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P074 Jumping translocation of 1q in a case of childhood acute lymphoblastic leukemia, type L3 with t(8;14)
S110
Posters, ISH EAD 2007, Budapest, Hungary, 29 August
Recognition of specific skin manifestations of acute myelogenous leukemia is important because they have prognostic value and always indicates poor prognosis. P074 Jumping translocation of 1q in a case of childhood acute lymphoblastic leukemia, type L3 with t(8;14) B. Bessenyei *, A. Ujfalusi, E. Balogh, Cs. Kiss, E. B´ ardi, ´. Ol´ I. Szegedi, E ah. Regional Genetic Laboratory, Dept. of Pediatrics, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary Burkitt’s type acute lymphoblastic leukemia (ALL), FAB type L3, represents a leukemic presentation of Burkitt’s lymphoma. This type of ALL is associated with translocation 8;14 and its variants t(8;22) and t(2;8). Besides these classical translocations, additional chromosomal changes, such as abnormalities of chromosome 1 have been reported. In a number of jumping translocations 1q arm is the most common donor chromosome. We report a case of a twelve years old patient with mature B-ALL of L3 type. Bone marrow from the patient was analysed with karyotyping using G-banding, interphase fluorescence in situ hybridization (i-FISH) with LSI IGH/MYC, CEP8 probe (Vysis) and Multicolor-FISH (M-FISH) analysis (Metasystems). Conventional cytogenetic analysis detected the following karyotype: 46, XY, +der(1), t(8;14), 21/47, XY, +i(1q), t(8;14). The occurence of t(8;14) was confirmed by i-FISH. The M-FISH study revealed the presence of three cell lines. All of them carried the specific translocation but had different 1q abnormalities besides the presence of two normal chromosome 1 resulting in trisomy 1q. In one of them a translocation of 1q to chromosome 21, in the other isochromosome 1q, and in the third cell line a translocation of 1q to the derivative chromosome 14 originated from t(8;14) were found. M-FISH analysis provides a useful complementary technique to routine conventional cytogenetics and i-FISH in the analysis of ALL with complex karyotypes. In our case M-FISH helped us not only to confirm, but clarify different chromosome 1 abnormalities. P075 Analysis of complex chromosome aberrations in childhood acute lymphoblastic leukemia (ALL) using multicolor fluorescent in situ hybridization (M-FISH) A. Ujfalusi *, E. Balogh, B. Bessenyei, Cs. Kiss, E. B´ ardi, I. Szegedi, E. Ol´ ah. Regional Genetic Laboratory, Dept. of Pediatrics, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary Introduction: Chromosomal changes are an independent prognostic factor in childhood ALL. Patients are classified into risk groups for treatment according to karyotype. Conventional cytogenetic technique has limitations in the identification of complex chromosomal aberrations. Multicolor fluorescence in situ hybridization (M-FISH) allows simultaneous visualization of all human chromosomes in different colors. M-FISH is useful for detection of cryptic genetic aberrations, identification of marker chromosomes and complex chromosomal rearrangements. We used this technique to refine the diagnosis of five pediatric ALL patients previously characterized by conventional karyotyping and identify the disease-specific genetic alterations more accurately. Materials and Methods: Cytogenetic examination was performed on G-banded metaphases at diagnosis. M-FISH was carried out using Metasystem’s 24Xcyte kit. Results: Using cytogenetic analysis in four of five patients primary specific translocations (t(1;19), t(2;8), t(8;14), t(9;22) and marker chromosomes were detected. In the fifth patient only marker chromsomes were observed. M-FISH confirmed the specific chromosome translocations in every four cases, identified the origin of marker chromosomes, revealed unbalanced translocations, isochromosome, insertion and trisomy.
2 September 2007
Conclusion: The confirmation of genetic alterations detected by conventional cytogenetic analysis and further characterization of marker chromosomes, clarification of complex chromosome aberrations, detection of cryptic rearrangements may provide useful information about the prognosis and influence the treatment of childhood ALL. P076 Identification of FLT3-ITD mutations in AML patients using different diagnostic methods P. Antal-Szalm´ as1 *, K. Koczok1 , I. Balogh1 , A. Kiss2 , M. Udvardy2 , J. Kappelmayer1 . 1 Department of Clinical Biochemistry and Molecular Pathology, 2 2nd Department of Internal Medicine, Debrecen, University of Debrecen, Medical and Health Science Center, Hungary Introduction: The internal tandem duplication (ITD) of exon 14 15 of the FLT3 gene is one of the most frequent genetic alteration associated with worse prognosis in the intermediate risk group of acute myeloid leukemia (AML). Materials and Methods: ITD was analyzed in DNA samples of 45 AML patients using a standard PCR+agarose gel electrophoresis method. In order to enhance the sensitivity of the detection the PCR products were also evaluated by polyacrylamide gel electrophoresis (PAGE), by an Agilent DNAon-chip and GeneScan analysis. Results: ITD-positivity could be detected by the standard method in 12 (26.7%) patients, and 1 more positive case could be identified by the other three assays. In 6 positive cases 1 or 2 additional large but very faint PCR products were seen on the agarose gel beside the wild type and the ITD bands. These extra bands were much clearly visible by PAGE and DNAon-chip, but were absent by GeneScan analysis. In 2 positive cases the PCR products of wild type, ITD and the large extra bands were separately isolated from the polyacrylamide gel and were sequenced. A 21 and a 33 bp insertion was identified in the ITD bands but both the wild type and the ITD sequences appeared when the large extra bands were analyzed. Mixing of the isolated wild type and ITD PCR products followed by a heating/cooling cycle could prove that these large fragments are heteroduplexes. Conclusions: The application of fluorescence primers combined with capillary electrophoresis of denatured PCR products provides the highest sensitivity and no interferences by heteroduplexes. Based on these data, GeneScan analysis seems the most appropriate method for FLT3-ITD detection. P077 NPM1 and FLT3 mutations in acute myeloid leukemia S. Nahajevszky1 *, A. Tordai2 , A. Szilvasi2 , N. Meggyesi2 , E. Adam1 , A. Kozma1 , S. Lueff1 , N. Lovas1 , B. Kapas1 , Z. Matrai1 , A. Sipos1 , T. Masszi1 , H. Andrikovics2 . 1 Dept. of Hematology and Stem Cell Transplantation, 2 Dept. of Molecular Diagnostics, National Medical Center, Budapest, Hungary Nucleophosmin (NPM) is a multifunctional, nucleocytoplasmic shuttling protein involved in ribosome biogenesis, in the maintenance of genomic stability and in the regulation of p53 tumor suppressor pathway. In acute myeloid leukaemia (AML) somatic mutations in NPM gene have recently been reported as one of the most common genetic alteration together with fms-like tyrosine kinase-3 (FLT3) internal tandem duplication (ITD) and tyrosine kinase domain (TKD affecting codon Asp835) mutations. In the present study, 120 consecutive adults with newly diagnosed AML (49 males and 71 females, age: 46±12 year) treated in our institute between January 2001 and December 2005 were enrolled. We analysed NPM mutation and FLT3 ITD by fluorescent PCR and TKD mutations by PCR-RFLP at the time point of diagnosis.
Posters, ISH EAD 2007, Budapest, Hungary, 29 August
Recognition of specific skin manifestations of acute myelogenous leukemia is important because they have prognostic value and always indicates poor prognosis. P074 Jumping translocation of 1q in a case of childhood acute lymphoblastic leukemia, type L3 with t(8;14) B. Bessenyei *, A. Ujfalusi, E. Balogh, Cs. Kiss, E. B´ ardi, ´. Ol´ I. Szegedi, E ah. Regional Genetic Laboratory, Dept. of Pediatrics, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary Burkitt’s type acute lymphoblastic leukemia (ALL), FAB type L3, represents a leukemic presentation of Burkitt’s lymphoma. This type of ALL is associated with translocation 8;14 and its variants t(8;22) and t(2;8). Besides these classical translocations, additional chromosomal changes, such as abnormalities of chromosome 1 have been reported. In a number of jumping translocations 1q arm is the most common donor chromosome. We report a case of a twelve years old patient with mature B-ALL of L3 type. Bone marrow from the patient was analysed with karyotyping using G-banding, interphase fluorescence in situ hybridization (i-FISH) with LSI IGH/MYC, CEP8 probe (Vysis) and Multicolor-FISH (M-FISH) analysis (Metasystems). Conventional cytogenetic analysis detected the following karyotype: 46, XY, +der(1), t(8;14), 21/47, XY, +i(1q), t(8;14). The occurence of t(8;14) was confirmed by i-FISH. The M-FISH study revealed the presence of three cell lines. All of them carried the specific translocation but had different 1q abnormalities besides the presence of two normal chromosome 1 resulting in trisomy 1q. In one of them a translocation of 1q to chromosome 21, in the other isochromosome 1q, and in the third cell line a translocation of 1q to the derivative chromosome 14 originated from t(8;14) were found. M-FISH analysis provides a useful complementary technique to routine conventional cytogenetics and i-FISH in the analysis of ALL with complex karyotypes. In our case M-FISH helped us not only to confirm, but clarify different chromosome 1 abnormalities. P075 Analysis of complex chromosome aberrations in childhood acute lymphoblastic leukemia (ALL) using multicolor fluorescent in situ hybridization (M-FISH) A. Ujfalusi *, E. Balogh, B. Bessenyei, Cs. Kiss, E. B´ ardi, I. Szegedi, E. Ol´ ah. Regional Genetic Laboratory, Dept. of Pediatrics, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary Introduction: Chromosomal changes are an independent prognostic factor in childhood ALL. Patients are classified into risk groups for treatment according to karyotype. Conventional cytogenetic technique has limitations in the identification of complex chromosomal aberrations. Multicolor fluorescence in situ hybridization (M-FISH) allows simultaneous visualization of all human chromosomes in different colors. M-FISH is useful for detection of cryptic genetic aberrations, identification of marker chromosomes and complex chromosomal rearrangements. We used this technique to refine the diagnosis of five pediatric ALL patients previously characterized by conventional karyotyping and identify the disease-specific genetic alterations more accurately. Materials and Methods: Cytogenetic examination was performed on G-banded metaphases at diagnosis. M-FISH was carried out using Metasystem’s 24Xcyte kit. Results: Using cytogenetic analysis in four of five patients primary specific translocations (t(1;19), t(2;8), t(8;14), t(9;22) and marker chromosomes were detected. In the fifth patient only marker chromsomes were observed. M-FISH confirmed the specific chromosome translocations in every four cases, identified the origin of marker chromosomes, revealed unbalanced translocations, isochromosome, insertion and trisomy.
2 September 2007
Conclusion: The confirmation of genetic alterations detected by conventional cytogenetic analysis and further characterization of marker chromosomes, clarification of complex chromosome aberrations, detection of cryptic rearrangements may provide useful information about the prognosis and influence the treatment of childhood ALL. P076 Identification of FLT3-ITD mutations in AML patients using different diagnostic methods P. Antal-Szalm´ as1 *, K. Koczok1 , I. Balogh1 , A. Kiss2 , M. Udvardy2 , J. Kappelmayer1 . 1 Department of Clinical Biochemistry and Molecular Pathology, 2 2nd Department of Internal Medicine, Debrecen, University of Debrecen, Medical and Health Science Center, Hungary Introduction: The internal tandem duplication (ITD) of exon 14 15 of the FLT3 gene is one of the most frequent genetic alteration associated with worse prognosis in the intermediate risk group of acute myeloid leukemia (AML). Materials and Methods: ITD was analyzed in DNA samples of 45 AML patients using a standard PCR+agarose gel electrophoresis method. In order to enhance the sensitivity of the detection the PCR products were also evaluated by polyacrylamide gel electrophoresis (PAGE), by an Agilent DNAon-chip and GeneScan analysis. Results: ITD-positivity could be detected by the standard method in 12 (26.7%) patients, and 1 more positive case could be identified by the other three assays. In 6 positive cases 1 or 2 additional large but very faint PCR products were seen on the agarose gel beside the wild type and the ITD bands. These extra bands were much clearly visible by PAGE and DNAon-chip, but were absent by GeneScan analysis. In 2 positive cases the PCR products of wild type, ITD and the large extra bands were separately isolated from the polyacrylamide gel and were sequenced. A 21 and a 33 bp insertion was identified in the ITD bands but both the wild type and the ITD sequences appeared when the large extra bands were analyzed. Mixing of the isolated wild type and ITD PCR products followed by a heating/cooling cycle could prove that these large fragments are heteroduplexes. Conclusions: The application of fluorescence primers combined with capillary electrophoresis of denatured PCR products provides the highest sensitivity and no interferences by heteroduplexes. Based on these data, GeneScan analysis seems the most appropriate method for FLT3-ITD detection. P077 NPM1 and FLT3 mutations in acute myeloid leukemia S. Nahajevszky1 *, A. Tordai2 , A. Szilvasi2 , N. Meggyesi2 , E. Adam1 , A. Kozma1 , S. Lueff1 , N. Lovas1 , B. Kapas1 , Z. Matrai1 , A. Sipos1 , T. Masszi1 , H. Andrikovics2 . 1 Dept. of Hematology and Stem Cell Transplantation, 2 Dept. of Molecular Diagnostics, National Medical Center, Budapest, Hungary Nucleophosmin (NPM) is a multifunctional, nucleocytoplasmic shuttling protein involved in ribosome biogenesis, in the maintenance of genomic stability and in the regulation of p53 tumor suppressor pathway. In acute myeloid leukaemia (AML) somatic mutations in NPM gene have recently been reported as one of the most common genetic alteration together with fms-like tyrosine kinase-3 (FLT3) internal tandem duplication (ITD) and tyrosine kinase domain (TKD affecting codon Asp835) mutations. In the present study, 120 consecutive adults with newly diagnosed AML (49 males and 71 females, age: 46±12 year) treated in our institute between January 2001 and December 2005 were enrolled. We analysed NPM mutation and FLT3 ITD by fluorescent PCR and TKD mutations by PCR-RFLP at the time point of diagnosis.