Perioperative change in neutrophil count predicts worse survival in esophageal squamous cell carcinoma

Abstract

Background

Numerous studies reported the prognostic significance of neutrophil count (or preferably NLR) in patients with esophageal squamous cell carcinoma (ESCC), while the relationship between perioperative change in neutrophil count and survival of patients with ESCC after surgery has not been assessed.

Methods

A total of 603 patients with newly diagnosed ESCC after the operation were enrolled in the study. Neutrophil change (Nc) (Nc = Post-Neutrophil–Pre-Neutrophil) was counted according to data within one week before surgery and two week after surgery. The median of Nc was selected to be the cut off value to evaluate the relationship between Nc and Overall survival (OS).

Results

105 (17.4%) patients had pathological stage 1a-1b, 205 (34.0%) patients had pathological stage 2a-2b, and 293 (48.6%) patients had pathological stage 3a-3c.There were 180 (29.9%) patients who had vessel invasive, and 423 (70.1%) patients without vessel invasive. There were 215(35.7%) patients who had nerve infiltration, and 388 (64.3%) without nerve infiltration. In univariate analysis, six parameters including neutrophil change(Nc)(≥ 2.60 vs.<2.60) (P = 0.010, HR = 0.694, 95% CI 0.526–0.915) significantly correlated to worse OS. Multivariate analysis revealed that neutrophil change(Nc)(≥ 2.60 vs.<2.60) was an independent prognostic marker for OS (P = 0.022, HR = 0.720, 95% CI 0.544–0.954). Kaplan-Meier curves shown that Nc < 2.60 was significantly associated with worse OS (P = 0.016).

Conclusions

Perioperative change in neutrophil count predicts worse survival in patients with ESCC after the operation.

Background

Esophageal cancer is one of the most aggressive and common carcinoma worldwide and the fourth most frequent cause of carcinoma-related mortality in China [1]. Esophageal squamous cell carcinoma (ESCC) remains the predominant pathological tissue type in China [2, 3]. The clinical outcome of ESCC patients remains unsatisfactory due to distant metastasis or a high recurrence rate. Although surgical intervention, chemotherapy, radiation and biologically targeted treatments have been improved, the five-year survival rate remains 20%-30% worldwide [4]. Therefore, it is urgent to identify reliable and conventional prognosis markers that may contribute to classify patients with high risk of death and may guide the perioperative management of these patients.

Accumulating evidence indicates neutrophils play a vital role in tumor progression by interacting with immune and cancer cells [5, 6]. Neutrophils are involve in anti- and pro-tumor activities including tumor cell killing, proliferation, aggressiveness, metastasis, angiogenesis, and participating in other immune responses[7–13]. In addition to their effect in tumor site, retrospective researches and functional analyses of neutrophil-to-lymphocyte ratio (NLR) indicate that neutrophils in blood could also play an important value in tumor progression. Oncologists systematically monitored neutrophil counts during cancer management, due to chemotherapy caused neutropenia that makes patients more susceptible to infection [14, 15]. Numerous reviews and meta-analyses reported the prognostic significance of neutrophil count (or preferably NLR) [16–20]. These findings focused on preoperative neutrophil count, while the relationship between perioperative change in neutrophil count and survival of patients with ESCC after the operation has not been assessed.

We hypothesized that the perioperative change in neutrophil count might reflect the efficacy of surgical treatment and predict the clinical outcome of patients with ESCC. Therefore, we investigated whether the perioperative change in neutrophil count might be an independent prognostic factor in patients with ESCC who received surgical intervention.

Methods

Results

Patient characteristics

There were 86 (14.3%) female and 517 (85.7%) male patients with newly diagnosed ESCC. There were 279 (46.3%) young patients whose age at first diagnosis≤60 years and 324 (53.7%) old patients (>60 years). 105 (17.4%) patients had pathological stage 1a-1b, 205 (34.0%) patients had pathological stage 2a-2b, and 293 (48.6%) patients had pathological stage 3a-3c.There were 180 (29.9%) patients who had vessel invasive, and 423 (70.1%) patients without vessel invasive. There were 215(35.7%) patients who had nerve infiltration, and 388 (64.3%) without nerve infiltration. 408 (67.7%) patients with surgery only, 137 (22.7%) patients who received postoperative chemotherapy, and 58 (9.6%) patients who received radiotherapy and chemotherapy after operation. The median value of the preoperative neutrophil count was 3.5, and the quartile interval is 2.8-4.5. The median value of the postoperative neutrophil count was 6.2, and the quartile interval is 5.0-7.6. Details of clinical features were shown in Table 1. Significant differences in the neutrophil count was observed prior to and following surgery (P<0.001) (Figure 1).

Relationship between neutrophil change and clinical features

The clinical data of patients in neutrophil change(Nc) <2.60 group and in neutrophil change(Nc) ≥2.60 group are listed in Table 1. There were no significant difference between the two groups in terms of clinical data including sex, age, depth of tumor, lymph node metastasis, pathological stage, pathology grade, vessel invasive, nerve infiltration, and treatment regimen. The median of Pre-Neutrophil and Post-Neutrophil were both higher in neutrophil change(Nc) ≥2.60 group than in neutrophil change(Nc) <2.60 group (both P <0.001).

Prognostic variables for OS

In univariate analysis, six parameters including neutrophil change(Nc)(≥2.60 vs.<2.60) (P=0.010), depth of tumor (P<0.05), lymph node metastasis (P<0.05), pathological stage (P<0.001), vessel invasive (absence vs. presence) (P<0.001), and nerve infiltration (absence vs. presence) (P<0.001) predict worse OS. Multivariate analysis revealed that neutrophil change(Nc)(≥2.60 vs.<2.60) (P=0.022), lymph node metastasis (P<0.05), and nerve infiltration (absence vs. presence) (P=0.005) could serve as independent prognostic markers of worse OS (Table 2). OS of patients with neutrophil change(Nc)(<2.60) group were notably worse compared to patients with neutrophil change(Nc)(≥2.60) group (P=0.016) (Figure 2).

Subgroup analysis according to other clinical features

To investigate the subgroups of patients with ESCC impacted by neutrophil change(Nc), we classified patients based on gender (male, n=517; female, n=86), age (≤60, n=279; >60, n=324), pathological stage (1a-1b, n=105; 2a-2b, n=205; 3a-3c, n=293), vessel invasive (Yes, n=180; No, n=423), and nerve infiltration(Yes, n=215; No, n=388). OS of male patients, age≤60 patients, patients without vessel invasive and patients without nerve infiltration were dramatically worse for those with neutrophil change(Nc)(<2.60) (P=0.013, P=0.002, P=0.004, and P=0.014), but OS did not differ in female patients, age>60 patients, patients with vessel invasive and patients with nerve infiltration (Figure 3- Figure 7). Patients with pathological stage 3a-3c tend to have worse OS in neutrophil change(Nc)(<2.60) group than in neutrophil change(Nc)(≥2.60), but do not reach statistical difference (P=0.050).

Discussion

Because chemotherapy caused neutropenia, oncologists always evaluated neutrophil counts during the process of chemotherapy treatment [14, 15]. Numerous retrospective studies and meta-analyses reported that neutrophil count (or preferably NLR) could serve as an independent prognostic factor in patients with ESCC [16–20]. However, these reports based on preoperative neutrophil count, the association between perioperative change in neutrophil count and clinical outcome of patients with ESCC undergoing curative surgery has not been investigated.

In the present study, all enrolled patients were divided into two groups including neutrophil change(Nc)(< 2.60) and neutrophil change(Nc)(≥ 2.60) according to the median of neutrophil change(Nc). Our study evaluated perioperative change in neutrophil count and the influence of Nc on survival in patients with ESCC. So far we firstly indicated that perioperative change in neutrophil count could be an independent prognostic factor in patients with ESCC. Based on subgroup analysis we shown that neutrophil change(Nc)(< 2.60) predicted worse OS in male patients, age ≤ 60 patients, patients without vessel invasive and patients without nerve infiltration, but OS did not differ in other subgroup. This finding indicated that neutrophil change(Nc) may serve as prognostic marker specially in these subgroup including male, age ≤ 60, with vessel invasive, and with nerve infiltration. Neutrophil change(Nc)(< 2.60) could assistant clinical doctors to predict and guide prognosis.

Even though the mechanisms of the association between Neutrophil change(Nc)(< 2.60) and worse OS remains unknown, the interpretation are as follows: (1) Neutrophils account for 50–70% of all circulating white blood cells in healthy adult, and they play an important role in the inflammatory reaction [21]. Neutropenia makes patients more susceptible to infection that belongs to the complication after surgery [22, 23]. In our findings, Neutrophil change(Nc)(< 2.60) group had lower Pre- Neutrophil that may be correlated with a preexisting hypoimmunity. (2) In addition, Neutrophil change(Nc)(< 2.60) group had weak Post-Neutrophil, which may lead to the infection and complication. Therefore Neutrophil change(Nc)(< 2.60) group with both lower Pre- Neutrophil and Post-Neutrophil predicted shorter OS.

An emerging pro-tumor ability of circulating neutrophils has recently been identified. Neutrophils are able to entrap circulating tumor cells (CTCs) by facilitating their extravasation thus promoting metastasis [24–28]. Moreover, neutrophils were described for their ability to infiltrate cancer tissue and are called tumor-associated neutrophils (TANs) [29]. TANs were shown to play a vital role in promoting tumor progression through proliferation, angiogenesis, and interacting with immune cells [30–34]. Although accumulating evidence indicates that TANs are key players in pro-tumor immunity, large researches suggest that contribute to anti-tumor. TANs were able to induce apoptosis through releasing cytotoxic molecules including ROS [35]. In addition, TANs play an important role in recruiting and activating cytotoxic CD8 + and intra-tumor CD4 + T cells [36]. Neutrophils were shown to kill cancer cells by a process termed trogoptosis, which was different from ferroptosis [37]. Therefore Neutrophils contribute to promote cytotoxic T cell reaction by eliminating resistant tumor clones [38].Neutrophil derived extracellular DNA (NETs) were shown to lead to cancer cell death [39]. Because of heterogeneity in neutrophil function, scientists hypothesize that different subgroups of TANs may be involved. The prognostic effect of TANs remains inconsistent because they have relationship with either a worse or better survival [40–45]. Due to the importance of neutrophils in the immunity upon bacterial infection, the neutrophil count in blood routine examination is utilized as a read-out during cancer management related toxicities. Neutrophil count is routine, cheap and reliable in clinical examination. The clinical application of neutrophil count could contribute to predict and guide the prognosis of patients with ESCC specially in these subgroup including male, age ≤ 60, with vessel invasive, and with nerve infiltration.

A beeswarm plot is a two-dimensional visualization technique in which the measured values are drawn in a fixed reference shaft and avoided the overlap of these values. It is good at presenting the experimental data points and the relative distribution of these data point. There were some shortcomings in this study: first, this retrospective design study did not include the validation set neither in our laboratory nor in multicenter hospital. The validation set could improve the suggestion of Neutrophil change(Nc)(< 2.60) may serve as an independent prognostic factor; Second, due to the low patients in the adjuvant treatments such as radiation and chemotherapy, this subgroup were not taken into account. Third, because of retrospective study, the neutrophil count could not be checked on specific days. In order to reduce the risk of stress response after surgery, the post-neutrophil count farthest within two weeks were collected. Despite these limitations, we first investigate the relationship between perioperative change in neutrophil count and clinical outcome in ESCC undergoing curative resection.

Conclusion

In conclusion, this study first indicate that Neutrophil change(Nc)(< 2.60) predict worse OS in patients with ESCC specially in these subgroup including male, age ≤ 60, with vessel invasive, and with nerve infiltration. Neutrophil change(Nc)(< 2.60) could contribute the oncologists to evaluate and predict prognosis based on clinical examination data. In the future, more prospective and multicenter studies are warranted to prove the relationship between them.

Abbreviations

ESCC: Esophageal squamous cell carcinoma; NLR: Neutrophil-to-lymphocyte ratio; Nc: Neutrophil change; OS: Overall survival; HR: Hazard ratio; CI: Confidence interval; CTCs: Circulating tumor cells; TANs: Tumor-associated neutrophils;

Declarations

Ethics approval and consent to participate

All procedures in our study were conducted in keeping with the ethical standards of the World Medical Association Declaration of Helsinki. All the patients provided written informed consent, the protocol was approved by ethics committee at Cancer Hospital of the University of Chinese Academy of Sciences.

Consent to publish

Not applicable.

Availability of data and materials

The data is available by contacting corresponding author.

Competing interests

No conflict of interest.

Funding

This study was funded by National Natural Science Foundation of China (contract/grant number: 81602615), Zhejiang Youth Talents Project (contract/grant number: 2019RC026), and General research program of Health Department of Zhejiang Province (contract/grant number: 2020KY480). The funders had no role in the design, analysis, or writing of this manuscript.

Authors' contributions

QS designed the study, and was a major contributor in writing the manuscript.; JW collected material and data and was involved in the statistical interpretation of the data.; SW collected material and data and performed statistical analysis,; SX drafted the manuscript.. All authors read and approved the final manuscript.

Acknowledgments

We thank the included individuals, including the oncologists, laboratory personnel, and patients in the present study.

Authors’ Information

¹Department of Clinical Laboratory, Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital); Institute of Cancer and Basic Medicine (ICBM), Chinese Academy of Sciences, Hangzhou, Zhejiang, People’s Republic of China;

²Cancer Research Institute, Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital); Institute of Cancer and Basic Medicine (ICBM), Chinese Academy of Sciences, Hangzhou, Zhejiang, People’s Republic of China.

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Tables

Table 1. Demographic and clinical data of 603 ESCC patients accroding to neutrophil change(Nc).

Abbreviations: S, surgery; C, chemotherapy; CRT, chemoradiotherapy. 

Table 2.Overall survival analyses according to neutrophil change(Nc) in 603 patients with ESCC.