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.
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.
Journal of Translational Medicine 10/2010; 8(1):107. DOI:10.1186/1479-5876-8-107 · 3.93 Impact Factor
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.
British Journal of Cancer 10/2005; 93(6):688-93. DOI:10.1038/sj.bjc.6602739 · 4.84 Impact Factor
Available from: Bradley Kropp
- "Based on this concept, several epithelial cell markers have been evaluated for detecting disseminated tumor cells in the blood circulation. Frequently used molecules include cytokeratins (CKs) 7, 19, and 20 [4-6], carcinoembryonic antigen (CEA) [7,8], epidermal growth factor receptor  including HER-2/neu , mucin-1 , β-subunit of human chorionic gonadotropin (β-hCG) , and α-fetoprotein . In prostate cancer (PCa) patients, the expression of prostate specific antigen (PSA) [14-16], prostate-specific membrane antigen (PSMA) [17,18], and human glandular kallikrein 2 (hK2)  along with other epithelial cell markers has been investigated individually or in combination  for their ability to detect CTCs in patients with localized and metastatic PCa. "
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ABSTRACT: Tumor metastasis and changes in host immunosurveillance are important components in cancer development. Tumor cell invasion into the bloodstream is an essential step for systemic metastasis. Currently, the detection of tumor cells in the circulation is mainly dependent upon the utilization of known epithelial cell markers. However, expression of these molecules is not limited to cancer patients; healthy people also have a small number of epithelial cells in their circulation. Utilizing these markers to detect circulating tumor cells (CTCs) cannot adequately explain the mechanisms of tumor cell survival or their development of metastatic potential in peripheral blood. The immune system can also evolve along with the cancer, actually promoting or selecting the outgrowth of tumor variants. Unfortunately, both metastasis and immunosurveillance remain mysterious and are debatable because we have yet to define the molecules that participate in these processes. We are interested in identifying the existence of expressed genes, or mRNA species, that are specifically associated with circulating cells of cancer-bearing patients using prostate cancer (PCa) as a model.
We established two comprehensive subtracted cDNA libraries using a molecular technique called suppression subtractive hybridization. This technique selectively amplifies transcripts that are specifically expressed in circulating cells of either PCa patients or healthy men. Following sequencing reaction, we showed that 17 out of 23 (73.9%) sequenced clones did not match any mRNAs in the GenBank database. This result suggests that genes associated with alterations in circulating cells of cancer-bearing patients are largely unknown. Semi-quantitative RT-PCR confirmed that two genes are up-regulated in circulating cells of PCa patients, whereas another two genes are down-regulated in the same patients.
The comprehensive gene expression analysis is capable of identifying differentially expressed genes in circulating cells of healthy men and PCa patients. We did not attempt to enrich specific cell types in this study because phenotypes of CTCs and subsets of leukocytes participating in immunosurveillance remain largely unknown. Continuous studies of these differentially expressed genes will eventually lead us to understand the mechanisms involved in tumor metastasis and immune modulation during cancer development.
Molecular Cancer 02/2005; 4(1):30. DOI:10.1186/1476-4598-4-30 · 4.26 Impact Factor
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