The Feasibility Study for Using FLT3/ITD as a Marker of the Minimal Residual Disease (MRD) in Patients with Acute Myeloid Leukemia (AML) by Serial Analysis in Diagnosis, Remission and Relapse.
Authors: Kim Yeo-Kyeoung , Kim Hee-Nam , Lee Il-Kwon , Lee Su-Mi , Shim Hyeok , Park Moo-Rim , Park Kyeoung-Soo , Yang Deok-Hwan , Lee Je-Jung , Kim Hyeoung-Joon 
Published: October 2019

Abstract
FLT3/ITD (internal tandem duplication) is the most frequent genetic marker in AML and has been associated with an increased relapse. However, since many studies reported on the instability of this marker during the course of the disease, it is still a matter of debate whether the FLT3/ITD play a role for the detection of MRD in AML patients or not. If so, it is remained to determine the best rapid and accurate method to detect this marker. We analyzed the FLT3/ITD in 42 patients who had available samples at presentation and at serial follow-up using PCR with argarose gel electroporesis (EP) and with genescan. There were 14, 27 and 1 patients belonging to the favorable, intermediate and poor risk group defined with cytogenetics, retrospectively. Before starting current study, we obtained FLT3/ITD PCR results from gel EP in 324 AML patients at presentation. PCR results from gel EP were positive in 83(26.7%) in all 324 AML patients and 31(26.7%) in 116 patients with normal cytogenetics, retrospectively. It was slightly lower than in western results but was similar in Japanese data. In this study, of total 42 patients, 10 patients (23.8%) carried FLT3/ITD mutation at presentation. 36 patients who achieved complete remission (CR) showed PCR negativity in samples at the time of remission. Among these 36 patients, eight who carried FLT/ITD at diagnosis showed conversion of PCR results from positive to negative in CR samples. 7 who failed to achieve CR, they showed well-matched PCR results in both samples at presentation and at the time of confirmation of remission induction failure. The FLT3/ITD mutation frequencies at relapse showed good correlation with the frequencies at presentation by gel EP (p=0.00). Among relapsed patients, only three showed contrary results to the presentation. There was a conversion from negative at diagnosis to positive at relapse in one patient, whereas conversion from positive to negative occurred in two patients by PCR results from gel EP. The negative PCR results of these 3 patients from gel EP revealed positivity on genescan analysis using same PCR products. However, mutant percentage of these 3 samples obtained by genescan analysis were less than 10%. Of relapsed patients, the PCR products in paired samples of 14 patients performed genescan analysis to verify the results from gel EP. The median mutant percentage was significantly higher in relapse samples. They were 0.61% (0~23.2) at presentation and 44.6% (0~82.1) at relapse, retrospectively. The revealed mutant sites at relapse were generally well matched with the sites at presentation, but three samples revealed different or additional mutation sites. As we expected, PCR results from gel EP showed acceptable false negativity but high false positivity (32%) in all samples. It might be a considerable problem in detection of MRD. Genescan analysis is known to be high power to detect the mutation which is able to distinguish a few differences of base-pair within samples. Therefore, these study suggest that PCR results from gel EP should be determined their power to detect MRD and genescan analysis is found to be a rapid and highly sensitive technique.