Immunoglobulin Heavy Chain Gene Research Articles

Inactivated whole influenza virus particle vaccines induce neutralizing antibodies with an increase in immunoglobulin gene subclones of B-lymphocytes in cynomolgus macaques

The All-Japan Influenza Vaccine Study Group has been developing a more effective vaccine than the current split vaccines for seasonal influenza virus infection. In the present study, the efficacy of formalin- and/or β-propiolactone-inactivated whole virus particle vaccines for seasonal influenza was compared to that of the current ether-treated split vaccines in a nonhuman primate model. The monovalent whole virus particle vaccines or split vaccines of influenza A virus (H1N1) and influenza B virus (Victoria lineage) were injected subcutaneously into naïve cynomolgus macaques twice. The whole virus particle vaccines induced higher titers of neutralizing antibodies against H1N1 influenza A virus and influenza B virus in the plasma of macaques than did the split vaccines. At challenge with H1N1 influenza A virus or influenza B virus, the virus titers in nasal swabs and the increases in body temperatures were lower in the macaques immunized with the whole virus particle vaccine than in those immunized with the split vaccine. Repertoire analyses of immunoglobulin heavy chain genes demonstrated that the number of B-lymphocyte subclones was increased in macaques after the 1st vaccination with the whole virus particle vaccine, but not with the split vaccine, indicating that the whole virus particle vaccine induced the activation of vaccine antigen-specific B-lymphocytes more vigorously than did the split vaccine at priming. Thus, the present findings suggest that the superior antibody induction ability of the whole virus particle vaccine as compared to the split vaccine is attributable to its stimulatory properties on the subclonal differentiation of antigen-specific B-lymphocytes.

IgG kappa multiple myeloma with isolated central nervous system relapse.

A 76-year-old male presented with acute renal failure and cast nephropathy was diagnosed with IgG kappa multiple myeloma (MM). His paraprotein was 40 g/L, kappa-free light chain level was 17,058 mg/L, kappa: lambda light chain ratio was 1445. An automated full blood count showed hemoglobin 90 g/L, white blood cells 2.96 × 109/L, neutrophil 1.7 × 109/L, and platelet 179 × 109/L. Bone marrow trephine sample was hypercellular with nearly 100% plasma cells, which were positive for CD138, CD56, and cyclin D1, and showed kappa light chain restriction. Fluorescence in situ hybridization analysis with dual color/dual probes for immunoglobulin heavy chain (IGH) and MYC genes (Cytocell) and whole genome screening using 8 × 60K oligonucleotide arrays (Agilent) performed on the CD138 enriched cells (CD138 microbeads Miltenyi Biotech) from the liquid bone marrow sample showed no evidence of IGH rearrangement, MYC rearrangement, 1q gain, 1p loss, 17p loss, or any clinically significant imbalance. He was treated with eight cycles of bortezomib, cyclophosphamide, and dexamethasone (VCD) to very good partial response. He relapsed 30 months after diagnosis with paraprotein of 36 g/L, kappa light chains of 10,000 mg/L, and creatinine of 498 μmol/L. He was treated with eight cycles of bortezomib, thalidomide, and dexamethasone (VTD) to paraprotein undetectable and kappa light chain 29 mg/L. Seven months post completion of VTD (4 years after myeloma diagnosis), he presented with abnormal gait, falls, and a left-sided facial droop. His paraprotein was 2 g/L and serum-free kappa light chain was 81 mg/L. Magnetic resonance imaging (MRI) head and whole spine showed multifocal diseases in spinal canal and intracranially, involving the cranial nerves most prominent the fifth, seventh, eighth, and hypoglossal nerve on the left, anterior surface of the inferior pons medulla (Figure 1A). There were multiple lesions in the cervical spine as well as in the thoracic spine (Figure 1B). Computerized tomography (CT) chest abdomen pelvis did not show the evidence of other new diseases. Plasma cells were detected in cerebral spinal fluid (CSF) (Figure 1C, May–Grünwald–Giemsa stain x100 objective). He was treated with pomalidomide and dexamethasone. Weekly cytarabine intrathecal (IT) treatment was given until the clearance of plasma cells from CSF samples for three times. Four months later, he achieved the complete remission on MRI head and whole spine with undetectable paraprotein and normal light chain ratio. Serial MRI scans confirmed ongoing remission. A year later, he relapsed again with worsening mobility and reappearance of myeloma cells in CSF. By flow cytometry, these cells were positive for CD138, CD38, CD56, CD117, and negative for CD19. MRI head showed leptomeningeal disease with a new 2.2 cm frontal lobe lesion. His paraprotein remained undetectable and kappa light chain remained at below 100 mg/L. He continued systemic pomalidomide and dexamethasone chemotherapy and received palliative whole brain radiotherapy 20 Gy in 5 fractions for symptom control. His disease continued progressing and he passed away 3 months later (5.5 years after myeloma diagnosis). Here, we reported a myeloma patient with isolated CNS relapse, who achieved 1 year of remission with combination of intrathecal chemotherapy and systemic pomalidomide and dexamethasone treatment, but sadly passed away 4 months after second CNS relapse. CNS MM is a rare form of extramedullary disease, but carries a very poor prognosis. The optimal approach to treatment of CNS MM is not currently known. The current approach includes systemic therapy with agents known to cross the blood brain barrier, intrathecal therapy, and CNS irradiation. Innovative approach to treatment is urgently needed. The authors received no specific funding for this work. Ke Xu and Lucy Kamuriwo wrote the manuscript. Ke Xu, Lucy Kamuriwo, Claire Anderson and Parag Jasani critically revised the final version of the manuscript. The authors declare they have no conflicts of interest. No written or verbal consent obtained from patient. Patient has passed away.

Landscape of immunoglobulin heavy chain gene repertoire and its clinical relevance to LPL/WM

AbstractLymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) is a heterogeneous disease in which the role of immunoglobulin heavy-chain genes (IGHs) remains unknown. To determine the clinical relevance of the IGH repertoire in patients with LPL/WM, we performed immunoglobulin gene rearrangement and complementarity determining region 3 (CDR3) analysis. The IGH variable gene (IGHV) repertoire was remarkably biased in LPL/WM. IGHV3-23, IGHV4-34, IGHV3-30, IGHV3-7, and IGHV3-74 accounted for one-half of the cohort's repertoire. Most cases (97.1%) were found to carry mutated IGHV genes, based on a 98% IGHV germline homology cutoff. IGHV3-30 was associated with long heavy chain CDR3, indicating there was specific antigen selection in LPL/WM. Patients with IGHV3-7 were significantly more likely to harbor the 6q deletion (P < .001) and an abnormal karyotype (P = .004). The IGHV hypermutation rate in patients with the MYD88 L265P mutation was significantly higher than that of wild-type patients (P = .050). IGHV3-23 and IGHV3-74 segments were more frequently detected in patients with MYD88-mutated LPL/WM (P = .050), whereas IGHV3-7 presented more frequently in MYD88 wild-type patients (P = .042). Patients with IGHV4, especially IGHV4-34, had higher levels of lactate dehydrogenase, and IGHV4 was a predictive marker of shorter progression-free survival. These results showed for the first time that the IGHV repertoire has clinical relevance in LPL/WM.

Human activation-induced deaminase lacks strong replicative strand bias or preference for cytosines in hairpin loops.

Activation-induced deaminase (AID) is a DNA-cytosine deaminase that mediates maturation of antibodies through somatic hypermutation and class-switch recombination. While it causes mutations in immunoglobulin heavy and light chain genes and strand breaks in the switch regions of the immunoglobulin heavy chain gene, it largely avoids causing such damage in the rest of the genome. To help understand targeting by human AID, we expressed it in repair-deficient Escherichia coli and mapped the created uracils in the genomic DNA using uracil pull-down and sequencing, UPD-seq. We found that both AID and the human APOBEC3A preferentially target tRNA genes and transcription start sites, but do not show preference for highly transcribed genes. Unlike A3A, AID did not show a strong replicative strand bias or a preference for hairpin loops. Overlapping uracilation peaks between these enzymes contained binding sites for a protein, FIS, that helps create topological domains in the E. coli genome. To confirm whether these findings were relevant to B cells, we examined mutations from lymphoma and leukemia genomes within AID-preferred sequences. These mutations also lacked replicative strand bias or a hairpin loop preference. We propose here a model for how AID avoids causing mutations in the single-stranded DNA found within replication forks.

Function of ATM and MSH2 during nonhomologous end joining in DNA repair

Abstract Class switch recombination (CSR) produces secondary immunoglobulin isotypes and requires AID-dependent DNA deamination of intronic switch (S) regions within the immunoglobulin heavy chain gene locus. Non-canonical repair of deaminated DNA by mismatch repair (MMR) or base excision repair (BER) creates DNA breaks that permit recombination between distal S regions. ATM-dependent phosphorylation of AID at serine-38 (pS38-AID) promotes its interaction with APE1, a BER protein, suggesting that ATM regulates CSR through BER. However, pS38-AID may also play a role in MMR during CSR, although the mechanism remains unknown. To examine whether ATM modulates BER- and/or MMR-dependent CSR, ATM−/− mice were bred to mice deficient for the MMR gene MSH2. Surprisingly, the predicted Mendelian frequencies of ATM−/−MSH2−/− adult mice were not observed. To obtain ATM- and MSH2-deficient B cells, ATM was conditionally deleted on an MSH2−/− background using a floxed ATM allele [ATMF] and B cell-specific Cre recombinase expression (CD23-cre) to generate a deleted ATM allele (ATMD). As compared to the ATMD/D and MSH2−/− mice and B cells, the ATMD/DMSH2−/− mice and B cells display a reduced CSR phenotype. Interestingly, Sμ-Sγ1 junctions from ATMD/DMSH2−/− B cells that were induced to switch to IgG1 in vitro revealed a significant loss of blunt end joins and an increase in insertions as compared to wildtype, ATMD/D, or MSH2−/− B cells. This data suggests that the absence of both ATM and MSH2 blocks NHEJ and leads to inefficient end joining and the reduced CSR. We identify complementary roles for ATM and MSH2 in NHEJ and A-EJ during CSR and propose a model whereby ATM and MSH2 function cooperatively to regulate end-joining during CSR through pS38-AID. This work was supported by The National Institute on Minority Health and Health Disparities (5G12MD007603), The National Cancer Institute (2U54CA132378), and The National Institute of General Medical Sciences (1SC1GM132035-01).

Aberrant HSP90 Expression in Lymphocytes and HSP90 Response to Anti-PD-1 Therapy in Lymphoma Patients.

HSP90 family of molecular chaperones has been shown to be implicated in various stages of tumor growth and development. Recent studies have highlighted the role of extracellular HSP90 in tumor immunology, however, the role that HSP90 plays in the regulation of immune responses and the impact of cancer immunotherapy, including immune checkpoint blockade, on HSP90 is still unclear. Here we assessed the surface and intracellular expression of constitutive cytosolic HSP90β isoform, mitochondrial HSP90 homolog TRAP1 and co-chaperone STIP1/HOP in T, NK, B and NKT cells derived from peripheral blood and bone marrow samples of patients with Hodgkin and B-cell Non-Hodgkin lymphomas. HSP90β and STIP1 were overexpressed in B lymphocytes, while TRAP1 expression was decreased in T, B, NK and NKT cells of lymphoma patients. HSP90 overexpression in B cells was not associated with malignant B cell clones, since no clonotypic B cells were detected by immunoglobulin heavy chain (IgH) gene rearrangements. PD-1 blockade was found to differently affect the intracellular and surface HSP90 in T, B, NK and NKT cells in patients with relapsed or refractory classical Hodgkin lymphoma. Modulating HSP90 was found to affect the NK cell degranulation response and IFNγ production in lymphoma patients. These findings provide the rationale to further explore HSP90 homologs for improving patient response to cancer immunotherapy.

Diagnostic Accuracy of Cell Block Preparations and Clinical Features Affecting It in Vitreoretinal Lymphoma.

Purpose: The purpose of this study was to examine the diagnostic accuracy of the cell block (CB) method and clinical features affecting it in patients with vitreoretinal lymphoma (VRL). Methods: This study enrolled 38 eyes in 33 VRL patients, and 7 eyes in 7 patients with idiopathic uveitis who underwent diagnostic vitrectomy. Medical records including the results of CB cytology, interleukin (IL)-10/-6 concentrations, and immunoglobulin heavy chain gene (IgH) rearrangement were retrospectively searched. Results: Patients with VRL comprised 16 women and 17 men, and the age of onset ranged from 44 to 85 years (mean: 70 years). CB preparations detected large malignant cells in 35 eyes (92%), whereas the other 3 VRL eyes were negative. Two of the latter three eyes showed subretinal infiltrates, which existed in 7 of 35 CB-positive eyes. Intravitreal IL-10 and -6 concentrations were 1866 ± 4088 pg/mL and 98 ± 139 pg/mL, respectively, and the rate of IL-10/-6 >1 was 86.9%. The presence of IgH monoclonality was 63.2%. In patients with uveitis, CB specimens revealed no atypical but small inflammatory cells. IL-6 concentration was 311.1 ± 240 pg/mL, whereas IL-10 was undetectable in six eyes, and the IL-negative rate was 85.7%. Six eyes (85.7%) with uveitis showed no IgH monoclonality. Conclusions: Diagnostic accuracy of CB preparations in VRL could achieve an equivalent outcome to IL ratio calculation and IgH monoclonality detection. The appearance of subretinal infiltrates may diminish the CB positivity.

Morphologic and molecular analysis of Richter syndrome in chronic lymphocytic leukaemia patients treated with ibrutinib or venetoclax.

Richter syndrome (RS) represents the development of high-grade lymphoma in patients with chronic lymphocytic leukaemia (CLL) or small lymphocytic lymphoma (SLL) and presents a diagnostic and therapeutic challenge with an adverse prognosis. The genetic background and morphology of RS in CLL patients treated with chemoimmunotherapy is extensively characterised; however, our knowledge about RS in patients treated with targeted oral therapies should be extended. To understand the morphologic and molecular changes leading to RS in CLL patients treated with the Bruton's tyrosine kinase inhibitor, ibrutinib, and the BCL2 inhibitor, venetoclax, sequential samples from six CLL/SLL patients undergoing RS were collected in both the CLL and RS phases. A detailed immunophenotypic analysis of formalin-fixed, paraffin-embedded tissue specimens of RS phase was performed, followed by extensive molecular characterisation of CLL and RS samples, including the immunoglobulin heavy chain gene (IGH) rearrangement, TP53 mutations, drug-induced resistance mutations in BTK and BCL2 genes and various copy number changes and point mutations detectable with multiplex ligation-dependent probe amplification (MLPA). Rare, non-diffuse large B-cell lymphoma phenotypes of RS were observed in 3/6 cases, including plasmablastic lymphoma and a transitory entity between diffuse large B-cell lymphoma and classical Hodgkin lymphoma. The majority of cases were clonally related and harboured an unmutated variable region of the immunoglobulin heavy chain gene. Abnormalities affecting the TP53 gene occurred in all patients, and every patient carried at least one genetic abnormality conferring susceptibility to RS. In the background of RS, 2/5 patients treated with ibrutinib showed a BTK C481S resistance mutation. One patient developed a BCL2 G101V mutation leading to venetoclax resistance and RS. In conclusion, our findings contribute to better understanding of RS pathogenesis in the era of targeted oral therapies. Rare phenotypic variants of RS do occur under the treatment of ibrutinib or venetoclax, and genetic factors leading to RS are similar to those identified in patients treated with chemoimmunotherapy. To our best knowledge, we have reported the first BCL2 G101V mutation in an RS patient treated with venetoclax.

Diagnosing Vitreoretinal Lymphomas-An Analysis of the Sensitivity of Existing Tools.

Simple SummaryDiagnostics of vitreoretinal lymphoma is very challenging, as the possibility of receiving false negative results is common. We retrospectively analyzed the sensitivity of the most commonly used diagnostic methods including ancillary immunohistochemistry, Myeloid Differentiation Factor 88 (MyD88) L256P mutation analysis, polymerase chain reaction (PCR) for monoclonal rearrangements of immunoglobulin heavy chain (IgH) and T-cell Receptor (TCR) genes, flow cytometry, and IL10 and IL6 analysis, to diagnose vitreoretinal lymphomas from published data in the literature. MyD88 mutation analysis caused by a hotspot mutation in MyD88 was the most sensitive and had the lowest coefficient of variation.Vitreoretinal lymphoma (VRL) is a rare ocular pathology that is notorious for mimicking chronic uveitis, which is a seemingly benign condition in comparison. The most common form of VRL is the diffuse large B-cell type, and there has been a high mortality rate. This dismal prognosis can be improved significantly if the disease is diagnosed early, but until now there is no consensus on an appropriate diagnostic algorithm. We conducted a retrospective search of PubMed Central® and analyzed results from thirty-three studies that were published between 2011–2021. The chosen studies incorporated some popular testing tools for VRL, and our analyses focused on comparing the average sensitivity of five diagnostic methods. The methods included cytology including ancillary immunohistochemistry, Myeloid Differentiation Factor 88 (MyD88) mutation analysis, polymerase chain reaction (PCR) for monoclonal rearrangements of immunoglobulin heavy chain (IgH) and T-cell Receptor (TCR) genes, flow cytometry, and IL10 and IL6 analysis. Across the varied diagnostic methods employed in thirty-three studies explored in this analysis, MyD88 mutation assay emerged as a strong contender given its sensitivity and low coefficient of variation. There is an imminent need for the introduction of newer assays that can further improve the sensitivity of identifying MyD88 mutation in cancer cells seen in the vitreous.

Diagnostic Accuracy and Prognostic Relevance of Immunoglobulin Heavy Chain Rearrangement and 18F-FDG-PET/CT Compared With Unilateral Bone Marrow Trephination for Detecting Bone Marrow Involvement in Patients With Diffuse Large B-Cell Lymphoma.

BackgroundIn diffuse large B-cell lymphoma (DLBCL), bone marrow involvement (BMI) has an important clinical implication as a component of staging and International Prognostic Index. This study aimed to determine whether molecular analysis of immunoglobulin heavy chain (IgH) genes and positron emission tomography-computed tomography (PET/CT) could overcome the limitation of defining morphologic BMI by trephination biopsy and could increase the diagnostic accuracy or prognostic prediction.MethodsA total of 94 de novo patients with DLBCL underwent PET/CT, polymerase chain reaction (PCR) test for detection of IgH gene rearrangement, and unilateral bone marrow (BM) trephination at diagnosis.ResultsA total of 9 patients (9.6%) were confirmed to present morphologic BMI (mBMI) based on trephination biopsy. On the other hand, 21 patients (22.3%) were confirmed to have IgH clonality (IgH BMI), while 16 (17.0%) were classified with BMI based on the assessment of PET/CT (PET BMI). Each IgH rearrangement PCR and PET/CT showed the high negative predictive value of detecting the BMI. However, the combined assessment of IgH rearrangement and PET/CT could increase the diagnostic accuracy and specificity with 87.2% and 97.0%, respectively. The survival outcome of patients with double positive PET BMI and IgH BMI was significantly worse than that with either single positive PET BMI or IgH BMI, and even less than patients with neither PET BMI nor IgH BMI (3-year PFS: 50.0% vs. 75.4% vs. 97.9%, P = 0.007, 3-year OS: 50.0% vs. 75.6% vs. 80.1%, P = 0.035, respectively).ConclusionThis study suggests that the combined evaluation of PET/CT and IgH rearrangement could give additional information for predicting therapeutic outcomes in patients with negative morphologic BMI as an important part of the prognosis.

Next-Generation Sequencing-Based Clonality Detection of Immunoglobulin Gene Rearrangements in B-Cell Lymphoma.

Immunoglobulin (IG) clonality assessment is a widely used supplementary test for the diagnosis of suspected lymphoid malignancies. The specific rearrangements of the immunoglobulin (IG) heavy and light chain genes act as a unique hallmark of a B-cell lymphoma, a feature that is used in clonality assessment. The widely used BIOMED-2/EuroClonality IG clonality assay, visualized by GeneScanning or heteroduplex analysis, has an unprecedented high detection rate because of the complementarity of this approach. However, the BIOMED-2/EuroClonality clonality assays have been developed for the assessment of specimens with optimal DNA quality. Further improvements for the assessment of samples with suboptimal DNA quality, such as from formalin-fixed paraffin-embedded (FFPE) specimens or specimens with a limited tumor burden, are required. The EuroClonality-NGS Working Group recently developed a next-generation sequencing (NGS)-based clonality assay for the detection of the IG heavy and kappa light chain rearrangements, using the same complementary approach as in the conventional assay. By employing next-generation sequencing, both the sensitivity and specificity of the clonality assay have increased, which not only is very useful for diagnostic clonality testing but also allows robust comparison of clonality patterns in a patient with multiple lymphoma's that have suboptimal DNA quality. Here, we describe the protocols for IG-NGS clonality assessment that are compatible for Ion Torrent and Illumina sequencing platforms including pre-analytical DNA isolation, the analytical phase, and the post-analytical data analysis.

Expression of lipoprotein lipase and SORL1 genes in patients with chronic lymphocytic leukemia

Introduction. Leukemic cells of patients with chronic lymphocytic leukemia (CLL) are characterized by high expression of the lipoprotein lipase (LPL) gene in unmutated (UM) status of the variable region of the immunoglobulin heavy chain (IGHV) genes and low expression of the SORL1 gene. SORL1 protein promotes the degradation of LPL in nervous cells in vitro that has been previously shown. Objective: to study SORL1 gene expression in CLL patients depending on LPL gene expression and mutational status of IGHV genes. Materials and methods. Analysis was performed in the group of 61 CLL patients. The IGHV gene mutational status was studied by polymerase chain reaction (PCR) followed by direct sequencing. LPL and SORL1 expression was evaluated by Quantitative Real-time PCR. Results. Relative LPL expression levels in CLL samples ranged from 0.5 to 119.5 (mean 23.65 ± 5.19) and correlated with IGHV mutational status (p &lt; 0.01). The average relative SORL1 expression level was 1.71 ± 0.55. No association between SORL1 expression and IGHV mutational status was found (p = 0.358). Among unmutated IGHV cases, negative correlation between LPL and SORL1 gene expression levels was identified (r = -0.764; p = 0.036). Conclusion. The obtained data support the involvement of SORL1 in the post-translational regulation of LPL levels in leukemic cells in CLL. Ketwords: chronic lymphocytic leukemia, lipoprotein lipase, SORL1.

Mcl-1 and Bcl-xL levels predict responsiveness to dual MEK/Bcl-2 inhibition in B-cell malignancies.

Most patients with chronic lymphocytic leukemia (CLL) initially respond to targeted therapies, but eventually relapse and develop resistance. Novel treatment strategies are therefore needed to improve patient outcomes. Here, we performed direct drug testing on primary CLL cells and identified synergy between eight different mitogen‐activated protein kinase kinase (MEK) inhibitors and the B‐cell lymphoma 2 (Bcl‐2) antagonist venetoclax. Drug sensitivity was independent of immunoglobulin heavy‐chain gene variable region (IGVH) and tumor protein p53 (TP53) mutational status, and CLL cells from idelalisib‐resistant patients remained sensitive to the treatment. This suggests that combined MEK/Bcl‐2 inhibition may be an option for high‐risk CLL. To test whether sensitivity could be detected in other B‐cell malignancies, we performed drug testing on cell line models of CLL (n = 4), multiple myeloma (MM; n = 8), and mantle cell lymphoma (MCL; n = 7). Like CLL, MM cells were sensitive to the MEK inhibitor trametinib, and synergy was observed with venetoclax. In contrast, MCL cells were unresponsive to MEK inhibition. To investigate the underlying mechanisms of the disease‐specific drug sensitivities, we performed flow cytometry‐based high‐throughput profiling of 31 signaling proteins and regulators of apoptosis in the 19 cell lines. We found that high expression of the antiapoptotic proteins myeloid cell leukemia‐1 (Mcl‐1) or B‐cell lymphoma‐extra large (Bcl‐xL) predicted low sensitivity to trametinib + venetoclax. The low sensitivity could be overcome by combined treatment with an Mcl‐1 or Bcl‐xL inhibitor. Our findings suggest that MEK/Bcl‐2 inhibition has therapeutic potential in leukemia and myeloma, and demonstrate that protein expression levels can serve as predictive biomarkers for treatment sensitivities.

Combined detection of lymphocyte clonality and MALT1 translocations in bronchoalveolar lavage fluid for diagnosing pulmonary lymphomas

Diagnosis of pulmonary lymphoma using small tissue samples is difficult and often requires surgical procedures; thus, a less invasive sampling method is desirable. We previously showed that pulmonary mucosa-associated lymphoid tissue (MALT) lymphoma can be diagnosed by detecting MALT lymphoma translocation gene 1 (MALT1) translocations in bronchoalveolar lavage fluid (BALF) cells. Analysis of B-cell clonality based on immunoglobulin heavy chain (IGH) gene rearrangements was also reportedly useful for diagnosing pulmonary lymphoma. The aim of this prospective multicenter study was to evaluate the yet unknown diagnostic potential of combined detection of MALT1 translocations and clonality using BALF. We analyzed B- and T-cell clonality based on IGH and T-cell receptor (TCR) rearrangements together with MALT1 translocations using BALF of patients with clinically suspected pulmonary lymphomas. In total, 39 patients were evaluated and categorized into three groups: B-cell lymphoma, lymphoproliferative disorders, and other diseases. IGH rearrangement detection for B-cell lymphoma diagnosis exhibited sensitivity and specificity of 88.9% and 90.0%, respectively. TCR rearrangements were not observed in patients with B-cell lymphomas. The presence of IGH rearrangements together with the absence of TCR rearrangements indicated 96.0% specificity for the diagnosis of B-cell lymphoma. The sensitivity and specificity of MALT1 translocations for diagnosing MALT lymphoma were 28.6% and 100%, respectively. The combined detection of lymphocyte clonality and MALT1 translocations using BALF is suitable for screening and diagnosis of B-cell lymphomas. Analysis of specific genes such as MALT1 should improve the precision of B-cell lymphoma diagnosis.

From IgZ to IgT: A Call for a Common Nomenclature for Immunoglobulin Heavy Chain Genes of Ray-Finned Fish

From IgZ to IgT: A Call for a Common Nomenclature for Immunoglobulin Heavy Chain Genes of Ray-Finned Fish

IRF4 Has a Unique Role in Early B Cell Development and Acts Prior to CD21 Expression to Control Marginal Zone B Cell Numbers.

Strict control of B lymphocyte development is required for the ability to mount humoral immune responses to diverse foreign antigens while remaining self-tolerant. In the bone marrow, B lineage cells transit through several developmental stages in which they assemble a functional B cell receptor in a stepwise manner. The immunoglobulin heavy chain gene is rearranged at the pro-B stage. At the large pre-B stage, cells with a functional heavy chain expand in response to signals from IL-7 and the pre-BCR. Cells then cease proliferation at the small pre-B stage and rearrange the immunoglobulin light chain gene. The fully formed BCR is subsequently expressed on the surface of immature B cells and autoreactive cells are culled by central tolerance mechanisms. Once in the periphery, transitional B cells develop into mature B cell subsets such as marginal zone and follicular B cells. These developmental processes are controlled by transcription factor networks, central to which are IRF4 and IRF8. These were thought to act redundantly during B cell development in the bone marrow, with their functions diverging in the periphery where IRF4 limits the number of marginal zone B cells and is required for germinal center responses and plasma cell differentiation. Because of IRF4’s unique role in mature B cells, we hypothesized that it may also have functions earlier in B cell development that cannot be compensated for by IRF8. Indeed, we find that IRF4 has a unique role in upregulating the pre-B cell marker CD25, limiting IL-7 responsiveness, and promoting migration to CXCR4 such that IRF4-deficient mice have a partial block at the pre-B cell stage. We also find that IRF4 acts in early transitional B cells to restrict marginal zone B cell development, as deletion of IRF4 in mature B cells with CD21-cre impairs plasma cell differentiation but has no effect on marginal zone B cell numbers. These studies highlight IRF4 as the dominant IRF family member in early B lymphopoiesis.

A Phase 1/1b Open-Label, Multicenter, Two-Part Study of SETD2 Inhibitor EZM0414 in Patients with Relapsed/Refractory Multiple Myeloma or Diffuse Large B-Cell Lymphoma

A Phase 1/1b Open-Label, Multicenter, Two-Part Study of SETD2 Inhibitor EZM0414 in Patients with Relapsed/Refractory Multiple Myeloma or Diffuse Large B-Cell Lymphoma

Normal B Cell-Derived iPSCs Capable of Inducing RAS Mutants and Aid to Explore Myeloma-Initiating Cells

Multiple myeloma (MM) cells are derived from mature B cells based on immunoglobulin heavy chain (IgH) gene analysis. The onset of MM is often caused by a reciprocal chromosomal translocation (cTr) between chr 14 with IgH and chr 11 with CCND1. We propose that mature B cells gain the potential to transform by reprograming, and the resulting chromosomal aberrations subsequently cause the development of abnormal B cells as a myeloma-initiating cell during B cell redifferentiation. To study myeloma-initiating cells, we previously established normal B cell-derived induced pluripotent stem cells (BiPSCs: BiPSC13 and MIB2-6); these BiPSCs have the same VDJ rearrangement of IgH as the original B lymphocytes and induce the expression of activation-induced cytidine deaminase (AID) by the Tet-off system (Sci Rep, 2017). Subsequently, we established two BiPSCs with reciprocal cTr t(11;14) using the CRISPR/Cas9 system; the cleavage site were located in the IgH Eμ region of either the VDJ rearranged allele or non-rearranged allele of IgH and the 5′-upsteam region of the CCND1 (two types of BiPSC13 with t(11;14) and MIB2-6 with t(11;14)). Furthermore, p53 was deleted using the CRISPR/Cas9 system in BiPSC13 with t(11;14) carrying the DJ-rearranged allele of IgH that reciprocally translocated with CCND1 (G28). All these BiPSCs differentiated into hematopoietic progenitor cells (HPCs) (Sci Res, 2021). Next, we created AID fused with the hormone-binding domain of the estrogen receptor T2 mutant (AID-ER) to analyze the function of AID. The ER sequence of pCAG-ERT2CreERT2 (Addgene # 13777) was amplified by PCR and cloned into the downstream part of the TREtight promoter of pRetroX-Tight-Hyg as an open reading frame using an In-Fusion HD cloning kit. The ER sequence was also cloned into the same vector as a negative control for the analysis of AID function. These vectors were packaged into a retrovirus using gp293 and pMD2.G, and the viruses were infected into HEK293 cells expressing Tet-off transcription factor and fluorescence-deficient EGFP, in which the artificially-introduced stop codon was at the 106 thcodon of the EGFP gene. The expression of AID-ER fusion protein was induced by removal of doxycycline three days before analysis. AID activity was induced by the addition of 4-hydroxytamoxifen, an agonist of ER mutants, one day before analysis. Therefore, AID activity was monitored by detecting the frequency of EGFP-positive cells, i.e., the recovery of fluorescence, by flow-cytometry. Mutation of the artificial stop codon (from TAG to TAC; from stop codon to Tyrosine) was confirmed by sequencing the EGFP DNA amplified by PCR from the sorted EGFP-positive cells. Subsequently, we examined the introduction of mutated KRAS and NRAS genes into G28, because the most frequently occurring mutations in patients with MM are in KRAS and NRAS. Mutated KRAS (G12A) and NRAS (G13D) were amplified by RT-PCR from poly (A) mRNA of MM.1S and KMM1 of MM cell lines, respectively. After confirming the sequence of these mutants, the mutant cDNA of KRAS or NRAS was cloned into pRetroX-Tight-Hyg in combination with AID-ER. These two vectors were packaged into retroviruses, following which the viruses were infected into BiPSC13 with t(11;14) expressing Tet-off transcription factors, respectively. These BiPSCs also differentiated into HPCs. However, unlike cord blood, these HPCs did not differentiate into B lymphocytes by co-culture with BM stromal cell (MS-5). Therefore, further ingenuity is required to differentiate the BiPSC-derived HPCs into B lymphocytes. DisclosuresNo relevant conflicts of interest to declare.

The Outcome of CLL Patients According to IGHV Mutational Status: An Israeli Perspective

Introduction: The prognostic significance of immunoglobulin heavy-chain variable region gene (IGHV) mutational status in chronic lymphocytic leukemia (CLL) is well established. Previous studies have shown that CLL patients with mutated IGHV (M-IGHV) have a better prognosis, manifested in a longer time-to first treatment (TTFT) and overall survival (OS). Here we present an analysis on the impact of IGHV mutational status in an Israeli cohort of patients with CLL.Methods: A total of 254 patients with CLL (diagnosed from 1991 to 2020), followed at the Tel-Aviv Sourasky Medical Center were included. The IGHV mutational status has been determined by next-generation sequencing (NGS) or cDNA Sanger. The sequences with a germline homology 98% or higher were considered unmutated, and those with a homology less than 98% as mutated CLL. IGHV subsets were analyzed using the ARResT/AssignSubsets website. All data were statistically analyzed by IBM SPSS Statistics 27 (IBM Corporation, Armonk, NY, USA), P-values were two-sided, and the significance level was determined at a<0.05 were considered significant.Results: Out of 254 patients, 132 (52.0%) had an unmutated IGHV gene (UM-IGHV), and 122 (48.0%) were mutated (M-IGHV). At diagnosis, most patients (Table 1) were ≤65 years of age (n=157, 61.8%), males (n=160, 63.0%) and had an absolute lymphocytic count equal to or less than (≤)15.0 x10 9/L (n=115, 52.5%). Advanced Binet stage was more commonly associated with UM-IGHV (n=44, 57.9%) (P=0.033). Among 240 patients (94.1%), the most frequently used VH gene segments (Figure 1) included; VH1-69 (n=35, 14.6%), VH4-34 (n=24, 10.0%), VH1-2 (n=17, 7.1%), and VH3-23 (n=16, 6.7%). Six major B-cell receptors (BCR) subsets (Figure 2) were identified in 21/180 of patients (11.7%), which most commonly included: CLL#1 (n=6, 28.6%), CLL#4 (n=5, 23.8%), and CLL#2(n=3, 14.3%). Patients with M-IGHV had a longer time to first treatment (TTFT) (P<0.001, hazard ratio (HR)=2.5, 95% confidence interval [1.8-3.5], Figure 3A) and a better overall survival (OS) (P=0.002, HR=2.7, [1.4-5.1]) compared to those with UM-IGHV gene (Figure 3B).In multivariate analyses of the entire cohort; for TTFT (Table 2A), males (P=0.002, HR=1.9, [1.3-2.9]), >65 years of age at diagnosis (P=0.008, HR=1.8, [1.2-2.7]), advanced Binet stage (P<0.001, HR=2.4, [1.5-3.8]) and UM-IGHV mutational status (P=<0.001, HR=2.0, [1.3-2.9]) were found to be significant predictors for shorter TTFT, while in a multivariate analysis for OS (Table 2B), only >65 years of age at diagnosis (P=0.010, HR=2.6, [1.3-5.5]) and UM-IGHV mutational status (P=0.004, HR=2.9, [1.4-5.9]) were found to be significant factors for shorter OS.In multivariate analyses performed separately for each IGHV mutational status group; males (P=0.021, HR=2.1, [1.1-4.0]), age >65 years (P=0.002, HR=3.2, [1.5-6.4]) and advanced Binet stage (P<0.001, HR=4.1, [1.9-8.7]) were found as independent predictors for a shorter TTFT in M-IGHV patients, while only males retained a statistically significance in UM-IGHV patients (P=0.017 HR=2.0, [1.1-3.5]). Furthermore, in a multivariate analysis for OS; age>65 years at diagnosis (P=0.039, HR=4.3, [1.1-17.0]) was the only independent predictor in M-IGHV patients, while no variable maintained its statistical significance in UM-IGHV cases.Conclusion: As previously studied, our cohort demonstrates that M-IGHV CLL patients have better TTFT and OS. However, the rate of BCR subsets in Israel appears to be lower than expected. A separated multivariate analysis for M-IGHV and UM-IGHV patients revealed different independent predictors for TTFT and OS to each of the IGHV mutational status groups. Further studies with larger cohorts are required to deeply study these differences and provide further clinical insight into the pathophysiology of CLL. [Display omitted] DisclosuresJoffe: AstraZeneca: Consultancy; Epizyme: Consultancy. Herishanu: AbbVie: Honoraria, Research Funding; Janssen: Honoraria; Roche: Honoraria; Medison: Honoraria.

The Landscape of Immunoglobulin Heavy Chain Gene Repertoire in Lymphoplasmacytic Lymphoma / Waldenström Macroglobulinemia

Introduction Immunoglobulin heavy-chain variable genes (IGHV) is critical for the defining epitope binding affinityand B cell differentiation. Lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) is a heterogeneous diseasewhose role of IGHV usage remains unknown. Besides, the clinical relevance of IGHV repertoire for LPL/WM remain largely unexplored. The aim of our study is to explore the IGH repertoire of LPL/WM in by far the largest series, and to evaluate the correlation between IGH rearrangements and genetic aberrations and clinical characteristics of LPL/WM patients.Methods A total of 162 patients with a diagnosis of LPL/WM were included in this study. Polymerase chain reaction (PCR)amplification of IGHV-IGHD-IGHJ was performed on genomic DNA or cDNA samples using the IGH Somatic Hypermutation Assay v2.0 (Invivoscribe, Technologies, San Diego, US). Sequences were aligned to IMGT (http://www.imgt.org/IMGT_vquest/vquest) and IGBLAST (https://www.ncbi.nlm.nih.gov/igblast/) databases. IGH gene repertoires, mutation status, IGHV CDR3 characteristics, genetic aberrations, MYD88 mutation status and clinical characteristics were collected to evaluate the relevance.Results Productive IGHV-D-J rearrangements were obtained in 136 out of 162 patients (84.0%). The IGHV gene repertoire was remarkably biased in LPL/WM. IGHV3-23 (15.4%), IGHV4-34 (10.3%), IGHV3-7 (8.1%), IGHV3-30 (7.4%) and IGHV3-74 (7.4%) were significantly overrepresented in LPL/WM(Figure 1). Among the 134 IGHD data, the most frequent segment was IGHD3-10 (21/134, 15.7%), followed by IGHD6-13 (18/134, 13.4%) (Figure 2). Among the 134 IGHJ data, IGHJ4 segment was selected in more than half of these rearrangements (70/136; 51.5%), followed by IGHJ6 (23/136; 16.9%) and IGHJ5 (21/136; 15.4%) (Figure 3). Most of the cases were mutated (97.0%) using a 98% IGHV germline homology cutoff. IGHV3-30 was associated with long heavy chain CDR3, indicating the specific antigen selection in LPL/WM. Patients with IGHV3-7 were significantly more likely to harbor 6q deletion (p<0.001)(Figure 4) and abnormal karyotype (p=0.004)(Figure 5).The IGHV hypermutation rate in patients with MYD88 L265P mutation was significantly higher than in wild-type patients (7.3% vs5.6%, p=0.009). IGHV3-23 and IGHV3-74 segments were more frequently detected in MYD88 mutated LPL/WM patients (25.7% vs. 4.3%, p=0.025). IGHV3-7 and IGHV4-59 were represented more in MYD88 wildtype patients (30.4% vs. 8.9%，p=0.005). Moreover, Patients with IGHV4 especially IGHV4-34 had higher level of LDH. IGHV4 was a prognostic marker of shorter progression-free-survival (Figure 6).Conclusion LPL/WM appears to be composed ofdifferent subgroups based on the IGHV repertoire. The mutational status and the IGHV CDR3 length indicated the role for antigenselection in LPL/WM development. The presence of IGHV4 genes proved to be a potential risk factor associated with outcome which deserved further study.These results showed for the first time that IGHV repertoire had clinical relevance in LPL/WM. [Display omitted] DisclosuresWang: AbbVie: Consultancy; Astellas Pharma, Inc.: Research Funding.