Simple, rapid and accurate molecular diagnosis of acute promyelocytic leukemia by loop mediated amplification technology

Orietta Spinelli, Alessandro Rambaldi, Francesca Rigo, Pamela Zanghì, Elena D'Agostini, Giulia Amicarelli, Francesco Colotta, Mariadomenica Divona, Claudia Ciardi, Francesco Lo Coco, Giulia Minnucci

Research output: Contribution to journalArticle

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 RTPCR 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.

Original languageEnglish
Pages (from-to)50-58
Number of pages9
JournalOncoscience
Volume2
Issue number1
DOIs
Publication statusPublished - 2015

Keywords

  • APL
  • LAMP
  • Molecular diagnosis
  • PML-RARA

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

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    Spinelli, O., Rambaldi, A., Rigo, F., Zanghì, P., D'Agostini, E., Amicarelli, G., Colotta, F., Divona, M., Ciardi, C., Lo Coco, F., & Minnucci, G. (2015). Simple, rapid and accurate molecular diagnosis of acute promyelocytic leukemia by loop mediated amplification technology. Oncoscience, 2(1), 50-58. https://doi.org/10.18632/oncoscience.114