Cytokine-induced killer cells in the treatment of patients with solid carcinomas: a systematic review and pooled analysis

Cytotherapy, 2012; 14: 483–493

Cytokine-induced killer cells in the treatment of patients with solid carcinomas: a systematic review and pooled analysis

YUE MA, ZAN ZHANG, LEI TANG, YING-CHUN XU, ZHI-MING XIE, XIAO-FENG GU & HONG-XIA WANG Department of Oncology, Shanghai Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Abstract Background aims. The aim of this study was to evaluate the efficacy and safety of cytokine-induced killer (CIK) cell therapy for solid carcinomas. Methods. We performed a computerized search of phase II/III clinical trial databases of CIK cell-based therapy using a combination of the terms ‘cytokine-induced killer cells’, ‘tumor’ and ‘cancer’. Results. Treatment with CIK cells was associated with a significantly improved half-year survival (P  0.003), 1-year survival (P  0.0005), 2-year survival (P  0.01) and mean survival time (MST) (P  0.001). Patients in the CIK group showed a prolonged half-year progressionfree survival (PFS) (P  0.01), 1-year PFS (P  0.01) and median time to progression (MTTP) (P  0.001). A favored disease control rate (DCR) was observed in patients receiving CIK cell therapy, while the objective response rate (ORR) was not altered (P  0.05) compared with the non-CIK group (P  0.007). CIK cell therapy could also reduce the adverse effects of grade III and IV leukopenia caused by chemotherapy (P  0.002) and diminish hepatitis B virus (HBV)-DNA content (P  0.01). However, the incidence of fever in the CIK therapy group was significantly higher than in the non-CIK group (P  0.02). The percentage of CD3 , CD4 , CD4 CD8 , CD3– CD56 and CD3 CD56T-lymphocyte subsets in the peripheral blood of cancer patients was significantly increased, whereas the percentage of CD8T-lymphocyte cells was significantly decreased in the CIK group compared with the non-CIK group (P  0.01). Conclusions. CIK cell therapy has demonstrated a significant superiority in prolonging the MST, PFS, DCR and quality of life (QoL) of patients. Key Words: cytokine-induced killer cells, meta-analysis, solid carcinomas

Introduction Cytokine-induced killer (CIK) cells, which are nonmajor histocompatibility complex (MHC)-restricted CD3 CD56T cells, were first described as having a marked ability to proliferate and an increased superiority over lymphokine-activated killer (LAK) cells in cytolytic activities against cancer by SchmidtWolf et al. (1). LAK cells are interleukin (IL)-2activating cytotoxic effector lymphocytes whose cytolytic activities are not restricted by MHC.The LAK cells are heterogeneous, consisting of CD3  CD56 natural killer (NK) cells, CD3 CD56 cells and CD3 CD56 T cells. Two subsets of LAK cells, CD3 CD56 T cells and CD3  CD56 NK cells, are the main contributors to the LAK cell cytolytic properties (2). CIK cells are generated by incubating mononuclear cells from peripheral blood, bone marrow or cord blood with various types of additions, such as CD3 monoclonal antibody, IL2, IL1 and interferon (IFN)γ. Like LAK cells, CIK cells are heterogeneous. The majority of them express

T-cell receptors, and others express NK cell markers. Eighty-eight per cent of CD3 cells co-express CD56, which have been proven to be the main effectors in CIK cells (3). CD3 CD56 cells are derived from CD3 CD56 T cells, expanding up to 1000fold in cultures and demonstrating enhanced cytotoxicity against various tumor cells compared with CD3 CD56  cells (4). The advantages of CIK cells, compared with LAK cells and primary cytotoxic T cells (CTL), include having a higher proliferation rate, increased efficacy with few side-effects, and non-MHC-restricted killing of tumor cells (5). Therefore, as an adoptive cellular immune therapy, CIK cell-based therapy is considered to be a promising method for removing minimal residual cancer cells. Treatment with autologous CIK cells has resulted in encouraging clinical prospects for many types of cancers, such as chronic myeloid leukemia, liver cancer, colon cancer, kidney cancer and breast cancer (6–8). The first clinical trial using CIK cell therapy for cancer

Correspondence: Hong-Xia Wang, MD, PhD, Department of Oncology, Shanghai Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200127, China. E-mail: [email protected] (Received 15 July 2011; accepted 6 Dec 2011) ISSN 1465-3249 print/ISSN 1477-2566 online © 2012 Informa Healthcare DOI: 10.3109/14653249.2011.649185

484

Y. Ma et al.

patients was reported in 1999 (9). Soon afterwards, CIK cells demonstrated encouraging results in several clinical trials. These studies showed that the immunotherapy of cancers with CIK cells may prevent recurrence, improve progression-free survival rates (PFS), and promote the quality of life (QoL) of cancer patients. However, clinical studies with CIK cells are still in their infancy and have only involved a relatively small number of patients in most of these reports. Therefore, we performed a systematic review and meta-analysis of randomized phase II and III clinical trials to assess the efficacy and tolerability of CIK cells in the treatment of patients with solid cancers. The aim of this review was to evaluate the impact of CIK-based therapy on the survival and QoL of cancer patients, as well as to assess the toxicity of such a treatment. Methods Study design, search strategy and eligibility criteria We searched for randomized studies in the databases of any language as of 30 August 2011, including the PubMed database, Cochrane Central Registry of Controlled Trials, Online Proceedings of the American Society of Clinical Oncology (ASCO) Annual Meetings and the European Cancer Conference (ECCO). A combination of keywords including ‘cytokine-induced killer cells’, ‘tumor’ and ‘cancer’ was used. We contacted experts in the field of CIK cell therapy to broaden our yield of potentially eligible trials. We also searched manually oncology journals known to publish a large number of clinical trials, and reviewed reference lists of published trials and relevant review articles. We considered all randomized trials as either one of two arms of therapeutic regimens: CIK-based therapy and non-CIK-based therapies in patients with solid cancers. To be included, the trials had to be randomized and not confounded by additional therapeutic regimens in either group. Data collection Data were independently extracted by two investigators (Lei Tang and Ying-Chun Xu). Discrepancies were discussed with a third investigator (Yue Ma). Yue Ma and Zan Zhang cross-checked the data collected against the original articles. For the trials included in the meta-analysis, we gathered the authors’ names, the journal name and year of publication, sample size per arm, performance status (PS) score, regimen used, median age of patients, and information pertaining to the study design (whether the trial reported the mode of randomization, allocation concealment, description of withdrawals per arm and blinding information). To assess

randomization integrity, we checked the patterns of treatment allocation and the balanced baseline characteristics by treatment group. Follow-up of surviving patients was checked to ensure that it was balanced by treatment group and was current. Definition of outcome measures The primary endpoints in the analysis were the median survival time (MST) and PFS. MST is the time at which half of the patients are expected to be alive, denoting how long patients survive with cancer in general or after a certain treatment. PFS was defined as the length of time during and after treatment for which a patient lived with a disease that did not worsen. The secondary endpoints were the objective response rate (ORR), defined as the sum of partial and complete response rates, and the disease control rate (DCR), defined as the sum of stable disease, partial response and complete response rates, defined according to World Health Organization (WHO) criteria. We also evaluated toxicity, QoL, hepatitis B virus HBV-DNA concentration and T-lymphocyte subsets in the peripheral blood of the cancer patients in the trials. Toxicity was graded according to the National Cancer Institute (NCI) Common Toxicity Criteria (CTC). QoL was assessed by the Karnofsky performance status (KPS) or Lung Cancer Symptom Scale (LCSS) (10,11). The results were obtained directly from the articles included or were calculated using the data in an article. Statistical analysis The analysis was performed using Review Manager Version 5.0 (Nordic Cochran Centre, Copenhagen, Denmark). Heterogeneity between the trials was assessed to determine which model should be used. To assess statistical heterogeneity between the studies, the Cochran Q-test was performed, with a predefined significance threshold of 0.1. The odds ratios (OR) were the principal measurements of effect and were presented with a 95% confidence interval (CI). P-values of  0.05 were considered to be statistically significant. All reported P-values resulted from twosided version tests of the respective tests. The analysis of Fail-safe number (NFS) was performed using a statistical analysis system (SAS 6.12) (12–14). Results Selection of trials One-hundred and sixty-two articles were identified initially as CIK-based therapy, 132 of which were secondarily considered ineligible for various reasons (nine for being review articles, 26 for using animal models and 97 for in vitro experimentation). The full

CIK cells for the treatment of solid carcinomas texts of 30 articles were retrieved for more detailed evaluation. Of these, 19 trials were excluded further (three for being phase I clinical trials, nine for being non-randomized controlled trials, two for insufficient data, four for blood cancer and one for dendritic cell CIK). The selection procedure of the clinical trials is shown in Figure 1. As a result, 11 articles reporting phase II and III clinical trials of CIK cell-based therapy were selected for meta-analysis. Characteristics of CIK cell-based therapy After the selection process, 11 eligible randomized trials with a total of 854 patients were included in present analysis. All of the trials were fully published. The clinical data of the trials are listed in Table I. Most of the patients in these studies had a good performance status (Child-Pugh A/B or KPS  70). In four of the trials (15–18), CIK therapy combined with transcatheter arterial chemoembolization (TACE), with or without radiofrequency ablation (RFA), was evaluated in patients diagnosed with hepatocellular carcinoma (HCC). In another trial (19), adjuvant CIK cell therapy was compared with surgery alone for HCC patients. In the remainder of the trials, CIK cell therapy was evaluated in patients with advanced solid malignancies such as non-small cell lung cancer (NSCLC), gastric cancer and renal cell cancer combined with traditional

Figure 1. Flow diagram of the study selection process.

485

systemic chemotherapy. IFN-γ, CD3 monoclonal antibody (McAB), interleukin-1a (IL-1a) and IL-2 were used in the CIK cell culture system in all of the trials analyzed. In eight trials, CIK cells were cultured in complete medium supplemented with human blood serum, and another three trials used serum-free medium to culture CIK cells. In addition, the CIK cells for 10 trials were prepared from peripheral blood and one trial used CIK cells derived from cord blood cells (CB-CIK). The number of CIK cells transfused into patients in these studies ranged from 1.0  109 to 5.0  1010/course. The patient information from two groups (CIK cell therapy and non-CIK cell therapy) of the trials, such as gender, age, clinicopathologic category and CIK cell dose, were analyzed by χ2 test (Table II). There was no statistically significant difference between groups, with all P-values being  0.05. The different article origins of the patient information in each group did not interfere with the results of the meta-analysis. However, other clinical information from the trials such as tumor diameter, tumor-node-metastasis (TNM) stage, performance status, serum alpha-fetoprotei (AFP) and HBV-DNA, were not analyzed because of insufficient data on assessments of tumor size and stages. Survival Patients in the CIK group were associated with prolonged MST compared with the non-CIK group (OR 6.51, 95% CI 8.52 to 4.5, P 0.001). Furthermore, the results of the pooled analysis showed that patients in the CIK group had a significantly improved half-year survival (OR 0.30, 95% CI 0.14–0.66, P  0.003), 1-year survival (OR 0.43, 95% CI 0.27–0.69, P  0.0005) and 2-year survival (OR 0.21, 95% CI 0.11–0.38, P 0.01) (Figure 2). However, the 3-year and 5-year survivals in the CIK group were not significantly different from those in non-CIK group (68.04% and 45.36%, respectively, in the CIK group, versus 60.38% and 37.74% in the non-CIK group; 3-year survival OR 0.72, 95% CI 0.36–1.42, P 0.35; 5-year survival OR 0.73, 95% CI 0.36–1.44, P 0.36). Concerning PFS, treatment with CIK cells was associated with a significantly prolonged half-year PFS (OR 0.28, 95% CI 0.15–0.51, P 0.01) and 1-year PFS (OR 0.28, 95% CI 0.18–0.45, P 0.01) (Figure 3). Patients in the CIK group also showed a prolonged median time to progression (MTTP) compared with the non-CIK group (OR –1.23, 95% CI 1.53 to 0.92, P 0.001). In Figure 3, each trial is represented by a square, the center of which gives the OR for that trial. The size of the square is proportional to the information in that trial. The ends of the horizontal bars denote a 95% CI. The black diamond gives the overall OR for the combined results of all trials. The center denotes the OR, and the extremities denote the 95% CI.

486

Y. Ma et al.

Table I. Clinical information of the eligible trials for the meta-analysis.

Trial reference 19

Tumor characteristics HCC

Number of patients 127

Regimens (per arm)

Number of patients (Male)

CIK (3 courses)

41 (31)

43 (32) 43 (34) 45 (31)

CIK regimens

Culture of CIK cell

1.0–2.0  1010/ course

CM, IFN-g, CD3 McAb, IL-1a, IL-2

1.0–1.5  1010/ course

CM, IFN-g, CD3 McAb, IL-1a, IL-2

1.1–1.5  1010/ course

CM, IFN-g, CD3 McAb, IL-1a, IL-2

18

HCC

85

CIK (6 courses) Surgery only TACE RFA CIK

17

HCC

64

TACE RFA TACE RFA

40 (29) 31 (29)

TACE RFA CIK TACE RFA CIK

33 (30) 42 (37)

 1.0  1010/course

CM, IFN-g, CD3 McAb, IL-1a, IL-2

16

HCC

83

15

HCC

146

TACE RFA TACE

41 (34) 74 (64)

1–5  1010/course

Serum-free culture medium, IFN-g, CD3 McAb, IL-1a, IL-2

40

NSCLC (III)

120

TACE CIK BAI

72 (65) 40 (23)

UK

Serum-free culture medium, IFN-g, CD3 McAb, IL-1a, IL-2

40 (23) 40 (26) 30 (27)

5  109/course

CM, IFN-g, CD3 McAb, IL-1a, IL-2

41

NSCLC (III–IV)

59

Chemo BAI CIK Chemo

42

GC (II–IV)

60

Chemo CIK Chemo

29 (24) 31 (21)

1.0  109/course

CM, IFN-g, CD3 McAb, IL-1a, IL-2

43

GC (IV)

57

Chemo CIK Chemo

29 (24) 25 (18)

5.0  109/course

CM, IFN-g, CD3 McAb, IL-1a, IL-2

28

Chemo CIK Chemo IL/INF

32 (21) 10 unknown (UK)

 1.0  1010/course

CM, IFN-g, CD3 McAb, IL-1a, IL-2

18 (UK)

40

Chemo IL/ interferon (INF) CIK Chemo

9  109/course

Serum-free culture medium, IFN-g, CD3 McAb, IL-2, IL-1a

Chemo CB-CIK

20 (UK)

44

45

RCC (I–II)

Advanced solid malignancies

20 (UK)

HCC, hepatocellular carcinoma; NSCLC, non-small cell lung cancer; GC, gastric cancer; RCC, renal cell cancer; RFA, radiofrequency ablation; BAI, bronchial arterial infusion; Chemo, vein chemotherapy; CM, complete medium (RPMI-1640 and gentamycin and 5% human blood AB serum); CB, cord blood.

Response rate The analysis of DCR also demonstrated favorable results for the CIK cell therapy arm, with the OR being 0.46 (95% CI 0.26–0.81, P 0.007). However, the ORR for the chemotherapy combined with CIK group was 53.69%, which did not differ significantly from the ORR

of 44.44% for the chemotherapy-alone group (OR 0.63, 95% CI 0.4–0.99, P 0.05) (Figure 4). Toxicity and HBV-DNA content The results revealed that fewer grade III and IV leukopenia, neutropenia, anemia and thrombocytopenia

CIK cells for the treatment of solid carcinomas Table II. Patient information for the eligible trials (χ2 tests).

Features Gender Male Female Age (years) 60  60 Category HCC Lung adenocarcinoma Lung nonadenocarcimoma Gastric cancer Renal cell cancer Other CIK cell number (per course) 1.0  109 5.0  109 9.0  109

Non-CIK cell therapy

CIK cell therapy

298 87

321 86

257 106

231 110

227 47

272 40

42

25

60 10 8

64 18 8

c2

P-vaZlue

0.249

0.05

0.773

0.05

10.14

4.334 31 75 258

29 61 307

0.05

0.05

487

occurred in the chemotherapy combined with CIK therapy group than in the non-CIK group. Therefore, CIK therapy can reduce the adverse effects of grade III and IV leukopenia caused by chemotherapy (OR 4.27, 95% CI 1.74–10.5, P  0.002). However, the adverse effects of anemia and thrombocytopenia in the CIK group were both 3.39%, which did not differ significantly compared with the non-CIK group (anemia 7.45%, OR 2.48, 95% CI 0.48–12.74, P  0.28; thrombocytopenia 6.38%, OR 1.99, 95% CI 0.37–10.59, P  0.42). In most trials, slight fever and chills could be seen, and the body temperature varied from 37.5°C to 39°C within 24 h after CIK cell transfusion. On the whole, the analysis showed that the incidence of fever in the CIK therapy group was significantly higher than in the non-CIK group (OR 0.13, 95% CI 0.02–0.77, P  0.02). The HBV-DNA content in the analysis was based on two HCC trials (12,13). Patients in the CIK group had lower HBV-DNA content than patients in the non-CIK group (OR 27.5, 95% CI 5.21–145.15, P  0.01).

Figure 2. Comparison of 0.5, 1- and 2-year overall survival (OS) between the non-CIK and CIK groups. The fixed-effects meta-analysis model (Mantel–Haenszel method) was used.

488

Y. Ma et al.

Figure 3. Comparison of 0.5- and 1-year PFS between the non-CIK and CIK groups.

Comparison of T-lymphocyte subsets in the peripheral blood of cancer patients The analysis showed that the ratio of CD3 , CD4 , CD4 CD8 , CD3 CD56 and CD3 CD56T cells was significantly increased in the CIK group compared with the non-CIK group, which was reflected by pooled OR of 5.79 for CD3 cells (95% CI 8.94 to 2.64, P  0.0003), 5.07 for CD4 cells (95% CI 7.54 to 2.6, P  0.001), 0.45 for CD4 CD8 cells (95% CI 0.50 to 0.40, P  0.001), 9.79 for CD3 CD56 cells (95% CI 11.81 to 7.77, P  0.001) and 2.61 for CD3 CD56 cells (95% CI 3.05 to 2.17, P  0.001). Furthermore, the percentage of CD8T cells was significantly decreased in the CIK group compared with the non-CIK group (OR 4.01, 95% CI 0.92–7.1, P  0.01) (Figure 5). Discussion CIK cells are the population of heterogeneous effector cells possessing enhanced cytotoxicity against tumor cells and a higher proliferation rate compared with LAK and tumor-infiltrating lymphocyte (TIL) cells. CIK cells are characterized by a rapid proliferation, potent anti-cancer capability, wide antineoplastic spectrum and sensitivity to multidrug resistance cancer cells. Current protocols to differentiate CIK cells are based on a combination of IFN-γ on day 1 of culture, followed by alpha-CD3, IL-2 and IL-1alpha 24 h later (20,21).

The first clinical trial of CIK therapy was reported in 1999 (9). In this report, autologous CIK cells electroporated with IL-2 genes were infused into 10 patients with metastatic renal carcinoma, colorectal cancer or lymphoma. Circulating CIK cells persisted for up to 2 weeks after infusion, and increases in serum IFN-γ, granulocyte–macrophage colony-stimulating factor GM-CSF and transforming growth factor (TGF)-β were observed, along with an increased cytotoxic activity of total peripheral blood lymphocytes. One patient with follicular lymphoma showed a complete response. No major side-effects were observed, except for three patients who developed mild fevers that spontaneously resolved. Subsequently, adoptive cellular immunotherapy treatment with CIK cells showed effectiveness in a variety of cancers, including acute leukemia, liver cancer, lung cancer, gastric cancer and renal cancer. In another study investigating CIK cell therapy for advanced renal carcinoma patients, a follow-up of 6–20 months showed one case of partial response, two cases of stable disease and one case of progressive disease in the four patients with measurable disease (22). In adjuvant therapy of acute leukemia, a study from China reported that 73.4% of the 19 patients who received one to four courses of autologous CIK cell infusions in combination with consolidation chemotherapy remained in continuous remission for a follow-up period of 4 years, compared with 27.3% in the chemotherapy-only group (23). As an

CIK cells for the treatment of solid carcinomas

489

Figure 4. Forest plot for ORR and DCR. Chemo, chemotherapy; Chemo CIK, chemotherapy and CIK therapy.

immunotherapeutic modality, infusion of CIK cells is most likely to show efficacy in a relatively low tumor burden stage or in an adjuvant setting rather than in high tumor burden diseases. Additional information about the clinical antitumor activity of CIK cells is available from autologous therapy trials. Our systematic review yielded several major findings. First, the analysis showed that CIK therapies were associated with significant prolonged 1-year and 2-year survivals, PFS and MTTP. In the subgroup analysis of HCC, the CIK cell therapy group also showed a favorable 1-year survival (OR 0.4, 95% CI 0.22–0.7, P  0.001), MTTP (OR 1.13, 95% CI 1.45 to 0.81, P  0.001), half-year PFS (OR 0.28, 95% CI 0.15–0.51, P  0.001) and 1-year PFS (OR 0.3, 95% CI 0.19– 0.49, P  0.001). However, the analysis of 3-year survival (P  0.35) and 5-year survival (P  0.36) showed no statistical significance, probably as a result of the following reasons. (i) Patients enrolled in these studies received 1–12 courses of CIK cell transfusion for the entire treatment. Theoretically, CIK cells die out gradually after transfusion termination. Patients receiving CIK therapy may need to be transfused in cycles to strengthen the immune response and maintain the efficacy of the CIK cells in clinical treatment. (ii) Most of the trials included in the analysis focused on the efficacy and short-term survival as the

endpoint of observation. Only two articles concerning HCC and renal carcinoma included 3- and 5-year survival analysis. Data regarding long-term toxicities and recurrence rates were unavailable in most of the trials. Second, the analysis of DCR demonstrated that patients receiving CIK cell therapy had better disease control compared with patients in the non-CIK group (P  0.01). Cytotoxicity by CIK cells does not rely on antibody-dependent cell cytotoxicity (ADCC) mechanisms. Conversely, the anti-tumor activity of CIK cells mainly relies on the engagement of NK group 2, member D (NKG2D) by NKG2D ligands on tumor cells, and on perforin-mediated pathways (24). CIK cells can kill cancer cells in three ways: CIK cells directly kill the cancer cells; the activated CIK cells secret numerous cytokines that can kill cancer cells; the CIK cells regulate the immune response directly by killing cancer cells. Interestingly, CIK cells have also been shown to be effective against multidrug-resistant and Fas ligand (FasL)positive malignant cells. FasL, which is affected by the Fas/FasL pathway, plays an important role in apoptosis of carcinoma cells inducted by CIK. Theoretically, CIK cells may kill the residual tumor cells that resisted the chemotherapy and may be beneficial for the patients (25, 26). The incidence of fever in the CIK therapy group was significantly higher than in the non-CIK group

490

Y. Ma et al.

Figure 5. Forest plot for the immunophenotype assessment.

(P  0.02). The body temperature varied from 37.5°C to 39°C within 24 h in most CIK groups and could be treated by simple allopathy. In most of the cases of biologic treatment, the occurrence of fever is a normal reaction of immune function and benefits the treatment (27). Third, the HBV-DNA levels decreased significantly in the CIK group (P  0.01) compared with the non-CIK group. Patients with hepatitis B virus infection can develop hepatic sclerosis and HCC. HCC patients with high levels of HBV-DNA have a poor

prognosis (28). CIK cell therapy also can reduce the AFP, Carcinoma Embryonic Antigen (CEA), carbohydrate antigen (CA) 199 and CA724 content more significantly than in the non-CIK group. The reduction of AFP and HBV-DNA content contributes to preventing the short-term recurrence of HCC and prolonging patient survival time. Fourth, the human immune response against a tumor is mainly dependent on cellular immunity. The ratios of T-lymphocyte subsets in peripheral blood are usually disordered in tumor patients (29). In the

CIK cells for the treatment of solid carcinomas present analysis, the percentage of CD3, CD4, CD4 CD8, CD3 CD56 and CD3 CD56 T cells was significantly increased in the CIK group compared with the non-CIK group (P  0.01). In contrast, the percentage of CD8 T cells was significantly decreased in the CIK therapy group (P  0.001). Tumor immunologic studies show that cellular immunity of cancer patients is closely related to the occurrence and development of cancers. For example, HCC patients are often found to have functional deficiencies in the host adaptive immune response and the innate immune response (30). The cellular immunity of HCC patients is significantly impaired by anticancer drugs for transcatheter arterial chemoembolization (TACE) (31). Many studies have shown that CIK cells possess strong cytotoxicity, can kill drugresistant HCC cells by inducing apoptosis, and can produce IL2, IL6, IFNγ and other antitumor cytokines (32,33). The percentages of CD4T cells and the ratio of CD4 /CD8 declined, whereas the percentages of CD8T cells increased significantly in recurrent HCC patients (18). These chemokines function together with IFN-γ, enabling them potentially to tilt the immune response in a helper T (TH)1 direction (34). The CD56 molecule (neural cell adhesion molecule) is a membrane glycoprotein belonging to the immunoglobulin superfamily expressed on neural and NK cells that also defines a major subclass of NK T cells. The function of NK T cells is to provide more rapid aid to the cellmediated immune response (IFN-γ) or antibodymediated response (IL-4) than the several days needed by conventional T-helper cells (35). Most studies show that serum-free culture medium (SFCM) is safer and can be used as an alternative to complete medium (CM). But there are different points of view regarding whether the culture of CIK cells amplifies more and produces more IFN-γ, IL-4 and IL-5 in CM than SFCM (36,37). Our analysis showed that 1-year survival, half-year PFS and 1-year PFS of CIK cells cultured in CM were 79.63%, 93.59% and 87.1%, which differed significantly from the 66.3%, 72.22% and 40.28% in CIK cells cultured in SFCM. The adverse effects of grade III and IV leukopenia was 3.39% in the SFCM group. This was significantly lower than in the CM group (34.48%). However, the ORR and DCR for the CM group was 53.33% and 84.44%, respectively, which did not differ from the 54.24% ORR and 84.44% DCR for the serum-free medium. Peripheral blood is not always a viable cell source, particularly for allogeneic transplantation, because of a lack of HLA-matched donor availability. Cord blood has several advantages, such as widespread availability, absence of donor risk, absence of donor attrition, low risk of transmissible infectious diseases in the cells,

491

decreased graft-versus-host disease (GvHD) without an increased incidence of relapse even in antigenmismatched situations, and, more importantly, a higher frequency of immune accessory and effector cell precursors such as dendritic cell precursors, natural killer cell precursors and T-cell precursors. Once activated, it can quickly produce a non-specific NK cell-like mechanism and induce CIK cells more easily to soluble tumor cells (38). CB-CIK mainly induce tumor cell necrosis and peripheral blood CIK (PBCIK) induce tumor cell apoptosis (39). However, our analysis showed that the 1-year survival and ORR of CIK cells derived from peripheral blood mononuclear cells (PBMC) was 76.67% and 57.36%, respectively, which differed significantly from the 45% and 30% of CIK cells derived from cord blood mononuclear cells (CBMC). But the DCR and adverse effects of grade III and IV leukopenia for the PBMC group were 86.05% and 16.18%, which did not differ significantly from those of 80% and 5% for the CBMC group. These data are insufficient to draw definite conclusions as only one article fulfilled the criteria for inclusion in our study, with the majority being used for the treatment of blood cancers. Because all 11 selected articles were from China, lacking multinational larger sample multicenter clinic research regarding CIK cell therapy for cancer, we used the NFS method to evaluate the published bias. NFS is the number of unpublished null studies needed to remove the significance from the findings of a meta-analysis. It does not provide a specific value, but represents a tendency compared with the number of research articles. The greater the NFS is, the more stable the outcome of the analysis. In our analysis, the NFS 0.05 of 1-year, 2-year and 3-year survivals, MST, MTTP, DCR and HBV-DNA content was 10.18, 17.51, 6.17, 239.52, 318.9, 9.21 and 13.67, respectively. The NFS 0.05 of T-lymphocyte subsets was from 35.12 to 447.81. However, the NFS 0.05 of 5-year survival and ORR were less than the number of research articles. This may cause bias and thus may affect the conclusion. In order to solve this problem, a larger scale, international multicenter clinical trial should be conducted in the near future. Taken together, the CIK cells were prepared after in vitro priming and were transfused into patients with solid tumors. These early results appear very promising, and the side-effects related to CIK cell transfusion have been few. It will hopefully lead to larger controlled clinical trials in these settings. CIK combined therapy has demonstrated significant superiority in prolonging half-year, 1-year and 2-year survivals and the MST, PFS, DCR and QoL of patients compared with non-CIK therapy. CIK therapy reduced the adverse effects of hematologic and

492

Y. Ma et al.

non-hematologic toxicities caused by chemotherapeutic agents when combined with chemotherapy for the treatment of cancer. These observations support further larger scale randomized controlled trial (RCT) to evaluate the efficacy of CIK cell therapy for the treatment of solid cancer with or without the combination of other therapeutic methods, including chemotherapy.

10.

Acknowledgments

11.

This study was supported by Shanghai Municipal Natural Science Foundation (project number 09ZR1417900), leading academic discipline project of Shanghai Municipal Education Committee (project number J50208), Shanghai Pujiang Program (project number 11PJ1406500) and the National Natural Science Funds (project number 81102015).

9.

12. 13.

14.

15.

Conflict of interest: The authors have declared that no conflict of interest exists. Author contributions: Lei Tang and Ying-Chun Xu performed the computerized search of the trials, contacted experts and participated in the trial selection. Yue Ma and Zan Zhang participated in the trial selection and performed the statistical analysis. Hong-Xia Wang conceived the study. All authors read and approved the final manuscript.

16.

17.

18.

References 1. Schmidt-Wolf IG, Negrin RS, Kiem HP, Blume KG, Weissman IL. et al. Use of a SCID mouse/human lymphoma model to evaluate cytokine-induced killer cells with potent antitumor cell activity. J Exp Med. 1991;174:139–49. 2. Dillman RO, Duma CM, Ellis RA, Cornforth AN, Schiltz PM, Sharp SL, et al. Intralesional lymphokine-activated killer cells as adjuvant therapy for primary glioblastoma. J Immunother. 2009;32:914–19. 3. Schmidt-Wolf IG, Lefterova P, Metha BA, Fernandez LP, Huhn D, Blume KG, et al. Phenotypic characterization and identification of effector cells involved in tumor cell recognition of cytokine-induced killer cells. J. Exp Hematol. 1993;21:1673–79. 4. Joshi PS, Liu JQ, Wang Y, Chang X, Richards J, Assarsson E, et al. Cytokine-induced killer T cells kill immature dendritic cells by TCR-independent and perforin-dependent mechanisms. J Leukoc Biol. 2006;80:1345–53. 5. Linn YC, Hui KM. Cytokine-induced NK-like T cells: from bench to bedside. J Biomed Biotechnol. 2010:1–8. 6. Hontscha C, Borck Y, Zhou H, Messmer D, Schmidt-Wolf IG. Clinical trials on CIK cells: first report of the international registry on CIK cells (IRCC). J Cancer Res Clin Oncol. 2010;137:305–10. 7. Bonanno G, Iudicone P, Mariotti A, Procoli A, Pandolfi A, Fioravanti D, et al. Thymoglobulin, interferon-γ and interleukin-2 efficiently expand cytokine-induced killer (CIK) cells in clinical-grade cultures. J Transl Med. 2010;7:129–143. 8. Introna M, Pievani A, Borleri G, Capelli C, Algarotti A, Micò C, et al. Feasibility and safety of adoptive immunotherapy

19.

20.

21.

22. 23.

24.

25.

with CIK cells after cord blood transplantation. Biol Blood Marrow Transplant. 2010;16:1603–7. Schmidt-Wolf IG, Finke S, Trojaneck B, Denkena A, Lefterova P, Schwella N, et al. Phase I clinical study applying autologous immunological effector cells transfected with the interleukin-2 gene in patients with metastatic renal cancer, colorectal cancer and lymphoma. Br J Cancer. 1999;81:1009–16. Yates JW, Chalmer B, McKegney FP. Evaluation of patients with advanced cancer using the Karnofsky performance status. Cancer. 1980;45:2220–24. Hollen PJ, Gralla RJ, Kris MG, Cox C. Quality of life during clinical trials: conceptual model for the Lung Cancer Symptom Scale (LCSS). Support Care Cancer. 1994;2:2213–22. Rosenthal R. The file drawer problem and tolerance for null results. Psychol Bull. 1979;86:638–41. Thornton A, Lee P. Publication bias in meta-analysis: its causes and consequences. J Clin Epidemiol. 2000;53: 207–16. Sutton JA, Song F, Gilbody MS, Abrams KR. Modelling publication bias in meta-analysis: a review. Stat Meth Med Res. 2000;9:421–45. Hao MZ, Lin HL, Chen Q, Ye YB, Chen QZ, Chen MS. Efficacy of transcatheter arterial chemoembolization combined with cytokine-induced killer cell therapy on hepatocellular carcinoma: a comparative study. Chin J Cancer. 2010:29:172–77. Pan CC, Huang ZL, Li W, Zhao M, Zhou QM, Xia JC, et al. Serum alpha-fetoprotein measurement in predicting clinical outcome related to autologous cytokine-induced killer cells in patients with hepatocellular carcinoma undergoing minimally invasive therapy. Chin J Cancer. 2010:29:596–602. Pugh RN, Murray-Lyon IM, Dawson JL, et al. Transection of the oesophagus for bleeding oesophageal varices. The British journal of surgery. 1973; 60:646–49. Zhao M, Wu PH, Zeng YX, Xia JC, Zhang FJ, Xian LJ, et al. Cytokine-induced killer cell fusion to lower recurrence of hepatocellular carcinoma after transcatheter arterial chemoembolization sequentially combined with radiofrequency ablation: a randomized trial. Zhonghua Yi Xue Za Zhi. 2006;86:1823–28. Weng DS, Zhou J, Zhou QM, Zhao M, Wang QJ, Huang LX, et al. Minimally invasive treatment combined with cytokineinduced killer cells therapy lower the short-term recurrence rates of hepatocellular carcinomas. J Immunother. 2008;31:63–71. Hui D, Qiang L, Jian W, Ti Z, Da-Lu K. A randomized, controlled trial of postoperative adjuvant cytokine-induced killer cells immunotherapy after radical resection of hepatocellular carcinoma. J Dig Liver Dis. 2009;41:36–41. Introna M, Borleri G, Conti E, Franceschetti M, Barbui AM, Broady R, et al. Repeated infusions of donor-derived cytokineinduced killer cells in patients relapsing after allogeneic stem cell transplantation: a phase I study. Haematologica. 2007;92: 952–59. Li Q, Chang AE. Adoptive T-cell immunotherapy of cancer. Cytokines Cell Mol Ther 1999; 5:105-17. Wang H, Zhou FJ, Wang QJ, Qin ZK, Huang LX, Liu ZW et al. Efficacy of autologous renal tumor cell lysate-loaded dendritic cell vaccine in combination with cytokineinduced killer cells on advanced renal cell carcinoma: a report of ten cases. Ai Zheng. 2006;25:625–30. Jiang H, Liu KY, Tong CR, Jiang B, Lu DP. The efficacy of chemotherapy in combination with auto-cytokine-induced killer cells in acute leukemia. Zhonghua Nei Ke Za Zhi. 2005;44:198–201. Verneris MR, Karami M, Baker J, Jayaswal A, Negrin RS. Role of NKG2D signaling in the cytotoxicity of activated and expanded CD8T cells. Blood. 2004;103:3065–72.

CIK cells for the treatment of solid carcinomas 26. Yu J, Zhang W, Jiang H, Li H, Cao S, Ren X. CD4 T cells in CIKs (CD4 CIKs) reversed resistance to fas-mediated apoptosis through CD40/CD40L ligation rather than IFN-gamma stimulation. Cancer Biother Radiopharm. 2008;23:342–54. 27. Li SY, Zhang CJ, Wang SY, Wu Qi, Zhao SQ, Pei CY. Study of inhibitory effects of CIK on Lewis lung carcinoma. Zhong Guo Xian Dai Yi Xue Za Zhi. 2007;21:2605–8. 28. Schmidt-Wolf GD, Negrin RS, Schmidt-Wolf IG. Activated T cells and cytokine-induced CD3 CD56 killer cells. Ann Hematol. 1997;74:51–6. 29. Michielsen P, Ho E. Viral hepatitis B and hepatocellular carcinoma. J. Acta Gastroenterol Belg. 2011;74:4–8. 30. Kastelan Z, Lukac J, Derezic D, Pasini J, Kusic´ Z, Sosic´ H, et al. Lymphocyte subsets, lymphocyte reactivity to mitogens, NK cell activity and neutrophil and monocyte phagocytic functions in patients with bladder carcinoma. Anticancer Res. 2003;23:5185–89. 31. Ladhams A, Schmidt C, Sing G, Butterworth L, Fielding G, Tesar P, et al. Treatment of non-resectable hepatocellular carcinoma with autologous tumor-pulsed dendritic cells. J Gastroenterol Hepatol. 2002;17:889–96. 32. Lu W, Li YH, He XF, Chen Y, Zeng QL, Qiu YR. Effect of dosage of anticancer agents during transcatheter arterial chemoembolization on T cell subsets in patients with hepatocellular carcinoma. J Di Yi Jun Yi Da Xue Xue Bao. 2002; 22:524–26. 33. Zhang YS, Yuan FJ, Jia GF, Zhang JF, Hu LY, Huang L, et al. CIK cells from patients with HCC possess strong cytotoxicity to multidrug resistant cell line. J World J Gastroenterol. 2005;11:3339–45. 34. Märten A, Ziske C, Schöttker B, Renoth S, Weineck S, Buttgereit P, et al. Interactions between dendritic cells and cytokine-induced killer cells lead to an activation of both populations. J Immunother. 2001;24:502–10. 35. Joshi PS, Lir JQ, Wang Y, Chang X, Richards J, Assarsson E, et al. Cytokine-induced killer T cells kill immature dendritic cells by Tcr-independent and perforin-dependent mechanisms. J Leukoc Biol. 2006;80:1345–53. 36. Saikh KU, Dyas B, Kissner T, Ulrich RG. CD56 T-cell responses to bacterial superantigens and immune recognition

37.

38.

39. 40.

41.

42.

43.

44.

45.

46.

493

of attenuated vaccines. Clin Diagn Lab Immunol. 2003; 10:1065–73. Cao JP, Jiang ZM, Zhang XC, Lin D, Chen W, Sun YH, et al. The proliferation, phenotype change and anti-tumor activity of cytokine induced killer cell. J Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2005;21:583–86. Kambe N, Kambe M, Chang HW, Matsui A, Min HK, Hussein M, et al. An improved proved procedure for the development of human mast cells from dispersed fetal liver cells in serum-free culture medium. J Immounol Methods. 2000;240:101–10. Gluckman E, Rocha V. Cord blood transplantation: state of the art. Hematologica. 2009;94:451–4. Li Y, Schmidt-Wolf IG, Wu YF, Huang SL, Wei J, Fang J, et al. Optimized protocols for generation of cord bloodderived cytokine-induced killer/natural killer cells. J Anticancer Res. 2010;30:3493–99. Zhao G, Huang Y, Ye L, Duan L, Zhou Y, Yang K, et al. Therapeutic efficacy of traditional vein chemotherapy and bronchial arterial infusion combining with CIKs on III stage non-small cell lung cancer. Zhongguo Fei Ai Za Zhi. 2009; 12:1000–4. Wu C, Jiang J, Shi L, Xu N. Prospective study of chemotherapy in combination with cytokine-induced killer cells in patients suffering from advanced non-small cell lung cancer. Anticancer Res. 2008;28:3997–4002. Jiang J, Wu C, Shi L, N Xu, H Deng, M Lu, et al. Side effects during treatment of advanced gastric carcinoma by chemotherapy combined with CIK-cell transfusion in elderly people. Clin Oncol Cancer Res. 2008;5:79–82. Jiang J, Xu N, Wu C, Deng H, Lu M, Li M, et al. Treatment of advanced gastric cancer by chemotherapy combined with autologous cytokine-induced killer cells. Anticancer Res. 2006;26:2237–42. Lei K, Wng J, Jia Y, Xiong Y, Yuan Z. The effects of CIK cells on the treatment of renal cell carcinoma. Chinese-German J Clinical Oncol. 2009;8:346–48. Niu Q, Wang W, Li Y, Qin S, Wang Y, Wan G, et al. Cord blood-derived cytokine-induced killer cells biotherapy combined with second-line chemotherapy in the treatment of advanced solid malignancies. J Int Immunopharmacol. 2011;11:449–56.