Replacing FISH with a comprehensive integrated approach for tumor genotyping and immune monitoring in multiple myeloma: The flagship study

Abstract Background: Several genomic research efforts in multiple myeloma (MM) have revealed clinically relevant molecular subgroups beyond conventional cytogenetic classifications, highlighting the need for a new generation of molecular diagnostic tools to inform clinical trial design and routine care. The current standard of care is based on fluorescence in situ hybridization (FISH), which is limited by its reliance on predefined probes, inability to detect single-nucleotide variants (SNVs) or novel structural variants (SVs), and dependence on viable tumor cells. In contrast, DNA-based next-generation sequencing (NGS) enables detection of SNVs, CNVs, and SVs, while RNA sequencing (RNA-seq) captures prognostic gene expression profiles. When applied to peripheral blood mononuclear cells (PBMCs), RNA-seq also supports immune cell deconvolution, offering insight into the immune microenvironment. We hypothesized that whole-exome sequencing (WES) with immunoglobulin heavy chain (IGH) capture on bone marrow tumor cells, combined with RNA-seq of tumor cells and PBMCs, would reproduce FISH-detectable abnormalities and reveal additional genomic and immune features. To test this, the FLAGSHIP study was designed to evaluate the feasibility of this integrative approach in 150 patients with MM. Methods: Bone marrow mononuclear cells were isolated via density gradient centrifugation and cryopreserved. CD138⁺ plasma cells were enriched by magnetic sorting, assessed by flow cytometry, and subjected to DNA/RNA extraction. Germline DNA was obtained from buccal swabs. WES augmented for IGH coverage was performed using BostonGene's platform, to achieve a targeted depth of 150X for tumor samples and 100X for matched normal samples. CNVs were inferred using FACETS, SVs were identified using GRIDSS, and SNVs were called using Mutect, Strelka, and Varsan. RNA-seq was performed on both tumor cells and PBMCs. Chimeric transcripts were detected using STAR. PBMC RNA-seq was analyzed with the Kassandra algorithm for immune cell deconvolution. MiXCR was used to characterize the clonal diversity of B and T-cell receptor repertoires. Results: WES and RNA-seq were performed on bone marrow CD138+ plasma cells from 49 patients (12 newly diagnosed, 37 relapsed), and PBMC RNA-seq was performed on 37 (7 newly diagnosed, 30 relapsed). Aspirate volumes ranged from 3–8 mL, with CD138⁺ plasma cell content ranging from 0.05% to 31.6%. While all samples were adequate for sequencing, FISH could not be performed in 8 cases due to insufficient tumor cells. WES was concordant with 100% of FISH-positive cases for del(17p)/TP53 (n = 10), amp(1q)/CKS1B (n = 15), and del(1p)/CDKN2C (n = 2). Combined WES and RNA-seq of tumor cells identified structural breakpoints in 100% of FISH-positive cases for t(11;14) (n = 7), t(4;14) (n = 5), and t(14;20) (n = 1). In three t(4;14) cases, breakpoints were localized within NSD2, a gene associated with poor prognosis. For t(14;16), NGS identified 1 of 3 FISH-positive cases. Of the two missed cases, one had a breakpoint detected by WES with only 2 supporting reads, indicating insufficient coverage, and both showed 10-fold elevated MAF expression relative to FISH-negative samples. WES further identified recurrent driver mutations, including NRAS, KRAS, BRAF, and TP53, and alterations in immunotherapy targets such as GPRC5D, that are not available in standard clinical FISH testing. NGS enabled the detection of prognostic CNVs (n = 6) and IGH translocation (n = 2) in cases where FISH was either unfeasible or yielded negative results. Kassandra-based immune profiling identified 20 PBMC subtypes and showed a strong correlation with clinically measured absolute lymphocyte counts (R² = 0.7, P 0.001). T-cell repertoire analysis revealed higher clonal skew in patients with low CD4:CD8 ratios, consistent with antigen-driven CD8+ expansion. Conclusion: Integrated bone marrow WES and RNA-seq recapitulated FISH-detectable lesions and revealed additional clinically actionable insights into somatic variants, structural alterations, and gene expression. This approach enabled robust genomic profiling even in low-tumor-content samples where FISH failed. PBMC RNA-seq supported immune deconvolution and T-cell repertoire analysis, enabling the integration of tumor–immune profiling. These findings support the feasibility of an NGS solution for tumor genotyping and immune monitoring in MM, offering broader diagnostic capabilities than FISH.