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Biomedical subjects

A Borkhardt

Publications and source records attributed to A Borkhardt.

At least 55 records · Page 3Linked to original sources
PubMed1995

Secondary acute myeloid leukemia with translocation (4;11) and MLL/AF4 rearrangement in a 15-year-old boy treated for common acute lymphoblastic leukemia 11 years earlier.

Secondary acute myeloid leukemia occurring in a 15-year-old boy 11 years after initial treatment of a common lymphoblastic leukemia (c-ALL) is described. Initial complete remission was terminated after 4 years by an isolated testicular relapse, followed by first bone marrow relapse within 18 months. After he achieved remission again, an allogeneic bone marrow transplantation from his HLA-identical brother was performed. Five years and 9 months later, the patient developed thrombocytopenia, leukopenia, and anemia, but bone marrow biopsies at this time demonstrated only myelofibrosis, with no blast cell population present. A polymerase chain reaction assay of a peripheral blood sample recognized the mRNA fusion region for the MLL/AF4 rearrangement, i.e., the molecular equivalent of the translocation (4;11)(q21,q23). Four weeks later, a blast cell population with AML-M1 morphology according to the FAB classification appeared in the bone marrow, and translocation (4;11) was detected by cytogenetics. Thus, secondary leukemias with chromosomal 11q23 rearrangement can develop after a long latency period and can be diagnosed earlier with the PCR technique.

Acute Disease↗
PubMed1995

Detection of chromosomal translocations in leukemia-lymphoma cells by polymerase chain reaction.

In recent years many chromosomal translocations involved in leukemia and lymphoma have been defined at the molecular level. In addition to advancing the understanding of pathological mechanisms underlying the transformation process, the cloning and sequencing of the genes altered by the translocations have provided new tools for diagnosis and monitoring of patients. In particular, the polymerase chain reaction (PCR) methodology yields rapid, sensitive and accurate diagnostic and prognostic information. As leukemias carrying certain translocations confer a higher risk of treatment failure, it is important to identify accurately all positive cases in order to give appropriate therapy. An important new initiative in the diagnostical setting and anti-leukemic therapy is the early detection of minimal residual disease (MRD). If MRD, implying an increased risk of relapse, is reliably detected during apparent clinical remission, alternative strategies could be applied early while the malignant cell burden is still minimal. The PCR assays are clearly more sensitive than other methods of MRD detection including morphology, immunophenotyping and cytogenetics; treatment failure is first detectable by PCR followed by cytogenetic relapse and finally clinical disease. PCR assays have been most often used in the MRD analysis of follicular lymphoma with t(14;18), chronic myeloid leukemia and acute lymphoblastic leukemia (ALL) with t(9;22), ALL with t(4;11), and acute myeloid leukemia (AML) with t(8;21) or t(15;17). PCR amplification is applicable to any other translocation provided the translocation is highly associated with the malignancy and the breakpoints are sufficiently clustered; a quickly increasing number of such specific molecular markers are now available for PCR assays. PCR still remains an experimental investigation for the detection of covert disease. However, the clinical relevance of MRD detection should be evaluated separately for each type of leukemia as significant prognostic differences between disease entities were found. This review describes the PCR assays available for the detection of leukemia cells with specific chromosomal translocations and summarizes the experience with the application of PCR techniques in monitoring patients during the course of the disease.

Biomarkers, Tumor↗
PubMed1995

Construction of RNA standards for high-resolution automatic product analysis in quantitative competitive RT-PCR.

The exponential character of PCR amplification may compromise quantitative assays because it multiplies minor sample-to-sample variations. To overcome these problems, several authors have used recombinant standard DNA or RNA molecules to be spiked into the samples in a dilution series of known copy numbers before co-amplification by PCR. To obtain an equal efficacy of reverse transcription and PCR amplification, standard and template molecules should be highly homologous. However, the limited resolution of commonly used agarose gel electrophoresis requires rather large differences in size and nucleotide sequence to separate both molecules from each other after PCR. Due to a much higher resolution, automatic post-PCR analyzing systems based on laser-induced fluorescence may help to overcome these difficulties. For using the capabilities of these systems in quantitative competitive RT-PCR, we developed a protocol to construct recombinant RNA standard molecules that only differ from the target sequence by a small deletion of 8 nucleotides. It is based on PCR-induced mutagenesis and solid-phase in vitro transcription. This protocol was applied to quantify multidrug resistance gene (MDRI) mRNA in malignant cells, but it can easily be adapted to any gene of interest.

Base Sequence↗
PubMed1995

Rapid synthesis of hybrid RNA molecules associated with leukemia-specific chromosomal translocations.

A number of gene arrangements have been described as characteristic abnormalities associated with different types of leukemia, and this list is still growing. In view of the biological, clinical and prognostic relevance of the pathological fusion products, techniques permitting their detection are of paramount importance in the clinical setting. In some instances, permanent leukemic cell lines carrying the abnormality of interest are available for the establishment and standardization of molecular assays. For a number of newly discovered gene rearrangements, however, this may not be the case. It is therefore of great interest for clinical laboratories to have alternative technical possibilities for the set-up of standardized molecular tests. This problem provided the stimulus to design a simple and rapid method for in vitro generation of chimeric RNA molecules corresponding to pathological fusion transcripts typical for chromosomal translocations in leukemias. Two separate fragments are generated in a four-primer multiplex PCR. Due to a PCR-generated overlap, a chimeric fragment can be synthesized in a second round of PCR. This PCR product is then purified with the help of magnetic beads. Due to the SP6 promotor sequence incorporated during the second round of PCR, transcription into RNA is easily facilitated while the template DNA is still bound to the solid phase. Following this strategy we were able to synthesize the fusion transcripts m-BCR/ABL, CBF beta/MYH11, and MLL/AFp1 which are the molecular equivalents of t(9;22)(q34,q11), inv16(p13;q22) and t(1;11)(p32;q23), respectively. The chimeric RNA will be useful as a control template in diagnostic RT-PCR strategies. It can also be further processed in translation systems leading to the corresponding chimeric oncoprotein. This approach can be easily used to create any hybrid RNA of interest.

Base Sequence↗
PubMed1995

Detection of four different 11q23 chromosomal abnormalities by multiplex-PCR and fluorescence-based automatic DNA-fragment analysis.

A nested polymerase chain reaction (PCR) protocol was developed for rapid detection of four different 11q23 abnormalities by a single PCR assay. During each of the two PCR rounds a sense primer located within exon 5 of the MLL gene at 11q23 was combined with four different antisense primers, each located within possible translocation partner genes at chromosomes 4, 6, 9, and 19, respectively. Except for the MLL primer all primers used during the second round of nested-PCR carried a characteristic fluorescence label at their 5'-end. Agarose gel analysis of the PCR products was sufficient to discriminate between the absence of any of the four MLL rearrangements and the presence of at least one of them. Discrimination of the four different MLL translocation partner genes was not possible by agarose gel analysis due to a molecular heterogeneity of the 11q23 breakpoints resulting in PCR products of variable size. For this reason, automatic fluorescence-based DNA-fragment analysis was used to exactly define the MLL translocation partner genes if a positive result had been obtained by agarose gel analysis. In patients with leukemia, this assay may enable a fast and highly sensitive detection of different 11q23 abnormalities, which usually correlate with poor clinical prognosis.

Base Sequence↗
PubMed1995

Recurrent chromosomal translocations and fusion genes in leukemia-lymphoma cell lines.

The occurrence of defined chromosomal translocations in specific subtypes of leukemia and lymphoma strongly suggests that these structural alterations play an important role in the process of tumorigenesis. Translocations may activate nearby cellular genes involved in the control of proliferation or differentiation. One mechanism by which genes become activated or overexpressed is by their juxtaposition with the genes coding for the lymphoid antigen receptors (immunoglobulins or T cell receptors). The majority of the translocated genes appear to encode transcription factors, the expression of which might become deregulated due to the influence of regulatory elements in the antigen receptor loci. A second general type of chromosomal translocation by which proto-oncogenes can be activated results in the synthesis of fusion genes. Here, chimera proteins containing portions of two different proteins are generated following exon-exon juxtaposition of two genes lying at the sites of translocation. The list of such tumor-associated fusion genes generated by specific translocations has greatly increased over the last years. A wide variety of leukemia-lymphoma cell lines have been established to act both as genetic resources and as model systems for the different disease types and stages providing an inexhaustible source of replicate material. For many translocations, DNA spanning the breakpoint was isolated by using cell lines carrying the particular abnormality. The cloning of the genes disrupted by the translocations has led to the availability of probes useful for diagnosis and monitoring of patients, eg with polymerase chain reaction (PCR) assays. Here, we review the cytogenetic and molecular data of chromosomal aberrations associated with leukemia-lymphoma and indicate a panel of human cell lines carrying such specific chromosomal abnormalities.

Cloning, Molecular↗
PubMed1994

Pre-pre-B acute lymphoblastic leukemia: high frequency of alternatively spliced ALL1-AF4 transcripts and absence of minimal residual disease during complete remission.

We used the polymerase chain reaction (PCR) to detect ALL1-AF4 rearrangements, the molecular hallmark of t(4;11) in a series of 46 pre-pre-B (CD19+, CD24+, CD10/CD20/cylgM/sIgM-) acute lymphoblastic leukemias (ALL). Eighteen patients (39%) exhibited fusion transcripts including 4 of 12 children and 14 of 34 adults. This genetic defect was associated with hyperleukocytosis (median leukocyte count 176 x 10(9)/L) and expression of myeloid-associated antigens (CDw65+). In contrast, only two patients from a group of 67 common (CD19/CD10+, cylgM/sIgM-) and pre-B ALLs (CD19/cylgM+, CD10 +/-, sIgM-) showed ALL1-AF4 mRNA. All PCR-positive cases showed multiple amplification products representing alternative splicing events. Moreover, reciprocal der (4)-derived AF4-ALL1 transcripts were observed in 65% of the cases analyzed. Eight of the 18 pre-pre-B ALL patients with an ALL1-AF4 recombination are currently in complete continuous remission for up to 54 months (median, 26 months). Twelve remission samples were available from seven cases, and all of them lacked evidence of minimal residual disease. Overall this study documents a similarly high incidence of ALL1-AF4 recombinations in children (infants excluded) and adults with pre-pre-B ALL and demonstrates the decline of the leukemic cell clone below the detection level of PCR in a remarkable proportion of patients under intense treatment protocols.

Adolescent↗
PubMed1994

[Detection of mycobacterial DNA from paraffin-embedded tissue in lung and lymphoid epithelial granulomas].

Standard techniques for the detection of mycobacteria in granulomatous diseases can be inadequate. We analysed 71 formalin fixed and paraffin-embedded tissue blocks from 68 non-immunocompromised patients with caseating, non-caseating, scarred, and miliary granulomas of lung and lymph nodes. A reamplification PCR protocol was established to detect a 123 bp product of the repetitive insertion sequence IS986/IS6110. After exclusion of 6 PCR-negative cases with clinical sarcoidosis 97% of lung tissue blocks with more than 10% caseating and non-caseating granulomas contained mycobacterial DNA. By routine microbiology mycobacteria could be detected in 78% of the patients. Scarred granulomas were PCR-negative. All miliary granulomas were PCR-positive. Lymph nodes showed comparable results. We think that this method facilitates aetiologic analysis of granulomatous diseases especially when the suspicous tissue is fixed and microbiology is not available.

DNA, Bacterial↗
PubMed1994

Spontaneous hematological remission in a boy with myelodysplastic syndrome and monosomy 7.

We report the case of a 14-month-old boy with myelodysplastic syndrome (refractory anemia with blast excess) and bone marrow monosomy 7. Within 2 years after presentation hematological remission gradually occurred without any chemotherapy. After the patient had received three transfusions within the first 4 months, red cell production normalized. However it took 18 more months for neutropenia to resolve. The patient is well 34 months after the first presentation. Molecular analysis of the bone marrow blasts showed no mutations of the ras genes or of the FLR-exon of the NF-1 gene. To our knowledge this is the first report of monosomy 7 syndrome with spontaneous hematological remission.

Base Sequence↗
PubMed1994

Molecular analysis of MLL-1/AF4 recombination in infant acute lymphoblastic leukemia.

We examined ten cases of acute lymphoblastic leukemia (ALL) in infants (less than 1 year of age) by RT-nested PCR for a MLL-1/AF4 rearrangement. Five patients revealed a positive result. The specific PCR product differed in size from approximately 380-670 bp indicating various splicing variants in the MLL-1/AF4 rearrangement. Three patients had a fusion between exon 6 of the MLL-1 gene and codon 362 of the known AF4 cDNA sequence. Moreover, in two patients more than one specific PCR product was detected, possibly due to alternative splicing. In the first case, sequencing of these products revealed a hybrid mRNA consisting of MLI-1 exon 7 or exon 8, respectively, fused to the AF4 gene at codon 348. In the second case with alternative splicing, again, exon 7 or 8 of the MLL-1 gene were fused to the AF4 gene as in case 1. The AF4 sequence involved in this patient, however, started at codon 362. The AF4 break was, therefore, identical to the three MLL-1/AF4 positive patients as described above. Moreover, we investigated all ten patients for the reciprocal mRNA transcript AF4/MLL-1 by a similar PCR approach. In none of these patients, including the five MLL-1/AF4 positive cases was a specific PCR product obtained. However, in the MV411 cell line bearing a t(4;11), which served as a positive control in our MLL-1/AF4-PCR assay, the reciprocal AF4/MLL-1 mRNA was detected. Our results indicate that a MLL-1/AF4 rearrangement occurs in about 50% of infants with ALL. In contrast, the reciprocal hybrid mRNA can only rarely be detected, if at all.

Base Sequence↗
PubMed1993

Clinical and immunological aspects of hepatitis B virus infection in children receiving multidrug cancer chemotherapy.

For two reasons hepatitis B virus infection is an important problem in patients with cancer. First, multidrug cancer chemotherapy may reactivate or worsen a previously benign chronic HBV infection. Second, patients undergoing cancer chemotherapy are at an increased risk of acquiring and spreading HBV which may result in an endemic infection. HBV reactivation may precipitate into a severe acute disease including fulminant hepatitis. In contrast, the acquisition of HBV during cancer chemotherapy commonly takes a mild clinical course but frequently leads to persistently high viremia. This state of immunotolerance to viral antigens allows viral replication without any sign of liver cell destruction. Withdrawal of chemotherapy does not cause significant changes if infection occurred during cytotoxic chemotherapy. Infection with HBV during cancer chemotherapy, therefore, may be considered as a model of an induced antigen-specific immunotolerance. In agreement with this hypothesis, vaccination against HBV during cancer chemotherapy does not prevent spread of HBV in oncology wards as it does not produce significant anti-HBs titers. Furthermore, vaccination even suppresses the immune response to later booster doses after chemotherapy has been withdrawn.

Antineoplastic Agents↗
PubMed1993

BCR-ABL rearrangement in a child with acute myelogenous leukaemia without a Philadelphia chromosome.

We describe a BCR/ABL rearrangement positive but Philadelphia chromosome negative status in a 9-year-old boy suffering from an acute myelogenous leukaemia (AML). This case was detected in a prospective PCR screening procedure including 21 children with newly diagnosed AML and 150 children with acute lymphoblastic leukaemia (ALL). We found a 5.4% incidence of BCR/ABL rearrangement positive cases in pre-B and c-ALL in childhood.

Child↗
PubMed1993

Quantification of leukaemic cells based on heteroduplex formation of tal-1 gene sequences after PCR coamplification.

Based on the high sensitivity of the polymerase chain reaction (PCR) several assays have already been described which can be applied to monitor minimal residual disease in patients with leukaemia. However, most of these approaches are only qualitative or semiquantitative at best. Moreover, the semiquantitative assays require rather large-scale procedures such as oligonucleotide hybridization or sampling of aliquots during the exponential phase of PCR amplification, which is time consuming and may be error prone. Using the deletion in the tal-1 gene as a clonal marker of malignant cells, which can be found in about 10-25% of T-cell acute lymphoblastic leukaemia, we have developed a rapid and simple PCR assay for the quantification of residual leukaemic cells. The assay is based on heteroduplex formation between standard and template DNA sequences after coamplification of both DNA sequences in the same PCR reaction. The sensitivity of this PCR approach allows the detection of neoplastic cells at frequency of at least 10(-4). Application of such an assay needs not to be restricted to patients harbouring a tal-1 gene deletion. It may easily be adapted to other clonal DNA markers of blast cells and therefore facilitate monitoring of minimal residual disease in many different kinds of leukaemia.

Base Sequence↗