ORIGINAL PAPER

Predictive value of circulating endothelial cells for efficacyof chemotherapy with Rh-endostatin in non-small cell lung cancer

Zhu-Jun Liu • Jing Wang • Xi-Yin Wei •

Peng Chen • Liu-Chun Wang • Li Lin •

Bao-Cun Sun • Kai Li

Received: 12 December 2011 / Accepted: 30 January 2012 / Published online: 14 February 2012

� Springer-Verlag 2012

Abstract

Purpose The present study was designed to elucidate the

fluctuation of activated CECs (aCECs) during different

therapies and to investigate their predictive value for effi-

cacy of anti-angiogenesis and chemotherapy in advanced

non-small cell lung cancer (NSCLC).

Methods Seventy-two patients were randomized into

three arms, treated with concomitant NP (vinorelbine and

cisplatin) and Rh-endostatin, Rh-endostatin followed

by NP, and single NP up to a maximum of six cycles.

Response, time to progression (TTP), and aCECs levels

were observed. The correlation between aCECs and effi-

cacy was analyzed.

Results We found that TTP was 8.5 months in concom-

itant NP and Rh-endostatin versus 5.3 months in NP

(p = 0.04) and 6.0 months in Rh-endostatin followed by

NP. aCECs fluctuated during the therapeutic period, with

a significantly high level from baseline on 8th day of

Rh-endostatin followed by NP regimen, that is, when single

Rh-endostatin was administered for 1 week, and upon

completion of therapy in cases of progressive disease in

each group (all p \ 0.05). When TTP was longer than

10 months, aCECs count difference (DaCECs, the differ-

ence in the aCECs by post-therapeutic amount minus pre-

therapeutic amount) was reversely correlated to TTP

(p = 0.003, r = -0.647).

Conclusions An improved synergistic effect was achieved

from concomitant NP and Rh-endostatin compared with

Rh-endostatin followed by NP and single NP. aCECs

increased when the disease was aggravated or single

Rh-endostatin treatment of Rh-endostatin was adminis-

tered, while they decreased when a clinical response to the

combined therapy was obtained. Our results suggest

DaCECs as an ideal marker to predict the response to

Rh-endostatin combined with chemotherapy.

Keywords Circulating endothelial cells � Non-small cell

lung cancer � Chemotherapy � Rh-endostatin

Introduction

Angiogenesis is essential for the development and metas-

tasis of malignancies (Folkman 1972) and is related with

prognoses of numerous cancers (Koukourakis et al. 2000).

Anti-angiogenic drugs for lung, colon, and renal carcino-

mas are effective (Hurwitz et al. 2004; Johnson et al. 2004;

Yang et al. 2003). However, these drugs are different from

chemotherapeutics in that shrinkage of tumor is rarely

observed briefly after treatment. The cytostatic nature of

anti-angiogenic drugs makes it difficult to evaluate tumor

response by WHO or Response Evaluation Criteria in

Solid Tumors (RECIST). There is unmet need to identify

new predictive markers to indicate tumor response to

anti-angiogenics. In previous studies, more attention has

been paid to tumor angiogenesis factors (TAFs), such as

Z.-J. Liu � J. Wang � P. Chen � L.-C. Wang � L. Lin � K. Li (&)

Department of Thoracic Oncology, Tianjin Lung Cancer Center,

Tianjin Cancer Institute and Hospital, Tianjin Medical

University, Tianjin 300060, People’s Republic of China

e-mail: likai5@medmail.com.cn

X.-Y. Wei

Central Laboratory of Oncology Department, Research Center

of Basic Medical Sciences, Tianjin Cancer Institute and

Hospital, Tianjin Medical University, Tianjin 300060,

People’s Republic of China

B.-C. Sun

Department of Pathology, Tianjin Lung Cancer Center,

Tianjin Cancer Institute and Hospital, Tianjin Medical

University, Tianjin 300060, People’s Republic of China

123

J Cancer Res Clin Oncol (2012) 138:927–937

DOI 10.1007/s00432-012-1167-5

vascular endothelial growth factor (VEGF), b-fibroblast

growth factor, and platelet-derived growth factor. Studies

clarifying the relationship of TAFs and efficacy of anti-

angiogenesis drugs have yielded conflicting results

(Koukourakis et al. 2000). The controversy may be because

the effects of TAFs are usually antagonized by endogenous

angiogenesis inhibitors and are easily degraded in serum.

Thus, it is challenging to indicate the acceleration or

deceleration of angiogenesis based only on TAFs levels.

Ideal and practical predictive markers for the efficacy of

anti-angiogenesis agents remain to be developed.

Circulating endothelial cells (CECs) are usually per-

ceived as markers that indicate the formation of new

micrangium when small vessels are injured. The CEC levels

of patients with carcinoma are significantly higher than those

in healthy volunteers, suggesting that CECs are involved in

angiogenesis induced by malignancies that provide tumor

vasculature. CECs comprise at least two groups, namely the

endothelial progenitor cells (EPCs) mobilized from the

marrow by TAFs and the mature CECs derived and dif-

ferentiated from EPC (Beaudry et al. 2005; Furstenberger

et al. 2006; Zhang et al. 2005) or shed from the wall of the

micrangium (Beerepoot et al. 2004). Early EPCs express

CD34 ? CD133 ? VEGFR-2 ? , and late EPCs express

CD133-VEGFR-2 ? CD105 ? CD62E ? CD31 ? CD146 ?

CD144 ? VWF ? . CD133 gradually decreases, while

CD62E, CD31, CD105, and CD146 emerge with cell dif-

ferentiation. The differentiated cells, which uptake the

acetylated low-density lipoprotein, in conjunction with ulex

europaeus agglutinin 1, can form a vascular structure (Duda

et al. 2006). In addition, TAFs can also activate endothelial

cells of micrangium around the tumor to move into circu-

lation and migrate into tumors to form neo-vasculature

(Lastres et al. 1996). Therefore, only late EPCs or mature

activated CECs (aCECs) can exert vascular formation

(Furstenberger et al. 2006). aCECs are positively correlated

to VEGF in serum (Beerepoot et al. 2004; Mancuso et al.

2001) and descend to normal range after resection of tumor

or chemotherapy (Mancuso et al. 2001). Thus, aCECs could

be considered potential ideal indicators of anti-angiogenic

therapeutic efficacy.

However, there has been a controversy on whether the

CECs level ascends or descends after effective anti-angio-

genic therapy, (Beaudry et al. 2005; Kawaishi et al. 2009)

and whether there is a correlation between CECs variation

and efficacy. Beaudry et al. (2005) reported that the CECs

level was remarkably elevated post-therapy along with

reduction of microvessel density (MVD) in the tumor and

shrinkage of tumor volume. His analysis suggested that this

condition resulted from an increase in the mature cells and

sloughing of endothelial cells from the microvessel in the

tumor. In contrast, a decrease in the aCECs was observed in

NSCLC cases after an effective combined therapy of

chemotherapy and Rh-endostatin by Wang et al. (2008).

Kawaishi et al. (2009) reported a similar result with carbo-

platin and paclitaxel treatment. Li et al. (2008) exhibited the

elevation of both CECs and its apoptotic subgroup after

docetaxel and thalidomide treatment.

Given that more than 50% of non-small cell lung can-

cers (NSCLC) are diagnosed at late stages (Bulzebruck

et al. 1992), platinum-based chemotherapy is considered as

a standard regimen. However, the efficacy of this treatment

is impossible to enhance through increasing its dosage

because of intolerable toxicity (Non-small Cell Lung

Cancer Collaborative Group 1995; Grilli et al. 1993).

Fortunately, anti-angiogenic agents have been proved to

increase tumor response to chemotherapy (Ramalingam

et al. 2008). Endostatin can bind to the VEGF receptor

(KDR/Flk-1) to prevent VEGF from entering endothelial

cells, and thereby induce their apoptosis. Additionally,

endostatin can restrain CEC migration and neo-vasculature

formation (Dhanabal et al. 1999; Hanai et al. 2002; Lee

et al. 2002). By the end of 1999, endostatin was finally

refolded successfully by adding nine amino acids to its

N-terminus to achieve stability. The Chinese State Food

and Drug Administration approved the application of the

endostatin clinical trial. After Phase I, II, and III trials, in

which synergistic efficacy with NP regimen was reported,

the drug was approved and marketed under the brand name

Endostar (human recombinated endostatin, Rh-endostatin).

However, similar to other anti-angiogenic agents, it is

difficult to predict the efficacy of Endostar during its early

administration. To reverify its efficacy and determine ideal

predictive markers for its efficacy, we participated in the

prospective Phase IV trial organized by the Tumor Hos-

pital of Chinese Academy of Medical Science. Regimens

included in the trial were single chemotherapy and com-

bination of chemotherapy and Rh-endostatin in order to

compare their efficacy. To elucidate potential connections

between aCECs and efficacy, we measured aCEC levels

during therapy. There has been an ambiguity on whether

CEC levels would become elevated or reduced after vari-

ous protocols (Beaudry et al. 2005; Li et al. 2008; Patterson

et al. 2006; Shaked et al. 2008). Thus, we performed two

administrative sequences as Rh-endostatin followed by NP

and concomitant NP and Rh-endostatin, in order to illus-

trate the variation in CEC levels in different sequences of

administration and explain the validity of this variation.

Materials and methods

Patients

The study was conducted at the Cancer Institute and

Hospital of Tianjin Medical University from March 2007

928 J Cancer Res Clin Oncol (2012) 138:927–937

123

to August 2009. All 72 patients were histologically or

cytologically documented as advanced NSCLC. Each

patient met the following criteria: (1) age range of

18–75 years old; (2) Stage III, IV, or recurrent NSCLC with

an ECOG performance status of 0–2; (3) naı̈ve endostatin

therapy; (4) naı̈ve, or previous chemotherapy allowed if

completed C4 weeks before enrollment; (5) at least one

lesion with measurable double diameters C20 mm identi-

fied by computed tomography (CT), or magnetic resonance

imaging (MRI), or C10 mm by helical CT scan; (6) no pre-

existing cardiovascular conditions, such as symptomatic

congestive heart failure, unstable angina pectoris, or cardiac

arrhythmia; (7) no history of gross hemoptysis; (8) no

concomitant diseases including ischemic heart diseases,

systemic vasculitis, pulmonary hypertension, or serious

complications including infectious disease or diabetes; (9)

no known central nervous system metastases as determined

by CT or MRI within 4 weeks before enrollment; (10) no

contraindication of chemotherapy, that is, WBC C 4.0 9

109/L, PLT C 80 9 109/L, Hb C 90 g/L; Cr B 2.0 9 UNL;

BIL B 2.0 9 UNL, ALT/AST B 5.0 9 UNL; and (11)

awareness and signing of information consent. Ethical

Committee approval number was E2007012.

Therapy Schedule

Patients were randomized into the combined or chemo-

therapy arms by central distribution table. In the chemo-

therapy group, regimen was designated based on the

National Comprehensive Cancer Network (NCCN) Clini-

cal Practice Guidelines in Oncology (2007) as NP (vino-

relbine 25 mg/m2 d1, 8; cisplatin 75 mg/m2/d2–4). In the

combined treatment group, 38 cases were matched into

two-paired arms undergoing regimens as NP ? Rh-endo-

statin (vinorelbine 25 mg/m2 d1,8; cisplatin 75 mg/m2/

d2–4; Rh-endostatin 7.5 mg/m2 d1–14) and Rh-endo-

statin ? NP (Rh-endostatin 7.5 mg/m2 d1–14, vinorelbine

25 mg/m2 d8, 15; cisplatin 75 mg/m2/d9–11), respectively.

The treatment in both groups was performed every 3–4

weeks until the patients met the criteria for progressive

disease (RECIST criteria), experienced unacceptable

toxicity, or completed six therapeutic cycles. Patients were

stratified based on sex, age, tumor pathological type, dis-

ease stage, previous therapy, and Eastern Cooperative

Oncology Group (ECOG) performance (Table 1).

Blood collection

Blood samples were obtained before treatment and 3 days

after completion of each cycle. In addition, blood samples

were obtained on the 8th day of every cycle in the

Rh-endostatin ? NP group, that is, 1 week after a single

Rh-endostatin administration. All blood samples were anti-

coagulated with EDTA and stored at 4�C before use.

Assay for aCECs

Flow cytometry (FCM) was used to identify aCECs

(CD45-CD146?CD105?). All antibodies were purchased

from Beckman Coulter (USA), except CD105, which was

from Chemicon (USA). Whole anti-coagulated peripheral

blood (100 lL) was added in the isotype control tube and

incubated for 30 min in the dark with 10 lL of fluorescein

isothiocyanate (FITC), phycoerythrin (PE), and PE-Cy5

IgG1 isotype control antibodies from mice. The same pro-

cedure was performed in the test tube incubated for 30 min

in the dark with 10 lL of CD45-PE-Cy5, CD146-PE, and

CD105-FITC antibodies, respectively. After incubation, red

blood cells were lysed with lysing solution A (purchased

from Beckman Coulter, USA) for 30 s and vortexed gently,

then with lysing solution B for 10 s and vortexed gently.

Afterward, cells were washed three times in phosphate-

buffered saline (PBS) by centrifugation. Using FS/SS gating

strategy, acquisition was performed by FCM (Beckman

Coulter, EPICS-XL) equipped with a 488-nm argon-ion

laser. A minimum of 100000 events were collected for each

sample.

Data from each sample were analyzed by Software-

System II (Beckman Coulter). aCECs (CD45-CD105?

CD146?) were identified using a sequential gating strategy.

Evaluation of efficacy

CT examinations were performed pre- and post-therapeu-

tically every two cycles or at any time during therapy, if

necessary. Efficacy was evaluated by CT scan at least

every two cycles according to the RECIST complete

response (CR), partial response (PR), stable disease (SD),

and progressive disease (PD). Time to progression (TTP),

time to failure (TTF), and progression-free survival (PFS)

were also documented. TTP is defined as the moment from

randomization of patients to tumor progression, whereas

TTF is the moment from randomization to treatment

termination in any situation, such as withdrawn consent or

violation of protocol. PFS is considered as the time from

randomization to tumor progression or death from any

cause.

Statistical analysis

All analyses were performed using statistical software

SPSS13.0. Results were expressed as median, P25-P75, and

mean ± SD for continuous variables as well as counts

and proportions for categorical variables. Response rates to

treatment between groups were compared using v2-test.

J Cancer Res Clin Oncol (2012) 138:927–937 929

123

Normalized aCEC counts (lnaCECs) between groups were

compared using F or t-test. Spearman’s correlation analysis

was performed to investigate the correlation between

aCECs count and TTP. The correlations between aCECs

baseline, aCECs count difference (DaCECs, difference of

aCECs by post-therapeutic amount minus post-therapeutic

amount), and efficacy were analyzed using the Kaplan–

Meier method. The log-rank test was used to assess the

survival difference between stratifications. Differences

were considered statistically significant at p \ 0.05 on two-

tailed test.

Results

A total of 72 patients were enrolled in the present study.

Characteristics of population are summarized in Table 1.

The median age was 55 years old (range 35–74) in NP ?

Rh-endostatin, 57.7 years (range 35–71) in Rh-endo-

statin ? NP, and 58.4 years (range 38–75) in NP. No sta-

tistical difference was found between the characteristics of

each group (all p [ 0.05).

Response to therapy

Out of 72 patients, 13 PD, 53 SD, 6 PR, and 0 CR were

found after 2 therapeutic cycles and confirmed 1 month

later with details shown in Table 2. No significant differ-

ence was found between groups (all p [ 0.05).

Time to progression

Patients were followed up in 26.5 months (range 5–31)

until January 2010. No significant difference in TTP was

found between combined and single chemotherapy groups

(p = 0.19). Among 19 patients treated by Rh-endo-

statin ? NP, 6 cases had TTF (5.6 months, range 4–7), and

13 had TTP (6.0 months, range 1–12) documented. 18 of 19

patients treated by NP ? Rh-endostatin had complete fol-

low-ups, and 7 had TTF (5.1 months, range 3–10 months),

and 11 had TTP (8.5 months, range 1–19 months) recorded.

Table 1 Baseline

characteristics of the patients

NP vnorelbine 25 mg/m2 d1, 8;

cisplatin 75 mg/m2/d2–4;

Rh-endostatin ? NPvinorelbine 25 mg/m2 d8, 15;

cisplatin 75 mg/m2/d9–11;

Rh-endostatin 7.5 mg/m2

d1–14; NP ? Rh-endostatinvinorelbine 25 mg/m2 d1, 8;

cisplatin 75 mg/m2/d2–4; and

Rh-endostatin 7.5 mg/m2 d1–14

Characteristics Combined treatment group (n = 38) Chemotherapy

group (n = 34)

NP ? Rh-endostatin Rh-endostatin ? NP NP

Number of cases (%) 19 (26.4) 19 (26.4) 34 (47.2)

Sex

Male 11 (57.9) 9 (47.4) 18 (52.9)

Female 8 (42.1) 10 (52.6) 16 (47.1)

Median age 55 57.7 58.4

Range 35–74 35–71 38–75

Histology

Adenocarcinoma 10 (52.6) 12 (63.2) 18 (52.9)

Squamous cell carcinoma 7 (36.8) 4 (21.0) 7 (20.6)

Others 2 (10.5) 3 (15.8) 9 (26.5)

TNM stage

IIIA 2 (10.5) 3 (15.8) 5 (14.7)

IIIB 6 (31.6) 4 (21.0) 8 (23.5)

IV 11 (57.9) 12 (63.2) 21 (61.8)

Prior therapy

No 11 (57.9) 12 (63.2) 24 (70.6)

Yes 8 (42.1) 7 (36.8) 10 (29.4)

ECOG

1 18 (94.7) 16 (84.2) 30 (88.2)

2 1 (5.3) 3 (15.8) 4 (11.8)

Table 2 Comparison of efficacy between chemotherapy and com-

bined treatment groups

Response

evaluation

Combined treatment group

number (%)

Chemotherapy

group number (%)

NP ? Rh-

endostatin

Rh-

endostatin ? NP

NP

CR 0 0 0

PR 1 (5.3) 4 (21.1) 1 (2.9)

SD 14 (73.7) 13 (68.4) 26 (76.5)

PD 4 (21.1) 2 (10.5) 7 (20.6)

Total 19 19 34

930 J Cancer Res Clin Oncol (2012) 138:927–937

123

TTP was not significantly different between patients

treated by Rh-endostatin ? NP and NP ? Rh-endostatin

(p = 0.23). Among 34 patients who underwent NP therapy,

2 had TTF (5.0 months, range 4–6 months), and 32 had

TTP (5.3 months, range 1–17 months) recorded. These

findings are similar to those treated by Rh-endostatin ? NP

(p = 0.53), but significantly lower than those treated by

NP ? Rh-endostatin (p = 0.04) (Table 3).

Given that no patient died from reasons other than

cancer deterioration in the present study, TTP and PFS

were consistent. PFS was compared among patients treated

by NP, NP ? Rh-endostatin, and Rh-endostatin ? NP. HR

for the three groups were 0.722 (0.513–1.017) for NP

versus NP ? Rh-endostatin (p = 0.044), 0.757 (0.396–

1.445) for NP versus Rh-endostatin ? NP (p = 0.36), and

0.399 (0.149–1.069) for Rh-endostatin ? NP versus NP ?

Rh-endostatin (p = 0.049) (Fig. 1).

Data of aCECs were firstly normalized logarithmically

since they represented non-normal distribution in order to F

or t-test can be introduced.

aCEC baseline levels

aCEC levels before treatment in the chemotherapy and

combined treatment group were (67, 25–335/105), lnaCECs

(4.4 ± 1.7) and (75, 35–390/105), lnaCECs (4.7 ± 1.5)

cells, respectively. No significant difference was found

between them. No correlation was found between aCEC

levels and sex, age, pathological type, previous treatment,

or clinical stage, or between aCECs and PFS (p = 0.84).

Variation in aCEC levels during chemotherapy

Mean therapeutic cycles of chemotherapy was 2.85 cycles

with 1 cycle in 3 cases, 2 in 13 cases, 3 in 6 cases, 4 in 10

cases, and 5 in 2 cases. Post-therapeutic aCEC numbers

were (105, 65–404/105), lnaCECs (4.5 ± 1.8); (175,

125–470/105), lnaCECs (5.0 ± 1.5); (254, 205–714/105),

lnaCECs (5.2 ± 1.9); and (110, 150–480/105), lnaCECs

(4.8 ± 1.9) cells after the 1st, 2nd, 3rd, and 4th cycles,

respectively. Post-therapeutic aCECs were significantly

higher after the 3rd cycle compared to baseline (p = 0.04).

Variation in aCEC levels during combined treatment

Mean therapeutic cycle numbers in Rh-endostatin ? NP

therapy was 3.32 with 1 cycle in 1 case, 2 in 3 cases, 3 in 4

cases, and 4 in 11 cases. Post-therapeutic aCEC numbers

were (121.5, 50.5–425.5/105), lnaCECs (5.0 ± 1.7); (46.5,

9.3–521.8/105), lnaCECs (4.6 ± 1.2); (181.5, 78.5–727.5/

105), lnaCECs (5.2 ± 1.5); (126.5, 61.5–256.3/105),

lnaCECs (5.0 ± 1.3); (322, 105.5–467/105), lnaCECs

(5.5 ± 0.9); (100.5, 44–131.3/105), lnaCECs (4.6 ± 1.3);,

(465, 117–851/105), lnaCECs (5.9 ± 1.2); and (180,

68–744/105), lnaCECs (5.3 ± 1.3) cells on 8th day, after

1st cycle, 29th day, after 2nd cycle, 50th day, after 3rd

cycle, 71st day, and after 4th cycle, respectively. aCEC

levels were significantly higher on the 8th day, 29th day,

after 2nd cycle, 50th day, 71st day, and after 4th cycle

compared to baseline. Among 54 blood samples collected

after single Rh-endostatin therapy, aCECs of 39 samples

(72.2%) were higher; whereas those of 15 samples (27.8%)

were lower than the baseline.

Mean therapeutic cycle numbers in NP ? Rh-endostatin

were 2.79 with 1 cycle in 2 cases, 2 in 8 cases, 3 in 2 cases,

4 in 6 cases, and 5 in 1 case. Post-therapeutic aCEC

numbers were (187, 67–360.8/105), lnaCECs (4.8 ± 1.6);

(144, 28–332.5/105), lnaCECs (4.8 ± 1.8); (157, 45–325/

105), lnaCECs (4.5 ± 1.9); and (67, 35–320/105), lnaCECs

(4.2 ± 1.7) cells after the 1st, 2nd, 3rd, and 4th cycles,

respectively. Post-therapeutic aCECs were not signifi-

cantly higher than the baseline. Details are illustrated in

Fig. 2.

Table 3 Time to progression of groups

Time Combined treatment group (n) Chemotherapy

group (n)

NP ? Rh-

endostatin

Rh-endostatin

? NP

NP

TTP (month) 8.5* (11) 6.0 (13) 5.3* (32)

TTF (month) 5.1 (7) 5.6 (6) 5.0 (2)

* NP ? Rh-endostatin versus NP (p \ 0.05)

Fig. 1 Progression-free survival probability of combined and che-

motherapy groups. NP ? Rh-endostatin exhibited the greatest pro-

gression of free survival probability

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123

Correlations between aCEC levels and efficacy

No significant difference in the aCECs was found between

the baselines in the cases of non-PD and PD in the che-

motherapy group (55, 25–290/105), lnaCECs (4.3 ± 1.4)

versus (75, 40–352/105), lnaCECs (4.2 ± 1.6) cells and

combined treatment group (65, 24–285/105), lnaCECs

(4.3 ± 1.8) versus (59, 25–110/105), lnaCECs (3.8 ± 1.7)

cells.

In patients of non-PD, compared to baseline, aCEC

levels increased after chemotherapy and combined therapy,

but the difference is not significant. However, among

patients of PD, aCEC levels increased significantly after

chemotherapy (p = 0.04) and combined therapy (p = 0.02)

compared to baseline (Fig. 3a).

In combined group, no significant difference was found

between aCECs at baseline of non-PD and PD cases in the

19 patients who underwent Rh-endostatin ? NP therapy

(80, 55–245/105), lnaCECs (4.5 ± 1.8) versus (65, 45–320/

105), lnaCECs (3.9 ± 1.4) cells and in the 19 patients who

underwent NP ? Rh-endostatin therapy (65, 40–190/105),

lnaCECs (4.2 ± 1.3) versus (75, 50–214/105), lnaCECs

(3.8 ± 1.5) cells. However, aCEC levels significantly

increased after therapy in PD patients compared with the

baseline in both Rh-endostatin ? NP (p = 0.044) and NP

?Rh-endostatin groups (p = 0.049) (Fig. 3b).

The amounts of DaCECs among the groups were com-

pared, and significant discrepancies were found between

those of non-PD and PD cases in combined group (40,

15–156/105), lnDaCECs (3.1 ± 1.5) versus (254, 85–456/

105), lnDaCECs (5.4 ± 1.4), p = 0.03, and (75, 20–114/

105), lnDaCECs (3.2 ± 1.4) versus (145, 70–345/105),

lnDaCECs (5.2 ± 1.3) in the chemotherapy group, p =

0.04 (Fig. 4).

Fluctuation of aCEC levels during therapy in cases

with different clinical outcomes

In the chemotherapy group, the mean therapeutic cycle

number of 7 PD cases was 2 with all 7 cases. The aCEC

levels after the 2nd cycle were (170, 150–570/105),

lnaCECs (5.3 ± 1.7) cells significantly increased from

the baseline (75, 40–352/105), lnaCECs (4.2 ± 1.6) cells

(p = 0.04). The mean therapeutic cycle number of 27 non-

PD cases were 3.22 with 1 cycle in 3 cases, 2 cycles in 5, 3

cycles in 6, 4 cycles in 10, 5 cycles in 2, and 6 cycles in 1

case. The aCEC levels were (55, 25–290/105), lnaCECs

(4.3 ± 1.4) cells on baseline and (65, 40–158/105),

Fig. 2 Fluctuation of lnaCEC

levels during NP,

NP ? Rh-endostatin, and

Rh-endostatin ? NP treatments.

*p \ 0.05 and **p \ 0.01

versus value of respective

baseline

Fig. 3 a LnaCEC levels of non-PD and PD cases at pre- and post-

therapies in the chemotherapy and combined treatment groups.

Significant increase was found in PD patients after therapy.*p \ 0.05.

b LnaCEC levels of non-PD and PD cases at pre- and post-therapies

in combined treatment groups. Significant increase was found in PD

patients after therapy *p \ 0.05

932 J Cancer Res Clin Oncol (2012) 138:927–937

123

lnaCECs (4.5 ± 1.3); (60, 30–254/105), lnaCECs (4.2 ±

1.2); (145, 45–248/105), lnaCECs (5.0 ± 1.7); and (45,

10–300/105), lnaCECs (4.6 ± 1.5) cells after the 1st, 2nd,

3rd, and 4th cycles, respectively. aCECs after the 3rd cycle

significantly increased compared to baseline (p = 0.03)

(Fig. 5).

In the combined treatment group, the mean therapeutic

cycle number of 6 PD cases was 2 with 1 cycle in 2 cases, 2

cycles in 3, and 4 cycles in 1. Amounts of aCECs were (59,

25–100/105), lnaCECs (3.8 ± 1.7) cells on baseline, (155,

55–300/105), lnaCECs (4.6 ± 1.6) cells after the 1st cycle,

and (240, 70–680/105), lnaCECs (5.2 ± 1.8) cells after the

2nd cycle, all of which were higher than the baseline

(p = 0.03). The mean therapeutic cycle number of 32 non-

PD patients was 3.31 with 1 cycle in 1 case, 2 cycles in 8, 3

cycles in 6, 4 cycles in 16, and 5 cycles in 1. Amounts of

aCECs were (65, 24–285/105), lnaCECs (4.3 ± 1.8) cells

on baseline and (70, 25–155/105), lnaCECs (4.4 ± 1.2);

(55, 40–251/105), lnaCECs (4.1 ± 1.9); (60, 34–325/105),

lnaCECs (4.0 ± 1.7); and (75, 15–310/105), lnaCECs

(4.5 ± 1.7) cells after the 1st, 2nd, 3rd, and 4th cycles,

respectively. There was no significant difference between

baseline and post-therapeutic aCECs (Fig. 5).

TTP was not significantly correlated with PS, patho-

logical type, stage, or with DaCEC for TTP \ 10 months

(p = 0.156, r = -0.25) (Fig. 6a) in 39 cases. Reverse

correlation was found between TTP and DaCECs when

TTP C 10 months (p = 0.003, r = -0.647) (Fig. 6b). In

17 cases, 4 (23.5%) underwent NP, 6 (35.29%) underwent

Rh-endostatin ? NP, and 7 (41.18%) underwent NP ?

Rh-endostatin. In 8 cases with TTP C 12 month, 2 patients

(25.0%) underwent NP, 1 (12.5%) underwent Rh-endo-

statin ? NP, and 5 (62.5%) underwent NP ? Rh-endo-

statin. In TTP C 10 months, the proportion of cases in the

combined treatment group was much higher than in the NP

group, but no statistical difference was found (p [ 0.05).

Furthermore, in TTP C 12 months, the proportion of cases

in the NP ? Rh-endostatin group was much higher than in

the NP and Rh-endostatin ? NP group, but no statistical

differences were found between them (p [ 0.05).

Discussion

Chemotherapy remains as the main therapy for advanced

NSCLC, despite response rates are only 30–40% and sur-

vival durations are 7–9 months (Baggstrom et al. 2007;

Belani et al. 2005; Kubota et al. 2004; Le Chevalier et al.

1994; Ohe et al. 2007; Schiller et al. 2002). Anti-angio-

genic agents remarkably extend overall survival beyond

1 year when combined with chemotherapy (Ramalingam

et al. 2008). However, it is a challenge to determine the

response to anti-angiogenic agents in a timely manner as

anti-angiogenics are generally cytostatic rather than cyto-

reductive. To evaluate the potential of aCECs as an early

predictive marker of patients’ response to anti-angiogenic

agents, and to determine aCEC levels during combined

anti-angiogenic and chemotherapeutic therapy, we com-

pared the efficacy of combined therapy versus single

chemotherapy, investigated aCEC levels during treatments,

and analyzed the relationship between aCECs and efficacy.

According to the rationale of anti-angiogenesis, the

preference of administration schedule may result from the

effects of which is the normalization and regression of

vasculature. Normalization enables a smooth blood flow

and allows additional anti-cancerous drugs to perfuse

easily in the tumor (Li et al. 2008). However, the nor-

malization window is very short, and the following

induction of apoptosis of endothelial cells by concurrent

chemotherapeutic drugs leads to vasculature regression and

Fig. 4 Amounts of lnDaCEC in the two groups, significant discrep-

ancies were found between those of non-PD and PD cases in both

groups (*p \ 0.05)Fig. 5 Fluctuation of the lnaCEC levels in PD and non-PD cases in

the two groups during therapy. *p = 0.03 after 1st and 2nd cycle

versus baseline of PD cases in combined treatment group and

p = 0.04 after 2nd cycle versus baseline of PD cases in chemotherapy

group. *p = 0.03 after third cycle versus baseline in chemotherapy

group non-PD cases

J Cancer Res Clin Oncol (2012) 138:927–937 933

123

inadequacy of blood in the tumor, which reduces drug

uptake. Our results suggest that administrating anti-angio-

genics prior to chemotherapy could not enhance the effi-

cacy of chemotherapy, which is supported by the facts

that TTP of NP ? Rh-endostatin was longer than those of

NP (8.5 vs. 5.3 months, p = 0.04) and Rh-endostatin ?

NP (8.5 vs. 6.0 months, p = 0.23). For example, out of 8

patients with TTP longer than 12 months, 62.5% (5

patients) underwent concurrent therapy, which was much

higher than those who underwent single NP (12.5%) and

sequential administration of Rh-endostatin ? NP (25.0%)

(p [ 0.05). It suggests that although Rh-endostatin could

lead to normalization, it could not suppress tumor growth,

while concurrent chemotherapeutic agents enhanced drug

uptake to reduce tumor size and secretion of TAFs.

Otherwise, it could also be ascribed to the fact that proper

‘normalization window’ could not be defined so exact that

chemotherapy followed Rh-endostatin could be adminis-

trated outside the window.

Our study provided insights on the usefulness of CEC

as a surrogate marker for anti-angiogenic agents. We

defined aCECs as CD45-CD146 ? CD105 ? CECs. CECs

are generally identified as CD45-CD146 ? Flk1 ? cells

(Beerepoot et al. 2004; Li et al. 2008). Beaudry et al.

(2005) employed CD117 to identify EPC, and Kawaishi

et al. (2009) chose CD105 to distinguish activated func-

tional CECs from total cells. Our previous study showed

that EPC levels were usually undetectable for its scarceness

in circulation (data not shown); instead, the correlation

between variation in CD105 ? CECs and efficacy was

discovered. Given that mature endothelial cells (negative

for the haematopoietic marker CD45) are viable and con-

tinues to exhibit proliferative capacity despite their termi-

nal differentiation, CD45-CD146 ? CD105 ? CECs was

developed as a reliable marker to identify aCECs in the

present study (Mancuso et al. 2001).

Our results helped to resolve the controversy of CEC

variations during anti-angiogenic and/or chemotherapeutic

therapy, and understand the corresponding mechanism.

Tong et al. revealed that dilated and tortuous vessels

around the tumor were constricted and stretched after

DC101 (VEGFR-2 antibody) treatment. In our study,

Rh-endostatin could enhance the expression of metallo-

proteinase inhibitors to suppress matrix metalloproteinase

(Sun et al. 2007). Consequently, the degradation of basal

membrane of vessels, as well as the seepage of fluid from

vessel and IFP, was reduced, leading to a series of events

reported by Tong et al. (2004). We perceived that the

diminution of the tumor vasculature area could induce the

shedding of endothelial cells from the blood vessel walls to

augment CEC population. In addition, the increase in the

CECs could be due to the mobilization of EPCs from

the bone marrow induced by increasing TAFs, which

resulted from insufficient treatment by single anti-angio-

genic agents. In contrast, subsequent or concomitant strong

chemotherapy abated CEC levels through apoptosis. It

suggests that aCECs exhibit general reducing tendency

with fluctuation when therapy is effective, in which inter-

mittent elevation means diminution of tumor vasculature

by normalization, while final reduction reflects apoptosis of

CECs, decrease of TAFs, and regression of vasculature in

tumor.

To test the hypothesis above, we investigated aCEC

levels after 7 days of single Rh-endostatin administration

in each therapeutic cycle of Rh-endostatin ? NP. Similar

to the results of Beaudry et al. (2005), significant elevation

of CECs from baseline was found at 39 of 54 collection

time points. Beaudry et al. (2005) reported a decrease of

MVD in xenograft tumors in accordance with the elevation

of CECs to indicate normalization of vessels. Due to eth-

ical issues, we examined results of perfusion CT imaging

that showed a reduction in the blood flow and area of

excess permeable micrangium (data not shown), suggesting

unenhanced angiogenesis during single Rh-endostatin

treatment, consistent with results in the clinical Phase II

trials (Yang et al. 2005).

Our present results indicated DaCECs as a predictive

marker of response in both single chemotherapy and

combined therapy. No significant difference in the aCECs

level between the pre- and post-therapies in non-PD cases

was found. However, there was significant elevation in the

PD cases in the two groups with discrepancies in DaCECs

Fig. 6 Scatter plot of the

differences in the amounts of

DaCECs between those at pre-

and post-therapies and TTP.

No correlation was found

between the amount of DaCECs

difference and TTP in patients

with TTP \ 10 months, and the

reverse correlation between

them was found in patients with

TTP C 10 months (p = 0.003,

r = -0.647)

934 J Cancer Res Clin Oncol (2012) 138:927–937

123

between non-PD and PD cases of both groups (p = 0.04

and 0.03). Although several peaks in the aCECs in

Rh-endostatin ? NP were discovered, aCECs in non-PD

cases still ultimately decreased so that the amount of aCECs

in non-PD cases of the entire combined therapy was kept at

a low level. The fluctuation of aCECs during treatment may

indicate the moving balance of vessel normalization and

CEC apoptosis. To clarify the variation in the subgroups of

CECs, the amount of apoptotic/dead and aCECs were

examined in our other study. Moreover, our results suggest

that imaging and clinical representation need to be taken

into consideration to determine further therapeutic regimen

considering temporary increase or decrease of CECs.

More PR/SD cases with longer PFS were reported to

have higher baseline CECs in the paclitaxel and carboplatin

therapy (Kawaishi et al. 2009), suggesting that anti-

angiogenic therapy may be effective in some patients with

more thriving angiogenesis prior to therapy. We did not

observe this phenomenon in the present study. Instead,

the reduction or elevation range, that is DaCECs, was

more correlated with longer TTP (p = 0.003, r = -0.647),

indicating this value could serve as a sensitive marker to

forecast long-term efficacy of combined anti-angiogenesis

and chemotherapy independent of administration sequence.

Our study showed no correlations between baseline

aCEC levels and pathological types, or clinical character-

istics, which may be due to the fact that aCECs could be

influenced by various factors related to angiogenesis, tumor

vasculature, and tumor localization (Kawaishi et al. 2009).

No correlation between TTP and pathological type of

NSCLC, PS, and stage was identified in the present study,

probably attributable to the limited case number.

In the present study, we found significantly higher ele-

vation in the aCECs in PD cases after combined therapy

than single chemotherapy, probably caused by the devia-

tion on the limited case number, or the acceleration of

malignancy growth due to the rebounce after insufficient

inhibition of angiogenesis.

In literature, elevation of CECs was found after single

taxane drug treatment (Li et al. 2008; Shaked et al. 2008;

Bijman et al. 2006). Our results exhibited a fluctuating

decreasing tendency of CECs after effective NP chemo-

therapy followed by Rh-endostatin, probably indicating

that this treatment is only strong enough for chemotherapy

of two drugs. With platinum, this treatment could probably

induce robust apoptosis of CECs, leading to its decrease,

which is consistent with the report in which CEC lev-

els significantly decrease after chemotherapy based on

anthracycline/taxane (Furstenberger et al. 2006).

In the present study, we investigated the correlations

between aCECs and the efficacy in different regimens and

clarified the controversy in previous reports about CEC

variation after anti-angiogenic therapies. Better synergistic

effects from concomitant chemotherapy and Rh-endostatin

treatment were achieved resulting in longer TTP. aCEC

levels fluctuated during effective therapies, but ultimately

descended and maintained at a low level. Our study sug-

gests that the trend of aCECs variations between post- and

pre-therapies (DaCECs) could be an ideal marker for the

efficacy of anti-angiogenesis combined with chemother-

apy, as well as for recurrence. Larger clinical trials are

needed to confirm these conclusions and to find the precise

window of vessel normalization.

Acknowledgments This work was supported by Grant from Tianjin

Science & Technology Project (No. 09ZCZDSF04400); CSCO Grant

(Y-X2011-001).

Conflict of interest We declare that we have no conflict of interest.

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