Molecular analysis of the PML/RAR alpha chimeric gene in pediatric acute promyelocytic leukemia.

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.

Multiplex RT-PCR assay for the differential diagnosis of alveolar rhabdomyosarcoma and Ewing's sarcoma.

Cytogenetic analysis has defined specific translocations associated with two of the most common small round cell tumors of childhood, t(11;22) in Ewing's sarcoma and t(2;13) in alveolar rhabdomyosarcoma. We and others have previously demonstrated the diagnostic utility of a reverse transcriptase polymerase chain reaction (RT-PCR) assay for the detection of the t(11;22) encoded EWS/FLI-1 chimeric message in Ewing's sarcoma. More recently, we have cloned the t(2;13)(q35;q14) translocation and have shown that it results in the fusion of the PAX3 gene on chromosome 2 to FKHR, a novel member of the fork-head family of transcription factors on chromosome 13. To define the morphological spectrum of childhood sarcomas that express the t(2;13) encoded PAX3/FKHR chimeric message, we have performed RT-PCR analysis on samples from 44 primary pediatric sarcomas and 8 sarcoma cell lines. PAX3/FKHR chimeric messages were detected in 24 of 27 alveolar, 2 of 12 embryonal, and 0 of 1 pleomorphic rhabdomyosarcoma and in 1 of 2 ectomesenchymomas. In contrast, none of 8 Ewing's sarcomas or 2 undifferentiated sarcomas expressed this message. Chimeric transcripts were detected in all cases with cytogenetic evidence of the (2;13) translocation, and in each case the chimeric PAX3/FKHR message had the identical junction sequence, suggesting that genomic chromosome breaks were clustered in a single intron in both genes. By combining the PAX3/FKHR RT-PCR assay with primers for detection of the Ewing's sarcoma t(11;22) encoded EWS/FLI-1 chimeric transcript, we have developed a multiplex RT-PCR reaction that allows the rapid and accurate identification of either translocation in a biopsy sample.

The der(11)-encoded MLL/AF-4 fusion transcript is consistently detected in t(4;11)(q21;q23)-containing acute lymphoblastic leukemia.

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.

Detection of the (11;22)(q24;q12) translocation of Ewing's sarcoma and peripheral neuroectodermal tumor by reverse transcription polymerase chain reaction.

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.

An AML1/ETO fusion transcript is consistently detected by RNA-based polymerase chain reaction in acute myelogenous leukemia containing the (8;21)(q22;q22) translocation.

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.