Evaluation of minimal residual disease in acute promyelocytic leucemia patients through”real time” quantitative pcr. comparision with conventional rt-pcr techniques

Introduction. Acute promyelocytic leukaemia (APL) is characterised by reciprocal translocation t(15; 17) involving promielocytic leukemia (PML) gene, by a transcription factor on chromosome 15q22 and by retinoic acid receptor alpha (RAR alpha) gene on chromosome 17q21. Quantitative evaluation of minimal residual disease, kind of fusion transcripts, could be useful on clinical practice. Aims of the study To compare the results obtained with quantitative reverse-transcriptase polymerase chain reaction (QRTPCR) with those obtained whit less sensitive conventional RT-PCR standardized by BIOMED-I protocol. To verify the presence of cryptic break cluster region 2 (bcr2) positive patients in qualitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). To evaluate, through, QRTPCR, minimal residual disease of APL patients in order to characterise kinetic of quantitative decrement of transcripts Materials and methods. This research involves 11 cases, 6 male and 5 female ( Mean age 46). Mean white blood count was 26.2 x103/microliter. All patients met morphologic criteria for the diagnosis of acute myelocytic leukemia [AML]- M3-variant (APL) according to the French – American – British classification system. Diagnosis of APL was confirmed by RT-PCR analysis for PML/RAR alpha rearrangements. Cells from bone marrow or peripheral blood were collected at exordium, induction and consolidation phase. Results In all cases avoid false-negative results, a housekeeping gene (Abelson) was assayed simultaneously during first-round PML/RAR alpha amplification. Seven cases Bcr1 positive (63%) and 4 Bcr3 positive (36%) were identifies. The “real-time” PCR did not revealed bcr2 cases cryptic among bcr1 positives cases. The pre-amplification methods by QRT-PCR reveals 1 false negative (14%) inside bcr1 negative patients respect RT-PCR method. The same procedure made for bcr3 cases, did not reveal difference. Different kinetics of decrement in the fusion transcripts have been evidence from quantitative PCR. Seven cases with Bcr1 positive isoforms were negative at first cycle of consolidation. While all cases with Bcr3 positive isoforms were negative after therapeutic induction. Conclusions. The pre-amplification techniques by QRTPCR reveals false negative isoforms respect RT-PCR method. The real-time PCR with pre-amplification, is a more sensible method respect conventional RT-PCR with a minor execution time and use of small reagents quantity.