Prediction of cancer progression in a group of 73 gastric cancer patients by circulating cell-free DNA
Pu et al. BMC Cancer
Prediction of cancer progression in a group of 73 gastric cancer patients by circulating cell-free DNA
Wang-Yang Pu 4
Rong Zhang 3
Li Xiao 2
Yong-You Wu 1
Wei Gong 1
Xiao-Dong Lv 1
Feng-Yun Zhong 1
Zhi-Xiang Zhuang 4
Xu-Ming Bai 5
Kai Li 0
Chun-Gen Xing 1
0 Department of Molecular Diagnostics and Biopharmaceutics, College of Pharmaceutical Science, Soochow University , Suzhou 215004 , China
1 Department of General Surgery, The Second Affiliated Hospital of Soochow University , Suzhou 215004 , China
2 Molecular Medicine Center, The Second Affiliated Hospital of Soochow University , Suzhou 215004 , China
3 Department of Obstetrics and Gynecology, The Second Affiliated Hospital of Soochow University , Suzhou 215004 , China
4 Department of Oncology, The Second Affiliated Hospital of Soochow University , Suzhou 215004 , China
5 Department of Interventional Radiology, The Second Affiliated Hospital of Soochow University , Suzhou 215004 , China
Background: Circulating cell-free DNA (ccf-DNA) in plasma may contain both specific and non-specific of tumor markers. The concentration and integrity of ccf-DNA may be clinical useful for detecting and predicting cancer progression. Methods: Plasma samples from 40 healthy controls and 73 patients with gastric cancers (two stage 0, 17 stage I, 11 stage II, 33 stage III, and 10 stage IV according to American Joint Committee on Cancer stage) were assessed respectively. qPCR targeting the Alu repeats was performed using two different sets of primers amplifying the long and short segments. DNA integrity was calculated as a ratio of the long to the short fragments of Alu repeats. Results: Plasma DNA concentration was significantly higher in patients with stage III and IV gastric cancers than in healthy controls (p = 0.028 and 0.029 respectively). The receiver operating characteristic (ROC) curve for discriminating patients with stage III and IV gastric cancers from healthy controls had an area under the curve (AUC) of 0.744 (95% CI, 0.64 to 0.85). Circulating cell-free DNA concentration increased within 21 days following surgery and dropped by 3 months after surgery. Conclusions: Concentration of ccf-DNA is a promising molecular marker for assessing gastric cancer progression.
Gastric cancer; Circulating cell-free DNA; DNA integrity; Alu-qPCR
Gastric cancer is the second most common cancer
and the fourth leading cause of cancer death . The
most significant prognostic indicator is TNM stage
. Diagnostic imaging methods such as endoscopic
ultrasound or computed tomography are effective for
detection of tumor depth, lymph node (LN) and distant
metastasis when they are relatively large. However, there
is no clinically established blood test which has the
predictive ability to determine gastric cancer for their
clinical stages. Therefore, the development of a
preoperative blood test for assessment of clinical stage
of gastric cancer is desired.
Circulating cell-free DNA in blood is a promising
biomarker for detection, monitoring, and prognostic
prediction of malignant tumor . Concentration of
ccf-DNA is influenced by the presence of cancers [4–6].
Methylated DNA detected in ccf-DNA has demonstrated
diagnostic and prognostic potential [7, 8]. Circulating
tumor DNA has been suggested correlation with surgical
resection and disease progression of gastric cancer . It
was showed that integrity of ccf-DNA calculated as a ratio
of the long to the short DNA fragments is higher in some
types of cancers [10–13]. In healthy individuals, ccf-DNA
is mainly originated from apoptotic cells which usually
release 185 to 200 base pair length of DNA fragments .
In contrast, ccf-DNA released from tumor cells varies in
size because of pathologic cell death in malignant tumors
. Therefore, ccf-DNA concentration and ccf-DNA
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integrity may have potential for tumor detection and
In this study, we measured the concentration of
ccfDNA by quantitative real-time PCR of Alu repeats on
113 plasma samples from gastric cancer patients and
healthy controls and analyze the relationship between
the ccf-DNA results and clinicopathological findings to
assess diagnostic and prognostic values of these genetic
markers of gastric cancer.
Collection of plasma samples and clinical information
Plasma samples from 73 patients suffering from gastric
cancers and 40 healthy volunteers were assessed. Blood
samples were draw before antitumor therapy, within 1–3
weeks after surgery, and during the follow-up period.
Staging was based on postoperative histopathology
findings for stage 0 to III, and imaging diagnoses were used
for stage IV. Patients included were those who had
treated in 2013 and 2014 at the Second Affiliated
Hospital of Soochow University. The eligibility criteria
included adenocarcinoma of stomach and age 18–78
years. Patients with second malignancies, connective
tissue disease, and pregnancy were excluded.
Plasma preparation for Alu-qPCR
Six ml of blood was collected in an EDTA-containing
tube, stored at 4 °C, and processed within 6 h. The blood
samples were centrifugation at 3000 rpm for 10 min at
room temperature to remove the cellular components.
Two ml of plasma were cryopreserved at −80 °C until
use. DNA was purified from 2 ml of plasma using
QIAamp® circulating nucleic acid kit according to
Quantitative PCR of Alu repeats
qPCR was performed using two different primer sets
for Alu repeats, amplifying 115 bp short and 219 bp
long products. The primer set for the 115 bp
amplicon (Alu115) amplified both the short and long DNA
fragments, and the primer set for the 219 bp
amplicon (Alu219) only amplified long DNA fragments.
The sequences of the Alu115 primers were forward:
5’-CCTGAGGTCAGGAGTTCGAG-3’ and reverse:
5’CCCGAGTAGCTGGGATTACA-3’ as described
previously , the sequences of the Alu219 primers were
forward: 5’- CACGCCTGTAATCCCAGCACTTT-3’
and reverse: 5’-CACGCCTGTAATCCCAGCACTTT-3’.
The primers Alu219 are newly designed. Alu115-qPCR
values represent the total amount of ccf-DNA.
Alu219qPCR results represent amounts of DNA released from
tumor cells. DNA integrity index is calculated as the ratio
of qPCR-results (Alu219-qPCR and Alu115-qPCR).
The reaction mixture for each Alu-qPCR consisted of
1 μl DNA template, 0.5 μl of the forward and reverse
primers, 12.5 μl Quantifast sybr green PCR mix (Qiagen,
Germany) and 10.5 μl RNase-free water in a total 25 μl
volume. Real-time PCR amplification was performed
with precycling heat activation of DNA polymerase at
95 °C for 5 min, followed 40 cycling of denaturation at
95 °C for 20 s, annealing and extension at 65 °C for 30 s
using Bio-Rad CFX96 PCR. The absolute equivalent
amount of DNA in each sample was calculated
according to a standard curve obtained with serial dilutions
(37.5 ng-0.375 pg) of prepared genomic DNA from
peripheral blood leukocytes of healthy controls. A negative
control (without template) was contained in each plate.
All qPCR assays were analyzed without knowledge of
specimen identity. Each assay was carried out in duplicates.
The Mann-whiney U-test was used to compare Alu115 or
Alu219/Alu115 between groups of gastric cancers and
healthy controls. Kruskal-wallis H-test was used for
multiple comparisons between the groups. Receiver-operating
characteristic curve and area under the ROC curve were
used to assess the diagnosis value of using Alu115 and
Alu219/Alu115 for gastric cancers. Statistical analysis
were performed using SAS software and results were
considered statistically significant at p < 0.05 (two tailed).
Evaluation of qPCR for measuring DNA concentration
To evaluate the performance of Alu-qPCR for
quantification of ccf-DNA, we tested the specificity of the two
primer sets for conventional PCR and then monitored the
efficiencies and qualities of amplification by real-time PCR
of a 10-fold serial dilution of genomic DNA exact from
peripheral blood leukocytes of healthy control. Agarose
gel electrophoresis of PCR products obtained with the
Alu115 and Alu219 primer sets conformed that the target
sequence was specifically amplified without major
aberrant bands (Additional file 1: Figure S1).
Clinical and pathologic characteristics of gastric cancers
The mean age was 49.68 ± 11.74 (standard deviation)
years for 40 healthy controls and 61.49 ± 10.18 years for
73 patients with gastric cancers. In 10 patients with
stage IV gastric cancer, one had liver and lung
metastases, two had lung metastases, two had peritoneal
metastases, two had para-aortic lymph node metastases, and
three had liver metastases. Table 1 shows AJCC stage
and histopathology characteristics of patients whose
plasma were sampled preoperatively.
AJCC regional lymph nodes
AJCC distant metastasis
Changes in ccf-DNA in plasma of patients with gastric
Circulating cell-free DNA in patients before antitumor
therapy was assessed for their levels and integrity. An
Alu115-qPCR value represents the total amount of
plasma DNA. The median (IQR 25–75) Alu115-qPCR
values in healthy controls and patients with stage 0-I, II,
III, and IV gastric cancer were 668.08 (401.18–1212.5),
1027.65 (408.2–1935), 1484.5 (400.7–1670), 1065.5
(676.7–1572.5), and 1666.5 (754.25–2479.5) ng ml−1,
respectively. The Alu115-qPCR values were significant
higher in patients with stage III and IV gastric cancer than
in healthy controls (p =0.028 and 0.029, respectively). A
trend of elevation in stage I and II cancer were observed
although it was not significant (p = 0.194 and 0.095,
respectively) (Additional file 2: Figure S2).
An Alu219-qPCR value represents ccf-DNA
concentration in longer sizes of plasma DNA. The median (IQR
25–75) Alu219-qPCR values in the controls and patients
with stage 0-I, II, III, and IV gastric cancer were 269.05
(149.85–425.35), 240.45 (184.9–582.1), 384.9 (188.85–
714.8), 355.15 (189.4–745.55), and 545.05 (165.4–961.6)
ng ml−1, respectively. The difference of Alu219-qPCR
values between healthy controls and patients with gastric
cancer was not significant (p =0.119).
The plasma DNA integrity was calculated as the ratio of
(Alu219-qPCR value/Alu115-qPCR value) of each sample.
The median (IQR 25–75) plasma DNA integrity in healthy
controls and patients with stage 0-I, II, III, and IV gastric
cancer was 0.43 (0.31–0.61), 0.34 (0.19–0.47), 0.39 (0.22–
0.47), 0.39 (0.28–0.53), and 0.38 (0.22–0.52), respectively.
There was no difference among these five groups.
The Alu115-qPCR values showed a trend that
increased with AJCC stage. The ROC curve of plasma
DNA concentration for discriminating patients with
stage III and IV gastric cancer from healthy controls had
an area under the curve value of 0.744 (95% CI, 0.64 to
0.85; Additional file 3: Figure S3). The ROC curve of
plasma DNA concentration for discriminating patients
with stage III and IV gastric cancer from patients with
stage 0-II gastric cancer had an area under the curve
value of 0.565 (95% CI, 0.43 to 0.70).
Plasma DNA concentration was independent of age
(p =0.3745). In 73 patients with gastric cancer, median
plasma DNA concentration was not correlated with depth
of tumor invasion (p =0.724), lymphovascular invasion
(p =0.960), and lymph node metastasis (p =0.289).
Fifty-eight patients with stage 0-III gastric cancer
(n = 2, 17, 11, 28, respectively) who had radical
surgery were followed up by telephone for 2 years. Tumour
metastasis was found in one patient with stage II and 10
patients with stage III. Spearman’s ρ coefficient of DFS
with Alu115-qPCR values was 0.007 (P = 0.95).
Circulating cell-free DNA dynamics in patients with gastric
cancers undergoing surgery
Postoperative ccf-DNA was assessed in 11 of 73
patients with gastric cancer. The distant metastasis was
found during surgery in 2 of 11 patients, and
palliative gastrectomy was performed on these two
patients. The other nine patients with postoperative
AJCC stage I to III (n = 3, 1, and 5, respectively)
received radical gastrectomy.
Alu115-qPCR values in plasma sampled preoperatively
from 11 patients varied from 400.7 to 18890 ng
ml−1(median = 1544 ng ml−1).
Within 3 weeks after surgery, Alu115-qPCR values
elevated median 109.9% (IQR 59.89–199.37%) than
preoperative in 10 of 11 patients. Only one patient
with stage III cancer was found with Alu115-qPCR
At 3 to 4 months after surgical intervention,
Alu115qPCR values dropped 19.16% (IQR 16.64–38.47) than
preoperative in 10 of the 11 patients (90.91%), and in
one patient with stage I cancer, Alu115-qPCR values
elevated 127.9% than preoperative. In two patients
received palliative gastrectomy, Alu115-qPCR values
dropped 1.19 and 24.81%, respectively. One patient
who had radical surgery developed peritoneal
dissemination in 25 months after operation, and Alu-qPCR
values elevated again (Additional file 4: Figure S4).
The present study analyzed ccf-DNA in a group of 73
patients with gastric cancers. Our data documented that
ccf-DNA levels were elevated in patients with late stage
cancers. Although dynamic changes of ccf-DNA levels
and integrities are highly useful in monitoring the effects
of therapy and progression of the cancer, limitation in
obtaining multiple blood samples and relatively high
individual variance shadowed its clinical application
presently. Our study identified the ccf-DNA elevated
significantly in one patient who had metastasis after
operation and adjuvant chemotherapy, suggesting that
longitudinal instead of comparing with normal controls
may be of more clinical impact. Technically, a new pair of
primers of Alu219 was tested with high reproducibility in
analysis of ccf-DNA in the present study.
Circulating cell-free DNA is a precious biomarker for
cancer not only because it contains the DNA released
from tumor cell but also pretty reliable and easy to
analyze . Studies have demonstrated that
concentration of ccf-DNA elevates in patients with lung cancer,
breast cancer, gastric cancer, colorectal cancer, urologic
tumor, head and neck tumor, etc. . Comparing to
concentration of ccf-DNA, concentration of circulating
tumor DNA (ctDNA) can reflect tumor burden more
accurately . However, detection of tumor-specific gene
mutation is limited by several factors. First, there must
be tumor-specific gene mutation genetically identified in
primary malignant tumor. Second, the low sensitivity and
complexity of assays make them less attractive for clinical
use, particularly for patients received surgical removal.
Alu repeats are short interspersed elements, typically
300 nucleotides . The Alu sequence is the most
abundant sequence in human genome with about one
million copies per genome. It is thus more sensitive for
Alu-qPCR method in assess the concentration of
In this study, we assessed the concentration of
ccfDNA by Alu-qPCR methods. We found that the level of
ccf-DNA of gastric cancer is higher than healthy
controls, especially in stage III and IV. This suggests the
potential application in preoperative stage and a
representative for cancer progression.
Some recent studies demonstrated that DNA integrity
elevated in many types of cancer [10–13]. However, the
study about DNA integrity didn’t show difference
between patients with gastric cancer and healthy controls
. Our result is identified with previously studies. DNA
integrity had no significant difference between patients
with gastric cancer and healthy controls. This may be
partially related to less ctDNA released to blood stream
rather than excreted through digestive track for gastric
cancers. Thus, DNA from dead tumor cell into blood
dropped. Another possibility is that the death pattern of
gastric cancers might be different from some other types
of cancers due to their environment of very low pH and
high level of proteinase, although this speculation needs
to be elucidated by experiments in animal models.
We assessed ccf-DNA dynamics in surgical gastric
cancer patients. In 21 days after surgery, in most of the
patients, ccf-DNA rised, until 3 months after surgery,
ccf-DNA was under the preoperative level, no matter
undergoing radical or palliative surgery. In one patient
developed distant metastasis after radical surgery, the
concentration of ccf-DNA was found to be elevated
again demonstrating that detection of change in
ccfDNA may be useful for monitoring of the burden of
gastric cancer in patients received surgery.
Technically, the present study tested a new pair of
primers to amplify the long fragment in Alu assay. This
pair of primers were screening from 6 primers. It
amplified 219 bp Alu fragments. This primers had higher
efficiency than the previous Alu247 primers . The new
high efficient pair of Alu219 primers may be applicable
for measurement of DNA integrity.
In conclusion, the present study observed that ccf-DNA
was higher in plasma from patients with advanced
gastric cancer than those from healthy controls. Circulating
cell free-DNA levels dropped after operation and
elevated again at tumor progression. Circulating cell-free
DNA may be valuable in monitoring progression and
prognosis of gastric cancer. In addition, We also tested a
new pair of Alu219 primers that have high efficiency and
may be applicable for measurement of DNA integrity.
Additional file 1: Figure S1. Results of agarose gel electrophoresis of
PCR products obtained with Alu115(0–4) and Alu219(5–9) primer sets.
Concentration of genomic DNA template from (0–4) and (5–9) is 0.00375,
0.0375, 0.375, 3.75 and 37.5 ng ml−1. (DOC 115 kb)
Additional file 2: Figure S2. Alu115-qPCR values in plasma from
healthy controls and patients with gastric cancer. Horizontal lines indicate
the median for each groups. The Alu115-qPCR values were significant
higher in patients with stage III and IV cancer than in healthy controls.
(DOC 210 kb)
Additional file 4: Figure S4. Circulating cell-free DNA dynamics in
patients with gastric cancers undergoing surgery. (A) Pre- and postoperative
plasma Alu115-qPCR values in patients with gastric cancer who underwent
radical surgery (n = 9) and palliative surgery (n = 2). (B) Alu115-qPCR values
in one patient who had radical surgery and developed peritoneal
dissemination at postoperative 25 months. (DOC 234 kb)
AJCC: American Joint Committee on Cancer; AUC: Area under ROC curve;
ccf-DNA: Circulating cell-free DNA; CI: Confidence interval; ct-DNA: Circulating
tumor DNA; IQR: Interquartile range; LN: Lymph node; ROC: Receiver operating
This study was supported by the Jiangsu Province’s clinical medical science
and technology program (No. BL2013016), the Chinese National 863 Major
Grant (No. 2012AA020905), and the National Natural Science Foundation of
China (No. 81301267).
Availability of data and materials
The datasets during and/or analysed during the current study available from
the corresponding author on reasonable request.
CGX, KL and WYP contributed to the design of the study and interpreted the
data. YYW, WG, XDL, FYZ, ZXZ and XMB provided the samples and clinical
data, and assisted in interpretation of the data. WYP and LX carried out the
PCR assays. WYP and RZ performed the statistical analyses. WYP drafted the
manuscript. All authors read and approved the final manuscript.
Consent for publication
Ethics approval and consent to participate
This study was approved by national drug clinical trial institutions ethics
committee of the second affiliated hospital of Soochow university. All
patients in this study gave written consent, and all relevant investigations
were performed according to the principles of the declaration of Helsinki.
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