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Results for “clonal analysis”

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At least 19 recordsLinked to original sources

PubMed1998

Efficacious diagnosis of primary and metastatic human carcinomas with a combination of p53 mutation analysis and clonality analysis of androgen receptor gene.

It is occasionally difficult to differentiate between multiple primary and metastatic tumors in case of multiple carcinomas. p53 gene mutation pattern by PCR-SSCP and a clonality analysis on the human androgen receptor gene (HUMARA) in multiple tumors were used to distinguish whether carcinomas were multiple primary tumors or a metastasis. Three female patients with multiple carcinomas, who had undergone surgical resection, were selected for these analyses as typical candidates from 11 cases tested. In the first case with bilateral breast cancer the combined analyses were performed on DNA extracted from tumor portions in paraffin sections of each tumor sample and the results indicated that the carcinomas consisted of a primary carcinoma and metastatic tumors. The carcinomas in the second patient consisted of multiple primary carcinomas and the tumors in the third patient consisted of two independent primary carcinomas with different clonal origins. Thus a combination of p53 gene mutation analysis and clonality analysis on HUMARA provided useful, reliable evidence which can help in discriminating multiple primary tumors from metastatic tumors.

Aged↗

PubMed1995

[Theory and practice of macrorestriction analysis for the clonal analysis of pathogens].

The macrorestriction analysis of bacterial genomes is a method that is generally applicable to the typing of bacteria. The chromosome is cleaved with a restriction endonuclease that cuts infrequently and subsequently separated by pulsed-field gel electrophoresis. The fragment pattern defines the genotype of the strain. The relatedness of strains is evaluated from the similarity of the fragment patterns. The quantitative comparison of fragment patterns allows the definition of the infrasubspecific rank of a clone. Macrorestriction analysis is a sensitive and specific method to trace the origin and spread of infections and to analyse the clonal structure of bacterial populations.

Base Sequence↗

PubMed2000

Methylation patterns in human androgen receptor gene and clonality analysis.

Tumor clonality, an important issue in tumor biology, has been analyzed using X-chromosome inactivation studies based on the differential methylation patterns of active and inactive alleles. Recently, a PCR-based androgen receptor gene (AR) analysis method was developed that takes advantage of highly polymorphic CAG repeats and nearby HpaII and HhaI sites in exon 1 of AR at the Xq13 region. However, the data from this assay, which is now widely used, are sometimes uninterpretable and irreproducible for some currently unclear reason. To determine that reason, we analyzed a panel of lung cancer cell lines, using HpaII or HhaI restriction enzymes, methylation-specific PCR, and bisulfite genomic sequencing of the polymorphic CAG repeat site of AR exon 1, including nearby CpG sites. We found direct evidence of a variable methylation pattern at the restriction sites that prevented proper enzyme cleavage in two lung cancer cell lines (NCI-H292 and NCI-H1944) obtained from female patients who had a polymorphic CAG repeat in AR exon 1. Our data suggest that methylation patterns at the CpG sites of AR exon 1 are complicated and vary among different individuals. Therefore, the reliability of the PCR-based clonality analysis may require further evaluation.

Alleles↗

PubMed2000

Clonality analysis of multifocal carcinoid tumours of the small intestine by X-chromosome inactivation analysis.

The clonality of intestinal carcinoids and the relationship between different tumour deposits of multiple intestinal carcinoids were investigated in this study. Six cases of multiple ileal carcinoids were selected for analysis and three independent carcinoid lesions from each case were microdissected. Clonality of the lesions was determined by polymerase chain reaction (PCR)-based X-chromosome inactivation of the human androgen receptor gene. Four out of six cases were heterozygous for microsatellite repeats within the androgen receptor gene and thus informative for the study. The results showed that all 12 lesions analysed had non-random X-chromosome inactivation (monoclonal) patterns, compared with the background normal intestinal mucosal tissues. This finding proves for the first time the monoclonal origin of human intestinal carcinoids, by X-chromosome inactivation analysis. More interestingly, identical X-chromosome inactivation patterns were found in different carcinoid lesions from each individual case. This evidence strongly indicates that multiple carcinoids of the small intestine were generated by metastasis of a primary tumour to different locations in the intestine, rather than being of multiple origin. This study provides an important insight into the carcinogenesis of intestinal carcinoids.

Carcinoid Tumor↗

PubMed1992

Proof of differentiative mode of action of all-trans retinoic acid in acute promyelocytic leukemia using X-linked clonal analysis.

Using X-linked clonal analysis, mechanism of action of all-trans retinoic acid (ATRA) was sought in a 16-year-old female with relapsed clonally evolved acute promyelocytic leukemia (APL), who achieved complete remission. On ATRA, metamorphosis of peripheral blood leukemic promyelocytes to mature neutrophils was observed, despite the persistence of t(15;17) in 100% of bone marrow metaphases. DNA was extracted from fractionated serial blood specimens, collected at diagnosis, in first complete remission (CR), relapse, and during ATRA treatment. Using a phosphoglycerokinase (PGK) probe, the patient was heterozygous for both Bgl I and Bst XI PGK polymorphisms. Methylation analysis showed monoclonal leukemic promyelocytes with a polyclonal first CR achieved by standard chemotherapy. Subsequent examination, in relapse, of granulocytes appearing during ATRA treatment showed these to be monoclonal, proving these were derived from the neoplastic clone. The X-linked clonal analysis methodology has provided in vivo evidence of cellular differentiation as the mechanism of action of ATRA. Parallel studies of cytogenetic and clonal analysis showed a regression of the t(15;17) cytogenetic abnormality and return of a polyclonal PGK methylation pattern in 5 weeks, indicating a repopulation of marrow by normal stem cells. As standard cytogenetic techniques are inappropriate for nondividing cells, X-linked clonal analysis provides a marker system to allow insight into mechanism of drug action in malignant hematologic disease.

Adolescent↗

PubMed1993

Clonal analysis by study of X chromosome inactivation in formalin-fixed paraffin-embedded tissue.

Analysis of clonality by X chromosome inactivation has proven to be a powerful strategy in the study of neoplastic and preneoplastic disorders (P. J. Fialkow, Biochim. Biophys. Acta, 458: 283-321, 1976; B. Vogelstein et al., Cancer Res., 47: 4806-4813, 1987). Recently, the gene for the androgen receptor has been shown to be a highly polymorphic locus in which methylation of DNA correlates with inactivation of one or the other X homologue (R. C. Allen et al., Am. J. Hum. Genet., 51: 1229-1239, 1992). Unlike other loci used for analysis of X inactivation, the methylation sites within the androgen receptor gene lie close to the region of DNA containing the polymorphism. Consequently, it should be possible to use methylation-sensitive restriction enzymes and polymerase chain reaction to study differential methylation among alleles of this gene in formalin-fixed and paraffin-embedded archival tissue specimens. To investigate this question, we performed clonal analysis on a variety of randomly selected, formalin-fixed, paraffin-embedded tumor tissues from 15 women. Thirteen of the women were found to be heterozygous for polymorphisms at the androgen receptor locus. Among these women, 11 tumors were clearly clonal in assays of methylation of the androgen receptor gene. Interpretation of results for the remaining two cases was complicated because of a skewed pattern of X chromosome inactivation found in normal control tissues. We conclude that analysis of methylation in the androgen receptor gene should allow study of clonality in most formalin-fixed, paraffin-embedded tissue specimens from women, including small preneoplastic lesions or rare conditions for which sufficient material is not available for analysis by other techniques.

Base Sequence↗

PubMed1994

Clonal analysis of human astrocytomas.

Clonal analysis of many human cancers have generally confirmed that they are monoclonal. Although astrocytic neoplasms are the most frequently occurring primary tumors in the central nervous system, their clonal composition has not been systematically studied. In this report, the clonal composition of 22 human astrocytomas of all histological grades (2 well-differentiated astrocytomas, 3 anaplastic astrocytomas and 17 glioblastoma multiforme) was determined by analysis of the pattern of X-chromosome inactivation. Leukocyte and non-tumor brain DNA were used as controls. In addition, specimens from different parts of four glioblastoma multiforme were analyzed to determine whether remote areas of the same tumor had the same clonal composition. Eighteen of nineteen informative astrocytomas had a monoclonal pattern of X-chromosome inactivation; one glioblastoma multiforme had loss of heterozygosity on the X chromosome. Specimens from different areas of the same tumor all had identical patterns of X-chromosome inactivation. Leukocytes and non-tumor brain used as controls uniformly had a polyclonal pattern of X-chromosome inactivation. Furthermore, loss of heterozygosity for chromosomes 10 or 17 p loci was found in 64% (9/14) of informative specimens and identical allelic patterns were observed in specimens from different areas of the same tumor. Our results demonstrate that human astrocytomas from low to high-grade are characterized by monoclonal cell populations. The presence of monoclonality in even low-grade neoplasms suggests that in astrocytic tumors the establishment of monoclonality occurs quite early. Also, the finding of a monoclonal pattern in intermediate- and high-grade astrocytomas further supports the hypothesis that clonal expansion underlies astrocytic tumor progression.

Adult↗

PubMed1998

Clonal analysis of sporadic pancreatic endocrine tumours.

The clonal composition of 34 benign and malignant sporadic pancreatic endocrine tumours (PETs) of female patients was studied using a sensitive polymerase chain reaction (PCR)-mediated non-isotopic clonality analysis, which is based on the inactivation patterns of polymorphic X-linked genes encoding the androgen receptor (AR) and phosphoglycerate kinase (PGK-1) proteins. Predigestion of DNA with the methylation-sensitive restriction endonuclease Hpa II permitted selective PCR amplification of the methylated (uncleaved) allele. Amplification was successful in 27 of 34 samples. Twenty patient samples were heterozygous for the AR microsatellite region or Bst XI polymorphic site of the PGK-1 gene, permitting analysis of clonality. A monoclonal pattern of X-chromosome inactivation was found in 7 of 20 PETs (35 per cent), since DNA pretreatment with Hpa II blocked amplification of one of the two AR or PGK-1 alleles. One additional tumour exhibited an oligoclonal inactivation pattern and two others a loss of heterozygosity (LOH) at the AR locus, indicative of monoclonality. A random pattern of X-chromosome inactivation and polyclonal cellular composition was observed in the remaining ten PETs (50 per cent). When comparing informative benign and malignant PETs, only 2/7 (29 per cent) benign tumours showed a monoclonal pattern and 8/13 (61 per cent) malignant tumours a monoclonal (5), oligoclonal (1), or LOH (2) pattern. The clonal composition of PETs was not associated with a particular growth pattern, proliferation index or immunohistochemical expression pattern. These findings suggest that PETs might initially represent poly-/oligoclonal neoplastic lesions which are eventually outgrown by a single, more aggressive cell clone with the potential for invasive growth and metastatic spread.

Adult↗

PubMed1993

Clonal analysis of fibroadenoma and phyllodes tumor of the breast.

Clonality of fibroadenoma and phyllodes tumor of the breast was analyzed by means of the polymerase chain reaction using small DNA samples prepared from cryostat sections. The method of clonal analysis was based on restriction fragment length polymorphism of X-chromosome-linked phosphoglycerokinase gene and on differential methylation of the gene. Specimens from 10 fibroadenomas and 5 phyllodes tumors heterozygous for the BstXI polymorphism of PGK gene were subjected to clonal analysis. It was found that fibroadenoma was polyclonal, but phyllodes tumor was made up of both monoclonal and polyclonal cell components. Since these tumors contained both epithelial and stromal components, clonality of each component was analyzed separately. Analysis of clonality of each cell component showed that both the epithelial and stromal cells were polyclonal in fibroadenoma and that the epithelial cells were polyclonal, but the stromal cells were monoclonal in phyllodes tumor. When DNA samples were prepared from widely separated sites of phyllodes tumors, every sample was found to contain a monoclonal stromal cell component. These results demonstrate that fibroadenoma is a hyperplastic lesion rather than a neoplasm, and that phyllodes tumor is a neoplasm of the stromal cells.

Adenofibroma↗

PubMed2003

A retrospective clonal analysis of the myocardium reveals two phases of clonal growth in the developing mouse heart.

Key molecules which regulate the formation of the heart have been identified; however, the mechanism of cardiac morphogenesis remains poorly understood at the cellular level. We have adopted a genetic approach, which permits retrospective clonal analysis of myocardial cells in the mouse embryo, based on the targeting of an nlaacZ reporter to the alpha-cardiac actin gene. A rare intragenic recombination event leads to a clone of beta-galactosidase-positive myocardial cells. Analysis of clones at different developmental stages demonstrates that myocardial cells and their precursors follow a proliferative mode of growth, rather than a stem cell mode, with an initial dispersive phase, followed by coherent cell growth. Clusters of cells are dispersed along the venous-arterial axis of the heart tube. Coherent growth is oriented locally, with a main axis, which corresponds to the elongation of the cluster, and rows of cells, which form secondary axes. The angle between the primary and secondary axes varies, indicating independent events of growth orientation. At later stages, as the ventricular wall thickens, wedge shaped clusters traverse the wall and contain rows of cells at a progressive angle to each other. The cellular organisation of the myocardium appears to prefigure myofibre architecture. We discuss how the characteristics of myocardial cell growth, which we describe, underlie the formation of the heart tube and its subsequent regionalised expansion.

Actins↗

PubMed1994

Clonal analysis of predominantly intraductal carcinoma and precancerous lesions of the breast by means of polymerase chain reaction.

Clonality of predominantly intraductal carcinoma (PIC) and precancerous lesions of the breast was analyzed by a method based on restriction fragment length polymorphism of the X-chromosome-linked phosphoglycerokinase gene and on random inactivation of the gene by methylation. The application of polymerase chain reaction to this method enabled clonal analysis of small lesions. In order to eliminate the contamination by normal stromal cells, intraductal components were microdissected from the frozen sections of PIC under a dissection microscope. Clonal analysis of the intraductal components from seven PICs revealed that all were monoclonal in origin. In three PICs with intraductal spreading of carcinoma cells over nearly a whole breast gland, the intraductal components were collected from eight widely separated sites in each case. Clonal analysis of these samples showed that every sample was monoclonal and the same allele of the phosphoglycerokinase gene was consistently inactivated in each case. These results suggest that PIC arises as a single monoclonal carcinoma and spreads through the ducts over the gland rather than having multicentric origins. Clonality of precancerous lesions such as atypical ductal hyperplasia and intraductal papilloma arising in the terminal ducts was also studied. Intraductal components were microdissected from the paraffin sections of these lesions and subjected to clonal analysis. Both atypical ductal hyperplasia and intraductal papilloma were found to be monoclonal in origin, suggesting that certain genetic changes had already occurred in the precancerous lesions. A further study is needed to elucidate these genetic changes, which would greatly help our understanding of the mechanism of carcinogenesis.

Base Sequence↗

PubMed1994

Clonal analysis of solitary intraductal papilloma of the breast by means of polymerase chain reaction.

Clonality of solitary intraductal papillomas of the breast was analyzed using a method based on restriction fragment length polymorphism of the X-chromosome-linked phosphoglycerokinase (PGK) gene and on random inactivation of the gene by methylation. The application of polymerase chain reaction to this method enabled clonal analysis of such a small intraductal lesion as papilloma. Clonal analysis of DNA samples obtained from the nine solitary intraductal papillomas and adjacent normal breast tissues showed that all of the papillomas were monoclonal and all the normal breast tissues were polyclonal in origin. When DNA samples were obtained from four widely separated sites in the papillomas, clonal analysis showed that all were monoclonal and, in addition, the same allele of PGK gene was inactivated in each case. These results demonstrate that solitary intraductal papilloma arises as a single monoclonal tumor and extends along the ducts rather than occurring as multicentric monoclonal tumors and merging together subsequently. Immunohistochemical staining of smooth muscle alpha-actin, a marker protein of myoepithelial cells, revealed that solitary intraductal papilloma was composed of approximately equal mixtures of luminal epithelial and myoepithelial cells. Since solitary intraductal papillomas were shown to be monoclonal in origin, it was suggested that this disease originates from a common precursor that could differentiate into both luminal epithelial and myoepithelial cells.

Base Sequence↗

PubMed1992

Clonal analysis of human breast cancer by means of the polymerase chain reaction.

Clonality of human breast cancer was analyzed in small DNA samples prepared from cryostat sections, by means of the polymerase chain reaction (PCR). The method used for clonal analysis was based on restriction fragment length polymorphism of X-chromosome-linked phosphoglycerokinase (PGK) gene and on the differential methylation of the PGK gene due to random inactivation of one of two X-chromosomes by methylation in females. All the 20 breast cancer samples analyzed by the PCR-based method were monoclonal in origin and adjacent normal breast tissues were polyclonal. When DNA samples were prepared from widely separated sites of cancers, every sample was found to be monoclonal, always exhibiting inactivation of the same X-chromosome in each tumor. The study on sensitivity showed that the PCR-based method for clonal analysis can detect the presence of monoclonal cells against a polyclonal background when the monoclonal cell population is 50% or more. These results demonstrate that clonal analysis by means of PCR offers a good method for studying the clonality in small DNA samples prepared from cryostat sections of tumors. This method could be applied to distinguish between benign (polyclonal) and malignant (monoclonal) breast lesions.

Alleles↗

PubMed1994

Differentiation of primary and secondary breast cancer with clonal analysis.

BACKGROUND: It is often difficult to draw a firm conclusion as to whether the second breast cancer is primary or secondary (metastasis from the initial breast cancer) in a patient with metachronous bilateral breast cancer. In this study we have applied clonal analysis of breast cancer to distinguish whether the second breast cancer is primary or secondary. METHODS: A 54-year-old woman underwent modified radical mastectomy of the right breast as a result of breast cancer. Five years later she had tumors in the right chest wall and left breast. Fine-needle aspiration cytologic examination revealed that both tumors were adenocarcinoma. To elucidate the origin of these tumors, clonal analysis was done on DNA samples prepared from cryostat sections of the initial right breast cancer and from fine-needle aspirates of the tumors in the right chest wall and left breast. The method for clonal analysis was based on restriction fragment length polymorphism of X chromosome-linked phosphoglycerokinase gene and on differential methylation of the gene caused by random inactivation of one of two X chromosomes. RESULTS: Clonal analysis revealed that clonal origin of the right breast cancer was different from that of the left breast cancer and identical to that of the chest wall tumor. Therefore it was concluded that the left breast cancer was primary and the chest wall tumor was concluded that the left breast cancer was primary and the chest wall tumor was a recurrence of the initial breast cancer. CONCLUSIONS: Clonal analysis appears to be a useful method in discriminating a primary from a secondary cancer.

Base Sequence↗

PubMed1994

Clonal analysis in the chicken retina reveals tangential dispersion of clonally related cells.

Development of the chicken retina was investigated through clonal analysis using retroviral vectors. Replication-incompetent retroviral vectors encoding either human placental alkaline phosphatase, beta-galactosidase, or a viral core protein were used in paired combinations to infect retinal progenitor cells from Embryonic Days 2.5-7.0. Labeled clones were analyzed late in embryonic development after retinal histogenesis was complete. The early accessibility of the chicken retina, combined with its large final size, resulted in the labeling of much larger clones than had been reported previously in other species. The clones were composed of many cell types, supporting previous conclusions from other vertebrates that the progenitor cells of developing retina are multipotent. The majority of clones derived from early infections consisted of multiple tightly clustered arrays of cells accompanied by dispersed individual cells. Clonal complexity and tangential dispersion were greater in peripheral than central retina and decreased considerably with increasing age of infection. These observations suggest that early during retinal development, shortly after optic cup formation, there is considerable mixing of progenitor cells.

Animals↗

PubMed2002

Improvements to B cell clonality analysis using PCR amplification of immunoglobulin light chain genes.

AIMS: Clonality analysis using polymerase chain reaction (PCR) amplification of the immunoglobulin heavy chain (IgH) gene is an important aid to the diagnosis of B cell lymphoproliferative diseases. However, the method has a relatively high false negative rate. In an attempt to improve detection rates simple PCR strategies for clonality analysis of B cell populations using amplification of Ig light chain genes have been developed. METHODS: Novel PCR protocols, designed to amplify Ig kappa and Ig lambda light chain genes, were evaluated using high molecular weight DNA samples from 28 selected cases of B cell lymphoma with known light chain expression and 12 reactive lymphoid specimens. Products were run on 10% polyacrylamide minigels using heteroduplex analysis. Conventional IgH PCR analysis was also performed. Twelve randomly selected formalin fixed, paraffin wax processed samples from cases submitted for molecular genetic analysis were also studied. RESULTS: Polyclonal products were seen in all reactive lymphoid samples. Using Ig kappa PCR, 24 of 28 lymphomas, including four of five IgH negative cases, displayed monoclonal patterns. Using Ig lambda PCR, eight of 12 Ig lambda expressing tumours, including two of five IgH negative cases, showed monoclonal patterns. Standard IgH PCR demonstrated monoclonality in 23 of 28 B cell lymphomas. The detection rate was improved to 27 of 28 lymphomas using heavy and light chain PCR. Efficient amplification was achieved using paraffin wax processed samples, seven of which showed monoclonality compared with eight using IgH PCR. CONCLUSIONS: Ig light chain PCR, used in conjunction with heavy chain analysis, enables improved detection of B cell monoclonality using routine histological specimens and can provide additional clone specific markers for the study of the biology of B cell tumours.

B-Lymphocytes↗

PubMed1994

[Clonal analysis of human breast tumors by means of polymerase chain reaction].

Clonal analysis has been done using the method based on restriction fragment length polymorphism of the X-chromosome-linked phosphoglycerokinase gene and on random inactivation of the gene by methylation. Introduction of polymerase chain reaction (PCR) of this method has enabled clonal analysis of minute lesions. Utilizing this PCR-based method, clonality of a variety of human breast tumors (breast carcinoma, fibroadenoma, phyllodes tumor, intraductal papilloma, and atypical ductal hyperplasia) was studied in the present investigation in order to obtain valuable information on the histogenesis of these tumors.

Base Sequence↗

PubMed2001

Clonal analysis in glioblastoma with epithelial differentiation.

Epithelial differentiation in glioblastomas (GBM) may be associated with circumscribed growth and focal keratin expression resembling carcinoma metastasis. Therefore these rare lesions can pose a diagnostic problem suggesting coincidental occurrence of two separate neoplasms. However molecular analysis should succeed in establishing a common origin of seemingly unrelated tumor samples. Five GBMs exhibiting epithelial differentiation were microdissected and analyzed for mutations in the TP53 gene. SSCP analysis of exons 5-8 was followed by direct sequencing of aberrantly migrating fragments. TP53 mutations were identified in tumors from two of five patients. A G-->T transversion in codon 176 was detected in a tumor, initially diagnosed as metastases of unknown origin, however, a later autopsy revealed GBM. In this lesion, the mutation was observed in both, areas of astrocytic differentiation and areas of epithelial differentiation. One tumor diagnosed as GBM with epithelial differentiation carried C-->T transition in codon 211 in both, areas of astrocytic and epithelial differentiation. Thus, molecular analysis proved clonality in two GBMs with epithelial differentiation, thereby excluding a collision tumor. The present data support the concept of clonal origin of these morphologically heterogeneous lesions.

Astrocytes↗