High-sensitivity CD19⁺ cell enrichment enables rare mutation detection and MRD-focused liquid biopsy in chronic lymphocytic leukemia

Background: Accurate TP53 mutation detection is critical for clinical trial enrollment and MRD monitoring in chronic lymphocytic leukemia (CLL), as TP53 disruption drives treatment resistance and guides eligibility and stratification in most CLL trials. Patients with TP53-aberrant disease are typically excluded from chemoimmunotherapy-based protocols and redirected to targeted-agent or high-risk studies, making reliable TP53 testing essential. However, targeted therapies can markedly suppress circulating CLL cells, reducing tumor fraction and compromising assay sensitivity. In line with the 2024 ERIC TP53 testing update recommending CD19⁺ B-cell enrichment for low-lymphocyte-count samples (≤10×10⁹/L), Predicine developed and validated an immunomagnetic CD19⁺ enrichment workflow optimized to isolate rare CLL circulating tumor cells from whole blood under low-burden conditions. Methods: Whole‑blood samples were processed using an anti‑CD19 immunomagnetic capture system. Because bead binding masks CD19 epitopes, enriched cells were quantified by flow cytometry using CD20 as a surrogate marker. Performance was evaluated across key analytical metrics, including cell viability, purity, ambient‑temperature stability, intra‑ and inter‑assay precision, and cross‑site concordance. The workflow's impact on mutation detection was assessed using MEC‑1 spike‑in controls and CLL patient samples by comparing TP53 variant allele frequencies (VAFs) before and after enrichment. Refinements were implemented to minimize cell loss and erythrocyte carryover, ensuring scalability for clinical trial environments. Results: The assay consistently yielded high-quality CLL cells with a median viability of 97.7% and substantially increased the tumor-cell fraction from 10.3% in PBMCs to 79.6% post-enrichment. B-cell enrichment was successful from whole blood stored at room temperature for at least four days. Precision studies demonstrated strong intra- and inter-assay reproducibility, and results were concordant across independent testing sites. Importantly, Enrichment significantly improved the sensitivity of mutation detection. In MEC-1 spike-in samples, the median TP53 VAF increased by 3.6-fold, while the CD19⁺ cell frequency increased by over 4-fold, highlighting robust tumor cell recovery and an enhanced signal-to-background ratio for NGS. Conclusions: Predicine's validated CD19⁺ enrichment workflow enables efficient, reproducible recovery of rare CLL CTCs and fully aligns with ERIC 2024 recommendations for tumor-cell enrichment at low lymphocyte counts. This approach supports accurate TP53 mutation assessment under low-burden conditions and provides a trial-ready liquid-biopsy solution for MRD monitoring, early relapse detection, and biomarker-guided clinical trial enrollment in CLL.