Detection of circulating tumor cells (CTCs) in patients with lung carcinoma by real-time fluorescent quantitative-PCR approach before and after chemotherapy

doi:10.4236/health.2009.13038

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Vol.1, No.3, 231-238 (2009)

doi:10.4236/health.2009.13038

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Detection of circulating tumor cells (CTCs) in patients with

lung carcinoma by real-time fluorescent quantitative-PCR

approach before and after chemotherapy

Ming-Jian Ge1, Qing-Chen Wu1, Mei Wang2, Li Li1, Xiao-Long Zhao1, Qiao-Min Huang1,

Liang-Bin Li1

1Department of Thoracic Surgery, the First Affiliated Hospital, Chongqing Medical University, Chongqing, China

mingjian_ge@hotmail.com

2Department of Clinical Laboratory, the First Affiliated Hospital, Chongqing Medical University, Chongqing, China

Received 18 September 2009; revised 9 October 2009; accepted 10 October 2009.

ABSTRACT

Circulating tumour cells (CTCs) are referred to the

tumour cells that disseminated from the primary

tumour and survive in circulating during the pro-

ceeding of tumour growth. As surgical treatment

evolves and local control has improved, the failure

of cancer treatment has largely remained the re-

sult of systemic metastasis. Selection of patients

most likely to benefit from adjuvant strategies

remains problematic. In order to develop a new

standard of curative effect, this study was de-

signed to track the number of CTCs in patients

with lung cancer during chemotherapy. Methods:

Samples of peripheral blood was taken from each

lung cancer patients (n=32) on the day before

chemotherapy as well as the third week after the

chemotherapy cycle. The samples were subjected

to real-time fluorescent quantitative reverse-tran-

scriptase polymerase chain reaction (fqRT-PCR).

Meanwhile the tumour size was determined by

chest X-ray or computed tomograghy. Results

Compared to that of pre-chemotherapy, the ex-

pression level of cytokeratin (CK) 19 in the pa-

tients significantly declined after chemotherapy

(t=4.659,P=0.000). The level of CK19 mRNA in pa-

tients with small cell lung cancer (SCLC) was

higher than that of patients with non-small cell

lung cancer (NSCLC) (t=1.944, P=0.061). The de-

crease of CK19 mRNA level correlated well with

the type during the treatment. Relatively the de-

crease of SCLC is more obvious (t=6.073,P=0.000).

The variation of CK19 mRNA level before and after

chemotherapy was positively related to the dis-

parity of tumour burden (r=0.593). There was also

a significant association between the type (NSCLC

vs. SCLC) and the change of tumour size (t=3.686,

P=0.001).The positive rate before chemotherapy

was 71.9% (23/32), while that after chemotherapy

was 37.5% (12/32), indicating that 11 patients con-

verted into negative after chemotherapy. Of the 16

patients which were in Ⅳ-stage, 11 cases were po-

sitive (11/16,68.8%). Surprisingly, of the remaining

16 patients which were Ⅱ/Ⅲ stage, 12 cases were

regarded as positive according to the criteria

(12/6,75%). Conclusions: The real-time flu- ores-

cent quantitative-PCR approach is useful for mea-

suring the relative number of CTCs in a patients’

peripheral blood to monitor the effectiveness of

treatment, and for designing more comprehensive

and reasonable therapeutic regimes at earlier

dates for patients. The treatment response can be

immediately assessed by serial quantitation of

CTCs after chemotherapy, and therefore this

method highlights an alternative approach to

rapidly access the patient’s response to treatment.

Keywords: Lung Neoplasm; Blood; Polymerase

Chain Reaction; Cytokeratin; Messenger RNA;

Chemotherapy

1. INTRODUCTION

Traditionally clinical response criteria for solid tumors

were defined according to the change of the mass during

therapy; briefly, complete response (CR) is defined as

complete disappearance of all measurable and available

clinical evidence of cancer; partial response (PR) is de-

fined as at least a 50% reduction in the size of all meas-

urable tumour areas, progressive disease (PD) is defined

as an increase of = or ＞ 25% (compared to baseline or

best response) in the size of all measurable tumour areas;

And stable disease (SD) is defined as neither sufficient

shrinkage to qualify for PR nor sufficient increase to

qualify for PD. If the mass was resected, how do we as-

Supported by grant from the Natural Science Foundation in China

(

No.30972961

M.J. Ge et al. / HEALTH 1 (2009) 231-238

232

Table 1. Clinicopathological featurse and CK19 mRNA expression in peripheral blood before and after chemotherapy for patients with

lung cancer.

Tumour size (cm2) CK19 mRNA*

Patient Gender

/Age Type Stage

Before After Before After

1＃ F/54 ADC Ⅱ 8.4 1.6 6.03 4.26

2＃ M/59 SCLC Ⅳ 19.8 9.6 5.19 4.05

3＃ F/48 SCLC Ⅳ 5.2 3.4 5.61 4.09

4＃ M/62 SCC Ⅲ 10.1 9.8 4.04 4.40

5＃ M/63 ADC Ⅳ 35.3 26.7 4.23 3.81

6＃ M/67 SCLC Ⅲ 34.4 15.8 4.86 2.28

7＃ M/68 SCC Ⅱ 5.2 3.4 4.22 3.40

8＃ M/60 SCC Ⅲ 8.3 6.4 3.51 4.30

9＃ F/53 ADC Ⅳ 5.1 5.0 3.62 3.78

10＃ M/58 ADC Ⅳ 9.3 8.7 3.55 3.50

11 ＃ M/45 ADC Ⅱ 19.6 12.6 4.60 3.89

12＃ M/63 SCC Ⅲ 22.5 10.4 6.15 4.75

13＃ M/62 SCC Ⅳ 14.4 8.7 3.31 3.29

14＃ M/49 SCC Ⅲ 36.1 33.2 3.93 3.73

15＃ F/54 ADC Ⅳ 42.6 33.6 5.90 4.74

16＃ F/47 SCLC Ⅳ 10.5 0.0 5.72 3.29

17＃ M/52 SCC Ⅲ 19.3 18.0 6.78 5.71

18＃ M/62 SCC Ⅲ 3.6 3.4 4.12 4.71

19＃ M/39 SCLC Ⅲ 60.8 11.9 5.99 3.98

20＃ M/33 SCC Ⅳ 16.5 17.2 5.06 5.08

21＃ F/50 SCC Ⅳ 10.9 11.8 4.64 4.26

22＃ M/60 SCLC Ⅲ 7.3 0.0 4.94 2.86

23＃ M/56 SCC Ⅲ 10.9 12.5 5.06 5.02

24＃ F/66 SCC Ⅳ 7.8 3.1 5.14 3.99

25＃ F/45 SCC Ⅳ 15.1 13.9 4.25 4.16

26＃ M/62 SCC Ⅳ 2.97 2.32 3.44 3.57

27＃ M/61 SCC Ⅱ 3.6 2.9 3.65 3.85

28＃ M/63 SCLC Ⅱ 11.9 2.9 5.41 3.30

29＃ M/47 SCLC Ⅱ 17.5 6.3 4.55 3.29

30＃ M/66 ADC Ⅳ 13.2 11.3 4.44 3.70

31＃ M/61 ADC Ⅳ 8.4 8.7 6.10 6.05

32＃ M/60 ADC Ⅳ 9.6 5.1 3.86 3.73

Abbreviation: F, female; M, male; ADC, adenocarcinoma; SCC, squamous cell carcinoma; SCLC, small cell lung cancer.

* The values of results were expressed as log10CK19 mRNA copies per millilitre serum.

sess efficacy of the treatmental strategy?

Clinical cancer care and study depends on accurately

staging the extent of disease to assess prognosis and de-

termine efficacy of treatment regimens. Staging criteria

for most solid tumours with which general surgeons

areconcerned are based on the tumour node metastasis

(TNM) system. But current approaches are limited in

adequately identifying individual patients who are at high

risk for future relapse and have the need for a more ag-

gressive therapy. This is mainly due to inaccuracy in the

identification of early stage metastasis. Furthermore the

diagnosis tools for therapy monitoring are restricted to

imaging techniques and the measurement of protein se-

rum markers. Whereas imaging techniques suffer in

general from their resolution of about 0.5 cm, the clinical

usefulness of tumour markers is questionable, leading to a

M.J. Ge et al. / HEALTH 1 (2009) 231-238

233

progressive reduction of their use in clinical practice

[1-6].

Since even small tumours already show dissemination

of cells, their disclosure has been considered as a tool to

improve early cancer detection. In addition to the ‘tumour

marker’ function of CTCs, these cells remain in the

lymphatic/systemic circulation after surgical removal of

the primary tumour and are responsible for the later re-

currence of the disease. Consequently this undetected cell

dissemination by conventional measures is of relevance

for cancer progression and has been already demonstrated

to be of prognostic impact in several malignancies [7-12].

CTCs are referred to the tumour cells that disseminated

from the primary tumour and survive in circulating during

the proceeding of tumour growth. There are no symptoms,

and current clinical procedures (i.e. radiography and

routine pathological examination) often fail to identify

the foci. Generally most of these cells are dormant (i.e. no

proliferation or a balance between cell growth and death).

If the host suffers from trauma or immunological im-

pairment, dormant tumour cells will proliferate continu-

ally and growth into a mass focus eventually. Therefore,

the detection of CTCs is important for evaluating the

prognosis and tailoring therapeutic strategy [13].

As surgical treatment evolves and local control has

improved, the failure of cancer treatment has largely

remained the result of systemic metastasis. There is

therefore much reason to develop effective adjuvant sys-

temic therapy. Identification of patients most likely to

benefit from adjuvant strategies remains problematic.

With the aim of aiding the development of a new standard

of curative effect, this study was designed to track the

number of CTCs in patients with lung cancer during

chemotherapy.

2. MATERIAL AND METHODS

2.1. Clinical Data

After obtaining informed consent, samples of 5ml pe-

ripheral blood(PB) were taken from each lung cancer

patient (n=32) on the day before chemotherapy as well as

the third week after the chemotherapy cycle. Meanwhile

the tumour size (cm2) was determined by chest X-ray or

computed tomography. The control population consisted

of healthy individuals who had no any evidence of solid

tumours or haematological malignancy (n=20).

All the cases were diagnosed for the first time and re-

ceived the first cycle of chemotherapy. They had never

received any other treatment (e.g. surgery or radiother-

apy). Each patient with SCLC included in this study re-

ceived the first cycle of chemotherapy consisting of eto-

poside 100mg/m2 per day on day(s) 1 to 3, cisplatin 50

mg/ m2 per day on day1. For patients with NSCLC, the

first cycle of chemotherapeutic strategy was composed by

mitomycin 10mg/m2 per day on day 1 and 8, vindesine

(VDS) 3mg/m2 per day on day 1 and cisplatin 80mg/m2

per day on day1.

The study population consisted of 32 patients with

histologically documented lung cancer. Staging proce-

dures included chest radiograph, bronchoscope, thoracic

computed tomography, sonography, bone scintigraphy

and biopsy of distant lesion (e.g. lymph node). The

clinicopathological features of the lung cancer patients

were summarized in Table1.

2.2. Procedure

2.2.1. RNA Isolation

The peripheral blood mononuclear cells (PBMNs) were

separated with Ficoll-Paque solution. As outlined by

manufacturer, the RNA was Isolated from PBMNs by

using Trizol reagent (Invitrogen). The concentration of

total RNA was quantitated by using UV spectropho-

tometer (Parmacia-Bitech). The integrity of extracted

RNA was verified by migration by gel electrophoresis.

2.2.2. CDNA Synthesis

Total RNA was denatured at 70℃ for 5min. Reverse tran-

scriptase reaction was carried out in 18µl 1×reverse tran-

scriptase buffer [50mM Tris-HCL(PH8.3), 75 mM KCL and

3mM MgCL2] with 0.5 mM deoxynucleotide triphosphates,

1µl of RNasin, and 200 units of Moloney murine leukaemia

virus reverse transcriptase(Promega). CDNA were synthesis

at 37℃ for 30min.

2.2.3. Primer/Probe Design

For the development of suitable combinations of Taqman

primers and probe, the Primer Express software (PE Ap-

plied Biosystems) was used. The resulting primer pair

produces a 202-bp fragment. Sequences (from 5’ to 3’) of

CK19 were as follow: upper primer GCA GAA CCG

GAA GGA TGC T; lower primer TCC GTT TCT GCC

AGT GTG TC. The Taqman probe was labelled at the 5’

end with the reporter dye molecular FAM (emission

wavelength 518nm) and at the 3’ end of the probe was

additionally phosphorylated to prevent extension during

PCR. The sequence of the probe is TGG TTC ACC AGC

CGG ACT GAA. The primer-probe set was selected so

that the primers were positioned over an intron-exon

junction, and were designed to differentiate between the

highly homologous pseudogenes.

2.2.4. Taqman PCR Reaction

PCR was conducted in 43µl 1× PCR buffer [10mM

Tris-HCL (PH8.4), 50mM KCL, and 1.5mM MgCL2]

with 0.2 mM deoxynucleotide triphosphates, 5 µl of

cDNAs, 2 units of Taq DNA polymerase (Promega), 0.4

µM of sense and antisense primers for CK19 respec-

tively. The optimised thermal profile was initiated with 5

min denaturation at 95℃, followed by 35 cycles of 95℃

for 30seconds, 62℃ for 20 seconds, and 72℃ for 20

seconds, and a final extension at 72℃ for 10 min. LC-5

M.J. Ge et al. / HEALTH 1 (2009) 231-238

234

Openly accessible at

RNA standards and multiple water blanks were analysed

in parallel with blood samples in each set of PCRs. All

reactions were performed in the ABI Prism 7000 Se-

quence Detection System.

The initial concentration of every sample could be

achieved according to the standard curves gained previ-

ously. In this study, the values of results were expressed

as log10CK19mRNA copies per millilitre serum. All reac-

tions were performed in the ABI Prism 7000 Sequence

Detection System (Perkin-Elmer Applied Biosystems),

which detects the signal from the fluorogenic probe dur-

ing PCR. The 7000 system has a built-in thermal cycler

and a laser directed via fiber optical cables to each of the

96 sample wells. A charge-coupled device camera col-

lects the emission from each sample and the data are

analysed automatically. The software accompanying the

7000 system calculates cycle threshold (Ct) and deter-

mines the starting copy number in the samples.

2.2.5. Spiking Experiment Using LC-5 Cells

LC-5 cells (a squamous cell line of lung cancer) from a

monolayer culture were harvested with trypsin-EDTA

(Sigma), washed in cold PBS and resuspended. Cell

densities were evaluated using a counting chamber and

viability by trypan blue staining (Sigma). Densities were

adapted to a total of 106 cells in 2 ml diehypyrocarbon-

ate-treated water. To simulate the presence of lung cancer

cells in the circulation of lung cancer patients, total RNA

was first extracted from 107 PBMNs from healthy sub-

jects and 106 LC-5 cells. Aliquots of total RNA from 106

PBMNs were mixed with LC-5 total RNA, corresponding

to 1,10,102,103,104 and 105 LC-5 cells. As for the negative

control, only RNA extracted from 106 PBMNs was used.

The RNA mixtures were then subject to fqRT-PCR for

construction of the calibration curves and receiver op-

erator characteristic (ROC) curves. Each sample was

measured in triplicate.

2.3. Statistical Analysis

The results were analysed with statistical software SPSS10.0

for Windows. The statistical assays included paired samples

t test, independent samples t test and bivariate correlate

analysis, with P＜0.05 indicating significance. The method

used to calculate the appropriate sample size in the study

was similar to that used to paired measurement data. Sup-

pose if TypeⅠerror is 0.05,the sample sizes for both pre-

and post-chemotherapeutical group were thirty, respectively.

The weighted sample size i.e. thirty-two, calculated by the

proposed method, preserved the power (1-β) above 0.9.

3. RESULTS

3.1. Evaluation of the CK19 Diagnostic Test

To rigorously define the value of the real time RT-PCR

1 - Specificity

1.00.75.50.250.00

Sensitivity

1.00

.75

.50

.25

0.00

Figure 1. ROC curve of CK19 diagnostic test. ROC curve analysis

is based on a plot of sensitivity as a function of 1-specificity. The

area under the curve in this study was 0.965, indicating high accu-

racy. In this study, the upper value of the confidence interval of the

median of the volunteer group was considered as the cut-off value,

which is 4.120.Under this standard, the sensitivity of the diagnostic

test was 90%, and the specificity was 84%.

Figure 2. Standard curve for LC-5cell line dilutions. The graph

shows the Ct value versus the log of the number of CK19 mRNA,

measured in triplicate. The standard curve shows 4 orders of

linear dynamic range.

analysis for the detection of CTCs, we performed a ROC

curve analysis using SPSS 10.0 for windows software. ROC

curve analysis is based on a plot of sensitivity as a function of

1-specificity. The area under the ROC curve in this study was

0.965 (95% confidence interval:0.916～1.014) (Figure 1).

In order to define the criteria to identify true positiv-

ity,the upper value of the confidence interval of the median

of the volunteer group was considered as the cut-off value,

M.J. Ge et al. / HEALTH 1 (2009) 231-238

235

Openly accessible at

which is 4.120. Under this standard, the sensitivity of the

diagnostic test was 90%, and the specificity was 84%.

The reproducibility of the technique was established

with the Ct value obtained for each dilution (105～108) of

the standard curve in different assays and within an assay:

the intraassay and interassay CVs of the threshold cycle

were 2.2 and 6.5%, respectively, on average.

Standard curve and amplification plots are showed in

Figure 2 and Figure 3, respectively.

3.2. Comparison of CK19 Mrna Level in PB

Before and after Chemotherapy

Compared to that of pre-chemotherapy, the expression

level of CK19 in peripheral blood significantly declined

post-chemotherapeutically (4.7323±0.9698 vs. 3.9864 ±

0.8309, t=4.659, P=0.000. Paired Samples T Test).

3.3. The Relationship Between CK19

MRNA Level in PB and Classic

Clinocopathological Features

The level of CK19 mRNA in patients with SCLC was

higher than that of patients with NSCLC (5.2853±0.4880

vs. 4.5480±1.0263, t=1.944, P=0.061, Independent Sam-

ples T Test, Figure 4). Nevertheless, there was no link

between value and other clinicopathological parameters

such as stage, gender and age etc. (P＞0.05).

The difference of CK19 mRNA level correlated well

with the type during the treatment. Relatively the dispar-

ity of SCLC is more obvious (1.8924 vs. 0.3637, t=6.073,

P=0.000, Independent Samples T Test). However, there

was no relationship between the difference of CK19

mRNA level and other parameters such as stage, gender

and age etc. (P＞0.05). The variation of CK19 mRNA

level before and after chemotherapy was positively re-

lated to the disparity of tumour burden (r=0.593, P=0.000,

Bivariate Correlation Analysis). There was also a sig-

nificant association between the type (NSCLC vs. SCLC)

and the difference of tumour size (3.0525 vs. 14.6875,

t=3.686, P=0.001, Independent Samples T Test). Figure 5

showed the difference of tumour volumes of different

types of tumour before and after treatment.

3.4. The Positive Rate of CK19 Gene

Expressive Level before and

after Chemotherapy

The upper value (4.120) of confidence interval of the

median (3.6647) of the healthy volunteer group was con-

sidered as the cut-off value. If lgCK19＞4.120, the case

would be classified as a positive one. The positive rate

before chemotherapy was 71.9% (23/32), while that after

chemotherapy was 37.5% (12/32), indicating that 11

patients converted into negative after chemotherapy. Of

the 16 patients who were in Ⅳ-stage,11 cases were posi-

tive(11/16,68.8%). Surprisingly, of the remaining 16

patients who were Ⅱ/Ⅲ stage,12 cases were regarded as

Figure 3. Typical amplification plot. The graph of the increment

of fluorescence reporter signal(ΔRn) versus cycle number

during PCR shows three stages: baseline, exponential phase, and

plateau. The Ct value is calculated by determining the point at

which the fluorescence exceeds an arbitrary threshold limit. For

each reaction tube, the fluorescence signal of the reporter dye

is divided by the fluorescence signal of the passive reference dye,

to obtain a ratio defined as the normalized reporter signal (Rn).

ΔRn represents the normalized reporter signal (Rn) minus the

baseline signal.

SCC/ADC SCLC

Pathological Type

Lg CK19mRNA

Figure 4. The comparison of CK19 mRNA in PB of patients

with SCLC and NSCLC before chemotherapy.

positive according to the criteria (12/16,75%).

4. DISCUSSION

Since traditional RT-PCR technology is at best semi-

-quantitative, it has been difficult to differentiate between

baseline level of gene expression in normal tissues and

increased level of gene expression associated with cancer,

rising the concerning for false-positive results. In this

study, the real time PCR was exploited to investigate the

possibility of using easily accessible body fluids as a

source for CTCs detection enabling longitudinal obser-

vation of the disease, therapy monitoring and initial di-

agnosis.

M.J. Ge et al. / HEALTH 1 (2009) 231-238

236

Openly accessible at

824824N =

Pathological Type

SCLCNSCLC

Variation of CK19 mRNA or Mass Volume

-5

-10

lgpre ck－lg po stck

prevol－postvol

Figure 5. The comparison of CK19 mRNA in PB and tumour

volumes of patients with SCLC and NSCLC before and after

treatment.

In this study, we could detect low level of CK19 ex-

pression in healthy volunteers, suggesting low expression

in non-malignant cells in peripheral blood. In order to

define criteria to identify true positivity, we have taken

this observation into account in the calculation of the

median and 95% CI of the median (i.e. the upper limit for

normal expression) of the volunteer group. In order to

define criteria to identify true positivity, the upper value

of the confidence interval of the median of the volunteer

group was considered as the “cut-off” value. Therefore,

the specificity of CK19 analysis could be restored by the

use of a quantitative assay with subtraction of background

transcription. The possibility of obtaining quantitative

information represents a milestone in the identification of

subtle changes in the expression profiles and numbers of

circulating tumour cells. The real-time PCR method of-

fers several advantages over other current quantitative

PCR methods [14].

The study demonstrated that, comparing with that of

pre-chemotherapy, the expression level of CK19 in PB of

patients with lung cancer declined dramatically after

chemotherapy (P=0.000). We believed that, due to anti-

cancer drugs, the number of cancer cells shedding from

primary tumour decrease greatly. Furthermore, these

drugs could directly kill cancer cells in the circulation.

The level of CK19 mRNA in PB of patients with SCLC

was higher than that of NSCLC (P=0.061). After one

cycle of chemotherapy, the dropping of CK19 mRNA

level in SCLC was much more obvious than that of

NSCLC (P=0.000). The phenomena could be due to two

group’s different sensitivity to chemotherapy, coordinat-

ing with previous studies. The traditional standard for

evaluating the curative effect of solid tumour depends on

the variation of the mass size. It belongs to magnificent

criterion. In clinical practise, in order to develop more

accurate standard, other factors such as cytology and

molecular ones should be taken into account. To improve

the current staging and effect of tumours, oncologists are

striving for new norm. This study showed that the dif-

ference in expression level of CK19 in PB was correlated

well with the variation of tumour size during chemo-

therapy, indicating that it could be considered as a brand

new criterion regarding assessing the result of therapy.

Blood can be sampled throughout the course of the dis-

ease and its collection is a minimally invasive procedure.

The permanent access to CTCs enables follow up studies

to be done to forecast prognostication of disease outcome,

therapy monitoring or drug targeting. For example, for

postoperative patients whose mass no longer exits, it

could be exploited to evaluate the animated therapeutic

results.

It was interesting that we found there was no correla-

tion between the existence of CTCs and the traditional

TNM staging system. Why were only 11 patients not all

who were in Ⅳ stage positive in this study? We insisted

that this phenomenon could be due to intermittent shed-

ding of tumour cells in the circulation. This problem

should be overcome by sequential sampling, defined as

the analysis of multiple blood samples at different time

points. False-negative results could also be due to

down-regulation of the target gene by therapy (e.g. hor-

monal treatment) or to the presence of poorly differenti-

ated subclones that do not express the tissue-specific

marker being tested. For example, PSA mRNA expres-

sion was shown to be decreased by antiandrogen therapy

[15] and in poorly differentiated prostatic carcinoma [16].

The use of multiple markers will help diminish these

types of false negative. On the other hand, it was sur-

prising that, of the 16 patients who were on Ⅱ/Ⅲ stage,

12 cases were positive! We believe that many cancers

have systemic spread at an early stage of their develop-

ment. The presence of CTCs in PB should be incorporated

in the UICC staging nomenclature by including isolated

tumour cells (Mi) in the M-stage of the Tumour - Node -

Metastases classification. Patients with occult dissemi-

nation of viable tumour cells are not cured by surgery

alone and may benefit from additional adjuvant therapy

[17].

The permanent access to CTCs enables longitudinal

studies to monitor therapy outcome. This aspect is sub-

stantially improved by recent quantitative methods (e.g.

FQ-PCR) allowing the detection of changes in the quality

and number of tumour cells [18]. In contrast to solid

metastatic tumours, micrometastatic tumour cells are an

appropriate target for intravenously applied agents be-

cause macromolecular and immunocompetent effectors

cells should have access to the tumour cells. Because the

majority of micrometastatic tumour cells may be non-

proliferative (G0 phase), standard cytotoxic chemothera-

pies aimed at proliferating cells may be less effective,

M.J. Ge et al. / HEALTH 1 (2009) 231-238

237

Openly accessible at

which might explain, in part, the failure of chemotherapy.

Thus, adjuvant therapies that are aimed at dividing and

quiescent cells, such as antibody-based therapies, are of

considerable interest [19].

Our study showed that quantitation of CTCs is possible

during the course of therapy. Because cancer cells express

different levels of CK19 mRNA, patients with higher

CK19 mRNA level do not necessarily have more CTCs in

peripheral blood than do patients with lower one. Nev-

ertheless, the real-time fluorescent quantitative-PCR ap-

proach is useful for measuring the relative number of

CTCs in a patients’ peripheral blood to monitor the ef-

fectiveness of treatment. In a limited number of patients,

we demonstrated that the relative number of CTCs cor-

related well with the tumour burden and therapeutic re-

sponse. This method highlights an alternative approach to

rapidly access the treatment response of patients. The

treatment response can be immediately assessed by serial

quantitation of CTCs after chemotherapy. This method

may, therefore, help to design more comprehensive and

reasonable therapeutic regimen at an earlier date for pa-

tients [20]. A large prospective study is needed to deter-

mine whether prognostic significance can be derived

from the presence of CTCs and whether this group of

patients might benefit from adjuvant treatment.

5. ACKNOWLEDGEMENTS

I extremely thank Dr Gaynor Bates, who works in Breast Cancer

Campaign in UK, for her suggestion concerning the revision for the

initial English draft of the study. I am indebted to all the members of the

Chongqing Lung Cancer Center who have cheerfully donated the sam-

ples.

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