Cytokine-induced killer cell/dendritic cell combined with cytokine-induced killer cell immunotherapy for treating advanced gastrointestinal cancer

Background This study aimed to investigate the efficacy and safety of cytokine-induced killer (CIK)/dendritic cell combined with CIK (DC–CIK) cell therapy in advanced gastrointestinal cancer (GIC). Methods The PubMed, Cochrane library, and Embase were searched to conduct a meta-analysis of clinical controlled trials to evaluate the efficacy and safety of CIK/DC–CIK cell therapy in advanced GIC. The pooled risk ratios (RRs) or weighted mean difference (WMD) with 95% confidence intervals (95% CIs) were calculated. Results A total of nine studies with 1113 patients were identified. The overall survival (RR = 1.84, 95% CI = 1.41–2.40, Pheterogeneity = 0.654, I2 = 0%), progression-free survival (RR = 1.99, 95% CI = 1.52–2.60, Pheterogeneity = 0.727, I2 = 0%), and quality of life (WMD = 16.09, 95% CI = 1.66–30.52, Pheterogeneity < 0.001, I2 = 98.8%) were significantly improved in patients who received chemotherapy combined with CIK/DC–CIK cells, and no severe adverse events were reported. Conclusion This meta-analysis suggested that the combination of CIK/DC–CIK immunotherapy and chemotherapy was safe and applicable for patients with advanced GIC. It is a feasible choice to prolong survival and improve quality of life.

Autoimmune therapy for malignant tumors is considered to be a feasible method, which mainly depends on the interference and inhibition effects of killer cells induced by the tumor, infiltrating lymphocytes and lymphokines, and CD3 monoclonal antibody [7,8]. Dendritic cells (DCs) have the ability to present antigens, making them an attractive vehicle for the delivery and development of therapeutic tumor vaccines [9] . Cytokine-induced killer cells (CIK) is a heterogeneous effector CD3 + CD56 + NKT cell population that can be expanded from peripheral blood mononuclear cells (PBMC) in vitro [10,11]. CIK cells have a strong MHC non-restrictive cytotoxicity, which inhibits both blood and solid malignancies, identifying and killing tumor targets without exposure or initiation. At present, CIK therapy or DC-CIK cell co-cultivation has been broadly applied in clinical trials for the treatment of GICs [12][13][14][15][16][17][18][19][20]; however, the results are not entirely consistent. Therefore, we conducted a systematic review and metaanalysis of published literature to evaluate the efficacy and safety of CIK/DC-CIK cells combined with chemotherapy for the treatment with advanced GIC cancer.

Literature search
The databases PubMed, Cochrane library, and Embase were searched for all relevant studies published in English. The search terms included "dendritic cells," "immunotherapy," "cytokine induced killer cells" OR "DC-CIK" combined with "colon OR rectal OR colorectal OR gastric cancer/tumor/carcinoma/neoplasm." The last research was updated on January 1, 2019. The reference lists of all retrieved studies and published reviews were searched manually for additional references, and all identified relevant studies were included.

Study selection
The inclusion criteria were as follows: (1) clinical controlled trials of patients with CRC or GC; (2) patients pathologically diagnosed with Tumor Node Metastasis (TNM) stages of II, III, and IV; and (3) patients in the experimental group treated using chemotherapy combined with CIK or DC-CIK immunotherapy, whereas patients in the control group treated using chemotherapy alone. The exclusion criteria were as follows: (1) reviews, conference abstracts, letter, or case reports; (2) multiple studies published on the same population (in which case the most recent and complete study was included); and (3) studies without available data for statistics. The studies meeting at least one of the aforementioned three criteria were excluded.

Data extraction and quality assessment
The following data from each study were extracted independently by two authors: authors, year of publication, sample size, chemotherapy regimen, follow-up period, curative effect, and adverse events of each eligible trial. The primary endpoints were OS and progression-free survival (PFS). The other endpoints were complete response (CR), partial response (PR), overall response rate (ORR), and quality of life (QOL). Safety analyses were also performed. Two reviewers independently extracted the studies. Any disagreements were resolved by consensus. Evaluation of the research quality was managed using the Cochrane Collaboration's tool for assessing risk of bias.

Statistical analysis
The meta-analysis was conducted using Stata 14.0 (Stata Corp., TX, USA). Risk ratios (RRs) or weighted mean difference (WMD) with 95% confidence intervals (CIs) were calculated as effect sizes. RR was the effect measurement for dichotomous outcomes, while WMD was applied for the continuous variables. The potential heterogeneity across studies was examined via Cochran's Q-statistic and I 2 statistics. The P value for heterogeneity < 0.1 indicated that the heterogeneity was statistically significant. Thus, the random-effects model was used to perform the analysis. Otherwise, the summary effect was computed using the fixed-effects model. In the sensitivity analysis, the influence of each study on the summary effect was analyzed by dropping one study at a time. The Begg's and Egger's tests were conducted to evaluate publication bias. The trim-and-fill method was used to determine the effect of potential publication bias on the pooled estimates. A P value less than 0.05 was considered to be statistically significant.

Literature search and study selection
A total of 466 studies were identified from PubMed, Cochrane library, and Embase. Then, three additional records were found by manually searching the reference lists of other studies. After deleting the duplications, 351 studies were selected. Then, 330 studies were discarded because of their irrelevance to the topic of interest. Of the remaining 21 studies, 3 were excluded for unavailability of data for statistics and four were excluded as they did not include controls. Besides, five studies did not focus on the advancedstage GIC. Finally, a total of nine studies, including 1113 patients, met the inclusion and exclusion criteria in this meta-analysis [11][12][13][14][15][16][17][18][19] . The flow diagram of the search process is shown in Fig. 1.

Study characteristics
The key characteristics of all included studies are summarized in Table 1. All the studies involved patients with advanced GIC followed up for at least 24 months. Nine studies from 2006 to 2017 compared CIK/DC-CIK plus chemotherapy with chemotherapy alone for treating advanced GIC. These nine studies were assessed using the Cochrane Collaboration's tool for the risk of bias. A graph and summary of the risk of bias are shown in Figs. 2 and 3. Four studies did not mention randomization, and three studies did not provide information of allocation concealment.

Meta-analyses Efficacy
Clinical responses were assessed in terms of the OS and PFS to evaluate the prognosis.

Efficacy
Efficacy was assessed in terms of CR, PR, and ORR. The analysis result is shown in Fig. 4; however, the RR of CR       Figure S1).

Subgroup analysis
This study probed the detailed results in subgroup analyses stratified by cancer type (GC or CRC). All subgroup results were quite consistent with the overall results. The results are summarized in Table 2.

Sensitivity analysis
Sensitivity analyses were performed to assess the stability of the results by sequentially removing each study. The removal of any single study did not change the overall statistical results, indicating that the results of this study were statistically robust (Fig. 5).

Publication bias
The outcome of OS, with the largest number of included studies, was chosen to test the publication bias. Visual examination of the funnel plot (Fig. 6a) revealed a considerable degree of asymmetry. In addition, publication bias was statistically significant by Egger's test or Begg's  Therefore, a sensitivity analysis was conducted using the trim-and-fill method (Fig. 6b) [21]. After imputing six unpublished studies, the trim and fill sensitivity analysis did not change the general result (RR = 0.255, 95% CI = 0.176-0.333, P < 0.01).

Discussion
GIC is still a fatal threat to human health due to metastasis, with recurrence inducing refractory advanced tumor stage and poor prognosis. Yan et al. [22] proved that the recurrence rate of patients with GC ranged from 40 to 65% due to distant metastasis and local recurrence. Adoptive cellular immunotherapy can be used in combination with standard therapy as adjuvant and palliative treatment after operation to improve the survival rate and QOL of patients with GIC. The CIK cells have shown the best efficacy in this treatment. Compared with lymphokine-activated killer cells, CIK cells are more readily available and show higher tumor-specific cytotoxic activity [23][24][25][26][27]. To date, several clinical trials have used chemotherapy plus DC-CIK immunotherapy to treat advanced GIC. However, in these trials, the clinical protocol applied was not standard, blurring the evaluation of treatment effects. In this study, a large number of comprehensive trials were investigated to achieve higher statistical reliability. This meta-analysis showed that chemotherapy combined with CIK/DC-CIK immunotherapy improved the OS, PFS, and QOL without causing serious adverse events. This study confirmed the safety of CIK/DC-CIK immunotherapy for advanced GIC patients, and the adverse events caused were tolerated by all patients. No significant difference was observed in terms of common adverse events, such as thrombocytopenia (RR = 1.04, 95% CI = 0.93-1.16, P heterogeneity = 0.497, I 2 = 0%), nausea, vomiting (RR = 0.67, 95% CI = 0.35-1.28, P heterogeneity = 0.003, I 2 = 82.9%), abnormal liver function (RR = 1.08, 95% CI = 0.95-1.23, P heterogeneity = 0.373, I 2 = 0%), neutropenia (RR = 0.55, 95% CI = 0.31-1.00, P heterogeneity = 0.955, I 2 = 0%), and myelosuppression (RR = 0.73, 95% CI = 0.48-1.11, P heterogeneity = 0.088, I 2 = 65.7%) in the CIK/DC-CIK immunotherapy group compared with the chemotherapyalone group. The CIK/DC-CIK immunotherapy enhanced Chemotherapy is thought to damage the immune system. Immunotherapy is a type of cancer treatment that helps the body's own immune system fight cancer, many clinical trials of immunotherapy have been conducted in multiple centers. DCs are an important immunotherapeutic cell type. Ishigami et al. [28] demonstrated that patients with GC having a high level of DC cell infiltration were less likely to have lymph node metastasis and a significantly increased 5-year survival rate. In addition, CIK cells exhibit strong cytotoxicity against a variety of tumor cell lines as well as newly isolated tumor samples (e.g. liver cancer [29], lung cancer [30], glioma [31], and GC [32]). Compared with other immune cells, CIK cells proliferate rapidly and exhibit strong antitumor activity and a broad spectrum of targeted tumors [14,33]. Therefore, CIK/DC-CIK cell-based immunotherapy is a promising treatment for a number of cancers. Zhao et al. [34] showed that "GP regimen combined with DC-CIK immunotherapy could reduce postoperative recurrence and prolong survival in patients with non-smallcell lung cancer." Ma et al. [35] demonstrated that CIK cell therapy had significant advantages in prolonging the median OS rate, PFS, DCR, ORR, and QOL of patients with hepatocellular carcinoma. Other studies of gastric cancer [13], advanced renal cancer [36], and metastatic nasopharyngeal carcinoma [37] demonstrated that DC-CIK cells could improve the prognosis of patients. CIK cells have become a promising immunotherapy method in tumor therapy due to their easy availability and strong antitumor activity, which is of great significance for tumor prognosis [38].
Heterogeneity is a problem with most meta-analyses. In the present meta-analysis, heterogeneity was found in overall analyses; thus, the random-effects model was used. Sensitivity analysis was performed to assess the stability of the results by sequentially removing each study. The removal of any single study did not change the overall statistical results, indicating that the results of this study were statistically robust. Furthermore, this study probed the detailed results in subgroup analyses stratified by cancer type (GC or CRC). Based on the data collected, this study suggested that the cancer type at least partly contributed to the between-study heterogeneity.
This meta-analysis had some limitations that might have affected the interpretation of results. First, the efficacy of CIK/DC-CIK immunotherapy was affected by many factors, such as injection mode, tumor stage, and metastasis cycle. Further, a detailed analysis needs to be carried out on the basis of complete literature, standardization of treatment options, and limitation of patient participation criteria. Second, some data on adverse events could only be used in two trials, which might have led to bias. Third, publishing bias existed in this study. Although the trim-and-fill method was used in this study to confirm the results, some negative data that were possibly omitted might have affected the results. Finally, the heterogeneity could not be completely eliminated in the analysis.

Conclusion
In summary, this meta-analysis confirmed that the combination of CIK/DC-CIK immunotherapy and chemotherapy was safe and applicable for patients with advanced GIC, and hence might serve a feasible choice to prolong survival and improve QOL. Therefore, CIK/ DC-CIK immunotherapy is an effective therapy for advanced GIC treatment.