Comprehensive DNA/RNA-based targeted sequencing for molecular karyotyping and clonality assessment in newly diagnosed multiple myeloma patients

Abstract

Abstract Multiple myeloma (MM) is a genetically heterogeneous malignancy marked by complex clonal dynamics and a range of cytogenetic alterations that impact prognosis and treatment response. While fluorescence in situ hybridization (FISH) remains a standard diagnostic tool, it is limited to known abnormalities and requires enriched plasma cells. Next-generation sequencing (NGS) offers comprehensive profiling in a single assay but is not yet widely adopted in routine clinical care. To evaluate the performance and clinical utility of a custom-designed, dual-input (DNA/RNA) targeted sequencing panel across multiple platforms (Ion Torrent and Illumina) for molecular karyotyping, clonality assessment, and transcriptomic profiling in newly diagnosed MM. Bone marrow samples from 85 patients enrolled in GEM clinical trials with high-risk FISH-defined cytogenetics were analyzed. DNA and RNA were extracted from CD138⁺ plasma cells and CD138⁻ germline controls. Targeted capture libraries were sequenced on Illumina and Ion Torrent platforms using a panel covering 2.735 Mb and 107 genes involved in MM pathogenesis. Bioinformatic analysis included variant calling (SNVs, indels), structural variant and CNV detection, and V(D)J rearrangement analysis via MiXCR. IG/TR clonality was calculated using normalized Shannon entropy. RNA-seq data were used for differential expression analysis. The sequencing panel achieved high coverage (median depth: 602× on Illumina; 291× on Ion Torrent) and uniformity. Molecular karyotyping revealed: mutations in 145 variants affecting 59 genes, with frequent alterations in KRAS, NRAS, ZFHX4, and TP53; translocations t(4;14), t(11;14), and t(14;16) were detected with 99% accuracy compared to FISH; and CNVs including del(1p), gain/amplification(1q), and del(17p) were accurately distinguished (PPV: 95%, sensitivity: 92%). Notably, NGS identified del(1p) events undetectable by CDKN2C FISH probes in 7% of cases and discriminated 1q gain vs amplification, potentially refining risk stratification. In parallel, RNA-seq revealed distinct expression clusters, including CCND1 and FGFR3-driven subgroups, and differential expression of SLAMF7, NF1, and ABCB4 associated with survival outcomes. IG/TR clonality, especially RNA-based IG clonality, was significantly associated with both overall and disease-free survival (p < 0.05). This dual-platform, dual-input targeted sequencing strategy enables a comprehensive molecular characterization of MM from limited material. It surpasses conventional techniques in detecting structural and numerical chromosomal abnormalities and provides actionable insights into clonal architecture and gene expression with prognostic significance. These findings support the integration of DNA/RNA-based NGS panels into routine MM diagnostic workflows to guide risk-adapted treatment strategies.