1/11. A real-time PCR assay for the quantification of residual malignant cells in B cell chronic lymphatic leukemia.Several new therapeutic approaches for the treatment of monoclonal B cell lymphomas are currently being investigated. In parallel with new therapeutic modalities, more sensitive diagnostic methods are needed. These methods should be highly sensitive in detecting very low amounts of malignant cells and should be specific for the malignant clone. In addition, these methods should allow the quantification of residual tumor cells. In this study a new real-time polymerase chain reaction (LightCycler) was evaluated to quantify residual tumor cells in monoclonal B cell malignancies. This technology combines the advantages of rapid cycling PCR with the online detection of PCR products using fluorescent dyes. Our assay is based on immunoglobulin heavy chain (IgVH)-specific PCR with allele-specific primers complementary to hypervariable CDRII and CDRIII regions. A set of framework region III (FRIII)-specific hybridization probes was used for detection of the specific amplification product, and IgVH copy number was quantified with the cloned IgVH sequence as an external standard. The approach was evaluated with the Hodgkin lymphoma cell line L428 in order to quantify L428 dilutions. L428 cells mixed with peripheral blood mononuclear cells (PBMNCs) were detected and quantified with a sensitivity of one cell within 1 x 10(5) PBMNCs. Sample dna from the peripheral blood and from the bone marrow of two patients with B-CLL was analyzed in the new set up at different time points before and after therapy. Statistically significant changes in IgVH copy numbers were documented in both patients. We conclude that this technology offers an additional opportunity to detect and quantify residual tumor cells in B-CLL over several log steps with a high sensitivity. The kinetics of residual tumor cell counts in B-CLL can be analyzed by this method.- - - - - - - - - - ranking = 1keywords = hybridization (Clic here for more details about this article) |
2/11. fluorescence in situ hybridization evaluation of minimal residual disease on stem-cell harvests.The usefulness of fluorescence in situ hybridization (FISH) analysis to detect minimal residual disease (MRD) in autologous bone marrow and peripheral blood stem-cell harvests has been tested in three patients with hematologic malignancies. Conventional cytogenetics and FISH were used to characterize the leukemic clones identifying the specific chromosomal abnormalities (monosomy 7 in a myelodysplastic patient and trisomy 8 in two acute myeloid leukemic patients). Such analysis was useful to monitor the MRD persistent after treating these patients with intensive chemotherapy. The myelodysplastic patient underwent eight peripheral blood-stem cell harvests in which FISH detected the persistence of monosomy 7 cells, precluding their use for autologous transplantation. This patient relapsed and died. In two acute myeloid leukemia patients who underwent an autologous marrow harvest, FISH did not show a significant proportion of trisomy 8 cells. Nevertheless, autologous transplantation was not performed, owing to an insufficient CD34 cell content in the harvests. One of these patients relapsed with the reappearance of trisomy 8 and died. The other patient, on the contrary, is alive in complete remission 3 years after the bone marrow harvest. The usefulness and applicability of MRD quantification in stem-cell harvests is discussed on the basis of the sensitivity of the methodology applied.- - - - - - - - - - ranking = 5keywords = hybridization (Clic here for more details about this article) |
3/11. Donor lymphocyte infusion followed by interferon-alpha plus low dose cyclosporine A for modulation of donor CD3 cells activity with monitoring of minimal residual disease and cellular chimerism in a patient with first hematologic relapse of chronic myelogenous leukemia after allogeneic bone marrow transplantation.A 15-year-old girl with Ph-positive chronic myelogenous leukemia in first chronic phase received bone marrow from her human leukocyte antigen matched brother. Twenty three months after bone marrow transplantation hematological relapse occured which was treated with two infusions of donor lymphocytes (DLI) (0.5x10(8) CD3/kg b.w./infusion). To enforce the graft-versus-leukemia effect (GvL), the first DLI was followed by administration of interferon-alpha (INF-alpha) 6x10(6) U/day for 30 days, whereas, after the second infusion INF-alpha was given at the same dose until hematological remission was achieved (80 doses). Minimal residual disease (MRD) was detected by conventional cytogenetics (Ph chromosome), fluorescence in situ hybridization (FISH) cytogenetics (BCR/ABL translocation) and reverse transcriptase-polymerase chain reaction (RT-PCR) Ecotropic virus integration site-1 (EVI-1 gene expression), whereas cellular chimerism was monitored by assessment of microsatellite markers PCR and Y-chromosomal dna content FISH. When hematological remission was achieved the pancytopenia was observed and the cytogenetic and molecular investigations revealed only partial remission and mixed chimerism, however, with predominance of donor origin hematopoiesis. To diminish the myelosupressive effect of donor CD3 cells without switching-off the GvL effect, a low dose of cyclosporine A was given. Further observation revealed significant improvement of hematopoiesis with parallel gradual decline of MRD and increase of donor hematopoiesis up to complete chimerism. Graft-versus-host disease was not observed at any stage of the treatment.- - - - - - - - - - ranking = 1keywords = hybridization (Clic here for more details about this article) |
4/11. Detection of minimal residual disease in peripheral blood stem cells from two acute myeloid leukemia patients with trisomy 8 predicts early relapse after autologous bone marrow transplantation.We report two cases of acute myeloid leukemia (AML) French-American-British M4 classification with trisomy 8 at diagnosis as the sole chromosome abnormality. Both patients were treated with the GIMEMA AML-10 protocol and underwent autologous bone marrow transplantation (ABMT) in hematologic remission. Peripheral blood stem cells (PBSC), and bone marrow in one patient, were collected after consolidation therapy and tested by fluorescence in situ hybridization (FISH) analysis with an alpha-satellite probe for chromosome 8. It revealed that all samples were positive for minimal residual disease (MRD) as the value of trisomic cells exceeded the mean 3 standard deviations of the controls. ABMT was done following a myeloablative regimen (busulphan/cyclophosphamide) and PBSC were reinfused. Both patients relapsed, 4 and 2 months, respectively, after autotransplant. Although more data are needed, these results suggest that the persistence of MRD, as detected by FISH, in stem cell collections, is associated with a poor outcome in AML patients with trisomy 8 undergoing ABMT.- - - - - - - - - - ranking = 1keywords = hybridization (Clic here for more details about this article) |
5/11. Combined analysis of morphology and fluorescence in situ hybridization in follow-up of minimal residual disease in a child with Philadelphia-positive acute lymphoblastic leukemia.The past decade has brought new technologies to the study of minimal residual disease (MRD) in leukemia. Each of them has limitations and is far from being accurate. Recently, a new multiparametric cell scanning system (Duet) was introduced to the field of MRD detection. This system has the advantage of automatically scanning large numbers of cells and performing combined analysis of morphology and fluorescence in situ hybridization (FISH) on the same cell. We used this system to characterize the lineage and degree of maturation of the cells carrying the minor m-BCR/ABL fusion, in a follow-up of an 8-year-old boy with Philadelphia-positive (Ph( )) acute lymphoblastic leukemia (ALL). The boy was treated using a high-risk protocol and was closely monitored with FISH analysis for cells carrying the m-BCR/ABL fusion. Consecutive analysis along 2.5 years from remission showed 0.2-4.5% m-BCR/ALB( ) cells in the peripheral blood (PB), which is within the accepted background range for this method. The combined analysis found that all the m-BCR/ABL( ) cells were mature lymphocytes. Because mature lymphocytes have a long life span in the circulation, this finding supports the fact that the patient is in remission. Moreover, since mature differentiated cells have a low proliferative capacity, there is a low risk for relapse.- - - - - - - - - - ranking = 5keywords = hybridization (Clic here for more details about this article) |
6/11. RT-PCR and FISH analysis of acute myeloid leukemia with t(8;16)(p11;p13) and chimeric MOZ and CBP transcripts: breakpoint cluster region and clinical implications.The translocation t(8;16)(p11;p13) is associated with acute myeloid leukemia displaying monocytic differentiation (AML FAB M4/5) and fuses the MOZ (also named MYST3) gene (8p11) with the CBP (also named CREBBP) gene (16p13). Detection of the chimeric rna fusions has proven difficult; only three studies have described successful amplification of the chimeric MOZ-CBP and CBP-MOZ fusions by reverse transcriptase-polymerase chain reaction (RT-PCR). We analyzed four cases of AML M4/5 with t(8;16)(p11;p13) by RT-PCR and fluorescence in situ hybridization (FISH) and characterized the reciprocal rna fusions from three cases. We cloned both genomic translocation breakpoints from one case by long-range PCR and successfully applied RT-PCR to monitor minimal residual disease (MRD) between clinical complete remission and relapse. In three cases, the genomic breakpoints occurred in MOZ intron 16 and CBP intron 2. In one case, no fusion transcript was detected. The available data suggest clustering of t(8;16)(p11;p13) breakpoints in these introns leading to reciprocal in-frame MOZ exon 16/CBP exon 3 and in-frame CBP exon 2/MOZ exon 17 chimeric transcripts in the majority of cases. The described RT-PCR strategy may be valuable both for the routine detection of the t(8;16)(p11;p13) as well as for monitoring of MRD in this prognostically unfavorable patient group.- - - - - - - - - - ranking = 1keywords = hybridization (Clic here for more details about this article) |
7/11. Biphenotypic acute leukemia with t(15;17).Biphenotypic acute leukemias (BAL) represent 5% of all acute leukemias. The most frequent cytogenetic abnormalities described are philadelphia chromosome and 11q23 involvement. Here we report a BAL case, with blasts showing lymphoblast morphology and positivity for myeloperoxidase (in 6% of the blast cells). Immunophenotype revealed the compromise of myeloid and B-lymphoid lineages. cytogenetic analysis showed the t(15;17) and 8 trisomy. PML/RARa rearrangement was detected by fluorescent in situ hybridization (FISH) on interphase nuclei while PML/RARa fusion transcript was detected in the bone marrow and peripheral blood by molecular biology studies (RT-PCR). This report describes a BAL case with an unfrequent cytogenetic abnormality, and highlights the importance of correlating the results of multiple diagnostic methods in order to establish a correct diagnosis and treatment in BAL patients.- - - - - - - - - - ranking = 1keywords = hybridization (Clic here for more details about this article) |
8/11. ABL1 amplification in T-cell acute lymphoblastic leukemia.ABL1 amplification, due to a cryptic episomal translocation NUP214/ABL1, is a novel finding in T-cell acute lymphoblastic leukemia (ALL). Here we report on the incidence and clinical features of this genetic defect in a series of 30 consecutive adult T-cell ALL patients. Multiple copies of the ABL1 gene were detected in two patients (6.6%), one with the karyotype 46,XY,t(1;3)(p36;p21),del(6)(q23)/46,XY and the other without analyzable metaphases. metaphase/interphase fluorescence in situ hybridization (FISH) detected multiple uncountable signals corresponding to ABL1 in mitotic cells and nuclei from both patients. In one patient, no signals corresponded with the 9p21 chromosomal region, which contains the p16INK4a gene, and in the other one signal was observed. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) demonstrated that in these patients ABL1 gene expression was 14- and 18-fold greater than in normal controls, and returned to normal levels only when complete remission was achieved. We reached the following conclusions: (1) FISH is the only technique that promptly identifies T-cell ALL patients with ABL1 amplification, (2) quick identification with FISH is fundamental in the clinic because this T-cell ALL subset is imatinib sensitive but may become resistant due to development of additional mutations, and (3) ABL1 quantitative RT-PCR may be easily applied to monitor minimal residual disease.- - - - - - - - - - ranking = 1keywords = hybridization (Clic here for more details about this article) |
9/11. Sequential interphase FISH analysis of m-BCR/ABL chimeric gene-positive cells in Ph-positive acute myeloid leukemia.We report a case of Philadelphia (Ph)-positive AML in which interphase fluorescence in situ hybridization (FISH) analysis was performed from diagnosis throughout the course of therapy using major (M-) breakpoint cluster region (BCR)/minor (m-) BCR and ABL cosmid probes. We also investigated the existence of the M-BCR or m-BCR at the rna or dna level by the reverse transcriptase polymerase chain reaction and Southern blot analysis, respectively. Complete remission with a normal karyotype was achieved after several regimens of chemotherapy and peripheral blood stem cell transplantation (PBSCT), but relapse occurred and his cells became 100% Ph-positive. We detected the m-BCR/ABL fusion gene by interphase FISH analysis using an m-BCR/ABL translocation probe, and found that FISH analysis was useful for classifying the BCR, identifying minimal residual disease, and for predicting imminent relapse after chemotherapy and PBSCT.- - - - - - - - - - ranking = 1keywords = hybridization (Clic here for more details about this article) |
10/11. Detection of minimal residual disease in an AML patient with trisomy 8 using interphase fish.patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) often exhibit clonal chromosomal abnormalities. Using a probe for the centromeric region of chromosome 8, fluorescence in situ hybridization (FISH) on interphase cells was used to detect trisomy 8 in an AML patient whose leukemia was characterised by the karyotype 47, XY, 8, del(9) (q21.1q32). We have demonstrated using FISH the presence of the trisomy at all stages of the patient's disease course (including remission, peripheral blood cell harvest and relapse), whereas conventional karyoptypic analysis was only able to detect the trisomy at diagnosis and clinical relapse. We have also shown using immunophenotyping, cell sorting and FISH, that the trisomic cells in this patient were restricted to the CD34 subset of blood and bone marrow and could not be found in the CD 34-, T or B cell compartment. overall we have shown FISH to be a rapid, quantitative method for the detection of cells with numerical chromosome abnormalities. FISH analysis of interphase cells provides valuable information on the status of the whole population, rather than just cycling cells, and can be applied successfully to monitor the level of leukemic cells.- - - - - - - - - - ranking = 1keywords = hybridization (Clic here for more details about this article) |
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