High-throughput sequencing for the identification ofNOTCH1mutations in early stage chronic lymphocytic leukaemia: biological and clinical implications

Introduction: Determination of the mutational status of rearranged immunoglobulin heavy chain variable (IgHV) genes in patients with Chronic Lymphocytic Leukaemia (CLL), is considered one of the most important prognostic factors: patients with unmutated IgHV (UM; ≥98% of identity to the germline) genes have a more aggressive disease course and develop more frequently unfavourable genetic deletions or mutations than patients with mutated IgHV (M; ≤98%). Mutational status, is currently determined by Sanger sequencing (Sseq) that allows the analysis of the major clone, however, international guidelines recommend caution in assigning mutational status in cases with "Borderline" IgHV identity (97-97.9%), and cases with double rearrangements with discordant mutational status. Objective: Analyze and determine the mutational status of the IgHV locus by High-throughput sequencing (HTS), in a cohort of CLL patients (n=51) with unclassifiable Sseq results: borderline status (n=22); double rearrangements (n=27) with discordant mutational status (n=2). Methods: We included 51 DNA samples extracted from peripheral blood of patients diagnosed of CLL according to the National Cancer Institute Working Group guidelines in our institution between 1986 and 2019 (median absolute lymphocytes 11.4x109/L [2,8-239,5x109/L]). Sseq amplification and analysis of IgHV rearrangements were performed on DNA conforming to the updated ERIC recommendations. In all the cases we were able to determinate the IGVH identity. To switch high-throughput sequencing to the clinical practice, we assessed the reliability of different library preparation methods to sequence IGH locus in patients with CLL. Amplification was performed using the Sequencing Multiplex Kit based on IGH FR (forward primers) and consensus JH (reverse primer) multiplex. PCR products were purified using Magsi-NGS Prep magnetic beads (Magnamedics Diagnostics), normalized and pooled to create a library for sequencing using a MiSeq equipment. To simplify and make automatic the analysis of the same we developed a specific bioinformatic pipeline that covers from preprocessing to final data summarization and interpretation. The backbone of the analysis includes read preprocessing, mapping against IMGT reference sequences, consensus IgHV reads pairwise alignment to determine mutational status and read classification into rearrangements. Results: This approach led to the identification of a dominant clone IgHV in all cases (n=51). Instead, the percentage of identity calculated by HTS analysis varies in: - 15/22 borderline cases whose mutational status could be recalculated into 10 MM and 5 UM. The rest 7 remaining in borderline group. - We could identify both clones in 29 double rearrangements cases, with concordant mutational status except 2/29 undetermined cases, included in UM group regarding HTS results. Our tool led to the identification of a dominant clonotypic IgHV in all cases, and when compared the HTS sequence/mutational status for the most abundant clone with Sseq and for the IgHV status determination, 15 out of 22 (68,18%), could be reclassified. This case showed a major clone with productive rearrangement mutated by Sseq but unmutated by HTS. Conclusions: Analyze and determine the mutational status of the IgHV locus by HTS, would potentially reveal multiple rearrangements and increase the prognostic precision of IgHV mutation analysis. IgHV-HTS classification is able to precisely classify patients with borderline status or/and multiple IgHV rearrangements for which Sseq is inconclusive. In this case, it has been possible to improved prognostication for 17 out of 24 patients. This is helping us to discover the advantages of the data obtained by HTS compared with current Sseq standard technique. Samples were provided by the INCLIVA Biobank. Funded by Gilead Felowship 257/17 Disclosures Terol: Abbvie: Consultancy; Janssen: Consultancy, Research Funding; Gilead: Research Funding; Roche: Consultancy; Astra Zeneca: Consultancy.

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Comprehensive pathogen detection in sera of Kawasaki disease patients by high-throughput sequencing: a retrospective exploratory study

BMC Pediatrics ◽

10.1186/s12887-020-02380-7 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Yuka Torii ◽

Kazuhiro Horiba ◽

Satoshi Hayano ◽

Taichi Kato ◽

Takako Suzuki ◽

...

Keyword(s):

Kawasaki Disease ◽

High Throughput ◽

High Throughput Sequencing ◽

Sequence Data ◽

Early Stage ◽

Rna Virus ◽

Systemic Vasculitis ◽

Human Herpesvirus ◽

Acute Stage ◽

Serum Samples

Abstract Background Kawasaki disease (KD) is an idiopathic systemic vasculitis that predominantly damages coronary arteries in children. Various pathogens have been investigated as triggers for KD, but no definitive causative pathogen has been determined. As KD is diagnosed by symptoms, several days are needed for diagnosis. Therefore, at the time of diagnosis of KD, the pathogen of the trigger may already be diminished. The aim of this study was to explore comprehensive pathogens in the sera at the acute stage of KD using high-throughput sequencing (HTS). Methods Sera of 12 patients at an extremely early stage of KD and 12 controls were investigated. DNA and RNA sequences were read separately using HTS. Sequence data were imported into the home-brew meta-genomic analysis pipeline, PATHDET, to identify the pathogen sequences. Results No RNA virus reads were detected in any KD case except for that of equine infectious anemia, which is known as a contaminant of commercial reverse transcriptase. Concerning DNA viruses, human herpesvirus 6B (HHV-6B, two cases) and Anelloviridae (eight cases) were detected among KD cases as well as controls. Multiple bacterial reads were obtained from KD and controls. Bacteria of the genera Acinetobacter, Pseudomonas, Delfita, Roseomonas, and Rhodocyclaceae appeared to be more common in KD sera than in the controls. Conclusion No single pathogen was identified in serum samples of patients at the acute phase of KD. With multiple bacteria detected in the serum samples, it is difficult to exclude the possibility of contamination; however, it is possible that these bacteria might stimulate the immune system and induce KD.

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