(PA-170) Whole Genome Sequencing of Cell-free DNA for Assessment of Minimal Residual Disease in High-risk Smoldering Multiple Myeloma

In multiple myeloma (MM), assessment of minimal residual disease (MRD) is limited by the need for serial bone marrow (BM) biopsies. Furthermore, single-site assessment of MRD (e.g., by flow cytometry) may be limited with extramedullary/systemic manifestation. We therefore performed a longitudinal analysis of MRD assessed by whole genome sequencing (WGS) of cell-free (cf)DNA from an interventional trial for high-risk smoldering MM.

Methods:

MRDetect is an ultra-sensitive tumor-informed detection approach for MRD. First, 80X WGS of baseline BM tumor samples (with 40x normal match) was performed on CD138+ plasma cells. Serial blood plasma (Streck tubes) from timepoints including pre-treatment baseline (BL), BM MRD assessment, and progression of disease (PD) were then subjected to 40x WGS and cfDNA reads supporting single nucleotide variants (SNV) corresponding to SNVs from BM WGS were used to estimate tumor fraction (TF) in blood. Patient-specific limits of detection (LOD) were computed by comparing detection rates in plasma to control SNV compendium detection rates derived from tumor-supporting reads in unrelated plasma samples. Results were compared to standard flow MRD in BM (10^-5).

Results:

We assayed 71 plasma samples with 25 BL BM samples from 25 patients (n=96 WGS; median follow-up 53 months). The median TF of BL plasma samples was 6x10^-4 (range; 1x10^-4- 9x10^-3). The median LOD across patients was 1.2x10^-4 (range 2x10^-4-9x10^-5) and 60/71 (84.5%) plasma samples had detectable disease. Tumors with genomic complexity (e.g., chromothripsis and APOBEC activity) had higher BL TF (p = 0.007). In line with this, a higher TF at BL was associated with biochemical/clinical PD (p = 0.01).

In longitudinal analysis, cfDNA WGS recapitulated MRD+ for 14/15 (93%) BM MRD+ assays. Representing a limitation with low mutational burden, one case with the lowest SNV count (~2000) could not be detected in plasma. However, 15/21 (71%) BM MRD- timepoints were seen to instead be MRD+ in plasma. Most discrepancies occurred in cases where BM MRD+ conversion or PD later occurred, or in cases where flanking MRD BM assessments were MRD+ (10/15, 75%), suggesting superiority of cfDNA over BM assessment.  
For cases with BM MRD+ conversion or PD, plasma collected three and six months prior detected early MRD resurgence in 4/6 (75%), representing a logistical advantage. Furthermore, incremental growth of TF in serial measurements foreshadowed PD. Finally, all patients with PD (n=5) had MRD+ in plasma at end of combination therapy, but only 2 were BM MRD+ at the same time point. No concordant plasma/BM MRD- patients experienced PD (HR; inf, p = 0.035).

Conclusions: MRD+ by cfDNA WGS recapitulates MRD+ BM flow cytometry. Despite the deeper LOD of localized BM flow, cfDNA WGS can detect MRD+ where BM flow did not, representing an advantage to the addition of this peripheral approach. LOD is expected to be deeper in newly diagnosed and relapsed MM, where SNV burden is higher.