scholarly journals Cytogenetic and molecular analysis in Philadelphia negative CML

Blood ◽  
1989 ◽  
Vol 73 (4) ◽  
pp. 1038-1044 ◽  
Author(s):  
DC van der Plas ◽  
AB Hermans ◽  
D Soekarman ◽  
EM Smit ◽  
A de Klein ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Chromosomal Rearrangements ◽  
Normal Karyotype ◽  
Break Point ◽  
Complex Chromosomal Rearrangements ◽  
Ph Chromosome ◽  
Polymerase Chain ◽  
Genomic Recombination

We studied the clinical, hematologic, cytogenetic and molecular biologic features in four patients with Philadelphia (Ph) negative chronic myeloid leukemia (CML). In all four cases the clinical and hematologic characteristics were indistinguishable from Ph positive CML. Cytogenetic analysis showed a normal karyotype in two patients and chromosomal translocations apparently not affecting chromosome 22 in the other two cases. Southern blot analysis using probes of the bcr region, demonstrated a bcr break-point in all four patients. In situ hybridization with bcr, c-abl, and c-sis probes showed unusual hybridization sites for 5′-bcr and c-abl indicating complex chromosomal rearrangements affecting three different chromosomes in the four patients investigated. Using polymerase chain reaction (PCR) followed by hybridization to oligonucleotide probes specific for the bcr-abl fusion region, the expression of a chimeric bcr-abl mRNA was detected. In these patients we demonstrated that (a) CML with a breakpoint in the bcr region without cytogenetically detectable Ph chromosome is characterized by the same genomic recombination of 5′-bcr and c-abl as CML with standard Ph translocation and (b) unusual localization of 5′- bcr and c-abl sequences caused by complex Ph translocation does not interfere with transcription of the bcr-abl fusion gene.

scholarly journals Cytogenetic and molecular analysis in Philadelphia negative CML

Blood ◽  
1989 ◽  
Vol 73 (4) ◽  
pp. 1038-1044 ◽  
Author(s):  
DC van der Plas ◽  
AB Hermans ◽  
D Soekarman ◽  
EM Smit ◽  
A de Klein ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Chromosomal Rearrangements ◽  
Normal Karyotype ◽  
Break Point ◽  
Complex Chromosomal Rearrangements ◽  
Ph Chromosome ◽  
Polymerase Chain ◽  
Genomic Recombination

Abstract We studied the clinical, hematologic, cytogenetic and molecular biologic features in four patients with Philadelphia (Ph) negative chronic myeloid leukemia (CML). In all four cases the clinical and hematologic characteristics were indistinguishable from Ph positive CML. Cytogenetic analysis showed a normal karyotype in two patients and chromosomal translocations apparently not affecting chromosome 22 in the other two cases. Southern blot analysis using probes of the bcr region, demonstrated a bcr break-point in all four patients. In situ hybridization with bcr, c-abl, and c-sis probes showed unusual hybridization sites for 5′-bcr and c-abl indicating complex chromosomal rearrangements affecting three different chromosomes in the four patients investigated. Using polymerase chain reaction (PCR) followed by hybridization to oligonucleotide probes specific for the bcr-abl fusion region, the expression of a chimeric bcr-abl mRNA was detected. In these patients we demonstrated that (a) CML with a breakpoint in the bcr region without cytogenetically detectable Ph chromosome is characterized by the same genomic recombination of 5′-bcr and c-abl as CML with standard Ph translocation and (b) unusual localization of 5′- bcr and c-abl sequences caused by complex Ph translocation does not interfere with transcription of the bcr-abl fusion gene.

scholarly journals Generation of a whole chromosome painting probe from monochromosomal hybrid cells by the alu-polymerase chain reaction

2007 ◽  
Vol 59 (2) ◽  
pp. 89-95 ◽  
Author(s):  
Danijela Drakulic ◽  
Gordana Nikcevic ◽  
Vesna Djordjevic ◽  
Milena Stevanovic
Keyword(s):  
Chromosome Painting ◽  
Fluorescent In Situ Hybridization ◽  
Chromosomal Rearrangements ◽  
Painting Probe ◽  
Complex Chromosomal Rearrangements ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Complex Chromosome ◽  
Whole Chromosome Painting

Fluorescent in situ hybridization (FISH) has become a widespread technique applicable in basic science and diagnostics. Chromosome painting represents a special application of FISH that has found increasing use in identification of complex chromosome rearrangements. Here we present a version of the Alu-PCR method modified to generate a whole chromosome painting probe (WCP) for human chromosome 19 using monochromosomal cell hybrids. In setting up conditions for this method, we established a cheap and fast approach to generation of WCPs for other human chromosomes that could be particularly useful for unambiguous identification of complex chromosomal rearrangements associated with cancer. .

scholarly journals BCR-ABL, ABL-BCR, BCR, and ABL genes are all expressed in individual granulocyte-macrophage colony-forming unit colonies derived from blood of patients with chronic myeloid leukemia

Blood ◽  
1995 ◽  
Vol 85 (8) ◽  
pp. 2171-2175 ◽  
Author(s):  
J Diamond ◽  
JM Goldman ◽  
JV Melo
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Pcr Amplification ◽  
Pcr Screening ◽  
Colony Forming Unit ◽  
Ph Chromosome ◽  
Polymerase Chain ◽  
Macrophage Colony ◽  
Positive Results

It has been suggested that the BCR-ABL gene of chronic myeloid leukemia (CML) is not uniformly expressed in Philadelphia (Ph)-positive cells, and that BCR-ABL gene expression precludes transcription of the normal BCR or ABL genes. Therefore, we have analyzed granulocyte-macrophage colony-forming unit (CFU-GM) colonies derived from peripheral blood of 11 CML patients by cytogenetic and by reverse transcriptase-polymerase chain reaction (PCR) amplification of BCR-ABL, ABL-BCR, BCR, and ABL. All CFU-GM colonies with analyzable metaphases were found to contain a Ph chromosome. In 2 patients, the initial PCR screening failed to detect BCR-ABL transcripts in 2 of 11 and 1 of 7 Ph-positive colonies. However, when amplification for BCR-ABL was repeated in quintuplicate, all but 1 colony from a single patient showed one or more positive results. Amplifications of the four genes in each colony showed that BCR-ABL, ABL-BCR, and the normal BCR and ABL were simultaneously expressed in the majority of CFU-GM colonies. Replicate PCR tests for BCR and for ABL in colonies initially scored as negative also uncovered previously undetected positive amplifications. We conclude that BCR-ABL expression does not suppress transcription from the normal BCR and ABL genes, and that Ph-positive, BCR-ABL-negative colonies derived from peripheral blood CFU-GM are rare or nonexistent.

scholarly journals Ph-negative chronic myeloid leukemia: molecular analysis of ABL insertion into M-BCR on chromosome 22

Blood ◽  
1990 ◽  
Vol 76 (9) ◽  
pp. 1812-1818 ◽  
Author(s):  
CM Morris ◽  
N Heisterkamp ◽  
MA Kennedy ◽  
PH Fitzgerald ◽  
J Groffen
Keyword(s):  
In Situ Hybridization ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Repetitive Sequences ◽  
Chromosome 9 ◽  
Leukemic Cells ◽  
Chromosome 22 ◽  
Chromosome 9Q34

Abstract Leukemic cells from a patient with Ph-negative chronic myeloid leukemia (CML) had a normal karyotype. M-BCR was rearranged and chromosome in situ hybridization showed an ABL insertion between 5′ and 3′ M-BCR on an apparently normal chromosome 22. The association of 5′ BCR and 3′ ABL at the 5′ junction of the chromosome 9 insert was typical of that found for the BCR-ABL fusion gene in other patients with the standard t(9;22) and CML. With an M-bcr-3′ probe, we cloned and characterized a 3′ junction fragment. Field inversion gel electrophoresis and chromosome in situ hybridization studies using a probe isolated from genomic DNA 5′ of the junction showed that 3′ M-BCR was joined to a region of chromosome 9q34 rich in repetitive sequences and lying some distance 3′ of ABL. The chromosome 9 insert was at least 329 kilobases long and included 3′ ABL and a larger portion of chromosome 9q34. Our results allowed us to exclude transposon- or retroviral-mediated insertion of ABL into chromosome 22. Instead, we favored a two- translocation model in which a second translocation reconstituted a standard t(9;22)(q34;q11) but left the chromosome 9 insert, including 3′ ABL, in chromosome 22.

Clinical Relevance of Complex Chromosomal Aberrations in Bone Marrow Cells of 107 Children with ETV6/RUNX1 Positive Acute Lymphoblastic Leukemia (ALL).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2278-2278 ◽  
Author(s):  
Zuzana Zemanova ◽  
Kyra Michalova ◽  
Libuse Babicka ◽  
Lenka Pavlistova ◽  
Marie Jarosova ◽  
...  
Keyword(s):  
Chromosomal Aberrations ◽  
Bone Marrow Cells ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Chromosomal Rearrangements ◽  
Chromosome 21 ◽  
Leukemic Cells ◽  
Rt Pcr ◽  
Cryptic Translocation ◽  
Complex Chromosomal Rearrangements

Abstract Cryptic translocation t(12;21)(p13;q22) which give origin to the ETV6/RUNX1 hybrid gene can be found by I-FISH in approximately 20–25% of children with B precursor ALL as the most frequent specific aberration. This translocation is generally associated with good outcome. Despite of its favorable prognostic value, late relapses may occur within this group of patients. One of the reasons could be the high instability of the genome of leukemic cells, which is manifested at the chromosomal level by additional aberrations and/or complex chromosomal rearrangements. The aim of the study was to evaluate the significance of the additional chromosomal aberrations for prognosis of children with ETV6/RUNX1 positive ALL. For the assessment of ETV6/RUNX1 fusion gene RT-PCR and/or double target interphase FISH with locus-specific probe (Abbott-Vysis, Des Plaines, Illinois, USA) were used (200 interphase nuclei analyzed, cut-off level 2.5% tested on controls, standard deviation ≤0.5%). Karyotypes were analyzed by conventional and molecular cytogenetic methods. Structural and/or complex chromosomal aberration were verified by FISH with whole chromosome painting probes (Cambio, Cambridge, UK) and/or by mFISH with the "24XCyte" probe kit (MetaSystems GmbH, Altlussheim, Germany). We performed prospective and retrospective study of 107 children with ALL and ETV6/RUNX1 fusion gene detected by RT-PCR and/or I-FISH. Patients were diagnosed between 1995 and 2006, age ranged between 15 months and 16.9 years (median 4.2 years). Relapse appeared in 19 children (17.8%), four of them died. In 64 children (59.8%) we found besides t(12;21)(p13;q22) additional chromosomal aberrations, the most frequently trisomy or tetrasomy of chromosome 21 (20 cases), deletion of non-translocated ETV6 allele (24 cases), deletion of 6q (7 cases) and/or rearrangements of the long arm of chromosome X (6 cases). Complex karyotypes were identified in 38 children (35.5%). In twelve of them variant translocations of chromosomes 12 and 21 with other partners were observed. Event-free survival (EFS) was significantly shorter in patients with additional structural and/or complex aberrations in ETV6/RUNX1 positive cells (p=0.01). In our cohort complex karyotypes indicated poor prognosis. Finding of complex chromosomal aberrations in leukemic cells is accompanied by higher risk of relapse even in those cases where the prognostically positive aberration is primarily present.

Variant Philadelphia Translocation with BCR/ABL Fusion Gene Site In 10q24 In a Case of Chronic Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4839-4839
Author(s):  
Rossana Bonomi ◽  
Pablo Lopez ◽  
Daniela Infante ◽  
Isabel Moro ◽  
Victoria Elizondo ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Chromosome 9 ◽  
Chromosome 22 ◽  
Fish Analysis ◽  
Signal Pattern ◽  
Ph Chromosome ◽  
Fusion Signal

Abstract Abstract 4839 Introduction. Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome (Ph) observed in more than 90% of patients with CML as a result of t(9;22)(q34;q11), leading to the formation of chimeric gene BCR/ABL encoding for proteins with abnormal tyrosine kinase activity. Cytogenetic variants of Ph chromosome can be identifed in 5 to 10% of CML patients, involving additional chromosomes other than 9 and 22. To explain the formation of variant translocations one-step, two-step and multi-step mechanisms have been proposed. Rarely, the variant Ph chromosome results from a BCR insertion on the ABL region and form a BCR/ABL fusion gene, generally mapping to 9q34, instead of the usual location at 22q11. In very few variant Ph cases, the insertion of the BCR/ABL product in a third chromosome was demonstrated. Case Report 28 year-old man, with bilateral central scotoma and gingivorragia. Physical examination: Grade 4 splenomegaly. Peripheral blood count showed hemoglobin concentration 11.5 g/dl, platelet count: 300.000/mm3, and white blood cell count 590.000/mm3. Blood smear: myelemia exhibiting 30% of myeloid blasts. Bone marrow biopsy: panmyelosis showing 20% of myeloid blasts. Cytogenetic analysis by G-banding performed in peripheral blood verified the following karyotype: 46, XY, t(9;22;10)(q34;q11;q24)[20] The analysis of the BCR-ABL fusion gene according to standard protocols detected the presence of the b3a2 isoform. Fluorescence in situ hybridization (FISH) studies using dual color dual fusion probes in metaphases showed a signal pattern 1F2G1R. The fusion signal mapped to 10q24, the red signal to 9q34, and the normal green signal to chromosome 22, while a second low intensity green signal mapped to the Ph chromosome. No signal was observed in der(9). Interphase FISH analysis in nuclei (n=200) presented the same signal pattern. Instead of using whole chromosome probes for 9 and 22, we hybridised probes used to detect DiGiorge syndrome. These probes detect gene control ARSA (spectrum green) localized at 22q13 and Tuple1 at 22q11 (spectrum orange). Two signals, green and orange were identified in normal chromosome 22. Ph chromosome showed the orange signal, whereas the green signal mapped to der(10). Discussion. The localization of the hybrid BCR/ABL gene on chromosomes other than 22q is a rare event wich can only be detected by FISH techniques. When these unusual translocation occurs, the hypothesis most often put forward is that several consecutive chromosome rearrangements have taken place. In the present case the interpretation of karyotypes, FISH data and molecular evidence lead to the following hypothesis: Insertion of the BCR sequence from chromosome 22 to chromosome 9 may have ocurred, producing a BCR/ABL fusion in der(9). The Ph chromosome detected by G-banding showed a different green fluorescence intensity in the metaphase FISH signal pattern with BCR/ABL dual color dual fusion probes, as a result of an insertion on chromosome 9. This first event was followed by the translocation between the derivative 9 and chromosome 10, being the final localization of the BCR/ABL gene in 10q24. FISH analysis using a DiGeorge syndrome probe, supports the hypothesis of a multistep mechanism underlying insertion and translocations events in the present case. The relocation of BCR/ABL fusion sequence on sites other than chromosme 22q11 represent a rare type of variant Ph translocation. At least 21 cases described in the literature, showed fusion gene BCR/ABL located at 9q24. Only 12 patients with variant Ph were reported bearing BCR/ABL on a third chromosome. All of them involved a masked Ph chromosome. To our best knowledge this is the first report showing a variant Ph chromosome detected by G-banding in a CML patient due to a BCR insertion on ABL sequences and exhibiting the fusion signal in a third chromosome. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Philadelphia-negative (Ph-) chronic myeloid leukemia (CML): comparison with Ph+ CML and chronic myelomonocytic leukemia. The Groupe Francais de Cytogenetique Hematologique

Blood ◽  
1991 ◽  
Vol 78 (1) ◽  
pp. 205-211 ◽  
Author(s):  
P Martiat ◽  
JL Michaux ◽  
J Rodhain
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Chronic Myelomonocytic Leukemia ◽  
Ras Mutation ◽  
Ph Chromosome ◽  
Polymerase Chain ◽  
Atypical Cml ◽  
Myelomonocytic Leukemia ◽  
Monocytic Lineage

To better understand the Philadelphia-negative (Ph-) chronic myeloid leukemia (CML) and its relationships with Philadelphia-positive (Ph+) CML and chronic myelomonocytic leukemia (CMML), a study was undertaken by the Groupe Francais de Cytogenetique Hematologique. Thirty-five Ph- CML patients were investigated and compared with 55 chronic phase Ph+ CML and 100 CMML patients. There were 12 M-BCR positive (M-BCR+) and 23 M-BCR negative (MBCR+) patients. No clinical or biologic differences were found between Ph+ and Ph-, M-BCR+ patients. In the Ph- group, M- BCR+ and M-BCR- patients differed significantly in age (47.7 +/- 6.6 v 67.0 +/- 6.1 years, respectively; P = .001), leukocytosis (153.4 +/- 135.1 v 58.5 +/- 37.7 10(9)/L, P = .002), relative monocytosis (1.8% +/- 1.2% v 5.6% +/- 1.4%, P = .048), absolute basophilia (8.5 +/- 9.7 v 0.9 +/- 1.5 10(9)/L, P = .001), percentage of immature myeloid precursors (IMP) in peripheral blood (29.0% +/- 9.5% v 15.3% +/- 8.1%, P = .001), and percentage of erythroblasts in bone marrow (BM) (6.5% +/- 3.5% v 14.6% +/- 3.6%, P = .001). Karyotypic abnormalities other than the Ph chromosome occurred in 0 of 12 M-BCR- at diagnosis and 7 of 23 M-BCR- Ph- CML (P = .033). None of the 13 investigated BCR- patients had detectable BCR/ABL transcripts using polymerase chain reaction (PCR) and none had an N-RAS mutation. Cytologic findings showed a marked morphologic difference between M-BCR+ and M-BCR- patients, especially in the monocytic lineage. Dysmyelopoietic features in CMML and M-BCR- patients were very similar, and the differences were of quantitative order only. Using four criteria (monocytosis, percentage of IMP, basophilia, and percentage of erythroblasts in BM), patients could be divided into typical and atypical CML and this classification correlated well with molecular findings. We conclude that, while Ph-, M- BCR+, and Ph+ CML are identical diseases, Ph-, M-BCR- CML, and CMML have many similarities and might be only different aspects of a same entity.

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