Combined pretreatment neutrophil-lymphocyte ratio and platelet-lymphocyte ratio predicts survival and prognosis in patients with non-metastatic nasopharyngeal carcinoma: a retrospective study

The clinical significance of the combination of neutrophil–lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) is unclear. This study investigated the predictive value of pretreatment NLR (pre-NLR) combined with pretreatment PLR (pre-PLR) for the survival and prognosis of nasopharyngeal carcinoma (NPC). A total of 765 patients with non-metastatic NPC from two hospitals were retrospectively analyzed. The pre-NLR-PLR groups were as follows: HRG, high pre-NLR and high pre-PLR. MRG, high pre-NLR and low pre-PLR or low pre-NLR and high pre-PLR. LRG, neither high pre-NLR nor high pre-PLR. Receiver operating characteristic (ROC) curves were used to identify the cutoff-value and discriminant performance of the model. We compared survival rates and factors affecting the prognosis among different groups. The 5-year overall survival (OS), local regional recurrence-free survival (LRRFS) and distant metastasis-free survival (DMFS) of NPC patients in HRG were significantly poorer than those in MRG and LRG. The pre-NLR-PLR score was positively correlated with T stage, clinical stage, ECOG, and pathological classification. Multivariate cox regression analysis showed that pre-NLR-PLR scoring system, ECOG, pre-ALB, pre-CRP and pre-LMR were independent risk factors affecting 5-year OS, LRRFS and DMFS. The ROC curve showed that area under the curve (AUC) values of pre-NLR-PLR of 5-year OS, LRRFS and DMFS were higher than those of pre-NLR and pre-PLR. pre-NLR-PLR is an independent risk factor for the prognosis of NPC. The pre-NLR-PLR scoring system can be used as an individualized clinical assessment tool to predict the prognosis of patients with non-metastatic NPC more accurately and easily.

Subsequently, multivariate cox regression analysis was conducted for the above variables.Variables for inclusion were carefully chosen, given the number of events available, to ensure parsimony of the final model.For example, because there was no significant difference between LRG and MRG in K-M curve of NPC patients, the pre-NLR-PLR score group was adjusted and included in the multivariate Cox regression analysis.Because the clinical stage was determined by stage T and stage N, we did not include it in the multivariate analysis.Analysis showed that pre-NLR-PLR scoring system, ECOG, pre-ALB, pre-CRP and pre-LMR were independent risk factors affecting 5-year OS, 5-year LRRFS and 5-year DMFS in NPC patients.Among them, Patients with pre-NLR-PLR score of 2 (HRG) were 1.905 times more likely to die within 5 years than those with score of 0-1 (LRG + MRG) (HR 1.905, 95% CI 1.340-2.709,p < 0.001), the risk of local recurrence was 1.864 times more likely than those with LRG + MRG (HR 1.864, 95% CI 1.109-3.133,p = 0.019), and the risk of distant metastasis was 1.631 times than LRG + MRG (HR: 1.631, 95% CI 1.104-2.410,p = 0.014).In addition, age, T(4) stage, N(3) stage and smoking history were independent risk factors for 5-year OS in NPC patients.T stage and N(3) stage were independent risk factors for DMFS at 5 years (Table 3).

Prognostic performance of pre-NLR-PLR scoring system
According to ROC curve analysis, it was concluded that the AUC values of pre-NLR-PLR of 5-year OS, LRRFS and DMFS in NPC patients were higher than those of pre-NLR and pre-PLR.Among them, the difference of the AUC values of pre-NLR-PLR and pre-PLR in 5-year OS and 5-year LRRFS was statistically significant, but not in 5-year DMFS.There was no statistical significance in the AUC values of 5-year OS, LRRFS and DMFS between pre-NLR-PLR and pre-NLR (Table 4).

Discussion
With the wide application of IMRT and the introduction of comprehensive treatment modalities such as chemoradiotherapy combined with immunotherapy and targeted therapy, the local control rate of NPC patients has been significantly improved, and the 5-year overall survival rate has reached more than 80% 2,12,13 .In spite of this, local recurrence and distant metastasis still remain the leading cause of NPC treatment failure, accounting www.nature.com/scientificreports/for about 70% of NPC specific deaths 14 .At present, the prognosis of NPC patients still depends on TNM staging system 15 .Nevertheless, although patients have the same clinical stage and receive the same treatment, there are still different treatment effects, which may be due to tumor heterogeneity, immune and inflammatory responses.TNM staging system is based on imaging and anatomy, and does not take into account the biological diversity of the tumor, so the stratified prognosis cannot be satisfactorily evaluated.Some scholars 16 proposed to use molecular genetic biomarkers, such as mRNA, as new prognostic predictors.However, the detection process of these markers is complicated and the detection cost is high.Many technologies are limited to the laboratory and cannot be promoted clinically.
Inflammation and immune response are involved in the occurrence and development of tumor, including initiation, promotion and metastasis 17 .Since the first reported by Virchow in 1863 18 , more and more studies have   www.nature.com/scientificreports/shown that inflammatory marker response plays a key role in tumor development and has shown independent prognostic value, such as NLR 5,17,19 , LMR 20 , PLR 21 .Malignant tumors induce systemic inflammatory responses by releasing cytokines and chemokines, which are manifested as elevated neutrophil and platelet counts and decreased lymphocytes 22 .Chen et al. 23 found in their study on the prognosis of 299 patients with limits-stage small cell lung cancer after surgery that the high levels of preoperative NLR and PLR indicated poor prognosis of patients with limits-stage small cell lung cancer after surgery, and the detection was simple, fast, and low-cost, which could be used as a reference for initial screening of patients who would benefit from immunotherapy.
Previous studies have shown that NLR and PLR are associated with the prognosis of NPC 24,25 .However, no comprehensive evaluation of the relationship between the combination of NLR and PLR and the prognosis of NPC patients has been reported.Therefore, based on the current research results that both NLR and PLR have an impact on the prognosis of NPC, our study combined these two hematological parameters and established pre-NLR-PLR scoring system, divided the NPC patients into groups according to different scores, investigated the correlation between hematological parameters and clinical characteristics, survival and prognosis of patients with NPC.In addition, in this study, NLR, PLR and other indicators closely related to OS, LRRFS, and DMFS were detected only by peripheral blood of patients with NPC, the method was simple, effective, reproducible, and practical.The level of NLR is closely related to the occurrence and development of tumor.It is speculated that the mechanism may be as follows: (1) neutrophils affect tumor microenvironment by releasing matrix metalloproteinase-9, vascular endothelial growth factor and other factors, promoting tumor neovascularization, and promoting tumor occurrence and development 26 ; (2) neutrophils promote tumor movement and migration by releasing enzymes, and promote tumor invasion and metastasis; (3) the rise of neutrophils can inhibit the immune cell activity of lymphocytes, natural killer cells and activated T cells, thus reducing the immunity of the body; (4) as the main members of tumor immunity, lymphocytes are involved in cell destruction and apoptosis.Lymphocytes such as CD4 + and CD8 + T cells can induce tumor cell apoptosis and inhibit tumor progression through immune-mediated cytotoxic activity 27 .In this study, pre-NLR was related to T stage, clinical stage, ECOG, pathological type, pre-HGB, pre-CRP, pre-LMR and pre-ALB of patients with NPC, which was similar to the conclusions of previous studies 28,29 .In addition, in this study, 5-year OS, LRRFS and DMFS of NPC patients with pre-NLR ≥ 3.29 were significantly lower than those with pre-PLR < 3.29, indicating that pre-NLR is a risk factor for survival and prognosis of NPC, high pre-PLR indicates poor prognosis.
Tumor growth requires abundant blood supply, platelets can promote angiogenesis and release growth factors, tumor cells mediate platelet aggregation, leading to the body in a hypercoagulable state.Platelet aggregation around tumor cells can protect them from NK cell killing, regulate the process of tumor micrometastasis by activating TGF-β signal transduction pathway, and promote tumor cell exosmosis 30,31 .Studies have shown that PLR can reflect not only the tumor-promoting state and inflammatory response in the body, but also the antitumor immune state.SUN et al. 32 found that PLR is a prognostic factor affecting PFS and OS in non-metastatic NPC, while some studies [33][34][35][36] believed that PLR is not significantly correlated with survival and prognosis of NPC, which may be attributed to tumor heterogeneity.In this study, pre-PLR was related to the gender, T stage, clinical stage, ECOG, pathological type, pre-HGB, pre-CRP, pre-LMR and pre-ALB of patients with NPC, which was consistent with the conclusion of JIANG et al. 31 who studied 247 patients with NPC who received IMRT.In addition, 5-year OS, LRRFS and DMFS of NPC patients with pre-PLR ≥ 196.74 were significantly lower than those with pre-PLR < 196.74, indicating that pre-PLR is a risk factor for survival and prognosis of NPC, high pre-PLR indicates poor prognosis.
Most previous studies evaluated NLR, PLR individually as well as their clinical significance in patients with various malignant tumors, including NPC.However, it is one-sided to rely solely on TNM staging and NLR or PLR to assess the prognosis.In the past, some studies have combined NLR and PLR to predict the prognosis of cancer, such as peritoneal metastasis cancer 37 , breast cancer 38 , gastric cancer 21 , etc., but no relevant study has carried out the combination of NLR and PLR to predict the prognosis of NPC.Therefore, this study combined these two hematological indicators to establish the pre-NLR-PLR scoring system innovatively.When comparing the baseline characteristics of patients, significant differences were found in T stage, N stage, clinical stage, ECOG, pathological type, pre-HGB, pre-CRP, pre-LMR and pre-ALB among different groups of patients.Survival analysis showed that 5-year OS, LRRFS, and DMFS in HRG (score of 2) patients were significantly worse than those in MRG (score of 1) and LRG (score of 0).Multivariate analysis showed that pre-NLR-PLR scoring system was an independent prognostic factor for NPC patients.Patients with pre-NLR-PLR score of 2 (HRG) were 1.905 times more likely to die within 5 years than those with score of 0-1 (LRG + MRG) (p < 0.001), the risk of local recurrence was 1.864 times more likely than those with LRG + MRG (p = 0.019), and the risk of distant metastasis www.nature.com/scientificreports/ was 1.631 times than LRG + MRG (p = 0.014).In ROC curve analysis, the AUC values of pre-NLR-PLR scoring system on OS, LRRFS and DMFS in 5 years were higher than those of pre-NLR and pre-PLR, which also indicated that pre-NLR-PLR scoring system had certain advantages in predicting the prognosis of patients with NPC.These results may suggest that the pre-NLR-PLR scoring system can discriminate patients with better prognosis after treatment from all patients, compared with pre-NLR or pre-PLR alone, and the pre-NLR-PLR scoring system is a potentially useful prognostic predictor that can be assessed before treatment.Those would be the greatest advantage of the pre-NLR-PLR score.In addition, the pre-NLR-PLR score can be easily determined by calculating the NLR and PLR with a small volume of blood (only 2 mL).Thus, assessment of the pre-NLR-PLR score is inexpensive.
To be honest, our study also has some limitations.Plasma EBV DNA has emerged as an important prognostic factor in contemporary studies, as a relatively new technique, the 5-year survival results of EBV DNA are not yet available at our center, so they were not included in our study.This study is a retrospective study, and all eligible patients have different treatment methods according to the choice of the doctors in charge, including chemotherapy regimen, chemotherapy cycle and drug dosage, which may affect the results of the study.some sample sizes are small, such as NKDC and KSCC, which may also affect the results of the study.In addition, without validation with other datasets, the NLR-PLR (cutoff scores of 3.29 and 196.74) is not sufficient for clinical use as a prognostic predictor of NPC.In the future, it is necessary to expand the sample size, collect the 5-year survival results of EBV DNA, further improve the classification of various clinical factors, control other factors that may affect the results, find and include more factors that may affect the outcome and prognosis, verify the usefulness of the NLR-PLR score by using a validation cohort, and explore a more perfect scoring system for better application in clinical diagnosis and treatment.

Conclusions
In summary, we established the pre-NLR-PLR scoring system innovatively, which is an independent risk factor for the prognosis of NPC.NPC patients with high pre-PLR and high pre-NLR have poor prognosis.The pre-NLR-PLR scoring system can be used as an individualized clinical assessment tool to predict the prognosis of patients with non-metastatic NPC more accurately and easily.

Patients
We retrospectively analyzed 765 patients with non-distant metastatic NPC treated with IMRT in two hospitals from December 2014 to December 2017 (the First Affiliated Hospital of Guangxi Medical University, 748 patients; the First Affiliated Hospital of University of South China, 17 patients).All patients met the following inclusion criteria: (1) confirmed by histopathology as NPC; (2) there was no distant metastasis before and during treatment; (3) had not received any antitumor therapy before; (4) denied the history related to other malignancies; (5) Eastern Cooperative Oncology Group(ECOG) score 0 ~ 1; (6) received radiotherapy or concurrent chemoradiotherapy with/without induction or adjuvant chemotherapy, and completed the entire treatment as planned; (7) complete clinical data, examination data and follow-up data were available.Our exclusion criteria included: (1) distant metastasis was found or could not be ruled out before and during treatment; (2) complicated with severe infection, underlying diseases; (3) previous or concurrent history of other malignant tumors; (4) unable to complete the treatment.
The data of all NPC patients' serum biomarkers and clinical characteristics were measured and collected within the two weeks before initiating treatment, including age, gender, pathological type, treatment regimen, smoking history, drinking history, ECOG score, radiotherapy technique and dose, chemotherapy regimen and dose, pretreatment (pre-) blood cell count (lymphocytes, neutrophils, monocytes, platelets, and white blood cells), pre-hemoglobin (pre-HGB), and pre-albumin (pre-ALB).We restaged all patients by the eighth edition of the AJCC/UICC TNM staging system.NLR was determined by dividing the absolute neutrophil count by the absolute lymphocyte count.PLR was determined by the absolute platelet count divided by the absolute lymphocyte count.The Ethics Committee at the First Affiliated Hospital of Guangxi Medical University approved the study, which analyzed anonymous information as well as waived the demand for informed consent (Approval Number: 2023-E207-01).The study was conducted in accordance with relevant guidelines and legislation.

Therapeutic schedule
In this study, TNM staging was performed according to the guidelines of the National Comprehensive Cancer Network (NCCN), and the standardized treatment plan was determined according to the TNM stage of the patient.Patients with stage I received radical radiotherapy.Patients in stage II were treated with radiotherapy or concurrent chemoradiotherapy combined with platinum.Patients with stage III-IVa were treated with concurrent induction chemotherapy or adjuvant chemotherapy.The patients were all treated with IMRT.The radiotherapy target areas of NPC include gross tumor volume of nasopharynx (GTVnx), metastatic cervical lymph node volume (GTVnd), Two clinical target volumes (CTV1 is high risk clinical target volume, CTV2 is low risk clinical target volume), GTV or CTV expansion of 3 to 5mm is the planned target volume (PTV).According to the nasopharyngeal primary focus, nasopharyngeal subclinical focus, cervical lymph node and cervical lymph drainage area, different prescription doses were given respectively.Prescription dosage of nasopharyngeal primary focus, PTV-GTVnx (68 ~ 76 Gy), PTV-CTV1 (60 ~ 64 Gy), PTV-CTV2 (50 ~ 54 Gy), 5 fractions/week for a total of 30-33 fractions.Prescription dosage of cervical lymph node, PTV-GTVnd (66 ~ 70 Gy), PTV-CTV2 (50 ~ 54 Gy), 5 fractions/week for a total of 30-33 fractions.For tissue and organ limit dose, refer to QUANTEC (2012 standard).Induction or adjuvant chemotherapy mainly included GP regimen (gemcitabine 1000 mg/ m 2 on days 1 and 8 + cisplatin 80 mg/m 2 on day 1), PF regimen (cisplatin 80 mg/m 2 on day 1 + 5-fluorouracil https://doi.org/10.1038/s41598-024-59131-2

Figure 1 .
Figure1.Kaplan-Meier curves of the different ratio of pre-NLR and pre-PLR.The 5-year overall survival rates of pre-NLR (A), locoregional recurrence-free survival rates of pre-NLR (B), distant metastasis-free survival rates of pre-NLR (C), 5-year overall survival rates of pre-PLR (D), locoregional recurrence-free survival rates of pre-PLR (E) and distant metastasis-free survival rates of pre-PLR (F).p values were calculated with the log-rank test.

Figure 2 .Figure 3 .
Figure 2.Kaplan-Meier curves of the different ratio of pre-LMR and pre-CRP.The 5-year overall survival rates of pre-LMR (A), locoregional recurrence-free survival rates of pre-LMR (B), distant metastasis-free survival rates of pre-LMR (C), 5-year overall survival rates of pre-CRP (D), locoregional recurrence-free survival rates of pre-CRP (E) and distant metastasis-free survival rates of pre-CRP (F).p values were calculated with the log-rank test.
In order to analyze the prognostic value of pre-NLR combined with pre-PLR, the pre-NLR-PLR scoring system was established in this study.Patients were divided into low-risk group (LRG), medium-risk group (MRG) and high-risk group (HRG), pre-NLR-PLR scoring criteria and grouping were as follows: HRG (n = 224, 29.3%), score of 2, high pre-NLR (≥ 3.29) and high pre-PLR (≥ 196.74).MRG (n = 152, 19.9%), score of 1, high pre-NLR and low pre-PLR or low pre-NLR and high pre-PLR.LRG (n = 389,50.8%),score of 0, neither high pre-NLR nor high pre-PLR.The correlation between different risk groups and the general clinical characteristics of patients was shown in Table

Table 2 .
Correlation between general clinical features and pre-NLR-PLR groups.HRG high risk group, MRG medium risk group, LRG low risk group.*Indicates a significant difference among groups with p < 0.05.

Table 3 .
Multivariable analysis of prognostic factors in NPC patients.

Table 4 .
Comparison of ROC curves of pre-NLR-PLR, pre-NLR and pre-PLR.*Indicates a significant difference among groups with p < 0.05.a Comparison between pre-NLR-PLR and pre-NLR.b Comparison between pre-NLR-PLR and pre-PLR.