Minimal residual disease (MRD) is the most important independent prognostic factor in acute lymphoblastic leukemia (ALL). Since it has been implemented into in treatment stratification strategies, cure rates have improved significantly for all age groups. Real time quantitative (RQ)-PCR of clonal immunoglobulin and T-cell receptor gene rearrangements using allele-specific primers is currently regarded as the gold standard for MRD analysis in ALL, as it is not only highly sensitive and specific but also provides accurate MRD quantification. Following recent advances in next-generation sequencing (NGS), much attention has been devoted to the development of NGS-based MRD assays. This new technique can enhance sensitivity provided that sufficient numbers of cells are analyzed. Recent reports have shown that NGS-MRD also tends to be more specific for relapse prediction than RQ-PCR. In addition, NGS provides information on the physiological B- and T-cell repertoire during and after treatment, which has been shown to be prognostically relevant. However, before implementation of NGS-MRD detection in clinical practice, several issues must be addressed and the whole workflow needs to be standardized, including not only the analytical phase (spike-in calibrators, quality controls) but also the pre-analytical (e.g. sample preparation) and the post-analytical phases (e.g. bioinformatics pipeline, guidelines for correct data interpretation). These topics are currently addressed by a European network, the EuroClonality-NGS Consortium. In conclusion, NGS is a promising tool for MRD detection with the potential to overcome most of the limitations of RQ-PCR and to become the new gold standard for MRD detection in ALL.

• MeSH
• Precursor Cell Lymphoblastic Leukemia-Lymphoma genetics   MeSH
• Humans MeSH
• Prognosis MeSH
• Receptors, Antigen, T-Cell genetics   MeSH
• Neoplasm, Residual MeSH
• Sequence Analysis, DNA methods   MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Analysis and interpretation of Ig and TCR gene rearrangements in the conventional, low-throughput way have their limitations in terms of resolution, coverage, and biases. With the advent of high-throughput, next-generation sequencing (NGS) technologies, a deeper analysis of Ig and/or TCR (IG/TR) gene rearrangements is now within reach, which impacts on all main applications of IG/TR immunogenetic analysis. To bridge the generation gap from low- to high-throughput analysis, the EuroClonality-NGS Consortium has been formed, with the main objectives to develop, standardize, and validate the entire workflow of IG/TR NGS assays for 1) clonality assessment, 2) minimal residual disease detection, and 3) repertoire analysis. This concerns the preanalytical (sample preparation, target choice), analytical (amplification, NGS), and postanalytical (immunoinformatics) phases. Here we critically discuss pitfalls and challenges of IG/TR NGS methodology and its applications in hemato-oncology and immunology.

• MeSH
• Alleles MeSH
• Gene Rearrangement MeSH
• Genes, Immunoglobulin MeSH
• Genes, T-Cell Receptor genetics   MeSH
• Hematology methods   MeSH
• Immunogenetics methods   standards   MeSH
• Immunologic Techniques * MeSH
• Humans MeSH
• Computational Biology methods   MeSH
• High-Throughput Nucleotide Sequencing * methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH