Prognostic Value of Preoperative Neutrophil-to -Lymphocyte Ratio in Adenoid Cystic Carcinoma of the Head and Neck


 BackgroundStudies in recent years have shown that the neutrophil-to-lymphocyte ratio (NLR) is associated with the survival and prognosis of patients with malignant tumors.However, the utility of the NLR in predicting outcomes in patients with adenoid cystic carcinoma (ACC) of the head and neck remains unclear. Here, we examined the prognostic value of the preoperative NLR in patients with ACC of the head and neck.MethodsThis study retrospectively analyzed 71 patients with a histopathological diagnosis of ACC from August 2005 to November 2020.The optimal cutoff value was obtained by receiver operating characteristic (ROC) analysis. The characteristics of the patients were evaluated using chi-squared tests.The value of the NLR used in predicting the outcomes of these patients was analyzed using Kaplan-Meier curves and Cox regression models.ResultsThe cutoff value of the NLR was 2.071. The chi-squared tests showed that a high NLR(>2.071)was associated with tumor stage and lymph node metastasis(LNM). Kaplan-Meier survival analysis showed that the NLR>2.071 group was correlated with a shorter Overall survival(P<0.05). In multivariate Cox regression analysis, high NLR (>2.071) and high age (>54 years) were independent risk factors predicting poorer outcomes in patients with ACC.ConclusionsWe suggest that the preoperative NLR is a useful biomarker for the prognosis of patients with ACC.


Abstract
Background Studies in recent years have shown that the neutrophil-to-lymphocyte ratio (NLR) is associated with the survival and prognosis of patients with malignant tumors.However, the utility of the NLR in predicting outcomes in patients with adenoid cystic carcinoma (ACC) of the head and neck remains unclear. Here, we examined the prognostic value of the preoperative NLR in patients with ACC of the head and neck.

Methods
This study retrospectively analyzed 71 patients with a histopathological diagnosis of ACC from August 2005 to November 2020.The optimal cutoff value was obtained by receiver operating characteristic (ROC) analysis. The characteristics of the patients were evaluated using chi-squared tests.The value of the NLR used in predicting the outcomes of these patients was analyzed using Kaplan-Meier curves and Cox regression models.

Results
The cutoff value of the NLR was 2.071. The chi-squared tests showed that a high NLR(>2.071)was associated with tumor stage and lymph node metastasis(LNM). Kaplan-Meier survival analysis showed that the NLR>2.071 group was correlated with a shorter Overall survival(P<0.05). In multivariate Cox regression analysis, high NLR (>2.071) and high age (>54 years) were independent risk factors predicting poorer outcomes in patients with ACC.

Conclusions
We suggest that the preoperative NLR is a useful biomarker for the prognosis of patients with ACC.

Background
Adenoid cystic carcinoma (ACC) is a rare malignant tumor with an annual incidence of 3-4.5 cases per million people 1 ,accounting for approximately 1% of all head and neck malignancies and 10% of all salivary gland tumors and is the most commonly reported malignancy of the minor salivary glands 2 .Unfortunately,ACCis still a poorly understood disease due to its unpredictability, the need for longterm follow-up and the paucity of available clinical trials 3 .Therefore,it is of great signi cance to correctly evaluate the prognosis of patients with ACC. Traditional prognostic indicators, including the presence of metastasis, tumor grade, histological subtype, and tumor volume, have gradually shown inaccuracy and inadequacy in clinical practice 4 . Identi cation of highly available and noninvasive prognostic factors could help us identify high-risk patients and provide them with the best therapy protocol, which may lead to further improvement of treatment outcomes 5 .
Many studies have illustrated a strong connection between cancer and in ammation 6 . In ammatory cells are signi cant tumor promoters early in the neoplastic process. Cancer-related in ammation enables cancer cells to form malignant biological behaviors, including proliferation, in ltration, angiogenesis, and metastasis 7 . In the past few years, many studies have shown that there are multiple in ammatory responses associated with poor outcomes in many types of malignancies, such as adrenocortical carcinoma,gastric cancer and ampullary carcinoma 8- 10 . In ammatory and hematological markers, including the neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and lymphocyte-tomonocyte ratio (LMR), have been used to guide treatment and predict prognosis in cancer patients.
The absolute value of neutrophils re ects the body's in ammatory response caused by tumors, while the absolute value of lymphocytes re ects the body's antitumor immune suppression level.The detection of the NLR as a systemic immune-in ammation index is useful to predict the prognosis of head and neck squamous cell carcinoma 11 ,and thus far, only two studies have examined the NLR in ACC 12,13 .In these studies, only 17 and 29 patients had available NLR data, respectively, so the conclusions of these studies were not quite convincing.Therefore,our study was designed to evaluate the NLR as a prognostic marker in a relatively larger group of patients with ACC. was usually performed within 4-6 weeks postoperatively. If patients had poor wound healing, radiotherapy was delayed appropriately(no more than 8 weeks). All patients were scanned with a 3 mm layer thickness, and the extent of the scan was determined by the location of the lesion, including at least 3 cm above the skull base to 3 cm below the clavicle. Clinical tumor volume (CTV) was outlined to include the preoperative primary tumor area and postoperative tumor bed area, as well as the neck subdivision where the metastatic lymph nodes were located. For those with de nite cranial nerve invasion, the CTV also involved the extent of cranial nerve travel to block the potential recurrence pathway. The planned target volume (PTV) is a three-dimensional extrapolation of 3 mm from the CTV; 66 Gy was prescribed for patients with positive margins and 54-60 Gy for the remaining patients, with fractional doses of 1.8 to 2.0 Gy/dose.Blood samples were obtained prior to the initiation of treatment. Neutrophil count, lymphocyte count and platelet count were used as serological indicators of in ammation. The NLR was calculated by dividing the absolute neutrophil count (count/µL) by the absolute lymphocyte count (count/µL),while the PLR was calculated by dividing the absolute platelet count (count/µL) by the absolute lymphocyte count (count/µL).Overall survival(OS) was de ned as the time from diagnosis to death, and disease-free survival (DFS) was de ned as the time from treatment start to the recurrence of the disease or death from any cause.

Statistical analysis
Time-dependent ROC curves were made by R program.The optimal cutoff value was determined by ROC analysis. The characteristics of the patients were analyzed using chi-squared tests. The survival curves were plotted using the Kaplan-Meier method, and the signi cance was determined through the log-rank test. Univariate and multivariate analyses were performed by the Cox proportional hazard model.Statistical analyses were performed using a statistical analysis software package (SPSS Statistics, version 25; IBM, Armonk, NY, USA), and P values <0.05 were considered signi cant.
As shown in Table 1, the median age was54± 25years. The tumors of these patients were located in a major salivary gland(29.58%), minor salivary gland(50.70%) and other sites(19.72%), respectively.Surgery alone was performed on 28(39.44%)patients.Surgery and adjuvant radiation were used on 43(60.56%)patients.
As presented in Table 1, the correlation between the preoperative NLR and clinical and pathologic characteristics was evaluated. Thirty-seven(52.11%) patients (NLR > 2.071) were included in the high NLR group,and 34 (47.89%) patients were distributed to the low NLR (NLR ≤ 2.071) group.
The preoperative NLR level was closely related to the TNM stage and LNM (P <0.05). No obvious correlations with age, sex, location,treatment or perineural invasion were observed (P >0.05). The relationship of the NLR with postoperative OS and DFS in patients with ACC Kaplan-Meier survival analysis showed that the NLR>2.071 group was correlated with a shorter OS (P<0.05), while the NLR had no relationship with DFS (Fig. 2).
As seen in the univariate analysis in Table 2

Discussion
In this analysis of 71 patients with ACC,we found that the preoperative NLR can be a predictive biomarker for OS in ACC and is related to TNM stage and the LNM.Participants were divided into two groups according to the cutoff value, and chi-squared tests were applied to evaluate the clinical and pathologic characteristics of patients with ACC.We found that the preoperative NLR l was closely related to the TNM stage and LNM (P <0.05). No obvious correlations with age, sex, location,treatment or perineural invasion were observed (P >0.05). We used Kaplan-Meier survival analysis to determine the relationship between the NLR and OS and the NLR and DFS. Patients with a NLR >2.071had a shorter OS (P <0.05), and the NLR was not associated with DFS (P >0.05).We used a Cox regression model and analyzed sex, age, TNM stage, nerve invasion, LNM and NLR of patients. We found that the NLR (HR 0.22, 95% CI [0.07-0.66]) and age(HR 0.31, 95% CI [0.12-0.81]) were independent risk factors for OS in patients with ACC. The nding that a high NLR (>2.071) was a strong indicator of OS in patients with ACC appears to be novel.
Neutrophils display a protumor phenotype that could be detrimental to the host. The tumor microenvironment controls neutrophil recruitment, and in turn, neutrophils can strengthen the biological behavior of the tumor, causing it to grow and metastasize 16  Higher neutrophil levels can upregulate the expression of growth factors, such as chemokines, which play an important role in tumor development and progression. The reduction in lymphocytes indicates that the body's immunity is reduced and its antitumor ability is weakened.An increased NLR indicates that the body's in ammatory response is increased, while the lymphocyte-mediated antitumor response is decreased, which can easily cause tumor deterioration and metastasis, ultimately leading to the poor prognosis of patients 15,16,17,23,24 .Takumi Hasegawa et al. reported that the NLR increased with the number of pathological lymph node metastases and shorter OS in patients with oral cancer, which was also an indication of a decrease in the lymphocyte count 25 . The results of a meta-analysis suggested that an elevated pretreatment NLR is a negative prognostic factor in patients with head and neck cancer 26 .
One of the results of our study showed that the NLR had a relationship with the TNM stage,which con rmed the ndings of Sibel Goksel,who was able to demonstrate that a higher NLR could be a robust predictor to distinguish advanced stages of lung cancers 27 . A high NLR implies relative lymphocytopenia and neutrophilic leukocytosis. The former indicates a decrease in lymphocyte-mediated anticancer effects 17 , and the latter indicates higher secretion of proangiogenic factors for tumor growth 23 ,which may partly explain the connection between the NLR and TNM stage.
One point worth mentioning is that an association between the LMN and NLR has been found in our research,which is the rst one that was illustrated in ACC. This relationship has already been demonstrated in many other cancers, such as esophageal squamous cellcarcinoma,endometrial cancer and resectable pancreatic neuroendocrine tumors 28-30 .This conclusion still needs to be further con rmed through more studies concerning the ACC to improve its validity.
Perineural invasion had no signi cant association with the preoperative NLR, which had been proved in published data 31 .
Chun-Ye Zhang et al. analyzed 218 cases of ACC of the head and neck(ACCHN)and reported that older age(>60 years)is a signi cant factor for predicting poor prognosis in Chinese patients with ACCHN 32 .
Yunsuk Choiet al. also reported that older people with ACCHN tend to haveshorterOS 33 . Our multivariate analysis indicated that older age(>54 years) is an independent predictor for the low survival of patients with ACC, which is largely in concordance with the results mentioned above.Comorbidities and worse performance status may lead to a poorer prognosis for elderly patients.
Kizuki Yuzaet al. found that the TNM stage clearly predicted outcomes of postoperative patients with gallbladder carcinoma. This is not in accordance with our results, which showed that patients with advanced TNM stage were prone to shorter OS,but it had no statisticalsigni cance 34 .Several studies have demonstrated a positive correlation between LNM and decreased survival in patients with ACC of the head and neck [35][36][37] .Although our univariate analysis showed that patients with LNM were prone to shorter OS, it had no statistical signi cance (Table 2). Therefore, more cases need to be enrolled to prove our ndings of patients with ACC.
Our present study has several limitations. First,because the optimal cutoff values for the NLR vary according to the clinical and pathological characteristics of participants and the number of participants,the cutoff value we obtained from the ROC curve is not generalizable to all patients with ACC.Second, because of the single-center design and rarity of ACC,our ndings involved a sample that was relatively small. Although multivariate analysis was performed to reduce the bias,caution should still be used when dealing with the results of our research.Therefore,further multicenter prospective studies are needed to validate the sensitivity and speci city of the NLR in evaluating the prognosis of ACC.
In conclusion,a high NLR was associated with advanced tumor stage and LNM, as well as correlated with a shorter OS. In multivariate Cox regression analysis, a high NLR and older age were independent risk factors predicting poorer outcomes in patients with ACC.Therefore,we hypothesized that the preoperative NLR was a useful biomarker for the prognosis of patients with ACC.
Abbreviations NLR Neutrophil-to-lymphocyte ratio Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
We wish to con rm that there are no known con icts of interest associated with this publication.

Funding
This study was supported by grants from the National Clinical Research Center for Oral Diseases (NCRCO-202101).
Authors' contributions XRL and JM conceived and designed the experiments. XRL, LZ, YXC and NL performed the experiments.
XRL and LZ analyzed the data. XRL and JM wrote the manuscript. All authors read and approved the nal manuscript.

Figure 1
The ROC curves of the NLR and PLR in patients with ACC. (A) The ROC area of NLR was 0.743(3 years).

Figure 2
Kaplan-Meier survival analysis indicates that patients with a NLR >2.071 have a shorter OS.