Inflammatory cytokines and change of Th1/Th2 balance as prognostic indicators for hepatocellular carcinoma in patients treated with transarterial chemoembolization

Tumor-associated immune response plays a critical role in cancer pathogenesis. This study evaluated clinical implications of T cell cytokines and regulatory T cells (Tregs) in HCC patients treated with TACE. Whole blood was obtained for analysis of T cell cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-17A, IL-22, IFN-γ, and TNF-α) and Tregs from 142 HCC patients. Patients with CTP class A had a significantly lower proportion of detectable IL-4 or IL-6, but a higher proportion of detectable IL-22 than patients with CTP class B/C. IL-6 level was well correlated with tumor stage and undetectable IL-17A was associated with extrahepatic metastasis. The overall survival rate was significantly higher in patients who had undetectable IL-6 or detectable IL-22 than patients who did not. IL-6 among cytokines remained independently predictive factor for survival. Increased IFN-γ/IL-10 ratio and no increase in IL-6 level following TACE were associated with prolonged survival, and baseline Tregs could affect Th1/Th2 balance. T cell cytokines are associated with a variety of clinical aspects of HCC, and IL-6 is the most significant predictor of survival. A shift toward increased Th1 response and no increase in IL-6 level exert favorable immunologic effects on HCC prognosis.

assessment of tumor immune cytokines is necessary to better understand the interplay between tumor and host immune-inflammatory responses that determine patient outcomes.
In this study, we comprehensively investigated the potential roles of multiple CD4+ T cell cytokines (Th1, Th2, and Th17 cell cytokines) and Tregs as factors representing tumor status and predicting prognosis in patients with HCC treated by transarterial chemoembolization (TACE). We also evaluated changes in the balance of CD4+ T cell subsets following TACE.

Baseline characteristics. The clinical characteristics and serum levels of multiple T cell cytokines and
Tregs (%) of 142 patients with HCC treated by TACE are shown in Table 1. Mean age was 60.4 ± 11.0 years, and HBV infection (71.1%) was the most prevalent cause of HCC. At enrollment, mean tumor size was 8.0 ± 6.5 cm, and nearly half of patients (49.3%) had a solitary tumor. Thirty-six (25.7%) patients had portal vein thrombosis (PVT), and 22 (15.7%) had extrahepatic metastasis. Eleven (7.7%) of 142 patients underwent TACE combined with radiotherapy while the others were treated only with TACE.
We then evaluated whether baseline Tregs influence changes in Th1/Th2 balance following TACE. Patients were divided into two groups based on median Treg levels at baseline, and the proportions of patients who showed an increase in at least 5 out of measured Th1/Th2 ratios were compared between the two groups. As a result, 24 (58.5%) of the 41 patients in the low Tregs group had an increase in Th1/Th2 ratio following first TACE, compared to 12 (34.3%) of the 35 patients in the high Tregs group (P = 0.041) (Fig. 4). This suggests that baseline Tregs level could affect the direction in which Th1/Th2 balance changes and is related to the prognosis of HCC patients.

Discussion
Although knowledge has accumulated regarding the role of immune cells and related cytokines in HCC, there is limited clinical use because each immune regulator is usually studied individually or from tumor tissues that are not easily accessible. We comprehensively analyzed the prognostic roles of multiple T cell cytokines and Tregs in patients with HCC treated by TACE using whole blood samples. IL-4 and IL-22 were related to hepatic reserve, while IL-17A was associated with extrahepatic metastasis. IL-6 was the most significant cytokine in this study related to liver function, tumor characteristics, and OS of HCC patients. Furthermore, increased IL-6 level after TACE significantly predicted poor OS. When Th1/Th2 balance shifted to Th1 dominance after TACE, outcomes were favorable. Baseline Tregs levels could affect the balance of Th1/Th2 cytokines.
IL-6 is a pro-inflammatory cytokine with deleterious effects in several malignancies and inflammatory disorders 12,13 . In a previous study of patients with HCC, serum IL-6 level was significantly associated with advanced tumor characteristics, including large tumor size, presence of PVT, and extrahepatic metastasis, and IL-6 has also been proven as a significant predictor for unfavorable outcome 14 . IL-6 has been suggested as a marker for HCC with higher efficacy than AFP 15 . This study also showed a close relationship between detectable IL-6 and tumor burden, and the mean serum level of IL-6 in patients with mUICC stage IV was significantly higher than that of patients with mUICC stage I-III. These features contribute to the function of IL-6 as an independent predictor of poor OS. Additionally, CTP or MELD score was higher in patients with detectable IL-6, indicating that IL-6 level is correlated with liver function. Senescent hepatocytes, which account for as many as 80% of hepatocytes in advanced chronic liver disease, might contribute to increased IL-6 expression 16,17 . www.nature.com/scientificreports www.nature.com/scientificreports/ In our analysis, cumulative OS was significantly higher in patients with detectable IL-22 than in patients with undetectable IL-22. The liver is an important target of IL-22, which generally demonstrates hepatoprotective properties through anti-fibrotic and regenerative effects during liver infection and damage 18 . However, IL-22 was previously demonstrated as a tumor promoting or negative prognostic factor in patients with HCC. A previous study showed that patients with HCC and high serum IL-22 concentration had poorer prognosis than patients with a low level of IL-22 19 . Sustained IL-22 elevation and subsequent STAT3 activation were found with up-regulation of downstream genes in HCC mouse models, promoting tumor development and metastasis 20 . IL-22 is secreted from Th22, Th17, and Th1 cells 21 , which can all act differently in HCC development. The role of IL-22 reportedly varied depending on the type or degree of liver disease 22,23 . Further study is needed to elucidate the function of IL-22 in hepatocarcinogenesis.   www.nature.com/scientificreports www.nature.com/scientificreports/ Although no single cytokine from Th1 or Th2 cells had prognostic value for OS in this study, we showed that dynamic changes of Th1/Th2 balance after TACE were associated with OS. An increase in IFN-γ/IL-10 ratio after TACE was independently predictive of prolonged OS, and increased IL-1/IL-10 ratio also tended to predict prolonged OS. These findings were consistent with previous studies in which Th2 response had a dominant role in tumor development and progression. Thus, tumor microenvironment shifted to Th1 dominance could have beneficial effects on tumor regression 24,25 . In patients with advanced HCC treated with intra-arterial chemotherapy, the proportion of Th2 cells was significantly higher both before and after treatment in patients with progressive tumor response than in patients with partial response or stable disease 26 .
In this study, a larger proportion of patients with low Tregs level at baseline changed to Th1 dominance after TACE compared to patients with a high baseline Tregs level. Tregs are functionally immunosuppressive and could directly or indirectly inhibit the proliferation of other CD4+ T cells via cytokines to further decrease local immunologic responses to tumor cells 27 . Similarly, a previous study of patients with colorectal cancer showed that Tregs suppressed effector CD4+ T cell responses to tumor-associated antigen before tumor resection, and the suppression was associated with tumor recurrence 28 .
This study has some limitations. Although a multiplex bead analysis used in our analysis allowed simultaneous measurement of T cell cytokines, the number of undetectable cytokines was relatively high with a high cut-off value, and Tregs levels (%) were relatively low compared to other studies. This might have lowered the sensitivity and prognostic power for T cell cytokines and Tregs to correlate with the clinical characteristics and prognosis of HCC. Changes in T cell cytokines following first TACE were insufficiently evaluated because subsequent measurements of cytokines were available in only a small number of patients. Nevertheless, this comprehensive analysis shows that T cell cytokines and Tregs have distinctive properties that correlate well with prognosis of patients with HCC.
In conclusion, T cell cytokines play an important role in hepatic function, tumor characteristics, and prognosis in patients with HCC. IL-6 was among the most significant predictive factors for poor OS in patients undergoing TACE. Baseline Tregs level could influence the direction of Th1 or Th2 response following treatment. A shift toward increased Th1 response and no increase in IL-6 level could exert favorable immunologic effects on HCC prognosis. This study showed the potential role of multiple cytokines as biomarkers for HCC, and the demand for multiple cytokine biomarkers will increase in the upcoming era of immunotherapy.

Methods
Patients. This study evaluated patients newly diagnosed with unresectable HCC who underwent TACE as the 1st treatment between March 2011 and December 2012 at a tertiary center in South Korea. In our center, TACE was mainly selected for patients with CTP class A or B who had unresectable HCC without main PVT. Additionally, patients who had small tumors but ineligible for radiofrequency ablation or percutaneous ethanol injection therapy were also considered to receive TACE. Some patients with PVT or extrahepatic metastasis were evaluated by a multidisciplinary team for TACE or TACE combined with radiotherapy if clinically warranted. Patients treated with other modalities including systemic therapy were excluded from the analysis. HCC was diagnosed by histologic evidence or typical radiologic findings and/or elevated AFP in patients with risk factors for HCC. Staging of HCC was complementarily determined based on mUICC and Barcelona Clinic Liver Cancer staging according to guidelines of The Korean Liver Cancer Association and National Cancer Center 29 . Patients were followed up until death or February 2017. Each patient provided informed consent to participate in the study. This study was approved by the Ethics Committees of The Catholic University of Korea in accordance with the 1975 Declaration of Helsinki.  www.nature.com/scientificreports www.nature.com/scientificreports/ Treatment and laboratory data. The intra-arterial chemotherapeutic agents used were doxorubicin (50 mg) or combined epirubicin (50 mg) and cisplatin (60 mg) in a mixture of lipiodol (5-10 mL). TACE sessions were repeated every 1-2 months until complete response by one experienced interventional radiologist. Clinical and laboratory data of enrolled patients were obtained at the time of diagnosis of HCC and routinely measured during the treatment period including gender, age, hematologic and biochemical parameters and tumor status.
Statistical analyses. All data were expressed as the mean ± SD or median (range). The chi-square test or Fisher's exact test was used to compare categorical variables. The cumulative rate of overall survival was estimated using the Kaplan-Meier method, and differences were evaluated with the log-rank test. Continuous variables were transformed into two-level categorical data, based on their median values. Univariate and multivariate analyses were performed using Cox proportional hazard model to identify prognostic factors for OS. A two-sided P value < 0.05 was considered statistically significant. Data were analyzed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA).

Data Availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. All data generated or analysed during this study are included in this published article.