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. 2014 Apr 22;22(6):1221–1229. doi: 10.1038/mt.2014.53

Multicenter Randomized Phase 2 Clinical Trial of a Recombinant Human Endostatin Adenovirus in Patients with Advanced Head and Neck Carcinoma

Wen Ye 1, Ranyi Liu 1, Changchuan Pan 2, Wenqi Jiang 1, Li Zhang 1, Zhongzhen Guan 1, Jiangxue Wu 1, Xiaofang Ying 1, Lixia Li 1, Su Li 1, Wen Tan 3, Musheng Zeng 1, Tiebang Kang 1, Qing Liu 1, George R Thomas 4, Manli Huang 5, Wuguo Deng 1, Wenlin Huang 1,6,*
PMCID: PMC4048902  PMID: 24662947

Abstract

A randomized, open-label, phase 2, multicenter clinical trial was conducted to evaluate the efficacy and safety of the addition of a recombinant human endostatin adenovirus (E10A) to cisplatin and paclitaxel in patients with advanced head and neck squamous cell carcinoma or nasopharyngeal carcinoma. Patients with locally advanced or metastatic head and neck squamous cell carcinoma or nasopharyngeal carcinoma not suitable for operation or radiotherapy were randomly assigned to receive E10A plus chemotherapy every 3 weeks for a maximum of six cycles or to receive chemotherapy only. One hundred and thirty-six eligible patients were randomly assigned. The addition of E10A did not significantly improve the objective response rate (29.9 versus 39.7%, P = 0.154). However, patients who received endostatin had longer progression-free survival (7.03 versus 3.60 months, P = 0.006; hazard ratio: 0.55). The combination of E10A with chemotherapy benefited prior chemotherapy-treated patients and those who received three to four treatment cycles (6.50 versus 3.43 months, P = 0.003; 8.27 versus 4.27 months, P = 0.018; respectively). The overall disease control rate significantly increased from 80.6% in the control group to 92.6% in the test group (P = 0.034). Except for fever, no adverse events were associated with the E10A treatment. In summary, E10A plus chemotherapy is a safe and effective therapeutic approach in patients with advanced head and neck squamous cell carcinoma or nasopharyngeal carcinoma.

Introduction

Angiogenesis, crucial for the development, invasion, and metastasis of tumors, depends mostly on the balance between angiogenic stimulators and inhibitors. The antitumor activity of endostatin, an endogenous angiogenic inhibitor derived from collagen XVIII, was first demonstrated in the Folkman Laboratory, where the administration of insoluble endostatin was found to suppress VEGF-stimulated endothelial cell proliferation, migration, and tumor angiogenesis with low toxicity and an absence of drug resistance.1

Gene therapy as a means of continuous endostatin delivery has attracted our interest. Our previous studies showed that E10A (Guangzhou Doublle Bioproducts, Guangzhou, China), a type 5 recombinant replication-deficient adenovirus vector carrying the human endostatin gene, can directly introduce the gene into tumors and continuously expresses the endogenous endostatin protein in host cells to limit vascularization and that this endostatin protein is metabolized in the liver.2,3 In vivo studies of multiple intratumoral administrations of E10A to nasopharyngeal carcinoma (NPC), gastric cancer, and hepatocellular carcinoma xenografts in nude mice demonstrated high-level endogenous endostatin expression with a relatively long half-life and a significant inhibition of tumor growth.4,5,6 E10A in combination with low-dose cisplatin induced high levels of apoptosis and significantly enhanced the tumor growth inhibitory effect of cisplatin in xenograft mouse models of head and neck squamous cell carcinoma (HNSCC) and lung cancer, respectively.7,8 Meanwhile, E10A combined with docetaxel inhibited prostate cancer growth and metastasis.9 A long-term, high-dose intramuscular administration of E10A in canines was proved not toxic and might be safe for clinical therapeutic use.10 In 2007, a dose-escalation phase 1 clinical trial on 15 patients with advanced solid tumors showed that no dose-limiting toxicity was developed from weekly intratumoral injections of E10A at doses ranging from 1 × 1010 virus particles to 1 × 1012 virus particles.11 A distribution study detected E10A in the patients' blood, throat, and injection site, but rarely in the urine and stool. Endostatin expression was increased throughout the period of treatment despite the presence of neutralizing antiadenovirus antibodies.12

Surgery, radiotherapy, and chemotherapy have generally been adopted as primary treatment modalities for advanced HNSCC and NPC in recent decades. However, locoregional recurrence and metastatic disease in these advanced cancer patients remain the main causes of death, with poor overall survival (OS; HNSCC: 7–10 months; NPC: 15–20 months). Therefore, a therapeutic approach that can effectively control head and neck cancers is urgently needed.13,14,15,16 We assumed E10A could benefit advanced cancer patients, and vascular-rich solid tumors on the body surface are applicable for the intratumoral administration of E10A and are straightforward for tumor observation/evaluation. Therefore, we used HNSCC and NPC patients as the target population of this trial.

To define the efficacy and safety of E10A, we performed a phase 2, open-label, multicenter clinical trial in 11 hospitals of China in patients with advanced HNSCC or NPC using paclitaxel and cisplatin with or without E10A in a randomized, controlled manner.

Results

From March 2008 to December 2010, 140 patients from 11 hospitals in China were consecutively enrolled. Three patients meeting the exclusion criteria were excluded, and one withdrew before randomization. In total, 136 patients were randomly assigned to two groups. One patient in the paclitaxel-cisplatin alone group (the control group) refused to receive the allocated treatment; thus, 67 patients in the control group and 68 patients in the paclitaxel-cisplatin plus E10A group (the E10A group) were included in the safety population and in the analysis set for efficacy and survival. The baseline characteristics were well balanced between the two groups (Table 1).

Table 1. Baseline characteristics of the study patients.

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Efficacy

The primary end point was the objective response rate (RR), defined as the proportion of patients who had a complete response (CR) or partial response (PR) at the target tumor lesion. The secondary end points were the objective disease control rate (DCR, or stable disease (SD) + PR + CR at the target tumor lesion), the overall RR, the overall DCR, OS, and progression-free survival (PFS).

The primary end point of objective RR did not meet statistical significance (29.9% in the control group, 39.7% in the E10A group, P = 0.154; odds ratio (OR) 0.65; Table 2). However, the administration of E10A benefited some subgroups of patients. In the HNSCC patients, the objective RR was 36.5% (15/41) with E10A administration, exhibiting a trend of exceeding the rate of 20.0% (7/35) in the control group (P = 0.090; OR: 0.43), whereas the objective RR was 44.4% (12/27) versus 40.6% (13/32) in the NPC patients (P = 0.487; OR: 0.86). Patients who had previously received chemotherapy in the E10A group had a 44.8% (12/29) objective RR, whereas patients in the control group had only a 22.6% objective RR (7/31; P = 0.06, OR: 0.36). In contrast, patients without previous chemotherapy had a similar RR in both groups (34.3 versus 39.4%; P = 0.426, OR: 1.25). There was no relationship between prior chemotherapy and the response of treated patients (Supplementary Table S1). Moreover, in the patients who received three to four treatment cycles, there were better objective/overall responses of the E10A group than the control group (P = 0.014, 0.026, respectively, Table 2). The overall DCR was 80.6% in the control group, significantly lower than the rate of 92.6% in the E10A group (P = 0.034; OR: 0.33; Table 2). Typical case presentations are shown in the Supplementary Figure S1.

Table 2. Tumor responses to treatment.

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As shown in Figure 1a and Table 3, the difference in the Kaplan–Meier estimates of PFS favored chemotherapy plus E10A, which resulted in a 3.43-month improvement. With a median follow-up of 10.47 months, the median PFS was 3.60 months (interquartile range: 2.60–7.63) in the control group and 7.03 months (interquartile range: 3.27–13.73) in the E10A group. As compared with the control group, the hazard ratio (HR) of progression was significantly lower in the E10A group (HR: 0.55, P = 0.006). Exploratory stratified analysis of PFS across key clinical subgroups supported the primary analysis, demonstrating the beneficial effects of adding E10A to cisplatin-paclitaxel chemotherapy significantly improved PFS in the subpopulations of males, patients older than 55 years, patients had received prior treatment, and those who finished three to four treatment cycles (P = 0.017, HR: 0.54; P = 0.016, HR: 0.45; P = 0.003, HR: 0.45; and P = 0.018, HR: 0.49; respectively), showing HRs that consistently favored the E10A-containg groups.

Figure 1.

Figure 1

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Kaplan–Meier estimates of survival for all patients by treatment groups. (a) Progression-free survival. (b) Overall survival. CI, confidence interval; HR, hazard ratio; PFS, progression-free survival; OS, overall survival.

Table 3. Stratified progression-free survival and overall survival analysis.

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The OS of the E10A group was relatively prolonged in different subgroups compared with the controls (e.g., 13.37 months versus 9.67 months in the HNSCC patients, 13.03 months versus 10.50 months in those who had received prior treatment; Figure 1b; Table 3), but these results did not translate into significantly superior survival.

Endostatin expression and antiangiogenic effects

The systemic endostatin level in serum showed that the gene-therapeutic adenovirus was actively producing the endostatin protein endogenously in patients. After the first administration of E10A, serum endostatin increased from 30.48 ± 16.59 ng/ml (baseline) to a peak of 51.69 ± 17.49 ng/ml (P < 0.001) 48 hours and then gradually decreased before the next E10A administration (day 8). During the subsequent treatment, serum endostatin maintained at a high level (77.69 ± 36.86 at day 21, 84.95 ± 41.48 at day 42, 86.44 ± 16.77 at day 63, and 85.52 ± 24.23 at day 84) thanks to the continuously expressed endostatin protein produced by repeated administration of E10A (Figure 2a; Table 4) Immunohistochemical assays of seven pairs of biopsy samples (before and after treatment of the same patient) showed that all of the patients had a significant increased of endostatin expression in injection site after treatment compared with before treatment (P = 0.004; Figure 2b,c). As a result of E10A treatment, the tumor microvessel density and the tumor tissue blood flow significantly decreased (Figure 2d–h). There was no difference in the serum concentration of endostatin over time in the subgroups of HNSCC versus NPC and chemotherapy-untreated versus -treated (P = 0.057, 0.057, respectively; Supplementary Figure S2).

Figure 2.

Figure 2

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Endostatin expression and antiangiogenic assessments. (a) Serum endostatin concentration during treatment; Pretreatment and posttreatment assessments of (b) immunohistochemical staining of endostatin in the biopsies of the E10A group. Bar = 100 µm; (c) H score of endostatin in the biopsies of the E10A group; (d) immunohistochemical staining of CD34 (marker of vascular formation). Bar = 100 µm; (e) microvessel density of tumors; (f) blood flow volume in tumors (evaluated by dynamic contrast-enhanced ultrasound); (g) echo-power of blood flow volume in the tumor of patient No. 01C075; (h) echo-power peaks of blood flow volume in eight tumors.

Table 4. Molecular markers in the E10A group serum.

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We measured eight molecular biomarkers (sVCAM-1, TSP-1, sE-selectin, VEGF, bFGF, angiopoietin-1, angiopoietin-2, and EGF) related to angiogenesis in the serum of some E10A patients before and after the first administration of E10A and grouped them according to their therapy responses into two categories: group CR + PR and group SD + progressive disease (PD; Table 4; Supplementary Figure S3). By performing a General Linear Model Analysis for Repeated Measures, we found that endostatin, bFGF, sVCAM-1, angiopoietin-2, and sE-selectin changed with time. VEGF ranged from 590 to 750 pg/ml in group CR + PR, which was the only biomarker that was significantly lower than in group SD + PD (895–992 pg/ml; P = 0.045, Supplementary Table S2).

An elevation of CD3+ T cells and CD19+ B cells infiltration in the posttreatment biopsies of the E10A group is shown in Figure 3.

Figure 3.

Figure 3

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Leukocyte infiltration in the E10A group tumor samples. Pretreatment and posttreatment immunohistochemical staining of (a) CD3+ T cells and (b) CD19+ B cells. Bar = 100 µm.

Safety

The overall incidence of adverse events was 92.6% (63/68) in the E10A group and 89.6% (60/67) in the control group (P = 0.905; Table 5). The most frequent adverse events in both groups were bone marrow suppression (leukopenia, neutropenia, and anemia), fever, and nausea. Fever was more frequent in the E10A group than in the control group (P = 0.002; OR: 4.02). Of the 68 patients who received E10A, 7 had grade 1 or 2 transient local reactions (local pyrexia, pain, swelling, hematoma, or hemorrhage). No significant differences were found between the groups with respect to the incidence of other adverse events.

Table 5. Common adverse events.

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Twelve serious adverse events occurred in eight patients: four in the E10A group and four in the control group (Supplementary Table S3). With timely treatment, four patients remitted without sequelae, and the other four died. Among the deaths, only one in the control group was considered likely to be chemotherapy-related, whereas no death was considered to be E10A-related.

Discussion

For patients with locoregional recurrence or metastatic HNSCC or NPC, extensive surgery and radiotherapy usually fail, and chemotherapy is integrated into the standard therapy in an attempt to improve disease control and prolong survival.17 Randomized clinical studies of patients with advanced HNSCC and NPC have shown that the combination of cisplatin and paclitaxel yields a DCR of up to 80%, an overall RR of 22–26%, and a median PFS of ~4 months.18,19 These findings are consistent with our results in the control group (29.9% RR, 80.6% DCR, and 3.60-month PFS) but are inferior to the outcomes of the E10A group (39.7% RR, 92.6% DCR, and 7.03-month PFS).

We failed to detect a significant difference in the primary end point between the groups, most likely due to our limited sample size to detect an objective RR raised from 29.9% in the control group and to 39.7% in the E10A group, which were higher than the 15–35% in the sample size calculation assumption. However, the trial still revealed a tendency for the administration of E10A to raise disease control in HNSCC patients and those who had previously received chemotherapy, compared with NPC patients and those without prior chemotherapy, respectively. We noticed the largely extended PFS in the E10A group by contrast to the control group in association with the nonspecific difference in objective RR, suggesting that the PFS difference was not driven by tumor shrinkage activity. The 45% reduction in the risk of progression with E10A as compared with the control group is clinically important, indicating that E10A acted as an adjuvant chemotherapy agent could enhance the tumor growth inhibitory effect of paclitaxel-cisplatin and postpone tumor progression despite its disability to reverse disease.

For an explorative phase 2 trial of a new drug, enrolling suitable patients regardless of their treatment history enabled us to locate a target population effectively. Based on the findings of this phase 2 trial, our phase 3 trial, which launched in November 2013, has selected previously treated HNSCC patients as the target population. Moreover, with more E10A treatments, patients had a better objective/overall RR compared with the control group. This finding encouraged us to further confirm this correlation in future applications, exploring the possibilities of increased administration frequency and prolonged medication period without significant adverse events.

The serum endostatin level peaked on the third day after the first injection, at a level that was ~21 ng/ml higher than the baseline, and then gradually dropped before the initiation of the next E10A administration. During the subsequent treatment, serum endostatin maintained at a high level (about 50 ng/ml higher than the baseline) thanks to the continuously expressed endostatin protein produced by E10A. The evidence about sustained and repeated cycles of endostatin expression proved that readministration of E10A was efficacious. Despite the intratumoral administration, the expression of long half-life endogenous endostatin with systemically sustained levels (instead of rising and dropping drastically within a short time) showed the advantage of this novel therapeutic adenovirus compared with exogenous endostatin. Some clinical trials with exogenous endostatin have been conducted, but they were disadvantages due to its very low yield and short bioactive half-life, which necessitate daily repeated administration.20,21,22,23,24 Gene therapy with E10A can produce endogenous, stable, and lasting endostatin with the same or even better tumor inhibitory effect than endostatin exogenously produced in prokaryotic cells.4,25 Our trial not only substantiates that E10A has a significant inhibitory effect on tumors but also provides a simplified therapeutic schedule.

We assessed eight molecular biomarkers related to angiogenesis, including the inhibitors TSP-1 and angiopoietin-2 and the activators VEGF, bFGF, sE-selectin, sVCAM-1, angiopoietin-1, and EGF, in the patients' serum before and after the first administration of E10A.26,27,28 The serum VEGF in the E10A patients was significantly lower in the CR + PR group than in the SD + PD group, which supports the hypothesis that endostatin suppresses VEGF-stimulated endothelial cell proliferation, migration, and tumor angiogenesis. Therefore, serum VEGF could be considered a predictive marker for treatment outcomes in future clinical applications, but this possibility needs further validation. Among the four biomarkers that changed with time, it is noteworthy that the proangiogenic factors were upregulated (bFGF, sVCAM-1, and sE-selectin) and angiopoietin-2 was downregulated. We may suppose that the inhibitory effect of endostatin caused a systemic negative feedback of angiogenesis-related pathways. As a result, the activators were upregulated, and angiopoietin-2 was downregulated. Kanazawa et al.27 reported that in the presence of high levels of VEGF, angiopoietin-2 promotes remodeling of the basal lamina, proliferation/migration of endothelial cells, and sprouting of new blood vessels. However, in the absence of VEGF, angiopoietin-2 induces vascular regression. This finding helps explain how angiopoietin-2 can inhibit angiogenesis when VEGF is downregulated, as occurred in our trial.

Except for fever, which was more frequent, but mild and transient, in the E10A group, the trial provided a convincing safety profile, with no other significant systemic toxicity. We suppose that in addition to the symptoms caused by chemotherapy, the systemic release of adenovirus caused fever, which has been the most common adverse event, reported in adenovirus-related clinical trials, with an incidence of 30–90%. In our phase 1 clinical trial, fever occurred in a dose-dependent manner.11,29,30,31

The antiadenovirus immune response induced by multiple administrations of recombinant adenovirus is usually considered to hinder the therapeutic efficiency. The significantly increased infiltration of CD3+ T cells and CD19+ B cells in target lesions posttreatment further indicated that E10A activated the immune system. In our preclinical experiments in an immune-competent mouse model, multiple intratumoral administrations of E10A did not lead to continuous increases of antiadenovirus neutralizing antibodies. Serum endostatin was slightly decreased but remained above the efficient therapeutic concentration to achieve an antiangiogenic effect.5 In our phase 1trial, 11 of the 14 patients showed an increased IgG titer posttreatment, and three patients had an increased IgM antibody titer after the administration of E10A. Despite the presence of neutralizing antibodies, endostatin transgene expression was detectable in most patients throughout the treatment. No correlation between antibody titer and adverse events or response was observed.12 Another report showed that the intratumoral injection of recombinant adenovirus activated the direct and indirect immune response to exert “bystander effects” for tumor inhibition in immune-competent individuals.32 Thus, coupled with the potential bystander effects induced by immunostimulation, the tumor inhibition of E10A was improved. Moreover, considering that cancer patients, especially advanced cases, are often immune-inhibited, the negative effects of neutralizing antibodies are miniscule compared with the benefits.

Overall, based on this pilot phase 2 study to assess the antiangiogenic effects of E10A, we have shown that E10A in combination with paclitaxel-cisplatin chemotherapy can increase disease control and prolong PFS in advanced head and neck carcinoma patients and that it has a favorable safety profile. In particular, patients with advanced HNSCC, prior chemotherapy or more E10A treatments had a better tumor response. These findings encourage us to conduct further studies to confirm the effects of this novel combination therapy.

Materials and Methods

Eligibility criteria. Patients older than 18 years with histologically or cytologically proven locoregionally advanced or metastatic HNSCC/NPC not suitable for operation or radiotherapy were eligible to participate. Patients were required to have at least one measurable (by imaging or photograph) lesion with the largest diameter ≥2 cm and suitable for the intratumoral injection of E10A, not received chemotherapy, radiotherapy, or biotherapy within 4 weeks, a life expectancy ≥12 weeks, an Eastern Cooperative Oncology Group performance status score of 0–2, and adequate bone marrow, renal, and liver functions. Known allergies to the study drug, the presence of important blood vessels/nerves or ulceration in the target lesion not suitable for injection, tumor relapse within 6 months after paclitaxel chemotherapy, severe coagulation disorders or bleeding tendency, severe uncontrolled medical conditions, a recent history of myocardial infarction, acute infection, pregnancy or lactation, or symptomatic brain metastases rendered patients ineligible.

From March 2008 to December 2010, 140 patients were consecutively enrolled in 11 hospitals of China. All patients provided written informed consent before enrollment.

Study design. This multicenter, open-label phase 2 clinical trial was approved by the State Food and Drug Administration of China and the ethics committee at each study hospital, and complied with the Declaration of Helsinki protocols and the provisions of the Good Clinical Practice guidelines.

The randomization assignment was prepared with SAS9.13 (SAS Institute, Cary, NC) software in a subblock randomization manner by a statistician not involved in the data management. The 140 random numbers were assigned to two groups in a 1:1 ratio and yielded into the patient allocation letters. If a patient enrolled successfully, he/she would be sequentially assigned a letter with a random number. As our study was an open-label trial, all patients and physicians were aware of the study group assignments once they unsealed the random letters.

According to the Response Evaluation Criteria in Solid Tumors, all therapeutic effects were classified into 4 grades as follows: CR, PR, SD, and PD. The primary end point was the objective RR (PR + CR). The secondary end points were the objective DCR (SD + PR + CR), the overall RR, the overall DCR, OS, and PFS.

The data were collected at each study hospital by principal investigators with the oversight of the project supervisor.

Treatment schedule. In the E10A group, patients received an E10A dose of 1.0 × 1012 virus particles intratumorally on day 1 and 8, paclitaxel 160 mg/m2 on day 3, and cisplatin 25 mg/m2 from day 3 to day 5. We chose the same single target lesion in which it was easiest to perform repeated intratumoral injection. If the target lesion obtained CR, E10A administration would be stopped. In the control group, the patients received the same chemotherapy as the E10A group but without E10A. This treatment regimen was repeated every 21 days for a scheduled treatment of two to six cycles. For the patients evaluated as PR, the cycle number could be up to six, whereas the maximum for SD patients was four cycles.

The principal investigators from each study hospital were trained in the standardized E10A administration procedure. First, E10A was diluted with 0.9% sodium chloride to appropriate dose according to the longest diameter of the target lesion (<2 cm: 1 ml; 2–4 cm: 2 ml; ≥4 cm: 3 ml). After local anesthesia, we penetrated the syringe under normal skin subcutaneously 5 mm into the tumor or vertically into the lymph node under direct visualization and withdrew it to confirm the absence of blood. Then, E10A was injected with one (≤3 cm2), two (3–5 cm2), or four (≥5 cm2) equal injections according to the area of the target lesion. Last, we applied local compression for 10 minutes and pasted a sterile sticker on the injection site to avoid bleeding (Supplementary Figure S4).

Tumor progression, treatment interval of more than 14 days, or request by the patient to withdraw due to intolerable side effects or the inability to comply with the trial protocol, the treatment would be terminated.

Patient assessment. Physical examination, assessment of hematologic/chemical variables, and monitoring of adverse events were routinely performed during the treatment. Adverse events were graded based on the NCI-CTC 3.0 guidelines. From the time of the first E10A administration to the latest follow-up, all adverse events were recorded regardless of their relevance to E10A (Supplementary Table S4).

The tumor evaluation was based on the assessment of target and nontarget lesions. At baseline, the end of every 2 treatment cycles, and every 3 months during follow-up until disease progression, tumor measurements were collected by computed tomography, magnetic resonance imaging, or photography. The CR or PR patients were reconfirmed after 4 weeks. During the entire trial process, the assessment of lesion size in any single patient had to be performed using the same method and the same investigator to ensure the comparability of the results. We adopted the best responses as effective end points during the period from cycle 2 to cycle 4 and calculated the survival during follow-up.

Angiogenesis-related molecular markers (sVCAM-1, TSP-1, sE-selectin, VEGF, bFGF, angiopoietin-1, angiopoietin-2, and EGF) in the serum were quantitatively evaluated on day 1, 3, 5, and 7 during the first treatment cycle. We performed ELISA assays to quantify the serum endostatin expression over time. The time points selected were day 1, 3, 5, and 7 of the first cycle, the fifth day after each subsequent E10A administration, and the last day of each treatment cycle. Patients in the E10A group at Sun Yat-sen University Cancer Center were randomly chosen to undergo biopsy of their target sites at baseline and 2 weeks after the final injection. Intratumoral expression of endostatin was scored by the following four categories based on the staining intensity: none (0), weak (1), medium (2) and strong (3). We applied a histoscore (H score) for each sample to calculate endostatin expression by multiplying the percentage of positively stained cells with each category of staining intensity.

Statistical analysis. The necessary sample size of 140 patients was calculated based on our hypothesis that we would observe an increase in the objective RR from 15% in the control group to no less than 35% in the E10A group, with a power of 80% to detect such an increase, provided with a one-sided χ2 test at the 5% significance level and a 20% drop-out rate. The estimated efficacy was based on our previous preliminary test of a small sample in the same population.

We analyzed the primary and secondary end points with a P value <0.05 was considered statistically significant. The Fisher's exact test or the Kruskal-Wallis H test was used to compare the rates between groups. The distributions of PFS and OS were estimated with Kaplan–Meier analysis and compared with the Mantel-Cox log-rank test. A General Linear Model Analysis for Repeated Measures was performed to assess the effects of E10A between the subgroups over time. We assumed that the addition of E10A would benefit patients on the basis of chemotherapy, thus one-sided tests were used when we calculated the sample size and compared the response rates in this trial of superiority test.

SUPPLEMENTARY MATERIAL Figure S1. Typical cases. Figure S2. Serum endostatin of the E10A subgroups. Figure S3. Molecular markers of angiogenesis in the serum of the E10A patients. Figure S4. Administration method of E10A. Table S1. Relationship of previous chemotherapy and patient responses. Table S2. General Linear Model Analysis for Repeated Measures of angiogenesis markers. Table S3. Serious Adverse Events (SAE). Table S4. Flow chart of trial.

Acknowledgments

We thank the participating patients and staff of the study hospitals: Zhiming Li, Yuhong Li (Sun Yat-sen University Cancer Center, Guangzhou, China); Wenchuan Zhao (Tianjin Medical University Cancer Institute and Hospital, Tianjin, China); Weimin Zhang (General Hospital of Guangzhou Military Command of PLA, Guangzhou, China); Qingquan Hua, Qibin Song (Renmin Hospital of Wuhan University, Wuhan, China); Yigang Li (Yue Bei People's Hospital, Shaoguan, China); Ping Li (West China Hospital of Sichuan University, Chengdu, China); Wenchao Liu (Xi Jing Hospital of the Fourth Military Medical University, Xi'an, China); Chunhong Hu (The second Xiangya Hospital of Central-South University, Changsha, China); Meizuo Zhong (The first Xiangya Hospital of Central-South University, Changsha, China); Yanbing Zhu (Bethune International Peace Hospital, Ha'erbin, China); Wankai Deng (Hubei Cancer Hospital, Wuhan, China). This work was supported by the National High Technology Research and Development Program of China (Project 863, No. 2012AA020803), National Major Scientific and Technological Special Project of China (No. 2012ZX09401015), National Key Basic Research Program of China (Project 973, No.2010CB529904), Guangdong Innovative Research Team Program (No. 2009010058), Guangdong Provincial Science and Technology Project (No. 2012A080205003; 2011A080502010), and Guangzhou Doublle Bioproducts. This trial is registered with ClinicalTrials.gov, number: NCT00634595. The authors declare no conflict of interest.

Supplementary Material

Supplementary Information
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