ASH 2025 | Diagnostic evaluation methods and prognostic impact of FLT3-ITD microclones in AML

As we know, capillary electrophoresis is the preferred detection method for FLT3-ITD mutation, but this technique requires a substantial expertise, especially to identify what we call FLT3-ITD microclones, namely those with an allelic ratio below 0.05. ELN recommendations do not state a clear-cut threshold of positivity, but in a large FLT3 inhibitors trial, this was set conventionally at 0.05 in the RATIFY, 0.03 in the QuANTUM-First. So together with the European Hematology Association AML specialized working group, we wanted to understand what’s the fate of these individuals with the FLT3-ITD microclones. 

So to gain more insight into this, we did a two-step study. In the first methodological part, we sent blindly almost 70 patient samples in seven European laboratories to test the interlaboratory reproducibility of results. And we have seen that actually, while for ITD length determination we had a very good intraclass correlation coefficient, for allelic ratio determination this was less satisfactory. So pointing that indeed capillary electrophoresis is a technique that requires a good amount of expertise, especially to identify these microclones. Then we also look at the reproducibility of the results in terms of AR determination with NGS comparison. And we have seen that indeed we found a 94% overlap with three out of 48 samples that were not detected. And indeed, who were these patients? Were those with an allelic ratio below 0.02 and those with a larger ITD that were detected by capillary electrophoresis and not by NGS. 

So besides this methodological issue, we then went on analyzing the clinical fate of these patients that are so far neglected by clinical trials, at least the ones that were registrative for the FLT3 inhibitors. Therefore, we gathered the information for 497 patients that were treated from 2017 to 2022 in all Europe and were FLT3-ITD positive and treated with intensive chemotherapy. And we have seen that there was no difference in terms of like outcome in between microclones and the patients with fully blown disease, and while NPM1 seemed to have a positive prognostic impact overall, this impact was lost in the microclone setting. Aand also other variables did not have any impact, including a NPM1 mutation, DNMT3A. And in the multivariable analysis, what we found was that increased WBC, NPM1, and age did have a prognostic impact, an independent prognostic impact, on overall survival. Instead, when looking at relapse feature, we found that there was no difference with regards to AR: 27% in both groups relapse at a median of eight months from disease onset. And what was interesting is that when we dissected the genomic architecture of relapse of these patients, we found that 91% recapitulated the same FLT3-ITD microclones that was present at onset. Some acquiring more clones, other losing clones, but there was the same inciting event that was present at the very beginning of their disease. And only 9% of cases had a completely new clone. So probably offering a sweet spot for FLT3 inhibition in these cases that needs to be evaluated in clinical trials.

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