Prognostic Signicance of Lymphocyte to Monocyte Ratio in Gallbladder Carcinoma

Background: Increasing evidence indicates cancer-associated inammatory biomarkers show great promise for predicting prognosis of cancer patients. The objective of this study aims to evaluate the prognostic signicance of the lymphocyte-to-monocyte ratio in patients with gallbladder carcinoma. Methods: Receiver operating characteristic curves was used to determine cut-off values for the LMR at detecting death. The primary outcome was overall survival, which was estimated by the Kaplan-Meier method. Univariate survival analysis was performed using a log rank test. Multivariate analysis using the Cox regression proportional hazard model was performed to identify the factors associated with the prognosis.A retrospective cohort of 80 GBC followed by operation was recruited between March 2008 and August 2014 at the Qingdao Municipal Hospital. Counts for absolute lymphocytes and monocytes were obtained and used to calculate the LMR. Results: For the LMR, the area under the ROC curve was 0.675 (95%CI: :0.530-0.820).The cut-off value for the LMR was determined to be 4.62. Patients in the high-LMR group experienced signicant improvements in median survival time compared with patients in the low-LMR group(P = 0.03).The univariate analysis demonstrated that LMR, differentiation degree, TNM stage,CA199, CEA, Resection margin and operative methods were associated with overall survival (P < 0.05). The multivariate analysis identied that Differentiation grade , TNM stage, and CRP as independent prognostic factors in the patients with GBC(P <0.05). Conclusion: Our study demonstrated that LMR is closely correlated with GBC prognosis and could be useful for the evaluation of prognosis of patients with GBC.


Background
Gallbladder carcinoma (GBC) is a common malignant tumor of the biliary tract .The Surveillance, Epidemiology, and End Results (SEER) program estimates the incidence of GBC at 2.5 per 100,000 persons [1].Despite the relatively low incidence rate, GBC-associated mortality is higher than that of other cancers [2].GBS constitutes a rare set of malignancies with dismal survival rate overall, which is due to early metastasis via lymphatic, perineural, and hematogenous routes, as well as direct invasion into the liver [3]. Although the tumor, node, and metastasis (TNM) staging system of the American Joint Committee on Cancer(AJCC) is the most widespread applied system, there is no global consensus on the optimal system or indicators for predicting the prognosis of GBC patients.Previous research has suggested that CA125 [4] and CA199 levels [5], and tumor-node-metastasis (TNM) stage [6] might affect the survival of these patients. Hence, there is an urgent need for exploring more speci c and sensitive factors for the prognosis of GBC.
It has been reported that systemic in ammation promotes tumor progression and metastasis via the inhibition of apoptosis, promotion of angiogenesis, and damaging of DNA [7] . The in ammatory response involves lymphocytes,monocyte,neutrophils, platelets, and acute-phase proteins, including albumin in peripheral blood.Lymphocyte and monocyte play an important role in in ammatory reaction, re ecting body immune status. Lymphocyte-to-monocyte ratio is a prognostic evaluation system based on the theory of tumor in ammation microenvironment [8].In recent years,a growing number of clinical researches show that pretreament of LMR in the patients with maligant tumor may have a certain value in evaluating the prognosis of cancer patients such as breast cancer [9], lung cancer [10], colorectal cancer [11],classical Hodgkin's lymphoma [12]and nasopharyngeal carcinoma [13,14].Thus, we hypothesized that LMR may also play an important role in GBC.
This study evaluated the correlations between LMR and clinicopathological features of GBC for the evaluation of their prognostic value in GBC patients.Moreover, we also investigated the optimal cut-off values of LMR for predicting prognosis.

Methods
Study population 80 collected patients with histologically con rmed gallbladder carcinoma, who have been operated between March 2008 and August 2014 at the Qingdao Municipal Hospital were included in this retrospective study. Patients diagnosed with gallbladder carcinoma, treated only with surgery, including radical operations, cholecystectomy, and palliative resection. The exclusion criteria were as follows:(1) patients who previously received preoperative chemotherapy or radiotherapy, (2) patients who were combined with other tumors , and (3) patients with clinical evidence of infection or other bone marrow, hematological,or autoimmune disease.

Laboratory data
As part of the physical examinations, Peripheral venous blood samples was collected within one week before treatment, and both peripheral lymphocytes and monocytes were counted. And the peripheral LMR was calculated by dividing the absolute lymphocyte count by the absolute monocyte count. Then we extracted information of total bilirubin, C-reactive protein (CRP), carbohydrate antigen (CA199) and carcinoembryonic antigen (CEA).Further, the following data were collected: age and gender, surgical margin, and operative methods, and the results of pathological reports. We adopted The TNM staging system (7th edition), based on the criteria of the American Joint Committee on Cancer [15].

Follow-up
Follow-up data were collected until August 2016 or death. Follow-up were put into effect every 2 months since start of treatment. Overall survival(OS) de ned as the time between operation and death, loss to follow-up or the last follow-up.

Statistical analysis
Receiver operating curve (ROC) curve analysis and area under the curve (AUC) was performed to select the optimal cut-off value of LMR to assess prognosis based on their utility as a marker for the clinically relevant binary outcome of death/survival. The OS was estimated by the Kaplan-Meier method, and compared using the log-rank test. Univariate survival analysis was performed using a log rank test. multivariate analysis using the Cox regression proportional hazard model was performed to determine the prognostic predictors for survival. These analyses were performed with SPSS software (version 22.0,SPSS Inc, Chicago, IL, USA). A P value less than 0.05 was considered statistically signi cant.

Results
Determination of the cut-off value for the LMR We used ROC curve analysis to determine the optimal cut-off value for LMR. The optimal cut-off value of the LMR was 4.62 with a sensitivity of 66.7% and a speci city of 64.6% by the Youden index. (Fig. 1). The area under the ROC curve (AUC) indicates the diagnostic power of LMR((AUC = 0.675, 95%CI:0.530-0.820,) There were 47 and 33 cases, respectively, in the low(LMR≤4.62) and high (LMR>4.62) LMR groups.

Patients' characteristics with GBC in the LMR group
The cohort included 35 men and 45 women, with a median age 63 years(range, 40-81 years). According to the LMR, We summarized clinicopathological characteristics(gender, age, differentiation grade, pTNM stage, Total bilirubin,CEA,CA199, CRP, surgical margin, and operative methods) in Table 1. As shown, there was no signi cant difference between the groups in age, gender, TNM stage, Total bilirubin, CEA, Resection margin and operative methods of the patients (all P>0.05). There was an obvious difference between the groups in the degree of differentiation (P =0.017) and C-reactive protein(P = 0.026).

Elevated LMR indicates better clinical outcome in GBC
Kaplan-Meier survival analysis and long-rank analysis were used to carry out the relationship between OS and LMR, which was statistically signi cant (log-rank P = 0.03) ( Figure 2). Furthermore, patients in the high-LMR group experienced signi cant improvements in median survival time [18 months (95% CI 1.1-34.853 ) vs. 6 months (95% CI 4.408-7.916 )] compared with patients in the low-LMR group.

Univariate and multivariate analysis of clinical and biochemical parameters
The univariate analysis was performed using the Kaplan-Meier method to assess the predictive parameters. The analysis showed that LMR(P =0.03),differentiation grade(P =0.03), TNM stage(p<0.001), CA199(p=0.028), CEA(p=0.012),CRP(p<0.001),Resection margin(p=0.011) and operative methods(p<0.001) were as signi cant tools for predicting OS in patients with GBC( Table 2). All parameters which were statistical signi cance in univariate analysis were included in multivariate analysis(Cox proportional hazard models ) . To assess the independent prognostic value of the LMR for OS , the multivariate analysis identi ed that Differentiation grade (P=0.011), TNM stage(P=0.001),and CRP(P = 0.008)as independent prognostic factors in the patients with GBC (Table 3). Whereas LMR is not(p=0.203),then it was a signi cant prognosis factor of GBC.

Discussion
In recent years, more and more studies have shown that chronic in ammation is closely related to malignant tumors. Cancer immunoediting points out that the tumor-promoting effect of In ammation has been well demonstrated [16][17][18].Systemic in ammation is a key component of tumor progression, because it can not only do great damage to cancer cells but also establish tumor microenvironment, which contributes to proliferation, migration and immune escape of malignant cells [19]. Literatures have reported hematological markers of systemic in ammation response, such as, LMR,NLR, and PLR might serve as independent prognostic markers of survival in various cancers.
Previous studies have demonstrated that several systemic in ammatory factors can be used to predict the prognosis of GBC patients, such as platelet-to-lymphocyte ratio [20] and neutrophil-to-lymphocyte ratio [20][21]. As one of the indexes re ecting in ammation,the correlation between LMR and survival has been covered [9][10][11][12][13][14]. As far as we know, this is the rst study to evaluate the prognostic signi cance of the LMR in patients with GBC.
In our study, Patients with low-LMR had a worse prognosis than those with high-LMR.The mechanism that LMR affects the prognosis of cancer patients is not yet clear, and further discussion is still needed.Low LMR often suggests that patients with low lymphocyte count, or higher monocyte count, which is often associated with the poor prognosis of the patients.The connection between the lymphocytes and monocytes may be explained by the suppression of the immune cells in ltrating in the tumor tissue or the growth of the tumor.LMR can re ect the immune status and load of the tumor.Tumorin ltrating lymphocytes(TILs) and tumor-associated macrophages(TAMs) are immune cells that have been found to play an important role in many malignant tumor tissues and can play a speci c role in predicting the prognosis. Lymphocyte is a kind of cell with anti-tumor and regulatory immunitiy functions, which plays an important role in immune surveillance in vivo [22], and the change of its content can re ect the immune function of organism.TILs control tumor progression by participating in cellular and humoral immunity. Low lymphocyte count may be a sign of poor immune function,which weakens the control of tumor immunity by immunization and leads to poor prognosis.Decrease of lymphocytes respectively positively with CD4+ cytotoxic T cells and CD8+ cytotoxic T cells,which is the contributory factor to the decrease of immunologic surveillance and immune clearance function.Protective immunity in LMR depended on CD4+ and CD8+ T cells. Actually, Nakakubo [23]suggested that a low presence CD4+ and CD8+ T-cells in the tumor correlate with poor prognosis after surgery for GBC. Recent studies have demonstrated that an lymphocyte count alone [24]are predictive of poor survival in patients with GBC.
On the other hand, when malignant tumors occurs, peripheral blood monocytes can be recruited into the tumor storma, where they gradually differentiate into Tumor-associated macrophages(TAMs) .
Tumor-associated macrophages (TAMs) were in ammatory cells [25],which promotes tumor cell invasion and migration [26], and their number is related to malignant degree and poor prognosis. TAMs are active in the secretion of tumor chemokines around tumor tissues. TAMs can also accelerate tumor progression by stimulating tumor angiogenesis and generate anti-immune responses through the production of growth factors and cytokines.Related studies have con rmed that patients with high TAMs in ltration have a poor prognosis [27][28]. Absolute peripheral blood monocytes can be used as a biomarker for the replacement of TAMs.Therefore, low LMR indicates de ciency of anti-tumor immunity and a tumor promoted in ammatory microenvironment.
Nevertheless, in our cohort, the LMR which is a simple combined index, was not identi ed as an independent prognostic factor. Univariate analysis showed that LMR was signi cantly correlated to the progosis(p<0.05).Multivariate analysis showed that LMR was not independent prognostic predictors(p>0.05). Interestingly, In ammatory markers such as C-reactive protein have been found to be a Independent predictors of prognosis in our research. Elevated CRP as a sensitive indicator of systemic in ammatory reaction, which is closely related to the prognosis of many cancers [29][30][31].
It's possible that factors not considered in the analyses could account for the results.First of all our study was obtained from data collected at a single center and a relatively small retrospective study of 80 patients, and there is a need for prospective, multicenter cohort studies to validate o prognostic value of LMR in gallbladder carcinoma. Moreover, this outcome could be partly explained by the difference among several differentiation grades of GBC, which leads to different effects of the standard prognostic variables. Further studies and analyses are needed on this issue by lamination design. Last but not least, Because of patients and their family members with poor medical compliance, our follow-up indicators did not include progression-free survival (PFS), local progression-free survival (LPFS) and distant metastases free survival (DMFS). Despite these limitations, we believe that our results provide valuable support for the prognostic role of LMR in GBC patients.Further large-scale studies are needed to clarify the prognostic value of preoperative LMR in GBC patients .

Conclusions
Taken together, LMR is closely correlated with GBC prognosis and could be useful for the evaluation of prognosis of patients with GBC. Low LMR was associated with poor overall survival. The LMR could be a widely available and low price prognostic biomarker of GBC in clinical practice.

Declarations
Ethics approval and consent to participate This study was approved by the ethics committee of the Qingdao Municipal Hospital. Written informed consent was obtained from all patients prior to treatment.

Consent for publication
Not applicable.

Availability of data and materials
The data sets generated and analysed during the current study are not publicly available due to privacy reasons, but anonymized data may be available from the corresponding author on reasonable request and after approval of the regional ethical committee. The optimal cut-off value of the LMR was 4.62 with a sensitivity of 66.7% and a speci city of 64.6% by the Youden index. (Fig. 1).