Prognostic Signicance of Globulin/Low-Density Lipoprotein Ratio In Patients With Hepatocellular Carcinoma After Local Ablative Therapy

Background: Low-density lipoprotein (LDL) and globulin have been found to be predictors for some malignant tumors, but their predictive value in hepatocellular carcinoma (HCC) has hardly to be elucidated. This study assessed the prognostic signicance of globulin to low-density lipoprotein ratio (GLR) in HCC patients before ablation. Materials and methods: This study analyzed 312 HCC patients hospitalized and underwent ablative treatment in Beijing You 'an Hospital, Capital Medical University, from January 1, 2012 to January 1, 2017. Cox regression analysis was used to assess the factors independently associated with recurrence and survival. The optimal cut-off value and prognostic role of GLR and other markers were evaluated via the receiver operating characteristic-ROC curves and the Youden index. Overall survival (OS) and recurrence-free survival (RFS) were calculated by Kaplan-Meier analysis, and compared between groups using the log-rank. Result: Univariate and multivariate analysis found that the tumor number (HR: 1.676;95%CI: 1.113-2.526), tumor size (HR: 1.967;95%CI: 1.251-3.092), GLR (HR: 1.028;95%CI: 1.004-1.052) were independent risk factors of relapse; while etiology (HR: 1.328;95%CI: 1.052-1.677), tumor number (HR: 1.615;95%CI: 1.015-2.570), tumor size (HR: 2.061; 95%CI: 1.243-3.418), Fib (HR: 0.73; 95%CI: 0.535-0.996) and GLR (HR: 1.031;95%CI: 1.003-1.06) were related to overall survival. We classied the patients into groups with high and low levels of GLR based on the optimal cut-off value of GLR identied by generating receiver operating characteristics (ROC) curve. The cumulative 1-, 3-, and 5-year RFS rates in the low GLR group were 76.4%, 53.8% and 43.4%, while those in the high GLR group were 71%, 31% and 22%, respectively (P <0.001). Concerning OS, the low GLR group showed a 1-, 3- and 5-year OS of 99.5%, 92.0% and 80.2% versus 98%, 73% and 63% for the high GLR group (P <0.001). Finally, patients were stratied by GLR and tumor size. The outcomes revealed that patients in group A (GLR<16.54 and tumor size ≤ 30mm) showed better prognosis than group B (GLR ≥ 16.54 and tumor size ≤ 30mm or GLR<16.54 and tumor size >30mm) and group C (GLR ≥ 16.54 and tumor size >30mm) (P <0.001).


Background
Hepatocellular cancer (HCC) is the sixth most common cancer in the world and the third leading cause of cancer mortality [1]. China reported 410,000 newly diagnosed cases of HCC and 390,000 deaths in 2020 [1]. First-line treatments for patients with early-stage HCC include and percutaneous ablation, surgical resection and liver transplantation. Ablative therapy has become the choice of more and more HCC patients and doctors, with the advantages of fewer adverse effects, shorter hospital stays, and shorter recovery time [2,3]. However, due to the high rate of postoperative recurrence and metastasis, the 5-year relapse rate of HCC is 70% [4].In China, the 5-year survival rate is only 12.1% [5]. Therefore, we should pay attention to the evaluation of clinical indicators for the prognosis in HCC patients [6].
The liver is a crucial organ that regulates lipid metabolism. Impaired liver function is standard in HCC patients, leading to the profound dysregulation of lipid and lipoprotein metabolism [7]. One study observed that the levels of LDL-C linked to an increased risk of cancer [8]. Similar ndings were suggested in another study that decreased LDL-C is an important prognostic factor in colorectal carcinoma [9].
Globulin is the main component of serum protein elevated levels of it indicate an overactive immune system that is often found in patients with chronic in ammation [10]. Previous studies demonstrated GLOB to be an independent risk factor for the incidence of colorectal and stomach cancers [11,12].
Thus far, few articles have investigated the prognostic prediction of serum lipid ratio to serum globulin HCC patients who receive ablation therapy. Therefore, this study was designed to investigate the prognostic value of GLR for HCC patients through clinical data.

Patient Enrollment
A total of 312 HCC patients who received local ablation at Beijing You 'an Hospital a liated to Capital Medical University from January 1, 2012 to January 1, 2017, were enrolled in this study. The diagnostic criteria for HCC is based on alpha fetoprotein (AFP), classic imaging features, and histological biopsy, which comes from the American Association for the Study of Liver Diseases (AASLD) [13]. Patients aged 18-75 years were treated with ablation to con rm complete ablation. Exclusion criteria include:1) history of other malignancies;2) Laboratory data, including globulin and LDL, were incomplete 3) Lymphocytic leukaemia, autoimmune diseases and other concomitant diseases that affected serum globulin levels;4) advanced stage of HCC;5) secondary liver cancer.
All information of the patient was kept con dential. Procedures consistent with the Declaration of Helsinki. The Ethics Committee of Beijing You 'an Hospital has granted informed consent exemptions for this study.

Data collection
Clinical data of all patients were collected for 7 days before treatment, which mainly

Follow-up
The patients were followed up in the outpatient department; standard physical examination, laboratory examinations and ultrasound each quarter, then enhanced CT/MRI examination every 6 months. The last follow update was June 30, 2020. When the typical HCC imaging pattern in the liver or extrahepatic tumors was detected, with or without elevated serum AFP levels, it was determined that the tumor had recurred. The primary endpoint was recurrence-free survival (RFS), which calculated from treatment initiation to cancer recurrence, while overall survival (OS) measured from treatment initiation to death or last follow-up. After con rmed recurrence patients were assessed and received TACE or radiofrequency ablation treatment.

Statistical analyses
Continuous data are presented as mean ±SD and categorical data as number and percentage. The comparisons of categorical data between groups were tested by Chi-square test. Using the Mann-Whitney U-test and Students t-test to analyze the comparisons of continuous variables between groups. Cox regression analysis was used to assess the factors independently associated with recurrence and survival. OS and RFS were calculated by Kaplan-Meier analysis, and compared between groups using the log-rank. The optimal cut-off value and prognostic role of GLR and other markers were evaluated via the receiver operating characteristic-ROC curves and the Youden index. The patients were classi ed into groups with high and low levels of GLR. P≤0.05 denoted statistical signi cance. A statistical software SPSS Version 26.0 (IBM, Armonk, NY) was performed for statistical calculations.

Prognostic factors related to OS
To further explore whether GLR was a predictive factor of OS, we used univariate analyses to evaluate the relationship between data and OS. Our results revealed that GLR, gender, antiviral, etiology, Child-Pugh classi cation, fractional ablation, tumor number, tumor size, viral load, AST, γ-GT and Fib were dramatically associated with OS. Multivariate analysis showed that that etiology (HR: 1.328;95%CI:  Table 3).

The prognostic value of GLR
According to the GLR cut-off value, all patients were divided into groups with high and low levels of GLR.
Kaplan-Meier survival curves found that the 1-, 3-, and 5-year RFS rates of the low GLR group were 76.4%,53.8% and 43.4%, respectively, with a median RFS of 43.1 months, while the 1-, 3-, and 5-year RFS rates of high GLR group were 71%,31% and 22%, respectively, with a median RFS of 19.3 months P<0.001 , which indicated that higher GLR values correlate with shorter recurrence time Figure 1 .
As for OS, the median OS of patients in the low GLR group was 59 months, and the OS rates at 1 year, 3 years, and 5 years were 99.5%,92.0% and 80.2%, respectively; and the median OS of high GLR group was 51 months, with 1-year, 3-year, and 5-year OS of 98%,73% and 63% P<0.001 , which illustrated that lower GLR values implied better survival Figure 2 .
Previous studies have noted that high serum globulin HCC patients were independent risk factors for poor survival [14]. Kaplan-Meier survival analysis was performed on patients with globulin < 35 g/L to exclude the effect of hyperglobulinemia. The results suggest that GLR remained a signi cant predictor for OS and RFS Figure 3 .

Associations between GLR and clinical data
To determine which clinical data were signi cantly associated with GLR, we produced a comparison of them. Eventually, we found etiology, Child-Pugh B, high AST levels, high ALP levels, low Fib levels and high Apolipoprotein A1/ B ratio were signi cantly associated with high GLR levels (Table 4), which demonstrated that high GLR levels represent the poor liver function.
Comparing the accuracy of predictions of GLR, Globulin and LDL It has been demonstrated that globulin can predict the prognosis of HCC patients undergoing surgical operation [14]. Meanwhile, LDL was associated with early recurrence of HCC [15]. Therefore, a ROC curve for GLR, globulin and LDL was plotted to determine whether the prediction e ciency of the composite indicator was better than that of the single indicator. Eventually, we found the area under the curve (AUC) for GLR was 0.600, which was superior to globulin (0.585) and LDL (0.416) levels alone Table 5 .

Stratify patients based on GLR and tumor size
We have previously demonstrated that high GLR levels re ect impaired hepatic functions in HCC patients, and the tumor size, which determined tumor burden was the independent risk factor for HCC relapse Table 2 . We further analyzed whether the indicator consisted of GLR and tumor size could further increase the predictive ability. Therefore, patients were classi ed into three groups, including group A (GLR<16.54 and tumor size ≤30mm), group B (GLR≥16.54 and tumor size ≤30mm or GLR<16.54 and tumor size >30mm) and group C (GLR≥16.54 and tumor size >30mm).The 5-year recurrence rate were 51% in group A, 73.7% in group B, 90% in group C( P <0.001 Figure 4 ,while 5-year OS for patients in group A, group B, group C were 84.1%,65.4% and 60%,respectively P <0.001 Figure 5 .

Discussion
To date, it is a great challenge to prolong the long-term survival in HCC patients. Despite recent advances in combination treatment, HCC, as the sixth most common cancer worldwide, has a limited survival bene t after the operation. Therefore, we must predict the risk of postoperative early-relapse in HCC patients to conduct early re-interventions in patients at high risk of relapse. While there are many prognostic markers for liver cancer, lack of some index scores with high sensitivity and high speci city recently to predict prognosis in HCC patients. Hence, we need to search for some robust predictive biomarkers to assess the risk of recurrence in HCC patients after ablation and guide individualized therapy.
Globulin, re ecting immune status, is a protein produced by immune organs that plays a vital role in immunity and in ammation. It can be detected as a regulator for the circulatory system to assist blood coagulation, transport proteins and indicate antibody levels [16]. Elevated globulin levels are involved in several in ammatory diseases that occur at speci c times during tumor progression, including initiation, promotion, malignant transformation, invasion, and metastasis [17]. The reason for those may is that cytokines released by in ammatory cells form an in ammation-associated tumor microenvironment that promotes the growth of tumor [18,19]. Meanwhile, in ammation could alter the biological characteristics of tumor cells and disrupt immune function, leading to poor prognosis of patients with malignant tumors [20]. Previous studies found that a high globulin level was associated with a poor prognosis in patients with colorectal cancer, non-small cell lung cancer, prostate cancer, ovarian cancer, and breast cancer [21][22][23][24][25].
Abnormal lipid metabolism plays an important role in the development of tumor by altering lipid metabolism pathways to sustain growth and proliferation, which would cause the change of relevant indicators [26]. Some studies have found that low LDL levels increase the risk of liver cancer in people infected HBV [27]. Lately, lots of studies, with the progression of tumor biology, have suggested that LDL are involved in various tumors development, including breast cancer, lung cancer and liver cancer [28][29][30]. A explanation is that the increased activity of LDL receptors accelerates LDL clearance from circulation, which reduces the risk of cancer [31]. Another interpretation is that hepatic lipase activity is inversely correlated with LDL [32]. Meanwhile, polymorphisms of hepatic lipase gene promoters were associated with HCC [33].
As the ratio between globulin and LDL, GLR has better predicting power through the proof of this study. Moreover, our study suggests that a high GLR level may represent a poor liver function by exploring the correlation between GLR and clinical data. Most importantly, our study demonstrates, for the rst time, the signi cance of the prognosis of GLR in HCC patients of various etiologies. Finally, multivariate Cox regression analysis proved GLR could predict OS and RFS outcomes in HCC patients undergoing complete ablation.
In the context of the high recurrence rate, it is essential to use combined indicators to predict the prognosis of HCC patients after ablation, then to further optimize treatment strategies and guidance. Some studies have found that the survival time of HCC patients with tumor size < 3 cm was signi cantly increased than other types of patients [34]. Our study found that the signi cance of the combination of GLR and tumor size on evaluating patient outcomes. By using combined indicators, patients will be divided into groups through preoperative evaluation. Patients, with higher recurrence risk and lower OS, should adjust the follow-up time to monitor the development of tumor and select appropriate treatment strategies, thus effectively prolonging the long-term survival of patients.
However, our study has some limitations. First of all, this was a retrospective, single-center study. Second, our sample size was limited. Therefore, it is necessary to validate these results by further large-scale multicenter randomized trials. In addition, our study did not provide evidence of the potential mechanism of GLR on tumor progression. Future studies, based on our results, will conduct further experiments to explore the mechanism.

Conclusions
In this study of patients with HCC of various etiologies followed up to 8 years, we identi ed the prognostic value of GLR. As a cheap, readily available, non-invasive biomarker, the globulin/low-density lipoprotein ratio can predict the prognosis of HCC patients who underwent complete ablation.

Availability of data and materials
Data to support the study ndings are available on request from the corresponding author.

Figure 4
The Kaplan-Meier analysis of recurrence (A) of the subgroup study strati cation of patients according to GLR and tumor size. Abbreviations: GLR, the globulin to LDL ratio.