|Year : 2018 | Volume
| Issue : 1 | Page : 40-45
Dynamic changes of T-cell subsets and their relation with tumor recurrence after microwave ablation in patients with hepatocellular carcinoma
Yan Zhou1, Xiaolin Xu2, Jianmin Ding1, Xiang Jing1, Fengmei Wang3, Yandong Wang1, Peng Wang4
1 Department of Ultrasound, Tianjin Third Central Hospital, Tianjin, China
2 Department of Ultrasound, The Third Affiliated Hospital of Tianjin Medical University, Tianjin, China
3 Department of Gastroenterology and Hepatology, Tianjin Third Central Hospital, Tianjin, China
4 Artificial Cell Engineering Technology Research Center of Public Health Ministry, Tianjin Institute of Hepatobiliary Disease, Tianjin Key Laboratory of Artificial Cell, Tianjin Third Central Hospital, Tianjin, China
|Date of Web Publication||8-Mar-2018|
Dr. Xiang Jing
No. 83 Jintang Rood, Hedong, Tianjin 300170
Source of Support: None, Conflict of Interest: None
Aims: Thermal ablation can evoke an immune response, which may have effects on the prognosis of patients with hepatocellular carcinoma (HCC). Our aim is to investigate the changes of circulating T-cell subsets after microwave ablation (MWA) and to explore the risk factors of tumor recurrence in patients with hepatitis B virus (HBV)-related HCC.
Methods: Thirty patients with HBV-related HCC were enrolled in this study. The blood samples were collected both before and after MWA (24 h, 72 h, and 1 month after MWA). The distributions of Th17 cells, regulatory Treg-cells, CD4+ T-cells, CD8+ T-cells, and CD3+ T-cells were determined by flow cytometer. The potential-related factors of tumor recurrence were analyzed by logistic regression.
Results: The levels of circulating T-cell subsets, except for Th17 cells, were relatively stable after MWA. The frequency of Th17 cells increased from 3.98% ± 2.40% before treatment to 5.53% ±3.27% 24 h after treatment. Eight of 30 patients had a tumor recurrence. The results of logistic regression suggested that among 11 candidates, only the level of Th17 cells was the risk factor of tumor recurrence. To remove the interference from other factors, seven patients with tumor(s) >3 cm or alpha-fetoprotein >400 ng/mL were excluded in another parallel logistic regression. The results of such regression clearly demonstrated that circulating Th17 cells is indeed a related factor of tumor recurrence.
Conclusions: Thermal ablation may evoke a transitional immune response by increasing the frequency of Th17 cells. Patients with high levels of baseline circulating Th17 cells, instead of the transient elevation of Th17 cells induced by MWA, are at the risk of tumor recurrence.
Keywords: Hepatitis B virus, hepatocellular carcinoma, immunology, microwave ablation, T lymphocytes, tumor recurrence
|How to cite this article:|
Zhou Y, Xu X, Ding J, Jing X, Wang F, Wang Y, Wang P. Dynamic changes of T-cell subsets and their relation with tumor recurrence after microwave ablation in patients with hepatocellular carcinoma. J Can Res Ther 2018;14:40-5
|How to cite this URL:|
Zhou Y, Xu X, Ding J, Jing X, Wang F, Wang Y, Wang P. Dynamic changes of T-cell subsets and their relation with tumor recurrence after microwave ablation in patients with hepatocellular carcinoma. J Can Res Ther [serial online] 2018 [cited 2021 Jun 24];14:40-5. Available from: https://www.cancerjournal.net/text.asp?2018/14/1/40/226762
| > Introduction|| |
The incidence of hepatocellular carcinoma (HCC) was ranked the sixth, and the mortality rate of HCC was ranked the second in the world among all cancers., In China, a majority of HCC developed from chronic viral infections, particularly hepatitis B virus (HBV). It has been reported that the changes of the immunity of patients resulting from HBV infection play an important role in the development of HBV-related HCC.,, Recent studies showed that local thermal ablation can evoke immune response in HCC patients with the changes of the proportions of T-cell subsets., However, a few studies also demonstrated that thermal ablation had no effect on the T-cell levels because of its minimal invasion. Therefore, it is of great importance to understand the effects of thermal ablation on T-cell subsets.
Most of the previous studies researched the changes of CD4+ T-cells, CD8+ T-cells, CD3+ T-cells and regulatory T (Treg) cells induced by local thermal ablation rather than helper T cells 17 (Th17). The latter, however, is a new subset of CD4+ T-cells. Different from Treg cells, playing a role in immune response inhibition, Th17 cells can promote host defense against infection and contribute to autoimmune diseases. Currently, Th17 cells have been recognized as an associated factor of tumor progression. Some studies found that the tumor infiltrating Th17 cells can be regarded as a predictor of tumor recurrence., However, the time evolution of the proportion of blood circulating Th17 cells after microwave ablation (MWA) and their effects on tumor progression were not clearly understood. Thus, we investigated the effects of thermal ablation on T-cell subsets by dynamically monitoring the changes of the proportion of CD4+ T-cells, CD8+ T-cells, CD3+ T-cells, Treg cells, and Th17 cells in the peripheral blood. We further explored the risk factors of the tumor recurrence after MWA.
| > Methods|| |
The study was approved by our Hospital Ethics Committee, and each specimen was taken with the patient's full consent. A total of 30 patients with HBV-related HCC undergoing percutaneous ultrasound-guided MWA therapy between April 2015 and October 2015 in Tianjin Third Central Hospital were enrolled in this study. These patients met the following inclusion criteria: (i) diagnosed as HBV-related HCC, (ii) meeting the Milan criteria, (iii) >18-year-old, (iv) receiving MWA treatment as first-line therapy, and (v) having Child-Pugh classification of A or B. All the diagnoses were confirmed by ultrasound-guided biopsy. The baseline characteristics of patients are shown in [Table 1].
|Table 1: Baseline characteristics of patients with hepatitis B virus-related hepatocellular carcinoma|
Click here to view
Peripheral blood collection and flow cytometric analysis
Blood samples of patients before and after MWA (including 24 h, 72 h, and 1 month after treatment) were collected. Red blood cell lysis buffer was used to split red cells and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation.
PBMCs were stained with the following antibodies: Fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (BD Biosciences, USA and Beckman Coulter, USA), phycoerythrin (PE)-cyanin 5.1 (PC-5)-conjugated anti-CD25 (Beckman Coulter, USA), PE-conjugated anti-Foxp3 (eBioscience, USA), PE-conjugated anti-interleukin (IL)-17a (BD Biosciences, USA), and FITC-conjugated anti-CD4/PE-conjugated anti-CD8/PC-5 conjugated anti-CD8 mixed reagent (Beckman Coulter, USA). For intracellular cytokine detection, cells were stimulated for 4–6 h with leukocyte activation cocktail (BD Biosciences, USA), stained with surface markers, fixed, and permeabilized with cytofix/cytoperm fixation/permeablilizaion solution (BD Biosciences, USA), and then stained with anti-IL-17a. For the analysis of FoxP3, the PE-conjugated anti-Foxp3 (eBioscience, USA) was used according to the manufacturer's instructions. For the analysis of CD4+ T-cells, CD8+ T-cells and CD3+ T-cells, FITC-conjugated anti-CD4/PE-conjugated anti-CD8/PC-5 conjugated anti-CD8 mixed reagent (Beckman Coulter, USA) was used according to the manufacturer's instructions. The proportions of T-cell subsets were detected by using Canto II flow cytometer (BD Biosciences, USA).
Enhanced computed tomogram (CT) or magnetic resonance imaging (MRI) scan was performed 1 month after MWA and every 2–3 months after the first scan. Tumor progression was defined as an abnormal arterial phase contrast enhancement and a washout in portal or delay phase in CT or MRI. Further, the tumor recurrence was evaluated by an experienced radiologist in our central.
Continuous variables were shown as a mean ± standard deviation. The proportion of Th17 cells, Treg cells, CD4+ T-cells, CD8+ T-cells, and CD3+ T-cells in peripheral blood at different times before and after MWA were studied using analysis of variance of repeated measures. The comparison of two mean values of two groups of numbers was performed using Chi-square test, t-test or Mann–Whitney U-test or two-tailed tests. Receiver operating characteristic (ROC) curve was used to determine the cutoff value. P < 0.05 indicates a statistically significant difference. Statistical analysis was performed by SPSS 21.0 Software package (IBM Corporation, USA).
| > Results|| |
Changes of circulating T-cell subsets after microwave ablation
The results of flow cytometry are shown in [Figure 1]. The proportions of blood circulating T-cell subsets, except for Th17 cells, of the patients before, 24 h after, 72 h after and 1 month after MWA treatment were relatively stable [Table 2]. The time evolutions of T-cell subsets are shown in [Figure 2]. The proportion of Th17 cells increased 24 h after the treatment (3.98% ± 2.40% vs. 5.53% ± 3.27%, P = 0.015) and returned to a similar level as that before treatment in 3 days (before vs. 72 h: [3.98% ± 2.40%] vs. [4.53% ± 1.82%], P = 0.384). Although the proportions of Treg, CD4+ T, CD8+ T, and CD3+ T-cells also fluctuated after treatment, there was no significant difference between these proportion and the corresponding values before treatment.
|Figure 1: Proportions of T cell subsets in peripheral blood of the patients. (a) The proportion of Treg cells; (b) the proportion of Th17 cells; (c-e) the proportions of CD4+ T, CD8+ T and CD3+ T cells, respectively|
Click here to view
|Table 2: Proportions of T-cell subsets before and after microwave ablation treatment|
Click here to view
|Figure 2: Time evolution of T-cell subsets in patients with hepatocellular carcinoma after microwave ablation treatment|
Click here to view
Results of univariate regression analysis on tumor recurrence
The median follow-up period was 16 months (ranging from 15 to 19 months) and 8 patients suffered from tumor recurrence. Univariate logistic regression was used to analyze the potential-related factors of tumor recurrence, including age, sex, smoking and drinking history, number of tumors, tumor size, Child-Pugh Classification, alpha-fetoprotein (AFP), the elevation of Th17 cells after 24 h, proportions of Th17 cells, and Treg cells before treatment. Sixteen patients had an elevation of Th17 cells after 24 h. The cutoff value of Th17 cells and Treg cells, 5.6% and 4.3%, respectively, were found by ROC curve. Seven patients were with a level of Th17 cells higher than the cutoff value and the rest 23 patients had a proportion lower than it. Eighteen patients were with a proportion of Treg cells higher than 4.3% while the frequency of the rest 12 patients lower than it. The results of univariate logistic regression were shown in [Table 3]. Among the 11 potential related factors, only the frequency of Th17 cells was found to be the risk factor of tumor recurrence. The level of Treg cells failed to be statistically significant.
Considering tumor size and AFP may have effects on the results, patients with tumor size >3 cm and AFP >400 ng/mL were excluded from the study. The rest 23 patients with six recurrences HCC were reanalyzed by logistic regression. The results showed that with high-risk coefficient, circulating Th17 cells was indeed the related factor of tumor recurrence [Table 3].
| > Discussion|| |
Th17 and Treg cells, two new subsets of CD4+ T-cells, play an important role in immune responses and disease progression in patients with chronic HBV infection. It is clear that in the early stage of HBV infection, the IL-10 and transforming growth factor-β produced by Tregs can inhibit a reaction of inflammation and prevent the liver from damage.,, On the contrary, Th17 can upregulate the expression of inflammatory cytokine to promote inflammatory response., Many studies demonstrated that patients with HBV infection had high frequencies of blood circulating Treg cells and Th17 cells and that the frequencies of these two subsets were correlated with the disease progression.,, In addition to their role in peripheral inflammation, Th17 and Treg were also associated with the progress and metastasis of HCC.
Thermal ablation, as a radical treatment of small HCC, was widely used in the treatment of liver malignancy., Previous studies demonstrated that the coagulated tumor tissue induced by thermal ablation can cause an inflammatory reaction, activate tumor-specific T-cells and change the number of T-cells and the level of cytokine in peripheral blood., This inflammatory reaction is due to the release of tumor antigens derived from coagulation necrosis tumor tissue. Some studies showed that thermal ablation can decrease the number of Treg cells and CD8+ T-cells, and increase the proportion of CD3+ T-cells, which may have a protective effect on the prognosis for patients.,, On the contrary, a few studies demonstrated that thermal ablation does not change the levels of T subsets cells because of its minimal invasion. Therefore, the effects of thermal ablation on T-cell subsets and the association of local immune response with disease progression were still not well studied. To clarify the immune response evoked by MWA, we detected the proportions of T-cells subsets before and after the treatment dynamically.
The results of our study show that the levels of T-cell subsets, including CD4+ T-cells, CD8+ T-cells, CD3+ T-cells and Treg cells, were relatively stable except for the proportion of Th17 cells. The proportion of Th17 cells increased from (3.98% ± 2.40%) (before treatment) to (5.53% ± 3.27%) 24 h after treatment. However, the high level of Th17 cell did not maintain for a long time. Instead, it recovered within three days. Our study indicates that MWA has a transitional effect on Th17 cells rather than Treg cells or other T-cell subsets, contrary to some previous studies suggesting that local thermal ablation may induce changes of T-cell subsets.,, We infer that there are two reasons for this difference. First, we used MWA only in this study while previous studies usually used radiofrequency ablation (RFA) or thermal ablation combined with transarterial chemoembolization. It is not unexpected that patients received two treatments suffered from a more severe injury than those experienced only one treatment. Besides, the inflammatory response caused by MWA was slighter than RFA because of the different mechanism of tissue coagulation necrosis. Compared with RFA, MWA has advantages of higher heating velocity, higher intratumoral temperature, and shorter duration. The high power and short duration of MWA may lead to less local inflammation and secondary responses. This hypothesis can be rationalized by several studies, which demonstrated that RFA can induce a significant inflammatory response while MWA can only evoke an extremely minimal immune response., Second, the tumor burden of patients in this study is slight, which result in a mild tumor immunosuppression.
With a median follow-up of 16 months, eight patients suffered from intrahepatic recurrence. We analyzed the risk factors of tumor recurrence after MWA. Eleven potential-related factors were assessed by univariate regression, and the results showed that the proportion of baseline circulating Th17 cells rather than the transient elevation of Th17 cells after 24 h was a risk factor of tumor recurrence. It is not unexpected that the marginal increase of Th17 cells for <3 days cannot significantly affect the consequent recurrence of tumors after MWA. Tumor size and AFP, which were confirmed to be the related factors of prognosis,, however, were not found to have any influence on tumor recurrence in this study. We infer that this inconsistency may be because all the patients included in this study met Milan criteria and were at a tumor stage of Barcelona Clinic Liver Cancer A. Besides, only five patients were with a size of tumor larger than 3.0 cm and two patients were with a level of AFP >400 ng/mL, which makes the tumor size and the proportion of AFP failed to be statistically significant. Considering that, the patients with a large tumor size or a high level of AFP may interfere the results of the logistic regression that Th17 cells were the risk factor of tumor recurrence because large tumor size usually accompanied by high level of Th17 cells, we excluded the patients with tumour size >3 cm or level of AFP >400 ng/mL for another logistic regression. The result of such regression showed that circulating Th17 cells was a “real”-related factor of tumor recurrence.
Some of the previous studies found that tumor infiltrating Treg or Th17 cells can be regarded as the predictor of prognosis in patients with HCC. For example, Liao et al. found that patients with a high expression of intratumoural IL-17 may be more likely to have a poor survival. Huang et al. also showed that patients with high frequencies of intratumoural Treg cells had a significantly lower survival rate than those with a small number of intratumoural Treg cells. The numbers of tumor-infiltrating T-cell subsets are usually detected from biopsy of liver tissues. The patient acceptance of this invasive inspection, however, is poor. Tumor-infiltrating T-cell subsets, therefore, are not the readily available markers for monitoring the prognosis of patients. Our results demonstrate that the proportion of blood circulating Th17 cells, similar to the tumor-infiltrating T-cell subsets, can be regarded as a predictor of tumor progression before MWA. Patients with high levels of Th17 cells before treatment are prone to suffering from tumor recurrence. However, we found that the level of Treg cells has no effect on tumor recurrence in this study. We infer this difference may be due to the distinct effects between Treg and Th17 cells on tumor progression. Treg cells display a suppressive function in the T-cell immunity and antitumour immune response. Treg cells, therefore, promote tumor progression by increasing the immune escape of tumor cells and then accelerating tumor growth., Th17 cells promote tumor growth and distant metastasis by fostering tumor angiogenesis, contributing more directly to the tumor progression.
This study has several limitations. First, the cytokines produced by T-cells were not studied. Further investigations are needed to study the effects of cytokines on the prognosis of patients with HCC. Second, overall survivals (OS) of patients were not observed because of the short-term follow-up. The impacts of T-cell subsets on the OS of patients, therefore, are not clarified.
| > Conclusions|| |
In summary, MW ablation has a transitional influence on Th17 cells but not Treg cells or other T-cell subsets. The proportion of Th17 cells increased and peaked 24 h after MWA. However, it recovered to a similar level as that before treatment within 3 days. The high proportion of blood circulating Th17 cells before treatment, instead of the transient elevation of Th17 cells 24 h after MWA, is an indicator for tumor progression. The level of Th17 cells can be used to predict the recurrence of tumor in patients with HCC.
The present work was supported by the Key Research Project of Tianjin Healthy Bureau, No. 13KG111 and Tianjin Science and Technology Commission funded key project (No. 13JCYBJC22500).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al.
Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 2015;136:E359-86.
Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A, et al.
Global cancer statistics, 2012. CA Cancer J Clin 2015;65:87-108.
Yan J, Liu XL, Xiao G, Li NL, Deng YN, Han LZ, et al.
Prevalence and clinical relevance of T-helper cells, th17 and th1, in hepatitis B virus-related hepatocellular carcinoma. PLoS One 2014;9:e96080.
Hammerich L, Heymann F, Tacke F. Role of IL-17 and th17 cells in liver diseases. Clin Dev Immunol 2011;2011:345803.
Zhang HH, Mei MH, Fei R, Liu F, Wang JH, Liao WJ, et al.
Regulatory T cells in chronic hepatitis B patients affect the immunopathogenesis of hepatocellular carcinoma by suppressing the anti-tumour immune responses. J Viral Hepat 2010;17 Suppl 1:34-43.
Zhou P, Liang P, Dong B, Yu X, Han Z, Xu Y, et al.
Phase I clinical study of combination therapy with microwave ablation and cellular immunotherapy in hepatocellular carcinoma. Cancer Biol Ther 2011;11:450-6.
Guan HT, Wang J, Yang M, Song L, Tong XQ, Zou YH, et al.
Changes in immunological function after treatment with transarterial chemoembolization plus radiofrequency ablation in hepatocellular carcinoma patients. Chin Med J (Engl) 2013;126:3651-5.
Gao HJ, Zhang YJ, Liang HH, Li P, Peng ZW, Pang XH, et al.
Radiofrequency ablation does not induce the significant increase of CD4+ CD25+ foxp3+ regulatory T cells compared with surgical resection in hepal-6 tumor model. Arch Immunol Ther Exp (Warsz) 2013;61:333-40.
Lafdil F, Miller AM, Ki SH, Gao B. Th17 cells and their associated cytokines in liver diseases. Cell Mol Immunol 2010;7:250-4.
Huang Y, Wang F, Wang Y, Zhu Z, Gao Y, Ma Z, et al.
Intrahepatic interleukin-17+T cells and foxP3+ regulatory T cells cooperate to promote development and affect the prognosis of hepatocellular carcinoma. J Gastroenterol Hepatol 2014;29:851-9.
Gu FM, Li QL, Gao Q, Jiang JH, Zhu K, Huang XY, et al.
IL-17 induces AKT-dependent IL-6/JAK2/STAT3 activation and tumor progression in hepatocellular carcinoma. Mol Cancer 2011;10:150.
Chisari FV, Isogawa M, Wieland SF. Iconography: Pathogenesis of hepatitis B virus infection. World J Gastroenterol 2007;13:82-90.
Peng G, Li S, Wu W, Sun Z, Chen Y, Chen Z, et al.
Circulating CD4+ CD25+ regulatory T cells correlate with chronic hepatitis B infection. Immunology 2008;123:57-65.
Wei HX, Chuang YH, Li B, Wei H, Sun R, Moritoki Y, et al.
CD4+ CD25+ foxp3+ regulatory T cells protect against T cell-mediated fulminant hepatitis in a TGF-beta-dependent manner in mice. J Immunol 2008;181:7221-9.
Stross L, Günther J, Gasteiger G, Asen T, Graf S, Aichler M, et al.
Foxp3+ regulatory T cells protect the liver from immune damage and compromise virus control during acute experimental hepatitis B virus infection in mice. Hepatology 2012;56:873-83.
Stockinger B, Veldhoen M. Differentiation and function of th17 T cells. Curr Opin Immunol 2007;19:281-6.
Wu W, Li J, Chen F, Zhu H, Peng G, Chen Z, et al.
Circulating th17 cells frequency is associated with the disease progression in HBV infected patients. J Gastroenterol Hepatol 2010;25:750-7.
Ding J, Jing X, Liu J, Wang Y, Wang F, Wang Y, et al.
Comparison of two different thermal techniques for the treatment of hepatocellular carcinoma. Eur J Radiol 2013;82:1379-84.
Ding J, Jing X, Liu J, Wang Y, Wang F, Wang Y, et al.
Complications of thermal ablation of hepatic tumours: Comparison of radiofrequency and microwave ablative techniques. Clin Radiol 2013;68:608-15.
Chu KF, Dupuy DE. Thermal ablation of tumours: Biological mechanisms and advances in therapy. Nat Rev Cancer 2014;14:199-208.
Hiroishi K, Eguchi J, Baba T, Shimazaki T, Ishii S, Hiraide A, et al.
Strong CD8(+) T-cell responses against tumor-associated antigens prolong the recurrence-free interval after tumor treatment in patients with hepatocellular carcinoma. J Gastroenterol 2010;45:451-8.
Fatourou EM, Koskinas JS. Adaptive immunity in hepatocellular carcinoma: Prognostic and therapeutic implications. Expert Rev Anticancer Ther 2009;9:1499-510.
Shaobin W, Yu X, Jiatian L, Zaizhong C, Luping D, Junhui C, et al.
Changes of CD4+ T-cell subsets after radiofrequency ablation in lung cancer and its significance. J Cancer Res Ther 2016;12:C166-70.
Ahmad F, Gravante G, Bhardwaj N, Strickland A, Basit R, West K, et al.
Changes in interleukin-1β and 6 after hepatic microwave tissue ablation compared with radiofrequency, cryotherapy and surgical resections. Am J Surg 2010;200:500-6.
Velez E, Goldberg SN, Kumar G, Wang Y, Gourevitch S, Sosna J, et al.
Hepatic thermal ablation: Effect of device and heating parameters on local tissue reactions and distant tumor growth. Radiology 2016;281:782-92.
Ding J, Jing X, Wang Y, Wang F, Wang Y, Du Z, et al.
Thermal ablation for hepatocellular carcinoma: A large-scale analysis of long-term outcome and prognostic factors. Clin Radiol 2016;71:1270-6.
Lam VW, Ng KK, Chok KS, Cheung TT, Yuen J, Tung H, et al.
Risk factors and prognostic factors of local recurrence after radiofrequency ablation of hepatocellular carcinoma. J Am Coll Surg 2008;207:20-9.
Liao R, Sun J, Wu H, Yi Y, Wang JX, He HW, et al.
High expression of IL-17 and IL-17RE associate with poor prognosis of hepatocellular carcinoma. J Exp Clin Cancer Res 2013;32:3.
Huang Y, Wang FM, Wang T, Wang YJ, Zhu ZY, Gao YT, et al.
Tumor-infiltrating foxP3+ tregs and CD8+ T cells affect the prognosis of hepatocellular carcinoma patients. Digestion 2012;86:329-37.
Numasaki M, Fukushi J, Ono M, Narula SK, Zavodny PJ, Kudo T, et al.
Interleukin-17 promotes angiogenesis and tumor growth. Blood 2003;101:2620-7.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]