Advanced breast cancer (ABC) is currently an incurable disease, and the purpose of treatment is prolongation of survival and maintenance or improvement of quality of life (1). Guidelines recommend several active agents as first-line treatments, which are based on previous therapy, differential toxicity, comorbidities, and patient preferences (1,2). At present, although many agents are effective against breast cancer and survival outcomes for patients with ABC have improved, it has been difficult to establish prognostic markers of overall survival (OS) benefit in patients with ABC (1).

Tumors have been reported to be associated with systemic inflammation, which can affect tumor development, progression, and response to treatment (3,4). As a result of inflammation, the tumor microenvironment contains macrophages, neutrophils, natural killer cells, T and B lymphocytes, and the surrounding stroma of cancer cells (3). Recently, systemic immune markers, which are surrogate indicators of the systemic inflammatory response, were reported to be prognostic markers in several cancers, including breast cancer (5-13). These markers are the neutrophil-to-lymphocyte ratio (NLR), absolute lymphocyte count (ALC), platelet-to-lymphocyte ratio (PLR), and lymphocyte-to-monocyte ratio (LMR).

However, most studies have demonstrated their utility as prognostic markers in early breast cancer (7,9,11), and few studies have focused on only patients with ABC (14). Little evidence on the usefulness of these markers at the time of ABC diagnosis is available. Here, we investigated the association between these markers at diagnosis and the clinical benefit in patients with ABC and confirmed them as prognostic markers of survival benefit in ABC.

Patients and treatments. We retrospectively analyzed patients diagnosed with ABC who received treatment at Fukuyama City Hospital between April 2009 and December 2020. We reviewed the medical records to determine patient backgrounds and outcomes. We defined the subtype based on estrogen receptor (ER) and human epidermal growth factor receptor-2 (HER2) expression: ER-positive was defined as ER positivity ≥1%, and HER2 positivity was defined according to the American Society of Clinical Oncology/College of American Pathologists guidelines (15). Clinical response was evaluated according to the Response Evaluation Criteria in Solid Tumors version 1.1 (16).

First-line and subsequent treatment regimens were determined based on previously published guidelines (1,2) and the physician’s judgment and/or patient preferences, as in routine clinical practice. Dose modifications, interruptions, and discontinuations were also determined. Treatment regimens included endocrine therapy, chemotherapy, and molecular targeted therapy.

All procedures involving human participants were performed in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. This study was conducted in full compliance of the law and after approval was obtained from the institutional review board of the center where the study was conducted (approval number: 595). Informed consent was obtained in the form of an opt-out option on the hospital website from all individual participants included in the study.

Measurements of systemic immune markers. Whole blood samples were obtained from ABC patients at diagnosis, and the neutrophil, lymphocyte, platelet, and monocyte counts were measured in a Sysmex XE-2100 or XE-5000 automated hematology system (Sysmex Co., Kobe, Japan) (17). The ALC, NLR, PLR, and LMR were calculated from blood cell counts, and the cut-off values of these parameters were defined according to previous studies (7,9,10,17): 1,500/μl for ALC, 3 for NLR, 150 for PLR, and 5 for LMR. All patients were divided into “low” and “high” groups according to the cut-off values.

Statistical analysis. We used Wilcoxon’s rank sum test to compare continuous variables and Fisher’s exact tests to compare categorical variables between groups. Survival was estimated using the Kaplan–Meier method and was compared using the log-rank test. We used Cox regression models for the univariate and multivariate analyses. The variates for which p<0.10 by univariate analysis were included in the multivariate analysis. Because the systemic immune markers (ALC, NLR, PLR, and LMR) were correlated with each other, we did not include these four markers simultaneously in the multivariate analysis. By contrast, these four markers were entered independently in each multivariate analysis for TTF and OS (17). In all statistical analyses, p<0.05 was considered significant. These analyses were performed using EZR software (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for the R software program (The R Foundation for Statistical Computing, Vienna, Austria) (18).

We defined TTF as the time from the administration of first-line treatment for ABC to the discontinuation of treatment for any reason, including disease progression, treatment-induced toxicity, patient/physician choice, and death from any cause. OS was defined as the time from ABC diagnosis to the date of death from any cause (17).

Patient characteristics and overall efficacy. We evaluated 331 ABC patients, including 213 patients (64.4%) with the luminal type (ER-positive and HER2-negative), 67 patients (20.2%) with the HER2 type (HER2-positive and ER-positive or ER-negative), and 51 patients (15.4%) with the triple-negative type (ER- and HER2-negative). The patient characteristics at baseline are shown in Table I. The median age was 61 years (range=26-100 years), 244 patients (73.9%) were postmenopausal, and 1 patient was male. Metastatic sites included bone in 154 patients (46.5%), lung in 126 patients (38.1%), and liver in 84 patients (25.4%). Overall, 100 patients (30.2%) received perioperative chemotherapy consisting of anthracycline and/or taxane-based regimens, and 194 patients (58.6%) had visceral metastases. Of the 331 patients, 139 (42.0%) received endocrine therapies and 192 patients (58.0%) received chemotherapy as first-line treatments, and these therapies were given in combination with molecular targeted agents (e.g., trastuzumab and cyclin-dependent kinase 4 and 6 inhibitors) if necessary.

Data cut-off was February 2021, and the median follow-up duration was 978 days. The median OS was 1,797 days (95%CI=1417-2384), and the median time to treatment failure (TTF) of first-line treatment was 308 days (95%CI=251-378).

Correlation between systemic immunity markers and OS. The OS was compared according to the systemic immune markers analyzed (Figure 1). Patients with high ALC, low NLR, and high LMR had significantly longer OS than those with low ALC, high NLR, and low LMR (high vs. low; ALC: 1,989 vs. 1,454 days; HR=0.70; 95%CI=0.51-0.96; log-rank p=0.025; NLR=1,207 vs. 1,989 days; HR=1.53; 95%CI=1.11-2.12; log-rank p=0.010; LMR: 2,453 vs. 1,158 days; HR=0.59; 95%CI=0.43-0.80; log-rank p<0.001) (Figure 1A, B, and D). PLR was not significantly associated with OS (Figure 1C).

We then performed univariate and multivariate analyses. Each of the four multivariate analyses indicated that high ALC and high LMR were independently associated with longer OS regardless of the subtype and the type of the first-line treatment (endocrine therapy or chemotherapy with/without molecular targeted therapy) (high vs. low; ALC: HR=0.69; 95%CI=0.50-0.94; p=0.020; LMR: HR=0.67; 95%CI=0.48-0.92; p=0.015) (Table II and Table III). Although a trend was observed for NLR, a significant difference in OS was not observed (HR=1.32; 95%CI=0.95-1.84; p=0.099).

Correlation between systemic immune markers and TTF after first-line treatment. The TTF values after first-line treatment were compared according to the systemic immune markers analyzed (Figure 2). Patients with high ALC, low NLR, and high LMR had significantly longer TTF than those with low ALC, high NLR, and low LMR (high vs. low; ALC: 378 vs. 217 days; HR=0.77; 95%CI=0.60-0.97; log-rank p=0.027; NLR: 203 vs. 378 days; HR=1.36; 95%CI=1.06-1.75; log-rank p=0.014; LMR: 378 vs. 250 days; HR=0.71; 95%CI=0.56-0.90; log-rank p=0.005) (Figure 2A, B, and D). By contrast, PLR did not show significant differences in TTF (Figure 2C).

We then performed univariate and multivariate analyses. Each of the four multivariate analyses indicated that high ALC was independently associated with longer TTF regardless of the subtype and the type of first-line treatment (high vs. low; HR=0.74; 95%CI=0.58-0.94; p=0.014; Table II and Table III).

The hypothesis that certain systemic immune markers predict a survival benefit in patients with malignancies has been investigated over the last decade (5-13). Specifically, a systematic review and meta-analysis demonstrated that high NLR was significantly associated with poorer OS in various tumors and at different disease stages (5,6). One study showed that low ALC was an independent prognostic factor for poorer OS in several tumor types (8). A systematic review and meta-analysis demonstrated that high PLR was associated with poorer OS in various solid tumor types and that high PLR was an independent prognostic marker in solid tumors (10). Another meta-analysis showed that low LMR was associated with poorer OS in patients with several types of solid tumors (12). Therefore, these systemic immune markers have been considered useful as prognostic markers in malignant tumors.

In breast cancer, an association between these systemic immune markers and survival benefits has also been reported (7,9,11,13). A meta-analysis that included 15 studies and a total of 8,563 patients demonstrated that high NLR was associated with poorer OS and that its prognostic value was consistent regardless of disease stage and subtype (7). Another study showed that low ALC was associated with poorer OS in patients treated with primary chemotherapy (9). Another meta-analysis revealed that high PLR was a poor prognostic marker (11). A retrospective cohort study showed that high LMR predicted a favorable response and prognosis in patients with locally advanced breast cancer (ABC) who received neoadjuvant chemotherapy (13). However, previous studies have primarily focused on patients with early breast cancer, and very few studies have focused on patients with ABC. One meta-analysis suggested that the association between high NLR and poorer OS tended to be greater in patients with metastatic disease than in those with nonmetastatic disease (6). Therefore, these immune markers may be more important in ABC. However, the utility of these parameters as prognostic markers in ABC remains uncertain. Our results showed that at diagnosis, these systemic immune markers were significantly associated with longer OS in patients with ABC and were considered useful as prognostic markers of better survival in ABC patients. A single institutional study that also solely focused on patients with ABC demonstrated that high NLR predicted poorer OS in ABC (14). Another study reported that according to a multivariate analysis, low ALC was an independent prognostic factor of poorer OS in patients with ABC (8). Given these findings, it is reasonable that these systemic immune markers are prognostic markers for OS not only in patients with early breast cancer but also in those with ABC.

Recently, several studies have reported that systemic immune markers may be predictive of benefits derived from certain treatment regimens in patients with ABC (17,19-23). High ALC and low NLR have also been found to be associated with improved progression-free survival (PFS) (19) and OS (20-22) in patients treated with eribulin and in those treated with paclitaxel plus bevacizumab (17,23). Because systemic immune markers may potentially reflect the tumor microenvironment, these markers may serve as predictive markers for certain treatment regimens that function by modulating the tumor microenvironment (19,20,23).

Furthermore, it has been reported that these markers predict the efficacy of neoadjuvant chemotherapy and that NLR is associated with pathological complete response (pCR) rates in early breast cancer (24,25). A retrospective study showed that pretreatment lower NLR was associated with higher pCR rates (24.5% vs. 14.3%, p<0.05) and that NLR was an important predictive factor of the response to neoadjuvant chemotherapy (24). A single-center retrospective study showed that patients with lower NLR achieved a significantly higher pCR rate compared with patients with higher NLR (56.9% vs. 28.6%, p<0.001) (25). Neoadjuvant chemotherapy is the standard treatment for locally ABC as it allows for curative surgery (26,27) and assessment of the chemosensitivity of tumors (26,28). Therefore, systemic immune markers can predict chemosensitivity of malignant breast tumors. By contrast, cancer cell heterogeneity, cancer-associated macrophages, and immune cell modulation are the primary causes of tumor chemoresistance, and these factors could lead to chemoresistance through modification of the tumor microenvironment during chemotherapy (29). Therefore, systemic immune markers may indirectly reflect the tumor microenvironment status and can predict drug sensitivity. Our results showed that the values of the systemic immune parameters at diagnosis were significantly associated with better TTF after first-line treatment in patients with ABC regardless of the subtype and the type of treatment (endocrine therapy or chemotherapy). Our results suggest that systemic immune markers could be indicators of drug sensitivity at the initiation of treatment for ABC.

Our study had several limitations. First, this was a single institutional retrospective study. Therefore, the possibility of unintended selection bias might not have been sufficiently excluded. Second, although the cut-off values that we used for systemic immune markers were based on previous reports, the optimal cut-off values in ABC remain unclear (7,9,10). Third, the mechanisms that underlie the association of systemic immune markers and outcomes of ABC patients are uncertain. However, the correlation of these markers with inflammation induced by immune cells helps in explaining this association (10). Further research is required to reveal the underlying mechanisms.

In conclusion, our study indicated that several systemic immune markers at the time of ABC diagnosis can predict not only a better OS but also a better TTF after first-line treatment. The values of these markers at diagnosis may reflect sensitivity to treatment. These markers are inexpensive and easily available and may allow clinicians to predict survival benefits at the time of ABC diagnosis.

Shogo Nakamoto has received lecture fees from Chugai Pharmaceuticals, Eisai CO. Ltd and Taiho Pharmaceuticals. Masahiko Ikeda has received lecture fees from AstraZeneca, Chugai Pharmaceuticals, Daiichi-Sankyo, Eisai, Eli-Lilly, Kyowa Kirin, Pfizer, Nippon Kayaku, Novartis, Mundipharma, Celltrion Healthcare Japan, and Sawai Pharmaceuticals outside the submitted work. The other Authors declare that they have no conflicts of interest in relation to this study.

All Authors contributed to the study conception and design. Shogo Nakamoto prepared the material preparation, collected the data, and performed the analysis. Shogo Nakamoto also wrote the first draft of the manuscript, and all Authors commented on the previous versions of the manuscript. All Authors read and approved the final manuscript.

The Authors would like to thank Enago for editing the draft of this manuscript.