Optical Genome Mapping for Cytogenetic Analysis in Multiple Myeloma: Real‐World Evidence

Multiple myeloma (MM) is a genetically heterogeneous plasma cell malignancy in which cytogenetic abnormalities are central to risk stratification and treatment decisions. 17p loss, 1p loss, 1q gain/amplification, and IGH rearrangements define high-risk disease [1, 2]. Conventional cytogenetic testing including fluorescence in situ hybridization (FISH) and karyotyping is limited: karyotyping needs dividing cells (only ~20%–30% diagnostic yield) and FISH tests only predefined targets/abnormalities, making it slow and costly [1-5]. Optical genome mapping (OGM) is a high-resolution technology that detects genome-wide structural variants (SVs), copy number variations (CNVs), and complex rearrangements in MM [5-8]. It has achieved high concordance with FISH and identified additional clinically significant lesions, increasing prognostic yield by ~30% [6]. OGM offers a comprehensive alternative to conventional cytogenetic testing [5-11]. However, before OGM transitions from a research tool to a primary clinical methodology, some practical questions must be answered. Therefore, the objectives of this study were to (1) determine whether OGM can replace conventional FISH and karyotyping for routine, clinical MM risk stratification and (2) delineate the practical requirements for the successful implementation of OGM testing, specifically addressing the minimum plasma cell percentage (%PC) required in a sample to ensure detection of all critical prognostic aberrations. Answering these questions is essential for realizing the full potential of OGM to improve diagnostic precision and ultimately, patient management. In a retrospective, multicenter study of 211 patients with MM from Johns Hopkins Hospital (JHH, n = 162) and MD Anderson Cancer Center (MDACC, n = 49), we compared OGM against standard-of-care methods (FISH, karyotyping, and next-generation sequencing [NGS] where available) to assess concordance for detecting clinically relevant abnormalities (e.g., del(17p), 1q gain/amp, 1p loss, MYC rearrangements, IGH translocations), quantify OGM's additional yield of significant SVs and complex genomic architectures, and determine the minimum plasma cell threshold for optimal sensitivity using receiver operating characteristic (ROC) curves based on tumor burden correlations from multiparameter flow cytometry (MFC), immunohistochemistry (IHC), and differential counts (Supporting Information Materials and Methods). Among the 211 participants (mean age 66.4 years, 51% male), FISH was performed in 209 (99%), karyotyping in 155 (74%), and OGM in 100 (47%). Cytogenetic abnormalities were detected in 55% (115/209), 37% (58/155), and 93% (93/100) of cases, respectively; OGM identified pathogenic abnormalities in 82% (14/17) of failed karyotyped cases (n = 17/56) and 92% (36/39) of normal karyotyped cases (n = 39/97; Figure 1a). ROC analysis of CD138-enriched JHH samples identified tumor burden thresholds (≥ 0.7% by MFC; ≥ 6.25% by IHC) for successful OGM detection (Figure 1b). Non-enriched MDACC samples, all above this threshold, consistently showed chromosomal abnormalities by OGM, confirming that high tumor burden enhances detection and that validating these cutoffs is clinically conservative. The diagnostic performance of OGM was evaluated against the FISH assay across eight loci in 100 samples. Taking FISH as the reference standard, OGM demonstrated high overall concordance (Figure 1c). The analysis revealed concordant results for all four IGH translocations (IGH::FGFR3, IGH::CCND1, IGH::MAF, IGH::MAFB) with sensitivity and specificity all at 100%. The assay effectively identified 1q gain (91.5% sensitivity, 100% specificity) and 1p loss (100% sensitivity, 86.5% specificity). The specificity for 1p loss was lower because OGM detected a wide distribution of breakpoints across the 1p arm that were undetectable by FISH (Figure 1d). Among the 12 patients analyzed, seven had 1p deletion breakpoints entirely outside the FISH probe coverage region, and four of the five remaining patients had breakpoints that only partially overlapped the FISH probe. One patient exhibited low-level 1p monosomy by OGM, below the FISH detection threshold. Consequently, FISH failed to detect 52.2% of 1p deletions. FISH, which uses locus-specific probes, lacks sensitivity for detecting 1p deletions in MM. Rather, variable breakpoints are better identified by OGM. OGM also showed strong performance for MYC rearrangements (93.3% sensitivity, 89.4% specificity) and TP53 loss (83.3% sensitivity, 96.6% specificity), supporting its utility in profiling clinically relevant loci. Seven loci tested positive by FISH but negative by OGM, including four cases of 1q gain and one case each of TP53 deletion, monosomy 17, and an atypical MYC signal pattern (Table S1). Six of these did not undergo CD138-enrichment before OGM analysis. In a CD138-enriched sample, the discordant case of low-level monosomy 17 by FISH showed a near-triploid clone with two normal chromosomes 17 by karyotyping, but OGM and SNP array revealed masked hyperdiploidy with two chromosomes 17, indicating a low-level subclonal event undetectable by DNA-based methods (Figure S1). For the other six cases, the variant allele frequency (VAF) for these alterations was all < 20%, suggesting their potential subclonal nature. Additionally, according to the latest International Myeloma Working Group (IMWG) criteria [2], only TP53 loss with a VAF > 25% is classified as a high-risk event. In our study, the two patients with OGM-negative results for TP53 loss had VAFs (6%–12.5%) well below the high-risk threshold. OGM revealed near-triploidy without MYC fusions in a patient with atypical FISH showing 5′ MYC gain. Beyond recapitulating canonical cytogenetic abnormalities routinely captured by FISH, OGM revealed a wide range of potentially clinically relevant genomic alterations. These included atypical MYC and IGH rearrangements and abnormalities not detectable by standard FISH probes, such as two involving CD38/4p, eight with GPRC5D/12p loss, 24 cases of 13q loss, two with TNFRSF17/16p gain (gene for BCMA), 40 cases of hyperdiploidy, 15 cases of hypodiploidy, 72 other CNVs, and 27 instances of chromoanagenesis. These findings highlight the broader genomic coverage of OGM and its ability to detect cryptic or complex SVs that may escape conventional cytogenetic methods. We identified MYC rearrangements in 25.8% (24/93) of abnormal OGM cases, including nine with immunoglobulin partners (4 with MYC::IGH, 4 with MYC::IGL, and 1 with MYC::IGKC), 10 with non-immunoglobulin partners (e.g., FOXO3, CRTC3, TXNDC5, PECAM1, TENT5C, NBEA), and five with intra-8q complex rearrangements. Complex and insertional MYC rearrangements occurred in 16.7% and 20.8% of these cases, respectively (Figure S2). Breakpoints for MYC rearrangements with immunoglobulin partners were clustered between 127.32 and128.18 Mb (mean ~ 127.78 Mb), those with non-immunoglobulin partners spanned 127.77–128.39 Mb (mean ~ 128.00 Mb), and intrachromosomal rearrangements showed a range of 127.74–128.60 Mb (mean ~ 128.01 Mb). This consistent clustering suggests a region of genomic fragility or regulatory importance in the centromeric region of the canonical MYC super-enhancer sites previously reported [13]. FISH missed 10 cases (41.7%), highlighting OGM's superior sensitivity for complex MYC SVs. We identified IGH rearrangements in 54.8% (51/93) of abnormal OGM cases, primarily with CCND1 (28 cases). Twelve FISH-identified cases involving the FGFR3/NSD2 locus were confirmed as IGH::NSD2 fusions by OGM with diverse breakpoints potentially associated with varying risk profiles (Figure 1e). The remaining cases comprised four IGH::MAF and two IGH::MAFB translocations, plus four atypical IGH rearrangements (IGH::HDAC9, IGH::MYCN, IGH::WWOX, and 13q33 intergenic). Thirteen cases also showed concurrent MYC rearrangements, indicating a potential double-hit profile. Among the 27 (29%) patients with OGM-identified chromoanagenesis, chromosome 12 was most frequently affected, followed by chromosomes 1, 2, 11, and 17; 15 cases involved a single chromosome and 12 involved 2 to 5 chromosomes. Analysis of 33 patients with chromoanagenesis (including prior cohorts [9]) showed hyperdiploidy and IGH rearrangements as equally common, accompanied by 1q gain (n = 15), 1p loss (n = 9), MYC rearrangements (n = 9), and hypodiploidy (n = 3; Figure 1f). Although the average number of SVs per patient was 119 (11 translocations, 42 deletions, 44 insertions, 17 inversions, 5 duplications), only 31 SVs involved cancer-related genes (2 translocations, 11 deletions, 13 insertions, 3 inversions, 2 duplications), suggesting that chromoanagenesis involves numerous novel genes and genomic regions whose clinical importance is yet to be explored and may not uniformly equate to adverse prognosis. To enhance and standardize diagnostic procedures for MM, we propose the following workflow based on %PC (Figure 2): use conventional methods for %PC < 0.70% (MFC) or < 6.25% (IHC) owing to OGM's limited feasibility in low-tumor samples, and prioritize OGM with NGS for %PC ≥ 0.70% (MFC) or ≥ 6.25% (IHC) to efficiently detect SVs and mutations, consistent with a 10% IHC threshold. Our non-CD138–enriched data indicate that OGM is effective without enrichment when %PC exceeds 40% (MFC) or 60% (IHC), reflecting average results, while false negatives (e.g., undetected 1q gains, TP53 losses with VAF < 20% in unenriched samples) support using a conservative approach that favors CD138-enrichment in MM. The latter approach elevates tumor DNA and effective VAF above OGM's detection threshold, minimizing missed subclonal alterations (VAF < 10%–20%). A 40% (MFC) or 60% (IHC) PC threshold, or enrichment, provides a safer margin than a 21% threshold [14], accounting for variable sample quality. OGM excels at detecting complex MM rearrangements, precisely mapping 1p breakpoints beyond FISH probe regions, uncovering atypical MYC and IGH translocation partners, and differentiating IGH::NSD2 (high-risk) from IGH::FGFR3 (less aggressive) translocations [12, 15]. Additionally, OGM reveals high frequencies of chromoanagenesis in 29% of patients with MM. This proportion far exceeds the < 5% detected by traditional methods and highlights extensive genomic instability. Integrating OGM delivers a comprehensive cancer genome profile, refines MM diagnostic workflows, and provides valuable insights into disease mechanisms and therapeutic targets. In summary, OGM matches conventional FISH and karyotyping while detecting ~30% more clinically significant genetic abnormalities, addressing critical WHO/ICC/IMWG genomic profiling needs. We show that %PC is a key factor in test selection, offer cautious guidance on enrichment thresholds, and emphasize the need for nuanced interpretation of complex events like 1p loss, MYC rearrangements, and chromoanagenesis. Together, these findings can contribute to more standardized and clinically useful genomic testing guidelines for MM. Conceptualization: S.L., G.T., Y.S.Z., and C.B.G. Methodology and analysis: S.L. and Y.S.Z. Acquisition, analysis, and interpretation of data: S.L., G.T., M.K., J.G., V.S., P.L., L.M., R.R.X., and Y.S.Z. Technical and materials support: M.K., J.G., V.S., P.L., and L.M. All authors contributed to the review, revision, and approval of the final paper. We thank the clinical cytogenetics and molecular pathology teams for their technical assistance and the patients who contributed samples to this study. This research received no external funding. The Johns Hopkins Cytogenomics Laboratory is an academic laboratory supported by the Johns Hopkins School of Medicine Department of Pathology. This study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of the Johns Hopkins School of Medicine (IRB00517357; date of approval: August 5, 2025). As this is a retrospective study, informed consent was waived. The authors declare no conflicts of interest. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Data S1: ajh70175-sup-0001-Supinfo.docx. Figure S1. Multiple myeloma with masked hyperdiploidy. (a) Karyogram showing a hyperdiploid clone with 63 chromosomes, described as 63,XY, +X, +3 + 3,+5,+5,+7,+7, +9, +9, +11, +11, +15, +15, +19, +19, +21, +21. (b) Whole-genome OGM plot revealing a masked hyperdiploidy with copy-neutral loss of heterozygosity (CN-LOH) involving chromosomes 1, 2, 4, 6, 8, 10, 12, 13, 14, 16, 17, 18, 20, and 22 (indicated by yellow bars) and tetrasomy with retained heterozygosity for chromosomes 3, 5, 7, 9, 11, 15, 19, and 21 (indicated by blue bars). (c). SNP array plot confirming CN-LOH for chromosome 17 with no loss of chromosome 17. (d) SNP array plot showing tetrasomy of chromosome 3 with retained heterozygosity. Figure S2. Various MYC rearrangements by OGM. (a) IGH::MYC gene fusion (case 59). (b) 8q deletion resulting in MYC::NSMCE2 fusion (case 25). (c) Insertional MYC rearrangement: insertion of 6p into 8q resulting in MYC::TXNDC5 fusion (case 31). (d) Complex intrachromosomal 8q rearrangements involving MYC gene (case 114). Table S1: Genetic Loci Positive by FISH but Negative by OGM. 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