Elevated preoperative platelet distribution width predicts poor prognosis in Esophageal Squamous Cell Carcinoma

Activated platelets play a multifaceted role in tumorigenesis and progression. Platelet distribution width (PDW) is generally applied platelet parameters from routine blood test. Preoperative PDW has been considered a prognostic factor in many cancers. Nevertheless, the prognostic value of PDW in esophageal squamous cell carcinoma (ESCC) remains unknown. The study aimed to investigate whether preoperative PDW could serve as a prognostic factor in patients with ESCC. A total of 495 patients with ESCC undergoing curative surgery were enrolled. The relationship between PDW and clinical features in ESCC was analyzed using chi-square tests. Receiver operating characteristic (ROC) curve was used to determine the optimal cut-off value. Overall survival (OS) and disease-free survival (DFS) stratified by PDW were evaluated by Kaplan–Meier method and log-rank test. Univariate and multivariate Cox regression were used to evaluate the prognostic effect of PDW. Of the 495 patients, elevated PDW was observed in 241(48.7%) of the patients, respectively. An elevated PDW was correlated with depth of tumor (T stage, P = 0.031), nerve infiltration (P = 0.016), hospital time after operation (P = 0.020), platelet (P < 0.001), red cell distribution width (P < 0.001), and aspartate transaminase (P = 0.001). Moreover, elevated PDW (PDW ≥ 13.4 fL) predicted a worse OS and DFS in patients with ESCC (both P < 0.001). Multivariate analyses revealed that PDW was independently associated with OS (hazard ratios 1.194; 95% confidence interval 1.120–1.273; P < 0.001) and DFS (hazard ratios 2.562; 95% confidence interval 1.733–3.786; P < 0.001). Our findings indicated that elevated PDW could serve as an independent worse survival in ESCC.


Results
patient characteristics. After screening, 495 patients (428 male and 67 female) with complete follow-up data were enrolled in the final study. The median age at diagnosis was 62 years (Interquartile range: 55-67 years). 38 (7.8%) with well differentiated pathology grade, 326 (67.1%) with middle differentiated pathology grade, 121 (24.9%) with poorly differentiated pathology grade, and 1 (0.02%) with undifferentiated pathology grade. In High pDW is a predictor of adverse pathological features. The areas under the ROC curves (AUCs) were 0.716 and 0.615 for OS and DFS, respectively (Fig. 1). The larger AUC of 0.716 acquired for OS was chose to be the optimal cut-off value of 13.4, with maximum specificity (81.0%) and sensitivity (59.49%) (Fig. 1A).
High pDW is related to poor oS and DfS. The Kaplan-Meier curves exhibited that patients with high PDW had a worse OS (P < 0.001, Fig. 3A) compared with low PDW group. In subgroup analysis according to lymph node metastasis and pathological stage, high PDW was related to worse OS for patients with or without lymph node metastasis (both P < 0.001) and less or more advanced stage (both P < 0.001) (Figs 4 and 5). In addition, univariate analysis shown that high PDW was correlated with worse OS (HR = 5.111, P < 0.001) ( Table 4). Using multivariate analysis, high PDW (HR = 1.194, P < 0.001), lymph node metastasis (P < 0.05), nerve infiltration (P = 0.004), and hospital time (P = 0.009) were notable related to worse OS (Table 4). By Kaplan-Meier analysis, the DFS was poor in the high PDW group (P < 0.001, Fig. 3B). Similarly, based on subgroup analysis, with lymph node metastasis (P < 0.001) and advanced stage (P < 0.001) could serve as predictors for short DFS in patients with ESCC, which was not observed in patients without lymph node metastasis (P = 0.291) and less advanced stage (P = 0.219) (Figs 4 and 5). In the univariate analysis, high PDW was a significant predictor of unfavorable DFS (HR = 2.302, P < 0.001) ( Table 5). After adjustment for confounders, high PDW (HR = 2.562, P < 0.001), lymph node metastasis (P < 0.05), and surgery (P = 0.047) were correlated with decreased DFS (Table 5). In a word, PDW was an independent prognostic factor for patients with ESCC undergoing surgery.

Discussion
Numerous researches showed that platelet activation play an important part in cancer progression. Thrombocytosis is related to worse clinical outcome in patients with various cancers, including ovarian cancer, colorectal cancer, and pancreatic cancer [20][21][22] . The PDW that is one of the platelet indices not merely check platelet volume heterogeneity, but also reactive platelet activity. Recently, several studies revealed that a high PDW is an unfavorable prognosis factor in melanoma patients, laryngeal cancer, and gastric cancer [23][24][25] . To the best of our knowledge, the prognostic value of the preoperative PDW in ESCC patients remains unknown.
This was the first retrospective research revealed that a PDW with a cut-off 13.4 fL was an independent prognostic factor for the OS and DFS in ESCC patients. Our findings reported that an elevated PDW was correlated     www.nature.com/scientificreports www.nature.com/scientificreports/ with depth of tumor, nerve infiltration, and hospital time after operation. Moreover, high PDW was an independent predictor for ESCC patients with lymph node metastasis according to further subgroup analyses.
Nevertheless, the potential mechanism by which PDW have an effect on cancer progression is unclear. One possible cause is that platelets facilitate the hypercoagulability in tumor. Activated platelets produce a procoagulant micro-environment and aggregate with tumor cell. Platelet-derived growth factor (PDGF) family members including PDGF-A, PDGF-B, PDGF-C and PDGF-D, play a vital role in cancer cell proliferation, apoptosis, transformation, invasion, metastasis and angiogenesis [26][27][28][29][30][31] . In esophageal cancer, PDGF-D expression is associated with clinical-pathological features and worse survival. Moreover, platelet-derived growth factor-D contributes to proliferation and invasion of esophageal squamous cell carcinoma by up-regulating NF-κB signaling pathways 32 . Consistent with previous studies, our findings indirectly suggested anti-platelet could serve as one part of cancer adjuvant therapy 33 .
Another possible mechanism is that bone marrow cells malfunction may be associated with the lower PDW. PDW reflects platelet heterogeneity, which is caused by heterogeneous demarcation of megakaryocytes 34 . Cytokines, including interleukin-6 (IL-6), macrophage colony stimulating factor (M-CSF), and granulocytes colony stimulating factor (G-CSF), have an effect on megakaryocytic maturation, platelet production, and platelet size 35 . IL-6 facilitates cancer cell proliferation, invasion, and metastasis. IL-6 is correlated with the prognosis and depression of cancer patients and is considered to the therapy target [36][37][38] . Moreover, G-CSF stimulates megakaryopoiesis and constrains tumor to proliferation. M-CSF was an important factor in the cancer microenvironment, involving in the interactions between tumor-infiltrated macrophages and tumor cells [39][40][41] . Those reports are in accord with the point that activated platelets participate in the pathogenesis of esophageal cancer.
There were several limitations of our study: first, this was the single-center design and retrospective study, which might have selection bias. Second, the biological mechanism of PDW affecting prognosis need to explored. Third, a controversial cut-off value determined by different ways, such as mean, ROC curve, and C index, could be the optimal predictor of clinical outcome in ESCC patients. In this study, we chose ROC curve to determine the cut-off value. Future studies with multi-center design and prospective trials are necessary to validate the prognostic value of PDW in ESCC patients.
An elevated preoperative PDW indicates a worse OS and DFS of patients with newly diagnosed ESCC undergoing surgery. Our finding may contribute to assess the prognosis of ESCC.

Methods patient recruitment and data collection. This retrospective study was approved by the Ethics
Committee of Zhejiang Cancer Hospital, and included 590 ESCC patients who were newly diagnosed between 2008 and 2013. 95 patients who met the following standard were excluded from the study: neoadjuvant chemotherapy or radiotherapy before surgery; loss to follow-up; data missing; concomitant disease that could interfere www.nature.com/scientificreports www.nature.com/scientificreports/ with platelet, including autoimmune disease, splenic disease, severe hypertension, and a history of blood transfusion; other factors that could affect the PDW, including megaloblastic anemia, acute myeloid leukemia, splenectomy, giant platelet syndrome, and thrombotic disease. The enrolled 495 patients completed written informed consent.
The pretreatment peripheral blood cell count was checked via a SYSMEX XE-2100 (Sysmex, Kobe, Japan) Automatic Blood Cell Analyzer. The PDW measurement is the first time of admission.
follow-up strategy. After surgery, patients were followed up every three months for the first year, six months during the second year and 12 months thereafter. Physical examination, blood routine examination, and medical history were achieved conventionally. Bone scans, chest/abdominal CT/MRI, and chest radiography were acquired when in cases of suspicious metastasis or recurrence. Statistical analysis. The PDW was analyzed as continuous variables and the clinical-pathological features were counted as categorical variables. The optimal cut-off value of PDW for predicting survival was determined by the ROC curve analysis. The relationship between PDW and clinical-pathological features in ESCC was analyzed by chi-square tests. The Kaplan-Meier method and the log-rank test were used for the overall survival (OS) and disease-free survival (DFS) analyses. The association between PDW and clinical-pathological features were investigated by logistic regression analysis. Clinical-pathological features with P < 0.01 were selected to be the subgroup factor. Subgroup analysis was based on lymph node metastasis and pathological stage. Whether the OS and DFS was an independent prognosis factor was determined by Cox proportional hazards regression models. Risk factors with P < 0.01 in univariate analysis were chosen to multivariate analyses. The SPSS software version 19.0 (IBM SPSS, Chicago, IL, USA) was utilized for statistical analysis. ethics approval and consent to participate. All Table 5. Disease-free survival analyses according to preoperative PDW in 495 patients with ESCC. Abbreviations: S, surgery; C, chemotherapy; CRT, chemoradiotherapy; PDW, platelet distribution width; RDW, red cell distribution width.