Improved Detection of Minimal Residual Disease in AML: Validation of IDH1/2 ddPCR Assays in the Perspective of Treatment with Target Inhibitors

Mutations in <i>IDH1/2</i> are frequent in Acute Myeloid Leukemia (AML), defining a molecularly distinct subgroup with therapeutic implications due to the availability of specific inhibitors. Accurate monitoring of treatment response is crucial and Droplet Digital PCR (ddPCR) offers a sensitive approach for quantifying mutational burden in <i>IDH</i>-mutated AML. This study aimed to optimize and validate ddPCR assays specific for <i>IDH1 R</i>132 and <i>IDH2 R</i>172/<i>R</i>140 mutations for future use in Minimal Residual Disease (MRD) monitoring. Four ddPCR assays were set to evaluate the trend of <i>IDH1/2</i> mutations in 191 diagnostic and follow-up samples. Each validation procedure included determining the limit of blank (LOB) and limit of detection (LOD) using titration series. Moreover, in AML harboring both <i>IDH</i> and <i>NPM1</i> mutations, we performed generalized estimating equations (GEE) to assess the association between <i>IDH</i> fractional abundance and <i>NPM1</i> RQ-Ratio across time points. Four <i>IDH1/2</i> ddPCR assays were validated, demonstrating high sensitivity with limits of detection of 0.07% for <i>IDH1</i> R132H, 0.1% for <i>IDH</i>2 R140Q and R172K, and 0.2% for <i>IDH1</i> R132C. The method also exhibited excellent intra-run reproducibility, providing consistent results for patient follow-up. Comparison of <i>IDH</i> and <i>NPM</i>1 trends during follow-up revealed a statistically significant positive correlation, both in raw (β = 0.079, <i>p</i> = 0.001) and ranked data (β = 0.99, <i>p</i> = 0.004), suggesting a co-dynamic pattern potentially useful for surrogate monitoring. While our study cannot yet define the clinical role of <i>IDH</i> mutation assessment by ddPCR due to the lack of comparative follow-up studies, it establishes a solid methodological foundation for standardizing minimal residual disease evaluation via ddPCR, paving the way for future prospective validation.