Elevated serum levels of vascular endothelial growth factor predict a poor prognosis of platinum-based chemotherapy in non-small cell lung cancer
OncoTargets and Therapy
elevated serum levels of vascular endothelial growth factor predict a poor prognosis of platinum-based chemotherapy in non-small cell
Jialan Zang 2
Yong hu 1 2
Xiaoyue Xu 1 2
Jie n i 1 2
Dali Yan 1 2
Jianzhong Wu 4
Jifeng Feng 1
0 Department of Oncology, The First h ospital of harbin city , harbin
1 Department of chemotherapy, n anjing Medical University affiliated cancer hospital , n anjing
2 The Fourth c linical school of n anjing Medical University
3 Department of Public health, southeast University , n anjing, People's republic of china
4 center of clinical l aboratory, n anjing Medical University affiliated cancer hospital
PowerdbyTCPDF(ww.tcpdf.org) *These authors contributed equally to this work Aim: This study was designed to investigate the predictive and prognostic values of serum vascular endothelial growth factor (VEGF) level in non-small cell lung cancer (NSCLC) patients treated with platinum-based chemotherapy. Methods: Patients' peripheral blood samples were collected prior to chemotherapy and after 1 week of the third cycle of combination chemotherapy. Serum VEGF levels were evaluated through Luminex multiplex technique. Between September 2011 and August 2015, a total of 135 consecutive advanced or recurrent histologically verified NSCLC patients were enrolled in the study. Moreover, all the patients received platinum-based combination chemotherapy as a first-line treatment. Results: No significant associations were found between pretreatment serum VEGF levels and clinical characteristics, such as sex (P=0.0975), age (P=0.2522), stage (P=0.1407), lymph node metastasis (P=0.6409), tumor location (P=0.3520), differentiated degree (P=0.5608), pathological (histological) type (P=0.4885), and response to treatment (P=0.9859). The VEGF load per platelet (VEGFPLT) levels were not correlated with sex, age, primary tumor site, and pathological type in NSCLC patients (all P.0.05). The median survival time of progressionfree survival (PFS) was 6.407 and 5.29 months in the low and high groups, respectively, when using 280 pg/mL VEGF level as the cutoff point (P=0.024). Conclusion: In conclusion, the serum VEGF levels were found to be a poor prognostic biomarker for the efficacy of platinum-based chemotherapy in terms of PFS, but it was not shown to be a suitable predictive marker for clinical response to platinum-based chemotherapy.
Video abstract; non-small cell lung cancer; VEGF; progression-free survival; platinum; chemo-
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Lung cancer is the leading cause of cancer-related death worldwide. Moreover,
non-small cell lung cancer (NSCLC) accounts for 80% of lung cancer.1 The majority
of the NSCLC patients are in advanced stage when diagnosed. Patients with a good
performance status (PS) in this stage can benefit from platinum-based chemotherapy,
particularly those without epidermal growth factor receptor mutation or anaplastic
lymphoma kinase translocation.2 However, NSCLC patients still show poor response to
chemotherapy because of the relatively short duration and rarity of complete remissions.
The median survival of advanced NSCLC patients treated with chemotherapy alone
is ~10 months.3 While selecting patients who may benefit from chemotherapeutic
modalities, identifying validated biomarkers associated
with chemotherapeutic response and prognosis of NSCLC
is essential. Moreover, stratifying NSCLC patients as
well as monitoring tumor progression and therapeutic
response may be useful. Being noninvasive and convenient,
blood-based markers have significant advantages over
Neoangiogenesis is critical in tumor growth and systemic
dissemination of cancer cells and may be related to the poor
survival in NSCLC patients. Vascular endothelial growth
factor (VEGF) is largely responsible for neoangiogenesis.4
VEGF signal pathways regulate endothelial cell migration,
proliferation, survival, and expression of downstream genes.5
Kondo et al first recognized the potential of VEGF as a
serum diagnostic marker for malignant diseases. They found
that the VEGF levels in the sera from cancer patients were
significantly higher than those without signs of cancer.6 Since
then, studies have focused on the predictive and prognostic
.vdoepw lsyeonu itmyppelsic.aTtihoensproefdciicrtciuvleatrionlgeVoEf
GciFrciunlNatSinCgLVCEaGndFoftohrerchcaenmcoer/ww lan therapy response was studied, and some results revealed
tsp rse that VEGF was not associated with chemotherapy response
trohm ropF in NSCLC patients.7,8 However, Lissoni et al reported that
fd the chemotherapy response was worse in patients of NSCLC
ade and colorectal carcinoma with higher levels of pretreatment
lnow VEGF.9 Similarly, investigations on the prognostic impact
yod of VEGF in chemotherapy revealed conflicting results. The
repa discrepancy between the studies might be explained by the
hT different methodologies used in assessing VEGF
concentradna tions and the different histological types, sample sizes, or
trsge treatment regimens used in the study populations. Therefore,
oaT improving detection techniques and classification criteria
cnO may help identify the predictive and prognostic values.
Serum marker detection was done traditionally using the
enzyme-linked immunosorbent assay (ELISA), which requires
a large sample volume and is expensive. Recently, the
FLEXMAP 3D™ system, which is based on lipid assay
technology, has been widely used for its multiplexed function
and enhanced sensitivity.10
We previously measured serum VEGF levels in NSCLC
patients using the Luminex® xMAP® and found that serum
VEGF levels were closely correlated with NSCLC
progression and metastasis.11 However, we did not explore the
relationship between VEGF concentrations and treatment
response. Following the abovementioned investigations,
we hypothesized that serum VEGF levels may be an
independent predictive and prognostic markers for NSCLC
patients. In this study, we estimated the predictive and
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prognostic values of serum VEGF level in platinum-based
Materials and methods
The study was approved by the Institutional Review Board
and the Research and Ethical Committee of Nanjing
Medical University Affiliated Cancer Hospital. Written informed
consents were obtained from all participants.
Between September 2011 and August 2015, a total of 135
consecutive advanced or recurrent NSCLC patients with
histologically verified were enrolled in the study. The 135 NSCLC
patients included 91 males and 44 females, with a mean age
of 58.5 years (range, 28–78 years). To be eligible for first-line
combination chemotherapy, the patients were required to meet
the following criteria: cytologically or histologically proven
lung cancer; measureable unresectable stage III–IV disease
or recurrence after surgery; an Eastern Cooperative Oncology
Group PS of 0 or 1; normal hepatic, renal and hematological
functions; and no concomitant or serious comorbidities. Stage
classification was based on the Union for International Cancer
Control–Tumor Node Metastasis classification. Patients with
other malignant neoplasms were ruled out. Written informed
consent was obtained from each patient prior to the start of the
study. Patient follow-ups were obtained through the hospital
records and direct patient contact.
All of the patients who received platinum-based
combination chemotherapy as a first-line treatment were enrolled
from September 2011 to August 2015. The chemotherapy
regimens were as follows: pemetrexed 0.5 g/m2 iv drip d1;
cisplatin 75 mg/m2 iv drip d1; q21d (PC regimen),
gemcitabine 1.25 g/m2 iv drip d1, 8; cisplatin 75 mg/m2 iv drip d1,
q21d (GP regimen), and docetaxel 75 mg/m2 iv drip d1;
cisplatin 75 mg/m2 iv drip d1, q21d (TP regimen), and the
GP regimen was continued for 4 cycles. PC or TP regimen
was administrated for 6 cycles. Pemetrexed or docetaxel was
maintained until disease progressed.
collection and preservation of blood
Patients’ peripheral blood samples were collected before
chemotherapy and after 1 week of the third cycle of conventional
chemotherapy. A total of 3 mL venous blood was extracted
from the fasting patients. The blood samples were immediately
collected in the endotoxin- and pyrogen-free test tubes. The
whole blood specimens were then shaken three times and left to
coagulate for 30 min at room temperature. The blood samples
were centrifuged at 1,000× g for 10 min at 4°C, and the serum
was transferred to Eppendorf tubes and stored at −80°C until
analysis. The sera of the participants were obtained following
the approval from the Ethics Committee of Nanjing Medical
University Affiliated Cancer Hospital (Nanjing, People’s
Republic of China). Written informed consent was obtained
from each patient.
Detection of serum VegF
The assay used a Luminex multiplex technique. The
FLEXMAP 3D system was supplied by Luminex Corporation
(Austin, TX, USA). Serum VEGF levels were examined
through human cytokine/chemokine panels (cat no
MPXHCYTO-60K). Assays were performed according to the
manufacturer’s instructions. All reagents were acclimatized to
room temperature before the main immunoassay procedure.
The placement of standards for VEGF was 0 (background),
3.2, 16, 80, 400, 2,000, and 10,000 pg/mL, control and test
specimens were then added to the plate (25 µL per well).
The specimens were shaken at room temperature for 16 h,
avoiding light. After washing twice, a 25-µL detection
antibody was added and the plates were shaken for 1 h at room
temperature. After incubating with agitation on a plate shaker
for 1 h at room temperature (20°C–25°C), 2-µL
streptavidin–phycoerythrin was added to each well. The plate was
then further incubated for 30 min and washed twice with
200 µL/well wash buffer, and 150-µL sheath fluid was added
to each well. The plate was run on the FLEXMAP 3D system,
and the median fluorescence intensity results were saved
and analyzed using a weighted five-parameter logistic method
to calculate the sample concentrations. The concentration of
VEGF load per platelet (VEGFPLT) (pg/mL) was calculated
as serum VEGF (pg/mL)/platelet count ×106, correcting for
the variations in platelet counts between patients.12
Statistical analysis was performed using Statistical Package
for the Social Sciences (SPSS) 18.0. Numeric values were
expressed as mean ± standard deviation (SD). The independent
sample t-test/Mann–Whitney U and chi-square tests/Fisher’s
exact test were used to compare values among different groups
for continuous variables and categorical variables,
respectively. Cox regression analysis was performed to assess
potential prognostic factors. We utilized Pearson’s/Spearman’s
correlation coefficient to evaluate the correlations between
VEGF concentration and clinical parameters. In all statistical
analyses, a P-value ,0.05 was considered significant.
general features of the patients
This study was conducted in 135 inoperable NSCLC patients.
The baseline characteristics of all the patients are summarized
in Table 1. Among the 135 patients, 91 were males and
44 were females. A total of 28 patients were pathologically
diagnosed with squamous cell carcinoma, 105 patients
with adenocarcinoma, and 2 were missing. Moreover, 61
patients (45.19%) were aged .60 years, 74 patients (54.81%)
were aged #60 years, and the median age was 59.5 years
(range, 28–78). During initial blood sample collection,
35.56% of the patients had stage III (48/135) and 64.44% had
stage IV cancers (87/135). On the basis of the tumor
location, 25.19% of the patients (34/135) had central pulmonary
tumors, 69.63% (94/135) exhibited peripheral pulmonary
tumors, and 5.18% were missing (7/135). Furthermore,
37 patients (27.41%) were histopathologically confirmed
to have poorly differentiated carcinomas, 10 (7.41%) had
moderately differentiated carcinomas, 2 (1.48%) had
welldifferentiated carcinomas, and 86 (63.7%) were missing.
A total of 35.56% (48/135) cases had no distant metastases
and 64.44% (87/135) cases had distant metastases.
correlation between serum VegF levels
and clinical pathological parameters
The relationship between serum VEGF and clinical
pathological parameters is shown in Table 2. The median serum
VEGF concentration in all patients was 134 pg/mL (range,
9.0–15,998 pg/mL). No significant associations were found
between pretreatment serum VEGF levels and clinical
characteristics, such as sex (P=0.0975), age (P=0.2522),
stage (P=0.1407), lymph node metastasis (P=0.6409), tumor
location (P=0.3520), differentiated degree (P=0.5608),
pathological (histological) type (P=0.4885), and response to
A significant amount of VEGF is stored within platelet
membranes, thus platelets may be a major source of serum
VEGF.13 Therefore, we examined VEGFPLT with an
expectation to minimize the variations due to the differences in
platelet counts among NSCLC patients, and this may be a
reliable method to examine the clinical effects of VEGF
inpatients. However, we failed to associate pretreatment
serum VEGFPLT and patients’ clinical characteristics.
The VEGFPLT levels showed no correlations with sex,
age, primary tumor site, and pathological type in NSCLC
patients (all P.0.05).
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Predictive and prognostic values of VegF
in nsclc patients
In this study, a total of 65 patients (48.15%) had complete or
partial response and 70 patients (51.85%) had stable or
progressive disease (PD) (Table 2). Moreover, pretreatment
concentrations of VEGF and first-line platinum-based combination
chemotherapy effects were not correlated (P=0.9859).
The median follow-up time was 7.0 months. During the
follow-up periods, disease progression was detected in 120
(88.89%) patients, 5 (3.70%) patients had died, 6 (4.44%)
patients had not progressed, and 4 (2.96%) patients were
missing. After 1 year, 100 (74.07%) patients were found to
have PD, 28 (20.74%) had not progressed, 3 (2.22%) had
died, and 4 (2.96%) were missing. One-year progression-free
survival (PFS) rate was 19.26%. Moreover, age, sex, stage,
treatment modality, response to treatment, and VEGF level
were evaluated for survival using univariate analysis. Among
the variables evaluated, VEGF .280 pg/mL was determined
as an independent factor for PFS. Results of multivariate
analysis are summarized in Table 3. After adjusting for age
and sex, high VEGF levels tended to be associated with a
poor prognosis (P=0.024). Patients with higher VEGF levels
($280 pg/mL) had shorter PFS (median, 5.29 months; 95%
CI, 2.293–8.186 months) versus lower VEGF levels (median
6.407 months; 95% CI, 5.068–7.745 months; Figure 1).
Neoangiogenesis is critical in tumor growth and systemic
dissemination of cancer cells14 and may be related to poor
survival among NSCLC patients.15 Studies showed that
the presence of neoangiogenesis is a significantly negative
prognostic factor for both overall and disease-free survival in
lung cancer.9,16–18 Compared with the immunohistochemical
evaluation of angiogenic factors in tumor tissues, assessing
these angiogenic factors in blood has theoretical advantages:
more available, cheaper, faster, more readily performed
preoperatively, easier to serially repeat, and less biased.19
Angiogenic factors secreted by tumor cells stimulate
endothelial cells to proliferate and form new blood vessels.
Among these factors, VEGF is the most important mediator20
because it regulates endothelial sprouting, increases vessel
permeability, and contributes to the mobilization and
recruitment of bone marrow-derived hematopoietic and endothelial
progenitor cells to tumors.21 Being a good reflector of tumor
angiogenic activity, circulating VEGF levels have been
related to patient tumor burden.22
Although VEGF in the serum may originate from the
tumor itself, it could come from peripheral blood cells as
well, especially platelets. A significant positive correlation
HR (95% CI)
Male 1.237 (0.843, 1.816)
age (years) 0.1956
.60 1.268 (0.885, 1.816)
TnM stage 0.932
iV 1.016 (0.700, 1.475)
Peripheral 0.941 (0.619, 1.430)
VegF (pg/ml) 0.0494 0.024
.280 1.539 (1.001, 2.365) 1.600 (1.063, 2.408)
PlT ( ×109/l) 0.4046
.300 0.815 (0.503, 1.319)
VegF PlT (×10−6 pg) 0.1288
.0.55 1.326 (0.921, 1.909)
ΔVegF PlT 0.7380
.0.005 1.064 (0.738, 1.534)
cr + Pr (pg/ml) 0.7390
VegF .280 1.112 (0.596, 2.074)
sD + PD (pg/ml) 0.0310 0.0224
VegF .280 1.935 (1.062, 3.527) 2.087 (1.110, 3.926)
Note: *adjusted for age and sex.
Abbreviations: ΔVegF PlT , the minus value of VegF PlT before the first cycle minus the value after the chemotherapy; CI, confidence interval; CR, complete response; HR,
hazard ratio; nsclc, non-small cell lung cancer; PD, progressive disease; PlT, platelets; Pr, partial response; sD, stable disease; TnM, tumor node metastasis; VegF, vascular
endothelial growth factor; VegF PlT , VegF load per platelet.
between serum VEGF and platelet counts was reported in
several types of malignancies, including NSCLC.23 However,
in this study, we failed to detect an association. Research
showed that besides platelets, serum VEGF may come from
malignant cells, leukocytes, and other cell types, and this
may have contributed to the inconsistent results regarding the
relationship between the serum VEGF level and the platelet
count.24 Moreover, different sample processing methods may
have led to the inconsistencies.
The predictive role of VEGF for chemotherapeutic response
in NSCLC was investigated with conflicting results.
Regarding platinum-based chemotherapy, Fu et al reported
that VEGFPLT levels in the gemcitabine plus cisplatin-sensitive
group decreased sharply after chemotherapy, but showed
opposite results in the gemcitabine plus cisplatin-resistant
group, indicating that VEGFPLT levels can be used as
surrogate biomarkers to determine chemotherapeutic response
in NSCLC.12 By contrast, Yazar et al concluded that VEGF
is not useful as a predictive and prognostic markers in
advanced NSCLC patients treated with cisplatin-containing
chemotherapy.7,8 Similarly, Ludovini et al concluded that
VEGF expression in cancerous tissues is not associated
with chemotherapeutic response and overall survival (OS)
in advanced NSCLC patients.25 Moreover, we found no
significant correlation between the baseline serum VEGF
levels and the efficacy of cisplatin-based combination
chemotherapy. Furthermore, serum VEGF levels between pre- and
post-treatment groups were not significantly different. The
decrease in VEGF levels in responders may be due to the effect
of cytotoxic drugs on tumor cells by killing them, thereby
decreasing the number of cells that synthesize and secrete
various angiogenic proteins, including VEGF.26 However,
certain chemotherapeutic agents may exert antiangiogenic
effects by affecting endothelial cells, thereby increasing the
mobilization of circulating endothelial progenitor cells, again
promoting tumor angiogenesis. Furthermore, as a response to
chemotherapy, VEGF produced by tumor cells can increase.
All of these may increase VEGF concentrations, even in
patients with response effects.27 However, inconsistencies
of the results prompt further study. Furthermore, different
histological types, stages, treatment strategies, and sample
processes may account for the disparities.
VEGF overexpression in NSCLC is correlated with
neoangiogenesis and poor prognosis,28 and increased VEGF levels
upon NSCLC diagnosis may predict poor survival.29 Herein
lay the controversial reports concerning the VEGF impact
on NSCLC patient prognosis. Jantus-Lewintre et al observed
that time to tumor progression (TTP) and OS were not
significantly associated with plasma VEGF-A concentrations
in NSCLC patients treated with cisplatin plus docetaxel,
although patients with higher pretreatment VEGF-A levels
tend to have shorter TTP and OS.30 Chakra et al also studied
451 NSCLC patients receiving conventional treatments and
found that the prognostic information from the high
circulating VEGF serum level is not an independent determinant
of survival in NSCLC.31 In contrast, Brattström et al
concluded that in NSCLC patients with normal platelet counts,
VEGF levels after radiotherapy significantly correlated to
good prognosis (P=0.023); however, VEGF levels during
radiotherapy indicated the same correlation (P=0.085),
indicating that serum VEGF is of clinical interest as a
prognostic factor.32 Moreover, Lissoni et al reported that
the chemotherapeutic response was worse in patients with
higher levels of pretreatment VEGF in patients of NSCLC
and colorectal carcinoma.9 In this study, patients with higher
VEGF levels ($280 pg/mL) had shorter PFS (median,
5.29 months; 95% CI, 2.293–8.186 months) compared with
those with lower VEGF levels (median, 6.407 months;
95% CI, 5.068–7.745 months). The discrepancy between
our study results and those of previous studies regarding
survival might be partially due to the differences among the
assay methods, heterogeneity of study populations, different
chemotherapy regimens, and sample size. Different from
most of the previous studies using a traditional ELISA as a
testing measure, we performed our examination using the
Luminex multiplex assay through which we previously found
that VEGF levels in NSCLC patients at stages III and IV were
higher than those with at stages I and II.11 Limitations of this
study should be considered including its retrospective feature.
Moreover, the PC regimen was administrated only among
patients with pathological type of adenocarcinoma and may
have resulted in statistical bias. Therefore, a large-scale
prospective validation study is needed to testify the value of
VEGF as a biomarker in platinum-based chemotherapy.
The findings of this study imply that pretreatment serum
VEGF levels may be a potential prognostic biomarker for the
anti-tumor efficacy of platinum-based chemotherapy in terms
of PFS. However, VEGF was not a useful marker to
anticipate the response to chemotherapy in patients with advanced
NSCLC. It needs further investigation to verify the prognostic
implications of serum VEGF in NSCLC patients.
The authors thank all the enrolled patients who contributed
to the study and Fei Deng, Haixia Cao, and Rong Ma for
data acquisition and technical support for this research. This
work was supported, in part, by the National Nature Science
Foundation of China (number 81372396).
The authors report no conflicts of interest in this work.
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