Primary liver carcinoma (PLC) is the third leading cause of cancer-related death worldwide. Liver carcinoma is also common in China, and its incidence is increasing, representing a serious threat to Chinese citizens’ health and wellbeing. The populations at highest risk for developing liver carcinoma in China include those with chronic hepatitis virus infection, those with a family history of tumors, and those with alcoholic cirrhosis.[2,3] Radical surgery is a major treatment method for liver carcinoma. However, the majority of patients have already developed advanced tumors at the time of diagnosis, and only a small number of patients have access to surgery. Therefore, achieving early diagnosis by screening populations at high risk for liver carcinoma is key to improving the efficacy of liver carcinoma treatment. Noninvasive diagnostic methods using imaging and serum tumor markers are still the most common approaches for PLC screening;[4–6] however, there is a high rate of missed diagnosis and misdiagnosis. So it is urgent and necessary for us to find new serum tumor markers that have higher diagnostic sensitivity and specificity in liver carcinoma.
Recent studies have shown that PLC is an inflammation-related disease, and that the vast majority of patients with PLC have a history of hepatitis B.[7–9] The occurrence and evolution of PLC are closely related to the tumor microenvironment in which PLC cells live. The tumor microenvironment is a complex immune regulatory network formed by the interaction of tumor cells, inflammatory immune cells, inflammatory factors, chemokines, cytokines, and other components. The tumor immune microenvironment plays an important role in helping tumor cells escape from immune system damage, inducing angiogenesis, inhibiting apoptosis, and promoting invasion and metastasis.[11,12] Therefore, it is important to investigate the role of inflammatory factors and immune factors in the development of liver carcinoma. Persistent infection with hepatitis virus and the associated production of cytokines and growth factors can generate a complex inflammatory microenvironment that is prone to tumor development.
The aim of this study was to identify a set of cytokines that would serve as sensitive indicators for monitoring the occurrence and development of PLC. We retrospectively evaluated changes in serum levels of cytokines including interleukin-1β (IL-1β), IL-2 receptor (IL-2R), IL-6, IL-8, IL-10, and tumor necrosis factor-α (TNF-α), in patients with chronic hepatitis B and patients with PLC and a history of hepatitis B virus infection to determine their clinical value in the predicting the occurrence and development of PLC.
Subjects and methods
Eight hundred and ninety-nine patients with PLC admitted to and treated at Changhai Hospita, Naval Military Medical University, China between March 2015 and June 2017 were included in this retrospective observational study. Informed consent was obtained from each participant. This study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Changhai Hospital, Naval Military Medical University, China (approval No. CHEC2020-080) on June 6, 2020. The hepatitis B control group (n = 30) and the healthy control group (n = 30) were also recruited from Changhai Hospital. At the same time, eight hundred and ninety-nine patients with PLC were divided into two subgroups (the HBsAg+, HBeAg+, HBcAb+ PLC group and the HBsAg+, HBeAb+, HBcAb+ PLC group). The hepatitis B control group included two subgroups (the HBsAg+, HBeAg+, HBcAb+ hepatitis group and the HBsAg+, HBeAb+, HBcAb+ hepatitis group). The characteristics of the patients and controls are given in Table 1. Both control and case studies are based on our existing data without previous sample size calculation. This is one of our limitations.
Table 1 -
Patient baseline characteristics
||PLC (n = 899)
||Hepatitis (n = 30)
||Healthy (n = 30)
|Sex (M/F, n)
|Age (mean ± SD, yr)
||56.4 ± 10.1
||44.7 ± 11.4
||53.8 ± 9.7
|HBsAg+, HBeAb+, HBcAb+ [n(%)]
|HBsAg+, HBeAg+, HBcAb+ [n (%)]
F = female, HBcAb = anti-hepatitis B virus core antibody, HBeAb = anti-hepatitis B virus e antibody, HBeAg = hepatitis B virus e antigen, HBsAg = hepatitis B virus surface antigen, M = male, PLC = primary liver carcinoma.
Patient inclusion and exclusion criteria
The inclusion criteria for the PLC group was as follows: final clinical diagnosis of early PLC including China liver cancer staging (CNLC) Ia and CNLC Ib (The maximum diameter of a single carcinoma node is less than 3 cm or the total maximum diameter of two carcinoma nodes is less than 3 cm by surgical pathology or imaging) based on the 2019 edition of Guidelines for diagnosis and treatment of primary liver carcinoma. The exclusion criteria were as follows: (1) patients who had diseases affecting other important organs such as the heart and kidneys, (2) patients who had recurrent liver carcinoma, (3) patients who had metastatic liver carcinoma, (4) patients who had combined acute or chronic infection, and (5) patients who also had other malignant tumors.
The inclusion criterion for the hepatitis group was as follows: patients who had chronic hepatitis B based on the 2019 edition of Guidelines for the prevention and treatment of hepatitis B (hepatitis B virus surface antigen (HBsAg) and/or hepatitis B virus (HBV) DNA positive for more than 6 months). The exclusion criteria were as follows: (1) patients who also had other acute or chronic viral or bacterial infections, (2) patients who had diseases affecting other important organs such as the heart and kidneys, and (3) patients who had blood diseases or endocrine diseases.
Detection of cytokines
Blood was collected from subjects using vacuum blood collection tubes according to a standard procedure in the clinical laboratory of outpatient service and ward. Fasting venous blood (3 mL) was collected in the morning and centrifuged to separate the serum, which was then collected and analyzed. For samples that could not be analyzed immediately, the serum was stored at −80°C, and analyses were conducted within 1 week.
A Siemens IMMULITE 1000 automatic chemiluminescent analyzing system (Siemens, Munich, Germany), with the corresponding reagents, was used for the detection of cytokines (IL-1β, IL-2R, IL-6, IL-8, IL-10, and TNF-α) via chemiluminescence. Detection was performed according to the instruction manuals for the reagents and the instrument. Quality control samples were purchased from Siemens.
An Abbott I2000SR automatic chemiluminescent analyzing system (Abbott Laboratories, Chicago, IL) was used for the detection of hepatitis B markers and alpha-fetoprotein (AFP) via chemiluminescence.
GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA) and SPSS 26.0 (IBM, Armonk, NY) statistical software were used to perform the statistical analyses. Data that conformed to a normal distribution are expressed as the mean ± SE. Data with non-normal distribution are expressed as median (quartile) [M (P25, P75)]. Multiple groups were compared using nonparametric test (Kruskal–Wallis test), and comparisons between two groups were performed using the Mann–Whitney U test. The correlation between each cytokine and the risk of developing PLC was assessed by binary Logistic regression. The predictive performance of all indicators was examined using receiver operating characteristic (ROC) curves. The maximum Youden index for each indicator was used to calculate the cutoff value for each cytokine in serum. The Spearman correlation coefficient was calculated. P
< 0.05 indicated that a difference was statistically significant.
Comparison of serum cytokine levels among the healthy group, the hepatitis group, and the PLC group
The serum levels of cytokines in the healthy group, the hepatitis B group, and the PLC group were statistically analyzed using nonparametric test (Kruskal–Wallis test). Compared with the healthy group, the IL-2R concentration in the PLC group was significantly higher (Fig. 1 and Table 2), and the concentrations of IL-1β, IL-8 and TNF-α in the PLC group were significantly lower (P < 0.05). Compared with the healthy group, the concentration of IL-1β and IL-8 in the hepatitis group was significantly higher (P < 0.05), and the TNF-α concentrations in the hepatitis group were significantly lower (P < 0.05). Compared with the hepatitis group, the IL-2R concentration in the PLC group was significantly higher (P < 0.05), and the concentrations of IL-1β and IL-8 in the PLC group were significantly lower (P < 0.05). The differences in IL-10, IL-6 among three groups were not statistically significant (P > 0.05).
Table 2 -
Comparison of serum cytokine levels among the healthy group, the hepatitis group, and the PLC group
||5 (5, 5)∗
||540 (394, 755)∗
||3.69 (2, 7.88)
||15.8 (7.33, 56.1)∗
||5 (5, 5)
||9.08 (6.9, 12.8)∗
||18.35 (5, 45.35)∗
||393 (334, 482)#
||2057 (1002, 3030)∗
||5 (5, 5)
||8.1 (6.9, 11.6)∗
||5 (5, 5)
||298.5 (250, 380.3)
||348.5 (95.4, 617.8)
||5 (5, 5)
||11.6 (7.02, 29.75)
Data are expressed as median (P25, P75). Nonparametric test (Kruskal-Wallis test) was used.IL = interleukin, PLC = primary liver carcinoma, TNF = tumor necrosis factor.
∗P < 0.05, vs healthy control group.
#P < 0.05, vs PLC group.
Comparison of all serum cytokine levels between PLC or hepatitis B subgroups
The cytokine concentrations at different disease stages (hepatitis or liver carcinoma) and in different infection states (HBsAg+, HBeAg+, HBcAb+ or HBsAg+, HBeAb+, HBcAb+) were compared. Mann–Whitney U test showed that the differences in cytokine concentrations (IL-1β, IL-2R, IL-6, IL-8, IL-10, TNF-α) between the HBsAg+, HBeAg+, HBcAb+ PLC group and the HBsAg+, HBeAb+, HBcAb+ PLC group were not statistically significant (P > 0.05) (Table 3). The differences in cytokine concentrations (IL-1β, IL-2R, IL-6, IL-8, IL-10, TNF-α) between the HBsAg+, HBeAg+, HBcAb+ hepatitis group and the HBsAg+, HBeAb+, HBcAb+ hepatitis group were not statistically significant (P > 0.05) (Table 4).
Table 3 -
Comparison of cytokine levels between the HBsAg+
PLC group and the HBsAg+
|HBsAg+, HBeAb+, HBcAb+
||5 (5, 5)
||11.67 ± 9.20
|HBsAg+, HBeAg+, HBcAb+
||5 (5, 5)
||5 (5, 5)
||12.31 ± 12.70
Data are expressed as median (P25, P75). Mann–Whitney U test was used. HBcAb = anti-hepatitis B virus core antibody, HBeAb = anti-hepatitis B virus e antibody, HBeAg = hepatitis B virus e antigen, HBsAg = hepatitis B virus surface antigen, IL = interleukin, PLC = primary liver carcinoma, TNF = tumor necrosis factor.
Table 4 -
Comparison of cytokine levels between the HBsAg+
hepatitis group and the HBsAg+
|HBsAg+, HBeAb+, HBcAb+
||16.7 (13, 26.5)
||379 (337, 467)
||4.49 (2.21, 10.3)
||2568 (1249, 3127)
||9.32 (7.29, 11.4)
|HBsAg+, HBeAg+, HBcAb+
||29.5 (13, 48.2)
||463 (315, 544)
||5.33 (2, 9.41)
||1264 (834, 2808)
||7.46 (6.65, 12.2)
Data are expressed as median (P25, P75). Mann–Whitney U test was used. HBcAb = anti-hepatitis B virus core antibody, HBeAb = anti-hepatitis B virus e antibody, HBeAg = hepatitis B virus e antigen, HBsAg = hepatitis B virus surface antigen, IL = interleukin, TNF = tumor necrosis factor.
The weight of each cytokine component in the diagnosis of PLC
Based on the Hosmer-Lemeshow test, 4 variables (IL-1β, IL-2R, IL-8, and TNF-α) were included and P = 0.907 (ie, P > 0.05), indicating that the model was an excellent fit for the data (Table 5). IL-1β, IL-2R, and TNF-α were all risk factors for PLC (P < 0.05), resulting in the following equation:
Table 5 -
Logistic regression analysis of cytokine expression levels in patients
IL = interleukin, TNF = tumor necrosis factor.
logit(P) = 0.086+0.01 × IL-2R−0.001 × IL-8−0.033 × TNF-α−0.041 × IL-1β (Table 5).
Diagnostic significance of serum cytokine concentrations in PLC
The ability of IL-1β, IL-2R, IL-8, and TNF-α levels to detect PLC was evaluated in 899 PLC patients using ROC curve analysis. The AUCs for IL-1β, IL-2R, IL-8, and TNF-α in PLC diagnosis were 0.781, 0.843, 0.873, and 0.625, respectively. The AUCs for IL-6 and IL-10 in PLC diagnosis were less than 0.6. Among the cytokines evaluated, IL-8 levels were the best at early prediction of PLC: AUC, 0.873; cutoff value, 153.5 pg/mL; sensitivity, 73.3%; specificity, 88.9%; positive predictive value, 87.0%; negative predictive value, 77.0%; and Youden index, 0.622. When a combination of 4 cytokines (IL-1β, IL-2R, IL-8, and TNF-α) was used, the values were as follows: AUC, 0.938; sensitivity, 79.2%; specificity, 96.7%; positive predictive value, 96.0%; negative predictive value, 82.0%; and Youden index, 0.759. The combination of these four cytokine levels showed a strong ability to diagnose PLC (Fig. 2 and Table 6).
Table 6 -
ROC curve analysis results for each cytokine in the diagnosis of patients in the PLC group
AUC = area under the receiver operating characteristic curve, CI = confidence interval, IL = interleukin, PLC = primary liver carcinoma, ROC = receiver operating characteristic, SE = Standard error, TNF = tumor necrosis factor.
∗Combination of four cytokines (IL-1β, IL-2R, IL-8, and TNF-α).
Correlation between IL-1β, IL-2R, IL-8, and TNF-α levels and AFP levels
AFP is the traditional tumor marker for PLC. To examine the reliability of the results described above, Spearman correlation analyses were performed. The results showed that the serum AFP concentration in patients with PLC correlated positively with IL-2R (r = 0.3502, P < 0.001), IL-8 (r = 0.1558, P
= 0.0273), and TNF-α (r = 0.2544, P < 0.001) levels, whereas there was no significant correlation between serum AFP and IL-1β concentrations in patients with PLC(r = −0.004, P = 0.4991) (Fig. 3).
This study showed that the combination of IL-1β, IL-2R, IL-8, and TNF-α serum levels is an important early marker for clinical diagnosis of the progression of chronic hepatitis B to liver carcinoma. Compared with detection of a single cytokine, the combined detection of four cytokines exhibited increased sensitivity, providing valuable reference for the clinical diagnosis of PLC. Serum IL-2R is mainly released by activated T lymphocytes and can be used as a marker of T lymphocyte activation.[16,17] The results in the current study showed that the IL-2R concentration was significantly higher in the PLC group than in the healthy group and the hepatitis group, which may be associated with T lymphocyte activation.[18–20] Detection of IL-2R levels can be used to monitor immune system function in patients with chronic hepatitis B and to evaluate disease progression, and thus has important clinical value in early prediction of the development of liver carcinoma.[21–23] The results from the current study showed that, compared with the healthy control group, the TNF-α level in the PLC group was significantly lower, indicating that the patients with PLC were immunosuppressed. Lower levels of TNF-α may be associated with a poor immune response to tumor antigens, which can lead to immune dysfunction and a significantly increased risk of malignancy.[25–27] IL-8 is usually described as a leukocyte chemotactic factor responsible for the maintenance of an hepatitis B virus (HBV)-associated inflammatory environment, and may play a role in tumor development. The results from the current study showed that IL-8 levels were significantly higher in the hepatitis group than in the healthy control group and significantly lower in the PLC group than in the healthy control and hepatitis groups, indicating that HBV plays an important role in activating IL-8 release. A previous study showed that HBV infection activates IL-29, IL-8, and cyclooxygenase-2 expression through an unknown mechanism. These three inflammatory factors are part of the same signaling cascade that is regulated by both positive and negative feedback. In liver carcinoma, disruption of IL-1β or TNF-α release due to immunosuppression could result in lower levels of IL-8.[30–32] The results from the current study showed that IL-1β levels were significantly higher in the hepatitis group than in the PLC and healthy control groups, indicating that IL-1β plays a very important role in chronic hepatitis caused by viral infection. High level of IL-2R secretion in patients with liver carcinoma may result in inhibition of IL-1β expression.[25,26] Furthermore, hepatitis B virus e antigen inhibits IL-1β-mediated NF-κB activation by interrupting K63-linked ubiquitination of NF-κB-essential modulator to enhance HBV replication and promote persistent infection.[33–35]
Logistic regression analysis resulted in the inclusion of 4 variables (IL-1β, IL-2R, IL-8, and TNF-α) in the best-fit equation. Changed serum levels of IL-1β, IL-2R, and TNF-α were all risk factors for PLC (P < 0.05). The equation that best fit the data was logit(P) = 0.086+0.01∗IL-2R−0.001∗IL-8−0.033∗TNF-α−0.041∗IL-1β. This equation was used to establish prediction models. When the predictive value was higher than 0.5, there was a chance that HBV would develop into PLC, whereas when the predictive value was lower than 0.5, there was a low probability of HBV developing into PLC. Thus, detecting IL-1β, IL-2R, IL-8, and TNF-α serum levels could facilitate early diagnosis of the progression of HBV to liver carcinoma. The AUCs for PLC diagnosis using IL-1β, IL-2R, IL-8, and TNF-α were 0.781, 0.843, 0.873, and 0.625, respectively. The diagnostic performance of the combination of cytokines was high. AFP is a liver carcinoma marker. To examine the reliability of the obtained results, Spearman correlation analyses were performed. The results showed that the serum AFP concentration was positively correlated with serum concentrations of IL-2R, IL-8, and TNF-α in patients with PLC. It is well known that the prognosis of patients with PLC depends on early diagnosis and treatment. At present, the pathology analysis of clinical biopsy samples is the gold standard for confirming a PLC diagnosis.
There may be some possible limitations in this study. First, our study is a single-center observational study. Second, our present work does not cover all the cytokines due to the clinical cytokine detection method. Third, our conclusion needed to be verified by a larger sample size. And whether our finding is consistent with the clinical outcome process from chronic hepatitis B to liver cancer still needs to be verified in future work.
Because the onset of PLC is insidious, the majority of patients have advanced disease at the time of diagnosis. Noninvasive diagnostic methods using imaging and serum tumor markers are still the most common approaches for PLC screening; however, there is a high rate of missed diagnosis and misdiagnosis. The results from this study showed the combined detections of IL-1β, IL-2R, IL-8, and TNF-α could help improve diagnostic sensitivity and specificity in liver carcinoma and facilitate diagnosis at an early stage of tumor development. The results from this study could help guide clinical work to improve virus control, limit and prevent progression of liver inflammation to liver carcinoma, and reduce mortality.
CH conceived and designed the study, interpreted and analyzed the data, and wrote the manuscript. QW, JZ, QQ, HW performed the study, interpreted and analyzed the data, and wrote the manuscript. SL revised the manuscript and approved the final manuscript. All authors approved the final version of the manuscript.
This work was supported by the State Key Program of National Natural Science Foundation of China (No. 82030073; to SL), the National Natural Science Foundation of China (No. 81501401; to CH), and Shanghai Science and Technology Committee (No. 18XD1405300; to SL).
Institutional review board statement
This study was conducted in accordance with Declaration of Helsinki and approved by the Institutional Review Board of Changhai Hospital, Naval Military Medical University, China (approval No. CHEC2020-080) on June 6, 2020.
Declaration of patient consent
The authors certify that they have obtained the patient consent forms. In the forms, patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity.
Conflicts of interest
The authors declare that they have no conflicts of interest.
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