Investigating effects of preoperative in�ammatory biomarkers on predicting survival outcomes of peripheral intrahepatic cholangiocarcinoma after curative resection.

Introduction: Intrahepatic cholangiocarcinoma (ICC) stands as the second most common malignant tumor in liver with poor patient prognosis. Increasing evidences have shown that in�ammation plays a signi�cant role in tumor progression, angiogenesis and metastasis. However, the prognosis signi�cance of in�ammatory biomarkers on recurrence-free survival (RFS) and overall survival (OS) in peripheral ICC patients is poorly recognized. Methods: Peripheral ICC patients who underwent curative hepatectomy and diagnosed pathologically were retrospectively analyzed. In�ammatory biomarkers, including neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), lymphocyte-to-monocyte ratio (LMR), systemic immune-inammation index (SII), were investigated. Results: Receiver operating characteristic (ROC) curves showed no signi�cance in NLR, PLR and LMR in RFS and OS, while signi�cant results were shown on SII in both RFS (P = 0.035) and OS (P = 0.034) with areas under ROC curve as 0.63 (95%CI: 0.52–0.74) and 0.62 (95%CI: 0.51–0.72), respectively. Kaplan-Meier curves revealed statistically signi�cant better survival data in SII-low groups on both RFS (P(cid:0)0.001) and OS (P(cid:0)0.001). The univariate and multivariate analyses revealed that higher level of SII was independently associated with both poorer RFS time and OS time. However, no signi�cant result was shown on NLR, PLR or LMR. Conclusion: SII is an effective prognostic factor for predicting the prognosis of peripheral ICC patient undergone curative hepatectomy, while NLR, PLR


Introduction
Intrahepatic cholangiocarcinoma (ICC) stands as the second common malignant hepatic neoplasms, however the incidence of ICC grows worldwide during past decades. 1,2 p to now, the best choice of curative treatments is surgical resection, while the treatments for unresectable ICC are very limited. 3ICC usually grows aggressively without symptom in early stage, resulting in a small proportion of ICC patients who can receive surgery.Furthermore, the prognosis of resectable ICC patient still remains poor and half of them will suffer from recurrence after surgery. 4ICC can be divided into two types according to the location: peripheral type and hilar type.Different mechanisms of oncogenesis and clinicopathologic characteristics were shown in two types according to multiple researches. 5,6 reasing evidences have shown that in ammation and in ammatory biomarkers are signi cant factors in tumor microenvironment, thus promoting proliferation, angiogenesis and metastasis by various in ammatory cells and cytokines. 7In recent years, multiple in ammatory biomarkers were investigated for predicting the prognosis of patients with various cancer, including neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), lymphocyte-to-monocyte ratio (LMR), systemic immune-in ammation index (SII).10] However, signi cance of these in ammatory biomarkers on prognosis of peripheral ICC patients underwent curative resection has not been fully understood.Therefore, the present study was performed for investigating the signi cance of various in ammatory biomarkers, including NLR, PLR, LMR and SII on patient prognosis in peripheral ICC after curative surgery.

Study cohort
Peripheral ICC patients underwent curative hepatectomy between January 2013 and December 2017 in Xiangya hospital, Central South University were retrospectively analyzed.Exclusion criteria were as followed: (1)
Patients were followed up every 3 month after surgery.Blood tests including liver function and serum alpha-fetoprotein level, and imaging examination were also performed during follow-up.Our primary end points were recurrence-free survival (RFS) and overall survival (OS).RFS was calculated from the rst day after hepatectomy to the recurrence of ICC or ICC-related death, while OS was calculated from the rst day after hepatectomy to the ICC-related death.
Statistical analysis SPSS 23.0 (SPSS Company, Chicago, IL) for Windows and Prism software (GraphPad Prism Software, La Jolla, CA) were used to analyze data and realize visualization.Independent-sample t test or Mann-Whitney U test was used to analyze the quantitative data expressed as mean±standard deviation (SD).
And Chi-square or Fisher exact test was used as appropriate to analyze the categorical data expressed as frequency (percentage).The cutoff values were calculated by receiver operating characteristic (ROC) curves.Kaplan-Meier curves were used to illustrate RFS and OS, while the log-rank test was used to detect the differences between groups.Meanwhile, the cox's proportional hazard regression was used to identify associated factors of RFS and OS.P < 0.05 was considered as statistically signi cant.

Results
Patient and tumor characteristics 128 ICC patients, including 70 males and 58 females, were nally included.The basic patient and tumor characteristics were shown in Table 1.29.7% of patients presented hepatitis B virus (HBV) infection, and 28.1% of patients presented multiple tumors.Proportions of patients with AJCC tumor stage I, II and III were 26.6%, 14.8% and 58.6%, respectively.35.2% of patients had liver cirrhosis, while 13.3% of patients had undermined liver function.The averages of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and albumin were (49.58±57.93)U/L, (42.51±31.27)U/L and (40.33±4.45)g/L.

Cutoff values of in ammatory biomarkers
ROC curves were performed to determine appropriate cutoff values of NLR, PLR, LMR and SII, and the results were shown in Figure 1.However, according to the ROC curves of RFS and OS, the results showed no signi cance in NLR, PLR or LMR, while signi cant results were shown on SII in both RFS (P=0.035) and OS (P=0.034) with areas under ROC curve as 0.63 (95%CI: 0.52-0.74)and 0.62 (95%CI: 0.51-0.72),respectively.Thus, the subsequent analyses were focused on SII with an ideal cutoff value as 1027 according to ROC curves and Youden index.

Survival analyses based on SII
Survival analyses were performed between SII-low group and SII-high group according to cutoff value of SII, and the results were shown in Figure 2. The median RFS times in SII-low group and SII-high group were 16.4 months and 5.7 months, and the median OS times were 25.2 months and 10.9 months, respectively.Statistically signi cant differences between two groups were revealed by Kaplan-Meier curves on both RFS (P 0.001) and OS (P 0.001), indicating potential prognostic value of SII.

Univariate and multivariate analyses
For further investigating risk factors affecting RFS and OS of ICC patients, the univariate and multivariate analyses were subsequently performed among available factors, with the results shown in Table 2.The analyses revealed that multiple tumors, higher AJCC tumor stage, poorer tumor differentiation, higher level of CEA and CA19-9, higher level of SII were independently associated with both poorer RFS time and OS time.However, no signi cant result was shown on NLR, PLR or LMR.

Discussion
It is widely recognized that the systemic in ammation involves in pathogenesis and progression of cancer by various mechanisms including cell proliferation, tissue in ltration and angiogenesis. 11,12 ltiple in ammatory biomarkers could effectively present the extent of in ammatory and immune response with high availability, therefore, were recommend as factors for predicting the prognosis of cancer patients.In the present study, we investigated the prognostic signi cance of in ammatory biomarkers in curative resected ICC patients.Our results suggested that SII could effectively predict the prognosis of peripheral ICC patients after curative hepatectomy, while NLR, PLR and LMR were not related with outcomes of these patients.
Extensive non-speci c in ammatory responses were usually led by allogeneic phenotype of cancer cell, followed by increasing of neutrophils and platelets, and deceasing of lymphocytes. 13Neutrophils could secrete TNF-alpha, VEGF and interleukin, thus to promote tumor cell proliferation and angiogenesis. 14anwhile, TGF-beta, VEGF and platelet derived factors could be secreted by platelets, accelerating differentiation and proliferation of cancer cells, and playing a signi cant role in adhesion and angiogenesis of tumor tissues.On the other hand, lymphocytes could mediate cytotoxicity and release cytokines, thus presenting antitumor effects as inhibiting growth, proliferation and metastasis of tumor cell. 15The decrease of lymphoctyes could lead to lower immune function, progression of tumor, and eventually poor prognosis of patients with tumor.Furthermore, studies showed that activity of lymphocytes could be suppressed by neutrophils. 16In addition, monocytes in tumor tissues can differentiate into tumor-associated macrophages, which place promoting effects on tumor growth, tumor cell in ltration and angiogenesis. 17Thus, the NLR, PLR, LMR and SII would theoretically be valuable biomarkers for predicting prognosis of cancer, considering all of them could be easily obtained from routine preoperative examinations.
In ICC, this study showed SII as the only independent risk factor on RFS and OS of patients.Two previous studies have also investigated the role of SII in OS among ICC patients. 18,19 heir results both indicated higher SII was associated with poorer patient survival in ICC, which was consistent with our results.However, one of them also showed that NLR had a better signi cance as a biomarker on ICC patient.The inconsistent results on NLR might be caused by different cohorts because they did not focus on the patients underwent a curative therapy but the whole ICC cohort.
The present study did contain a few limitations.Firstly, this was a retrospective study with not large sample size.Further prospective, multicenter clinical studies with large cohorts should be performed to validated the values of these in ammatory biomarkers in ICC.Secondly, these in ammatory biomarkers were assessed by single measurements during admission, which might cause uncontrolled bias.Thirdly, some factors which could make an impact on these in ammatory biomarkers, such as smoking and alcoholic, were not fully under control.

Conclusion
In summary, our study shows SII can effectively predict the prognosis of peripheral ICC patient undergone curative hepatectomy, while NLR, PLR and LMR are not related with clinical outcomes of these patients.

Ethics approval and consent to participate
The study was approved by the ethics committee of Xiangya Hospital of Central South University.Patient consent was not required to review their medical records by the ethics committee of Xiangya Hospital of Central South University because of its retrospective design, and exemption from informed consent did not adversely affect the health and rights of subjects.This study kept con dentiality of patient data and strictly complied with the Declaration of Helsinki and its later amendments or comparable ethical standards.Better survival data were showed in SII-low group on both RFS (P<0.001) and OS (P<0.001).SII, systemic immune-in ammation index; RFS, recurrence free survival; OS, overall survival.
pathology did not support the diagnosis of ICC; (2) recurrence of ICC; (3) received an antitumor therapy before resection; (4) suffering from infectious diseases before resection; (5) suffering from autoimmune diseases or immunode ciency diseases; (6) patients who died of postoperative complications or reasons other than ICC; (7) R1 or R2 resection; (8) hilar type of ICC; (9) incomplete clinical data.The ethics committee of Xiangya Hospital of Central South University approved this study.
Abbreviations ICC, Intrahepatic cholangiocarcinoma 19. Tsilimigras DI, Moris D, Mehta R, et al.The systemic immune-in ammation index predicts prognosis in intrahepatic cholangiocarcinoma: an international multi-institutional analysis.HPB: the o cial journal of the International Hepato Pancreato Biliary Association.2020.

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Table 2 .
Univariate and multivariate analyses of risk factors with RFS and OS in ICC patients.