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

Numerous studies reported the prognostic signicance 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. 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).

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][8][9][10][11][12][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 signi cance of neutrophil count (or preferably NLR) [16][17][18][19][20]. These ndings 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 re ect the e cacy 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.

Patient selection
A total of 603 patients who were newly diagnosed ESCC and radical esophagectomy were enrolled in the present study at Cancer Hospital of the University of Chinese Academy of Sciences, from 2008 to 2014.
All tumor specimens were pathologically con rmed as ESCC after surgery. The blood routine was examined within one week before surgery. If the preoperative neutrophil count was performed on the rst day and on the fourth day before surgery, the closest experimental data was selected. The postoperative neutrophil count was checked within two weeks after operation. In order to reduce the risk of stress response after surgery, the neutrophil count farthest within two weeks were collected. The unit of neutrophil count was per L in our clinical laboratory. Patients with incomplete clinical and laboratory data were excluded. Patients with active infection or any simultaneous hematological diseases or other tumors before the operation were excluded. Patients who received neoadjuvant chemotherapy were excluded. Because the preoperative neutrophil counts may be in uenced by these diseases or treatments.
The present study was approved by the Ethics Committee of Cancer Hospital of the University of Chinese Academy of Sciences (Hangzhou, China). The study was conduct in accordance with the Declaration of Helsinki. In the end, all patients received written informed consent.

Statistical analysis
The preoperative neutrophil counts and the postoperative neutrophil counts were counted as continuous variables, which do not conform to the normal distribution. Continuous data was represented by median and quartile intervals. The clinical characteristics of ESCC patients were analyzed as categorical variables that were presented as numbers and percentage. The chi-square test was used to compare categorical variables. Overall survival (OS) was calculated from the date of surgery to the date of death and the last follow-up. Overall survival curve was estimated by the log-rank test and the Kaplan-Meier method. The overall survival curve was plotted by GraphPad Prism 7 software. COX regression analyses were used to evaluate independent prognostic markers. All statistical analysis was calculated by SPSS, version 19.0 (SPSS, Chicago, IL, USA). The software CRAN-R (version 3.3.0.) was used to illustrate the beeswarm plot. P less than 0.05 were considered as statistical signi cance.

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 signi cant 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 in ltration, 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).

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][17][18][19][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 in uence of Nc on survival in patients with ESCC. So far we rstly 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 in ltration, but OS did not differ in other subgroup. This nding indicated that neutrophil change(Nc) may serve as prognostic marker specially in these subgroup including male, age ≤ 60, with vessel invasive, and with nerve in ltration. 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 in ammatory reaction [21]. Neutropenia makes patients more susceptible to infection that belongs to the complication after surgery [22,23]. In our ndings, 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 identi ed. Neutrophils are able to entrap circulating tumor cells (CTCs) by facilitating their extravasation thus promoting metastasis [24][25][26][27][28]. Moreover, neutrophils were described for their ability to in ltrate 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][31][32][33][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][41][42][43][44][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 in ltration.
A beeswarm plot is a two-dimensional visualization technique in which the measured values are drawn in a xed 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: rst, 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 speci c 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 rst investigate the relationship between perioperative change in neutrophil count and clinical outcome in ESCC undergoing curative resection.

Conclusion
In conclusion, this study rst 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 in ltration. 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. 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 con ict of interest. 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 nal manuscript.
40. Wikberg ML, Ling A, Li X, Oberg A, Edin S, Palmqvist R. Neutrophil in ltration is a favorable prognostic factor in early stages of colon cancer. Human pathology.  Figure 1 Differences in the neutrophil count prior to and following surgery was illustrated by beeswarm plots. The horizontal line in the middle indicates the median, the horizontal line above represents the 25th-percentile, and the horizontal line below represents the 75th-percentile.   Overall survival analysis in patients with pathological stage 1a-1b, patients with pathological stage 2a-2b, and patients with pathological stage 3a-3c according to perioperative change in neutrophil count (A, B, C).   Overall survival analysis in all 603 patients with ESCC according to perioperative change in neutrophil count.