Gerhard, M, Juhl, H, Kalthoff, H, Schreiber, HW, Wagener, C & Neumaier, MSpecific detection of carcinoembryonic antigen-expressing tumor cells in bone marrow aspirates by polymerase chain reaction. J Clin Oncol 12: 725-729
Abteilung für Klinische Chemie, Universitäts-Krankenhaus Eppendorf, Hamburg, Germany. Journal of Clinical Oncology
(Impact Factor: 18.43).
To establish a sensitive assay for the specific detection of carcinoembryonic antigen (CEA)-expressing tumor cells in the bone marrow of patients with colorectal cancer and other CEA-positive carcinomas.
A CEA-specific nested reverse transcriptase (RT) polymerase chain reaction (PCR) assay was developed and optimized using limiting dilutions of a CEA-positive cancer cell line mixed with normal bone marrow cell specimens. The optimized test was then used to examine bone marrow samples obtained from 15 patients with abdominal carcinomas (colorectal, n = 10; pancreatic, n = 3; gastric, n = 2) and six patients with breast cancer. Specificity was assessed by examination of 56 negative controls (malignant hematologic disease, n = 28; nonmalignant disease conditions, n = 5; healthy bone marrow donors, n = 8; normal peripheral-blood samples, n = 15). For 11 patients with abdominal carcinomas, immunostaining evaluations were performed using an anti-CEA and an anticytokeratin antibody, and the results compared with the nested PCR assay.
In the sensitivity calibration system, single CEA-expressing tumor cells were detected in 2 to 5 x 10(7) normal bone marrow cells. All 56 control samples failed to amplify. This demonstrates that mRNAs coding for highly homologous CEA-related antigens expressed by various lineages of blood cells do not interfere. Bone marrow samples from 10 of 15 patients with abdominal cancers and four of six breast cancer patients scored positive, indicating micrometastatic bone disease. Four of 11 samples from the gastrointestinal cancer patients were found to be positive by the PCR method, but were negative with the immunocytology method.
Since approximately 30% of the colorectal carcinoma patients that score negative in immunocytology staining of bone marrow samples have been reported to relapse, earlier diagnosis of the presence of malignant cells is needed. Our result that samples scoring positive in the described CEA-specific PCR test remained negative by two immunostaining methods suggests a higher sensitivity. We conclude that PCR amplification of CEA mRNA may lead to an earlier diagnosis of micrometastatic bone disease in patients with CEA-expressing carcinomas.
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- "New methods like immunohistochemicaland flow cytometrymethod using the monoclonal antibodies to detect tumor-related antigens could partly improve the sensitivity and specificity over routine cytology. In the 1990s, the extremely high sensitivity of RT-PCR technique made it possible to diagnose micrometastasis on the cancer tissue–specific mRNA expression in peripheral vein, bone marrow, and lymph nodes.In 1997, Nakanishi etresearches adopted this RT-PCR–based method for detecting IFCCs because of its higher sensitivity than conventional cytology. Moreover, the use of multiple biomarkers like CEA, CK20, CK19, and MMPfor RT-PCR assay could greatly improve both the sensitivity and the specificity of detecting IFCCs and make it a reliable way for clinical use. "
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ABSTRACT: OBJECTIVE: To assess the effect of hyperthermic intraperitoneal chemotherapy (HIPEC) to eradicate intraperitoneal free cancer cells and to explore the feasibility of cytological cure for peritoneal carcinomatosis (PC). METHODS: The peritoneal lavage fluid (or ascites) from 50 PC patients was collected before and after intraoperative HIPEC, respectively, for conventional cytology test, and conventional and real-time quantitative reverse transcript polymerase chain reaction detecting carcinoembryonic antigen (CEA) mRNA and cytokeratin-20 (CK20) mRNA. The blood samples 3 days before and 7 days after intraoperative HIPEC were also collected for detecting the serum tumor markers, including CEA, carbohydrate antigen (CA) 125, and CA19-9. RESULTS: The positive rate of conventional cytology test before HIPEC versus after HIPEC was100.0% versus 22.0% (P = .000). The positive rates of CEA mRNA and CK20 mRNA before HIPEC versus after HIPEC were 100.0% versus 86.0% (P = .012) and 100.0% versus 96.0% (P = .495), respectively. Moreover, after HIPEC, 18 (36.0%) patients had a decline in CEA mRNA (P = .000), and 17 (34.0%) patients had a decline in CK20 mRNA (P = .000). The positive rates of serum CEA, CA125, and CA199 before HIPEC versus after HIPEC were 52.0% versus 28.0% (P = .014), 52.0% versus 44.0% (P = .423), and 40.0% versus 28.0% (P = .205), respectively. CONCLUSION: HIPEC could effectively eradicate intraperitoneal free cancer cells and partially achieve cytological cure for PC.
Available from: Miao-Zhen Qiu
- "A CEA-specific oligonucleotide primer was designed based on the report by Gerhard et al. . The sequences were: 5'-TGTCGGCATCATGATTGG-3' (sense) and 5'-GCAAATGCTTTAAGGAAGAAGC-3' (antisense). "
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ABSTRACT: The existence of circulating tumor cells (CTCs) in peripheral blood as an indicator of tumor recurrence has not been clearly established, particularly for gastric cancer patients. We conducted a retrospective analysis of the relationship between CTCs in peripheral blood at initial diagnosis and clinicopathologic findings in patients with gastric carcinoma.
Blood samples were obtained from 123 gastric carcinoma patients at initial diagnosis. mRNA was extracted and amplified for carcinoembryonic antigen (CEA) mRNA detection using real-time RT-PCR. Periodic 3-month follow-up examinations included serum CEA measurements and imaging.
The minimum threshold for corrected CEA mRNA score [(CEA mRNA/GAPDH mRNA) × 106] was set at 100. Forty-five of 123 patients (36.6%) were positive for CEA mRNA expression. CEA mRNA expression significantly correlated with T stage and postoperative recurrence status (P = 0.001). Recurrent disease was found in 44 of 123 cases (35.8%), and 25 of these (56.8%) were positive for CEA mRNA. Of these patients, CEA mRNA was more sensitive than serum CEA in indicating recurrence. Three-year disease-free survival of patients positive for CEA mRNA was significantly poorer than of patients negative for CEA mRNA (P < 0.001). Only histological grade and CEA mRNA positivity were independent factors for disease-free survival using multivariate analysis.
CEA mRNA copy number in peripheral blood at initial diagnosis was significantly associated with disease recurrence in gastric adenocarcinoma patients. Real-time RT-PCR detection of CEA mRNA levels at initial diagnosis appears to be a promising predictor for disease recurrence in gastric adenocarcinoma patients.
Available from: Sumiya Ishigami
- "Some authors have proven that RT – PCR can detect lymph node micrometastasis in gastrointestinal tract or breast cancers (Mori et al, 1995; Matsumoto et al, 2002). Circulating cancer cells in the bone marrow or peripheral blood and free cancer cells in the peritoneal lavage fluid have been detected by RT – PCR (Gerhard et al, 1994; Burchill et al, 1995; Marutsuka et al, 2003). Thus, RT – PCR-based techniques can detect a minuscule number of occult cancer cells. "
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ABSTRACT: The monoclonal antibody D2-40 is a specific lymphatic endothelial markers and D2-40 staining have been applicable to evaluate lymphatic invasion in various malignant neoplasms. In the present study, we investigated lymph node micrometastasis determined by immunohistochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR) in all dissected lymph nodes obtained from 80 patients with node-negative gastric cancer, and analysed the relationship between micrometastasis and clinicopathological findings including lymphatic invasion of the resected primary tumour using D2-40 immunohistochemical staining. The incidence of micrometastasis determined by IHC and RT-PCR was 11.3% (nine out of 80) and 31.3% (25 out of 80), respectively. Although haematoxylin-eosin (HE) staining revealed lymphatic invasion in 11.3% (nine out of 80) of patients, D2-40 staining uncovered new invasion in 23.8% (19 out of 80) of patients. In the diagnosis of HE and D2-40 staining, the incidence of micrometastasis was significantly higher in patients with lymphatic invasion than in those without lymphatic invasion (P=0.0150 and P<0.0001, respectively). Micrometastasis correlated more closely with D2-40 than with HE staining. We demonstrated a high incidence of micrometastasis and lymphatic invasion and a correlation between them even in pN0 gastric cancer. When planning less invasive treatment, the presence of such occult cancer cells should be considered.
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