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Results for “clonal analysis”

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PubMed2000

Clonality analysis of various hematopoietic disorders in cats naturally infected with feline leukemia virus.

The clonality analysis of the bone marrow cells was carried out by detecting the integrated proviruses of feline leukemia virus (FeLV) to understand the pathogenesis of FeLV-associated hematopoietic disorders in cats. Bone marrow cells from 4 cases with acute myeloid leukemia (AML), 9 cases with myelodysplastic syndromes (MDS), 2 cases with pure red cell aplasia (PRCA) and 3 healthy carriers infected with FeLV were subjected to Southern blot analyses using an exogenous FeLV probe. Clonal hematopoiesis was found in all the cases with AML and in 6 of the 9 cases with MDS, but not in the cases with both PRCA and healthy carriers infected with FeLV. In the 2 cases with MDS, it was thought that the same clones of the hematopoietic cells might proliferate before and after the progression of the disease irrespective of the changes of the hematological diagnoses by cytological examination. This study indicates that MDS in cats is a disease manifestation as a result of clonal proliferation of hematopoietic cells and can be recognized as a pre-leukemic state of AML.

Animals↗

PubMed1995

Clonal analysis of benign and malignant human breast tumors by means of polymerase chain reaction.

Clonal analysis was conduced on a variety of benign and malignant human breast tumors using the method based on restriction fragment length polymorphism (RFLP) of the X 120 chromosome-linked phosphoglycerokinase gene and on random inactivation of the gene by methylation. Breast carcinoma was shown to be monoclonal in origin, consistent with a somatic mutational theory. Precancerous lesions such as atypical ductal hyperplasia and multiple intraductal papilloma were also found to be monoclonal, indicating that certain genetic changes had been accumulated in these lesions. Solitary intraductal papilloma was found to be monoclonal. Since this tumor is composed of two types of cells, luminal epithelial cells and myoepithelial cells, it was suggested that the origin of solitary intraductal papilloma is a precursor cell which is capable of differentiating into both luminal and myoepithelial cells. The fact that fibroadenoma is polyclonal indicates that this tumor is not neoplasia but hyperplasia of a lobule. Epithelial component of phyllodes tumor was found to be polyclonal but stromal component was found to be monoclonal. Thus, phyllodes tumor is considered to be a neoplasm of stromal cells but not of epithelial cells.

Base Sequence↗

PubMed1997

Clonality in nevocellular nevus and melanoma: an expression-based clonality analysis at the X-linked genes by polymerase chain reaction.

The true nature of nevocellular nevus is still unknown and it has been ambiguously classified as a neoplasm or a hamartoma. We studied the clonality of nevocellular nevus and melanoma (malignant melanoma), using an expression-based clonality analysis at the X-linked genes by means of polymerase chain reaction. DNA was extracted from cryostat sections of 20 nevocellular nevi (10 compound and 10 intradermal type) and five melanomas from female patients. A polymorphic portion of the inactivated X-linked gene was amplified after selective digestion of the active X-chromosome with a methylation-sensitive restriction enzyme, Hpa II. Paternal- and maternal-derived fragments were resolved with electrophoresis using the polymorphic restriction endonuclease (BstX I) site for the phosphoglycerate kinase assay, and using the difference of CAG repeats for the human androgen-receptor gene assay. Both assays revealed that all informative nevocellular nevi were polyclonal in origin and all melanomas were monoclonal. Results of the clonality were independent of either the histologic type of nevocellular nevus or whether the nevocellular nevus was of congenital or acquired origin. Thus, nevocellular nevus, congenital or acquired, may be a hamartomatous rather than a neoplastic lesion. The analysis of clonality could be applied to the differential diagnosis of benign melanocytic disease and melanomas.

Adolescent↗

PubMed1986

Developmental origins of skeletal muscle fibers: clonal analysis of myogenic cell lineages based on expression of fast and slow myosin heavy chains.

A clonal analysis was used to show that skeletal muscle myoblasts are committed to distinct cell lineages during development. Myoblasts taken from embryonic chicken hindlimb muscles of different ages were cultured at clonal density. The content of fast and slow classes of the myosin heavy chain isoforms in the myotubes of the resulting muscle colonies was determined immunocytochemically with specific monoclonal antibodies that served as markers for the different fiber types. The muscle colonies formed by cloning myoblasts from early hindlimbs (days 4-6 in ovo) were of three types: the most numerous type, in which all myotubes in a colony contained only the fast class of myosin heavy chain; a less numerous type, in which all myotubes in a colony contained both the fast and slow classes of myosin heavy chain isoforms; and a rare type, in which all myotubes in a colony contained only the slow class of myosin heavy chain. The muscle colonies formed by cloning myoblasts from later hindlimbs (days 10-12 in ovo) were, however, all of one type, in which every myotube in a colony contained only fast myosin heavy chain. Thus, myoblasts in the early embryo (days 4-6 in ovo) were a heterogeneous population committed to three myogenic lineages: fast, mixed fast/slow, and slow, whereas myoblasts from the later embryo (days 10-12 in ovo) were only in the fast myogenic lineage. These results suggest that muscle fiber formation is rooted in two developmental phases--an early phase in which diverse fiber types are formed from intrinsically diverse populations of myoblasts and a later phase in which fibers are formed from a single population of myoblasts.

Age Factors↗

PubMed1981

A clonal analysis of the IgE response and its implications with regard to isotope commitment.

In a clonal analysis of the IgE response, it was found that a small proportion of primary or nonimmune B cells in spleen and mesenteric lymph nodes can be stimulated by antigen to produce IgE-secreting clones. In addition, there appears to be no substantial difference in the frequency of such cells between the classical low and high IgE responder strains. An analysis of immune, or memory, B cells revealed substantial increases in the frequency of B cells secreting IgE as compared with primary B cells, although the actual proportion of B cells secreting IgE remained relatively low. When the IgE-secreting clones derived from either primary or secondary B cells were reanalyzed for the presence of other isotypes, it was found that all clones secreting IgE were secreting at least one other isotype, with the majority secreting two or three other isotypes in addition to IgE. This demonstrates that there is no distinct subpopulation of B cells committed to IgE expression per se.

Animals↗

PubMed1979

[Clonal analysis of a transplantable rat seminoma].

The study of rat testis seminoma by the method of clonal analysis and ectopic transplantations has shown that typical and anaplastic types are different stages of tumor progression in the course of which atypical and anaplastic features of the seminoma cell elements increase. It does not seem inconceivable that the spermacytic form, never encountered during our investigation of 400 tumors, may be an independent seminoma form incapable of transforming into a typical one. A spindle-cell form of seminoma has been described most frequently occurring during transplantation of a typical seminoma into testis and during cloning. The obtained evidence permits to consider the small dark cells of the seminoma as being cambial cells of the tumor which occur abundantly in growing tumors, and are absent in tumors with ceased proloferating. At later stages of the seminoma development, the small dark cells may be absent, their function being performed practically by all tumor cells. Acinar structures found in part of clones from lungs and in the majority of transplants from eye anterior chamber suggest a histogenetic relationship between the typical seminoma and embryocarcinoma, and thus enable us to regard the latter as the most differentiated variants of the seminoma.

Anaplasia↗

PubMed2001

[Clonal analysis of the heterogeneity of neoplastic hepatocytes].

The heterogeneity of hepatoma MH-22a cell line was studied by clonal analysis on the basis of the following factors: splenocyte-induced apoptosis of tumor hepatocytes, tumor hepatocyte-induced apoptosis of splenocytes and profile of B1-associated DNA fragments. Induction of apoptosis of tumor hepatocytes by splenocytes and of splenocytes--by tumor hepatocytes was carried out in the main population of hepatocytes and in five clonal lines of hepatoma MH-22a. Genetic heterogeneity was studied using the same material and PCR primed for murine B1-elements. It was shown that apoptosis of hepatocytes of the main population and two clonal lines of hepatoma MH-22a was induced by splenocytes. The ability to induce apoptosis of splenocytes was observed in the same clonal lines and population of tumor hepatocytes. The latter involved enhanced genetic heterogeneity which was identified by B1-PCR analysis. Thus, a correlation was established in tumor hepatocytes between apoptosis-related characteristics and frequency of genome rearrangements.

Animals↗

PubMed2005

Improved clonality analysis of multifocal bladder tumors by combination of histopathologic organ mapping, loss of heterozygosity, fluorescence in situ hybridization, and p53 analyses.

The clonality status of multifocal bladder tumors is still controversially discussed with experimental evidence for both monoclonality and field cancerization. Methodologically, loss of heterozygosity (LOH) and genomic sequencing analyses are widely used in clonality analysis of malignant tumors. In the present study, we used LOH analysis and genomic sequencing in combination with fluorescence in situ hybridization (FISH) and extensive histopathologic whole-organ mapping to determine the clonal relationship of multifocal bladder cancer disease. Tissue sections (1 cm(2)) covering the entire urothelial lining were systematically dissected from 2 cystectomy specimens (cystectomy 1, no urothelial lesions, bladder infiltration by a leiomyosarcoma of the vaginal wall; cystectomy 2, multifocal pT3G3 tumors). The location of each sample was documented (bladder mapping). Urothelial cells were microdissected for LOH (chromosomes 9, 17p) and FISH analysis (CDKI2 (9p21), FACC (9q22), p53 (17p13.1), and centromeric probes for corresponding chromosome). Exons 5 to 9 of the p53 gene were sequenced in all tumor samples. No chromosomal alterations were detected in the cystectomy specimen without urothelial malignancies. The tumor-bearing bladder showed an increasing frequency of deletions with increasing malignancy of the investigated lesions. LOH analysis detected deletions only on chromosomes 9p and 17p. In contrast, FISH analysis revealed deletions of all investigated genes at chromosomes 9p, 9q, and 17p in all samples analyzed (preneoplastic and neoplastic tissue). An identical p53 mutation in codon 281 was found in all 7 analyzable tumor samples. Combination of molecular data with histopathologic bladder mapping suggested a monoclonal development of the multifocal lesions mostly via intraurothelial migration. Our data strengthen the results from recently published studies that patients with advanced urothelial carcinoma seem to have a monoclonal panurothelial disease in most cases. FISH showed a much higher sensitivity for detection of chromosomal losses than classical LOH analysis, especially in preneoplastic and small lesions. Combining 3 molecular approaches together with histopathologic organ mapping presents a valuable tool to determine the clonality status of multifocal bladder tumors.

Chromosomes, Human, Pair 17↗

PubMed1983

Direct demonstration of the clonogenic potential of every human peripheral blood T cell. Clonal analysis of HLA-DR expression and cytolytic activity.

In an attempt to determine the clonogenic properties of human peripheral blood T cells, we have developed a limiting dilution microculture system using phytohemagglutinin (PHA) as T cell activator and supernatant from PHA-stimulated spleen cultures as a source of T cell growth factors. The frequencies of cells capable of extensive proliferation under these culture conditions were 0.52-0.73, 0.98-1.11, and less than 0.02 in peripheral blood mononuclear, E-rosette-positive, and E-rosette-negative cell populations, respectively. The clonogenic potential of virtually all T cells was confirmed in experiments using single cells isolated by micromanipulation. Clone size ranged between 5 and 30 X 10(4) cells on day 14 of culture. The same microculture system was used to determine the precursor frequency of all cytolytic T lymphocytes (CTL-P). As assessed by a lectin-dependent 51Cr release assay, the CTL-P frequency in purified T cell populations ranged between 0.30 and 0.34. In comparison, the precursor frequency of T cells capable of lysing K562 target cells was ranging between 0.14 and 0.16. Parallel analysis of individual clonal cultures for both lytic activities showed that 50% of the clones exhibiting lectin-dependent lysis were also active against K562 target cells. All of the proliferating clones expressed HLA-DR antigens, although to a varying degree as assessed by flow cytofluorometry. Given the high cloning efficiency of this culture system, it appears now possible to determine the precursor frequencies of the various classes of functional cells in T cell populations.

Cells, Cultured↗

PubMed1990

Clonal analysis of joint fluid T lymphocytes in patients with juvenile rheumatoid arthritis.

Synovial fluid (SF) lymphocytes from 4 patients with pauciarticular juvenile rheumatoid arthritis (JRA) and 4 patients with polyarticular JRA were examined for their phenotypic and functional characteristics. In all 8 patients there was a high proportion of activated SF T cells, together with an increased proportion of CD2+CD3- and the presence of CD3+CD4-CD8-WT31- lymphocytes. The functional analysis at the clonal level in 5 patients (427 clones) showed a relevant proportion of cytotoxic T cell clones, which were not confined to typically cytolytic phenotypes, but were also present among CD3+CD4+CD8- cultures. Compared to those with pauciarticular JRA, patients with polyarticular disease had a significantly higher proportion of T cell clones with cytotoxic activity. Although derived from a limited number of patients, our data suggest a direct involvement of T cells in the pathogenetic mechanisms that originate and maintain the articular damage, and the possibility of different or more pronounced T cell reactivities in the clinically more diffuse JRA types.

Adolescent↗

PubMed1997

Clonality analysis of hematopoiesis in essential thrombocythemia: advantages of studying T lymphocytes and platelets.

Essential thrombocythemia (ET) is a myeloproliferative disorder characterized by a sustained elevation of the platelet count in the absence of other causes of thrombocytosis. ET is difficult to diagnose, and the demonstration of clonal hematopoiesis may be of value. However, clonality analysis of hematopoietic cells based on the study of the X-chromosome inactivation pattern is complicated by the observation that some normal females present skewed lyonization. Moreover, DNA methylation of X-linked genes in hematopoietic cells may differ from that in other tissues. Appropriate controls for skewed lyonization are therefore critical for the study of clonality. We developed two techniques based on X-chromosome inactivation and polymerase chain reaction (PCR) analysis of polymorphisms, to study clonality in ET patients. Reverse transcriptase-PCR analysis of IDS, P55, and G6PD mRNAs was used to examine the different hematopoietic cell lineages including platelets in patients heterozygous for these polymorphisms and analysis of the HUMARA gene methylation pattern permitted us to study clonality in all nucleated cell fractions of the other patients. Using both types of assay and T lymphocytes as a control tissue for lyonization, clonal hematopoiesis was demonstrated in 28 patients. In 14 patients, the granulocytes were polyclonal; among these patients, platelets were monoclonal in 3 cases, polyclonal in 7 cases, and in the remaining 4 cases this fraction could not be studied because the patients were homozygotes for all RNA markers. No conclusion about clonality could be drawn in 6 cases. Polyclonal hematopoiesis was found in all the cases of reactive thrombocytosis. These findings confirm the high frequency of monoclonal hematopoiesis in ET, the utility of studying platelets, and the possibility of using T lymphocytes as a control tissues for X-chromosome inactivation patterns.

Adolescent↗

PubMed2005

Clonality analysis for normal and cancerous colon tissues with human androgen receptor gene polymerase chain reaction.

We assessed the feasibility of clonality analysis with human androgen receptor gene polymerase chain reaction in terms of the sensitivity and specificity for normal and cancerous colonic tissues taken from fourteen informative cases selected from 22 women with colonic adenocarcinoma. Ten crypts microdissected from each 10-microm-thick cryostat sections and whole tissues were used as samples. DNA was extracted from the samples and amplified with and without prior enzyme digestion. These products were analyzed by capillary electrophoresis for clonality. Of the whole-tissue DNA, none of the normal tissues and seven (50.0%) of the cancerous tissues showed monoclonality. Of the microdissected samples, monoclonality was found in 88.4% (107/121) of normal crypts and 95.9% (117/122) of cancerous crypts. Samples composed of crypts with short and long alleles were found in eight of the 14 normal colonic mucosae, but in none of the cancerous tissues. We concluded that the sensitivity of this method is limited for both whole-tissue DNA and microdissected-tissue DNA, because monoclonality from small samples does not always indicate monoclonality of the entire lesion. The high specificity of this method, however, allows polyclonal results in whole tissues to be confirmed by additional analysis of microdissected tissues.

Adenocarcinoma↗

PubMed1995

Clonal analysis of haemopoietic cells in essential thrombocythaemia.

Essential thrombocythaemia (ET) is a myeloproliferative disease (MPD) predominantly involving the platelet lineage, with a diagnosis often difficult to establish in practice. The demonstration of a clonal haemopoiesis can contribute to diagnosis. The clonal origin of blood cells can be assessed in female patients using X-chromosome-linked polymorphisms, assuming a random inactivation of the X chromosome. The human androgen receptor gene HUMARA has been used for this purpose, taking advantage of a highly polymorphic trinucleotide repeat in the first exon. The close proximity of the polymorphic trinucleotide repeat to four methylation sites permits a clonal analysis based on the polymerase chain reaction. We have used this technique to study the clonality of haemopoietic cells in 32 patients with ET and 23 age-matched control heterozygotes for the HUMARA polymorphism. A monoclonal pattern of haemopoiesis was detected in unfractionated nucleated blood cells from 22 patients, suggesting that haemopoiesis is essentially monoclonal in a majority of cases in this disease. In some patients a monoclonality of granulocytes was documented, whereas the mononuclear fraction had a polyclonal pattern of X-inactivation. Finally, in two patients for whom a polyclonality had been found in unfractionated blood, analysis of G6PD transcripts in platelets revealed a clonal megakaryocytopoiesis. These findings confirm the heterogeneity of haematological abnormalities in ET patients and the potential utility of a multifaceted laboratory approach to investigate these patients.

Adult↗

PubMed1991

In vitro clonal analysis of quail cardiac neural crest development.

The developmental potentials of cardiac neural crest cells were investigated by in vitro clonal analysis. Five morphologically distinct types of clones were observed: (1) "pigmented" clones contained melanocytes only; (2) "mixed" clones consisted of pigmented and unpigmented cells; (3) "unpigmented dense" clones consisted of flattened, closely aligned unpigmented cells; (4) "unpigmented loose" clones consisted of a few loosely arranged, flattened cells; and (5) "unpigmented large" clones included a large number of small, stellate cells that were highly proliferative. The binding patterns of antibodies against lineage-specific markers showed that cells in the different clones expressed characteristic phenotypes. The following phenotypes were expressed in addition to pigment cells: smooth muscle cells, connective tissue cells, chondrocytes, and cells in the sensory neuron lineage. Mixed clones expressed all five phenotypes. Unpigmented dense clones contained smooth muscle cells, connective tissue cells, chondrocytes, and sensory neurons. Unpigmented loose clones exclusively consisted of smooth muscle cells, whereas unpigmented large clones contained chondrocytes and sensory neuron precursors. Based on these results, the following conclusions can be drawn: (1) Pigmented and unpigmented loose clones are most likely formed by precursors that are committed to the melanogenic and myogenic cell lineages, respectively. (2) Mixed and unpigmented dense clones are derived from pluripotent cells with the capacity to give rise to four or five phenotypes. (3) Unpigmented large clones originate from progenitor cells that appear to have a partially restricted developmental potential, that is, these cells are capable of generating two phenotypes in clonal cultures. Thus, the data indicate that the early migratory cardiac neural crest is a heterogeneous population of cells, consisting of pluripotent cells, cells with a partially restricted developmental potential, and cells committed to a particular cell lineage.

Animals↗

PubMed2006

Rapid emergence of enfuvirtide resistance in HIV-1-infected patients: results of a clonal analysis.

OBJECTIVES: To study the dynamics of enfuvirtide (T-20) resistance development in HIV-1-infected subjects. PATIENTS AND METHODS: Clonal analysis of gp41 sequences was performed on serial samples obtained from HIV-1-infected subjects with early virologic failure of T-20-based regimens. RESULTS: Enfuvirtide resistance mutations at codons 36 to 45 in the first heptad repeat of gp41 emerged within 2 weeks in most subjects and were associated with the return of plasma HIV-1 RNA level toward baseline by weeks 4 to 8. Mutations at codons 36 (G36E, G36D, or G36S) and 38 (V38A, V38G, or V38M) were the most commonly detected resistance mutations at week 2. Mutations at codons 40 (Q40H) and 43 (N43D) were more prevalent at week 4 than at week 2 and seemed to emerge more slowly than mutations at codons 36 and 38. CONCLUSIONS: The rapid emergence of mutations associated with T-20 resistance in the absence of a fully suppressive antiretroviral regimen demonstrates a low genetic barrier to resistance and underscores the importance of combining T-20 with other active drugs when constructing regimens for highly treatment-experienced patients.

Amino Acid Sequence↗

PubMed1998

Establishment of a novel B cell clonality analysis using single-strand conformation polymorphism of immunoglobulin light chain messenger signals.

The remarkable diversity of the complementarity determining region (CDR) 3 of the immunoglobulin (Ig) heavy (H) chain gene rearrangements has been exploited to identify the clonal populations of B cells in B cell malignancies. However, when B cell malignancies of different categories were examined, the overall detection rate was found to be approximately 70%. The development of a simple clonality analysis using Ig light (L) chain CDR3 diversity has been hampered due to the sparseness of knowledge regarding the sequence of Vkappa and Vlambda gene segments and the restriction of L chain CDR3 length. Based on the recently reported Vkappa and Vlambda gene sequences, we designed Vkappa and Vlambda framework 3 consensus primers. We combined the reverse transcriptase polymerase chain reaction (RT-PCR) of IgL chain transcripts with a single-strand conformation polymorphism (SSCP) analysis and then analyzed samples from patients with B cell malignancies. Clonal B cell populations were detected as discrete bands, and identical clones showed a similar mobility in a RT-PCR SSCP analysis. This method was thus found to be a useful supplement to the previously described approach of VH gene amplification for detecting clonal B cell populations. By using SSCP, we were able to determine the clonal identities of B cell expansion in different samples.

Arthritis, Rheumatoid↗

PubMed1997

Arbitrarily primed polymerase chain reaction fingerprinting and clonal analysis of oral Fusobacterium nucleatum isolates.

F. nucleatum is the most commonly isolated microorganism from subgingival plaque, but the role of this microorganism in periodontal diseases remains undefined. Arbitrarily primed polymerase chain reaction (AP-PCR) was evaluated as a method for fingerprinting F. nucleatum isolates and for use in clonal analysis. Pulsed field gel electrophoresis was used to further differentiate F. nucleatum isolates, with identical AP-PCR patterns. Extremely heterogeneous AP-PCR fingerprints were observed among the 98 F. nucleatum isolates, with 36 different genotypes observed with primer C1 and 30 different genotype detected with primer C2. Combining the results of the AP-PCR genotype analysis from C1 and C2 primer amplifications revealed that up to 7 different genotypes could be distinguished from isolates from the same oral cavity and that up to 4 different genotypes were observed within a single site. An intense amplicon at approximately 450 bp generated in AP-PCR amplification with primer C2 was associated with F. nucleatum subsp. nucleatum (ATCC 25586) and with 15 F. nucleatum isolates from diseased sites and 2 isolates from healthy sites. Pulsed field gel electrophoresis confirmed the AP-PCR genotypes and demonstrated increased discriminatory power over AP-PCR. The results indicated that AP-PCR and pulsed field gel electrophoresis provide a simple and sensitive means for differentiating oral F. nucleatum isolates and further demonstrate the heterogeneity of this species. These techniques may serve as useful tools in the clonal and epidemiological analysis of F. nucleatum isolates, which may help define the role of these microorganisms in periodontal diseases.

Bacterial Typing Techniques↗

PubMed1990

Clonal analysis in the ultrastructure of cell-to-cell interaction between a human glioma cell line and autologous tumor-specific cytotoxic T lymphocytes.

The clonal analysis in the ultrastructure of tumor-lymphocyte interaction was carried out in order to investigate the precise mechanism responsible for CTL-mediated cytolysis of tumor cells. A glioma-derived cell line (GI-1) and autologous tumor-specific cytotoxic T lymphocyte (CTL) clones were established. The CTL lines were composed of the morphologically homogeneous lymphocytes with intracytoplasmic electron-dense secretory granules. After the stimulation by GI-1, the size of the CTLs increased, and the intracytoplasmic organellas were developed. It was noted that the intracytoplasmic secretory granules markedly increased in number and size, and many of them exhibited an "immature" appearance. On the other hand, the tumor cells underwent a progressive degeneration. In contrast, the stimulation by other antigens caused only small morphological changes in the CTLs. It is suggested, therefore, that the secretory function of tumor-specific CTLs is activated by the stimulation of the specific antigen, and that soluble factors in the secretory granules in the CTLs may be closely associated with the mechanism of target cell lysis.

Cell Communication↗