Paper Details

ABSTRACT

Background: Risk stratification (R2-ISS, IMS-IMWG) in multiple myeloma (MM) increasingly depends on genomic profiling, typically by fluorescence in situ hybridization (FISH) on CD138+ bone marrow plasma cells (BMPCs). However, FISH can be incomplete in up to 48% of patients (Slade et al. ASH 2025). Whole-genome sequencing (WGS) offers comprehensive genomic characterization, but data supporting its clinical implementation remains limited. We evaluated clinical grade WGS by comparing risk classification with standard FISH and assessing its ability to identify therapeutically relevant genomic alterations. 

Methods: WGS (target coverage 50x) was performed on CD138+ BMPCs from 39 MM patients with sufficient cell counts and enriched for genomic variants on paired FISH, including 1q gain (1q+), deletion of 17p (del 17p), t(4;14), t(11;14), t(14;16), and deletion of 1p32 (del 1p32). WGS data was analyzed for standard MM risk stratification and therapeutically actionable and target-altering mutations. Risk assessment was compared between WGS and FISH per R2-ISS and IMS-IMWG.  

Results: Among 39 patients, 38 (97.4%) had adequate WGS coverage and were analyzed. 29 patients (76%) had newly diagnosed MM (NDMM) and 9 (24%) had relapsed/refractory MM. Median age at diagnosis was 60.5 years, and the cohort was 87% White, 13% Black, and 47% female. 

Mean WGS coverage was 75.2x (range: 45-105). FISH was limited by testing prior to initiation of 1p/1q probes (n=17) and insufficient cells for analysis (n=4). FISH/WGS discordance was 8.8% (n=15/171) in loci evaluated by both assays. Accounting for insufficient-cell unknowns as negative to reflect real-world FISH limitations, overall discordance was 8.3% (n=15/181). By alteration, discordance was highest in 1q+ (30%, n=6/20: 4 FISH+/WGS-, 2 FISH-/WGS+), followed by del 17p (10.5%, n=4/38: 2 FISH+/WGS-, 2 FISH-/WGS+) and t(4;14) (8.1%, n=3/37: 2 FISH+/WGS-, 1 FISH-/WGS+). Relative to FISH, WGS sensitivity was 77% - primarily limited by 1q+ (50%) and del 17p (67%) - and specificity was 96%. WGS also identified 2 patients with TP53 mutations, which cannot be detected by standard FISH. 

FISH/WGS discrepancies affected risk classification (n=35, 3 patients with missing data). Accounting for loci tested by both assays and insufficient-cell unknowns, 7 patients (20%) had discordant R2-ISS by WGS (5 down-staged, 2 up-staged). 4 patients (11.4%) had discordant IMS-IMWG by WGS (3 down-staged, 1 up-staged). Overall, 7 unique patients (20%) were re-staged by WGS with IMS-IMWG and/or R2-ISS, which was primarily driven by FISH+/WGS- calls (4 1q+, 2 del 17p, 2 t(4;14) calls with FISH). 

Among 16 patients (1 patient missing data) tested pre-1p/1q probe initiation, classifying unknown FISH loci as negative resulted in WGS up-staging 8 (50%) patients and down-staging 1 (6.3%) with R2-ISS, while WGS up-staged 5 (31.3%) patients with IMS-IMWG. Discordance was primarily driven by FISH unknown/WGS+ calls (9 1q+, 2 monoallelic del 1p32, 2 t(4;14) calls with WGS), suggesting WGS up-staged risk classification when resolving unknown loci status.  

WGS also identified actionable genomic alterations. MAPK-pathway hotspot mutations targetable by BRAF/MEK inhibition were found in 14 patients (36.8%), with BRAF V600E (n=3, 7.9%), activating NRAS mutation (G12, G13, or Q61) (n=7, 18.4%), and activating KRAS mutation (G12, G13, or Q61) (n=5, 13.2%). One patient had a targetable IDH2 R140Q mutation. 8 patients (21.1%) had MYC rearrangement and 4 (10.5%) had MYC copy number gain, targetable by BET/CDK9 inhibition. Immunotherapy targets alterations primarily occurred in NDMM (80%) and included loss of GPRC5D (n=6, 15.8%), CS1 (SLAMF7) (n=2, 5.3%), and FCRL5 (n=2, 5.3%). No TNFRSF17 (BCMA) loss occurred, but 2 patients (5.3%) had copy-number gain, which may affect CAR-T/bispecific target availability. 

Conclusion: We demonstrate the feasibility of clinical grade WGS for MM risk stratification, with relatively low discordance but differences in sensitivity relative to FISH. In patients with incomplete genomic profiling by FISH, use of WGS resulted in up-staging risk classification, suggesting WGS may better accommodate contemporary risk schema. WGS also identified actionable and target-altering mutations that cannot be assessed by FISH. These findings support clinical grade WGS as a comprehensive assay for cytogenetic risk stratification and therapeutic target profiling, though prospective outcome studies are needed.