Med Oncol (2015) 32:139 DOI 10.1007/s12032-015-0583-1
ORIGINAL PAPER
Pseudomonas aeruginosa preparation plus chemotherapy for advanced non-small-cell lung cancer: a randomized, multicenter, double-blind phase III study Jianhua Chang1 • Yunpeng Liu2 • Baohui Han3 Caicun Zhou4 • Chunxue Bai5 • Jin Li1
•
Received: 13 February 2015 / Accepted: 14 March 2015 Ó Springer Science+Business Media New York 2015
Abstract Pseudomonas aeruginosa preparation (PAP) has shown activity in inhibiting the metastasis of cancer and can improve the immune function of cancer patients during chemotherapy. This study aimed to investigate whether PAP can improves the efficacy of chemotherapy in patients with advanced non-small-cell lung cancer (NSCLC). A total of 72 subjects with stage IIIB/IV NSCLC were randomized into PAP arm (n = 36) or control arm (n = 36) at a 1:1 ratio. Subjects had vinorelbine and cisplatin combined with 0.5 ml PAP/placebo on d1, followed by 1 ml PAP/placebo injected subcutaneously three times a week. The primary end point was the objective response rate (ORR) and secondary endpoints were time to progression (TTP), overall survival (OS), safety and quality of life (QOL). Sixty-six patients were included in intent-to-treat analysis. After two cycles of treatment, there was a borderline statistically significant improvement in
ORR of PAP arm (46.88 vs. 23.53 %, P = 0.0532), and after four cycles of treatment, the ORR in PAP arm was 31.25 versus 14.71 % in control arm (P = 0.1110). Median TTP and OS were 160 and 454 days in PAP arm, 196 and 388 days in control arm, P = 0.4609 and 0.6587, respectively. The 1-year survival rate in PAP and control arms was 53.55 and 50.15 %, respectively. PAP did not result in increased toxicity and or had negative impact on QOL. The results demonstrate the therapeutic potential of PAP for advanced NSCLC. PAP can be used with chemotherapy to improve the response rate. Long-term follow-up might help define whether the combination therapy can result in survival benefit.
& Jin Li [email protected]
Introduction
1
Department of Medical Oncology, Fudan University Shanghai Cancer Center, No. 270 Dong’an Road, Shanghai 200032, China
2
Department of Medical Oncology, The First Affiliated Hospital of China Medical University, No. 155 North Nanjing Road, Shenyang 110001, China
3
Department of Pulmonary, Shanghai Chest Hospital, Shanghai Jiaotong University, No. 241 West Huaihai Road, Shanghai 200030, China
4
Department of Oncology, Shanghai Pulmonary Hospital, Tongji University, No. 507 Zhengmin Road, Shanghai 200433, China
5
Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, No. 180 Fenglin Road, Shanghai 200032, China
Keywords Non-small-cell lung cancer Cisplatin Vinorelbine Pseudomonas aeruginosa injection Randomized study
Carcinoma of lung is the leading cause of cancer death worldwide, and non-small-cell lung cancer (NSCLC) is the most frequent type of all lung cancers making up about 85 % of all new diagnoses [1]. Surgery, radiation therapy and chemotherapy are the three modalities commonly used to treat patients with NSCLC; however, the treatment options for advanced inoperable NSCLC (stage III–IV) are limited. Platinum-based chemotherapies remain the cornerstone of therapy in the first-line setting in NSCLC and have reached a plateau in effectiveness. Many of the platinumdoublet combinations produce a similar objective response and survival. In spite of the development of new chemotherapy regimens, the prognosis for advanced NSCLC is
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poor. New treatment methods, such as immunotherapy, may help improve the poor prognosis and outcome of patients suffering from advanced NSCLC. Currently, a wide variety of immunotherapeutic agents based on different strategies are being tested in clinical trials for advanced NSCLC. Cetuximab (Erbitux), a monoclonal antibody targeting the epidermal growth factor receptor (EGFR), proved in a phase III trial its potential to prolong median survival time of EGFR-positive advanced NSCLC patients treated with platinum-based chemotherapy [2]. L-BLP25 (Stimuvax), a liposome vaccine targeting the MUC1 protein, was found in a phase IIB trial to extend the survival time by 4.4 months for patients with stage IIIB and IV NSCLC [3]. Another cancer vaccine, MAGE-A3, aims at melanoma-associated antigen E-3 (MAGE-3), a tumor-specific antigen expressed in 35 % NSCLC patients. A randomized phase II study shows a positive trend for activity of MAGE-A3 vaccine treatment in MAGE-A3positive stage IB/II NSCLC patients with a relative improvement of DFI and DFS of 27 % [4]. Unlike antigen-specific strategies described above, nonspecific immunotherapeutic agents which are not limited by the expression status of cancer antigen and can induce both the innate and adaptive immune response might provide clinical benefits for more patient populations. In a case–control study for NSCLC patients who underwent radical surgery, BCG-CWS, which is developed with bacillus Calmette–Guerin (BCG) cell wall skeleton, was reported with higher 5- and 10-year survival rates of 71 and 61 % compared with 63 and 43 % for the control group [5]. A randomized phase II trial demonstrated that addition of PF-3512676 (CpG 7909), a Toll-like receptor 9 (TLR9) agonist, to taxane plus platinum chemotherapy for first-line treatment of stage IIIB–IV NSCLC improves objective response and may improve survival; further phase III trials are still ongoing [6]. PA-MSHA is a biologically established Pseudomonas aeruginosa (PA) strain characterized by rich mannosesensitive type 1 fimbriae compared with the wild strain. As a bacterium with abundant pathogen-associated molecular patterns (PAMPs), PA-MSHA can induce host immune responses through pattern recognition receptors (PRRs). It has been reported recently that the adhesion portion of type 1 fimbriae is a functional ligand for Toll-like receptor 4 (TLR4) [7]. Previous studies showed that, both in vivo and in vitro, PA-MSHA can effectively activate human natural killer cells (NKs) and mediate a Th1 response, both of which are important for immune surveillance against tumors. Interestingly, our latest study found that PA-MSHA directly induces the apoptosis of various tumor cells (breast cancer [8], liver cancer [9], gastric cancer [10]) through the inhibition of EGFR signaling mediated by type 1 fimbriae, although this mechanism has not been confirmed in
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Med Oncol (2015) 32:139
humans [10]. PA injection was a biological product developed from a chemically inactivated PA-MSHA strain and was approved by the State Food and Drug Administration (SFDA), People’s Republic of China, in 1998 for adjuvant therapy for human malignant melanoma and infectious diseases. A decade of clinical application has proven that PA injection alone or in combination with chemotherapy and radiotherapy provides a safe and effective modality for cancer patients, including lymphoma, breast cancer and lung cancer [11, 12]. To investigate whether PA injection can improve the response and survival of advanced NSCLC treated with platinum-based chemotherapy, a multicenter, randomized, double-blind placebo-controlled clinical trial was conducted, and 72 patients with stage III–IV inoperable NSCLC were enrolled at six institutions in China. The primary end point was objective response rate (ORR) and secondary endpoints were time to tumor progression (TTP), overall survival (OS), toxicity and quality of life (QOL).
Patients and methods The procedures followed have been approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. Patient selection Eligibility criteria included patients with histologically and/or cytologically confirmed advanced NSCLC, age range of 18–75 years, clinical stage III or IV (with no indications or intention to receive curative surgery or radiotherapy), at least one measurable or assessable lesion according to RECIST criteria (any lesion which had been treated with local radiotherapy should not be included), adequate organ function evidenced by absolute neutrophil count (ANC) C1.5 9 109/l, platelet count (PLT) C80 9 109/l, total bilirubin levels (TBIL) and serum creatinine (Cr) no higher than the upper limit of normal range (ULN), hepatic enzyme (ALT, AST, ALP) levels B2.59 ULN (B59 ULN when liver metastasis), Eastern Cooperative Oncology Group (ECOG) performance status (PS) of B2, expected survival [3 months and written informed consent. Exclusion criteria included pregnancy or lactation, previous or concurrent malignant diseases (except conebiopsied carcinoma in situ of the cervix or adequately treated basal or squamous cell carcinoma of the skin), severe uncontrolled internal medical diseases or active infection, any indications of brain metastases, or a history of allergy to various drugs.
Med Oncol (2015) 32:139
Treatment schedule Patients were randomly assigned to one of the two treatment groups (VC or VCP). Random assignment was conducted by an independent research organization using computer-generated lists. Both the VC and VCP groups received the identical regimen of chemotherapy, 75 mg/m2 of cisplatin intravenous injection on day 1 and 25 mg/m2 of vinorelbine intravenous injection on days 1 and 8, and the cycle were repeated every 3 weeks. In addition to chemotherapy, the patients in VCP group received 1.0 ml (0.5 ml for the first time) of PA (Wanter Ltd., Beijing, China) subcutaneous injection, three times a week, until the end of chemotherapy. As a placebo control, the patients in the VC group received 1.0 ml of saline subcutaneous injection instead with exactly the same treatment schedule as the PA injection. Each treatment was repeated for four cycles unless the patient withdrew consent, met the criteria for progressive disease or experienced unacceptable toxicity. Response, survival and toxicity criteria The tumor response was evaluated twice according to the response evaluation criteria in solid tumors (RECIST 1.1) after two and four cycles of treatment. Time to progression (TTP) was defined as the time from random assignment to first documentation of progressive disease. OS was defined as the time from the date of random assignment to date of death. The toxicity grading criteria in National Cancer Institute Common Toxicity Criteria version 2.0 (NCI-CTC 2.0) were used to evaluate toxicity. QOL evaluation QOL was evaluated based on the criteria of QOL assessment (score range 0–60) issued by China Anti-Cancer Association in 1990.
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36 were allocated to VCP group. The median follow-up was longer than 2 years and was similar between the two groups. Six patients did not receive even one response evaluation due to withdrawing consents (3), adverse events (2) or death (1). Sixty-six patients (intent-to-treat set) were assessable for efficacy and 72 patients were assessable for toxicity. Age, gender, PS, histology and cancer staging were well balanced between groups (Table 1). Response Tumor response data are presented in Table 2. The investigator-assessed complete and partial responses include both confirmed and unconfirmed responses. After two cycles of treatment, one complete response and 14 partial responses occurred in 32 assessable patients in the VCP group, for an ORR of 46.88 %. In the VC group, there were only eight (23.53 %) partial responses in 34 assessable patients; the difference between groups was not significant (P = 0.0532). After four cycles of treatment, two complete responses (CRs) and eight partial responses occurred in the VCP group, for an ORR of 31.25 %, and five partial responses occurred in VC group, for an ORR of 14.71 % (P = 0.1110). Median TTP for the VCP group was 160 days (95 % CI 94–202) compared with 196 days (95 % CI 135–224) in VC group (P = 0.4609). Survival Kaplan–Meier estimates of OS are shown in Fig. 1. Median survival time for patients in the VCP group was 454 days (95 % CI 311–511) compared with 388 days (95 % CI 307–838) for patients in the VC group. The test for superiority using a nonparametric log-rank test resulted in P = 0.6587. One-year survival rates were 53.55 % for the VCP group and 50.15 % for VC group. Toxicity
Statistical analysis OS and TTP were estimated by Kaplan–Meier curve and tested by log-rank test or Wilcoxon test. Response rate, survival rate and frequency of severe toxicity were tested by Chi-squared test or Fisher’s exact test.
Results Patient characteristics From June 2006 to May 2008, a total of 72 patients were registered by six hospitals in China. Of the 72 patients registered, 36 were allocated to the VC group and the other
No significant differences were seen between the two groups with regard to grade 3 and grade 4 toxicities. Hematologic toxicity occurred more frequently than non-hematologic toxicity (Table 3). Neutropenia was the most common hematologic toxicity in both groups. The incidence of grade 3 and grade 4 neutropenia was 58.33 % in VCP group and 52.78 % in VC group. Grade 3 and grade 4 anemia and leukopenia occurred in 13.89 % and 11.11 % of the patients in the VCP group and 8.33 and 8.33 % in the VC group. Grade 3 thrombocytopenia occurred in 2.78 % of patients in both groups. Grade 3 febrile neutropenia was only found in the VC group with an incidence of 8.33 %. Non-hematologic toxicity was mainly observed in the gastrointestinal system. Grade 3 and grade 4 nausea
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Med Oncol (2015) 32:139
Table 1 Baseline patients and disease characteristics
VCP (n = 32)
VC (n = 34)
No. of patients
%
No. of patients
%
Age Median Range
56
58
39–73
27–74
Sex Male
21
65.63
20
58.82
Female
11
34.38
14
41.18
0
5
15.63
5
14.71
1
25
78.13
29
85.29
2
2
6.25
0
ECOG PS
0
Histology Adenocarcinoma
12
37.50
12
35.29
Squamous cell
4
12.50
5
14.71
16
50.00
17
50.00
III
10
31.25
14
41.18
IV
22
68.75
20
58.82
Radiotherapy
1
3.13
0
0
Chemotherapy
0
0
1
2.94
Others Stage
Prior therapy
ECOG Eastern Cooperative Oncology Group, PS performance status
Table 2 Tumor response of intention-to-treat population
Two cycles
Four cycles
VCP (n = 32)
VC (n = 34)
Patients
%
Patients
RR
15
46.88
P value
0.0532
CR
1
3.13
0
0
2
6.25
0
0
PR
14
43.75
8
23.53
8
25.00
5
14.71
SD
9
28.13
18
52.94
7
21.88
16
47.06
PD
8
25.00
8
23.53
15
46.88
13
38.24
8
VCP (n = 32)
VC (n = 34)
%
Patients
%
Patients
23.53
10
31.25
5
% 14.71
0.1110
occurred in 11.11 % of the patients in the VCP group and in 8.33 % in the VC group. Three patients in the VCP group experienced grade 3 vomiting (incidence of 8.33 %). Vomiting was not seen in VC group.
QOL of VCP-treated patients was not significantly lower than that of the VC patients at any treatment cycle.
Discussion Quality of life QOL was evaluated at randomization (baseline) and after each cycle of treatment (Table 4). Mean baseline QOL scores of the VCP and VC groups were similar, and all the QOL scores were about 50. No statistically significant difference of QOL was observed between groups after any cycle of treatment compared with baseline values. The
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In humans, immunosurveillance against tumors mainly relies on the innate immune response by NKs and the subsequent tumor-specific response by cytotoxic T cell (CTL) [13]. Unfortunately, such responses are usually absent or weak in cancer patients, and the direct recognition of tumor-associated antigens (TAAs) by T cells usually induces tolerogenic state [14]. The activation of NK and T
Med Oncol (2015) 32:139
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Fig. 1 Time to tumor progression and overall survival
Table 3 Toxicity
VCP (n = 36)
VC (n = 36)
Grade (%)
Grade (%)
3
4
3
4
Allergic reaction
0
0
0
0
Fatigue
0
0
0
0
Fever
0
0
0
0
Infection
0
0
0
0
Febrile neutropenia
0
0
3 (8.33)
0
Injection site reaction
0
0
0
0
Anemia
4 (11.11)
1 (2.78)
3 (8.33)
0
Leukopenia
4 (11.11)
0
3 (8.33)
0
Neutropenia
12 (33.33)
9 (25.00)
13 (36.11)
6 (16.67)
Thrombocytopenia
1 (2.78)
0
1 (2.78)
0
Nausea
4 (11.11)
0
3 (8.33)
0
Vomiting
3 (8.33)
0
0
0
Diarrhea
0
0
0
0
Constipation Neuropathy (motor/sensory)
0 0
0 0
0 0
0 0
Hyperglycemia
0
0
0
0
Bilirubin
0
0
0
0
AKP
0
0
0
0 0
AST
0
0
0
ALT
0
0
1 (2.78)
0
Creatinine
0
0
0
0
Hematuria
0
0
0
0
Proteinuria
0
0
0
0
cells is essentially dependent on the assistance of antigenpresenting cells (APCs), particularly dendritic cells (DCs) [15]. There is growing evidence that the maturation of DCs
is actually dependent on the activation of Toll-like receptors (TLRs) by pathogen-specific molecular patterns (PAMPs) which are derived from microbes such as bacteria
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and viruses [16, 17]. For cancer patients, tumor cells do not provide PAMPs, so exogenous PAMPs are needed to ensure the effective maturation of DCs [17]. Microbial components used for the adjuvant immunotherapy of cancer began about 100 years ago when Dr. William Coley invented a mixed vaccine of streptococcal and staphylococcal bacteria known as Coley’s toxin [18]. BCG has been successfully used to treat bladder cancer since the first half of the last century [19]. In recent years, many attempts with bacterial components for the adjuvant immunotherapy of NSCLC have been made and desired survival benefits in clinical researches achieved, for example the BCG-CWS for NSCLC patients who undergo radical surgery [5] and PF-3512676 (CpG 7909) for stage IIIB–IV metastatic NSCLC [6]. In this study, we used PA injection, a novel biological product licensed by SFDA in 1998, as adjuvant immunotherapy and investigated whether it can result in both response and survival advantages for the advanced NSCLC patients who were treated with conventional adjuvant chemotherapy. PA injection is developed from a biologically established and chemically inactivated PA strain called PA-MSHA which is characterized by rich mannose-sensitive type 1 fimbriae [8–10]. The PA-MSHA has far lower toxicity than the wild strain and has been confirmed more suitable for human application. It has been proven that PA-MSHA has excellent immune stimulatory activity and can not only activate human DC, NK and T cell, but induces the production of Th1 cytokines including IFN-c and IL-2 [12]. Although the exact receptor recognition mechanism is not clear, recent studies have proven that type 1 fimbriae are functional ligands for TLR4 which is widely expressed on various innate and adaptive immune cells, such as DCs and B cells [7]. In addition, as integral gram-negative bacteria, it is believed that PA-MSHA has a variety of PAMPs and the potential to activate the majority of TLRs on immune cells other than TLR4 [8]. To evaluate whether PA injection can improve the therapeutic efficacy of conventional chemotherapy for advanced NSCLC, a randomized, double-blind, placebocontrolled trial was conducted in China. From June 2006 to May 2008, a total of 72 patients who suffered from stage III–IV NSCLC were enrolled by six institutions and randomly assigned to the experimental group (VCP) and placebo control group (VC). The patients in both groups received four cycles of adjuvant chemotherapy of cisplatin and vinorelbine. Along with the chemotherapy, the patients in VCP group received 1.0 ml of PA injection, three times a week, until the end of chemotherapy unless the disease becomes progressive. The patients in VC group received 1.0 ml of saline, three times a week, as placebo control. The result of tumor response evaluation showed some benefits for the patients in VCP group although without
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Med Oncol (2015) 32:139 Table 4 Quality of life VCP
VC
P value
Baseline
50.56 ± 6.36
50.53 ± 5.90
Cycle 1
50.13 ± 5.19
49.79 ± 6.53
0.8525
Cycle 2
50.13 ± 5.07
50.18 ± 6.58
0.9633
Cycle 3
49.41 ± 5.03
49.94 ± 6.98
0.6631
Cycle 4
49.16 ± 5.51
49.09 ± 6.16
0.9307
significant differences from the VC group. After two cycles of treatment, the ORR was 46.88 % for VCP group and 23.53 % for VC group. The ORR after four cycles was 31.25 % for VCP group and 14.71 % for VC group. It was worth mentioning that only two CRs occurred in the VCP group. Although there was an improved tumor response in the VCP group, long-term survival benefits were not observed by Kaplan–Meier analysis. Median TTP and OS were 160 and 454 days for VCP group and 196 and 388 days in the VC group. One-year survival rate was 53.55 % in VCP group and 50.15 % in VC group. Presumably, the short course of application of PA injection might be a reason for not obtaining survival benefits in the VCP group. In addition, there was no significant difference of QOL and toxicity between the two groups, which at least indicates the safety and reliability for PA injection to treat advanced NSCLC. In many reports on the immunotherapy for NSCLC, relative long-term intervention was essential and recommended regardless of specific or non-specific immunotherapy strategies [5, 6], especially for adjuvant immunotherapy since the innate immune system does not exhibit memory response, and therefore needed to be stimulated repeatedly and continuously. Regrettably, as the first attempt for advanced NSCLC patients, PA injection was used only to the end of four cycles of chemotherapy, so it cannot be concluded that PA injection cannot provide additional survival benefit for NSCLC patients on the basis of adjuvant chemotherapy. For advanced NSCLC, a reasonable therapeutic regimen for PA injection should include at least two phases, an intensive phase and a maintenance phase. In the intensive phase, PA injection should be administered aggressively, for example three or four times a week. Other therapeutic methods, such as chemotherapy and radiotherapy, might also be used in this phase. The main goal of intensive phase treatment is to make the tumor shrink as far as possible or at least achieve stable disease. In the maintenance phase, PA injection will be used alone to maintain the therapeutic efficacy and prolong the survival of patients. The dosing interval in maintenance phase should be extended appropriately, for example two or four times a month, and, if
Med Oncol (2015) 32:139
possible, the dosing should be continued until disease progression. In addition to the regimen, the sensitivity of patient should also be considered and some early indicators may provide information for the doctor to understand the patient’s sensitivity to treatment and determine whether the patient should receive additional or long-term treatment. In Kodama’s BCG-CWS study, IFN-c induction tests had been performed at the start of treatment to evaluate the immunotherapeutic effects of BCG-CWS on NSCLC patients [5]. Regrettably, in this trial, the immunological indicators, such as IFN-c, IL-12 and NK activity, were not used as endpoints, and these should be included in future studies. Conflict of interest
Page 7 of 7
6.
7.
8.
9.
The authors declare no conflict of interest.
Ethical standard The study has been approved by the appropriate ethics committee and has therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. All persons gave their informed consent prior to their inclusion in the study. Details that might disclose the identity of the subjects under study have been omitted.
10.
11.
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ORIGINAL PAPER
Pseudomonas aeruginosa preparation plus chemotherapy for advanced non-small-cell lung cancer: a randomized, multicenter, double-blind phase III study Jianhua Chang1 • Yunpeng Liu2 • Baohui Han3 Caicun Zhou4 • Chunxue Bai5 • Jin Li1
•
Received: 13 February 2015 / Accepted: 14 March 2015 Ó Springer Science+Business Media New York 2015
Abstract Pseudomonas aeruginosa preparation (PAP) has shown activity in inhibiting the metastasis of cancer and can improve the immune function of cancer patients during chemotherapy. This study aimed to investigate whether PAP can improves the efficacy of chemotherapy in patients with advanced non-small-cell lung cancer (NSCLC). A total of 72 subjects with stage IIIB/IV NSCLC were randomized into PAP arm (n = 36) or control arm (n = 36) at a 1:1 ratio. Subjects had vinorelbine and cisplatin combined with 0.5 ml PAP/placebo on d1, followed by 1 ml PAP/placebo injected subcutaneously three times a week. The primary end point was the objective response rate (ORR) and secondary endpoints were time to progression (TTP), overall survival (OS), safety and quality of life (QOL). Sixty-six patients were included in intent-to-treat analysis. After two cycles of treatment, there was a borderline statistically significant improvement in
ORR of PAP arm (46.88 vs. 23.53 %, P = 0.0532), and after four cycles of treatment, the ORR in PAP arm was 31.25 versus 14.71 % in control arm (P = 0.1110). Median TTP and OS were 160 and 454 days in PAP arm, 196 and 388 days in control arm, P = 0.4609 and 0.6587, respectively. The 1-year survival rate in PAP and control arms was 53.55 and 50.15 %, respectively. PAP did not result in increased toxicity and or had negative impact on QOL. The results demonstrate the therapeutic potential of PAP for advanced NSCLC. PAP can be used with chemotherapy to improve the response rate. Long-term follow-up might help define whether the combination therapy can result in survival benefit.
& Jin Li [email protected]
Introduction
1
Department of Medical Oncology, Fudan University Shanghai Cancer Center, No. 270 Dong’an Road, Shanghai 200032, China
2
Department of Medical Oncology, The First Affiliated Hospital of China Medical University, No. 155 North Nanjing Road, Shenyang 110001, China
3
Department of Pulmonary, Shanghai Chest Hospital, Shanghai Jiaotong University, No. 241 West Huaihai Road, Shanghai 200030, China
4
Department of Oncology, Shanghai Pulmonary Hospital, Tongji University, No. 507 Zhengmin Road, Shanghai 200433, China
5
Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, No. 180 Fenglin Road, Shanghai 200032, China
Keywords Non-small-cell lung cancer Cisplatin Vinorelbine Pseudomonas aeruginosa injection Randomized study
Carcinoma of lung is the leading cause of cancer death worldwide, and non-small-cell lung cancer (NSCLC) is the most frequent type of all lung cancers making up about 85 % of all new diagnoses [1]. Surgery, radiation therapy and chemotherapy are the three modalities commonly used to treat patients with NSCLC; however, the treatment options for advanced inoperable NSCLC (stage III–IV) are limited. Platinum-based chemotherapies remain the cornerstone of therapy in the first-line setting in NSCLC and have reached a plateau in effectiveness. Many of the platinumdoublet combinations produce a similar objective response and survival. In spite of the development of new chemotherapy regimens, the prognosis for advanced NSCLC is
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poor. New treatment methods, such as immunotherapy, may help improve the poor prognosis and outcome of patients suffering from advanced NSCLC. Currently, a wide variety of immunotherapeutic agents based on different strategies are being tested in clinical trials for advanced NSCLC. Cetuximab (Erbitux), a monoclonal antibody targeting the epidermal growth factor receptor (EGFR), proved in a phase III trial its potential to prolong median survival time of EGFR-positive advanced NSCLC patients treated with platinum-based chemotherapy [2]. L-BLP25 (Stimuvax), a liposome vaccine targeting the MUC1 protein, was found in a phase IIB trial to extend the survival time by 4.4 months for patients with stage IIIB and IV NSCLC [3]. Another cancer vaccine, MAGE-A3, aims at melanoma-associated antigen E-3 (MAGE-3), a tumor-specific antigen expressed in 35 % NSCLC patients. A randomized phase II study shows a positive trend for activity of MAGE-A3 vaccine treatment in MAGE-A3positive stage IB/II NSCLC patients with a relative improvement of DFI and DFS of 27 % [4]. Unlike antigen-specific strategies described above, nonspecific immunotherapeutic agents which are not limited by the expression status of cancer antigen and can induce both the innate and adaptive immune response might provide clinical benefits for more patient populations. In a case–control study for NSCLC patients who underwent radical surgery, BCG-CWS, which is developed with bacillus Calmette–Guerin (BCG) cell wall skeleton, was reported with higher 5- and 10-year survival rates of 71 and 61 % compared with 63 and 43 % for the control group [5]. A randomized phase II trial demonstrated that addition of PF-3512676 (CpG 7909), a Toll-like receptor 9 (TLR9) agonist, to taxane plus platinum chemotherapy for first-line treatment of stage IIIB–IV NSCLC improves objective response and may improve survival; further phase III trials are still ongoing [6]. PA-MSHA is a biologically established Pseudomonas aeruginosa (PA) strain characterized by rich mannosesensitive type 1 fimbriae compared with the wild strain. As a bacterium with abundant pathogen-associated molecular patterns (PAMPs), PA-MSHA can induce host immune responses through pattern recognition receptors (PRRs). It has been reported recently that the adhesion portion of type 1 fimbriae is a functional ligand for Toll-like receptor 4 (TLR4) [7]. Previous studies showed that, both in vivo and in vitro, PA-MSHA can effectively activate human natural killer cells (NKs) and mediate a Th1 response, both of which are important for immune surveillance against tumors. Interestingly, our latest study found that PA-MSHA directly induces the apoptosis of various tumor cells (breast cancer [8], liver cancer [9], gastric cancer [10]) through the inhibition of EGFR signaling mediated by type 1 fimbriae, although this mechanism has not been confirmed in
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humans [10]. PA injection was a biological product developed from a chemically inactivated PA-MSHA strain and was approved by the State Food and Drug Administration (SFDA), People’s Republic of China, in 1998 for adjuvant therapy for human malignant melanoma and infectious diseases. A decade of clinical application has proven that PA injection alone or in combination with chemotherapy and radiotherapy provides a safe and effective modality for cancer patients, including lymphoma, breast cancer and lung cancer [11, 12]. To investigate whether PA injection can improve the response and survival of advanced NSCLC treated with platinum-based chemotherapy, a multicenter, randomized, double-blind placebo-controlled clinical trial was conducted, and 72 patients with stage III–IV inoperable NSCLC were enrolled at six institutions in China. The primary end point was objective response rate (ORR) and secondary endpoints were time to tumor progression (TTP), overall survival (OS), toxicity and quality of life (QOL).
Patients and methods The procedures followed have been approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. Patient selection Eligibility criteria included patients with histologically and/or cytologically confirmed advanced NSCLC, age range of 18–75 years, clinical stage III or IV (with no indications or intention to receive curative surgery or radiotherapy), at least one measurable or assessable lesion according to RECIST criteria (any lesion which had been treated with local radiotherapy should not be included), adequate organ function evidenced by absolute neutrophil count (ANC) C1.5 9 109/l, platelet count (PLT) C80 9 109/l, total bilirubin levels (TBIL) and serum creatinine (Cr) no higher than the upper limit of normal range (ULN), hepatic enzyme (ALT, AST, ALP) levels B2.59 ULN (B59 ULN when liver metastasis), Eastern Cooperative Oncology Group (ECOG) performance status (PS) of B2, expected survival [3 months and written informed consent. Exclusion criteria included pregnancy or lactation, previous or concurrent malignant diseases (except conebiopsied carcinoma in situ of the cervix or adequately treated basal or squamous cell carcinoma of the skin), severe uncontrolled internal medical diseases or active infection, any indications of brain metastases, or a history of allergy to various drugs.
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Treatment schedule Patients were randomly assigned to one of the two treatment groups (VC or VCP). Random assignment was conducted by an independent research organization using computer-generated lists. Both the VC and VCP groups received the identical regimen of chemotherapy, 75 mg/m2 of cisplatin intravenous injection on day 1 and 25 mg/m2 of vinorelbine intravenous injection on days 1 and 8, and the cycle were repeated every 3 weeks. In addition to chemotherapy, the patients in VCP group received 1.0 ml (0.5 ml for the first time) of PA (Wanter Ltd., Beijing, China) subcutaneous injection, three times a week, until the end of chemotherapy. As a placebo control, the patients in the VC group received 1.0 ml of saline subcutaneous injection instead with exactly the same treatment schedule as the PA injection. Each treatment was repeated for four cycles unless the patient withdrew consent, met the criteria for progressive disease or experienced unacceptable toxicity. Response, survival and toxicity criteria The tumor response was evaluated twice according to the response evaluation criteria in solid tumors (RECIST 1.1) after two and four cycles of treatment. Time to progression (TTP) was defined as the time from random assignment to first documentation of progressive disease. OS was defined as the time from the date of random assignment to date of death. The toxicity grading criteria in National Cancer Institute Common Toxicity Criteria version 2.0 (NCI-CTC 2.0) were used to evaluate toxicity. QOL evaluation QOL was evaluated based on the criteria of QOL assessment (score range 0–60) issued by China Anti-Cancer Association in 1990.
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36 were allocated to VCP group. The median follow-up was longer than 2 years and was similar between the two groups. Six patients did not receive even one response evaluation due to withdrawing consents (3), adverse events (2) or death (1). Sixty-six patients (intent-to-treat set) were assessable for efficacy and 72 patients were assessable for toxicity. Age, gender, PS, histology and cancer staging were well balanced between groups (Table 1). Response Tumor response data are presented in Table 2. The investigator-assessed complete and partial responses include both confirmed and unconfirmed responses. After two cycles of treatment, one complete response and 14 partial responses occurred in 32 assessable patients in the VCP group, for an ORR of 46.88 %. In the VC group, there were only eight (23.53 %) partial responses in 34 assessable patients; the difference between groups was not significant (P = 0.0532). After four cycles of treatment, two complete responses (CRs) and eight partial responses occurred in the VCP group, for an ORR of 31.25 %, and five partial responses occurred in VC group, for an ORR of 14.71 % (P = 0.1110). Median TTP for the VCP group was 160 days (95 % CI 94–202) compared with 196 days (95 % CI 135–224) in VC group (P = 0.4609). Survival Kaplan–Meier estimates of OS are shown in Fig. 1. Median survival time for patients in the VCP group was 454 days (95 % CI 311–511) compared with 388 days (95 % CI 307–838) for patients in the VC group. The test for superiority using a nonparametric log-rank test resulted in P = 0.6587. One-year survival rates were 53.55 % for the VCP group and 50.15 % for VC group. Toxicity
Statistical analysis OS and TTP were estimated by Kaplan–Meier curve and tested by log-rank test or Wilcoxon test. Response rate, survival rate and frequency of severe toxicity were tested by Chi-squared test or Fisher’s exact test.
Results Patient characteristics From June 2006 to May 2008, a total of 72 patients were registered by six hospitals in China. Of the 72 patients registered, 36 were allocated to the VC group and the other
No significant differences were seen between the two groups with regard to grade 3 and grade 4 toxicities. Hematologic toxicity occurred more frequently than non-hematologic toxicity (Table 3). Neutropenia was the most common hematologic toxicity in both groups. The incidence of grade 3 and grade 4 neutropenia was 58.33 % in VCP group and 52.78 % in VC group. Grade 3 and grade 4 anemia and leukopenia occurred in 13.89 % and 11.11 % of the patients in the VCP group and 8.33 and 8.33 % in the VC group. Grade 3 thrombocytopenia occurred in 2.78 % of patients in both groups. Grade 3 febrile neutropenia was only found in the VC group with an incidence of 8.33 %. Non-hematologic toxicity was mainly observed in the gastrointestinal system. Grade 3 and grade 4 nausea
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Table 1 Baseline patients and disease characteristics
VCP (n = 32)
VC (n = 34)
No. of patients
%
No. of patients
%
Age Median Range
56
58
39–73
27–74
Sex Male
21
65.63
20
58.82
Female
11
34.38
14
41.18
0
5
15.63
5
14.71
1
25
78.13
29
85.29
2
2
6.25
0
ECOG PS
0
Histology Adenocarcinoma
12
37.50
12
35.29
Squamous cell
4
12.50
5
14.71
16
50.00
17
50.00
III
10
31.25
14
41.18
IV
22
68.75
20
58.82
Radiotherapy
1
3.13
0
0
Chemotherapy
0
0
1
2.94
Others Stage
Prior therapy
ECOG Eastern Cooperative Oncology Group, PS performance status
Table 2 Tumor response of intention-to-treat population
Two cycles
Four cycles
VCP (n = 32)
VC (n = 34)
Patients
%
Patients
RR
15
46.88
P value
0.0532
CR
1
3.13
0
0
2
6.25
0
0
PR
14
43.75
8
23.53
8
25.00
5
14.71
SD
9
28.13
18
52.94
7
21.88
16
47.06
PD
8
25.00
8
23.53
15
46.88
13
38.24
8
VCP (n = 32)
VC (n = 34)
%
Patients
%
Patients
23.53
10
31.25
5
% 14.71
0.1110
occurred in 11.11 % of the patients in the VCP group and in 8.33 % in the VC group. Three patients in the VCP group experienced grade 3 vomiting (incidence of 8.33 %). Vomiting was not seen in VC group.
QOL of VCP-treated patients was not significantly lower than that of the VC patients at any treatment cycle.
Discussion Quality of life QOL was evaluated at randomization (baseline) and after each cycle of treatment (Table 4). Mean baseline QOL scores of the VCP and VC groups were similar, and all the QOL scores were about 50. No statistically significant difference of QOL was observed between groups after any cycle of treatment compared with baseline values. The
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In humans, immunosurveillance against tumors mainly relies on the innate immune response by NKs and the subsequent tumor-specific response by cytotoxic T cell (CTL) [13]. Unfortunately, such responses are usually absent or weak in cancer patients, and the direct recognition of tumor-associated antigens (TAAs) by T cells usually induces tolerogenic state [14]. The activation of NK and T
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Fig. 1 Time to tumor progression and overall survival
Table 3 Toxicity
VCP (n = 36)
VC (n = 36)
Grade (%)
Grade (%)
3
4
3
4
Allergic reaction
0
0
0
0
Fatigue
0
0
0
0
Fever
0
0
0
0
Infection
0
0
0
0
Febrile neutropenia
0
0
3 (8.33)
0
Injection site reaction
0
0
0
0
Anemia
4 (11.11)
1 (2.78)
3 (8.33)
0
Leukopenia
4 (11.11)
0
3 (8.33)
0
Neutropenia
12 (33.33)
9 (25.00)
13 (36.11)
6 (16.67)
Thrombocytopenia
1 (2.78)
0
1 (2.78)
0
Nausea
4 (11.11)
0
3 (8.33)
0
Vomiting
3 (8.33)
0
0
0
Diarrhea
0
0
0
0
Constipation Neuropathy (motor/sensory)
0 0
0 0
0 0
0 0
Hyperglycemia
0
0
0
0
Bilirubin
0
0
0
0
AKP
0
0
0
0 0
AST
0
0
0
ALT
0
0
1 (2.78)
0
Creatinine
0
0
0
0
Hematuria
0
0
0
0
Proteinuria
0
0
0
0
cells is essentially dependent on the assistance of antigenpresenting cells (APCs), particularly dendritic cells (DCs) [15]. There is growing evidence that the maturation of DCs
is actually dependent on the activation of Toll-like receptors (TLRs) by pathogen-specific molecular patterns (PAMPs) which are derived from microbes such as bacteria
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and viruses [16, 17]. For cancer patients, tumor cells do not provide PAMPs, so exogenous PAMPs are needed to ensure the effective maturation of DCs [17]. Microbial components used for the adjuvant immunotherapy of cancer began about 100 years ago when Dr. William Coley invented a mixed vaccine of streptococcal and staphylococcal bacteria known as Coley’s toxin [18]. BCG has been successfully used to treat bladder cancer since the first half of the last century [19]. In recent years, many attempts with bacterial components for the adjuvant immunotherapy of NSCLC have been made and desired survival benefits in clinical researches achieved, for example the BCG-CWS for NSCLC patients who undergo radical surgery [5] and PF-3512676 (CpG 7909) for stage IIIB–IV metastatic NSCLC [6]. In this study, we used PA injection, a novel biological product licensed by SFDA in 1998, as adjuvant immunotherapy and investigated whether it can result in both response and survival advantages for the advanced NSCLC patients who were treated with conventional adjuvant chemotherapy. PA injection is developed from a biologically established and chemically inactivated PA strain called PA-MSHA which is characterized by rich mannose-sensitive type 1 fimbriae [8–10]. The PA-MSHA has far lower toxicity than the wild strain and has been confirmed more suitable for human application. It has been proven that PA-MSHA has excellent immune stimulatory activity and can not only activate human DC, NK and T cell, but induces the production of Th1 cytokines including IFN-c and IL-2 [12]. Although the exact receptor recognition mechanism is not clear, recent studies have proven that type 1 fimbriae are functional ligands for TLR4 which is widely expressed on various innate and adaptive immune cells, such as DCs and B cells [7]. In addition, as integral gram-negative bacteria, it is believed that PA-MSHA has a variety of PAMPs and the potential to activate the majority of TLRs on immune cells other than TLR4 [8]. To evaluate whether PA injection can improve the therapeutic efficacy of conventional chemotherapy for advanced NSCLC, a randomized, double-blind, placebocontrolled trial was conducted in China. From June 2006 to May 2008, a total of 72 patients who suffered from stage III–IV NSCLC were enrolled by six institutions and randomly assigned to the experimental group (VCP) and placebo control group (VC). The patients in both groups received four cycles of adjuvant chemotherapy of cisplatin and vinorelbine. Along with the chemotherapy, the patients in VCP group received 1.0 ml of PA injection, three times a week, until the end of chemotherapy unless the disease becomes progressive. The patients in VC group received 1.0 ml of saline, three times a week, as placebo control. The result of tumor response evaluation showed some benefits for the patients in VCP group although without
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Med Oncol (2015) 32:139 Table 4 Quality of life VCP
VC
P value
Baseline
50.56 ± 6.36
50.53 ± 5.90
Cycle 1
50.13 ± 5.19
49.79 ± 6.53
0.8525
Cycle 2
50.13 ± 5.07
50.18 ± 6.58
0.9633
Cycle 3
49.41 ± 5.03
49.94 ± 6.98
0.6631
Cycle 4
49.16 ± 5.51
49.09 ± 6.16
0.9307
significant differences from the VC group. After two cycles of treatment, the ORR was 46.88 % for VCP group and 23.53 % for VC group. The ORR after four cycles was 31.25 % for VCP group and 14.71 % for VC group. It was worth mentioning that only two CRs occurred in the VCP group. Although there was an improved tumor response in the VCP group, long-term survival benefits were not observed by Kaplan–Meier analysis. Median TTP and OS were 160 and 454 days for VCP group and 196 and 388 days in the VC group. One-year survival rate was 53.55 % in VCP group and 50.15 % in VC group. Presumably, the short course of application of PA injection might be a reason for not obtaining survival benefits in the VCP group. In addition, there was no significant difference of QOL and toxicity between the two groups, which at least indicates the safety and reliability for PA injection to treat advanced NSCLC. In many reports on the immunotherapy for NSCLC, relative long-term intervention was essential and recommended regardless of specific or non-specific immunotherapy strategies [5, 6], especially for adjuvant immunotherapy since the innate immune system does not exhibit memory response, and therefore needed to be stimulated repeatedly and continuously. Regrettably, as the first attempt for advanced NSCLC patients, PA injection was used only to the end of four cycles of chemotherapy, so it cannot be concluded that PA injection cannot provide additional survival benefit for NSCLC patients on the basis of adjuvant chemotherapy. For advanced NSCLC, a reasonable therapeutic regimen for PA injection should include at least two phases, an intensive phase and a maintenance phase. In the intensive phase, PA injection should be administered aggressively, for example three or four times a week. Other therapeutic methods, such as chemotherapy and radiotherapy, might also be used in this phase. The main goal of intensive phase treatment is to make the tumor shrink as far as possible or at least achieve stable disease. In the maintenance phase, PA injection will be used alone to maintain the therapeutic efficacy and prolong the survival of patients. The dosing interval in maintenance phase should be extended appropriately, for example two or four times a month, and, if
Med Oncol (2015) 32:139
possible, the dosing should be continued until disease progression. In addition to the regimen, the sensitivity of patient should also be considered and some early indicators may provide information for the doctor to understand the patient’s sensitivity to treatment and determine whether the patient should receive additional or long-term treatment. In Kodama’s BCG-CWS study, IFN-c induction tests had been performed at the start of treatment to evaluate the immunotherapeutic effects of BCG-CWS on NSCLC patients [5]. Regrettably, in this trial, the immunological indicators, such as IFN-c, IL-12 and NK activity, were not used as endpoints, and these should be included in future studies. Conflict of interest
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6.
7.
8.
9.
The authors declare no conflict of interest.
Ethical standard The study has been approved by the appropriate ethics committee and has therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. All persons gave their informed consent prior to their inclusion in the study. Details that might disclose the identity of the subjects under study have been omitted.
10.
11.
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