The Blood Neutrophil-to-lymphocyte Ratio Predicts Survival in Patients with Advanced Hepatocellular Carcinoma Receiving Sorafenib

Hepatocellular carcinoma (HCC) is one of the leading malignancies worldwide. Despite the increaseing use of surveillance programs, a large proportion of patients with HCC are diagnosed at an advanced stage (Forner et al., 2012). Moreover, a number of patients with earlyto intermediate-stage HCC eventually progress to the advanced stage of the disease. Only palliative treatment options are available for these patients (Forner et al., 2012). Sorafenib is a multikinase inhibitor with effects on tumor proliferation and angiogenesis and was shown to prolong survival of patients with advanced HCC in two placebo-controlled randomized trials (Llovet et al., 2008; Cheng et al., 2009). Many investigators have tried to identify baseline or on-treatment predictive factors for sorafenib because sorafenib does not show the same effect in all HCC patients and the response rate of sorafenib in HCC was modest (Llovet et al., 2008). In an Italian study, younger age, extrahepatic tumor spread and ECOG performance status were associated with


Introduction
Hepatocellular carcinoma (HCC) is one of the leading malignancies worldwide.Despite the increaseing use of surveillance programs, a large proportion of patients with HCC are diagnosed at an advanced stage (Forner et al., 2012).Moreover, a number of patients with earlyto intermediate-stage HCC eventually progress to the advanced stage of the disease.Only palliative treatment options are available for these patients (Forner et al., 2012).
Sorafenib is a multikinase inhibitor with effects on tumor proliferation and angiogenesis and was shown to prolong survival of patients with advanced HCC in two placebo-controlled randomized trials (Llovet et al., 2008;Cheng et al., 2009).Many investigators have tried to identify baseline or on-treatment predictive factors for sorafenib because sorafenib does not show the same effect

RESEARCH ARTICLE
The Blood Neutrophil-to-lymphocyte Ratio Predicts Survival in Patients with Advanced Hepatocellular Carcinoma Receiving Sorafenib You-Bing Zheng 1,2 , Wei Zhao 1,2 , Bing Liu 1,2 , Li-Gong Lu 1 *, Xu He 1 , Jian-Wen Huang 1 , Yong Li 1 , Bao-Shan Hu 1   an increased likelihood of non-response after sorafenib (Iavarone et al., 2011).In addition, the side effects caused by sorafenib could also be used as clinical predictive parameters in patients with HCC.Early hand-foot reaction or diarrhea during sorafenib therapy was associated with prolonged TTP or OS than did those without skin toxicity (Vincenzi et al., 2010;Cho et al., 2013).
In the last decade, research on the intersections between inflammation and cancer pathogenesis has blossomed, and the inflammation can be considered as an enabling characteristic for its contributions to the acquisition of core hallmark capabilities of cancer (Hanahan et al., 2011).Recent attention has focused on the systemic inflammatory state as a surrogate for tumor biology in multiple solid tumors.In particular, the ratio of absolute neutrophil count to absolute lymphocyte count (neutrophil-lymphocyte ratio; NLR) has been evaluated as a predictor of recurrence and survival in various malignancies.The NLR is particularly useful, as it can be easily measured by routine complete blood work.Published data also suggest that an elevated NLR may correlate with a worse prognosis in patients with HCC who underwent transcatheter arterial chemoembolization (TACE) radiofrequency ablation (RFA) resection or orthotopic liver transplantation (OLT) (Gomez et al., 2008;Chen et al., 2012;Limaye et al., 2012;McNally et al., 2013).To date, there have been no reports regarding NLR in HCC patients treated with sorafenib.The aim of this study was to investigate the prognostic significance of NLR at the time of diagnosis of patients with advanced HCC who received sorafenib monotherapy.

Patients
A database of 65 patients with advanced HCC admitted to our Hospital between January 2011 and December 2012 was evaluated retrospectively.Written informed consent was obtained from patients before treatment and the study was approved by the Ethical Committee of our hospital.All patients satisfied the diagnostic criteria for HCC based on radiologic or histologic grounds according to the American Association for the Study of the Liver guidelines (Bruix et al., 2011) and were not suitable candidates for locoregional treatment (resection, OLT, TACE and RFA).All patients received contrast enhanced computed tomography (CT) or magnetic resonance (MR) imaging at baseline as well as blood samples including complete blood routine examination, liver function, and AFP.
NLR was defined as the absolute neutrophil count divided by the absolute lymphocyte count.Serum complete blood count levels were used to calculate NLR at baseline.NLR measurements were obtained without demonstrable infection.Patients were divided into a high NLR group (>4) and a normal NLR group (≤4) according to the level of NLR before treatment (Motomura et al., 2013).

Treatments and methods
The initial sorafenib dose was 400 mg orally twice daily.Dose reductions of sorafenib and drug interruptions were allowed for toxicities, according to the National Cancer Institute's Common Terminology Criteria for Adverse Events (CTCAE) version 4.0.Treatment was continued until unacceptable toxicity, radiological or clinical progression, death or patient refusal.Patients were seen every 4 weeks for toxicity management and clinical assessment.CT or MR imaging was performed at baseline and every 2-4 months.Tumor response was assessed according to response evaluation criteria in solid tumors (RECIST).as complete response (CR) partial response (PR) stable disease (SD) and progressive disease (PD) (Therasse et al., 2000).

Statistical Analyses
Data were evaluated using Statistical Package for Social Sciences, v.13.0.Continuous variables were expressed as mean ± standard deviation, and compared using t test.Categorical variables were expressed as frequency and compared using Chi-square test.The Kaplan-Meier method was used for survival analysis.A multivariate analysis was performed by Cox regression for significant variables on univariate analysis.All p-values were based on two-sided testing and a p-value <0.05 was considered as significant.

Patient Characteristics
A total of sixty-five patients were consecutively treated at Guangdong General Hospital, China, between January 2011 and December 2012.78.5% were male and mean age was 55±12 years.All of the patients were BCLC stage C. 75.4 percent of the patients were in PS 0-1, and 69.2% had a well preserved liver function (CP-A).A large proportion of patients had highly advanced disease with vascular invasion (61.5%) and extrahepatic metastases (52.3%).Detailed patient characteristics are shown in Table 1.

Tolerability and Safety Aanlysis
The median duration of sorafenib treatment was 123 days (range: 48-424 days).The median maximum administered dose of sorafenib was 800 mg/d (range: 400-800 mg/d).There were no treatment-related deaths.Dose reduction was applied in 28 patients (43.1%) because of side effects related to sorafenib.Dose decreased to 400 mg/d and 400 mg every other day in 21 patients (32.3%) and 7 patients (10.8%) respectively.Sorafenib was discontinued permanently because of tumor progression in 36 patients (55.4%) and side effects in 11 (16.9%).13 (21%) patients accepted drug withdrawal after dose reduction.Table 2 showed the details of the side effects secondary to sorafenib observed based on CTCAE 4.0.

NLR≤4 and NLR>4
The most common adverse effects related to sorafenib were diarrhea (46.2%) rash (27.7%) and hand-foot skin reaction (20%) most of which were mild to moderate.
Response evaluations 44 patients (67.7%) completed at least 3 months of sorafenib therapy and were evaluable for assessment of tumor response.There were no complete responders.4 patients (6.2%).had a partial response, 15 (23.1%) had stable disease, and 25 (38.5%).had progressive disease.The objective response rate (complete and partial responses) was 6.2%.

Discussion
Currently, sorafenib is the first and only systemic agent to be approved for the treatment of HCC and to have demonstrated an overall survival (OS) benefit in advanced HCC.It can target both cell surface tyrosine kinase receptors and downstream intracellular serine/ threonine kinases in the Ras/MAPK cascade (Wilhelm et al., 2002;et al., 2006).In this retrospective study, we show sorafenib therapy is safe and effective with no unexpected side effects in advanced HCC.In the literature, TTP and OS in sorafenib treated advanced patients were reported as 2.8-7 months and 5.4-12 months, respectively.(Abou-Alfa et al., 2006;et al., 2008;Cheng et al., 2009;Hsu et al., 2010;Prete et al., 2010).The survival rate in this study is considerably similar to those in We also found that an elevated NLR, a biomarker comparing tumor inflammation and host immunity, could predict outcome of advanced HCC patients initially treated with sorafenib.The median OS of patients with NLR >4 was much shorter when compared with those with a normal NLR (≤4) 6.5 and 12.5 months, respectively.This is consistent with the above studies which associated high NLR with poor outcome in HCC.(Gomez et al., 2008;et al., 2012;Limaye et al., 2012;McNally et al., 2013).
Despite significant progress with the advent of sorafenib as a treatment option for advanced HCC, this disease is still a great clinical challenge.Given the heterogeneity of the disease and its causes, there is still much to learn about to whom and when these therapies should be applied.Many prognostic and predictive factors have been developed in HCC patients receiving cytokine and targeted therapy, such as clinical staging systems, the presence of adverse effects, molecular profiling, genetic and epigenetic information (methylation and miRNA profiling) (Minguez et al., 2011).However, the detection of most biomarkers are expensive and not available in clinical practice.While NLR is a cost effective indicator, which can be determined by routine complete blood count (CBC).analyzers.
Recently, a number of studies indicated the prognostic value of inflammatory markers in many cancers.Systemic inflammatory responses have been shown to reflect the promotion of angiogenesis, DNA damage and tumor invasion through upregulation of cytokines (Jaiswal et al., 2000;Coussens et al., 2002).Though measured easily, NLR is more complicated due to its special feature as a combined factor of inflammation and host immune reaction.
An elevated NLR can promote the progression of HCC not only via angiogenesis, but also via some sort of inflammatory microenvironment (Motomura et al., 2013).Motomura et al reported a correlation between elevated NLR and upregulation of IL-17 production in both peritumoral regions of the liver and peripheral blood.IL-17 is a proinflammatory cytokine that promotes the growth of HCC.IL-17 is an initiator of neutrophil recruitment by CXC chemokines, such as CCL2 released from IL-17-producing T cells.
On the other hand, high NLR reflects relatively depleted lymphocytes, exhibiting a poor lymphocyte-mediated immune response to malignancy because of decreasing T4/T8 ratio (Chew et al., 2010;Ding et al., 2010).Weaker immune reaction due to relative lymphocytopenia and elevated NLR could explain for depletion of tumorinfiltrating lymphocytes that are independently predictive of cancer specific survival (Ohtani, 2007).Lymphocytes have pivotal roles in cytotoxic cell death and cytokines production that inhibit proliferation and metastatic activity of tumor cells (Ding et al., 2010b).Consequently, patients with weaker lymphocytic infiltration within tumor would have a worse prognosis (Chew et al., 2010b).
There are some limitations of this study.The relative small size and the nature of the retrospective design are main limitations.Besides, there is no universal consensus regarding the threshold that defines high NLR, and this impeded the direct comparison between different studies.
In summary, a high NLR may be used as an available and cheap biomarker for predicting poor survival in advanced HCC patients who received sorafenib.However, concrete molecular mechanisms are still unknown and logical further studies are needed.

Table 2 . Adverse Effects During Sorafenib Therapy
ALT, alanine transaminase; AST, aspartate transaminase; Adverse effects were analysed according to the National Cancer Institute's Common Terminology Criteria for Adverse Events version 4.0