Aurélie Caillault

Fast multiclonal clusterization of V(D)J recombinations from high-throughput sequencing

BackgroundV(D)J recombinations in lymphocytes are essential for immunological diversity. They are also useful markers of pathologies. In leukemia, they are used to quantify the minimal residual disease during patient follow-up. However, the full breadth of lymphocyte diversity is not fully understood.ResultsWe propose new algorithms that process high-throughput sequencing (HTS) data to extract unnamed V(D)J junctions and gather them into clones for quantification. This analysis is based on a seed heuristic and is fast and scalable because in the first phase, no alignment is performed with germline database sequences. The algorithms were applied to TR γ HTS data from a patient with acute lymphoblastic leukemia, and also on data simulating hypermutations. Our methods identified the main clone, as well as additional clones that were not identified with standard protocols.ConclusionsThe proposed algorithms provide new insight into the analysis of high-throughput sequencing data for leukemia, and also to the quantitative assessment of any immunological profile. The methods described here are implemented in a C++ open-source program called Vidjil.

Minimal residual disease monitoring in t(8;21) acute myeloid leukemia based on RUNX1-RUNX1T1 fusion quantification on genomic DNA

Although acute myeloid leukemia (AML) with t(8;21) belongs to the favorable risk AML subset, relapse incidence may reach 30% in those patients. RUNX1‐RUNX1T1 fusion transcript is a well‐established marker for minimal residual disease (MRD) monitoring. In this study, we investigated the feasibility and performances of RUNX1‐RUNX1T1 DNA as MRD marker in AML with t(8;21). In 17/22 patients with t(8;21)‐positive AML treated in the French CBF‐2006 trial, breakpoints in RUNX1 and RUNX1T1 were identified using long‐range PCR followed by next‐generation sequencing. RUNX1‐RUNX1T1 DNA quantification was performed by real‐time quantitative PCR using patient‐specific primers and probe. MRD levels were evaluated in 71 follow‐up samples from 16 patients, with a median of four samples [range 2–7] per patient. RUNX1 breakpoints were located in intron 5 in all cases. RUNX1T1 breakpoints were located in intron 1b in 15 cases and in intron 1a in two cases. RUNX1‐RUNX1T1 MRD levels measured on DNA and RNA were strongly correlated (r = 0.8, P < 0.0001). Discordant MRD results were observed in 10/71 (14%) of the samples: in three samples from two patients who relapsed, RUNX1‐RUNX1T1 was detectable only on DNA, while RUNX1‐RUNX1T1 was detectable only on RNA in seven samples. MRD monitoring on genomic DNA is feasible, but with sensitivity variations depending on the patient breakpoint sequence and the qPCR assay efficiency. Although interpretation of the results is easier because it is closely related to the number of leukemic cells, this method greatly increases time, cost and complexity, which limits its interest in routine practice. Am. J. Hematol. 89:610–615, 2014.

Multi-loci diagnosis of acute lymphoblastic leukaemia with high-throughput sequencing and bioinformatics analysis.

High‐throughput sequencing (HTS) is considered a technical revolution that has improved our knowledge of lymphoid and autoimmune diseases, changing our approach to leukaemia both at diagnosis and during follow‐up. As part of an immunoglobulin/T cell receptor‐based minimal residual disease (MRD) assessment of acute lymphoblastic leukaemia patients, we assessed the performance and feasibility of the replacement of the first steps of the approach based on DNA isolation and Sanger sequencing, using a HTS protocol combined with bioinformatics analysis and visualization using the Vidjil software. We prospectively analysed the diagnostic and relapse samples of 34 paediatric patients, thus identifying 125 leukaemic clones with recombinations on multiple loci (TRG, TRD, IGH and IGK), including Dd2/Dd3 and Intron/KDE rearrangements. Sequencing failures were halved (14% vs. 34%, P = 0.0007), enabling more patients to be monitored. Furthermore, more markers per patient could be monitored, reducing the probability of false negative MRD results. The whole analysis, from sample receipt to clinical validation, was shorter than our current diagnostic protocol, with equal resources. V(D)J recombination was successfully assigned by the software, even for unusual recombinations. This study emphasizes the progress that HTS with adapted bioinformatics tools can bring to the diagnosis of leukaemia patients.

High-throughput sequencing in acute lymphoblastic leukemia: Follow-up of minimal residual disease and emergence of new clones

Minimal residual disease (MRD) is known to be an independent prognostic factor in patients with acute lymphoblastic leukemia (ALL). High-throughput sequencing (HTS) is currently used in routine practice for the diagnosis and follow-up of patients with hematological neoplasms. In this retrospective study, we examined the role of immunoglobulin/T-cell receptor-based MRD in patients with ALL by HTS analysis of immunoglobulin H and/or T-cell receptor gamma chain loci in bone marrow samples from 11 patients with ALL, at diagnosis and during follow-up. We assessed the clinical feasibility of using combined HTS and bioinformatics analysis with interactive visualization using Vidjil software. We discuss the advantages and drawbacks of HTS for monitoring MRD. HTS gives a more complete insight of the leukemic population than conventional real-time quantitative PCR (qPCR), and allows identification of new emerging clones at each time point of the monitoring. Thus, HTS monitoring of Ig/TR based MRD is expected to improve the management of patients with ALL.

A novel type of NPM1 mutation characterized by multiple internal tandem repeats in a case of cytogenetically normal acute myeloid leukemia

The NPM1 gene, mapped on chromosome 5q35, encodes a nucleolar phosphoprotein composed of 294 amino acids. The NPM1 protein continuously shuttles between the nucleus and the cytoplasm (with predominant nucleolar localization) and is involved in several cellular processes, including centrosome