title: Simple, rapid and accurate molecular diagnosis of acute promyelocytic leukemia by loop mediated amplification technology.
abstract: The diagnostic work-up of acute promyelocytic leukemia (APL) includes the cytogenetic demonstration of the t(15;17) translocation and/or the PML-RARA chimeric transcript by RQ-PCR or RT-PCR. This latter assays provide suitable results in 3-6 hours. We describe here two new, rapid and specific assays that detect PML-RARA transcripts, based on the RT-QLAMP (Reverse Transcription-Quenching Loop-mediated Isothermal Amplification) technology in which RNA retrotranscription and cDNA amplification are carried out in a single tube with one enzyme at one temperature, in fluorescence and real time format. A single tube triplex assay detects bcr1 and bcr3 PML-RARA transcripts along with GUS housekeeping gene. A single tube duplex assay detects bcr2 and GUSB. In 73 APL cases, these assays detected in 16 minutes bcr1, bcr2 and bcr3 transcripts. All 81 non-APL samples were negative by RT-QLAMP for chimeric transcripts whereas GUSB was detectable. In 11 APL patients in which RT-PCR yielded equivocal breakpoint type results, RT-QLAMP assays unequivocally and accurately defined the breakpoint type (as confirmed by sequencing). Furthermore, RT-QLAMP could amplify two bcr2 transcripts with particularly extended PML exon 6 deletions not amplified by RQ-PCR. RT-QLAMP reproducible sensitivity is 10(-3) for bcr1 and bcr3 and 10(-)2 for bcr2 thus making this assay particularly attractive at diagnosis and leaving RQ-PCR for the molecular monitoring of minimal residual disease during the follow up. In conclusion, PML-RARA RT-QLAMP compared to RT-PCR or RQ-PCR is a valid improvement to perform rapid, simple and accurate molecular diagnosis of APL.
authors: Hematology and Bone Marrow Transplant Unit, Azienda Ospedaliera Papa Giovanni XXIII, Bergamo, Italy.
authors: Orietta
authors: O
authors: Spinelli
authors: Hematology and Bone Marrow Transplant Unit, Azienda Ospedaliera Papa Giovanni XXIII, Bergamo, Italy.
authors: Alessandro
authors: A
authors: Rambaldi
authors: DiaSorin SpA, Gerenzano (VA), Italy.
authors: Francesca
authors: F
authors: Rigo
authors: Hematology and Bone Marrow Transplant Unit, Azienda Ospedaliera Papa Giovanni XXIII, Bergamo, Italy.
authors: Pamela
authors: P
authors: Zanghì
authors: DiaSorin SpA, Gerenzano (VA), Italy.
authors: Elena
authors: E
authors: D'Agostini
authors: DiaSorin SpA, Gerenzano (VA), Italy.
authors: Giulia
authors: G
authors: Amicarelli
authors: DiaSorin SpA, Gerenzano (VA), Italy.
authors: Francesco
authors: F
authors: Colotta
authors: Department of Biomedicine and Prevention, University Tor Vergata, Roma, Italy.
authors: Mariadomenica
authors: M
authors: Divona
authors: Department of Biomedicine and Prevention, University Tor Vergata, Roma, Italy ; Fondazione Santa Lucia, Rome, Italy.
authors: Claudia
authors: C
authors: Ciardi
authors: Department of Biomedicine and Prevention, University Tor Vergata, Roma, Italy ; Fondazione Santa Lucia, Rome, Italy.
authors: Francesco Lo
authors: FL
authors: Coco
authors: DiaSorin SpA, Gerenzano (VA), Italy.
authors: Giulia
authors: G
authors: Minnucci
keywords: APL; LAMP; PML-RARA; molecular diagnosis