Japanese Journal of Clinical Oncology 30:499-503 (2000)
© 2000 Foundation for Promotion of Cancer Research
Detecting Circulating Cancer Cells Using Reverse Transcriptase-Polymerase Chain Reaction for Cytokeratin mRNA in Peripheral Blood from Patients with Gastric Cancer
1Department of Surgery I and 2Department of Parasitology, National Defense Medical College, Tokorozawa, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Background: Cytokeratins (CKs) 19 and 20 have been used as targets for detecting cancer cells. We attempted to detect circulating cancer cells in patients with gastric cancer using a high-sensitivity reverse transcriptase-polymerase chain reaction (RT-PCR) assay for CK transcripts.
Methods: RT-PCR for CK 19 and CK 20 was performed on peripheral blood samples obtained from 52 patients with gastric cancer, from 24 of whom blood samples were collected on three occasions. Fourteen healthy volunteers served as controls.
Results: CK 19 and CK 20 were positive in five (9.6%) of 52 patients with gastric cancer. Of these five, four were classified into stage IV and the other stage I, according to the TNM Classification. In gastric cancer patients, three (12.5%) were positive in the 24 cases examined three times and two (7.1%) were positive in 28 cases examined only once. Among the stage IV cancer, positive cases for CK showed significantly lower survival rates than those negative for CK. Between CK 19 and CK 20 in the 24 cases examined three times, CK 19 was found to be more sensitive in detecting cancer cells. CK 20 was detected in one (7.0%) of 14 healthy volunteers, whereas CK 19 was not detected.
Conclusions: We conclude that repeated blood sampling may be desirable to detect circulating cancer cells in peripheral blood, even in patients with advanced gastric cancer; CK 19 may be superior to CK 20 in detecting these cells. The clinical significance of detecting occult cancer in peripheral blood remains to be determined.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cytokeratins (CKs), proteins of the intermediate filaments of epithelial cells, have been used as specific markers for tumor cells of epithelial origin (1,2). Among some 20 different isotypes, CK 19 and CK 20 are expressed more selectively by mucosal epithelial cells than the others (3).
In previous studies attempting to detect circulating cancer cells in peripheral blood, the reported positive rates varied widely although the same targets were used (4,5). Differing polymerase chain reaction (PCR) assay design may be one reason for the discrepancies, as may the sensitivity of the assay between CK 19 and CK 20 (6–9). In addition to these analytical factors, biological factors such as the possibility that cancer cells may be released from the primary cancer into the circulation intermittently have not been investigated. Comparing CK 20 with CK 19, CK 20 is likely to be the more useful target for the detection of circulating epithelial-derived tumor cells, because the presence of pseudogenes for CK 19 complicates the interpretation of data and CK 20 is more specifically expressed in the gastrointestinal tract than CK 19 (4,10,11). However, sensitivity and specificity have not yet been compared between CK 19 and CK 20 in the same PCR assays.
The aim of this study was to determine whether CK 19 and CK 20 can be used as markers for cancer cells in peripheral blood.
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MATERIALS AND METHODS |
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Blood Samples
Peripheral blood was obtained preoperatively from 52 patients with gastric cancer. Blood samples were collected three times at an interval of 1–2 weeks in 24 patients and only once in the remaining 28 patients. As controls, peripheral blood samples were taken from 14 healthy volunteers three times at intervals of 1–2 weeks. A venous catheter was inserted into a superficial vein and blood samples were collected; the initial 10 ml were discarded to reduce possible contamination with epidermal cells and the following 10 ml, drawn using a new syringe, were used for RNA extraction. The clinical stage of the patients’ cancer was assigned according to the International Union Against Cancer’s Tumor, Nodes, Metastasis (TNM) Classification (1997). The survival curves were generated using the Kaplan–Meier method and the significance of the difference in survival rate was determined by the generalized Wilcoxon test. A p value of <0.05 was considered significant. Informed consent for this study was obtained from all patients and healthy volunteers.
RNA Extraction
Total RNA was extracted from each whole blood sample by the acid guanidinium–phenol–chloroform technique using ISOGEN (Nippon Gene, Tokyo, Japan), according to the manufacturer’s protocol. RNA was dissolved in 20 ml of RNase-free water. The recovery and purity of RNA were determined by optical density measurements at 260 and 280 nm. The extracted RNA was stored at –80°C until use.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
First-strand cDNA was synthesized using Superscript II and an oligo(dT)12–18 primer (Life Technologies, Eggenstein, Germany). Aliquots (1 mg) of total RNA were preincubated with 1 mg of oligo (dT)12–18 primer in 24 ml of solution for 10 min at 65°C. After chilling on ice, 8 ml of 5-fold synthesis buffer, 1 ml of RNase inhibitor, 5 ml of 10 mM dNTP mixture (all from Boehringer Mannheim, Mannheim, Germany) and 4 ml (200 U/ml) of Superscript RT (Life Technologies) were added. The reaction mixture was then incubated for 80 min at 37°C. The reaction was terminated by heating at 70°C for 10 min and this reaction mixture was stored at –80°C.
PCR was carried out as described previously (12). The sequences of the primers used are shown in Table 1. To distinguish from contaminating genomic DNA, we selected both upstream primer and downstream primer at different exons. Integrity of the isolated RNA was demonstrated by RT-PCR analysis of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PCR products were visualized after electrophoresis and ethidium bromide staining on a UV transilluminator. The nucleotide sequence of the RT-PCR product was confirmed by the chain termination method. Briefly, after the amplified cDNAs had been subcloned into pBluescript II KS+ (Stratagene, La Jolla, CA), sequences were analyzed using an ABI Prism 310 DNA Sequencer (Perkin-Elmer, Norwalk, CT) using cycle sequencing with M13 primers.
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Each sample was examined in duplicate. CK expression in each patient or healthy volunteer was deemed positive when at least one sample showed a positive result.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sensitivity of the RT-PCR Assay for CKs
MKN-28 gastric cancer cells were diluted 10–1–10–6 in mononuclear cells obtained from healthy volunteers. RNA was extracted and subjected to reverse transcription. The assay used in this study detected one cancer cell in 106 mononuclear cells when either CK 19 mRNA or CK 20 mRNA was used as a target for RT-PCR (Fig. 1). DNA sequences of RT-PCR products in this study were consistent with those of GenBank (CK 19, Y00503; CK 20, X73501) and no mutation was found.
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Expression of CK 19 and CK 20 mRNA in Peripheral Blood Samples from Healthy Volunteers
A total of 42 RNA samples extracted from 14 healthy volunteers were subjected to RT-PCR. CK19 expression was not detected in any sample. However, CK20 expression was observed in two of the three samples from one healthy donor (7.1%).
Expression of CK 19 and CK 20 mRNA in Peripheral Blood Samples from Gastric Cancer Patients
Five (9.6%) of 52 patients with gastric cancer were positive for both CK 19 and CK 20 (Table 2). Among the 24 patients from whom blood samples were collected three times at intervals of 1–2 weeks, three (12.5%) were positive for CK 19 and CK 20. Two (7.1%) of the remaining 28 patients, from whom only a single blood sample was collected, were positive for CK 19 and CK 20 (Table 2). On a sample basis, the positive rates for CK 19 and CK 20 were 9.0% (9/100 samples) and 5.0% (5/100 samples), respectively. Among the three patients positive for CK, in whom blood samples were collected three times, CK 19 was detectable in two or three samples, whereas CK 20 was detected in only one sample (Table 3).
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Characteristics and Prognoses of Patients Positive for CK
Four of the five patients positive for CK had tumors of the diffuse type. Four patients had advanced gastric cancer at stage IV. All patients with liver metastasis in this study were positive for CK and one of four patients with peritoneal metastasis was also positive for CK. In the 11 patients at stage IV, follow-up studies concentrating on survival (Kaplan–Meier calculation) were performed. The four patients positive for CK had a significantly lower survival rate than the seven patients negative for CK (Fig. 2; p < 0.05). The mean survival times for those positive and those negative for CK were 3.67 ± 1.20 and 12.4 ± 2.99 months, respectively. No significant differences in background factors existed between the patients positive and negative for CK (Fisher’s exact probability test, Table 4). CK 19 and CK 20 mRNA were detected in one patient with stage IB cancer; the patient had a tumor invading into the submucosa, measuring 65 x 60 mm, that had metastasized to the lymph nodes along the greater curvature (Table 3). This patient was negative for both CK 19 and CK 20 mRNA in peripheral blood at 6 months after resection and is alive without any sign or symptom of recurrence at 18 months after surgery.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, specificity of the RT-PCR assay used to detect CK transcripts was enhanced by the following measures: the initial 10 ml of blood were discarded to reduce possible contamination with epidermal cells, the selection of a target gene without known pseudogenes and the selection of primer locations to distinguish from contaminating genomic DNA. This system showed a sensitivity of 1:10–6 in cell dilutions and the identification of amplification products was confirmed by sequence analysis. Results were reliably reproduced on several occasions.
Peripheral blood samples obtained from five (7.7%) of 52 patients with gastric cancer showed positive results for CK 19 and CK 20 in the RT-PCR assay. All three patients with liver metastasis also showed positive results.
There have been several reports attempting to detect the existence of occult cancer cells in peripheral blood by an RT-PCR assay detecting CK transcripts. Soeth et al. (13,14) detected CK 20 in a stage-dependent manner in patients with colorectal cancer. Wyld et al. (15) detected CK 20 mRNA only in samples from patients with stage IV colorectal cancer. Aihara et al. (8) reported that CK 19 mRNA was not detected in patients with gastric cancer even with it in an advanced stage. The difference in the positive rate among the studies may be explained by the sensitivity of the RT-PCR assays and target genes used. In this study, the positive rate for CK was 12.5% when blood samples were collected three times at intervals of 1–2 weeks in each patient, whereas it was 7.1% when blood samples were collected only once. The increased rate could be the result of intermittent release of tumor cells from the primary tumor, which may also explain the differences in positive rates among the previous reports. Another explanation is that all gastric cancer cells might not express the CK genes. The possibility of false-negative results should be considered.
CK 19 tended to be more sensitive than CK 20 as a target for cancer cells in the peripheral blood in this study. One of 14 healthy volunteers was positive for CK 20 by the RT-PCR assay, whereas no positive result was obtained for CK 19. Contamination or amplification of DNA seems unlikely, because two of three samples were positive for CK 20 for this patient. CK was reported to be transcribed under certain conditions, such as chronic inflammation or cytokine stimulation (16). He might have suffered from occult disease or chronic inflammation. Wyld et al. (15) also detected CK 20 mRNA in one of 12 normal blood samples. Destruction of epithelial cells should be considered to be one of the possibilities of false-positive results. According to a recent study, CK 20 expression is not limited to epithelium and detection of CK 20 mRNA depends not only on the PCR sensitivity but also background transcription (17–19).
The patients positive for CK showed a lower survival rate than those negative for CK in stage IV cancer. The patient with stage IB cancer was positive for CK 19 and CK 20; both were negative after tumor resection. The significance of the CK expression in this patient is still to be determined. Semi-quantitative RT-PCR for CK genes that estimate the number of cancer cells might speculate their prognosis among the CK-positive patients.
We conclude that repeated blood sampling may be desirable to detect circulating cancer cells in peripheral blood even in patients with advanced gastric cancer. CK 19 might be superior to CK 20 for the detection of cancer cells in peripheral blood. It should be considered when detecting circulating cancer cells in peripheral blood that CK gene transcripts may not always express cancer cell origin. The clinical significance of detecting occult cancer in peripheral blood remains to be determined.
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FOOTNOTES |
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+ For reprints and all correspondence: Takashi Majima, Department of Surgery I, National Defense Medical College, Tokorozawa 359-8513, Japan. E-mail: majima@ndmc.ac.jp
Abbreviations: ck, cytokeratin; RT-PCR, reverse transcriptase-polymerase chain reaction
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REFERENCES |
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Received May 12, 2000; accepted September 1, 2000.
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