Encyclopedia of CLL Subsets - a Unique Bioinformatics Tool and Database for Analysis of Subsets of Stereotypical B-Cell Receptors in CLL.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is clinically and biologically highly variable disease which is closely related with multiple cellular and molecular markers, including sequence motifs of B-cell receptors. These motifs are highly similar (stereotyped) within one third of CLL patients and create homogeneous groups called stereotyped CLL subsets. The homogeneity is reflected also in clinical and biological characteristics of the disease. To facilitate access to the information about individual subsets, we have created a publicly available web-based tool Encyclopedia of CLL Subsets. MATERIALS AND METHODS: The Encyclopedia of CLL subsets belongs to our bioinformatics platform Antigen Receptor Research Tool (ARResT) developed for analysis, clustering, and annotation of immunoglobulin sequences. To gather primary knowledge about the subsets, we have analyzed a dataset of 7,500 CLL patients published by Agathangelidis et al in 2012 [1]. We have created an overview of major stereotyped subsets and their characteristics. Additional clinical and cytogenomic information about individual subsets has been obtained by machine text processing of available literature from server PubMed and is regularly updated. RESULTS: We have created a unique web-based application Encyclopedia of CLL Subsets available from http: //arrest.tools/subsets for an interactive access to the information about stereotyped CLL subsets. A user can obtain and compare basic information about the major subsets including their clinical and cytogenomic characteristics. These have been manually curated from machine processed results from PubMed database by experts in CLL research. Through the Encyclopedias user interface, user can also directly use our published tool ARResT/AssignSubsets to assign new immunoglobulin sequences to the major subsets. CONCLUSION: The Encyclopedia of CLL Subsets is a publicly available online tool facilitating access to the most recent research knowledge about stereotyped CLL subsets and enabling analysis of own data and interpretation of the results. This gives the Encyclopedia a great potential for its use in clinical routine. This work was supported by Czech Ministry of Health grant No. 34272A. All rights reserved. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 1. 3. 2019 Accepted: 4. 3. 2019.

GvL effects in T-prolymphocytic leukemia: evidence from MRD kinetics and TCR repertoire analyses.

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.

Lack of common TCRA and TCRB clonotypes in CD8(+)/TCRαß(+) T-cell large granular lymphocyte leukemia: a review on the role of antigenic selection in the immunopathogenesis of CD8(+) T-LGL.

Clonal CD8(+)/T-cell receptor (TCR)αß(+) T-cell large granular lymphocyte (T-LGL) proliferations constitute the most common subtype of T-LGL leukemia. Although the etiology of T-LGL leukemia is largely unknown, it has been hypothesized that chronic antigenic stimulation contributes to the pathogenesis of this disorder. In the present study, we explored the association between expanded TCR-Vß and TCR-Vα clonotypes in a cohort of 26 CD8(+)/TCRαß(+) T-LGL leukemia patients, in conjunction with the HLA-ABC genotype, to find indications for common antigenic stimuli. In addition, we applied purpose-built sophisticated computational tools for an in-depth evaluation of clustering of TCRß (TCRB) complementarity determining region 3 (CDR3) amino-acid LGL clonotypes. We observed a lack of clear TCRA and TCRB CDR3 homology in CD8(+)/TCRαß(+) T-LGL, with only low level similarity between small numbers of cases. This is in strong contrast to the homology that is seen in CD4(+)/TCRαß(+) T-LGL and TCRγδ(+) T-LGL and thus underlines the idea that the LGL types have different etiopathogenesis. The heterogeneity of clonal CD8(+)/TCRαß(+) T-LGL proliferations might in fact suggest that multiple pathogens or autoantigens are involved.

Over 30% of patients with splenic marginal zone lymphoma express the same immunoglobulin heavy variable gene: ontogenetic implications.

We performed an immunogenetic analysis of 345 IGHV-IGHD-IGHJ rearrangements from 337 cases with primary splenic small B-cell lymphomas of marginal-zone origin. Three immunoglobulin (IG) heavy variable (IGHV) genes accounted for 45.8% of the cases (IGHV1-2, 24.9%; IGHV4-34, 12.8%; IGHV3-23, 8.1%). Particularly for the IGHV1-2 gene, strong biases were evident regarding utilization of different alleles, with 79/86 rearrangements (92%) using allele (*)04. Among cases more stringently classified as splenic marginal-zone lymphoma (SMZL) thanks to the availability of splenic histopathological specimens, the frequency of IGHV1-2(*)04 peaked at 31%. The IGHV1-2(*)04 rearrangements carried significantly longer complementarity-determining region-3 (CDR3) than all other cases and showed biased IGHD gene usage, leading to CDR3s with common motifs. The great majority of analyzed rearrangements (299/345, 86.7%) carried IGHV genes with some impact of somatic hypermutation, from minimal to pronounced. Noticeably, 75/79 (95%) IGHV1-2(*)04 rearrangements were mutated; however, they mostly (56/75 cases; 74.6%) carried few mutations (97-99.9% germline identity) of conservative nature and restricted distribution. These distinctive features of the IG receptors indicate selection by (super)antigenic element(s) in the pathogenesis of SMZL. Furthermore, they raise the possibility that certain SMZL subtypes could derive from progenitor populations adapted to particular antigenic challenges through selection of VH domain specificities, in particular the IGHV1-2(*)04 allele.

MeSHy: Mining unanticipated PubMed information using frequencies of occurrences and concurrences of MeSH terms.

MOTIVATION: PubMed is the most widely used database of biomedical literature. To the detriment of the user though, the ranking of the documents retrieved for a query is not content-based, and important semantic information in the form of assigned Medical Subject Headings (MeSH) terms is not readily presented or productively utilized. The motivation behind this work was the discovery of unanticipated information through the appropriate ranking of MeSH term pairs and, indirectly, documents. Such information can be useful in guiding novel research and following promising trends. METHODS: A web-based tool, called MeSHy, was developed implementing a mainly statistical algorithm. The algorithm takes into account the frequencies of occurrences, concurrences, and the semantic similarities of MeSH terms in retrieved PubMed documents to create MeSH term pairs. These are then scored and ranked, focusing on their unexpectedly frequent or infrequent occurrences. RESULTS: MeSHy presents results through an online interactive interface facilitating further manipulation through filtering and sorting. The results themselves include the MeSH term pairs, along with MeSH categories, the score, and document IDs, all of which are hyperlinked for convenience. To highlight the applicability of the tool, we report the findings of an expert in the pharmacology field on querying the molecularly-targeted drug imatinib and nutrition-related flavonoids. To the best of our knowledge, MeSHy is the first publicly available tool able to directly provide such a different perspective on the complex nature of published work. IMPLEMENTATION AND AVAILABILITY: Implemented in Perl and served by Apache2 at http://bat.ina.certh.gr/tools/meshy/ with all major browsers supported.

A restricted clonal T-cell receptor αß repertoire in Sézary syndrome is indicative of superantigenic stimulation.

BACKGROUND: Sézary syndrome (SS) is a cutaneous T-cell lymphoma characterized by erythroderma, lymphadenopathy and malignant clonal T cells in the skin, lymph nodes and peripheral blood. A role for superantigens in the pathogenesis of SS has been postulated before. OBJECTIVES: To investigate a putative involvement of chronic (super-)antigenic stimulation in driving T-cell expansion in SS. METHODS: Antigenic specificity of the T-cell receptor (TCR) was assayed by molecular analysis of the TCRA (n=11) and TCRB (n=28) genes, followed by detailed in silico analysis. RESULTS: Sequence analysis of clonally rearranged TCRB genes showed over-representation of Vß8, Vß13, Vß17, Vß21 and Vß22, and under-representation of Vß2 and Jß1.1 when compared with healthy controls. No similarity was detected in amino acid motifs of the complementarity determining region 3 (CDR3). Analysis of TCRA rearrangements showed that there was no common Vα or Jα gene usage, and that TCRA CDR3 amino acid motifs were not highly similar. CONCLUSIONS: The lack of clear stereotypic TCRA and TCRB CDR3 amino acid motifs would argue against involvement of a single common antigen in the pathogenesis of SS. Nevertheless, the skewing of Vß and Jß gene usage does seem to point to a restricted TCR repertoire, possibly as a result of superantigenic selection prior to neoplastic transformation.

MASiVE: Mapping and Analysis of Sirevirus Elements in plant genome sequences.

UNLABELLED: We present MASiVE, an expertly built tool for the large-scale, yet sensitive and highly accurate, discovery, preliminary analysis and insertion age estimation of intact Sirevirus LTR-retrotransposons in plant genomic sequences. Validation was based on the recently available and annotated large maize chromosome one. Results show a considerable improvement in the annotation of Sireviruses, and support our approach as an important addition to the bioinformatics toolbox of plant biologists. AVAILABILITY: PERL source code and essential files are available online at http://bat.ina.certh.gr/tools/masive/. The freely available Vmatch, LTRharvest, Wise2, and MAFFT algorithms are required.

Intraclonal diversification of immunoglobulin light chains in a subset of chronic lymphocytic leukemia alludes to antigen-driven clonal evolution.

The study of intraclonal diversification (ID) in immunoglobulin (IG) genes offers valuable insight into the role of ongoing interactions with antigen in lymphomagenesis. We recently showed that ID in the IG heavy chain genes of patients with chronic lymphocytic leukemia (CLL) was generally limited; however, intense ID was evident in selected cases, especially those expressing stereotyped IGHV4-34 rearrangements and assigned to subset 4. Here, we report results from a large-scale subcloning study of IG light variable genes, in a total of 1008 subcloned sequences from 56 CLL cases. Multiple analogies were noted between heavy and light chains regarding the occurrence and molecular features of ID. More specifically, the impact of ID on the clonotypic light chains was generally low, with the significant exception of subset 4. Similar to the IGHV4-34 heavy chains of this subset, their partner IGKV2-30 light chains were affected by an active and precisely targeted ID process. Altogether, these findings strengthen the argument that stereotypy in subset 4 extends to stereotyped ID patterns for both heavy and light chains through persistent antigenic stimulation. Furthermore, they strongly suggest that light chains have an active role in the antigen selection process, at least for certain subsets of CLL cases.

A different ontogenesis for chronic lymphocytic leukemia cases carrying stereotyped antigen receptors: molecular and computational evidence.

Chronic lymphocytic leukemia (CLL) is uniquely characterized by the existence of subsets of cases with quasi-identical, 'stereotyped' B-cell receptors (BCRs). Herein we investigate this stereotypy in 2662 patients with CLL, the largest series yet, using purpose-built bioinformatics methods based on sequence pattern discovery. Besides improving the identification of 'stereotyped' cases, we demonstrate that CLL actually consists of two different categories, based on the BCR repertoire, with important biological and ontogenetic differences. The first ( approximately 30% of cases) shows a very restricted repertoire and is characterized by BCR stereotypy (clustered cases), whereas the second includes cases with heterogeneous BCRs (nonclustered cases). Eleven major CLL clusters were identified with antigen-binding sites defined by just a few critically positioned residues, regardless of the actual immunoglobulin (IG) variable gene used. This situation is closely reminiscent of the receptors expressed by cells participating in innate immune responses. On these grounds, we argue that whereas CLL cases with heterogeneous BCRs likely derive from the conventional B-cell pool, cases with stereotyped BCRs could derive from progenitor cells evolutionarily adapted to particular antigenic challenges, perhaps intermediate between a true innate immune system and the conventional adaptive B-cell immune system, functionally similar to what has been suggested previously for mouse B1 cells.

Molecular evidence for EBV and CMV persistence in a subset of patients with chronic lymphocytic leukemia expressing stereotyped IGHV4-34 B-cell receptors.

The chronic lymphocytic leukemia (CLL) immunoglobulin repertoire is uniquely characterized by the presence of stereotyped B-cell receptors (BCRs). A major BCR stereotype in CLL is shared by immunoglobulin G-switched cases utilizing the immunoglobulin heavy-chain variable 4-34 (IGHV4-34) gene. Increased titers of IGHV4-34 antibodies are detected in selective clinical conditions, including infection by B-cell lymphotropic viruses, particularly Epstein-Barr virus (EBV) and cytomegalovirus (CMV). In this context, we sought evidence for persistent activation by EBV and CMV in CLL cases expressing the IGHV4-34 gene. The study group included 93 CLL cases with an intentional bias for the IGHV4-34 gene. On the basis of real-time PCR results for CMV/EBV DNA, cases were assigned to three groups: (1) double-negative (59/93); (2) single-positive (CMV- or EBV-positive; 25/93); (3) double-positive (9/93). The double-negative group was characterized by heterogeneous IGHV gene repertoire. In contrast, a bias for the IGHV4-34 gene was observed in the single-positive group (9/25 cases; 36%). Remarkably, all nine double-positive cases utilized the IGHV4-34 gene; seven of nine cases expressed the major BCR stereotype as described above. In conclusion, our findings indicate that the interactions of CLL progenitor cells expressing distinctive IGHV4-34 BCRs with viral antigens/superantigens might facilitate clonal expansion and, eventually, leukemic transformation. The exact type, timing and location of these interactions remain to be determined.

PuReD-MCL: a graph-based PubMed document clustering methodology.

MOTIVATION: Biomedical literature is the principal repository of biomedical knowledge, with PubMed being the most complete database collecting, organizing and analyzing such textual knowledge. There are numerous efforts that attempt to exploit this information by using text mining and machine learning techniques. We developed a novel approach, called PuReD-MCL (Pubmed Related Documents-MCL), which is based on the graph clustering algorithm MCL and relevant resources from PubMed. METHODS: PuReD-MCL avoids using natural language processing (NLP) techniques directly; instead, it takes advantage of existing resources, available from PubMed. PuReD-MCL then clusters documents efficiently using the MCL graph clustering algorithm, which is based on graph flow simulation. This process allows users to analyse the results by highlighting important clues, and finally to visualize the clusters and all relevant information using an interactive graph layout algorithm, for instance BioLayout Express 3D. RESULTS: The methodology was applied to two different datasets, previously used for the validation of the document clustering tool TextQuest. The first dataset involves the organisms Escherichia coli and yeast, whereas the second is related to Drosophila development. PuReD-MCL successfully reproduces the annotated results obtained from TextQuest, while at the same time provides additional insights into the clusters and the corresponding documents. AVAILABILITY: Source code in perl and R are available from http://tartara.csd.auth.gr/~theodos/