Absolute Counts of Peripheral Lymphocyte Subsets as Potential Immune Impairment Markers in Patients With Breast Cancer

Purpose This study was to evaluate clinic value of absolute counts of lymphocyte subsets (ACL) as potential blood biomarkers in progression and prognosis in breast cancer (BC) patients. Methods A total of 237 BC patients and 55 age-matched female normal healthy donors (normal cantrals, NCs) were enrolled in this study. The absolute counts (AC) and percentages of CD3 + , CD3 + CD4 + , CD3 + CD8 + , B and NK cells were determined by ow cytometry. The clinicopathological parameters inuencing disease progression were determined by binary logistic regression. The progression-free survival (PFS) was evaluated by Kaplan-Meier. Univariable and multivariable analyses were performed using log-rank test and proportional hazard regression models, respectively. Results Compared with NCs, the ACL in BC patients decreased signicantly, while the percentages of lymphocytes showed no change. Of them, AC of CD3 + CD4 + cells was closely related to clinical stages. The ACL, especially CD3 + CD4 + cells, were affected by different treatments. Analysis of logistic regression showed that the cut-off value of CD3 + CD4 + cells ≥ 451 cells/μL was the favorable prognostic factor. Multivariate analysis of prognostic factors of PFS showed CD3 + CD4 + and CD3 + CD8 + cells were independent factors for predicting PFS. Conclusions The AC of CD3 + , CD3 + CD4 + , CD3 + CD8 + , B, and NK cells in BC patients were impaired obviously and can be as potential susceptive indications to evaluate the patient's immune states. The higher level of AC of CD3 + CD4 + and CD3 + CD8 + cells contributed to longer PFS and favorable outcome, and could help to adopt appropriate treatment strategies in clinic.

According to the latest statistics in 2018, patients with BC in 185 countries and regions in the world increase by 2.089 million every year, with 627000 deaths [1]. In recent years, the incidence rate of BC is increasing and getting younger, but BC is a type of cancer with a good prognosis [2]. At present, the main treatments of BC are surgery, chemotherapy, endocrine, and other comprehensive therapy. For patients with early and mid-term BC, postoperative adjuvant chemotherapy can improve the local control rate, and reduce the recurrence rate of local and regional lymph nodes [3].
It has been found that the recurrence and metastasis of tumors are closely related to the decline of immune function, which leads to the tumor immune escape [4]. The classical lymphocyte subsets include CD3 + , CD3 + CD4 + , CD3 + CD8 + , B, and nature killer (NK) cells,which are crucial in regulating immunity and speci c killing of tumor cells [5]. Although their compositions are simple, the roles in immune responses are diverse, encompassing both innate and adaptive immunity, cell and humoral immunity simultaneously. CD3 + CD4 + cells can activate other lymphocyte subsets by releasing cytokines and suppress tumor development by directly killing tumor cells expressing adequate levels of major histocompatibility complex (MHC) class II molecules [6]. CD3 + CD8 + cells, a kind of cytotoxic cells, are able to recognize tumor cells expressing MHC class I molecules presented by antigen presenting cells and produce IFN-γ, perforin, granzyme B for targeting and killing tumor cells [7]. B cells play an important role in humoral immunity, which can not only present antigen to T cells, participate in the immune response of T cells, but also can recognize different antigen epitopes, secrete immunoglobulin and participate in the humoral immune response [8]. NK cells are indispensable effector cells in the innate immune system, and they are the rst line of defense for host immune defense against cancer and pathogens [9]. Therefore, the analysis of peripheral lymphocyte subsets is the most signi cant and convenient way to assess the functions of immune including the percentages of lymphocytes (PL) and the absolute count of lymphocytes (ACL). Especially, the ratio of CD4 + /CD8 + is an indicator of the balance of the immune system, which is maintained by the proportion and the number of peripheral blood lymphocytes [10].
Traditionally, the ACL was measured by dual-platform technology by which the percentages of lymphocyte subsets measured by ow cytometry was combined with the absolute lymphocyte count measured by an automatic hematology analyzer,which produced obvious errors among different laboratories [11][12][13][14]. Here, we adopted the single-platform technology to carry out the detection entirely on the ow cytometer, which signi cantly improved the accuracy of the analysis. Published studies showed that lymphopenia, or low peripheral blood lymphocyte count, may predict higher mortality and increase the risk of recurrence after primary surgery and neoadjuvant therapy, whereas higher absolute lymphocyte counts predict lower mortality from early-stage triple-negative BC [15,16]. But, these studies only analyzed the total number of lymphocytes, and did not further analyze the in uence of the ACL. However, the correlations of prognosis and the ACL in patients with BC remain unclear.
The aim of this study was to evaluate the clinic value of PL and ACL in BC progression and prognosis, and analyze the importance and susceptivity of ACL served as biomarkers of immune impairment.

Materials And Methods
Clinical data

E cacy evaluation
Based on RECIST, version 1.1 [17], the e cacy evaluation was divided into complete response (CR), partial response (PR), stable disease (SD) and progress disease (PD). CR + PR were considered to be effective, while SD + PD were considered ineffective.

The main reagents and instruments
The assay of lymphocyte subsets was performed by using a lyse/no-wash procedure based on asingleplatform technique by ten-color ow cytometry (BD FACS Canto II: U6573380-00541

Sample collection
Two milliliters of fresh whole blood were drawn from NCs and patients respectively and were stored in the EDTA-anticoagulant blood collecting tubes.

Cellular staining and analyzing
The whole blood of the 292 participants in this study were collected between January 2018 and October 2020 and performed by ow cytometry. The manipulation was referred to BD operating instruction. in brief, for each sample, two trucount tubes were labeled with letters A and B to distinguish them from each other; added 20µL of BD Multitest CD3/CD4/CD8/CD45 and CD3/CD16 + CD56 + /CD45/CD19 reagents into the bottom of each tube A and B, respectively; then added 50µL of well-mixed whole blood into the bottom of every tube; nally, added 450µL of lysing solution into every tube, mixed and incubated for 15 min in dark at room temperature for analysis.

Statistics
The normality of statistical data was evaluated by the Shapiro-Wilk test. Using two independent sample ttest to analyze the differences in PL and ACL between patients with BC and NCs. The differences among three or more groups of continuous numerical variables with normal distribution were analyzed by oneway ANOVA (Bonferroni or Tamhane's test). Progression-free survival (PFS) was de ned as the time from the date of enrollment to disease progression, recurrence, or death, and the follow-up deadline was October 31, 2020. Categorical variables were analyzed using the chi-squared test. Factors affecting disease progression were analyzed by binary logistic regression. Calculating survivals were with the method of Kaplan-Meier. Univariate and multivariate analyses were performed to assess the relationship between clinicopathologic parameters and PFS, for which the Log-Rank test and proportional hazard regression model were used, respectively. Variables with a P-value < 0.05 by a univariable analysis were entered for multivariable analysis. Odds ratios (ORs) were reported with 95% con dence intervals (CIs).
Values of P < 0.05 were considered statistically signi cant. Data were analyzed by SPSS 25.0(IBM Corporation). Figures were prepared using Graph Pad Prism version 9.0 software (San Diego, USA).

Analysis of lymphocyte subsets between patients and NCs
The principle of ACL detected by the single-platform was that the known total number of uorescent microbeads were used as the standard internal parameters and uorescent-labeled antibodies added into the trucount tubes, then applied acquisition and analysis software in the ow cytometry to calculate data.
Using the method, we primitively compared both the PL and AC of CD3 + , CD3 + CD4 + , CD3 + CD8 + , B, and NK cells in all patients with BC and NCs. There was no difference in percentages between the two groups (P > 0.05, Fig. 1a). But the AC of CD3 + , CD3 + CD4 + , CD3 + CD8 + , B and NK cells were signi cantly decreased in patients with BC (P < 0.001, Fig. 1b). The results suggested that it's ACL but not PL decreased in patients with BC. To our knowledge, PL represents the proportion or composition of each subsets, indicating the development and differentiation of lymphocytes, while ACL demonstrates the exact number of peripheral lymphocyte subsets, indicating the proliferation of lymphocyte progenitor. The results showed that the proliferation ability of lymphocytes was impaired distinctly.
In addition, compared with NCs,the PL in patients with BC at stage -and -showed no signi cant difference(P > 0.05, Fig. 1c, e), but signi cant differences were observed in the ACL (P < 0.001, Fig. 1d, f). The results suggested that signi cant decrease of ACL was one of important characteristics of immune impairment or one sign of immunodepletion.

ACL in patients with different clinical stages
There was no signi cant difference in PL of CD3 + , CD3 + CD4 + , CD3 + CD8 + , B and NK cells among patients at different clinical stages (Fig. 2f). Compared with the AC of CD3 + , CD3 + CD4 + , CD3 + CD8 + , B and NK cells in patients at different clinical stages, we discovered that all of them had varying degrees of decline (Fig.   2). The AC of CD3 + in patients at stage IV was lower than patients at stage I and II(P 0.001), but patients at stage III had no signi cant difference compared with patients at stage I and II (Fig. 2a). With the progression of clinical stages, the decline of AC of CD4 + was particularly signi cant in BC patients.
Compared with patients at stage I, II and III, the AC of CD4 + in patients at stage IV decreased most strikingly (P 0.001), then followed by stage III (P 0.01), stage II (P 0.01), and stage I (P 0.001). However, there was no obvious difference of AC between stage I and stage II in patients (Fig. 2b).
Compared to patients at stage I, the AC of CD8 + was decreased in patients at stage II (P < 0.05) and stage III-IV (P < 0.01) (Fig. 2c). Compared with patients at different stages, the variation of B cells AC was only observed in patients at stage I and IV (P < 0.01) (Fig. 2d). Meanwhile, the difference in NK cell AC was only found in patients at stage II and IV (P < 0.001) (Fig. 2e). In a word, our data showed that the decrease of CD4 + and CD8 + AC was closely related to clinical stages, which indicated that it declined upon the exacerbations of BC.

ACL between CR + PR and SD + PD group
To further study the relationship of ACL and e cacy, we divided the patients into two groups according to curative effect, one was effective group (CR + PR), and the other was ineffective group(SD + PD).
Independent sample t-test revealed that AC of CD3 + , CD3 + CD4 + , CD3 + CD8 + , B, and NK cells in CR + PR group were signi cantly higher than that in SD + PD group (P < 0.001, Fig. 3a, b, d, e; P < 0.01, Fig. 3c). In brief, the results indicated that ACL, especially AC of CD3 + CD4 + was closely related to the curative effect, the higher the ACL, the better the e ciency.

ACL in different treatments
To further study the relationship of ACL and different treatments, we divided the patients in effective(CR + PR) and ineffective group(SD + PD) into surgery, chemotherapy and endorinotherapy according to curative ways. The common characteristics of effective and ineffective groups were that AC of CD3 + , CD3 + CD4 + , CD3 + CD8 + , B, and NK cells were all the highest treated by endocrinotherapy, then followed by surgery and chemotherapy (Fig. 4). The differences were that mean numbers of ACL of every treatment in effective group were higher than that in ineffective group. Strikingly, the mean value of AC of CD3 + CD4 + showed the most obvious difference with 518 cells/uL in effective group and 393 cells/uL in ineffective group (Fig. 4b), suggesting AC of CD3 + CD4 + was closely related to the e cacy, and may be served as potent blood biomarker to evaluate the prognosis.

Effect of ACL on the progression of BC
In order to ascertain whether ACL can in uence the progression of BC (freedom from any events as follows: other newly diagnosed tumors; distant organ metastasis; concurrent infection), we further conducted binary logistic regression analysis to determine the in uencing factors of disease progression.

Discussion
The study showed that it was much more convenient and accurate to apply the single-platform ow cytometry to analyze the PL and ACL in BC patients. Currently, the PL are mostly used to evaluate the immune function of tumor patients in clinical, but the detection of ACL are relatively insu cient, which may lead to serious impairment of the immune function of cancer patients being ignored.
The immune system normally protects our body from the invasion of pathogens and malignant tumors, mainly including cellular immunity and humoral immunity. Studies have shown that cellular immunity plays a major role in the process of anti-tumor immunity [18][19]. CD3 + cells mainly consists of CD3 + CD4 + and CD3 + CD8 + cells, CD3 + CD4 + cells play an immunomodulation role, which can assist B cells to produce antibodies and meanwhile secrete cytokines to enhance the CD3 + CD8 + cells to kill tumors [20].
Thus, CD3 + CD4 + cells can target tumor cells in various ways, either directly by eliminating tumor cells through cytolytic mechanisms or indirectly by modulating the tumor microenvironment [21]. The increase in the number of CD3 + CD4 + cells indicates that the immune response is improved and the anti-tumor activity is strengthened [22]. CD3 + CD8 + cells can be classi ed into cytotoxic T lymphocytes (CTLs) and inhibitory cells. CTLs are the preferred tool to target tumors, as they detect extracellular antigens that are presented by MHC class I molecules expressed by all tumor cell types [23]. CD19 + , as a surface marker of B cells, is mainly involved in the humoral immunity, and the level of its expression can measure the strength of the humoral immunity [24]. CD16 + CD56 + is a speci c marker of NK cells which are a group of cells with special properties and do not have antigen-recognition receptors on the surface [25]. NK cells are the rst activated immune cells in anti-tumor immunity and important cytotoxic cells in the innate immunity, which can be activated and exert their effector functions without antigenic stimulation [26]. The decreased number of NK cells indicates that the immune system is less capable of monitoring, killing, and clearing tumor cells [27]. Hence, the detection of the ACL in tumor patients is of great signi cance for understanding the changes of patients' condition and prognosis [28].
The research suggested that ACL could be served as a potential peripheral blood immune impairment markers in patients with BC to monitor the immune function, and predict the prognosis and therapeutic effect. It is the rst time to report the clinic value of peripheral ACL in BC, though some research suggested that total number of peripheral blood lymphocytes was associated to the prognosis and therapeutic effect [13,14,15,29,30]. Firstly, just as our study showed, there was no signi cant difference in the percentages of CD3 + , CD3 + CD4 + , CD3 + CD8 + , B, and NK cells between NCs and BC patients.  [32]. Therefore, more attention should be paid to AC of CD3 + CD4 + and CD3 + CD8 + cells in anti-tumor immunity.
The increased numbers of CD3 + CD4 + and CD3 + CD8 + cells signify a good prognosis in BC, which is consistent with some previous study [33][34]. CD3 + CD4 + cells can promote the activation of CD8 + CTLs, boost the effector and memory function of CTLs, and reduce the immunosuppression of CTLs,which helps T cells to amplify their response to tumor-associated antigens without generating an autoimmune response [35]. Additionally, antigen-speci c contacting with CD3 + CD4 + cells enables dendritic cells to optimize antigen presentation and deliver speci c cytokines and co-stimulation signals to CD3 + CD8 + cells, so as to promote their cloning, ampli cation, and differentiation into effector or memory T cells [36]. In brief, the ACL in BC patients were low, indicating that the immune function of patients was impaired.
The patient's impaired immune system could not indeed keep tumor from progressing [37]. Therefore, it is essential to enhance the immune function of patients and improve their anti-tumor ability during the clinical therapy. CD3 + CD4 + cells contribute to initiating a gene expression program of CD3 + CD8 + cells via multiple molecular mechanisms to enhance the function of CD8 + CTLs and overcome the obstacle of antitumor immunity [38].
Consequently, the ACL not only re ects the immune status of the body, but also plays a pivotal role in the prediction of disease prognosis and the curative effect [31], therefore we should pay more attention to the change of ACL in clinic, so as to provide more reference value for predicting the patient's condition and clinical treatment.

Conclusion
Page 17/23 The ACL and PL have different clinical signi cance, it was ACL, not PL appeared signi cant impairment in BC patients, and correlated to progression of tumor, PFS, e cacy, it is necessary and pivotal to detect ACL in clinic. ACL can be as potential peripheral blood biomarkers to monitor the immune impairment, and predict the prognosis and therapeutic effect.