An entity evolving into a community: defining the common ancestor and evolutionary trajectory of chronic lymphocytic leukemia stereotyped subset #4.

Work was completed during previous post-I joined DMU on January 2016Patients with chronic lymphocytic leukemia (CLL) assigned to stereotyped subset #4 express highly homologous B-cell receptor immunoglobulin (BcR IG) sequences with intense intraclonal diversification (ID) in the context of ongoing somatic hypermutation (SHM). Their remarkable biological and clinical similarities strongly support derivation from a common ancestor. We here revisited ID in subset #4 CLL to reconstruct their evolutionary history as a community of related clones. To this end, using specialized bioinformatics tools we assessed both IGHV-IGHD-IGHJ rearrangements (n = 511) and IGKV-IGKJ rearrangements (n = 397) derived from eight subset #4 cases. Due to high sequence relatedness, a number of subclonal clusters from different cases lay very close to one another, forming a core from which clusters exhibiting greater variation stemmed. Minor subclones from individual cases were mutated to such an extent that they now resembled the sequences of another patient. Viewing the entire subset #4 data set as a single entity branching through diversification enabled inference of a common sequence representing the putative ancestral BcR IG expressed by their still elusive common progenitor. These results have implications for improved understanding of the ontogeny of CLL subset #4, as well as the design of studies concerning the antigenic specificity of the clonotypic BcR IGs

High-Resolution Description of Antibody Heavy-Chain Repertoires in Humans

Antibodies' protective, pathological, and therapeutic properties result from their considerable diversity. This diversity is almost limitless in potential, but actual diversity is still poorly understood. Here we use deep sequencing to characterize the diversity of the heavy-chain CDR3 region, the most important contributor to antibody binding specificity, and the constituent V, D, and J segments that comprise it. We find that, during the stepwise D-J and then V-DJ recombination events, the choice of D and J segments exert some bias on each other; however, we find the choice of the V segment is essentially independent of both. V, D, and J segments are utilized with different frequencies, resulting in a highly skewed representation of VDJ combinations in the repertoire. Nevertheless, the pattern of segment usage was almost identical between two different individuals. The pattern of V, D, and J segment usage and recombination was insufficient to explain overlap that was observed between the two individuals' CDR3 repertoires. Finally, we find that while there are a near-infinite number of heavy-chain CDR3s in principle, there are about 3–9 million in the blood of an adult human being

Immunological aspects in chronic lymphocytic leukemia (CLL) development

Chronic lymphocytic leukemia (CLL) is unique among B cell malignancies in that the malignant clones can be featured either somatically mutated or unmutated IGVH genes. CLL cells that express unmutated immunoglobulin variable domains likely underwent final development prior to their entry into the germinal center, whereas those that express mutated variable domains likely transited through the germinal center and then underwent final development. Regardless, the cellular origin of CLL remains unknown. The aim of this review is to summarize immunological aspects involved in this process and to provide insights about the complex biology and pathogenesis of this disease. We propose a mechanistic hypothesis to explain the origin of B-CLL clones into our current picture of normal B cell development. In particular, we suggest that unmutated CLL arises from normal B cells with self-reactivity for apoptotic bodies that have undergone receptor editing, CD5 expression, and anergic processes in the bone marrow. Similarly, mutated CLL would arise from cells that, while acquiring self-reactivity for autoantigens—including apoptotic bodies—in germinal centers, are also still subject to tolerization mechanisms, including receptor editing and anergy. We believe that CLL is a proliferation of B lymphocytes selected during clonal expansion through multiple encounters with (auto)antigens, despite the fact that they differ in their state of activation and maturation. Autoantigens and microbial pathogens activate BCR signaling and promote tolerogenic mechanisms such as receptor editing/revision, anergy, CD5+ expression, and somatic hypermutation in CLL B cells. The result of these tolerogenic mechanisms is the survival of CLL B cell clones with similar surface markers and homogeneous gene expression signatures. We suggest that both immunophenotypic surface markers and homogenous gene expression might represent the evidence of several attempts to re-educate self-reactive B cells

Different spectra of recurrent gene mutations in subsets of chronic lymphocytic leukemia harboring stereotyped B-cell receptors.

We report on markedly different frequencies of genetic lesions within subsets of chronic lymphocytic leukemia patients carrying mutated or unmutated stereotyped B-cell receptor immunoglobulins in the largest cohort (n=565) studied for this purpose. By combining data on recurrent gene mutations (BIRC3, MYD88, NOTCH1, SF3B1 and TP53) and cytogenetic aberrations, we reveal a subset-biased acquisition of gene mutations. More specifically, the frequency of NOTCH1 mutations was found to be enriched in subsets expressing unmutated immunoglobulin genes, i.e. #1, #6, #8 and #59 (22-34%), often in association with trisomy 12, and was significantly different (P<0.001) to the frequency observed in subset #2 (4%, aggressive disease, variable somatic hypermutation status) and subset #4 (1%, indolent disease, mutated immunoglobulin genes). Interestingly, subsets harboring a high frequency of NOTCH1 mutations were found to carry few (if any) SF3B1 mutations. This starkly contrasts with subsets #2 and #3 where, despite their immunogenetic differences, SF3B1 mutations occurred in 45% and 46% of cases, respectively. In addition, mutations within TP53, whilst enriched in subset #1 (16%), were rare in subsets #2 and #8 (both 2%), despite all being clinically aggressive. All subsets were negative for MYD88 mutations, whereas BIRC3 mutations were infrequent. Collectively, this striking bias and skewed distribution of mutations and cytogenetic aberrations within specific chronic lymphocytic leukemia subsets implies that the mechanisms underlying clinical aggressiveness are not uniform, but rather support the existence of distinct genetic pathways of clonal evolution governed by a particular stereotyped B-cell receptor selecting a certain molecular lesion(s

Immunoglobulin sequence analysis and prognostication in CLL: guidelines from the ERIC review board for reliable interpretation of problematic cases

prognostication in chronic lymphocytic leukemia (CLL) and the definition of standardized procedures has allowed reliable and reproducible results. Occasionally, a straightforward interpretation of the sequences is not possible because of the so-called 'problematic sequences' that do not fit the 'classic' interpretation and pose scientific questions at the cross-road between hematology and immunology. Thanks to a dedicated effort within the European Research Initiative on CLL (ERIC), we have now the possibility to present such cases, offer a scientific explanation and propose recommendations in terms of prognostication. Leukemia (2011) 25, 979-984; doi:10.1038/leu.2011.49; published online 1 April 201

Immunoglobulin genes in multiple myeloma: Expressed and non-expressed repertoires, heavy and light chain pairings and somatic mutation patterns in a series of 101 cases

Background and Objectives. The available data on the immunoglobulin gene (IG) repertoire in multiple myeloma (MM) derive mainly from heavy chains; considerably less is known about light chains. We assessed in parallel IGH and IGK/IGL rearrangements in 101 MM patients so as to gain insight into: (i) IG repertoires; (ii) antigen impact; (iii) the role of receptor editing. Design and Methods. Bone marrow aspirates were collected from all cases. IGHV-(D)-J and IGLV-J rearrangements were amplified by reverse transcriptase polymerase chain reaction (PCR). In all cases, IGKV-J rearrangements were analyzed in parallel on cDNA/genomic DNA. IGKV-KDE and IGKJ-C-INTRON-KDE were also amplified by DNA-PCR. RT-PCR products were directly sequenced. Results. IGHV3 genes predominated; the IGHV4-34 gene was used in only one case. Five IGKV and five IGLV genes accounted fcr the majority of in-frame, transcribed IGKV-J or IGLV-J rearrangements. Taking IGKV-J, IGKV-KDE and IGKJ-C-INTRON-KDE rearrangements together, biallelic IGK locus rearrangements were detected in 22/43 κ-MM cases. In λ-MMM, 36/42 cases had at least one rearranged IGK allele; 8/19 IGKV-J rearrangements in λ-MM were in-frame. All in-frame, transcribed IGH/IGK/IGL sequences were mutated; parallel heavy/light chain analysis demonstrated a comparable impact of somatic hypermutation. Interpretation and Conclusions. Biases in IG repertoire did not seem disease-related but followed a similar pattern to that of the normal repertoire. The under-representation of the IGHV4-34 gene provides an explanation for the paucity of autoimmune phenomena in MM. Somatic mutation patterns indicate the complementary role of MM IGH/IGK/IGL sequences in antigen recognition. Finally, the frequent inactivation of productive IGKV-J joints by secondary rearrangements in MM suggests active receptor editing. ©2006 Ferrata Storti Foundation