Abstract Body

Myelodysplastic syndromes (MDS) are a heterogeneous group of chronic hematological malignancies characterized by dysplasia, ineffective hematopoiesis and a risk of progression to acute myeloid leukemia (AML). During the last decades, the great diversity in MDS characteristics has been shown to be supported by a variety of underlying cytogenetic abnormalities and an increasing number of mutations in genes involved in RNA-splicing (SF3B1, U2AF1, SRSF2, ZRSR2), chromatin modification (ASXL1, EZH2, BCOR, STAG2), DNA methylation (DNMT3A, TET2, IDH1/2), transcriptional regulation (RUNX1, GATA2), tumor suppressing (TP53) and signal transduction (CBL, JAK2, N/KRAS). Those alterations have been shown to be interconnected and dynamic, with some of them defining critical steps in disease progression and thus identified as potential prognostic markers and targets for new therapies.

This great diversity of alterations has made challenging the establishment of recommendations for appropriate MRD markers and/or time points in patients with MDS. Quantitative PCR (qPCR) and droplet digital PCR (ddPCR) remain the most sensitive, standardized, cost-effective, and time-efficient molecular technologies available in most clinical laboratories. They rely on the design and optimization of lesion-specific primers, limiting their use to frequent recurrent mutations without broad applicability in MDS. However, they are a technology of choice for specific markers, particularly in the context of targeted therapies to measure the eradication of a clone (e.g. IDH1/2 mutational hotspots). Besides gene mutations, monitoring of WT1 mRNA expression, which correlates with the blast population, could represent an alternative in patients with high risk disease treated with intensive therapies.

In this context, high-throughput sequencing (NGS) has emerged as an attractive tool, especially in patients with high-risk diseases receiving chemotherapy or undergoing stem cell transplantation. NGS allows the tracking of all variants at once and its repetition at multiple time points allows the appreciation of clonal hierarchy and dynamics and possibly the emergence of resistance and/or progression subclones with appropriate pipelines. However, its implementation in clinical practice still suffers from significant limitations. It requires standardization and important bioinformatic supports and remains expensive if used as a prospective tool. Sensitivity remains highly variable, depending on bioinformatic pipelines, sequencing technologies, as well as the type and location of variants. NGS achieves a sensibility of about 10^-2 with usual pipelines, although a threshold of 10^-4 is attainable with error-corrected sequencing using unique molecular identifiers (UMI). Additionally, many aspects have to be considered regarding result interpretation. Optimal targets and variant allele frequencies for decision making must be defined. Somatic mutations in hematological malignancy-free people, defining age-related clonal hematopoiesis, are found in at least 10% to 20% of people by age 60 years with current NGS technologies and may be identified in all individuals with more sensitive techniques. The most commonly mutated genes include DNMT3A and TET2 (by far the most common) and, to a latter extend, ASXL1, RNA-splicing genes, CBL or TP53. These mutations may accompany (distinct clone) or precede (part of the clone) the acquisition of MDS-driving mutations and may persist after tumor cells clearance. On the other hand, if the persistence of signaling mutations (RAS, CBL, NF1, FLT3) may be considered as poor prognostic signal when positive, their negativity should not lead to false reassurance, especially when the mutation is therapeutically targeted. Finally, germline predisposition to MDS and AML (DDX41, RUNX1, GATA2) may affect up to 10% of patients with high-risk diseases are, by definition, non-informative for MRD monitoring.

In conclusion, molecular MRD assessment is promising regarding new therapeutic strategies offered to patients with MDS. Considering the heterogeneity of molecular landscape, NGS holds great promise but recommendations and standardization are challenging. Furthermore, prospective trials are necessary to evaluate its efficacy in this regard.