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PubMed2004

Serial monitoring of BCR-ABL transcripts in chronic myelogenous leukemia (CML) treated with imatinib mesylate.

Survival among chronic myelogenous leukemia (CML) patients can be linked to the reduction in leukemic cell burden. Treatment with imatinib mesylate results in a high frequency of complete cytogenetic response, which can be further stratified using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). We have serially monitored peripheral blood and bone marrow BCR-ABL transcripts using qRT-PCR in CML patients commencing imatinib therapy, and compared the results with bone marrow cytogenetics. Seventeen patients (aged 25-74 yr) with Philadelphia chromosome positive CML in first chronic phase were treated with imatinib targeting a dose of 400 mg/d. The median follow up is 30 mo (range 9-33 mo). Every third month the product of the BCR-ABL fusion gene was evaluated in both blood and bone marrow specimens by real-time RT-PCR using the TaqMan probe system. In 113 simultaneously obtained blood and bone marrow samples, the BCR-ABL transcript values agreed well with cytogenetic data. Blood and bone marrow specimens gave comparable values for BCR-ABL transcripts. Before start of imatinib therapy there was a considerable variation in BCR-ABL transcripts among the patients, ranging approximately one log (base 10). Similarly, patients with a complete cytogenetic response following imatinib therapy had variable BCR-ABL transcript levels, ranging at least three logs (base 10). The major decline in BCR-ABL transcripts occurred within 6 mo after start of imatinib therapy. The decline in BCR-ABL transcripts, following imatinib therapy, appears to level off at 12-15 mo. Two late responders were identified with a still decreasing level in BCR-ABL transcripts after 24 mo of treatment. It is concluded that BCR-ABL mRNA quantification in peripheral blood is suitable for routine monitoring of the response to treatment and long-term disease status in CML, especially in patients who have achieved a complete cytogenetic response. A plateau in BCR-ABL transcripts seems to have been reached after 12-15 mo of imatinib treatment; however, some "late responders" are seen.

Adult↗
PubMed2005

Chronic myelocytic leukemia--Part II: Approaches to and molecular monitoring of therapy.

DATA SYNTHESIS: Chronic myelocytic leukemia (CML) was initially described in 1845 and is considered one of the first leukemias discovered. Effective approaches to therapy were not instituted until arsenic was first administered in 1865. Since then, four major therapeutic milestones have been achieved; the development of alkylating agents like busulphan and 6-thioguanine in 1953, alpha interferon in 1983, bone marrow transplantation in 1986, and tyrosine kinase inhibitors in 1998. The discovery that the protein product of this fusion gene expresses constitutive tyrosine kinase activity prompted the synthesis of a designer drug, imatinib mesylate, which binds the fusion protein and neutralizes the tyrosine kinase activity. Molecular methods of detecting BCR-ABL transcripts are showing promise in confirming drug resistance and predicting patient outcomes in response to imatinib mesylate therapy. Evidence of drug resistance can guide physicians in selecting alternative therapeutic approaches early in the course of the disease to potentially rescue non responders. Although the success of clinical trials has been dramatic, drug resistance and disease relapse are issues to be considered. CONCLUSION: The discovery that the BCR/ABL fusion protein exhibits increased and constitutive tyrosine kinase activity led investigators to develop an inhibitor to this activity. The synthesis of imatinib mesylate, currently marketed as Gleevec(TM) or Glivec(R), is in stage III clinical trials and has proven to be the most successful antileukemic drug to date. As in CML, an understanding of the leukemogenic mechanisms involved in other leukemias will provide the groundwork for the development of therapeutic interventions tailored to the specific molecular defects identified, eventually rendering obsolete the shotgun approaches to massive cell killing produced by chemotherapy.

Antineoplastic Agents↗
PubMed2003

Contribution of fluorescence in situ hybridization analyses to the characterization of masked and complex Philadelphia chromosome translocations in chronic myelocytic leukemia.

Bone marrow samples from 112 patients with chronic myelocytic leukemia were investigated using cytogenetic methods. Fluorescent in situ hybridization (FISH) with whole-chromosome paints and BCR-ABL probes was used to confirm and/or complete the banding findings when a variant or a masked Philadelphia chromosome (Ph) translocation was found. Eight variant Ph translocations were identified. Three-way Ph translocations were found in seven patients. Chromosome 4 was involved in two of these cases and chromosomes 3, 11, 14, 17, and 16 in one case each; in the patient with chromosome 16 involvement, a ring of the translocated chromosome 9 was identified, that is r(9)t(9;16;22). The eighth patient had a five-way Ph translocation: t(2;9;16;22;22). The BCR-ABL fusion gene was detected on the Ph chromosome in all eight cases; two cases presented also a deletion of the 5' ABL region on the derivative chromosome 9. In the five-way translocation, the 3' DNA sequence of the ABL oncogene was fused with the 5' DNA sequence of the BCR gene on the Ph chromosome and the 5' end of ABL was inserted into the other chromosome 22. A masked Ph chromosome was identified in one of the 112 patients; it involved the insertion of the 3' ABL into BCR on an apparently normal chromosome 22, resulting in the BCR-ABL fusion gene. In conclusion, FISH analyses allowed not only a more accurate characterization of complex Ph translocations with subtle abnormalities and the identification of cryptic rearrangements, but also the recognition of deletion of the 5' ABL region, which could carry with it a poor prognosis.

Chromosome Painting↗
PubMed2005

Tyrosine kinase inhibitor STI571 (Imatinib) cooperates with wild-type p53 on K562 cell line to enhance its proapoptotic effects.

In order to ascertain whether p53 has a role in chronic myeloid leukemia hematopoietic progenitor response to the innovative tyrosine kinase inhibitor STI571 (Imatinib), we overexpressed a wild type (wt) p53 construct in the K562 cell line, generated from a human blast crisis and lacking endogenous p53. Wt p53 overexpression was associated with a significant reduction of bcr-abl expression levels resulting, at least in part, from post-transcriptional events affecting the stability of p210 bcr-abl fusion protein. Moreover, we demonstrated that p53 overexpression enhances the commitment to the apoptotic death fate of K562 following its in vitro exposure to 1 microM STI571. Multiple mechanisms are involved in p53 impact on K562 survival: Most importantly, we found that a greater reduction of bcr-abl transcription by STI571 was associated with the overexpression of wt p53. Further studies are required to elucidate the mechanisms involved in the transcriptional repression of bcr-abl by STI571 and p53 and in their synergic effects on the clonal hematopoiesis of chronic myeloid leukemia.

Apoptosis↗
PubMed1987

Multiple molecular abnormalities in Ph1 chromosome positive acute lymphoblastic leukaemia.

The Ph1 chromosome is present in 95% of patients with chronic myelogenous leukaemia (CML). The Ph1 chromosome also occurs in 5-25% of children and adults with acute lymphoblastic leukaemia (ALL). This observation raises questions as to whether these diseases are similar or identical. In patients with CML the c-abl and bcr genes are translocated and abnormally expressed. We studied molecular events related to bcr and c-abl in five patients with ALL to determine its relationship to CML. Four had the Ph1 chromosome; the fifth a probable Ph1 chromosome. c-abl and bcr abnormalities identical to CML were detected in four suggesting a common molecular basis. One patient with the Ph1 chromosome and c-abl translocation lacked these molecular changes but had abnormal c-abl gene transcription apparently unrelated to bcr. These data suggest that Ph1 chromosome positive ALL is heterogeneous; in some patients the molecular abnormality is identical to CML; in others c-abl is likewise involved but via a different mechanism.

Adolescent↗
PubMed1995

Multi-unit ribozyme-mediated cleavage of bcr-abl mRNA in myeloid leukemias.

Chronic myelogenous leukemia is characterized by the Philadelphia chromosome, which at the molecular level results from the fusion of the bcr gene on chromosome 22 and the abl gene on chromosome 9. The bcr-abl fusion gene encodes a novel tyrosine kinase with transforming activity. In this study, we have synthesized a multi-unti ribozyme that targets bcr-abl mRNA. In vitro ribozyme cleavage reactions show increased cleavage efficiency of this multi-unit ribozyme compared with single or double ribozymes. The multiunit ribozyme was then transfected into murine myeloblasts transformed with the bcr-abl gene (32D cells). Ribozyme transfection was accomplished either by liposomes or using follic acid-polylysine as a carrier. Multi-unit ribozyme transfection reduced the level of bcr-abl mRNA 3 logs when transfected via folate receptor-mediated uptake into transformed 32D cells. These results suggest that a multi-unit ribozyme could be an effective therapeutic agent for the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia.

Animals↗
PubMed1996

Very low level of major BCR-ABL expression in blood of some healthy individuals.

The nested reverse transcriptase polymerase chain reaction (RT-PCR) provides a powerful tool for detection of minimal residual disease in CML. The RT-PCR used in the present study for detection of the major bcr-abl fusion gene, the hallmark and presumably the cause of CML, was optimized by: (a) increasing the amount of total RNA involved in the reverse transcription reaction to correspond to total RNA extracted from 10(8) cells; (b) using a specific abl primer in this reverse transcriptase reaction, and (c) reamplifying 10% of the RT-PCR product in a nested amplification. This optimized RT-PCR permitted to detect up to 1 copy of RNA bcr-abl synthesized in vitro, mixed with yeast RNA in a quantity equivalent to 10(8) white blood cells (WBC). Using the highly sensitive RT-PCR, a systematic study of the possible expression of bcr-abl RNA in WBC of healthy adults, children and umbilical cord blood (UCB) revealed the presence of bcr-abl transcripts in blood cells of 22/73 adults, 1/22 children but not in 22 samples of UCB. The comparison of these three groups indicated a significant tendency for the anomaly to increase in frequency with age.

Adolescent↗
PubMed2003

Novel translocations that disrupt the platelet-derived growth factor receptor beta (PDGFRB) gene in BCR-ABL-negative chronic myeloproliferative disorders.

The BCR-ABL-negative chronic myeloproliferative disorders (CMPD) and myelodysplastic/myeloproliferative diseases (MDS/MPD) are a spectrum of related conditions for which the molecular pathogenesis is poorly understood. Translocations that disrupt and constitutively activate the platelet-derived growth factor receptor beta(PDGFRB) gene at chromosome band 5q33 have been described in some patients, the most common being the t(5;12)(q33;p13). An accurate molecular diagnosis of PDGFRB-rearranged patients has become increasingly important since recent data have indicated that they respond very well to imatinib mesylate therapy. In this study, we have tested nine patients with a CMPD or MDS/MPD and a translocation involving 5q31-33 for disruption of PDGFRB by two-colour fluorescence in situ hybridization (FISH) using differentially labelled, closely flanking probes. Normal control interphase cells gave a false positive rate of 3% (signals more than one signal width apart). Six patients showed a pattern of one fused signal (from the normal allele) and one pair of signals separated by more than one signal width in > 85% of interphase cells, indicating that PDGFRB was disrupted. These individuals had a t(1;5)(q21;q33), t(1;5)(q22;q31), t(1;3;5)(p36;p21;q33), t(2;12;5)(q37;q22;q33), t(3;5) (p21;q31) and t(5;14)(q33;q24) respectively. The remaining three patients with a t(1;5)(q21;q31), t(2;5)(p21;q33) and t(5;6)(q33;q24-25) showed a normal pattern of hybridization, with > or = 97% interphase cells with two fusion signals. We conclude that two-colour FISH is useful to determine the presence of a PDGFRB rearrangement, although, as we have shown previously, this technique may not detect subtle complex translocations at this locus. Our data indicate that several PDGFRB partner genes remain to be characterized.

Chromosomes, Human, Pair 1↗
PubMed2006

Diagnosis and monitoring of chronic myeloid leukemia by qualitative and quantitative RT-PCR.

Real-time quantitative reverse-transcription polymerase chain reaction (RQ-PCR) methods for the quantitation of BCR-ABL mRNA in the blood of patients with chronic myeloid leukemia (CML) has become the predominant molecular monitoring technique. The BCR-ABL fusion gene is expressed in over 95% of patients with CML, and RQ-PCR provides a reliable, high-throughput method to accurately assess the level of treatment response and provides an early indication of emerging drug resistance. The ABI Prism 7700 Sequence Detection System uses TaqMan fluorogenic probes to quantitate specific nucleic acid sequences using RQ-PCR. The analyzer monitors an increase in fluorescence during the PCR cycle, which is proportional to the amount of accumulated product. The starting copy number is calculated relative to a series of standards. The copy number is normalized to a control gene that compensates for variations in the efficiency of the RT step and for the degree of RNA degradation. In our experience, reliable and consistent RQ-PCR requires thorough validation of all aspects of the procedure, including the selection of an appropriate control gene, careful assay design to avoid polymorphisms in primer or probe binding sites and to exclude the amplification of contaminating DNA, and monitoring the performance of the RQ-PCR by the use of quality control samples.

Cloning, Molecular↗
PubMed2000

Complex chromosome 9, 20, and 22 rearrangements in acute lymphoblastic leukemia with duplication of BCR and ABL sequences.

Cytogenetic analysis was performed on bone marrow cells from a 28-year-old woman who was diagnosed with acute lymphoblastic leukemia (ALL). Her karyotype was: 46,XX,t(9;22)(q34;q11)[6]/47, XX,+8,t(9;22)(q34;q11)[4]/47,XX,+8,t(9;22)(q34;q11),del(20)(q11)[2]/46, XX,t(9;22)(q34;q11),del[20](q11)[7]/45,XX,der(9)t(9;22)(q34;q11),-20,-22 , +mar1[8]/45,XX,der(9)t(9;22)(q34;q11),-20,-22,+mar2[3]. Both marker chromosomes are dicentric and have the same size and banding pattern but different primary constrictions. Fluorescence in situ hybridization (FISH) demonstrated that both markers were derived from chromosomes 9, 20, and 22. FISH with the bcr/abl probe showed fusion of the BCR gene with the ABL gene; however, this fusion signal was present in duplicate on both marker chromosomes. To our knowledge, duplication of the BCR/ABL fusion signal on a single chromosome arm has not been reported before, except for the extensive amplification of BCR/ABL fusion signals in the leukemic cell line K-562. These data demonstrate that the marker chromosomes are the result of complex genomic rearrangements. At the molecular level, the BCR/ABL fusion gene encodes the p190 fusion protein. Similar findings have never been observed in any case of ALL.

Adult↗
PubMed2003

Novel mutation in the gene encoding c-Abl-binding protein SH3BP2 causes cherubism.

Cherubism is a rare autosomal dominant inherited condition caused by mutations in the c-Abl-binding protein SH3BP2. It is characterized by multiple cystic giant cell lesions of the jaw appearing in early childhood with stabilization and remission after puberty. In the present study, we used direct sequence analysis of the SH3BP2 gene of several individuals from a family with cherubism to search for additional SH3BP2 mutations resulting in cherubism. In affected relatives, we found a previously unreported G to A transition in exon 9 leading to a Gly to Arg substitution at amino acid position 420. G420R has been reported previously with a G to C transversion. To date there have been no disease causing mutations outside exon 9. Therefore, the amino acid sequence from positions 415 to 420 may represent a specific protein domain which, when disrupted, leads to the cherubism phenotype.

Adaptor Proteins, Signal Transducing↗
PubMed1987

Cis- and trans-acting elements responsible for the cell-specific expression of the human alpha 1-antitrypsin gene.

The 5' flanking region of the human alpha 1-antitrypsin (alpha 1-AT) gene contains cis-acting signals for liver-specific expression and, when fused to a reporter gene, is able to drive the expression of this gene specifically in liver cells. Here we report the results of a functional dissection of the alpha 1-AT regulatory region. The expression of the bacterial chloramphenicol-transacetylase (CAT) gene, fused to a set of alpha 1-AT 5' flanking regions shortened by progressive deletions or mutated by base pair substitutions, has been compared by transfection in HepG2 (hepatocyte) and HeLa (non-hepatocyte) human cell lines. A minimal tissue-specific element has been identified between the nucleotides -137 and -37 (from the transcriptional start site). This DNA segment activates the heterologous SV40 promoter in hepatoma cell lines but not in HeLa cells. This element contains at least two regions referred to as the A (-125/-100) and B (-84/-70) domains, both essential for transcription. There are at least two other regulatory domains located upstream of the 'minimal element'; the most active of these is located between positions -261 and -210 from the cap site. These upstream elements activate the heterologous SV40 early promoter both in hepatoma cell lines and in HeLa cells. Upon fractionation of rat liver nuclear extracts two proteins have been identified, alpha 1TF-A and alpha 1TF-B, which bind specifically to the A and B domains respectively. Transcriptionally inactive A and B domain mutants are not able to bind these proteins.

Base Sequence↗
PubMed1992

Initiation and progression of chronic myelogenous leukemia.

Different aspects related to initiation of chronic myelogenous leukemia by the t(9;22) translocation and progression of the disease were investigated. Computer search indicated that the repeat within BCR exon I has significant sequence homology to the long terminal repeats of three retroviruses, to two transposons and to the Alu family. This raises the possibility that the BCR repeat is involved in the t(9;22) as well as in generation of the BCR-related loci. Possible involvement of the p53 gene in clinical transition to acute phase was studied. In six patients and cell lines where one allele of the gene was deleted, the other allele was inactivated by loss of transcription, point mutation or rearrangement. The majority of patients, however, have both p53 alleles; detailed analysis of the p53 gene in several of them indicated normal transcription and amino acid sequence.

Base Sequence↗