Abstract
Purpose
To assess the activity and safety of postoperative adjuvant immunotherapy with transfusion of cytokine-induced killer (CIK) cells combined with chemotherapy in patients with colorectal cancer.
Methods
We retrospectively studied 96 consecutive patients with colorectal cancer who were treated with resection between January 2010 and December 2012 as well as adjuvant chemotherapy. Twenty-one of these patients accepted at least 1 cycle of CIK cell transfusion for immunotherapy (CIK group). Disease free survival (DFS), immune cells and treatment related side effects were assessed. The patients were followed up until May 2013.
Results
By the end of follow-up, 10 patients (10.42 %) had died. Eighteen patients (18.75 %) had withdrawn. All the patients in the CIK group are still alive, and only 1 patient had withdrawn. Patients in the CIK group had significantly longer DFS than those in the control group [HR = 0.28, 95 % CI (0.09, 0.91), p = 0.034]. The 2-year DFS rates of patients in the CIK group and the control group were 59.65 ± 24.80 % and 29.35 ± 6.39 %, respectively. The CD4+/CD8+ ratios were significantly lower during the period of chemotherapy than those before chemotherapy (p = 0.0038), while the ratios were significantly higher during the period of CIK cell transfusion than those before CIK therapy (p = 0.0484). There were no immediate adverse reactions to the CIK cell transfusions.
Conclusion
Adjuvant transfusion of CIK cells prolongs DFS in patients with colorectal cancer.
Keywords: Colorectal cancer, Cytokine-induced killer cells, Adjuvant immunotherapy, Adjuvant chemotherapy
Introduction
Colorectal cancer is the forth most common cancer worldwide [1]. For resectable colorectal patients, resection-based multidisciplinary treatment is the preferred strategy. Adjuvant chemotherapy is recommended for patients with high-risk stage II or more advanced disease. However, most of the relapses happened within 2 years after surgery [2]. It has been reported that the immune status of patients with cancer were suppressed, including colorectal cancer. The immunosuppression may be more serious after surgery and adjuvant chemotherapy [3]. Thus, therapies to recover the immunity may decrease the recurrence rate after surgery.
Cytokine-induced killer cells are generated by in vitro expansion of peripheral blood lymphocytes (PBLs) using anti-CD3 antibodies, interferon γ (INF-γ), interleukin-2 (IL-2) and IL-1 [4]. In early studies, PBLs were cultured with IL-2, which generated lymphokine -activated killer (LAK) cells [5]. However, LAK cells were not used successfully in clinical practice, due to limited LAK cells, low cytolytic activity and high toxicity of infusion of IL-2 in vivo. This problem was solved by adding INF-γ, anti-CD3 antibodies and IL-1 in the culture, which can achieve more than 1,000-fold expansion of CIK cells, about 20–30 % of which are CD3+CD56+ T cells [4]. The cytotoxicity mediated by CD3+CD56+ T cells depends on binding to and formation of cellular conjugation with tumor cells and production of perforin and granzyme B, but not depend on major histocompatibility complex (MHC) [6, 7]. As we all know, most of the tumor cells do not express MHC or human leukocyte antigen (HLA), which helped tumor cells to escape from the immune system [8]. Thus, CIK cells have the potent to treat cancers as an immunotherapy.
Studies have shown that CIK cells possess cytotoxic activities against a number of tumor cell lines or freshly isolated tumor samples, including hematopoietic cancer cells [9] and solid tumors [10], such as liver cancer [11], gastric cancer [12], lung cancer [13], and colorectal cancer [14]. In animal studies, the CIK cells not only can kill tumor cells directly, but also increase host cellular immune function by stimulating and restoring the immune system to recognize and kill tumor cells [15, 16]. In clinical studies, CIK cells have been evaluated as an immunotherapy for advanced solid cancer patients, including renal cancer, colorectal cancer, lung cancer, gastric cancer and lymphoma [17–21].
Therefore, CIK cells have the potent to eradicate residual cancer cells and prevent recurrence after surgery. However, few studies reported the adjuvant effect of CIK cells in cancer. In this study, we compared the effect of adjuvant immunotherapy with CIK cells and chemotherapy in patients with colorectal cancer who were treated with resection.
Materials and methods
Patients and treatment
We retrospectively studied all the colorectal patients who were admitted in Department of Oncology of Guangdong Provincial Hospital of Chinese Medicine and accepted colectomy or rectectomy between January 2010 and December 2012 as well as adjuvant chemotherapy. Patients with resectable metastatic disease who underwent resection of metastasis were also eligible for this study. Patients who relapsed within 1 month after surgery were defined as non-radical resection and were excluded from this study.
A total of 96 patients were enrolled in this study. All of these patients were not enrolled in any clinical trials before. Twenty-one patients accepted at least 1 cycle of CIK cell transfusion for immunotherapy who were defined as ‘CIK group.’ The other 75 patients were defined as ‘control group.’ All the data used in this study were from the electronic medical record system of our hospital. Patients were followed up according to the NCCN guideline. Briefly, patients’ chest/abdominal/pelvic CT scan every 3–6 months and colonoscopy every 6–12 months for the first 2 years and then CT scan every 6–12 months and colonoscopy every 1 year up to a total of 5 years were followed up. The patients were followed up until May 2013.
This was a retrospective study, so we have required to the ethics committee of our hospital for waiver of written informed consent for this study, declaring that we respect and will protect patients’ privacy. However, all the patients have provided written informed consent to accept chemotherapy or CIK transfusion before each cycle of treatment. This study, as well as the requirement for waiver of informed consent, was approved by the Ethics Committee of Guangdong Provincial Hospital of Chinese Medicine.
Preparation of CIK cells
CIK cells were prepared by the cell laboratory of ZMKS International Cancer Therapy Biotechnologies Co., LTD (Shenzhen City, Guangdong Province, China) according to previous reports [10, 20]. Briefly, the peripheral blood mononuclear cells (PBMCs) were collected using Ficoll density gradient (Tianjin Haoyang Biological manufacture CO., LTD, Tianjin, China), rinsed twice by saline solution and then suspended in AIM-V® medium CTS™ (Therapeutic grade, GIBCO®, Invitrogen Co., USA). After 3–4 h of incubation in the atmosphere with 5 % CO2 at 37 °C,the non-adherent cells were removed by aspiration and the cell density was adjusted to 2 × 106/ml using AIM-V medium containing 1,000 U/ml IFN-γ (Shanghai Kelong Biological manufacture CO., LTD, Shanghai, China). After 24 h of incubation in the atmosphere with 5 % CO2 at 37 °C, 1,000 U/ml IL-1 (PeproTech Inc., USA), 1,000 U/ml IL-2 (Beijing SL Pharmaceutical CO. LTD., Beijing, China) and 50 ng/ml monoclonal antibody against human CD3 (PeproTech Inc., USA) were added. IL-2 and AIM-V medium were added everyday according to the cell growth. After 14–16 days of culture, the CIK cells were mature.
Before infusion, the viability of CIK cells was tested by the dye exclusion with no less than 90 % viable cells. The biological activity of the CIK cells was tested by Flow Cytometry with no less than 20 % CD3+CD56+ cells. Bacterial contamination was tested every 3–4 days during the culture and within 48 h before transfusion. The endotoxin was tested by tachypleus amebocyte lysate (TAL) with <0.06 EU. For patients who accepted CIK transfusion during the period of adjuvant chemotherapy, the CIK cells were infused at the intervals of at least 3 days before or after chemotherapy. For those treated with CIK cells after the period of adjuvant chemotherapy, the CIK cells transfusions were started 2–4 weeks after last chemotherapy; 3 × 109 CIK cells were transfused into patients within 1 h on days 1–3 that was a total of 9 × 109, which was defined as 1 cycle. The subsequent cycle was started at the interval of at least 4 weeks from last transfusion [19].
Statistical analysis
Differences in demographic and clinical characteristics between the two groups were evaluated using the t test or χ2 test, with an alpha <0.05. Disease free survival (DFS) of patients was the only end point for assessing the effect of CIK transfusion. It was defined as the time elapsed from the date of surgery to either the date of recurrence or the date of last follow-up information. The patients were followed up until May 2013. Patients who were recurrence free at the end of study or lost to follow-up were censored. DFS curves were calculated using the Kaplan–Meier method. The Cox’s model for hazard ratio (HR) and 95 % CI was performed for comparison of the two groups. Data were documented using EpiData software (version 3.1, The EpiData Association, Odense, Denmark) and analyzed using Stata software (version 10.0, StataCorp LP, College Station, USA).
Results
Patient characteristics
The demographic and clinical characteristics were shown in Table 1. All patients’ ECOG performance status was 0–1. There were no significant differences in demographic or clinical characteristics between the 2 groups except that patients in the CIK group were older than those in the control group (p = 0.0015). The treatment strategies were also similar between the 2 groups except the CIK transfusion. Only a few of the patients accepted neoadjuvant chemotherapy or radiotherapy. Six patients accepted only 1 cycle of CIK cell transfusion and 5 patients accepted 2 cycles. 10 patients accepted at least 3 cycles of CIK cell transfusion. One patient accepted CIK transfusion during the period of adjuvant chemotherapy, while 3 patients accepted CIK transfusion during and after the period of adjuvant chemotherapy. The rest 17 patients accepted CIK transfusion after the period of adjuvant chemotherapy.
Table 1.
Demographic, clinical and therapeutic information of 96 patients
| Characteristic | Total (n = 96) | Control (n = 75) | CIK (n = 21) | p |
|---|---|---|---|---|
| Sex, male/female | 55/41 | 43/32 | 12/9 | 0.988 |
| Age(year), mean ± SE | 56.86 ± 1.44 | 54.49 ± 1.63 | 65.33 ± 2.31 | 0.0015 |
| Location of tumor | 0.161 | |||
| Colon cancer | 59 | 47 | 12 | |
| Rectal cancer | 36 | 28 | 8 | |
| Rectosigmoid junction | 1 | 0 | 1 | |
| Histologic differentiation | 0.642 | |||
| Well differentiation | 2 | 2 | 0 | |
| Moderate differentiation | 73 | 57 | 16 | |
| Poorly differentiation | 12 | 8 | 4 | |
| Undifferentiation | 1 | 1 | 0 | |
| Unknown | 8 | 7 | 1 | |
| UICC stage | 0.694 | |||
| I | 6 | 5 | 1 | |
| II | 27 | 23 | 4 | |
| III | 51 | 38 | 13 | |
| IV | 7 | 5 | 2 | |
| Unknown | 5 | 4 | 1 | |
| Tumor deposits (+/−/unknown) | 21/15/60 | 16/11/48 | 5/4/12 | 0.845 |
| Perineural invasion (+/−/unknown) | 13/6/77 | 9/5/61 | 4/1/16 | 0.516 |
| Sampled lymph node (<12/≥12/unknown) | 31/43/22 | 26/31/18 | 5/12/4 | 0.235 |
| Lymphovascular invasion (+/−/unknown) | 14/9/73 | 9/5/61 | 5/4/12 | 0.675 |
| Chemotherapy regimen | 0.214 | |||
| Oxaliplatin + Fluorouracil | 73 | 59 | 14 | |
| Irinotecan + Fluorouracil | 9 | 5 | 4 | |
| Fluorouracil | 11 | 8 | 3 | |
| Other or unknown | 3 | 3 | 0 | |
| Chemotherapy cycle | 0.253 | |||
| <3 | 22 | 18 | 4 | |
| 4–6 | 58 | 47 | 11 | |
| >6 | 16 | 10 | 6 | |
| Adjuvant radiotherapy (yes/no) | 1/95 | 1/74 | 0/21 | 0.595 |
| Neoadjuvant chemotherapy (yes/no) | 6/90 | 3/72 | 3/18 | 0.085 |
| Neoadjuvant radiotherapy (yes/no) | 2/94 | 1/74 | 1/20 | 0.331 |
DFS and OS
The median follow-up period was 19 months (range, 5–42 months). By the end of follow-up, 10 patients (10.42 %) had died. Eighteen patients (18.75 %) had withdrawn. All the patients in the CIK group are still alive, and only 1 patient had withdrawn. The 1-, 2- and 3-year survival rate in the control group was 97.26 ± 1.91 %, 85.36 ± 4.94 % and 75.88 ± 8.02 %, respectively. No statistically significant difference was found in the overall survival between the two groups.
The DFS curves for the CIK group and the control group are presented in Fig. 1. The 1-year DFS rates of patients in the CIK group and the control group were 89.47 ± 7.04 % and 64.84 ± 5.82 %, and the 2-year DFS rates were 59.65 ± 24.80 % and 29.35 ± 6.39 %, respectively. Patients in the CIK group had significantly longer DFS than those in the control group [HR = 0.28, 95 % CI (0.09, 0.91), p = 0.034].
Fig. 1.
Kaplan–Meier estimates for DFS for patients in the two groups
However, this is a retrospective cohort study, where selection bias is the most important bias for this study. Most of the patients in the CIK group have accepted 4–6 cycles of adjuvant chemotherapy before CIK therapy, and the median time from surgery to first CIK cell transfusion was 5 months. However, some of the patients recurred within 5 months during the postoperative chemotherapy and were defined as the control group. Thus, we then compared the DFS of patients with longer than 5-month DFS in the control group (68 patients) to those in the CIK group (21 patients). Significantly longer DFS was still found in the CIK group [Fig. 2. HR = 0.31, 95 % CI (0.10, 0.99), p = 0.049].
Fig. 2.
Kaplan–Meier estimates for DFS for patients in the CIK group and those with longer than 5-month DFS in the control group
T lymphocytes subset
The T lymphocytes subsets of only 21 patients, including 14 patients in the CIK group, were evaluated by flow cytometry in peripheral blood before and during the adjuvant chemotherapy or immunotherapy (Table 2). Although no significant differences were found between the average percentages of T lymphocytes subsets during chemotherapy or CIK therapy and those before therapy, we still found a tendency that the CIK therapy may increase the percentages of CD4+ and decrease the CD8+ cells, while the chemotherapy affected contrarily on the lymphocyte subsets. The minimum percentages of CD4+ lymphocytes and CD4+/CD8+ ratios during the period of chemotherapy were significantly lower than those before chemotherapy (p = 0.0374 for CD4+, p = 0.0038 for CD4+/CD8+), while the maximum percentages of CD8+ lymphocytes were significantly higher than those before chemotherapy (p = 0.0220). The maximum ratios of CD4+/CD8+ during the period of CIK therapy were significantly higher than those before CIK therapy (p = 0.0484), while the minimum percentages of CD8+ lymphocytes were significantly lower than those before CIK therapy (p = 0.0185). The maximum percentages of CD4+ lymphocytes and CD4+/CD8+ ratios and the minimum percentages of CD8+ lymphocytes during chemotherapy were similar to those before chemotherapy. The minimum CD4+/CD8+ ratios and the maximum percentage of CD8+ lymphocytes during the period of CIK therapy were similar to those before CIK therapy. Effects on CD3+ lymphocytes, natural killer cells (NK cells) of chemotherapy or CIK therapy and effects on CD4+ lymphocytes of CIK therapy were not found in our study.
Table 2.
T lymphocytes subset before and during the period of chemotherapy or CIK therapy
| Lymphocyte phenotype (mean ± SE) | Before chemotherapy (%) | During chemotherapy (%) | Before CIK therapy (%) | During CIK therapy (%) |
|---|---|---|---|---|
| CD3 + | ||||
| Maximum | 56.55 ± 5.39 | 67.08 ± 4.27 | 57.28 ± 11.50 | 60.53 ± 10.48 |
| Minimum | 56.95 ± 5.68 | 50.55 ± 17.00 | ||
| Average | 61.89 ± 4.12 | 57.26 ± 12.20 | ||
| CD3 + CD4 + | ||||
| Maximum | 35.55 ± 3.25 | 39.2 ± 3.49 | 29.65 ± 4.52 | 35.00 ± 6.39 |
| Minimum | 29.88 ± 3.07* | 28.03 ± 9.78 | ||
| Average | 34.88 ± 3.25 | 32.32 ± 7.25 | ||
| CD3 + CD8 + | ||||
| Maximum | 20.2 ± 1.99 | 26.05 ± 0.92* | 25.13 ± 7.36 | 26.38 ± 4.33 |
| Minimum | 18.73 ± 1.45 | 21.03 ± 7.55§ | ||
| Average | 22.65 ± 0.97 | 23.36 ± 5.78 | ||
| NK cells | ||||
| Maximum | 19.43 ± 7.65 | 21.45 ± 6.98 | 24.36 ± 7.45 | 29.58 ± 12.23 |
| Minimum | 11.83 ± 3.55 | 16.6 ± 7.37 | ||
| Average | 15.75 ± 4.71 | 25.08 ± 9.39 | ||
| CD4 + /CD8 + | ||||
| Maximum | 1.77 ± 0.13 | 1.97 ± 0.09 | 1.48 ± 0.47 | 1.78 ± 0.45§ |
| Minimum | 1.23 ± 0.11* | 1.07 ± 0.26 | ||
| Average | 1.58 ± 0.11 | 1.56 ± 0.26 | ||
* p < 0.05 compared to before chemotherapy
§ p < 0.05 compared to before CIK therapy
Side effects
Only 59 patients recorded the side effect of chemotherapy. The most common side effects were bone marrow suppression (64.4 %), vomiting (42.9 %), numbness (36.2 %), hepatic dysfunction (10.7 %), diarrhea (7.1 %), renal dysfunction (1.79 %) and fever (1.79 %). Grade 3–4 side effects were bone marrow suppression(11.8 %), vomiting (5.3 %), diarrhea (3.57 %), and hand and foot syndrome (3.45 %). There were no immediate adverse reactions to the CIK cells transfusions.
Discussion
Adjuvant chemotherapy and radiotherapy after surgery is the standard treatment modality for stage III and part of stage II or stage IV patients. However, most of the relapses happened within 2 years after surgery [2]. Presently, immunotherapy has become the fourth treatment modality for malignant tumors and may be a promising approach to represent recurrence after surgery [22]. However, many tumors have lost expression of antigens or MHC molecules, which may restrict the effect of immunotherapy, such as the tumor infiltrating lymphocytes (TIL)-based therapy [8]. Thus, there is a potential utility of non-MHC-based therapy.
CIK cells are generated by in vitro expansion of peripheral blood lymphocytes (PBLs) using anti-CD3 antibodies, IL-2 and IL-1. Theses cells are non-MHC-restricted CD3+CD56+ T cells. In this study, we assessed the adjuvant activity of adoptive immunotherapy with CIK cells combined with adjuvant chemotherapy in colorectal cancer patients after resection. Patients in the CIK group had significantly longer PFS than those in the control group. No statistically significant difference was found in the overall survival. To our knowledge, only two studies of adjuvant efficacy of CIK cells, for hepatocellular carcinoma (HCC) [23] and gastric cancer [24], respectively, have been reported. In the first study, disease-free survival rates were significantly higher in 3-cycle CIK group (p = 0.001) and 6-cycle CIK group (p = 0.004) than in the control group for HCC. In the other study, an improvement of 17.9 % in 5-year DFS rate (p = 0.044) in the patients receiving adjuvant immunotherapy compared to the control group was found for gastric cancer. No statistically significant difference was found in the overall survival among the groups in the two studies. The results of these studies were similar, which indicate that adjuvant transfusion of CIK cells prolongs DFS not OS for solid tumors after resection.
Studies in vitro have shown that CIK cells possess cytotoxic activities against a number of tumor cell lines or freshly isolated tumor samples, including hematopoietic cancer cells and solid tumors [10]. In mice, the CIK cells can be detected in lung, liver and spleen within 16 h after CIK infusion, and majority of CIK cells were found in tumor by 72 h and remain for more than 9 days. In addition, these CIK cells were demonstrated to possess antitumor effect [25]. Thus, the clinical benefit of CIK cells may be due to the direct tumor killing activity. In fact, CIK cells are composed of a mixture of effector and central memory T cells, and the immediate cytotoxic activity was due to the effector T cells. However, the effect of those central memory T cells is still not clear. Studies have shown that the CIK cells also produce some cytokines, such as INF-γ and tumor necrosis factor α (TNF-α), and express high level of NKG2D that is prone to apoptosis and promote T cell proliferation, which may also be responsible to clinical benefit [10]. Thus, response of host immune system to CIK cells transfusion in vivo should also be studied to help improve this therapy.
However, there were no standard clinical markers to indicate the immune status of hosts. In some study, the measurement of T lymphocyte subsets was used as a clinical indicator of immune status in tumors [26]. Milasiene V reported that patients with stage III colorectal and gastric cancer had better survival rates when absolute number of CD3+, CD4+ and CD8+ lymphocytes were greater [27]. However, the results on CD8+ of Cho MY were different. They reported that in gastric cancer patients, values of CD3+ and CD4+ T cells, and CD4+/CD8+ ratios before surgery and 1, 3, 6 months after surgery were consistently lower in the recurrence group, indicating that the higher CD4+ lymphocytes percentages and lower CD8+ lymphocytes percentages were associated with better survivals [28]. One reason for this difference may be that the time for T lymphocyte subset test in these studies is different. Shi L found that the CD3+ and CD4+ T cells, and CD4+/CD8+ ratios were higher 1 week after first CIK cells transfusion than those before CIK therapy, but returned to baseline after 2 months [24]. Jiang J and Wu C also found that in advanced gastric cancer and lung cancer patients, CD4+/CD8+ ratio was significantly increased in CIK group [18, 19]. In our study, although no significant differences were found between the average percentages of T lymphocytes subsets during chemotherapy or CIK therapy and those before therapy, we still found a tendency that the CIK therapy may increase the percentages of CD4+ and decrease the CD8+, while the chemotherapy affected contrarily on the lymphocyte subsets. Nevertheless, the results are confusing, for when PBLs are cultured under CIK conditions; more than 90 % of the cells expanded are CD3+ cells, most of which were CD8+ cells (about 70 %). Following culture, CD3+CD8+ but not CD3+CD4+ cells express high levels of CD56+ [6], which indicate that increased CD8+ should be detected in patients after CIK transfusion. Our explanation for this phenomenon was that the CD8+ and CD56+ cells, as the effector cells with cytotoxic activity, may be detected and increased immediately after CIK transfusion. In these studies, as well as our study, the T lymphocyte subsets were tested at least from 1 week to 1 month after CIK transfusion, when the cytokines produced by CIK cells and the central memory T cells have affected on hosts’ immune system. For example, the IFN-γ and TNF-α also promote Th1 cells [10]. To our knowledge, the effect of CIK cells on hosts’ immune system is still unknown and may need more researches on this field.
This is a retrospective study, and there are some limitations in this study. First, selection bias was the most important bias for this study, because this is a single-center hospital-based study. Most of the patients in the CIK group accepted surgery as well as adjuvant chemotherapy in our hospital, while some of the patients in the control group had accepted surgery and chemotherapy in the other hospitals and came to our hospital due to unsatisfied efficacy or side effect. Second, some of the patients in this study did not accept standard adjuvant chemotherapy regimens and thus cannot represent the patient population, although the differences between the two groups were not significant. Therefore, larger and well-designed studies are needed to confirm our findings. In fact, we have designed a prospective randomized controlled trial (RCT) to assess the efficacy of postoperative adjuvant immunotherapy with CIK cells combined with standard chemotherapy in patients with colorectal cancer, which has been supported by the Wu Jieping Medical Foundation.
In conclusion, this study showed that postoperative adjuvant transfusion of CIK cells prolongs DFS in patients with colorectal cancer. However, considering the selection bias, the conclusions cannot be considered as definitive, and a well-designed prospective RCT is needed.
Conflict of interest
The authors declare that they have no conflict of interest.
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