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    Myeloma Genomics and Microenvironment and immune profiling

    Category: Myeloma Genomics and Microenvironment and immune profiling

    Next-Generation Molecular Techniques as an Alternative to Conventional FISH in Newly Diagnosed Multiple Myeloma: A Pilot Study

    (PA-256) Next-Generation Molecular Techniques as an Alternative to Conventional FISH in Newly Diagnosed Multiple Myeloma: A Pilot Study

    Friday, September 19, 2025

    Introduction:      Risk stratification in newly diagnosed multiple myeloma (NDMM) has traditionally relied on conventional fluorescence in situ hybridization (FISH). However, novel next-generation sequencing (NGS) technologies offer a more precise and comprehensive molecular characterization of the disease. Incorporating these advanced approaches is critical for improving risk stratification and enabling measurable residual disease (MRD) tracking.

    Methods:   

    NDMM patients (2023–2024) were evaluated using conventional FISH and NGS to detect somatic mutations, chromosomal translocations, and copy number variations (CNVs). Genomic DNA (~20 ng) was extracted from purified CD138+ plasma cells. NGS was performed using the EuroClonality-NDC assay on the AVITI sequencing system (Element Biosciences), enabling simultaneous detection of clonal IG/TR gene rearrangements, SNPs, and chromosomal translocations. CNVs were assessed by DigitalMLPA (SALSA® digitalMLPA™ Probemix D006), which combines traditional MLPA with NGS to analyze up to 644 genomic targets simultaneously. Sequencing was performed on an Illumina MiniSeq, and data were analyzed with Coffalyser software. FISH, NGS and digitalMLPA results were compared, focusing on high-risk features: 1q21 gain/amplification, del(1p32), t(4;14), and del(17p).

    Results:   

    FISH and NGS data were available for 34 NDMM patients. FISH failed in one case, and two samples lacked sufficient material for MLPA (though mutation analysis was successful). For 1q21 gain/amplification, FISH detected 13 positives versus 17 by NGS (81% concordance, 25/31 pairs). Del(1p32) was found in 3/33 cases by FISH and 6/32 by NGS (77% concordance, 24/31). Translocation t(4;14) was identified in 4/33 by FISH and 3/33 by NGS (97% concordance, 30/31), and del(17p) in 3/33 by FISH and 4/32 by NGS (97% concordance, 30/31). NGS identified HR features in 10 patients missed by FISH, while FISH detected 4 HR features not captured by NGS. Additionally, NGS revealed TP53 mutations in 3/32 cases, further upgrading patient risk. Beyond structural abnormalities, somatic mutations were frequently identified (e.g., KRAS in 15/33, NRAS in 4/33, BRAF in 3/33). Clonotypic CDR3 sequences were identified in 33/34 patients (97%), supporting potential MRD monitoring.

    Conclusions:   

    NGS combined with DigitalMLPA offers a robust and comprehensive molecular assessment of NDMM, often outperforming conventional FISH. These techniques enable enhanced risk stratification by incorporating key somatic mutations such as TP53 and offer the additional advantage of identifying CDR3 sequences for MRD tracking. Where available, molecular approaches should be preferred over FISH for initial evaluation of NDMM.

    Supported by Ministry of Health of the Czech Republic, grant nr. NW24-03-00062.