The prognostic value of neutrophil-to-lymphocyte ratio in de novo stage IV breast cancer: a retrospective cohort study

Introduction

Breast cancer (BC) is the most common tumor in women and is a leading cause of cancer-related death worldwide (1). About 5–10% of patients with BC are diagnosed with de novo stage IV cancer (2). With advances in systemic therapy, the mortality rate for patients with stage IV BC is decreasing, but overall survival (OS) remains unsatisfactory (3). This is partly due to the fact that stage IV BC has a heterogeneous prognosis ranging from a few months to many years with variations in tumor biology and patient characteristics. These factors, such as age, race, performance status, molecular subtype, clinical stage, metastatic sites, number of metastatic sites, and previous medical treatments, affect the prognosis of patients (4-6). An accurate estimation of survival is critical to patient treatment decisions (7).

Neutrophils and lymphocytes are important components of the human immune system. Many studies have examined the prognostic role of neutrophils, particularly the neutrophil-to-lymphocyte ratio (NLR) and the association with clinical outcomes in many cancer types, including BC (8,9). Several studies on early-stage BC have shown that the baseline NLR predicts survival outcomes (9,10). In addition, a meta-analysis systematically analyzed the published data on NLR and treatment outcomes. NLR was found to be an independent prognostic factor for survival in most of studies on the early-stage BC. However, no significant correlation was found between survival and NLR for advanced BC patients (11). Analysis of prognostic factors in patients with metastatic BC (MBC) is confounded by changes that cancer cells develop at the time of distant relapse, as well as responses to treatment. Therefore, patients with de novo stage IV BC with no prior treatment are more suitable for studying prognostic factors.

The primary aim of this study was to analyze the influence of NLR’s relationship with other biological and clinical factors on the survival of patients with de novo stage IV BC upon initial diagnosis. The secondary aim was to evaluate factors associated with baseline NLR. We present the following article in accordance with the REMARK reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-22-5612/rc).

Methods

We retrospectively identified 128 female patients who were diagnosed with de novo stage IV BC without prior treatment at Peking University Cancer Hospital between January 2011 and December 2020, without infectious diseases, auto immune diseases, concurrent hematological disorders, or other malignancies. All cases were histologically confirmed as invasive BC. Treatment efficacy was evaluated in accordance with the Response Evaluation Criteria in Solid Tumors (RECIST) criteria, version 1.1 (12). In this study, estrogen receptor (ER) and/or progesterone receptor (PR) positivity was defined as ≥1%. Human epidermal growth factor receptor 2 (HER2) was defined as negative with an immunohistochemical score of 0, 1+ or 2+ with fluorescence in situ hybridization (FISH) (−). All patients were followed up by telephone every 6 months. This study was approved by the Ethics Committee of the Peking University Cancer Hospital (No. 2017KT40) and was conducted in accordance with the Declaration of Helsinki (as revised in 2013). Individual consent for this retrospective analysis was waived.

Overall response rate (ORR) was defined as the proportion of patients who achieved complete response (CR) or partial response (PR). Clinical benefit rate (CBR) was defined as the percentage of patients who achieved CR, PR, or stable disease (SD). Progression-free survival (PFS) was the time from the start of treatment to disease progression, death, or loss to follow-up. The OS was defined as the time from the beginning of treatment to death or loss to follow-up.

NLR

Peripheral blood was collected within 7 days prior to the first line therapy. The differential counts of the white blood cells were determined using a hemocytometer. The percentages of the differential counts were determined using a BC-6900 Hematology Analyzer (Mindray, Shenzhen, China). The NLR calculations were made by dividing the serum neutrophil count by the lymphocyte count.

T-lymphocyte subtype detection in peripheral blood

Whole blood samples (200 µL) were incubated with conjugated antibodies as indicated in the dark for 10 minutes at room temperature before red blood cells were lysed. Samples pellets were re-suspended in 500 µL phosphate-buffered saline (PBS) after centrifugation for 5 minutes at 1,300 g at room temperature and analyzed by flow cytometry.

Conjugated antibodies used in this study were purchased from Beckman Coulter (Brea, CA, USA) and included CD3-PC5/CD4-FITC/CD8-PE (IM1650), CD3-FITC/(CD16⁺/CD56)-PE (A07735), CD(14+16)-FITC/CD85k(ILT3)-PE/CD33-PC5 (A23413), CD4-FITC (A007750), CD8-FITC (A07756), CD19-PC5 (A07771), and CD25-PE (A07774).

Beckman-Coulter FC500 (Beckman Coulter, USA) and CXP analysis software (Beckman Coulter) was used for flow cytometry. There are 10,000 gated events in every analysis. Lymphocytes subtypes were selected according to physical characteristics including volume and transmissivity. The level of T lymphocyte subtype was expressed as percentage of the total number of lymphocytes.

Stromal tumor infiltrating lymphocytes (TILs) evaluation

Evaluation of TILs was performed by independent pathologists on a whole section of needle biopsy stained by hematoxylin and eosin (HE), based on guidelines of the International TILs Working Group (4). In short, TILs were expressed as the percentage of immune cells in the stroma within the tumor. The number of TILs was a continuous measurement and two categories were applied in the study: low TILs (≤10%) and high TILs (>10%).

Statistical analysis

The software SPSS 22.0 (IBM Corp., Armonk, NY, USA) was used for the statistical analysis. The baseline characteristics of patients and treatment were described as mean ± standard deviation for continuous variables. Student’s t-tests were used to compare normally distributed variables and Mann-Whitney U tests were used for non-normally distributed variables. Receiver operating characteristic (ROC) curve analysis was used to determine the optimal cutoff for NLR. The chi-square test (or Fisher’s exact test when necessary) was performed to compare clinicopathological characteristics between high NLR and low NLR groups. Survival was estimated using the Kaplan-Meier method. Log-rank test was used to compare PFS or OS between the different subgroups. NLR and other factors relevant to survival were tested by univariable and multivariable Cox proportional hazard models. All tests were two-tailed, and P<0.05 was considered to indicate a statistically significant difference.

Results

Patient characteristics

A total of 168 women were diagnosed with stage de novo stage IV BC between January 2011 and December 2020, of whom 128 patients were enrolled in our study based on the inclusion and exclusion criteria. The clinical characteristics of patients are presented in Table 1. The median age of cases was 53 (range, 29–76) years. At initial diagnosis, 70 (54.7%) cases were hormone receptor (HR)-positive/HER2-negative, 79 (61.7%) patients had visceral metastasis and 67 (52.3%) patients had more than 2 metastatic sites. A total of 116 (90.6%) cases received first line chemotherapy; 33 (73.3%) cases received anti-HER2 target therapy among the 45 patients with HER2 positive BC.

NLR and clinical characteristics in de novo stage IV BC patients

The optimal NLR cutoff value of 2.9 was determined by ROC curve analysis (Figure 1). The area under the ROC curve (AUC) was 0.684, with sensitivity at 0.59, and specificity at 0.75. Totals of 77 and 51 patients were classified into the NLR-low (≤2.9) group and NLR-high (>2.9) group, respectively. Cases in the NLR-high group had significantly higher proportion of T4 stage tumor than those in NLR-low group (P=0.002). The NLR did not show an association with other clinicopathological features, including molecular subgroups and treatment regimen (Table 1).

Figure 1 ROC curves assessing the cutoff point of NLR. AUC, area under the ROC curve; ROC, receiver operating characteristic; NLR, neutrophil-to-lymphocyte ratio.

NLR and clinical outcome in de novo stage IV BC patients following first line treatments

After a median follow-up of 32 (range, 2.6–169.2) months, 110 patients progressed, and 51 deaths were reported. The ORR was 43.8% (56/128), with CR 0.8% (1/128), PR 43.0% (55/128), SD 46.1% (59/128), and progressive disease (PD) 10.2% (13/128). The NLR levels were not associated with ORR (45.5% in NLR-low vs. 41.2% in NLR-high, P=0.633) but were associated with CBR (94.8% in NLR-low vs. 82.4% in NLR-high, P=0.022) (Table 2).

To investigate whether an elevated NLR is associated with the clinical outcome of BC, univariate and multivariate analyses were performed. Kaplan-Meier analysis revealed that patients with NLR-high had significantly shorter PFS than those with NLR-low (7.2 vs. 14.8 months, P=0.004) (Figure 2A). Univariate analysis identified liver metastasis, visceral metastasis, and NLR as prognostic factors (Table 3). A multivariate Cox proportional hazard model including NLR, liver metastasis, or visceral metastasis was established and NLR was identified as an independent prognostic factor for PFS [hazard ratio =1.791; 95% confidence interval (CI): 1.213–2.644; P=0.003]. When stratifying different subgroups, NLR-high was only associated with PFS in the HER2-positive and triple negative BC (TNBC) subgroups, but not in the HR⁺HER2⁻ subgroup (Figure 2B-2D).

Figure 2 Kaplan-Meier plots of PFS in de novo stage IV BC patients according to baseline NLR. (A) PFS according to baseline NLR in the whole population. (B) PFS according to baseline NLR in HR-positive subgroup. (C) PFS according to baseline NLR in HER2-positive subgroup. (D) PFS according to baseline NLR in TNBC subgroup. NLR, neutrophil-to-lymphocyte ratio; PFS, progression-free survival; BC, breast cancer; HR, hormone receptor; HER2, human epidermal growth factor receptor 2; TNBC, triple negative breast cancer.

The median OS of all patients was 56.0 (95% CI: 29.7–82.4) months. The OS had no significant difference between NLR-low (≤2.9) and NLR-high (>2.9) group in univariate survival analysis (64.1 vs. 56.0 months, P=0.980) (Figure 3A). The NLR also had no significant prognostic value in different molecular subtypes (Figure 3B-3D). In the multivariate analysis, we included all of the factors that might affect OS, including age, pathologic grade, molecular subtype, Ki-67, visceral metastasis, number of metastasis sites, first line therapy, and NLR. Visceral metastasis was a significant prognostic factor for OS in the univariate analysis (P=0.006) and multivariate analysis (hazard ratio =2.425; 95% CI: 1.008–5.833; P=0.048) (Table 4).

Figure 3 Kaplan-Meier plots of OS in de novo stage IV BC patients according to baseline NLR. (A) OS according to baseline NLR in the whole population. (B) O according to baseline NLR in HR-positive subgroup. (C) OS according to baseline NLR in HER2-positive subgroup. (D) OS according to baseline NLR in TNBC subgroup. NLR, neutrophil-to-lymphocyte ratio; OS, overall survival; BC, breast cancer; HR, hormone receptor; HER2, human epidermal growth factor receptor 2; TNBC, triple negative breast cancer.

NLR and peripheral blood T lymphocyte subtype

The distribution of lymphocyte subtype in the peripheral blood could partially represent the immune status of the patients. We compared the distribution pattern of different peripheral blood lymphocyte subtypes between the NLR-low (≤2.9; 52 cases) and NLR-high (>2.9; 35 cases) groups. The patients with NLR-low had higher levels of CD3⁺ T cells (P=0.028) and lower levels of CD4⁺CD25⁺ regulatory T (Treg) cells (P=0.041) than those with NLR-high (Figure 4, Table 5).

Figure 4 Peripheral blood total CD3⁺ T cells (A) and CD4⁺CD25⁺ T cell (B) percentage in de novo stage IV BC patients according to baseline NLR. Data are presented as the percentage of cells mean of n=52 and n=35 patients for the NLR-high and NLR-low groups, respectively. NLR, neutrophil-to-lymphocyte ratio; BC, breast cancer.

NLR and TILs

We also explored the association of the TILs level with NLR level. The TILs in 30 cases were assessed. The median value of TILs was 7.5% (1–80%). A total of 12 (40%) cases had higher TILs levels (>10%) and 18 (60%) cases had low TILs levels (≤10%) With 15 cases with NLR-low and 15 cases with NLR-high, the NLR-low patients had higher levels of TILs (P=0.025; Figure 5).

Figure 5 TILs in de novo stage IV BC patients according to baseline NLR. (A) The TILs ratio according to baseline NLR in 15 cases with NLR-low (≤2.9) and 15 cases with NLR-high (>2.9). (B) Immunohistochemistry stained by HE of high TILs with low NLR. (C) Immunohistochemistry stained by HE of low TILs with high NLR. Magnification times: ×400. “*”, “°”, special high value of TIL ratio. TILs, tumor infiltrating lymphocytes; NLR neutrophil-to-lymphocyte ratio; BC, breast cancer; HE, hematoxylin and eosin.

Discussion

The predictive value of NLR has been shown in a variety of solid tumor types, including MBC (9,10,13-21). Although stage IV and recurrent MBC share molecular characteristics and biological behaviors, recurrent MBC patients tend to have a worse prognosis compared to stage IV MBC patients (22), which may be related to the fact that stage IV MBC, with its treatment-naïve status, is more sensitive to systemic treatments. Whether NLR has different roles between the two groups remains mostly unknown. In addition, metastatic patterns have also been associated with survival in MBC. Patients with visceral metastasis were shown to have shorter survival than those with non-visceral metastasis. The number of metastatic sites is also correlated with survival (23). Our study showed that NLR-high was associated with a shorter PFS in de novo MBC patients, without a significant role in OS. We found no association between NLR and metastatic patterns, except for T4 stage tumors, which demonstrated higher levels of NLR and were often associated with a large tumor mass or tumor rupture.

Previous results on the role of NLR in different MBC molecular subtypes have been contradictory (9,24-28). In our cohort, we found that NLR was associated with a poor prognosis in patients with HER2-positive or TNBC, but not in patients with HR⁺HER2⁻. TNBC and HER2⁺ BCs with aggressive biological behavior demonstrate higher levels of both genomic instability and tumor mutation burden (TMB), which may promote the presentation of new antigens and result in a potential sensitization in response to immunotherapies in these patients (29).

We then investigated the relationship between NLR and immunological factors, including peripheral lymphocyte classification and the TILs. Our results demonstrated that NLR-low was associated with higher levels of CD3⁺ T cells, lower levels of CD4⁺CD25⁺ Treg cells, and higher levels of TILs. Correlation of NLR with immunological factors is consistent with an immune active status in NLR-low patients. Neutrophils inhibit the immune response by inhibiting the activity of immune cells [lymphocytes, activated T cells, and natural killer (NK) cells]. Lymphocytes play a key role in tumor immune monitoring by inducing cytotoxicity and apoptosis in tumor cells (30,31). The CD4⁺ T helper (Th) cells play multiple roles in the induction of immune responses against tumor cells. The effector CD4⁺ T cells are classified into subsets including Th1, Th2, Th17, and Treg lymphocytes based on the synthesis of a specific cytokine profile (32). Tregs, which typically express CD25, are naturally present in the immune system, accounting for 5–10% of CD4⁺ T cells; they are important in the control of immune responses by suppressing T cell proliferation and cytokine production and serve as regulatory factors in the tumor microenvironment (TME) by inhibiting CD8⁺ T, NK, B, and antigen-presenting cells (APCs) (33-35). Tregs also secrete immunomodulatory cytokines [interleukin (IL)-10, transforming growth factor (TGF)-β, and IL-35], express granzyme/perforin, consume IL-2, and degrade adenosine triphosphate (ATP) (36,37).

With its endogenous antitumor response, higher levels of TILs were associated with a longer OS in early-stage BC independent of other prognostic clinicopathological parameters (38). The immune landscape of MBC has remained largely unexplored. As mentioned above, the TME in metastatic tumors appears to be in an inert status, compared to early-stage tumors, by showing lower TILs levels and depleted immune functions (downregulation of immune-activating molecules and the upregulation of those with immunosuppressive properties) (39-41). In our study, we found significantly higher levels of TILs in the NLR-low group (P=0.025).

Conclusions

In conclusion, our results suggest that the baseline NLR is a prognostic factor for PFS in de novo stage IV BC with first line treatment, especially in HER2 positive and TNBC subtypes. A NLR-low status may indicate immune activation at the baseline with activated immune profiles (high CD3⁺ T cells, low CD4⁺CD25⁺ Tregs and high TILs). These results offer evidence for taking NLR into consideration when making treatment decisions in the first line setting in de novo stage IV BC patients. Future prospective and larger scale studies will help to validate these findings.

Acknowledgments

Funding: None.

Footnote

Reporting Checklist: The authors have completed the REMARK reporting checklist. Available at https://atm.amegroups.com/article/view/10.21037/atm-22-5612/rc

Data Sharing Statement: Available at https://atm.amegroups.com/article/view/10.21037/atm-22-5612/dss

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-22-5612/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was approved by the Ethics Committee of the Peking University Cancer Hospital (No. 2017KT40) and was conducted in accordance with the Declaration of Helsinki (as revised in 2013). Individual consent for this retrospective analysis was waived.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: J. Jones)