Rapid and reproducible karyotyping with long-read sequencing in patients with AML

Abstract Acute myeloid leukemia (AML) is characterized by recurrent chromosomal abnormalities that form the basis of the European LeukemiaNet risk classification and serve as essential determinants of prognosis and therapeutic decision-making. Conventional metaphase karyotyping remains the diagnostic gold standard for detecting these abnormalities; however, its utility is limited by longer turnaround times, often delaying critical clinical management. Here, we present a long-read, low-coverage whole-genome sequencing (lcWGS) approach using Oxford Nanopore Technology as a rapid and scalable alternative for cytogenetic profiling. A total of 100 diagnostic AML samples were analyzed, comprising 50 retrospectively selected patients with known adverse-risk cytogenetics and 50 prospectively enrolled patients with clinically defined de novo AML. LcWGS demonstrated robust analytical performance, identifying chromosomal aberrations with 93% sensitivity, specificity, and overall accuracy, respectively. Complex karyotypes were reliably detected, with an area under the curve of 0.971. Reproducibility was validated through replicate sequencing at 2 independent laboratories (R = 0.99). LcWGS-derived estimates of clone size showed moderate correlation with conventional cytogenetic assessments (R = 0.54). Patients with complex karyotypes identified by lcWGS exhibited significantly shorter overall and relapse-free survival, closely mirroring outcomes defined by conventional karyotyping and underscoring the value of lcWGS for risk stratification. The median turnaround time from sample receipt to bioinformatics interpretation was ∼34 hours, enabling delivery of actionable karyotype results within 72 hours. These findings establish lcWGS as a rapid, reproducible, and accurate platform for detecting clinically relevant chromosomal abnormalities, addressing a critical need for timely risk stratification and treatment initiation in AML.