Technical Notes
PML/RARa translocation t (15; 17) for Minimal Residual Disease (MRD)
Clinical Significance:
RARa/RARa is the molecular equivalent of t (15; 17) that is highly characteristic of acute promyelocytic leukemia
(AML-M3), about 95% of acute promyelocytic leukemias harbor this translocation (1).It is important to identify leukemias harboring PML/RARa translocation because they have distinct clinical features and therapeutic management options (2).
The translocation involves the PML gene on chromosome 15 and the Retinoic acid receptor alpha (RARa) gene on chromosome 17. The altered retinoic acid receptor structure renders it less sensitive to the effects of retinoic acid, resulting in uncontrolled cell proliferation and failure of promyelocytes to differentiate. The defect can be overcome by treating with high doses of retinoic acid thus underlining the importance of sensitive detection of this translocation. The mRNA for PML/RARa transcript can be distinguished by a reverse transcriptase PCR assay (3).
As the presence of PML/RARa transcript correlates with the proliferative activity of the malignant clone, quantitation of the transcript can be utilized to predict relapse and MRD (4, 5, 6). The Real Time RT-PCR based assay provides quantitative data over a wide dynamic detection range of over five orders of magnitude and has a sensitivity of detecting 1 leukemic cell with the translocation in 105 to 106 normal cells (7).
Methodology:
Minor groove binder (MGB) with Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) has been utilized to develop a quantitative, Real Time RT-PCR based assay for the ultrasensitive detection and quantitation of PML/RARa mRNA.The assay has been validated by a positive control reaction using RNA of confirmed AML-M3 patients.
References:
1) Melnick A., Licht J.D. (1999) Blood 93, 3167.2) Grignani F., Fagioli M., Alcalay M. et al (1994) Blood 83, 10.
3) Miller W.H. Jr., Kakizuka A., Frankel S.R. et al (1992) Proc. Natl. Acad. Sci. USA, 89, 2694.
4) Diverio D., Pandolfi P.P., Biondi A. et al (1993) Blood 85, 3556.
5) Perogo R.A., Marenco p., Bianchi C. et al (1996) Leukemia 10, 207.
6) Cassinat B., Zassadowski F., Balitrand N. et al (2000) Leukemia 14 (2), 324.
7) Kutyavin I.V., Afonina I.A., Mills A. et al (2000) Nuc. Acid Res. 28, 655.