Effect of microwave ablation treatment against hepatic malignance on serum cytokine level.

Microwave ablation (MWA) is widely used to treat unresectable primary and secondary malignancies of the liver, and a limited number of studies indicate that ablation can cause not only necrosis at the in situ site but also an immunoreaction of the whole body. This study aimed to investigate the effects of MWA on cytokines in patients who underwent MWA against a hepatic malignancy. Methods between June 2015 and February 2019 were selected. Peripheral blood was collected from patients with a hepatic malignancy treated with MWA. The levels of cytokines (IL-2, IFN-γ, TNF-α, IL-12 p40, IL-12 p70, IL-4, IL-6, IL-8, IL-10, and vascular endothelial growth factor (VEGF)) were detected with a MILLIPLEX ® MAP Kit. The comparison times were as follows: before ablation, 24 hours after ablation, 15 days after ablation, and 30 days after ablation. Data were analyzed using a paired sample t-test and Spearman’s correlation analysis. A total of 43 were differences IL-12 IL-1β, IL-8, and TNF-α at 24 after (>2-fold vs. before were and after Elevated and levels were correlated

Effect of microwave ablation treatment against hepatic malignance on serum cytokine level. Introduction Primary and secondary malignancies of the liver have a substantial impact on morbidity and mortality worldwide. In China, hepatocellular carcinoma (HCC) ranks second in the mortality rate of malignancies [1]. The treatment of primary and secondary hepatic malignancies via interventional imaging therapy is undertaken by investigators in the eld of interventional radiology and possibly by a smaller group of practitioners known as interventional oncologists, whose major focus is cancer care via minimally invasive approaches [2,3]. Recently, percutaneous ablation therapy has been widely accepted as one of the radical treatment methods for HCC, and its ve-year survival rate is similar to that of resection [4]. Microwave ablation (MWA) is widely used to treat unresectable HCC and recurrent HCC with the advantages of minimal invasion, a good curative effect, and no radiation or chemotherapy side effects. Immune checkpoint inhibitors(ICI) such as PD-1/PD-L1 and CTLA4 antibodies were widly applicated in several cancers, researches indicated that ICI treatment could enhance the effect of ablation [5]. Evidence indicated that hyperthermia destruction caused release of a large population of heterogenous tumor antigens and in ammatory cytokines may play crucial roles [6]. Cytokines are mediators that regulate a broad range of processes involved in the pathogenesis of cancer. Several cytokines, which can arise from either tumor cells or immunocytes [7], such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, IL-10, and vascular endothelial growth factor (VEGF), have been linked with cancers and can either promote or inhibit tumor development. The serum levels of cytokines differ during cancer development. Although cytokines have found to be altered after anticancer treatment, such as chemotherapy and radiotherapy [8,9], few investigations have focused on cytokines before and after MWA. It is still unknown whether the above cytokines changed before and/or after MWA in patients with a hepatic malignancy. In this study, we investigated the effects of MWA on the serum levels of cytokines in patients with hepatic malignancies.
Material And Methods

Patients and samples
The patient population examined in this study was derived from the First A liated Hospital of Soochow University. Patients were admitted to the Oncology Department between June 2015 and February 2019. The total number of patients was 43, with 37 liver metastases and 6 primary liver cancers. The inclusion criterion was a tumor located at a hepatic site (either primary or metastases). All patients with metastastic hepatic malignances should be underwent systemetic treatments (chemotherapy or target therapy) and get at least stable disease (SD) or particial partial response (PR) for more than 45 days. Informed consent for blood draw and the relevant therapy was obtained from all patients. The protocol was approved by the Human Ethics Committee of the First A liated Hospital of Soochow University and was conducted in accordance with the Declaration of Helsinki. Whole blood (4 mL) was drawn into EDTA anticoagulant tubes on days -3 to 0 before and 24 hours, 15 days, and 30 days after ablation, mostly on the last day of the course, for cytometry and cytokine analyses.

Ablation procedure
The ablation procedure used in this research was MWA. The puncture site and pathway were determined under the guidance of a computed tomography (CT) scan. Local in ltration anesthesia was achieved by using 0.5% lidocaine. The placement of microwave ablation probes was guided by CT scan or ultrasonic device, all probes were placed at the maximum diameter layer. Double probes were employed when the maximum diameter of tumor was up to 3cm. The power and time of ablation were designed for each patient in the range of 40~70 W and 5~20 min, respectively, based on the size, number, and position of the tumor. The boundaries of ablation zones were designed as extended 1cm upon tumor site.

Statistical analysis
IBM SPSS Statistics 20.0 software was used for the statistical analysis, along with GraphPad Prism 8 for gure creations. Normally distributed numerical data are expressed as the mean ± standard deviation (SD), and nonnormally distributed numerical data are expressed as the median and 95% con dence interval (95% CI). Cytokines at different times were compared using a one-tailed paired t-test. Spearman's correlation analysis was executed to analyze the correlation between clinical indexes and cytokine levels. p<0.05 indicated a signi cant difference.

Clinical characteristics of the patients enrolled
As shown in table 1, a total of 43 patients with tumors located on the liver (37 liver metastases, 6 primary liver cancers) were analyzed. The patients' cytokine levels were compared according to time: before treatment, 24 hours after treatment, 15 days after treatment, and 30 days after treatment.

Discussion
Local therapies such as surgical resection play a critical role in liver cancer. However, surgical operations are limited by an insu cient patient condition or tumor metastasis. As technology continues to develop, other types of local therapy, such as radiotherapy, chemical ablation and hyperthermal ablation, for primary and metastatic liver cancer are increasingly being used. MWA for liver malignances is reserved for patients who are not suitable for surgical removal or whose other treatments have failed [10]. A consensus guideline was recently developed to address indications for MWA in these patients. Thermal ablation is a process that heats the target tissue to a temperature that causes immediate coagulative necrosis (usually over 100°C). Terminal treatment requires that a necrotic area surrounds the target site with an additional 5-10-mm margins [11]. However, in the liver, high tissue perfusion and large blood vessels can cause a "heat sink effect" around the ablation zone, making it di cult to achieve terminal ablation [12]. The heat sink effect can lead to sublethal temperatures and the retention of malignant cells, thereby increasing the likelihood of local tumor progression (LTP) [13]; however, an incompletely ablated zone containing immune cells and cancer cells, as well as functional vessels, could establish a serious in ammatory site that may provide tumor-speci c antigens, cytokines, and activated immune cells.
In recent years, ablation-induced systemic effects, such as the tumor-associated immune response, have attracted wide attention [14]. de Baere T rst reported two cases of the spontaneous regression of multiple pulmonary metastases occurring after the radiofrequency ablation of a single lung metastasis [15]. Although growing evidence suggests that thermal ablation can induce spontaneous regression of the socalled "abscopal effect" on distant tumors, the incident rate of such an effect is rare, probably due to uncontested immunological activation caused by one ablation treatment and the lack of immunoampli cation management. In 2004, it was described that in situ tumor destruction can provide a useful antigen source for the induction of antitumor immunity [16]; however, clinical studies could not su ciently utilize such an effect until the development of immune checkpoint blockade reagents [17,18].
Ablation therapy can mediate antitumor immunity, as tumor tissue necrosis caused by ablation may release a scale of antigens that eventually form a kind of "in situ vaccination" [19]. Moreover, ablative therapy cannot only directly kill cancer cells in situ but also regulate immune cells and promote the immune function of patients with liver cancer [20,21]. Many immunoregulatory cytokines are released or expressed after thermal ablation. It is important to note that the cytokines released after thermal ablation can regulate the positive and negative aspects of the cancer immune cycle. Previously, researchers demonstrated that proin ammatory cytokines such as IL-1, IL-6, IL-8, IL-18, and TNF-α were increased several hours or days after thermal ablation [22][23][24]. In our investigation, we employed a concept of "energy" to evaluate the relationship between the increased levels of cytokines and MWA treatment. Our ndings indicated that IL-2 and IL-6 were signi cantly evaluated after the ablation procedure and positively correlated with MWA energy. IL-2 is commonly derived from activated T cells, primarily Th1 cells. IL-2 can stimulate T cells to proliferate and differentiate, activate natural killer (NK) cells and macrophages, and enhance the functions of cytotoxic T lymphocytes (CTLs) [25]. Our ndings suggest that IL-2 is signi cantly increased at 24 h after MWA, indicating that IL-2 may induce a nonspeci c immune response after MWA. The increase IL-2 did not last as long, but it decreased 24 h post-MWA in our study, suggesting that the IL-2-induced immune response may not last as long.
Immune checkpoint inhibitors(ICI) such as PD-1/PD-L1 and CTLA4 antibodies were widly applicated in several cancers, researches indicated that ICI treatment could enhance the effect of ablation [5]. Evidence indicated that hyperthermia destruction caused release of a large population of heterogenous tumor antigens and in ammatory cytokines may play crucial roles[6]. However, opposite evidence indicated that by incomplete radiofrequency ablation-induced in ammation could accelerates tumor progression and hinders PD-1 immunotherapy[26], suggesting there could be a mechanism that ablation treatment may promote tumor progression. Our data demonstrated that IL-6 was signi cantly increased after MWA treatment, IL-6 is derived from monocytes, macrophages, Th2 cells and sometimes cancer cells, and it plays a key role in T cell proliferation and survival [27]. Evidence indicates that IL-6 plays an indispensable role in T cell in ltration to the tumor site, which could bene t immunomodulatory therapy. However, IL-6 can increase myeloid-derived suppressor cells (MDSCs) [28], inhibit the development and maturation of dendritic cells (DCs) [29], and inhibit the polarization of Th1 cells [30], eventually illustrating a negative immunomodulatory function. According to Muneeb Ahmed's work, the adjuvant use of a nanoparticle small interfering RNA (siRNA) can be successfully used to target the IL-6-mediated locoregional and systemic effects of thermal ablation. IL-6 knockout via a nanoparticle anti-IL-6 siRNA in mice could decrease the local VEGF level at the ablation site [31]. Therefore, how to utilize the positive effect of IL-6 while avoiding the negative effect after MWA needs further investigation. Preclinical research indicated that IL-6 and PD-L1 blockade combination therapy reduced tumour progression in animal models [32,33]. Thus, anti-IL-6 strategy after ablation should be consiterable while combined with ICI therapy. Previous studies and ours demonstrate that most cytokine levels returned to pretreatment levels 30 days after ablation. This result suggests that 24 hours to 15 days after ablation may be optimal timing for additional immunomodulatory therapy.