An eight cytokine signature identified from peripheral blood serves as a fingerprint for hepatocellular cancer diagnosis

Background Hepatocellular carcinoma is an aggressive disease in Asia and Africa with poor prognosis partially due to lack of disease-specific biomarkers. Objectives The aim of this study was to assess the concentrations of different cytokines and chemokines in peripheral blood of patients with hepatocellular carcinoma and identify the potential biomarkers that would help in clinical assessment. Methods Profiling of 14 cytokines, chemokines and growth factors was performed in peripheral blood of 78 patients and 78 healthy controls using Bio-Plex Human 15-plex assay kit. Results The results showed that patients had significantly higher levels of IL-1β (p=0.034), IL-6 (p=2.13e-06), IL-10 (p=0.013), IL-17A (p=0.017), IL-22 (p=0.00276), IL-25 (p=0.0005), but lower levels of IL-4 (p=0.00341) and IL-33 (p=0.00982) in peripheral blood. Conclusion We identified a unique eight-peripheral blood cytokines signature for hepatocellular carcinoma detection. This work will serve as the basis for further studies about the clinical value of peripheral blood cytokines in forecasting prognosis


Introduction
Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancers in Asia and Africa with high mortality rate 1 . Although regular surveillance with alpha fetal protein (AFP) and ultrasound scan is recommended, it is debatable if this strategy improves survival 2 . AFP, the most widely available tumor marker, only has a sensitivity of 41-65% for HCC 3 . New biomarkers for earlier diagnosis of HCC and identification of high risk groups are required.
In the past two decades, several studies have investigated the relevance of cytokines alterations in the multifactorial pathogenesis of hepatocellular carcinoma 4 . Some cytokines mainly present during hepatocellular carcinoma development and can possibly be considered as state markers, such as IL-6 and transforming growth factor-β (TGF-β) 5,6 . It has been reported that high levels of pro-inflammatory cytokines in hepatocellular carcinoma might be related to an over-activation of Th17, such as IL-7A, IL-17F and IL-22 7 . However, variations in peripheral blood or plasma cytokines levels might not directly reflect the activity of peripheral blood immune cells. Indeed, peripheral blood and plasma cytokines levels may partly derive from the vessel walls or from other lymphoid or non-lymphoid cells, such as hepatocytes or adipocytes 8 . For these reasons, studying cytokine expression level in peripheral blood might present a more reliable method to investigate the specific activity of immune cells and their degree of activation.
To ascertain whether a peripheral blood cytokine expression signature can distinguish hepatocellular carcinoma from cancer-free controls, we conducted peripheral blood cytokine expression profiling by Bio-Plex Human 15-plex assay kit and extensively evaluated peripheral blood cytokine expression. By statistical analysis, we obtained a profile of eight peripheral blood cytokines, which can serves as a biomarker for hepatocellular carcinoma detection. The correlation between peripheral blood cytokine and hepatocellular carcinoma progression need to be further assessed.

Materials and methods Study design, patients and control subjects
A multi-stage, case-control study was designed to identify a peripheral blood cytokine profile as a biomarker for hepatocellular carcinoma.

Quantification of Cytokines, Chemokines
Fresh heparinized blood samples (4 mL) of venous blood which was collected on an empty stomach early morning. The plasma samples (HCC patients and the corresponding controls) were centrifuged at 3000rpm for 10 minutes and then stored at -80°C for further analysis. Subsequently, the samples were analyzed in duplicate using Bio-Plex Human 15-plex assay kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's instructions. The complete list of cytokines (IL-1β, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, IFN-γ, sCD40L and TNFα) was quantified in these cohorts, and their detection limits and reproducibility were provided in the product manual. The Bio-Plex Protein Array System (Bio-Rad) was employed to distinguish the fifteen distinct sets of fluorescently dyed beads. The detection of 15 cytokine profiling had a high sensitivity and broad dynamic range (0-32,000pg/ml).

Statistical analysis
Statistical analysis was performed using the Statistical Analysis System software (v.9.1.3; SAS Institute, Cary, NC). Data is presented as the median±SD. Non-parametric Mann-Whitney U test was used to compare the difference of peripheral blood cytokines between the cancer and healthy group. P<0.05 was considered statistically significant.

Description and clinical characteristics of the patients
We employed 78 hepatocellular carcinoma patients (diagnosed by two independent pathologic examinations) and 78 healthy people to investigate the potential biomarkers of hepatocellular carcinoma. The clinical characteristics of the patients and healthy samples were represented in Table 1. There was no significant difference in the distribution of smoking (p=0.749), alcohol consumption (p=0.729), age (p=0.059) and gender (p=0.357) between the cancer patients and healthy cases. Among 78 patients, there were 5 patients (6.41%) classified as stages I or II, 56 (71.8%) classified as stages III, 17(21.8%) classified as stages IV, respectively (data not shown). Moreover, elevated levels of AFP (>100ng/ml) were found in 31(39.7%) patients (data not shown).   Figure 1).

Discussion
Hepatocellular carcinoma usually has a poor prognosis mostly due to an advanced stage at the time of diagnosis 9,10 . So developing early diagnostic methods at an earlier stage may help to improve prognosis. However, early diagnostic methods with accurate performance and high-throughout biomarkers for hepatocellular carcinoma are not yet available. In the quest for cancer biomarkers, systemic inflammations is frequently highlighted as a potential confounding factor, as cancer development and inflammation have been reported to be associated 11 .
In addition, it was known that the identified peripheral blood immune signatures could be considered as snapshots of the immunologic activity in a patient at the time of sampling. Hence, these fingerprints reflect a combination of indirect systemic effects in response to the cancer, as well as factors secreted by the tumor 6 .
On the basis of the notion that immune-regulation is a particular phenomenon in hepatocellular carcinoma, we thus performed the Bio-plex Human cytokines assay to detect the peripheral blood cytokines. The result showed African Health Sciences Vol 18 Issue 2, June, 2018 that levels of cytokines IL-1β, IL-6, IL-10, IL-17A, and IL-25 are elevated, but cytokines IL-4, IL-22 and IL-33 were decreased in patients' peripheral blood. Moreover, we employed ROC curve analysis to investigate the potential values of the eighty cytokine for hepatocellular carcinoma. The result revealed that the AUC of the eight were all greater than 0.7 which indicated they represented a high diagnostic value. Furthermore, the high sensitivity and specificity of the 8 cytokines demonstrated their potential role for diagnosis which was consistent with the AUC result. However, there were some limitations in the present study. We identified a unique eight-peripheral blood cytokines signature for hepatocellular carcinoma detection but the cytokines are many and this makes it difficult to be widely used in clinics. The prominent cytokines among these eight cytokines need to be selected in the further studies to provide a wide use for the pathogenesis of HCC. As well, the effect of staging of HCC on the levels of cytokines should be investigated in the further studies. In sum, we identified a unique eight-peripheral blood cytokines signature for hepatocellular carcinoma detection. This work will serve as the basis for further studies about the clinical value of peripheral blood cytokines in forecasting prognosis.