Evaluation Of Minimal Residual Disease (MRD) In The Acute Myeloid Leukemia(AML-M2) By Real-time Quantitative Reverse Transcription Polymerase Chain Reaction

Objective: This study is aimed to set up the standard substance for detecting the AML1/ETO fusion gene by real-time quantitative reverse transcription polymerase chain reaction(RQ-RT-PCR), monitored of minimal residual disease(MRD) in the acute myeloid leukemia(AML-M2) though this approach, and analyzed the clinical prognostic value.Method:1. Extracted the total RNA from the samples of the patients and obtained the AML1/ETO fusion gene by the qualitative RT-PCR with a pair of specific primers, confirmed the objective gene by the 2% agarose electrophoresis. 2. The amplified fragment after recovering was ligated into cloning vector PMD-18T and transformed into E.coil competent cell JM-109. 3. The plasmids of the positive colony were extracted after being incubated on the AIX plates. 4. The plasmids ligated successfully were confirmed by PCR. 5. The plasmids were sequenced to confirm the expected sequence by TAKARA in dalian and linearized with incision enzyme HindⅢ. 6. Detected the concentration of RNA and gained 1010 copies as RNA standard substance after transcribing in vitro. 7. The function of the standard substance and the samples were tested by RQ-RT-PCR, and we followed the 5 patients. 8. Measuring apoptosis-related protein expression such as Bcl-2, Bcl-xL, Bax, and the leukemia- associated immunophenotype by the flow cytometry. 9. Followed the patient with the negative of the AML1/ETO fusion gene by the leukemia-associated immunophenotype.Results: 1. The length of AML1/ETO fusion gene from the patients by RT-PCR was 402bp which was sequenced to confirm the expected gene. 2.The standard curve displayed a linear correlation of the Ct and the log of the RNA concentration of each standard dilution step, the equation was Y=-3.477×LOG(X)+41.63, the coefficient of amplification was 93.9%, the melting temperature was 83℃±1℃, had a good specificity. 3. The sensitivity of RQ-RT-PCR reached 10-5, the coefficient variation(CV) of inter-batch and intra-batch is 10% and 6% respectively. 4. The average of the relative level of AML1/ETO fusion gene in the patients at diagnosis and the patients relapsed was higher than the patients in ongoing complete remission(CR), P<0.05. 5. The relative level of AML1/ETO fusion gene of the follow-up patients was higher at diagnosis, lower in ongoing CR, then heightened when the patient relapsed. The patients who the relative level of AML1/ETO fusion gene in ongoing CR decreased 2 log more than at diagnosis had a lower risk of relapse. If the relative level of AML1/ETO fusion gene kept increasing, the patients would have a poor prognosis. 6. The Bcl-2/Bax ratio was increased in patient who didn't achieve complete remission. 7. CD13 and HLA-DR were expressed stably in the patients and as a supplementary tool for detecting MRD of the patients with the negative of the AML1/ETO fusion gene. 8. Immunophenotype shifted in No. 21 patient during induction therapy.Conclusion: The RQ-RT-PCR is a sensitive, specific, reliable and reproducible approach for monitoring AML1/ETO fusion gene. The change of the level of AML1/ETO fusion gene was correlated with the development of patient's condition. In short, that application of RQ-RT-PCR to detect AML1/ETO fusion gene for monitoring MRD in AML-M2 was helpful to evaluating leukemia burden, assessing response to treatment estimating the risk of relapse, as an important tool for confirming the time to therapy strongly and prolonging the time of complete remission. The immunological method was a supplementary for the patients with the negative of the special gene.