Sensitivity of assays designed for the detection of disseminated epithelial tumor cells is influenced by cell separation methods.
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Bone Marrow↗
Biomedical subjects
C Wagener
Publications and source records attributed to C Wagener.
Redefined nomenclature for members of the carcinoembryonic antigen family.
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Alternative Splicing↗
Detection of micrometastasis by cytokeratin 20 RT-PCR is limited due to stable background transcription in granulocytes.
The reverse transcription polymerase chain reaction (RT-PCR) amplification of cytokeratin 20 (CK20) mRNA is considered a promising candidate method for the detection of circulating tumour cells in bone marrow and peripheral blood of cancer patients. In this study we have investigated the diagnostic specificity of the CK20 mRNA detection in samples from healthy donors (HD; n = 33), intensive care units patients (ICU; n = 20) and bone marrow obtained from patients suffering from chronic inflammatory diseases (CID; n = 14). RNAs purified from stabilized lysates showed positive results in 24% of the HD group (8/33), 35% of the ICU group (8/20) and in 40% of the CID group (5/14). The use of Ficoll gradients to separate nucleated cells completely restored the specificity of this CK20 RT-PCR assay. The CK20-expressing cells are positively identified to belong to the granulocyte fraction of leucocytes, which appear to express the gene on a background level. Our results demonstrate for the first time that CK20 mRNA expression is not limited to epithelium. Its occurrence in normal granulocytes has to be considered in tests designed to detect circulating cancer cells or micrometastases.
Biomarkers, Tumor↗
Enrichment of mutant alleles by chromatographic removal of wild type alleles: a new principle for the detection of alleles with unknown point mutations at excess of wild type alleles.
In human carcinomas, mutations that alter tumour genes such as the KRAS, P53, or APC genes, are mostly point mutations. The detection of mutant alleles of tumour genes in specimens such as urine, pancreatic juice, sputum, and stool holds great promise for an early diagnosis of cancer. In addition, the detection of mutant tumour genes in tissue samples, such as lymph nodes or resection margins, may allow a sensitive diagnosis of residual malignant disease. However, the reliable detection of mutant alleles in excess of wild type alleles remains an unresolved analytical problem when the mutations are not known a priori. In the present communication, a new approach is described which makes possible the detection of unknown point mutations in tumour genes at excess of wild type alleles. The method is based on the removal of wild type alleles by hybridisation to immobilised complementary oligonucleotides. Using this approach, an enrichment of mutant KRAS, P53 and APC alleles of one mutant in up to 10(3) normal alleles has been achieved. Parallel miniaturised separation units with oligonucleotides complementary to defined sequences of a wild type allele should allow the detection of unknown point mutations as well as small insertions or deletions which occur in the sequence range covered by the oligonucleotides.
Alleles↗
E2F-6: a novel member of the E2F family is an inhibitor of E2F-dependent transcription.
The E2F family of transcription factors are essential for the regulation of genes required for appropriate progression through the cell cycle. Five members of the E2F family have been previously reported, namely E2F1-5. All five are key elements in transcriptional regulation of essential genes, and they can be divided into two functional groups, those that induce S-phase progression when overexpressed in quiescent cells (E2Fs 1-3), and those that do not (E2Fs 4-5). Here, we describe the identification of a novel member of this family, which we refer to as E2F-6. E2F-6 shares significant homology with E2Fs 1-5, especially within the DNA binding, heterodimerization and marked box domains. Unlike E2Fs 1-5, E2F-6 lacks a transactivation and a pocket protein binding domain, hence, forms a unique third group within the E2F family. E2F-6 is a nuclear protein that can form heterodimers with the DP proteins (both DP-I and DP-2) in vitro and in vivo. Our results show that the complex formed between E2F-6 and the DP proteins, possesses high DNA binding activity, displaying a preference for a TTTCCCGC E2F recognition site, which is slightly different to the E2F consensus site derived from the E2 promoter (TTTCGCGC). In contrast to the other members of the E2F family, ectopic expression of E2F-6 inhibits transcription from promoters possessing E2F recognition sites rather than activating transcription. In addition, overexpression of E2F-6 suppresses the transactivational effects of coexpression of E2F-1 and DP-1. The inhibitory effect of E2F-6 is dependent on its DNA binding activity and its ability to form heterodimers with the DPs. Interestingly, ectopic expression of E2F-6 leads to accumulation of cells in S-phase. Our data suggest that E2F-6 expression delays the exit from S-phase rather than inducing S-phase, which further emphasizes the functional difference between E2F-6 and the previously known E2F family members.
Amino Acid Sequence↗
Specificity of reverse transcriptase polymerase chain reaction assays designed for the detection of circulating cancer cells is influenced by cytokines in vivo and in vitro.
Several reverse transcriptase polymerase chain reaction (RT-PCR) assays have been described for the detection of circulating tumour cells in blood and bone marrow. Target mRNA sequences for this purpose are the cytokeratins (CK) 19 and 20, the carcinoembryonic antigen (CEA), and the prostate-specific antigen messages. In this study, we investigated biological factors influencing the specificity of the CK19 and CEA RT-PCR assays. Bone marrow, granulocyte colony-stimulating factor (G-CSF)-mobilized blood stem cells and peripheral blood samples obtained from healthy volunteers (n = 15; CEA n = 7), from patients with epithelial (n = 29) and haematological (n = 23) cancer and from patients with chronic inflammatory diseases (n = 16) were examined. Neither CEA nor cytokeratin 19 messages could be amplified from bone marrow samples from healthy subjects and from patients with haematological malignancies. In contrast, specimens from patients with inflammatory diseases scored positive up to 60%. To investigate the influence of inflammation on target mRNA expression, haemopoietic cells were cultured with and without cytokine stimulation in vitro. CK19 messages could be easily detected in cultured marrow cells without further stimulation, CEA messages only after gamma-interferon (gamma-INF) stimulation. In contrast, G-CSF-mobilized peripheral blood stem cells were positive for CK19 messages only after stem cell factor (SCF) or interleukin stimulation. We conclude that transcription of so-called tissue-specific genes is inductible in haemopoietic tissues under certain conditions. These factors have to be considered in future applications of RT-PCR for the detection of minimal residual disease.
Abdominal Neoplasms↗
External quality assessment of molecular biology-based methods used in laboratories of clinical chemistry and human genetics.
The Reference Institute of Bioanalysis of the German Society of Clinical Chemistry has performed the first external assessment of molecular genetics methods used in medical diagnosis. The following procedures were tested: (I) DNA preparation from whole blood, (II) PCR amplification using "standard" primers, and (III) submarine agarose gel electrophoresis. Out of 50 participants, 45 returned samples for evaluation.
Chemistry, Clinical↗
Fundamentals of quality assessment of molecular amplification methods in clinical diagnostics. International Federation of Clinical Chemistry Scientific Division Committee on Molecular Biology Techniques.
The increasing interest in molecular biology diagnostics is a result of the tremendous gain of scientific knowledge in genetics, made possible especially since the introduction of amplification techniques. High expectations have been placed on genetic testing, and the number of laboratories now using the relevant technology is rapidly increasing--resulting in an obvious need for standardization and definition of laboratory organization. This communication is an effort towards that end. We address aspects that should be considered when structuring a new molecular diagnostic laboratory, and we discuss individual preanalytical and analytical procedures, from sampling to evaluation of assay results. In addition, different means of controlling contamination are discussed. Because the methodology is in constant change, no general standards can be defined. Accordingly, this publication is intended to serve as a recommendation for good laboratory practice and internal quality control and as a guide to troubleshooting, primarily in amplification techniques.
Chemistry, Clinical↗
Dysregulated expression of CD66a (BGP, C-CAM), an adhesion molecule of the CEA family, in endometrial cancer.
CD66a (BGP, C-CAM) is an adhesion molecule of the carcinoembryonic antigen family that has been shown to be down-regulated in colorectal, prostate, and breast cancers. The purpose of the present study was to determine its expression pattern in the normal human endometrium and in endometrial neoplasia. For this purpose, we performed immunohistochemistry using the 4D1/C2 monoclonal antibody on a series of 24 normal endometrial samples and 47 endometrial carcinomas. Strong CD66a expression was observed in glandular and luminal epithelial cells of the normal endometrium with a consistent localization at the apical poles of these cells throughout the cycle. In late secretory (premenstrual) phase, loss of cellular polarity resulted in a membranous expression pattern in some glandular cells. In the analyzed tumor samples increasing areas with a complete loss of expression were observed with increasing malignancy grade. The apical expression pattern of the normal epithelium was changed to a membranous all-around pattern in 55% of the tumors, mostly in solid areas. This change correlated with malignancy grade and could be observed in 3 of 15 G1 tumors, 4 of 12 G2 tumors, 11 of 12 G3 tumors, and 8 of 8 serous-papillary carcinomas. Areas with membranous expression pattern could be observed along with areas with a normal apical expression pattern in lower grade carcinomas and with areas with complete loss of expression in high grade tumors. Northern blot analysis showed a loss of mRNA expression in tumor samples and HEC-1B endometrial adenocarcinoma cells. Loss of protein expression in the tumor samples was also observed by Western blot. In conclusion, CD66a protein expression is dysregulated in endometrial carcinomas, showing reduction or loss of expression with increasing malignancy grade and a change from the apical to a membranous localization.
Antigens, CD↗
Expression of CD66a (human C-CAM) and other members of the carcinoembryonic antigen gene family of adhesion molecules in human colorectal adenomas.
Among the members of the carcinoembryonic antigen (CEA) family, CD66a (human C-CAM) and CGM2 (CEA gene family member 2) mRNAs are frequently down-regulated in colorectal cancer. In contrast, nonspecific cross-reactive antigen (NCA) mRNA is overexpressed in the majority of these carcinomas. In animal models, the rodent homologues of CD66a have been shown to act as tumor suppressors, suggesting an important role in carcinogenesis. Here we investigate the mRNAs of CD66a, CGM2, and NCA in 22 human colorectal adenomas and the respective normal mucosa specimens by Northern blots. The expression of both CD66a and CGM2 changed in a concomitant fashion. Using oligonucleotides specific for the N-terminal domains, two CD66a transcripts 3.9 and 1.5 kb in size were identified. These showed a greater than 50% down-regulation in 20 of 22 and 18 of 22 adenomas, respectively. Reduction of the CGM2 message was observed in 21 of 22 cases. Complete or near-complete losses of the CD66a 3.9-kb mRNA and the CGM2 message were found in 13 of 22 and 15 of 22 of the tumors, respectively. The medians of CD66a and CGM2 expressions were between 0.3 and 0.0, respectively. The tumor:normal ratio of NCA mRNA expression was increased up to 2.4-fold in 11 of 22 adenomas. Altogether, these results compare well to the changes reported previously for colorectal carcinomas. The high frequency and early appearance of dysregulation of members of the carcinoembryonic antigen family during colorectal tumorigenesis suggests that these changes may be important for the development of the malignant phenotype.
Adenoma↗
Molecular diagnostics.
Nucleic acid technology has assumed an essential role in various areas of in vitro diagnosis. Its major applications include the genomic characterization of mutations and polymorphisms, amplification of nucleic acids by the polymerase chain reaction, analysis of gene expression patterns at the mRNA level, specific detection of mutant proteins, and engineering of proteins used in ligand binding assay. Manipulating the expression of genes in cells and experimental animals allows detailed analysis of processes leading from genomic alterations to disease manifestations and may ultimately yield refinements in diagnostic strategies. The addition of DNA and RNA to the conventional diagnostic in vitro targets and novel approaches to the study of disease processes and manifestations characterize a diagnostic strategy referred to as "molecular diagnostics." This contribution describes some of essential ways in which molecular diagnostics differs from conventional diagnostic approaches. Applications such as differential diagnosis, prenatal diagnosis, early diagnosis, and diagnosis of disease susceptibility are well established in single-gene disorders, and the diagnostic impact of DNA polymorphisms is steadily increasing in multifactorial diseases. The high sensitivity of the polymerase chain reaction makes possible the detection of single infectious agents or tumor cells. With increasing knowledge of expressed gene sequences the expression pattern of mRNA will reflect the biological state of cells with high precision. Mutant proteins can be analyzed based on their structural or biological properties. Use of the appropriate expression systems makes it possible to design proteins for diagnostic in vitro applications such as ligand binding assays.
Aging↗
Nuclear localization and increased levels of transcription factor YB-1 in primary human breast cancers are associated with intrinsic MDR1 gene expression.
Breast cancers are either primarily resistant to chemotherapy (intrinsic resistance), or respond to chemotherapy but later recur with a multidrug-resistant phenotype because of overexpression of the multidrug transporter P-glycoprotein. The MDR1 gene encoding P-glycoprotein may be transcriptionally regulated by a Y-box transcription factor. We now report that, in multidrug-resistant MCF-7 breast cancer cells, nuclear localization of YB-1 is associated with MDR-1 gene expression. In drug-sensitive MCF-7 cells, however, YB-1 was localized to the cytoplasm. Regulated overexpression of YB-1 in drug-sensitive diploid breast epithelial cells induced MDR-1 gene expression and multidrug resistance. In 27 out of 27 untreated primary breast cancers, YB-1 protein was expressed in the cytoplasm although it was undetectable in normal breast tissue of these patients. In a subgroup of tumors (9/27), however, YB-1 was also localized to the nucleus and, in these cases, high levels of P-glycoprotein were present. These results show that in a subset of untreated primary breast cancers, nuclear localization of YB-1 protein is associated with intrinsic multidrug resistance. Our data show that YB-1 has an important role in controlling MDR1 gene transcription and this finding provides a basis for the analysis of molecular mechanisms responsible for intrinsic multidrug resistance in human breast cancer.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Differential expression of CD66a (BGP), a cell adhesion molecule of the carcinoembryonic antigen family, in benign, premalignant, and malignant lesions of the human mammary gland.
CD66a, also known as biliary glycoprotein (BGP), is a member of the carcinoembryonic antigen (CEA) family and the human homologue of the rat cell-CAM. There is evidence that aberrant expression or loss of CD66a in tumor tissue is of biological significance. No data about its expression in breast carcinoma cells and only sparse information about the expression of CD66a in normal breast are available thus far. In this study we used monoclonal antibodies to analyze the expression of CD66a and CEA in normal tissue, benign lesions, and in noninvasive and invasive carcinomas of the mammary gland. In normal tissue and benign lesions, CD66a was consistently expressed at the apical sites of epithelial cells and in myoepithelia, whereas CEA was absent or was restricted only to some apical membranes within the ductal tree. The specific staining of myoepithelia was most evident in pseudoinfiltrative radial scars and sclerosing adenosis. However, the apical expression of CD66a disappeared with the development of the malignant phenotype in noninvasive and invasive carcinomas, and changed gradually from low- to high-grade noninvasive carcinomas into a predominant uniform membrane staining all around the atypical cells. CEA expression was irregular in intensity and distribution. The native apical CD66a staining was partially preserved in some highly differentiated invasive carcinomas with a better prognosis, such as tubular and papillary carcinomas. These findings indicate that loss of CD66a expression rather than a change in staining patterns coincides with the development of the malignant phenotype.
Antibodies, Monoclonal↗
Reversal of RT-PCR inhibition observed in heparinized clinical specimens.
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Anticoagulants↗
Methods for detection of point mutations: performance and quality assessment. The IFCC Scientific Division, Committee on Molecular Biology Techniques.
We give an overview of current methods for the detection of point mutations as well as small insertions and deletions in clinical diagnostics. For each method, the following characteristics are specified: (a) principle, (b) major modifications, (c) maximum fragment size that can be analyzed, (d) ratio and type of mutations that can be detected (e) minimum ratio of mutant to wild-type alleles at which mutations can be detected, and (j) detection methods. Special attention is paid to the possibilities of quality assessment and the potential for standardization and automation.
Genetic Techniques↗
Quality management and influential factors for the detection of single metastatic cancer cells by reverse transcriptase polymerase chain reaction.
The sensitive and specific detection of micrometastasis holds great promise for earlier staging of cancer patients. By amplification of tissue-specific gene expression, the reverse transcriptase polymerase chain reaction (rtPCR) readily detects single tumour cells in different tissues. An increasing number of rtPCR assays with possible relevance for routine laboratory diagnostic procedures is currently being reported in the literature. Interestingly, when used in the clinical setting, assays for the same target mRNA perform very differently, despite comparable sensitivities and specificities in-vitro. Using rtPCRs specific for carcinoembryonic antigen (CEA), prostate-specific antigen (PSA) and cytokeratin 18 (CK 18), we have started to systematically investigate, both experimentally and in clinical specimens, a number of factors that contribute to the varying and seemingly implausible test results. Here we have concentrated on sample collection modalities, assay stability and test reproducibility at the sensitivity limit. Our results demonstrate in detail that, at the maximum sensitivity required for micrometastasis detection, preanalytical and statistical influences increasingly become important for the consistency of the assay results. We conclude that the prerequisite for translating the results from highly sensitive and specific rtPCR assays into clinically relevant data is the thorough definition of assay procedures and the number of tests performed on a sample. Addressing questions of standardization and quality control management is a central aspect yet to be emphasized in assay development and application of routine laboratory rtPCR tests in oncology.
Carcinoembryonic Antigen↗
Methods for detection of point mutations: performance and quality assessment. IFCC Scientific Division, Committee on Molecular Biology Techniques.
We give an overview of current methods for the detection of point mutations as well as small insertions and deletions in clinical diagnostics. For each method, the following characteristics are specified: (a) principle, (b) major modifications, (c) maximum fragment size that can be analyzed, (d) ratio and type of mutations that can be detected, (e) minimum ratio of mutant to wild-type alleles at which mutations can be detected, and (f) detection methods. Special attention is paid to the possibilities of quality assessment and the potential for standardization and automation.
DNA↗
Dysregulation of carcinoembryonic antigen group members CGM2, CD66a (biliary glycoprotein), and nonspecific cross-reacting antigen in colorectal carcinomas. Comparative analysis by northern blot and in situ hybridization.
Genes coding for CD66a (biliary glycoprotein), carcinoembryonic antigen (CEA) group member 2 (CGM2), and nonspecific cross-reacting antigen (NCA) are members of the human CEA gene subgroup. We investigated a series of 11 colorectal carcinomas by Northern blot and isotopic in situ hybridization (ISH), demonstrating underexpression of CD66a and CGM2 in the majority of the carcinomas as compared with the normal mucosa, whereas NCA was overexpressed. ISH for CD66a and CGM2 mRNA revealed that large areas of the carcinomas remained without or with only faint hybridization signals. However, in every carcinoma, at least some positive foci were observed, indicating remaining cell populations that actively transcribe CD66a and CGM2. In contrast, ISH for NCA displayed strong and extensive autoradiographic signals. By analysis of step sections, foci of CD66a and CGM2 expression were shown to co-localize. Furthermore, these foci contained relatively few nuclei immunohistochemically positive for the proliferation-associated nuclear antigen Ki-67. Our data indicate a dysregulation of the three genes possibly with a common transcriptional control for CD66a and CGM2 and a different control for NCA. The focal expression of CD66a and CGM2 could be interpreted as due to a focal, incomplete, and abortive differentiation or, alternatively, as a consequence of genetic heterogeneity with foci of slow-proliferating subclones.
Antigens, CD↗