Leveraging a spectrum of cytogenomics methods for profiling complex karyotypes in chronic lymphocytic leukemia.

BACKGROUND: A highly complex karyotype (high-CK) is a key biomarker of poor prognosis in chronic lymphocytic leukemia (CLL). While conventional methods lack the resolution to fully characterize complex structural variants (SVs), emerging technologies such as short-read WGS (sr-WGS), nanopore sequencing (ONT), optical genome mapping (OGM), and chromatin conformation capture (Micro-C) offer powerful tools for high-resolution SVs analysis, illuminating the genomic architecture underlying CLL. METHODS: We selected nine CLL cases bearing diverse genomic alterations. Each underwent routine diagnostic evaluation via chromosome banding analysis (CBA), multicolor fluorescence in situ hybridization (mFISH), and chromosomal microarray (CMA) and was further analyzed using sr-WGS, ONT, OGM, and Micro-C. We performed a comprehensive comparison of copy number variants (CNVs) and SVs across all methods. RESULTS: Across five methods (CMA, ONT, OGM, sr-WGS, Micro-C), 56.3% (179/318) of CNVs were consistently detected. A high-confidence CNV set, defined as those identified by ≥ 3 methods, comprised 70.4% (224/318). SV detection varied by genome complexity: 2910 unique breakends (BNDs) were identified, with only 10.9% (320/2910) supported by all methods. A high-confidence SV set, supported by ≥ 3 methods, included 20.4% (595/2910) of BNDs. Dicentric chromosomes (DICs) and complex derivative chromosomes (CDERs), particularly those involving BNDs near centromeric or telomeric regions, were the most difficult to resolve. Micro-C fully confirmed 71.4% (5/7) of CDERs and all ten DICs. Overall, Micro-C aligned best with classical cytogenetics, confirming 85.5% (47/55) of aberrations, followed by OGM (65.5%) and both ONT and sr-WGS (56.4%). CONCLUSION: Each technology offers unique insights into the leukemia genome. Combining classical cytogenetics with high-throughput methods improves the detection of structural complexity and clinically relevant alterations.