Prognostic role of lipids, apolipoproteins in patients with resectable hepatocellular carcinoma

Background: To further clarify the association between abnormal levels of serum lipid components as the main features of dyslipidaemia and hepatocellular cancinima, which remains unclear. Methods: We examined the serum level of lipids and apolipoproteins pattern in 471 patients undergoing curative resection for HCC, 193 patients with chronic liver disease, and 104 patients with benign liver diseases. We performed uni- and multivariate analyses to evaluate the predictive roles of lipids and apolipoproteins for recurrence and survival of HCC in a training cohort of 242 patients and then validated in a cohort of 229 patients. Results: The majority circuling lipid and apolipoprotein levels such as ApoA1, HDL, LDL in chronic liver disease and HCC were slight to significantly decreased as compared with those in benign lesion. But no significant differential expression patterns of lipids and apolipoproteins were observed between chronic liver hepatitis and HCC. Multivariable analysis identiﬁed ApoA1 as a key parameter related to recurrence and survival in both training and validation cohorts. Moreover, we further demonstrated that low ApoA1 was an independent prognostic factor of poor early recurrence in two cohorts. Conclusions: Although the alterations of circulating lipids and apolipoproteins were observed in HCC, none of lipids and apolipoproteins could serve as a diagnostic marker. Serum ApoA1 merits consideration as a novel prognostic marker for patients with HCC undergoing surgery, since it predicts early recurrence and survival, especially for early stage patients and may improve the prognostic stratiﬁcation of patients for clinical management and promote HCC clinic outcomes.


Introduction
Hepatocellular carcinoma (HCC) is the most prevalent cancer with a dismal prognosis worldwide [1].
Until now, surgical resection has remained the primary treatment to cure this disease, but the prognosis of patients with HCC remains poor, mainly because most patients are diagnosed at advanced stages and because of a high incidence of tumor recurrence post-surgery [2]. Although HCC patients who receive curative therapy are at a very early/early disease stage, early recurrence is considered an important adverse prognostic factor after curative therapy for HCC [3,4]. Therefore, sensitive and effective biomarkers are urgently needed for early diagnosis and the accurate prognostic prediction of patients undergoing curative resection. The identification of patients who are at high risk of early recurrence after curative surgery allows clinicians to provide active surveillance to detect recurrent HCC at its earliest stage and to improve the prognosis of this population when curative therapy may still be feasible.
Many epidemiological studies have consistently linked dyslipidemia with increased incidence and death from diverse types of cancer [5][6][7]. The liver plays a key role in the metabolism of lipids and lipoproteins. The majority of patients with HCC are frequently affected by liver cirrhosis, and chronic hepatitis B and C are the major etiological agents [8]. Therefore, liver function is clearly impaired in HCC, which leads to a distinctly abnormal pattern of serum lipid and lipoprotein expression. It has been shown that high-density lipoprotein (HDL) and its major apolipoproteins, ApoA1 and ApoA2, are frequently reduced in patients with HCC [9,10]. Studies have also demonstrated that plasma triglycerides (TG) and total cholesterol (TC) are decreased in patients with HCC [11]. In contrast, Alsabti [12] reported that serum TG and TC were increased in HCC patients compared with serum TG and TC in patients with cirrhosis. Ooi et al [13] reported that the plasma TG levels in HCC patients were not significantly different compared with those in controls. A potential explanation for these conflicting results could be the sole focus on the level of lipids or lipoproteins without an extensive list of potential confounders that are not always controlled such as body mass index (BMI) and glucose metabolism agents. Ahaneku et al[14] analyzed lipid profiles in cirrhosis patients with or without HCC and found that plasma HDL-C, HDL-PL and HDL-C/HDL-PL were significantly lower in HCC patients than in controls. It is suggested that variations in the level of plasma lipids and apolipoproteins may assist in describing the nature of these two forms of liver disease. More striking is the discovery, by mass spectrometric analysis, that ApoA1 was differentially expressed between chronic hepatitis C virus (HCV) infection and HCC patients, which suggested that ApoA1 might be a candidate biomarker for early diagnosis, prognosis, and monitoring of HCC [15]. A similar result was found by Steel et al [16] in that ApoA1 had a diagnostic role in patients with HCC compared with hepatitis B virus-positive individuals. These results suggested that the plasma lipid profile might be a potential candidate that can be used to differentiate HCC from other liver diseases and from normal liver. The plasma lipid profile also acted as a diagnostic biomarker of HCC. However, the limitations of the studies mentioned above are obvious, such as the small number of cases enrolled and the techniques used, which are unsuitable for large-scale analysis in clinical application. Therefore, the accuracy and reliability of the experimental results mentioned above have yet to be verified.
Thus far, few studies have investigated the use of serum lipids as biomarkers for diagnosing HCC and predicting the recurrence or survival of patients with HCC. In this study, we investigated the expression pattern of blood lipids and apolipoproteins including ApoA1, ApoB, ApoE, TC, TG, HDL, and low-density lipoprotein (LDL) in HCC and other liver diseases including chronic hepatitis, cirrhosis and benign disease. We also explored their plausibility as novel diagnostic biomarker candidates for HCC. Furthermore, we analyzed the associations of the circulating lipid and apolipoprotein levels with the clinicopathological characteristics and prognosis of patients with HCC.

Study population
In all, 471 patients with primary HCC who underwent curative liver resection between December 2010 and January 2012 at the Liver Cancer Institute, Zhongshan Hospital, Fudan University were included in this retrospective study. All patients had histologically confirmed HCC. None of the patients had received presurgical cancer treatment and none had a recurrence of HCC or any other known malignancy. The patients were randomly divided into a training cohort(n=242) and a validation cohort(n=229). In addition, 140 patients with chronic hepatitis, 53 with cirrhosis, and 104 with benign liver diseases, including hepatic hemangioma, hepatic cyst and focal nodular hyperplasia, were also enrolled in this study. In this cohort, patients with benign diseases had normal liver function relative to normal, age-matched donors.
Lipid and apolipoprotein profiles were studied descriptively in relation to potential confounders of metabolism including BMI, blood concentration of C reactive protein (CRP) as a biomarker for systemic inflammation, fasting blood glucose (GLU), hemoglobin A1C as well as insulin resistance (C peptide, insulin). As an index of insulin resistance, we used the homeostatic model assessment of insulin resistance (HOMA2-IR). Clinical tumor stages were determined according to the TNM classification system of the International Union Against Cancer.Tumor differentiation was assigned according to the Edmondson-Steiner classification.
Follow-up procedures were described in our previous study [17]. Overall survival (OS) was defined as the interval between surgery and either the time of death or the last follow-up. Disease-free survival (DFS) was defined as the interval between surgery and the time of recurrence or the date of the last follow-up without recurrence. The overall median follow-up time was 70 months (range, 7-79months).

Laboratory measurements
All baseline blood samples were collected prior to surgery from patients who underwent surgery and from nonsurgery participants during the first week after admission. TC and TG were quantitatively determined by a colorimetric method, while HDL and LDL were measured using enzymatic methods.
ApoA1, ApoB and ApoE were determined by turbidimetric methods. All the above measurements were performed in an automated biochemistry analyzer (Hitachi 7600). C-peptide and insulin were examined by an electrochemiluminescent immunoassay using an Roche Cobas 602 autoanalyzer.

Statistical analysis
SPSS version 20 (SPSS Inc, Chicago, IL) was used to analyze the data.Continuous variables were compared using the Mann-Whitney U-test or One-way ANOVA. Categorical variables were compared using the χ 2 test or Fisher's exact test. The Spearman rank correlation test was performed to determine any associations between variables. The overall survival and disease-free survival were computed using the Kaplan-Meier method and were calculated using the log-rank test. A Cox regression hazard model was utilized for the multivariate analysis. To avoid colinearity bias, the lipids and apolipoproteins were preliminarily tested in a multivariate model that included the individual variables comprising theTNM stage. All statistical tests were two-sided, and p-values <0.05 were interpreted as significant.

Measurement of the expression pattern of serum lipids and apolipoproteins in liver disease
We first compared the serum lipid and apolipoprotein levels in the four different groups. In patients with HCC, the serum level of ApoA1 was statistically lower than that in patients with benign liver disease but was higher than that in the liver cirrhosis group. No significant difference was observed between HCC and the hepatitis group. In addition, the expression pattern of HDL was similar to that of ApoA1. The serum ApoE level was significantly higher in the HCC group compared with the chronic liver hepatitis group, whereas the level did not differ significantly between the HCC group and the other two groups. Compared with the liver cirrhosis group, the TG and LDL levels were significantly elevated in the HCC, liver hepatitis and benign liver disease groups, whereas no significant differences were shown in the expression levels among these three groups. Moreover, we did not find any significant differences in the serum ApoB and TC levels among these four groups ( Figure 1 and Figure S1).

Correlations between the lipid and apolipoprotein levels and clinical characteristics
As shown in Table 1, no significant differences were found in the relationships between lipid and apolipoprotein levels and clinical background factors such as age, liver cirrhosis, alanine aminotransferase (ALT), albumin (ALB), alpha-fetoprotein (AFP), and serum total bilirubin (TB).
However, ApoA1, ApoE and HDL were closely associated with gender. With regard to metabolic profiles, TG and HDL had positive correlations with BMI, whereas LDL and ApoB were associated with glycometabolic indexes such as blood glucose and hemoglobin A1C (HbA1C).TG had a close relationship with HbA1C and the insulin resistance index HOMA2-IR. However, most lipids and apolipoproteins were not closely related to metabolic parameters in HCC patients. Finally, with respect to tumor-related covariates, we revealed that ApoA1 was significantly related to vascular invasion and that LDL was associated with tumor size. However, no significant relationships were observed between most lipids and tumor-related factors.

Prediction of Tumor Recurrence and Patient Survival in the training cohort
We next explored the prognostic significance of lipids and apolipoproteins in patients with HCC. The baseline characteristics of the patients were not significantly different between the training and validation cohorts (Table S1). Using X-tile 3.6.1 software, the optimal cutoff values of each lipid and apolipoprotein were established to separate patients in the training cohort into low and high groups [18]. Divisions of the validation cohort were also based on these numerical standards. A univariate analysis indicated that a lower ApoA1 level was significantly correlated with poor prognosis of HCC patients. The cumulative OS and DFS rates of patients in the low ApoA1 group were significantly lower than those of patients in the high ApoA1 group  Table 2 and Table S2).

Prognostic value of lipid and apolipoprotein levels in the validation cohort
Lipids and apolipoproteins were further assessed for their prognostic power in an independent cohort of 229 patients. We found that HCC patients with lower serum ApoA1 levels had a significantly worse prognosis than those with higher serum ApoA1 levels. In the univariate analysis of OS, ApoA1 and ApoE were univariate predictors of OS (P=0.01; P=0.02, for ApoA1 and ApoE, respectively)( Figure 2 and Figure S4). A Cox regression analysis demonstrated that a lower serum ApoA1 level was an independent indicator of OS (HR: 0.418; 95% CI: 0.234-0.747; P=0.003). In addition, a higher serum ApoE level was also an independent risk factor for OS (HR:1.897; 95% CI: 1.123-3.204; P=0.02). In the univariate analysis of DFS, a lower ApoA1 level was a significant predictor of poor prognosis (P=0.007), and a Cox regression analysis showed that ApoA1 remained an independent prognostic factor for DFS (HR:0.609; 95% CI: 0.409-0.908; P=0.02) (Table S3 and Table S4). However, other lipids and apolipoproteins including TC, TG, HDL, LDL and ApoB did not show any statistically significant associations with OS and DFS( Figure S4 and S5).

Serum ApoA-1 levels are correlated with early recurrence in the training and validation cohorts
Furthermore, the prognostic significance of the serum ApoA-1 level in patients with AFP levels ≤400 ng/mL and/or in patients with early recurrence was investigated. Among patients with an AFP concentration ≤400 ng/mL, the OS and DFS rates in low ApoA1 patients were significantly lower than In addition, it is worth noting that, for early recurrence in the training cohort, the serum ApoA-1 level was significantly associated with recurrence in the univariate analysis. In the multivariate analysis, the serum ApoA-1 level was an independent indicator of early recurrence (HR: 0.51; 95% CI: 0.297-0.875; P=0.02). Similar results were confirmed in the validation cohort: lower ApoA-1 was an independent predictor of early recurrence (HR, 0.478; 95% CI: 0.278-0.881; P=0.008) (Figure 3, Table   3 and Table S5). Previous studies have shown that predictive factors for early recurrence are mainly tumor-related clinicopathologic factors (i.e., tumor size, tumor number, tumor markers, microscopic vascular invasion) [19,20]. The results of the multivariate analysis in the present study also show that early recurrence was associated with larger tumor size, vascular invasion, and advanced TNM stage.
Further stratification analyses were conducted in the subgroups based on AFP, tumor size, number, vascular invasion, and TNM stage. We found that low ApoA1 was remarkably related to early recurrence in patients with an AFP concentration≤400 μg/L, a tumor size ranging from 2cm to 5cm, TNM stage I and solitary tumor in both the training and validation cohorts. By contrast, no significant associations were observed between the ApoA1 level and the early recurrence rate in the vascular invasion-negative subgroup ( Figure S7 and Figure S8).

Discussion
Until now, the association between lipids and apolipoproteins and HCC has not been thoroughly investigated, and limited data concerning their clinical significance in relation to HCC have been comprehensively reported. In this study, we analyzed the serum lipid and apolipoprotein levels in patients with benign lesions, chronic hepatitis, liver cirrhosis and HCC, and found that most circulating lipid and apolipoprotein levels in chronic liver disease and HCC were slightly to significantly decreased compared with those in benign lesions. This result was in accordance with some previous studies [9,[21][22][23].
As far as we know, the currently available serological biomarkers, such as AFP, are insufficiently sensitive or specific for use in diagnostic assays [24,25], and thus novel serum markers capable of precisely detecting HCC at an early stage as well as accurate monitoring of HCC over a long period are needed. Proteomic technology has revealed that certain apolipoproteins, such as ApoA1, can distinguish serum from patients with cancer and healthy control subjects with high sensitivity and specificity. Mustafa et al [15] discovered proteomic alterations in ApoA1 in HCC-derived serum samples and a decreased ApoA1 level relative to HCV. Similar to the result described above, Liu et al[26] found a great discriminatory value of ApoA1 between the two patient populations with HCC and liver cirrhosis and suggested that ApoA1 had diagnostic ability in detecting HCC. According to the above mentioned results, it seems that the conclusion that ApoA1 is an effective diagnostic biomarker for HCC is valid. Although our results showed that the levels of most lipids and apolipoproteins are apparently different between HCC and the normal benign groups, no significant differential expression patterns of lipids and apolipoproteins were observed between chronic liver hepatitis and HCC in our study. Furthermore, the circulating ApoA1, HDL and LDL levels were substantially lower in the liver cirrhosis group compared with the HCC group, which did not contradict results of previous studies [10,22,28]. A possible explanation for this is that most previous studies included patients with more advanced stages of liver cancer accompanied by more severe liver dysfunction, which inevitably led to a lower level of lipids and apolipoproteins compared with our patients. Collectively, we considered that ApoA1 was not a sufficiently sensitive or specific biomarker to separate HCC from other chronic liver diseases. In addition, some data have suggested that ApoA1 could provide diagnostic utility to distinguish patients with bladder cancer, gastric cancer and breast cancer from healthy controls. The more important concern is that these results emphasize that changes in lipid and apolipoprotein profiles may imply a relationship with the presence and/or risk of cancer, although the underlying biological mechanisms are not fully understood.
The majority of HCC patients have an underlying chronic liver disease, and therefore, hepatic function is obviously damaged, which significantly influences lipid and apolipoprotein metabolism [27].
Consequently, it has been suggested that plasma levels of lipids and apolipoproteins in HCC reflect the hepatic cellular impairment status. We revealed that the majority of lipids and apolipoproteins were positively correlated with prealbumin and gamma-glutamyl transferase, two hepatic protein synthetic ability indices, and suggested that the pattern of changes in lipid and apolipoprotein levels may be a good indicator of the hepatic protein synthetic ability and liver injury during the perioperative period after hepatectomy. Many studies have demonstrated that liver injury parameters are closely related to poor prognosis in HCC [28,29], and hence, we hypothesize that circulating lipids and apolipoproteins could serve as indicators of prognosis after curative hepatectomy in routine clinical practice. In fact, the correlation of lipid and apolipoprotein levels in peripheral blood with the prognosis of HCC has never been investigated. Our study has revealed that although most of the lipids and apolipoproteins were not significantly associated with overall survival and tumor recurrence in either the training or validation cohorts, a lower serum ApoA1 level was a negative prognostic factor for OS and DFS in patients with HCC in two independent cohorts. Therefore, serum ApoA1 might be a powerful and noninvasive biomarker that can predict the prognosis of HCC patients who undergo curative surgery. Our results also suggest that ApoA1 could also serve as a prognostic indicator in patients with lower AFP concentrations. Therefore, this marker may help surgeons identify surgical candidates at high risk of recurrence and poor prognosis before surgery. Furthermore, this would be a clinical monitoring indicator for patients with lower AFP concentrations after surgery since AFP is not feasible as a monitoring indicator, while ApoA1 is significantly correlated with the prognosis of these patients.
Notably, the current study demonstrated no progressive relationship between ApoA1expression and postoperative survival in patients with an AFP concentration ≥ 400 µg/L, which is generally associated with advanced stage and tumor progression. This suggests that ApoA1 may be an efficient predictor of early prognostic status for patients. Various reports have suggested that patients with early recurrence generally have a worse prognosis than those with late-phase recurrence [30,31]. Since the clinical outcome of patients who experience early recurrence is extremely dismal[32], identifying patients at high risk for early recurrence may help improve the prognosis of this population by rational adjuvant treatment. Therefore, we next investigated the prognostic value of ApoA1 to identify its correlations with the early recurrence of HCC. Here, we further demonstrated that low ApoA1 was an independent prognostic factor for early recurrence according to the multivariate analysis in two cohorts. As we know, HCCs larger than 5 cm always have a poor prognosis[33, 34], while small HCCs less than 2 cm are always associated with a good prognosis. We then stratified patients into three subgroups according to tumor size. We observed a statistically significant difference in the curves for early HCC recurrence-free survival: patients with low ApoA1 had a higher recurrence rate when their tumor size ranged from 2 cm to 5 cm. It is also worth noting that in patients with an AFP concentration < 400 µg/L and TNM stage I, Kaplan-Meier survival curves showed that patients with lower ApoA1 levels in both cohorts had a poor early recurrence rate. Consequently, we consider that ApoA1 might be utilized to identify a certain potentially manageable portion of HCC patients at high risk for early recurrence who are good candidates to receive more intensive surveillance and aggressive treatment post-surgery, especially for those with early-stage disease and an negative AFP concentration.
To date, the precise mechanisms by which serum lipids and apolipoproteins contribute to the initiation and/or progression of cancer are not completely understood. Dyslipidemia is known to be associated with the induction of oxidative stress as well as the chronic inflammatory response [35,36] and is closely related to insulin resistance Our study has some limitations to consider. First, all enrolled participants with HCC underwent surgery. Since most patients who underwent hepatic resection had well-compensated liver function, the serum lipid and apolipoprotein levels might not have been significantly influenced by HCC compared with those with chronic liver diseases. This partly explained the lack of a significant difference in the lipid level that was revealed between the HCC and chronic liver disease groups according to our data. Second, the number of participants with chronic liver disease was relatively small compared with the number of participants with HCC, and thus the power to detect a statistical difference might be weakened. Last, the present study was a retrospective single-centric study, and further multicentric prospective studies are warranted to confirm our results.

Conclusions
The current study showed alterations in lipid and apolipoprotein patterns in patients with resectable HCC, chronic infection and benign liver disease. Among these factors, although ApoA1 was not a suitable diagnostic biomarker for HCC, it was confirmed to be an independent prognostic factor for stage HCC. Further trials are therefore necessary to clarify the mechanisms underlying the anti-tumor effects of ApoA1.

Availability of data and materials
The datasets that were analyzed during the current study are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate
This study was performed in accordance with the World Medical Association Declaration of Helsinki and was approved by the Ethics Committee of Zhongshan Hospital of China Fudan University.     Figure 1 The

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download.