Molecular genetic demonstration of the diverse evolution of Richter's syndrome (chronic lymphocytic leukemia and subsequent large cell lymphoma)

Paired samples of chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) and the subsequent diffuse large cell lymphoma (DLL) of six cases of Richter's syndrome were investigated to establish the clonal relationship between the CLL/SLL and the DLL components and to define the oncogene and/or tumor-suppressor gene alterations involved in the morphologic transformation of CLL/SLL. Southern blot hybridization analysis showed identical clonal immunoglobulin (Ig) gene-rearrangement patterns in the CLL/SLL and DLL components in four cases and different Ig gene-rearrangement patterns in two cases. Polymerase chain reaction (PCR) amplification, cloning, and DNA sequencing of complementary determinant region 3 (CDR3) of the Ig-heavy chain gene of one of the two cases in which the Ig gene- rearrangement patterns were different showed nonidentical sequences in the CLL/SLL and DLL components. In the other case, monomorphic Epstein- Barr virus (EBV) genome integration was detected in the DLL but not in the CLL, suggesting that the CLL and DLL components in this case of Richter's syndrome also represent unrelated clones. Single-strand conformation polymorphism (SSCP) analysis and sequencing of exons 5 through 9 of the p53 tumor-suppressor gene showed a mutation in codon 176 of the DLL but not in the CLL/SLL component in one case where the CLL/SLL and DLL represented different clones. The p53 mutation probably played a role in the development of the lymphoma rather than morphologic transformation of the CLL/SLL in this case. SSCP analysis and sequencing also showed identical mutations in codon 282 in both the CLL/SLL and DLL components in a case where the CLL and DLL represented identical clones. Thus, this p53 gene mutation was present both before and after morphologic transformation, and therefore, probably did not play a primary role in this process. Southern blot hybridization analysis failed to show evidence of bcl-1, bcl-2, c-myc proto-oncogene or retinoblastoma (Rb) tumor-suppressor gene rearrangements in these six cases of Richter's syndrome. In conclusion, the original CLL/SLL and the subsequent DLL in Richter's syndrome may or may not be derived from identical clones, and the well-known proto-oncogenes and tumor- suppressor genes do not appear to play an obvious and consistent role in the morphologic transformation of CLL/SLL to DLL.     

Concurrent chromosomal alterations at 3q27, 8q24 and 18q21 in B-cell lymphomas

Recurrent chromosomal translocations in malignant lymphomas most commonly involve 18q21(bcl-2), 8q24 (c-myc) and 3q27 (bcl-6), with an incidence of 27%, 11% and 6%, respectively. Individual cases concurrently harbouring two of these three rearrangements have been previously reported. This report describes four patients with cytogenetic alterations affecting all three loci, which was confirmed by molecular analysis in one case. Clinically, each patient had aggressive B-cell lymphoma with disseminated disease often involving the central nervous system, poor response to chemotherapy and short survival. Activation of c-myc in association with deregulation of bcl-2, bcl-6 or both confers high-grade disease with a poor prognosis.     

Molecular features of primary mediastinal B-cell lymphoma: involvement of p16INK4A, p53 and c-myc

Primary mediastinal B-cell lymphoma (PMBL) shows chromosome 9p anomalies in 50% of cases. Based on reports that p16INK4A gene, located on this chromosomal arm, is frequently altered in aggressive lymphomas, we analysed for alterations of this gene in 27 cases of PMBL, which were part of a series of 32 PMBL cases that have been characterized for alterations in c-myc, p53, N-ras, bcl-1, bcl-2, bcl-6 and for Epstein-Barr virus (EBV) infection. Four cases showed p16INK4A gene anomalies, including three with promoter methylation and one homozygous deletion. Eight PMBLs showed c-myc rearrangements. Three additional cases showed sequence variations in the c-myc P2 promoter, two of which consisted of the same germline variation involving a novel polymorphic XhoI site. Four tumours contained p53 gene mutations and three had clonal EBV infection. One case had a bcl-6 rearrangement. In conclusion, our study shows that p16INK4, c-myc and p53 alterations occur in 15%, 25% and 13% of PMBLs, respectively. EBV monoclonality was found in 9% of cases, whereas no abnormality was detected in bcl-1, bcl-2 and N-ras. Thus, none of the common genetic aberrations seen in other types of non-Hodgkin's lymphomas appears to be stringently involved in the pathogenesis of this unique lymphoma type.     

Establishment and characterization of acute B-cell lymphocytic leukemia cell line showing (8;14) and (14;18) chromosome translocation

Cell line KHM-2B expressing two oncogene products, c-myc and bcl-2, was established from a patient with acute lymphocytic leukemia with an 8;14 and 14;18 chromosome translocation. Surface marker studies of the cell line showed that the cells were positive for HLA-DR, CALLA (CD10), B1 (CD20) and B4 (CD19), but negative for T11 (CD2). The fresh cells from peripheral blood of the patient had no surface immunoglobulins, whereas KHM-2B cells were positive for mu.lambda type surface immunoglobulin. A cytogenetic analysis of the cell line revealed two translocations, t (8;14) (q24;q32) and t(14;18)(q32;q21). Rearrangement of the c-myc and bcl-2 genes was detected by Southern blot analysis of the KHM-2B DNA. Northern blot analysis revealed production of c-myc and bcl-2 mRNAs. These results indicated that two oncogenes were activated by two translocations to immunoglobulin genes.     

Bendamustine + rituximab chemoimmunotherapy and maintenance lenalidomide in relapsed,refractory chronic lymphocytic leukaemia and small lymphocytic lymphoma: A Wisconsin Oncology Network Study

Bendamustine + rituximab (BR) has demonstrated high response rates in relapsed/refractory (R/R) chronic lymphocytic leukaemia (CLL) and small lymphocytic lymphoma (SLL). However, progression‐free survival (PFS) after BR is <18 months. This study was designed to determine if maintenance lenalidomide after BR induction could improve PFS in R/R CLL/SLL. Thirty‐four patients with R/R CLL/SLL who had received 1–5 prior chemotherapy regimens were treated with 6 cycles of BR induction. Patients achieving at least a minor response received twelve 28‐d cycles of lenalidomide 5–10 mg/d. The primary endpoint was PFS. The median age was 67 years, with a median of 2 prior therapies. Eleven patients had confirmed presence of 17p and/or 11q deletions. Twenty‐five (74%) completed 6 cycles of induction BR (response rate 56%). Nineteen (56%) patients received maintenance lenalidomide; only 6 patients completed the intended 12 cycles, highlighting the limited feasibility of lenalidomide in this setting, primarily due to haematological and infectious toxicities. The observed median PFS of 18·3 months is not significantly different from that of BR induction in R/R CLL/SLL without maintenance therapy (15·2 months). It is possible that lenalidomide maintenance may be more feasible and effective in the front‐line setting, which is being tested in an ongoing trial (NCT01754857).     

Dissecting aggressive B-cell lymphoma through genomic analysis – What is clinically relevant?

The aggressive B-cell lymphomas are a diverse collection of cancers grouped together based on clinical behavior and derivation from B lymphocytes. Genomic analyses on these tumours are now translating into improved classification systems and identification of underpinning targetable biology. Simple karyotyping revealed key translocations involving MYC, BCL2, and BCL6 that have impacted lymphoma classification in the World Health Organization classification scheme. Subsequently, gene expression profiling identified molecular subgroups within the most common lymphoma, diffuse large B-cell lymphoma (DLBCL): activated B-cell-like and germinal centre B-cell-like. Finally, next generation sequencing has revealed a modest number of frequently mutated genes and a long list of infrequent mutations. The mutational landscapes involve diverse genes associated with dysregulated signalling, epigenetic modification, blockade of cellular differentiation, and immune evasion. These mutational “signatures” are enriched in the different aggressive lymphoma subtypes impacting phenotypes and identifying therapeutic targets. Challenges to implementing genomic assays into clinical practice remain.     

苦参素注射液联合顺铂对人肝癌SMMC-7721细胞凋亡相关基因c-myc、bcl-2和bax表达的影响

目的探讨苦参素注射液(MI)联合顺铂(DDP)对人肝癌SMMC-7721细胞凋亡相关基因c-myc、bcl-2和bax表达的影响。方法分别采用MI、顺铂及MI联合顺铂干预SMMC-7721细胞。TUNEL法检测细胞凋亡率,半定量RT-PCR法检测c-myc、bcl-2和bax mRNA表达,二步法免疫组化检测c-myc、bcl-2和bax蛋白表达。结果苦参素组、顺铂组和联合用药组细胞凋亡率显著上升,与对照组(不干预)比较差异有统计学意义(P<0.05或P<0.01),联合用药具有单纯相加或协同作用;联合用药组的细胞凋亡率显著增加,bax mRNA和蛋白表达显著升高,c-myc、bcl-2 mRNA和蛋白表达显著减少,与DDP单药组比较差异有统计学意义(P<0.05或P<0.01)。结论苦参素注射液联合顺铂具有单纯相加或协同诱导肝癌SMMC-7721细胞凋亡作用,其机制可能与bax基因表达上调和c-myc、bcl-2基因表达下调有关。     

Histogenetic correlations between subcategories of small noncleaved cell lymphomas.

To assess the biologic relevance of the morphologic distinctions between subtypes of small noncleaved cell lymphomas (SNCL), ie, the sporadic Burkitt's type (sBT) and the non-Burkitt's type (nBT), we have examined the molecular organization of several lymphomagenic oncogenes (c-myc, bcl-1, bcl-2) and the potential pathogenetic contribution of the Epstein-Barr virus (EBV). Twenty-nine cases of SNCL, not associated with immunodeficiency syndromes, were reviewed and classified as sBT (18 cases) or nBT (11 cases) without knowledge of the clinical or molecular data. Southern blot analysis of 18 sBTs found 17 to contain c-myc rearrangements. Fifteen of these comigrated with an Ig heavy-chain gene segment, indicating t(8;14) translocation. Chromosome 8 breakpoints were clustered in the first exon and the first intron of the c-myc gene. Chromosome 14 breakpoints mapped to the JH locus in three tumors, the S mu locus in nine tumors, and the S alpha locus in the remaining three tumors. Cases involving the S alpha locus appeared to have a more rapid clinical course. All sBTs possessed germline bcl-2 and bcl-1 gene fragments. In contrast, Southern blot analysis of 11 nBTs found none with c-myc rearrangements. Rather, three of 10 evaluable nBTs had bcl-2 rearrangements. The remaining seven showed no evidence of involvement by any of the lymphoma-associated oncogene/breakpoint regions studied. EBV genome was detected in two sBTs and in one nBT, and thus was not a distinguishing feature. These results indicate that the subtle histologic differences that distinguish subcategories of SNCL are significant biologically and reflect distinct molecular mechanisms of lymphomagenesis. Furthermore, the data suggest that the nBTs comprise a heterogeneous group with respect to their molecular genetic composition and confirm the remarkable molecular genetic homogeneity of the sBT group.     

A prospective study of 728 cases of non-Hodgkin lymphoma from a single laboratory in Shanghai,China

The frequency of subtypes of lymphoid neoplasms was determined in a prospective series of 831 patients presenting at 29 Shanghai hospitals over a 4-year period. Diagnosis and classification was established in a single laboratory according to the 2001 WHO classification system. The frequency of non-Hodgkin lymphoma was 87.6% (n = 728) and Hodgkin lymphoma was 12.4% (n = 103). The most prevalent NHL subtypes diagnosed using WHO criteria were diffuse large B cell lymphoma (DLBCL), precursor B lymphoblastic leukemia/lymphoma and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Although a low incidence has been reported in some Asian populations, CLL/SLL was commonly encountered, indicating that chronic lymphoid neoplasms are not rare in Shanghai. Consistent with previous reports, our findings indicate a decrease in the frequency of follicular lymphoma and an increase in T cell neoplasms compared to the West. Precursor T lymphoblastic leukemia/lymphoma, anaplastic large T cell lymphoma, aggressive NK cell leukemia, angioimmunoblastic T cell lymphoma and peripheral T cell lymphoma were prominent subtypes of T cell NHL.     

IDEC-C2B8 anti-CD20 (rituximab) immunotherapy in patients with low-grade non-Hodgkin's lymphoma and lymphoproliferative disorders: evaluation of response on 48 patients

This study focused on the efficacy of IDEC-C2B8 (chimeric anti-CD20) immunotherapy relative to specific subtypes of low-grade lymphoproliferative disorders/non-Hodgkin's lymphomas (LPD/NHL). Forty-eight patients with resistant or relapsed disease completed the IDEC-C2B8 infusion schedule of 375 mg/m2/wk x 4 wk. The LPD/NHL subtypes included: (a) follicular centre cell lymphoma (FCC) in 22 patients; (b) mantle cell lymphoma (MCL) in 10; (c) 1 diffuse large cell lymphoma (DLCL); and (d) the category of small lymphocytic lymphoma/chronic lymphocytic leukaemia (SLL/CLL) and related disorders in 15 patients. No patient obtained a complete remission. Ten patients (21%) achieved partial remission: 6 FCC, 2 MCL, 1 DLCL and 1 patient from the SLL/CLL group. Twenty-eight patients had stable disease and 10 progressed during immunotherapy. In patients with CLL and MCL in leukaemic phase, there was no correlation between the marked decrease in circulating neoplastic cells following antibody infusions and amelioration of the tumour burden. The results suggest that the subtype of LPD/NHL and the intensity of CD20 on the tumour cells influence the effectiveness of IDEC-C2B8. The antibody was most efficacious against FCC lymphoma. The efficacy (at the dose schedule of 375 mg/m2/wk x 4) against MCL and SLL/CLL appeared to be limited, however.     

幽门螺杆菌感染与胃癌及癌前病变中c-myc、p53、C-erbB-2及bcl-2蛋白表达关系的研究

目的 观察胃癌、癌前病变中幽门螺杆菌(Hp)感染情况及其与c-myc、p53、c-erbB-2、bcl-2在胃癌及癌前病变中的表达关系,探讨Hp在胃癌、癌前病变发生及发展中的作用以及探索从癌前病变到癌变过程中的基因变化规律.方法 放大内镜及超声内镜检查收集103例胃黏膜标本,用免疫组织化学染色方法检测Hp感染及不同组织间c-myc、p53、c-erbB-2、bcl-2的表达.结果 c-myc、p53、c-erbB-2和bcl-2阳性表达率在胃癌组及癌前病变组中呈过度表达,与正常对照组相比有显著性差异(P<0.05).此外,Hp感染组c-myc、p53、c-erbB-2及bcl-2同时表达者为5例(9.4%);与无Hp感染组相比有显著性差异(P<0.01).结论 胃癌及癌前病变中组织存在c-myc、p53、c-erbB-2、bel-2多个表达,与Hp感染密切相关.     

Etiologic heterogeneity among non-Hodgkin lymphoma subtypes

Understanding patterns of etiologic commonality and heterogeneity for non-Hodgkin lymphomas may illuminate lymphomagenesis. We present the first systematic comparison of risks by lymphoma subtype for a broad range of putative risk factors in a population-based case-control study, including diffuse large B-cell (DLBCL; N = 416), follicular (N = 318), and marginal zone lymphomas (N = 106), and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL; N = 133). We required at least 2 of 3 analyses to support differences in risk: (1) polytomous logistic regression, (2) homogeneity tests, or (3) dichotomous logistic regression, analyzing all 7 possible pairwise comparisons among the subtypes, corresponding to various groupings by clinical behavior, genetic features, and differentiation. Late birth order and high body mass index (>/= 35) kg/m(2)) increased risk for DLBCL alone. Autoimmune conditions increased risk for marginal zone lymphoma alone. The tumor necrosis factor G-308A polymorphism (rs1800629) increased risks for both DLBCL and marginal zone lymphoma. Exposure to certain dietary heterocyclic amines from meat consumption increased risk for CLL/SLL alone. We observed no significant risk factors for follicular lymphoma alone. These data clearly support both etiologic commonality and heterogeneity for lymphoma subtypes, suggesting that immune dysfunction is of greater etiologic importance for DLBCL and marginal zone lymphoma than for CLL/SLL and follicular lymphoma.     

High expression of the bcl-x gene in Reed-Sternberg cells of Hodgkin's disease

The expression of bcl-x protein, a bcl-2-related protein present in cortical thymocytes, activated lymphocytes, and plasma cells of reactive lymph nodes, was investigated in 44 cases of Hodgkin's disease (HD) in parallel with bcl-2 and Epstein-Barr virus (EBV) status. Eighty- six percent of the cases were positive for bcl-x, among them 27% with a strong signal in more than 75% of the Reed-Sternberg cells. Positivity for bcl-x was found in, respectively, 100% and 92% of the nodular sclerosis and mixed cellularity subtypes, although 4 cases of lymphocyte predominance subtype were negative. This finding was in contrast with the weaker positivity for bcl-2 staining in 44% of the cases. EBV small RNAs were detected in 43% of the cases by using in situ hybridization. Of interest, 100% of the EBV-positive samples were positive for bcl-x, whereas only 38% of these cases were bcl-2 positive. Our findings show that the bcl-x gene expression is high in HD, suggesting that bcl-x may have a role in the pathogenesis of at least some cases of HD via apoptosis regulation.     

Association of bcl-1 rearrangements with lymphocytic lymphoma of intermediate differentiation

Previous studies using classical cytogenetics have demonstrated the presence of the t(11;14) (q13;q32) chromosomal translocation in some cases of lymphocytic lymphoma of intermediate differentiation (IDL), a distinct type of low grade B-cell lymphoma. This finding suggested that the bcl-1 region (located at band q13 of chromosome 11) might be involved in this neoplasm. Using a genomic probe from the major breakpoint area of the bcl-1 locus, we identified rearrangements of the bcl-1 region in 10 of 19 cases, 2 of which comigrated with a rearranged allele of the immunoglobulin heavy chain gene joining region. In contrast, bcl-1 rearrangements were not found in other types of low grade B-cell lymphoma, specifically in 36 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and 27 cases of follicular lymphoma (FL). To further assess the molecular pathology of IDL, we analyzed these cases for rearrangements of the bcl-2 proto-oncogene, which is associated primarily with follicular lymphomas. None of the 19 cases of IDL had rearrangements. Furthermore, none of the 36 cases of CLL/SLL showed bcl-2 rearrangements, whereas, as expected, 21 of 27 cases of FL had rearrangements of the bcl-2 locus. Our findings demonstrate an association between a rearranged bcl-1 region with approximately 50% of IDLs and suggest that abnormalities of this locus may be important in the pathogenesis of IDL.     

The bcl-2 gene is rearranged in many diffuse B-cell lymphomas

Southern blotting was used to detect rearrangement of the bcl-2 gene in 104 cases of non-Hodgkin's lymphoma subclassified by the Working Formulation, 24 cases of B cell chronic lymphocytic leukemia (B-CLL) and 14 cases of T cell malignancy. Earlier workers reported rearrangement of this gene (located on chromosome 18) in a major fraction of follicular lymphomas, lymphomas in which a 14;18 chromosome translocation is frequently observed. In the present study, bcl-2 was rearranged in 30% (11 of 37) of follicular lymphomas and 19% (11 of 58) of diffuse lymphomas of follicle center cell lineage. In 18 of 19 samples studied, the rearranged bcl-2 fragment also hybridized with a probe for the joining region of the immunoglobulin heavy chain gene located on chromosome 14, indicating a 14;18 translocation. In lymphomas not derived from follicle center cells, ie, diffuse lymphomas of small B lymphocytes, B-CLL and T cell neoplasms, the bcl-2 gene was always in germline configuration. The frequent rearrangement of bcl-2 in a variety of B cell lymphomas of diffuse morphology (small cleaved cell, large cell, small noncleaved cell and immunoblastic) is noteworthy.     

Apoptosis and its relationship with cell proliferation, p53, Waf1p21, bcl-2 and c-myc in esophageal carcinogenesis studied with a high-risk population in northern China

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Differential expression of LFA-1 molecules in non-Hodgkin's lymphoma and lymphoid leukemia

We investigated the expression of adherence molecules lymphocyte function-associated antigens-1 alpha and -beta (LFA-1 alpha, -beta) and p150, 95 in 103 well-characterized non-Hodgkin's lymphomas (NHLs) and lymphoid leukemias (LLs). We found that NHLs and LLs differentially express LFA-1 molecules according to their lineage derivation, degree of clinical aggressiveness, and anatomic site of involvement. Specifically, (a) T-cell neoplasms nearly always express these molecules; (b) diffuse aggressive B-cell NHLs and mature LLs often lack LFA-1 alpha molecules; and (c) B-cell chronic lymphocytic leukemia (CLL) is often LFA-1 alpha-negative while B-cell small lymphocytic lymphomas (SLLs) are nearly always LFA-1 alpha-positive. Furthermore, the low expression of LFA-1 alpha in CLL is related to the low degree of homotypic lymphocyte adhesion after tumor promoter antigen stimulation that does not modulate the expression of LFA-1 alpha in vitro. The differential expression of LFA-1 by B-cell CLL and SLL and their degree of homotypic lymphocyte adhesion may account for the distinct anatomic compartmentalization and characteristic clinical behavior of these two morphologically and immunologically similar lymphoid malignancies.     

低Se低VE诱导肝细胞凋亡及相关基因所起的作用

目的 研究低硒（Ｓｅ）低维生素Ｅ（ＶＥ）能否诱导大鼠肝细胞凋亡及相关基因ｐ５３、ｂｃｌ－２和ｃ－ｍｙｃ所起的作用。方法 以天然的和人工半合成的低Ｓｅ低ＶＥ饲料喂养大鼠１７周,采用末端脱氧核苷酸转移酶介导的缺口末端标记（ＴＵＮＥＬ）检测肝细胞凋亡,采用免疫组化法检测肝细胞ｐ５３、ｂｃｌ－２和ｃ－ｍｙｃ蛋白。结果 与补Ｓｅ和ＶＥ组大鼠相比,低Ｓｅ低ＶＥ组大鼠肝细胞凋亡显著增加;ｐ５３和ｃ－ｍｙｃ蛋白增     

Inverse expression of bcl-2 protein and Fas antigen in lymphoblasts in peripheral lymph nodes and activated peripheral blood T and B lymphocytes

To examine the regulatory mechanism of apoptosis in lymphoid cells, expression of both bcl-2 protein and Fas antigen was investigated in reactive lymph nodes, in resting lymphocytes from peripheral blood (PBLs), and in PBLs stimulated with pokeweed mitogen, interleukin-4 (IL- 4) + anti-IgM antibody, IL-2 + anti-CD3 antibody, phytohemagglutinin + phorbol myristate acetate using immunohistochemistry and flow cytometry. Germinal center cells expressed a large amount of Fas antigen, which is associated with the induction of apoptosis in lymphoid cell lines, in contrast to the lack of bcl-2 protein as an apoptosis inhibitor. On the other hand, mantle zone lymphocytes expressed a high level of bcl-2 protein and less Fas antigen. This inverse expression of bcl-2 protein and Fas antigen was also shown in activated T and B lymphocytes from peripheral blood. These lymphoblasts fell into apoptosis dose-dependently in the presence of anti-Fas monoclonal antibody, but resting lymphocytes that expressed both bcl-2 protein and Fas antigen did not undergo apoptosis. These findings suggest that bcl-2 expression prevents the apoptosis of lymphoid cells induced by the Fas antigen-dependent mechanism and that apoptosis of lymphocytes is exquisitely controlled, at least in part, by regulation of the bcl-2 and Fas genes.