Chain Gene Rearrangement Research Articles

Dual Mechanism of Rag Gene Repression by c-Myb during Pre-B Cell Proliferation.

Developing B lymphocytes undergo clonal expansion following successful immunoglobulin heavy chain gene rearrangement. During this proliferative burst, expression of the Rag genes is transiently repressed to prevent the generation of double-stranded DNA (dsDNA) breaks in cycling large pre-B cells. The Rag genes are then reexpressed in small, resting pre-B cells for immunoglobulin light chain gene rearrangement. We previously identified c-Myb as a repressor of Rag transcription during clonal expansion using Abelson murine leukemia virus-transformed B cells. Nevertheless, the molecular mechanisms by which c-Myb achieved precise spatiotemporal repression of Rag expression remained obscure. Here, we identify two mechanisms by which c-Myb represses Rag transcription. First, c-Myb negatively regulates the expression of the Rag activator Foxo1, an activity dependent on M303 in c-Myb's transactivation domain, and likely the recruitment of corepressors to the Foxo1 locus by c-Myb. Second, c-Myb represses Rag transcription directly by occupying the Erag enhancer and antagonizing Foxo1 binding to a consensus forkhead site in this cis-regulatory element that we show is crucial for Rag expression in Abelson pre-B cell lines. This work provides important mechanistic insight into how spatiotemporal expression of the Rag genes is tightly controlled during B lymphocyte development to prevent mistimed dsDNA breaks and their deleterious consequences.

Establishment of induced pluripotent stem cells from normal B cells and inducing AID expression in their differentiation into hematopoietic progenitor cells

B cell derived induced pluripotent stem cells (BiPSCs) were recently established from peripheral blood B cells by the simultaneous transfection of Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) and C/EBPα using a Sendai virus vector. Here, using a different method, we established BiPSCs with immunoglobulin heavy chain (IgH) gene rearrangement from normal B cells purified from lymph nodes. The critical points of our method are pre-stimulation of B cells with IL-21 and CD40-ligand (CD40L), followed by consecutive transfection of highly concentrated Yamanaka factors using a retroviral vector. Following each transfection the cells were centrifuged onto a retronectin coated plate and the activated by IL-4, IL-2, and CD40L. Furthermore, we established BiPSCs (BiPSC-A) in which activation-induced cytidine deaminase (AID) could be induced using the doxycycline-controlled. Both the parental BiPSC and BiPSC-A showed the capability of differentiating into hematopoietic progenitor cells (HPCs) based on confirmation of CD34 expression and colony-formation from CD34-positive cells. The findings that BiPSC-A can differentiate into HPCs suggest that there is a possibility that induction of AID expression would result in chromosomal translocations in the process of differentiation from BiPSCs, and therefore that these BiPSCs could be useful in elucidating the tumor origin of abnormal B cells in myelomagenesis.

Detection of Immunoglobulin Heavy Chain Gene Clonality by Next-Generation Sequencing for Minimal Residual Disease Monitoring in B-Lymphoblastic Leukemia.

Minimal residual disease (MRD) following B-lymphoblastic leukemia (B-ALL) treatment has gained prognostic importance. Clonal immunoglobulin heavy chain (IGH) gene rearrangement is a useful follow-up marker in B-ALL owing to its high positivity rate. We evaluated the performance and clinical applicability of a next-generation sequencing (NGS) assay for IGH rearrangement in B-ALL MRD monitoring. IGH rearrangement was tested by using fluorescence PCR-fragment analysis and the NGS assay in eight B-ALL patients. The NGS assay was run on two platforms: the Ion Torrent PGM (Thermo Fisher Scientific, USA) (18 samples from 1st to 7th patients) and the MiSeq system (Illumina, USA) (four samples from 8th patient). All initial diagnostic samples and four follow-up samples were positive for clonal IGH rearrangement with fluorescence PCR-fragment analysis and the NGS assay, and six follow-up samples were positive only with NGS. In one case with BCR-ABL1 translocation, BCR-ABL1 quantitative PCR was negative but the NGS IGH assay was positive just prior to full-blown relapse, suggesting the high sensitivity and clinical utility of the NGS assay. The NGS assay is proposed for MRD monitoring in B-ALL Additional studies are needed to confirm the clinical implications of cases showing positive results only in NGS.

A Small Case Series of Intravascular Large B-Cell Lymphoma with Unexpected Findings: Subset of Cases with Concomitant Extravascular Central Nervous System (CNS) Involvement Mimicking Primary CNS Lymphoma.

BackgroundIntravascular large B-cell lymphoma (IVLBCL) is a rare type of extranodal lymphoma with growth mainly in the lumina of vessels. We studied a small series of IVLBCL and focused on its central nervous system (CNS) involvement. MethodsSearching the medical records of Northwestern Memorial Hospital, we identified five cases of IVLBCL from January 2007 to January 2015. Clinical information, hematoxylin and eosin stained histologic slides and immunohistochemistry studies were reviewed for all cases. Polymerase chain reaction (PCR) analysis for the immunoglobulin (Ig) heavy and light chain gene rearrangement was performed on all five cases.ResultsThree of the five cases of IVLBCL were autopsies. Patients’ age ranged from 56 to 84. CNS involvement was present in two cases—in both patients, the CNS involvement showed an extravascular pattern with confluent sheet-like formation. PCR analysis confirmed that in one case the systemic intravascular and CNS extravascular components were clonally identical. ConclusionsIn a small case series of IVLBCL, we observed that CNS involvement by IVLBCL often has an extravascular morphology, but is clonally identical to the intravascular counterpart by PCR analysis. As IVLBCL can have a rapidly progressing poor outcome, it should be kept in the differential diagnoses for patients presenting with lymphoma of the CNS. The presence of extravascular growth patterns in the CNS should not exclude IVLBCL as a diagnosis.

Extranodal marginal zone lymphoma of the uterine cervix with concomitant copy number gains of the MALT1 and BCL2 genes: A case report.

Extranodal marginal zone lymphoma (EMZL) of mucosa-associated lymphoid tissue (MALT) of the uterus is rare, and the etiology, pathophysiology and cytogenetic features remain unknown at present. The present study reports a case of a 71-year-old female with EMZL of the uterine cervix that was 80 mm in diameter and invaded directly into the rectal serosa. Complete remission was successfully induced by 6 courses of immunochemotherapy with rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisolone. Although the metaphase spread of the tumor cells was unavailable for whole cytogenetic analysis, fluorescence in situ hybridization (FISH) detected triple signals for MALT1 and B-cell lymphoma 2, located at chromosome 18q21, and the centromere of chromosome 18, which was suggestive of trisomy 18, and in combination with previous studies, suggested a possible association between trisomy 18 and the large tumor at initial presentation in the present patient. In addition, FISH examination detected immunoglobulin heavy chain gene rearrangement, although the translocation partner was unconfirmed. A total of 18 previously-studied patients with EMZL of the uterus, including that of the present study, were reviewed with respect to their clinical features and treatment and cytogenetic abnormality. In the evaluation of the English scientific literature, this is the first reported patient with EMZL of the uterus with partly determined cytogenetic abnormalities.

Ig Light Chain Precedes Heavy Chain Gene Rearrangement during Development of B Cells in Swine.

The current mammalian paradigm states that 1) rearrangements in the IgH locus precede those in IgL loci, 2) IgLλ genes rearrange only when IgLκ genes are consumed, and 3) the surrogate L chain is necessary for selection of productive IgH gene rearrangements. We show in swine that IgL rearrangements precede IgH gene rearrangements, resulting in the expression of naked IgL on a surface of precursor B cells. Findings also suggest that there is no dependency on the surrogate L chain, and thus the authentic IgL proteins may be used for selection of the IgH repertoire. Although rearrangement starts with IgLκ genes, it is rapidly replaced by IgLλ rearrangement. Fast replacement is characterized by occurrence of IgLλloIgLκlo dual-expressing precursors in which IgLκ expression is a remnant of a previous translation. Most IgLκ+ B cells are then generated later, indicating that there are two waves of IgLκ synthesis in different developmental stages with IgLλ gene rearrangements in between. In the absence of stromal cells, the stepwise order of rearrangements is blocked so that IgLλ gene rearrangements predominate in early B cell development. To our knowledge, this is the first evidence that some mammals can use an inverted order of Ig loci rearrangement. Moreover, a situation in which the generation of BCR-bearing IgLκ is delayed until after IgLλ becomes the dominant isotype may help explain the extreme deviations in the IgLκ/IgLλ ratios among mammals.

Fulminant Mycosis Fungoides with Tissue Eosinophilia: A Unique Presentation of Two Cases with Acro-Periorbital Ulceration and An Aggressive Clinical Course.

We describe two unique cases of fulminant mycosis fungoides with remarkably similar and aggressive clinical courses resulting in death. Both cases demonstrated ulcerated palmar and periorbital plaques and marked tissue eosinophilia, which was confirmed by T-cell receptor γ chain gene rearrangement studies to display identical monoclonality at temporally and anatomically distinct sites. Dense eosinophilic infiltrates on biopsy led to misdiagnosis of inflammatory dermatoses in both instances.While mycosis fungoides may be challenging to diagnose histologically, the presence of eosinophils in progressive disease may herald a poor prognosis and should not exclude the diagnosis.

Molecular Monitoring after Autologous Stem Cell Transplantation and Preemptive Rituximab Treatment of Molecular Relapse; Results from the Nordic Mantle Cell Lymphoma Studies (MCL2 and MCL3) with Median Follow-Up of 8.5 Years

The main objectives of the present study were to monitor minimal residual disease (MRD) in the bone marrow of patients with mantle cell lymphoma (MCL) to predict clinical relapse and guide preemptive treatment with rituximab. Among the patients enrolled in 2 prospective trials by the Nordic Lymphoma Group, 183 who had completed autologous stem cell transplantation (ASCT) and in whom an MRD marker had been obtained were included in our analysis. Fresh samples of bone marrow were analyzed for MRD by a combined standard nested and quantitative real-time PCR assay for Bcl-1/immunoglobulin heavy chain gene (IgH) and clonal IgH rearrangements. Significantly shorter progression-free survival (PFS) and overall survival (OS) was demonstrated for patients who were MRD positive pre-ASCT (54 patients) or in the first analysis post-ASCT (23 patients). The median PFS was only 20 months in those who were MRD-positive in the first sample post-ASCT, compared with 142 months in the MRD-negative group (P < .0001). OS was 75% at 10 years and median not reached in the MRD-negative group, compared with only 35 months in the MRD-positive group (P < .0001). Of the 86 patients (47%) who remained in continuous molecular remission, 73% were still in clinical remission after 10 years. For all patients, the median time from ASCT to first molecular relapse was 55 months, with a continuous occurrence of late molecular relapses. Fifty-eight patients who experienced MRD relapse received rituximab as preemptive treatment on 1 or more occasions, and in this group, the median time from first molecular relapse to clinical relapse was 55 months. In most cases, rituximab converted patients to MRD negativity (87%), but many patients became MRD-positive again later during follow-up (69%). By multivariate analysis, high-risk Mantle Cell Lymphoma International Prognostic Index score and positive MRD status pre-ASCT predicted early molecular relapse. In conclusion, preemptive rituximab treatment converts patients to MRD negativity and likely postpones clinical relapse. Molecular monitoring offers an opportunity to select some patients for therapeutic intervention and to avoid unnecessary treatment in others. MRD-positive patients in the first analysis post-ASCT have a dismal prognosis and thus are in need of novel strategies.

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.

Establishment of Induced Pluripotent Stem Cells from Lymph Node B Cells and Induction of Aid Expression in Their Differentiation into Hematopoietic Stem Cells

Induced pluripotent stem cells (iPSCs) have been established from a variety of somatic cells not only for regenerative medicine but also for studies of the pathogenesis of inherited genetic diseases or neoplasms. In iPSCs established from T cells, the rearrangement of the T cell receptor (TCR) of the established T cell-derived iPSCs (TiPSCs) took over that of the original T cells. If B cell-derived iPSCs (BiPSCs) could be similarly established from mature B cells or plasma cells, the rearrangement of the B cell receptor (BCR) of the BiPSCs would take over that of the original B cell. BiPSCs were recently established from peripheral blood B cells by the simultaneous transfection of Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) together with ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-α (C/EBPα) using a Sendai virus vector (Bueno C, et al. Leukemia 2016).We were able to also establish BiPSCs with immunoglobulin heavy chain (IgH) gene rearrangement from normal B cells purified from lymph nodes using a method different from the above. The main points of our method are consecutive transfection of a high concentration of Yamanaka factors into B cells, which were pre-stimulated with IL-21 and CD40L, using a retrovirus vector, and centrifugations of the cells after their activation by IL-4, IL-2, and CD40-ligand (CD40L) on a retronectin coated plate. We further established doxycycline-controlled (Tet-off system) activation-induced cytidine deaminase (AID)-induced BiPSCs (BiPSCs-A). AID is an enzyme that initiates somatic hypermutation (SHM) and class-switch recombination (CSR) in B cells. We hypothesized that the origin of a myeloma cell is a reprogrammed mature B cell, in which reciprocal chromosome translocation occurs by double stranded breakage (DSB) of DNA induced by AID activation in the nonproductive (nonfunctional) allele of chromosome 14. First, we did not detect an increase in dicentric chromosome (DIC) formation, which is evidence of DSB of DNA, in the BiPSCs-A.We next analyzed the ability of these BiPSCs to differentiate into hematopoietic stem cells (HSCs). Both the parental BiPSCs and BiPSCs-A were capable of differentiating into HSCs as judged by confirmation of CD34 expression and colony-formation of macrophages, granulocytes, and erythrocytes from CD34-positive cells. However, these cells were negative for CD38, CD43, and CD45; we therefore think that these CD34+/CD38-/CD43-/CD45- cells might be hematoendothelial cells as Maxim proposed previously (Vodyanik MA, et al. Blood 2006). Furthermore, both BiPSCs with induced AID expression and BiPSCs without induced AID expression were capable of differentiating into HSCs. Based on the findings regarding the differentiation of BiPSCs-A into HSCs and their retention of the IgH gene rearrangement, there is a possibility that the induction of AID expression might induce chromosomal translocations in the process of differentiation of these BiPSCs into HSCs and further into B cells in experiments using mouse. Thus these BiPSCs might be useful in elucidating the tumor origin of abnormal B cells in B cell tumor formation. These cells might be especially useful in understanding multiple myeloma, which is thought to originate from germinal center (GC) or post-GC B cells and has a productive (functional) allele with IgH gene rearrangement that produces M-protein, and another nonproductive (nonfunctional) allele of chromosome 14. DisclosuresNo relevant conflicts of interest to declare.

Allogeneic Stem Cell Transplantation Versus B-Cell-Receptor Inhibitors in 17p Deletion and/or Refractory Chronic Lymphocytic Leukemia: A Retrospective Comparative Analysis of 'Real Life' Approaches to High Risk Patients, on Behalf of Rete Ematologica Lombarda (REL) and Gruppo Italiano Trapianto Di Midollo Osseo (GITMO)

Introduction: chronic lymphocytic leukemia (CLL) is an indolent disease, but 17p deleted (del) and/or patients who experience early relapse or not responding to chemoimmunotherapy have a very poor outcome. In these patients an allogeneic stem cell transplantation (alloSCT) is indicated, whenever possible. B-cell receptor inhibitors (BCRi) have shown high efficacy with a low toxicity, making the choice of an alloSCT challenging even in high risk patients. The aim of the study is to highlight the outcome of different clinical approaches available in the era of the new drugs.Method: this is a multicenter retrospective analysis on 88 high risk patients treated in 8 Italian Centers. Inclusion criteria were: i) age ≤ 70 years; ii) responding to one of the EBMT (European Bone Marrow Transplantation) criteria for elegibility to alloSCT: relapse within one year after purine-analogues or two years after chemioimmunoterapy or autologous-SCT (ASCT); de novo or acquired 17p del; iii) alloSCT from 2001. Patients who received BCRi before alloSCT or ineligible to alloSCT due to comorbidities were excluded. Patients were assigned to alloSCT or BCRi based on physician and/or patient choice or unavailability of a donor. The analysis started from the transplant date or the start date of BCRi therapy.Results: 50 patients (M/F: 42/8) received an alloSCT, and 38 (M/F 30/8) were treated with BCRi (ibrutinib n=28, rituximab-idelalisib n=10). Median age was 55 (range, 34-68) in alloSCT and 60 years (42-69) in BCRi (p=0.06). Time from diagnosis to alloSCT or BCRi was 59,4 (range, 5-210) and 86,3 months (1-211) (p=0.15), respectively. Fluorescence in situ hybridization (FISH) data were available in 34/50 (68%) alloSCT patients: 17p del was positive in 21 (62%) (de novo n=12, acquired n=8, unknown n=1). Elegibility criteria for alloSCT group were: early relapse n=11, refractory n= 7, ASCT relapse n=11, 17p del n=21 (of which 11 were also chemorefractory). FISH data were available in all BCRi patients: 17p del was positive in 26 (68%) (de novo n=8, acquired n=14, unknown n=4). Elegibility criteria for BCRi group were: early relapse n=2, refractory n= 9, ASCT relapse n=1, 17p del n=26 (of which 16 were also chemorefractory). AlloSCT group had more patients in early relapse or with a failed ASCT (p=0.03 and p=0.01). Heavy chain gene rearrangement was available in 26/50 (52%) alloSCT and 35/38 (92%) BCRi patients and was unmutated in 87% and 86%, respectively. The median number of previous therapy was 2 in both groups (alloSCT: range 1-7; BCRi: range 0-8, p=0.25). Reduced-intensity conditioning regimen was used in 48/50 alloSCT patients, and donor type was sibling in 19, matched unrelated in 26 and haploidentical in 5 cases. Disease status before alloSCT was complete remission (CR)=20, partial remission (PR)=17, stable/progressive disease (SD/PD)=13. The median follow-up was 33 months (1-134) and 14 months (3-32), respectively for alloSCT and BCRi group (p=0.0008). Two-year OS was 59% vs 79% in alloSCT and BCRi, respectively (p=0.32). The cumulative incidence of relapse at 2 years was 30% in alloSCT and 23% in BCRi, with a non-relapse mortality of 20% after alloSCT. Median PFS was 18,6 months (range 1-134) and 12,6 (range 3-24,6) in alloSCT and BCRi, respectively. Two-year PFS was 54% in alloSCT and 77% in BCRi (p=0.19). The main cause of treatment failure was disease progression. In the BCRi group, 8 patients achieved CR at the last follow-up, 5 patients relapsed and died of progressive disease (2 with Richter's transformation), 1 patient died of second cancer. In the alloSCT group, 17 patients were alive and in CR, 11 CR patients died of transplant-related-mortality (6 graft-versus-host disease, 2 infections, 1 embolism, 2 second cancers), 19 patients died in SD/PD at the last follow-up.Conclusions: these retrospective data showed that so far no significant difference in the outcome of 17p del and/or refractory CLL patients have been observed after either alloSCT or BCR inhibitors. The significant different follow-up of the two groups implies some limits: i) BCR inhibitors still have to show long term responses; ii) alloSCT results may improve over the next future by a better selection of eligible patients and the improvement of the transplant procedures. Hopefully, the combination of the two strategies will increase the chance of cure of poor risk CLL patients. DisclosuresReda:Roche: Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding.

Characterization of a PCR-based lymphocyte clonality assay as a complementary tool for the diagnosis of feline lymphoma.

Differentiation between resident mature lymphocyte populations and small cell lymphoma cannot be made by cytological review alone and remains challenging in histopathological review. These cases warrant application of complementary tools like PCR-based immunoglobulin (IG) and T-cell receptor (TCR) clonality testing for confirmation. In this prospective study, diagnostic sensitivity and specificity of different primer sets for routine diagnosis of feline TCR gamma (TCRG) and complete IG heavy chain (IGH) gene rearrangements were assessed. Fine needle aspirates from 20 feline lymphoma cases and lymph node material from 10 cats without hematopoietic neoplasia were subjected to clonality testing. Feline lymphoma cell lines and previously confirmed patient material served as positive control. Detection limits for clonal populations within a polyclonal background was 90% for B-cells and 50% for T-cells. Diagnostic sensitivity and specificity of the clonality assay were 70% and 90%. Overall diagnostic accuracy was 77%, positive predictive value 93% and negative predictive value 60%.

Hepatic MALTOMA in a Case of Chronic Hepatitis C

Primary hepatic lymphoma (PHL) must be considered in any patient who presents with a liver mass or infiltration picture with cholestatic liver abnormality. The presence of constitutional symptoms and significant lymphadenopathy with Hepatitis C infection should invoke a search for PHL by liver biopsy (LB) as it is treatable. A 59-yr male presented with epigastric discomfort, fatigue, 30 lb. weight loss. Minimal alcohol use, 30-pack year smoking, no IVDU, blood transfusion but had a tattoo. He was afebrile, anicteric and had mild hepatomegaly with no lymphadenopathy. Laboratory work up showed elevated AST 112 U/L, ALT 333 U/L, with normal ALP 111 U/L, bilirubin 0.6 mg/dL, LDH 151 U/L, platelets 152,000/mm3, PT 11.1 sec, and INR 1.1. Iron studies, alpha 1 antitrypsin levels, ANA, AMA, and ASMA were normal. However, HCV was positive with HCV RNA of 2.3 million with genotype 2. Subsequent CT abdomen showed multiple enlarged lymph nodes within retroperitoneal and portacaval regions and a 1 cm hypo density in gall bladder (GB) wall. MRCP showed a contracted normal GB. LB confirmed mild chronic hepatitis, with lymphocytic portal inflammation with minimal fibrosis. EUS with biopsy confirmed benign intra-abdominal lymphadenopathy. Bone marrow biopsy showed no evidence of lymphoma. Immunohistochemistry on LB showed portal-based lymphocytic infiltrate with immunophenotype: CD20+ / BCL2+ / CD5- / CD10- / BCL6- / CD23- / cyclin D1-, with monoclonal pattern on immunoglobulin heavy chain gene rearrangement. A diagnosis of extra nodal marginal zone lymphoma (MZL) of mucosa-associated lymphoid tissue (MALT) was made. We treated with sofosbuvir and ribavirin for 12 weeks and he achieved SVR 12. Repeat LB after 3 months after achieving SVR 12 confirmed reduced lymphocytic infiltration and our oncologist decided to observe him with no chemotherapy but his LFT's are normal and he was asymptomatic. PHL is a rare malignancy (0.016% of all non-Hodgkin lymphomas) and is often misdiagnosed. Among PHLs, low-grade marginal B-cell lymphoma (MZL) of MALT is extremely rare. Etiology of MZL is unclear HCV, HBV and EBV are implicated. Hepatic MZL affect middle-aged men and present with constitutional symptoms, hepatomegaly and cholestatic jaundice. LB compatible with lymphoma with no lymphoproliferative disease outside the liver is the presentation. Treating the primary infection and Chemotherapy with rituximab is standard.

Significant Differences in Physicochemical Properties of Human Immunoglobulin Kappa and Lambda CDR3 Regions.

Antibody variable regions are composed of a heavy and a light chain, and in humans, there are two light chain isotypes: kappa and lambda. Despite their importance in receptor editing, the light chain is often overlooked in the antibody literature, with the focus being on the heavy chain complementarity-determining region (CDR)-H3 region. In this paper, we set out to investigate the physicochemical and structural differences between human kappa and lambda light chain CDR regions. We constructed a dataset containing over 29,000 light chain variable region sequences from IgM-transcribing, newly formed B cells isolated from human bone marrow and peripheral blood. We also used a published human naïve dataset to investigate the CDR-H3 properties of heavy chains paired with kappa and lambda light chains and probed the Protein Data Bank to investigate the structural differences between kappa and lambda antibody CDR regions. We found that kappa and lambda light chains have very different CDR physicochemical and structural properties, whereas the heavy chains with which they are paired do not differ significantly. We also observed that the mean CDR3 N nucleotide addition in the kappa, lambda, and heavy chain gene rearrangements are correlated within donors but can differ between donors. This indicates that terminal deoxynucleotidyl transferase may work with differing efficiencies between different people but the same efficiency in the different classes of immunoglobulin chain within one person. We have observed large differences in the physicochemical and structural properties of kappa and lambda light chain CDR regions. This may reflect different roles in the humoral immune response.

A novel canine B-cell leukaemia cell line. Establishment, characterisation and sensitivity to chemotherapeutics.

We established a new B-cell leukaemia cell line CLB70 from a dog with chronic lymphocytic leukaemia. This cell line is positive for CD20, CD45, CD79a, MHC class II, IgG, IgM; weakly positive for CD21; and negative for CD3, CD4, CD5, CD8, CD14, CD34, CD117. PCR for antigen receptor gene rearrangement (PARR) analysis revealed a biclonal immunoglobulin heavy chain (IgH) gene rearrangement and negative result for TCRγ. Western blot analysis of anti- and pro-apoptotic proteins showed increased expression of Bcl-2, Mcl-1, NF-kB, and Ras, and decreased expression of p53. CLB70 cells grow rapidly in vitro and are tumourigenic in nude mice. The CLB70 line is highly sensitive to doxorubicin, less sensitive to etoposide and imatinib, and resistant to piroxicam, celecoxib and dexamethasone. Our results indicate that CLB70 cells are derived from mature B-cells and they may be a useful tool for the development of new therapeutic strategies for both dogs and humans.

Characterization of the T-cell receptor gamma chain gene rearrangements as an adjunct tool in the diagnosis of T-cell lymphomas in the gastrointestinal tract of cats

Feline alimentary lymphoma is the most common hematopoietic neoplasia in cats. It affects mainly the small intestines and is most frequently of T-cell origin. Evaluation of a fine needle aspirate is often the first step in the diagnostic work-up. Differentiation between a resident mature lymphocyte population as encountered in inflammatory bowel disease and small cell lymphoma cannot be achieved by cytology alone. Even full thickness biopsies evaluated by histopathology can be inconclusive. These cases warrant the application of complementary tools like PCR-based T-cell receptor (TCR) clonality testing for confirmation.The aim of this study was to optimize the DNA extraction protocol for formalin fixed and paraffin embedded tissues (FFPE) and to establish a heteroduplex analysis to enhance resolution of the PCR fragments of the T-cell receptor gamma (TCRG) V-J gene. The new protocols resulted in improved quantity and quality of the extracted DNA. Heteroduplex analysis of the samples improved the resolution of the electrophoresis results so that rules for interpretation of the different patterns could be established. Application of this improved setup detected clonal rearrangements in at least one TCRG primer reaction in 31 of 36 of our feline intestinal lymphoma samples after DNA quality testing.

Spleen Histologic Appearance in Common Variable Immunodeficiency: Analysis of 17 Cases.

Histologic and phenotypic analyses of splenectomy samples from 17 patients with common variable immunodeficiency (CVID) showed the following nonspecific, evocative, white-pulp lesions: white-pulp hyperplasia (WPH) with reactive follicles, giant follicles (GFs), marginal zone hyperplasia, periarteriolar T-zone hyperplasia (PATH) and/or granulomas, which enabled us to discern 2 groups: the first (n=6) composed of WPH with reactive follicles without granulomas, and the second (n=9) characterized by the presence of granulomas with or without WPH. All specimens were Epstein-Barr virus negative by in situ hybridization. Molecular analyses revealed a polyclonal immunoglobulin heavy chain gene (IGH) rearrangement (n=12). WPH-only patients were mostly female individuals and younger at CVID onset, diagnosis, and splenectomy, but their interval between the first symptom and diagnosis was longer; they had more associated infectious events, autoimmune disease, pulmonary complications, and liver regenerative nodular hyperplasia; their IgG, IgA, and IgM concentrations were also higher. Granuloma-group patients were mostly male individuals; were older at CVID onset, diagnosis, and splenectomy; had disseminated granulomatous disease, but infectious events, autoimmune disease, pulmonary complications, and liver regenerative nodular hyperplasia were less common; their immunoglobulin concentrations were lower. Histologic comparisons between the WPH-only and granuloma groups showed more intense WPH and more intense marginal zone hyperplasia and fewer GFs in the former versus more developed PATH and more common GFs in the latter. The results of this novel comparative study of the histologic patterns of 17 CVID patients' evocative splenic lesions suggested different biological and clinical profiles.

MiRNAs are critical for the regulation of RAG expression and secondary Ig rearrangement in peripheral B lymphocytes

Abstract The differential expression of miRNAs throughout B cell development suggests that these ncRNAs contribute to stage-specific regulation of the intricate transcriptional program during B cell development. Conditional ablation of Dicer or deletion of the miR-17~92 cluster in early pro-B cells, revealed a critical role of miRNAs in B cell differentiation. In the present study we compare the phenotypes of mice in which enzymes critical for miRNA biogenesis, Dicer, Drosha and DGCR8 are conditionally ablated in B lymphocytes, allowing us to definitively explore the role of miRNAs in B cell development and function. Global ablation of miRNAs in B lymphocytes lead to an early block in B cell development. Rescue of B cell surviva by overexpression of the anti-apopotic factor Bcl2 revealed that in the absence of miRNAs, B cells in the periphery expressed low levels of Ig heavy chain without expressing light chain. We demonstrate that miRNA-deficient B cells fail to regulate recombination machinery in the periphery, resulting in ongoing Ig light chain gene rearrangement. In addition to the upregulation of RAG1/2 in peripheral B cells, we demonstrated ongoing DNA double strand breaks at Ig light chain loci and upregulation of surrogate light chain components. We show that these events occur downstream of deregulated PI3K signaling and we recapitulate many of these defects in wild-type B lymphocytes by targeting individual components of PI3K signaling network. Furthermore, we achieve complete rescue of miRNA deficient B cells when we introduce a pro-survival Bcl2 transgene along with an Ig transgene resistant to light chain editing. Our data highlight an important and novel role for miRNAs in the maintenance of a mature phenotype in peripheral B cells.

An Immunogenetic Signature of Ongoing Antigen Interactions in Splenic Marginal Zone Lymphoma Expressing IGHV1-2*04 Receptors.

Prompted by the extensive biases in the immunoglobulin (IG) gene repertoire of splenic marginal-zone lymphoma (SMZL), supporting antigen selection in SMZL ontogeny, we sought to investigate whether antigen involvement is also relevant post-transformation. We conducted a large-scale subcloning study of the IG rearrangements of 40 SMZL cases aimed at assessing intraclonal diversification (ID) due to ongoing somatic hypermutation (SHM). ID was identified in 17 of 21 (81%) rearrangements using the immunoglobulin heavy variable (IGHV)1-2*04 gene versus 8 of 19 (40%) rearrangements utilizing other IGHV genes (P= 0.001). ID was also evident in most analyzed IG light chain gene rearrangements, albeit was more limited compared with IG heavy chains. Identical sequence changes were shared by subclones from different patients utilizing the IGHV1-2*04 gene, confirming restricted ongoing SHM profiles. Non-IGHV1-2*04 cases displayed both a lower number of ongoing SHMs and a lack of shared mutations (per group of cases utilizing the same IGHV gene). These findings support ongoing antigen involvement in a sizable portion of SMZL and further argue that IGHV1-2*04 SMZL may represent a distinct molecular subtype of the disease.

Diagnostic efficacy of cell block method for vitreoretinal lymphoma.

BackgroundVitreoretinal lymphoma (VRL) is a life- and sight-threatening disorder. The aim of this study was to analyze the usefulness of the cell block method for diagnosis of VRL.MethodsSixteen eyes in 12 patients with VRL, and 4 eyes in 4 patients with idiopathic uveitis presenting with vitreous opacity were enrolled in this study. Both undiluted vitreous and diluted fluids were isolated during micro-incision vitrectomy. Cell block specimens were prepared in 19 eyes from diluted fluid containing shredding vitreous. These specimens were then submitted for HE staining as well as immunocytological analyses with antibodies against the B-cell marker CD20, the T-cell marker CD3, and cell proliferation marker Ki67. Conventional smear cytology was applied in 14 eyes with VRL using undiluted vitreous samples. The diagnosis of VRL was made based on the results of cytology, concentrations of interleukin (IL)-10 and IL-6 in undiluted vitreous, and immunoglobulin heavy chain gene rearrangement analysis.ResultsAtypical lymphoid cells were identified in 14 out of 15 cell block specimens of VRL (positive rate: 93.3 %), but in 5 out of 14 eyes in conventional smear cytology (positive rate: 35.7 %). Atypical lymphoid cells showed immunoreactivity for CD20 and Ki67. Seven cell block specimens were smear cytology-negative and cell block-positive. The cell block method showed no atypical lymphoid cells in any patient with idiopathic uveitis.ConclusionsCell block specimens using diluted vitreous fluid demonstrated a high diagnostic sensitivity and a low pseudo-positive rate for the cytological diagnosis of VRL. The cell block method contributed to clear differentiation between VRL and idiopathic uveitis with vitreous opacity.Electronic supplementary materialThe online version of this article (doi:10.1186/s13000-016-0479-1) contains supplementary material, which is available to authorized users.