ABSTRACT
Acute promyelocytic leukemia (APL) is characterized cytogenetically by the t(15;17)(q22;q11-21) translocation. To compare molecular events among pediatric and adult APL cases, we designed two sets of oligonucleotide primers using published cDNA sequence for PML/RAR alpha fusion transcripts, and undertook reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of 22 US pediatric cases of APL. PML/RAR alpha fusion transcripts were detected in all APL cases, including two cases lacking cytogenetic evidence of t(15;17). Breakpoint usage in PML was determined using a combination of PCR amplification with differing 5' primers, junction-specific probes, and sequence analysis in selected cases. Consistent with previously published data, case analysis demonstrated fusion products resulting from three breakpoint cluster regions (bcr) in PML, and a single breakpoint region in intron 2 of RAR alpha. Transcripts resulting from breakpoints in bcr1 were detected in 59 percent of cases, bcr2 in 27 percent and bcr3 in 14 percent. This distribution is dissimilar to that observed in adults, where bcr2 comprises a lesser and bcr3 a greater portion of cases. These results suggest that the pathogenesis of the t(15;17) in APL may differ among patient sets. RT-PCR with these primer sets is a reliable method for detecting PML/RAR alpha chimeric transcript in t(15; 17)-containing APL.
Subject(s)
Chimera , Chromosomes, Human, Pair 15 , Chromosomes, Human, Pair 17 , Leukemia, Promyelocytic, Acute/genetics , Neoplasm Proteins , Nuclear Proteins , Protein-Tyrosine Kinases , Proto-Oncogene Proteins , Receptors, Retinoic Acid/genetics , Transcription Factors/genetics , Translocation, Genetic , Adult , Amino Acid Sequence , Base Sequence , Child , Chromosome Mapping , DNA Primers , Exons , Genes, abl , Humans , Introns , Molecular Sequence Data , Oligonucleotide Probes , Oncogene Proteins/genetics , Polymerase Chain Reaction , Promyelocytic Leukemia Protein , Proto-Oncogene Proteins c-bcr , Receptors, Retinoic Acid/biosynthesis , Retinoic Acid Receptor alpha , Transcription Factors/biosynthesis , Transcription, Genetic , Tumor Suppressor ProteinsABSTRACT
The t(4;11)(q21;q23) is the most common translocation involving band 11q23 and is found predominantly in acute lymphoblastic leukemias (ALLs) of infants. Recent studies have shown that this translocation involves the MLL gene on chromosome 11 and the AF-4 gene on chromosome 4. Using oligonucleotide primers derived from these genes, we established reverse transcription-polymerase chain reaction (RT-PCR) assays for the detection of the fusion transcripts from both the der(11) and der(4) chromosomes. Using these assays we analyzed 23 pediatric cases of t(4;11) containing ALL. RT-PCR analysis for the der(11)-derived MLL/AF-4 fusion transcript resulted in its detection in every case at a sensitivity of greater than 1 leukemic cell in 10(5) cells. Sequence analysis of MLL/AF-4 PCR products demonstrated fusion mRNAs resulting from breaks in MLL introns 6, 7, or 8, with alternative splicing to one of three exons in the AF-4 gene. In contrast, analysis for the der(4)-derived transcript resulted in the detection of this chimeric mRNA in only 84% of the cases analyzed. These data suggest that the critical chimeric gene product involved in the establishment of the leukemic clone is derived from the der(11) chromosome. Moreover, these data demonstrate the utility of the RT-PCR assay for the der(11)-encoded message both for diagnosing t(4;11)-containing leukemia and for monitoring patients for minimal residual disease.
Subject(s)
Chromosomes, Human, Pair 11 , Chromosomes, Human, Pair 4 , DNA-Binding Proteins/genetics , Nuclear Proteins/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Proto-Oncogenes , Transcription Factors , Translocation, Genetic , Adolescent , Amino Acid Sequence , Base Sequence , Child , Child, Preschool , Female , Histone-Lysine N-Methyltransferase , Humans , Infant , Infant, Newborn , Male , Molecular Sequence Data , Myeloid-Lymphoid Leukemia Protein , RNA, Messenger/analysis , Recombinant Fusion Proteins/genetics , Transcriptional Elongation FactorsABSTRACT
Ewing's sarcoma and the related primitive neuroectodermal tumor (PNET) share a unique and specific t(11;22)(q24;q12) chromosomal translocation. The breakpoints have recently been cloned and shown to involve the EWS gene on chromosome 22 and the FLI-1 gene on chromosome 11. Translocation results in the fusion of these genes on the der(22) chromosome, resulting in the production of a novel chimeric EWS/FLI-1 message. Using oligonucleotide primers derived from EWS and FLI-1 complementary DNAs, we were able to amplify a specific fusion transcript from 18 of 18 cases containing t(11;22) and 10 of 14 cases of Ewing's sarcoma/PNET that had unsuccessful cytogenetics. No EWS/FLI-1 fusion transcripts were detected in five cell lines derived from cases of pediatric sarcomas having a histological diagnosis other than Ewing's sarcoma/PNET. The sensitivity and specificity of this PCR analysis demonstrates the usefulness of this approach for the primary diagnosis of t(11;22)-containing Ewing's sarcoma/PNET and for the detection of metastatic or residual disease.
Subject(s)
Chromosomes, Human, Pair 11 , Chromosomes, Human, Pair 22 , Neuroectodermal Tumors, Primitive, Peripheral/genetics , Sarcoma, Ewing/genetics , Translocation, Genetic/genetics , Amino Acid Sequence , Base Sequence , Child , Humans , Molecular Sequence Data , Polymerase Chain Reaction/methods , Transcription, Genetic , Tumor Cells, CulturedABSTRACT
The 8;21 translocation is one of the most common chromosomal translocations in acute myelogenous leukemia (AML), accounting for 40% of pediatric AML with French-American-British (FAB)-M2 morphology. The chromosomal breakpoints have recently been identified at the molecular level and shown to involve the AML1 gene on chromosome 21 and the ETO gene on chromosome 8. Translocation results in the consistent fusion of these genes on the der(8) chromosome, resulting in the production of a novel chimeric gene and message. Using oligonucleotide primers derived from the AML1 and ETO cDNAs, we were able to amplify a specific fusion transcript from 26 of 26 patients with t(8;21) by a reverse transcriptase polymerase chain reaction (PCR) approach. DNA fragments of identical size were generated from each case including two with complex translocations. Studies on the sensitivity and specificity of this approach show that PCR analysis can be used as a rapid, accurate, and sensitive means for detecting this chromosomal abnormality, and for following the patients' response to therapy.
Subject(s)
Chromosome Aberrations/diagnosis , DNA-Binding Proteins , Leukemia, Myeloid, Acute/genetics , Proto-Oncogene Proteins , Transcription Factors , Translocation, Genetic , Adolescent , Adult , Base Sequence , Child , Child, Preschool , Chromosome Disorders , Chromosomes, Human, Pair 21 , Chromosomes, Human, Pair 8 , Core Binding Factor Alpha 2 Subunit , Female , Gene Expression , Humans , Infant , Male , Molecular Sequence Data , Neoplasm Proteins/genetics , Oligodeoxyribonucleotides/chemistry , Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Neoplasm/genetics , Recombinant Proteins/geneticsABSTRACT
The authors reviewed 250 consecutive children with ALL to determine if the periodic acid-Schiff (PAS) score was a useful, independent predictor of time to failure. PAS stains were scored from 0 to 400 and divided into low- and high-score groups using a variance-ratio test (F test) to optimize any effect of PAS on prognosis. Although the effect of PAS score considered alone approached significance for time to failure, the PAS score lost all significance when the patients were divided into standard-risk and high-risk groups on the basis of peripheral white count, central nervous system involvement, mediastinal mass, or E-rosette positivity at diagnosis. A Cox regression analysis was performed on a subgroup of 198 patients for whom cytogenetic studies were also available. The PAS score again approached the level of significance when considered alone but was of no significance after the effects of peripheral white count, pseudodiploidy, mediastinal mass, and E-rosette positivity were removed. The authors conclude that the PAS stain has no independent prognostic significance in childhood ALL.