title: Improved rapid detection of the PML/RARalpha fusion gene in acute promyelocytic leukemia.
abstract: Acute promyelocytic leukemia (APL) is a medical emergency which requires rapid diagnosis and tailored treatment. Detection of the PML/RARalpha fusion gene in APL blasts is critical to start promptly the specific therapy with all-trans retinoic acid (ATRA). APL lacking this genetic lesion have been reported as being ATRA resistant. Reverse-transcription polymerase chain reaction (RT-PCR) has been extensively used to detect the PML/RARalpha cDNA. The reported PML/RARalpha amplification techniques are laborious and time consuming, and include conventional RNA extraction, cDNA synthesis and a two-round (nested) PCR. We hereby describe a few variations of the commonly adopted RNA extraction and PML/RARalpha RT-PCR protocols which allow a molecular diagnosis of APL to be carried out in less than 5 h. Processing of small volumes of leukemic cell lysate (0.5 ml) in a microfuge allows extraction of good quality RNA in 1 h. After reverse transcription to obtain cDNA, a 'hot start' PCR procedure was adopted which enabled us to amplify clearly visible and specific products after a single (not nested) amplification round. The PML/RARalpha fusion gene was detected in the blasts of six consecutive APL at diagnosis, and an APL-tailored protocol including ATRA was started in each case within 6 h of admission. On repeated experiments, the assay proved highly specific and sensitive for the rapid detection of all PML/RARalpha transcript types. Our data should encourage the use of this rapid procedure for the diagnosis of both typical APL and, particularly, less typical cases awaiting urgent therapeutic intervention.
authors.affiliation: Hematology, Department of Human Biopathology, University La Sapienza, Rome, Italy.
authors.forename: D
authors.initials: D
authors.lastname: Diverio
authors.forename: R
authors.initials: R
authors.lastname: Riccioni
authors.forename: A
authors.initials: A
authors.lastname: Pistilli
authors.forename: S
authors.initials: S
authors.lastname: Buffolino
authors.forename: G
authors.initials: G
authors.lastname: Avvisati
authors.forename: F
authors.initials: F
authors.lastname: Mandelli
authors.forename: F
authors.initials: F
authors.lastname: Lo Coco
mesh_terms: D001483:Base Sequence; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000235:genetics*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics*; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D014158:Transcription, Genetic
chemical_list: D009363:Neoplasm Proteins; D015514:Oncogene Proteins, Fusion; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein