Abstract LB101: New assay technology and utility of peripheral blood vs bone marrow for molecular MRD in MDS
Authors: Bosaeus Linus , Chen Lei , Tobiasson Magnus , Lindholm Caroline , Cavelier Lucia , Lindberg Eva Hellström 
Published: April 2024

Abstract
Background Rare tumor-specific mutations in patient samples serve as excellent markers to monitor the course of malignant disease and responses to therapy. However, improved assay techniques are needed for broad adoption in routine clinic. We describe herein - superRCA assays - providing rapid and highly specific detection of DNA sequence variants present at very low frequencies in DNA samples using a standard flow cytometer for readout. We demonstrate and compare the assay against ddPCR for precise, ultra-sensitive detection of single-nucleotide mutant sequences from malignant cells to follow the course of MDS patients following allogeneic stem cell transplantation, both in bone marrow and peripheral blood.
Methods This study is based on patient material from the Nordic MDS study NMDSG14B (NCT02872662). The patients in this retrospective study have previously undergone sequencing (TruSight, Illumina) upon diagnosis, after which patient specific driver mutations (1-4 per patient) has been chosen and analyzed longitudinally to monitor molecular MRD using ddPCR (Bio-Rad). These longitudinal samples were re-analyzed using superRCA mutation assays (Rarity Bioscience). Both quarterly bone marrow aspirates and monthly peripheral blood samples post allogeneic stem cell transplantation were included to investigate to what extent frequent blood derived molecular MRD gives equivalent relapse information as bone marrow. For each mutation, sensitivity was assessed through serial dilution and wild type control, with the limit of detection calculated as Mean(wt)+3*SD(wt). Samples were analyzed in duplicates or triplicates and sample to sample variation was calculated as SD/Mean.
Results A total of 22 relapse patients and 21 different mutations were analyzed, covering single nucleotide variants, insertions, deletions, as well as mutations with very high GC in ASXL1 and SRSF2. The analysis demonstrating lower background and limit of detection for the superRCA assays compared to the equivalent ddPCR data. With spike-in dilution series, we demonstrated the superRCA assay could detect single point mutations as low as 1 in 100,000 with the flow cytometer readout. Mean background for superRCA was 0,0016% in wild type controls, and mean limit of detection was 0,0023%. From available serial dilutions, the mean sample to sample variation (CV) at 0,016% was 22,87% and 29,32% at 0,001% dilution.In longitudinal sample analysis of relapse patients, superRCA assay detected remaining mutations after initial treatment and clearly revealed the remaining malignant clone, subsequently leading to a relapse. The superRCA assay also demonstrated the feasibility of detecting corresponding leukemia mutations in the PBMCs when such mutations were present in bone marrow.
Conclusions The data shows comparable mutant allele frequency results between superRCA and ddPCR upon diagnosis and relapse. The data further demonstrates superRCA mutation assays for the selected driver mutations can reach to very low limit of detection and increasing mutations could be detected in both blood and bone marrow predicting the relapse of MDS post transplantation in clinical samples. To further study the utility of using peripheral blood for molecular MRD in MDS patients, more data is needed and studies are ongoing.
Citation Format: Linus Bosaeus, Lei Chen, Magnus Tobiasson, Caroline Lindholm, Lucia Cavelier, Eva Hellström Lindberg. New assay technology and utility of peripheral blood vs bone marrow for molecular MRD in MDS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB101.