Characterization of a New Human B Cell Line (Bonna-12) with Trisomy 9 and Trisomy 12 Chromosomal Abnormality

A new EBV positive human B-cell line, BONNA-12 was established from splenic cells of a patient with a hairy cell leukemia (HCL). BONNA-12 cells grew spontaneously and formed colonies in semisolid media. Although the BONNA-12 cell line was identical with the patient's spleen cells by HLA analysis and Southern blot examination of minisatellite DNA patterns, the immunoglobulin heavy and light chain rearrangement patterns differed from the original HCL. Cytogenetic analysis of the BONNA-12 cell line demonstrated in the major cell clone a 47, X, -Y, +9, +12 karyotype. Trisomy 12 is a characteristic abnormality in chronic lymphocytic leukemia that also rarely occurs in HCL. The BONNA-12 cell line is of potential value in the study of trisomy 12 in chronic B cell malignancies.     

EHMT2 (G9a) activation in mantle cell lymphoma and its associated DNA methylation and gene expression

Objective:The function of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) has been studied in several cancers; however, little is known about its role in mantle cell lymphoma (MCL). Thus, this study aimed to characterize the significance and function of EHMT2 in MCL.Methods:EHMT2 expression in MCL and reactive hyperplasia (RH) were investigated by immunohistochemistry. Genome-wide analysis of DNA methylation was performed on EHMT2 + MCL samples. The function of EHMT2 was determined by CCK8, flow cytometry, and western blot assays. Gene expression profile analysis was performed before and after EHMT2 knockdown to search for EHMT2-regulated genes. Co-immunoprecipitation (Co-IP) experiments were conducted to identify the proteins interacting with EHMT2.Results:EHMT2 was expressed in 68.57% (24/35) of MCLs but not in any RHs. Genome-wide analysis of DNA methylation on EHMT2 + MCLs revealed that multiple members of the HOX, FOX, PAX, SOX, and CDX families were hypermethylated or hypomethylated in EHMT2 + MCLs. BIX01294, a EHMT2 inhibitor, inhibited MCL cell growth and stalled cells in the G1 phase. Additionally, BIX01294 downregulated the expressions of cell cycle proteins, cyclin D1, CDK4, and P21, but upregulated the expressions of apoptosis-related proteins, Bax and caspase-3. Co-IP experiments revealed that EHMT2 interacted with UHRF1, HDAC1, and HDAC2 but not with HDCA3. After EHMT2 knockdown, multiple genes were regulated, including CD5 and CCND1, mostly enriched in the Tec kinase signaling pathway. In addition, several genes (e.g., MARCH1, CCDC50, HIP1, and WNT3) were aberrantly methylated in EHMT2 + MCLs.Conclusions:For the first time, we determined the significance of EHMT2 in MCL and identified potential EHMT2-regulated genes.     

Differential diagnosis of chronic lymphocytic leukemia/small lymphocytic lymphoma and other indolent lymphomas,including mantle cell lymphoma

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) accounts for approximately 1% of all lymphomas in our department. In this article, we describe the differential diagnosis of CLL/SLL from other indolent lymphomas, with special reference to follicular lymphoma, marginal zone B-cell lymphoma, lymphoplasmacytic lymphoma, and mantle cell lymphoma, although the latter is considered to be aggressive. CLL/SLL often exhibits proliferation centers, similar to follicular lymphoma. Immunohistological examination can easily distinguish these two lymphomas. The most important characteristic of CLL/SLL is CD5 and CD23 positivity. Mantle cell lymphoma is also CD5-positive and there are some CD23-positive cases. Such cases should be carefully distinguished from CLL/SLL. Some marginal zone lymphomas are also positive for CD5 and such cases are often disseminated. Lymphoplasmacytic lymphoma should also be a differential diagnosis for CLL/SLL. It frequently demonstrates MYD88 L265P, which is a key differential finding. By immunohistological examination, the expression of lymphoid enhancer-binding factor 1 is specific for CLL/SLL and can be a good marker in the differential diagnosis.     

Concomitant high expression of Toll‐like receptor (TLR) and B‐cell receptor (BCR) signalling molecules has clinical implications in mantle cell lymphoma

Mantle cell lymphoma (MCL) is an aggressive disease with frequent relapse. Targeted therapies against B‐cell receptor (BCR) molecules have demonstrated improved outcomes in relapsed cases. However, clinical responses are slow and selective, with failure to attain complete remission in a significant subset of patients. Complex interaction of BCR signal transduction with toll‐like receptor (TLR) and other pathways in MCL remains unknown, thus averting progress in development of targeted therapies. We have performed detailed digital quantification of BCR/TLR signalling molecules and their effector pathways in a cohort (n = 81) of MCL patients and correlated these data with overall survival. Hierarchical clustering model based on BCR/TLR genes revealed two distinct (BCR^high and BCR^low) subsets of patients (n = 32; 40%) with significant differences in expression (>1.5‐fold change; p < 0.05). Higher levels of BTK/SYK/BLNK/CARD11/PLCG signalosome and lower expression of MALT1/BCL10 genes suggested tonic pattern of BCR activation. Amplified expression of TLR6/TLR7/TLR9 was noted in concert with hyper‐responsiveness of BCR machinery. MYD88, a key TLR adaptor molecule, was not upregulated in any of these clusters, which may suggest a ‘cross‐talk’ between BCR and TLR pathways. In sync with BCR/TLR signalling, we recorded significantly enhanced expression of genes associated with NF‐kB pathway in BCR^high subset of MCL patients. On univariate analysis, the BCR^high patients showed a trend towards inferior clinical response to a standardized treatment protocol, compared with the BCR^low group (log rank, p = 0.043). In conclusion, we have identified hyperactive BCR/TLR signalling pathways and their effector downstream targets in a subset of MCL patients and associated it with poor clinical outcomes. Our study provides quantitative evidence at RNA expression level of possible concomitant collaboration between TLR and BCR signalling molecules in MCL. These data will provide further insights for future functional studies and, hence, development of targeted therapies for MCL patients. Copyright © 2015 John Wiley & Sons, Ltd.     

The ultrastructural study of non-Hodgkin’s lymphoma cell,chronic lymphocytic and hairy cell leukemia

Non-Hodgkin’s lymphoma cell leukemia (NHLCL), chronic lymphocytic leukemia (CLL) and hairy cell leukemia (HLC) are the diseases very similar to each other. The differential diagnosis is very difficult, especially when there are small lymphoid cells in peripheral blood and bone marrow under light microscope. We have observed 34 cases with electron microscope. The studies were correlated with clinical manifestation, cytology, pathology and immunologic histochemistry. Ultrastructural features strongly indicated the difference in three various diseases, although all the immunologic markers showed B-cell type. It is concluded that electron microscopic examination is of a definite significance in the diagnosis and successful treatment.     

Patient with acute B-cell leukemia of burkitt's type (L3) and marker chromosomes including an (8;14) translocation

A 20-year-old man with acute B-cell leukemia of Burkitt's type (L3) presenting unusual début symptoms with jaw involvement is reported. The leukemic cells revealed chromosomal abnormalities including four marker chromosomes [1q+, 6q?, t(8;14)].     

Veterans with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) have a markedly increased rate of second malignancy, which is the most common cause of death

Patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) have an increased incidence of high-grade lymphoid malignancy. The risk of non-lymphoid second malignancy in this population is not well-defined to date. To test the hypothesis that patients with CLL/SLL have an increased risk of second malignancy, we studied the rate of second malignancy in 132 CLL/SLL patients and compared it to the rate of malignancy (excluding non-melanomatous skin cancer) in the Central Arkansas Veterans Healthcare System population of approximately 38,000 veterans over a period of 11.5 years. The rate of second malignancy, diagnosed concomitantly or after CLL/SLL, and the age-adjusted rate of malignancy calculated from tumor registry reports and demographic data, were used to calculate a Standardized Morbidity Ratio (SMR) with 95% confidence interval (CI). Twenty-one (16%) of the CLL/SLL patients had second malignancies (19 non-lymphoid, 1 Richter's transformation and 1 Hodgkin's disease), which were fatal in 15 (71%) patients. The SMR for the CLL/SLL population was 2.97 (95% CI 1.84 - 4.55) for second malignancy and 2.69 (95% CI 1.62 - 4.21) for non-lymphoid second malignancy. This study of a well-defined CLL/SLL population shows a significantly increased risk of second malignancy, which was the primary cause of death for 9% of all CLL/SLL patients (34% of all patient deaths).     

An unusual case of indolent B-cell lymphoma with distinct chronic lymphocytic leukemia and marginal zone differentiation according to the site of involvement

The immunological profile of lymphoproliferative disorders is usually conserved whatever the involved site, thus allowing a reliable diagnosis from peripheral blood analysis, especially in small lymphocytic lymphoma/chronic lymphocytic lymphoma (SLL/CLL). Here we present a case wherein the cytology and immunophenotype of blood specimen and bone marrow argue in favor of SLL/CLL with a typical Matutes score (5/5), whereas the cyto-histology and immunophenotype of spleen specimen led to the diagnosis of splenic marginal zone B-cell lymphoma (SMZL). Moreover genomic analysis showed that the splenic cells displayed a SMZL signature. Whereas these data suggested the presence of 2 B-cell clones, the study of the mutational status of IgV_H gene in blood and spleen demonstrated the presence of a single clone, which likely developed simultaneously along two distinct ways of differentiation according to the anatomic site suggesting here the predominant role of a micro-environmental factor in cell differentiation. Although rare, this kind of event must be kept in mind as a cause of discrepancies between diagnoses from different sites.     

Factors affecting toxicity, response and progression-free survival in relapsed patients with indolent B-cell lymphoma and mantle cell lymphoma treated with rituximab: a Japanese phase II study.

BACKGROUND: The aim of the study was to determine factors affecting the toxicity and efficacy of rituximab monotherapy in relapsed patients with indolent B-cell lymphoma and mantle cell lymphoma (MCL). PATIENTS AND METHODS: A total of 90 patients were enrolled and treated with rituximab infusions at 375 mg/m2 once weekly for 4 weeks. Central pathology review revealed that histologically, 81 patients had indolent B-cell lymphoma or MCL: 59 with follicular lymphoma, 17 with MCL, four with marginal zone lymphoma and one with lymphoplasmacytoid lymphoma. Of these, four were ineligible due to violation of other eligibility criteria. Pre-treatment variables affecting toxicities were analyzed for all 90 patients, and those affecting response and progression-free survival (PFS) were analyzed for 77 eligible patients with confirmed indolent B-cell lymphoma or MCL. The relationship between serum rituximab levels and efficacy was also analyzed for 66 eligible patients. RESULTS: Hematological toxicities (grade > or =3) occurred more frequently in females (P <0.05), and thrombocytopenia and leukopenia were more frequent in patients with high lactate dehydrogenase (LDH) levels (P <0.05). Non-hematological toxicities (grade > or =2) were more frequent in patients with extranodal disease or bone marrow involvement. The overall response rate (ORR) in patients receiving one prior chemotherapy regimen was higher than those receiving two or more regimens (P <0.05). The median PFS was shorter in MCL patients, in those with extranodal disease, or in those receiving two or more prior chemotherapy regimens (P <0.01). The PFS intervals of patients with higher serum rituximab levels (> or =70 microg/ml) immediately before the third infusion were longer than in other patients (P <0.01). CONCLUSIONS: Several prognostic factors and serum rituximab levels are useful for predicting the toxicity and efficacy of rituximab monotherapy.     

Erroneous class switching and false VDJ recombination: Molecular dissection of t(8;14)/MYC-IGH translocations in Burkitt-type lymphoblastic leukemia/B-cell lymphoma

The chromosomal translocation t(8;14)(q24;q32) with juxtaposition of MYC to enhancer elements in the immunoglobulin heavy chain (IGH) gene locus is the genetic hallmark of the majority of Burkitt lymphoma and a subset of Diffuse large B-cell lymphoma patients. Around 3% of adult B-lineage acute lymphoblastic leukemia (ALL) patients show this aberration. Flow cytometry mostly reveals a “mature B-ALL” or “Burkitt-type” ALL immunophenotype. Using long-distance PCR for t(8;14)/MYC-IGH fusion, we investigated bone marrow, peripheral blood and a few other samples with suspected Burkitt-ALL or mature B-ALL and identified 133 MYC-IGH-positive cases. The location of the chromosomal breaks in the IGH joining and the 8 different switch regions was determined using a set of long-distance PCRs. The chromosomal breakpoints with the adjacent MYC regions on 8q24 were characterized by direct sequencing in 49 cases. The distribution of chromosomal breaks among the IGH joining and switch regions was the following: JH 23.3%, M 21.8%, G1 15.0%, G2 7.5%, G3 3.8%, G4 4.5%, A1 12.8%, A2 3.8%, E 7.5%. Two breakpoint clusters near MYC were delineated. There was no clear correlation between the degree of somatic hypermutation and the chromosomal break locations. Epstein Barr virus was detected in 5 cases (4%). This detailed and extensive molecular analysis illustrates the molecular complexity of the MYC-IGH translocations and the detected distribution of breakpoints provides additional evidence that this translocation results from failed switch and VDJ recombinations. This study may serve as a model for the analysis of other IGH translocations in B-cell lymphoma.     

Pharmacokinetic profile and first preliminary clinical evaluation of bendamustine in Taiwanese patients with heavily pretreated indolent B‐cell non‐Hodgkin lymphoma and mantle cell lymphoma

Prior studies found bendamustine is efficacious in patients with indolent B‐cell non‐Hodgkin lymphoma (NHL). To date, no studies have reported the efficacy of bendamustine in a Chinese population. This multicentre phase II trial evaluated the pharmacokinetics (PK), safety and efficacy of bendamustine monotherapy in Chinese patients in Taiwan with pretreated indolent B‐cell NHL or mantle cell lymphoma (MCL). For PK assessments, patients were randomized (n = 16; 11 with indolent B‐cell NHL and five with MCL) to 90 or 120 mg/m² of bendamustine for the first cycle. Plasma levels of bendamustine and its two metabolites were analyzed. For efficacy and safety evaluations, bendamustine 120 mg/m² was given to all patients every 3 weeks starting at cycle 2 for a minimum of a total of six cycles. The median age of patients was 61.7 years, and the majority were men (75%). The median number of prior treatments was 4 (range, 1–9 regimens), and all patients were previously treated with rituximab. Bendamustine plasma concentration peaked near the end of infusion and was rapidly eliminated with a mean elimination half‐life (t_1/2) of 0.67–0.8 h. Of the evaluable patients (n = 14), the overall response rate was 78.6%, including 7.2% of patients having a complete response. Mean progression‐free survival was 27.5 weeks. The most common grade 3–4 adverse events were leucopenia (56.3%), neutropenia (56.3%) and thrombocytopenia (25%). In conclusion, bendamustine was efficacious and well tolerated in Taiwanese patients with indolent NHL and MCL with a similar PK profile to that of other populations. Copyright © 2014 John Wiley & Sons, Ltd.     

Frequency and impact of grade three or four toxicities of novel agents on outcomes of older patients with chronic lymphocytic leukemia and non-Hodgkin lymphoma (alliance A151611)

Objective

Older patients with cancer suffer from chemotherapy-related toxicities more frequently than younger patients. As novel agents are being used more commonly in chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL), toxicities of these agents in older patients have not been well studied. Further, impact of these toxicities on outcomes in the elderly is unknown. This study aimed to answer both questions.

CD5-positive, cyclinD1-negative mantle cell lymphoma with a translocation involving the CCND2 gene and the IGL locus

Distinguishing mantle cell lymphoma (MCL), from low-grade B-cell lymphoma is important because MCL is clinically more aggressive and is treated differently. Though most MCL overexpress cyclinD1 (CCND1) and have a t(11;14)(q13;q32), MCL that are negative for CCND1 exist. Some have translocations involving cyclinD2 (CCND2) and either the immunoglobulin heavy chain or kappa light chain locus. We present a CD5-positive, CCND1-negative B-cell lymphoma with a novel translocation involving CCND2 and the immunoglobulin lambda (IGL) gene. A 64-year-old male underwent resection of a polypoid mass of the ileum. Histology showed atypical, medium-sized lymphoid cells positive for CD20, CD5, CD43, and CCND2 by immunohistochemistry, and negative for CCND1, CCND3, and p27. Fluorescence in situ hybridization was negative for CCND1 abnormalities, but demonstrated a CCND2/IGL fusion. Clinical workup revealed stage IV disease. Current diagnostic criteria are insufficient for subclassifying this case, highlighting the need for additional studies on CCND2-translocated B-cell lymphomas to guide therapy appropriately.     

Inhibition of poly(ADP‐ribose) polymerase (PARP) and ataxia telangiectasia mutated (ATM) on the chemosensitivity of mantle cell lymphoma to agents that induce DNA strand breaks

There is a high incidence of genomic aberration of ataxia telangiectasia mutated (ATM) and genes encoding proteins involved in the ATM pathway in mantle cell lymphoma (MCL). It has been shown that poly(ADP‐ribose) polymerase inhibitor (PARPi) strongly enhances the cytotoxicity of agents, causing single‐strand DNA breaks in cells with impaired homologous recombination repair. Here, we show that PARPi AG14361 potentiates the cytotoxicity induced by topotecan treatment in MCL cell lines, which was not dependent on either TP53 or CHEK2 status. Inhibition and/or knockdown of ATM and BRCA2 did not potentiate the cytotoxic effect of treatment with PARPi and topotecan. With loss of function of ATM, other kinases can still mediate activation of ATM substrates as demonstrated by continued phosphorylation of CHEK2 (Thr‐68), although attenuated and delayed. These results suggest that PARPi may enhance the therapeutic efficacy of DNA damaging agents on MCL through TP53‐independent mechanisms without requiring the inhibition of either ATM or BRCA2. Copyright © 2011 John Wiley & Sons, Ltd.     

Congenital MLL-positive B-cell acute lymphoblastic leukemia (B-ALL) switched lineage at relapse to acute myelocytic leukemia (AML) with persistent t(4;11) and t(1;6) translocations and JH gene rearrangement

Congenital acute leukemia is a rare form of childhood leukemia, in which lineage conversion at relapse is very rarely reported. Here we describe a case of congenital B-cell acute lymphoblastic leukemia (B-ALL) with t(4;11) and t(1;6) translocations, which at relapse underwent a switch to monocytic lineage with persistence of the original cytogenetic translocations and clonal rearrangement of the JH gene. Similar to the other described cases of congenital acute leukemia with lineage conversion, our case had a MLL gene rearrangement and followed an aggressive clinical course.     

弥漫性大B细胞淋巴瘤t(14;18)易位和c-myc基因重排研究进展

 弥漫性大B细胞淋巴瘤（DLBCL）是一组B淋巴细胞来源最常见的、且具有异质性的非霍奇金淋巴瘤（NHL）。就DLBCL中t(14;18)易位和c-myc基因重排及其诊断分型、治疗和预后评价方面的最新研究进展进行综述。