Meta-analysis of chemotherapy and dendritic cells with cytokine-induced killer cells in the treatment of non-small-cell lung cancer

Abstract

Background: Non-small-cell lung cancer (NSCLC) is one of the most fatal cancers, which leads to large number of people dead. Followed by surgery, chemotherapy and radiotherapy, chemotherapy combined dendritic cells with cytokine-induced killer cells (DC-CIK) immunotherapy has been applied in NSCLC for some time, but little consistent beneficial results are provided. So, it is essential to weigh the pros and cons of the new therapeutic method. Methods: We searched the randomized controlled trials of NSCLC mainly by PubMed database. Terms combination of “cytokine-induced killer cells”, “tumor” and “cancer” were used. After evaluating the heterogeneity of selected studies, then we performed the meta-analysis. Pooled risk ratios (RRs) were estimated and 95% confidence intervals (CIs) were calculated using a fixed-effect model. Sensitivity analysis was also performed. Results: Six eligible trials were enrolled. Efficiency and safety of chemotherapy followed by DC-CIK immunotherapy (experimental group) and chemotherapy alone (control group) were compared. 1-year overall survival (OS) (P=0.02) and progression free survival (PFS) (P=0.005) in the experimental group were significantly increased compared with the control. Disease control rate (DCR) (P=0.006) rose significantly in experimental group. However, no significant differences between the two groups were observed in 2-year OS (P=0.21), 2-year PFS (P=0.10), overall response rate (ORR) (P=0.76) and partial response (PR) (P=0.22). Temporary fever, anemia, leukopenia and nausea were the four major adverse events (AEs) treated by chemotherapy. The incidence of anemia, leukopenia and nausea in the experimental group was obviously lower than the control group. Temporary fever rate was higher in experimental group than that in the control, but could be alleviated by taking sufficient rest. Conclusions: Chemotherapy combined with DC-CIK immunotherapy showed superiority in DCR, 1-year OS and PFS, and no more AEs appeared, however, there was no significant improvement in ORR, PR, 2-year OS and PFS. As a whole, the combination therapy is safer but modest in efficacy for advanced NSCLC patients.

Introduction

Lung cancer, especially NSCLC, has been considered one of most commonly diagnosed and devastating disease in the world [1]. NSCLC was regarded as one of the leading killers in the causes of mortality worldwide and possess about 80% to 85% of all lung cancer cases [2].

So far, the most widely applied cancer treatments are still surgery, radiation and chemotherapy; however, these methods have some side effects and often fail to completely remove minimal residual cancer cells, including small lesions and metastatic cells. What’s worse, five-year survival rate keeps very poor [3], then drug resistance and adverse effects still remain, thus, the more effective and safer treatments are urgently required to improve the quality and duration of life [4]. Then immunotherapy appears. Dendritic cells with cytokine-induced killer cell (DC-CIK) therapy has been widely used as an important component of cancer treatments [5].

The CIK cell is developed on the basis of LAK (Lymphokine-activated killer, LAK). LAK cells are IL-2-activating cytotoxic effector lymphocyte, and CIK cells areaquired by incubating mononuclear cells from peripheral blood, bone marrow or cord blood with additions, such as interleukin (IL)-2, IL-1, CD3 monoclonal antibody and interferon (IFN)-γ [6]. CIK and LAK are both heterogeneous. LAK cells mainly consist of CD3^-CD56⁺ natural killer (NK) cells, and CD3⁺CD56^- T cells while CIK cells are mainly have 88% CD3⁺CD56⁺ T cells [7]. The main components of them are the main contributors to the cytolytic properties respectively. They were first described as higher proliferation and more advantages in antitumor activities than LAK cells by Schmidt Wolf et al. [8]. Though CD3⁺CD56⁺ cells are derived from CD3⁺CD56^- T cells, they were demonstrated that they had enhanced cytotoxicity against various tumor cells compared with CD3⁺CD56^- cells and expanded up to 1000-fold in cultures [9].

CIK cells have been proven to take advantages over LAK cells and primary cytotoxic T cells (CTL), like higher proliferation rate, greater efficacy but few side-effects, and non-MHC-restricted killing [10]. Therefore, CIK cell-based therapy shows more promising clinical prospects for many types of cancers in clearing minimal residual cancer cells. More and more current clinical trials have proved this point. Since the first clinical trial of CIK immunotherapy for cancer patients was reported in 1999 [11], CIK cells therapy was widely used in various types of solid tumors, such as breast cancer, liver cancer, kidney cancer, colorectal cancer and non-small-cell lung cancer (NSCLC). Several clinical trials have shown encouraging results. They demonstrated that CIK immunotherapy may prevent recurrence, improve progression-free survival rates (PFS), and promote the quality of life (QoL) [12,13]. However, clinical studies of CIK immunotherapy are still at its start and lack a certification provided by multicenter studies with large samples. Therefore, we performed a systematic review and meta-analysis of randomized phase II and III clinical trials to assess the efficacy and safety of DC-CIK immunotherapy in the treatment of patients with NSCLC. We aim to evaluate the effect of CIK-based therapy on the survival and QoL of cancer patients, as well as the toxicity.

Methods

Search strategy

Articles published in English from PubMed, EMBASE, and Web of Science were collected for studies which could be enrolled in this meta-analysis from 2003 to 2014. Keywords like “Dendritic Cells with Cytokine-Induced Killer Cells” or “DC-CIK immunotherapy”, “Non-Small-Cell Lung Cancer” or “NSCLC”, and “chemotherapy” were used in conjunction.

Inclusion criteria and study selection

All the randomized studies of patients with NSCLC as one of two arms of therapeutic regimens: DC-CIK-based immunotherapy and chemotherapy alone were considered.

The study selection was as follows: (1) studies were published in English and performed in human (2) studies should be randomize controlled trials (3) all the trials had not to be confounded by other treatments in either group.

Data collection

To collect the potential relevant articles, all the full articles and available abstracts were scanned and analyzed by two independent reviewers (Chenhong Zheng and Ganjun Yu). Divergences were discussed with a third investigator (Hui Wang). The following information was cross-checked and collected: the first and last authors’ names, the published year and journal, the place of the performed study, the study design (mode of randomiation, description of withdraws, blinding information), the number of sample size, the median age of patients, the treatments that patients received, the study period and the NSCLC stages. The randomization integrity were assessed by checking the patterns of treatment allocation and the balanced baseline characteristics. To ensure that the follow-up of surviving patients was balanced by treatment group and was current, we checked all the available data.

Measures of curative outcome

Clinical responses such as the median survival time (MST), the overall survial (OS) and progression free survival (PFS) were evaluated in prognosis. Partial response (PR), overall response rate (ORR) and disease control rate (DCR) were assessed in treatment efficacy. 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. OS was defined as the time from the start of the treatment to the time of death from any case. PFS was defined as the length of time during and after treatment for which a patient lived with a disease that did not worsen. ORR was defined as the sum of partial and complete response rates, while the DCR as the sum of stable disease, partial response and complete response rates. All the criteria were defined according to the World Health Organization (WHO). Then we evaluated the toxicity, QoL and subsets of the T-lymphocyte in patients of the studies. Toxicity was assessed in accordance with the criteria of the National Cancer Institute (NCI) Common Toxicity Criteria (CTC). QoL was assessed on the basis of the Karnofsky performance status (KPS) or Lung Cancer Symptom Scale (LCSS). The results were provided by the articles included or calculated according to the data in an article.

Statistical analysis

We did Statistical analysis with Review Manager Version 5.2 (Cochrane Collaboration). P<0.05 was considered statistically significant. All reported P-values resulted from two sided version tests of the respective tests. Heterogeneity between the trials was assessed to determine which model might be most suitable. The Cochran Q-test was performed when the homogeneity of the results of studies was considered at a predefined significance threshold of P>0.1 or I²<50%. The odds ratios (OR) were the principal measurements of effect and were presented with a 95% confidence interval (CI).

Results

Selection results

Sixteen publications were finally considered eligible by referring to pr-defined inclusion criteria. The reasons for study inclusion/exclusion are graphically described in Figure 1.

Figure 1.

Flow diagram of the study selection process Table 1 showed the characteristics of the whole selected trials.

The characteristics and quality of the studies are presented in Tables 1 and 2, respectively.

Table 1.

Clinical information of the eligible trails for the meta-anlysis

First author	Nation	Journal	Study type	Sample size
				Chemo-DC-CIK	Chemo
Hui Li [14]	China	Cytotherapy	Case control	42	42
Lili Yang [15]	China	Cancer Immunol Immunother	Case control	61	61
Min Zhao [16]	China	Experimental and Therapeutic Medicine	Case control	79	78
Runbo Zhong [17]	China	Cancer Immunol Immunother	Case control	14	14
Shengbin Shi [18]	China	Tumori	Case control	30	30
Junping Zhang [19]	China	Chinese-German Journal of Clinical Oncology	Case control	50	50

Chemo, chemotherapy; Chemo-DC-CIK, chemotherapy and DC-CIK therapy. NR not reported.

Table 2.

Patient information for the eligible trials (X² tests) (P>0.05)

Features	Chemo-	Chemo-DC-CIK	X2	P-value
Gender	225	226	0.0043	0.9479
Male	148	149
Female	77	77
Category			0.2797	0.8695
Adenocarcinoma	158	164
Squamouscinoma	95	92
Large-cell carcinoma	11	13
Stage			0.0078	0.9961
IIIa	45	44
IIIb	45	44
IV	47	47

Bias of the included studies are presented in Figure 2.

Figure 2.

Funnel plot of comparison: 1 OS, outcome: 1.3 OS 2 Y.

Risk of bias summary: review authors’ judgements about each risk of bias item for each included study are presented in Figure 3.

Figure 3.

Risk of bias summary: review authors’ judgements about each risk of bias item for each included study.

After strict selection, the clinical trials included in the paper is shown in Figure 1. So, 6 articles reporting clinical trials of DC-CIK cell-based immunotherapy and chemotherapy for NSCLC were used in our meta-analysis.

All patients in the immuno-CT- and CT-treated groups received for cycles of chemotherapy after surgery, except for stage I patients. In the immuno group, patients were treated with CIK cells that were activated by Ag-loaded DC after chemotherapy. PBMC were collected from NSCLC patients after surgery and chemotherapy. For culture of CIK cells, PBMC were cultured with anti-CD3 antibody (Ab) to stimulate CIK cell growth, recombinant human interleukin (IL)-1α and recombinant human interferon (IFN)-γ and recombinant human IL-2 and IFN-γ-containing medium was added to the culture system. IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were cultured for DC, to facilitate DC growth. To stimulate DC, autologous tumor lysate was added and co-cultured with DC. For CIK cell activation, after CIK cell culture, CIK cells were co-cultured with DC loaded with tumor Ag.

Efficacy assessment: DCR, ORR

The analysis of DCR also demonstrated favorable results for the CIK cell therapy arm, with the RR being 1.3 (95% CI 1.11-1.55, P=0.002) (Figure 4). However, the ORR for the chemotherapy combined with CIK group was 53.69%, which did not differ signifcantly from the ORR of 44.44% for the chemotherapy-alone group (RR 1.15, 95% CI 0.80-1.65, P=0.45) (Figure 5).

Figure 4.

Forest plot of the comparison of disease control rate (DCR). P values are from P for the effect modification evaluation of heterogeneity within or across the groups of regimens. CI, confidence interval; RR, risk ratio; DC/CIK, DC-CIK immunotherapy; Chemo, chemotherapy; Con, control group; Exp, experimental group. A fixed-effect meta-analysis model (Mantel-Haenszel method) was used.

Figure 5.

Forest plot of the comparison of overall response rate (ORR), P values are from P for the effect modification evaluation of heterogeneity within or across the groups of regimens. CI, confidence interval; RR, risk ratio; DC/CIK, DC-CIK immunotherapy; Chemo, chemotherapy;Con, control group; Exp, experimental group. A fixed-effect meta-analysis model (Mantel-Haenszel method) was used.

Prognosis evaluation: OS and TPP

Patients in the CIK group were associated with prolonged 1 year TTP compared with the non-CIK group (OR 1.87, 95% CI 1.04-2.69, <0.00001) (Figure 6). Furthermore, the results of the pooled analysis showed that patients in the CIK group had a significantly improved half-year survival (RR 1.16, 95% CI 1.04-1.31, P=0.01), 1-year survival (RR 1.57, 95% CI 1.02-2.04, P=0.04) and 2-year survival (RR 1.30, 95% CI 1.10-1.53, P=0.002) and the 3-year survival (RR 1.51, 95% CI 1.19-1.91, P=0.0007) (Figure 7).

Figure 6.

Forest plot for 1 year TTP. Chemo, chemotherapy; Chemo-DC-CIK, chemotherapy and DC-CIK therapy.

Figure 7.

Forest plot of the comparison of overall survival (OS). 0.5, 1-, 2-year and 3-year overall survival (OS) between the non-DC-CIK and DC-CIK groups. P values are from P for the effect modification evaluation of heterogeneity within or across the groups of regimens. CI, confidence interval; RR, risk ratio; DC/CIK, DC-CIK immunotherapy; Chemo, chemotherapy; Con, controlgroup; Exp, experimental group. The fixed effects meta-analysis model (Mantel-Haenszel method) was used.

Toxicity analysis

In most trials, nausea and peripheral nerve toxicity could be seen within 24 h after CIK cell transfusion. On the whole, the analysis showed that the incidence of Nausea (RR 0.63, 95% CI 0.44-0.88, P=0.008). However, the Peripheral nerve toxicity group. (RR 1.43, 95% CI 0.57-3.59, P=0.45) for the chemotherapy combined with CIK group did not differ significantly from in the chemotherapy group (Figure 8).

Figure 8.

Forest plot for the toxicity. Comparison of the toxicity (Nausea and Peripheral nerve toxicity) between the non-DC-CIK and DC-CIK groups. P values are from P for the effect modification evaluation of heterogeneity within or across the groups of regimens. CI, confidence interval; RR, risk ratio; DC/CIK, DC-CIK immunotherapy; Chemo, chemotherapy; Con, control group; Exp, experimental group. A fixed-effect meta-analysis model (Mantel-Haenszel method) was used.

Immuno response

The analysis showed that the ratio of CD3, CD4, NK, CD3⁺CD56⁺ cells was significantly increased in the CIK group compared with the non-CIK group, which was reflected by pooled R of -0.45 for CD3 cells (95% CI-0.73 to -0.18, P=0.001), CD4 R of -7.18 (95% CI-7.94 to -6.42, P<0.001), NK R of -4.99 (95% CI-6.91 to -3.06, P<0.001), CD3⁺CD56⁺ cells R of -6.32 (95% CI-7.36 to -5.28, P<0.001). Furthermore, the percentage of CD8 T cells was significantly decreased in the CIK group compared with the non-CIK group (OR 5.97, 95% CI 5.00-6.94, P<0.001) (Figure 9).

Figure 9.

Forest plot for the immunophenotype assessment. Pre-DC-CIK-chemotherapy and post-DC-CIK-chemotherapy. P values are from P for the effect modification evaluation of heterogeneity within or across the groups of regimens. CI, confidence interval; DC/CIK, DC-CIK immunotherapy; Chemo, chemotherapy; Con, control group; Exp, experimental group. A fixed-effect meta-analysis model (Mantel-Haenszel method) was used.

Sensitivity analysis

We performed sensitivity analyses to examine the stability of the results of this meta-analysis. The pooled ORs were not altered qualitatively when each study was excluded, suggesting our results are statistically reliable.

Discussion

Immunotherapy has become the most promising approach for cancer therapy, as the Science has commented that cancer immunotherapy is the top 1 of the ten scientific and technological breakthroughs in 2013 [20]. Due to CIK (Cytokine-induced killer) cell treatment play a central role in cell-mediated immunity, anti-tumor cells activity included, which is a subset of immunotherapy, has attracted more and more attention and made gratifying achievements.

The effect of cellular immunotherapy based on CIK cells, which generates form non-major histocompatibility complex (MHC) restricted cytotoxic lymphocytes through peripheral blood lymphocytes plus interleukin (IL)-2, IL-1, anti-CD3 monoclonal antibody, and interferon gamma (IFN-γ). Dendritic cells (DC) specialized antigen-presenting cells can be found in body, which exists in most tissues. When countering with antigens, dendritic cells can be show its constitutive patrol activities from immune-organs, which could kill cells. Accumulated studies have been shown that DC (Dendritic cell) -activated CIK (Cytokine-induced killer) cell treatment play a critical role in anti-tumor therapeutic strategies [21,22]. Compared with the CIK treatment, DC-CIK treatment have demonstrated that which could be increasing the anti-tumor activities than CIK treatment both in vivo and in vitro [23,24]. Furthermore, More and more studies have proven that DC-CIK has powerful anti-tumor properties and a good potential for clinical application [25]. Clinic studies also have proven that DC-CIK treatment display more immune response, safety, and survival and quality of life outcomes than common chemotherapies alone in colorectal cancer [26,27]. Assessment of safety and efficacy of this approach for the treatment of other types of tumors is under way.

This meta-analysis focus on the chemotherapy combined with DC-CIK in NSCLC treatment. Until up-to now, the number of DC-CIK therapy clinical trials about NSCLC are published for about 20 articles. Due to the quality of publish reports may influence confidence and bias of the meta-analysis, we enrolled six reports in our meta-analysis.

Our analysis demonstrated that DCR had favorable results for the CIK cell therapy arm, But the ORR for the chemotherapy combined with CIK did not differ significantly from the chemotherapy-alone group. Patients in the CIK group were associated with prolonged 1 year TTP compared with the non-CIK group. Furthermore, the results of the pooled analysis showed that patients in the CIK group had a significantly improved half-year survival, 1-year survival, and 2-year survival, and the 3-year survival. Nausea and peripheral nerve toxicity for the chemotherapy combined with CIK group did not differ significantly from in the chemotherapy group.

There are advantages over the conventional treatments using DC-CIK cells immunotherapy as follows: Firstly, It is more accurate to kill tumor cells. Compared with chemotherapy and radiotherapy killing all cells together, DC-CIK therapy is more like “precision-guided missiles” that can accurately kill tumor cells without killing innocent cells. Therefore, the side effects of the treatment to the patients are relatively small. Secondly, the possibility of developing drug-resistance is less, so it could be applied to clinics for a long time [28]. Last, but the most important, it could still play an important role in immune surveillance after killing tumor cells, and protecting the body all his lifetime.

In contrast to other tumor biotherapies, DC-CIK cells therapy is of broad applicability, slight side effects and stability. Though the approach currently has some limitations, such as low levels of cell number persisted in vivo after infusion, low affinity for tumor-specific antigen and relatively long time frame required to isolate and expand target T cell clones, some clinical trials has made it a potential candidate for cancer therapy. Here we introduce and summarize the studies about DC-CIK cells applying to cancer immunotherapy.

Disclosure of conflict of interest

None.