A rapid RT-PCR screening assay incorporating multiplexed validated control genes for CBF rearrangements at diagnosis in AML.
OBJECTIVE
Our objective was to establish a multiplexed assay using the Biomed 1 primers to detect AML1-ETO transcripts and 10 different CBFB-MYH11 transcripts, using BCR and ABL transcripts as controls.


METHODS
Control genes were systematically tested for characteristics of optimal controls. The final assay was validated on 50 AML patient samples.


RESULTS
Testing confirmed that the designated control gene criteria were fulfilled. Of 50 patient samples tested, four RT-PCR results were discordant with the cytogenetic result. In three cytogenetically negative cases, RT-PCR detected cryptic CBF rearrangements (one AML1-ETO and two CBFB-MYH11). The fourth case was inv(16) positive but negative by RT-PCR; however, the control gene result revealed suboptimal RNA quality.


CONCLUSIONS
We have described a robust multiplex RT-PCR assay that incorporates experimentally validated control genes that are important for accurate interpretation. The assay is more sensitive than cytogenetics in the detection of CBF AML. Application to large patient cohorts will determine the prognostic significance of cryptic CBF rearrangements compared with their cytogenetic counterparts.
Saal
Russell J
RJ
Queensland Health Pathology and Scientific Services, Department of Haematology, Princess Alexandra Hospital, Brisbane, Queensland 4101, Australia. russell_saal@health.qld.gov.au
Marlton
Paula V
PV
Timson
Georgina
G
Gill
Devinder S
DS
Waugh
Marcus
M
Springall
Francisca
F
Iland
Harry
H
D000208:Acute Disease; D050676:Core Binding Factor Alpha 2 Subunit; D050660:Core Binding Factors; D020732:Cytogenetic Analysis; D015321:Gene Rearrangement; D006801:Humans; D007951:Leukemia, Myeloid / Q000175:diagnosis* / Q000235:genetics*; D008403:Mass Screening / Q000379:methods*; D009363:Neoplasm Proteins / Q000235:genetics*; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000302:isolation & purification; D000075142:RUNX1 Translocation Partner 1 Protein; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000235:genetics* / Q000302:isolation & purification
C107844:AML1-ETO fusion protein, human; C095514:CBFbeta-MYH11 fusion protein; D050676:Core Binding Factor Alpha 2 Subunit; D050660:Core Binding Factors; D009363:Neoplasm Proteins; D015514:Oncogene Proteins, Fusion; D000075142:RUNX1 Translocation Partner 1 Protein; D014157:Transcription Factors