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Clinical Cancer Research 13, 2992-2997, May 15, 2007. doi: 10.1158/1078-0432.CCR-06-2072
© 2007 American Association for Cancer Research
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Cancer Therapy: Clinical

Circulating Tumor Cells in Perioperative Esophageal Cancer Patients: Quantitative Assay System and Potential Clinical Utility

Zhian Liu1, Ming Jiang2, Jianhua Zhao2,3 and Huangxian Ju1

Authors' Affiliations: 1 MOE Key Laboratory of Analytical Chemistry for Life Science, Department of Chemistry, Nanjing University; 2 Tumor Hospital of Jiangsu Province; and 3 Center of Clinical Laboratory Science and Department of Surgery, Nanjing, P.R. China

Requests for reprints: Huangxian Ju, MOE Key Laboratory of Analytical Chemistry for Life Science, Department of Chemistry, Nanjing University, Nanjing 210093, P.R. China. Phone: 86-25-83593593; Fax: 86-25-83593593; E-mail: hxju{at}nju.edu.cn.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: To establish a quantitative system for evaluating the role of circulating tumor cells (CTC) in peripheral blood samples in patients who undergo surgery for treatment of esophageal cancer.

Experimental Design: One hundred fifty-five peripheral blood samples from 53 esophageal cancer patients were collected before surgery (B-1), immediately after surgery (B0), and on the 3rd day postoperatively (B+3). Eighty-nine samples from 22 benign patients who underwent thoracotomy and 30 healthy volunteers were obtained as controls. A real-time reverse transcription-PCR quantitative analysis system based on carcinoembryonic antigen (CEA) mRNA gene expression was designed for detection of CTC.

Results: This developed system can detect CEA mRNA-positive cells down to 3 cells per milliliter of peripheral blood. The cells in negative control groups were lower than the detection limit. The medians of 188 [95% confidence interval (95% CI), 155-498], 1513 (95% CI, 660-7,974) and 707 (95% CI, 737-3,005) CEA mRNA-positive cells per mL with the CEA-positive rates of 28.3%, 60.4%, and 42.9% in B-1, B0, and B+3 peripheral blood samples were obtained, respectively. There was statistically significant difference between B-1 and B0 (P = 0.0001) and between B-1 and B+3 (P = 0.0209). Fifty percent of patients with R > 0.4 showed metastasis in 1 year after surgery, whereas the probability was only 14.3% for patients with R < 0.4 (where R is CTC ratio of B+3 to B0, P = 0.043).

Conclusions: Esophageal cancer operation results in tumor cells dissemination and significant increase of CTC in peripheral blood, which is related to the developed metastasis. CTC are helpful for evaluating micrometastasis and have the potential for predicting recurrence in esophageal cancer.

Esophageal cancer is one of the lethal cancers with a poor prognosis. Its survival rate is only 15% at 5 years (1) and remains less than 20% to 30% at 2 years even after surgical resection (2). Long-term observation suggests that the micrometastasis in esophageal cancer patients might exist before surgery, which is also enhanced upon surgical manipulation due to the shedding of tumor cells into bloodstream in animal studies (3–5). Although surgery is the current gold standard for treatment of this aggressive cancer, and the postoperative mortality has also been declined by improving the complete resection and using current treatment strategies such as adjuvant chemotherapy and radiotherapy after complete surgery, the presence of disseminated tumor cells in lymph nodes (6) or blood circulation (7, 8) is related to metastatic relapse, which remains the most frequent cause of cancer-related deaths (9). Thus, an accurate specific system for quantification of circulating tumor cells (CTC) in esophageal cancer patients is desired for monitoring the dissemination of tumor cells in peripheral blood, which is helpful to assess the extent of tumor cells dissemination in peripheral blood and the development of distant metastasis.

The micrometastasis of esophageal cancer patients has been studied using classic reverse transcription-PCR (RT-PCR) coupled with gel-electrophoresis measurement to detect the tumor cells in bone marrow (10), lymph node (11–14), and peripheral blood (15–17). However, this technique can only obtain the positive or negative result and is unable to reflect quantitatively the dissemination status of tumor cells. This work uses a real-time quantitative RT-PCR method for analysis of carcinoembryonic antigen (CEA) mRNA expression to achieve the quantification of CTC in esophageal cancer patients. As well known, real-time quantitative RT-PCR is a sensitive, quantitative, and contamination-free technique with numerous advantages over classic RT-PCR and relative quantitative PCR based on 2–{Delta}{Delta}Ct method (18). It has been extensively applied to detect micrometastasis in breast (19–21), colorectal (22), prostatic (23), and lung cancers (24). Owing to the high efficiency of reverse transcription and PCR amplification, real-time quantitative RT-PCR could be extremely easily affected by various factors during manipulation, leading to possible conflicting results of CTC detection even if the used procedures are similar or the same. Thus, it is crucial to choose appropriate controls and standardize all the uncertain factors in each step for minimizing the systematic errors in quantification.

The gene encoding CEA is one of the most widely expressed genes in tumor cells. The CEA mRNA expression occurs in 92% to 95% of carcinomas of gastrointestinal tract (25). The CEA-specific RT-PCR method has been widely used to detect CTC in blood from cancer patients since established in 1994 (26). This study established a standardized quantitative assay system, with which the numbers of tumor cells in peripheral blood during surgical manipulation could be detected accurately and directly. The results indicated the dissemination of tumor cells in circulation during and after surgery could be monitored, which could be further developed for evaluating micrometastasis and predicting recurrence in esophageal cancer.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients. According to the rules of the local ethical committee, 155 peripheral blood samples were collected from 53 patients without distant metastasis of esophageal squamous carcinoma (mean age of 58.1) during September to December 2005 in the Tumor Hospital of Jiangsu Province. These samples were collected at three time points, before surgery (B-1), immediately after surgery (B0), and the 3rd day postoperatively (B+3), by central venipuncture after discarding the first 5 mL of blood to avoid epithelial contamination from venipuncture.

Fifty-nine peripheral blood samples were collected from 22 patients without any evidence of malignancy, who underwent thoracotomy over the same period (seven peripheral blood samples missed at B+3 time point) to compose an ideal negative control group. Thirty peripheral blood samples from 30 healthy volunteers formed another negative control group.

Eight tissue samples were obtained from these patients during surgery and shock-frozen in liquid nitrogen for later RNA extraction as positive quality controls for real-time quantitative PCR.

Cell line and cell culture. The human esophageal squamous carcinoma cell line TE-10 expressing CEA gene was obtained from Shanghai Institute for Biological Sciences and cultured in RPM1 1640 containing 2 mmol/L glutamine and supplemented with 15% fetal serum at 37°C in air containing 5% CO2.

To assess the sensitivity and get a calibration curve of the system for CEA mRNA detection, TE-10 cells grown in monolayer were collected by digesting the monolayer with PBS (pH 7.4) containing 0.25% trypsin and 0.53 mmol/L EDTA for 5 min at 37°C. After the digested monolayer was washed with PBS, the obtained cells were counted, and their viability was assessed by trypan blue dye exclusion. Finally, the TE-10 cells were serially diluted with PBS and mixed with 2 mL blood obtained from healthy volunteers to form a concentration gradient of tumor cells ranging from 1 to 105 cells per milliliter of blood.

Sample processing and cDNA synthesis. Five milliliters of each peripheral blood sample were divided as follows: 1.0 mL for counting monocytes, 2.0 mL for RNA extraction, and 2.0 mL for repetition. The samples for RNA extraction were processed immediately after collection and distribution to avoid RNA degradation. Mononuclear blood cells were firstly harvested by density centrifugation (1,800 rpm for 20 min) with lymphocytes separation medium and washed with PBS (800 rpm for 10 min). They were then counted. The total RNA in these cells was extracted using TRI REAGENT (Molecular Research Center, Inc.), and its purity and quality were measured by UV-visible spectrophotometer (Bio-Tek); 1% agarose gel electrophoresis was used to assess the integrity of the obtained RNA. cDNA with a total volume of 20 µL was synthesized using commercially available RevertAid First Strand cDNA Synthesis kit #k1622 containing reverse transcriptase (Fermentas) according to the recommended protocol by the manufacturer.

Real-time quantitative PCR. Real-time quantitative PCR of the target CEA gene and GAPDH as internal control was carried out with icycler iQ Multicolor Real-time PCR Detection System (Bio-Rad Laboratories, Inc.). According to their sequences, amplification primers and Taqman probes of the two genes were synthesized (DaAn Gene Co. Ltd.) as follows: CEA, forward primer 5'-CCGCATACAGTGGTCGAGAGA-3', reverse primer 5'-GTGTAGGGTGTAGAATCCTGTGTCA-3', probe 5'-CCCAATGCATCCCTGCTGATCCAG-3'; GAPDH, forward primer 5'-CCACCCATGGCAAATTCC-3', reverse primer 5'-TGGGATTTCCATTGATGACAAG-3', probe 5'-TGGCACCGTCAAGGCTGAGAACG-3'.

The probes were labeled at the 5'-end with reporter dye molecule FAM and the 3'-end with quencher dye TAMRA. The primers were designed to extend across at least one intron so that eventual DNA contamination would not pose a significant problem. Corresponding length of PCR products of CEA and GAPDH mRNA was 92 and 69 bp, respectively. PCR was done with 400 ng of total RNA.

Calculation of results and experimental controls. PCR product of CEA mRNA in positive control was purified and cloned into pMD-18T. The recombination plasmids were further purified and quantified with UV-visible spectrometry. The obtained solution was diluted at different dilutions to prepare the standard solutions (N1, copies per microliter). A curve showing the linear correlation between the cycle threshold (Ct) value and logarithm of plasmids or CEA mRNA copies of the quantified vectors was then obtained. This calibration curve was modified for each run to eliminate the errors caused by the difference in PCR efficiency. The calibration curve showing the correlativity between the total CEA mRNA copies (N2) and the number of CEA mRNA-positive cells per milliliter was obtained with the serial diluted suspensions of TE-10 cells as standard samples.

The number of CEA mRNA-positive cells was calculated with the obtained Ct value and the two calibration curves obtained above. N1 was first obtained from the Ct value. N2 was then calculated according the equation: Formula, where 20 was the total volume of cDNA synthesized in microliters, V1 was the total volume of total RNA extracted from 2.0 mL peripheral blood sample, and V2 was the volume corresponding to 2.0 µg total RNA for reverse transcription, which was ascertained by UV-visible spectrometry at A260.

The reliability of the results was assessed by comparing the expression of GAPDH or CEA mRNA with those from preliminary experiments. Total RNA was extracted from 10 healthy volunteers for GAPDH mRNA and from positive quality control for CEA mRNA. Real-time quantitative PCR was done for 10 times with 400 ng of total RNA each time, from which the average Ct values (Ct) and deviations (d) for GAPDH and CEA mRNA were calculated, respectively. If the obtained Ct value of GAPDH mRNA was beyond the range of Ct ± 4d, or that of CEA mRNA in each run was not in the range of Ct ± SD, we ran the sample again.

Statistics. Data analysis was carried out with Stata statistical packages. Mann-Whitney test was used to compare the CEA mRNA levels in peripheral blood between (a) B-1 and B0 and (b) B0 and B+3. When all results of CEA mRNA expression were negative at B-1, B0, and B+3, the data would be abandoned in statistics. When one or two of three results were positive, the other expression would be treated as 1 for statistical convenience. The relationship between the amount of CEA mRNA-positive cells in peripheral blood sample and classic prognostic factors, such as the tumor location, size, tumor-node-metastasis stage, and pathologic stage, was investigated using Man-Whitney test (for comparison between two groups) or Kruskal-Wallis test (for comparison among three groups), respectively. The relationship between expression of CEA mRNA and classic prognostic factors was analyzed using the {chi}2 test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Evaluation of the system. In preliminary experiments, CEA and GAPDH mRNA extracted from TE-10 cells were amplified. No amplification product was detectable in absence of RT enzyme, indicating the primers were appropriately designed to avoid the contamination from DNA derived from the processed pseudogene. All eight tissue samples showed positive results after real-time quantitative RT-PCR of CEA mRNA. The Ct and SD values of target CEA mRNA in the positive control were 29.7 and 0.91 (n = 10, inter-assess), whereas these values of GAPDH mRNA from healthy volunteers were 14.8 and 0.33 (n = 10, inter-assess), respectively.

The calibration curves for detection of mRNA-positive cells were shown in Fig. 1 . The minimum amount of 3 CEA mRNA-positive cells per milliliter of peripheral blood could be detectable, which was used as the detection limit of the proposed method. To assess the effect of leukocyte concentration in peripheral blood on the detection, 2 x 103 TE-10 cells were added to 2.0 mL peripheral blood containing different concentrations of leukocytes (5 x 106 to 1 x 107 per milliliter). The results of detectable TE-10 cells gave a coefficient of variation of 9.5% (n = 10, inter-assess).


Figure 1
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  		Fig. 1. Calibration curves for quantification. A, CEA mRNA copies. B, CEA mRNA-positive cells. 95% confidence interval. N1, CEA mRNA copies per microliter; N2, standardized CEA mRNA copies.

 
The cells expressing CEA mRNA were lower than the detection limit in either the samples from 22 patients without any evidence of malignancy who underwent thoracotomy at three time points or the samples from 30 healthy volunteers. Although the scale of the control group was not large due to the limited amount of such patients in our hospital during the period, data from these two groups could be regarded as competent controls.

Detection of CEA mRNA-positive cells in peripheral blood samples from esophageal cancer patients. One hundred fifty-five peripheral blood samples from 53 esophageal cancer patients (four samples of B+3 were absent because of sampling failure) were analyzed. The medians of CEA mRNA-positive cells at B-1, B0, and B+3 were 188 (95% confidence interval, 155-498), 1,513 (95% confidence interval, 660-7,974), and 707 (95% confidence interval, 737-3,005) cells per milliliter of peripheral blood, respectively. The change in the number of CEA mRNA-positive cells upon surgery was shown in Fig. 2A .


Figure 2
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  		Fig. 2. Distributions of CEA mRNA-positive cells numbers and developed metastasis. A, B0 to B-1; B, B+3 to B0. If cells number was lower than the detection limit, its value was treated as 2 for calculating convenience. The percentages were the proportion of the patient number with marked ratios in (A) 53 patients and (B) 14 patients. P values were calculated using {chi}2 test.

 
Using the minimum amount of detectable cells as a cutoff, 28.3% (15 of 53), 60.4% (32 of 53), and 42.9% (21 of 49) of B-1, B0, and B+3 peripheral blood samples were considered CEA mRNA positive, respectively. The CEA mRNA expression status in different samples was shown in Table 1 . The distribution of CEA mRNA-positive cells at different time points was illustrated in Fig. 3 .


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  		Table 1. Results of the real-time quantitative PCR for CEA expression

 

Figure 3
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  		Fig. 3. Box-whisker comparative plots of the numbers of CEA mRNA-positive cells per milliliter of peripheral blood of esophageal cancer patients before surgery (B-1; n = 53), immediately after surgery (B0; n = 53), and on the 3rd day after surgery (B+3; n = 49).

 
Clinical correlation. With different classic prognostic factors of gender, age, tumor location, size, depth, pathologic stage, and lymph node status, the mean numbers of detectable CEA mRNA-positive cells in peripheral blood collected at the same time points did not show statistical difference except the mean numbers at B0 for the ages <60 and >60. However, the positivity of CEA mRNA-expressed cells was statistically related to some factors, such as lymph node status and pathologic stage (Table 2 ). Patients with positive lymph nodes, who were known to have a worse survival, showed higher positivity of CEA mRNA-expressed cells than those with negative lymph nodes; and patients with stage III disease, who had a worse outcome than those with stage II, seemed to have a high positivity of CEA mRNA-expressed cells.


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  		Table 2. Correlation between CEA mRNA-positive cells detectable at B-1, B0, or B+3 and classic prognostic factors in 53 esophageal cancer patients undergoing resection

 
When 155 peripheral blood samples from 53 esophageal cancer patients were considered as three groups according to the time points, the median of CEA mRNA-positive cells showed statistically significant difference between B-1 and B0 (P = 0.0001) and between B-1 and B+3 (P = 0.0209), but no statistically significant difference occurred between B0 and B+3 (P = 0.4396). When the patients were divided into different subgroups according to each classic prognostic factor, statistically significant difference from B-1 to B+3 could be observed except two when the tumors were at the upper location and in the pathologic stage II (Table 3 ).


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  		Table 3. Difference of the amounts of peripheral blood CEA mRNA-positive cells of esophageal cancer patients from B-1 to B+3 classified with classic prognostic factors

 
Follow-up. The follow-up of 45 patients who were successfully sampled at B+3 was available in 1 year. Thirteen patents showed the developed metastasis, and three of them died of the metastasis. In these 13 patients, four came from those showing undetectable CTC at all three sampling points, and nine came from 28 patients who showed detectable CTC at B0. The developed metastasis was statistically irrelative to the ratio of B0/B-1 CTC (Fig. 2A). To assess the correlation between the CTC level and metastasis possibility, another variable R was defined as the ratio of CTC values at B+3 to B0. Fifty percent (7 of 14) of patients who had the R > 0.4 produced the developed metastasis, which was significantly higher than 14.3% (2 of 14) of patients who showed the R < 0.4 (P = 0.043), as shown in Fig. 2B.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Since its establishment, real-time quantitative RT-PCR has been considered as a sensitive method to detect CTC and applied in various carcinomas (27–29). It provides a formidable tool for cancer therapy personalization (30–32). However, experimental variation of this technique is easily brought due to the complicated sample processing and highly susceptive amplification (33, 34). The obtained results indicated acceptable specificity of the proposed procedure. No evidence indicated that the stimulating factors in thoracotomy manipulation could change the CEA mRNA expression, although cytokines, steroids, or environmental factors could modulate the expression of some tissue-specific genes such as CK19 (35); 59.2% (29 of 49) of patients at B0 and 42.9% (21 of 49) of patients at B+3 showed CEA mRNA expression, and 20 patients (20 of 53, 40.8%) had no evidence of measurable CTC at B-1, B0, and B+3. These results were similar to those of 51.9% immediately after resection (17) and 36.8% in follow-up period (36). Such positive detection rate of CTC was due to the CTC level lower than the detection limit or absent CEA expression in esophageal cancer tumor cells, which could be overcome using multiple-marker detection (37).

This method could detect CEA mRNA-positive cells down to 3 cells per milliliter of peripheral blood, indicating high sensitivity due to the high RNA extraction efficiency and the standardized detection procedure. The leukocytes showed little effect on CEA mRNA detection. The coefficient of variation of 9.5% for CEA mRNA-positive cell detection at different concentrations of leukocytes indicated that the effect of leukocyte change during surgery was negligible. It means that the proposed calibration curve method was superior to relative quantitative PCR, and its sensitivity and specificity were enough to determine the CTC for elucidating the effect of esophageal cancer surgery on CTC in the peripheral blood.

The results showed that the peripheral blood samples from nine esophageal cancer (9 of 49, 18.4%) patients (5/4 for N0/N1 of lymph node status, 3/0/6 for I/II/III of pathologic stage, and 3/4/2 for pT1/pT2/pT3 of tumor depth) without preoperative CTC were found to contain tumor cells at both B0 and B+3. Five of them showed the developed metastasis. Twelve esophageal cancer patients (12 of 49, 24.5%) showed peripheral blood–positive results of CEA mRNA expression at all three time points; moreover, the number of CEA mRNA-positive cells in peripheral blood samples was significantly elevated (P = 0.0003) from the median value of 209 to that of 4,062 following the tumor removal manipulation. The median value of 1,178 at B+3 was still higher than that before surgery, which did not significantly decrease when compared with the number of CEA mRNA-positive cells at B0. Although six patients (4/2 for N0/N1 of lymph node status, 3/1/2 for I/II/III of pathologic stage, and pT1/pT2/pT3 of tumor depth) showed negative results of CEA mRNA expression in peripheral blood samples collected at both B-1 and B+3, all of them showed positive results at B0. The tumor resection led to significant increase of CEA mRNA-positive cells in peripheral blood samples. The facts supported the hypothesis that surgical manipulation would cause tumor cells to shed into peripheral blood. The disappearance of CEA mRNA-positive cells in peripheral blood samples collected from these six patients at B+3 might be attributed to the functions of body immune system because significant increases of T-cell counts were also observed in these patients (data not shown).

Pantel et al. (38, 39) suggested that the detection and characterization of disseminated tumor cells in patients with cancer could provide important information about the cascade of metastatic events, and it was important to know how many disseminated epithelial cells were actually of neoplastic origin. The extent of CEA mRNA-positive cells increased upon surgery revealed the proportion of CEA mRNA-positive cells entering into peripheral blood. Five patients showed a consecutive increase from B-1 to B+3 in the number of CEA mRNA-positive cells, and three of them had lymph node–positive disease, suggesting that tumor cells originated from other minimal focus might also enter into peripheral blood by the stimulation of surgical operation in these patients. The positivity of patients with lymph node–negative disease was 26.3%, 52.6%, and 34.2% at B-1, B0, and B+3, respectively, whereas it was 33.3%, 80%, and 53.3% for patients with lymph node–positive disease, respectively. Furthermore, the median value of CEA mRNA-positive cells at B+3 in the patients with the lymph node metastasis beside tumor tissue was 2,689 (n = 9), and the value in the patients with the lymph node metastasis at other location was 163 (n = 6). These results implied that the regional lymph nodes, particularly the lymph node metastasis beside tumor tissue, might lead to higher positivity of CEA mRNA cells or more tumor cells dissemination in peripheral blood. This information would be complementary with those reported with pathologically lymph node–negative patients (11, 40).

The survival status of CTC in peripheral blood was still unknown. Mehes et al. (41) reported that apoptotic CTC in peripheral blood had a significant percentage in breast cancer, whereas Meng et al. (42) observed the CTC could remain for as long as 22 years. Here, CEA mRNA-positive cells increased significantly at B0 and maintained at a higher expression level at B+3 than B-1. It suggested that the tumor cells not only shed into the bloodstream by esophageal cancer operation manipulation but also existed to some extent at B+3. This would increase the possibility of neoplasia. The data from follow-up in 1 year showed the increasing possibility. When the ratio of CTC values at B+3 to B0 was >0.4, 50% of patients produced the developed metastasis, whereas it occurred in only 14.3% of patients who showed the ratio < 0.4. At the same time, 69% of patients (9 of 13) with developed metastasis had detectable CTC at both B0 and B+3. Only 4 of 13 patients with no detectable CTC showed the developed metastasis due to other factors, which needed some new techniques for further study.

Another interesting observation was that the extent of tumor cells dissemination into peripheral blood by surgical manipulation was larger in esophageal cancer than those in breast cancer (11). The surgical manipulation of breast cancer showed the relative increase of median of CK19-positive cells in peripheral blood from 109 to only 179. These phenomena might be helpful to reveal the reason of high recurrence and poor survival in esophageal cancer patients. To testify this, further investigations with these patients would be carried out.


   Footnotes
 
Grant support: The National Science Funds for Distinguished Young Scholars grant 20325518, Creative Research Groups grant 20521503, National Natural Science Foundation of China Key Program grant 20535010, Science Foundation of Jiangsu grants BS2006006 and BS2006074, and Tumor Hospital of Jiangsu Province/Scientific Research Foundation grant ZQ200402.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: J. Zhao and H. Ju, as well as their institutes, contributed equally to this work.

Received 8/18/06; revised 3/ 1/07; accepted 3/19/07.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2006. CA-Cancer J Clin 2006;56:106–30.[Abstract/Free Full Text]
  2. Bancewicz J, Clark PI, Smith DB, et al. Surgical resection with or without preoperative chemotherapy in oesophageal cancer: a randomised controlled trial. Lancet 2002;359:1727–33.[CrossRef][Medline]
  3. Romsdal MM, McGrath RG, Hoppe E, et al. Experimental model for the study of tumor cells in the blood. Acta Cytol 1965;9:141–5.[Medline]
  4. Liotta LA, Kleinerman J, Sadiel GM. Quantitative relationship of intravascular tumor cells, tumor vessels and pulmonary metastases following tumor implantation. Cancer Res 1974;34:997–1004.[Abstract/Free Full Text]
  5. Nishizaki T, Matsumata T, Kanematsu T, et al. Surgical manipulation of VX2 carcinoma in the rabbit liver evokes enhancement of metastases. J Surg Res 1990;49:92–7.[CrossRef][Medline]
  6. Izbicki JR, Hosch SB, Pichlmeier U, et al. Prognostic value of immunohistochemically identifiable tumor cells in lymph nodes of patients with completely resected esophageal cancer. N Engl J Med 1997;337:1188–94.[Abstract/Free Full Text]
  7. Weitz J, Kienle P, Lacroix J, et al. Dissemination of tumor cells in patients undergoing surgery for colorectal cancer. Clin Cancer Res 1998;4:343–8.[Abstract/Free Full Text]
  8. Eschwege P, Dumas F, Blanchet P, et al. Haematogenous dissemination of prostatic epithelial cells during radical prostatectomy. Lancet 1995;346:1528–30.[CrossRef][Medline]
  9. Schumacher K, Haensch W, Röefzaad C, et al. Prognostic significance of activated CD8+ T cell infiltrations within esophageal carcinomas. Cancer Res 2001;61:3932–6.[Abstract/Free Full Text]
  10. Inoue H, Kajiyama Y, Tsurumaru M. Clinical significance of bone marrow micrometastases in esophageal cancer. Dis Esophagus 2004;17:328–32.[CrossRef][Medline]
  11. Godfrey TE, Raja S, Finkelstein SD, et al. Prognostic value of quantitative reverse transcription-polymerase chain reaction in lymph node-negative esophageal cancer patients. Clin Cancer Res 2001;7:4041–8.[Abstract/Free Full Text]
  12. Kijima F, Natsugoe S, Takao S, et al. Detection and clinical significance of lymph node micrometastasis determined by reverse transcription-polymerase chain reaction in patients with esophageal carcinoma. Oncology 2000;58:38–44.[CrossRef][Medline]
  13. Yoshioka S, Fujiwara Y, Sugita Y, et al. Real-time rapid reverse transcriptase-polymerase chain reaction for intraoperative diagnosis of lymph node micrometastasis: clinical application for cervical lymph node dissection in esophageal cancers. Surgery 2002;132:34–40.[CrossRef][Medline]
  14. Luketich JD, Kassis ES, Shriver SP, et al. Detection of micrometastases in histologically negative lymph nodes in esophageal cancer. Ann Thorac Surg 1998;66:1715–8.[Abstract/Free Full Text]
  15. Koike M, Hibi K, Kasai Y, et al. Molecular detection of circulating esophageal squamous cell cancer cells in the peripheral blood. Clin Cancer Res 2002;8:2879–82.[Abstract/Free Full Text]
  16. Kaganoi J, Shimada Y, Kano M, et al. Detection of circulating oesophageal squamous cancer cells in peripheral blood and its impact on prognosis. Br J Surg 2004;91:1055–60.[CrossRef][Medline]
  17. Nakashima S, Natsugoe S, Matsumoto M. Clinical significance of circulating tumor cells in blood by molecular detection and tumor markers in esophageal cancer. Surgery 2003;133:162–9.[CrossRef][Medline]
  18. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(–Delta Delta C) method. Methods 2001;25:402–8.[CrossRef][Medline]
  19. Ismail MS, Wynendaele W, Aerts JLE, et al. Detection of micrometastatic disease and monitoring of perioperative tumor cell dissemination in operable breast cancer patients using real-time quantitative reverse transcription-PCR. Clin Cancer Res 2004;10:196–201.[Abstract/Free Full Text]
  20. Aerts J, Wynendaele W, Paridaens R, et al. A real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to detect breast carcinoma cells in peripheral blood. Ann Oncol 2001;12:39–46.[Abstract/Free Full Text]
  21. Bieche I, Nogues C, Paradis V, et al. Quantitation of hTERT gene expression in sporadic breast tumors with a real-time reverse transcription-polymerase chain reaction assay. Clin Cancer Res 2000;6:452–9.[Abstract/Free Full Text]
  22. Ito S, Nakanishi H, Hirai T, et al. Quantitative detection of CEA expressing free tumor cells in the peripheral blood of colorectal cancer patients during surgery with real-time RT-PCR on a LightCycler. Cancer Lett 2002;183:195–203.[CrossRef][Medline]
  23. Patel K, Whelan PJ, Prescott S, et al. The use of real-time reverse transcription-PCR for prostate-specific antigen mRNA to discriminate between blood samples from healthy volunteers and from patients with metastatic prostate cancer. Clin Cancer Res 2004;10:7511–9.[Abstract/Free Full Text]
  24. Saintigny P, Coulon S, Kambouchner M, et al. Real-time RT-PCR detection of CK19, CK7 and MUC1 mRNA for diagnosis of lymph node micrometastases in non small cell lung carcinoma. Int J Cancer 2005;115:777–82.[CrossRef][Medline]
  25. Goldenberg DM, Sharkey RM, Primus FJ. Carcinoembryonic antigen in histology: immunoperoxidase staining of conventional tissue section. J Natl Cancer Inst 1976;57:11–22.[Medline]
  26. Gerhard M, Juhl H, Kalthoff H, et al. Neumaier M. Specific detection of carcinoembryonic antigen-expressing tumor cells in bone marrow aspirates by polymerase chain reaction. J Clin Oncol 1994;12:725–9.[Abstract]
  27. Bustin SA, Gyselman VG, Williams NS, et al. Detection of cytokeratins 19/20 and guanylyl cyclase C in peripheral blood of colorectal cancer patients. Br J Cancer 1999;79:1813–20.[CrossRef][Medline]
  28. de Vries TJ, Fourkour A, Punt CJA, et al. Reproducibility of detection of tyrosinase and MART-1 transcripts in the peripheral blood of melanoma patients: a quality control study using real-time quantitative RT-PCR. Br J Cancer 1999;80:883–91.[CrossRef][Medline]
  29. Kammula US, Lee KH, Riker AI, et al. Functional analysis of antigen-specific T lymphocytes by serial measurement of gene expression in peripheral blood mononuclear cells and tumor specimens. J Immunol 1999;163:6867–75.[Abstract/Free Full Text]
  30. Mocellin S, Keilholz U, Rossi CR, et al. Circulating tumor cells: the ‘leukemic phase’ of solid cancers. Trends Mol Med 2006;12:130–9.[CrossRef][Medline]
  31. Mocellin S, Rossi CR, Pilati P, Nitti D, Marincola FM. Quantitative real-time PCR: a powerful ally in cancer research. Trends Mol Med 2003;9:189–95.[CrossRef][Medline]
  32. Cristofanilli M, Hayes DF, Budd GT, et al. Circulating tumor cells: a novel prognostic factor for newly diagnosed metastatic breast cancer. J Clin Oncol 2005;23:1420–30.[Abstract/Free Full Text]
  33. Giulietti A, Overbergh L, Valckx D, et al. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 2001;25:386–401.[CrossRef][Medline]
  34. Niesters HGM. Quantitation of viral load using real-time amplification techniques. Methods 2001;25:419–29.[CrossRef][Medline]
  35. Jung R, Kruger W, Hosch S, et al. Specifi-city of reverse transcriptase polymerase chain reaction assays designed for the detection of circulating cancer cells is influenced by cytokines in vivo and in vitro. Br J Cancer 1998;78:1194–8.[Medline]
  36. Setoyama T, Natsugoe S, Okumura H, et al. Carcinoembryonic antigen messenger RNA expression in blood predicts recurrence in esophageal cancer. Clin Cancer Res 2006;12:5972–7.[Abstract/Free Full Text]
  37. Sher YP, Shih JY, Yang PC, et al. Prognosis of non-small cell lung cancer patients by detecting circulating cancer cells in the peripheral blood with multiple marker genes. Clin Cancer Res 2005;11:173–9.[Abstract/Free Full Text]
  38. Pantel K, Brakenhoff RH. Dissecting the metastatic cascade. Nat Rev Cancer 2004;4:448–56.[CrossRef][Medline]
  39. Pantel K, Muller V, Auer M, et al. Detection and clinical implications of early systemic tumor cell dissemination in breast cancer. Clin Cancer Res 2003;9:6326–34.[Abstract/Free Full Text]
  40. Sanchez-Cespedes M, Esteller M, Hibi K, et al. Molecular detection of neoplastic cells in lymph nodes of metastatic colorectal cancer patients predicts recurrence. Clin Cancer Res 1999;5:2450–4.[Abstract/Free Full Text]
  41. Mehes G, Witt A, Kubista E, et al. Circulating breast cancer cells are frequently apoptotic. Am J Pathol 2001;159:17–20.[Abstract/Free Full Text]
  42. Meng SD, Tripathy D, Frenkel EP, et al. Circulating tumor cells in patients with breast cancer dormancy. Clin Cancer Res 2004;10:8152–62.[Abstract/Free Full Text]




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