Cryoablation combined with allogenic natural killer cell immunotherapy improves the curative effect in patients with advanced hepatocellular cancer

In this study, the clinical efficacy of cryosurgery combined with allogenic natural killer cell immunotherapy for advanced hepatocellular cancer was evaluated. From October 2015 to March 2017, we enrolled 61 patients who met the enrollment criteria and divided them into two groups: 1) the simple cryoablation group (Cryo group, n = 26); and 2) the cryoablation combined with allogenic natural killer cells group (Cryo-NK group, n = 35), the safety and short-term effects were evaluated firstly, then the median progression-free survival, response rate and disease control rate were assessed. All adverse events experienced by the patients were recorded, and included local (e.g., pain, pleural effusion, and ascites) and systemic (e.g., chills, fatigue, and fever) reactions, fever was more frequent. Other possible seriously side effects (e.g., blood or bone marrow changes) were not detected. Combining allogeneic natural killer cells with cryoablation had a synergistic effect, not only enhancing the immune function, improving the quality of life of the patients, but also reducing the expression of AFP and significantly exhibiting good clinical efficacy of the patients. After a median follow-up of 8.7 months (3.9 –15.1months), median progression-free survival was higher in Cryo-NK (9.1 months) than in Cryo (7.6 months, P = 0.0107), median progression-free survival who received multiple natural killer was higher than who just received single natural killer (9.7 months vs.8.4 months, P = 0.0011, respectively), the response rate in Cryo-NK (60.0%) was higher than in Cryo (46.1%, P < 0.05), the disease control rate in Cryo-NK (85.7%) was higher than in Cryo group (69.2%, P < 0.01). Percutaneous cryoablation combined with allogeneic natural killer cell immunotherapy significantly increased median progression-free survival of advanced hepatocellular cancer patients. Multiple allogeneic natural killer cells infusion was associated with better prognosis to advanced hepatocellular cancer.


INTRODUCTION
Hepatocellular carcinoma (HCC), which is the most common type of primary liver cancer, due to usually discovered late and has a poor prognosis [1]. Although surgical resection is the best option to HCC, but many patients suffer bad factors such as cirrhosis, multiple tumors or other diseases, so they are unsuitable for surgical Research Paper www.impactjournals.com/oncotarget resection [2,3]. Up to now, systemic chemotherapy dose not obviously increase survival advanced HCC patients [4,5]. Radiation has toxicity to the normal liver, so it is limited method to treat HCC [6,7]. Hence, the newer and more effective therapies for advanced HCC patients are needed.
Percutaneous cryoablation is a new method which can induce tumor necrosis, it is currently considered the best option for treating unresectable HCC [8]. Cryosurgery has many advantages which carried out successfully in many solid tumors (e.g., prostate cancer and RR) [9,10], and also emerged as a new therapy pattern for HCC [11,12].
There are some reports have stated that cancer occurence and development in HCC patients are affected by the tumor immune [13][14][15][16][17][18]. However, due to downregulating the expression of MHC, tumor cells often immune escape [19] . Manipulating the immune system for therapeutic benefit in HCC patients has been studied for many decades [20][21][22]. NK cells are belonged to the innate immune system and play a importment role against cancer in the early time [23,24]. With progress in the NK cell biology field and in understanding NK function, adoptive NK cell transfer has promising anti-tumor effects on various cancers [25][26][27][28][29], including liver cancer [30][31][32][33][34].
In our research, we prospectively investigated the clinical response of cryosurgery combining with NK cell immunology for advanced HCC, to elucidate a potential therapeutic strategy.

Identification of NK cells in the enrolled patients
In line with the protocol described in Figure 1B, the peripheral blood of the enrolled patients' kinsfolk was separated in the laboratory. Prior to the cell culture, the median fraction of the CD3 -CD56 + population was 6.69% (range: 4.3% -14.9%). Following the cell culture and expansion, the viable cells exceed 90%. The median proportion of CD56+ cells was 86.1% (range: 71.6% -96.1%). The median percentage, viable cells and the median proportion of CD56+CD3-cells were similar to our prior report [35]. The representative results are presented in Figure 2.

Safety and clinical efficacy evaluation
All adverse events were recorded, including local (e.g., pain, pleural effusion, and ascites) and systemic (e.g., chills, fatigue, and fever) reactions, fever was more frequent.
The adverse events was compared using a chi-square test; they were no differences (P = 0.8481; Figure 3A).
The pre-treatment immune test data of were merged and compared with post-treatment (Table 2). Aimed to the counts, all cells were exceed in the Cryo-NK group following treatment (P < 0.01), especially to NK, the number in Cryo-NK group was significantly higher (P < 0.001). Considering lymphocyte functionality, the level of Th1 cytokines was also improved in the cryo-NK group, the level of Th2 cytokine levels remained invariability.
AFP expression was higher than normal at 1 day pre-treatment and decreased gradually at day 3, and 1 month and 3 months post-treatment in the two groups ( Figure 3B). There was no difference between the two groups at post-treatment day 3 (P > 0.05), but at 1 month and 3 months post-treatment, AFP expression was obviously lower in the Cryo-NK ( Figure 3B, P < 0.01), even there were 13patients within normal range who received Cryo-NK therapy.
The pre-treatment KPS of the Cryo-and Cryo-NK groups was 68.7 ± 6.5 and 70.1 ± 3.7, respectively, 78.6 ± 4.2 and 82.9 ± 5.1 one month post-treatment, respectively, and 81.2 ± 4.7 and 90.1 ± 1.6 at three months posttreatment, respectively. Compared with the pre-treatment KPS, the scores of both groups were obviously improved post-treatment ( Figure 3C; P < 0.01); however, the KPS was higher in the Cryo-NK group both one month and three months post-treatment ( Figure 3D; P < 0.05).
The clinical efficacy between the two groups was observed three months post-treatment. The maximum transverse diameters are listed in Table 3. The tumor volume was obviously decreased post-treatment in both two groups; however, three months post-treatment, the maximum tumor diameter in cryo-NK was smaller than Cryo (P < 0.01). In Table 4, CT value was obviously decreased post-treatment, the CT value three months posttreatment in the Cryo-NK group was lower than that of the Cryo group (P < 0.05). During the follow-up period, there was no patient died; the number of CR in Cryo was 5 but 9 in cryo-NK (Table 5). In addition, the RR in cryo-NK was 60.0% higher than 46.1% in Cryo (P < 0.01), similarly, DCR in cryo-NK was obviously higher than patients who just received Cryo (85.2% vs. 69.5%, P < 0.01). The representative results are shown in Figure 4.

DISCUSSION
For the majority of patients with advanced HCC, tumor is not suitable to resect. With many advances and www.impactjournals.com/oncotarget  Note: All cell subsets and cytokines were analyzed via a Dunnett's multiple comparison test (one-way ANOVA). NK cell, natural killer cell; IL, interleukin; TNF, tumor necrosis factor; IFN, interferon *P < 0.05; **P < 0.01; ***P < 0.001.    [39,40]. The recent success of immunotherapy has highlighted the potential of immune-based therapy approaches for advanced HCC treatment [21,41,42]. Our previous research of Cryo-DC/CIK treatment with in metastatic pancreatic cancer [43] and HCC [44] has displayed a good clinical outcome. After cryoablation, account of continued antigen released, which could stimulate the immune system sequentially, but CIK could not kill the low tumor burden directly, so we brought in NK cells technology by understanding NK function. In our study, we attempted to investigate the safety and clinical efficacy of percutaneous cryoablation combined with allogeneic natural killer cell therapy for treating advanced HCC, then the median PFS, RR and DCR were assessed. To our surprise was that allogeneic NK cells combined with cryosurgical for advanced HCC exhibited a synergistic effect, significantly enhanced the immune function of patients ( Table 2). The increasing number of NK cells after allogeneic NK cells immunotherapy may be related to ectogenic NK cells amplified, because NK cells were infused in the period of logarithmic phase which owned the best activity and amplified sequentially. Moreover, combined treatment can markedly improve the  median PFS prolonger, and the median PFS was higher in patients who received multiple NK than who just underwent only one course NK, also to RR and DCR (both P < 0.05). Facts proved that this comprehensive therapy was also safety and efficacy. For decades, natural killer (NK) NK cells existed as "non-specific" killer cells were different from CTL or other immunocytes identified the target. We have learned that NK cells are trained to recognize "non-self" histocompatibility antigens (human leukocyte antigen, HLA) on the surface of cells through their killer cell immunoglobulin-like (KIR) receptors. Recent discoveries that better explain how NK cells recognize and kill their targets and their ability to produce immune-active cytokines have made them more attractive tools for immunotherapy. In view of this, we brought NK cells into this clinical research. In our previous report [45], showed a good outcome for allogenic NK cell immunotherapy to advanced renal cell cancer and metastasis pancreatic cancer [35]. Therefore, we used allogeneic NK cell adoptive therapy in this study. Facts proved that it was tolerant and efficacy. But whether the current number and purity of NK cells would be the optimal dose or not which is worthy us considering, maybe, we will carry out the dose grope in the future.
In conclusion, in this single-center prospective study, we provided evidence that allogeneic NK cell therapy combined with percutaneous cryoablation exhibited a favorable outcome for advanced HCC patients, indicating a potential novel therapeutic strategy.

Ethics
This study was approved by the Ethics Committee of Guangzhou Fuda Cancer Hospital. In accordance with the Declaration of Helsinki, written informed consent effusion, and ascites were nearly remitted compared with the patients oral at pre-treatment following symptomatic treatment; (B) There was no difference between the two groups at post-treatment day 3 (P > 0.05), but at 1 month and 3 months post-treatment, AFP expression was obviously lower in the cryo-NK group than in the cryo group; (C) Compared with the pre-treatment KPS, the scores of both groups were obviously improved post-treatment; (D) The KPS was higher in the cryo-NK group both one month and three months post-treatment. *P < 0.05; **P < 0.01. www.impactjournals.com/oncotarget

Percutaneous cryoablation
The percutaneous puncture point was determined by computed tomography (CT). All cryoablations were performed with the commercially available Cryocare Surgical System (Endocare, Irvine, CA, USA) using argon and helium gas as the cryogen. Cryoprobes (3, 5 or 8 mm) were inserted into the center of the tumor mass under CT guidance and two freeze/thaw cycles were performed, each reaching a temperature between −125°C and −150°C for 15 min followed by rewarming at 15-20°C. A margin of at least 1 cm of normal breast tissue was frozen circumferentially around the tumor. For masses larger than 5 cm in diameter, two or three cryoprobes were placed within the center and periphery of the tumor, to ensure freezing of the entire mass. After cryoablation, antibiotics and haemostatic agents were administered and vital signs such as blood pressure, pulse, respiration and blood oxygen saturation were monitored routinely.

CT and MRI examination
HCC patients confirmed by pathology were required to undergo dynamic CT or MRI one week pre-treatment, with a follow-up at one and three months post-treatment. The maximum diameter and CT value were measured and compared between the pre-and post-treatment values. A comprehensive analysis combined with the clinical data was completed together by two experienced nuclear medicine physicians who had multiple years of experience in making CT diagnoses.

NK cell therapy
Clinical-grade NK cells were cultured using clinical-grade reagents and under good manufacturing practice conditions. The human high activity NK cell in vitro preparation kit was used (Hank Bioengineering Co. Ltd, Shenzhen, China) that contained chimeric active cellular factors on K562 cell membranes [46], plasma treatment fluid, lymphocyte culture fluid additives, serum-free medium additives and cell infusion additives. This kit is intended for expanding and activating NK cells in peripheral blood mononuclear cells in vitro to prepare NK cells of higher quantity, purity and activity, namely highly activated NK (HANK) cells [47]. Blood samples from the patient and donors were analyzed using the TIANamp Blood DNA kit (Tiangen Biotech Co., Ltd., Beijing, China) and KIR/HLA-C allotypes Genotyping Low Resolution kit (Tianjin Super Biotechnology Developing Co., Ltd., Tianjin, China). Approximately 8-10 billion HANK cells may be harvested after culture from 80 ml of peripheral blood using NK cell serumfree medium and culture bags (Tianjin Haoyang Biological Manufacture Co., Ltd, Tianjin, China). Cell counting and quality control inspection are commonly performed on day 12 of culture, and the quality indicators include ≥8 billion total cells with ≥ 90% living cells, ≥ 85% CD3−/CD56+ cells [47], ≤ 1 EU/ml endotoxin, ≥ 80% cell killing activity against K562 target cells [47], and bacteria-, fungi-and mycoplasma-negative culture. After 12 days of cell culture, the NK cells were divided into three groups and intravenously infused into the patients from Day 13 to 15. Each patient must two cycles NK therapy continuously as one course.
NK cells were generated according to previously published protocols [47], 80 ml peripheral blood from allogenic donors was drawn 7 days before cryoablation and the immunotherapy was given 3days after cryoablation.
For donor selection, the killer cell immunoglobulinlike receptors (KIRs) genotyping should be mismatched to the human leukocyte antigen (HLA) class I molecules of the patient [47][48][49][50][51]. We used PCR-SSP to detect the KIR/ HLA-Cw which can get the result on the day.

Cryo-NK therapy procedure
All of the enrolled patients' kinsfolk were informed, and the peripheral blood was collected for NK cell isolation seven days before cryoablation. Cryoablation was carried out on Day 9 and Day 12, and the cultured NK cells were infused intravenously from Days 13 to 15 ( Figure 1B). www.impactjournals.com/oncotarget

Safety and curative effect evaluation index Adverse events
The most common adverse reactions were recorded and included local (e.g., pain, pleural effusion, and ascites) and systemic (e.g., chills, fatigue, and fever) reactions.

Quality of life (QOL)
By karnofsky score standard as an index, after treatment, KPS increased ≥ 10 divided into QOL improved, increased < 10 divided into QOL stable, KPS reduced ≥ 10 divided into QOL lower.

AFP
Using electrochemical luminescence immunity analyzer and accessory kit (Roche Cobase 411) to detect serum AFP concentration at 1 day pre-treatment and at day 3, 1 month, 3 months post-treatment, carried out in accordance with the instruments and reagents. AFP normal range: 0-20 ng/ml.

Imaging change
The WHO first published the evaluation standards for the tumor curative effect for the main study objective of observing tumor changes [52]. According to the degree of change in the largest transverse diameter, the therapeutic effect is divided into a complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD). To accurately observe the therapeutic effects, the total area of all tumors before and after treatment was compared. The recent curative effect must be maintained for more than four weeks, and CR+PR represented the effective rate (RR).

Follow-up
The patients were required to undergo plain CT and enhanced CT at 1 week pre-treatment, and followed at 1 month and 2 months post-treatment. Imaging procedures were conducted every 3 months thereafter. The endpoints of interest were progression-free survival (PFS). PFS was defined as the interval between Cryo and local relapse, distant metastasis, or death, whichever occurred first.

Evaluation and statistical analysis
Complications were recorded and classified in accordance with the Common Terminology Criteria of Adverse Events v4.0. Radiographic local tumor control was assessed using image-guided tumor ablation criteria [53]. The basic characteristics of the two groups were compared using the chi-square test; immunity detection result data are presented as the mean ± standard deviation; the changes of imaging were compared using the Student's t-test; local and systemic adverse events were marked in the nursing records and compared using the Chi-square test. Significant differences were indicated by P < 0.05, P < 0.01 or P < 0.001. All analyses were conducted using GraphPad software (GraphPad, San Diego, CA, USA).

ACKNOWLEDGMENTS AND FUNDING
We would like to thank the native English speaking scientists of Elixigen Company for editing our manuscript. This work was supported by the Science and Technology Program of Tianhe District, Guangzhou, China [grant number 201504KW008].