Unique molecular assay (UMA): a next-generation sequencing targeted panel for efficient and comprehensive genomic profiling and risk stratification of multiple myeloma

Abstract

Multiple myeloma (MM) is characterized by genetic abnormalities in plasma cells, requiring precise genomic characterization for effective risk stratification and treatment. This study presents the Unique Molecular Assay (UMA) panel, a targeted DNA-sequencing approach designed to capture critical genomic aberrations in MM, including canonical immunoglobulin heavy chain translocations (t-IgH), copy number alterations (CNA), and mutations in 82 genes. The UMA panel is the first MM sequencing panel validated against traditional methods like fluorescence in situ hybridization (FISH) and SNP arrays across two laboratories for clinical-grade accuracy and reproducibility. The study included 150 patients whose DNA samples were analyzed using the UMA panel, achieving a median coverage of 233X with a requirement of ≥4 million reads per sample. The UMA panel demonstrated high concordance with FISH in detecting both CNA and t-IgH, achieving a balanced accuracy of over 93%. Moreover, inter-laboratory validation confirmed the robustness and reliability of the panel on genomic alteration calls. Importantly, the UMA panel enabled precise risk stratification based on the Second Revision of the International Staging System (R2-ISS), identifying high-risk features such as TP53 mutations and genome-wide CNA. This comprehensive and cost-effective genomic profiling tool supports clinical decision-making and personalized treatment strategies in MM. The validated performance and scalability of the UMA panel suggest its potential to complement traditional diagnostic methods, offering detailed insights into the genomic landscape of MM.

Figure 1.

Efficiency of multiple myeloma targeted next-generation sequencing panels in our literature review. All the considered papers are represented along the X axis, while the Y axis quantifies the sequenced Mbp per sample (target dimension multiplied by target coverage). The detected alteration types of panels are color coded. It is noticeable that the Unique Molecular Assay (UMA) panel stands out for its efficiency among panels which detect the complete set of alteration types (translocation that involves IGH gene [t-IgH], mutations, and copy number alterations [CNA]). MM: multiple myeloma; NGS: next-generation sequencing.

Figure 2.

Unique Molecular Assay panel summary heatmap. Columns represent newly diagnosed multiple myeloma (NDMM) patients included in the main cohort (Bologna [BO], N=130), while rows represent all detected genomic alterations by the Unique Molecular Assay (UMA) panel. To avoid excessive matrix size, only mutations with count >1 are showed. Columns’ heatmap sections correspond to the Second Revision of the International Staging System (R2-ISS) patients’ class, while rows’ heatmap sections correspond to the different alteration types detectable with the UMA panel (translocation that involves IGH gene [t-IgH], hyperdiploidy [HD], broad and focal copy number alterations [CNA], and gene mutations). avail.: available; F: Female; freq.: frequency; M: Male; Mil: millions; VAF: variant allele frequency.

Figure 3.

Intra-laboratory validation of the Unique Molecular Assay panel’s calls in the Bologna cohort compared with gold-standard fluorescence in situ hybridization calls. The Bologna cohort, N=129 patients. (A) Stacked histogram which summarizes the overall number of concordant and discordant copy number alteration (CNA) calls between the Unique Molecular Assay (UMA) panel and fluorescence in situ hybridization (FISH), over the four considered target genes. In the nested table, all the relevant performance metrics are reported (using FISH data as reference). (B) Stacked histogram which summarizes the overall number of concordant and discordant translocations (t) that involve IGH gene (t-IgH) calls between UMA and FISH, over the five considered t-IgH. In the nested table, all the relevant performance metrics are reported (using FISH data as reference). Neg Concord: negative concordance; Pos Concord: positive concordance.

Figure 4.

Bologna-Milano interlab validation results. (A) The plot displays the copy number (CN) profiles of the Bologna-Milano (BO-MI) cohort DNA, overlaying the BO sample (top line) on the MI replicate (bottom line). Red indicates amplifications above 40% clonality, while blue shows deletions below 40%. (Right) Frequencies of events in each profile are shown; amplifications and deletions found in the replicate are colored red and blue, respectively, with discordances marked in orange and petrol blue. (Left) Concordance rate of the pairwise matching analysis at the segment level (BO-MI CN matching) is presented. Additionally, the samples were compared using single-nucleotide polymorphism array (SNParray) (SNParray concordance – MI and SNParray concordance – BO). Concordance percentages are displayed on a scale from purple to yellow. (B) Translocations (t) involving the IGH-region (t-IgH) were evaluated: t(4;14), t(11;14), t(14;16), t(6;14), and t(14;20), and compared with fluorescence in situ hybridization (FISH) results. Again in correspondence to the sample code, the top line represents the BO sample and the bottom line represents the MI replicate. For t(4;14) and t(11;14), replicates showed 100% concordance, with 5 and 7 patients testing positive, respectively. Three patients were positive for the t(14;16) event, although this translocation was not detected in the external replicate DNA 35. No translocations were detected for t(6;14) and t(14;20), all consistent with negative FISH results. The UMA panel calls were colored according to the number of tools used, on a scale of blue (negative, only Manta, only Delly, and both tools), while fluorescence in situ hybridization (FISH) results were shown as green for positive cases and gray for negative. Cases without FISH data are not included. (C) Pearson correlation test among the variant allele frequency (VAF) of the variants identified in the replicates. DNA were colored based on the reason for discordance with the replicate after manual review. (D) Pie charts showing the genes’ variant frequencies in each cohort. The internal circle represents the frequency of variants in the BO cohort, while the external circle represents the variants in the MI cohort. (E) Pie chart showing the total variants concordance between cohorts. Variants called in both BO and MI samples in blue; variants called only in the BO samples in red or only in the MI samples in orange. CNA: copy number alteration; Amp C: concordant amplification; Amp D: discordant amplification; Del C: concordant deletion; Del D: discordant deletion.

Figure 5.

Bologna-Milano interlab validation results. (A) The plot displays the copy number (CN) profiles of the Bologna-Milano (BO-MI) cohort DNA, overlaying the BO sample (sequenced with the the Unique Molecular Assay [UMA] panel, top line) on the MI replicate (sequenced with ultra-low pass whole genome sequencing [ULP-WGS], bottom line). Amplifications above 40% clonality are shown in red, while deletions below 40% are in blue. (Left) Concordance rate of the pairwise matching analysis at the segment level (ULP-UMA CN matching). Concordance percentages are displayed on a scale from purple to yellow. (B) Translocations (t) involving the IGH-region (t-IgH) were evaluated: t(4;14), t(11;14), t(14;16), and compared with MI fluorescence in situ hybridization (FISH). Cases without MI FISH data are not included. (C) Pearson correlation test among the variant allele frequency (VAF) of the variants identified in the replicates. Samples were colored based on the reason for discordance with the replicate after manual review. (D) Pie chart showing the total variants concordance between the UMA panel and MI panel; variants called in both the UMA panel and MI panel samples in blue, variants called only in the MI samples in red. (E) Pie chart showing that 18 variants were detected by both panels and, of these, 8 are pathogenic (affecting TP53 and NRAS); the group of single-nucleotide variants (SNV) found only by the MI panel are shown in light blue, those shared with the UMA panel in green, and pathogenic variants in light green. CNA: copy number alteration.

Figure 6.

Unique Molecular Assay panel usage for multiple myeloma risk stratification. (A) Dotplot summarizing the main genomic risk factors identified by the Unique Molecular Assay (UMA) panel in the Bologna (BO) cohort, broken down by Second Revision of the International Staging System (R2-ISS) classes (only patients [pts] with available R2-ISS are showed, N=98). A histogram describing the classes’ frequencies is to be found above the dotplot. (B) Risk factors-patients matrix which illustrates the co-occurrence of risk factors within single patients. It is possible to observe the overall increase of detected risk-factors in higher risk classes. MM: multiple myeloma; del: deletion; Int: Intermediate, t: translocation.