Abstract
INTRODUCTION Multiple Myeloma (MM) is a genetically complex blood cancer marked by diverse mutations, structural rearrangements, and chromosomal abnormalities that drive disease progression and treatment resistance. Traditional genomic approaches often miss critical alterations due to technical limitations in resolving complex regions. Oxford Nanopore long-read sequencing offers unprecedented insight by generating ultra-long reads capable of spanning difficult genomic landscapes. ONT's innovative Adaptive Sampling technology enables high-resolution detection of clinically relevant mutations and structural variants without the need for laborious capture or amplification steps, opening new avenues for comprehensive and rapid MM genomic profiling.
METHODS We designed a custom adaptive sampling panel covering ∼6% of the genome, targeting key genes, structural hotspots, and translocation-prone regions relevant to cancer. Genomic DNA from CD138+ cells of 17 MM patients was processed using ONT protocols and sequenced on the PromethION (P2i) platform with Adaptive Sampling enabled. We assessed enrichment efficiency, coverage, and variant detection. All patients had been previously characterized using gold standard methods, including FISH for del(17p), del(1p), amp(1q), t(4;14), t(14;16), and a targeted 38-gene NGS panel for multiple myeloma.
RESULTS This technology enabled molecular karyotyping of all 17 patients through whole-genome characterization. Using adaptive sampling, we performed low-coverage whole-genome sequencing, which facilitated the detection of large-scale structural variants and copy number alterations across the entire genome. Among the 17 patients, 2 showed no major chromosomal alterations; 4 were classified as hyperdiploid, each presenting trisomy's in various odd-numbered chromosomes. One patient exhibited an isolated del(1p), while another showed an isolated amp(1q). The remaining 7 patients displayed diverse combinations of genomic abnormalities, including del(1p), amp(1q), monosomy 8, monosomy 13, del(17p), and hyperdiploidy. Across 51 comparable probes (3 CNV targets per patient), 94.1% (48/51) showed concordant results, while the 3 discordant cases corresponded to small deletions in the short arm of chromosome 1 (1p), likely undetected by conventional FISH probes.
We initially focused on rearrangements involving the IGH locus on chromosome 14, given their relevance in multiple myeloma. All patients had previously been evaluated by FISH for t(4;14) and t(14;16) as stablished prognostic markers and were negative for both. However, long-read sequencing identified IGH rearrangements in two distinct patients: one involving t(14;20) and the other t(11;14), both of which have been previously reported in MM. In addition, 88% [15/17] of the patients exhibited other chromosomal translocations that warrant further characterization to assess their potential biological and clinical significance in the disease.
Regarding SNVs, the short-read targeted NGS panel did not detect variants in 5 out of 17 patients. In contrast, long-read sequencing identified pathogenic variants with variant allele frequencies (VAFs) above 5% in all 17 patients, suggesting that exome sequencing with long-read technology enhances the detection of clinically relevant alterations. Furthermore, the use of a clinical exome enables the identification of variants in genes associated with other diseases or with potential pharmacogenomic implications, as it is not limited to a predefined set of targets.
CONCLUSIONS Preliminary results indicate that the proposed technology enables the simultaneous detection of the main genomic alterations required for a comprehensive diagnostic workup in patients with multiple myeloma (MM), using a single, unified assay. Its agnostic design, based on broad genomic coverage without pre-selection of specific regions, allows for an unbiased analysis of the tumor genome. This approach not only facilitates the integration of multiple diagnostic markers into a single workflow but also supports potential scalability to other hematologic malignancies or solid tumors, as it is not restricted to a predefined set of genomic loci. The flexibility, robustness of the method and the short turnaround time for comprehensive characterization of MM patients position it as a promising tool for precision oncology.
