Prognostic significance of platelet-to-albumin ratio in patients with esophageal squamous cell carcinoma receiving definitive radiotherapy

Accumulating evidence indicates that inflammation and nutrition status are associated with clinical outcomes in patients with various malignancies. This study aimed to evaluate the prognostic significance of the pretreatment platelet to albumin ratio (PAR) in esophageal squamous cell carcinoma (ESCC) patients undergoing definitive radiotherapy. A total of 470 patients who underwent definitive radiotherapy with or without chemotherapy were enrolled. The optimal cut-off values of PAR and other indicators were determined by the X-tile. The Kaplan–Meier method, multivariate analyses Cox regression were conducted to identify the association between those indicators and the survival outcomes. The median follow-up time was 23.5 months. The optimal cut-off value of PAR was 5.7 × 109 and patients were stratified as the low PAR group and the high PAR group. In the univariate analysis, a low overall survival rate was significantly associated with T stage (P = 0.005), TNM stage (P < 0.001), Adjuvant chemotherapy (P = 0.007), neutrophil to lymphocyte ratio (NLR) (P = 0.006), platelet to lymphocyte ratio (P < 0.001), systemic immune-inflammation index (P < 0.001), prognostic nutritional index (P < 0.001) and platelet to albumin ratio (PAR) (P < 0.001). Patients with high PAR were associated with poorer OS and PFS than patients with low PAR. On multivariate analysis, TNM stage (P = 0.001), adjuvant chemotherapy (P < 0.001), and PAR (P = 0.033) were independent prognostic factors in ESCC treated with definitive radiotherapy. PAR is a novel, convenient, and inexpensive prognostic indicator for patients with ESCC undergoing definitive radiotherapy. Future validation from prospective larger-scale studies is warranted.

Treatment protocol. Radiotherapy was delivered by two-dimensional conventional radiotherapy (2D-CRT), three-dimensional conformal radiation therapy (3D-CRT), or intensity-modulated radiation therapy (IMRT) technique in this study. The gross tumor volume (GTV) was determined by the contrast-enhanced CT, barium swallow, endoscopic examination, or PET-CT, which contained both the primary tumor and the positive regional lymph nodes. The clinical target volume (CTV) is composed of subclinical lesions (GTV extending 0.5-1.0 cm in axial direction and 3 cm in longitudinal direction) and the relative mediastinal lymphatic drainage field. The CTV plus a 0.5 cm expansion margin in all directions was defined as the planning target volume (PTV). Patients who received two-dimensional conventional radiotherapy (2DRT) used anterior and posterior opposing techniques. The irradiated field included the primary tumor and a distal and proximal margin of 3 cm and a 0.5-1.0 cm radial margin around the tumor.
Definition of the NLR, PLR, SII, PNI and PAR. The baseline neutrophil, lymphocyte, platelet counts, and serum albumin levels were collected from routine test reports 7 days prior to the first treatment. The definitions of NLR, PLR, SII, PNI and PAR are calculated as follows: NLR = absolute neutrophil count/absolute lymphocyte count; PLR = platelet counts/absolute lymphocyte count; SII = platelet counts* absolute neutrophil count/absolute lymphocyte count; PNI = serum albumin level (g/L) + 5* absolute lymphocyte count; PAR = platelet counts/serum albumin level (g/L). The optimal cutoff values for the NLR, PLR, SII, PNI and PAR were determined by X-tile software (http:// www. tissu earray. org/ rimml ab) 19 .
Follow-up. During treatment, patients were assessed weekly to screen the treatment toxicities, including blood routine, biochemical test and physical examination. After treatment, Follow-up examinations were conducted 1 month after finishing radiotherapy, and then every 3 months in the first year, every 6 months over the next 2 years, and once a year thereafter. The routine examination items included physical examination, laboratory tests, tumor markers, thoracic CT scanning, and esophageal barium. The followed up lasted until death or the last contact. The primary endpoint was overall survival (OS), which was defined as the period from treat-  Figs. 1 and 2) Then patients were divided into the low PAR group (PAR < 5.7 × 10 9 ) and the high PAR group (PAR ≥ 5.7 × 10 9 ) for further analyses.
Furthermore, we explored the prognostic value of PAR in a subgroup analysis which was stratified by AJCC TNM stage. The result showed the high-PAR was significant shorter OS in stages III (P = 0.002), Fig. 4B). Although there was no statistical significance in the subgroup analyses of OS in stages II (P = 0.385, Fig. 4A) and stage IV (P = 0.295, Fig. 4C), the trend of worse prognosis in high-PAR patients was consistent.

Discussion
Inflammation in the microenvironment of tumors is known to promote both carcinogenesis and disease progression and could reduce the efficacy of systemic treatments 21 . Platelet, as a critical component in hemostasis, is also observed to be associated with systemic inflammation and the immune system 22,23 . Nutritional status is also a critical part of cancer management as malnutrition is frequent in cancer patients and is correlated with poor prognosis [24][25][26] . Previous studies have demonstrated albumin-related malnutrition was significantly associated with poor prognosis in ESCC patients 27,28 . Hence, a novel inflammation-based indicator PAR was established, representing both inflammation and nutritional status.
The current study first evaluated the prognostic value of a pretreatment PAR in ESCC patients who received definitive radiotherapy ± adjuvant chemotherapy. Our results showed that patients with a low pretreatment PAR(< 5.7 × 10 9 ) had a significantly better prognosis in both PFS and OS than those with a high pretreatment PAR(≥ 5.7 × 10 9 ). A high PAR was more likely to associate with an younger age, a more advanced TNM stage, suggesting that PAR could reflect tumor progression in patients with ESCC. Multivariable analyses also validated the PAR as an independent indicator of PFS and OS. Taken together, our results demonstrated that pretreatment PAR is an independent prognostic factor for patients with ESCC who received definitive radiotherapy. www.nature.com/scientificreports/ Comparing to PAR, the existing inflammation-based indicators, including NLR, PLR, SII and PNI, showed no statistical significance as an independent prognostic factor in the multivariable cox regression analysis. Previous studies showed that high pretreatment of NLR and PLR were independent prognostic markers for OS in esophageal cancer patients. In the current study, NLR and PLR showed a correlation with OS but failed to maintain significance in multivariable cox analysis. The findings were consistent with Geng et al. research 29 . Likewise, SII also demonstrated no independent prognostic value in this study.
It's been known that inflammation not only participates in tumorigenesis, malignant progression, and metastasis but also interplay with antitumor immunity 30 . A number of studies have revealed that high platelet counts can affect tumor development and result in thrombocytosis, which is an unfavorable factor for patients' clinical Table 1. Baseline clinical variables of the study participants stratified by pretreatment PAR. PAR platelet to albumin ratio, NLR neutrophil to lymphocyte ratio, PLR platelet to lymphocyte ratio, SII systemic immuneinflammation index, PNI prognostic nutritional index, 2DRT two-dimensional conformal radiation therapy, 3DRT three-dimensional conformal radiation therapy, IMRT intensity-modulated radiation therapy.

Variables
Total, n(%) Low PAR (N = 172) High PAR (N = 298) P-value www.nature.com/scientificreports/ outcomes 31 . It has been hypothesized that platelet was activated by cytokines secreted by tumor cells, such as platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1). Those cytokines are components of platelet and play a critical role in tumor development, including tumor proliferation, angiogenesis, and metastasis 32 . Moreover, platelet could shield peripheral circulating tumor cells and interfere with natural killer cells for recognition of tumor cells, which enhanced their metastatic potential 33 . Most recently, more emphasis has been put on the platelets and their complicated interplay with leukocytes and tumor cells in the tumor microenvironment, which could help us to better understand the mechanism behind the commonly used antiplatelet therapy and shed light on novel cancer therapy 34 . Low serum albumin concentration could also lead to a high PAR. Previous studies have demonstrated that pretreatment serum albumin is associated with short life expectancy in cancer patients 35 . Malnutrition is a significant problem in digestive cancer patients, especially in those with locally advanced esophageal cancer undergoing definitive radiotherapy. The tumor-caused restriction of oral intake and radiation-induced esophagitis are the main reasons that account for undernutrition. Patients with an undernutrition status before treatment were demonstrated to be associated with poor treatment response and prognosis 36 . In our study, we found that patients with high serum albumin concentration had a better survival outcome than those with low serum albumin concentration (as shown in Supplementary Fig. 3), which is consistent with previous findings. Moreover, www.nature.com/scientificreports/ albumin synthesis was suppressed by the systemic inflammation. Consequentially the immune system functions were impaired due to hypoalbuminemia, and tumor cells could progress more easily due to immune suppression. Therefore, risk stratification based on inflammation-nutritional indicators is of great significance and will help the clinical physician to provide timely and effective nutritional intervention. Although our study demonstrated the prognostic significance of PAR in patients with ESCC, several limitations in our study still need to be noted. First, this is a retrospective study from a single center, which may lead to selection bias. Second, platelet counts and serum albumin levels could be influenced by other factors such as

Conclusion
Our study demonstrated that the PAR, a novel independent risk predictor, had the potential for predicting prognosis of ESCC patients undergoing definitive radiotherapy. Measurement of the PAR is convenient, inexpensive, and reliable in the routine clinical practice. Anti-inflammation therapy and/or nutritional interventions should be considered for patients with low pretreatment PAR levels. Therefore, PAR measurement will help the clinical decision-making according to the individual difference. Future validation from prospective larger-scale studies is warranted.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.