Rafael Alonso Fernandez

Measurable residual disease assessment in bone marrow (BM‐MRD) is a crucial prognostic factor in multiple myeloma (MM), but its clinical use has some caveats, such as invasiveness, poor spatial representativity, and serial sampling. Novel minimally invasive approaches for monitoring peripheral residual disease (PRD) could facilitate its clinical use. We aimed to investigate a sensitive method for detecting PRD in patients with MM. This study included 84 patients enrolled in PETHEMA/GEM clinical trials, and a total of 292 longitudinal samples were analyzed. BM‐MRD in these patients was assessed using EuroFlow‐based next‐generation flow cytometry (NGF) and PRD was simultaneously examined in cell‐free DNA (cfDNA) using AltumTrackSeq®, a highly sensitive next‐generation sequencing (NGS) method based on patient‐specific multiplexed amplicon mini‐panels that target somatic mutations identified at diagnosis. Our findings revealed that automated isolation methods enabled standardization and yielded cfDNA levels comparable to manual protocols. A high median cfDNA concentration of 393.2 ng was obtained, with a range from 17.3 to 6300 ng. Furthermore, we demonstrated that compared with traditional BM‐MRD by NGF, our ctDNA‐NGS approach showed a higher positive predictive value (PPV, 88.9% vs. 34.5%) and specificity (Sp, 98.4% vs. 70.3%) for predicting relapse during the follow‐up period. Detectable ctDNA was associated with a high risk of progression and/or death (HR, 11.5; 95% CI, 2.66–49.4), and this was confirmed in the multivariate analysis. In conclusion, this assay offers a method for detecting imminent relapse risk in the peripheral blood of MM patients.

DNA promoter methylation of a subset of proteasome subunit genes. Heatmap comparing methylation pattern of 35 MM patients and 30 PBMC samples. The methylation degree is given by a color code: red – methylated state, blue – unmethylated state. Each line represents an individual sample, each column a single CpG. The methylation degree is encoded by color with blue for an unmethylated and red for a methylated state. Artificially methylated controls were included. For PSMD5, hypermethylation was only present in MM cells, not in PBMCs. For PSMC2, hypermethylation occurred in both, MM cells and PBMCs, indicating a physiological state probably independent of disease development and progression. In the remaining proteasome subunit genes, no aberrant promoter methylation was detected.

Raw data measurements of the dual-luciferase reporter assay in the L363 cell line. All measurements were performed in duplicates on 20.000 cells.

Patient-derived mutations impair PI response to Carfilzomib and Ixazomib. Cytotoxicity assay and IC50 values of RPMI-8226 MM cell lines harboring two different stably expressed patient-derived mutations in proteasome subunits. The mutants were more resistant to the proteasome inhibitors Carfilzomib and Ixazomib compared to the controls (*) harboring lentivirally-encoded WT sequence of PSMC6/PSMD1. Shown are the means and standard deviations of three independent experiments (biological replicates) and three technical replicates in each experiment. The PSMC6 and PSMD1 expression levels were equivalent for the mutants and the controls.

Genetic variants in the Würzburg dataset.

Primers used for luciferase reporter assay.

Cell survival of DNMTi pretreated KMS11 cells under BTZ exposure. AlamarBlue assay after 72 hours of incubation with varying concentrations of BTZ (0.5 nM to 4 nM) on DNMTi pretreated cells (48 h 500 nM Aza, 72 h 100 nM Dec) and PI naïve KMS11. No resensitizing effect to PI treatment was observed.

Quality check of DBS assays with artificially methylated standard DNAs. DNA standards of 0%, 25%, 50%, 75% and 100% methylation were sequenced with the PSMB5, PSMC2, PSMC5, PSMC6, PSMD1 and PSMD5 DBS assays to confirm assay sensitivity. Results for all investigated DBS amplicons correlated well with the expected curves (P < 0.001) (A). The sequencing depths of the PSMD5 DBS assay was 10-3 (B).