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: D Diverio, R Riccioni, A Pistilli, S Buffolino, G Avvisati, F Mandelli, F Lo Coco
• Journal: Leukemia (Leukemia) Vol. 10 Issue 7 Pg. 1214-6 (Jul 1996) ISSN: 0887-6924 [Print] England
• PMID: 8684004 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Neoplasm Proteins
• Oncogene Proteins, Fusion
• promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein

Topics

• Base Sequence
• Humans
• Leukemia, Promyelocytic, Acute (genetics)
• Molecular Sequence Data
• Neoplasm Proteins (genetics)
• Oncogene Proteins, Fusion (genetics)
• Polymerase Chain Reaction (methods)
• Transcription, Genetic