Ann Thorac Surg 2002;73:927-932
© 2002 The Society of Thoracic Surgeons
Original article: general thoracic
Clinical significance of telomerase activity in peripheral blood of patients with esophageal squamous cell carcinoma
Kazuo Koyanagi, MDa,
Soji Ozawa, MDa,
Nobutoshi Ando, MDa,
Yuko Kitagawa, MDa,
Masakazu Ueda, MD*a,
Masaki Kitajima, MDa
a Department of Surgery, School of Medicine, Keio University, Tokyo, Japan
Accepted for publication October 20, 2001.
* Address reprint requests to Dr Ueda, Department of Surgery, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
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Abstract
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Background. The presence of tumor cells in the blood stream is considered evidence of a high risk of distant organ metastasis. We examined the usefulness of telomerase activity in peripheral blood polymorphonuclear cells as an indicator of distant metastasis in patients with esophageal squamous cell carcinoma.
Methods. Telomerase activity was measured in the peripheral blood mononuclear cell and polymorphonuclear cell fractions obtained from blood samples of healthy volunteers mixed with squamous cell carcinoma cell lines, and cell distribution was analyzed by flow cytometry. Then telomerase activity of forty-two polymorphonuclear cell fractions obtained from esophageal squamous cell carcinoma patients was measured.
Results. Telomerase activity was detected in polymorphonuclear cell fractions and cell distribution analysis revealed the presence of esophageal squamous cell carcinoma cells. Organ metastasis was detected in 7 (78%) of the 9 patients with telomerase-positive polymorphonuclear cell fractions as opposed to only five (15%) of the 33 with telomerase-negative cases, and there was a significant positive correlation between telomerase activity and organ metastasis (p < 0.0008).
Conclusions. Measurement of telomerase activity in the polymorphonuclear cell fractions is useful for identifying a high risk group for distant organ metastasis in patients with esophageal squamous cell carcinoma.
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Introduction
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Esophageal squamous cell carcinoma (ESCC) is an aggressive cancer with a poor survival rate because of a strong tendency to spread through the blood stream or lymphatic system to form metastatic lesions, and the outcome of patients with distant organ metastasis is miserable [1]. Early relapse of the disease is observed in many patients after radical operation. Conventional clinicopathologic examinations cannot indicate the high-risk group of patients with ESCC for distant organ metastasis, but if it were possible, an appropriate therapeutic strategy could be selected.
The process of distant organ metastasis involves a series of steps in which tumor cells are released from the primary lesion and disseminate to distant organs where they proliferate to form new tumor foci [2, 3]. The presence of surviving tumor cells in blood is an essential of distant organ metastasis, however most tumor cells in the blood stream are thought to be dead [2–4]. Recently, developments in molecular biology such as immunochemistry and reverse transcriptase–polymerase chain reaction have enabled detection of circulating tumor cells in peripheral blood or bone marrow, and the presence of tumor cells in peripheral blood could indicate a high risk of organ metastasis [5–8].
Telomerase is a ribonucleoprotein enzyme that adds telomeric repeats onto the ends of chromosomes, compensating for an end replication problem [9, 10]. Telomerase activity has been detected in almost all immortal cancer cells, but not in many normal cells, except germline cells and peripheral blood mononuclear cells [11–14]. We also have demonstrated that ESCC cells have telomerase activity, but not in normal esophageal squamous cells [12, 13]. These studies suggest that the expression of telomerase activity in cancer cells is important to overcoming cellular senescence and maintaining chromosomal stabilization, and telomerase activity is considered a useful indicator of cancer cells [11–13].
Peripheral blood mononuclear cells have weak telomerase activity, and thus cancer cells must be separated from them if we use the telemetries activity to detect circulating cancer cells in peripheral blood. In this study, we established a new method of detecting the cancer cells in peripheral blood by examining for telomerase activity. We then measured telomerase activity in samples of peripheral blood obtained from patients with ESCC, and assessed the correlation between telomerase activity and organ metastasis to investigate the usefulness of telomerase activity as an indicator of the high-risk group for distant organ metastasis of ESCC.
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Material and methods
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Telomerase activity in peripheral blood
As a control, we measured telomerase activity in peripheral blood samples obtained from 10 healthy volunteers. The peripheral blood mononuclear cell (PBMC) fraction and peripheral blood polymorphonuclear cell (PBPC) fraction were isolated from heparinized blood by Polymorphprep (Daiichi-kagaku, Tokyo, Japan) density-gradient centrifugation at 450 x g for 30 minutes [15], and each sample was snap frozen in liquid nitrogen and stored at -80°C until used for telomerase assay. We provided the squamous cell carcinoma (SCC) cell lines from the esophagus (TE 1, TE 2, TE 3, TE 5, and TE 8) and vulva (A431), and mixed 1 x 106 of the cells with 10 mL of heparinized peripheral blood obtained from a healthy volunteer. The PBMC and PBPC fractions were isolated, and telomerase activity of each fraction was measured.
To analyze the sensitivity of telomerase activity, from 1 x 102 to 105 TE 8 cells were added to 20 mL of heparinized peripheral blood containing the 1 x 1011 normal hematopoietic cells obtained from a healthy volunteer, and the PBPC fraction was isolated and its telomerase activity was assayed. The ratios of cancer cells to normal hematopoietic cells ranged from 1:1 x 106 to 1:1 x 109.
Analysis of SCC cell distribution in peripheral blood
To analyze the distribution of SCC cells in peripheral blood, 1 x 106 TE 8 cells and A431 cells, which overexpress epidermal growth factor receptor (EGFR) were mixed with 10 ml of heparinized peripheral blood obtained from a healthy volunteer [16]. The PBMC and PBPC fractions were then isolated by Polymorphprep density-gradient centrifugation and washed with PBS, and samples of 1 x 106 cells were prepared. The mononuclear cells and polymorphonuclear cells were stained with fluorescein isothiocyanate conjugated (FITC)-labeled antilymphocyte antibody (CD3) and Cy3-labeled anti-EGFR antibody. Cell distribution was analyzed by flow cytometry (FACScan, Becton Dickinson Immunocytometry Systems, San Jose, CA). Then TE 8 was directly mixed with Cy3-labeled anti-EGFR antibody and FACScan analyzed its relative fluorescence.
Clinical samples
A total of 42 samples of peripheral blood were obtained from 42 consecutive patients with ESCC in Keio University Hospital between 1996 and 1997 (median age, 64 years; range, 45 to 84; 39 men and 3 women). Blood sampling of peripheral blood was performed before treatment. Informed consent was obtained from all patients who were included in the study. The PBMC and PBPC fractions were isolated and telomerase activity of each sample was measured. Roentgenogram, ultrasonography, and computed tomography studies were performed routinely to detect distant organ metastasis before treatment. Ultimately 22 patients were treated by radical operations and 20 patients received chemotherapy or radiation therapy, or both. Stage grouping was made according to the TNM classification for the esophagus [17]. The staging was made by histopathological findings in 22 patients that underwent operations and clinicopathologic studies in another 20 patients. After treatment, periodic examinations were performed to check for new metastatic lesions. In 3 patients distant organ metastasis was detected at the time blood samples were collected. The maximum patient follow-up period was 47 months, and the mean observation period in 39 patients without synchronous metastasis was 22 months after treatment.
Telomerase assay
Telomerase activity of each fraction was assayed as previously described (telomeric repeat amplification protocol) [11]. Samples were suspended in ice-cold wash buffer Cl2(10 mmol/L Hepes-KOH [pH 7.5], 1.5 mmol/L MgCl2, 10 mmol/L KCl, 1 mmol/L dithiothreitol), pelleted at 10,000 x g for 5 minutes at 4°C, washed and repelleted, and then homogenized and resuspended in 200 µL of ice-cold lysis buffer (10 mmol/L tris-HCl [pH 7.5], 1 mmol/L MgCl2, 1 mmol/L EGTA, 0.1 mmol/L 4-[2-aminoethyl] benzenesulfonyl fluoride hydrochloride, 5 mmol/L ß-mercaptoethanol, 0.5% 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate, 10% glycerol). The suspension was incubated for 30 minutes at 4°C, the lysate was centrifuged at 16,000 x g for 20 minutes at 4°C, and the supernatant was removed, rapidly frozen on dry ice, and stored at -80°C. Protein concentrations were measured with a Micro Bicinchoninic Acid Protein Assay Reagent kit (Pierce Chemical Co, Rockford, IL), and extracts were adjusted to 6 µg of protein for each assay. For ribonuclease treatment, 5 µL extracts were incubated with 1 µg of ribonuclease A for 20 minutes at 37°C. Extracts were assayed in 50 µL telomeric repeat amplification protocol reaction solutions containing 20 mmol/L tris-HCl (pH 8.3), 1.5 mmol/L MgCl2, 63 mmol/L KCl, 0.005% Tween 20, 1 mmol/L EGTA, 50 µmol/L of each deoxynucleotide triphosphate, 0.1 µg deoxyoligonucleotide TS primer (5'-AATCCGTCGAGCAGAGTT-3'), 1 µg T4 gene 32 protein (Boehringer Mannheim, Mannheim, Germany), 0.1 mg/mL bovine serum albumin, 2 units of AmpliTaq DNA polymerase (Perkin Elmer, Branchburg, NJ), 0.4 µL of (
-32P) deoxycytidine triphosphate and (-32P) thymidine triphosphate, in a 0.5 mL tube that contained 0.1 µg deoxyoligonucleotide CX primer (5'-CCCTTACCCTTACCCTTACCCTTA-3') on the bottom of the tube sealed in 7 µL molten wax (Ampliwax PCR Gem 100; Perkin Elmer). After 30 minutes of incubation at 23°C to allow telomerase-mediated extension of the TS primer, the reaction mixtures were heated at 90°C for 90 seconds to inactivate the telomerase. Assay tubes were then transferred to 31 polymerase chain reaction cycles at 94°C for 30 seconds, 50°C for 30 seconds, and 72°C for 50 seconds. The CX primer was liberated when the wax barrier melted at 70°C. The polymerase chain reaction products were analyzed by electrophoresis in 0.5 x tris-borate ethylenediaminetetraacetic acid on 10% polyacrylamide nondenaturing gels at 200 volts for 2 hours. The gels were then scanned with a Bioimage Analyzer (BAS 2000; Fuji, Tokyo, Japan), and the extracts that produced 6-base pair DNA ladders from 40-base pair, and were also sensitive to ribonuclease A, were considered telomerase-positive.
Statistical analysis
Patient groups were compared by using the 
2 test, and p values less than 0.05 were considered statistically significant.
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Results
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Telomerase activity in peripheral blood
No telomerase activity was detected in any of the PBPC fractions in the samples of peripheral blood obtained from 10 healthy volunteers, but it was detected in all PBPC fractions isolated from peripheral blood mixed with SCC cell lines (TE 1, TE 2, TE 3, TE 5, TE 8, and A431) (data not shown). Telomerase activity was detected in all PBMC fractions from both the healthy volunteers and peripheral blood mixed with SCC cell lines, and the activity was detected even with 0.6 µg of protein (data not shown).
The sensitivity of the analysis showed detection of telomerase activity in the PBPC fraction of peripheral blood mixed with 1 x 104 of TE 8. The ratio of cancer cells to normal hematopoietic cells was 1:1 x 107.
SCC cell distribution
We analyzed the PBPC and PBMC fractions of peripheral blood mixed with FITC-labeled antilymphocyte antibody (CD3) and Cy3-labeled anti-EGFR antibody. The FACScan revealed that the PBPC fraction showed strong fluorescence with the Cy3-labeled anti-EGFR antibody and no fluorescence with antilymphocyte antibody. In contrast, PBMC fraction showed strong fluorescence with FITC-labeled antilymphocyte antibody and no fluorescence with anti-EGFR antibody (Fig 1).
The FACScan also revealed the same results for A431 as for TE 8. The FACScan study demonstrated that TE 8 could be detected by the Cy3-labeled anti-EGFR antibody (Fig 2).
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Fig 1. Relative fluorescence of peripheral blood mononuclear cell (PBMC) and peripheral blood polymorphonuclear cell (PBPC) fraction obtained from peripheral blood mixed with squamous cell carcinoma cells that over express epidermal growth factor receptor (EGFR). (a) and (b) PBMC fraction. (a) Strong fluorescence with fluorescein isothiocyanate conjugated (FITC)-labeled anti-lymphocyte antibody, (b) but no fluorescence with anti-EGFR antibody. (c) and (d) PBPC fraction. (c) No fluorescence with anti-lymphocyte antibody, but strong fluorescence with Cy3-labeled anti-EGFR antibody. The thin arrows in panels (a) and (c) refer to the position of the relative fluorescence of FITC-labeled antilymphocyte antibody. The thick arrows in panels (b) and (d) refer to the position of the relative fluorescence of Cy3-labeled EGFR antibody. Therefore, lymphocytes are detected in only PBMC fraction, and SCC cells are detected in PBPC fraction.
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Fig 2. Relative fluorescence of TE 8 conjugated with Cy-3 labeled antiepidermal growth factor receptor antibody (arrow).
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Telomerase activity in clinical samples and correlation with distant organ metastasis
Telomerase activity was positive in nine (21%) of the 42 PBPC fractions obtained from patients with ESCC (Fig 3).
Distant organ metastasis was found in 7 (78%) of the 9 telomerase-positive patients; 2 patients had metastasis to both lung and liver, 4 to lung alone, and 1 to the liver (Table 1).
In 3 of these 7 patients, the metastatic lesions were detected before treatment, whereas in the other 4 patients there were no metastatic lesions before treatment, and the distant organ metastases were detected at 3, 3, 7, and 11 months after treatment, respectively. One of the 2 telomerase-positive patients without metastasis died of severe pneumonia within 5 months, and the other patient is alive with no recurrence for 40 months. Distant metastasis was not detected in any of the 33 patients with negative telomerase activity before treatment, but 5 (15%) of them had new metastatic lesions develop during the subsequent period (in the bone in 4 patients and in the skin in 1 patient at 6, 10, 23, 33, and 6 months after treatment, respectively). There was a significant positive correlation between telomerase activity and the presence of distant organ metastasis (p < 0.0008). Each factor in the TNM classification diagnosed by imaging (roentgenogram, ultrasonography and computed tomography), endoscopy, or surgical exploration, or a combination thereof, was compared in the telomerase-positive and telomerase-negative cases (Table 2).
Patients with positive telomerase activity in PBPC fractions had a higher incidence of T3 and T4 than those with negative telomerase activity, although the increases were not statistically significant, and the differences in the regional lymph nodes between the telomerase-positive and telomerase-negative groups were not significant. There were three cases of organ metastasis before treatment, and in the other 39 cases there was no significant difference in stage grouping between the telomerase-positive and telomerase-negative groups. We compared subsequent treatment in the two groups, but found no significant differences.
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Fig 3. Telomerase activity of the peripheral blood mononuclear cell (PBMC) and peripheral blood polymorphonuclear cell (PBPC) fraction obtained from patients with esophageal squamous cell carcinoma. Telomerase activity was detected in all PBMC fractions. In patients 1 and 2, telomerase ladders were negative in the PBPC fraction and distant organ metastasis was not detected. In patients 3 and 4, telomerase ladders were positive, and distant organ metastasis was detected. (M = PBMC fraction; P = PBPC fraction.)
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Table 1. Detection of Telomerase Activity and Distant Organ Metastasis in Peripheral Blood Polymorphonuclear Cell Obtained From Esophageal Squamous Cell Carcinoma Patients
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Table 2. Association Between Histopathologic Findings and Telomerase Activity in Peripheral Blood Polymorphonuclear Cells
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All samples of PBMC fractions obtained from patients with ESCC were positive for telomerase activity.
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Comment
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Distant organ metastasis is highly correlated with survival rate in patients with carcinoma of the esophagus [1]. The process of distant organ metastasis involves a sequence of steps in which tumor cells are released from the primary lesion and disseminated to distant organs. Based on the rationale that dissemination of the tumor cells is essential to the formation of metastatic lesions, the presence of tumor cells in the bloodstream increases the possibility of distant organ metastasis. Recent polymerase chain reaction studies of K-ras or p53 mutations and cytokeratins have demonstrated a small number of tumor cells in lymph nodes and peripheral blood [7, 18, 19]. However, the use of K-ras or p53 mutations is restricted to cases in which the primary tumor has a mutation. In addition, these methods may detect DNA in both viable and nonviable cancer cells [20]. Most circulating tumor cells tend to perish easily because of the mechanical trauma and host immune response [2, 3, 4], and it is necessary to detect viable tumor cells in peripheral blood and investigate the correlation with distant organ metastasis.
The negativity for telomerase activity in samples of the PBPC fraction obtained from healthy volunteers showed that they contained no immortalized cells. However, telomerase activity was detected in samples of the PBPC fraction isolated from peripheral blood mixed with SCC cell lines. Analysis by flow cytometry revealed that SCC cells and lymphocytes could be easily separated by the antibody used. The PBPC fraction showed strong fluorescence with the Cy3-labeled anti-EGFR antibody and no fluorescence with anti-lymphocyte antibody. The reverse was true for the PBMC fraction in which there was no fluorescence with anti-EGFR antibody but strong fluorescence with anti-lymphocyte antibody. Because SCC cells over express EGFR, these cells can be detected by the Cy3-labeled anti-EGFR antibody and will be found in the PBPC fraction. Telomerase activity in the PBPC fraction obtained from patients with ESCC was attributable to immortalized cells, such as cancer cells, and not to peripheral lymphocytes, which have weak telomerase activity. Therefore, the measurement of telomerase activity in the PBPC fraction is useful for detecting tumor cells in peripheral blood. Our method can detect one SCC cell in the background of 1 x 107 normal blood cells. It was more sensitive than immunocytochemistry and almost as sensitive as the reverse transcriptase–polymerase chain reaction method [7, 8, 21].
There were 3 patients with both distant organ metastasis and positive telomerase activity in the PBPC fraction synchronously. We followed another 39 patients after treatment; 6 patients had positive telomerase activity and 33 had negative telomerase activity, and 4 of the 6 telomerase-positive patients, as opposed to only 5 of the 33 telomerase-negative patients had metastatic lesions develop. These results showed that there was a significant positive correlation between telomerase activity in the PBPC fraction and the development of distant organ metastasis. These findings also demonstrated that telomerase activity in the PBPC fraction meant an increased possibility of distant organ metastasis and enabled us to predict the high-risk group for distant organ metastasis in patients with ESCC.
Two telomerase-positive PBPC patients did not develop distant metastasis. It was thought that 1 patient died before occurrence of metastasis. There must be a "fertile soil" for attachment and subsequent growth of circulating tumor cells. This theory may largely explain the result of another patient [2]. Five telomerase-negative PBPC patients had metastatic lesions develop during the following period, and there are three reasons to explain this phenomenon: (1) intermittent shedding of tumor cells and one-point sampling of peripheral blood [5]; (2) numbers of cancer cells present below lower limits of the assay; and (3) the possibility of tumor cell dissemination doing surgical manipulation. All 5 patients underwent radical operations after sampling. Tumor cell dissemination during an operation has been focused on, and several studies have disclosed this phenomenon [22, 23].
There was a clear tendency to metastasize to the lung or liver, or both, in the telomerase-positive PBPC patients. In contrast, metastasis was to bone and skin in telomerase-negative PBPC patients. We cannot explain the difference of this mode of metastasis, and other factors besides the bloodstream may have been affected.
Several investigators have reported that the presence of cancer cells in peripheral blood is a very useful indicator of distant organ metastasis [8, 24]. Our results showed that telomerase activity in the PBPC fraction was correlated with the development of metastatic lesions. Telomerase activity in the PBPC fraction of patients with ESCC indicates a high-risk group for development of distant organ metastasis. Such a group deserves more intensive staging before any planned resection and the need for close follow-up, and it may be a useful future marker indicating the need for adjuvant therapy.
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Acknowledgments
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We thank Ms S. Matsuda and Ms Y. Inaba for their technical assistance.This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan, Ministry of Health and Welfare of Japan, and a National Grant-in-Aid for the establishment of a high-tech research center in a private university of Japan.
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References
|
|---|
-
Roder J.D., Stein H.J., Siewert J.R. Oesophageal carcinoma. In: Hermanek P., Gospodarowicz M.K., Henson D.E., Hutter R.V.P., Sobin L., eds. Prognostic factors in cancer. Berlin: Springer-Verlag, 1992:37-46.
-
Fidler I.J., Gersten D.M., Hart I.R. The biology of cancer invasion and metastasis. In: Klein G., Weinhouse S., eds. . Advances in cancer research. New York: Academic Press Inc, 1978:149-250.[Medline]
-
Poste G., Fidler I.J. The pathogenesis of cancer metastasis. Nature 1980;283:139-146.[Medline]
-
Hanna N., Fidler I.J. Role of natural killer cells in the destruction of circulating tumor emboli. J Nat Cancer Inst 1980;65:801-809.
-
Glaves D., Huben R.P., Weiss L. Haematogenous dissemination of cells from human renal adenocarcinomas. Brit J Cancer 1988;57:32-35.[Medline]
-
Smith B., Selby P., Southgate J., Pittman K., Bradley C., Blair G.E. Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction. Lancet 1991;338:1227-1229.[Medline]
-
Kruger W., Krzizanowski C., Holweg M., et al. Reverse transcriptase/polymerase chain reaction detection of cytokeratine-19 mRNA in bone marrow and blood of breast cancer patients. J Cancer Res Clin Oncol 1996;122:679-686.[Medline]
-
Mori M., Mimori K., Ueo H., et al. Clinical significance of molecular detection of carcinoma cells in lymph nodes and peripheral blood by reverse transcription-polymerase chain reaction in patients with gastrointestinal or breast carcinomas. J Clin Oncol 1998;16:128-132.[Abstract/Free Full Text]
-
Levy M.Z., Allsopp R.C., Futcher A.B., Greider C.W., Harley C.B. Telomere end-replication problem and cell aging. J Mol Biol 1992;225:951-960.[Medline]
-
Greider C.W., Blackburn E.H. A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature 1989;337:331-337.[Medline]
-
Kim N.W., Piatyszek M.A., Prowae K.R., et al. Specific association of human telomerase activity with immortal cells and cancer. Science 1994;266:2011-2015.[Abstract/Free Full Text]
-
Koyanagi K., Ozawa S., Ando N., Takeuchi H., Ueda M., Kitajima M. Clinical significance of telomerase activity in non-cancerous epithelial region of oesophageal squamous cell carcinoma. Br J Surg 1999;88:116-122.
-
Koyanagi K., Ozawa S., Ando N., et al. Telomerase activity as an indicator of malignant potential in iodine-nonreactive lesion of the esophagus. Cancer 2000;88:1524-1529.[Medline]
-
Hiyama K., Hirai Y., Kyoizumi S., et al. Activation of telomerase activity in human lymphocytes and hematopoietic progenitor cells. J Immunol 1995;155:3711-3715.[Abstract]
-
Ferrante A., Thong Y.H. Optimal conditions for simultaneous purification of mononuclear and polymorphonuclear leucocytes from human blood by the hypaque-ficoll method. J Immunol Methods 1980;36:109-117.[Medline]
-
Yamamoto T., Kamata N., Kawano H., et al. High incidence of amplification of the epidermal growth factor receptor gene in human squamous carcinoma cell lines. Cancer Res 1986;46:414-416.[Abstract/Free Full Text]
-
International Union Against Cancer. TNM classification of malignant tumours, 5th edition New York: Wiley-Liss Inc, 1997.
-
Hayashi N., Ito I., Yanagisawa A., et al. Genetic diagnosis of lymph-node metastasis in colorectal cancer. Lancet 1995;345:1257-1259.[Medline]
-
Tamagawa T., Ueda M., Takahashi S., et al. Pancreatic lymph nodal and plexus micrometastases detected by enriched polymerase chain reaction and nonradioisotopic single-strand conformation polymorphism analysis: a new predictive factor for recurrent pancreatic carcinoma. Clin Cancer Res 1997;3:2143-2149.[Abstract]
-
Krismann M., Todt B., Schroder J., et al. Low specificity of cytokeratin 19 reverse transcriptase-polymerase chain reaction analysis for detection of hematogenous lung cancer dissemination. J Clin Oncol 1995;13:2769-2775.[Abstract]
-
Osborne M., Wong G.Y., Asina S., Old L.J., Cote R.J., Rosen P.P. Sensitivity of immunocytochemical detection of breast cancer cells in human bone marrow. Cancer Res 1991;51:2706-2709.[Abstract/Free Full Text]
-
Liotta L.A., Kleinerman J., Saidel G.M. The significance of hematogenous tumor cell clumps in the metastatic process. Cancer Res 1976;36:889-894.[Abstract/Free Full Text]
-
Choy A., McCulloch P. Induction of tumour cell shedding into effluent venous blood breast cancer surgery. Br J Cancer 1996;73:79-82.[Medline]
-
Mellado B., Colomer D., Castel T., et al. Detection of circulating neoplastic cells by reverse-transcriptase polymerase chain reaction in malignant melanoma: association with clinical stage and prognosis. J Clin Oncol 1996;14:2091-2097.[Abstract/Free Full Text]
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Abstract
Introduction
