P218 | Quantification of Atypical Bcr::abl1 Transcripts by Digital Pcr: a Eutos Project
Year: 2025
Type: article
Abstract: Introduction: Molecular response (MR) assessment in chronic myeloid leukemia (CML) is based on BCR::ABL1 transcript quantification, crucial for evaluating treatment efficacy and eligibility for tyrosine kinase inhibitor (TKI) discontinuation. While quantification of common BCR::ABL1 transcripts e13a2 and e14a2 is standardized, about 2% of CML patients harbor rare variants like e13a3, e14a3 or e19a2 lacking standardized quantification methods. Real-time quantitative PCR (RT-qPCR) is the gold standard for MR evaluation, but droplet digital PCR (ddPCR) offers enhanced accuracy without needing standard curves. This study investigates ddPCR use for MR monitoring in CML patients with atypical transcripts. Methods and Results: We analyzed 29 peripheral blood samples from 7 CML patients with e13a3, e14a3 or e19a2 fusion transcripts using RT-qPCR and multiplex ddPCR. EUTOS-recommended primers and probes were used, with β-Glucuronidase (GUSB) as internal control. We confirmed specificity, linearity, and efficiency of primers and estimated a limit of detection (LoD) of 10⁻⁴ (%BCR:ABL1/GUSB: 0.001%). Samples were analyzed in duplicate, and target concentrations were expressed as the ratio of BCR::ABL1/GUSB in percent. MR was assessed by comparing follow-up (FU) and diagnostic (Dx) samples, calculating individual MR (IMR) via RT-qPCR and digital IMR (dIMR) via ddPCR (Figure) using the formula: log((BCR::ABL1)/GUSB% at Dx) - log((BCR::ABL1)/GUSB% at FU). Follow-up monitoring showed that dIMR generally corresponded with IMR, although in 8 out of 29 samples IMR exceeded 4 in RT-qPCR but not in ddPCR, suggesting that ddPCR may have greater sensitivity in quantifying low disease levels. Discussion: TKIs have transformed CML treatment, enhancing long-term survival. The focus now is on maintaining quality of life, minimizing toxicities, and achieving treatment-free remission (TFR). To qualify for TFR, patients must achieve a deep molecular response (DMR), defined as MR levels greater than MR4 on an international scale. However, MR evaluation is not performed for atypical transcripts due to lack of standardized assays and reference scales. Our results demonstrate that ddPCR is effective for monitoring CML patients with atypical transcripts, and that EUTOS-recommended primers and probes are also suitable for ddPCR, enabling the detection of low BCR::ABL1 levels. Future efforts will focus on multi-center validation and defining thresholds for safe therapy discontinuation.
Authors: A.C. Danzero, E. Gottardi, D. Cilloni, J. Petiti, R. Vacca, A. Ferrando, S. Grano, V. Bonuomo, F. Castagnetti, M.C. Miggiano, S. Galimberti, F. Muriano, G. Saglio, A. Hochhaus, T. Ernst, C. Fava
Language: English
Subfield: Hematology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W7093122187
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