SEARCH · PubMed Health

Results for “clonality”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 109 records · Page 6Linked to original sources

PubMed1998

Clonal analysis by ribotyping of Fusobacterium nucleatum isolates obtained from healthy young adults with optimal plaque control.

Fusobacterium nucleatum is a Gram-negative anaerobic rod implicated in the pathogenesis of periodontal disease. However, this organism has also been frequently identified in high numbers in healthy adults. These observations suggest that the species may comprise different clonal types, some of which may participate in disease. The purpose of the present investigation was to use restriction endonuclease analysis (REA) and ribotyping to characterize F. nucleatum clonal types isolated from healthy young adults with optimal plaque control and investigate the stability of some of these clonal types. A group comprising 11 dental students and 11 dental outpatients with optimal plaque control was sampled. Clonal stability was investigated by sampling the dental student group at baseline and at 16 months. One hundred and thirty-two clinical isolates of F. nucleatum were successfully recovered from 15/22 individuals. For the positive subjects, 29 different clonal types were identified by REA and ribotyping, each subject and site being colonized by 1-4 clonal types. For the dental students, 9 and 15 different clonal types were identified at baseline and 16 months, respectively. None of the students harboured identical clonal types at both sampling times. Our results show that ribotyping is a useful technique for monitoring the distributions of F. nucleatum clonal types and indicate that healthy individuals with optimal plaque control can be colonized by more than one F. nucleatum clonal type and that these clonal types appear to be unstable.

Adult↗

PubMed2003

Determination of clonality of metastasis by cell-specific color-coded fluorescent-protein imaging.

It has been thought that metastases are clonal and originate from rare cells in primary tumors that are heterogeneous in genotype and phenotype. Recent studies using DNA array analysis challenge this hypothesis and suggest the genetic background of the host is the important determinant of metastatic potential implying that metastases are not necessarily clonal. Previous methods to determine clonality of metastasis used karyotype or molecular analysis that were complicated, thereby limiting the number of metastatic colonies analyzed and the conclusions that could be drawn. We describe here the use of green fluorescent protein-labeled or red fluorescent protein-labeled HT-1080 human fibrosarcoma cells to determine clonality by simple fluorescence visualization of metastatic colonies after mixed implantation of the red and green fluorescent cells. Resulting pure red or pure green colonies were scored as clonal, whereas mixed yellow colonies were scored as nonclonal. In a spontaneous metastasis model originating from footpad injection in severe combined immunodeficient mice, 95% of the resulting lung colonies were either pure green or pure red, indicating monoclonal origin, whereas 5% were of mixed color, indicating polyclonal origin. In an experimental lung metastasis model established by tail vein injection in severe combined immunodeficient mice, clonality of lung metastasis was dependent on cell number. With a minimum cell number injected, almost all (96%) colonies were pure red or green and therefore monoclonal. When a large number of cells were injected, almost all (87%) colonies were mixed color and therefore heteroclonal. We conclude that spontaneous metastasis may be clonal because they are rare events, thereby supporting the rare-cell clonal origin of metastasis hypothesis. The clonality of the experimental metastasis model depended on the number of input cells. The simple fluorescence method of determining clonality of metastases described here can allow large-scale clonal analysis in numerous types of metastatic models.

Animals↗

PubMed2005

Molecular clonality determination of ipsilateral recurrence of invasive breast carcinomas after breast-conserving therapy: comparison with clinical and biologic factors.

We established clonality relationships between invasive ipsilateral breast failures (IBFs; local recurrences) and initial invasive carcinomas using a molecular polymerase chain reaction loss of heterozygosity (LOH) assay for 26 patients treated with breast-conserving therapy for invasive carcinoma with no distant metastases (DMs) before the IBE LOH was +/- 50% allelic loss. Eighteen IBFs (69%) were related clonally to initial carcinomas; 8 (31%) were clonally distinct, second primary carcinomas. IBFs and initial invasive carcinomas were morphologically similar in 6 (75%) of 8 clonally different cases. Clinical IBF classification and molecular assay results differed in 11 cases (42%). The mean intervals to IBF were 4.7 years in related and 8.7 years in different cases (P = .013). In 6 patients, DMs developed; 5 had related IBFs. In related IBF cases, the mean increase in fractional allelic loss (FAL) of IBFs associated with DMs was 18.9% compared with 7.6% in cases unassociated with DMs (P = .004). Molecular assays can accurately establish the clonality of most IBFs. Morphologic comparison and clinical IBF classification are unreliable methods of determining clonality. Clonally related IBFs occurred sooner than clonally different IBFs. Patients with clonally related IBFs are the main pool in which DMs occur Not all clonally related IBFs have the same DM association; those with large FAL gains were associated with DMs. Molecular clonality assays may provide a reliable means of identifying patients who might benefit from systemic chemotherapy at the time of IBF.

Adenocarcinoma↗

PubMed1995

Quantitative non-radioactive clonality analysis of human leukemic cells and progenitors using the human androgen receptor (AR) gene.

Clonal analysis of FACS-purified primitive hematopoietic stem cells and of their progeny as assessed by the progenitors obtained from long-term cultures requires PCR-based approaches, mainly because of the low number of cells available. We have developed a non-radioactive androgen receptor (AR) assay which allows a simple and quantitative evaluation of the clonality of hematopoietic cells and progenitors. In this approach 5' AR primer is labelled by fluorescein and the amplified product is run on a sequencing gel which allows evaluation of the intensity of the fluorescent peaks generated. A computer software then analyzes the reduction of the intensity of the peaks on HpaII-digested samples. In order to determine the feasibility of the technique, we analyzed the clonality of leukemic cells from a patient with an acute-phase CMML which showed a typical clonal pattern of her leukemic DNA sample (WBC = 300 x 10(9)/I) using phosphoglycerate kinase (PGK) analysis. The same sample was then analyzed with either radioactive- or fluorescein-labelled AR primers, showing a typical clonal pattern (complete disappearance of one allele after HpaII digestion). A short-term clonogenic assay was then set up on methylcellulose and clonogenic progenitors were individually analyzed. All 24 colonies tested showed a typical clonal pattern with the disappearance of the same allele on each sample after HpaII digestion, indicating that they all derived from the same leukemic stem cell. Using this approach we then analyzed 94 patients with several hematologic malignancies and quantification of their fluorescent peaks. Fifty-four percent of the patients were clearly heterozygous (ie, a difference of > or = 2 CAG repeats was present between the two copies of the gene) and could be analyzed in an automatic sequencer using the fluorescent primers. Bone marrow mononuclear cells from all patients with acute myeloid leukemia (AML) showed a clonal or oligoclonal pattern at diagnosis whereas a polyclonal pattern was seen when remission was obtained. Similarly, out of 21 patients with a diagnosis of myelodysplastic syndrome (MDS), a clonal pattern was demonstrated in 10 whereas an oligoclonal or non-clonal pattern was shown in 11. These results show that this non-radioactive and safe technology can now be used on a large scale to evaluate the clonality of highly purified hematopoietic stem cells and their progenitors in hematopoietic malignancies and this might allow new insights into the targets of clonal amplification.

Acute Disease↗

PubMed1998

Clonal diversity of Ig and T-cell-receptor gene rearrangements identifies a subset of childhood B-precursor acute lymphoblastic leukemia with increased risk of relapse.

Current prognostic indicators such as age, sex, and white blood cell count (WBC) fail to identify all children with more aggressive forms of B-precursor acute lymphoblastic leukemia (ALL), and a proportion of patients without poor prognostic indicators still relapse. Results obtained from an analysis of 65 pediatic B-precursor ALL patients indicated that subclone formation leading to clonal diversity, as detected by Ig and T-cell receptor (TCR) gene rearrangements, may represent a very useful prognostic indicator, independent of age, sex, and WBC. Disease-free survival was significantly shorter in those patients showing clonal diversity at presentation. Furthermore, clonal diversity was detected not only in the majority of high-risk patients who relapsed but was also associated with a high probability of relapse in standard-risk patients. Sixty-five percent (13/20) of standard-risk patients who also showed clonal diversity subsequently relapsed, whereas the percentage of relapses among standard-risk patients without clonal diversity was much lower at 19% (7/36). Continued clonal evolution during disease progression is an important feature of aggressive B-precursor ALL. All 5 patients with clonal diversity who were followed up in our study showed a change in the pattern of clonality between presentation and relapse. This implies an important role for clonal diversity as a mechanism of disease progression through the process of clonal variation and clonal selection.

Adolescent↗

PubMed2001

An approach to the sensitivity of temperature-gradient gel electrophoresis in the detection of clonally expanded T-cells in cutaneous T-cell lymphoma.

Detection of T-cell clonality by polymerase chain reaction (PCR) and high-resolution electrophoresis facilitates differentiation of early stages of cutaneous T-cell lymphoma (CTCL) from benign T-cell-rich dermatoses. However, data regarding the sensitivity of the various electrophoresis techniques differ remarkably. In the present study, the capacity of heteroduplex (HD)-loaded temperature-gradient gel electrophoresis (TGGE) to detect clonally expanded T-cells was assessed systematically and modifications to the procedure were defined. Using our standard protocol, HD-TGGE detected clonal T-cell receptor (TCR)-gamma PCR products, generated from the Jurkat cell line, down to a total of 2 ng/microL (14 ng) DNA. However, slowly migrating single strands of the clonal PCR product reduced the amount of the clonality indicating homoduplices. To overcome this single-strand formation, thus decreasing the detection limit, the urea concentration in the gel and the temperature ramp for the HD-formation were altered, as well as the temperature gradient in the gel. Application of the modified protocol resulted in a tenfold lower detection limit of 0.15 ng/microL (1.05 ng) DNA in the clonal band. The sensitivity of the adapted HD-TGGE was investigated by dilution experiments using the well established T-cell lines Jurkat, Molt-4, MyLa and SeAx. By these approaches clonal PCR products diluted in nonclonal PCR products were detectable down to concentrations of 5-10%. Comparably, in the case of mixtures of clonal in nonclonal DNA the detection limit reached 5-10% clonal DNA. However, by dilution of clonal cells in nonclonal peripheral blood mononuclear cells, which corresponds to in vivo conditions, a lower detection limit of approximately 1-5% was observed.

DNA, Neoplasm↗

PubMed2004

Clonal B cell populations in the blood and liver of patients with chronic hepatitis C virus infection.

OBJECTIVE: The association of hepatitis C virus (HCV) infection with type II mixed cryoglobulinemia is well established, but the role of HCV in B cell lymphoma remains controversial. The objective of this study was to determine the frequency of circulating and liver-infiltrating monoclonal B cells in patients with HCV infection. METHODS: One hundred sixty patients were studied prospectively, including 115 HCV-positive patients and 45 HCV-negative patients with other nonimmune chronic liver disease(s). B cell clonality was determined by DNA amplification of the IgH rearrangements, followed by polyacrylamide gel electrophoresis. RESULTS: A clonal B cell population was detected in the blood of 21 (26%) of 81 HCV-positive patients whose cryoglobulin status was known, including 12 of 25 patients with type II cryoglobulinemia, 2 of 12 patients with type III cryoglobulinemia, and 7 of 44 patients without cryoglobulins. A clonal IgH rearrangement was detected in 26 (32%) of 81 liver biopsy specimens from HCV- infected patients, including 16 patients with a circulating clonal population. A clonal B cell population was not observed in the blood of 40 patients with non-HCV liver diseases and was present in only 1 (3%) of 30 liver biopsy specimens. Logistic regression analysis showed that HCV-infected patients with clonal B cell proliferation in both the blood and liver were older (P = 0.004) and had longer duration of HCV infection (P = 0.009), higher serum cryoglobulin levels (P = 0.001) that were more frequently symptomatic (P < 0.03), and liver disease that was more severe than that in patients without a clonal B cell population in the blood or liver (P = 0.05). In 4 of 16 patients with a clonal B cell population in both the blood and liver, a definite B cell malignancy was finally diagnosed. CONCLUSION: Clonal B lymphocytes are frequently detected in the blood and liver of patients with chronic HCV infection, in the absence of overt B cell malignancy. These clones are usually, but not always, associated with the presence of type II cryoglobulins. A high percentage of patients with B cell clonality in both the blood and liver were finally diagnosed as having a definite B cell malignancy.

Adult↗

PubMed2000

Cytokines can reduce clonal, CD34-positive cells in acute myeloid leukemia in vitro.

We studied the influence of cytokine mixes on the survival of acute myeloid leukemia (AML) bone-marrow (BM) cells in a 14-day culture assay in vitro. Southern-blot analysis using a panel of different probes in combination with densitometry and flow cytometry were used to detect and compare the amount of clonal or CD34-positive BM cells before and after the culturing procedure. A significant reduction of CD34-positive cells after incubation with a cytokine mix [interleukin (IL)-1beta, IL-3, IL-6, stem cell factor (SCF), erythropoietin (EP) with granulocyte macrophage/colony-stimulating factor (GM-CSF, Cytok1) could be achieved in all 16 cases with a CD34-positive blast phenotype studied at diagnosis (P<0.001), in 3 of 10 cases at relapse, and in 8 of 18 cases in complete remission. In healthy donors, an increase of CD34-positive cells was demonstrated in 5 of 5 samples. A reduction of clonal DNA through incubation with Cytok1 was achieved in 5 of 5 (100%) cases studied at diagnosis, in 1 of 4 (25%) cases at relapse, and in 7 of 9 cases (78%) in complete remission. Cytokine cocktails with GM-CSF (Cytok1) were more efficient in reducing (clonal) CD34-positive cells than cocktails without GM-CSF (Cytok2). AML patients at diagnosis and in complete remission had a better survival probability if their CD34-positive or clonal cells could be reduced in vitro by cytokine cultivation (P<0.05). Vitality of BM cells was not influenced by 14-day cytokine treatment; however, the total cell count could be increased by Cytok1 and Cytok2 by 55-174%, but not by the control medium. Our data show that: (1) clonal cell populations can be regularly detected at diagnosis, during complete remission, and at relapse; (2) CD34-positive cells in AML can be demonstrated to be clonal, gene-rearranged cells; (3) incubation of AML BM-cells with Cytokl leads to a reduction of the CD34-positive, clonal cell load in all cases at diagnosis and in 78% of the cases in complete remission of AML, but in only 25% of the cases at relapse; (4) in all healthy BM samples, proportions of 'healthy' CD34-positive cells were increased. Moreover, absolute cell counts were increased by cytokine incubation of cells obtained at diagnosis, relapse, or complete remission of AML and from healthy donors indicating a selective stimulation of healthy, but not of leukemic CD34-positive cells; (5) cytokine cocktails containing GM-CSF are more efficient in reducing leukemic cells than cocktails without GM-CSF; and (6) in vitro reactivity of clonal or CD34-positive BM cells against Cytokl has clinical relevance. We conclude, that Southern-blot analysis and flow cytometry are suitable methods to detect and quantify leukemic disease and to distinguish between clonal or non-clonal CD34-positive cells. The ex vivo or clinical application of specific combinations of cytokines might be a feasible and successful application of immunotherapy in AML that merits further investigations.

Acute Disease↗

PubMed2005

Sexual reproduction, clonal diversity and genetic differentiation in patchily distributed populations of the temperate forest herb Paris quadrifolia (Trilliaceae).

Clonal plant species have been shown to adopt different strategies to persist in heterogeneous environments by changing relative investments in sexual reproduction and clonal propagation. As a result, clonal diversity and genetic variation may be different along environmental gradients. We examined the regional and local population structure of the clonal rhizomatous forest herb Paris quadrifolia in a complex of forest fragments in Voeren (Belgium). Relationships between population size (the number of shoots), shoot density (the number of shoots per m2) and local growth conditions were investigated for 47 populations. Clonal diversity and genetic variation within and among 19 populations were investigated using amplified fragment length polymorphism markers. To assess the importance of sexual reproduction, seed set, seed weight and germination success were determined in 18 populations. As predicted, local growth conditions largely affected population distribution, size and density of P. quadrifolia. Populations occurring in moist and relatively productive sites contained significantly more shoots. Here, shoots were also much more sparsely distributed compared to populations occurring in dry and relatively unproductive sites, where shoots showed a strongly aggregated distribution pattern. Clonal diversity was relatively high, compared with other clonal species (G/N ratio=0.43 and Simpson's D=0.81). Clonal diversity significantly (P<0.01) decreased with increasing shoot density while molecular genetic variation was significantly (P<0.01) affected by population size and local environmental conditions. Lack of recruitment and out-competition of less-adapted genotypes may explain the decreased genetic variation in dry sites. Analysis of molecular variance revealed significant genetic variation among populations (PhiST=0.42, P<0.001), whereas pairwise genetic distances were not correlated to geographic distances, suggesting that gene flow among populations is limited. Finally, the number of generative shoots, the number of seeds per fruit and seed weight were significantly and positively related to population size and local growth conditions. We conclude that under stressful conditions populations of clonal forest plant species can slowly evolve into remnant populations characterized by low levels of genetic variation and limited sexual reproduction. Conservation of suitable habitat conditions is therefore a prerequisite for effective long-term conservation of clonal forest plant species.

Magnoliopsida↗

PubMed2006

Wotherspoon criteria combined with B cell clonality analysis by advanced polymerase chain reaction technology discriminates covert gastric marginal zone lymphoma from chronic gastritis.

BACKGROUND AND AIMS: Gastric mucosa associated lymphoid tissue lymphoma is a well defined B cell lymphoma yet often impossible to distinguish from severe chronic gastritis on morphological grounds alone. Therefore, it was suggested to use the clonality of the immunoglobulin (Ig) heavy chain (H) genes, as detected by polymerase chain reaction (PCR), as a decisive criterion. However, there is controversy as to whether B cell clonality also exists in chronic gastritis, hence rendering this approach futile at present. METHODS: An expert panel re-examined the histology and immunohistochemistry of a total of 97 cases of gastric biopsies, including clearcut marginal zone lymphoma, chronic gastritis, and ambiguous cases, applying the Wotherspoon criteria on the basis of haematoxylin-eosin and CD20 immunostainings. In addition, a new and advanced PCR system for detection of clonal IgH gene rearrangements was independently applied in two institutions in each case. RESULTS: The overall IgH clonality assessments of both institutions were in total agreement. Overt lymphoma (Wotherspoon score 5) was clonal in 24/26 cases. Chronic gastritis (Wotherspoon scores 1 and 2) was not clonal in 52/53 cases; the clonal case being Wotherspoon score 2. Of 18 cases with ambiguous histology (Wotherspoon scores 3 and 4) four were clonal. CONCLUSIONS: Using advanced PCR technology, clonal gastritis is extremely rare, if it exists at all. Thus B cell clonality in Wotherspoon 3 and 4 cases is regarded as suitable for definitively diagnosing gastric marginal zone lymphoma.

Algorithms↗

PubMed1992

Clonal analysis of human tumors with M27 beta, a highly informative polymorphic X chromosomal probe.

The clonality of human tumors can be studied by X inactivation/methylation analysis in female patients heterozygous for X-linked DNA polymorphisms. We present a detailed study on clonal tumor analysis with M27 beta, a highly informative probe detecting a polymorphic X chromosomal locus, DXS255. The polymorphism detected at this locus is due to variable numbers of tandem repeats. The rate of constitutional heterozygosity detected by M27 beta was 88%. Normal tissue from gastrointestinal mucosa and thyroid showed random, hence polyclonal, patterns. Nonrandom clonal X inactivation was detected in all 22 malignant neoplasms that had been shown to be clonal by other DNA markers, such as antigen receptor gene rearrangements or clonal loss of heterozygosity at 17p and other loci. 16/48 normal blood leukocyte samples (33%) showed considerably skewed X inactivation patterns. Comparison of blood leukocytes and normal tissue indicated that in a given individual, X inactivation patterns may be tissue specific. M27 beta was used to study the clonal composition of 13 benign thyroid nodules from 12 multinodular goiters with rapid recent growth, traditionally termed "adenomas." Nine of them were clonal, whereas four nodules and tissue from a case of Graves' goiter were not, indicating that some, but not all, such thyroid nodules may represent true clonal neoplasms. The M27 beta probe permits one to study the clonal composition by the X inactivation approach of a wide variety of solid tumors from most female patients. As a control, normal tissue homologous to the tumor type of interest is preferable to DNA from blood leukocytes, since the latter may show nonrandom X inactivation patterns in a fairly high proportion of cases. M27 beta may, therefore, be of limited use for the clonal analysis of neoplasms derived from hematopoietic cells.

Clone Cells↗

PubMed1998

Suppression of cytogenetic clonal evolution with interferon alfa therapy in patients with Philadelphia chromosome-positive chronic myelogenous leukemia.

PURPOSE: To investigate whether cytogenetic clonal evolution can be suppressed with interferon alfa (IFN-alpha) therapy in patients with chronic myelogenous leukemia (CML). PATIENTS AND METHODS: Ninety patients with CML and cytogenetic clonal evolution who received IFN-alpha-based regimens were analyzed. Clonal evolution was defined as the presence of karyotypic abnormalities in addition to the Philadelphia (Ph) chromosome. Patients were evaluated for the suppression of cytogenetic clonal evolution after therapy, the cytogenetic response, and survival. RESULTS: The median age of the population was 39 years (range, 15 to 70 years), median time from diagnosis to clonal evolution 14 months (range, 0 to 145 months), and median percentage of abnormal metaphases 18% (range, 4% to 100%). Fifty six patients (62%) achieved some suppression of cytogenetic clonal evolution; in 41 patients (46%), the suppression was complete. The overall median survival was 51 months, with 43% alive at 5 years. Patients who achieved a complete suppression of cytogenetic clonal evolution had a median survival of 66 months, with 51% alive at 5 years. Characteristics associated with a better response include a lower percentage of abnormal metaphases, time to cytogenetic clonal evolution of 24 months or less, and absence of other features of accelerated disease. A prognostic classification for cytogenetic clonal evolution defined three groups with complete response (CR) rates of 85%, 34%, and 0% (P < .0001) and median survival times of 58, 31, and 30 months, respectively (P=.02). CONCLUSION: Patients with cytogenetic clonal evolution can respond to IFN-alpha therapy, and this response is associated with longer survival. A previously described prognostic model separates patients into subsets with different probabilities of response to IFN-alpha and survival.

Adolescent↗

PubMed1999

Chromosome aberrations of clonal origin in irradiated and unexposed individuals: assessment and implications.

Chromosome painting has proven useful for the detection of chromosomal rearrangements, although the presence of cells containing clonal aberrations can have an effect on the outcome of cytogenetic analyses (e.g. aberration frequency and chromosomal distribution studies). Cells with clonal chromosomal changes have been found in studies of both radiation-exposed Chernobyl cleanup workers ("liquidators") and healthy unexposed human subjects. We have used a simple statistical method to aid in the identification of individuals from distinct Chernobyl radiation-exposed and unexposed control populations who may possess cells containing clonal rearrangements. A chi2 value determined from the observed number of aberrations and the expected number based on chromosome length that corresponds to a probability less than 0.005 appears to be an indicator of clonality. These selected individuals can be analyzed further for clonality, thereby sparing detailed examination of the entire population. Here we present an analysis of individuals possessing clonal aberrations to assess the influence of clonality on the results of cytogenetic studies. Our results show that the subtraction of clonal events from the chi2 calculation for the "outliers" results in nearly all of these values losing their statistically significant deviation from proportionality. These adjustments can also be made to prevent the overestimation of frequencies of chromosome aberrations for biodosimetry. The frequency of clonal aberrations appears to increase as a function of age in control subjects, whereas an age effect was not evident in Chernobyl liquidators. This suggests that spontaneous and radiation-induced clonal expansion are occurring in control subjects and liquidators, respectively.

Adult↗

PubMed2001

[The Detection of Clonal IgH Gene Rearrangement in Bone Marrow and Peripheral Blood from B-NHL Patients and Its Clinic Significance]

To evaluate the significance of bone marrow (BM) and peripheral blood (PB) cells with clonal gene rearrangement of the third complementary determining region of immunoglobulin heavy chain (IgHCDR3) in the diagnosis, clinical staging, determination of treatment effects and prediction of relapse in B-NHL, clonal IgH gene rearrangement of BM from 46 and PB from 38 cases with B-NHL were tested by semi-nested polymerase chain reaction (SnPCR) and polyacrylamide gel electrophoresis before treatment, and ten of them were tested in complete remission after treatment. Results showed that this method was applicable to detecting one clonal IgHCDR3 gene rearrangement positive cell from up to 1 000 normal cells. Specificity of detection was 97%. Clonal IgHCDR3 rearrangement was shown in all 3 cases of BM and 2 of PB specimens with morphologic involvement. The clonal IgHCDR3 was detected in 65.1% of the BM and 44.4% of the PB without morphologic involvement in untreated patients with B-NHL, independent of Ann Arbor staging and systemic symptoms. In 10 cases of B-NHL with clonal IgHCDR3 rearrangement in diagnostic tissues, the molecular marker became negative in 7 patients who entered and remained in complete remission. Two cases relapsed in whom clonal IgHCDR3 rearrangements were detected in serial samples of BM or PB after autologous PBSCT. One patient in whom clonal IgHCDR3 rearrangement was detected at 10 months post-PBSCT remained in complete remission up to now. It was concluded that clonal IgHCDR3 gene rearrangements were found in BM and PB from B-NHL patients without morphologic abnormality. Persistence of molecular marker-positive may be associated with relapse for patients in complete remission, and the patients without clonal IgHCDR3 rearrangement will be in continuous complete remission. Little is known about a few patient who was a long-term disease-free survivor despite the presence of PCR-IgH rearrangement in the marrow.

Journal Article↗

PubMed1996

Clonal circulating cells are common in plasma cell proliferative disorders: a comparison of monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, and active myeloma.

The blood of most patients with active multiple myeloma (MM) contains cells related to the bone marrow tumor. However, identifying clonal cells in the blood of patients with monoclonal gammopathy of undetermined significance (MGUS) has been difficult. In this study, we analyzed blood mononuclear cells (BMNCs) from 16 patients with MGUS, 2 with amyloidosis, 8 with smoldering MM (SMM), 2 with indolent MM (IMM), and 15 with active MM using three different methods to detect and quantitate clonal cells, ie, immunofluorescence microscopy (IM) for monoclonal plasma cells, three-color flow cytometry (FC) for CD38(+)CD45- CD45(dim) cells, and the allele-specific oligonucleotide polymerase chain reaction (ASO-PCR). Using ASO-PCR, we were able to detect clonal cells in the blood in 13 of 16 patients with MGUS, 2 of 2 with amyloid, 6 of 8 with SMM, 2 of 2 with IMM, and 13 of 15 with MM. In 9 of the 13 patients with MGUS with blood involvement, the number of clonal cells was very small ( < 0.04% of the BMNCs). The median percentage of clonal cells as determined by ASO-PCR was 0.02 for MGUS, 0.02 for SMM, and 0.24 for MM. Clonal plasma cells or CD38+CD45-CD45(dim) cells were identified by IM or FC in 6 of 16 MGUS patients, 4 of 8 with SMM, and 11 of 15 with MM. In all cases in which IM or FC detected clonal cells, the ASO-PCR was positive. This study shows that, by using ASO-PCR, clonal cells can be found at very low levels in the blood in most patients with MGUS. However, the number of clonal cells in the blood of MGUS patients is less than those with overt MM (P = .006). In contrast to MGUS, patients with active MM are more likely to have identifiable clonal circulating plasma cells (P = .05).

Alleles↗

PubMed2024

A rapid and simple clonality assay for bovine leukemia virus-infected cells by amplified fragment length polymorphism (AFLP) analysis.

UNLABELLED: Enzootic bovine leukosis (EBL), although eradicated in some European countries, is still the most common neoplastic disease of cattle, caused by the bovine leukemia virus (BLV). During the progression of EBL, BLV-infected cells clonally expand, and some of which result in tumor onset. The clonality of BLV-infected cells is generally evaluated with NGS or Sanger sequencing. Although these methods clearly distinguish EBL from non-EBL cases, the procedures are complex and not practical for routine veterinary diagnosis. In this study, we developed an amplified fragment length polymorphism (AFLP) analysis for BLV clonality assay (BLV-AFLP). This analysis uses restriction enzyme digestion to amplify the chimeric regions of BLV 3' linear transcribed region (LTR) and host genome through conventional polymerase chain reaction (PCR) and visualizes the results by gel-electrophoresis. The method was established using cattle samples representing different stages of the disease: BLV-uninfected, non-EBL, and EBL cattle. Non-EBL cattle showed smeared bands, indicating polyclonal proliferation, while EBL cattle showed distinct bands, indicating clonal expansion. The results of BLV-AFLP correlated well with those of previously reported methods, suggesting its efficacy in detecting clonal proliferation. The validation using blood samples of non-EBL cattle and tumor samples of EBL cattle confirmed that BLV-AFLP could effectively identify clonal proliferation in EBL samples. Moreover, the emergence of dominant clones in the tumor at later stages was successfully detected before EBL onset in some cattle, highlighting its sensitivity and potential for early detection. Overall, BLV-AFLP is suitable for practical use in the field, improving BLV management strategies and minimizing economic losses. IMPORTANCE: Enzootic bovine leukosis (EBL) is routinely diagnosed based on external manifestations at the farm, such as the presence of tumors and/or general lymph node enlargement. However, due to the nonspecific clinical manifestations of EBL, over half of EBL cases are unrecognized at the farm, with most cases being diagnosed during postmortem inspection at the slaughterhouse. Early detection and monitoring of clonal expansion are necessary for managing EBL and reducing economic losses. In this study, we developed BLV-AFLP that represents a significant advancement in the diagnosis of EBL in cattle. This method can rapidly assess the clonal proliferation of BLV-infected cells, crucial for distinguishing between asymptomatic and EBL cattle. Additionally, tracking clonal dynamics offers insights into the disease's progression, potentially providing strategies for avoiding economic losses. Overall, as BLV-AFLP is a simple and rapid test for detecting EBL, it is feasible and efficient for routine veterinary practice.

Leukemia Virus, Bovine↗

PubMed1999

Microsatellite instability and clonal heterogeneity of MDR1 messenger RNA expression in trimetrexate-resistant human leukemia MOLT-3 cells developed in thymidine.

Various gene alterations are involved in the drug resistance of leukemia cells. To understand the mechanism that underlies the emergence of cells with such gene alterations in human leukemia, we performed clonal analysis of the gene expression of mutant dihydrofolate reductase (DHFR) and mdr1 in trimetrexate-resistant human leukemia MOLT-3 cells. Trimetrexate-resistant (70- and 60-fold) sublines were developed in the presence or absence of an exogenous supply of thymidine (MOLT-3/TMQ70/Th+, MOLT-3/TMQ60/Th-, respectively). Ten clonal lines were isolated by methyl cellulose cloning from each of the 2 trimetrexate-resistant MOLT-3 sublines. All the clonal lines from the 2 sublines expressed mutated DHFR mRNA, with a base change (T --> C) at the second position of codon 31, as well as the wild-type mRNA, in accordance with cross-resistance to methotrexate. On the other hand, mdr1 mRNA expression was demonstrated by reverse-transcription polymerase chain reaction only in clonal lines from MOLT-3/TMQ70/Th+ cells. mdr1 mRNA expression in clonal lines from MOLT-3/TMQ70/Th+ cells and subclonal lines subsequently obtained from the 3 clonal lines with different mdr1 mRNA expression levels was heterogeneous, and its high expression levels were correlated with acquisition of the multidrug resistance (MDR) phenotype. Polymerase chain reaction-based assay for separate microsatellites, mfd27 and mfd41, demonstrated genomic instability among clonal and subclonal lines of MOLT-3. The clonal analysis of polymorphic microsatellites also suggested that emergence of MDR in trimetrexate-resistant MOLT-3 cells in thymidine was not only heterogeneous but also progressively expanding among clones. Genomic instability may play a role in the establishment and clonal evolution of drug resistance in leukemia cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Detection of clonally rearranged T-cell-receptor gamma chain genes from T-cell malignancies and acute inflammatory rheumatic disease using PCR amplification, PAGE, and automated analysis.

Clonal expansions of T cells carrying identical T-cell-receptor (TCR) genes are the hallmark of T-cell malignancies, but they can also result from a strong immune reaction to a dominant epitope. The basis for the molecular detection of clonal T cells is amplification of the V-(D)-N-J region of the TCR gene. We evaluated PCR amplification of the rearranged gamma TCR from genomic DNA extracted from peripheral blood and subsequent polyacrylamide gel electrophoresis (PAGE) in an automated DNA sequencer. We determined the sensitivity for the detection of clonal T cells and propose a standardized evaluation procedure for the electrophoretic profiles generated by the DNA sequencer. The sensitivity of our method was 0.6-1.25% of clonal T cells within a polyclonal background. Sixteen patients with T-cell malignancies, ten with acute inflammatory rheumatic diseases, and twelve healthy controls were examined. Among the systemic T-cell malignancies, all but one patient with T-PLL (8/ 9) revealed a clonal PCR signal. No clonal signal was detectable in any patient in clinical complete remission (5/5) or in either of the two patients with lymphomas limited to cutaneous sites. However, clonal T cells were detected in one patient with polymyalgia rheumatica and in one with reactive arthritis. A polyclonal signal was found in the remaining eight patients with acute inflammatory rheumatic diseases and in 12 healthy controls. Taking our results together, the PCR/PAGE assay is able to reliably distinguish clonal from polyclonal T-cell populations. However, although the sensitivity is limited to approximately 1%, clonal T cells can be found in the peripheral blood of some patients with autoimmune diseases and not only in T-cell malignancies.

Adult↗