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J Harbott

Publications and source records attributed to J Harbott.

At least 37 records · Page 2Linked to original sources
PubMed2001

Absence of point mutations within the AML-1 gene in patients with MDS/AML and loss of chromosome 5q or 7.

There is increasing evidence that the acute myeloid leukemia 1 (AML-1) gene plays a versatile role in hematopoiesis, and its inactivation has been described in various hematopoetic disorders, e.g., leukemia or familial thrombocytopenia. AML-1 can be affected by various mechanisms, such as chromosomal translocations or point mutations. On the other hand, the specific underlying molecular lesions in myelodysplastic syndromes (MDS) or leukemias with aberrations of chromosomes 5q or 7, respectively, are largely unknown. Despite extraordinary scientific effort no specific genes on chromosome 5q or 7, which act as tumor suppressors, have definitely been identified. Therefore, it has recently been speculated that the AML-1 gene, even if distantly located on chromosome 21q22, may be involved in leukemogenesis in patients with aberrations at chromosome 5q or monosomy 7 [2]. Therefore, we sequenced all exons of the AML-1 gene in 15 patients with MDS/AML and deleted chromosome 5q or 7q, respectively. None of the patients analyzed had any AML-1 mutation.

Adolescent↗
PubMed2001

Detection of translocations involving the HOX11/TCL3-locus in 10q24 by interphase fluorescence in situ hybridization.

The t(10;14)(q24;q11) and its variant t(7;10)(q35;q24), which are recurrent in acute T-cell leukemia, lead to activation of the HOX11/TCL3-gene in chromosomal region 10q24 by juxtaposing this gene to one of the T-cell receptor loci. In the present study, we established a diagnostic assay for detecting these translocations by interphase fluorescence in situ hybridization (FISH). BAC clones flanking the HOX11/TCL3-locus were obtained from a fingerprinted BAC-contig of chromosomal region 10q24. BAC clones located proximal and distal of the HOX11/TCL3-locus were differently labeled and applied to interphase-FISH in seven normal controls and eight T-cell neoplasms with t(10;14)(q24;q11) or t(7;10)(q35;q24). In over 1600 nuclei of controls, a considerable split defined as separation of each one signal for the proximal and distal probe by more than three times the signal diameter was observed in only one cell. In contrast, all T-cell neoplasms with t(10;14) or t(7;10) contained at least 47% of nuclei with a signal split indicating a breakpoint in the HOX11/TCL3-locus. Thus, the established double-color FISH approach provides a new reliable and routinely applicable tool for diagnosing breakpoints in the HOX11/TCL3-locus.

Adult↗
PubMed2001

Impact of CD133 (AC133) and CD90 expression analysis for acute leukemia immunophenotyping.

BACKGROUND AND OBJECTIVES: AC133 is a novel monoclonal antibody (Moab) reacting with a population of immature/primitive or granulo-monocytic committed CD34+ve cells. Up to now, only few studies with small numbers of cases have examined AC133 (recently designated CD133) expression in acute leukemia. To determine the value of this Moab for acute leukemia immunophenotyping, we investigated a large series of leukemic cell samples for their reactivity with Moab AC133. DESIGN AND METHODS. A total of 298 cell samples from patients with de novo acute myeloid leukemia (AML) (n=142), acute lymphoblastic leukemia (ALL) (n=119), CD34+ve biphenotypic acute leukemia (n=13), and CD34+ve CML blast crisis (=BC; 21 myeloid BC/3 lymphoid BC) were investigated by flow cytometry for Moab AC133 reactivity.CD133 expression was compared with CD90(Thy-1) expression, another CD34-associated antigen. RESULTS: Fifteen (5%) samples expressed CD90, whereas 114 (38%) samples were positive for Moab AC133 (20% cut-off level). No significant differences in CD133 and CD90 expression levels between AML and ALL were observed. In AML, but not ALL, CD133 was more often expressed in CD34+ve cases than in CD34-ve ones (p<0.00001). However, CD133 expression was not restricted to CD34+ve leukemic cells in individual cell samples. All 8 pro-B-ALL cell samples with 11q23-anomalies and MLL (mixed lineage leukemia) gene translocations were positive for CD133, whereas only 2 of 9 pro-B-ALL without MLL gene translocations expressed CD133 (p<0.002). In contrast, none of the 5 AML cell samples with a t(9;11) and MLL gene translocation reacted with Moab AC133. CD34+ve CML cells in myeloid BC were less often positive for CD133 than CD34+ve de novo AML cells (p<0.0001). INTERPRETATION AND CONCLUSIONS: CD133 and CD90 expression analysis is not helpful for lineage determination in acute leukemia immunophenotyping. However, MoabAC133 may be an informative marker for the detection and further characterization of immature AML cells, as well as pro-B-ALL cells with MLL gene translocations, by flow cytometry.

AC133 Antigen↗
PubMed2000

The human GRAF gene is fused to MLL in a unique t(5;11)(q31;q23) and both alleles are disrupted in three cases of myelodysplastic syndrome/acute myeloid leukemia with a deletion 5q.

We have isolated the human GRAF gene (for GTPase regulator associated with the focal adhesion kinase pp125(FAK)). This gene was fused with MLL in a unique t(5;11)(q31;q23) that occurred in an infant with juvenile myelomonocytic leukemia. GRAF encodes a member of the Rho family of the GTPase-activating protein (GAP) family. On the protein level, it is 90% homologous to the recently described chicken GRAF gene that functions as a GAP of RhoA in vivo and is thus a critical component of the integrin signaling transduction pathway. The particular position of the human GRAF gene at 5q31 and the proposed antiproliferative and tumor suppressor properties of its avian homologue suggest that it also might be pathogenetically relevant for hematologic malignancies with deletions of 5q. To investigate this possibility, we sequenced 4-5 individual cDNA clones from 13 cases in which one allele of GRAF was deleted. We found point mutations within the GAP domain of the second GRAF allele in one patient. In two additional patients we found an insertion of 52 or 74 bp within the GRAF cDNA that generates a reading frame shift followed by a premature stop codon. GRAF maps outside the previously defined commonly deleted 5q31 region. Nevertheless, inactivation of both alleles in at least some cases suggests that deletions and mutations of the GRAF gene may be instrumental in the development and progression of hematopoeitic disorders with a del(5q).

Artificial Gene Fusion↗
PubMed2000

TEL/AML1 gene fusion is related to in vitro drug sensitivity for L-asparaginase in childhood acute lymphoblastic leukemia.

The t(12;21) translocation resulting in TEL/AML1 gene fusion is present in approximately 25% of patients with precursor B-lineage pediatric acute lymphoblastic leukemia (ALL). Studies suggest an association with a good prognosis; however, relapse can occur. We studied the relation between t(12;21), determined by fluorescence in situ hybridization or polymerase chain reaction, and in vitro drug resistance, measured by the MTT assay, in childhood B-lineage ALL at diagnosis. A total of 180 ALL samples were tested, 51 (28%) of which were positive for t(12;21). The median LC(50) values did not differ significantly between TEL/AML1-positive and -negative samples for prednisolone, dexamethasone, daunorubicin, thiopurines, epipodophyllotoxins, and 4-HOO-ifosfamide. However, the TEL/AML1-positive patients were relatively more sensitive to L-asparaginase (ASP; 5.9-fold; P =.029) and slightly but significantly more resistant to vincristine (1.5-fold; P =.011) and cytarabine (1.5-fold; P =.014). After matching for unevenly distributed patient characteristics-that is, excluding patients younger than 12 months, patients with CD10-negative immature B-lineage ALL, patients with Philadelphia chromosome, and patients who were hyperdiploid (more than 50 chromosomes) from the TEL/AML1 negative group-the only remaining difference was a relative sensitivity for ASP in the TEL/AML1-positive samples (10.8-fold; P =. 012). In conclusion, the presence of TEL/AML1 gene fusion in childhood precursor B-lineage ALL does not seem to be associated with a high in vitro drug sensitivity, except for ASP, indicating that these patients could benefit from treatment schedules with significant use of this drug.

Antineoplastic Agents↗
PubMed2000

Improved outcome in childhood acute lymphoblastic leukemia despite reduced use of anthracyclines and cranial radiotherapy: results of trial ALL-BFM 90. German-Austrian-Swiss ALL-BFM Study Group.

Trial ALL-BFM 90 was designed to improve outcome in patients with childhood acute lymphoblastic leukemia (ALL) by using a reduced treatment regimen. Patients were stratified into a standard-risk group (SRG), a medium-risk group (MRG), both defined by adequate early treatment response; and a high-risk group (HRG), defined by inadequate response to the cytoreductive prednisone prephase, induction failure, or Philadelphia-chromosome-positive ALL. Four treatment modifications were evaluated: dose intensification in induction by a more rapid drug sequence; administration of L-asparaginase during consolidation therapy in the MRG (randomized); enforced consolidation by rotational elements in the HRG; and reduction in the dose of anthracyclines and use of only 12-Gy preventive cranial radiotherapy in the MRG and HRG, with the aim of avoiding toxicity. Among all 2178 patients (</= 18 years of age), the 6-year event-free survival (EFS) rate (+/- SE) was 78% +/- 1%, with a median observation time of 4.8 years. EFS was 85% +/- 2% in the SRG (n = 636) and 82% +/- 1% in the MRG (n = 1299). L-asparaginase did not improve outcome in the MRG: the event-free interval was 83% +/- 2% with L-asparaginase (n = 528) and 81% +/- 2% without it (n = 557). Because there were more systemic relapses in the HRG (n = 243), EFS was 34% +/- 3%, an outcome inferior to that in the HRG in a previous trial, ALL-BFM 86, in which EFS was 47% +/- 5% (P =.04). The rates of isolated central nervous system relapse in the MRG and HRG were 0.8% and 1.6%, respectively; thus, the 12-Gy preventive cranial radiotherapy regimen apparently provided sufficient central nervous system prophylaxis. The overall improvement over the results in ALL-BFM 86 (6-year EFS, 72%; P =. 001) was based on fewer recurrences among patients in the MRG with B-cell-precursor ALL, indicating an advantage of more condensed induction therapy. In multivariate analysis, inadequate in vivo response emerged as the strongest adverse prognostic variable.

Adolescent↗
PubMed2000

t(7;12)(q36;p13), a new recurrent translocation involving ETV6 in infant leukemia.

The ETV6 gene is rearranged as a result of translocations involving a wide variety of chromosomal partners. To date, 12 partner genes for ETV6 have been cloned, and a further 23 chromosomal regions have been described. We previously identified a cryptic t(7;12) with ETV6 involvement in two cases of infant leukemia. The finding of a third case of t(7;12), also in an infant, prompted a more focussed search based on the common features found in these patients and those reported in the literature. The selection criteria were age at diagnosis < 20 months and the presence of +19 and/or +8 in the karyotype; cases with abnormalities of 7q and/or 12p were also considered. FISH studies using whole chromosome paints and probes for the ETV6 gene revealed a t(7;12) in 10 out of 23 cases studied. Seven of these had evidence of ETV6 rearrangement. Of those with ETV6 involvement, six had a 7q36 and one a 7q22 breakpoint. Importantly, in three cases the 7q36 breakpoint was within the same PAC, suggesting the existence of a new nonrandom translocation. However, in at least one patient the 7q36 breakpoint was different. The identification of the 7q partner genes will determine whether it is the disruption of ETV6 alone, or the formation of fusion genes, that is important for leukemogenesis in these patients. As both 7q36 and 7q22 are critical regions of gene loss in del(7q) leukemias, the identification of partner genes from these regions may also be important in understanding the pathogenesis of these diseases.

Chromosome Breakage↗
PubMed2000

Detection of acute leukemia cells with mixed lineage leukemia (MLL) gene rearrangements by flow cytometry using monoclonal antibody 7.1.

Translocations involving 11q23 are among the most common genetic abnormalities in hematologic malignancies, occurring in approximately 5-10% of acute lymphoblastic leukemia (ALL) and 5% of acute myeloblastic leukemia (AML). In 11q23 translocations, the mixed lineage leukemia (MLL) gene on chromosome 11, band q23, is usually disrupted. The human homologue of the rat NG2 chondroitin sulfate proteoglycan molecule, as detected by the monoclonal antibody (moab) 7.1, was shown to be expressed on leukemic cells with MLL rearrangements of children with acute leukemia. We further investigated the reactivity of the moab 7.1 on 533 cell samples of adults (n = 215) and children (n = 318) with acute leukemias (271 AML, 217 B-lineage ALL, 37 T-lineage ALL, eight CD7+ CD56+ myeloid/natural killer cell precursor acute leukemias) by flow cytometry. In AML, 38 samples were positive for moab 7.1 ('20%-cut-off-level'). These moab 7.1-positive AML cases revealed a myelomonocytic-differentiated immunophenotype with coexpression of the NK cell marker CD56 in 33 of 38 cases. Two of eight cell samples of the recently described CD7+ CD56+ myeloid/natural killer cell precursor acute leukemia entity reacted with moab 7.1. In ALL, 35 samples mostly of the pro-B-ALL subtype (33 pro-B-ALL, one common-ALL, one pre-B-ALL) were positive for moab 7.1. 58 (81%) of 72 samples with MLL rearrangements were positive for moab 7.1 including 28/31 with a t(4;11), 16/17 with a t(9;11), 3/5 with a t(11;19), and 2/6 with a del(11)(q23). All moab 7.1-positive ALL (n = 34) and childhood AML (n = 17) cases revealed MLL rearrangements as detected by Southern blot analysis and RT-PCR. However, 11 adults with AML, and one adult with moab 7.1-positive CD7+ CD56+ myeloid/natural killer cell precursor acute leukemia were negative for MLL rearrangements as proved by Southern blot analysis. We conclude that moab 7.1 is a sensitive but not entirely specific marker for the identification of 11q23-associated AML and ALL by flow cytometry in children and adults.

Acute Disease↗
PubMed2000

Long-term results of four consecutive trials in childhood ALL performed by the ALL-BFM study group from 1981 to 1995. Berlin-Frankfurt-Münster.

Four thousand, four hundred and forty eligible children of up to 18 years of age were treated in four consecutive trials between 1981 and 1995 with the treatment protocols of the Berlin-Frankfurt-Münster (BFM) study group for childhood acute lymphoblastic leukemia (ALL). The probability for event-free survival (pEFS) at 8 years improved from 65.8% in study ALL-BFM 81 to 75.9% in study ALL-BFM 90. The cumulative incidence of recurrences with CNS involvement was 10.1% and 9.3% in studies ALL-BFM 81 and 83, but was reduced to less than 5% in study ALL-BFM 90 (for isolated CNS relapses from 5.3% in study ALL-BFM 81 to 1.1% in study ALL-BFM 90). Four major findings were derived from this series of trials performed by 37 to 96 centers in Germany, Austria, and Switzerland: (1) Reintensification is a crucial part of treatment, even in low risk patients; (2) presymptomatic cranial radiotherapy can be safely reduced to 12 Gy, or even be eliminated if it is replaced by early intensive systemic and intrathecal methotrexate applied; (3) maintenance therapy given a total of 24 months from diagnosis provides a lower rate of systemic relapses than treatment for 18 months; (4) inadequate response to an initial 7-day prednisone window (combined with one intrathecal injection of methotrexate on day 1) defines about 10% of the patients with a very high risk of relapse. For patients with adequate early response (90% of all) an 8-year pEFS of 80% has been achieved in the most recent trial ALL-BFM 90. While it has proven so far to be impossible to improve the outcome for the small group of high risk patients, the number of recurrences could be effectively reduced for the large group of patients responding adequately to the prednisone in vivo sensitivity test. Apart from inadequate prednisone response, patients with hyperleukocytosis, age <1 year, or the presence of the Philadelphia-chromosome (Ph+ ALL) are at a particularly high risk of failure.

Adolescent↗
PubMed2000

Constitutive expression levels of CD95 and Bcl-2 as well as CD95 function and spontaneous apoptosis in vitro do not predict the response to induction chemotherapy and relapse rate in childhood acute lymphoblastic leukaemia.

CD95 (Fas/APO-1) expression and function and Bcl-2 expression, as well as spontaneous apoptosis in vitro, have been shown to be predictive markers for the in vivo response to chemotherapy in acute myeloid leukaemia (AML). To determine the clinical significance of apoptosis-regulating factors in acute lymphoblastic leukaemia (ALL), we investigated cell samples of children with ALL who had been included in the German ALL Berlin-Frankfurt-Münster (BFM) study using flow cytometry for constitutive expression levels of CD95 (n = 110) and Bcl-2 (n = 110). Furthermore, we determined the extent of spontaneous apoptosis in vitro (n = 102) and susceptibility to anti-CD95-induced apoptosis (CD95-sensitivity) (n = 97). We correlated these findings with the functional activity of the multidrug resistance (MDR)-associated P-glycoprotein (P-gp), as detected by the rhodamine123 efflux test, immunophenotype, cytogenetics and clinical data of the patients examined. Good responders to initial prednisone therapy ('prednisone response') revealed significantly higher Bcl-2 expression levels [5.4 +/- 3.4 relative fluorescence intensity (RFI), n = 68] than poor responders (3.7 +/- 2.6 RFI, n = 42; P = 0.002). There was no significant correlation between the other investigated parameters and prednisone response. Moreover, neither the CD95 and Bcl-2 expression levels nor the extent of spontaneous apoptosis in vitro, CD95 sensitivity or P-gp function were correlated with the response to induction chemotherapy or relapse rate, either for B-cell precursor ALL or T-cell ALL. No consistent pattern of change in CD95 (n = 10) and Bcl-2 expression (n = 9) was noted in cases studied at both initial diagnosis and relapse. In conclusion, our findings underline the different cell biological features of primary AML and ALL cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Clinical significance of P-glycoprotein expression and function for response to induction chemotherapy, relapse rate and overall survival in acute leukemia.

BACKGROUND AND OBJECTIVES: A multidrug-resistance (MDR) phenotype mediated by P-glycoprotein (P-gp) contributes to chemotherapy failure in acute leukemia. However, the exact prognostic significance of this resistance mechanism is still unclear, mostly due to methodologic problems in P-gp detection. We therefore investigated, whether P-gp expression levels or functional P-gp activity better predict response to induction chemotherapy, relapse rate and overall survival in acute leukemia. DESIGN AND METHODS: We examined cell samples of 121 adults with de novo acute myeloid leukemia (AML) and 102 children with newly diagnosed acute lymphoblastic leukemia (ALL) for P-gp expression and functional P-gp activity by flow cytometry. P-gp function was determined by the rhodamine 123 (rh123)-efflux test (AML n=121, ALL n=102) and P-gp expression levels using the P-gp specific monoclonal antibodies (moabs) MRK-16 (AML n=51, ALL n=31), 4.E3 (AML n=35, ALL n=32), or UIC-2 (AML n=68, ALL n=50). We correlated our findings with the immunophenotype, FAB morphology, cytogenetics and clinical data of the examined patients. RESULTS: P-gp expression levels as detected by MRK-16 and 4.E3 were very low and did not differ between AML and ALL as estimated using relative fluorescence intensity (RFI) values and D-values by Kolmogorow-Smirnov (KS) statistics. For moab UIC-2, P-gp expression levels were higher in AML than in ALL. Within AML, moab UIC-2 mainly reacted with myelomonocytic-differentiated leukemic cells of the FAB M4/5 subtypes. No correlation between P-gp expression levels as detected by MRK-16, 4.E3 or UIC-2 and the response to induction chemotherapy or relapse rate, both in AML and ALL, was observed. However, a prognostic impact of P-gp expression levels on overall survival in AML was seen for moab MRK-16. Moreover, within AML, P-gp function was higher in immature blast cells as defined by immunophenotype and FAB morphology and correlated with response to induction chemotherapy, relapse rate, overall survival as well as cytogenetic risk groups. In ALL, the overall functional P-gp activity was lower than in AML and did not correlate with immunophenotypical subgroups, response to induction chemotherapy, relapse rate or overall survival. INTERPRETATION AND CONCLUSIONS: Our data demonstrate a prognostic impact of P-gp in AML but not ALL and indicate that the functional rh123-efflux assay should be preferred for flow-cytometric P-gp evaluation in acute leukemia compared with P-gp expression analysis by monoclonal antibodies.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

A DNA damage repair mechanism is involved in the origin of chromosomal translocations t(4;11) in primary leukemic cells.

Some chromosomal translocations involved in the origin of leukemias and lymphomas are due to malfunctions of the recombinatorial machinery of immunoglobulin and T-cell receptor-genes. This mechanism has also been proposed for translocations t(4;11)(q21;q23), which are regularly associated with acute pro-B cell leukemias in early childhood. Here, reciprocal chromosomal breakpoints in primary biopsy material of fourteen t(4;11)-leukemia patients were analysed. In all cases, duplications, deletions and inversions of less than a few hundred nucleotides indicative of malfunctioning DNA repair mechanisms were observed. We concluded that these translocation events were initiated by several DNA strand breaks on both participating chromosomes and subsequent DNA repair by 'error-prone-repair' mechanisms, but not by the action of recombinases of the immune system.

Adolescent↗
PubMed1999

A new recurrent translocation, t(5;11)(q35;p15.5), associated with del(5q) in childhood acute myeloid leukemia. The UK Cancer Cytogenetics Group (UKCCG)

Partial deletion of the long arm of chromosome 5, del(5q), is the cytogenetic hallmark of the 5q-syndrome, a distinct subtype of myelodysplastic syndrome-refractory anemia (MDS-RA). Deletions of 5q also occur in the full spectrum of other de novo and therapy-related MDS and acute myeloid leukemia (AML) types, most often in association with other chromosome abnormalities. However, the loss of genetic material from 5q is believed to be of primary importance in the pathogenesis of all del(5q) disorders. In the present study, we performed fluorescence in situ hybridization (FISH) studies using a chromosome 5-specific whole chromosome painting probe and a 5q subtelomeric probe to determine the incidence of cryptic translocations. We studied archival fixed chromosome suspensions from 36 patients with myeloid disorders (predominantly MDS and AML) and del(5q) as the sole abnormality. In 3 AML patients studied, this identified a translocation of 5q subtelomeric sequences from the del(5q) to the short arm of an apparently normal chromosome 11. FISH with chromosome 11-specific subtelomeric probes confirmed the presence of 11p on the shortened 5q. Further FISH mapping confirmed that the 5q and 11p translocation breakpoints were the same in all 3 cases, between the nucleophosmin (NPM1) and fms-related tyrosine kinase 4 (FLT4) genes on 5q35 and the Harvey ras-1-related gene complex (HRC) and the radixin pseudogene (RDPX1) on 11p15.5. Importantly, all 3 patients with the cryptic t(5;11) were children: a total of 3 of 4 AML children studied. Two were classified as AML-M2 and the third was classified as M4. All 3 responded poorly to treatment and had short survival times, ranging from 10 to 18 months. Although del(5q) is rare in childhood AML, this study indicates that, within this subgroup, the incidence of cryptic t(5;11) may be high. It is significant that none of the 24 MDS patients studied, including 11 confirmed as having 5q-syndrome, had the translocation. Therefore, this appears to be a new nonrandom chromosomal translocation, specifically associated with childhood AML with a differentiated blast cell phenotype and the presence of a del(5q).

Adult↗
PubMed1999

Mapping of the breakpoints on the short arm of chromosome 17 in neoplasms with an i(17q).

Isochromosomes are monocentric or dicentric chromosomes with homologous arms that are attached in a reverse configuration as mirror images. With an incidence of 3-4%, the i(17q) represents the most frequent isochromosome in human cancer. It is found in a variety of tumors, particularly in blast crisis of chronic myeloid leukemia (CML-BC), acute myeloid leukemia (AML), non-Hodgkin's lymphoma (NHL), and medulloblastoma (MB), and indicates a poor prognosis. To determine the breakpoints on the molecular genetic level, we analyzed 18 neoplasms (six CML, four AML, one NHL, and seven MB) with an i(17q) and two MB with a pure del(17p) applying fluorescence in situ hybridization (FISH) with yeast artificial chromosome (YAC) clones, P1-artificial chromosome (PAC) clones, and cosmids from a well-characterized contig covering more than 6 Mb of genomic DNA. We identified four different breakpoint cluster regions. One is located close to or within the centromere of chromosome 17 and a second in the Charcot-Marie-Tooth (CMT1A) region at 17(p11.2). A third breakpoint was found telomeric to the CMT1A region. The fourth, most common breakpoint was detected in MB, AML, and in CML-BC specimens and was bordered by two adjacent cosmid clones (clones D14149 and M0140) within the Smith-Magenis syndrome (SMS) region. These results indicate that the low copy number repeat gene clusters which are present in the CMT and SMS regions may be one of the factors for the increased instability that may trigger the formation of an i(17q).

Adolescent↗
PubMed1999

Multiplex PCR--a rapid screening method for detection of gene rearrangements in childhood acute lymphoblastic leukemia.

Chromosomal rearrangements in childhood acute lymphoblastic leukemia (ALL) play an important role in the identification of clinical relevant subgroups. For rapid and easy detection of the clinically most important gene rearrangements, a nested multiplex reverse transcriptase polymerase chain reaction (multiplex PCR) was developed. This multiplex PCR enables the detection of M-BCR/ABL, m-BCR/ABL, TEL/ AML1, and MLL/AF4 fusion transcripts in one PCR reaction. However, the existence of splicing variants and different breakpoints on the DNA level hampers the discrimination of the rearrangements by their fragment size on an agarose gel. Therefore, one of the internal primers of each translocation (ABL-2, TEL-2, AF4-2) was labeled with a characteristic fluorescent dye, and an automatic fluorescence-based DNA fragment analysis was performed. The sensitivity of this multiplex PCR is in the same range as that of the corresponding single PCR reaction and allows a fast screening for the detection of therapy-relevant rearrangements, with a high turnover of samples.

Base Sequence↗