Valuation of the Safety and Efficacy of Combination of Cryoablation and Dendric Cell/Cytokine-induced Killers Cells Treatment for Advanced Liver Cancers

The treatment of liver cancer needs integrated medical strategies, including surgery, chemotherapy, target therapy, radiotherapy, and immunotherapy. According to the patient's condition to develop a personalized and best treatment plan. Cryoablation can produce osmotic shock through repeated freeze-thaw to cause tumor cell necrosis, and release tumor antigens to activate anti-tumor immune responses. Immune cell therapy is an emerging field across cancer types in current cancer treatment. This study desired to combine cryotherapy and cellular immunotherapy to achieve the effect of tumor control. In recent years, cancer treatment studies have showed that cryoablation combined with immune cell therapy can play a good auxiliary effect and improve the cancer treatment efficacy significantly.

This trial study is a single center, single-arm, non-blind open-label human clinical trial. To explore the therapeutic effect and safety of cryoablation combined with autologous DC-CIK (through hepatic artery infusion, HAI) for patients with advanced liver cancers. The DC-CIK biologics are provided by BOHUI Biotech company. Their core technology (including clinical treatment and cell culture techniques) was transferred from Dr. Hasumi who is a Japanese clinician and this technical founder.

The World Health Organization's International Agency for Research on Cancer (IARC) has released the latest global cancer burden data for 2020, which shows that 19.3 million new cancer cases happened and nearly 10 million deaths occurred in 2020. Currently, one in five people of the world will have cancer in their lifetime, and one in eight men and one in 11 women will die of cancer. According Taiwan Cancer registry introduction from Ministry of Health and Welfare in 2017, the number of new cancer cases was 111,684, 5,852 more people than in 2015. Liver cancer, in particular, ranks fourth in the cancer rankings. The main causes of liver cancer are hepatitis B, hepatitis C, or cirrhosis of the liver caused by alcohol. Other causes include Aspergillus flavus toxin, non-alcoholic fatty liver disease and liver aspirates etc.

Immune cell therapy is an emerging field in the treatment of advanced liver cancer. Many types of immune effector cells such as lymphokine-activated killer (LAK) cells, dendritic cells (DC), tumor infiltrating lymphocyte (TIL) cells, cytokine-induced killer (CIK) cells, and lymphocyte-activated lymphocyte (LAK) cells are used to treat advanced liver cancer. CIK cells have been developed for immunotherapy applications. CIK cells such as CD3+CD56+ cells, CD3+CD56- cells and CD3-CD56+ cells, have the ability to kill tumors directly by demonstrating the non-restrictive cytolytic effect of MHC, similar to the function of general T cells. CIK cells bind to LFA-1 ligands on tumor cells through LFA-1, resulting in tumor cell killing, and then binding of the receptor to its ligand on NK cells initiates a downstream cell signaling pathway and further activates CIK cells for tumor cytotoxicity. Finally, CIK cells activate the signaling pathway via Fas-FasL and induce apoptosis in cancer cells. DCs are differentiated from hematopoietic stem cells in the bone marrow, often in the form of monocytes, which differentiate into mature dendritic cells in response to cytokine stimulation. After tumor-associated antigen (TAA) phagocytosis, antigens are treated by two pathways, the cytosolic pathway and vacuolar pathway, by which they are processed into peptides and loaded onto the major histocompatibility complex class I(MHC I). Then the TAA-MHC I complex is delivered to the DC surface. When DC-presenting TAAs migrate and reach the lymph nodes, they are able of priming T cells and prompting anti-tumor immunity. Dendritic cells are efficient activators of naïve T cells and are potent APCs. They play a central role in immune-mediated cancer elimination through antigen presentation and T-cell priming. More recently, they have been combined to form DC-CIK cell therapy, which is a combination immunotherapy where DC and CIK cells are cultured together. In terms of therapeutic mechanism, dendritic cells are specialized antigen-presenting cells, and mature dendritic cells can present tumor antigens via the MHC-I pathway to effectively counteract the immune escape mechanism of tumor cells. The specific tumor-killing effect of CIK is further enhanced. Many studies have shown that treating cancer patients with DC-CIK cells significantly prolong the survival time and enhance the immune function of the patient.

Recent trends in cancer treatment have confirmed that when cryoablation of tumors is combined with immune cell therapy, it can have a good complementary effect and significantly improve the efficacy of cancer treatment. It is believed that cryotherapy causes necrosis of tumor cells through the physical changes of osmotic shock produced by repeated freezing and thawing, which in turn releases tumor antigens and activates anti-tumor immune responses. A retrospective study conducted in 2013 in patients with stage IV lung cancer investigated the efficacy of cryoablation combined with immune cell DC-CIK and showed that cryoablation with DC-CIK significantly prolonged the overall survival (OS) of patients. The results of the 2017 clinical trial showed that cryoablation with NK cell therapy improved the clinical response rate and disease control rate (DCR) of patients with advanced lung cancer, with the proportion of patients with PR (63.3% vs. 43.3%, P\<0.01) and disease control rate ( The results of the clinical trial in advanced hepatocellular carcinoma showed that among 61 patients admitted from 2015 to 2017, cryoablation with NK immunotherapy significantly prolonged the progression free survival (PFS) of patients (9.5%). (PFS) (9.1 months vs. 7.6 months, P=0.0107), response rate (60% vs. 46.1%, P\<0.05) and disease control rate (85.7% vs. 69.2%, P\<0.05).

The trial was designed as a single-arm, uncontrolled, open-label, non-random assignment design. The efficacy and safety of hepatic artery infusion with autologous DC-CIK immune cells before and after ablation of liver tumors using cryoablation technique in patients with advanced liver cancer was investigated. The DC-CIK immune cell therapy technology was developed in collaboration with Bohui Biotech, and the core cell technology was transferred from Dr. Hasumi autologous immune cell therapy in Japan. The program will track the changes in tumor size and tumor index before and after treatment, and conduct safety assessment to evaluate the safety and efficacy of this trial treatment.

During the clinical trial, peripheral blood will be collected from patients for exploratory index analysis to complement the clinical evaluation results. According to the results of the clinical study in 2013, patients with stage IV solid cancer who had failed standard treatment had increased CD8+CD56+ cell populations after autologous immune cell therapy by Dr. Hasumi in Japan and continued to be followed up until one year later. In 2019, a study also showed that disease control was lower in the group of patients with liver cancer with high free DNA concentrations treated with sorafenib than in the group with low free DNA concentrations. The whole-genome sequencing of cell-free DNA (cfDNA) to obtain copy number alteration (CNA) profiles can be used as predictors of sorafenib treatment outcome. Therefore, in this clinical trial, blood samples were taken from patients before and after treatment, and the distribution of immune cell populations and changes in cell-free DNA (cfDNA) in blood were analyzed separately by separating the buffy coat and plasma using a density gradient centrifuge, and using the Qubit 2.0 Fluorometer. The qubit dsDNA High Sensitivity assay kit (Life Technology, Carlsbad, CA, USA) was used to quantify the amount of free DNA and to detect the copy number of free DNA, and to analyze the free DNA to find out the biomarkers that can be used for treatment effect tracking.

Eligibility Criteria:
Inclusion Criteria:

1. Patients aged between 20 and 80 years old.
2. Patients with advanced stage hepatocellular carcinoma or advanced stage hepatic metastasis derived from extrahepatic primary carcinoma, who are judged as advanced stage by clinical criteria (The Eighth Edition AJCC Cancer Staging Manual) through pathology and imaging reports.
3. Patients who have been evaluated by a physician for benefits and risks and are no longer considered suitable for previous third-line therapies will be included in this trial.
4. There is at least one target lesion larger than 1.0 cm that can be measured by MRI or CT and is appropriately located for cryoablation by imaging.
5. Child-Pugh class (A or B).
6. Adequate bone marrow function (white blood cell count \> 2x109/L, platelet count \> 5x1010/L).
7. coagulation function: prothrombin time (PT), activated partial thromboplastin time (APTT) ≤ 1.5 times the upper limit of normal
8. renal function: blood creatinine concentration (creatinine) \< 1.5 mg/dL .
9. Karnofsky Performance Status (KPS) score70 -10.
10. maximum tumor diameter ≤ 5 cm
11. number of tumors ≤ 5
12. Survival period greater than 3 months as predicted by the physician.

Exclusion Criteria:

1. Tumor size \>5 cm in diameter or tumor number \>5.
2. not suitable for cytarabine or cryoablation as determined by the trial physician.
3. Blood screening for any of the following viral infections: human immunodeficiency virus, human T-lymphotropic virus, syphilis, hepatitis B virus, hepatitis C virus, cytomegalovirus IgM antibody positive, etc.
4. Previous treatment prior to screening: Chemotherapy or radiation therapy must be completed before 3 weeks of screening to avoid systemic immune responses interfering with the immune cell therapy. If participating in clinical trials of other biologic agents or immunotherapy, at least 4 weeks of screening is required.
5. Patient is acutely or chronically infected or in acute cytomegalovirus attack at the time of screening.
6. Patients with level 3 hypertension, or patients with severe coronary disease.
7. patients with autoimmune disease.
8. patients who are pregnant, breastfeeding, or unable to use effective contraception.