Heavy Chain Variable Region Research Articles

Serum Proteomic Analysis by Nanoflow LC-MS/MS-Based Proteomics in IgA Chronic Kidney Disease.

IgA nephropathy (IgAN) is the most frequently occurring primary glomerulonephritis. A lack of specific biomarkers hinders the early diagnosis and treatment of this disease. This study analyzes and validates potential serum biomarkers using mass spectrometry proteomics. Global proteomics profiles of serum from 60 patients with IgAN and 43 healthy control subjects were compared to identify significantly changed proteins. These proteins were validated with targeted proteomics using parallel reaction monitoring (PRM) in an independent validation set consisting of samples from 67 different stage IgAN patients and 60 healthy controls. A total of 37 significantly changed proteins were found in the discovery set, among which 18 proteins were identified as potential biomarkers for IgAN through PRM assays in the validation set. Of these 18 proteins, IgGFc-binding protein, MS-A1 light chain variable region, transthyretin, ficolin-3, and myosin-reactive immunoglobulin light chain variable region were up-regulated in different IgAN stages, B cell receptor heavy chain variable region, rheumatoid factor RF-ET6, heavy chain Fab, cryocrystalglobulin CC1 heavy chain variable region, FLJ94213, lumican, and Q68CN4 (uncharacterized protein) were down-regulated in different IgAN stages. These proteins support previous findings that CKD is accompanied by altered immune response. This study lays the groundwork for additional research using biomarkers to clinically diagnose IgAN. These proteins are potential molecular markers that could help us understand the potential molecular mechanism of IgAN.

OBINUTUZUMAB FOR THE TREATMENT OF CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS: SINGLE CENTER EXPERIENCE

Monoclonal antibodies (mAbs) targeting CD20 molecule on B lymphocyte are of great importance in the treatment of B-cell malignancies. In recent years a great effort has been put in developing novel mAbs that can provide greater efficacy than the well-known rituximab. A second class of mAbs obinutuzumab has been presented as a more powerful tool in the treatment of this group of patients. In this retrospective study 70 patients with symptomatic CLL were included. CLL patients were diagnosed and treated at the University Clinic for Hematology between January 2018 and January 2022. All patients were evaluated for traditional clinical and laboratory prognostic factors and newer prognostic factors including IGHV mutation status and CLL prognostic and predictive genetic abnormalities. Most of the patients treated with obinutuzumab had Binet B stage (56%). Mutational status of the immunoglobulin variable region heavy chain (IGHV) in most of CLL patients treated with obinutuzumab was unmated IGHV gene. The most frequently encountered adverse events were tumor lysis syndrome and leukopenia. The analysis of the initial results of the application of obinutuzumab-based therapy allows us to conclude that this therapeutical modality is not associated with severe adverse events that would limit the administration of therapy.

EXPRESSION OF IMMUNOGLOBULIN SEQUENCES HOMOLOGOUS TO ANTI-SARS-CoV-2 ANTIBODIES AND HIV IN CHRONIC LYMPHOCYTIC LEUKEMIA CASES.

Identification of epitopes recognized by leukemic B cells could provide insights into themolecular mechanisms of B cell transformation in chronic lymphocytic leukemia (CLL). Theaim of this paper was to compare nucleotide sequences of immunoglobulin heavy chain variable region (IGHV) genes in CLL with known sequences directed against antigens of different origins available in public databases. Analysis was performed in thegroups of 412unselected CLL patients with productive IGHV gene using polymerase chain reaction followed by direct sequencing. Homology between CLL Ig sequences and antibodies directed against autoantigens was found in 12patients (2.9%), homology between CLL Ig sequences and antiviral antibodies- in 35patients (8.5%). Most of these sequences belonged to stereotypical clusters. Among thesequences that have homology to antiviral antibodies, themost prevalent were cases homologous with antibodies against HIV (14cases, 3.4%) and SARS-CoV-2antigens (10cases, 2.4%). None of thepatients in our cohort was HIV-infected and thestudy was conducted before theemergence of SARS-CoV-2virus. Suggestions could be made about thepossible impact of past infection of SARS-CoV-2virus on thepathogenesis of CLL. In particular, an increase in theproportion of CLL cases with theexpression of some stereotyped BCR and/or an increase of CLL risk in thelong-term period after SARS-CoV-2virus infection is not excluded. This assumption needs to be verified by epidemiological data.

Humanized V(D)J-rearranging and TdT-expressing mouse vaccine models with physiological HIV-1 broadly neutralizing antibody precursors

Antibody heavy chain (HC) and light chain (LC) variable region exons are assembled by V(D)J recombination. V(D)J junctional regions encode complementarity-determining-region 3 (CDR3), an antigen-contact region immensely diversified through nontemplated nucleotide additions ("N-regions") by terminal deoxynucleotidyl transferase (TdT). HIV-1 vaccine strategies seek to elicit human HIV-1 broadly neutralizing antibodies (bnAbs), such as the potent CD4-binding site VRC01-class bnAbs. Mice with primary B cells that express receptors (BCRs) representing bnAb precursors are used as vaccination models. VRC01-class bnAbs uniformly use human HC VH1-2 and commonly use human LCs Vκ3-20 or Vκ1-33 associated with an exceptionally short 5-amino-acid (5-aa) CDR3. Prior VRC01-class models had nonphysiological precursor levels and/or limited precursor diversity. Here, we describe VRC01-class rearranging mice that generate more physiological primary VRC01-class BCR repertoires via rearrangement of VH1-2, as well as Vκ1-33 and/or Vκ3-20 in association with diverse CDR3s. Human-like TdT expression in mouse precursor B cells increased LC CDR3 length and diversity and also promoted the generation of shorter LC CDR3s via N-region suppression of dominant microhomology-mediated Vκ-to-Jκ joins. Priming immunization with eOD-GT8 60mer, which strongly engages VRC01 precursors, induced robust VRC01-class germinal center B cell responses. Vκ3-20-based responses were enhanced by N-region addition, which generates Vκ3-20-to-Jκ junctional sequence combinations that encode VRC01-class 5-aa CDR3s with a critical E residue. VRC01-class-rearranging models should facilitate further evaluation of VRC01-class prime and boost immunogens. These new VRC01-class mouse models establish a prototype for the generation of vaccine-testing mouse models for other HIV-1 bnAb lineages that employ different HC or LC Vs.

Updates in the treatment of mantle cell lymphoma: A Canadian expert framework

Mantle cell lymphoma (MCL) is a B cell Non-Hodgkin Lymphoma that develops in the mantle zone of the lymph node. It is more common in men and is usually diagnosed at an advanced stage with involvement of lymph nodes, bone marrow, and potentially the gastrointestinal tract. MCL accounts for 5–10% of all new NHL cases per year in Canada, which is estimated at 11,400 for 2022. While most patients respond to initial treatment, relapses occur early and MCL generally shows a variable response to subsequent treatments, often with limited duration of benefit. Two main subtypes of MCL can be distinguished that arise from in situ MCL lesions. The most common subtype, classic MCL, arises from these cells with limited or no immunoglobulin heavy chain variable region (IGHV) mutations. Cells from this subtype express SOX11, are genetically unstable, and have naïve B cell-like characteristics. Classic MCL is more often nodal and extranodal and may eventually progress to aggressive blastoid or pleomorphic MCL. The other subtype, leukemic non-nodal MCL, arises from cells that have undergone IGHV somatic hypermutations, do not express SOX11, and exhibit characteristics of memory B cells. This subtype can have an indolent clinical behavior for a long time, often several years, but frequently acquires TP53 and other mutations and progresses to a more aggressive subtype. Treatment options have expanded significantly over the past decades, with improvements in both overall survival (OS) and progression-free survival (PFS) compared to earlier treatment eras. This Canadian expert framework aims to discuss the management considerations for patients with MCL, and will present both front-line treatment options as well as those for relapsed and refractory disease.

Selecting initial therapy in CLL.

Targeted therapy is a powerful treatment option in chronic lymphocytic leukemia (CLL) that has outperformed conventional chemoimmunotherapy in most clinical settings. Except for selected young, fit patients with a mutated immunoglobulin heavy chain variable region gene, most patients benefit from targeted therapy with either a continuous BTK inhibitor or 1-year fixed-duration venetoclax-obinutuzumab as first-line treatment of CLL. Treatment selection is driven by patient-, treatment-, and disease-related factors, encompassing patient preference, concomitant medications, comorbidities, safety profile of the regimen, and TP53 aberration. Clinical trials are actively investigating the simultaneous inhibition of Bruton's tyrosine kinase (BTK) and B-cell lymphoma 2 (BCL-2) proteins with or without a CD20 monoclonal antibody, which can achieve deep response in most patients (52%-89% undetectable minimal residual disease in bone marrow).

Prognostic markers in patients with chronic lymphocytic leukaemia on targeted therapy, chemoimmunotherapy with anti-CD20 monoclonal antibody: a systematic review and meta-analysis of prognostic factors

Combination chemoimmunotherapy (CIT) consisting of anti-CD20 has improved the progression-free survival (PFS) and overall survival (OS) of patients with chronic lymphocytic leukaemia (CLL). We performed a comprehensive synthesis of prognostic factors in patients with CLL on combined CIT with anti-CD20 antibodies compared with standard chemotherapy alone or targeted therapy.We searched the MEDLINE and academic search complete electronic databases as well as clinicaltrials.gov (from inception up to 01 August 2022) for randomised controlled trials examining chemoimmunotherapy and targeted therapy in patients with CLL. The risk of bias and the quality of evidence was assessed using the quality in prognostic studies tool (QUIPS).A total of 10 prognostic factors were identified and evaluated in patients with CLL on anti-CD20 antibody-containing CIT. The predictive value of the following prognostic factors was confirmed and associated with poor patient outcomes; deletion 17p (HR = 3.39), Immunoglobulin heavy chain variable region gene mutation status (HR = 0.96) and β2-microglobulin (HR = 1.41).Conventional predictive factors may have retained prognostic value and could be useful in the stratification of patients who may be non-responsive to CIT.Trial registration: International Prospective Register of Systematic Reviews (PROSPERO) registry (CRD42021218997).

Hyper N-Glycosylated SEL1L3 As B-Cell Receptor Autoantigen of Primary Vitreoretinal Lymphoma

Chronic antigenic stimulation of B lymphocytes through their B-cell receptor (BCR) is thought to play an important role in the genesis of B-cell lymphomas. SAMD14/neurabin-I has been described as auto-antigenic target of primary central nervous system lymphoma (PCNSL) BCRs. Primary vitreoretinal lymphoma (PVRL) is a rare subtype of DLBCL and can progress to PCNSL. Besides manifestation in an immunologically privileged site, PVRL and PCNSL share frequent recurrent genetic alterations like MYD88 and CD79B mutations. Both PVRL and PCNSL are considered being related to the MCD subtype of DLBCLs and may depend on chronic BCR signaling through antigenic stimulation. To investigate the role of chronic antigenic stimulation in PVRL, we cloned and expressed BCRs from PVRL patients as antibody fragments (Fabs) and tested for specific binding against common antigens. All cases with corresponding BCR sequences were previously reported by Belhouachi et al. (Blood Adv. 2020;4(7):1357-1366). For 20 of 55 PVRL BCR sequences both immunoglobulin heavy and light chain variable region (V) genes were available. DNA clones comprising an ApaLI restriction site, the complete light chain V gene, a κ- or λ-constant region gene, a ribosome binding site, a signal sequence, the heavy chain V gene and a BstEII restriction site were ordered from GeneCust® (GeneCust, 5690 Ellange, Luxembourg) in a pUC57 vector. DNA fragments were cloned in front of a γ-1 constant region gene and a Histidine-tag into a modified pCES-1 vector for expression as Fabs. PVRL-BCRs were screened on protein macroarrays representing clones of UniPEx human cDNA expression libraries expressed in E. coli (Bioscience, Dublin, Ireland). SEL1L3 was identified as the auto-antigenic target of the B-cell receptors in 3 out of 20 PVRL cases. None of the PVRL Fabs bound to LRPAP1 (common BCR antigen of mantle cell lymphomas), RpoC (Moraxella catarrhalis bacterial antigen and target of nodular lymphocyte-predominant Hodgkin lymphoma cells) or galectin-3 (member of the lectin family). SEL1L3 is a 1132 amino acid long protein with multiple glycosylation and phosphorylation sites (uniprot.org). This is especially important since previously identified BCR antigens from different lymphoma entities showed post-translational modifications such as hyper-N-glycosylation or hypo-phosphorylation. Furthermore, SEL1L3 is highly expressed in the pigmented layer of the retina. Western Blot analysis of the blood of a PVRL patient with serum antibodies against SEL1L3 showed an increased molecular weight of SEL1L3 as compared to SEL1L3 of controls. After treatment with N-acetyl-β-d-glucosaminidase or PNGase F, this difference in molecular weight disappeared, indicating a hyper N-glycosylation of SEL1L3 in PVRL patients with SEL1L3-reactive BCRs as trigger for its' immunogenicity. SEL1L3 induced proliferation of aggressive lymphoma cell lines transfected with SEL1L3-reactive BCRs. Moreover, the BCR binding epitope of SEL1L3 (aa 560 - 585) conjugated to a truncated form of Pseudomonasaeruginosa exotoxin A, killed exclusively lymphoma cells expressing BCRs with the respective reactivity. Additionally, BCRs of a locally treated PVRL patient with serum antibodies against SEL1L3 were cloned from biopsies of the vitreous body at initial diagnosis and of a lymphoma manifestation in the breast at relapse. Both BCRs showed binding to SEL1L3 suggesting a continued dependence of the BCR on antigen stimulation. These results suggest an important role of chronic antigenic stimulation by the post-translationally modified auto-antigen SEL1L3 in the genesis of a subgroup of patients with PVRL, might give an explanation for the selective tropism of PVRLs and provide the basis for a new treatment approach with high specificity.

Chronic Lymphocytic Leukemia in African Ancestry Population Is Characterized By Increased Telomere Erosion

Background: Chronic lymphocytic leukemia (CLL), a neoplastic disease of relatively mature B-cells, is characterized by a variable clinical course. Although initial studies suggesting more aggressive disease phenotype and inferior clinical outcomes among CLL patients of African ancestry (AA) were published a decade ago, biological features contributing towards this augmented disease severity remain largely unknown. Telomere erosion in a cohort of CLL patients of European Ancestry (EA) has been previously associated with high-risk molecular markers including unmutated immunoglobulin heavy-chain variable region (u-IGHV) status, and genetic alterations such as del(17p) and del(11q) by FISH. Here we characterized telomere erosion in AA CLL patients compared to CLL patients of EA. Methods: We extracted DNA from CD5+/CD19+ cells peripheral blood mononuclear cells and utilized it to measure the average telomere length through quantitative-PCR (qPCR). Briefly, primers for telomere and the single copy gene (SCG) HBB were used for amplification of 20 ng of DNA in a total volume of 20 µL in triplicates using the KAPA SYBR® FAST optimized for LightCycler®480 and analyzed on a LightCycler®480. Absolute telomere lengths were then obtained by generating standard curves using synthetic oligonucleotide PCR standards for telomere and the SCG gene. The qPCR results were validated by terminal restriction fragment length (TRF) analysis and Southern hybridization (N=28; r=0.8531; p<0.0001) using the TeloTTAGGG Telomere Length Assay. TRF was used as the gold standard of telomere length quantification; however, the high quantity of genomic DNA required for the assay (2µg) limited its broad use. Five CLL samples with known telomere lengths through TRF analysis were included in every qPCR plate in order to perform a linear regression to account for variability between qPCR plates as described previously. Results: Telomere erosion was evaluated in a cohort of 133 AA (mean age at sample collection 62.7 years ±13.6) and 147 age-matched EA CLL patients (mean age at sample collection 61.6 years ±10.2). The AA samples were collected from 95 (71%) male patients and 71% presented u-IGHV, while EA samples were collected from 110 (77%) male patients and 66% u-IGHV. As expected, we observed shorter telomere length in u-IGHV CLL (median 3.7 kb; range 0.8-44.9) compared to mutated immunoglobulin heavy-chain variable region (m-IGHV) CLL (median 6.8 kb; range 1.8-44.7; p<0.0001) in the full cohort. AA CLL presented shorter telomere lengths (median 4.2 kb; range 0.6-13.1) when compared to EA CLL (median 4.7 kb; range 1.1-44.9; p=0.005; Figure 1A). We highlight that the differences in telomere length observed were restricted to the CD5+/CD19+ cells, with no difference observed in germline samples collected from AA and EA patients. When stratifying by IGHV mutation status, shorter telomere length was observed in u-IGHV when compared to m-IGHV among both AA CLL (median 3.5 kb and 6.3, respectively; p<0.0001) and EA CLL (median 3.9 kb and 7.5, respectively; p<0.0001; Figure 1B). The significant shorter telomere lengths in AA CLL remained significant (p=0.006) after accounting for IGHV status, age, sex, number of mutated CLL driver genes, and Rai stage in multivariate analyses. Conclusions: Our results demonstrated increased telomere erosion in AA CLL compared to EA CLL, even after accounting for other known prognostic factors. Telomere length has been described as an independent prognostic factor in multiple cancers. In CLL, telomere shortening can lead to a selective pressure for loss of function of DNA damage checkpoint genes, becoming permissive to further cell proliferation and telomere shortening. Future studies are needed to see if these findings contribute towards the severe clinical outcomes in AA CLL compared to EA CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Proximally Biased V(D)J Recombination and Evolution of Non-Productive Clones in B-Cell Precursor Acute Lymphocytic Leukemia with KMT2A::AFF1 Fusion Genes

Background: B-cell precursor acute lymphocytic leukemia (BCP-ALL) carrying KMT2A::AFF1 rearrangements is an aggressive disease with unsatisfactory response to treatment and poor prognosis. KMT2A is a histone methyl transferase involved in the establishment of epigenetic memory. In the fusion protein encoded by the KMT2A::AFF1 fusion gene, the catalytic SET domain of KMT2A mediating H3K4 trimethylation is lost. Instead, the fusion protein interacts with DOT1L and mediates H3K79 methylation. The subversion of the catalytic activity of KMT2A arrests affected cells in an immature stage of development that may precede V(D)J recombination. Aim: Investigate the immune repertoire of KMT2A::AFF1 rearranged BCP-ALL and evaluate the feasibility of measurable residual disease (MRD) detection using immunoglobulin (Ig) gene rearrangements. Patients and Methods: The cohort includes 290 adult and 89 pediatric BCP-ALL patients. The diagnosis was established following WHO classification. WTS (5x107 reads, 2x101bp) data were generated on HiSeq and NovaSeq instruments. Ig gene rearrangements were analyzed using MiXCR and NCBI-IgBlast. Results: We analyzed Ig heavy-chain variable region (IGHV) gene rearrangements in BCP-ALL samples. We observed that the immune repertoire of leukemic cells carrying KMT2A::AFF1 is less diverse than that of other BCP-ALL samples. We investigated the expression levels of IGHV genes using WTS data. We found that IGHV genes located proximally to the IGHD/IGHJ cluster of genes on chr14 were more likely to be expressed in samples carrying KMT2A::AFF1 (Fig 1A). This difference was particularly significant (p<10-18) for IGHV6-1, the IGHV gene closest to the IGHD/IGHJ cluster (Fig 1B). Proximally biased V(D)J recombination is indicative of incomplete Ig locus contraction. Locus contraction is regulated by PAX5, which suppresses WAPL, a negative regulator of chromatin loop extrusion. Chromatin looping of IGHV genes ensures a balanced immune repertoire by bringing distant IGHV genes in proximity to the V(D)J recombination center. We found that in samples with KMT2A::AFF1 fusion genes WAPL expression levels are significantly (p<10-10) higher than in other subtypes of BCP-ALL. Analysis of the Ig gene rearrangements in KMT2A::AFF1 samples revealed abundant expression of alleles encoding non-productive V(D)J rearrangements or IGHV alleles in germline configuration. Only 35% of complementarity determining region 3 covering reads encoded productive rearrangements in KMT2A::AFF1 samples while in other types of BCP-ALL 67% of reads are productive (p<10-6). In samples with chronic lymphocytic leukemia, a tumor of post-germinal center B cells, this percentage is generally 90% or more. Among the non-productive V(D)J rearranged alleles, in-frame stop codons were equally overrepresented as out-of-frame V-J fusions when compared to their abundance in other types of BCP-ALL (p<10-15 in both cases). As light chain rearrangements require signaling from a productive heavy chain, the significantly (p<9x10-7) lower expression of light chain alleles detectable in KMT2A::AFF1 samples supports the notion that the abundant expression of non-productive alleles is not an artefact. Clonal expansion as measured by the Gini index for the number of reads per clonotype is distributed uniformly between 0 (polyclonality) and 1 (monoclonality). Monoclonality was observed in only 10% of cases. Furthermore, some DJ-rearrangements with identical non-templated linker sequences were found to be recombined with different IGHV genes, indicating ongoing V(D)J recombination and clonal evolution. Conclusions: Our data suggest that the KMT2A::AFF1 rearrangement locks the cell of origin at a point of ongoing V(D)J recombination. V(D)J recombination appears to be induced prematurely in these cells. Poor locus contraction could explain the proximally biased V(D)J recombination pattern. The non-sense mediated decay pathway involved in down regulating the expression of non-productive alleles seems to be poorly functional. Consequently, the cells express unusually high amounts of non-productive alleles or alleles in germline configuration that are clonally evolving and derived frequently from IGHV6-1. These features distinguish KMT2A::AFF1 BCP-ALL from other BCP-ALL subtypes and make MRD detection in KMT2A::AFF1 BCP-ALL based on specific Ig gene rearrangements highly error prone. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mutational patterns along different evolution paths of follicular lymphoma.

Follicular lymphoma (FL) is an indolent disease, characterized by a median life expectancy of 18-20 years and by intermittent periods of relapse and remission. FL frequently transforms into the more aggressive diffuse large B cell lymphoma (t-FL). In previous studies, the analysis of immunoglobulin heavy chain variable region (IgHV) genes in sequential biopsies from the same patient revealed two different patterns of tumor clonal evolution: direct evolution, through acquisition of additional IgHV mutations over time, or divergent evolution, in which lymphoma clones from serial biopsies independently develop from a less-mutated common progenitor cell (CPC). Our goal in this study was to characterize the somatic hypermutation (SHM) patterns of IgHV genes in sequential FL samples from the same patients, and address the question of whether the mutation mechanisms (SHM targeting, DNA repair or both), or selection forces acting on the tumor clones, were different in FL samples compared to healthy control samples, or in late relapsed/transformed FL samples compared to earlier ones. Our analysis revealed differences in the distribution of mutations from each of the nucleotides when tumor and non-tumor clones were compared, while FL and transformed FL (t-FL) tumor clones displayed similar mutation distributions. Lineage tree measurements suggested that either initial clone affinity or selection thresholds were lower in FL samples compared to controls, but similar between FL and t-FL samples. Finally, we observed that both FL and t-FL tumor clones tend to accumulate larger numbers of potential N-glycosylation sites due to the introduction of new SHM. Taken together, these results suggest that transformation into t-FL, in contrast to initial FL development, is not associated with any major changes in DNA targeting or repair, or the selection threshold of the tumor clone.

Mutational features of immunoglobulin heavy chain variable region gene in patients with chronic lymphocytic leukemia

Objective: To investigate the mutational features of the immunoglobulin heavy chain variable region (IgHV) gene in patients with chronic lymphocytic leukemia (CLL) using immunophenotypic and molecular genetic methods. Methods: The laboratory results of 266 CLL patients who underwent IgHV gene examination at Sino-US diagnostics laboratory from February 2020 to February 2021 were analyzed for the IgVH mutational status and presence of specific IgVH fragments. In addition, their immunophenotypic, molecular, chromosomal karyotypic, and FISH profiles were investigated and correlated with the IgVH mutational status. Results: Among 266 patients, 172 were male and 94 were female, with a media age of 67 years (20-82 years).There were more patients with mutated IgHV (m-IgHV) than unmutated IgHV (un-IgHV) (69.2%∶30.8%). There was association of VH family and the presence of gene fragments: the overall incidence of VH families including VH3 family (142/266, 53.4%), VH4 family (75/266, 28.2%), and VH1 family (34/266, 12.8%) was about 95%, among which the proportion of VH4-34 (26/266, 9.8%), VH3-23 (25/266, 9.4%), VH3-7 (24/266, 9.0%), and VH4-39 (16/266, 6.0%) was about 35%. VH3-20 and VH3-49 only occurred in un-IgHV (P<0.05). In addition, the expression rates of CD38 (26.3% vs. 3.0%), CD79b (71.1%∶45.5%) and 11q deletion (25.5%∶5.3%) were higher in un-IgHV, and single trisomy 12 (37.9%∶5.6%) were more commonly found in m-IgHV (P<0.05). MYD88 was one of the major mutation genes in m-IgHV, while ATM had the highest mutation rate in un-IgHV. Conclusion: CLL patients have differential expression in terms of IgHV gene mutations, correlating to their immunophenotype and genetics characteristics.

Autoimmune cytopenias in chronic lymphocytic leukemia: a growing challenge in targeted therapies?

As the world’s population ages, the incidence of chronic lymphocytic leukemia (CLL) will continue to increase. CLL-related autoimmune cytopenias (AICs) are becoming a growing challenge in daily clinical practice. AICs occur in c.10% of CLL patients, and include autoimmune hemolytic anemia, immune thrombocytopenia, pure red cell aplasia, and autoimmune granulocytopenia. The complication can appear at any disease stage, both in treated and previously untreated patients. In some cases, AICs precede the diagnosis of the underlying disease. The diagnostic process of autoimmune complications is often difficult. First of all, however, it requires differentiation from bone marrow infiltration resulting from underlying disease progression. A properly established diagnosis has prognostic and therapeutic implications. Autoimmune cytopenias are more often associated with high-risk CLL (with 17p deletion, 11q deletion, wild-type variant of gene encoding immunoglobulin heavy chain variable region) and show a complex pathogenesis. CLL cells and the surrounding microenvironment are involved in autoimmune mechanisms. Treatment of AICs depends on CLL stage. In the case of isolated symptoms of AICs, without accompanying features of CLL progression, glucocorticosteroids or rituximab are recommended in the first line treatment. No response or a suboptimal response to treatment implies further therapeutic decisions. According to significant advances in the treatment of chronic lymphocytic leukemia, therapeutic strategies for autoimmune cytopenias also need to be optimized. The widespread introduction of ibrutinib, idelalisib, and venetoclax has highlighted the need for understanding the interplay between targeted therapies and AICs.

Glycosphingolipids form characteristic-sized liposomes that correlate with their antibody-inducing activities in mice

Immunization of mice with liposomes consisting of dipalmitoylphosphatidylcholine, cholesterol, lipid A, and glycosphingolipids (GSLs) can efficiently induce the production of antibodies that recognize specific GSLs. Here, we analyzed the effect of GSL species on the particle sizes of GSL-containing liposomes. We prepared liposomes containing Gb4Cer/globoside, GM3, and several artificial GSLs, and analyzed their particle sizes in phosphate-buffered saline by dynamic light scattering. The particle sizes of liposomes were significantly altered by the addition of GSLs, and they formed 65- to 1737-nm particle sizes depending on their constituent GSL species. We compared the sizes of each GSL-containing liposome with the IgM- or IgG-inducing activity of these liposomes in mice, and found a positive correlation between increasing liposome size and IgG-inducing activity. We also determined the nucleotide sequences of the heavy and light chain variable regions of anti-Gb4Cer IgM and IgG3 obtained from the Gb4Cer-containing liposome-immunized mice, and found that they were composed of different gene segments. This result indicates that the GSL-containing liposomes induce the production of IgG3 through an immune pathway different from that of IgM, rather than efficiently inducing class switching.

Unravelling soluble immune checkpoints in chronic lymphocytic leukemia: Physiological immunomodulators or immune dysfunction.

Chronic lymphocytic leukemia (CLL) is a lymphoid neoplasm characterized by the accumulation of mature B cells. The diagnosis is established by the detection of monoclonal B lymphocytes in peripheral blood, even in early stages [monoclonal B-cell lymphocytosis (MBLhi)], and its clinical course is highly heterogeneous. In fact, there are well-characterized multiple prognostic factors that are also related to the observed genetic heterogenicity, such as immunoglobulin heavy chain variable region (IGHV) mutational status, del17p, and TP53 mutations, among others. Moreover, a dysregulation of the immune system (innate and adaptive immunity) has been observed in CLL patients, with strong impact on immune surveillance and consequently on the onset, evolution, and therapy response. In addition, the tumor microenvironment is highly complex and heterogeneous (i.e., matrix, fibroblast, endothelial cells, and immune cells), playing a critical role in the evolution of CLL. In this study, a quantitative profile of 103 proteins (cytokines, chemokines, growth/regulatory factors, immune checkpoints, and soluble receptors) in 67 serum samples (57 CLL and 10 MBLhi) has been systematically evaluated. Also, differential profiles of soluble immune factors that discriminate between MBLhi and CLL (sCD47, sCD27, sTIMD-4, sIL-2R, and sULBP-1), disease progression (sCD48, sCD27, sArginase-1, sLAG-3, IL-4, and sIL-2R), or among profiles correlated with other prognostic factors, such as IGHV mutational status (CXCL11/I-TAC, CXCL10/IP-10, sHEVM, and sLAG-3), were deciphered. These results pave the way to explore the role of soluble immune checkpoints as a promising source of biomarkers in CLL, to provide novel insights into the immune suppression process and/or dysfunction, mostly on T cells, in combination with cellular balance disruption and microenvironment polarization leading to tumor escape.

Gastric DLBCL clonal evolution as function of patient age.

Diffuse large B cell lymphoma (DLBCL) is the most common type of NHL, accounting for about 40% of NHL cases, and is one of the most aggressive lymphomas. DLBCL is widespread in individuals aged more than 50 years old, with a maximum incidence in the seventh decade, but it may also occur in younger patients. DLBCL may occur in any immune system tissue, including those around the gastrointestinal tract, and even in the stomach, though gastric DLBCL has yet to be sufficiently investigated. This study aimed to understand changes in gastric Diffuse Large B cell lymphoma (gastric DLBCL) development with age. Immunoglobulin (Ig) heavy chain variable region genes were amplified from sections of nine preserved biopsies, from patients whose age varied between 25 and 89 years, sequenced and analyzed. We show first that identification of the malignant clone based on the biopsies is much less certain than was previously assumed; and second that, contrary to expectations, the repertoire of gastric B cell clones is more diverse among the elderly DLBCL patients than among the young.

Molecular map of chronic lymphocytic leukemia and its impact on outcome.

Recent advances in cancer characterization have consistently revealed marked heterogeneity, impeding the completion of integrated molecular and clinical maps for each malignancy. Here, we focus on chronic lymphocytic leukemia (CLL), a B cell neoplasm with variable natural history that is conventionally categorized into two subtypes distinguished by extent of somatic mutations in the heavy-chain variable region of immunoglobulin genes (IGHV). To build the 'CLL map,' we integrated genomic, transcriptomic and epigenomic data from 1,148 patients. We identified 202 candidate genetic drivers of CLL (109 new) and refined the characterization of IGHV subtypes, which revealed distinct genomic landscapes and leukemogenic trajectories. Discovery of new gene expression subtypes further subcategorized this neoplasm and proved to be independent prognostic factors. Clinical outcomes were associated with a combination of genetic, epigenetic and gene expression features, further advancing our prognostic paradigm. Overall, this work reveals fresh insights into CLL oncogenesis and prognostication.

Clonally unrelated Richter syndrome are truly de novo diffuse large B-cell lymphomas with a mutational profile reminiscent of clonally related Richter syndrome.

SummaryRichter syndrome (RS) is mostly due to the direct transformation of the chronic lymphocytic leukaemia (CLL) clone, as documented by the same immunoglobulin heavy‐chain variable region (IGHV) rearrangement in both CLL and RS cells. In rare cases characterized by a better outcome, the RS clone harbours a different IGHV rearrangement compared to the CLL phase. We investigated the CLL phase of clonally unrelated RS to test whether the RS clone was already identifiable prior to clinicopathologic transformation, albeit undetectable by conventional approaches. CLL cells of eight patients with unrelated RS were subjected to an ultra‐deep next‐generation sequencing (NGS) approach with a sensitivity of 10−6. In 7/8 cases, the RS rearrangement was not identified in the CLL phase. In one case, the RS clone was identified at a very low frequency in the CLL phase, conceivably due to the concomitance of CLL sampling and RS diagnosis. Targeted resequencing revealed that clonally unrelated RS carries genetic lesions primarily affecting the TP53, MYC, ATM and NOTCH1 genes. Conversely, mutations frequently involved in de novo diffuse large B‐cell lymphoma (DLBCL) without a history of CLL were absent. These results suggest that clonally unrelated RS is a truly de novo lymphoma with a mutational profile reminiscent, at least in part, of clonally related RS.

Takotsubo cardiomyopathy or hidden cardiotoxic event by rituximab

Rituximab is a genetically engineered chimeric mouse/human monoclonal antibody representing a glycosylated immunoglobulin (Ig) with human IgG1 constant regions and murine light-chain and heavy-chain variable region sequences. The antibody is produced by mammalian cell suspension culture and purified

Characterization of the Intra-tumoral B Cell Immunoglobulin Repertoire Is of Prognostic Value for Esophageal Squamous Cell Carcinoma.

Esophageal Squamous Cell carcinomas (ESCC) is a highly heterogeneous malignancy that is among the leading cause of cancer-related death worldwide. B cells play pivotal roles in the immune defense system and cancer progression and regression, yet the repertoire of tumor infiltrating B cells (TIBs) and its association with clinical outcome remains unexplored in ESCC. Here we collected bulk RNA-seq sequencing data from 119 ESCC tumors and matched adjacent normal samples to delineate the B cell repertoire. We found that ESCC is more heavily infiltrated by B cells and plasma cells compared to activated T cells. The immunoglobulin heavy chain variable region (IGHV) gene usage was remarkably biased and IGHV3-74 was under-represented in ESCC tumors. The TIBs showed a more oligoclonal profile along with widespread clonal expansion and IgG subclass switch events (CSRs). Survival analysis revealed several unexpected associations between tumor infiltrating B cells and prognosis. Higher levels of immunoglobulin expression (IGH), CD138 expression, IGH to MS4A1 ratio, CSR events and clone diversity are all associated with better survival. Notably, we found that the abundance of CD20-negative IgG2-producing plasma cells has a strong positive effect on overall survival with a hazard ratio (HR) of 0.40 (log-rank p: 0.002). Combing molecular subtyping, the IgG2-producing plasma cells could stratify high-risk patients more accurately with a HR of 0.253 (log-rank p: 0.0006). The direct link between protective B cell populations and ESCC prognosis provides biomarkers for high-risk patient selection and holds great promise for developing strategies for immunotherapy targeting B cells in ESCC patients.