Droplet Digital PCR With Higher Sensitivity To CML Than RT-qPCR Of Detecting BCR/ABL(P210) Minimal Residual Disease And Monitoring Progression

Objective:Chronic myeloid leukemia?CML?has achieved unprecedented improvement through the standard treatment of tyrosine kinases inhibition?TKI?.Regular monitoring of the expression of BCR/ABL?P210?in CML patients is the precondition for ensuring clinical efficacy.At present,the Real-time fluorescent quantitative PCR?RT-qPCR?widely used has the limitation of detection sensitivity and reference gene.The droplet digital PCR?dd-PCR?is a new method of higher detection sensitivity,which can achieve the absolute quantification of target gene.The study intended to establish a dd-PCR for monitoring minimal residual disease?MRD?in patients with BCR/ABL?P210?-positive CML,thereby achieving deep-level monitoring of tumor load and determining the efficacy for guided clinically individualized treatment.Methods:1.Using dd-PCR and RT-qPCR,two cell suspensions were obtained from K562 cell and Peripheral blood mononuclear cell?PBMC?by gradient dilution and were measured at the cellular level.Detection the limit of the two methods and the correlation between the two results.2.At peripheral blood?PB?level,61 cases with CML-chronic phase?CML-CP?were obtained after TKIs treatment and regular follow-ups.By RT-qPCR,BCR/ABL?P210?fusion gene was undetectable in PB after three successive analyses,which were performed once every three months.At the same time,dd-PCR was performed simultaneously with the last equal amount of cDNA.Ten CML patients with MR4.5 were followed up by the two methods.Results:1.From detection and analysis at the cellular level,results showed that minimum detectable concentration for RT-qPCR was 10^-5,whereas that for dd-PCR reached 10^-6.consistency of results of dd-PCR and RT-qPCR reached R²?0.99,with conversion equation of Y=33.148X^1.222?Y:dd-PCR results;X:RT-qPCR results?.2.In the dd-PCR test,11 of the 61 CML patients?18.03%?tested positive and showed statistically significant difference?P<0.05?.In the follow-up of 10 CML patients who were in MR4.5,10 patients loss of MR4.0,and 4 patients were tested positive by dd-PCR 3months earlier than by RT-qPCR.Conclusion:1.In contrast with RT-qPCR,dd-PCR is more sensitive.2.The conversion equation enabling accurate conversion of dd-PCR results into internationally standard RT-qPCR results.3.The higher sensitivity dd-PCR is used to achieve a deeper molecular biology-based stratification of BCR/ABL?P210?,predicting disease progression earlier than RT-qPCR,and guiding clinical medication.