Clinical significance of bone marrow involvement by immunoglobulin gene rearrangement in de novo diffuse large B-cell lymphoma: a multicenter retrospective study.

Background: Bone marrow (BM) involvement is an indicator of a poor prognosis in diffuse large B-cell lymphoma (DLBCL); however, few studies have evaluated the role of immunoglobulin gene rearrangement (IgR) in detecting BM involvement. Methods: We evaluated the clinical characteristics and treatment outcomes of patients with DLBCL based on histological BM involvement or positive BM IgR using polymerase chain reaction or next-generation sequencing. We also investigated the role of consolidative upfront autologous hematopoietic stem cell transplantation (ASCT) in patients with DLBCL and BM involvement. Results: Among 624 patients, 123 (19.7%) with histological BM involvement and 88 (17.5%) with positive IgR in histologically negative BM had more advanced disease characteristics. Overall (OS) and progression-free (PFS) survival was better for patients with negative BM histology and negative IgR than that in patients with histological BM involvement (P = 0.050 and P < 0.001, respectively) and positive IgR with negative BM histology (P = 0.001 and P = 0.005, respectively). Survival rates did not differ among 82 (13.1%) patients who were treated with upfront ASCT and had histological BM involvement or positive IgR with negative BM histology. The survival outcomes were worse for patients who were not treated with upfront ASCT and for those with histological BM involvement or positive IgR, than for those with negative BM histology and negative IgR. Conclusion: Patients diagnosed with DLBCL and BM involvement based on histology or IgR had aggressive clinical features and poor survival. Upfront ASCT mitigated poor prognosis due to BM involvement.

The spliceosome component Usp39 controls B cell development by regulating immunoglobulin gene rearrangement.

The spliceosome is a large ribonucleoprotein complex responsible for pre-mRNA splicing and genome stability maintenance. Disruption of the spliceosome activity may lead to developmental disorders and tumorigenesis. However, the physiological role that the spliceosome plays in B cell development and function is still poorly defined. Here, we demonstrate that ubiquitin-specific peptidase 39 (Usp39), a spliceosome component of the U4/U6.U5 tri-snRNP complex, is essential for B cell development. Ablation of Usp39 in B cell lineage blocks pre-pro-B to pro-B cell transition in the bone marrow, leading to a profound reduction of mature B cells in the periphery. We show that Usp39 specifically regulates immunoglobulin gene rearrangement in a spliceosome-dependent manner, which involves modulating chromatin interactions at the Igh locus. Moreover, our results indicate that Usp39 deletion reduces the pre-malignant B cells in Eµ-Myc transgenic mice and significantly improves their survival.

Temporally uncoupled signal and coding joint formation in human V(D)J recombination.

In vertebrate antigen receptor gene rearrangement, V(D)J recombination events can occur by deletion or by inversion. For deletional events, the signal joint is deleted from the genome. Nearly half of the immunoglobulin light chain genes undergo V(D)J recombination in an inversional manner, and both signal and coding joint formation must occur to retain chromosomal integrity. But given the undetermined amount of pre-B and pre-T cell death that occurs during V(D)J recombination, the efficiency with which both joints are completed is not known, nor is the relative efficiency (balance) of signal versus coding joint formation. Signal joint formation only requires Ku and XRCC4:DNA ligase 4 of the nonhomologous DNA end joining repair pathway. Coding joint formation requires these proteins as well, but in addition requires Artemis and DNA-dependent protein kinase to open the hairpin DNA coding ends, which the RAG complex generated; and further processing is required because the hairpin opening generates incompatible 3' overhangs. Mutations in some of the end processing enzymes affect one, but only minimally the other joint. We have devised a precise cellular assay that does not have any cellular, enzymatic or biochemical selective bias to assess signal and coding joint formation independently, and it can detect intermediates for which one joint has formed but not the other. We find that intermediates with only one completed joint are more abundant than molecules with both joints completed. This indicates that either joint can form independent of the other and joint formation can be a relatively slow process.

DNA-PKcs chemical inhibition versus genetic mutation: Impact on the junctional repair steps of V(D)J recombination.

Spontaneous DNA-PKcs deficiencies in animals result in a severe combined immunodeficiency (SCID) phenotype because DNA-PKcs is required to activate Artemis for V(D)J recombination coding end hairpin opening. The impact on signal joint formation in these spontaneous mutant mammals is variable. Genetically engineered DNA-PKcs null mice and cells from them show a >1,000-fold reduction in coding joint formation and minimal reduction in signal joint formation during V(D)J recombination. Does chemical inhibition of DNA-PKcs mimic this phenotype? M3814 (also known as Nedisertib) is a potent DNA-PKcs inhibitor. We find here that M3814 causes a quantitative reduction in coding joint formation relative to signal joint formation. The sequences of signal and coding junctions were within normal limits, though rare coding joints showed novel features. The signal junctions generally did not show evidence of resection into the signal ends that is often seen in cells that have genetic defects in DNA-PKcs. Comparison of the chemical inhibition findings here with the known results for spontaneous and engineered DNA-PKcs mutant mammals is informative for considering pharmacologic small molecule inhibition of DNA-PKcs in various types of neoplasia.

Current insights into the mechanism of mammalian immunoglobulin class switch recombination.

Immunoglobulin (Ig) class switch recombination (CSR) is the gene rearrangement process by which B lymphocytes change the Ig heavy chain constant region to permit a switch of Ig isotype from IgM to IgG, IgA, or IgE. At the DNA level, CSR occurs via generation and joining of DNA double strand breaks (DSBs) at intronic switch regions located just upstream of each of the heavy chain constant regions. Activation-induced deaminase (AID), a B cell specific enzyme, catalyzes cytosine deaminations (converting cytosines to uracils) as the initial DNA lesions that eventually lead to DSBs and CSR. Progress on AID structure integrates very well with knowledge about Ig class switch region nucleic acid structures that are supported by functional studies. It is an ideal time to review what is known about the mechanism of Ig CSR and its relation to somatic hypermutation. There have been many comprehensive reviews on various aspects of the CSR reaction and regulation of AID expression and activity. This review is focused on the relation between AID and switch region nucleic acid structures, with a particular emphasis on R-loops.

Eosinophilic dermatosis of hematologic malignancy: Correlation of molecular characteristics of skin lesions and extracutaneous manifestations of hematologic malignancy.

BACKGROUND: Skin diseases are frequent in patients with chronic lymphocytic leukemia (CLL) and other hematological neoplasias. Eosinophilic dermatosis (ED) of hematologic malignancy has long been considered a nonspecific cutaneous reaction pattern. Recently neoplastic cells have been shown to be present in ED, thus challenging the classification as a nonspecific dermatosis. METHODS: We report five patients with ED in association with CLL. We further investigated the presence of neoplastic B-cells in the skin infiltrate by immunohistochemistry and immunoglobulin heavy chain rearrangement and compared these to extracutaneous manifestations of CLL. RESULTS: The phenotype of the lymphocytic infiltrate was predominately CD3+ (range: 60%-90%). CD20+ and CD79a+ lymphocytes were less frequent, accounting for up to 15% (range: absent - 15%). CD23+ lymphocytes represented up to 20% (range: absent - 20%) of the infiltrate. The analysis of the immunoglobulin heavy chain rearrangement in the skin specimens showed clonal rearrangements in 4/5 patients and in three of these four patients clones were identical to extracutaneous CLL manifestations. CONCLUSION: Our data show that neoplastic B-cells are very frequently found in ED when systematically evaluated. This findings support the hypothesis that leukemic cells play a pathogenetic role in ED of hematologic malignancy.

Molecular assessment of clonality in lymphoid neoplasms.

Molecular clonality assays in B- and T-cell lymphoproliferative disorders often provide critical information in establishing a diagnosis of a lymphoproliferative disorder. These assays rely on the unique genetic structures that serve as assay targets, created in the process of generating immunoglobulin and T-cell receptors during B- and T-cell development. Molecular clonality assays are generally used when flow cytometry or immunohistochemistry has not sufficiently clarified the benign or malignant nature of a lymphoid proliferation. Additionally, since molecular clonality assays are tumor specific, they allow the clinician to distinguish recurrences from second tumors, and have the sensitivity to monitor minimal residual disease. In this review, we discuss the principles underlying these tests, the current approaches to clonality testing, some of the pitfalls in their interpretation, and the future applications of next generation sequencing technology to clonality testing.

A BTB-ZF protein, ZNF131, is required for early B cell development.

Members of the BTB-ZF transcription factor family play important roles in lymphocyte development. During T cell development, ZNF131, a BTB-ZF protein, is critical for the double-negative (DN) to double-positive (DP) transition and is also involved in cell proliferation. Here, we report that knockout of Znf131 at the pre-pro-B cell stage in mb1-Cre knock-in mouse resulted in defect of pro-B to pre-B cell transition. ZNF131 was shown to be required for efficient pro-B cell proliferation as well as for immunoglobulin heavy chain gene rearrangement that occurs in the proliferating pro-B cells. We speculate that inefficient gene rearrangement may be due to loss of cell proliferation, since cell cycle progression and immunoglobulin gene rearrangement, which would occur in a mutually exclusive manner, may be interconnected or coupled to avoid occurrence of genomic instability. ZNF131 suppresses expression of Cdk inhibitor, p21cip1, and that of pro-apoptotic factors, Bax and Puma, targets of p53, to facilitate cell cycle progression and suppress unnecessary apoptosis, respectively, of pro-B cells. There results demonstrate the essential roles of ZNF131 in coordinating the B cell differentiation and proliferation.

The functional 3'-end of immunoglobulin heavy chain variable (IGHV) genes.

Inference of antibody gene repertoires using transcriptome data has emerged as an alternative approach to the complex process of sequencing of adaptive immune receptor germline gene loci. The diversity introduced during rearrangement of immunoglobulin heavy chain variable (IGHV), diversity, and joining genes has however been identified as potentially affecting inference specificity. In this study, we have addressed this issue by analysing the nucleotide composition of unmutated human immunoglobulin heavy chains-encoding transcripts, focusing on the 3ö most bases of 47 IGHV germline genes. Although transcripts derived from some of the germline genes predominately incorporated the germline encoded base even at position 320, the last base of most IGHV genes, transcripts originating in other genes presented other nucleotides to the same extent at this position. In transcripts derived from two of the germline genes, IGHV3-13*01 and IGHV4-30-2*01, the predominating nucleotide (G) was in fact not that of the gene (A). Hence, we suggest that inference of IGHV genes should be limited to bases preceding nucleotide 320, as inference beyond this would jeopardize the specificity of the inference process. The different degree of incorporation of the final base of the IGHV gene directly influences the distribution of amino acids of the ascending strand of the third complementarity determining region of the heavy chain. Thereby it influences the nature of this specificity-determining part of the antibody population. In addition, we also present data that indicate the existence of a common so far un-recognized allelic variant of IGHV3-7 that carries an A318G difference in relation to IGHV3-7*02.

The mTOR-Bach2 Cascade Controls Cell Cycle and Class Switch Recombination during B Cell Differentiation.

The transcription factor Bach2 regulates both acquired and innate immunity at multiple steps, including antibody class switching and regulatory T cell development in activated B and T cells, respectively. However, little is known about the molecular mechanisms of Bach2 regulation in response to signaling of cytokines and antigen. We show here that mammalian target of rapamycin (mTOR) controls Bach2 along B cell differentiation with two distinct mechanisms in pre-B cells. First, mTOR complex 1 (mTORC1) inhibited accumulation of Bach2 protein in nuclei and reduced its stability. Second, mTOR complex 2 (mTORC2) inhibited FoxO1 to reduce Bach2 mRNA expression. Using expression profiling and chromatin immunoprecipitation assay, the Ccnd3 gene, encoding cyclin D3, was identified as a new direct target of Bach2. A proper cell cycle was lost at pre-B and mature B cell stages in Bach2-deficient mice. Furthermore, AZD8055, an mTOR inhibitor, increased class switch recombination in wild-type mature B cells but not in Bach2-deficient cells. These results suggest that the mTOR-Bach2 cascade regulates proper cell cycle arrest in B cells as well as immunoglobulin gene rearrangement.

The t(8;14)(q24.1;q32) and its variant translocations: A study of 34 cases.

BACKGROUND: The t(8;14)(q24.1;q32) and its variants - the t(2;8)(p12;q24.1) and t(8;22)(q24.1;q11.2) are associated with B-cell neoplasia and result in MYC/immunoglobulin (IG) gene rearrangement. PATIENTS AND METHODS: We correlated the cytogenetic, molecular and clinico-pathological findings of patients with 8q24 translocations seen in the Department of Haematology, Christian Medical College, Vellore, from January 2003 to December 2015. RESULTS: There were 34 patients with 8q24 translocations (31, ALL and three myeloma). The t(8;14) was seen in 25 patients, t(8;22) in seven and t(2;8) in two. The salient findings were as follows: 85% males; 79% adults, median age 37 years; L3 morphology in 61%; mature B immunophenotype in 77%; extra-medullary disease in 41%; additional abnormalities in 28 (85%), notably, structural abnormalities of chromosome 1q (41%) and 13q (9%) and monosomy 13 (15%); complex karyotypes in 68%. There were two double-hit lymphoma/leukemia, one with a t(14;18)(q32;q21) and the other with a t(3;14)(q27;q11.2), associated with nodal high grade B cell lymphoma and dermal leukemic infiltrates respectively. Only 13 samples were processed for DNA PCR and all these samples were positive for MYC-IgH (c-gamma type) rearrangement. Only in one patient, in addition to c-gamma, c-alpha rearrangement was also detected. CONCLUSION: The frequency (1.7%) and distribution of these translocations in our series and the association with 1q and 13q abnormalities is similar to the literature. Trisomies 7 and 12 were seen in less than 10% of our patients.

Application of immunoglobulin gene rearrangement-derived real-time quantitative polymerase chain reaction in monitoring minimal residual disease of B-cell lymphoblastic leukemia / 白血病·淋巴瘤

Objective To establish a real-time quantitative polymerase chain reaction (qPCR) assay for B-cell lymphoblastic leukemia according to individualized and specific immunoglobulin gene rearrangements in leukemia cells, and to use it for the monitoring of minimal residual disease (MRD) of B-cell lymphocytic leukemia. Methods The immunoglobulin gene rearrangements of bone marrow samples from 15 cases of B-cell lymphoblastic leukemia were analyzed with a validated European BIOMED-2 system, and the individualized and specific qPCR-based quantification of leukemic immunoglobulin gene rearrangements was established. Results Unique and specific gene rearrangements of immunoglobulin light and heavy chains were identified in 14 cases and Ig-qPCR based on these gene rearrangements had a sensitivity of 10-5 and high specificity which met the international criteria in 10 patients. Leukemia MRD quantification with immunoglobulin gene rearrangement-based qPCR was similar as compared with other MRD detection methods. Conclusion Immunoglobulin gene rearrangement-based leukemia MRD quantification is feasible, sensitive, specific, precise and much valuable for clinical decision of treatments in B-cell lymphoblastic leukemia.

The Importance of an In-depth Study of Immunoglobulin Gene Rearrangements When Ascertaining the Clonal Relationship between Concomitant Chronic Lymphocytic Leukemia and Multiple Myeloma.

Chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) are hematological disorders that occur at different stages of B-cell development. It has been shown that CLL B-cells can differentiate into plasma cells in vitro and in vivo. CLL is the most frequent adult leukemia in the western world. It is a heterogeneous disease, characterized by clonal proliferation and the accumulation of mature CD5+ B lymphocytes (1). MM is a clonal plasma cell malignancy that accounts for more than 10% of all hematologic cancers (2). Although secondary cancers [particularly solid tumors (3-5)] can occur with CLL and MM, the concomitant occurrence of these two disorders in the same patient is rare [for a review of the few reported cases, see Ref. (6)]. The clonal relationship between these diseases has not always been clarified but is important in terms of understanding the pathogenesis and optimizing treatment. The clonal relationship between CLL and MM can be evaluated by (i) analyzing immunoglobulin (Ig) heavy chain and light chain (Ig kappa light chain and Ig lambda light chain) gene rearrangement, (ii) identifying and comparing somatic mutations, and (iii) studying chromosomic aberrations. Nevertheless, Ig rearrangements must always be interpreted in the light of specific phenomena such as allelic exclusion, B-cell receptor (BCR) revision (VH and DH gene replacement), BCR editing, and somatic mutations-events that were not considered in previous studies. These issues can be addressed by sequencing the rearranged Ig genes from sorted populations and interpreting the generated data. In the present study, we evaluated the putative clonal relationship between the two diseases by combining DNA copy number analysis with an assessment of Ig gene rearrangements [clonality assessment, V(D)J sequencing, and somatic hypermutation analysis] in highly enriched CD19+ CD5+ (CLL) and CD38+ CD138+ (MM) cell populations. Array comparative genomic hybridization data suggested a possible phylogenic progression from CLL to MM. Moreover, V(D)J sequencing indicated that both CLL and MM cells used the same VH and JH genes but different DH genes. However, in-depth analysis and interpretation of Ig gene rearrangements ultimately suggested that the two diseases had distinct clonal origins.

Deep sequencing reveals myeloma cells in peripheral blood in majority of multiple myeloma patients.

INTRODUCTION: The evaluation of myeloma cells in multiple myeloma (MM) patients has generally been limited to the assessment of bone marrow involvement because of the sensitivity limitations of traditional minimal-residual-disease-detection methods. MATERIALS AND METHODS: We developed a sequencing-based method to identify myeloma cells in bone marrow (BM) and peripheral blood (PB) samples, based on their unique immunoglobulin gene rearrangements, that can detect cancer clones at levels well below 1 in 1 million leukocytes (0.0001%). In this multisite study, we used this sequencing method to determine the fraction of patients with myeloma cells in their PB at diagnosis and posttreatment time points. RESULTS: Using this sequencing approach, we detected myeloma cells in the PB in the vast majority of MM patients (44/46, 96%). We demonstrated a clear correlation (R(2) = 0.57) between myeloma clone levels in paired BM and PB samples, and noted that PB clone levels were approximately 100-fold lower than levels in BM samples. The sequencing assay demonstrated a clear sensitivity advantage in the BM compartment and at least equivalent sensitivity in the PB compared with that of monoclonal-protein results. CONCLUSION: This study highlights the promise of a blood-based, sequencing minimal-residual-disease assay that can be used to measure MM disease burden at different time points and various disease stages.

Whole-genome amplification for the detection of molecular targets and minimal residual disease monitoring in acute lymphoblastic leukaemia.

Accurate genomic characterization requires sufficient amounts of optimal quality DNA. An approach for increasing the DNA amount is the whole-genome amplification (WGA) method. We applied WGA to the molecular quantification and minimal residual disease (MRD) evaluation of acute lymphoblastic leukaemia (ALL), aiming to compare the results obtained from genomic DNA and amplified DNA with WGA, and to evaluate the applicability and the reliability of WGA-DNA. Twenty paired samples from adult ALL patients were sequenced to identify the functional germline V-D-J segment at diagnosis; real-time quantitative polymerase chain reaction (RQ-PCR) quantitative analysis was performed both at diagnosis and follow-up. Genomic DNA and WGA-DNA screening identified equivalent 87 rearrangements. At diagnosis, the quantitative evaluation of genomic DNA samples showed 1 logarithm difference to WGA-DNA samples; these levels are comparable, being within the degree of acceptability and confidence. In the follow-up samples, RQ-PCR analysis on genomic DNA and WGA showed concordant MRD results in 16/18 samples, while 2/18 were MRD-positive outside the quantitative range by RQ-PCR (i.e. <5 × 10(-5)) on genomic DNA and MRD-negative on WGA-DNA. WGA-DNA enables: (i) the design of accurate targets for MRD evaluation in ALL patients, (ii) accurate disease quantification at diagnosis, (iii) MRD quantification comparable to genomic DNA.

The DG75 B-cell lymphoma line exhibits biclonal immunoglobulin gene rearrangement.

Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement (GR) studies have been successfully employed to investigate the clonality and cell lineage of various lymphoid malignancies. Several lymphoma cell lines, such as BJAB, RAJI, DG75 and Jurkat cell lines, were often used as the positive controls in GR detection assays. Of those, the DG75 B-cell lymphoma line was found to exhibit biclonality [two or more homoduplex and heteroduplex bands in a polymerase chain reaction (PCR) product of clonality assay] in the PCR of GR detection assays. To further explore these characteristics of the biclonal phenomenon, the PCR products were purified and cloned into a pEGM-T clone vector. The sequences were analyzed using DNA analysis software. The results demonstrated that the two bands originated from two forms of GR of DG75 cell lines, i.e., DG75 is a biclonal cell line in Ig GRs, which has not been reported before.

Cutaneous B-Cell Pseudolymphoma: Report of Two Cases

Cutaneous pseudolymphoma (CPL) has a microscopic appearance that resembles that of cutaneous lymphoma, but shows a clinically benign course. The differential diagnosis of CPL with cutaneous lymphoma is very important because clinical outcomes of them are quite different. We herein describe two cases of B-cell pseudolymphoma, which were difficult to differentiate from cutaneous B-cell lymphlma. All of two cases, Polymerase chain reaction of immunoglobulin heavy chain gene rearrangement showed polyclonal pattern.

Primary Cutaneous B Cell Lymphoma

We report a case of B-cell lymphoma primarily involving the skin in a 12-year-old boy. The histopathologic findings were compatible with those of small lymphocytic type of non-Hodgkin's lymphoma. A cutaneous lesion was the sole manifestation of his disease without any other organ involvement. Immunophenotypic studies and immunoglobulin gene rearrangement with Southern blot analysis determined its lineages and monoclonality with result of B-cell lineage neoplasm, i. d. CD20⁺, C1323⁺, CD35⁻ and rearranged band on JH probe. We treated him with surgical excision and CVP regimen of chemotherapy (cyclophosphamide, vincristine, prednisolone). There is no recurrence or metastasis during the last six months.

Cellular and Molecular Characterization of Two Cases of Castleman's Disease, Plasma Cell Variant.

We report the cellular and molecular characterization of two cases of Castleman's disease, plasma cell variant, that differed in their clinical presentation and course. Patient 1 had Castleman's disease in association with Kaposis's sarcoma unrelated to human immunodeficiency virus (HIV) infection and died while he was receiving an aggressive chemotherapeutic regimen for Kaposi's sarcoma. Patient 2 had an isolated retroperitoneal lymphoid mass with an adjacent enlarged limph nodes and his symptoms disappeared completely following the surgical removal of both. Pathologic and immunohistochemical analyses in both cases, revealed that there was a massive infiltration of polyclonal plasma cells in the interfollicular areas of the lymph nodes. Immunoglobulin gene rearrangement studies confirmed the polyclonal nature of B-lineage cells in the involved lymph nodes. Southern blot experiments failed to demonstrate the presence of EBV genome copies in the same lymph nodes. These paradigmatic cases lend further support to the notion that Castleman's disease is an extremely heterogeneous entity.