Clinical trials on CIK cells: first report of the international registry on CIK cells (IRCC)

Abstract

Purpose

Recently, a subset of natural killer T lymphocytes termed “cytokine-induced killer (CIK) cells” has been described. To build an international registry, we collected the clinical data and treatment of patients with cancer using CIK cells from the literature and the respective investigators. This registry is expected to set a new set of standards on the reporting of results from clinical trials using CIK cells. A standardized reporting system will accelerate discoveries and allows us to improve treatment to benefit the patients.

Methods

We searched in PubMed for “CIK cells clinical trials”.

Results

Within the 867 matches found, 11 clinical trials with CIK cells were identified. Within these trials, 426 patients were treated, of which 313 were male, and 113 were female. Most trials included male patients with hepatocellular carcinoma, gastric cancer, and Hodgkin or non-Hodgkin disease. In 10 of 11 studies, autologous CIK cells were used. The total number of CIK cells injected ranged from 21.9 × 10⁷ to 5.2 × 10¹⁰. The number of CIK cells used per infusion ranged from 7.2 × 10⁶ to 2.1 × 10¹⁰. Patients were treated with up to 40 infusions of CIK cells. Of the 384 patients, where a clinical response was reported, 24 patients showed a complete response, 27 patients showed a partial response, 40 patients showed a minor response. The total response rate (RR) was 91/384 reported patients, 161 patients had a stable disease, 129 patients had progressive disease. A decrease in tumor volume was only described in three patients. Side effects of CIK cell treatment were minor. Interestingly, a reduction of hepatitis B virus load was described in patients undergoing treatment with CIK cells. Disease-free survival rates were significantly higher in patients treated with CIK cells than in a control group without CIK treatment.

Conclusion

Adjuvant immunotherapy with cytokine-induced killer cells may prevent recurrence and improve quality of life and progression-free survival rates in patients with cancer.

Introduction

Many patients with advanced malignant disease cannot be cured by standard forms of cancer therapy. Surgery, radiation, and chemotherapy have many side effects and often fail to remove the tumor completely. Small lesions and metastatic cells remain and cause recurrence of disease.

Immunotherapy is a promising treatment option for various cancers. It takes advantage of body’s natural abilities to eliminate tumor cells by stimulating and restoring the immune system to recognize and kill tumor cells.

A subset of natural killer T lymphocytes called “cytokine-induced killer (CIK) cells” is able to eradicate or reduce tumors. Especially cells transfected with cytokine genes possess an improved proliferation rate and a higher cytotoxic activity when compared to untransfected cells.

CIK cells are generated in vitro by incubation of peripheral blood lymphocytes with anti-CD3 monoclonal antibody, IL-2, IL-1 alpha, and interferon-gamma. Compared to standard lymphokine-activated killer (LAK) cells, CIK cells possess enhanced cytotoxic activity (Lu and Negrin 1994; Margolin et al. 1997). They have a higher proliferation rate and efficacy with low toxicity and few side effects. The higher anti-tumor activity of CIK cells is mainly due to the higher proliferation rate of CD3 and CD56 double-positive cells (Schmidt-Wolf et al. 1999).

To collect an international registry about the treatment of patients with advanced malignant tumors using CIK cells, we drew up a list of indices and created an international registry form. At the homepage www.cik-info.org, new and missing trials can also be registered. The registry form can also be found on this site. The goal of this type of registry is to collect as much information on studies with CIK cells worldwide as possible and build up a standard process in treatment of patients with advanced malignant tumors with CIK cells.

Materials and methods

Study accrual

We entered in PubMed the keywords “CIK cells clinical trials” and looked for human trials, where patients with malignant tumors were treated with cytokine-induced killer (CIK) cells. We evaluated 11 studies where the results of adjuvant immunotherapy with CIK cells were presented. The studies were printed in the following scientific journals: Digestive and Liver Disease, Chinese Journal of Cancer (4), Anticancer Research, World Journal of Gastroenterology (2), Haematologica, British Journal of Cancer, Biology of Blood, and Marrow Transplantation. In some studies, abstracts were only available in Chinese.

Generation of registry form

The following data were collected: name and address including e-mail, title, journal, phase; cell entity (autologous or allogeneic), tumor entity, number of patients (males and females), median- and age range, stage of disease, inclusion and exclusion criteria, total number of CIK cells (plus number of CIK cells per infusion and number of infusions), HLA type of patient’s CIK cells, storage (fresh or frozen), clinical and immunologic responses, toxicity (hematologic/non-hematologic toxicity), and follow-up (time period of follow-up, duration of responses).

Evaluation of studies

For each trial, one form was filled out. After evaluation of 11 studies, we filled out a summary form, where indices of the registry of each study were listed and compared. Many studies had interesting points in common and we were able to work out means and standard deviation. A special emphasis was placed on survival, improvement of quality of life, and progression-free survival rates.

Contacting the authors

When the registry form was filled out, we tried to get in contact with the first and last authors of all 11 clinical trials. We sent them the enclosed form and asked them to check for errors, fill out the missing parts in the form, and return it back to us.

Furthermore, we asked if they had any further studies concerning CIK cell treatment, which had not been published yet and asked to get information on them as well. In addition, we asked if they knew of other people who did clinical trials with CIK cells.

Summary and comparison

When the study accrual and evaluation were finished, we filled out a summary form encompassing the 11 studies, where all information concerning the indices was collected. So that the absolute numbers of patients, male and female, etc. could be determined.

From the published numbers (e.g., the increase of CD-positive receptor cells), the mean and standard deviation could be calculated.

Statistical analysis

Student’s T-test was used to analyze for statistical significance. A P value <0.05 was considered significant.

Results

Here, we present a summary of the current data present in our database. By applying the above mentioned registry form and evaluation of the 11 studies, we obtained the following results.

Assessment of patient characteristics

In 11 trials, 426 patients were treated, 313 men and 113 women. The age range was between 15 and 79 years. In 9 of 11 studies, the median age was exactly given. In study no. 10, 14 patients in the study group and 10 patients in the control group were older than 60 years and 31 patients in the study group and 30 in the control group were younger than 60 years. In study no. 11, 81 patients were older or exactly 50 years old and 46 patients were younger than 50 years.

Patients with different malignancies underwent treatment. In Table 1, we present tumor entity, male-to-female ratio, and the observed therapeutic effects in the collected 11 studies. Most trials enrolled male patients with hepatocellular carcinoma, gastric cancer, and Hodgkin or non-Hodgkin disease.

Table 1.

Tumor entity with number of respective patients, male-to-female ratio, and therapeutic effects

Tumor entity	Male	Female	Total	Therapeutic success
Primary HCC	72	26; 15 unknown	113
Recurrent HCC post-surgery	16	1	17
HCC after resection	97	30	127
Hodgkin’s disease	7	3; 3 unknown	13	High
Non-Hodgkin lymphoma	2	–; 19 unknown	21	High
Metastatic renal cell carcinoma	8	3	11
Colorectal carcinoma	6	1	7
Lymphoma	2	–	2	High
Acute myelogenous leukemia	1	3	4
Chronic myelomonocytic leukemia	–	1	1	High
Pre-B-acute lymphoblastic leukemia	–	1	1
Myelodysplasia	1	1	2	High
Gastric cancer	39	18; 14 unknown	71
Esophageal squamous carcinoma	Unknown	Unknown	11
Lung cancer	Unknown	Unknown	6
Breast cancer	Unknown	Unknown	5
Other malignant tumors	Unknown	Unknown	12
Rhabdomyosarcoma	Unknown	Unknown	2
Total number of patients	313	113	426

We have no information about the absolute numbers, which tumor entity had most therapeutic effects. We defined the tumor entity, of which we have concrete statements of cases of complete remission as “high therapeutic effect”. All other clinical responses as progressive disease, stable disease, partial remission and minor remission were not defined as “high therapeutic effect”

In 8 studies, patients with advanced stage of disease were enrolled. In study no. 1, two patients with early stage were enrolled. In study no. 9, no statement concerning the stage of disease was provided. In 3 studies, we obtained the stage of disease, recurrence post-operation, relapsed tumor burden, tumor diameter 2–13 cm, or the differentiation tumor ≥ or <5 cm combined with statements to Child–Pugh classification or vascular invasion.

The inclusion criteria are listed in Table 2. Age, metastases, and ECOG/Karnofsky score describing the general state of the patient were important criteria. On the other hand, inadequate renal or hepatic function, Karnofsky score less than 70, and metastases or other severe diseases were listed as exclusion criteria in most studies.

Table 2.

Indications and contraindications of CIK immunotherapy in 11 clinical trials

Inclusion criteria (9 of 11 studies)	Exclusion criteria
State of health
Hepatocirrhosis with more than 20 years of chronic HBV infection	Inadequate renal or hepatic function
Metastatic colon carcinoma, renal carcinoma, melanoma and lymphoma	Karnofsky score less than 70
Leukocyte count at least 3000/μl before apheresis	Decompensated heart insufficiency
Platelet count at least 100,000/μl	Ventricular rhythm disorders
Leukocyte count at least 3,000/μl before apheresis	Severe psychiatric disease
Platelet count at least 100,000/μl	Active hepatitis A, B, C or HIV
Karnofsky score 70–100	CML
ECOG 0–2	Acute graft-versus-host disease more than grade 2
ECOG 0–1	Metastasis in portal vein
Without metastasis in portal vein	Active lesions
CHILD A/B;	Alcohol abuse after surgery
Solitary tumor	Other malignant tumor
No preoperative transfusion	Immunosuppressive disease
Tumor diagnosis confirmed by pathology	Hypersusceptibility and/or adverse effects
Resection margin more than 1 cm
No tumor fracture and hemorrhage
No distant metastases
Treatment course
Hematologic malignancies with relapse after allogeneic HSCT (including loss or insufficient lymphoid chimerism)	Time interval between chemotherapy and CIK treatment less than 28 days
Gastric cancer stage 4, palliative gastrectomy, 3 cycles of chemotherapy with FOLFOX 4	Chemo- or radiotherapy before CIK treatment
	Patient refused to participate
	Previous treatment with CIK cells
Personal indices
15–79 years old

Assessment of cells, studies, numbers, and infusions

In 10 of 11 studies, autologous CIK cells were used for infusion. We received information on cell storage on 9 of 11 studies. Seven studies used fresh CIK cells and 2 studies used CIK cells (study no. 2: used frozen cell in most assays, except for flow cytometric analysis where fresh cells were used).

Seven of 11 studies were phase I studies and 4 were phase II studies. Information on the total number of CIK cells used was only available in 8 studies and ranged from 21.9 × 10⁷ to 5.2 × 10¹⁰.

In 9 of 11 studies, we received the information that patients were treated with up to 40 infusions. In 10 of 11 studies, we received information about the CIK cell number per infusion, which ranged from 7.2 × 10⁶ to 2.1 × 10¹⁰. One study 3 dose levels were used which ranged from 1 × 10⁹ to 5 × 10⁹ up to 1 × 10¹⁰.

Assessment of immunologic response

Overall the serum levels of tumor bio-markers, especially AFP, ALT, CEA, MG-7 Ag, CA72-4, CA19-9 decreased after treatment. In two studies (study 1 and 7), the serum level of AFP decreased in 15 patients (study 1: 6 and study 7: 9), but remained elevated in 1 patient and ALT levels decreased in 9 patients, but increased in 2 patients. In 3 studies, we obtained information on the serum levels of CEA. In patients with elevated CEA serum levels prior to treatment, 14 cases showed a decrease in serum CEA and 1 case remained elevated after the treatment. In one study, MG-7 Ag, CA72-4, CA19-9, and CEA also decreased significantly.

Furthermore, the absolute numbers of CD3+, CD56+, CD3+ CD56+, CD3+ CD8+, CD25+, CD4+, CD8+, CD4+ CD8 + T cells increased. No significant increase in the number of CD3+ cells was reported in patients that were treated with CIK cells (P = 0.88; Fig. 1).

Fig. 1.

Comparison of CD3, CD3CD56, CD8 and CD4-positive cells before and after treatment with CIK cells as described in the “Results” section. Comparison of the values in percent pre/post-treatment of CD3+, CD3+ CD56+, CD3+ CD8+, and CD8+ is shown. Results are presented as mean plus standard deviation

In one study, a reduction of the viral load was described. In study no. 1, the average viral load decreased from 1.9 × 10⁶ copies/ml to 1.4 × 10⁵ copies/ml after 3 months. This statement refers to chronic HBV infection.

Treatment toxicity and adverse effects

The toxicity was highest on days 22 and 43. On these days, hematologic side effects were found: In study no. 3, one case of anemia was reported on days 22 and 43, one case of increased thrombocyte counts on days 22 and 43, one case of increased potassium on days 4 and 22, one case of increased AP, and one case of increase of CRP on days 25 and 43 were described.

Further side effects such as mild hypotension, fever (7 of 11 studies), thrombocytopenia, chill, headache, nausea, and vomiting were also described. In study no. 4, four patients showed acute GVHD grade 1 and 2, which progressed into chronic GVHD in two patients.

Assessment of clinical response and patient outcome

In 7 of 11 studies we obtained information on clinical response after CIK cell treatment: Of the 384 patients where a response was reported, 24 patients had a complete response, 27 patients had a partial response, and 40 patients had a minor response. The total response rate (RR) was 91/384 reported patients. 161 patients had a stable disease and 129 patients had progressive disease. A decrease in tumor volume was described for only 3 patients.

Interestingly, the log rank test showed higher progression-free survival rates in group 1, which was treated with 3 courses (P = 0.001) and group 2, which was treated with 6 courses (P = 0.004) than in the control group without CIK treatment (Fig. 2). No statistical significance was found between group 1 (treated with 3 courses) and 2 (treated with 6 courses) (P = 0.345). The overall survival remained similar (study no. 11).

Fig. 2.

The 1-, 3- and 5-year disease-free survival rates in patients receiving CIK cells (groups 1 and 2) when compared to no CIK cell treatment (127 patients); (trial 11). Group 1 was treated with 3 courses of CIK cells, group 2 was treated with 6 courses of CIK cells and the control group (group 3) received no CIK cell treatment for comparison

Furthermore, we obtained information that CIK treatment ameliorated symptoms:

Patients had increased appetite, improved sleep, gained body weight, and had pain relief.

In study 1, the improvements mentioned above were shown in most patients. In study 6, an improved quality of life was described in both groups.

In study 7, 51 cases of improved appetite, 32 cases of improved sleep, and 13 cases of pain relief were described. Overall, CIK treatment improved the quality of life.

We cannot make a firm statement on the overall survival rate because of incomplete reporting.

In study 4, seven patients died after a median of 520 days after autologous stem cell transplantation (ASCT) and four patients were alive after a median of 910 days after ASCT. In study 6, all patients died after 5 years. In study 11, the 1-, 3-, and 5-year overall survival rates were not significantly different among the three groups.

Discussion

Malignant cells often survive traditional treatment strategies such as surgery, radiation, and chemotherapy and the latter two treatments carry many side effects. Most importantly, small lesions and metastatic cells often remain and cause recurrence of disease.

Immunotherapy is a new and promising treatment for a number of cancers. It has the advantage of stimulating and restoring the body’s natural abilities of the immune system which can recognize and kill tumor cells. Natural killer T lymphocytes called cytokine-induced killer (CIK) cells are able to eradicate tumors or reduce tumor mass with only few side effects. Thus, CIK cell-based immunotherapy is a promising new treatment modality with the potential to kill a wide spectrum of tumor cells.

An increasing amount of studies dealing with the treatment of patients with cancer with CIK cells has been published, with many studies having been performed in Asia. Unfortunately, these studies are often published only in Chinese. It would be beneficial to take advantage of the worldwide experiences of those studies. One step in this direction would be to apply an international standard in publication and a closer exchange between research groups dealing with CIK cells.

Therefore, we established a registry of clinical trials with CIK cells (www.cik-info.org). Here, we collected 11 studies that include a variety of cancers in our registry. Eight of these studies were published by Chinese authors. In a summary form, the study parameters of the registry of each study were compared. Many studies had interesting points in common.

Unfortunately, the statements in the 11 trials were heterogeneous. For example: we could either find only a few statements or different parameters with regard to the immunologic response or HLA type of patients.

In our registry, we found that adjuvant immunotherapy with cytokine-induced killer cells may prevent recurrence, improve progression-free survival rates, and improve the quality of life. We have not been given information on the overall survival rate and thus cannot make a statement. However, we feel that this information should be reported in the future.

The observations made in patients enrolled in clinical trials were similar to results reported in preclinical animal studies, which showed strong anti-tumor effects of CIK cells. Kim et al. (2007) showed anti-tumor activity of CIK cells against human non-small cell lung cancer in vitro and in an in vivo using nude mouse xenograft models. Interestingly, CIK cells injected intravenously twice a week at dose ranges from 0.3 to 30 × 10⁶ cells per mouse inhibited tumor growth by 32, 67, and 77% with few side effects. Desirable effects such as gained body weight have also been described.

Chan et al. (2006) showed promising results in treating mice with ovarian cancer using CIK cells alone or in combination with bispecific antibodies against the cancer antigen-125 and Her2. Mice that underwent adoptive transfer of CIK cells redirected with bispecific antibodies showed a significant reduction in tumor burden and prolonged survival compared to mice treated with CIK cells alone.

It is likely that combination treatments will further improve the survival rates. More studies are needed to evaluate the efficacy of such treatment strategies.

Furthermore, an interesting observation was the effect of CIK cell treatment on the HBV viral load in patients with HCC. Shi et al. (2004) showed a measurable decrease in viral load after already 1 month after CIK treatment. CIK treatment of patients with HBV infection could also be a promising treatment strategy. This is of particular interest since up to now active hepatitis A, B, or C were exclusion criteria for CIK treatment.

Finally, we want to emphasize that adjuvant immunotherapy with CIK cells may prevent recurrence and improve quality of life and progression-free survival rates. Our goal is to use our registry to publish a standardized list of parameters and initiate a new set of standards for future studies and publications that will allow for better comparison among studies and thus for better data analysis and faster improvement of this treatment approach. We suggest an international standard in treatment and an international registry. The promising results of CIK cell treatment must also be critically evaluated. The long-term therapeutic efficacy is not really researched yet. In the trials, we have very different and partially superficial statements concerning patients, tumor response, and survival rates. A reliable statement concerning the therapeutic efficacy of reaction of special tumors cannot yet be made.

To further collect information on the use of CIK cells in clinical trials, we would be happy to obtain further information. Therefore, we invite every reader to contact us in case of further studies with CIK cell treatment, which have not been published yet (Ingo.Schmidt-Wolf@ukb.uni-bonn.de). Furthermore, we invite you to visit our homepage www.cik-info.org. The standardized registry form can also be found at the flowing site: http://www.cik-info.org/index.php?kat=brot. In summary, to improve medical and scientific conclusions from clinical trials and thus speed up the process of identifying the best treatment conditions using CIK cells, the following parameters should be reported: Publication details, title, journal, phase of clinical trial, use of autologous or allogeneic cells, tumor entity (diagnosis), number of patients, sex of patients, median age, age range, stage of disease, inclusion criteria, exclusion criteria, total number of CIK cells, HLA type of patients’ CIK cells, storage of CIK cells: Frozen or fresh CIK cells, number of CIK cell per infusions, CIK cell number per infusion, hematologic and non-hematologic toxicity, clinical response, results of follow-up, time period of follow-up in months, duration of responses, survival status of patients, and immunologic responses. In addition, we would like to collect future clinical trials using CIK cells.

Open questions that need to be addressed in the future include survival rates of patients, combinations with standard treatment options, and targeting of CIK cells to tumor cells.