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Ig gene (IG) clonality analysis has an important role in the distinction of benign and malignant B-cell lymphoid proliferations and is mostly performed with the conventional EuroClonality/BIOMED-2 multiplex PCR protocol and GeneScan fragment size analysis. Recently, the EuroClonality-NGS Working Group developed a method for next-generation sequencing (NGS)–based IG clonality analysis. Herein, we report the results of an international multicenter biological validation of this novel method compared with the gold standard EuroClonality/BIOMED-2 protocol, based on 209 specimens of reactive and neoplastic lymphoproliferations. NGS-based IG clonality analysis showed a high interlaboratory concordance (99%) and high concordance with conventional clonality analysis (98%) for the molecular conclusion. Detailed analysis of the individual IG heavy chain and kappa light chain targets showed that NGS-based clonality analysis was more often able to detect a clonal rearrangement or yield an interpretable result. NGS-based and conventional clonality analysis detected a clone in 96% and 95% of B-cell neoplasms, respectively, and all but one of the reactive cases were scored polyclonal. We conclude that NGS-based IG clonality analysis performs comparable to conventional clonality analysis. We provide critical parameters for interpretation and discuss a first step toward a quantitative scoring approach for NGS clonality results. Considering the advantages of NGS-based clonality analysis, including its high sensitivity and possibilities for accurate clonal comparison, this supports implementation in diagnostic practice.

B-cell neoplasms represent a clinically heterogeneous group of hematologic malignancies with considerably diverse genomic architecture recently endorsed by next-generation sequencing (NGS) studies. Because multiple genetic defects have a potential or confirmed clinical impact, a tendency toward more comprehensive testing of diagnostic, prognostic, and predictive markers is desired. This study introduces the design, validation, and implementation of an integrative, custom-designed, capture-based NGS panel titled LYmphoid NeXt-generation sequencing (LYNX) for the analysis of standard and novel molecular markers in the most common lymphoid neoplasms (chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma). A single LYNX test provides the following: i) accurate detection of mutations in all coding exons and splice sites of 70 lymphoma-related genes with a sensitivity of 5% variant allele frequency, ii) reliable identification of large genome-wide (≥6 Mb) and recurrent chromosomal aberrations (≥300 kb) in at least 20% of the clonal cell fraction, iii) the assessment of immunoglobulin and T-cell receptor gene rearrangements, and iv) lymphoma-specific translocation detection. Dedicated bioinformatic pipelines were designed to detect all markers mentioned above. The LYNX panel represents a comprehensive, up-to-date tool suitable for routine testing of lymphoid neoplasms with research and clinical applicability. It allows a wide adoption of capture-based targeted NGS in clinical practice and personalized management of patients with lymphoproliferative diseases.

Assessment of clonality, marker identification and measurement of minimal residual disease (MRD) of immunoglobulin (IG) and T cell receptor (TR) gene rearrangements in lymphoid neoplasms using next-generation sequencing (NGS) is currently under intensive development for use in clinical diagnostics. So far, however, there is a lack of suitable quality control (QC) options with regard to standardisation and quality metrics to ensure robust clinical application of such approaches. The EuroClonality-NGS Working Group has therefore established two types of QCs to accompany the NGS-based IG/TR assays. First, a central polytarget QC (cPT-QC) is used to monitor the primer performance of each of the EuroClonality multiplex NGS assays; second, a standardised human cell line-based DNA control is spiked into each patient DNA sample to work as a central in-tube QC and calibrator for MRD quantification (cIT-QC). Having integrated those two reference standards in the ARResT/Interrogate bioinformatic platform, EuroClonality-NGS provides a complete protocol for standardised IG/TR gene rearrangement analysis by NGS with high reproducibility, accuracy and precision for valid marker identification and quantification in diagnostics of lymphoid malignancies., This work was supported by Ministry of Health of the Czech Republic, grant no. 16-34272A; computational resources were provided by the CESNET LM2015042 and the CERIT Scientific Cloud LM2015085, provided under the programme “Projects of Large Research, Development, and Innovations Infrastructures”. Analyses in Prague (JT, EF and MS) were supported by Ministry of Health, Czech Republic, grant no. 00064203, and by PRIMUS/17/MED/11. Analyses in the Monza (Centro Ricerca Tettamanti, SS, AG and GC) laboratory were supported by the Italian Association for Cancer Research (AIRC) and Comitato Maria Letizia Verga.

International audience; One of the hallmarks of B lymphoid malignancies is a B cell clone characterized by a unique footprint of clonal immunoglobulin (IG) gene rearrangements that serves as a diagnostic marker for clonality assessment. The EuroClonality/BIOMED-2 assay is currently the gold standard for analyzing IG heavy chain (IGH) and κ light chain (IGK) gene rearrangements of suspected B cell lymphomas. Here, the EuroClonality-NGS Working Group presents a multicentre technical feasibility study of a novel approach involving next-generation sequencing (NGS) of IGH and IGK loci rearrangements that is highly suitable for detecting IG gene rearrangements in frozen and formalin-fixed paraffin-embedded tissue specimens. By employing gene-specific primers for IGH and IGK amplifying smaller amplicon sizes in combination with deep sequencing technology, this NGS-based IG clonality analysis showed robust performance, even in DNA samples of suboptimal DNA integrity, and a high clinical sensitivity for the detection of clonal rearrangements. Bioinformatics analyses of the high-throughput sequencing data with ARResT/Interrogate, a platform developed within the EuroClonality-NGS Working Group, allowed accurate identification of clonotypes in both polyclonal cell populations and monoclonal lymphoproliferative disorders. This multicentre feasibility study is an important step towards implementation of NGS-based clonality assessment in clinical practice, which will eventually improve lymphoma diagnostics.

MotivationSanger sequencing remains the reference method for sequence variant detection, especially in a clinical setting. However, chromatogram interpretation often requires manual inspection and in some cases considerable expertise. Additionally, variant reporting and nomenclature is typically left to the user, which can lead to inconsistencies.ResultsWe introduce GLASS, a tool built to assist with the assessment of gene variations in Sanger sequencing data. Critically, it provides a standardized variant output as recommended by the Human Genome Variation Society.AvailabilityThe program is freely available online at http://bat.infspire.org/genomepd/glass/.Contactnikos.darzentas@gmail.com, malcikova.jitka@fnbrno.czSupplementary informationSupplementary data are available at Bioinformatics online.

Allogeneic stem cell transplantation (alloSCT) is used for treating patients with T-prolymphocytic leukemia (T-PLL). However, direct evidence of GvL activity in T-PLL is lacking. We correlated minimal residual disease (MRD) kinetics with immune interventions and T-cell receptor (TCR) repertoire diversity alterations in patients after alloSCT for T-PLL. Longitudinal quantitative MRD monitoring was performed by clone-specific real-time PCR of TCR rearrangements (n=7), and TCR repertoire diversity assessment by next-generation sequencing (NGS; n=3) Although post-transplant immunomodulation (immunosuppression tapering or donor lymphocyte infusions) resulted in significant reduction (>1 log) of MRD levels in 7 of 10 occasions, durable MRD clearance was observed in only two patients. In all three patients analyzed by TCR-NGS, MRD responses were reproducibly associated with a shift from a clonal, T-PLL-driven profile to a polyclonal signature. Novel clonotypes that could explain a clonal GvL effect did not emerge. In conclusion, TCR-based MRD quantification appears to be a suitable tool for monitoring and guiding treatment interventions in T-PLL. The MRD responses to immune modulation observed here provide first molecular evidence for GvL activity in T-PLL which, however, may be often only transient and reliant on a poly-/oligoclonal rather than a monoclonal T-cell response.

Introduction: Development of mature B cells depends upon the expression of a functional B cell receptor (BCR) and of its immunoglobulin (Ig) heavy (H) and light (L) chains. The molecular hallmark of B cell development, somatic recombination of the genes that encode the IgH (V, D and J) and IgL (V and J) chains, takes place in B cells in primary B lymphopoiesis sites throughout development (i.e. fetal liver (FL), fetal bone marrow (FBM) and adult BM). However, the spatiotemporal relationship between the IgH repertoire in FL with that in FBM, and the impact of the fetal Ig repertoire on the long-term repertoire present in post-natal life, as well as the link between this and the development of disease, are largely unknown. Aim: To gain insights into the ontogeny of the human innate B cell repertoire by performing high resolution analysis of the IgH-Cmu repertoire of human FL, FBM and post-natal B cells. Methods: CD34negCD19pos B cells from human 2nd trimester FL and FBM, child peripheral blood (cPB) and adult peripheral blood (aPB) were isolated by flow sorting. For initial assessment of the IgM repertoire ontogeny in FL and FBM B cells, we performed CDR3 fragment length analysis of VH1-VH6 families in the B cells. To gain further insights into the ontogeny of IgH diversification, we employed 454 next generation sequencing (NGS)-based analysis of the IgVH-Cmu mRNA repertoire in FL, FBM and postnatal B cells. In total, 20 libraries generated from 17 individual, flow-sorted CD34negCD19pos B cell samples were sequenced: 5 FL (4 performed in independent duplicate libraries; GA 14-18 weeks), 3 FBM (GA 13-17 weeks), 3 cPB (age 4mo-4yrs) and 5 aPB (age 29-53 yrs) samples. Bioinformatic analysis was performed with the ARResT/Interrogate immunoprofiler, IMGT tools, and appropriate statistics. Results: Spectratyping and NGS data showed that the molecular mechanisms responsible for VDJ recombination-dependent repertoire diversification, such as convergent recombination, are active in early B cell development and as efficient as postnatal B cells in both FL and FBM. Comparably diversified B cell lymphopoiesis exists contemporaneously in FL and FBM. However, analysis of the top 100 most abundant clonotypes in each developmental stage showed that their mean abundance in FBM B cells was 10 times lower (0.12%) than in FL B cells (1.2%; p Conclusions: The lack of mature, antigen-responsive B cells in FBM and the pronounced clonotypic expansions in FL suggest that FL is the main source of IgM natural immunity during the 2nd trimester. Our results are consistent with persisting fetal IgM+ B cells being an important source of the natural IgM repertoire also in adult life. Further, the origins of specific stereotypic IgM+ BCR associated with CLL, can be traced back to fetal B cell lymphopoiesis, suggesting that persisting fetal B cells can be subject to malignant transformation late in life. Overall, these novel data provide unique insights into the ontogeny of physiological and malignant B lymphopoiesis that spans the human lifetime. Disclosures No relevant conflicts of interest to declare.

Introduction: It is widely acknowledged that acute lymphoblastic leukemia (ALL) is caused by acquisition of mutations, with a central role for chromosomal translocations. Therefore, genomic profiling of ALL has the potential to identify important new prognostic markers and druggable fusion genes. Aim: (i) setting up a Next Generation Sequencing-Targeted Capture (NGS-TC) panel focused on ALL, identifying both novel and conventional fusion genes; (ii) providing a user-friendly but powerful, knowledge-based bioinformatics platform for its analysis. Methods: NGS-TC approaches on MiSeq platform (Illumina) were evaluated: (i) on DNA, by Nextera Rapid Capture Custom panel (Illumina), 17 genes; (ii) on RNA, by comparing alternative panels: the Ovation Fusion Panel Target Enrichment System (Nugen, pre-designed 500 or custom 77 genes) and the TruSight Pan-Cancer (Illumina, pre-designed 1385 genes). Both panels include probes for genes involved in B-cell leukemogenesis (such as ABL1, JAK2, EBF1, PAX5). DNA datasets were analyzed by our purpose-built bioinformatics platform, 'BreakingPoint'. RNA results were analyzed by both 'BreakingPoint' and other similar tools available on 'Illumina BaseSpace' Cloud, such as 'TopHat'. Results: 'BreakingPoint' detects fusion genes from datasets derived from any sequencing technology and any biological material (DNA/RNA). Its distinctive feature is that it initially aligns reads on targets to efficiently and effectively find candidate breakpoints, then merges relevant reads into longer (thus more informative) consensus sequences, which are then studied across the genome to confirm the breakpoints and identify fusion partners. 'BreakingPoint' also uses an ever-expanding user database to filter out potential false positives and highlights known or novel true fusions. Firstly, we targeted 17 genes on N=65 DNA of ALL patients and we evaluated 'BreakingPoint' on a subset of 10 samples with 7 known rearrangements: 6 of them were detected with almost no false positives. P2RY8/CRLF2 known fusion was not detected mainly due to a non-homogenous target coverage which affected all the DNA NGS-TC experiments: coverage analyses showed high variability among different genes and in a sample-specific manner. Nevertheless, we detected N=6 novel fusions: 3 involving PAX5 and 3 involving ABL1. Secondly, the two candidate RNA protocols were transversally analyzed in a subset (N=5) of patients with known fusion genes. In this setting, we compared library preparation flowcharts in term of amount of RNA (Ovation kit 200ng vs. PanCancer kit 100ng), timeline and man-effort (2 up to 3 days), cost (similar), single run output (v2 vs. v3 MiSeq reagent kit with a mean of 15M reads/8samples and 25M reads/8samples, respectively) and capturing: both methods efficiently detected known fusion genes. Subsequently, the Ovation Fusion Panel was used to analyze N=59 ALL patients, enrolled consecutively to the protocol to mimic the diagnostic practice. From routinely RT-PCR screening, 14/59 patients carried a previously known fusion gene. By Ovation Nugen NGS-TC, they were all confirmed, such as BCR/ABL1 (N=1), ETV6/RUNX1 (N=8), TCF3/PBX1 (N=4), and P2RY8/CRLF2 (N=1). More interestingly, N=1 gene fusion involving TCF3 and N=2 cases carrying NUP214/ABL1 were detected for the first time. The TruSight Pan-Cancer panel was used to process N=42 newly enrolled cases. Previously known fusion genes were confirmed (N=2 ETV6/RUNX1, N=3 BCR/ABL1, N=3 TCF3/PBX, N=2 P2RY8/CLRF2). Moreover, we detected N=7 previously unknown fusions, mostly confirmed by RT-PCR (validation in progress): N=1 MLL/MLLT10, N=1 EBF1/PDGFRB, N=1 TCF3/OAZ1, N=2 JAK2-fusions, N=1 PAX5-fusion, and N=1 involving MLL with a new partner. Importantly, and in contrast to DNA capture, both RNA-based approaches were successful in detecting the P2RY8/CRLF2 cases. Conclusion: Our results show that RNA NGS-TC assays offer the most feasible solution towards a routine diagnostic protocol and that target coverage is the most important condition for success. Consequently, we have built our bioinformatics approach, 'BreakingPoint', to sensitively and reliably detect fusion genes even in cases of low coverage, irrespective of biological material or protocol. We consider our approach complete and clinically-relevant for identifying known and novel targetable fusion genes in pediatric ALL. Disclosures Biondi: Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Cellgene: Other: Advisory Board; BMS: Membership on an entity's Board of Directors or advisory committees.

Motivation The study of immunoglobulins and T cell receptors using next-generation sequencing has finally allowed exploring immune repertoires and responses in their immense variability and complexity. Unsurprisingly, their analysis and interpretation is a highly convoluted task. Results We thus implemented ARResT/Interrogate, a web-based, interactive application. It can organize and filter large amounts of immunogenetic data by numerous criteria, calculate several relevant statistics, and present results in the form of multiple interconnected visualizations. Availability and Implementation ARResT/Interrogate is implemented primarily in R, and is freely available at http://bat.infspire.org/arrest/interrogate/ Supplementary information Supplementary data are available at Bioinformatics online.