtypes and Benign Breast Disease: The TCLSIH Cohort Study

Inflammation plays a crucial role in the formation of benign breast disease. Given the limited study to explore the association between leukocyte as an indicator of immune system and benign breast disease, we used data from a large cross-sectional study to investigate association between leukocyte and its subtypes and benign breast disease among women in the general population. The data were derived from baseline data of the Tianjin chronic low-grade systemic inflammation and health (TCLSIH) cohort study during 2014 and 2016. Breast thickness and nodules status were assessed by using ultrasonography. Leukocyte and its subtype counts were carried out using the automated hematology analyzer. Multiple logistic regression analysis was used to examine the association between leukocyte and its subtypes and prevalence of benign breast disease. In the present study, the prevalence of benign breast disease was 20.9%. After adjustments for potentially confounding factors, the odds ratios (95% confidence interval) for benign breast disease across lymphocyte quintiles were as follows: 1.00 (reference), 0.99 (0.82, 1.2), 0.85 (0.69, 1.04), 0.84 (0.68, 1.02), and 0.75 (0.61, 0.92) (P for trend = 0.002). An inverse association between lymphocyte counts and benign breast disease was found, but leukocyte and other subtypes have nothing to do with benign breast disease. Further prospective studies are needed to determine the findings.


Introduction
Breast cancer (BC) is a commonly diagnosed malignancy and the leading cause of cancer death in women worldwide [1]. Benign breast disease (BBD), the hallmark of which is epithelial proliferation, is a putative BC precursor [2]. BBD refers to a galaxy of pathophysiologic lesions resulting from progressive/regressive changes involving the component mammary structures, i.e., ducts, acini, stroma, and fat tissue [3]. e etiology of BBD is poorly characterized. Increasing evidence suggests that cancer-related inflammatory response plays a critical role in the development and progression of several malignancies. For instance, impairment of adaptive immune responses during chronic inflammation may favor tumor growth, angiogenesis, and cancer cell survival [4][5][6][7][8]. Previous studies have also indicated that inflammation pathways may be associated with BBD, from which early stage BC may develop [2,9]. e primary response cells to inflammation are leukocytes. Leukocytes are the main building blocks of the immune system. e immune defense against tumors is largely a cell-mediated one and relies on the integrity of T cell subsets [9]. Meanwhile, B cells can also mount humoral immune response with antitumor effects [9]. Furthermore, different leukocyte counts reflect different levels of inflammation [10]. In past studies, researchers have found that different leukocyte counts are strongly associated with different types of cancer, such as endometrial cancer, lung cancer, endometrial cancer, and BC [11][12][13]. However, to the best of our knowledge, no previous studies have investigated the association between leukocyte and its subtypes and BBD in adult women. erefore, we designed a cross-sectional study to investigate how leukocyte and its subtypes are associated with the prevalence of BBD among women in the general population.

Study Population.
e study sample was taken from participants in the Tianjin chronic low-grade systemic inflammation and health (TCLSIH) cohort, details of which have been published elsewhere [14]. is study conformed to the ethical guidelines of the 1975 Declaration of Helsinki. e protocols and procedures of the study were approved by the Institutional Review Board of Tianjin Medical University with the reference number of TMUhMEC 201430, and written informed consent was obtained from each participant.
During the research period, 5,954 participants, who received health examinations including body mass index (BMI), waist, a series of blood parameters, and breast ultrasonography and had completed questionnaires regarding their smoking and drinking habits and disease history over the course of January 2014 to December 2016, were recruited. We excluded participants who did not undergo leukocyte counts (n � 23). Participants with a history of cancer (n � 89) were also excluded. After these exclusions, the final cohort study population comprised 5, 842 participants ( Figure 1).

Assessment of Leukocyte and Its Subtype Counts.
Fasting blood samples were gained from all the participants by venipuncture of the cubital vein in the morning and immediately mixed with EDTA. Leukocyte and its subtype counts were carried out using the automated hematology analyzer XE-2100 (Sysmex, Kobe, Japan) and expressed as × 1, 000 cells/mm 3 . e test for blanks was ≤0.2 × 10 9 cells/L; the intra-and interassay coefficients of variation (CV) were ≤2.0%; and the cross-contamination rate was ≤0.5%. In order to investigate how the leukocyte and its subtypes are associated with prevalence of BBD, we divided participants into quintiles based on leukocyte and its subtype counts.

Assessment of BBD.
For the early screening and diagnosis of breast disease, both breasts were examined by breast ultrasound in the present study [15]. Breast ultrasonography was performed by trained sonographers using an iU22 ultrasonography system (Royal Philips) equipped with a 50 mm linear array transducer and a bandwidth of 7-12 MHz to measure breast gland thickness and status of breast nodule (position, size, and nature) [16]. All participants were asked to stay in the supine position and exposed breast. Arms are naturally lifted that bilateral breast and axillaries were fully exposed. Bilateral breasts were measured in turn. Measured indicators include breast thickness and breast nodule size. e lesions were classified as simple cyst, complicated cyst, fibrocystic changes, sonographically benign solid lesion (mass), and ductal ectasia. If an ultrasonographic image owned the features of more than one of any of the abovementioned lesions, then it was named as a mixed lesion [17]. Imaging results were also categorized into five levels using the Breast Imaging Reporting and Data System (BI-RADS) diagnostic category [18]. e intra-and inter-measure coefficients of variability (CV) were <2.9%.

General Examination.
e anthropometric indicators (height, body weight, and waist circumference (WC)) were measured by well-trained investigators using a standard protocol. Waist circumference was measured at the umbilical level with subjects standing and breathing normally. e thresholds used to determine abdominal obesity were defined as WC ≥ 80 cm for Chinese women. BMI was calculated as weight/height 2 (kg/m 2 ) [19]. Obesity was defined as BMI ≥ 28 kg/m 2 according to the criteria of the Working Group on Obesity in China (WGOC) [19]. e sociodemographic variables including age, disease history, family history as well as current medication, menstrual and reproductive history (age at menarche, parity, age at first full-term birth, and menopausal status), smoking (defined as 'current smoker,' 'ex-smoker,' and 'nonsmoker'), and drinking status (defined as 'everyday,' 'sometimes,' 'exdrinker,' and 'nondrinker') were noted by self-reported form on a questionnaire. If women were ex-smokers or exdrinkers, they were asked age at which they had ceased smoking or drinking. Ex-smokers or ex-drinkers were defined as not smoking or drinking in a month prior to baseline, but used to be drinker and smoker. Occasional drinkers were defined as except for daily drinkers and drinking frequency more than one time in the past month e history of surgery was assessed according to the responses to relevant questions and the personal health records.
All blood tests were performed after an overnight. As for lipids, total cholesterol (TC) and triglycerides (TG) were measured by enzymatic methods. Low-density lipoprotein (LDL) was measured by the polyvinyl sulfuric acid precipitation method and high-density lipoprotein (HDL) was measured by the chemical precipitation method. Levels of fasting blood glucose (FBG) were measured by glucose oxidase method. All these measurements were conducted by using appropriate kits on a Cobas 8000 automatic biochemistry analyzer (Roche, Mannheim, Germany). Blood pressure (BP) was measured twice in the right arm using an automatic device (TM-2655P, A&D Company, Ltd., Tokyo, Japan) after 5 minutes of rest in a seated position. e mean of two measurements was taken as the BP value.

Statistical Analysis.
All statistical analyses in the present study were performed using the Statistical Analysis System 9.3 edition for Windows (SAS Institute). In this study, all continuous variables were not normally distributed by using the Kolmogorov-Smirnov test. e log transformation for those nonnormal distribution variables was applied before the statistical analysis. e continuous variables are presented as the geometric means and 95% confidence interval (CI) after logarithmic transforming, and categorical variables are shown as percentages. For analysis, the BBD statuses were used as dependent variables, and the quintiles of leukocyte and its subtype counts were used as independent variables. For characteristics analysis, differences among BBD statuses were examined using ANCOVA for continuous variables or using multiple logistic regression analysis for categorical variables after adjustment for age. Associations between different leukocyte and its subtypes counts and BBD were examined using logistic regression in three different models, and odds ratio (OR) and 95% CI were calculated. Analysis was performed without any adjustment in model 1; the analysis was adjusted for age and BMI in model 2; model 3 additionally adjusted for smoking status, drinking status, history of surgery, menopausal status, hypertension, hyperlipidaemia, diabetes, and the metabolic syndrome as well as for family history of CVD (cerebrovascular disease). e median value of each leukocyte subtype quintile was used to calculate the P values for linear trends. Interactions between different leukocyte subtypes counts, menopausal status, and confounders of BBD were tested by addition of cross-product terms to the regression model. e analysis applied a two-tailed significance test and considered P < 0.05 as an indication of statistical significance.

Results
Among 5,842 individuals, 1,221 participants (20.9%) met the criteria of BBD. e characteristics of the study participants according to no BBD and BBD status are

Discussion
is study was designed to assess the association between leukocyte and its subtypes and BBD in a general women population. e results suggested that higher lymphocyte counts are independently associated with the lower prevalence of BBD. To our knowledge, this is the first large-scale study concerning the topic of lymphocyte counts and prevalence of BBD.
To date, only a nested case-control study has investigated the association between several inflammatory markers and BBD, and its results suggested that serum C-reactive protein (CRP) levels were independently associated with BBD, and adiponectin levels had an inverse association with BBD in the general women population [2]. Although leukocyte and its subtypes are simple and inexpensive markers of inflammation and associated with many diseases [20][21][22], no previous studies have assessed the association between leukocyte and its subtypes and BBD. e current study demonstrated that higher lymphocyte counts are independently associated with lower prevalence of BBD, whereas leukocyte and other subtypes did not demonstrate any association with BBD. More studies are needed to confirm whether these results can be observed in other populations.
Lymphocytes, especially tumor-infiltrating lymphocytes, play an important role in tumor-derived inflammatory responses [23][24][25]. Lymphocytes exert their antitumor activity by inducing cytotoxic cell death and inhibiting tumor proliferation [26]. Several studies have shown that increased infiltration of lymphocytes in tumor tissue predicted better survival outcomes in cancer patients [27,28]. erefore, low lymphocyte counts might result in an insufficient immunological reaction, which leads to inferior survival in multiple cancers [29,30]. ese mechanisms may partially explain our results. Further studies should be conducted to mechanistically describe the association between leukocyte and its subtypes and BBD. e reliability and validity of these findings are strengthened by the population-based design, the large sample size, and the fact that we performed a detailed evaluation of an extensive set of potential confounders including sociodemographic, lifestyle, and clinically relevant variables. Previous studies have shown that surgery may affect the immunological and inflammatory response [31][32][33], resulting in a difference in leukocyte counts. Besides, the menopausal status can be an effect modifier or confounding factor for the association between breast tumor and WBC subtypes [34]. In order to avoid the influence of these variables, we adjusted them as confounding factors. Several limitations of the present study should be noted. First, this is a cross-sectional study, so we could not infer the causality between leukocyte subtypes and BBD. erefore, further cohort studies and intervention trials should be conducted to confirm the association between leukocyte subtypes and BBD. Second, although numerous confounding factors were adjusted for during the analysis, the study cannot eliminate the potential effects of other unmeasured factors completely.
ird, the use of oral contraceptives has been reported to influence the development of benign breast disease [35]. However, the present study did not collect the information on the use of oral contraceptives.
us, the effect of oral contraceptives on the association between lymphocyte counts and benign breast disease cannot be ruled out. Finally, since we did not have specific pathological types of BBD, we are unable to assess the association between leukocyte subtypes and specific types of BBD. A more detailed study on different subtypes of BBD is needed to better understand the association observed between leukocyte subtypes and BBD.
In conclusion, the present study firstly showed that a higher lymphocyte count is associated with a lower prevalence of BBD. is finding suggests that lymphocyte counts might be a novel predictive factor or treatment target for developing BBD. Further prospective or clinical studies are necessary to confirm these preliminary findings.

BC:
Breast cancer BBD: Benign breast disease TCLSIH: Tianjin chronic low-grade systemic inflammation and health BMI: Body mass index CV: Coefficients of variation WC: Waist circumference WGOC: Working Group on Obesity in China TC: Total cholesterol TG: Triglycerides LDL: Low-density lipoprotein HDL: High-density lipoprotein FBG: Fasting blood glucose BP: Blood pressure CI: Confidence interval OR: Odds ratio CVD: Cerebrovascular disease CRP: C-reactive protein.

Journal of Oncology 5
Data Availability e raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.

Ethical Approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
is article does not contain any studies with animals performed by any of the authors.

Consent
Informed consent was obtained from all individual participants included in the study.

Conflicts of Interest
No potential conflicts of interest were disclosed.