Clinical Validation of Optical Genome Mapping in Multiple Myeloma Without Plasma Cell Enrichment

Cytogenetic alterations are important in risk stratification for multiple myeloma (MM). Translocations involving the immunoglobulin heavy chain (IGH), such as t(4;14), t(14;16), as well as del(17p) and gain(1q), are recognized as high-risk cytogenetic markers in staging systems [1]. Fluorescence in situ hybridization (FISH) is the primary method for detecting these genetic alterations. However, cytogenetic testing in MM is challenging owing to the lower proportion of plasma cells in bone marrow (BM) aspirates, which may arise from sample variability or suboptimal sample quality. To address these challenges, clinical laboratories employ CD138+ plasma cell enrichment procedures, such as cytoplasmic immunoglobulin FISH or cell sorting using either flow cytometry or magnetic beads. Although these techniques can increase the analytical sensitivity of FISH, they also come with drawbacks, such as the need for additional steps, associated costs, the time required, and the need for larger volumes of BM samples.

Optical genome mapping (OGM) is an emerging cytogenetic technology that offers advantages in detecting genome-wide structural variations and copy number variants with high sensitivities in hematologic malignancies [2]. In MM, OGM has revealed promising results compared with the conventional cytogenetic methods, such as karyotyping and FISH [3, 4]. Moreover, with its ability to perform high-resolution, genome-wide analysis, OGM facilitates the classification and detection of genetic alterations not identified by conventional methods, including those involving the MYC gene [3, 5]. Compared with whole-genome sequencing, OGM may be more cost-effective while achieving higher coverage, directly improving the detection of variants with low variant allele frequencies (VAFs). With the 300× coverage, OGM is reported to be capable of detecting VAF ≥ 5%.

In this study, we evaluated the clinical application of OGM for detecting cytogenetic alterations, which are routinely performed using FISH. Based on promising results from a pilot study using OGM on BM aspirate samples with a plasma cell percentage > 50% without CD138+ plasma cell enrichment [3], we aimed to evaluate the performance of OGM in samples with varying plasma cell percentages without CD138+ plasma cell enrichment. We also aimed to identify optimal plasma cell percentages to enable routine application of OGM in clinical settings.

This study included 25 BM aspirate samples obtained from patients with newly diagnosed MM between January 2023 and June 2024 at the Guro Hospital, Korea University (Table S1). All patients had ≥ 10% plasma cells in BM aspirates. Samples with concurrent results of plasma cell percentages obtained using flow cytometry and FISH results were included. This study was approved by the Institutional Review Board of Korea University (2024GR0240), Guro Hospital, and conducted in accordance with the Declaration of Helsinki.

OGM was performed using a previously reported procedure [5]. Briefly, ultra-high molecular weight DNA from BM aspirates was labeled using Standard Direct Label Enzyme 1 reaction and loaded onto a Saphyr chip (Bionano Genomics, San Diego, CA, USA). Images obtained using a Saphyr instrument were analyzed using Bionano Solve/Access software and the Rare Variant analysis pipeline (Bionano Genomics, San Diego, CA, USA). All procedures were performed according to the manufacturer's protocols, achieving approximately 300× effective reference coverage. Detailed methods are provided in the Data S1.

Interphase FISH analysis was performed using CD138+ plasma cells isolated using magnetic-assisted cell sorting (Miltenyi Biotech, Bisley, UK). Probes used were as follows: LSI IGH/FGFR3, IGH/MAF, IGH/CCND1 dual-color probe, LSI TP53 (17p13.1)/CEP 17 dual-color probe, LSI 13 (D13S319) 13q14.3 single-color probe (MetaSystems, MA, USA), and LSI 1q21/1p32 dual-color probe (Cytocell Inc., Cambridge, UK). A minimum of 200 cells were counted for each probe. The cutoff values were 1.0% for translocations, 2.5% for amplification, 3.8% for deletions, and 2.5% for break-apart signals. To directly compare OGM and FISH data, we estimated the allelic frequency (AF) (termed herein “estimated AF”) of cytogenetic alterations using the plasma cell percentage obtained from the microscopic examination of BM aspirate. The estimated AF was calculated by multiplying the FISH percentage by the plasma cell percentage and then dividing the result by 10 000. As OGM detects variants with a VAF ≥ 5%, cytogenetic alterations identified using FISH with an estimated AF < 5%, but not by OGM, were not considered discordant.

We also explored using plasma cell percentages from flow cytometry to identify optimal thresholds for routine OGM application in clinical settings. We calculated the estimated AF using flow cytometry and microscopic examination-derived plasma cell percentages. However, as expected, the flow cytometry consistently yielded lower plasma cell percentages than the microscopic examination [6] (Figure S1). Given this discrepancy, and the well-established role of morphology in clinical assessment, we primarily used the estimated AF based on microscopic plasma cell percentages for our analysis.

Among the 25 cases with BM plasma cell percentage data enumerated using microscopic examination and flow cytometry, 38 aberrations were detected using FISH, including 13 canonical translocations involving IGH and 25 copy number variations (CNVs) (Figure 1A). Among the aberrations, 10.5% (n = 4) had an estimated AF below 5%. When considering aberrations with an estimated AF of ≥ 5% (34 aberrations), OGM exhibited a concordance of 98.2% (n = 168/171) across the 171 loci tested using FISH. For the loci tested using FISH that were included in the revised international staging system (R-ISS) as high-risk associated chromosomal abnormalities (t(4;14), t(14;16), and del(17p)), the concordance between FISH and OGM was 100% (n = 74/74). For the loci included in the second revision of ISS (R2-ISS), t(4;14), gain(1q), and del(17p), the concordance was 98.6% (n = 72/73).

FIGURE 1

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(A) Concordance of cytogenetic abnormalities between FISH and OGM. In the Plasma cell percentage section, the value in bold represents the lowest value of plasma cell percentage at which all cytogenetic alterations could be detected. (B) Detection of CNVs and translocations by OGM based on allelic fraction estimated using BM plasma cell percentages, derived from flow cytometry (top) and morphology (bottom). The black horizontal line represents the estimated AF threshold of 5%.

Compared with the FISH results, OGM revealed 91.2% sensitivity (n = 31/34) and 100% specificity (n = 137/137) for cytogenetic alterations with estimated AF ≥ 5%. Of the 11 translocations detected using FISH (five cases with t(4;14) and six cases with t(11;14)), OGM identified all except for one case with t(11;14). This case showed a 72.3% abnormality by FISH and had a relatively low estimated AF of 7.6%, due to low levels of plasma cells in the sample (BM plasma cell 10.5%). Among the 23 CNVs (10 cases with 1q gain, two cases with del(1p), 10 cases with del(13q), and one case with del(17p)), OGM could not detect CNV alterations in one case with both 1q gain and del(1p). In contrast to previous findings, additional analysis using the de novo assembly pipeline could not retrieve these undetected alterations [4].

To explore the application of OGM in MM without plasma cell enrichment, we evaluated the optimal estimated AF and BM plasma cell percentage by comparing the results to FISH. Our analysis revealed that OGM detected all translocations with an estimated AF ≥ 10.0% (flow cytometry AF ≥ 1.4%). For CNVs, OGM detected all alterations with an estimated AF ≥ 13.8% (flow cytometry AF ≥ 2.2%) (Figure 1B). These results indicate that OGM could detect translocations at a lower AF threshold compared with CNVs, suggesting higher sensitivity for translocations. Although the estimated AF was the primary factor in detecting sensitivity, plasma cell percentage also influenced the results. OGM detected all alterations with an estimated AF ≥ 5% when the morphological plasma cell percentage was ≥ 21.0% (flow plasma cell percentage ≥ 3.6%). Conversely, alterations with relatively high estimated AF were not detected in a sample with a plasma cell percentage of 20.6% (flow plasma cell percentage of 3.3%), suggesting improved sensitivity at higher percentages. We did not evaluate the full range of plasma cell percentages, and the values represent the lowest percentages of plasma cells in a sample where all alterations were detected. Both AF and plasma cell percentages should be considered for optimal detection.

Our study reports the following significant findings: (1) OGM demonstrated the clinical potential for detecting cytogenetic alterations in MM without plasma cell enrichment, particularly for variants with a VAF ≥ 5%, achieving an overall concordance with FISH of 98.2%; (2) OGM could detect translocations at a lower estimated AF threshold compared with CNVs (an estimated AF ≥ 10.0% vs. an estimated AF ≥ 13.8%); (3) Based on our pilot study, the threshold of the morphological plasma cell percentage ≥ 21.0% could serve as minimal plasma cell percentages for OGM to reliably detect alterations with an estimated AF of 5%.

Genome-wide analysis using OGM facilitates the genetic classification of MM beyond the abnormalities targeted by FISH. Of the five samples identified with hyperdiploidy, OGM identified hyperdiploidy in four samples, detecting hyperdiploidy at an estimated AF of ≥ 15.0% (3 of 20 metaphases) but missing one sample with an AF of 10.0% (2 of 20 metaphases) (data not shown). These results suggest that the AF threshold for detecting hyperdiploidy is comparable to those for other abnormalities identified in this study using FISH. Regarding genome-wide analysis, compared with whole-genome sequencing, OGM may offer a more cost-effective solution to achieve high coverage, improving the detection of alterations with low VAF in clinical settings.

Future analytical validation studies, including repeated measurements of samples across a range of VAFs and a larger number of clinical samples with an estimated AF of approximately 5%, are needed to fully establish the sensitivity of OGM. While our findings highlight OGM's potential as a complementary or initial screening tool to FISH in routine clinical settings, careful consideration of AF and plasma cell percentages is essential to optimize its application.