Cellular immunotherapy as maintenance therapy prolongs the survival of the patients with small cell lung cancer
Ding et al. Journal of Translational Medicine
Cellular immunotherapy as maintenance therapy prolongs the survival of the patients with small cell lung cancer
Xiao Ding 0 1
He Cao 0 1
Xiao Chen 0 1
Haofan Jin 1
Ziling Liu 1
Guanjun Wang 1
Lu Cai 2
Dan Li 1
Chao Niu 1
Huimin Tian 1
Lei Yang 1
Yuguang Zhao 1
Wei Li 1
Jiuwei Cui 1
0 Equal contributors
1 Cancer Center, the First Hospital of Jilin University , No. 71. Xinmin Street, Changchun 130021 , China
2 Kosair Children's Hospital Research Institute, Department of Pediatrics, the University of Louisville , Louisville, KY 40202 , USA
Background: Small cell lung cancer (SCLC) relapses rapidly after the initial response to chemotherapy and shows drug-resistance. This study was to investigate the efficacy and safety of cellular immunotherapy (CIT) with autologous natural killer (NK), γδT, and cytokine-induced killer (CIK) cells as maintenance therapy for SCLC patients. Methods: A pilot prospective cohort study was conducted with SCLC patients who had responded to initial chemotherapy. Patients elected to receive either CIT as maintenance therapy (study group), or to be followed-up without further treatment (control group). Progression-free survival (PFS), overall survival (OS), and adverse effects were investigated. Results: We recruited 58 patients (29 in each group). The patient characteristics of the 2 groups were well balanced. PFS was not significantly different between the groups, but OS was significantly longer in the study group than the control (20 vs. 11.5 months, P = 0.005; hazard ratio [HR], 0.434, 95 % confidence interval [CI], 0.236-0.797, P = 0.007). Among patients with limited-stage disease, there was no difference in PFS between the groups, but OS was longer in the study group compared to the control (26.5 vs. 11.8 months, P = 0.033; HR, 0.405, 95 % CI, 0.169-0.972, P = 0.043). Among patients with extensive-stage disease, both PFS and OS were longer in the study group than the control (5 vs. 2.7 months, P = 0.037; HR, 0.403, 95 % CI, 0.162-1.003, P = 0.051, and 14.5 vs. 9 months, P = 0.038; HR, 0.403, 95 % CI, 0.165-0.987, P = 0.047, respectively). No significant adverse reactions occurred in patients undergoing CIT. Conclusions: CIT maintenance therapy in SCLC prolonged survival with only minimal side effects. Integrating CIT into current treatment may be a novel strategy for SCLC therapy, although further multi-center randomized studies are needed.
Small cell lung cancer; Cellular immunotherapy; Maintenance therapy
Lung cancer is the most commonly diagnosed cancer
and also the leading cause of cancer death globally .
Small cell lung cancer (SCLC) is usually reported to
comprise about 15 % of all lung cancer cases recorded
. Despite a high initial response rate to first-line
therapy, most patients die rapidly from recurrent,
drugresistant disease. Even with more advanced
chemotherapeutic agents and molecularly targeted drugs, the
prognosis of this disease remains poor due to limited
treatment efficacy [3–5]. Recently, maintenance therapy
in advanced non-small cell lung cancer (NSCLC) has
been found to be an acceptable treatment paradigm to
improve progression free survival (PFS) . However, a
meta-analysis of published randomized clinical trials 
showed that both maintenance and consolidation therapy
failed to improve the outcomes of SCLC, and in some
cases even caused severe side effects or toxic death. Thus,
there is no recommendation for maintenance therapy in
current SCLC treatment guidelines. Given its high
recurrence rate and mortality, new therapeutic strategies are
urgently needed to improve the outcome of this disease.
Immune escape plays an important role in cancer
recurrence and metastasis [8, 9]. With an improved
mechanistic understanding of immune response and
immune escape, several immunotherapies were investigated
in SCLC. Some of them were failed, such as the dendritic
cell-based p53 vaccine , but some of them obtained a
certain effect, such as phased ipilimumab (an antibody
against cytotoxic T-lymphocyte antigen-4 [CTLA-4]) with
paclitaxel/carboplatin exhibited improved immune-related
PFS (irPFS) . It indicated that immunotherapy might
have the potential to improve the prognosis of SCLC.
Besides, it also suggested that different patterns of
immunotherapy combined with chemotherapy might
have an influence on the prognosis of SCLC. Therefore,
increasing attention has been paid to the possibility of
immunotherapy for SCLC patients in recent years.
SCLC patients have often been found to have a
functional deficiency in a variety of immunocytes [12–14],
implying that adoptive transfusion of ex vivo-activated
and expanded immunocytes may be a feasible and
effective therapy. Cellular immunotherapy (CIT) has been
shown to be effective for a variety of cancers [15–17],
but its use in SCLC patients has not been reported.
Activated immune cells can reach the lungs within
minutes of intravenous injection and selectively enter
malignant tissue. Consequently, a substantial number of
these cells can accumulate at cancer sites within 24 hr of
treatment [18, 19]. We postulated that CIT would provide
an anti-caner effect and prolong the survival of SCLC
patients. However, it is not yet known which cell types are
needed in CIT for performing anti-cancer effects optimally.
Several specific immunotherapies to induce cytotoxic
T lymphocyte (CTL) for SCLC have been tried, such as
the dendritic cell-based p53 vaccine , few of them
have lengthened survival, partly due to the complexity of
the immune escape mechanism in this malignancy.
Decreased expression of HLA-class I antigen has been
reported in SCLC, which may be one of the mechanisms
of SCLC cells to escape CTL attack . Natural killer
(NK) and γδT cells are effector cells of innate immunity,
and both can exert anti-cancer effects in a
non-MHCrestricted manner. Cytokine-induced killer (CIK) cells
are ex vivo-activated lymphocytes, and represent a
heterogeneous cell population, including CD3+CD56+, CD3
+CD56− (typical T) and CD3−CD56+ (NK) cells. The
anti-cancer activity of CIK cells is mainly due to the
CD3+CD56+ cells, which show an NK-like,
non-MHCrestricted cytolytic activity against cancer cells. CIT
based on any of these cell types has proved to be
effective against a variety of cancers [15–17]. SCLC cells were
also found to be susceptible to NK or γδT cell-mediated
cytotoxicity in preclinical studies [20, 21]. In addition,
γδT cells can induce the NK cell-mediated killing of
cancers that are usually resistant to NK cytolysis  and
cross-present tumor antigens to CD8+ CTLs to mediate
adaptive immune responses . These findings suggest
that the combined application of NK, γδT and CIK cells
may provide significantly synergistic anti-cancer effects
via different mechanisms, and could provide effective
CIT for SCLC patients. We therefore conducted this
pilot prospective cohort study to evaluate the efficacy
and safety of combined NK, γδT, and CIK cell based
CIT as a maintenance therapy for SCLC patients, with
the objective of consolidating remission and prolonging
survival in patients who responded to first-line therapy.
Patients and study design
A pilot prospective cohort study was conducted to
evaluate the efficacy and safety of combined NK, γδT, and CIK
cell based CIT as a maintenance therapy for SCLC
patients. All patients with SCLC that met the following
criteria at the First Hospital of Jilin University were
included in this study after June 1, 2009.
Eligibility: Patients had to (i) have been diagnosed as
SCLC and have completed first-line therapy, (ii) have
achieved stable disease (SD), partial remission (PR) or
complete remission (CR) after the first-line treatment, (iii)
be at least 18 years old, (iv) have an Eastern Cooperative
Oncology Group (ECOG) performance status ≤ 2, (v) have
normal kidney, liver, and bone marrow function and be
free of cardiac arrhythmias, congestive heart failure or
severe coronary artery disease, and (vi) have a life
expectancy ≥ 3 months. Exclusion criteria included (i)
autoimmune disease, (ii) a clinically serious infection,
(iii) for women, pregnancy or lactation, (iv) a history of
organ transplantation, and (v) the administration of
another immunotherapy. This study was conducted in
accordance with the Declaration of Helsinki and was
reviewed and approved by the Ethical Committee of the
First Hospital of Jilin University [ID #: 2009–020].
Written informed consent was obtained from all
patients before their enrollment into the study.
Treatment plan: The treatment regimens of SCLC
patients in this study were based on the Small Cell Lung
Cancer NCCN Guidelines – version 2.2009 . The
firstline therapy regimen for limited stage SCLC (LS-SCLC)
patients was platinum based chemotherapy, e.g.:
etoposide/cisplatin (EP) regimen or etoposide/carboplatin (EC)
regimen for 4–6 cycles plus concurrent chest radiotherapy
(1.8–2 Gy once daily to 60–70 Gy) . The initial
chemotherapy for extensive stage SCLC (ES-SCLC)
patients was the same as those for LS-SCLC patients.
After first-line therapy, patients either received CIT (at
least 1 course) as maintenance treatment (study group), or
were followed up without further treatment (control
group). The decision whether to undergo CIT or to be
entered into the control group was made by the patient. For
the study group, autologous peripheral blood mononuclear
cells (PBMCs) were collected by apheresis on D0, and were
induced into NK, γδT, or CIK cells. The expanded
immunocytes were then infused back into the patients 14 days
later (D14) as the initial transfusion. There were for 6
consecutive transfusion days (D14–D20) with 2 kinds of
immune cells for each infusion, and each CIT course was
completed within 3 weeks after apheresis. The second
course of PBMCs collection was started 1 to 3 weeks after
the end of the first course. The treatment schedule is
summarized in Fig. 1. Maintenance treatment was continued
unless there was progressive disease (PD) or the patient
refused to undergo further CIT. In cases of PD, the patient
was given either a best supportive treatment or a second or
even a third-line chemotherapy regimen, depending on
their general health status and/or preference.
The second-line chemotherapy regimens were selected
based on the time of relapse. If patients experienced
relapse within 6 months after the first-line therapy,
patients would be treated with topotecan or irinotecan;
if patients relapsed more than 6 months after the
completion of first-line therapy, they would receive the
original regimen. The third-line chemotherapy regimens
were selected based on the previous chemotherapy.
Patients could be given irinotecan, topotecan or paclitaxel
that had not been used in the previous chemotherapy.
Follow up: In general, follow-up was required every
3 months. Each follow-up included a complete physical
examination, basic serum chemistry, and computed
tomography (CT) of the chest and abdomen. Brain magnetic
resonance imaging (MRI) and a technetium bone scan
were performed as clinically indicated.
Preparation of immune cells
All procedures for preparing the autologous immune cells
were carried out under Good Manufacturing Practice
(GMP) conditions (Certificate ID: A20090047) which were
approved by the Jilin Provincial Center for Sanitation
Inspection and Test. The preparation of and quality
control for each type of immunocytes was performed in
strict accordance with the standard operating procedure
(SOP). Immune cells were prepared as described in our
previous study , with some modifications based upon
previously published procedures [26, 27]. Briefly, about
1.5 × 109 (1.0–2.0 × 109) PBMCs were collected from the
patient using a Cobe Spectra Apheresis System (Gambro
BCT, Inc. USA). These PBMCs were separated into 2
parts and placed in 50 ml centrifuge tubes that were
centrifuged for 10 min at 3000 rpm. The supernatant was
then removed, and the cell pellets were re-suspended in
30 ml PBS, and placed on top of a 15 ml Ficoll-Hypaque
(Amersham Biosciences, Uppsala, Sweden) in a sterile
50 ml tube. Lymphocytes were isolated from PBMCs using
Ficoll-Hypaque density centrifugation (Ficoll separation).
Approximately 1.3 × 109 (1.0 − 1.8 × 109) mononuclear
cells were obtained after these procedures. The cells were
then separated into 3 pools to induce NK, γδT and CIK
cells using different cytokines.
For expansion of NK cells, PBMCs were cultured in
AIM-V medium (Invitrogen, Carlsbad, CA, USA), 700 U/
mL IL-2 (Miltenyi, Cologne, German) and 1 ng/mL
OK432 (Shandong Lu Ya Pharmaceutical, Jining, China)
for 24 hr at 37 °C, in a mouse anti-human CD16
monoclonal antibody (mAb; Beckman Coulter, Marseille, France)
coated flask. The cultured cells were then centrifuged, and
the supernatant was discarded. The cells were again
cultured in AIM-V medium and 700 U/mL IL-2 at 37 °C for
2–3 weeks. IL-15 (Miltenyi, Cologne, Germany) was
added to the culture medium to promote the growth of
NK cells [25, 26]. To generate γδT cells, PBMCs were
stimulated with 1 μM zoledronate (Zometa®, Novartis,
Beijing, China) in AIM-V medium containing 400 U/mL
human IL-2. Fresh medium and IL-2 supplement at 400
U/mL were added every 3 days [25, 27]. To prepare CIK
cells, PBMCs were cultured in AIM-V medium and 1000
Fig. 1 The schedule for autologous cellular immunotherapy (CIT). Autologous peripheral blood mononuclear cells (PBMCs) were collected by
apheresis on D0, and were induced into NK, γδT, or CIK cells. The expanded immunocytes were then infused back into patients 14 days later
(D14) as the initial transfusion. There were for 6 consecutive transfusion days (D14–D20) with 2 kinds of immune cells for each infusion, and each
CIT course was completed within 3 weeks of apheresis. The second course of PBMCs collection was started 1 to 3 weeks after the end of the
U/mL IFN-γ (Miltenyi, Cologne, German) at 37 °C for
24 hr. Then, 100 ng/mL mouse anti-human CD3 mAb
(Peprotech, Rocky Hill, NJ, USA), 1000 U/mL IL-2 and
1000 U/mL IL-1α (Miltenyi, Cologne, German) were
added to the media. Fresh complete medium and IL-2
supplement at 1000 U/mL were added every 3 days .
Before transfusion, a fraction of cells was collected
so that they could be enumerated, evaluated for
viability and phenotype, and checked for possible
Phenotypic analysis of immunocytes before infusion
The phenotype of immunocytes were determined on the
basis of their specific cell surface markers using 4-color
flow cytometry performed on a FACSCalibur (BD
Biosciences, San Diego, CA, USA) with directly conjugated
mAbs against the markers. Briefly, cultured NK cells
were collected, washed, and incubated with mouse mAbs
against human CD3-PerCP, CD69-PE, and CD56-APC
(BD Biosciences) for 15 min. The γδT cells were
incubated with Vγ9-FITC and CD3-APC (BD Biosciences),
and the CIK cells were incubated with CD3-PerCP,
CD4-FITC, CD8-PE and CD56-APC (BD Biosciences).
Isotype-matched antibodies were used as controls.
Administration of immune cells
The dye-exclusion test was used to assess the viability of
the final cell suspension. Possible contamination of the
immune cells was tested using a PCR-based assay for
mycoplasma, as well as assays for endotoxins, bacteria,
and fungi, 24 hr before and on the day of product
release. The immunocytes could not be used for patients
if they failed to meet the following release criteria: (i) a
viability of more than 95 %, (ii) no contamination by
bacteria, fungi, endotoxins, or mycoplasma in either of
the 2 assessments, (iii) at least 1.2 − 2.0 × 109 of each
type of cell per infusion; and (iv) more than 50 % of the
cells had the NK (CD3−CD56+) or γδT (CD3+Vγ9+)
phenotype, and more than 20 % of cells had the CD3
+CD56+ CIK phenotype in NK, γδT and CIK cell culture
system respectively, as detected by flow cytometry.
Before reinfusion, immunocytes were washed 3 times
with normal saline and re-suspended in 50 mL of
normal saline. The cells were then administered to patients
via an intravenous drip in 30 min. The number of cells
in each transfusion ranged from 2.4 − 4.0 × 109.
Cytotoxic effects of immune cells in vitro
The cytotoxicity of immune cells was examined by
measuring lactate dehydrogenase (LDH) levels in the
culture medium, according to the manufacturer’s
instructions for the CytoTox 96 Non-Radioactive
Cytotoxicity Assay Kit (Promega, Madison, WI). The
target cells used for this assay were the
SCLCderived cell line NCI-H446, and 5 × 104 cells were used
to examine the cytotoxic effects of immune cells in vitro.
The tested ratios of effector cells to target cells (E/T) were
25:1, 12:1, 6:1, and 3:1. In order to investigate the
synergistic effect of the different kinds of the immune cells, we
combined the 3 types of immunocytes at a ratio of 1:1:1 to
examine their cytotoxic effect on target cells.
Overall survival (OS) was the primary end point, and the
secondary end points included PFS, safety of CIT and
clinical benefit rate (CBR) of the second-line chemotherapy.
Response was determined based on the National Cancer
Institute’s Response Evaluation Criteria in Solid Tumors
(RECIST 1.1) guidelines . OS was defined as the
period from the day on which first-line treatment was
completed to death from any cause. PFS was defined as
the period between the completion of first-line treatment
and the onset of PD or death from any cause. CBR was
defined as the percentage of patients with CR + PR + SD.
Proportion of the immune cells in peripheral blood
In order to detect the alteration of the proportion of the
immune cells of the patients, two milliliters of peripheral
blood was collected before the apheresis and 1 week
after the end of one CIT course. T cells (CD3+CD4+,
CD3+CD8+), NK cells (CD3−CD56+), NKT cells (CD3
+CD56+), B cells (CD19+), regulatory T (Treg) cells
(CD4+CD25+Foxp3+) and monocytes (CD14+)
populations were analyzed with FACSCalibur (BD Biosciences).
All antibodies were purchased from BD Biosciences.
All calculations were performed using SPSS 17.0
software (SPSS, Chicago, IL). PFS and OS were assessed
according to the Kaplan-Meier method and compared
between groups using the log-rank test. The
multivariate Cox proportional hazard model was applied to
analyze factors found to be statistically significant by
univariate analysis. The Mann–Whitney test was used
to compare medians, and Fisher’s exact test was used
to compare binary outcomes. P ≤ 0.05 was considered
to be statistically significant.
A total of 58 eligible patients were recruited between
June 1, 2009 and November 1, 2012, with 29 patients
in each group. Follow-up of all patients was ended on
December 30, 2013, with a median follow-up time of
13 months (range, 3–54 months). Of the 29 patients
in each group, 17 patients had LS-SCLC and 12 had
ES-SCLC. Two ES-SCLC patients in CIT group had
one solitary pulmonary lesion highly suspected as
lung cancer under the CT scan. Their abdominal CT and
brain MRI were normal. They underwent surgery
after these examinations, and pathologically
confirmed SCLC. Considering the properties of invasion
and metastasis of SCLC, technetium bone scan was
performed after surgery for clinical stage. Bone scan
showed that one patient had lumbar metastasis, and
the other patient had pelvic metastasis. Therefore,
they were diagnosed as ES-SCLC. The patient
demographics were well balanced between the groups,
including characteristics such as sex, smoking index,
ECOG performance status, chemotherapy courses,
chemotherapy responses, radiotherapy and surgery
(Table 1). The only exception was age, as ES-SCLC
patients in the CIT group were on average older
than those in the control group (P < 0.05).
Quality of the cultured immune cells
The viability of each type of immune cell in our culture
system was found to exceed 95 %, none of the cultured
immune cells were found to be contaminated, and all of
the preparations met the release criteria. The
percentage of NK (CD3−CD56+), γδT (CD3+Vγ9+) and CIK
(CD3+CD56+) cells before and after induction was
8.01 % (range, 4.12–17.35 %) vs. 85.32 % (range, 61.33–
99.61 %), 4.22 % (range, 2.79–11.26 %) vs. 82.63 %
(range, 63.72–98.21 %) and 4.51 % (range, 1.62–7.96 %)
vs. 34.52 % (range, 27.25–57.28 %), respectively
(Table 2). Thus, each of these cell types was significantly
more prevalent after induction. Induction also resulted
in a significant increase in the proportion of activated
NK cells (CD56+CD69+). Representative results from a
single patient are shown in Fig. 2, and the numbers of
Table 1 Clinical characteristics of patients in the treatment and control groups
Chemotherapy responses *
Abbreviations: ECOG, Eastern Cooperative Oncology Group; CR, complete remission; PR, partial remission; SD, stable disease; LS-SCLC, limited stage small cell lung
cancer; ES-SCLC, extensive stage small cell lung cancer
Note: *The responses for the first-line therapy
Table 2 Summarized data of the percentage of NK, γδT and CIK
cells before and after induction
Before (median, range)
8.01 % (4.12–17.35 %)
4.22 % (2.79–11.26 %)
4.51 % (1.62–7.96 %)
After (median, range)
85.32 % (61.33–99.61 %)
82.63 % (63.72–98.21 %)
34.52 % (27.25–57.28 %)
Abbreviations: NK, natural killer; CIK, cytokine-induced killer
the 3 types of immunocytes infused into each patient
are shown in Additional file 1: Table S1.
Cytotoxic effect of immune cells on cancer cells
The cytotoxic effect of immune cells was examined by
measuring LDH release levels. All 3 types of immune
cells exhibited a significant cytotoxic effect on
NCIH446 cells when used alone, but a greater cytotoxic
effect was achieved when they were combined, and this
cytotoxicity increased further as the E/T cell ratio
increased. At an E/T cell ratio of 25:1, the median
cytotoxicity level of NK, γδT and CIK cells was 75.5 %
(range, 59.1–90.7 %), 70.6 % (range, 48.3–80.0 %), and
49.0 % (range, 27.4–68.9 %), respectively. The combined
immune cells showed a synergistic anti-cancer effect with
a median cytotoxicity level of 85.5 % (range, 63.4–96.5 %)
at an E/T cell ratio of 25:1 (Additional file 2: Figure S1).
PFS and OS
The median PFS did not differ significantly between the
groups (hazard ratio [HR], 0.667; 95 % confidence
interval [CI], 0.380–1.169; P = 0.157). However, the median
OS in the study group was significantly longer than that
in the control group (20 vs. 11.5 months, P = 0.005; HR,
0.434, 95 % CI, 0.236–0.797, P = 0.007) (Fig. 3A).
Patients were divided into two subgroups according to
disease stage: LS-SCLC and ES-SCLC. Among the
former, there was no significant difference in PFS (HR,
0.751; 95 % CI, 0.349–1.618; P = 0.465); however, the OS
of the study group was longer than that of the control
group (26.5 vs. 11.8 months, P = 0.033; HR, 0.405, 95 %
CI, 0.169–0.972, P = 0.043) (Fig. 3B). Among the ES-SCLC
patients, both the PFS and OS of the study group were
longer than those of the control group (5 vs. 2.7 months,
P = 0.037; HR, 0.403, 95 % CI, 0.162–1.003, P = 0.051, and
14.5 vs. 9 months, P = 0.038; HR, 0.403, 95 % CI, 0.165–
0.987, P = 0.047, respectively) (Fig. 3C, D).
Potential factors influencing the outcome of CIT
In the multivariate analysis, we found that sex, age,
smoking history, ECOG performance status,
chemotherapy courses, chemotherapy responses, radiotherapy and
surgery had no effect on the prognosis of SCLC patients
receiving CIT (P > 0.05). We then investigated the
Fig. 2 The percentage of NK, γδT and CIK cells before and after induction. Representative results from a single patient are shown. The percentage
of NK cells (A), γδT cells (B) and CIK cells (C) before and after induction was 13.3 % vs. 85.9 %, 2.79 % vs. 80.5 % and 4.04 % vs. 49.9 %, respectively.
CD56+CD69+ cells were considered to be activated NK cells
Fig. 3 Progression free survival (PFS) and overall survival (OS) in both groups. (A) OS of all patients. The OS in the study group was significantly
longer than that in the control group (20 vs. 11.5 months; P = 0.005). (B) OS of the limited-stage patients. OS in the study group was significantly
longer than in the control group (26.5 vs. 11.8 months; P = 0.033). (C) PFS of the extensive-stage patients. PFS in the study group was longer than
the control group (5 vs. 2.7 months; P = 0.037). (D) OS of the extensive-stage patients. OS in the study group was significantly longer than in the
control group (14.5 vs. 9 months; P = 0.038)
influence of the CIT frequency on the prognosis of
patients in the study group, in which the median frequency
of CIT was 3 courses (range, 1–8 courses). Patients were
divided into two subgroups: CIT ≥ 3 courses and < 3
courses. The characteristics of the patients in these
subgroups were well balanced (Table 3). The median PFS in
the CIT ≥ 3 courses group (n = 17) was longer than that
of the CIT < 3 courses group (n = 12) (9.5 vs. 2 months),
but this difference was not significant (P = 0.057) (HR,
0.465; 95 % CI, 0.204–1.063; P = 0.070) (Fig. 4A).
However, the median OS of the CIT ≥ 3 courses group was
significantly longer than that of the CIT < 3 courses
group (23 vs. 9 months, P = 0.020; HR, 0.335, 95 % CI,
0.125–0.893, P = 0.029) (Fig. 4B).
Response to second-line chemotherapy
A total of 18 patients accepted second-line therapy in the
study group, and 17 patients received second-line therapy
in the control group. The median second-line
chemotherapy courses in the study and control group were 3 (range,
1–6) and 2 (range, 1–6), respectively, but there was no
significant difference between two groups (P > 0.05). None
Table 3 Clinical characteristics of patients who received CIT
for ≥ 3 courses or < 3 courses
CIT ≥ 3 courses
Chemotherapy responses *
Abbreviations: CIT, cellular immunotherapy; ECOG, Eastern Cooperative
Oncology Group; CR, complete remission; PR, partial remission; SD,
Note: *The responses for the first-line therapy
of the patients in two groups achieved a CR after
secondline treatment. Eight patients got a PR or SD with a CBR of
44.4 % in the study group, and 5 patients got a PR or SD
with a CBR of 29.4 % in the control group, but there was
no significant difference between the groups (P = 0.489).
Side effects of CIT infusion
Two patients reported mild fatigue after CIT infusion.
One patient had a transient fever of 37.8 °C after one
infusion, but recovered after 1 hr. No other significant side
effects were observed.
Proportion of the immune cells in peripheral blood
before and after CIT
The proportion of T, NK, NKT, B, Treg cells and
monocytes were analyzed before and after CIT. However, there
was no significant change in these cells. There were
relatively less Treg cells at some time points after CIT, but
these differences were not significant (data not shown).
Immune escape in SCLC patients is closely associated
with the recurrence of the disease, and may contribute
to poor patient survival [8, 9, 12–14]. However, the
findings of several studies suggest that maintenance and/or
consolidation using cytokines and other biological agents
fail to improve SCLC outcomes . The therapeutic
effects of CIT in SCLC have rarely been reported in
either preclinical or clinical studies. Meanwhile, cancers
employ multiple mechanisms to evade an immune
response [29–31], and thus CIT with only one type of
immune cells is unlikely to achieve an optimal
anticancer effect. NK and γδT cells were shown to have a
synergistic anti-cancer effect in a preclinical study .
Besides, chemotherapy could not only alleviate immune
suppression by reducing the tumor burden [32, 33], but
also up-regulate the expression of the NKG2D ligands
on cancer cells, thus sensitizing them to lysis mediated
by NKG2D-expressing lymphocytes . In this study,
we applied CIT with a combination of NK, γδT and CIK
cells as maintenance therapy after systemic
chemotherapy, and showed survival advantage for SCLC patients
for the first time. At the same time, another study
conducted by our group also confirmed the efficacy of
the CIT with combination of these immunocytes in
improving the outcome of patients with hepatocellular
carcinoma after radiofrequency ablation (RFA) . Thus,
CIT may be a novel treatment for SCLC patients who
respond to first-line therapy, and could provide a novel
strategy for the devastating disease.
OS is widely used as a primary end point for
immunotherapy in many cancers, such as melanoma [35, 36] and
prostate cancer . SCLC is a highly malignant tumor
associated with short survival and limited chemotherapy
regimens, and hence there are less confounding factors
in the observation of OS. In our study, OS was
significantly longer after CIT both in LS-SCLC and ES-SCLC
patients. There are several possible reasons for the
prolonged survival of SCLC patients with CIT. Cancer stem
cells (CSCs) have been reported to be responsible for
cancer progression, metastasis, and the development of
drug resistance [38, 39]. They have also been shown to
be susceptible to immunocyte-mediated toxicity,
suggesting that CIT may be useful in eliminating minimal
residual disease (MRD) and thus reducing cancer
recurrence [40–42], which may explain why CIT maintenance
therapy prolongs PFS and consequently prolongs
survival of SCLC patients. Although PFS has recently been
shown to be a good predictor of OS in SCLC , but
PFS did not differ significantly between the groups in
Fig. 4 The influence of cellular immunotherapy (CIT) courses on small cell lung cancer (SCLC) patients’ prognosis. (A) The median progression
free survival (PFS) in the CIT ≥ 3 courses group was longer than that of the CIT < 3 courses group (9.5 vs. 2 months), although this difference was
not significant (P = 0.057). (B) The median overall survival (OS) in the CIT ≥ 3 courses group was significantly longer than that of patients in the
CIT < 3 courses group (23 vs. 9 months; P = 0.020)
LS-SCLC patients. It is noteworthy though that another
study exploring the relationship between PFS and OS in
SCLC found no significant relationship between them
, suggesting that OS might be affected by other
factors, such as second or third-line therapy.
It has been reported that patients who receive cancer
vaccines respond better to subsequent chemotherapy
than those who do not, suggesting that immunotherapy
might sensitize cancer cells to cytotoxic drugs [45, 46].
The CBR of the relapsed patients to second-line
treatment was also detected in our study. Patients with CIT
tended to have higher CBR than the control, although
there was no significant difference between two groups,
indicating that CIT might enhance the sensitivity of
recurrent SCLC to second-line chemotherapy. This is a
potential mechanism by which OS could be extended by
CIT, although the exact mechanistic basis for this needs
to be further explored.
The quality of the cultured immunocytes is of primary
importance in CIT. Both the purity and viability of the
immune cells in our study were acceptable, and indeed
they were actually better than those in previously
reported expansion methods [47, 48]. The percentage of
activated NK cells (CD56+CD69+) was also significantly
increased after induction, indicating that the activity of
NK cells was remarkably improved. Each kind of
immunocytes had strong cytotoxicity when used alone, but the
highest cytotoxic effect was achieved when they were
combined, reflecting a strong synergy between these
cells. Safety is another important factor determining the
application of CIT. We did not observe any significant
side effects after CIT, and only 2 patients felt mild
fatigue and 1 patient had a transient fever during 1 of
the infusions. All the symptoms diminished without
treatment. Thus, CIT was well tolerated in our study,
and it may be a good candidate for maintenance therapy
when side effects need to be minimized.
Potential factors influencing the outcome of CIT were
also detected in this study. Using a multivariate
analysis, we found that sex, age, smoking history, ECOG
performance status, chemotherapy courses,
chemotherapy responses, radiotherapy and surgery were not
related to the efficacy of treatment. However, the OS of
patients who received more than 3 courses of CIT was
significantly longer than that of patients who received
less than 3 courses of CIT. PFS also tended to be longer
in patients who received more than 3 CIT courses,
although this was not statistically significant. It was
previously demonstrated that NSCLC patients who
received more than 7 courses of CIK cell treatment had
a significantly better prognosis than those who received
fewer courses . Taken together, our findings and
those of a previous report  suggest that a greater
number of CIT courses improves patient outcome.
However, the optimal number of treatment courses and
the duration of treatment are yet to be determined.
Besides, bias might exist in the analysis of the influence
of the CIT frequency on the prognosis of patients. Even
though the characteristics of the patients in these
subgroups were well balanced, some patients stopped CIT
treatment in CIT < 3 courses group because of disease
progression. Therefore, the shorter PFS and OS might
be affected not only by less frequency of CIT treatment,
but also by other factors, such as the worse features of
the disease status in this subgroup of patients. However,
these results could provide an instructive reference for
subsequent clinical trials.
There is no consensus on the best indicators for
assessing immune response after CIT. In this study, T, NK,
NKT, B, Treg cells and monocytes populations in the
peripheral blood were analyzed before and after CIT.
However, there was no significant change in these cells.
There were relatively less Treg cells at some time points
after CIT, but these differences were not significant. This
might reflect the relatively few CIT courses and the
small number of patients involved. However, it may also
be that the proportion of immune cells in the peripheral
blood does not relate to the response to CIT, and this
needs to be addressed in a further study.
CIT could improve the quality of life of cancer patients
as reported . However, the patients enrolled in this
study had a good performance status (ECOG 0–2) before
disease progression, and the status of the patients showed
no significant difference between the 2 groups. Therefore,
it was difficult to determine whether CIT affected the
quality of life in this study.
In our study, ES-SCLC patients who underwent CIT
were on average older than those who did not undergo
therapy. However, the multivariate analysis showed that
age had no effect on the prognosis of these patients. It has
also been reported that effector cell function decreases in
older people, and might be associated with reduced
antitumor immunity in these patients . Additionally,
patients in the study group had longer PFS and OS,
indicating that CIT may effectively reduce
immunosuppression, provide an anti-cancer effect and prolong OS, even
in elderly patients.
This study was designed as a prospective cohort study.
The patients were enrolled to each group of the study
based on their choice of the therapeutic options.
Therefore, this design allowed us to collect valuable data on the
efficacy of CIT, and better reflects a practical clinical
settings. At the same time, although innovative cancer
treatments might bring benefits to the cancer patients,
cost effectiveness is an important aspect that must be
considered in the clinical practice. Nowadays, maintenance
therapy for SCLC is very limited. CIT as a maintenance
therapy has shown its potential to prevent disease
recurrence and prolong the survival of SCLC patients with
minimal side effects in our study. Besides, the methods of
the preparation of immune cells are very simple compared
with the complicated preparation methods of other CIT
therapies, such as sipuleucel-T, which is used in prostatic
cancer and chimeric antigen receptor T-cell
immunotherapy (CAR-T) applied in B cell leukemia. And its price
might be no more than those of other available
maintenance therapies for NSCLC such as erlotinib, bevacizumab
and pemetrexate. Therefore, CIT as maintenance therapy
may be a novel cost effective strategy for SCLC therapy,
and warrants further investigation.
CIT as a maintenance therapy after first-line treatment is
well tolerated, and it might prevent disease recurrence
and prolong the survival of SCLC patients. We also
found that more frequent CIT administration might
improve treatment outcome. The exact mechanism
through which CIT extends PFS and OS in SCLC
patients remains unclear and a multi-center randomized
clinical trial is needed to further verify its efficacy.
Additional file 1: Table S1. The number of the 3 types of immunocytes
infused into each patient.
Additional file 2: Figure S1: The cytotoxic effect of immune cells on
NCI-H446 cells. All 3 types of immune cells exhibited a significant cytotoxic
effect on NCI-H446 cells when used alone, but a greater cytotoxic effect
was achieved when they were combined, and this cytotoxicity increased
further as the E/T cell ratio increased. At an E/T cell ratio of 25:1, the median
cytotoxicity level of NK, γδT and CIK cells was 75.5 % (range, 59.1–90.7 %),
70.6 % (range, 48.3–80.0 %), and 49.0 % (range, 27.4–68.9 %), respectively. The
combined immune cells showed a synergistic anti-cancer effect with a median
cytotoxicity level of 85.5 % (range, 63.4–96.5 %) at an E/T cell ratio of 25:1.
CBR: Clinical benefit rate; CI: Confidence interval; CIK: Cytokine-induced killer;
CIT: Cellular immunotherapy; CR: Complete remission; CT: Computed
tomography; CSCs: Cancer stem cells; CTL: Cytotoxic T lymphocyte;
CTLA-4: Cytotoxic T-lymphocyte antigen-4; EC: Etoposide/carboplatin;
ECOG: Eastern Cooperative Oncology Group; EP: Etoposide/cisplatin;
ES-SCLC: Extensive stage small cell lung cancer; GMP: Good Manufacturing
Practice; HR: Hazard ratio; irPFS: immune-related progression free survival;
LS-SCLC: Limited stage small cell lung cancer; LDH: Lactate dehydrogenase;
MRD: Minimal residual disease; MRI: Magnetic resonance imaging; NK: Natural
killer; NSCLC: Non-small cell lung cancer; OS: Overall survival;
PBMCs: Peripheral blood mononuclear cells; PD: Progressive disease;
PFS: Progression free survival; PR: Partial remission; RFA: Radiofrequency
ablation; SCLC: Small cell lung cancer; SD: Stable disease; SOP: Standard
The authors declare that they have no competing interests.
XD participated in the acquisition of data, analysis and interpretation of data
and the drafting of the manuscript and/or its critical revision. HC and XC
participated in acquisition of data and analysis and interpretation of data. HJ,
ZL, GW, LC, DL, CN, HT, LY and YZ participated in the acquisition of data. WL
and JC participated in the conception, design and coordination of this study,
acquisition of data, analysis and interpretation of data and helped to draft
the manuscript or revise it critically. All authors read and approved the final
This work was supported by National Major Scientific and Technological
Special Project for Significant New Drugs Development during the Twelfth
Five-year Plan Period (2013ZX09102032), the Chinese Ministry of Science and
Technology (2012CB911100), the Provincial Science Foundation of Jilin
Provincial Department of Finance (20140414014GH) and the Ministry of
Education Key Project of Science and Technology (311015).
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